ISSN:
1040-452X
Keywords:
Trypsin-like
;
Proteinase inhibitors
;
Testes
;
Sperm
;
Male reproductive tract
;
Life and Medical Sciences
;
Cell & Developmental Biology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
Notes:
Three guinea pig testicular, low-molecular-weight, acid-stable inhibitors specific for typsin-like proteinases were isolated, purified, and characterized. The procedure comprised acid extraction of testicular acetone powder, pH precipitation of the extract, gel filtration of the supernatant on Sephadex G-100 and G-50, ion-exchange chromatography on SP-Sephadex, followed by QAE-Sephadex. Final purification was by rechromatography on Sephadex G-50 superfine gel. The three proteinase inhibitors were labeled A, B, and Cnb, the latter to denote nonbinding of Cnb to the QAE-Sephadex. Components A and Cnb showed competitive, whereas B showed noncompetitive, inhibition against trypsin. All three inhibitors were active against trypsin but were ineffective against chymotrypsin. The inhibition constants, Ki, were obtained using trypsin-catalyzed hydrolysis of the N-benzyloxycarbonyl-L-arginyl amide of 7-amino-4-trifluoro-methylcoumarin (CBZ-Arg-AFC) at pH 8.0. The values were calculated to be, for A, 1.5 × 10-8 M; for B, 1.5 × 10-8 M; and, for Cnb, 2.2 × 10-7 M. The Ki values calculated from inhibition of trypsin-catalyzed hydrolysis of the active site titrant 4-methylumbelliferyl-p-guanidinobenzoate (MUGB) using Easson-Stedman plots were, for A, 7.7 × 10-9 M; for B, 6.7 × 10-9 M; and, for Cnb, 1.4 × 10-7 M. The MrS as determined by active site titration with MUGB were A, 11.2 kDa; B, 10.5 kDa; Cnb, 17.0 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave Mr values for A of 11 kDa, for B of 4 kDa, and for Cnb of 19 kDa. The discrepancy in Mr values for A and 11 kDa, for B of 4 kDa, and for Cnb of 19 kDa. The discrepancy in Mr values for B indicates that it may function as a dimer or trimer in the active state.
Additional Material:
8 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/mrd.1080290106
Permalink