Studies on ram acrosin. Isolation from spermatozoa, activation by cations and organic solvents, and influence of cations on its reaction with inhibitors.
Studies on ram acrosin. Isolation from spermatozoa, activation by cations and organic solvents, and influence of cations on its reaction with inhibitors.
复制标题
公羊顶体蛋白的研究。
作者：
C. Brown;Z. Andani;E. Hartree
1.本文介绍了一种从绵羊精子中分离出一种水溶性的顶体酶（一种类似胰蛋白酶的酶）的简单方法。它不含位于精子中的顶体酶抑制剂。2.在N-α-苯甲酰基-L-精氨酸乙酯的水解中，Ca（2+）和其它阳离子对顶体酶的激活程度取决于抑制剂的污染程度。在50 mm-Tris-HCl缓冲液（pH8.2）中，Ca（2+）对顶体酶的激活不超过40%，但部分抑制的顶体酶可激活高达300%，这是由于阳离子介导的顶体酶对抑制剂的保护作用。3.增加缓冲液浓度（如Tris）也能激活顶体酶，但在高于一定缓冲液浓度时，Ca（2+）不再发挥激活作用，可能会成为抑制性的。Ca（2+）在加入到含有具有螯合特性的阴离子缓冲液的测定系统中时也具有抑制作用。这是由于pH值下降。上述结果表明，在处理Ca（2+）对顶体酶活性的影响的论文中，结论相互矛盾的原因。5. Kunitz胰蛋白酶抑制剂对顶体酶的抑制作用在加入Ca（2+）后增强。顶体酶抑制剂和Kunitz抑制剂对胰蛋白酶的抑制作用对Ca（2+）不敏感。6.与胰蛋白酶一样，顶体蛋白酶可被2-甲基-丙-2-醇、二甲基亚砜和其他一些水溶性溶剂活化高达60%。阳离子和溶剂的作用往往是加和的，并且在测定系统中，不同浓度的溶剂、盐和缓冲液可以实现共同的最大顶体酶活性。当存在低浓度的顶体酶抑制剂时，溶剂的活化增加。7.当底物是N-α-苯甲酰基-dl-精氨酸2-萘酰胺时，盐和溶剂对顶体酶的激活作用更明显。8.公羊顶体酶（约0.2mm）的K（m）值远高于胰蛋白酶，K（cat.）值略高于胰蛋白酶的值。考虑离子和二甲基亚砜对顶体酶和胰蛋白酶的活性和动力学常数的影响表明，构象变化的因素主要负责报道的顶体酶的激活。9.得出以下结论。(a)顶体酶在精子穿透卵细胞而不脱离顶体膜中起作用。(b)该酶是一种外周膜蛋白，可归类为组织蛋白酶。(c)可溶性顶体酶活性对阳离子和溶剂的敏感性表明其为柔性分子，即缺乏与顶体膜结合（推测为离子）所施加的构象限制的分子。
1. A simple method is given for isolating from ram spermatozoa a water-soluble form of acrosin (a trypsin-like enzyme) which is about 25% pure. It is free from an acrosin inhibitor which is located in the spermatozoa. 2. In the hydrolysis of N-alpha-benzoyl-l-arginine ethyl ester the degree of activation of acrosin by Ca(2+), and by some other cations, is dependent on the extent of contamination by the inhibitor. In 50mm-Tris-HCl buffer (pH8.2) activation by Ca(2+) did not exceed 40%, but acrosin that is partially inhibited may be activated by up to 300%: this is due to cation-mediated protection of acrosin against the inhibitor. 3. Increasing concentrations of buffers (e.g. Tris) also activate acrosin but at above certain buffer concentrations Ca(2+) no longer exerts an activating effect and may become inhibitory. Ca(2+) is also inhibitory when added to assay systems involving anionic buffers with chelating properties. This is due to a fall in pH. 4. The above results suggest reasons for conflicting conclusions in papers dealing with the effects of Ca(2+) on acrosin activity. 5. Inhibition of acrosin by the Kunitz pancreatic trypsin inhibitor is increased on addition of Ca(2+). Inhibitions of trypsin by the acrosin inhibitor and by the Kunitz inhibitor are insensitive to Ca(2+). 6. Like trypsin, acrosin is activated, up to 60%, by 2-methyl-propan-2-ol, dimethyl sulphoxide, and some other water-miscible solvents. Effects of cations and solvents tend to be additive and a common maximum acrosin activity can be achieved with various concentrations of solvent, salts and buffer in the assay system. Activation by solvents is increased when low concentrations of the acrosin inhibitor are present. 7. Activations of acrosin by salts and by solvents are more pronounced when the substrate is N-alpha-benzoyl-dl-arginine 2-naphthylamide. 8. K(m) values for ram acrosin (about 0.2mm) are much higher than those for trypsin, and k(cat.) values are slightly higher than those for trypsin. Considerations of the influences of ions and dimethyl sulphoxide on the activities and kinetic constants of acrosin and trypsin suggest that conformational changes are the factors mainly responsible for the reported activations of acrosin. 9. The following conclusions are reached. (a) Acrosin plays a role in the penetration of the sperm cell into the egg without becoming detached from the acrosomal membrane. (b) The enzyme is a peripheral membrane protein which may be classed as a cathepsin. (c) The susceptibility of the activity of soluble acrosin to cations and solvents points to a flexible molecule, i.e. one lacking conformational restraints imposed by association (presumably ionic) with the acrosomal membrane.