Purification and characterization of intact cytochrome b5 from yeast microsomes

The inhibition of an oligomycin sensitive ATPase prepared from bovine heart submitochondrial particles (J.A. Berden and M.M. Voorn-Brouwer, 1978, Biochim. Biophys. Acta 501, 424-439) by a number of cationic dyes has been compared in order to develop a structure-function relationship. Two generalizations emerge from this comparison. First, the most effective dyes have net positive charge at neutral pH; and second, those dyes containing alkyl substituted secondary and tertiary amino groups are more effective than analogs with primary aromatic amino groups. Some of the cationic dyes exhibit uncoupling activity when added to intact rat liver mitochondria, stimulating both State 4 respiration and the latent ATPase activity. The order of effectiveness and concentrations for maximal stimulation of respiration are: coriphosphine (0.3 microM), Nile blue A (0.5 microM), pyronin Y (0.8 microM), and acridine orange (10 microM). Atypically, oligomycin inhibits the stimulation of respiration by these cationic acid uncouplers. The order of effectiveness and concentrations for maximal stimulation of the latent ATPase are: Nile blue A (2 microM), pyronin Y (8 microM), acridine orange (25 microM), and coriphosphine (75 microM). At concentrations greater than those shown for maximal stimulation, the uncoupling dyes inhibited respiration and the latent ATPase. The cationic dyes tested that were not uncouplers are inhibitors of respiration and the latent ATPase of intact mitochondria at all concentrations tested.     

The binding of cytochrome b5 to phosphatidylcholine vesicle

Cytochrome b₅ was isolated from rabbit liver by a detergent procedure and by a proteolytic procedure. Only cytochrome b₅ isolated by the detergent procedure would bind to phosphatidylcholine vesicles and the cytochrome b₅ was not removed by 1 M KCl. The E_o′ and visible absorption spectrum of the cytochrome b₅ and its rate of reduction by NADH plus NADH-cytochrome b₅ reductase did not change appreciably upon binding. These data indicate that cytochrome b₅ is bound to phospholipid by a hydrophobic interaction which leaves the heme portion in the aqueous environment.     

NADPH-cytochrome P-450 reductase of yeast microsomes.

NADPH-cytochrome c reductase of yeast microsomes was purified to apparent homogeneity by solubilization with sodium cholate, ammonium sulfate fractionation, and chromatography with hydroxylapatite and diethylaminoethyl cellulose. The purified preparation exhibited an apparent molecular weight of 83,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The reductase contained one molecule each of flavin-adenine dinucleotide and riboflavin 5′-phosphate, though these were dissociative from the apoenzyme. The purified reductase showed a specific activity of 120 to 140 μmol/min/mg of protein for cytochrome c as the electron acceptor. The reductase could reduce yeast cytochrome P-450, though with a relatively slow rate. The reductase also reacted with rabbit liver cytochrome P-450 and supported the cytochrome P-450-dependent benzphetamine N-demethylation. It can, therefore, be concluded that the NADPH-cytochrome c reductase is assigned for the cytochrome P-450 reductase of yeast. The enzyme could also reduce the detergent-solubilized cytochrome b₅ of yeast. So, this reductase must contribute to the electron transfer from NADPH to cytochrome b₅ that observed in the yeast microsomes.     

Detection of phenobarbital-induced cytochrome P-450 in rat hepatic microsomes using an enzyme-linked immunosorbent assay

