Mouse-liver glutathione reductase. Purification, kinetics, and regulation.

Glutathione reductase from the liver of DBA/2J mice was purified to homogeneity by means of ammonium sulfate fractionation and two subsequent affinity chromatography steps using 8-(6-aminohexyl)-amino-2'-phospho-adenosine diphosphoribose and N6-(6-aminohexyl)-adenosine 2',5'-biphosphate-Sephadex columns. A facile procedure for the synthesis of 8-(6-aminohexyl)-amino-2'-phospho-adenosine diphosphoribose is also presented. The purified enzyme exhibits a specific activity of 158 U/mg and an A280/A460 of 6.8. It was shown to be a dimer of Mr 105000 with a Stokes radius of 4.18 nm and an isoelectric point of 6.46. Amino acid composition revealed some similarity between the mouse and the human enzyme. Antibodies against mouse glutathione reductase were raised in rabbits and exhibited high specificity. The catalytic properties of mouse liver glutathione reductase have been studied under a variety of experimental conditions. As with the same enzyme from other sources, the kinetic data are consistent with a 'branched' mechanism. The enzyme was stabilized against thermal inactivation at 80 degrees C by GSSG and less markedly by NADP+ and GSH, but not by NADPH or FAD. Incubation of mouse glutathione reductase in the presence of NADPH or NADH, but not NADP+ or NAD+, produced an almost complete inactivation. The inactivation by NADPH was time, pH and concentration dependent. Oxidized glutathione protected the enzyme against inactivation, which could also be reversed by GSSG or other electron acceptors. The enzyme remained in the inactive state even after eliminating the excess NADPH. The inactive enzyme showed the same molecular weight as the active glutathione reductase. The spectral properties of the inactive enzyme have also been studied. It is proposed that auto-inactivation of glutathione reductase by NADPH and the protection as well as reactivation by GSSG play in vivo an important regulatory role.     

Purification of thioredoxin, thioredoxin reductase, and glutathione reductase by affinity chromatography.

A scheme is described for the large scale purification of thioredoxin, thioredoxin reductase, and glutathione reductase. The scheme is based on an initial separation of thioredoxin from the two reductases by affinity chromatography on agarose-bound N6-(6-aminohexyl)-adenosine 2',5'-bisphosphate (agarose-2',5'-ADP). The two reductases were then separated by hydrophobic chromatography and purified separately to homogeneity. Thioredoxin was purified to homogeneity by immunoadsorption to agarose containing immobilized goat anti-thioredoxin. Overall yields for thioredoxin, thioredoxin reductase, and glutathione reductase exceeded 80% in each case. Both reductases exhibit an absorption band at approximately 320 nm which appears due to a residual amount of tightly bound NADP. Presence of this absorption band has no apparent effect on the specific activity of either enzyme.     

2-[(4-Bromo-2,3-dioxobutyl)thio]- and 2-[(3-bromo-2-oxopropyl)thio]adenosine 2'5'-bisphosphate: new nucleotide analogues that act as affinity labels of nicotinamide adenine dinucleotide phosphate specific isocitrate dehydrogenase

Two new reactive adenine nucleotide analogues have been synthesized and characterized: 2-[(4-bromo-2,3-dioxobutyl)thio]adenosine 2',5'-bisphosphate (2-BDB-TA-2',5'-DP) and 2-[(3-bromo-2-oxopropyl)thio]adenosine 2',5'-bisphosphate (2-BOP-TA-2',5'-DP). Starting with NADP+, 2'-phospho-adenosine 5'-(diphosphoribose) (PADPR) was generated enzymatically and was converted to PADPR 1-oxide by reaction with m-chloroperoxybenzoic acid. Treatment with NaOH followed by reaction with carbon disulfide yielded 2-thioadenosine 2',5'-bisphosphate (TA-2',5'-DP). Condensation of TA-2',5'-DP with 1,4-dibromobutanedione or 1,3-dibromo-2-propanone gave the final products 2-BDB-TA-2',5'-DP and 2-BOP-TA-2',5'-DP, respectively. The structure of these new reagents was determined by UV, 1H NMR, 31P NMR, and 13C NMR spectroscopy as well as by bromide and phosphorus analysis. Both of these reagents exhibit properties expected for an affinity label of the coenzyme site of NADP+-dependent isocitrate dehydrogenase. With both reagents, biphasic kinetics of inactivation are observed that can be described in terms of a fast initial phase of inactivation resulting in partially active enzyme of 6-7% residual activity, followed by a slower phase leading to total inactivation. The inactivation rate constants for both reagents exhibit a nonlinear dependence on reagent concentration, consistent with the formation of a reversible complex with the enzyme prior to irreversible modification. The enzyme incorporates both reagents to a limited extent and is protected against inactivation by NADP+ and NADPH. The reaction of these new nucleotide analogues with isocitrate dehydrogenase is compared to the much slower inactivation caused by bromoacetone, indicating the importance of the nucleotide moiety in the functioning of the affinity labels. It is likely that 2-BDB-TA-2',5'-DP and 2-BOP-TA-2',5'-DP will have general applicability as affinity labels for other NADP+ binding enzymes.     

