Molecular cloning and expression in Escherichia coli of a cellodextrinase gene from Bacteroides succinogenes S85.

A DNA fragment coding for a cellodextrinase of Bacteroides succinogenes S85 was isolated by screening of a pBR322 gene library in Escherichia coli HB101. Of 100,000 colonies screened on a complex medium with methylumbelliferyl-beta-D-cellobioside as the indicator substrate, two cellodextrinase-positive clones (CB1 and CB2) were isolated. The DNA inserts from the two recombinant plasmids were 7.7 kilobase pairs in size and had similar restriction maps. After subcloning from pCB2, a 2.5-kilobase-pair insert which coded for cellodextrinase activity was isolated. The enzyme was located in the cytoplasm of the E. coli host. It exhibited no activity on carboxymethyl cellulose, Avicel microcrystalline cellulose, acid-swollen cellulose, or cellobiose but hydrolyzed p-nitrophenyl-beta-D-cellobioside and p-nitrophenyl-beta-D-lactoside. The Km (0.1 mM) for the hydrolysis of p-nitrophenyl-cellobioside by the enzyme expressed in E. coli was similar to that reported for the purified enzyme from B. succinogenes. Expression of the cellodextrinase gene was subjected to catabolite repression by glucose and was not induced by cellobiose. The origin of the DNA insert from B. succinogenes was confirmed by Southern blot analysis. Western blotting (immunoblotting) using antibodies raised against the purified B. succinogenes cellodextrinase revealed a protein with a molecular weight of approximately 50,000 in E. coli clones which comigrated with the native enzyme isolated from B. succinogenes. These data indicate that the cellodextrinase gene expressed in E. coli is fully functional and codes for an enzyme with properties similar to those of the native enzyme.     

Calmodulin-linked equilibria in smooth muscle myosin light chain kinase

Competition experiments using 9-anthroylcholine, a fluorescent dye that undergoes calmodulin-dependent binding by smooth muscle myosin light chain kinase [Malencik, D. A., Anderson, S. R., Bohnert, J. L., & Shalitin, Y. S. (1982) Biochemistry 21, 4031], demonstrate a strongly stabilizing interaction between the adenosine 5'-triphosphate and myosin light chain binding sites operating within the enzyme-calmodulin complex but probably not in the free enzyme. The interactions in the latter case may be even slightly destabilizing. The fluorescence enhancement in solutions containing 5.0 microM each of the enzyme and calmodulin is directly proportional to the maximum possible concentration of bound calcium on the basis of four calcium binding sites. Evidently, all four calcium binding sites of calmodulin contribute about equally to the enhanced binding of 9-anthroylcholine by the enzyme. Fluorescence titrations on solutions containing 1.0 microM enzyme plus calmodulin yield a Hill coefficient of 1.2 and K = 0.35 +/- 0.08 microM calcium. Three proteolytic fragments of smooth muscle myosin light chain kinase, apparent products of endogenous proteolysis, were isolated and characterized. All three possess calmodulin-dependent catalytic activity. Their interactions with 9-anthroylcholine, in both the presence and absence of calmodulin, are similar to those of the native enzyme. However, the stabilities of their complexes with calmodulin vary. The corresponding dissociation constants range from 2.8 nM for the native enzyme and 8.5 nM for the 96K fragment to approximately 15 nM for the 68K and 90K fragments [0.20 N KCl, 50 mM 3-(N-morpholino)propanesulfonic acid, and 1 mM CaCl2, pH 7.3, 25 degrees C]. A coupled fluorometric assay, modified from a spectrophotometric assay for adenosine cyclic 3',5'-phosphate dependent protein kinase [Cook, P. F., Neville, M. E., Vrana, K. E., Hartl, F. T., & Roskoski, R. (1982) Biochemistry 21, 5794], has provided the first continuous recordings of myosin light chain kinase phosphotransferase activity. The results show that smooth muscle myosin light chain kinase is a responsive enzyme, whose activity adjusts rapidly to changes in solution conditions.     

Subunit interactions control protein phosphatase 2A. Effects of limited proteolysis, N-ethylmaleimide, and heparin on the interaction of the B subunit.

