1 00:00:00,500 --> 00:00:02,820 The following content is provided under a Creative 2 00:00:02,820 --> 00:00:04,360 Commons license. 3 00:00:04,360 --> 00:00:06,660 Your support will help MIT OpenCourseWare 4 00:00:06,660 --> 00:00:11,020 continue to offer high quality educational resources for free. 5 00:00:11,020 --> 00:00:13,650 To make a donation or view additional materials 6 00:00:13,650 --> 00:00:17,600 from hundreds of MIT courses, visit MIT OpenCourseWare 7 00:00:17,600 --> 00:00:18,550 at ocw.mit.edu. 8 00:00:25,740 --> 00:00:29,160 JOANNE STUBBE: Where we were at at the end of the last lecture 9 00:00:29,160 --> 00:00:30,810 was trying to figure out what do we 10 00:00:30,810 --> 00:00:34,580 do with the fact that cholesterol-- 11 00:00:34,580 --> 00:00:37,530 its solubility is five micromolar. 12 00:00:37,530 --> 00:00:42,670 Yet if you look inside your blood, 13 00:00:42,670 --> 00:00:45,120 the levels would be 5 millimolar. 14 00:00:45,120 --> 00:00:47,580 And so the question is, how does it gets transported? 15 00:00:47,580 --> 00:00:53,280 And it gets transported in a complex fashion. 16 00:00:53,280 --> 00:00:56,130 We need to deal with that with any kind of very 17 00:00:56,130 --> 00:00:59,430 insoluble lipophilic materials. 18 00:00:59,430 --> 00:01:04,709 And I briefly introduced you to lipoproteins, 19 00:01:04,709 --> 00:01:08,060 which are mixtures of different kinds of lipids, 20 00:01:08,060 --> 00:01:11,700 triacylglycerols, phospholipids, cholesterol, 21 00:01:11,700 --> 00:01:14,310 cholesterol esters. 22 00:01:14,310 --> 00:01:17,670 And the key question we learned in the first couple 23 00:01:17,670 --> 00:01:20,780 lectures that cholesterol could be biosynthesized. 24 00:01:20,780 --> 00:01:23,760 And what we started focusing on in the last lecture 25 00:01:23,760 --> 00:01:25,703 was that it can be taken up by the diet. 26 00:01:25,703 --> 00:01:27,120 That's what we're focusing on now. 27 00:01:27,120 --> 00:01:29,970 And then after we do a little more background, 28 00:01:29,970 --> 00:01:33,480 then how is it taken up and then how is this all regulated? 29 00:01:33,480 --> 00:01:37,560 How do you control biosynthesis versus cholesterol 30 00:01:37,560 --> 00:01:38,190 from the diet. 31 00:01:38,190 --> 00:01:39,930 What are the sort of major mechanisms? 32 00:01:39,930 --> 00:01:42,810 So at the end of the last lecture 33 00:01:42,810 --> 00:01:44,670 I'd given you a second picture. 34 00:01:44,670 --> 00:01:47,600 And the PowerPoint-- the original PowerPoint 35 00:01:47,600 --> 00:01:48,600 didn't have this figure. 36 00:01:48,600 --> 00:01:50,910 This is taken out of a new Voet and Voet-- 37 00:01:50,910 --> 00:01:53,220 the newest Voet and Voet-- which I think better 38 00:01:53,220 --> 00:01:55,003 describes what's going on. 39 00:01:55,003 --> 00:01:56,670 But really sort of what you need to know 40 00:01:56,670 --> 00:01:58,860 is you form these particles, chylomicrons, 41 00:01:58,860 --> 00:02:00,270 if you look at the handout I gave 42 00:02:00,270 --> 00:02:06,390 you have lots of proteins, all kinds of lipids, cholesterol. 43 00:02:06,390 --> 00:02:11,220 And they get into the bloodstream 44 00:02:11,220 --> 00:02:16,170 and they pass off as they go through adipocytes 45 00:02:16,170 --> 00:02:18,780 or as they go through muscle. 46 00:02:18,780 --> 00:02:23,010 The surface of these cells have lipases, phospholipases 47 00:02:23,010 --> 00:02:25,080 that can clip off the fatty acids 48 00:02:25,080 --> 00:02:28,390 that you need for metabolism at most cells. 49 00:02:28,390 --> 00:02:32,610 And what happens is the size of these particles just change. 50 00:02:32,610 --> 00:02:36,870 And so in the end, you remove the triacylglycerols 51 00:02:36,870 --> 00:02:38,880 and you remove phospholipids. 52 00:02:38,880 --> 00:02:42,600 And what you're left with is more of a cholesterol. 53 00:02:42,600 --> 00:02:47,350 And that-- and so what happens is the chylomicrons change 54 00:02:47,350 --> 00:02:47,850 size. 55 00:02:47,850 --> 00:02:49,770 They call them the remnants. 56 00:02:49,770 --> 00:02:53,700 And there are receptors on liver cells, which 57 00:02:53,700 --> 00:02:58,020 can take up these remnants, these lipoprotein remnants. 58 00:02:58,020 --> 00:03:03,080 And then they repackage them into other lipoproteins. 59 00:03:03,080 --> 00:03:05,310 And again, the differences in the lipoproteins 60 00:03:05,310 --> 00:03:08,480 we talked about very briefly, we have an outline. 61 00:03:08,480 --> 00:03:10,580 Somebody measured these with a-- 62 00:03:10,580 --> 00:03:14,640 again, they're variable, but they're based on density. 63 00:03:14,640 --> 00:03:19,980 And so the liver repackages these things 64 00:03:19,980 --> 00:03:24,090 to a particle that's very low density, lipoprotein. 65 00:03:24,090 --> 00:03:28,260 And then again, they can dump off components 66 00:03:28,260 --> 00:03:33,540 into the tissues where you can use the lipids to do 67 00:03:33,540 --> 00:03:37,590 metabolism, changing the size, intermediate density, 68 00:03:37,590 --> 00:03:40,260 eventually low density lipoprotein which 69 00:03:40,260 --> 00:03:41,610 is what we're focused on now. 70 00:03:41,610 --> 00:03:43,500 And then today what we're focused 71 00:03:43,500 --> 00:03:48,540 on is how does the low density lipoprotein get taken up 72 00:03:48,540 --> 00:03:50,100 by the liver? 73 00:03:50,100 --> 00:03:54,640 And also, can it get taken up by other kinds of cells? 74 00:03:54,640 --> 00:03:58,200 And if you have excess cholesterol produced 75 00:03:58,200 --> 00:04:01,350 in any of these extrahepatic cells, 76 00:04:01,350 --> 00:04:05,110 it can be taken up to form particles called high density 77 00:04:05,110 --> 00:04:06,600 lipoproteins. 78 00:04:06,600 --> 00:04:08,590 And they can come back. 79 00:04:08,590 --> 00:04:10,830 So they act as cholesterol scavengers, 80 00:04:10,830 --> 00:04:15,030 come back and deliver it back into the liver 81 00:04:15,030 --> 00:04:18,006 by a mechanism that is really different from what we're going 82 00:04:18,006 --> 00:04:19,089 to be talking about today. 83 00:04:19,089 --> 00:04:21,209 So that's the overview picture. 84 00:04:21,209 --> 00:04:26,700 And so what I want to do now is focus 85 00:04:26,700 --> 00:04:28,950 on the question, why do we care about cholesterol 86 00:04:28,950 --> 00:04:31,650 and what was the motivator for Brown 87 00:04:31,650 --> 00:04:38,130 and Goldstein's discovery of the low density lipoprotein 88 00:04:38,130 --> 00:04:39,130 receptor. 89 00:04:39,130 --> 00:04:44,160 So this is the motivator. 90 00:04:44,160 --> 00:04:47,550 They were seeing when they were at medical school, 91 00:04:47,550 --> 00:04:52,850 a number of children that presented at an early age. 92 00:04:52,850 --> 00:04:55,170 These guys were six and eight. 93 00:04:55,170 --> 00:04:58,020 And the way they present, if they turn out 94 00:04:58,020 --> 00:05:01,110 to have both genes, both copies of the gene 95 00:05:01,110 --> 00:05:04,200 are messed up for low density lipoprotein 96 00:05:04,200 --> 00:05:09,780 receptor, that's called familial hypercholesterolemia. 97 00:05:09,780 --> 00:05:14,730 The way they present is they have these little xanthomases 98 00:05:14,730 --> 00:05:16,620 that are apparently yellow. 99 00:05:16,620 --> 00:05:20,010 And what they are is they're full of cholesterol. 100 00:05:20,010 --> 00:05:24,600 OK, and so if you have someone that's heterozygous 101 00:05:24,600 --> 00:05:26,350 rather than homozygous-- 102 00:05:26,350 --> 00:05:28,420 these guys are homozygous-- 103 00:05:28,420 --> 00:05:30,250 you still see these but you see it 104 00:05:30,250 --> 00:05:34,940 at a much later time in their life. 105 00:05:34,940 --> 00:05:38,750 And so again, what it is, it's a function of the fact 106 00:05:38,750 --> 00:05:40,400 that you have too much cholesterol 107 00:05:40,400 --> 00:05:41,420 and this is the way-- 108 00:05:41,420 --> 00:05:44,010 one of the ways-- it manifests itself. 109 00:05:44,010 --> 00:05:45,740 The second way it manifests itself 110 00:05:45,740 --> 00:05:49,970 is if you look at the concentration of low density 111 00:05:49,970 --> 00:05:51,830 lipoprotein and the plasma, which 112 00:05:51,830 --> 00:05:56,900 is given in milligrams for 100 mils, what you see 113 00:05:56,900 --> 00:05:59,240 is the concentrations of cholesterol 114 00:05:59,240 --> 00:06:02,300 are actually 5 to 10 times higher. 