Treatment Of Microsomal Fraction Research Articles

A Rare UGT2B7 Variant Creates a Novel N-Glycosylation Site at Codon 121 with Impaired Enzyme Activity.

The UDP glucuronosyltransferase (UGT) superfamily comprises glycoproteins that reside in the endoplasmic reticulum membranes and that undergo post-translational modifications (PTMs). UGT2B7 is of particular interest because of its action on a wide variety of drugs. Most studies currently survey common variants and examine only a small fraction of the genetic diversity; however, rare variants (frequency <1%) might have a significant effect because they are predicted to greatly outnumber common variants in the human genome. We discovered a rare single nucleotide UGT2B7 variant of potential pharmacogenetic relevance that encodes a nonconservative amino acid substitution at codon 121. This low-frequency variation, found in two individuals of a population of 305 healthy volunteers, leads to the translation of an asparagine instead of an aspartic acid (UGT2B7 p.D121N). This amino acid change was predicted to create a putative N-glycosylation motif NX(S/T) subsequently validated upon endoglycosidase H treatment of microsomal fractions and inhibition of N-glycosylation of endogenously produced UGT2B7 with tunicamycin in human embryonic kidney (HEK293) cells. The presence of an additional N-linked glycan on the UGT2B7 enzyme, likely affecting proper protein folding, resulted in a significant decrease of 49% and 40% in the formation of zidovudine and mycophenolic acid glucuronides, respectively. A systematic survey of the Short Genetic Variations database uncovered 32 rare, naturally occurring missense variations predicted to create or disrupt N-glycosylation sequence motifs in the other UGT2B enzymes. Collectively, these variants have the potential to increase the proportion of variance explained in the UGT pathway resulting from changes in PTMs, such as N-linked glycosylation with consequences on drug metabolism.

Occurrence of glucocorticoid binding sites in solubilized microsomes from rat liver.

Recent studies suggested the presence of specific glucocorticoid binding sites on rat liver microsomal membranes. We report here a new solubilization procedure which allows the physicochemical characterization of the microsomal glucocorticoid binding sites. Solubilization was achieved with 2 mM CHAPS in the presence of 5 mM benzamidine. Binding of [3H]cortisol showed a high affinity (Kd = 5.1 x 10(-9) M) and a limited capacity (0.72 pmol/mg of protein). The binding activity was abolished by elevated temperature and pronase. Competition experiments revealed that natural glucocorticoids and progesterone were highly potent competitors whereas dexamethasone and triamcinolone acetonide did not compete. Chromatography on DEAE Trisacryl and heparin Ultrogel confirmed that the solubilized protein is different from corticosteroid binding globulin and the cytosolic glucocorticoid receptor. Treatment of microsomal fractions with phosphatidyl inositol phospholipase C promoted the release of specific binding activity suggesting a putative glycosylphosphatidyl anchor for this protein. This finding may have interesting implications concerning the mechanism of glucocorticoid hormone action.

The influence of cholesterol on the activity, on the isothermic kinetics and on the temperature-induced kinetics of 3-hydroxy-3-methylglutaryl coenzyme a reductase

The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase was assayed in liver microsomal preparations from rats fed cholesterol, cholestyramine or the standard diet, without and after treatment with digitonin. The activity of the enzyme in the microsomal fraction was high in the preparations from rats fed cholestyramine and low in the preparations from rats fed cholesterol as compared with the activity of the enzyme in preparations from rats fed the standard diet. The concentration of free cholesterol and of cholesterol esters was different in the microsomal fraction from the rats in the three experimental conditions. Treatment of the microsomal fraction with digitonin resulted in all three cases in solubilization of free cholesterol and of cholesterol esters, but the specific activity of hydroxymethylglutaryl-CoA reductase was higher in the digitonin-treated preparation than in the corresponding microsomal fraction. There was no detectable activity of hydroxymethylglutaryl-CoA reductase in the supernatant obtained after treatment of the microsomal fraction with digitonin. Isothermic kinetics showed that hydroxymethylglutaryl-CoA reductase in the microsomal fraction from rats fed cholesterol has an affinity for substrate that is many times higher than that in the microsomal fraction from rats fed cholestyramine and that the enzyme in microsomes from rats fed the standard diet has an intermediate affinity for substrate. In all three experimental conditions enzyme activity in the microsomal fraction was inhibited at high substrate concentrations. Treatment of the microsomal fraction with digitonin has decreased the apparent K m, in all three conditions, as compared with the value observed with the corresponding untreated microsomes. In all three experimental conditions, preincubation of the enzyme present in the microsomal fraction or in digitonin-treated microsomal preparation, resulted in an increased V, but the apparent K m value obtained with the preincubated microsomal fraction was similar to the value obtained with the corresponding microsomal fraction assayed without preincubation. Preincubation revealed hydroxymethylglutaryl-CoA reductase activity in the supernatant obtained from the treatment of the microsomal fraction with digitonin, in all three experimental conditons. The soluble enzymes, from cholestyramine-fed rats and from rats fed the standard diet, had identical apparent K m values. The differences in the Arrhenius plots of hydroxymethylglutaryl-CoA reductase in the microsomal fractions from rats in the three experimental conditions and the changes brought about by the treatment of the various microsomal fractions with digitonin are consistent with the possibility that such differences are due to changes in the free cholesterol concentration in the membrane of the various microsomal preparations. It is suggested that there is a pool of free cholesterol in the immediate lipid environment of hydroxymethylglutaryl-CoA reductase and that the size of this pool is important for the intracellular regulation of hydroxymethylglutaryl-CoA reductase.

Interactions of hormones with thymus-cell fractions

The binding of cortisol, progesterone and testosterone to thymus-cell fractions was studied by the equilibrium dialysis procedure. The hormones appear to follow the “polarity rule”, progesterone binding to the greatest extent, cortisol the least. Ribosomal particles seem to have a higher capacity for binding hormone than the pH 5.1 precipitate or the material in the supernatant resulting from treatment of microsomal fraction with deoxycholate.The possibility is discussed that progesterone is binding to a single component in the ribosomal fraction while cortisol and testosterone bind to more than one component.