Enzymatic synthesis of the neuroexcitatory amino acid quisqualic acid by cysteine synthase

Purification of cysteine synthase from the leaves of Quisqualis indica var. villosa reveals the presence of two forms of this enzyme, separated by chromatography on DEAE-Sephadex A-50. Isoenzyme A was purified 10 000-fold and had a specific activity of 10.8 U/mg protein. Isoenzyme B was purified 460-fold with a specific activity of 0.49 U/mg protein. Both isoenzymes have the same M,s (58 000) and dissociate into identical subunits (M_r 29 000). The K_m value of isoenzyme A is 1.9 mM for O-acetyl-L-serine and 59 μM for sulphide, while that of isoenzyme B is 7.1 mM for O-acetyl-L-serine and 4.0 mM for 3,5-dioxo-1,2,4-oxadiazolidine. Both isoenzymes catalyse the formation of cysteine from O-acetyl-L-serine and hydrogen sulphide, but only isoenzyme B catalyses the formation of L-quisqualic acid. Other significant differences occur in the substrate specificity of the two isoenzymes. Some properties of the purified cysteine synthase isoenzymes are also described.     

Regulation of phospholipase D from human hepatocarcinoma cell line by purine nucleotides and protein kinase A

The regulation of phosphatidylcholine-specific phospholipase D by purine nucleotides and protein kinase A were studied in vitro using an enzyme preparation partially purified from the membranous fraction of 7721 hepatocarcinoma cells. It was found that the enzyme activity was elevated by low concentrations of some purine nucleotides, but the activating effects were decreased when the concentrations of the nucleotides were higher. The optimal concentrations of GTP, GTP[S] , GDP and ATP for maximal activation were 0.1mM, 5M,1 mM and 1 mM respectively. The activation caused by 1mM ADP was lower. The enzyme was not activated by 1mM AMP, but significant activation was observed by the addition of 1mM cAMP. The latter was mediated by protein kinase A, as a specific inhibitor of protein kinase A ablished the activation. There were synergic effects between ATP and GTP, ATP and PIP₂, but not between ATP and GTP[S] , or PIP₂ and GTP[S]. The activating effects of GTP and ATP were abolished by neomycin, a PIP₂ scavenger. These results suggest that phospholipase D is regulated by GTP-binding protein and the presence of PIP₂ is required for the activation induced by GTP. Protein kinase A may be another protein kinase in addition to protein kinase C and protein tyrosine kinase which regulate the activity of phospholipase D, when the intracellular concentration of cAMP is increased.     

Cherry fruit development: Indoleacetic acid oxidase isoenzymes in the seed

Two anionic indoleacetic acid oxidase isoenzymes were separated by polyacrylamide gel electrophoresis from an acetate buffer (0.2 M, pH 4.0) extract of sour cherry ( Prunus cerasus L. cv. Montmorency) seed. One isoenzyme migrated to Rf 0.25 (I1) and the other to Rf 0.78 (I₂). Isoenzyme I, exhibited hyperbolic kinetics and was found during all three stages of fruit development with the highest levels during early stage II. The isoenzyme I₂ showed sigmoidal kinetics and was found only during stages II and III of fruit growth with highest levels during stage III. The activities of both isoenzymes were markedly enhanced by addition of Mn2+ and 2,4-dichloro-phenol to the reaction mixture. Isoenzyme I, showed higher affinity for indoleacetic acid than isoenzyme I₂. The significance of these isoenzymes in cherry fruit growth is discussed.     

Organ distribution of rat histidine-pyruvate aminotransferase isoenzymes.

The organ distribution of rat histidine-pyruvate aminotransferase isoenzymes 1 and 2 was examined by using an isoelectric-focusing technique. Isoenzyme 1 (pI8.0) is present only in the liver and its activity is increased by the injection of glucagon, whereas isoenzyme 2 (pI5.2) is distributed in all tissues (liver, kidney, brain and heart) tested, and is not affected by glucagon injection. Isoenzyme 2 of the liver, kidney, brain and heart was purified by the same procedure and characterized. Isoenzyme 2 preparations from these four tissues were nearly identical in physical and enzymic properties. These properties differed from those previously found for the highly purified isoenzyme 1 preparation of rat liver. Isoenzyme 2 was active with pyruvate but not with 2-oxoglutarate as amino acceptor. Amino donors were effective in the following order of activity: tyrosine greater than histidine greater than phenylalanine greater than kynurenine greater than tryptophan. Very little activity was found with 5-hydroxytryptophan. The apparent Km for histidine was about 0.45 mM. The Km for pyruvate was about 4.5 mM with histidine as amino donor. The amino-transferase activities of isoenzyme 2 towards phenylalanine and tyrosine were inhibited by histidine. The ratio of aminotransferase activities towards these three amino acids was constant through gel filtration, electrophoresis, isoelectric focusing and sucrose-density-gradient centrifugation of the purified isoenzyme 2 preparations. These results suggest that these three activities are properties of the same enzyme protein. Sephadex G-150 gel filtration and sucrose-density-gradient centrifugation yielded mol.wts. of approx. 95000 and 92000 respectively. The pH optimum was between 9.0 and 9.3.     