Protein phosphatase 2A consists of a heterotrimeric complex composed of a catalytic subunit (C) and two associated subunits (A and B). Limited tryptic digestion of the heterotrimeric ABC form resulted in the selective degradation of the Mr = 55,000 B subunit to a 48-kDa polypeptide. The cleavage sites were determined to be within a 3-7-kDa region of the COOH terminus. Proteolysis led to dissociation of the B subunit from the enzyme complex and correlated with an increase in cardiac myosin light chain, smooth muscle myosin light chain peptide, and Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) phosphatase activity. Purification of the digestion products and native gel electrophoresis indicated that dissociation of the B subunit was responsible for the increase in phosphatase activity. Kinetic analyses with several substrates revealed that dissociation of the B subunit resulted in a 2-7-fold increase in Vmax and a 1.6-5 fold increase in Km. Proteolytic dissociation of the B subunit increased the sensitivity of protein phosphatase 2A to inhibition by okadaic acid. Inhibition of the trypsinized enzyme was very similar to that observed for the purified AC form of protein phosphatase 2A. Incubation of the ABC complex with N-ethylmaleimide resulted in dissociation of the C subunit and generation of an AB complex. Selective release of the C subunit indicated that the B subunit interacts directly with the A subunit and that one or more free sulfhydryls are required to maintain the heterotrimeric structure of protein phosphatase 2A. Treatment of the enzyme with heparin resulted in an increase in specific activity that was due to the release of the B subunit from the complex. These results provide evidence that the B subunit binds directly to the A subunit to modulate enzyme activity and substrate specificity and that the COOH-terminal region of this protein is important for interaction with the AC complex. Dissociation of the B subunit by polyanionic substances related to heparin may represent a mechanism for regulating the activity of this enzyme.     

Enzyme activity of the cytochrome P-450 monooxygenase system in the presence of single chain lipid molecules

The influence of single chain lipids on the 7-ethoxycoumarin O-deethyase activity of the reconstituted binary protein complex of isolated cytochrome P450 and NADPH-cytochrome P450 reductase has been examined. The enzyme activity of this binary enzyme complex has been shown to be influenced by (i) altering the complexation process of both proteins, (ii) by altering the catalytic cycle time of the active binary protein complex and (iii) by altering the fraction of substrate molecules at the catalytic center of the enzyme. Competitive inhibition was measured for all single chain molecules. The following dissociation coefficients of substrate and lipids used for the catalytic center of the protein were obtained: 110 microM 7-ethoxycoumarin (substrate), 1.1 microM MOG (1-monooleoyl-rac-glycerol), 0.3 microM SPH (D-sphingosine), 1.5 microM OA (oleic acid), 3.0 microM LPC (L-alpha-lysophosphatidyl-choline), 15.5 microM MSG (1-monostearoyl-rac-glycerol), 9.5 microM AA (arachidonic acid), 9.0 microM PaCar (palmitoyl-L-carnitine), 3.5 microM MPG (2-monopalmitoyl-glycerol), 1.5 microM LPI (L-alpha-lysophosphatidyl-inositol), 50 microM LA (lauric acid), 60 microM MA (myristic acid), 85 microM PA (palmitic acid), >100 microM SA (stearic acid). Only competitive inhibition with the substrate molecule 7-ethoxycoumarin was observed for the single chain lipids LA, MA, PA, SPH, SA, and OA. Non-competitive effects were observed for MPG (-0.03 microM(-1)), PaCar (-0.02 microM(-1)), MSG (-0.023 microM(-1)), LPC (-0.03 microM(-1)), AA (-0.03 microM(-1)), and MOG (+0.04 microM(-1)). The negative sign indicates that the cycle time of the working binary complex is enlarged. The positive sign indicates that the formation of the binary complex is enhanced by MOG.     

Phage-enzymes: expression and affinity chromatography of functional alkaline phosphatase on the surface of bacteriophage.

