Biochemical study of a kazal type trypsin inhibitor from mouse seminal vesicle secretion

• Main Authors: Lai,Min-Long, 賴明龍
• Other Authors: Chen,Yee-Hsiung
• Format: Others
• Language: zh-TW
• Published: 1993
• Online Access: http://ndltd.ncl.edu.tw/handle/03063349080939818375

博士 === 國立臺灣大學 === 生化科學研究所 === 81 === A Kazal-type trypsin inhibitor purified from mouse seminal vesicle secretion. It was shown to be a weak basic protein with an isoelectric point of 8.7 and it contained no carbohydrate. The protein had a specific activity of 184 U/ miligram protein. Analysis of the kinetic data revealed that the protein was a competitive inhibitor with an inhibitory constant (Ki) of 0.15 nana Mol. The molcular mass of the protein was determined to be 7000. Results of direct amino acid sequence determinations indicated that this protein corresponded to the reading frame of MP12 cDNA manipulated from mouse prostate (Mills et al., 1987a). E. coli cells were transformed with an expression vector constructed by inserting the DNA sequence encoded for this inhibitor into pGEX-2. The chimeric polypeptide could be partially purified, was digested further with thrombin and a recombinant trypsin inhibitor was isolated. Antibody induced from the chimeric polypeptide could recognize native inhibitor specifically. The trypsin inhibitor bound to the acrosomal cap region of sperm head which appeared as a crescent-shaped fluorescence as the cells were stained by indirect immunofluorescent technique. Immunoaggregation of the bound trypsin inhibitor on the sperm surface resulted in acrosome reaction. The trypsin inhibitor showed caltrin activity. It could suppress the calcium uptake of the capacitated spermatozoa. Cleavage at the reactive site Arg-Ile of the protein lost the inhibitory effect on trypsin activity but retained the caltrin activity even stronger than the native protein. This trypsin inhibitor may function during the early stages of sperm transit through the female reproductive tract to prevent a premature onset of the acrosome reaction by inhibition of the calcium transport prior to acrosome reaction.