INTERACTIONS OF LIPIDS WITH A MEMBRANE STRUCTURAL PROTEIN FROM MYELIN
INTERACTIONS OF LIPIDS WITH A MEMBRANE STRUCTURAL PROTEIN FROM MYELIN
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DOI：
10.1021/bi00839a014
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发表时间：
1969-01-01
期刊：
影响因子：
2.9
通讯作者：
RADIN, NS
中科院分区：
文献类型：
--
作者：
BRAUN, PE;RADIN, NS
*来自密西根大学心理健康研究所，密西根州安娜堡48104。1969年4月16日收到。部分由美国公共卫生服务国家神经疾病和中风研究所拨款NB-03192支持。f现地址：宾夕法尼亚大学生物化学系，宾夕法尼亚州费城，19104胆固醇和脑苷)和卵磷脂与蛋白质形成非沉淀络合物，这可以通过在蔗糖密度梯度中离心来证明。这些脂质也与蛋白质-阴离子脂质复合物结合。该蛋白的琥珀酰化大大减少了与脂质的相互作用，并消除了形成不溶性复合物的能力。这些观察结果讨论了不同种类的脂质在髓磷脂中可能承担的结构作用。牛肉脑和冷冻储存。根据Autilio et al.（1964）的程序，使用Beckman B-14分区转子进行大规模髓鞘分离。经过渗透破裂和多次水洗去除轴突碎片后，最终制备成致密的浆料颗粒。含蛋白脂质蛋白。Tenenbaum和Folch（1966）对这一过程进行了修改，提供了目前所有研究中使用的髓磷脂蛋白。将新制备的髓磷脂溶解于19体积氯仿-甲醇（2:1,v/v）中。按照Folch et al.（1957）的方法，将清液用水分割，将含有所有可溶性蛋白脂的下相转移到氯仿-甲醇提取透析管中。在氯仿-甲醇中透析7天后，透析液酸化至0。用浓HC1。此时出现的沉淀物通过离心（20,000 g， 15分钟）除去。将清上清液返回新管中，对氯仿-甲醇-盐酸进行透析。通过在增加甲醇和水的溶液中连续透析，最后在单独的水中透析，将脱去的蛋白质转移到水溶液中。在100，-OOOg下离心60分钟，除去少量悬浮物质。最终，清澈的蛋白质水溶液的pH值接近中性。用稀释的NH4OH渗透蒸发或沉淀（pH 9-10）将蛋白质溶液浓缩至1-10 mg/ml，然后用极稀的醋酸溶解并透析。由于蛋白质脂质蛋白是通过冻干或冷冻和解冻而变性的（Tenenbaum和Folch， 1966），因此蛋白质溶液在4℃下储存，并加入几滴氯仿作为防腐剂。将氯仿-甲醇-盐酸不溶性蛋白在氯仿-甲醇中再溶解。将这种蛋白质转移到水溶液中是通过上述程序完成的。由于这种蛋白是可透析的，因此发生了相当大的损失。这很可能是以前通过其他方法分离的低分子量碱性髓磷脂蛋白（Lowden et al.）。1966年;Roboz Einstein etal。, 1968;Eng等等。, 1968)。髓磷脂浆（436 g；髓磷脂16.1 g，干重）产生1.5 g水溶性蛋白脂蛋白和0.8 g碱性蛋白。在溶胶-过程中，第三个蛋白质部分留在界面上
* From the Mental Health Research Institute, University of Michigan, Ann Arbor, Michigan 48104. Received April 16, 1969. Supported in part by Grant NB-03192 from the NationalInstitute of Neurological Diseases and Stroke, U. S. Public Health Service. f Present address: Department of Biochemistry, University of Pennsylvania, Philadelphia, Pa. 19104. lesterol and cerebroside) and lecithin form nonprecipitating complexes with the protein which can be demonstrated by centrifugation in sucrose density gradients. These lipids also bind to protein-anionic lipid complexes. Succinylation of the protein greatlyreduces interaction with lipids andabolishes the capacity to form insoluble complexes. These observations are discussed with respect to the structural role which different kinds of lipids might assume in myelin. beef brains and stored frozen. Large-scale myelin isolations were carried out with the Beckman B-14 zonal rotor according to the procedure of Autilio et al.(1964). The final preparation, following osmotic rupture and numerous water washes to re-move axonal fragments, consisted of a dense slurry pellet. Proteolipid Protein. A modification of the procedure de-scribed by Tenenbaum and Folch (1966) provided the myelin protein used in all thepresent studies. Freshly prepared myelin was dissolved in 19 volumes of chloroform-methanol (2: 1, v/v). The clear solution was partitioned with water according to Folch et al.(1957) and the lower phase, containing all the soluble proteolipid, was transferred to chloroform-methanol extracted dialysis tubing. Following dialysis for 7 days in chloroform-methanol the dialysate was acidified to0. 04 n with concentrated HC1. The precipitate which appeared at this point was removed by centrifugation (20,000 g, 15 min). The clear supernatant fluid was returned to fresh tubing and dialyzed against chloroform-methanol-HCl. The transfer of the delipidatedprotein toaqueous solution was per-formed by successive dialysis in solutions of increasing methanol and water and finally in water alone. A smallamount of suspended material was removed by centrifugation at 100,-OOOg for 60 min. The pH of the final, clearaqueous solution of protein was close to neutrality. Protein solutions were con-centrated to 1-10 mg/ml by either pervaporation or precipita-tion (pH 9-10) with dilute NH4OH followed by resolution in very dilute acetic acid and dialysis. Since proteolipid protein is denatured by lyophilization or by freezing and thawing (Tenenbaum and Folch, 1966), protein solutions were stored at 4 with a few drops of chloroform added as a preservative. The chloroform-methanol-HCl-insoluble protein was re-dissolved inchloroform-methanol. Transfer of this protein to aqueous solution was accomplished by the above procedure. Considerable losses of this protein occurred, as it is dialyzable. This is most probably the low molecular weight basic myelin protein isolated previously by other procedures (Lowden et al.. 1966; Roboz Einstein etal., 1968; Eng etal., 1968). Myelin slurry (436 g; 16.1 g of myelin, dry weight) yielded 1.5 g of water-soluble proteolipid protein and 0.8 g of basic protein. A third protein fraction remained at the interface during sol-