Mitochondrial nicotinamide nucleotide transhydrogenase: active site modification by 5'-[p-(fluorosulfonyl)benzoyl]adenosine

Membrane-bound and purified mitochondrial energy-linked nicotinamide nucleotide transhydrogenase (TH) was inhibited by incubation with 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA), which is an analogue of TH substrates and their competitive inhibitors, namely, 5'-, 2'-, or 3'-AMP. NAD(H) and analogues, NADP, 5'-AMP, 5'-ADP, and 2'-AMP/3'-AMP mixed isomers protected TH against inhibition by FSBA, but NADPH accelerated the inhibition rate. In the absence of protective ligands or in the presence of NADP, FSBA appeared to modify the NAD(H) binding site of TH, because, unlike unmodified TH, the enzyme modified by FSBA under these conditions did not bind to an NAD-affinity column (NAD-agarose). However, when the NAD(H) binding site of TH was protected in the presence of 5'-AMP or NAD, then FSBA modification resulted in an inhibited enzyme that did bind to NAD-agarose, suggesting FSBA modification of the NADP(H) binding site or an essential residue outside the active site. [3H]FSBA was covalently bound to TH, and complete inhibition corresponded to the binding of about 0.5 mol of [3H]FSBA/mol of TH. Since purified TH is known to be dimeric in the isolated state, this binding stoichiometry suggests half-of-the-sites reactivity. A similar binding stoichiometry was found earlier for complete inhibition of TH by [14C]DCCD [Phelps, D.C., & Hatefi, Y. (1984) Biochemistry 23, 4475-4480]. The active site directed labeling of TH by radioactive FSBA should allow isolation of appropriate peptides for sequence analysis of the NAD(H) and possibly the NADP(H) binding domains.     

Expression of the Escherichia coli pntA and pntB genes, encoding nicotinamide nucleotide transhydrogenase, in Saccharomyces cerevisiae and its effect on product formation during anaerobic glucose fermentation.

We studied the physiological effect of the interconversion between the NAD(H) and NADP(H) coenzyme systems in recombinant Saccharomyces cerevisiae expressing the membrane-bound transhydrogenase from Escherichia coli. Our objective was to determine if the membrane-bound transhydrogenase could work in reoxidation of NADH to NAD+ in S. cerevisiae and thereby reduce glycerol formation during anaerobic fermentation. Membranes isolated from the recombinant strains exhibited reduction of 3-acetylpyridine-NAD+ by NADPH and by NADH in the presence of NADP+, which demonstrated that an active enzyme was present. Unlike the situation in E. coli, however, most of the transhydrogenase activity was not present in the yeast plasma membrane; rather, the enzyme appeared to remain localized in the membrane of the endoplasmic reticulum. During anaerobic glucose fermentation we observed an increase in the formation of 2-oxoglutarate, glycerol, and acetic acid in a strain expressing a high level of transhydrogenase, which indicated that increased NADPH consumption and NADH production occurred. The intracellular concentrations of NADH, NAD+, NADPH, and NADP+ were measured in cells expressing transhydrogenase. The reduction of the NADPH pool indicated that the transhydrogenase transferred reducing equivalents from NADPH to NAD+.     