We have demonstrated that an active enzyme can be expressed on the surface of a bacteriophage. The gene encoding alkaline phosphatase from Escherichia coli was cloned upstream of gene 3, which encodes a minor coat protein of the filamentous bacteriophage, fd. A fusion protein of the correct size was detected from viral particles by Western blotting. Ultrafiltration confirmed that the enzyme fusion behaves as part of a larger structure as would be expected of an enzyme fused to a viral particle. Both wild-type alkaline phosphatase (Arg166) and an active site mutant (Ala166) expressed in this way retain catalytic activity and have qualitatively similar kinetic properties to free enzyme. Values were obtained for Km of 72.7 and 1070 microM respectively whilst relative kcat for the mutant was 36% of that for wild-type. Phage particles expressing alkaline phosphatase were bound to an immobilized inhibitor (arsenate-Sepharose) and eluted with product (20 mM inorganic phosphate). In this way, the functional enzyme is co-purified with the DNA encoding it. This may permit a novel approach to enzyme engineering based on affinity chromatography of mutant enzymes expressed on the phage surface.     

Human pyridoxal kinase: overexpression and properties of the recombinant enzyme

Pyridoxal kinase catalyses the phosphorylation of the vitamin B6. A human brain pyridoxal kinase cDNA was isolated, and the recombinant enzyme was overexpressed in E. coli as a fusion protein with maltose binding protein (MBP). Pure pyridoxal kinase exhibits a molecular mass of about 40 kDa when examined by SDS-PAGE and FPLC gel filtration. The recombinant enzyme is a monomer endowed with catalytic activity, indicating that the native quaternary structure of pyridoxal kinase is not a prerequisite for catalytic function. Zn2+ is the most effective divalent cation in the phosphorylation of pyridoxal, and the human enzyme has maximum catalytic activity in the narrow pH range of 5.5-6.0. The Km values for two substrates pyridoxal and ATP are 97 microM and 12 microM, respectively. In addition, the unfolding processes of the recombinant enzyme were monitored by circular dichroism. The values of the free energy change of unfolding (AGo = 1.2 kcal x mol(-1) x K(-1)) and the midpoint transition (1 M) suggested that the enzyme is more stable than ovine pyridoxal kinase against denaturation by guanidine hydrochloride. Intrinsic fluorescence spectra of the human enzyme from red-edge excitation and fluorescence quenching experiments showed that the tryptophanyl residues are not completely exposed and more accessible to neutral acrylamide than to the negatively charged iodide. The first complete set of catalytic and structural properties of human pyridoxal kinase provide valuable information for further biochemical studies on this enzyme.     

A stable binary complex between Leishmania major thymidylate synthase and the substrate deoxyuridylate. A slow-binding interaction

The thymidylate synthase (TS) activity in Leishmania major resides on the bifunctional protein thymidylate synthase-dihydrofolate reductase (TS-DHFR). We have isolated, either by Sephadex G-25 chromatography or by nitrocellulose filter binding, a binary complex between the substrate deoxyuridylate (dUMP) and TS from L. major. The kinetics of binding support a "slow binding" mechanism in which dUMP initially binds to TS in a rapid, reversible pre-equilibrium step (Kd approximately 1 microM), followed by a slow first-order step (k = 3.5 X 10(-3) s-1) which results in the isolable complex; the rate constant for the dissociation of dUMP from this complex was 2.3 X 10(-4) s-1, and the overall dissociation constant was approximately 0.1 microM. The stoichiometry of dUMP to enzyme appears to be 1 mol of nucleotide bound/mol of dimeric TS-DHFR. Binary complexes between the stoichiometric inhibitor 5-fluorodeoxyuridylate (FdUMP) and TS, and between the product deoxythymidylate (dTMP) and TS were also isolated by nitrocellulose filter binding. Competition experiments indicated that each of these nucleotides were binding to the same site on the enzyme and that this site was the same as that occupied by the nucleotide in the FdUMP-cofactor X TS ternary complex. Thus, it appeared that the binary complexes were occupying the active site of TS. However, the preformed isolable dUMP X TS complex is neither on the catalytic path to dTMP nor did it inhibit TS activity, even though the dissociation of dUMP from this complex is several orders of magnitude slower than catalytic turnover (approximately 3 s-1). The results suggest that dUMP binds to one of the two subunits of the native protein in a catalytically incompetent form which does not inhibit activity of the other subunit.     

