Novel bifunctional probe for radioisotope-free photoaffinity labeling: compact structure comprised of photospecific ligand ligation and detectable tag anchoring units.

A novel method for radioisotope-free photoaffinity labeling was developed, in which a bifunctional ligand is connected to a target protein by activation of a photoreactive group, such as an aromatic azido or 3-trifluoromethyl-3H-diazirin-3-yl group, and identification of the ligated product is achieved by anchoring of a detectable tag through the Staudinger-Bertozzi reaction with an alkyl azido moiety that survives photolysis. The chemical ground of this method was confirmed using model compounds with the bifunctional group under photoirradiation in the presence of trapping agents for reactive intermediates. The utility of the method has been demonstrated by specific labeling of the catalytic portion of human HMG-CoA reductase.

Ionizing radiation alters hepatic cholesterol metabolism and plasma lipoproteins in Syrian hamster.

PURPOSE: The investigation of the effects of ionizing radiation on hepatic cholesterol metabolism and the concentration and composition of plasma lipoproteins in the male Syrian hamster. MATERIALS AND METHODS: After sublethal whole-body 60Co gamma-irradiation (8 Gy, 1 Gy/min), plasma lipoproteins were separated by density-gradient ultracentrifugation. Activities of hydroxymethylglutarylCoA (HMGCoA) reductase and of cholesterol 7alpha-hydroxylase were measured in hepatic microsomes and the low-density lipoprotein (LDL) receptor mass was determined in hepatic total membranes. Lipid peroxidation in LDL was assessed in vitro as the formation of conjugated dienes at 234 nm. A group of pair-fed animals served as controls as the food intake was markedly decreased with exposure to radiation. RESULTS: Plasma lipid concentrations decreased 2 days post-irradiation and then markedly increased by day 6 post-irradiation; plasma cholesterol was increased by 77% and triglycerides by +207%. LDL accumulated in plasma while high-density lipoprotein (HDL) levels decreased. HDL contained significant amounts of apo SAA, the acute phase apolipoprotein. The activities of hepatic HMGCoA reductase, the rate-limiting enzyme for cholesterol synthesis, increased (+125%, p=0.06); hepatic cholesterol 7alpha-hydroxylase, the rate-limiting enzyme for bile acid synthesis, decreased (-85%); and the hepatic LDL receptor mass also decreased (-44%). The susceptibility of LDL to oxidation was also increased when animals were exposed to radiation. CONCLUSIONS: Lipoprotein modifications that appeared following radiation exposure may result from an induced inflammatory state and may further contribute to vascular damage.

In situ determination of the functional size of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase by radiation inactivation analysis.

Radiation inactivation analysis of liver pieces yielded a target size of 210 kDa for hepatic 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase [S)-mevalonate:NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34) from rats fed a normal diet. Feeding a diet containing mevinolin and colestipol, which causes a marked increase in enzyme activity, resulted in a reduction of the target size to 120 kDa. These results are consistent with those obtained by radiation inactivation and immunoblotting analysis of isolated microsomes and suggest that the increase in HMG-CoA reductase activity caused by these dietary agents is accompanied by a change from a dimer to a monomer form of the enzyme.

Sulfhydryl/disulfide forms of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase.

Using radiation inactivation and immunoblotting techniques, evidence for functionally active forms of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase with molecular weights of about 100,000 and 200,000 was obtained. In liver microsomes isolated from rats fed both mevinolin and colestipol, the Mr 100,000 form was the predominant species, whereas in microsomes from animals fed only colestipol, the Mr 200,000 species was the major form. This Mr 200,000 form could be converted to the Mr 100,000 form by addition of dithiothreitol or beta-mercaptoethanol. Although both forms appear to possess catalytic activity, the Mr 200,000 species displays sigmoidal kinetics with respect to the concentration of NADPH, whereas the Mr 100,000 form exhibits typical hyperbolic kinetics.

Functional size of rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase as determined by radiation inactivation.

The functional molecular weight of rat liver 3-hydroxy-3-methylglutaryl-CoA reductase was determined by radiation inactivation. Both isolated hepatic microsomes and primary hepatocytes were irradiated with high energy electrons at -135 degrees C, and the residual microsomal enzyme activity was subsequently determined. The loss of enzyme activity in both irradiated microsomes and microsomes isolated from irradiated hepatocytes followed a single exponential decay which corresponded to a molecular mass of 200 kDa. This minimal molecular size of the functional enzyme was unaffected by either addition of cholestyramine to the rat diet or addition of 25-hydroxycholesterol plus mevalonate to the isolated rat hepatocytes. In addition, surviving enzyme protein was determined by immunoprecipitation of radiolabeled enzyme from hepatocytes that had been incubated with [35S]methionine before irradiation. The target size for loss of the monomer subunits was 98 kDa. The simplest interpretation of these results is that rat liver 3-hydroxy-3-methylglutaryl-CoA reductase in situ is a noncovalently linked dimer of the Mr = 97,200 enzyme subunit.