牛肉脑皮质腺苷酸环化酶的氟化物刺激成分与非氟化物刺激成分在钙离子、乙二醇双（β-氨基乙醚）N，N'-四乙酸和曲拉通X-100作用下的分化。

Differentiation of fluorides-stimulated and non-fluoride-stimulated components of beef brain cortex adenylate cyclase cy calcium ions, ethyleneglycol-bis-(beta-aminoethyl ether) N,N'-tetraacetic acid and Triton X-100.

作者信息

MacDonald I A

出版信息

Biochim Biophys Acta. 1975 Jul 27;397(1):244-53. doi: 10.1016/0005-2744(75)90197-7.

Abstract

Beef brain cortex adenylate cyclase (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) activity is 84--88% inhibited by 5 - 10(-5) M ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-tetraacetic acid in the absence of F- but only 50--60% inhibited by 5 - 10(-5) M ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-tetraacetic acid in the presence of F-. In either case, further increase in EGTA concentration did not alter the degree of inhibition. The inhibition can be completely reversed in both cases by addition of 3 - 10(-5) M Ca2+, (yielding a [free Ca2+] of approximately 2 - 10(-6) M) and 5 - 10(-5) M Mn2+ or Co2+ and partially by 5 - 10(-5) M Sr2+ but not by addition of 5 - 10(-5) M Ba2+, Zn2+, Ni2+ or Fe2+. A [free Ca2+] of 7.2 - 10(-5) M markedly inhibited cyclase activity in the presence of F-. Solubilization by 1.8% Triton X-100 resulted in an enzyme preparation no longer stimulated by NaF and 100% inhibited by the addition of 5 - 10(-5) M ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-tetraacetic acid either in the absence or presence of NaF. However, in contrast to ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-TETRAACETIC ACID, EDTA had no measurable effect on adenylate cyclase either in the presence or absence of NaF and ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-tetraacetic acid did not affect ATPase or phosphodiesterase activities. The data is rationalized by the postulation of two independent enzyme components in brain cortex: one component is about six-fold activated by NaF and the NaF effect is enhanced by low concentrations of Ca2+ and Mg2+. A second component is totally Ca2+ dependent and inhibited by high concentrations of F-. Mn2+, Co2+ and Sr2+ appear to be in vitro Ca2+ substitutes for both enzyme systems. On this basis, Triton X-100 treatment results in about a three-fold increase in specific activity of the Ca2+ dependent cyclase component but a complete abolition of the NaF stimulated component.

摘要

在没有氟离子（F-）的情况下，5×10⁻⁵ M的乙二醇双（β-氨基乙醚）N，N'-四乙酸（EGTA）可抑制牛脑皮层腺苷酸环化酶（ATP焦磷酸裂解酶（环化），EC 4.6.1.1）活性的84% - 88%；而在有氟离子存在时，5×10⁻⁵ M的EGTA仅能抑制其活性的50% - 60%。在这两种情况下，进一步增加EGTA浓度并不会改变抑制程度。在两种情况下，加入3×10⁻⁵ M的钙离子（Ca²⁺）（使游离钙离子浓度约为2×10⁻⁶ M）、5×10⁻⁵ M的锰离子（Mn²⁺）或钴离子（Co²⁺）可使抑制作用完全逆转，加入5×10⁻⁵ M的锶离子（Sr²⁺）可部分逆转，而加入5×10⁻⁵ M的钡离子（Ba²⁺）、锌离子（Zn²⁺）、镍离子（Ni²⁺）或铁离子（Fe²⁺）则不能逆转。在有氟离子存在时，7.2×10⁻⁵ M的游离钙离子可显著抑制环化酶活性。用1.8%的 Triton X - 100进行增溶处理后，酶制剂不再受氟化钠（NaF）刺激，并且在有无NaF的情况下，加入5×10⁻⁵ M的EGTA均可使其活性被100%抑制。然而，与EGTA不同，乙二胺四乙酸（EDTA）在有无NaF的情况下对腺苷酸环化酶均无明显影响，且EGTA不影响ATP酶或磷酸二酯酶的活性。通过假设脑皮层中存在两种独立的酶成分可对这些数据作出合理解释：一种成分可被NaF激活约6倍，低浓度的Ca²⁺和Mg²⁺可增强NaF的作用。第二种成分完全依赖Ca²⁺，并受到高浓度F-的抑制。Mn²⁺、Co²⁺和Sr²⁺似乎在体外可替代两种酶系统中的Ca²⁺。在此基础上，Triton X - 100处理可使依赖Ca²⁺的环化酶成分的比活性增加约3倍，但会使受NaF刺激的成分完全丧失活性。