Cytoplasmic location of linoleoyl-CoA desaturase in microsomal membranes of rat liver

The intramembrane localization of linoleoyl-CoA desaturase in rat liver microsomes was examined by various methods, such as digestion by proteases, effect of detergents, and inhibition by the antibodies against purified terminal desaturase. Exposure of the desaturase on the surface of microsomal vesicles was suggested by the fact that the enzyme activity in the intact microsomes was susceptible to tryptic digestion, and considerably inhibited by anti-desaturase antibodies. When microsomes were previously treated with trypsin, the enzyme became more susceptible to the antibodies. Furthermore, it was demonstrated that the protein fragments cleaved from microsomal membranes by tryptic digestion formed a single precipitin line with the antibodies by the double-immunodiffusion test. These findings suggest the presence of linoleoyl-CoA desaturase on the cytoplasmic surface in the endoplasmic reticulum, since tryptic digestion liberates only the protein components situated on the surface area of membranes. In addition, desaturase activity in the intact microsomes was not stimulated by addition of the detergent, indicating the further outside location of the active site of the enzyme in microsomal vesicles. The pretreatment of microsomes with a low concentration (0.05%) of sodium deoxycholate, which destroys the permeability barrier for macromolecules without membrane disassembly, did not increase the susceptibility to tryptic digestion and the antibodies. These results show that linoleoyl-CoA desaturase is not present in a latent state in the membrane.     

The metabolism of benzo[a]pyrene phenols by rat liver microsomal fractions

The oxidative metabolism of benzo[a]pryrene (B[a]P) phenols catalyzed by liver microsomes in vitro leads to multiple products. High-pressure liquid chromatography analysis of the organic-soluble products formed indicates that regardless of the animal pretreatment regime, 3-hydroxy-B[a]P is metabolized to the 3,6-quinone and to a hydroxylated derivative tentatively identified as 3,9-dihyroxy-B[a]P. However, the distribution of products obtained with 9-hydroxy-B[a]P varied with animal pretreatment. A maximum of three distinct metabolites was obtained when the 9-phenol was metabolized in vitro with microsomes from phenobarbital-pretreated rats and the tentative 3,9-dihydroxy derivative was a common metabolite for all pretreatment regimes. Physical characterization, including mass spectrometry, indicates that all three products have an extra oxygen atom incorporated into their molecular structure from molecular oxygen. Studies utilizing specific inhibitors of the cytochrome P-450-dependent monooxygenase clearly suggest that the formation of dihydroxy or phenol-oxide derivatives is catalyzed by the hemoprotein, cytochrome P-450. These metabolites of the benzo[a]pyrene phenols are most likely related to the putative phenol-oxides of benzo[a]pyrene which have been demonstrated to alkylate DNA and protein. Repetitive scan difference spectrophotometric analysis of incubation mixtures containing rat liver microsomes, 3- or 9-hydroxy-B[a]P, NADPH, and oxygen shows the conversion of the phenols into products which absorb in the region from 400 to 500 nm. During and after the steady state of the reaction, it can be seen that certain of the hydroxy compounds produced are in equilibrium with their respective quinone form and may be involved in an oxygen-coupled redox cycle.     

Lipid peroxidation increases the molecular order of microsomal membranes

The effect of enzymatic lipid peroxidation on the molecular order of microsomal membranes was evaluated by ESR spectroscopy using the spin probes 5-, 12-, and 16-doxyl-stearic acid. Rat liver microsomal membranes were peroxidized by the NADPH-dependent reaction in the presence of the chelate ADP-Fe3+. Peroxidation resulted in a preferential depletion of polyenoic fatty acids and an increase in the percentage composition of shorter fatty acyl chains. There was no change in the cholesterol/phospholipid ratio of the peroxidized microsomes. The molecular order of both control and peroxidized membranes decreased toward the central region of the bilayer, and the order parameter (S) of each probe was temperature dependent. Peroxidation of the microsomal membrane lipids resulted in an increase in the order parameter determined with the three stearic acid spin probes. Of the three probes, 12-doxylstearic acid was the most sensitive to the changes in membrane organization caused by peroxidation. These data indicate that ESR spectroscopy is a sensitive method of detecting changes in membrane order accompanying peroxidation of membrane lipids.     

Vitamin E dependent reduced glutathione inhibition of rat liver microsomal lipid peroxidation

Effects of reduced glutathione (GSH) were investigated on $\text{in}$$\text{vitro}$ lipid peroxidation of hepatic microsomes obtained from Long-Evans Hooded rats fed chemically defined, purified diets containing adequate or documented deficiencies of vitamin E (E), selenium (Se) or both. Glutathione inhibited lipid peroxidation mediated by both NADPH-dependent enzymatic and ascorbate-dependent non-enzymatic systems. The inhibitory effect of GSH was observed in microsomes obtained from E supplemented groups whereas it had no effect on microsomes from E deficient animals. Selenium status had no effect on GSH inhibition. Glutathione was found to be specific for the E dependent inhibition of lipid peroxidation and could not be substituted by other sulfhydryl compounds tested. Also, GSH did not inhibit non-enzymatic lipid peroxidation of heat-denatured microsomes from either E-supplemented groups or any of the other dietary regimens.     

