Microsomal enzymes of cholesterol biosynthesis from lanosterol. Characterization, solubilization, and partial purification of NADPH-dependent delta 8,14-steroid 14-reductase

The membrane-bound enzyme of microsomes that catalyzes NADPH-dependent reduction of the 14-double bond of conjugated delta 8,14- and delta 7,14-sterols has been studied both as collected in microsomes from broken cell preparations of rat liver and after solubilization. Optimal incubation conditions for assay of the membrane-bound enzyme have been determined, and properties of the microsomal enzyme have been established with respect to cofactor requirements, kinetics, pH, addition of inhibitors, addition of glycerol phosphatides, and sterol substrate specificity. The 14-reductase is readily solubilized with a mixture of octylglucoside and taurodeoxycholic acid. The solubilized enzyme has been enriched by precipitation with polyethylene glycol and chromatography on DEAE-Sephacel and hydroxylapatite columns. The resulting partially purified enzyme has been obtained free of other microsomal enzymes of cholesterol biosynthesis: 4-methyl sterol oxidase, delta 5,7-sterol 7-reductase, delta 8,24-sterol 24-reductase, 3-ketosteroid reductase, and steroid 8----7-ene isomerase, plus microsomal cytochrome P-450, cytochrome P-450 reductase, cytochrome b5 reductase, and cytochrome b5. The partially purified enzyme is stimulated by addition of phospholipids. All of the properties exhibited by partially purified 14-reductase are consistent with the suggestion that the solubilized and enriched enzyme catalyzes the microsomal reduction of the 14-double bond of the sterol-conjugated dienes. However, presence of the enzyme does not prove that the sterol-conjugated dienes are obligatory precursors of cholesterol.     

Purification and comparative properties of the glycoprotein nicotinamide adenine dinucleotide glycohydrolase from rat liver microsomal and plasma membranes.

NAD glycohydrolase, or NADase (NAD+ glycohydrolase, EC 3.2.2.5) was solubilized with porcine pancreatic lipase from isolated fractions of microsomes and plasma membranes obtained from rat livers. The enzyme from each organelle was further purified by DEAE-cellulose chromatography, gel filtration and isoelectric focusing. The solubilized, partially purified enzymes had similar molecular weights, pH-activity profiles and Km values. Marked charge heterogeneity was observed for the microsomal enzyme on isoelectric focusing between pH 6 and 8 with maximum activity focusing at pH 8.0. Plasma membrane NADase displayed a single peak at pH 6.7. Treatment of the partially purified microsomal or plasma membrane enzyme with neuraminidase resulted in a single peak of activity on isoelectric focusing (pH 3.5--10) with a pI of 9.2. Polyacrylamide gel electrophoresis of either NADase revealed a periodate-Schiff positive band which was coincident with enzyme activity. Compositional analyses of the microsomal enzyme focusing at pH 8.0 confirmed the presence of hexoses, hexosamines and sialic acid. Differences in carbohydrate composition might be important in determining the subcellular distribution of this enzyme.     

Identification, characterization, and partial purification of 4 alpha-carboxysterol-C3-dehydrogenase/ C4-decarboxylase from Zea mays.

A microsomal preparation from seedlings of Zea mays catalyzed the NAD+-dependent oxidative decarboxylation of several substrates, including 4alpha-carboxy-cholest-7-en-3beta-ol, synthesized according to a new procedure, giving the first in vitro evidence for this enzymatic activity in a higher plant. A GC assay has been developed to detect the Delta7-cholestenone produced and the kinetic parameters of the microsomal system have been established. 4alpha-Carboxysterol decarboxylation shows an exclusive requirement for an oxidized pyridine nucleotide, with NAD+ being more efficient than NADP+. The decarboxylation reaction is independent of molecular oxygen. 4alpha-Carboxysterol-C3-dehydrogenase/C4-decarboxylase (4alpha-CD) is a microsome-bound protein which can be efficiently solubilized by detergents, including Brij W-1 and sodium cholate. The Brij W-1-solubilized enzyme was partially purified 290-fold by a combination of DEAE anion-exchange chromatography, Cibacron blue 3GA-agarose dye chromatography, and gel permeation. The apparent molecular mass of 4alpha-CD in sodium cholate was estimated to be 45 kDa. These results support the contention that demethylation at C4 of plant sterols is composed of two separate processes: an oxygen- and NAD(P)H-dependent oxidation of the 4alpha-methyl group to produce the 4alpha-carboxysterol metabolite (S. Pascal et al., J. Biol. Chem. 268, 11639, 1993) followed by oxygen-independent dehydrogenation/decarboxylation to produce an obligatory 3-ketosteroid.     

