Kenneth R. Cutroneo

Induction of benzpyrene hydroxylase by flavone and its derivatives in fetal rat liver explants

Abstract Fetal rat liver organ culture affords a system for the comparison of the mechanism(s) of induction∗ of benzpyrene (BP) hydroxylase by flavones and 3-methyl-cholanthrene(3-MC). Flavone and β-naphthoflavone (BNF) were equally potent inducers at 10−5 M. 4′-halogenated flavone derivatives proved even more effective in this regard; flavanone and the naturally occuring flavone, tangeretin, were inactive. Although BP hydroxylase activity was inhibited by BNF, 3-MC or 4′-bromoflavone when added to homogenates of fetal rat liver explants, no interference by the inducer was observed under the experimental conditions employed in this study. When equal amounts of the induced and control enzyme were mixed, no less than additive enzyme activity was observed. Flavone derivatives and 3-MC appear to act by a similar mechanism in elevating BP hydroxylase, since combinations of inducers at unsaturating levels resulted in an additive effect, while at saturating levels, enzyme induction was no greater than that of the most potent agent alone.

Collagen lysyl hydroxylation occurs within the cisternae of the rough endoplasmic reticulum

Abstract Resolution of the heavy microsomal fraction of lung tissue by Ficoll density gradient centrifugation yielded a rough endoplasmic reticulum microsomal fraction containing the highest specific activity of detergent-released lysyl hydroxylase. This same microsomal fraction was previously shown to contain the highest specific activity of detergent-released prolyl hydroxylase activity. When hydroxylation was inhibited during the biosynthesis of collagen, this microsomal fraction contained lysine-rich, hydroxylysine-deficient, collagenase-digestible substrate that could be hydroxylated in the absence of detergent. The results indicate coordinate localization of both prolyl and lysyl hydroxylation reactions within the cisternae of the rough endoplasmic reticulum.

Association of prolyl hydroxylase activity with membranes

Abstract Addition of ionic and nonionic detergents to whole homogenates of liver, kidney and lung prepared by a mild homogenization technique resulted in a two- to three-fold increase of prolyl hydroxylase activity. After subcellular fractionation of whole homogenates of liver, particulate and supernatant fractions were incubated in the presence and absence of triton X-100 and assayed for prolyl hydroxylase activity. All particulate fractions tested were able to release significant amounts of prolyl hydroxylase activity in the presence of triton. The release of enzyme activity by triton was observed with the 1000 × g and 17,000 × g supernatants but not with the 105,000 × g supernatant; thus indicating that detergent does not activate soluble enzyme nor make the substrate more accessible to hydroxylation by the enzyme during incubation. Rigorous homogenization of the 17,000 × g particulate fraction with the Polytron ST system resulted in a substantial loss of the amount of prolyl hydroxylase activity released by treatment with triton. These data suggest that a significant amount of prolyl hydroxylase activity is associated with membranes under physiological conditions.

Vitamin A prevention of triamcinolone acetonide effects on granuloma growth: lack of effect on prolyl hydroxylase.

Summary Vitamin A administered concurrently with triamcinolone acetonide prevented the effects of steroid on granuloma wet weight, vascularity, hydroxyproline, protein, and DNA. Although vitamin A blocked the general inhibitory effects of glucocorticoid treatment on granuloma growth, decreases of both prolyl hydroxylase activity and proteinaceous hydroxyproline formation were observed. These results indicate that vitamin A did not prevent the specific effects of glucocorticoids on prolyl hydroxylase activity and hydroxyproline formation.SummaryVitamin A administered concurrently with triamcinolone acetonide prevented the effects of steroid on granuloma wet weight, vascularity, hydroxyproline, protein, and DNA. Although vitamin A blocked the general inhibitory effects of glucocorticoid treatment on granuloma growth, decreases of both prolyl hydroxylase activity and proteinaceous hydroxyproline formation were observed. These results indicate that vitamin A did not prevent the specific effects of glucocorticoids on prolyl hydroxylase activity and hydroxyproline formation.

The diurnal variation of peptidyl proline hydroxylase activity in different tissues of the rat.

Abstract The activity of peptidyl proline hydroxylase undergoes diurnal fluctuations in the aorta, heart, liver, lung and skin of the rat. Miximal enzyme activity was noted during the dark period in skin and during the light period in the other tissues examined. Further differences between skin and liver were noted in their responses to adrenalectomy and to hydrocortisone treatment. The data implicate adrenal steroids in the rhythmicity of peptidyl proline hydroxylase in skin but not in liver. Furthermore, this system offeres a model to determine the relationship between glucocorticoid induced alterations of peptidyl proline hydroxylase activity and collagen biosynthesis.

In vitro synthesis of collagen peptides on fetal and neonatal rat skin polysomes by rabbit reticulocyte initiation factors

Abstract Salt-washed polysomes isolated from fetal or neonatal rat skin were approximately 50% as active as rabbit reticulocyte salt-washed polysomes (per A 260 unit) in the incorporation of [ 14 C]leucine in a protein-synthesizing system derived entirely from rabbit reticulocytes. Rabbit reticulocyte IF-M 3 stimulated protein synthesis directed by homologous saltwashed polysomes (4- to 5-fold) and by rat skin salt-washed polysomes (3-fold). Aurintricarboxylic acid significantly inhibited protein synthesis by both types of polysomes. Incubation of the reaction products with prolyl hydroxylase from neonatal rat skin resulted in hydroxylation of [3,4- 3 H]peptidyl proline, as determined by both Dowex chromatography and the formation of 3 H 2 O. Digestion of [ 3 H]proline-labeled products with purified collagenase indicates that collagen peptides were synthesized.