Purification of ornithine transcarbamylase from rat liver by affinity chromatography with immobilized transition-state analog

Ornithine transcarbamylase (EC 2.1.3.3) was purified to homogeneity from rat liver. The basis of the method is the chromatography of a high-speed supernatant fraction of a homogenized rat liver on an affinity column consisting of the transition-state analog of ornithine transcarbamylase, δ-N-(phosphonacetyl)-l-ornithine, immobilized on epoxy-activated Sepharose 6B through the α-amino group. The enzyme was eluted from the column using a gradient of the substrate, carbamyl phosphate, and further purified by gel filtration. The enzyme elutes with a constant specific activity of 250 to 260 μmol min^?1 mg^?1 at pH 8.5, 37°C, and is free of contaminating proteins on sodium dodecyl sulfate gel electrophoresis. Determination of the molecular weight of the purified enzyme by centrifugation (98,000) and by gel electrophoresis in the presence of sodium dodecyl sulfate (35,300) indicates that the enzyme from rat liver is a trimer. The enzyme exhibits conventional Michaelis-Menten kinetics at pH 7.4 and in this respect differs from the enzyme prepared by other methods.     

Biochemical properties of short- and long-chain rat liver microsomal trans-2-enoyl coenzyme A reductase

This study describes the biochemical properties of the rat hepatic microsomal NADPH-specific short-chain enoyl CoA reductase and NAD(P)H-dependent long-chain enoyl CoA reductase. Of the substrates tested, crotonyl CoA and trans-2-hexenoyl CoA are reduced by the short-chain reductase only in the presence of NADPH. The trans-2-octenoyl CoA and trans-2-decenoyl CoA appear to undergo reduction to octanoate and decanoate, respectively, catalyzed by both enzymes; 64% conversion of the C8:1 is catalyzed by the short-chain reductase, while 36% conversion is catalyzed by the long-chain enzyme. For the C10:1 substrate, 45% is converted by the short-chain reductase, while 55% is reduced by the long-chain reductase. trans-2-Hexadecenoyl CoA is a substrate for the long-chain enoyl CoA reductase only. Reduction of C4 and C6 enoyl CoA's was unaffected by bovine serum albumin (BSA), whereas BSA markedly stimulated the conversion of C10 and C16 enoyl CoA's to their respective saturated product. Reduction rates as a function of microsomal protein concentration, incubation time, pH, and cofactors are reported including the apparent Km and Vmax for substrates and cofactors. In general, the apparent Km's for the substrates ranged from 19 to 125 microM. The apparent Vmax for the short-chain enoyl CoA reductase was greatest with trans-2-hexenoyl CoA, having a turnover of 65 nmol/min/mg microsomal protein, while the apparent Vmax for the long-chain enzyme was greatest with trans-2-hexadecenoyl CoA, having a turnover of 55 nmol/min/mg microsomal protein. With respect to electron input, NADPH-cytochrome P-450 reductase, either alone, mixed with phospholipid, or incorporated into phospholipid vesicles, possessed no enoyl CoA reductase activity. Cytochrome c did not affect the NADPH-dependent conversion of the trans-2-enoyl CoA. In addition, anti-NADPH-cytochrome P-450 reductase IgG did not inhibit the reduction of trans-2-hexadecenoyl CoA in hepatic microsomes. Finally, the NADPH-specific short-chain and NAD(P)H-dependent long-chain enoyl CoA reductases were solubilized and completely separated from NADPH-cytochrome P-450 reductase by employing DE-52 column chromatography. These studies demonstrate the noninvolvement of NADPH-cytochrome P-450 reductase in either the short-chain (13) or long-chain enoyl CoA reductase system. Thus, the role of NADPH-cytochrome P-450 reductase in the microsomal elongation of fatty acids appears to be at the level of the first reduction step.     

Solubilization of ligand-stabilized vasopressin receptors from plasma membranes of bovine kidney and rat liver

The solubilization of vasopressin receptors from plasma membranes of bovine kidney and rat liver by different detergents was investigated. A prerequisite for the extraction of vasopressin receptors retaining binding affinity for their ligand was the stabilization of the receptors by the prior formation of the membrane-bound hormone-receptor complexes. The vasopressin-receptor complexes from both kidney and liver membranes were solubilized in a high yield with dodecyl-beta-D-maltoside and 3-laurylamido-N,N'-dimethylpropylaminoxide. Several other nonionic detergents including octyl-beta-D-glucopyranoside effectively extracted the hepatic vasopressin receptor. For the hormone-receptor complex solubilized from bovine kidney with dodecyl-beta-D-maltoside, a Stokes' radius of 5.8 nm was determined.     

