Siekevitz P, Palade G E
J Cell Biol. 1966 Sep;30(3):519-30. doi: 10.1083/jcb.30.3.519.
Amylase distribution was studied in guinea pig pancreas microsomes fractionated by centrifuging, for 2 hr at 57,000 g in a linear 10 to 30% sucrose gradient, a resuspended high speed pellet obtained after treating microsomes with 0.04% deoxycholate (DOC).(1) Amylase appeared in the following positions in the gradient: (a) a light region which contained approximately 35% of total enzymic activity and which coincided with a monomeric ribosome peak; (b) a heavy region which contained approximately 10% of enzymic activity in a sharp peak but which had very little accompanying OD(260) absorption; (c) a pellet at the bottom of the centrifuge tube which contained approximately 20% of the enzymic activity. After 5 to 20 min' in vivo labeling with leucine-1-C(14), radioactive amylase was solubilized from these three fractions by a combined DOC-spermine treatment and purified by precipitation with glycogen, according to Loyter and Schramm. In all cases, the amylase found in the pellet had five to ten times the specific activity (CPM/enzymic activity) of the amylase found in the light or heavy regions of the gradient. The specific radioactivity (CPM/mg protein) of the proteins or peptides not extracted by DOC-spermine was similar for all three fractions. Hypotonic treatment of the fractions solubilized approximately 80% of the total amylase in the fraction from the heavy region of the gradient, but only approximately 20% of the amylase in the monomer or pellet fraction. Electron microscope observation indicates that the monomer region of the gradient contained only ribosomes, that the heavy region of the gradient contained small vesicles with relatively few attached ribosomes, and that the pellet was composed mostly of intact or ruptured microsomes with ribosomes still attached to their membranes. It is concluded from the above, and from other evidence, that most of the amylase activity in the monomer region is due to old, adsorbed enzyme; in the heavy region mostly to enzyme already inside microsomal vesicles; and in the pellet to a mixture of newly synthesized and old amylase still attached to ribosomes. Furthermore, the ribosomes with nascent, finished protein still bound to them are more firmly attached to the membranes than are ribosomes devoid of nascent protein.
通过在含有10%至30%线性蔗糖梯度的溶液中以57,000 g离心2小时，对豚鼠胰腺微粒体进行分级分离，这些微粒体是在用0.04%脱氧胆酸盐（DOC）处理后重新悬浮的高速沉淀产物。（1）淀粉酶在梯度中的以下位置出现：（a）一个轻区，含有约35%的总酶活性，与单体核糖体峰重合；（b）一个重区，在一个尖锐峰中含有约10%的酶活性，但伴随的OD(260)吸收很少；（c）离心管底部的沉淀，含有约20%的酶活性。在用亮氨酸-1-C(14)进行5至20分钟的体内标记后，根据洛伊特和施拉姆的方法，通过DOC-精胺联合处理从这三个部分中溶解放射性淀粉酶，并通过糖原沉淀进行纯化。在所有情况下，沉淀中发现的淀粉酶的比活性（CPM/酶活性）是梯度轻区或重区中发现的淀粉酶的五至十倍。对于所有三个部分，未被DOC-精胺提取的蛋白质或肽的比放射性（CPM/mg蛋白质）相似。对这些部分进行低渗处理可溶解梯度重区部分中约80%的总淀粉酶，但单体或沉淀部分中仅约20%的淀粉酶。电子显微镜观察表明，梯度的单体区仅含有核糖体，梯度的重区含有附着核糖体相对较少的小泡，沉淀主要由完整或破裂的微粒体组成，核糖体仍附着在其膜上。从上述内容以及其他证据可以得出结论，单体区中的大部分淀粉酶活性是由于陈旧的、吸附的酶；重区中的大部分是由于已经在微粒体小泡内的酶；沉淀中的是新合成的和仍然附着在核糖体上的陈旧淀粉酶的混合物。此外，带有新生、已完成蛋白质的核糖体比没有新生蛋白质的核糖体更牢固地附着在膜上。