Purification of pyruvate kinase isoenzymes type M1 and M2 from dog (Canis familiaris) and comparison of their properties with those from chicken and rat

Pyruvate kinase isoenzymes type M1 and M2 from dog muscle, lung and tumor have been isolated. The K0.5 for phosphoenol pyruvate have been determined to be 0.04 mM for dog muscle type M1, 0.24 mM for lung type M2 and 0.28 mM for tumor type M2 isoenzymes. The activator constant ka of L-serine is 240 nM from lung and 70 nM from tumor isoenzyme. Consistent with the assumption of a special form of pyruvate kinase M2 in dog tumor cells different isoelectric points and amino acid compositions have been found for the isoenzymes of lung and tumor.     

Lactate dehydrogenase isoenzymes of human semen

1. A lactate dehydrogenase isoenzyme present in human spermatozoa and semen was isolated and characterized biochemically in term of its pH for optimum activity and by means of K(m) values for lactate, NAD(+) and NAD analogues. The results were compared with those obtained with the human heart-type and the liver-type lactate dehydrogenase isoenzymes. 2. The enzyme was characterized by its resistance to digestion with different proteolytic enzymes. The time for 50% digestion in terms of residual dehydrogenase activity was compared with times obtained for the H(4)- and M(4)-types.     

Purification and characterization of two glutamate dehydrogenase isoenzymes from Brassica napus

Glutamate dehydrogenase (L-glutamate: NAD⁺ oxidoreductase, EC 1.4.1.2) was purified from Brassica napus leaves. Isoenzyme 1 (GDH1), with the lowest, and isoenzyme 7 (GDH7) with the highest electrophoretic mobility were characterized. The native GDH was estimated to have a molecular mass of about 239 kDa and consisted of six identical 41.4-kDa subunits for GDH1 and 42.4-kDa subunits for GDH7. The pH optima of both isoenzymes in amination and deamination reactions were 9.0 and 9.5, respectively. At optimum pH, the K_m values for ammonium, 2-oxoglutarate, NADH, NAD and glutamate did not differ between the two isoenzymes. Addition of 10 mM EGTA inhibited the amination activity of GDH1, but that of GDH7 remained at about 30 %. Cellular fractionation experiments showed that both GDH1 and GDH7 localized in mitochondria with a loose association with the mitochondrial membrane.     

Location and properties of two isoenzymes of cardiac adenylate kinase.

Adenylate kinase catalyses the equilibrium 2ADP = ATP + AMP. There are two isoenzymes of adenylate kinase in bovine ventricular tissue, one cytoplasmic, the other mitochondrial. Mitochondrial subfractionation locates this isoenzyme between the mitochondrial membranes with fatty acid-CoA ligase. The cytoplasmic and mitochondrial isoenzymes are distributed in ratio 3:2, and both forms were purified to homogeneity. They differ principally by charge, Km values for ATP, ADP and AMP, pH-stability and -activity profiles, and susceptibility to the inhibitor adenosine pentaphosphoadenosine. The forward and reverse reactions show similar energies of activation for the cytoplasmic enzyme, but differ for the mitochondrial enzyme. The molecular weights are indistinguishable. An integrated mechanism is formulated whereby one isoenzyme suppresses the activation of fatty acid and the other enhances carbohydrate utilization in hypoxic myocytes.     

Stimulation of the thiol-dependent ADP-ribosyltransferase and NAD glycohydrolase activities of Bordetella pertussis toxin by adenine nucleotides, phospholipids, and detergents

Pertussis toxin catalyzed ADP-ribosylation of the guanyl nucleotide binding protein transducin was stimulated by adenine nucleotide and either phospholipids or detergents. To determine the sites of action of these agents, their effects were examined on the transducin-independent NAD glycohydrolase activity. Toxin-catalyzed NAD hydrolysis was increased synergistically by ATP and detergents or phospholipids; the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) was more effective than the nonionic detergent Triton X-100 greater than lysophosphatidylcholine greater than phosphatidylcholine. The A0.5 for ATP in the presence of CHAPS was 2.6 microM; significantly higher concentrations of ATP were required for maximal activation in the presence of cholate or lysophosphatidylcholine. In CHAPS, NAD hydrolysis was enhanced by ATP greater than ADP greater than AMP greater than adenosine; ATP was more effective than MgATP or the nonhydrolyzable analogue adenyl-5'-yl imidodiphosphate. GTP and guanyl-5'-yl imidodiphosphate were less active than the corresponding adenine nucleotides. Activity in the presence of CHAPS and ATP was almost completely dependent on dithiothreitol; the A0.5 for dithiothreitol was significantly decreased by CHAPS alone and, to a greater extent, by CHAPS and ATP. To determine the site of action of ATP, CHAPS, and dithiothreitol, the enzymatic (S1) and binding components (B oligomer) were resolved by chromatography. The purified S1 subunit catalyzed the dithiothreitol-dependent hydrolysis of NAD; activity was enhanced by CHAPS but not ATP. The studies are consistent with the conclusion that adenine nucleotides, dithiothreitol, and CHAPS act on the toxin itself rather than on the substrate; adenine nucleotides appear to be involved in the activation of toxin but not the isolated catalytic unit.     