The major phenobarbital-inducible form of cytochrome P-450 (cytochrome P-450 PB) was purified to homogeneity from rat liver microsomes and rabbit antibodies prepared against the purified enzyme. Using these antibodies, an enzyme-linked immunosorbent assay (ELISA) was developed for the detection of cytochrome P-450 PB in microsomes which was sensitive at the nanogram level. The content of cytochrome P-450 PB was determined in hepatic microsomes from rats treated with various xenobiotics. Phenobarbital and Aroclor 1254 pretreatments resulted in several-fold increases in immunoreactive cytochrome P-450 PB over control levels. ELISA measurements of cytochrome P-450 PB were also carried out over a 48-h time course of phenobarbital induction in liver microsomes. Significant increases over control levels were seen at 16 h and beyond. Measurements of ELISA-detectable cytochrome P-450 PB were made in microsomes following the administration of CCl4 to phenobarbital-pretreated rats. Immunoreactive cytochrome P-450 PB was observed to decrease less rapidly than the spectrally detectable enzyme in the microsomal membranes. Inhibition of heme synthesis was carried out by the administration of 3-amino-1,2,4-triazole (AT) to rats. Concomitant pretreatment with phenobarbital and AT resulted in levels of ELISA-detectable cytochrome P-450 PB which were significantly increased over control levels, while spectrally detectable levels of total holoenzyme remained unchanged. These results support the idea that this cytochrome P-450 may exist, at least partly, in the microsomal membrane in an inactive or apoprotein form.     

Inactivation of yeast fatty acid synthetase by modifying the beta-ketoacyl reductase active lysine residue with pyridoxal 5'-phosphate

Treatment of yeast fatty acid synthetase with pyridoxal 5'-phosphate inhibited the enzyme. Assays of the partial activities of the pyridoxal phosphate-treated synthetase showed that only the beta-ketoacyl reductase was significantly inhibited. NADPH prevented inactivation of the enzyme by pyridoxal phosphate, indicating that pyridoxal modifies a residue near or in the beta-ketoacyl reductase site. The pyridoxal-treated synthetase shows a fluorescence spectrum with a maximum of 426 nm after uv irradiation at 325 nm. Binding of the pyridoxal phosphate to the synthetase is reversible as shown by the disappearance of the fluorescence band after dialysis of pyridoxal-treated enzyme. Reduction with NaBH4 of the pyridoxal-treated enzyme eliminates this fluorescence maximum and causes the appearance of a new band at 393 nm. These observations suggest that pyridoxal phosphate interacts with the synthetase by forming a Schiff base with lysine residue at the beta-ketoacyl reductase site. Amino acid analyses of the HCl hydrolysates of the borohydride-reduced, pyridoxal-treated synthetase showed the presence of 6 mol of N6-pyridoxal derivative of lysine per mole of fatty acid synthetase, indicating the presence of six sites of beta-ketoacyl reductase in the native enzyme. Autoradiography of sodium dodecyl sulfate-polyacrylamide gels of the pyridoxal phosphate enzyme reduced with NaB3H4 indicates that the alpha subunit contains the beta-ketoacyl reductase domain. These findings are consistent with the proposed structure of the alpha 6 beta 6 complex required for palmitoyl-CoA synthesis.     

Role of the carbohydrate part of yeast acid phosphatase

Acid phosphatase, purified from the yeast Saccharomyces cerevisiae, was completely deglycosylated by endo-beta-N-acetylglucosaminidase H or by HF treatment. Three protein bands were obtained on sodium dodecyl sulfate (SDS)-electrophoresis, with molecular weights of 73,000, 71,000 and 61,500. The released carbohydrate chains varied in size from 12 to 142 mannose units. To study the role of carbohydrate chains in the structure and function of acid phosphatase, a comparison of the properties of the partially deglycosylated enzyme with the native one was performed. The 60% deglycosylated enzyme retained the original activity, and CD and fluorescence spectra showed that the native conformation of the enzyme was preserved. The 90% deglycosylated enzyme showed a pronounced loss of enzyme activity, accompanied by the disruption of the three-dimensional structure. The partially deglycosylated enzyme was less soluble and more susceptible to denaturing effects of heat, pH, urea, and guanidine hydrochloride. Under conditions of electrophoresis, the partially deglycosylated enzyme dissociated, indicating a possible role of carbohydrate chains in maintaining the dimeric structure of the enzyme. Susceptibility of acid phosphatase toward proteolysis was drastically increased by deglycosylation.     