Molecular cloning and expression in Escherichia coli of a cellodextrinase gene from Bacteroides succinogenes S85

A DNA fragment coding for a cellodextrinase of Bacteroides succinogenes S85 was isolated by screening of a pBR322 gene library in Escherichia coli HB101. Of 100,000 colonies screened on a complex medium with methylumbelliferyl-beta-D-cellobioside as the indicator substrate, two cellodextrinase-positive clones (CB1 and CB2) were isolated. The DNA inserts from the two recombinant plasmids were 7.7 kilobase pairs in size and had similar restriction maps. After subcloning from pCB2, a 2.5-kilobase-pair insert which coded for cellodextrinase activity was isolated. The enzyme was located in the cytoplasm of the E. coli host. It exhibited no activity on carboxymethyl cellulose, Avicel microcrystalline cellulose, acid-swollen cellulose, or cellobiose but hydrolyzed p-nitrophenyl-beta-D-cellobioside and p-nitrophenyl-beta-D-lactoside. The Km (0.1 mM) for the hydrolysis of p-nitrophenyl-cellobioside by the enzyme expressed in E. coli was similar to that reported for the purified enzyme from B. succinogenes. Expression of the cellodextrinase gene was subjected to catabolite repression by glucose and was not induced by cellobiose. The origin of the DNA insert from B. succinogenes was confirmed by Southern blot analysis. Western blotting (immunoblotting) using antibodies raised against the purified B. succinogenes cellodextrinase revealed a protein with a molecular weight of approximately 50,000 in E. coli clones which comigrated with the native enzyme isolated from B. succinogenes. These data indicate that the cellodextrinase gene expressed in E. coli is fully functional and codes for an enzyme with properties similar to those of the native enzyme.     

Expression and purification of pheophorbidase, an enzyme catalyzing the formation of pyropheophorbide during chlorophyll degradation: comparison with the native enzyme

Formation of pyropheophorbide (PyroPheid) during chlorophyll metabolism in some higher plants has been shown to involve the enzyme pheophorbidase (PPD). This enzyme catalyzes the conversion of pheophorbide (Pheid) a to a precursor of PyroPheid, C-13(2)-carboxylPyroPheid a, by demethylation, and then the precursor is decarboxylated non-enzymatically to yield PyroPheid a. In this study, expression, purification, and biochemical characterization of recombinant PPD from radish (Raphanus sativus L.) were performed, and its properties were compared with those of highly purified native PPD. Recombinant PPD was produced using a glutathione S-transferase (GST) fusion system. The PPD and GST genes were fused to a pGEX-2T vector and expressed in Escherichia coli under the control of a T7 promoter as a fusion protein. The recombinant PPD-GST was expressed as a 55 kDa protein as measured by SDS-PAGE and purified by single-step affinity chromatography through a GSTrap FF column. PPD-GST was purified to homogeneity with a yield of 0.42 mg L(-1) of culture. The protein purified by this method was confirmed to be PPD by measuring its activity. The purified PPD-GST fusion protein revealed potent catalytic activity for demethylation of the methoxycarbonyl group of Pheid a and showed a pH optimum, substrate specificity, and thermal stability quite similar to the native enzyme purified from radish, except for the Km values toward Pheid a: 95.5 microM for PPD-GST and about 15 microM for native PPDs.     

Cloning, expression, and characterization of Bacillus sp. snu-7 inulin fructotransferase

A gene encoding inulin fructotransferase (di-D-fructofuranose 1,2': 2,3' dianhydride [DFA III]-producing IFTase, EC 4.2.2.18) from Bacillus sp. snu-7 was cloned. This gene was composed of a single, 1,353-bp open reading frame encoding a protein composed of a 40-amino acid signal peptide and a 410-amino acid mature protein. The deduced amino acid sequence was 98% identical to Arthrobacter globiformis C11-1 IFTase (DFA III-producing). The enzyme was successfully expressed in E. coli as a functionally active, His-tagged protein, and it was purified in a single step using immobilized metal affinity chromatography. The purified enzyme showed much higher specific activity (1,276units/mg protein) than other DFA III-producing IFTases. The recombinant and native enzymes were optimally active in very similar pH and temperature conditions. With a 103-min half-life at 60 degrees C, the recombinant enzyme was as stable as the native enzyme. Acidic residues and cysteines potentially involved in the catalytic mechanism are proposed based on an alignment with other IFTases and a DFA IIIase.     