Solubilization, partial purification, and immunodetection of squalene synthetase from tobacco cell suspension cultures

Squalene synthetase, an integral membrane protein and the first committed enzyme for sterol biosynthesis, was solubilized and partially purified from tobacco (Nicotiana tabacum) cell suspension cultures. Tobacco microsomes were prepared and the enzyme was solubilized from the lipid bilayer using a two-step procedure. Microsomes were initially treated with concentrations of octyl-β-d-thioglucopyranoside and glycodeoxycholate below their critical micelle concentration, 4.5 and 1.1 millimolar, respectively, to remove loosely associated proteins. Complete solubilization of the squalene synthetase enzyme activity was achieved after a second treatment at detergent concentrations above or at their critical micelle concentration, 18 and 2.2 millimolar, respectively. The detergent-solubilized enzyme was further purified by a combination of ultrafiltration, gel permeation, and Fast Protein Liquid Chromatography anion exchange. A 60-fold purification and 20% recovery of the enzyme activity was achieved. The partially purified squalene synthetase protein was used to generate polyclonal antibodies from mice that efficiently inhibited synthetase activity in an in vitro assay. The apparent molecular mass of the squalene synthetase protein as determined by immunoblot analysis of the partially purified squalene synthetase protein separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 47 kilodaltons. The partially purified squalene synthetase activity was optimal at pH 6.0, exhibited a K_m for farnesyl diphosphate of 9.5 micromolar, and preferred NADPH as a reductant rather than NADH.     

The effect of Hg2+ on rabbit hepatic and pulmonary solubilized,partially purified N,N-dimethylaniline N-oxidases

Solubilization and partial purification of the rabbit pulmonary and hepatic N,N-dimethylaniline N-oxidases were carried out in order to study the effect of Hg²⁺ in vitro observed previously in the microsomal enzymes. Rabbit lung microsomal N,N-dimethylaniline (DMA) N-oxidase activity was stimulated 1.5–2 times by 0.1 mM Hg²⁺ added in vitro. This concentration of mercury inhibited hepatic microsomal N-oxidase by 50%. Upon solubilization and partial purification of the lung N-oxidase enzyme, stimulation of the N-oxidase activity by 0.1 mM Hg²⁺ was lost. It was found that the concentration of Hg²⁺ that would stimulate the partially purified pulmonary N-oxidases was 25 μM or less. Stimulation by 0.1 mM Hg²⁺ of the partially purified N-oxidase from lung was restored by addition of flavins (FMN or FAD) or a heat-stable (NH₄)₂SO₄ precipitated fraction obtained during the purification of the N-oxidase from solubilized pulmonary or hepatic microsomes. However, addition of the flavins or the solubilized, heat-stable fraction from liver or lung microsomes did not reverse inhibition by 0.1 mM Hg²⁺ of the N-oxidase in hepatic microsomes or in partially purified preparations from these hepatic microsomes. Kinetic data suggest that flavins and the heatstable factor isolated from microsomes lower the concentration of free Hg²⁺.The determination of kinetics of Hg²⁺ inhibition (liver) and activation (lung) with the partially purified N-oxidases showed that the pulmonary and hepatic DMA N-oxidase enzymes are markedly different with respect to their in vitro response to Hg²⁺. This suggests that the N-oxidases from liver and lung may be different enzymes.     

The role of sterol carrier protein2 and other hepatic lipid-binding proteins in bile-acid biosynthesis.