3,4,5,3',4'-Pentachlorobiphenyl as a useful inducer for purification of rat liver microsomal cytochrome P448.

An easy purification of rat liver microsomal cytochrome P448 was performed by using 3,4,5,3′,4′-pentachlorobiphenyl as an inducer. The cytochrome P448, a high spin form, was purified to 18.1 nmoles/mg protein with a good yield by ω-aminooctyl Sepharose 4B column chromatography followed by a hydroxyapatite column chromatography. This hemoprotein cross-reacted with antibody to cytochrome P448 from β-naphthoflavone-treated rats, but not with antibody to cytochrome P450 from phenobarbital-treated rats at all. The results of amino acid analyses suggested that this cytochrome P448 is similar to cytochrome P448 of 3-methylcholanthrene-treated rats.     

Immunochemical evidence for a role of cytochrome P-450 in liver microsomal ethanol oxidation

Antibodies to cytochrome P-450 isozyme 3a, the ethanol-inducible isozyme in rabbit liver, were used to determine the role of this enzyme in the microsomal oxidation of alcohols and the p-hydroxylation of aniline. P-450 isozymes, 2, 3b, 3c, 4, and 6 did not crossreact with anti-3a IgG as judged by Ouchterlony double diffusion, and radioimmunoassays indicated a crossreactivity of less than 1%. Greater than 90% of the activity of purified form 3a toward aniline, ethanol, n-butanol, and n-pentanol was inhibited by the antibody in the reconstituted system. The catalytic activity of liver microsomes from control or ethanol-treated rabbits was unaffected by the addition of either desferrioxamine (up to 1.0 mM) or EDTA (0.1 mM), suggesting that reactions involving the production of hydroxyl radicals from H2O2 and any contaminating iron in the system did not make a significant contribution to the microsomal activity. The addition of anti-3a IgG to hepatic microsomes from ethanol-treated rabbits inhibited the metabolism of ethanol, n-butanol, n-pentanol, and aniline by about 75, 70, 80, and 60%, respectively, while the inhibition of the activity of microsomes from control animals was only about one-half as great. The rate of microsomal H2O2 formation was inhibited to a lesser extent than the formation of acetaldehyde, thus suggesting that the antibody was acting to prevent the direct oxidation of ethanol by form 3a. Under conditions where purified NADPH-cytochrome P-450 reductase-catalyzed substrate oxidations was minimal, the P-450 isozymes other than 3a had low but significant activity toward the four substrates examined. The residual activity at maximal concentrations of the antibody most likely represents the sum of the activities of P-450 isozymes other than 3a present in the microsomal preparations. The results thus indicate that the enhanced monooxygenase activity of liver microsomes from ethanol-treated animals represents catalysis by P-450 isozyme 3a.     

NH2-terminal sequence analyses of four rat hepatic microsomal cytochromes P-450

Cytochromes P-450f, P-450g, P-450h, and P-450i are four hepatic microsomal hemoproteins that have been purified from adult rats. Whereas cytochromes P-450g and P-450h appear to be male-specific hemoproteins, cytochrome P-450i is apparently a female-specific enzyme purified from untreated adult female rats. Cytochrome P-450f has been purified from adult male and female rats with equivalent recoveries. Amino-terminal sequence analyses of the first 15-20 amino acid residues of each of these cytochromes P-450 has been accomplished in the current investigation. Each protein possesses a hydrophobic leader sequence consisting of 65-87% hydrophobic amino acids, and only one charged amino acid (Asp) in the amino-terminal region. Although differences in the amino-terminal sequences of cytochromes P-450f, P-450g, P-450h, and P-450i are identified, these hemoproteins all begin with Met-Asp, and marked structural homology is observed among certain of these enzymes. Cytochromes P-450g and P-450h, two male-specific proteins, have 11-12/15 identical residues with cytochrome P-450i, a female-specific isozyme. Cytochromes P-450f and P-450h have 16/20 identical amino-terminal residues. Only limited sequence homology is observed between the amino-terminal sequences of cytochromes P-450f-i compared to rat liver cytochromes P-450a-e. The results demonstrate that cytochromes P-450f, P-450g, P-450h, and P-450i are isozymic to each other and five additional rat hepatic microsomal cytochrome P-450 isozymes (P-450a-e).     