C1-Tetrahydrofolate synthase from rabbit liver. Structural and kinetic properties of the enzyme and its two domains

C1-Tetrahydrofolate synthase is a multifunctional enzyme which catalyzes three reactions in 1-carbon metabolism: 10-formyltetrahydrofolate synthetase; 5,10-methenyltetrahydrofolate cyclohydrolase; 5,10-methylenetetrahydrofolate dehydrogenase. A rapid 1-day purification procedure has been developed which gives 40 mg of pure enzyme from 10 rabbit livers. The 10-formyltetrahydrofolate synthetase activity of this trifunctional enzyme has a specific activity that is 4-fold higher than the enzyme previously purified from rabbit liver. Conditions have been developed for the rapid isolation of a tryptic fragment of the enzyme which contains the methylenetetrahydrofolate dehydrogenase and methenyltetrahydrofolate cyclohydrolase activities. This fragment is a monomer exhibiting a subunit and native molecular weight of 36,000 in most buffers. However, in phosphate buffers the native molecular weight suggests that the fragment is a dimer. Conditions are also given whereby chymotryptic digestion allows the simultaneous isolation from the native enzyme of a large fragment containing the 10-formyltetrahydrofolate synthetase activity and a smaller fragment containing the dehydrogenase and cyclohydrolase activities. The large fragment is a dimer with a subunit molecular weight of 66,000. The small fragment retains all of the dehydrogenase and cyclohydrolase activities of the native enzyme. The large fragment is unstable but retains most of the 10-formyltetrahydrofolate synthetase activity. Km values of substrates for the two fragments are the same as the values for the native enzyme. The 10-formyltetrahydrofolate synthetase activity of the native enzyme requires ammonium or potassium ions for expression of full catalytic activity. The effect of these two ions on the catalytic activity of the large chymotryptic fragment is the same as with the native enzyme. We have shown by differential scanning calorimetry that the native enzyme contains two protein domains which show thermal transitions at 47 and 60 degrees C. Evidence is presented that the two domains are related to the two protein fragments generated by proteolysis of the native enzyme. The larger of the two domains contains the active site for the 10-formyltetrahydrofolate synthetase activity while the smaller domain contains the active site which catalyzes the dehydrogenase and cyclohydrolase reactions. Replacement of sodium ion buffers with either ammonium or potassium ions results in an increase in stability of the large domain of the native enzyme. This change in stability is not accompanied by a change in the quaternary structure of the enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

The enzyme defect in bovine protoporphyria. Studies with purified ferrochelatase

Ferrochelatase was purified from the livers of normal and protoporphyria cattle by chromatography on Blue Sepharose CL-6B in order to investigate the enzyme defect in this disorder. The increase in specific activity (up to 2900-fold) indicated that the normal and protoporphyria enzymes were purified to a similar degree. The mutant enzyme had catalytic activity which was 10 to 15% of normal ferrochelatase, although the Michaelis constants for protoporphyrin and iron were similar. The molecular mass of the normal and protoporphyria enzyme protein was 40 kDa as evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In the presence of 15 mM sodium cholate, gel filtration demonstrated a similar size. However, at a lower concentration of sodium cholate (4 mM) the molecular mass was about 240 kDa, suggesting that the purified enzymes aggregate under this condition. Polyvalent antibodies were raised in rabbits using as antigens purified normal native enzyme and normal 40-kDa protein which had been further purified by preparative SDS-PAGE. In Western blots these antibodies complexed with both the normal and mutant 40-kDa proteins. The amount of 40-kDa protein in normal and protoporphyria mitochondrial fractions was also similar as evaluated by Western blots. These studies indicate that the ferrochelatase defect in bovine protoporphyria probably results from a point gene mutation that causes a minor change in enzyme structure.     