115 00:06:02,300 --> 00:06:03,980 So that's the manifestation. 116 00:06:03,980 --> 00:06:08,540 And children that manifest at this early age 117 00:06:08,540 --> 00:06:13,400 die of heart attacks by the time they're 30. 118 00:06:13,400 --> 00:06:16,040 And so this was the motivator. 119 00:06:16,040 --> 00:06:19,190 They were trying to figure out what is the basis 120 00:06:19,190 --> 00:06:22,690 or bases for this disease. 121 00:06:22,690 --> 00:06:25,910 So that's what I said. 122 00:06:25,910 --> 00:06:30,410 This is a dominant effect. 123 00:06:30,410 --> 00:06:34,160 At the time, the gene or genes responsible for this 124 00:06:34,160 --> 00:06:36,320 were not known. 125 00:06:36,320 --> 00:06:40,610 It turns out from the data that I've gotten from some paper, 126 00:06:40,610 --> 00:06:43,970 one in 500 people are heterozygotes. 127 00:06:43,970 --> 00:06:46,160 That's quite prevalent, actually. 128 00:06:46,160 --> 00:06:48,200 But the ones that manifest themselves 129 00:06:48,200 --> 00:06:51,410 in this really terrible way early on 130 00:06:51,410 --> 00:06:54,650 is something like one in a million. 131 00:06:54,650 --> 00:06:58,523 And so-- but even the heterozygotes, 132 00:06:58,523 --> 00:07:00,440 Brown and Goldstein study all of these people, 133 00:07:00,440 --> 00:07:02,450 also manifest in this way. 134 00:07:02,450 --> 00:07:05,150 They have elevated cholesterol levels. 135 00:07:05,150 --> 00:07:09,350 And so this was is a huge problem. 136 00:07:09,350 --> 00:07:12,110 And so they decided they wanted to really devote 137 00:07:12,110 --> 00:07:12,980 their life to it. 138 00:07:12,980 --> 00:07:15,690 And I think they didn't know this in the beginning, 139 00:07:15,690 --> 00:07:19,850 but it's really associated with one gene. 140 00:07:19,850 --> 00:07:23,270 Most diseases are much more complicated than that. 141 00:07:23,270 --> 00:07:27,020 And so I think because of the, quote, "simplicity" unquote, 142 00:07:27,020 --> 00:07:30,140 you'll see it's not so simple, they 143 00:07:30,140 --> 00:07:31,490 were able to make progress. 144 00:07:31,490 --> 00:07:34,340 And these experiments were carried out really 145 00:07:34,340 --> 00:07:35,480 sort of in the-- 146 00:07:35,480 --> 00:07:37,920 started in the 1970s. 147 00:07:37,920 --> 00:07:43,640 So I think Brown and Goldstein-- we talked about the cholesterol 148 00:07:43,640 --> 00:07:45,800 biosynthetic pathway. 149 00:07:45,800 --> 00:07:47,630 And we talked about what was rate limiting. 150 00:07:47,630 --> 00:07:50,990 So hopefully you all know that the rate limiting step 151 00:07:50,990 --> 00:07:57,410 is the reduction of hydroxymethylglutaryl CoA down. 152 00:07:57,410 --> 00:08:00,790 So the CoA is reduced all the way down to an alcohol 153 00:08:00,790 --> 00:08:02,882 and that product is mevalonic acid. 154 00:08:02,882 --> 00:08:04,340 And if you can't remember this, you 155 00:08:04,340 --> 00:08:08,540 should pull out the biosynthetic pathway. 156 00:08:08,540 --> 00:08:11,180 And that was proposed to be by other people working 157 00:08:11,180 --> 00:08:14,030 in this field to be the rate limiting 158 00:08:14,030 --> 00:08:16,460 step in this overall process. 159 00:08:16,460 --> 00:08:19,160 And when you take an introductory course 160 00:08:19,160 --> 00:08:22,323 in biochemistry, you talk about regulation. 161 00:08:22,323 --> 00:08:23,990 I guess it depends on who's teaching it, 162 00:08:23,990 --> 00:08:26,090 how much you talk about regulation. 163 00:08:26,090 --> 00:08:29,390 But of course, one of the major mechanisms of regulation 164 00:08:29,390 --> 00:08:32,450 that's sort of easy to understand in some fashion, 165 00:08:32,450 --> 00:08:37,039 is that oftentimes the end product of a pathway 166 00:08:37,039 --> 00:08:39,830 can come back way at the beginning 167 00:08:39,830 --> 00:08:42,000 and inhibit the pathway. 168 00:08:42,000 --> 00:08:45,650 So that's called feedback inhibition. 169 00:08:45,650 --> 00:08:50,355 We saw that cholesterol biosynthesis was 30 steps. 170 00:08:50,355 --> 00:08:52,730 And if you go back and you look at the pathway, you know, 171 00:08:52,730 --> 00:08:54,250 I think this is step four or five. 172 00:08:54,250 --> 00:08:56,180 I can't remember which one it is. 173 00:08:56,180 --> 00:08:58,610 And so the model was-- 174 00:08:58,610 --> 00:09:01,528 and there was some evidence that suggested that 175 00:09:01,528 --> 00:09:03,320 from what had been done in the literature-- 176 00:09:03,320 --> 00:09:08,630 that cholesterol was potentially acting as a feedback inhibitor. 177 00:09:08,630 --> 00:09:12,800 And that's what their original working hypothesis was. 178 00:09:12,800 --> 00:09:16,430 So the hypothesis was-- 179 00:09:16,430 --> 00:09:18,470 this is how they started it out. 180 00:09:18,470 --> 00:09:22,150 And what we'll do is just look at a few experiments 181 00:09:22,150 --> 00:09:24,710 of how they were trying to test their hypothesis 182 00:09:24,710 --> 00:09:28,400 and then how they change their hypothesis to come up 183 00:09:28,400 --> 00:09:32,330 with a new model for cholesterol regulation. 184 00:09:32,330 --> 00:09:36,410 So you start out with acetyl CoA and you 185 00:09:36,410 --> 00:09:41,390 go through mevalonic acid. 186 00:09:41,390 --> 00:09:42,740 And then we get to cholesterol. 187 00:09:42,740 --> 00:09:45,740 And so the model was that-- 188 00:09:45,740 --> 00:09:51,931 this is HMG reductase-- that this was a feedback inhibitor. 189 00:09:55,160 --> 00:09:58,350 And that it inhibited by allosteric regulation. 190 00:09:58,350 --> 00:10:00,290 And that's true of many pathways. 191 00:10:00,290 --> 00:10:03,590 And often, that's one out of many mechanisms 192 00:10:03,590 --> 00:10:08,160 that are involved in regulation. 193 00:10:08,160 --> 00:10:12,110 So the first problem they faced-- 194 00:10:12,110 --> 00:10:14,030 and for those of you who want to read 195 00:10:14,030 --> 00:10:16,970 about this in more detail, the original experiments, 196 00:10:16,970 --> 00:10:21,485 I'm just going to present a few simple experiments 197 00:10:21,485 --> 00:10:23,610 and I'm going to present them in a simple way. 198 00:10:23,610 --> 00:10:26,870 OK, everything with human cells is more complicated 199 00:10:26,870 --> 00:10:28,820 than the way I'm presenting it. 200 00:10:28,820 --> 00:10:34,190 But for those of you would like to read a little bit more 201 00:10:34,190 --> 00:10:36,200 about the actual experiments, there 202 00:10:36,200 --> 00:10:40,220 are two papers that I think are particularly compelling. 203 00:10:40,220 --> 00:10:43,520 And in previous years, I've actually used these papers 204 00:10:43,520 --> 00:10:45,450 in recitation. 205 00:10:45,450 --> 00:10:46,670 OK, so this is one of them. 206 00:10:46,670 --> 00:10:49,640 I'll put the other one up later on so 207 00:10:49,640 --> 00:10:51,620 that you can look at the detail, more 208 00:10:51,620 --> 00:10:52,940 about the experimental details. 209 00:10:52,940 --> 00:10:56,420 And I think in these particular experiments, what you're 210 00:10:56,420 --> 00:11:02,690 being introduced to, which most students don't experience, 211 00:11:02,690 --> 00:11:04,700 is the fact that you have-- 212 00:11:04,700 --> 00:11:08,570 all you do with these insoluble membrane-like proteins 213 00:11:08,570 --> 00:11:10,460 and how do you deal with membrane proteins. 214 00:11:10,460 --> 00:11:13,400 Most of us-- I haven't had any experience with this at all. 215 00:11:13,400 --> 00:11:16,380 So this week's recitation, for example, 216 00:11:16,380 --> 00:11:19,580 sort of shows you what they had to go through 217 00:11:19,580 --> 00:11:22,310 to be able to answer these questions. 218 00:11:22,310 --> 00:11:23,750 And it's complicated. 219 00:11:23,750 --> 00:11:26,220 And I think reading the experimental details 220 00:11:26,220 --> 00:11:28,470 in the end, if you're going to do something like this, 221 00:11:28,470 --> 00:11:32,180 this provides a nice blueprint of how you try-- 222 00:11:32,180 --> 00:11:34,910 how you try to design experiments. 223 00:11:34,910 --> 00:11:36,620 And you'll see some of the complexity 224 00:11:36,620 --> 00:11:40,510 from the few experiments I'm just going to briefly describe. 225 00:11:40,510 --> 00:11:45,260 OK, so what they needed was a model system. 