Structure-guided engineering of the coenzyme specificity of Pseudomonas fluorescens mannitol 2-dehydrogenase to enable efficient utilization of NAD(H) and NADP(H)

The structure of Pseudomonas fluorescens mannitol 2-dehydrogenase with bound NAD+ leads to the suggestion that the carboxylate group of Asp(69) forms a bifurcated hydrogen bond with the 2' and 3' hydroxyl groups of the adenosine of NAD+ and contributes to the 400-fold preference of the enzyme for NAD+ as compared to NADP+. Accordingly, the enzyme with the Asp(69)-->Ala substitution was found to use NADP(H) almost as well as wild-type enzyme uses NAD(H). The Glu(68)-->Lys substitution was expected to enhance the electrostatic interaction of the enzyme with the 2'-phosphate of NADP+. The Glu(68)-->Lys:Asp(69)-->Ala doubly mutated enzyme showed about a 10-fold preference for NADP(H) over NAD(H), accompanied by a small decrease in catalytic efficiency for NAD(H)-dependent reactions as compared to wild-type enzyme.     

Affinity labeling of a glutamyl peptide in the coenzyme binding site of NADP+-specific glutamate dehydrogenase of Salmonella typhimurium by 2-[(4-bromo-2,3-dioxobutyl)thio]-1,N6-ethenoadenosine 2',5'-bisphosphate

NADP+-specific glutamate dehydrogenase from Salmonella typhimurium, cloned and expressed in Escherichia coli, has been purified to homogeneity. The nucleotide sequence of S. typhimurium gdhA was determined and the amino acid sequence derived. The nucleotide analogue 2-[(4-bromo-2,3-dioxobutyl)thio]-1,N6-ethenoadenosine 2',5'-bisphosphate (2-BDB-T epsilon A-2',5'-DP) reacts irreversibly with the enzyme to yield a partially inactive enzyme. After about 60% loss of activity, no further inactivation is observed. The rate of inactivation exhibits a nonlinear dependence on 2-BDB-T epsilon A-2',5'-DP concentration with kmax = 0.160 min-1 and KI = 300 microM. Reaction of 200 microM 2-BDB-T epsilon A-2',5'-DP with glutamate dehydrogenase for 120 min results in the incorporation of 0.94 mol of reagent/mol of enzyme subunit. The coenzymes, NADPH and NADP+, completely protect the enzyme against inactivation by the reagent and decrease the reagent incorporation from 0.94 to 0.5 mol of reagent/mol enzyme subunit, while the substrate alpha-ketoglutarate offers only partial protection. These results indicate that 2-BDB-T epsilon A-2',5'-DP functions as an affinity label of the coenzyme binding site and that specific reaction occurs at only about 0.5 sites/enzyme subunit or 3 sites/hexamer. Glutamate dehydrogenase modified with 200 microM 2-BDB-T epsilon A-2',5'-DP in the absence and presence of coenzyme was reduced with NaB3H4, carboxymethylated, and digested with trypsin. Labeled peptides were purified by high performance liquid chromatography and characterized by gas phase sequencing. Two peptides modified by the reagent were isolated and identified as follows: Phe-Cys(CM)-Gln-Ala-Leu-Met-Thr-Glu-Leu-Tyr-Arg and Leu-Cys(CM)-Glu-Ile-Lys. These two peptides were located within the derived amino acid sequence as residues 146-156 and 282-286. In the presence of NADPH, which completely prevents inactivation, only peptide 146-156 was labeled. This result indicates that modification of the pentapeptide causes loss of activity. Glutamate 284 in this peptide is the probable reaction target and is located within the coenzyme binding site.     

Biospecific affinity chromatography of an adenosine 3′:5′-cyclic monophosphate-stimulated protein kinase (protamine kinase from trout testis) by using immobilized adenine nucleotides