Properties of a cGMP-dependent monomeric protein kinase from bovine aorta

A form of cGMP-dependent protein kinase (cGK) that was different from previously described cGK was purified from bovine aorta smooth muscle. The partial amino-terminal sequencing of this enzyme indicated that it was derived by endogenous proteolysis of the type I beta isozyme of cGK. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this form migrated as a smaller protein (Mr = 70,000) than the parent cGK (Mr = 80,000), and since the calculated nondenatured Mr was approximately 89,000 compared to Mr = 170,000 for the dimeric native enzyme, it represented a monomeric form of cGK. The monomer bound approximately 2 mol of [3H]cGMP per mol of monomer, although it had only one rapid component in [3H]cGMP dissociation assays as compared to one rapid and one slow component for the native cGK. The specific catalytic activity of the kinase was similar to that of the native enzyme, suggesting that the catalytic domain was essentially intact. The monomeric cGK incorporated significant 32P when incubated with Mg2+ and [gamma-32P]ATP in the presence of cGMP, although the phosphorylation proceeded at a slower rate than that obtained with native cGK. In contrast to previous reports of monomeric forms of cGK, this monomer was highly cGMP-dependent, although it had a slightly higher Ka (0.8 microM) for cGMP than that of the native enzyme (0.4 microM) and a low Hill coefficient of 1.0 (1.6 for the native enzyme). The cGMP dependence of the monomer did not decrease with dilution, implying that the cGMP dependence was not due to monomer-monomer interactions in the assay. The results indicated that the catalytic domain, cGMP binding domain(s), and inhibitory domain of cGK interact primarily within the same subunit rather than between subunits of the dimer as previously hypothesized for dimeric cGK.     

Structure and enzymatic functions of thioredoxin refolded by complementation of two tryptic peptide fragments.

The physicochemical and catalytic properties of thioredoxin-T' are described. This complemented protein structure consists of a 1:1 complex between the inactive fragments thioredoxin-T-(1--73) and thioredoxin T-(74--108). These are generated by selective trypsin cleavage at Arg-73 in lysine-modified and denatured Escherichia coli thioredoxin. Thioredoxin-T' was a slowly formed but stable complex with an apparent KD below 10(-8) M. The tryptophan fluorescence spectrum and the CD spectrum were very similar to those of native thioredoxin; some conformational differences were detected by gel chromatography and radioimmunoassay. Thioredoxin-T'-S2 was a substrate for NADPH and thioredoxin reductase and had 1--2% of the activity of native thioredoxin. This low relative activity was the result of a major increase in the Km value. Thioredoxin-(SH)2 was a hydrogen donor for E. coli ribonucleotide reductase with about 3% relative activity. These results for thioredoxin-T' are correlated with the known three-dimensional structure of thioredoxin. The microenvironment around Arg-73 that is close to the active disulfide appears to be of critical importance for the interactions of thioredoxin with thioredoxin reductase and ribonucleotide reductase.     

Purification and properties of deacetoxycephalosporin C synthase from recombinant Escherichia coli and its comparison with the native enzyme purified from Streptomyces clavuligerus

A putatively rate-limiting synthase (expandase) of Streptomyces clavuligerus was stabilized in vitro and purified 46-fold from cell-free extracts; a major enriched protein with a Mr of 35,000 was further purified by electrophoretic elution. Based on a 22-residue amino-terminal sequence of the protein, the synthase gene of S. clavuligerus was cloned and expressed in Escherichia coli (Kovacevic, S., Weigel, B.J., Tobin, M.B., Ingolia, T.D., and Miller, J. R. (1989) J. Bacteriol. 171, 754-760). The synthase protein was detected mainly from granules of recombinant E. coli. The recombinant synthase was solubilized from the granules by urea, and for the first time a highly active synthase was purified to near homogeneity. The synthase was a monomer with a Mr of 34,600 and exhibited two isoelectric points of 6.1 and 5.3. Its catalytic activity required alpha-ketoglutarate, Fe2+, and O2, was stimulated by dithiothreitol or ascorbate but not by ATP, and was optimal at pH 7.0 in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer and at 36 degrees C. The Fe2+ requirement was specific, and at least one sulfhydryl group in the purified enzyme was apparently essential for the ring expansion. The Km values of the enzyme for penicillin N and alpha-ketoglutarate were 29 and 18 microM, respectively, and the Ka for Fe2+ was 8 microM. The recombinant synthase was indistinguishable from the native synthase of S. clavuligerus by those biochemical properties. In addition to the enzymic ring expansion of penicillin N to deacetoxycephalosporin C, the recombinant synthase catalyzed a novel hydroxylation of 3-exomethylenecephalosporin C to deacetylcephalosporin C.     

