Purification and Some Properties of Membrane Guanylate Cyclase in Rat Brain.

1.我们在突触质膜上发现了一种膜结合型鸟苷环化酶，该酶由Ca~(2+)和不饱和脂肪酸激活。环化酶的激活需要GTP或其类似物的共存。在保留了被钙、GTP和不饱和脂肪酸激活的特性的基础上，对环化酶进行了纯化。经离子交换、GTP亲和、疏水亲和、WGA亲和、凝胶过滤等步骤，得到高纯度的环化酶。最终纯化的酶被上述激活剂刺激，但幅度小于膜中的激活剂。用最终纯化的酶与丙烯酰胺凝胶中的不饱和脂肪酸、GTP和Ca^-lt；2-gt；进行结合，以确定参与活性的酶的亚基。利用计算机程序分析了GTP、Ca^&lt；2&gt；和不饱和脂肪酸激活的动力学特性。在测试的几种化合物中，有一组化合物被发现选择性地抑制环化酶。这一发现有助于阐明该循环酶在细胞功能中的生理作用。研究了不饱和脂肪酸在激活过程中的专一性。观察到不饱和脂肪酸-过氧化氢是无效的。

1. We found a membrane bound type of guanylate cyclase activated by Ca^<2+> and unsaturated fatty acid in synaptic plasma membranes. The activation of the cyclase required co-existence of GTP or its analogues. The cyclase was tried to be purified, under retaining the properties of being activated by Ca^<2+>, GTP and unsaturated fatty acid. After several steps of chromatographies ; ion exchange, GTP-affinity, hydrophobic affinity, WGA-affinity and gel filtration, the highly purified cyclase was obtained. The finally purified enzyme was observed to be stimulated by the activators above, but in less extent than that in the membranes.2. The binding of Ca^<2+>, GTP and unsaturated fatty acid to bands in acrylamide gels from SDS-PAGE with the finally purified enzyme was examined to identify sub-units of the enzyme which participated in the activation.3. Kinetic properties of the activation by GTP, Ca^<2+> and unsaturated fatty acid were analyzed by using computer programs.4. Among several chemical compounds tested, a group of compounds was found to inhibit the cyclase selectively. This finding is useful in elucidating a physiological role of this cyclase in cell function.5. Specificity of unsaturated fatty acid in the activation was studied. Unsaturated fatty acid-peroxide was observed to be ineffective.