1. Two adenine nucleotides, 8-(6-aminohexyl)aminoadenosine 3':5'-cyclic monophosphate and 8-(6-aminohexyl)amino-AMP, were synthesized. Their structures were established in particular by using mass spectroscopy. 2. Free cyclic AMP and 8-(6-aminohexyl)amino cyclic AMP both stimulate protamine kinase activity at low concentrations, but are inhibitory at concentrations above 0.1mm. AMP is an inhibitor of enzymic activity, whereas neither 8-(6-aminohexyl)amino-AMP nor the earlier synthesized N(6)-(6-aminohexyl)-AMP is inhibitory. 3. The nucleotides were coupled to Sepharose 4B and used for biospecific chromatography of partially purified protamine kinase. Enzyme applied at high buffer concentrations to the cyclic AMP-Sepharose material was retarded and thereby purified tenfold. At low buffer concentrations the enzyme was adsorbed to the affinity material, and was subsequently released by a pulse of the inhibitor AMP, yielding a 50-100-fold purification. Enzyme applied to immobilized 8-(6-aminohexyl)amino-AMP or N(6)-(6-aminohexyl)-AMP was eluted together with the main protein peak in the void volume. 4. Protamine kinase eluted from 8-(6-aminohexyl)amino cyclic AMP-Sepharose was no longer activated by cyclic AMP. Results from sucrose gradient centrifugation suggest that a dissociation of the enzyme took place on the immobilized nucleotide. 5. Further information on the mass spectroscopy has been deposited as Supplementary Publication SUP 50026 at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5.     

Purification and properties of 5'-nucleotidase from the membrane of Vibrio costicola, a moderately halophilic bacterium.

Two different Mg2+-dependent adenosine 5'-triphosphate-hydrolyzing activities were detected in membranes of Vibrio costicola, a novel 5'-nucleotidase and an N,N'-dicyclohexylcarbodiimide-sensitive adenosine triphosphatase. The former and the latter had different requirements for Mg2+ and were selectively assayed in the membranes by using, respectively, 20 and 2 mM Mg2+. The two enzymes were extracted with a combination of Triton X-100 and octylglucoside, separated on a diethylaminoethyl cellulose column, and purified on glycerol gradients. The purified 5'-nucleotidase consisted of one major polypeptide of 70,000 daltons when analyzed on polyacrylamide gels in the presence of sodium dodecyl sulfate. The purified 5'-nucleotidase was similar in substrate specificities, divalent cation specificities, and pH profiles to the membrane-bound N,N'-dicyclohexylcarbodiimide-insensitive nucleotide-phosphohydrolyzing activity. The enzyme hydrolyzed nucleoside 5'-tri, 5'-di, and 5'-monophosphates at comparable rates. Inorganic pyrophosphate, p-nitrophenyl phosphate, glucose 6-phosphate, beta-glycerophosphate, adenosine 5'-diphosphate glucose, adenosine 3'-monophosphate, and cyclic adenosine 3',5'-monophosphate were not hydrolyzed, either im membranes or by the purified 5'-nucleotides. Actions of NaCl and KCl on the activity of the 5'-nucleotidase were studied. The enzyme was activated by both NaCl and KCl; the activation profiles however, were different for the membrane-bound and purified 5'-nucleotidase. The purified enzyme, unlike the membrane-bound enzyme, was markedly stimulated by high concentrations of NaCl (up to 3 M).     

The effects of several ion channel blockers and calmodulin antagonists on fertilization-induced acid release and 45Ca2+ uptake in sea urchin eggs

The effects of calcium antagonists, diltiazem and verapamil, and calmodulin antagonists, chlorpromazine, N-(6-aminohexyl)-1-naphthalenesulfonamide hydrochloride (W-5) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), were tested on two responses of the sea urchin egg to insemination: (1) H+ release; (2) Ca2+ uptake. It was found that calcium antagonists inhibited both processes, while calmodulin antagonists only inhibited H+ release but not Ca2+ uptake. Verapamil and diltiazem were effective to inhibit H+ release when added to the egg suspension up to 120 sec and W-7 was effective around 150 sec after insemination. Calcium antagonists became ineffective earlier than W-7 in inhibiting H+ release. A calmodulin-dependent step may thus occur linking the Ca2+ uptake and H+ release. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an anion channel blocker, also inhibited both Ca2+ uptake and H+ release. This result suggests that an uptake of anion(s) occurs along with Ca2+ uptake.     

Inosine 5''-monophosphate dehydrogenase of Escherichia coli. Purification by affinity chromatography, subunit structure and inhibition by guanosine 5''-monophosphate.

