Six variants of P12, a Kazal-type trypsin inhibitor in the secretion of male mouse accessory sexual glands, were made using single-site mutations including R19L, Y21V, D22G, R43G, K44S, and R45T, based on one-letter-code mutation of amino acids. The other two variants, Nd10 and Cd8, were made using the deletion of 10 and 8 residues from the N-and C-terminals, respectively. Their CD profiles revealed maintenance of the P12 conformation in the seven variants, excluding Cd8, which became unfolded.

more »

... Only R19L entirely lost the ability while the other variants were as strong as P12 in inhibiting the trypsin digestion of N-benzoyl-Phe-Val-Arg 7-amido-4-methylcoumarin. The immunocytochemical results demonstrated that D22G and Cd8 failed to bind to sperm, Y21V very weakly did so, and the other variants retained their sperm-binding abilities. Concomitantly, the immunocytochemical stainability of each ligand was parallel to its inhibitory effect on 125 I-P12-sperm binding, and a synthetic oligopeptide corresponding to residues 18-24 of P12 was able to inhibit P12-sperm binding. The data together concluded that R 19 was essential for protease inhibition and D 22 and/or Y 21 mainly being responsible for the binding of P12 to sperm. The steric arrangement of R 19 , Y 21 , and D 22 on the tertiary structure of P12 is discussed. gamete biology, male reproductive tract, male sexual function, seminal vesicles, sperm