The ability of different lipid-binding proteins in liver cytosol to affect enzyme activities in bile-acid biosynthesis was studied in whole microsomes (microsomal fractions) and mitochondria and in purified enzyme systems. Sterol carrier protein2 stimulated the 7 alpha-hydroxylation of cholesterol and the 12 alpha-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha-diol in microsomes and the 26-hydroxylation of cholesterol in mitochondria 2-3-fold. It also stimulated the oxidation of 5-cholestene-3 beta, 7 alpha-diol into 7 alpha-hydroxy-4-cholesten-3-one in microsomes. The stimulatory effect of sterol carrier protein2 was much less with purified cholesterol 7 alpha- and 26-hydroxylase systems than with microsomes and mitochondria. No stimulatory effect of sterol carrier protein2 was observed with purified 12 alpha-hydroxylase and 3 beta-hydroxy-delta 5-C27-steroid oxidoreductase. Sterol carrier protein (fatty-acid-binding protein), 'DEAE-peak I protein' [Dempsey, McCoy, Baker, Dimitriadou-Vafiadou, Lorsbach & Howards (1981) J. Biol. Chem. 256, 1867-1873], ligandin (glutathione transferase B) and serum albumin had no marked stimulatory effects in either crude or in purified systems. The results suggest that sterol carrier protein2 facilitates the introduction of the less-polar substrates in bile-acid biosynthesis to the membrane-bound enzymes in crude systems in vitro. The broad substrate specificity appears, however, not to be consistent with a specific regulatory function for sterol carrier protein2 in bile-acid biosynthesis.     

Solubilization and Purification of NAD(P)H Dehydrogenase of Cucurbita Microsomes

An NAD(P)H dehydrogenase stimulated by quinone (P Pupillo, V Valenti, L de Luca, R Hertel 1986 Plant Physiol 80: 384-389) was solubilized from washed microsomes of zucchini squash hypocotyls (Cucurbita pepo L.) by use of 1% Triton X-100. The solubilized enzyme remained in solution in aqueous buffer and could be purified by a combination of Sepharose 6B chromatography and Blue Ultrogel chromatography. Of the three peaks of activity eluted from the latter column with a salt gradient, peak 3 had 50% or more of the activity and was almost pure enzyme. The preparation examined in SDS-gel electrophoresis consisted of two types of subunits, a (molecular weight 39,500) and b (37,000) in equal amounts. Peak 2 was less pure but had a similar polypeptide pattern. The active protein is proposed to be a heterotetramer (a₂b₂) having a molecular weight of about 150,000, as found by gel exclusion chromatography. The purified enzyme can reduce several quinones, DCPIP, cytochrome c, and with best efficiency ferricyanide, and is therefore a diaphorase. The kinetics for the substrates are negatively cooperative with Hill coefficients n_H = 0.55 ± 0.05 for NADPH and 0.22 ± 0.04 for duroquinone. A weak inhibition by p-hydroxymercuric benzoate and mersalyl (stronger with microsomal preparations) suggests the presence of essential sulfhydryl group(s). The possibility is discussed that the dehydrogenase is an NAD(P)H-P450 reductase or similar flavoprotein, and that it is responsible for the NADPH-cytochrome c reductase activity of plant microsomes.     

Microsomal enzymes of cholesterol biosynthesis from lanosterol. Solubilization and purification of steroid 8-isomerase

Steroid-8-ene isomerase that catalyzes isomerization of delta 8- to delta 7-sterols has been solubilized from rat liver microsomes with a mixture of two detergents, octylglucoside and sodium taurodeoxycholic acid. During a 40-fold enrichment of the solubilized enzyme, other enzymes of cholesterol biosynthesis, endogenous lipids, and electron carriers are removed. A comparison of properties of the solubilized and partially purified isomerase with the membrane-bound enzyme shows they are essentially identical with respect to pH profile, effect of inhibitors and cofactors, substrate specificity, and Km values. Addition of phospholipid to the partially purified enzyme stimulates activity as much as 1.8-fold over control rates. Although the relative rate of isomerization of cholesta-8,24-dien-3 beta-ol is six times that observed with cholest-8-en-3 beta-ol, the delta 8 to delta 7 ratio at equilibrium is approximately equal. The reversibility of the reaction has been demonstrated by the direct conversion of cholest-7-en-3 beta-ol to cholest-8-en-3 beta-ol; at equilibrium the delta 7-isomer is predominant (19/1). The purified enzyme does not catalyze isomerization of cholesta-8,14-dien-3 beta-ol and cholest-8(14)-en-3 beta-ol under conditions that result in equilibrium mixtures of isomers from cholest-8(9)-en-3 beta-ol. These results are consistent with the earlier suggestion that delta 8(14)-sterols are neither formed nor metabolized by the same microsomal enzymes that catalyze transformation of lanosterol to cholesterol.     