Escherichia coli IMP dehydrogenase (EC 1.2.1.14) was purified by affinity chromatography on immobilized nucleotides. The enzyme binds to agarose-bound 8-(6-aminohexyl)-AMP, N6-(6-aminohexyl)-AMP and 8-(8-amino-octyl)-IMP but not to immobilized NAD+ or Cibacron Blue F3G-A. AMP proved to be an effective eluent. A large-scale purification scheme in which 8-(6-aminohexyl)-AMP-agarose was used resulted in a homogeneous preparation of IMP dehydrogenase. The enzyme was also purified by immunoprecipitation with monospecific antisera. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, N-terminal amino acid analysis and tryptic 'finger-printing' demonstrated that IMP dehydrogenase comprises identical subunits of mol.wt. 58000. Trypsin and Pronase cleave the 58000-mol.wt. subunit into peptides of mol.wts. 42000 and 14000, with a concomitant decrease in enzyme activity. These observations rationalize much of the contradictory data on the subunit composition of the enzyme found in the literature. GMP appears to be a competitive inhibitor with respect to IMP, with no evidence for regulatory behaviour being found. The two purification procedures were also used to purify inactive mutant enzymes from guaB mutant strains of E. coli.     

Coupling of "malic" enzyme and NADPH:NAD transhydrogenase in the energetics of Hymenolepis diminuta (Cestoda)

Acetylpyridine NADP replaced NADP in promoting the Mn2+ ion-requiring mitochondrial "malic" enzyme of Hymenolepis diminuta. Disrupted mitochondria displayed low levels of an apparent oxaloacetate-forming malate dehydrogenase activity when NAD or acetylpyridine NAD served as the coenzyme. Significant malate-dependent reduction of acetylpyridine NAD by H. diminuta mitochondria required Mn2+ ion and NADP, thereby indicating the tandem operation of "malic" enzyme and NADPH:NAD transhydrogenase. Incubation of mitochondrial preparations with oxaloacetate resulted in a non-enzymatic decarboxylation reaction. Coupling of malate oxidation with electron transport via the "malic" enzyme and transhydrogenase was demonstrated by polarographic assessment of mitochondrial reduced pyridine nucleotide oxidase activity.     

Properties of enzyme fraction A from Chlorella and copurification of 3' (2'), 5'-biphosphonucleoside 3' (2')-phosphohydrolase, adenosine 5'phosphosulfate sulfohydrolase and adenosine-5'-phosphosulfate cyclase activities.

Enzyme fraction A from Chlorella which catalyzes the formation of adenosine 5'-phosphosulfate from adenosine 3'-phosphate 5'-phosphosulfate is further characterized. Fraction A is found to contain an Mg2+ -activated and Ca2+ -inhibited 3' (2')-nucleotidase specific for 3' (2'), 5'-biphosphonucleosides. This activity has been named 3' (2), 5'-biphosphonucleoside 3' (2')-phosphohydrolase. The A fraction is also found to contain an activity which catalyzes the formation of adenosine 3':5'-monophosphate (cyclic AMP) from adenosine 5'-phosphosulfate (adenosine 5'-phosphosulfate cyclase). Under the same conditions of assay, 5'-ATP and 5'-ADP are not substrated for cyclic AMP formation. Unlike the 3' (2'), 5'-biphosphonucleoside 3' (2')-phosphohydrolase activity, the adenosine 5'-phosphosulfate cyclase activity does not require Mg2+, requires NH+4 or Na+, and is not inhibited by Ca2+. The A fraction also contains an adenosine 5'-phospho sulfate sulfohydrolase activity which forms 5'-AMP and sulfate. The three activities remain together during purification and acrylamide gel electrophoresis of the purified preparation yields a pattern where only one protein band has all three activities. The phosphohydrolase can be separated from the other two activities by affinity chromatography on agarose-hexyl-adenosine 3'n5'-bisphosphate yielding a phosphohydrolase preparation showing a single band on gel electrophoresis. The adenosine 5'-phosphosulfate cyclase may provide an alternate route of cyclic AMP formation from sulfate via ATP sulfurylase, but its regulatory significance in Chlorella, if any, remains to be demonstrated. In sulfate reduction, the phosphohydrolase may serve to provide a readily utilized pool of adenosine 5'-phosphosulfate as needed by the adenosine 5'-phosphosulfate sulfotransferase. The cyclase and sulfohydrolase activities would be regarded as side reactions incidental to this pathway, but may be of importance in other metabolic and regulatory reactions.     