Trans-resveratrol modulates the catalytic activity and mRNA expression of the procarcinogen-activating human cytochrome P450 1B1

The present study was performed to determine if trans-resveratrol (3,5,4'-trihydroxy-trans-stilbene) modulates the catalytic activity and gene expression of cytochrome P450 1B1 (CYP1B1). In vitro, trans-resveratrol decreased human recombinant CYP1B1-catalyzed 7-ethoxyresorufin O-dealkylation activity, with an IC50 value of 1.4 +/- 0.2 microM (mean +/- SEM). Enzyme kinetic analysis indicated that trans-resveratrol inhibited CYP1B1 enzyme activity by a mixed-type inhibition and the apparent Ki was 0.75 +/- 0.06 microM. To determine if trans-resveratrol modulates constitutive CYP1B1 gene expression, cultured MCF-7 human breast carcinoma cells were treated with trans-resveratrol. As indicated by RT-PCR analysis, treatment of MCF-7 cells with 10 microM trans-resveratrol decreased relative CYP1B1 mRNA levels after 5 h, but not after 1.5 or 3 h, of exposure. trans-Resveratrol treatment at 5, 7.5, 10, or 20 microM for 5 h produced a concentration-dependent decrease in CYP1B1 mRNA levels. The extent of suppression was approximately 50% at 20 microM concentration. The suppressive effect was not a consequence of a toxic response to the compound as assessed by a cell proliferation assay. Overall, our novel finding that trans-resveratrol inhibits the catalytic activity and suppresses the constitutive gene expression of CYP1B1 leads to the possibility that this nutraceutical confers protection against toxicity and carcinogenicity induced by compounds that undergo CYP1B1-catalyzed bioactivation.     

Inactivation of liver aldolase in fasting rabbits: Evidence for the accumulation of two different immunoreactive forms

During prolonged starvation the activity of aldolase in crude rabbit liver extracts decreases to less than one-half the value observed in extracts of livers from fed animals. The specific activity of the enzyme purified by adsorption on phosphocellulose and elution with substrate is also approximately one-half that of the purified native enzyme. Both the level of enzyme activity and the specific activity are restored to normal within 36 h of refeeding. After removal of active aldolase from the liver extracts by adsorption on phosphocellulose an additional immunoreactive protein can be isolated by adsorption on antialdolase-Sepharose and elution with 4 m MgCl₂. This protein is devoid of catalytic activity and in livers of fasted rabbits accounts for nearly 40% of the total immunoreactive material. It has also been detected in extracts prepared from livers of fed rabbits, where it accounts for 10–20% of the total protein adsorbed by antialdolase-Sepharose. The low-activity enzyme isolated from livers of fasted rabbits cannot be reactivated by sulfhydryl compounds; it shows similar sensitivity to heat and denaturing agents as the enzyme isolated from livers of fed rabbits. The activity ratios with fructose 1,6-bisphosphate, fructose 1-phosphate, and triose phosphate are similar to those observed for the native liver enzyme.     

Competitive cobalt for zinc substitution in mammalian methionine sulfoxide reductase B1 overexpressed in E. coli: structural and functional insight

Expression of the mammalian enzyme methionine sulfoxide reductase B1 (MsrB1) in Escherichia coli growing in cobalt-containing media resulted in the reproducible appearance of the stable cobalt-containing protein MsrB1-Co. NMR studies and biocomputing using the programs AnisoFit and Amber allowed us to generate a structure of MsrB1-Co sharing the overall fold with the native zinc-containing protein MsrB1-Zn. Our data suggest that the N-terminus containing resolving cysteine tends to be closer to the protein’s catalytic center than was previously reported. It is argued that this proximity supports the proposed catalytic mechanism and ensures high catalytic efficiency of MsrB1. Functional studies showed that both MsrB1-Zn and MsrB1-Co exhibit similar levels of activity, in agreement with the structural studies performed. The proposed metal ion substitution approach may have a methodological significance in determining whether methionine sulfoxide reductase B proteins contain a metal ion.     