Purification of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

This paper describes a rapid purification procedure for 3-hydroxy-3-methylglutaryl coenzyme A reductase, the major regulatory enzyme in hepatic cholesterol biosynthesis. A freeze-thaw technique is used for solubilizing the enzyme from rat liver microsomal membranes. No detergents or other stringent conditions are required. The purification procedure employs Blue Dextran-Sepharose-4B affinity chromatography, and purification can be carried out from microsomal membranes to purified enzyme in 8 to 10 hours. The purified enzyme has a specific activity of 517 nmoles/min/mg protein, and it is 975-fold purified with respect to the original microsomal membrane suspension. SDS polyacrylamide gel electrophoresis of the purified enzyme shows only trace impurities; the subunit molecular weight for the enzyme measured by this technique is 47,000.     

In vitro phosphorylation of 3-hydroxy-3-methylglutaryl coenzyme a reductase: Analysis of 32P-labeled,inactivated enzyme

Rat liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase was inactivated with Mg²⁺ and [γ-³²P]ATP, then solubilized and purified to homogeneity. The ³²P radioactivity was precipitated by antibody to homogeneous rat liver reductase and comigrated with nonprecipitated, homogeneous reductase on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under nondenaturing conditions, ³²P radioactivity comigrated with reductase protein and activity on polyacrylamide gels. These results provide direct support for the concept that the enzyme is covalently phosphorylated during the in vitro incubation of microsomes with Mg²⁺ and ATP.     

Characterization of the microsomal steroid-8-ene isomerase of cholesterol biosynthesis

Rat liver microsomes contain an enzyme that catalyzes the isomerization of the nuclear double bond of steroids from the 8(9) position to the 7(8) position. The enzyme is most active with zymosterol, 5alpha-cholesta-8,24-dien-3beta-ol, which is a precursor of cholesterol. Properties of the microsomal isomerase have now been studied, and preliminary data are reported on both regulation of enzymic activity and first steps in the solubilization of the enzyme from membranes. After a brief lag period, the velocity of isomerase is relatively constant for about 5 min of incubation, and then isomerization subsides. The apparent Michaelis constant (52-70 micro M) is difficult to determine accurately, due to these complex kinetic changes. V(max) is 4.0-4.7 nmol/min per mg of microsomal protein. The apparent specific activity is more than ten times that of liver microsomal methyl sterol oxidase. The maximal specific activity of microsomal isomerase is approximately doubled when rats are fed an intestinal bile acid sequestrant, cholestyramine. Changes in specific activity of isomerase parallel changes in activities of other microsomal enzymes of cholesterol biosynthesis, such as 3-hydroxy-3-methylglutaryl-CoA reductase and 4-methyl sterol oxidase. Isomerase activity is destroyed by phospholipase A digestion, high concentration of bile salts, and solvent extraction, all of which are known either to remove phospholipid or to alter microsomal membrane integrity. On the other hand, isomerase remains active in the presence of a mild, nonionic detergent, Triton WR-1339; thus, solubilization with nonionic detergents is under study.     

TheNeurospora crassa erg3 gene encodes a protein with sequence homology to both yeast sterol C-14 reductase and chicken lamin B receptor

Theerg3 gene ofNeurospora crassa was sequenced (EMBL accession no. X77955) and found to encode a protein of 490 amino acid residues with significant homology to the yeast sterol biosynthetic enzyme C-14 reductase (39% identity) and also to the C-tenninal region in the sequence reported for the chicken lamin B receptor (41% identity). The possibility that a single protein may possess both lamin B receptor and sterol C-14 reductase functions might account for non-sterol-biosynthetic effects of mutations in sterol biosynthesis genes and of inhibitors of sterol biosynthetic enzymes.     