Conformational features of bovine heart mitochondrial transhydrogenase

Both purified and functionally reconstituted bovine heart mitochondrial transhydrogenase were treated with various sulfhydryl modification reagents in the presence of substrates. In all cases, NAD+ and NADH had no effect on the rate of inactivation. NADP+ protected transhydrogenase from inactivation by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in both systems, while NADPH slightly protected the reconstituted enzyme but stimulated inactivation in the purified enzyme. The rate of N-ethylmaleimide (NEM) inactivation was enhanced by NADPH in both systems. The copper-(o-phenanthroline)2 complex [Cu(OP)2] inhibited the purified enzyme, and this inhibition was substantially prevented by NADP+. Transhydrogenase was shown to undergo conformational changes upon binding of NADP+ or NADPH. Sulfhydryl quantitation with DTNB indicated the presence of two sulfhydryl groups exposed to the external medium in the native conformation of the soluble purified enzyme or after reconstitution into phosphatidylcholine liposomes. In the presence of NADP+, one sulfhydryl group was quantitated in the nondenatured soluble enzyme, while none was found in the reconstituted enzyme, suggesting that the reactive sulfhydryl groups were less accessible in the NADP+-enzyme complex. In the presence of NADPH, however, four sulfhydryl groups were found to be exposed to DTNB in both the soluble and reconstituted enzymes. NEM selectively reacted with only one sulfhydryl group of the purified enzyme in the absence of substrates, but the presence of NADPH stimulated the NEM-dependent inactivation of the enzyme and resulted in the modification of three additional sulfhydryl groups. The sulfhydryl group not modified by NEM in the absence of substrates is not sterically hindered in the native enzyme as it can still be quantitated by DTNB or modified by iodoacetamide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human platelet secretion and aggregation induced by calcium ionophores. Inhibition by PGE1 and dibutyryl cyclic AMP

Ca2+, Mg2+-ionophores X537A and A23,187 (10(-7)-10(-6) M) induced the release of adenine nucleotides adenosine diphosphate (ADP, adenosine triphosphate (ATP), serotonin, beta-glucuronidase, Ca2+, and Mg2+ from washed human platelets. Enzymes present in the cytoplasm or mitochondria, and Zn2+ were not released. The rate of ATP and Ca2+ release measured by firefly lantern extract and murexide dye, respectively, was equivalent to that produced by the physiological stimulant thrombin. Ionophore-induced release of ADP, and serotonin was substantially (approximately 60%) but not completely inhibited by EGTA, EDTA, and high extracellular Mg2+, without significant reduction of Ca2+ release. The ionophore-induced release reaction is therefore partly dependent upon uptake of extracellular Ca2+ (demonstrated using 45Ca), but also occurs to a significant extent due to release into the cytoplasm of intracellular Ca2+. The ionophore-induced release reaction and aggregation of platelets could be blocked by prostaglandin E1 (PGE1) or dibutyryl cyclic AMP. The effects of PGE1, and N6, O2-dibutyryl adenosine 3':5'-cyclic monophosphoric acid (dibutyryl cAMP) were synergistically potentiated by the phosphodiesterase inhibitor theophylline. It is proposed that Ca2+ is the physiological trigger for platelet secretion and aggregation and that its intracellular effects are strongly modulated by adenosine 3':5'-cyclic monophosphoric acid (cyclic AMP).     

N5-(L-1-carboxyethyl)-L-ornithine:NADP+ oxidoreductase from Streptococcus lactis. Purification and partial characterization