226 00:11:48,230 --> 00:11:51,860 And of course, you can't do experiments on humans. 227 00:11:51,860 --> 00:11:54,220 So what they wanted to do was have some kind 228 00:11:54,220 --> 00:11:56,680 of tissue culture system. 229 00:11:56,680 --> 00:11:58,735 So they wanted a model system. 230 00:12:01,630 --> 00:12:06,250 And there was some evidence in the literature 231 00:12:06,250 --> 00:12:13,870 that human fibroblast skin cells were actually 232 00:12:13,870 --> 00:12:16,970 able to biosynthesize cholesterol. 233 00:12:16,970 --> 00:12:19,690 So they wanted to ask the question, 234 00:12:19,690 --> 00:12:22,690 do these skin cells recapitulate what 235 00:12:22,690 --> 00:12:28,480 people had seen from the biological studies in humans? 236 00:12:28,480 --> 00:12:31,030 And so the first experiments I'll show you, 237 00:12:31,030 --> 00:12:33,280 does recapitulate that. 238 00:12:33,280 --> 00:12:34,810 It didn't have to. 239 00:12:34,810 --> 00:12:37,000 But then this became their model, 240 00:12:37,000 --> 00:12:39,640 human fibroblast cells became the model 241 00:12:39,640 --> 00:12:42,730 for which they're carrying out all of these experiments 242 00:12:42,730 --> 00:12:46,300 that we're going to very briefly look at. 243 00:12:46,300 --> 00:12:50,440 OK, so the experiments, I think, are simple, 244 00:12:50,440 --> 00:12:51,550 at least on the surface. 245 00:12:51,550 --> 00:12:53,590 Although I think it wasn't so easy to figure out 246 00:12:53,590 --> 00:12:55,810 how to do these experiments. 247 00:12:55,810 --> 00:13:00,890 So what they wanted to do, they had patients-- 248 00:13:00,890 --> 00:13:01,390 whoops. 249 00:13:01,390 --> 00:13:02,490 I didn't want to do that. 250 00:13:02,490 --> 00:13:05,960 Anyhow, sorry I'm wasting time. 251 00:13:05,960 --> 00:13:08,650 OK, this patient is JD. 252 00:13:08,650 --> 00:13:11,390 And all of the experiments I'm going to show you is JD. 253 00:13:11,390 --> 00:13:14,650 But they had 25 other patients. 254 00:13:14,650 --> 00:13:17,830 And what you'll see is they all manifest themselves 255 00:13:17,830 --> 00:13:18,955 in different ways. 256 00:13:18,955 --> 00:13:20,830 And we're going to see that that, in the end, 257 00:13:20,830 --> 00:13:25,120 becomes important in sorting out really what was going on. 258 00:13:29,050 --> 00:13:31,580 OK, so the first set of experiments they did 259 00:13:31,580 --> 00:13:32,690 was the following. 260 00:13:32,690 --> 00:13:35,690 So they had some kind of normal control. 261 00:13:35,690 --> 00:13:37,310 And then so we have a normal-- 262 00:13:39,860 --> 00:13:42,440 so we have skin cells from a normal person. 263 00:13:42,440 --> 00:13:45,620 And this is the control. 264 00:13:45,620 --> 00:13:53,070 And then you have the FH patient, JD. 265 00:13:53,070 --> 00:13:55,170 And in the two papers I'm going to reference, 266 00:13:55,170 --> 00:14:01,950 they did a lot of experiments on JD's fibroblasts. 267 00:14:01,950 --> 00:14:05,960 And so they did some simple experiments. 268 00:14:05,960 --> 00:14:08,280 And remember, the rate limiting step 269 00:14:08,280 --> 00:14:13,620 is proposed to be hydroxymethyl-- 270 00:14:13,620 --> 00:14:16,320 HMG CoA reductase. 271 00:14:16,320 --> 00:14:18,480 And so they wanted to first ask what 272 00:14:18,480 --> 00:14:22,470 happens if you treat the cells, so you have them growing. 273 00:14:22,470 --> 00:14:28,320 OK, and you let them grow for a certain period of days. 274 00:14:28,320 --> 00:14:31,020 And then what you do is you take the media, 275 00:14:31,020 --> 00:14:35,460 change it, and remove low density lipoproteins 276 00:14:35,460 --> 00:14:36,278 from the media. 277 00:14:36,278 --> 00:14:38,070 I don't know whether they removed them all. 278 00:14:38,070 --> 00:14:39,600 They said they removed 5%. 279 00:14:39,600 --> 00:14:42,277 I don't know what the percent that was there. 280 00:14:42,277 --> 00:14:43,860 And so we're going to do that for both 281 00:14:43,860 --> 00:14:45,960 the experiment and the control. 282 00:14:45,960 --> 00:14:48,880 So this is the experiment. 283 00:14:48,880 --> 00:14:50,940 This is the normal person. 284 00:14:50,940 --> 00:14:55,080 This is the experiment, the FH patient. 285 00:14:55,080 --> 00:14:59,040 And if you look at the axis in measuring HMG CoA 286 00:14:59,040 --> 00:15:00,660 reductase activity. 287 00:15:00,660 --> 00:15:06,240 So what they're going to do is look at plus or minus LDL. 288 00:15:06,240 --> 00:15:11,130 So in this panel, they've removed the LDL, OK? 289 00:15:11,130 --> 00:15:15,180 And if we remove the LDL, you remove the cholesterol, 290 00:15:15,180 --> 00:15:21,180 what might you expect to happen to the normal HMG CoA reductase 291 00:15:21,180 --> 00:15:23,890 levels or activities? 292 00:15:23,890 --> 00:15:28,240 If you remove the cholesterol from the plasma, 293 00:15:28,240 --> 00:15:31,907 what might you expect to happen to the activity? 294 00:15:31,907 --> 00:15:32,990 What would you want to do? 295 00:15:32,990 --> 00:15:34,198 Would you want to turn it on? 296 00:15:34,198 --> 00:15:35,570 Would you want to turn it off? 297 00:15:35,570 --> 00:15:35,990 STUDENT: Turn on. 298 00:15:35,990 --> 00:15:37,400 JOANNE STUBBE: Turn it on, right. 299 00:15:37,400 --> 00:15:40,530 So that's what they're going to be assaying. 300 00:15:40,530 --> 00:15:44,440 They remove it and if you look at the normal patient, 301 00:15:44,440 --> 00:15:46,190 the normal control, what's going to happen 302 00:15:46,190 --> 00:15:50,000 is the biosynthesis is turned on. 303 00:15:50,000 --> 00:15:53,630 So it'll look at this, then, you need to have-- 304 00:15:53,630 --> 00:16:01,250 and this goes back to the things we've talked about a little bit 305 00:16:01,250 --> 00:16:02,600 about in class-- 306 00:16:02,600 --> 00:16:04,790 and in fact, the original recitation 307 00:16:04,790 --> 00:16:10,120 that we had on radioactivity was completely 308 00:16:10,120 --> 00:16:13,330 focused on Brown and Goldstein's work. 309 00:16:13,330 --> 00:16:16,330 So we're going to see that they use a lot of radioactivity 310 00:16:16,330 --> 00:16:19,030 and all the assays I'm going to be describing today. 311 00:16:19,030 --> 00:16:23,680 So what we're going to be doing is revisiting radioisotopes. 312 00:16:23,680 --> 00:16:27,130 They couldn't have done that without these radioisotopes. 313 00:16:27,130 --> 00:16:32,620 And this is converted to this. 314 00:16:32,620 --> 00:16:36,740 OK, what's the cofactor for this reaction? 315 00:16:36,740 --> 00:16:38,670 So, I'm not going to draw up the rest of this. 316 00:16:38,670 --> 00:16:41,700 This is mevalonic acid. 317 00:16:41,700 --> 00:16:43,635 What's the cofactor required for this process? 318 00:16:52,300 --> 00:16:54,340 Any DPH. 319 00:16:54,340 --> 00:16:56,810 So you have any DPH. 320 00:16:56,810 --> 00:16:58,420 OK, so how would you assay this? 321 00:17:02,540 --> 00:17:05,050 So we're doing this now in tissue culture systems. 322 00:17:05,050 --> 00:17:07,750 That's what-- we are doing this in fibroblast cells in tissue 323 00:17:07,750 --> 00:17:09,099 culture. 324 00:17:09,099 --> 00:17:10,630 So we don't have very much material. 325 00:17:10,630 --> 00:17:12,730 You might have a plateful of cells. 326 00:17:12,730 --> 00:17:15,026 How would you do the assay? 327 00:17:15,026 --> 00:17:17,109 So this is the first thing you have to figure out. 328 00:17:17,109 --> 00:17:19,359 And I would say, almost everything in this class, 329 00:17:19,359 --> 00:17:20,859 when you're studying the biology, 330 00:17:20,859 --> 00:17:24,339 first thing you have to do is figure out a robust assay. 331 00:17:24,339 --> 00:17:26,630 This case, I think it turned out to be quite easy. 332 00:17:26,630 --> 00:17:29,482 But it's not necessarily easy in many cases. 333 00:17:29,482 --> 00:17:30,940 So this is something, as a chemist, 334 00:17:30,940 --> 00:17:33,080 you bring a lot to the table. 335 00:17:33,080 --> 00:17:33,628 Yeah? 336 00:17:33,628 --> 00:17:35,170 STUDENT: You would measure the change 337 00:17:35,170 --> 00:17:38,740 in the absorption at 340. 338 00:17:38,740 --> 00:17:39,820 JOANNE STUBBE: 340. 