Activities of 3-hydroxyl-3-methylglutaryl-CoA reductase and acetyl-CoA carboxylase and the rate of mevalonic acid, squalene, sterol and fatty acid biosynthesis from [1-14C]acetyl-CoA and [2-14C]malonyl-CoA in rat liver: effects of Triton WR 1339, starvation and cholesterol diet

The effects of Triton WR 1339, starvation and cholesterol diet on the activities of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) and acetyl-CoA carboxylase and on the rates of mevalonic acid (MVA) biosynthesis from acetyl-CoA and malonyl-CoA in the soluble (140 000 g) and microsomal fractions of rat liver, on the rate of incorporation of these substrates into squalene, cholesterol and lanosterol in the rat liver postmitochondrial fraction and on the rate of fatty acid biosynthesis was studied. The administration of Triton WR 1339 (200 mg per 100 g of body weight twice) stimulated the activity of HMG-CoA reductase and MVA biosynthesis from acetyl-CoA and malonyl-CoA in the intact and solubilized microsomal fractions and had no effect on these parameters in the soluble fraction. Starvation for 36 hrs did not cause inhibition of the reductase activity or MVA biosynthesis from both substrates in the soluble fraction. Alimentary cholesterol significantly increased the activity of HMG-CoA reductase, had no effect on the rate of MVA biosynthesis from acetyl-CoA and stimulated the malonyl-CoA incorporation in to MVA in the soluble fraction. Starvation an alimentary cholesterol inhibited the HMG-CoA reductase activity and MVA biosynthesis from both substrates in the solubilized microsomal fraction. Triton WR 1339 stimulated 4--19-fold the lipid formation in the total unsaponified fraction and its components i.e. squalene, lanosterol, cholesterol, from acetyl-CoA and only insignificantly (1,2--1,7-fold) increased malonyl-CoA incorporation into these compounds. Starvation and alimentary cholesterol repressed lanosterol and cholesterol biosynthesis from acetyl-CoA, decreased malonyl-CoA incorporation into these sterols and had no influence on squalene biosynthesis from the two substrates. Triton WR 1339 and starvation inhibited the acetyl-CoA carboxylase activity, unaffected by alimentary cholesterol. No significant changes in the rate of fatty acid biosynthesis from the substrates were observed. The data obtained provide evidence for the existence of autonomic pathways of MVA biosynthesis localized in the soluble and microsomal fractions of rat liver. The pathway of MVA biosynthesis in the soluble fraction is less sensitive to regulatory factors. Sterol biosynthesis from malonyl-CoA is also more resistant to regulatory effects than sterol biosynthesis from acetyl-CoA. This suggests that HMG-CoA reductase localized in the soluble fraction takes part in MVA and sterol biosynthesis from malonyl-CoA.     

Solubilization of the 97-kDa native form of liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase

Liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase was partially purified from cholestyramine-fed rats by sequential extraction of the membrane with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and polyethylene glycol nonylphenyl ether (Triton N-101) and solubilized by incorporation of the resulting insoluble protein preparation into a detergent mixture of Triton N-101 and sodium N-lauroylsarcosinate (Sarkosyl) in the presence of high salt. The purification procedure resulted in approximately a 3-4-fold increase in specific activity compared with the microsomal fraction, and the enzyme was recovered with yields as high as 63%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a blotting experiment using antiserum to the purified 53,000-dalton reductase fragment showed that the major immunoreactive polypeptide had a Mr of 97,000, that expected for the native intact form of the enzyme (Chin, D. J., Gil, G., Russell, D. W., Liscum, L., Luskey, K. L., Basu, S. K., Okayama, H., Berg, P., Goldstein, J. L., and Brown, M. S. (1984) Nature 308, 613-617). In addition, the effect of various detergents on the activity and stability of the membrane-bound and the partially purified enzyme was determined, and a method for protection of the reductase from inactivation caused by the addition of anionic detergents to the assay mixture is described.     

