ABSTRACT
BACKGROUND: Prostate-specific antigen (PSA) is a serine protease that can cleave insulin-like growth factor-binding protein-3 (IGFBP3), thereby decreasing its affinity for insulin-like growth factor-I (IGF-I). Dissociation of the IGF-I-IGFBP3 complex renders IGF-I available to bind to its receptor and stimulates cellular proliferation. We evaluated the potential for PSA to modulate the effects of IGF-I and IGFBP3 on the proliferation of human benign prostatic hyperplasia (BPH)-derived fibromuscular stromal cells in primary cultures. METHODS: We cultured BPH-derived stromal cells for 48 hours in serum-free RPMI-1640 medium supplemented with 0.2% bovine serum albumin and studied the effects of IGF-I, IGFBP3, PSA, and ZnCl(2) at varying concentrations. Differences in cell growth between control and treated cultures were evaluated by use of Dunnett's test. Concentration-related trends were evaluated by linear regression of log-transformed concentrations of test reagents on BPH-derived stromal cell number responses. Statistical tests were two-sided. RESULTS: We observed a concentration-dependent proliferative response of BPH-derived stromal cells to IGF-I. IGFBP3 inhibited this response in a concentration-dependent fashion. IGFBP3 alone had no effect on stromal cell proliferation. When stromal cells were incubated with PSA alone or with PSA, IGF-I, and IGFBP3, an increase in stromal cell numbers that was dependent on PSA concentration was evident in both instances. Zinc, an endogenous inhibitor of PSA enzymatic activity, was able to attenuate the stimulatory effect of PSA at intraprostatic physiologic concentrations. CONCLUSIONS: These results are consistent with the idea that PSA can modulate in vitro interactions between IGF-I and IGFBP3 and suggest that PSA may play a role in the regulation of human prostatic fibromuscular cell growth.
Subject(s)
Chlorides/metabolism , Insulin-Like Growth Factor Binding Protein 3/metabolism , Insulin-Like Growth Factor I/metabolism , Prostate-Specific Antigen/metabolism , Prostate/growth & development , Prostate/metabolism , Prostatic Hyperplasia/metabolism , Zinc Compounds/metabolism , Cell Division , Cells, Cultured , Chymotrypsin/metabolism , Humans , Male , Recombinant Proteins/metabolismABSTRACT
The ketone cinnamoyl-(1-13C-Phe)-CGly-Pro-Pro [(4-13C-5-cinnamido-4-oxo-6-phenylhexanoyl)-Pro-Pro 2] competitively inhibits a mixture of collagenases from Clostridium histolyticum with a Ki of 40 +/- 6 nM. 13C-nmr spectroscopy of the ketone in the presence of this collagenase shows a bound 13C resonance at 102.6 ppm and the resonance of the free ketone at 212 ppm. Ketone alone shows no trace (less than 0.5%) of a resonance in the region around 100 ppm. The bound resonance is displaceable by another competitive inhibitor. This ketone is thus a transition state analog which is rehybridized from trigonal planar to tetrahedral upon binding to collagenase.
Subject(s)
Clostridium/enzymology , Dipeptides/pharmacology , Ketones/pharmacology , Microbial Collagenase/antagonists & inhibitors , Binding Sites , Binding, Competitive , Kinetics , Structure-Activity Relationship , Substrate SpecificityABSTRACT
(dl)-2-Benzyl-4-oxo-5,5,5-trifluoropentanoic acid is a strong transition state analog inhibitor of the zinc protease carboxypeptidase A. 19F NMR spectroscopy of the aqueous solution of this inhibitor shows the hydrate of the ketone carbonyl to be the major species, with a shift of -9.95 ppm. As the pH is varied from 4.9 to 13.1, a 1.53 ppm downfield shift occurs, giving a pK alpha of 11.10. When excess inhibitor is added to the enzyme, a new, bound peak appears at -8.84 ppm, in addition to the free hydrate peak. Spectra taken at pH's from 4.90 to 9.15 show no change in the position of the bound resonance; from 9.15 to 12.15, a 0.26 ppm upfield shift occurs. The interpretation is that the monoanion of the hydrate is the form that binds to the enzyme.