339 00:17:39,820 --> 00:17:41,890 So that's the way chemists would do that. 340 00:17:41,890 --> 00:17:43,560 Why can't you do that here? 341 00:17:43,560 --> 00:17:47,180 STUDENT: You have to isolate the HMG CoA reductase 342 00:17:47,180 --> 00:17:49,270 or somehow be able to parse it from everything-- 343 00:17:49,270 --> 00:17:50,645 JOANNE STUBBE: Well, you might be 344 00:17:50,645 --> 00:17:53,380 able to do it in crude extracts if you had a lot of it. 345 00:17:53,380 --> 00:17:54,520 But it's tough. 346 00:17:54,520 --> 00:17:59,070 NADPH is used in hundreds of reactions. 347 00:17:59,070 --> 00:18:04,210 It's a great assay because the absorption change is removed 348 00:18:04,210 --> 00:18:06,190 from where most of the material absorbs, 349 00:18:06,190 --> 00:18:10,540 which is, you know, 280, 260, 280. 350 00:18:10,540 --> 00:18:11,620 It's not that sensitive. 351 00:18:11,620 --> 00:18:16,690 The extinction coefficient is 6,300 molar inverse centimeter 352 00:18:16,690 --> 00:18:17,622 inverse. 353 00:18:17,622 --> 00:18:19,330 And the bottom line is if you look at it, 354 00:18:19,330 --> 00:18:21,130 it's nowhere near sensitive enough. 355 00:18:21,130 --> 00:18:24,250 So if it's not sensitive enough, then what do you need to go to? 356 00:18:27,150 --> 00:18:28,680 That's what-- the radioactivity. 357 00:18:28,680 --> 00:18:31,340 So what you're going to be doing here is-- 358 00:18:31,340 --> 00:18:34,110 so you could use either 14c-- 359 00:18:34,110 --> 00:18:38,130 hopefully you remember that's a beta emitter, which then gets 360 00:18:38,130 --> 00:18:40,710 converted into mevalonic acid. 361 00:18:40,710 --> 00:18:44,400 And then you need a way of separating starting material 362 00:18:44,400 --> 00:18:46,350 from products. 363 00:18:46,350 --> 00:18:48,340 And there are many ways that one could do that. 364 00:18:48,340 --> 00:18:52,890 But in the original paper, they use TLC. 365 00:18:52,890 --> 00:18:55,620 And that's how they monitored their reactions. 366 00:18:55,620 --> 00:18:58,550 And you need to have material that's 367 00:18:58,550 --> 00:19:02,693 of hot enough radioactivity so you can see these 368 00:19:02,693 --> 00:19:03,360 into conversion. 369 00:19:03,360 --> 00:19:07,920 So that's the assay that they used. 370 00:19:07,920 --> 00:19:15,090 And so in the PowerPoint, I decided not to draw out. 371 00:19:15,090 --> 00:19:21,000 So if you PowerPoint, you look at the data, what do you see? 372 00:19:21,000 --> 00:19:25,800 What you see is that if you look at the experiment 373 00:19:25,800 --> 00:19:28,980 where they removed the low density 374 00:19:28,980 --> 00:19:31,300 lipoprotein from the media-- 375 00:19:31,300 --> 00:19:32,560 so they've taken it out. 376 00:19:32,560 --> 00:19:35,260 They've grown the cells they have HMG CoA 377 00:19:35,260 --> 00:19:36,870 reductase activity. 378 00:19:36,870 --> 00:19:39,250 What do you see immediately-- 379 00:19:39,250 --> 00:19:44,930 and the control and the patient's cells 380 00:19:44,930 --> 00:19:47,740 are growing exactly the same way. 381 00:19:47,740 --> 00:19:50,230 What do you see immediately? 382 00:19:50,230 --> 00:19:53,490 You see a huge difference in the amount of activity. 383 00:19:53,490 --> 00:19:55,430 So this is 2. 384 00:19:55,430 --> 00:19:57,720 This 150 or something. 385 00:19:57,720 --> 00:20:02,370 And so there could be a number of reasons for all of that. 386 00:20:02,370 --> 00:20:05,370 And so the question is, what is the basis for this increase 387 00:20:05,370 --> 00:20:08,130 in activity due to increased huge amount, 388 00:20:08,130 --> 00:20:10,100 the amount of HMG CoA reductase. 389 00:20:10,100 --> 00:20:11,150 Has the activity changed? 390 00:20:11,150 --> 00:20:13,830 Is there a mutation that changes the activity? 391 00:20:13,830 --> 00:20:16,680 There are lots of explanations. 392 00:20:16,680 --> 00:20:19,920 And so what they then did, when they remove this, 393 00:20:19,920 --> 00:20:23,580 they started doing assays over 24 hours. 394 00:20:23,580 --> 00:20:28,140 And they crack open the cells and do this radioactive assay. 395 00:20:28,140 --> 00:20:33,180 And then they looked at the rate of formation of mevalonic acid. 396 00:20:33,180 --> 00:20:38,860 And so what do you see with the normal control? 397 00:20:38,860 --> 00:20:40,920 You see exactly what you might predict. 398 00:20:40,920 --> 00:20:42,990 So if the cholesterol levels become low, 399 00:20:42,990 --> 00:20:46,620 you might want to biosynthesize it. 400 00:20:46,620 --> 00:20:53,070 But then what do you see with a homozygote, the JD patient? 401 00:20:53,070 --> 00:20:55,710 What you see is the levels start out 402 00:20:55,710 --> 00:21:00,450 high you have complete absence of regulation 403 00:21:00,450 --> 00:21:02,427 by changing the concentration of cholesterol. 404 00:21:02,427 --> 00:21:03,510 That's what you're seeing. 405 00:21:03,510 --> 00:21:05,520 So it seems like a simple experiment. 406 00:21:05,520 --> 00:21:08,410 It is a simple experiment. 407 00:21:08,410 --> 00:21:12,390 The basis for these observations is still open to debate. 408 00:21:12,390 --> 00:21:16,030 But the experiment turned out to be straightforward. 409 00:21:16,030 --> 00:21:19,810 Then what they did is at 24 hours, 410 00:21:19,810 --> 00:21:25,240 they then started adding low density lipoprotein back 411 00:21:25,240 --> 00:21:28,210 into the media. 412 00:21:28,210 --> 00:21:31,060 So they start over here, they removed it. 413 00:21:31,060 --> 00:21:32,580 They add it back. 414 00:21:32,580 --> 00:21:33,850 Here's with non. 415 00:21:33,850 --> 00:21:36,070 Here's with two micrograms per mL. 416 00:21:36,070 --> 00:21:38,170 Here's with 20 micrograms per mL. 417 00:21:38,170 --> 00:21:41,500 And what do you see with a normal patient? 418 00:21:41,500 --> 00:21:42,610 With a normal control? 419 00:21:42,610 --> 00:21:47,040 What you see with the normal control is a loss of activity. 420 00:21:47,040 --> 00:21:51,600 So that's exactly what you would expect that cholesterol-- you 421 00:21:51,600 --> 00:21:54,930 have a lot of cholesterol, you don't need to make it anymore. 422 00:21:54,930 --> 00:21:58,410 So this data, then, this simple data told you-- 423 00:21:58,410 --> 00:22:05,890 the control told you that minus LDL, 424 00:22:05,890 --> 00:22:13,210 you increased HMGR activity. 425 00:22:16,630 --> 00:22:24,390 And plus LDL, you decreased activity. 426 00:22:24,390 --> 00:22:26,710 And what about the patient? 427 00:22:26,710 --> 00:22:30,400 The FH JD patient? 428 00:22:30,400 --> 00:22:36,310 So here what you see is that removing cholesterol 429 00:22:36,310 --> 00:22:38,600 from the plasma has no effect. 430 00:22:38,600 --> 00:22:40,570 What about adding it back? 431 00:22:40,570 --> 00:22:42,400 Has no effect. 432 00:22:42,400 --> 00:22:46,470 So somehow the patient is-- 433 00:22:46,470 --> 00:22:50,200 the patient's cells is oblivious to the presence or absence 434 00:22:50,200 --> 00:22:51,800 of cholesterol. 435 00:22:51,800 --> 00:22:59,140 So in this case, plus or minus LDL had no effect. 436 00:23:02,460 --> 00:23:09,380 So we say loss of cholesterol regulation, which 437 00:23:09,380 --> 00:23:12,890 could be due to feedback inhibition, 438 00:23:12,890 --> 00:23:14,390 it could be due to something else. 439 00:23:14,390 --> 00:23:18,060 We'll see it is due to something else. 440 00:23:18,060 --> 00:23:21,050 And so this was consistent with what they predicted. 441 00:23:21,050 --> 00:23:25,700 And they furthermore learned that these fibroblast cells 442 00:23:25,700 --> 00:23:28,550 might be a good model for actually studying 443 00:23:28,550 --> 00:23:29,810 what's going on in the liver. 444 00:23:29,810 --> 00:23:31,268 I mean, you always have this issue. 445 00:23:31,268 --> 00:23:34,130 You have to figure out what you can study as a model system 446 00:23:34,130 --> 00:23:36,320 since we don't work on the humans. 447 00:23:36,320 --> 00:23:38,630 And so you always have to worry about how 448 00:23:38,630 --> 00:23:42,110 that extrapolates to humans. 449 00:23:42,110 --> 00:23:51,390 So basically, you're looking at cholesterol in the media. 450 00:23:51,390 --> 00:23:55,050 You're looking at cholesterol not in the media. 451 00:23:55,050 --> 00:23:58,720 And these are the experiments we just described. 