Closing the gap: identification of human 3-ketosteroid reductase,the last unknown enzyme of mammalian cholesterol biosynthesis

The protein encoded by the HSD17B7 gene was originally described as a prolactin receptor-associated protein and as 17beta-hydroxysteroid dehydrogenase (HSD) type 7. Its ability to synthesize 17beta-estradiol in vitro has been reported previously. However, we demonstrate that HSD17B7 is the ortholog of the yeast 3-ketosteroid reductase Erg27p and converts zymosterone to zymosterol in vitro, using reduced nicotinamide adenine dinucleotide phosphate as cofactor. Expression of human and murine HSD17B7 in an Erg27p-deficient yeast strain complements the 3-ketosteroid reductase deficiency of the cells and restores growth on sterol-deficient medium. A fusion of HSD17B7 with green fluorescent protein is located in the endoplasmic reticulum, the site of postsqualene cholesterogenesis. Further critical evidence for a role of HSD17B7 in cholesterol metabolism is provided by the observation that its murine ortholog is a member of the same highly distinct embryonic synexpression group as hydroxymethyl-glutaryl-coenzyme A reductase, the rate-limiting enzyme of sterol biogenesis, and is specifically expressed in tissues that are involved in the pathogenesis of congenital cholesterol-deficiency disorders. We conclude that HSD17B7 participates in postsqualene cholesterol biosynthesis, thus completing the molecular cloning of all genes of this central metabolic pathway. In its function as the 3-ketosteroid reductase of cholesterol biosynthesis, HSD17B7 is a novel candidate for inborn errors of cholesterol metabolism.     

NADH-Ferricyanide Reductase of Leaf Plasma Membranes : Partial Purification and Immunological Relation to Potato Tuber Microsomal NADH-Ferricyanide Reductase and Spinach Leaf NADH-Nitrate Reductase

Plasma membranes obtained by two-phase partitioning of microsomal fractions from spinach (Spinacea oleracea L. cv Medania) and sugar beet leaves (Beta vulgaris L.) contained relatively high NADH-ferricyanide reductase and NADH-nitrate reductase (NR; EC 1.6.6.1) activities. Both of these activities were latent. To investigate whether these activities were due to the same enzyme, plasma membrane polypeptides were separated with SDS-PAGE and analyzed with immunoblotting methods. Antibodies raised against microsomal NADH-ferricyanide reductase (tentatively identified as NADH-cytochrome b₅ reductase, EC 1.6.2.2), purified from potato (Solanum tuberosum L. cv Bintje) tuber microsomes, displayed one single band at 43 kilodaltons when reacted with spinach plasma membranes, whereas lgG produced against NR from spinach leaves gave a major band at 110 kilodaltons together with a few fainter bands of lower molecular mass. Immunoblotting analysis using inside-out and right-side-out plasma membrane vesicles strongly indicated that NR was not an integral protein but probably trapped inside the plasma membrane vesicles during homogenization. Proteins from spinach plasma membranes were solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio] 1-propane-sulfonate and separated on a Mono Q anion exchange column at pH 5.6 with fast protein liquid chromatography. One major peak of NADH-ferricyanide reductase activity was found after separation. The peak fraction was enriched about 70-fold in this activity compared to the plasma membrane. When the peak fractions were analyzed with SDS-PAGE the NADH-ferricyanide reductase activity strongly correlated with a 43 kilodalton polypeptide which reacted with the antibodies against potato microsomal NADH-ferricyanide reductase. Thus, our data indicate that most, if not all, of the truly membrane-bound NADH-ferricyanide reductase activity of leaf plasma membranes is due to an enzyme very similar to potato tuber microsomal NADH-ferricyanide reductase (NADH-cytochrome b₅ reductase).     

Fungal metabolism of biphenyl.

