alpha-chymotrypsin and 7-amino-4-trifluoromethylcoumarin

The proteasome displays three distinct proteolytic activities. Currently, proteasome inhibitors are evaluated using specific fluorescent substrates for each of the individual active sites. However, the photophysical properties of the commonly used fluorophores are similar and thus, the simultaneous monitoring of the three proteolytic activities is not possible. We have developed a bimodal fluorescent fluorinated substrate as a novel tool to study the chymotrypsin-like (ChT-L) proteolytic activity and its regulation by inhibitors and by substrates of trypsin-like (T-L) and caspase-like sites (PA). We demonstrate that this substrate is reliable to evaluate the ChT-L inhibitory activity of new molecules either by fluorescence or (19)F NMR spectroscopy. We have found that the ChT-L activity is dramatically reduced in the presence of T-L and PA substrates. This work provides a proof of concept that the fluorinated substrate enables investigation of the allosteric regulation of the ChT-L activity.

Topics: Allosteric Regulation; Animals; Chymotrypsin; Coumarins; Fluorine; Hydrazines; Inhibitory Concentration 50; Magnetic Resonance Spectroscopy; Oligopeptides; Peptides; Proteasome Endopeptidase Complex; Rabbits; Substrate Specificity

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.

Topics: Animals; Chromatography, Ion Exchange; Chymotrypsin; Coumarins; Electrophoresis, Polyacrylamide Gel; Guinea Pigs; Hymecromone; Kinetics; Male; Protease Inhibitors; Spectrophotometry, Ultraviolet; Sperm Maturation; Substrate Specificity; Testis; Trypsin; Trypsin Inhibitors