452 00:23:58,720 --> 00:24:01,350 And so one of the questions you can ask, 453 00:24:01,350 --> 00:24:06,990 then, is what happens now? 454 00:24:06,990 --> 00:24:09,900 Another thing that can happen is what if cholesterol 455 00:24:09,900 --> 00:24:11,590 can't get into the cell? 456 00:24:11,590 --> 00:24:14,850 So what they did is another experiment where they-- 457 00:24:14,850 --> 00:24:18,300 they did two things to look at the HMG CoA reductase 458 00:24:18,300 --> 00:24:25,620 activity in the normal control and in the FH patient. 459 00:24:25,620 --> 00:24:29,010 And one of them was they repeated this experiment 460 00:24:29,010 --> 00:24:30,810 in the presence of ethanol, where 461 00:24:30,810 --> 00:24:32,550 they dissolved the cholesterol. 462 00:24:32,550 --> 00:24:35,700 And apparently that allows the cholesterol 463 00:24:35,700 --> 00:24:37,730 to get across the membrane. 464 00:24:37,730 --> 00:24:39,900 OK, so we're bypassing what we now 465 00:24:39,900 --> 00:24:42,030 know is going to be a receptor. 466 00:24:42,030 --> 00:24:47,640 So they did a second experiment and they 467 00:24:47,640 --> 00:24:52,330 used ethanol cholesterol. 468 00:24:52,330 --> 00:24:54,100 And it goes across membrane. 469 00:24:58,260 --> 00:25:02,340 And then they looked at the HMG CoA reductase activity. 470 00:25:02,340 --> 00:25:06,660 And the activity of both the patient and the normal controls 471 00:25:06,660 --> 00:25:07,980 was the same. 472 00:25:07,980 --> 00:25:18,360 OK, so the activity, HMGR activity the same. 473 00:25:18,360 --> 00:25:21,030 They don't report the details of this experiment. 474 00:25:21,030 --> 00:25:23,160 But another way you could do this is you 475 00:25:23,160 --> 00:25:26,610 could pull out the protein or partially purify 476 00:25:26,610 --> 00:25:28,920 the protein in crude extracts and try to measure 477 00:25:28,920 --> 00:25:31,800 the activity using this assay. 478 00:25:31,800 --> 00:25:35,520 And if you have a good measure of the amount of protein, which 479 00:25:35,520 --> 00:25:38,780 is key, so you can measure specific activity, 480 00:25:38,780 --> 00:25:41,160 micromoles of product or nanomoles of product produced 481 00:25:41,160 --> 00:25:43,890 per minute per milligram of protein, 482 00:25:43,890 --> 00:25:49,140 you could actually see that the HMG CoA reductase 483 00:25:49,140 --> 00:25:55,850 activity was the same in the wild type in the normal 484 00:25:55,850 --> 00:25:56,700 and in the patient. 485 00:25:56,700 --> 00:26:02,770 So you could also measure this using assay. 486 00:26:02,770 --> 00:26:06,690 And again, the result was that they 487 00:26:06,690 --> 00:26:10,110 were the same in both the normal and the patient. 488 00:26:10,110 --> 00:26:12,960 So then the elevated levels could be-- 489 00:26:12,960 --> 00:26:15,895 elevated levels, you saw in the very beginning of the HMG CoA 490 00:26:15,895 --> 00:26:17,770 reductase activity, could be due to the fact, 491 00:26:17,770 --> 00:26:20,040 they had a huge amount of protein, 492 00:26:20,040 --> 00:26:22,440 more so than you do with the fibroblasts. 493 00:26:22,440 --> 00:26:25,120 And so, there's no reason to think a prior 494 00:26:25,120 --> 00:26:29,430 if you looked at that previous slide, that the control, 495 00:26:29,430 --> 00:26:31,080 that normal control in the wild type-- 496 00:26:31,080 --> 00:26:34,200 I don't know what the scatter is in the data for HMG reductase 497 00:26:34,200 --> 00:26:36,730 activities, but that's something you need to think about. 498 00:26:36,730 --> 00:26:40,320 But a 60-fold change is a huge change. 499 00:26:40,320 --> 00:26:43,290 So this data, the initial set of data 500 00:26:43,290 --> 00:26:46,320 said that, yeah, cholesterol may be 501 00:26:46,320 --> 00:26:48,450 acting as a feedback inhibitor. 502 00:26:48,450 --> 00:26:50,820 But here, we can get cholesterol into the cell 503 00:26:50,820 --> 00:26:52,680 and the activities are the same. 504 00:26:52,680 --> 00:26:57,980 So they needed to come up with an alternative hypothesis. 505 00:26:57,980 --> 00:27:03,120 OK, so they then, using these two sets of data, 506 00:27:03,120 --> 00:27:07,780 came up with an alternative hypothesis. 507 00:27:07,780 --> 00:27:18,250 So they concluded that it's not cholesterol feed back 508 00:27:18,250 --> 00:27:19,030 regulated. 509 00:27:22,310 --> 00:27:27,000 And so then they set out to do a second set of experiments 510 00:27:27,000 --> 00:27:28,810 based on a new hypothesis. 511 00:27:34,220 --> 00:27:36,900 And the new hypothesis is that there 512 00:27:36,900 --> 00:27:40,530 would be some protein that might be involved 513 00:27:40,530 --> 00:27:44,540 in taking up the LDL particle, which has 514 00:27:44,540 --> 00:27:46,420 a cholesterol into the cell. 515 00:27:46,420 --> 00:27:51,900 So the new hypothesis was there is an LDL receptor, 516 00:27:51,900 --> 00:27:53,340 so r is receptor. 517 00:27:53,340 --> 00:27:56,760 That's how I'm going to abbreviate it. 518 00:27:56,760 --> 00:28:07,980 That's key to taking up LDL. 519 00:28:07,980 --> 00:28:12,120 And so that's what's shown here. 520 00:28:12,120 --> 00:28:16,200 And so then the question is, what sets of experiments 521 00:28:16,200 --> 00:28:17,110 do they do next. 522 00:28:17,110 --> 00:28:20,880 So this is a second set of experiments 523 00:28:20,880 --> 00:28:25,620 that was done in a paper that's also quite interesting. 524 00:28:25,620 --> 00:28:27,180 And so, for those of you who want 525 00:28:27,180 --> 00:28:39,150 to look at the details of this, this was published in 1976. 526 00:28:39,150 --> 00:28:41,730 And so this is where the data that I'm going to show you 527 00:28:41,730 --> 00:28:43,260 on this slide came from. 528 00:28:43,260 --> 00:28:46,590 Because I think they actually put it in one of the two review 529 00:28:46,590 --> 00:28:48,230 articles I gave you to read. 530 00:28:48,230 --> 00:28:49,980 But if you want to read the original data, 531 00:28:49,980 --> 00:28:51,510 the papers aren't that long. 532 00:28:51,510 --> 00:28:54,150 And they go through the details of the rationale of how 533 00:28:54,150 --> 00:28:57,030 they design their experiments. 534 00:28:57,030 --> 00:28:59,910 OK, so what we want to do now is test 535 00:28:59,910 --> 00:29:05,840 the idea that to get cholesterol into the cell, 536 00:29:05,840 --> 00:29:07,965 there is an LDL receptor. 537 00:29:12,290 --> 00:29:15,380 And that that's going to play a key role in controlling 538 00:29:15,380 --> 00:29:16,400 cholesterol levels. 539 00:29:16,400 --> 00:29:18,340 That was the working hypothesis. 540 00:29:18,340 --> 00:29:22,350 OK, so how would you go about testing this experimentally? 541 00:29:22,350 --> 00:29:26,180 So these are the results of the experiments. 542 00:29:26,180 --> 00:29:28,460 And the question is, how would you 543 00:29:28,460 --> 00:29:30,650 go about testing this experimentally 544 00:29:30,650 --> 00:29:33,030 if this were your hypothesis? 545 00:29:33,030 --> 00:29:38,150 And so if you think about it, you might like to know, 546 00:29:38,150 --> 00:29:42,050 does the LDL particle bind to the surface of the cell? 547 00:29:42,050 --> 00:29:42,980 Does it bind? 548 00:29:42,980 --> 00:29:46,130 OK, so that would be one thing you could do. 549 00:29:46,130 --> 00:29:48,920 And in fact, Brown and Goldstein were 550 00:29:48,920 --> 00:29:50,330 treating many, many patients. 551 00:29:50,330 --> 00:29:54,320 So they had fibroblasts for many patients, 20 to 25 patients. 552 00:29:54,320 --> 00:29:56,300 They all had different phenotypes. 553 00:29:56,300 --> 00:29:57,710 And again, these were differences 554 00:29:57,710 --> 00:29:59,870 in the phenotypes actually helped them 555 00:29:59,870 --> 00:30:03,380 to try to dissect this process. 556 00:30:03,380 --> 00:30:06,290 And so could it bind? 557 00:30:06,290 --> 00:30:08,922 And so we can ask the question, how would we look at binding? 558 00:30:08,922 --> 00:30:10,380 I'm going to ask you that question. 559 00:30:10,380 --> 00:30:12,380 We're going to have a recitation on binding, 560 00:30:12,380 --> 00:30:16,550 I think, not this week, but next week. 561 00:30:16,550 --> 00:30:19,520 Then it gets into the cell. 562 00:30:19,520 --> 00:30:21,530 OK, so how do you know it gets into the cell? 563 00:30:24,270 --> 00:30:26,190 And so that's another question. 564 00:30:26,190 --> 00:30:27,720 Inside, outside. 