Comparisons of warfarin metabolism by liver microsomes of rats treated with a series of polybrominated biphenyl congeners and by the component-purified cytochrome P-450 isozymes

R- and S-warfarin metabolite profiles (regio- and stereoselectivity) have been determined with hepatic microsomes from untreated rats and rats treated with nine individual polybrominated biphenyl (PBB) congeners, a commercial mixture of PBBs, and, for comparison with phenobarbital and 3-methylcholanthrene. The metabolic rates have been correlated with cytochrome P-450 (P-450) isozyme concentrations in the microsomes determined by immunochemical quantitation techniques (G. A. Dannan, F. P. Guengerich, L. S. Kaminsky, and S. D. Aust, (1983) J. Biol. Chem., 258, 1282–1288). The warfarin hydroxylase activities of the P-450 isozyme components of the various microsomal preparations (F. P. Guengerich, G. A. Dannan, S. T. Wright, M. V. Martin, and L. S. Kaminsky (1982) Biochemistry, 21, 6019–6030) were multiplied by the corresponding isozyme concentrations to obtain an assessment of the potential warfarin hydroxylase capacity of the microsomes, and the results were compared with actual activities. The results of these studies and comparisons indicate that substrate regio- and stereoselectivities of microsomal-bound P-450s are essentially retained on purification of the isozymes to homogeneity and reconstitution, that warfarin metabolism by microsomal preparations can be used to predict microsomal P-450 isozyme compositions, and that microsomal warfarin hydroxylase activity is greater than would be predicted based on the approx 20:1 ratio of P-450 to NADPH-P-450 reductase in the microsomes and on the known activities of constituent isozymes. Two P-450 isozymes which are induced by treatment of rats with phenobarbital appear to be more tightly linked to NADPH-P-450 reductase than does an isozyme induced by β-naphthoflavone.     

Changes in the activities of the protoheme-synthesizing system during the growth of yeast under different conditions.

The levels of some enzymatic activities involved in protoheme synthesis have been measured in subcellular fractions obtained at different stages of the growth of the yeast Saccharomyces cerevisiae grown anaerobically and aerobically with glucose (50 or 6 g/ liter), and ethanol (20 g/liter) as the carbon source. The degree of repression of the respiratory system is estimated by the respiratory capacity of whole cells, by the activities of succinate-cytochrome c reductase and cytochrome c oxidase of the mitochondrial particles, and by the cytochrome spectra. The results show that (i) the more porphyrins (cytochromes) that are synthesized by the cells, the lower is the specific activity of δ-aminolevulinic acid (ALA) synthetase and the higher is the specific activity of ALA dehydratase, the activity ratio ALA synthetase/ALA dehydratase decreasing at least 10-fold compared to the repressed cells; (ii) the amount of intracellular ALA found under all conditions tested (from 0.05 to 1.5 mm in the cell sap) correlates well with the measured ALA synthetase activity; its presence argues against a rate-limiting function for ALA synthetase and rather favors such a role for the ALA dehydratase in the formation of heme in yeast; (iii) the rate of porphyrin synthesis measured in vitro is higher in the case of cells with high cytochrome contents; and (iv) the specific activities of succinyl CoA synthetase and protoheme ferrolyase are always present in nonlimiting amounts. Some experiments are described showing that the values of the activities which are calculated from these in situ and in vivo experiments compare well with the values measured in vitro in the acellular extracts. The results concerning the enzymatic activities, together with (i) the excretion of coproporphyrin(ogen) and the accumulation of protoporphyrin + Zn-protoporphyrin in anaerobiosis, (ii) the presence of protoporpho(di)methene (P503) in anaerobic and repressed cells, and (iii) the presence of intracellular ALA under all growth conditions, are discussed in terms of possible control(s) of heme synthesis in yeast.     

