Purification and preliminary characterization of guinea pig testicular proteinase inhibitors.
Three guinea pig testicular, low-molecular-weight, acid-stable inhibitors specific for trypsin-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 x 10(-8) M; for B, 1.5 x 10(-8) M; and, for Cnb, 2.2 x 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 x 10(-9) M; for B, 6.7 x 10(-9) M; and, for Cnb, 1.4 x 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 B indicates that it may function as a dimer or trimer in the active state.
Falase
E A
EA
Department of Obstetrics and Gynecology, University of Pennsylvania School of Medicine, Philadelphia.
Storey
B T
BT
Teuscher
C
C
D000818:Animals; D002852:Chromatography, Ion Exchange; D002918:Chymotrypsin / Q000378:metabolism; D003374:Coumarins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006168:Guinea Pigs; D006923:Hymecromone / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D008297:Male; D011480:Protease Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013079:Sperm Maturation / Q000502:physiology; D013379:Substrate Specificity; D013737:Testis / Q000737:chemistry*; D014357:Trypsin / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
C017532:4-methylumbelliferylguanidinobenzoate; D003374:Coumarins; D011480:Protease Inhibitors; D014361:Trypsin Inhibitors; D006923:Hymecromone; C055467:7-amino-4-trifluoromethylcoumarin; D002918:Chymotrypsin; D014357:Trypsin