Characterization and Biosynthesis of Soluble and Membrane‐Bound Carbonic Anhydrase in Brain
Characterization and Biosynthesis of Soluble and Membrane‐Bound Carbonic Anhydrase in Brain
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脑中可溶性膜结合碳酸酐酶的表征和生物合成
DOI：
10.1111/j.1471-4159.1983.tb13563.x
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发表时间：
1983
影响因子：
4.7
通讯作者：
J. M. Gilbert
中科院分区：
文献类型：
--
作者：
V. Sapirstein;P. Strocchi;M. Wesołowski;J. M. Gilbert
摘要：从大鼠前脑细胞质和膜中提取碳酸酐酶，对其酶活性、免疫反应性和体外生物合成进行了表征。提出了一种快速纯化膜结合型和可溶性脑碳酸酐酶的方法，可以保留充分的酶活性。当测定CO2水合活性时，发现两种形式的酶都显示出约5500单位/毫克蛋白质的特定活性。此外，当用对硝基苯乙酸测定时，它们表现出相似的酯酶活性。膜结合形式，虽然需要洗涤剂从膜中提取，但纯化后可自由溶解于水缓冲液中。可溶性碳酸酐酶和膜结合碳酸酐酶的分子量均为30,000道尔顿，混合实验没有显示出在大小方面有任何显着差异。这两种形式也表现出7.2的等电点。然而，这两种蛋白质被发现在两个方面有所不同。补体固定表明，针对可溶性碳酸酐酶的抗体对可溶性形式的亲和力高于膜结合形式。脉冲追踪研究未能观察到这两种蛋白质之间的任何前体-产物关系以及碳酸酐酶样蛋白质在自由多体和粗内质网上合成的建立表明，这些蛋白质是由两种不同的机制合成的。用不同的抗体和不同的分析程序，用两种独立的方法测定了游离和结合多体的体外合成。讨论了这些发现的基础及其生理学意义。
Abstract: Carbonic anhydrase from both the cytoplasmic and membrane fractions of the forebrains of rats was characterized with respect to enzymatic activity, immunoreactivity, and in vitro biosynthesis. A procedure for the rapid purification of both membrane‐bound and soluble brain carbonic anhydrase is presented that permits retention of full enzymatic activity. Both forms of the enzyme were found to show specific activities of approximately 5500 Units/mg protein when CO2 hydrating activity was determined. In addition, they exhibited similar esterase activity when assayed with p‐nitrophenyl acetate. The membrane‐bound form, although requiring detergent for extraction from membranes, was freely soluble in aqueous buffers after purification. The molecular weights of both soluble and membrane‐bound carbonic anhydrase are 30,000 daltons, and mixing experiments failed to show any significant differences with respect to size. The two forms also exhibit isoelectric points of 7.2. However, the two proteins were found to differ in two respects. Complement fixation indicated that antibodies to soluble carbonic anhydrase had a higher affinity for the soluble form than for the membrane‐bound form. The failure to observe any precursor‐product relationship between these two proteins with pulse chase studies and the establishment that carbonic anhydrase‐like proteins are synthesized on both free polysomes and the rough endoplasmic reticulum indicated that these proteins are synthesized by two separate mechanisms. In vitro synthesis on both free and bound polysomes was determined by two independent methods using different antibodies and different analytical procedures. The basis for these findings and their physiologic importance are discussed.