Characteristics of b-type cytochromes in brain microsomes: Comparison with liver microsomes

Biochemical aspects of b-type cytochromes in swine cerebral microsomes were different from those of cytochrome b₅ in liver microsomes, as well as the difference in absorption spectra. First, the kinetic constants, K_m and V_max, in rotenone-insensitive NADH-cytochrome c reductase activity were different from those of liver microsomes, and the activity of cerebral microsomes was higher than that of liver microsomes. Second, midpoint potentials (E_m) of b-type cytochromes in cerebral microsomes were measured and compared with liver microsomal cytochrome b₅. In cerebral microsomes two components of b-type cytochromes were resolved, and showed E_m's of ?30 and +50 mV, respectively, in the presence of 2 mm KCN. On the other hand, the E_m of liver microsomal cytochrome b₅ was ?6 mV. The high-potential component of cerebral microsomal b-type cytochromes was identified as brain-b′₅ [S. Yoshida, T. Yubisui, and M. Takeshita (1983)Biochem. Int. 7, 291–298] and the low-potential component as brain-b₅. The significance of the difference between cerebral and liver microsomal b-type cytochromes was discussed.     

Colchicine inhibition of insulin induction of stearoyl-CoA desaturase and fatty acid synthetase in cultured avian liver explants

Chicken embryo liver explants cultured in chemically defined medium in the absence of serum provide a unique system to probe into the mechanism of insulin induction of lipogenic enzymes. Colchicine at concentrations of 0.2 and 1 microM in the culture medium caused inhibition of insulin induction of stearoyl-CoA desaturase and fatty acid synthetase by 50 and 90%, respectively. As measured by immunochemical techniques, the inhibition of the induction of these two enzyme systems resulted from the decreased content of the delta 9-terminal desaturase component of the stearoyl-CoA desaturase and the fatty acid synthetase. Colchicine, however, had no effect on the general protein synthesis, nor did it affect the malic enzyme, which is induced by triiodothyronine but not by insulin. Also, colchicine had no influence on the binding of 125I-insulin to isolated plasma membrane. Pretreatment of liver explants with insulin for 0.5-1 h and subsequent incubation in insulin-free media for 48 h resulted in induction of the desaturase and fatty acid synthetase. However, inclusion of colchicine in the media for 3 h subsequent to the treatment with insulin completely abolished the inductive effect of insulin, suggesting that colchicine affects events occurring subsequent to insulin binding to the cell surface membranes. Since lumicolchicine, an inactive isomer of colchicine, had no effect on insulin action, it is suggested that the inhibition of insulin induction of the desaturase and synthetase is related to the depolymerizing action of colchicine. Therefore, in eliciting long-term responses to insulin, microtubular integrity of the cell may be required for the transfer of a putative from cell surface insulin receptor to intracellular sites.     

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.     

Assay of phosphoenolpyruvate carboxykinase in crude yeast extracts.

The applicability of a spectrophotometric assay of phosphoenolpyruvate car?ykinase to crude yeast extracts has been studied. The assay measured oxalacetate production by coupling to the malate dehydrogenase reaction (phosphoenolpyruvate + ADP + bicarbonate → oxalacetate + ATP; oxalacetate + NADH → malate + NAD). Disappearance of NADH depended strictly on the presence of phosphoenolpyruvate, bicarbonate, ADP, and Mn²⁺. Furthermore, the disappearance of NADH was shown to be accompanied by stoichiometric accumulation of malate. Addition of 10 mm quinolinate, which is a known inhibitor of liver phosphoenolpyruvate car?ykinase, completely prevented phosphoenolpyruvate-dependent NADH disappearance. These observations demonstrated that the assay provides a quantitative measure of phosphoenolpyruvate car?ykinase activity in crude extracts. The assay could be applied to crude extracts from yeast cells grown under laboratory conditions but not to extracts from commercially produced baker's yeast, because of an extremely high rate of endogeneous oxidation of NADH in the latter extracts. With the spectrophotometric assay, optimal activity was observed at pH 7.0 with both crude extracts and a 15-fold-purified preparation.     

