Lee D M, Singh S
Lipoprotein and Atherosclerosis Research Program, Oklahoma Medical Research Foundation, Oklahoma City 73104.
Biochim Biophys Acta. 1988 May 22;960(2):148-56. doi: 10.1016/0005-2760(88)90060-4.
The purpose of this study was to investigate the molecular forms of apolipoprotein B (ApoB) in human chylomicrons under well-preserved conditions. To this end, plasma and serum were collected from the same normal subjects after ingestion of a fatty meal. The samples were divided into three or four aliquots before the addition of various preservative mixtures, including antibiotics, antioxidants and proteinase inhibitors. The chylomicrons were isolated immediately, and all steps were carried out at or below 4 degrees C. Changes in the molecular weight of ApoB in chylomicrons were followed by a time study using 3.3% polyacrylamide gel electrophoresis containing SDS. ApoB from chylomicrons analyzed within 5 h of blood collection showed a single band with mobility identical to that of ApoB (ApoB-100) in low-density lipoproteins. When analyzed after 1-2 days, satellite bands smaller than ApoB-100 were observed, and a very faint band with Mr 200,000 appeared, which comigrated with intestinal ApoB (ApoB-48). Upon storage, the molecular weight of ApoB was smaller in chylomicrons subjected to a higher number of reflotations than those in chylomicrons washed less frequently, suggesting that purified chylomicrons degrade faster. A longer storage time at 4 degrees C (i.e., 7 or 14 days) revealed a stepwise degradation of ApoB, yielding Mr 200,000 band as the prominent form. The degradation of ApoB-100 was slower when both proteinase inhibitors, leupeptin and epsilon-amino caproic acid, were employed, and the appearance of Mr 200,000 band was quicker when the chylomicrons were processed at higher temperature (15-25 degrees C) in the absence of a proteinase inhibitor. Immunoblotting shows that the segment removed from ApoB-100 was the carboxyl-terminal portion. These results suggest the possible presence of a proteinase(s), which copurified with chylomicrons, and which converts ApoB-100 from a large to a smaller molecular form. Although the stop codon has been discovered recently in intestinal ApoB mRNA, which explains the mechanism for direct synthesis of ApoB-48, apparently ApoB-100 is also synthesized in the intestine of all eight subjects studied here, and the ApoB-100 degrades to a form which is ApoB-48-like.
本研究的目的是在保存良好的条件下，研究人乳糜微粒中载脂蛋白B（ApoB）的分子形式。为此，在正常受试者摄入高脂餐后，采集其血浆和血清。在添加包括抗生素、抗氧化剂和蛋白酶抑制剂在内的各种保存混合物之前，将样本分成三或四份。立即分离乳糜微粒，所有步骤均在4℃或4℃以下进行。采用含SDS的3.3%聚丙烯酰胺凝胶电泳，通过时间研究追踪乳糜微粒中ApoB分子量的变化。在采血后5小时内分析的乳糜微粒中的ApoB显示出一条迁移率与低密度脂蛋白中的ApoB（ApoB-100）相同的单带。在1-2天后分析时，观察到比ApoB-100小的卫星带，并且出现了一条非常淡的分子量为200,000的带，其与肠ApoB（ApoB-48）共迁移。储存时，与洗涤频率较低的乳糜微粒相比，经过更多次再漂浮的乳糜微粒中ApoB的分子量更小，这表明纯化的乳糜微粒降解更快。在4℃下较长时间的储存（即7天或14天）显示ApoB逐步降解，产生分子量为200,000的带作为主要形式。当同时使用蛋白酶抑制剂亮抑酶肽和ε-氨基己酸时，ApoB-100的降解较慢，并且当在没有蛋白酶抑制剂的情况下在较高温度（15-25℃）下处理乳糜微粒时，分子量为200,000的带出现得更快。免疫印迹显示从ApoB-100去除的片段是羧基末端部分。这些结果表明可能存在一种与乳糜微粒共纯化的蛋白酶，它将ApoB-100从大分子形式转化为小分子形式。尽管最近在肠ApoB mRNA中发现了终止密码子，这解释了直接合成ApoB-48的机制，但显然在这里研究的所有八名受试者的肠道中也合成了ApoB-100，并且ApoB-100降解为类似ApoB-48的形式。