565 00:30:27,720 --> 00:30:30,240 And then the next question is, what is LDL? 566 00:30:30,240 --> 00:30:33,360 Hopefully you remember it's a lipoprotein that has 567 00:30:33,360 --> 00:30:36,360 a single protein on it, apoB. 568 00:30:36,360 --> 00:30:39,320 And then it's full with cholesterol, cholesterol 569 00:30:39,320 --> 00:30:41,400 esters, and phospholipids. 570 00:30:41,400 --> 00:30:44,610 What happens to that stuff once it's inside the cell? 571 00:30:44,610 --> 00:30:47,760 OK, so those are the questions in this experiment 572 00:30:47,760 --> 00:30:49,650 that they set out to ask. 573 00:30:49,650 --> 00:30:53,070 OK, so what I want to do-- 574 00:30:53,070 --> 00:31:10,160 so binding, internalization, and then 575 00:31:10,160 --> 00:31:15,600 the fate of LDL inside the cell. 576 00:31:15,600 --> 00:31:18,050 So that's what they were focused on. 577 00:31:18,050 --> 00:31:23,270 So what I want to do is show you the tools 578 00:31:23,270 --> 00:31:26,630 that they developed to try to answer these questions. 579 00:31:26,630 --> 00:31:28,760 OK, I'm going to show you a few things because this 580 00:31:28,760 --> 00:31:32,100 isn't such an easy set of experiments to carry out. 581 00:31:32,100 --> 00:31:34,970 And then what they observed on the normal cells 582 00:31:34,970 --> 00:31:37,110 and the patient cells. 583 00:31:37,110 --> 00:31:44,660 OK, so the tools that I want to talk about are the following. 584 00:31:44,660 --> 00:31:48,440 OK, so we just talked about the fact that, to do the assay, 585 00:31:48,440 --> 00:31:50,240 we needed radioactivity. 586 00:31:50,240 --> 00:31:52,910 We needed to be sensitive enough. 587 00:31:52,910 --> 00:31:56,108 If you're going to be looking at binding on the surface, 588 00:31:56,108 --> 00:31:56,900 how do you do that? 589 00:31:56,900 --> 00:31:58,670 Do you think there are a lot of receptors? 590 00:31:58,670 --> 00:32:00,980 Are there a few receptors? 591 00:32:00,980 --> 00:32:02,370 So you might not know that. 592 00:32:02,370 --> 00:32:05,700 But in general, there aren't huge numbers of receptors. 593 00:32:05,700 --> 00:32:08,840 So measuring binding to the surface of the cell usually 594 00:32:08,840 --> 00:32:11,510 requires a very sensitive assay. 595 00:32:11,510 --> 00:32:14,060 So the first thing they needed to do 596 00:32:14,060 --> 00:32:16,340 was they decided that they needed 597 00:32:16,340 --> 00:32:19,910 to make the LDL radial labels. 598 00:32:19,910 --> 00:32:22,220 And if you go back and you look through your notes 599 00:32:22,220 --> 00:32:26,660 in recitation three where we talked about radioactivity, 600 00:32:26,660 --> 00:32:29,780 we saw that we have beta, c14 beta, which 601 00:32:29,780 --> 00:32:31,070 is what they used up there. 602 00:32:31,070 --> 00:32:35,810 But they also used i125, which is a gamma, which 603 00:32:35,810 --> 00:32:37,590 is much more sensitive. 604 00:32:37,590 --> 00:32:40,670 And so what they decided they needed to make 605 00:32:40,670 --> 00:32:44,630 was i125 labeled LDL. 606 00:32:44,630 --> 00:32:46,970 So if you haven't radio labeled, can you 607 00:32:46,970 --> 00:32:50,240 somehow see it sitting on the surface of the cell? 608 00:32:50,240 --> 00:32:53,150 So the question is, how can you do that? 609 00:32:53,150 --> 00:32:54,980 Well, we talked about the composition 610 00:32:54,980 --> 00:32:58,070 of the LDL particle. 611 00:32:58,070 --> 00:32:59,710 There is cholesterol. 612 00:32:59,710 --> 00:33:04,010 There's cholesterol esters, phospholipids, and one protein. 613 00:33:04,010 --> 00:33:06,830 And so what they're doing to put the iodine in 614 00:33:06,830 --> 00:33:09,970 is putting it into only the protein. 615 00:33:09,970 --> 00:33:15,110 OK, so what they use is a method called 616 00:33:15,110 --> 00:33:23,120 Bolton Hunter, which uses radial level iodide and a reagent. 617 00:33:23,120 --> 00:33:24,502 I'm not going to go through-- 618 00:33:24,502 --> 00:33:26,210 you can look it up if you're interested-- 619 00:33:26,210 --> 00:33:28,310 I'm not going to go through the details. 620 00:33:28,310 --> 00:33:32,430 And what it does is it takes a protein-- 621 00:33:32,430 --> 00:33:34,950 this is still actually widely used. 622 00:33:34,950 --> 00:33:35,890 So this would be apoB. 623 00:33:38,910 --> 00:33:43,070 And it iodinates at the ortho position. 624 00:33:43,070 --> 00:33:53,960 So what you end up with, then, is iodinated apoB. 625 00:33:53,960 --> 00:33:56,120 So that's going to be your handle. 626 00:33:56,120 --> 00:34:00,560 You can make this a very high, specific activity. 627 00:34:00,560 --> 00:34:03,140 OK, so that's one thing that they needed to do. 628 00:34:03,140 --> 00:34:06,860 OK, the second thing that they needed to do 629 00:34:06,860 --> 00:34:11,588 is if they're going to look for binding to the surface, 630 00:34:11,588 --> 00:34:13,130 how would you design that experiment? 631 00:34:13,130 --> 00:34:15,830 What might you need to do to figure out 632 00:34:15,830 --> 00:34:19,280 how you're going to look at binding only and not 633 00:34:19,280 --> 00:34:22,710 binding and uptake? 634 00:34:22,710 --> 00:34:26,580 What parameter could you change that would help you do that? 635 00:34:26,580 --> 00:34:27,150 Temperature. 636 00:34:27,150 --> 00:34:29,850 So everything-- and you'll see this also 637 00:34:29,850 --> 00:34:34,420 in experiments this week-- the temperature is really critical. 638 00:34:34,420 --> 00:34:34,920 Why? 639 00:34:34,920 --> 00:34:39,389 Because hopefully you all know lipid bilayers are very fluid. 640 00:34:39,389 --> 00:34:40,889 And if you cool the temperature, you 641 00:34:40,889 --> 00:34:42,940 prevent uptake and other things. 642 00:34:42,940 --> 00:34:44,190 You have to test all this out. 643 00:34:44,190 --> 00:34:46,750 They did a huge number of controls. 644 00:34:46,750 --> 00:34:49,400 So the second thing that they wanted to do 645 00:34:49,400 --> 00:34:52,900 is they used temperature. 646 00:34:52,900 --> 00:34:58,110 So four degrees, they're going to use to look at binding. 647 00:34:58,110 --> 00:35:00,750 Or if they're looking at a time course and they 648 00:35:00,750 --> 00:35:03,720 want to stop the reaction, and the reaction is normally 649 00:35:03,720 --> 00:35:05,420 done at 37 degrees-- 650 00:35:05,420 --> 00:35:14,280 so uptake experiments would be at 37 degrees. 651 00:35:14,280 --> 00:35:17,700 OK, so again, temperature is the key parameter. 652 00:35:17,700 --> 00:35:19,440 You could, if you wanted to a time course 653 00:35:19,440 --> 00:35:22,620 and stop the reaction, you could cool with down to four degrees. 654 00:35:22,620 --> 00:35:27,720 I mean, this was a hypothesis they had. 655 00:35:27,720 --> 00:35:33,210 And so that's the second tool that they're going to use. 656 00:35:33,210 --> 00:35:37,380 And the third tool, which I think isn't necessarily so 657 00:35:37,380 --> 00:35:40,440 intuitive, is if you're looking at something binding 658 00:35:40,440 --> 00:35:42,540 on the surface, you have to always worry 659 00:35:42,540 --> 00:35:44,430 about non-specific binding. 660 00:35:44,430 --> 00:35:46,710 You'll talk about that in the recitation. 661 00:35:46,710 --> 00:35:48,560 On this that's always a problem. 662 00:35:48,560 --> 00:35:51,130 You're using really hot, iodine-labeled materials, 663 00:35:51,130 --> 00:35:53,670 so you could get neuron specific binding. 664 00:35:53,670 --> 00:35:56,280 And so how do you-- so you need to wash it. 665 00:35:56,280 --> 00:36:03,300 So if the LDL particle bound loosely to the LDL receptor, 666 00:36:03,300 --> 00:36:05,220 that makes the problem extremely challenging 667 00:36:05,220 --> 00:36:08,250 because when you're trying to wash away the excess as you 668 00:36:08,250 --> 00:36:10,860 change the concentration of the LDL, 669 00:36:10,860 --> 00:36:12,152 you're going to start to lose-- 670 00:36:12,152 --> 00:36:13,610 you're going to have an equilibrium 671 00:36:13,610 --> 00:36:15,830 and you're going to start to lose binding. 672 00:36:15,830 --> 00:36:17,960 It binds really tightly so they had 673 00:36:17,960 --> 00:36:19,330 to have some kind of a wash. 674 00:36:19,330 --> 00:36:21,670 So they figured out and optimized a wash. 675 00:36:21,670 --> 00:36:22,795 So you need to have a wash. 676 00:36:25,470 --> 00:36:28,950 So if you have a wash and then you're 677 00:36:28,950 --> 00:36:34,290 still looking at the receptor with the particle bound-- 678 00:36:34,290 --> 00:36:37,890 so that's the LDL-LDL receptor-- then the question is-- 679 00:36:37,890 --> 00:36:39,360 and it's tight binding-- 680 00:36:39,360 --> 00:36:42,180 how do you get that off? 681 00:36:42,180 --> 00:36:46,590 And remember, you're also going to have 682 00:36:46,590 --> 00:36:50,100 LDL that's been internalized. 