N5-(L-1-Carboxyethyl)-L-ornithine:NADP+ oxidoreductase (EC 1.5.1.-) from Streptococcus lactis K1 has been purified 8,000-fold to homogeneity. The NADPH-dependent enzyme mediates the reductive condensation between pyruvic acid and the delta- or epsilon-amino groups of L-ornithine and L-lysine to form N5-(L-1-carboxyethyl)-L-ornithine and N6-(L-1-carboxyethyl)-L-lysine, respectively. The five-step purification procedure involves ion-exchange (DE52 and phosphocellulose P-11), gel filtration (Ultrogel AcA 44), and affinity chromatography (2',5'-ADP-Sepharose 4B). Approximately 100-200 micrograms of purified enzyme of specific activity 40 units/mg were obtained from 60 g of cells, wet weight. Anionic polyacrylamide gel electrophoresis revealed a single enzymatically active protein band, whereas three species (pI 4.8-5.1) were detected by analytical electrofocusing. The purified enzyme is active over a broad pH range of 6.5-9.0 and is stable to heating at 50 degrees C for 10 min. Substrate Km values were determined to be: NADPH, 6.6 microM; pyruvate, 150 microM; ornithine, 3.3 mM; and lysine, 18.2 mM. The oxidoreductase has a relative molecular mass (Mr = 150,000) as estimated by high pressure liquid chromatography exclusion chromatography and by polyacrylamide gradient gel electrophoresis. Conventional gel filtration indicated an Mr = 78,000, and a single protein band of Mr = 38,000 was revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is composed of identical subunits of Mr = 38,000, which may associate to yield both dimeric and tetrameric forms. Polyclonal antibody to the purified protein inhibited enzyme activity. The amino acid composition of the enzyme is reported, and the sequence of the first 37 amino acids from the NH2 terminus has been determined by stepwise Edman degradation.     

Effects of spermine on activity and stability of calcium-dependent guanosine 3',5'-monophosphate phosphodiesterase.

Spermine in micromolar concentrations decreased the basal activity of a guanosine 3',5'-monophosphate (cGMP) phosphodiesterase from bovine brain but had no effect in the presence of Ca2+ plus the calcium-dependent regulatory protein (CDR) which increased the activity of the enzyme 4- to 6-fold. Similar effects of spermine were observed on the enzyme at several stages of purification. Spermidine and putrescine were also inhibitory but higher concentrations were required. In the absence of Ca2+ and CDR, the enzyme exhibited two apparent Km values for cGMP (2.5 and 20 microM) which were unaltered by spermine. In the presence of Ca2+ and CDR (when spermine had no effect on activity), a single Km (3.5 microM) was observed. Enzyme purified by chromatography on CDR-Sepharose was rapidly inactivated during incubation at 30 degrees C in 5 mM potassium phosphate buffer (pH 7.0) with EDTA and ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA). Spermine (20 microM) partially stabilized enzyme activity under these conditions, although it was somewhat less effective than 2 mM MgCl2. The inhibitory effects of spermine (or other polyamines) on basal phosphodiesterase activity, which can be overcome by Ca2+ and CDR, could be important in the regulation of cellular cyclic nucleotide content.     

Conformations of the coenzymes and the allosteric activator, ADP, bound to NAD(+)-dependent isocitrate dehydrogenase from pig heart

NAD(+)-dependent isocitrate dehydrogenase from pig heart is an allosteric enzyme that is activated by ADP and is inhibited by NADPH in the presence of NADH. Transferred nuclear Overhauser effect measurements, made at a range of times to ensure that observed effects are due to direct dipole-dipole transfer and not to spin diffusion, were used to determine the conformations of pyridine nucleotide coenzymes and of the allosteric effector ADP. For NAD+, significant effects were observed on the N2 proton (on the nicotinamide ring) when the N1' proton (on the nicotinamide ribose) was saturated and on the N6 proton when the N2' proton was saturated, indicating that the conformation of the nicotinamide-ribose moiety is anti. The anti conformation is expected because of the stereospecificity of NAD(+)-dependent isocitrate dehydrogenase and is the same as for NADP(+)-dependent isocitrate dehydrogenase. For the adenosine moiety of NAD+, the predominant nuclear Overhauser effect on the A8 proton is found when the A2' proton is saturated. This result implies that the adenine-ribose bond is anti with respect to the ribose. Previous kinetic and binding studies of ADP activation have shown an influence of divalent metal ions. The conformation of bound ADP, in the presence of Mg2+ and/or Ca2+, is found to be anti about the adenine-ribose bond. The 3'H-8H distance increases when Ca2+ is added to the Mg-ADP-enzyme complex. Changes in the 4'H-1'H distance upon addition of isocitrate are indicative of interactions between the ADP activator site and the isocitrate site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and properties of the H(+)-nicotinamide nucleotide transhydrogenase from Rhodobacter capsulatus.

