人血清碱性核糖核酸酶的纯化及性质

Purification and properties of alkaline ribonuclease from human serum.

Akagi K, Murai K, Hirao N, Yamanaka M

Biochim Biophys Acta. 1976 Sep 6;442(3):368-78. doi: 10.1016/0005-2787(76)90311-7.

PMID:9140

Abstract

Five alkaline ribonucleases (EC 3.1.4.22) were purified about 140- to 1900-fold from human serum by phosphocellulose and DEAE-cellulose chromatographies and Sephadex G-75 filtration, with a total recovery of 22%. These were designated as RNAases 1-5. 2. Optimum activities were observed at pH 8.5-8.7 for RNAases 1-4, and at pH 7.5 for RNAase 5. The molecular weights of these enzymes were estimated by gel filtration as 45 000, 32 000, 20 000, 13 000 and 8500, respectively. 3. These RNAases were found to be heat-labile proteins but are markedly stabilized with bovine plasma albumin. The reaction was activated by Na+, K+, Mg2+ and Ca2+, and inhibited by Co2+, Fe2+, Cu2+ and Zn2+. EDTA had little effect on the velocity of the reaction. Spermine caused 2- to 7-fold activation. 4. Among the substrates examined, these RNAases preferentially hydrolyzed pyrimidine bodies and except for RNAase 5 had a higher affinity for poly(C) than poly(U) as substrate. Each enzyme was free from other nucleolytic enzymes and hydrolyzed only RNA.

摘要

通过磷酸纤维素和二乙氨基乙基纤维素色谱法以及葡聚糖凝胶G - 75过滤，从人血清中纯化出5种碱性核糖核酸酶（EC 3.1.4.22），纯化倍数约为140至1900倍，总回收率为22%。这些酶被命名为核糖核酸酶1 - 5。2. 核糖核酸酶1 - 4在pH 8.5 - 8.7时观察到最佳活性，核糖核酸酶5在pH 7.5时观察到最佳活性。通过凝胶过滤估计这些酶的分子量分别为45000、32000、20000、13000和8500。3. 发现这些核糖核酸酶是热不稳定蛋白，但与牛血浆白蛋白一起时能显著稳定。该反应被Na⁺、K⁺、Mg²⁺和Ca²⁺激活，被Co²⁺、Fe²⁺、Cu²⁺和Zn²⁺抑制。EDTA对反应速度影响很小。精胺引起2至7倍的激活。4. 在检测的底物中，这些核糖核酸酶优先水解嘧啶体，除核糖核酸酶5外，作为底物时对聚（C）的亲和力高于聚（U）。每种酶都不含其他核酸分解酶，且仅水解RNA。