Convenient procedure for the isolation of highly enriched,cytochrome P-450-containing microsomal fraction from Candida tropicalis

The suitability of Ca²⁺ ions for the precipitation of the microsomal fraction from the hydrocarbon-grown yeast Candida tropicalis was evaluated. In the final procedure the microsomes were precipitated by the addition of 16 mm CaCl₂. Crude extracts obtained from cells via spheroplast lysis were centrifuged at 12,000g for 15 min and at 25,000g for 15 min prior to precipitation. The cytochrome P-450 content of the fraction was between 0.22 and 0.35 nmol mg^?1 protein. The isolated microsomes exhibited both hexadecane hydroxylation activity and NADPH-cytochrome c reductase activity.     

Fatty acid distribution of the normal sciatic nerve lipids in the rat

We isolated and purified the main lipids of the rat sciatic nerve. After methanolysis, fatty acids were isolated and purified by thin layer chromatography, and studied by gas chromatography. C 16 and C 18 fatty acids are the most abundant. Among the long-chain fatty acids, arachidonic acid (20:4) is present in some lipids; highly desaturated fatty acids in C 22 and C 24 are also present. In general, the fatty acids are highly saturated; cholesterol esters and ethanolamine phosphoglyceride fatty acids are highly unsaturated.     

Extra proton translocation and membrane potential generation--universal properties of cytochrome bc1/b6f complexes reconstituted into liposomes

Isolated cytochrome complexes from different sources like beef heart mitochondria, spinach chloroplasts, cyanobacteria, and photosynthetic bacteria were incorporated into liposomes by sonication as revealed by sucrose density gradient centrifugation and electron microscopy. The reconstituted cytochrome complexes show suppressed rates of quinol-cytochrome c/plastocyanin oxidoreduction which can be stimulated by ionophores and uncouplers. In addition, extra proton translocation out of the vesicles and membrane potential generation during electron transport were observed, suggesting a universal mechanism of electron and proton transport through all the tested cytochrome complexes.     

Fatty acid and sterol specificity in the biosynthesis of steryl esters by enzyme preparations from spinach leaves

Acetone powders prepared from the 20,000g participate fraction of spinach (Spinacia oleracea L.) leaves catalyzed the formation of steryl esters from free sterol and 1,2-diacylglycerol as the acyl donor. There was no sterol specificity when cholesterol, sitosterol, and campesterol were compared. When rates of sterol ester biosynthesis were compared using different 1,2-diacylglycerols it was found that the shorter chain fatty acids and the more unsaturated fatty acids were preferred. When the substrate concentration of diacylglycerol was varied, the maximal velocities obtained with the different substrates were dipalmitoleoyl- >dilinolenoyl- >dioleoyl- >dilinoleoyl-glycerol. It was demonstrated by silver nitrate thin-layer chromatography that the fatty acids of the supplied diacylglycerols were transferred to the sterol. When diacylglycerol mixtures were supplied, it was found that unsaturated diacylglycerols greatly stimulated conversion of saturated diacylglycerols to saturated steryl esters. For an equimolar mixture of dipalmitoyl-, dioleoyl-, dilinoleoyl-, and dilinolenoyl-glycerol, about equal amounts of the four steryl ester species were synthesized.     

Fatty acid composition of the lipids of Puffinus tenuirostris (Temminck) in relation to its diet

The fatty acid composition of lipids isolated from the depot fat, stomach contents, and proventricular oil of adult and chick Puffinus tenuirostris (Temminck) has been analysed. The diet of both adults and chicks is almost exclusively derived from the euphausiid Nyctiphanes australis Sars, and an attempt was made to determine whether dietary lipid affects the composition of depot fat, and whether individual fatty acids in the stomachs and proventricular oil can be used as markers for the origin of the diet. An apparent selectivity in the deposition of fatty acids in the fat depots can be explained by the conversion of fatty alcohols, derived from the euphausiid wax ester, into fatty acids of equivalent chain length and unsaturation. Hexadecadienoic acid appeared to be the only possible marker fatty acid from the euphausiid, but wide variations in its level limits its usefulness as a reliable index of the diet of Puffinus tenuirostris.