Bovine liver monoamine oxidase: A modified purification procedure and preliminary evidence for two subunits and one FAD

Bovine liver mitochondrial monoamine oxidase was isolated in a more active state and in higher yields by an improved purification method which utilized β-mercaptoethanol and which contained several other important modifications. The subunit structure of the purified enzyme components was investigated by chemical and enzymatic methods. The subunit molecular weight of the three enzyme components isolated was estimated to be 52,000 by sodium dodecyl sulfate disc electrophoresis and by exclusion-diffusion chromatography on Biogel A-5m with 6 m guanidine HCl as the solvent. The number of peptides observed in the peptide map of the tryptic digest of the S-β-carboxymethylcysteine derivative of the enzyme also showed that the subunit molecular weight was about 52,000. Since it was previously reported that the monomer molecular weight of the enzyme was about 110,000, the active enzyme is made up of two subunits. The NH₂-terminus of the enzyme of both subunits is blocked since Edman degradation and aminopeptidase failed to release an NH₂-terminal amino acid. The COOH-terminal amino acid of both subunits was shown to be leucine by carboxypeptidase digestion of the enzyme since it was liberated quantitatively. From the FAD content of the enzyme and the subunit data, it is proposed that the enzyme probably consists of two subunits which differ possibly in that only one subunit contains 8-α-cysteinyl FAD.     

The effect of temperature on the activity of the adenylate cyclase system of liver plasma membranes

The rat liver adenylate cyclase system shows a discontinuity in the Arrhenius plots at 20°C in the nonstimulated activity (basal) with activation energies of 16 and 28 Kcal/mole. The discontinuity disappears when the enzyme is stimulated either by glucagon, sodium fluoride, 5′ guanylyl-imidodiphosphate or glucagon plus 5′ guanylyl-imidodiphosphate and the energy of activation was the same with all the compounds tested. If the activator was initially in contact with the membranes at 0°C the energy of activation was similar to that observed below the break (26 Kcal/mole) but it changed to that above the break if the compound contacted the membranes at temperatures above the break (22–24°C). We discuss the possibility of two different conformations of the enzyme; both conformations can be “frozen” by any of the compounds tested, “isolating” the enzyme from any subsequent physical change of the membrane due to temperature.     

Characterization of microsomal glycosyl-transferases of rat pancreas and influence of diets

Four rat pancreatic microsomal glycosyl-transferases (fucosyl-, galactosyl-, mannosyl- and N-acetylglucosaminyl-transferases) are studied and characterized for their optimal conditions and their relation with interfering reaction (glycosyl-nucleotide pyrophosphatases, osidases and proteinases). Dietary treatments of the rats induce modification: for all the transferase activities, the highest levels are found in a high-starch diet and the lowest one in a high-fat diet. The activities found in the standard diet are at the level of the high-starch or of the high-fat diet depending on the enzyme studied. The observed modifications are not explained by alterations in physico-chemical parameters of the enzymes or by intervention of glycosyl-nucleotide pyrophosphatases, osidases or proteolytic enzymes. The modifications observed for the mannosyl-transferase are predominantly found in a lipid fraction extracted by chloroform-methanol ($\text{2}\text{1}$).     

Isolation of copper thionein from rat liver

The inducible Cu-binding protein from adult rat liver previously referred to as Cu-chelatin has been purified and shown to be Cu-thionein. The Cu-protein was purified to homogeneity by gel filtration and thiopropyl-Sepharose chromatography. The Cu-thionein exhibited an amino acid composition similar but not identical to that of the two forms of rat liver Cd,Zn-thionein. The polypeptide-chain molecular weight of Cu-thionein was indistinguishable from that of Cd,Zn-thionein. The identification of the Cu-protein as metallothionein was substantiated by the complete immunological cross-reactivity with antisera prepared against purified rat liver Cd,Zn-thionein. Purified Cu-thionein bound 9–11 g atoms of Cu per mole of protein in an electron paramagnetic resonance nondetectable form. The $\text{Cu}\text{Zn}$ ratio of the protein is about 100. Ion-exchange chromatography resolved the Cu-protein into three polymorphic forms which differed from the polymorphism of Cd,Zn-thionein.     

Cell-cell adhesion in the embryonic chick: Partial purification of liver adhesion molecules from liver membranes

Polyspecific xenoantisera specific for embryonic chick liver cells have been used to develop an assay for the detection of embryonic chick liver adhesion molecules (LAM). The neutralization of the antiaggregation immune sera and subsequent semiquantitative assessment of the remaining activity is the basis for this assay. Embryonic chick liver membrane proteins have been solubilized using a number of detergents and the LAM activity has been partially purified using sucrose-gradient sedimentation, isoelectric focusing, gel filtration, affinity chromatography, and preparative polyacrylamide gel electrophoresis procedures. The isolated LAM appears to be an intrinsic, trypsin-resistant, membrane glycoprotein of approximately 65,000 daltons. The 65K preparation of LAM is an effective immunogen in generating an aggregation blocking xenoantiserum specific for embryonic chick liver cells. Purification of LAM to homogeneity has been hampered by an apparent lability by self-association or loss by adsorption of the more purified LAM antigen(s).