Benau D A, Storey B T
Department of Obstetrics and Gynecology, University of Pennsylvania School of Medicine, Philadelphia 19104.
Biol Reprod. 1988 Sep;39(2):235-44. doi: 10.1095/biolreprod39.2.235.
There are at least two binding sites for the mouse egg zona pellucida on the surface of mouse sperm: a site with galactosyltransferase (GT) activity inhibitable by uridine-5'-diphosphate-dialdehyde (UDPd) and alpha-lactalbumin, and a trypsin inhibitor-sensitive (TI) site that hydrolyzes guanidinobenzoate (GB) esters. Characterization of GT activity gave the Km for UDP galactose as 37 microM with N-acetylglucosamine as galactose acceptor, and Vmax as 0.37 pmol/min/10(6) sperm. UDP galactose from 12.5-100 microM inhibited sperm binding to zona-intact eggs in a concentration-dependent manner with close correlation to GT activity (r = 0.95). To assess the independence and spatial relationship of the two types of site, cross-perturbation studies were performed. p-Nitrophenyl-GB, a low molecular mass inhibitor specific for the TI site, had no effect on the enzyme activity of the GT site. Conversely, UDPd, a specific inhibitor of GT, had no effect on GB hydrolysis. Weak inhibitions were found when soybean trypsin inhibitor (SBTI) was included with the GT assay and when GB hydrolysis was assayed in the presence of alpha-lactalbumin or asialo-agalacto-(alpha 1-acid glycoprotein). Acid-solubilized zona protein (ASZP) weakly inhibited the GT reaction, while stronger inhibition was seen with chymotrypsin-solubilized zona protein (CSZP). ASZP inhibited sperm binding to zonae with the same concentration dependence associated with inhibition of GB hydrolysis, but the inhibition of GT enzyme activity was on the same order as that found with SBTI, indicating that ASZP was only binding to the TI site under enzyme assay conditions. The results support the hypothesis that the two types of site are independent in binding their specific zona ligands, but are close enough for steric perturbation of the enzyme activity of one site by macromolecules bound to the other. The different interactions of solubilized zona preparations with the GT site under enzyme assay conditions are an indication that conditions which favor the enzyme activity of the site may interfere with the physiological binding functions of the site.
一个具有半乳糖基转移酶（GT）活性的位点，其活性可被尿苷-5'-二磷酸二醛（UDPd）和α-乳白蛋白抑制；另一个是对胰蛋白酶抑制剂敏感（TI）的位点，可水解胍基苯甲酸酯（GB）。对GT活性的表征显示，以N-乙酰葡糖胺作为半乳糖受体时，UDP半乳糖的Km为37μM，Vmax为0.37 pmol/min/10⁶精子。12.5 - 100μM的UDP半乳糖以浓度依赖的方式抑制精子与完整透明带卵的结合，且与GT活性密切相关（r = 0.95）。为评估这两种位点的独立性和空间关系，进行了交叉干扰研究。对TI位点具有特异性的低分子量抑制剂对硝基苯基-GB对GT位点的酶活性没有影响。相反，GT的特异性抑制剂UDPd对GB水解没有影响。当大豆胰蛋白酶抑制剂（SBTI）与GT测定一起使用时，以及当在α-乳白蛋白或去唾液酸-去半乳糖基-（α1-酸性糖蛋白）存在的情况下测定GB水解时，发现有微弱的抑制作用。酸溶性透明带蛋白（ASZP）对GT反应有微弱抑制，而胰凝乳蛋白酶溶性透明带蛋白（CSZP）的抑制作用更强。ASZP以与抑制GB水解相关的相同浓度依赖性抑制精子与透明带的结合，但对GT酶活性的抑制程度与SBTI相当，这表明在酶测定条件下ASZP仅与TI位点结合。结果支持这样的假设，即这两种位点在结合其特定的透明带配体时是独立的，但彼此距离足够近，以至于与一个位点结合的大分子会对另一个位点的酶活性产生空间干扰。在酶测定条件下，可溶性透明带制剂与GT位点的不同相互作用表明，有利于该位点酶活性的条件可能会干扰该位点的生理结合功能。