Purification and properties of NADP-isocitrate dehydrogenase from the unicellular cyanobacterium Synechocystis sp. PCC 6803.

NADP-dependent isocitrate dehydrogenase activity has been screened in several cyanobacteria grown on different nitrogen sources; in all the strains tested isocitrate dehydrogenase activity levels were similar in cells grown either on ammonium or nitrate. The enzyme from the unicellular cyanobacterium Synechocystis sp. PCC 6803 has been purified to electrophoretic homogeneity by a procedure that includes Reactive-Red-120-agarose affinity chromatography and phenyl-Sepharose chromatography as main steps. The enzyme was purified about 600-fold, with a yield of 38% and a specific activity of 15.7 U/mg protein. The native enzyme (108 kDa) is composed of two identical subunits with an apparent molecular mass of 57 kDa. Synechocystis isocitrate dehydrogenase was absolutely specific for NADP as electron acceptor. Apparent Km values were 125, 59 and 12 microM for Mg2+, D,L-isocitrate and NADP, respectively, using Mg2+ as divalent cation and 4, 5.7 and 6 microM for Mn2+, D,L-isocitrate and NADP, respectively, using Mn2+ as a cofactor. The enzyme was inhibited non-competitively by ADP (Ki, 6.4 mM) and 2-oxoglutarate, (Ki, 6 mM) with respect to isocitrate and in a competitive manner by NADPH (Ki, 0.6 mM). The circular-dichroism spectrum showed a protein with a secondary structure consisting of about 30% alpha-helix and 36% beta-pleated sheet. The enzyme is an acidic protein with an isoelectric point of 4.4 and analysis of the NH2-terminal sequence revealed 45% identity with the same region of Escherichia coli isocitrate dehydrogenase. The aforementioned data indicate that NADP isocitrate dehydrogenase from Synechocystis resembles isocitrate dehydrogenase from prokaryotes and shows similar molecular and structural properties to the well-known E. coli enzyme.     

Isolation and properties of glycerol-3-phosphate oxidoreductase from human placenta

Glycerol-3-phosphate oxidoreductase (sn-glycerol 3-phosphate: NAD+ 2-oxidoreductase, EC 1.1.1.8) from human placenta has been purified by chromatography on 2,4,6-trinitrobenzenehexamethylenediamine-Sepharose, DEAE-Sephadex A-50 and 5'-AMP-Sepharose 4B approximately 15800-fold with an overall yield of about 19%. The final purified material displayed a specific activity of about 88 mumol NADH min-1 mg protein-1 and a single protein band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The native molecular mass, determined by Ultrogel AcA 44 filtration, was 62000 +/- 2000 whereas the subunit molecular mass, established on polyacrylamide gel in the presence of 0.1% sodium dodecyl sulphate, was 38000 +/- 500. The isoelectric point of the enzyme protein, determined by column isoelectric focusing, was found to be 5.29 +/- 0.09. The pH optimum of the placental enzyme was in the range 7.4-8.1 for dihydroxyacetone phosphate reduction and 8.7-9.2 for sn-glycerol 3-phosphate oxidation. The apparent Michaelis constants (Km) for dihydroxyacetone phosphate, NADH, sn-glycerol 3-phosphate and NAD+ were 26 microM, 5 microM, 143 microM and 36 microM respectively. The activity ratio of cytoplasmic glycerol-3-phosphate oxidoreductase to mitochondrial glycerol-3-phosphate dehydrogenase in human placental tissue was 1:2. The consumption of oxygen by human placental mitochondria incubated with the purified glycerol-3-phosphate oxidoreductase, NADH and dihydroxyacetone phosphate was similar to that observed in the presence of sn-glycerol 3-phosphate. The possible physiological role of glycerol-3-phosphate oxidoreductase in placental metabolism is discussed.