1. H(+)-transhydrogenase from Rhodobacter capsulatus is an integral membrane protein which, unlike the enzyme from Rhodospirillum rubrum, does not require the presence of a water-soluble component for activity. 2. The enzyme from Rb. capsulatus was solubilised in Triton X-100 and subjected to ion-exchange, hydroxyapatite and then gel-exclusion column chromatography. SDS/PAGE of the purified enzyme revealed the presence of two polypeptides with apparent Mr 53,000 and 48,000. Other minor components which were stained on the electrophoresis gels or which were revealed on Western blots exposed to antibodies raised to total membrane proteins, were probably contaminants. 3. Antibodies raised to the 53-kDa and 48-kDa polypeptides cross-reacted with equivalent polypeptides in Western blots of solubilised membranes from Rb. capsulatus, Rhodobacter sphaeroides and Rhs. rubrum. The significance of this finding is discussed in the context of the hypothesis [Fisher, R.R. & Earle, S.R. (1982) The pyridine nucleotide coenzymes, pp. 279-324, Academic Press, New York] that the soluble component associated with H(+)-transhydrogenase from Rhs. rubrum is an integral part of the catalytic machinery. Antibodies against the 48-kDa and 53-kDa polypeptides of the Rb. capsulatus enzyme cross-reacted with equivalent polypeptides in solubilised membranes of Escherichia coli. 4. The dependence of the rate of H- transfer by purified H(+)-transhydrogenase on the nucleotide substrate concentrations under steady-state conditions, the effects of inhibition by nucleotide products and the inhibition by 2'-AMP and by 5'-AMP suggest that the reaction proceeds by the random addition of substrates to the enzyme with the formation of a ternary complex. 5. In conflict with this conclusion, the reduction of acetylpyridine adenine dinucleotide (AcPdAD+) by NADH in the absence of NADP+ by bacterial membranes was earlier taken as evidence for the existence of a reduced enzyme intermediate [Fisher, R.R. & Earle, S.R. (1982) The pyridine nucleotide coenzymes, pp. 279-324, Academic Press, New York]. However, it is shown here that although chromatophore membranes of Rb. capsulatus catalysed the reduction of AcPdAD+ by NADH, the reaction was not associated with the purified H(+)-transhydrogenase. Moreover, in contrast with the true transhydrogenase reaction, the reconstitution of AcPdAD+ reduction by NADH (in the absence of NADP+) in washed membranes of Rhs. rubrum with partially purified transhydrogenase factor, was only additive.     

Mitochondrial nicotinamide nucleotide transhydrogenase: NADPH binding increases and NADP binding decreases the acidity and susceptibility to modification of cysteine-893

The mitochondrial nicotinamide nucleotide transhydrogenase is a dimeric enzyme of monomer Mr 110,000. It is located in the inner mitochondrial membrane and catalyzes hydride ion transfer between NAD(H) and NADP(H) in a reaction that is coupled to proton translocation across the inner membrane. The amino acid sequence and the nucleotide binding sites of the enzyme have been determined [Yamaguchi, M., Hatefi, Y., Trach, K., & Hoch, J.A. (1988) J. Biol. Chem. 263, 2761-2767; Wakabayashi, S., & Hatefi, Y. (1987) Biochem. Int. 15, 915-924]. N-Ethylmaleimide, as well as other sulfhydryl group modifiers, inhibits the transhydrogenase. The presence of NADP in the incubation mixture suppressed the inhibition rate by N-ethylmaleimide, and the presence of NADPH greatly increased it. NAD and NADH had little or no effect. The NADPH effect was concentration dependent and saturable, with a half-maximal NADPH concentration effect close to the Km of the enzyme for NADPH. Study of the effect of pH on the N-ethylmaleimide inhibition rate showed that NADPH binding by the enzyme lowers the apparent pKa of the N-ethylmaleimide-sensitive group by 0.4 of a pH unit and NADP binding raises this pKa by 0.4 of a pH unit, thus providing a rationale for the effects of NADP and NADPH on the N-ethylmaleimide inhibition rate. With the use of N-[3H]ethylmaleimide, the modified sulfhydryl group involved in the NADP(H)-modulated inhibition of the transhydrogenase was identified as that belonging to Cys-893, which is located 113 residues upstream of the tyrosyl residue modified by [p-(fluorosulfonyl)benzoyl]-5'-adenosine at the putative NADP(H) binding site of the enzyme (see above references).(ABSTRACT TRUNCATED AT 250 WORDS)