683 00:36:50,100 --> 00:36:53,430 So the creative approach they used 684 00:36:53,430 --> 00:36:55,790 was to use the molecule heparin. 685 00:36:55,790 --> 00:36:57,212 OK, so heparin-- 686 00:36:57,212 --> 00:36:58,920 I'm not going to draw out the structure-- 687 00:36:58,920 --> 00:37:01,485 but this is a third tool and this was key. 688 00:37:05,280 --> 00:37:09,480 And so they have heparin-sensitive 689 00:37:09,480 --> 00:37:11,020 and heparin-resistant. 690 00:37:14,130 --> 00:37:15,130 And what does this mean? 691 00:37:15,130 --> 00:37:19,630 Heparin turns out to-- it's a sugar. 692 00:37:19,630 --> 00:37:22,960 Many of you have probably heard about it. 693 00:37:22,960 --> 00:37:25,240 It plays a key role in blood coagulation. 694 00:37:25,240 --> 00:37:27,490 But anyhow, from the point of view of today's lecture, 695 00:37:27,490 --> 00:37:29,160 you just need to know it's a sugar 696 00:37:29,160 --> 00:37:31,460 and it's got sulfates all over the outside of it. 697 00:37:31,460 --> 00:37:33,130 So it's negatively charged. 698 00:37:33,130 --> 00:37:36,910 So heparin is a sulfated sugar. 699 00:37:42,270 --> 00:37:46,680 So basically, you have something like this with SO3 minuses 700 00:37:46,680 --> 00:37:48,600 on the outside. 701 00:37:48,600 --> 00:37:51,930 And so what happens is if-- 702 00:37:51,930 --> 00:37:55,170 what you want to do is release the LDL particle 703 00:37:55,170 --> 00:37:57,780 from the receptor. 704 00:37:57,780 --> 00:38:01,590 And apparently, treatment with heparin at certain levels-- 705 00:38:01,590 --> 00:38:03,510 I think they tried a lot of things-- 706 00:38:03,510 --> 00:38:10,020 was able to release the surface LDL. 707 00:38:10,020 --> 00:38:16,760 So this is involved in release of surface bound. 708 00:38:23,170 --> 00:38:26,230 So then what you have left after you release this, 709 00:38:26,230 --> 00:38:31,000 is you could still have radio label that's been internalized. 710 00:38:31,000 --> 00:38:35,260 So that then becomes heparin-resistant. 711 00:38:35,260 --> 00:38:36,450 And so you can count that. 712 00:38:39,220 --> 00:38:42,850 And so then you have bound and internalized. 713 00:38:42,850 --> 00:38:46,990 Now, if you're studying this as a function of time, 714 00:38:46,990 --> 00:38:51,010 what can happen to-- once you internalize the LDL particle, 715 00:38:51,010 --> 00:38:56,050 what can happen to the iodinated LDL particle? 716 00:38:56,050 --> 00:38:56,770 What can happen? 717 00:39:00,343 --> 00:39:01,760 So this is something else you need 718 00:39:01,760 --> 00:39:04,010 to think about in these assays. 719 00:39:04,010 --> 00:39:14,500 So now we have internalized LDL, i125 label. 720 00:39:14,500 --> 00:39:18,775 What can happen-- if you remember from recitation 721 00:39:18,775 --> 00:39:21,025 this past week, you remember what happened to the LDL? 722 00:39:24,840 --> 00:39:25,720 So you got protein. 723 00:39:25,720 --> 00:39:26,470 You got lipids. 724 00:39:26,470 --> 00:39:27,260 What's going to happen? 725 00:39:27,260 --> 00:39:28,552 You might not know the details. 726 00:39:28,552 --> 00:39:30,040 That's what this whole-- 727 00:39:30,040 --> 00:39:31,870 that's what Brian and Goldstein uncovered, 728 00:39:31,870 --> 00:39:36,130 which we're going to talk about in the next few minutes. 729 00:39:36,130 --> 00:39:37,760 But LDL, you have a protein. 730 00:39:37,760 --> 00:39:39,850 What can happen to proteins? 731 00:39:39,850 --> 00:39:41,290 They can get degraded. 732 00:39:41,290 --> 00:39:47,350 So if you have the apoB, what can happen is inside 733 00:39:47,350 --> 00:39:51,010 the cells-- so this is inside-- 734 00:39:51,010 --> 00:39:56,170 you could have proteases that degrade this down to peptides. 735 00:39:56,170 --> 00:40:00,340 This happens in a lysosol where you still 736 00:40:00,340 --> 00:40:03,810 have iodinated tyrosine. 737 00:40:03,810 --> 00:40:10,050 Or it can be broken down all the way to just iodinated tyrosine. 738 00:40:10,050 --> 00:40:12,140 So if you're breaking this down all the way here, 739 00:40:12,140 --> 00:40:16,728 the iodinated tyrosine could likely exit the cell. 740 00:40:16,728 --> 00:40:18,270 So you need to really think-- so what 741 00:40:18,270 --> 00:40:23,490 do you do to control for this aspect of the metabolism? 742 00:40:23,490 --> 00:40:27,970 What happens to the LDL inside the cell? 743 00:40:27,970 --> 00:40:33,660 And so to do this, how would you distinguish LDL itself from, 744 00:40:33,660 --> 00:40:36,240 say-- 745 00:40:36,240 --> 00:40:39,840 as a chemist, what could you do to distinguish LDL protein 746 00:40:39,840 --> 00:40:45,220 from LDL on small peptides or LDL as an amino acid? 747 00:40:45,220 --> 00:40:48,930 So the key question was, what sort of bulk method 748 00:40:48,930 --> 00:40:52,470 do you use to try to distinguish between these two things. 749 00:40:52,470 --> 00:40:55,350 So then you can incorporate that into the analysis, which 750 00:40:55,350 --> 00:40:57,660 is what's on the slide here. 751 00:40:57,660 --> 00:41:01,980 So what happens if you treat proteins in general with acid? 752 00:41:06,630 --> 00:41:07,140 They what? 753 00:41:07,140 --> 00:41:08,970 They hydrolize? 754 00:41:08,970 --> 00:41:11,640 So peptide bonds are really strong. 755 00:41:11,640 --> 00:41:14,190 If you want to break a peptide bond, 756 00:41:14,190 --> 00:41:17,880 you have to heat it for 16 hours at 100 degrees. 757 00:41:17,880 --> 00:41:19,350 So that's not going to happen. 758 00:41:19,350 --> 00:41:21,750 So that's not an option. 759 00:41:21,750 --> 00:41:22,800 But what else happens? 760 00:41:22,800 --> 00:41:27,210 What do you do when you put a protein into acid? 761 00:41:27,210 --> 00:41:30,070 What happens to the protein? 762 00:41:30,070 --> 00:41:31,900 It what? 763 00:41:31,900 --> 00:41:33,160 Yeah, it crashes out. 764 00:41:33,160 --> 00:41:36,490 So proteins in general, not all proteins, most proteins 765 00:41:36,490 --> 00:41:41,350 precipitate, but these kinds of things would be soluble. 766 00:41:44,020 --> 00:41:46,160 So they've been able to take advantage-- 767 00:41:46,160 --> 00:41:49,120 so you have to, again, treat the cells in a certain way so 768 00:41:49,120 --> 00:41:51,010 that you can look at what's still 769 00:41:51,010 --> 00:41:58,300 retained in the LDL versus what's undergone degradation. 770 00:41:58,300 --> 00:42:00,930 OK, we're going to see that's key to the model we're 771 00:42:00,930 --> 00:42:01,870 going to come up with. 772 00:42:01,870 --> 00:42:06,200 OK, so those are the tools that they needed to develop. 773 00:42:06,200 --> 00:42:10,710 And so the question is, then, what did they observe? 774 00:42:10,710 --> 00:42:12,630 OK, so we're doing these same experiments. 775 00:42:12,630 --> 00:42:15,840 We're looking for binding on the outside, internalization, 776 00:42:15,840 --> 00:42:16,490 and breakdown. 777 00:42:16,490 --> 00:42:18,690 That's what we're looking for. 778 00:42:18,690 --> 00:42:25,290 And so here is the patient and here is the control. 779 00:42:25,290 --> 00:42:30,400 So if we look at here, these guys are the binding. 780 00:42:30,400 --> 00:42:33,970 So Brown and Goldstein, in this particular paper, 781 00:42:33,970 --> 00:42:37,300 which is underneath here but in the cell paper, 782 00:42:37,300 --> 00:42:38,960 looked at 22 patients. 783 00:42:38,960 --> 00:42:44,950 And out of the 22 patients, most of them were binding deficient. 784 00:42:44,950 --> 00:42:47,240 They could see no binding at all. 785 00:42:47,240 --> 00:42:51,220 Some of them were binding modified. 786 00:42:51,220 --> 00:42:55,150 That is, they had lower levels of binding. 787 00:42:55,150 --> 00:43:00,040 And this one patient, JD, had normal binding. 788 00:43:00,040 --> 00:43:02,770 So in this experiment, we're looking at here-- so 789 00:43:02,770 --> 00:43:11,380 in the PowerPoint, this is one of 22 patients 790 00:43:11,380 --> 00:43:14,450 they had normal binding. 