gamma-Glutamyl phosphate reductase, the second enzyme of proline biosynthesis, catalyses the formation of l-glutamic acid 5-semialdehyde from gamma-glutamyl phosphate with NAD(P)H as cofactor. It was purified 150-fold from crude extracts of Pseudomonas aeruginosa PAO 1 by DEAE-cellulose chromatography and hydroxyapatite adsorption chromatography. The partially purified preparation, when assayed in the reverse of the biosynthetic direction, utilized l-1-pyrroline-5-carboxylic acid as substrate and reduced NAD(P)(+). The apparent K(m) values were: NAD(+), 0.36mm; NADP(+), 0.31mm; l-1-pyrroline-5-carboxylic acid, 4mm with NADP(+) and 8mm with NAD(+); P(i), 28mm. 3-(Phosphonoacetylamido)-l-alanine, a structural analogue of gamma-glutamyl phosphate, inhibited this enzyme competitively (K(i)=7mm). 1-Pyrroline-5-carboxylate reductase (EC 1.5.1.2), the third enzyme of proline biosynthesis, was purified 56-fold by (NH(4))(2)SO(4) fractionation, Sephadex G-150 gel filtration and DEAE-cellulose chromatography. It reduced l-1-pyrroline-5-carboxylate with NAD(P)H as a cofactor to l-proline. NADH (K(m)=0.05mm) was a better substrate than NADPH (K(m)=0.02mm). The apparent K(m) values for l-1-pyrroline-5-carboxylate were 0.12mm with NADPH and 0.09mm with NADH. The 3-acetylpyridine analogue of NAD(+) at 2mm caused 95% inhibition of the enzyme, which was also inhibited by thio-NAD(P)(+), heavy-metal ions and thiol-blocking reagents. In cells of strain PAO 1 grown on a proline-medium the activity of gamma-glutamyl kinase and gamma-glutamyl phosphate reductase was about 40% lower than in cells grown on a glutamate medium. No repressive effect of proline on 1-pyrroline-5-carboxylate reductase was observed.     

Purification and immunochemical characteristics of NADPH-cytochrome P-450 oxidoreductase from tobacco cultured cells

NADPH-cytochrome P-450 oxidoreductase (EC 1.6.2.4) was purified from the microsomal fraction of tobacco (Nicotiana tabacum) BY2 cells by chromatography on two anion-exchange columns and 2′,5′ ADP-Sepharose 4B column. The purified enzyme showed a single protein band with a molecular weight of 79 kDa on SDS-PAGE and exhibited a typical flavoprotein redox spectrum, indicating the presence of an equimolar quantity of FAD and FMN. This enzyme followed Michaelis-Menten Kinetics with K_m values of 24 μM for NADPH and 16 μM for cytochrome c. An in vitro reconstituted system of the purified reductase with a partially purified tobacco cytochrome P-450 preparation showed the cinnamic acid 4-hydroxylase activity at the rate of 14 pmol min ⁻¹nmol⁻¹ P-450 protein and with a purified rabbit P-450_2C14 catalyzed N-demethylation of aminopyrine at the rate of 6 pmol min⁻¹ lnmo⁻¹ P-450 protein. Polyclonal antibodies raised against the purified reductase reacted with tobacco reductase but not with yeast reductase on Western blot analysis. Anti-yeast reductase antibodies did not react with the tobacco reductase. This result indicate that the tobacco reductase was immunochemically different from the yeast reductase. The anti-tobacco reductase antibodies totally inhibited the tobacco reductase activity, but not the yeast reductase. Also, Western blot analyses using the anti-tobacco reductase antibodies revealed that leaves, roots and shoots of Nicotiana tabacum plants contained an equal amount of the reductase protein. From these results, it was suggested that there are different antibody binding sites, which certainly participate in enzyme activity, between tobacco and yeast reductase.     

Kinetic characterization of an oxidative,cooperative HMG-CoA reductase from Burkholderia cenocepacia

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is a key enzyme in endogenous cholesterol biosynthesis in mammals and isoprenoid biosynthesis via the mevalonate pathway in other eukaryotes, archaea and some eubacteria. In most organisms that express this enzyme, it catalyzes the NAD(P)H-dependent reduction of HMG-CoA to mevalonate. We have cloned and characterized the 6x-His-tagged HMGR from the opportunistic lung pathogen Burkholderia cenocepacia. Kinetic characterization shows that the enzyme prefers NAD(H) over NADP(H) as a cofactor, suggesting an oxidative physiological role for the enzyme. This hypothesis is supported by the fact that the Burkholderia cenocepacia genome lacks the genes for the downstream enzymes of the mevalonate pathway. The enzyme exhibits positive cooperativity toward the substrates of the reductive reaction, but the oxidative reaction exhibits unusual double-saturation kinetics, distinctive among characterized HMG-CoA reductases. The unusual kinetics may arise from the presence of multiple active oligomeric states, each with different V_max values.