大鼠脑中磷脂酰肌醇磷酸二酯酶活性与微管蛋白特定亚基之间的关联。

The association between phosphatidylinositol phosphodiesterase activity and a specific subunit of microtubular protein in rat brain.

作者信息

Quinn P J

出版信息

Biochem J. 1973 Jun;133(2):273-81. doi: 10.1042/bj1330273.

DOI:10.1042/bj1330273

PMID:4353236

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1177696/

Abstract

Supernatant proteins from rat brain were separated into two fractions containing phosphatidylinositol phosphodiesterase activity by chromatography on DEAE-Sephadex A-50. 2. The first fraction sediments in linear sucrose density gradients in two bands corresponding to molecular weights of 66000 and 36000. There was presumptive evidence that the lighter protein constituted the monomeric form of the enzyme. The second fraction sediments predominantly as a single protein of molecular weight 86000. 3. Treatment of rat brain supernatant with [(3)H]colchicine abolished the second DEAE-Sephadex peak and removed the lighter protein from the first peak. These proteins emerged in the same position as the protein binding [(3)H]colchicine at high salt concentration; phospholipase activity was recovered from linear sucrose density gradients in positions corresponding to molecular weights 88000 and 43000, together with an aggregate of molecular weight 140000. Electrophoresis on sodium dodecyl sulphate-urea-polyacrylamide gels of this fraction revealed only three proteins: the alpha and beta-subunits of microtubular protein, of molecular weights 56000 and 52000 respectively, and a protein of molecular weight 38000. 4. A sample of microtubular protein from mouse, labelled in vivo with [(3)H]proline and (32)P(i), was added to rat brain supernatant together with an equal amount of the same microtubular protein treated with cyclic AMP and [gamma-(32)P]ATP and the mixture subsequently characterized by ion-exchange chromatography. Some phospholipase activity characteristic of the second peak from DEAE-Sephadex was associated with one fraction of added microtubular protein. This fraction was identified on the basis of the (3)H:(32)P ratio as the beta subunit of the protein treated with ATP and cyclic AMP. The subunit of added microtubular protein untreated with nucleotides was not associated with phospholipase activity.

摘要

通过在DEAE-葡聚糖A-50上进行层析，将大鼠脑的上清液蛋白分离成两个含有磷脂酰肌醇磷酸二酯酶活性的组分。2. 第一个组分在线性蔗糖密度梯度中沉降为两条带，分别对应分子量为66000和36000。有初步证据表明较轻的蛋白质构成了该酶的单体形式。第二个组分主要以分子量为86000的单一蛋白质形式沉降。3. 用[³H]秋水仙碱处理大鼠脑上清液消除了第二个DEAE-葡聚糖峰，并从第一个峰中除去了较轻的蛋白质。这些蛋白质在高盐浓度下与结合[³H]秋水仙碱的蛋白质出现在相同位置；从线性蔗糖密度梯度中回收的磷脂酶活性对应于分子量为88000和43000的位置，以及分子量为140000的聚集体。对该组分在十二烷基硫酸钠-尿素-聚丙烯酰胺凝胶上进行电泳，仅显示三种蛋白质：微管蛋白的α和β亚基，分子量分别为56000和52000，以及一种分子量为38000的蛋白质。4. 将用[³H]脯氨酸和³²P(i)在体内标记的小鼠微管蛋白样品，与等量的用环磷酸腺苷和[γ-³²P]三磷酸腺苷处理的相同微管蛋白一起加入大鼠脑上清液中，随后通过离子交换层析对混合物进行表征。DEAE-葡聚糖第二个峰特有的一些磷脂酶活性与添加的微管蛋白的一个组分相关。根据³H:³²P比率，该组分被鉴定为用三磷酸腺苷和环磷酸腺苷处理的蛋白质的β亚基。未用核苷酸处理的添加微管蛋白的亚基与磷脂酶活性无关。