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T A Bicsak

Publications and source records attributed to T A Bicsak.

23 records · Page 2Linked to original sources
PubMed1986

Hormonal regulation of granulosa cell inhibin biosynthesis.

The hormonal regulation of inhibin production by cultured granulosa cells from immature hypophysectomized, estrogen-treated rats was examined using a specific RIA which detects the N-terminal portion of the inhibin alpha-chain. The RIA measured bioactive inhibin of Mr about 32,000 in granulosa cell conditioned media fractionated by fast protein liquid chromatography. In the presence of 10(-7) M androstenedione, FSH stimulated inhibin production in a dose-dependent manner during a 2-day culture. Inclusion of a phosphodiesterase inhibitor decreased the EC50 for FSH from 2.6 to 0.8 ng/ml (n = 3). The stimulatory effect of FSH could be mimicked with forskolin (an adenyl cyclase activator) and with a cAMP analog, (Bu)2cAMP, consistent with FSH action mediated through a cAMP dependent pathway. Intracellular levels of inhibin were unmeasureable, suggesting that inhibin is not stored to any great extent by the granulosa cells. This finding was consistent with in vivo studies which showed that whereas FSH treatment for 2 days doubled serum inhibin levels when compared with basal levels, there was no increase in the concentration of extractable inhibin in ovarian tissue. Granulosa cells which had been exposed to 20 ng/ml FSH for 2 days to induce LH receptors produced inhibin in response to both LH and human CG during the subsequent 2-day culture, with the levels of inhibin equalling the amount inducible by FSH. In contrast, neither PRL nor terbutaline, a beta 2-adrenergic agonist, had any effect on inhibin production even though receptors for these hormones are also induced by FSH. GnRH was found to inhibit the FSH-stimulated production of inhibin (IC50, 10(-7) M), consistent with previous observations that GnRH can act at the ovarian level to inhibit granulosa cell differentiation. This inhibition by GnRH could be reversed by inclusion of a specific GnRH antagonist. On the other hand, another regulatory peptide, vasoactive intestinal peptide, slightly stimulated inhibin production. The effect of several growth factors was also tested. Insulin-like growth factor I raised not only FSH-stimulated inhibin levels, but basal levels as well. Insulin was also effective, but only at 100-fold higher concentration. Epidermal growth factor inhibited FSH-stimulated inhibin production (IC50 = 0.1 ng/ml), whereas fibroblast growth factor had no effect. Thus, granulosa cell inhibin secretion is regulated by FSH and LH but not by PRL, presumably via a cAMP-mediated pathway.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Purification of nonspecific protease-free collagenase from Clostridium histolyticum.

The collagenase (EC 3.4.24.3) produced by the bacterium Clostridium histolyticum has been purified free from nonspecific protease contaminants by a two-step procedure. The crude culture medium is chromatographed over heparin-Sepharose and Sephacryl S-200, and the resulting preparation has no activity versus noncollagenous proteins or N alpha-benzoyl-L-arginine ethyl ester, yet cleaves native thermally reconstituted collagen fibrils quite efficiently (specific activity, 3000 units/mg). The purification described may be useful for those investigators requiring substantially purified collagenase for applications such as cell culture or collagen quantitation in protein mixtures.

Arginine↗
PubMed1985

Chemical and kinetic characterization of tadpole back skin collagenase.

The purified collagenase from tadpole (Rana catesbiana) back skin was studied with respect to its activation energy using soluble and fibrillar type I collagen, as well as a synthetic peptide substrate, DNP-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg. The activation energy appeared to be independent of the nature of the substrate, ranging between 28 and 35 kcal/mol. The peptide was cleaved at the Gly-Ile bond and proved to be a poor substrate (kcat/Km, 1.21 h-1 microM-1) when compared with native type I collagen in solution (kcat/Km, 40.6 h-1 microM-1), consistent with the enzyme's low activity versus gelatin [T. A. Bicsak and E. Harper (1984) J. Biol. Chem. 259, 13145]. The amino acid composition of the collagenase was shown to be high in glycine and glutamic acid, and the preparation was shown not to be contaminated with collagen by digestion with bacterial collagenase. The enzyme was not inhibited by iodoacetic acid or 2-hydroxy-5-nitrobenzyl bromide, suggesting the lack of essential cysteinyl and tryptophanyl residues, but was inhibited by micromolar concentrations of ZnCl2, consistent with the presence of essential histidine(s). Ethoxyformic anhydride irreversibly inhibited the collagenase suggesting the presence of essential lysyl residues.

Amino Acids↗
PubMed1984

Purification and characterization of tadpole back-skin collagenase with low gelatinase activity.

A collagenase secreted by tadpole (Rana catesbiana) back-skin explants in culture has been purified to electrophoretic homogeneity by successive chromatography on sulfopropyl Sephadex, Sephacryl S-200, collagen Sepharose, and heparin Sepharose. The purified enzyme has a molecular weight of approximately 49,000 and an isoelectric pH of 5.0. The enzyme is more active versus soluble collagen than reconstituted fibrils and exhibits very low activity against gelatin (specific activities: Type I collagen, 7660 units/mg; Type I gelatin, 66 units/mg). The collagenase obeys simple Michaelis-Menten kinetics using soluble type I collagen (Km), 0.35 microM; Vm, 1380 units/mg, at 25 degrees C and pH 7.4) and is inhibitable by chelating agents specific for transition metals. Methylene blue catalyzes the photoinactivation of this collagenase, suggesting the presence of essential histidine, tryptophan, tyrosine, or methionine residues.

Animals↗