791 00:43:17,060 --> 00:43:18,890 And the others-- and that's because we'll 792 00:43:18,890 --> 00:43:22,280 see that there are multiple ways you can have 793 00:43:22,280 --> 00:43:24,380 defects in your LDL receptor. 794 00:43:24,380 --> 00:43:26,110 We'll come back to that in a minute. 795 00:43:26,110 --> 00:43:32,300 But you can have deficient binding 796 00:43:32,300 --> 00:43:33,540 or you could have no binding. 797 00:43:37,020 --> 00:43:39,780 Or you could have normal binding. 798 00:43:39,780 --> 00:43:41,610 So those are all possible. 799 00:43:41,610 --> 00:43:44,850 And the one that we've taken the data for here 800 00:43:44,850 --> 00:43:48,115 and that's described in the paper, is normal binding. 801 00:43:48,115 --> 00:43:49,490 And they did a lot of experiments 802 00:43:49,490 --> 00:43:52,260 I'm not describing to try to show you 803 00:43:52,260 --> 00:43:55,650 that this experiment, which suggests normal binding, 804 00:43:55,650 --> 00:43:56,910 is in fact normal binding. 805 00:43:56,910 --> 00:43:57,990 They looked at off rates. 806 00:43:57,990 --> 00:44:02,513 They looked at competition with HDL and LDL. 807 00:44:02,513 --> 00:44:03,930 And so if you look at that, if you 808 00:44:03,930 --> 00:44:06,570 look at the levels of binding, they really 809 00:44:06,570 --> 00:44:11,500 aren't very different between the experiment and the control. 810 00:44:11,500 --> 00:44:14,970 And so now what happens, if you look 811 00:44:14,970 --> 00:44:19,200 at the normal, what happens is with time, 812 00:44:19,200 --> 00:44:21,700 the LDL on the surface goes away. 813 00:44:21,700 --> 00:44:24,840 And that's because it's becoming internalized. 814 00:44:24,840 --> 00:44:26,970 Whereas down here, what happens? 815 00:44:26,970 --> 00:44:30,750 You started out the same, but now you can see over-- 816 00:44:30,750 --> 00:44:33,510 this is hours down here, it really 817 00:44:33,510 --> 00:44:35,700 hasn't changed very much. 818 00:44:35,700 --> 00:44:37,830 It's not becoming internalized. 819 00:44:37,830 --> 00:44:39,870 And so then they wanted to use their method 820 00:44:39,870 --> 00:44:43,620 to look at internalized LDL. 821 00:44:43,620 --> 00:44:48,540 And so internalized LDL, using the heparin-resistant 822 00:44:48,540 --> 00:44:52,230 versus heparin-sensitive, that's the assay they used, 823 00:44:52,230 --> 00:44:55,920 what you see is as the surface binding at least early 824 00:44:55,920 --> 00:45:00,480 on decreases, the amount internalized increases. 825 00:45:00,480 --> 00:45:04,120 But what happens over here to the patient? 826 00:45:04,120 --> 00:45:07,460 With the patient, you get nothing internalized. 827 00:45:07,460 --> 00:45:11,120 And the other question is, what happens to the LDL-- 828 00:45:11,120 --> 00:45:13,250 and it's labeled on the protein-- 829 00:45:13,250 --> 00:45:14,520 does that get degraded? 830 00:45:14,520 --> 00:45:16,880 And so using a method with TCA, they 831 00:45:16,880 --> 00:45:19,760 used a couple of different methods, what they see 832 00:45:19,760 --> 00:45:26,510 is that you slowly degrade the protein into small pieces. 833 00:45:26,510 --> 00:45:30,030 And again, with the patient, it's not internalized 834 00:45:30,030 --> 00:45:34,650 so you can't get degradation. 835 00:45:34,650 --> 00:45:38,660 So this type of experiment with this particular patient 836 00:45:38,660 --> 00:45:45,950 and also with the other patients that I talked about, one 837 00:45:45,950 --> 00:45:50,030 through 21, they drew a strong conclusion 838 00:45:50,030 --> 00:45:54,590 that there are two things that have to happen for cholesterol 839 00:45:54,590 --> 00:45:55,520 to get into the cell. 840 00:45:55,520 --> 00:45:57,320 Number one, it has to bind. 841 00:45:57,320 --> 00:45:59,480 And number two, there's got to be some mechanism 842 00:45:59,480 --> 00:46:01,010 for internalization. 843 00:46:01,010 --> 00:46:08,980 So the conclusions from this is we need binding, 844 00:46:08,980 --> 00:46:11,760 which is consistent with the LDL receptor. 845 00:46:11,760 --> 00:46:17,310 And then we need, in some way, internalization. 846 00:46:17,310 --> 00:46:22,200 And of course, JD was the only one out of all of these 847 00:46:22,200 --> 00:46:25,670 patients where they can study internalization 848 00:46:25,670 --> 00:46:28,320 because in the other patients they didn't-- they had really 849 00:46:28,320 --> 00:46:31,560 poor binding or no binding at all. 850 00:46:31,560 --> 00:46:34,080 So they needed to have this spectrum of patients 851 00:46:34,080 --> 00:46:38,210 to be able to start to sort out what was going on 852 00:46:38,210 --> 00:46:39,210 in these experiments. 853 00:46:39,210 --> 00:46:42,433 So I think on the surface, the experiments look pretty-- 854 00:46:42,433 --> 00:46:44,850 you'll look at them, they look like they're really simple. 855 00:46:44,850 --> 00:46:47,580 But technically, they're not so simple. 856 00:46:47,580 --> 00:46:50,460 And if you care about the technical details, which we'll 857 00:46:50,460 --> 00:46:53,040 see again in this week's recitation dealing 858 00:46:53,040 --> 00:46:55,860 with these membrane proteins and stickiness, 859 00:46:55,860 --> 00:46:58,890 becomes the key how creative you can be. 860 00:46:58,890 --> 00:47:03,363 And usually, we're not really plugged into that. 861 00:47:03,363 --> 00:47:05,280 And you usually don't do experiments like that 862 00:47:05,280 --> 00:47:07,080 unless you work in a lab that is focused 863 00:47:07,080 --> 00:47:11,940 on membrane-bound proteins. 864 00:47:11,940 --> 00:47:14,960 So this resulted in the model. 865 00:47:14,960 --> 00:47:19,980 So this kind of experiment and many other experiments 866 00:47:19,980 --> 00:47:31,890 resulted in the model for receptor mediated endocytosis. 867 00:47:31,890 --> 00:47:34,140 And you've seen this before. 868 00:47:34,140 --> 00:47:37,170 You saw this in recitation last week 869 00:47:37,170 --> 00:47:42,360 because we saw interference with the PCSK9 870 00:47:42,360 --> 00:47:44,850 with receptor mediated endocytosis. 871 00:47:44,850 --> 00:47:46,860 So we're back where we started last week. 872 00:47:46,860 --> 00:47:49,980 And the first slide I showed you was this slide. 873 00:47:49,980 --> 00:47:52,110 And so what is the model? 874 00:47:52,110 --> 00:47:54,690 So there are many, many more experiments 875 00:47:54,690 --> 00:47:56,610 that have gone into coming up with this model. 876 00:47:56,610 --> 00:47:59,670 And the model is really still incomplete. 877 00:47:59,670 --> 00:48:02,640 I have a cartoon here, the whole process, 878 00:48:02,640 --> 00:48:06,300 every step along the pathway, how you go here and there 879 00:48:06,300 --> 00:48:09,420 and what the kinetics are, it's all complicated. 880 00:48:09,420 --> 00:48:12,730 But this is the working hypothesis. 881 00:48:12,730 --> 00:48:17,580 And so the first thing is you make the LDL receptor. 882 00:48:17,580 --> 00:48:21,010 It's a membrane protein, has a single transmembrane spanning 883 00:48:21,010 --> 00:48:21,510 region. 884 00:48:21,510 --> 00:48:23,670 Is made in the ER. 885 00:48:23,670 --> 00:48:26,450 And because of this transmembrane spanning region, 886 00:48:26,450 --> 00:48:29,850 it's got to be transported to the surface. 887 00:48:29,850 --> 00:48:34,170 And it's done so in little coded vesicles, 888 00:48:34,170 --> 00:48:36,000 which keeps things soluble. 889 00:48:36,000 --> 00:48:38,670 And it does this by passing through the Golgi stacks, which 890 00:48:38,670 --> 00:48:41,550 we talked about at the very beginning. 891 00:48:41,550 --> 00:48:43,980 Eventually, it gets to the surface. 892 00:48:43,980 --> 00:48:46,650 These little things here are the LDL receptors. 893 00:48:49,620 --> 00:48:51,480 You can go home and sleep on this 894 00:48:51,480 --> 00:48:53,500 and look at it again because I'm over. 895 00:48:53,500 --> 00:48:57,150 And it just seems like I just started and it's already over. 896 00:48:57,150 --> 00:48:58,070 I'm sorry. 897 00:48:58,070 --> 00:48:59,617 OK, I must have spent too much time 898 00:48:59,617 --> 00:49:01,950 talking about something I wasn't supposed to talk about. 899 00:49:01,950 --> 00:49:04,380 But anyhow, hopefully you now all 900 00:49:04,380 --> 00:49:06,697 can go back and look at this and think 901 00:49:06,697 --> 00:49:09,030 about this, because we're going to be talking about this 902 00:49:09,030 --> 00:49:12,770 again in recitation this week.