Modulation of guanine deaminase

1. Guanine deaminases purified from the 15000g supernatant fraction of iso-osmotic sucrose homogenates of rat and mouse liver and brain were tested for the influence of GTP and allantoin. 2. The suffixes A and B were assigned to the isoenzyme fractions eluted from DEAE-cellulose with the lower and the higher molarity of eluent respectively. Isoenzyme A from rat liver, the activity of which showed a sigmoid dependence on substrate saturation, was activated by GTP and inhibited by allantoin. Isoenzyme B, which had a hyperbolic substrate-saturation curve, was not influenced by GTP or allantoin. 3. Isoenzyme A from rat brain, the activity of which had a sigmoid dependence on substrate concentration, was stimulated by GTP. Isoenzyme B, which showed classical Michaelis-Menten kinetics, was inhibited by allantoin. 4. Mouse liver guanine deaminase was not influenced by either GTP or allantoin. 5. Isoenzyme A from mouse brain, which had a hyperbolic substrate-saturation curve, was not influenced by GTP or allantoin but isoenzyme B, with sigmoidal kinetics, was inhibited by allantoin. 6. Mg(2+) activated, or inhibited or did not have an effect on guanine deaminase, depending on the source of the enzyme. 7. The bearing of the above findings on the possible regulation of guanine deaminase activity in vivo is discussed.     

Pyruvate kinase isoenzymes in chromatin extracts of Ehrlich ascites tumour, Morris hepatoma 7777 and normal mouse and rat livers

A pyruvate kinase (EC 2.7.1.40) variant inhibited by L-cysteine has been found in Ehrlich ascites tumour and Morris hepatoma 7777, but not in normal mouse and rat livers used for comparison. Chromatin extracts of all materials studied contained three pyruvate kinase isoenzymes (alpha, beta, gamma) which showed the greatest electrophoretic mobility in normal mouse and rat livers. The isoenzyme mobility diminished in both tumour chromatin extracts, and the slow migrating gamma isoenzyme exhibited sensitivity to L-cysteine inhibition. This gamma isoenzyme sensitive to L-cysteine might be considered as a tumour marker. All tumour pyruvate kinase isoenzymes were insensitive to normal signal molecules, i.e., to ATP and fructose 1,6-diphosphate, which regulate liver pyruvate kinase activity. It was, however, noted that the binding of pyruvate kinase isoenzymes to DNA is connected with a diminution in their catalytic activity.     

Lactate dehydrogenase isoenzymes in the eye, cardiac and skeletal muscles of several decapods]

Properties of lactate dehydrogenase (LDH) in the eye, heart and muscles of Hemigrapsus sanguineus, Paralithodes camtschatica, Erimacrus isenbeckii, Pandalus latirastrus, Pagurus brachiomastus have been studied with acrylamide gel electrophoresis and kinetics analysis. LDH in all the tissues of all the representatives studied was found to be specific for L-pyruvate and lactate; it migrated in electrophoresis as a single band revealing low mobility towards anode. The isoenzyme from P. camtschatica and P. latirastrus differed from the isoenzymes of other animals studied by higher mobility towards anode that reflected higher negative value of its total charge. The LDH isoenzymes in all the animals studied resembled the A4 (LDH5) of the vertebrates being unstable to the denaturing action of high temperature and being unaffected by high concentrations of pyruvate up to 1.0.10-3M. On the other hand, in conrast to the A4 of mammals, the LDH in question displayed enhancement of the reaction rate and decrease of the Km values upon increase in the NAD+ and NAD.H concentrations both in the presence of high or low lactate and pyruvate concentrations. The isoenzymes displayed catalytic activity also in the presence of NADP, the Km values for pyruvate in the presence of equimolar (2.25 mM) concentrations of NAD.H or NADP.H were practically identical and were found to be within the limits of 14-26.10-5 M. Molecular weight of the LDH studied assessed by the gel filtration method was found to be 130-140,000. It is suggested that the LDH isoenzyme from the representatives of the decapod crayfish studied is homologous in its certain properties to the homotetrameric A4 form of the vertebrates.     

Ion-pair reverse-phase high-performance liquid chromatography. Application to the study of chicken liver NAD+ kinase

An ion-pair, reverse-phase, high-performance liquid chromatography method of assay was developed and used in a series of rate studies carried out with the enzyme chicken liver NAD+ kinase (ATP:NAD+ 2'-phosphotransferase, EC 2.7.1.23). Complete separation of all products and reactants was achieved within 15 min. ATP, NAD+, ADP, and NADP+ were monitored at 260 nm as they eluted from a Zorbax (Dupont) ODS (4.6 X 250-mm) column using an acetonitrile and 0.01 mM NH4(H2PO4)/0.005 M tetrabutylammonium phosphate (pH 7.0) gradient. The enzyme shows a marked preference for ATP (and dATP) and Mg2+ (or Mn2+) relative to other trinucleotides and divalent metal ions. It exhibits residual adenylate kinase and ATPase activity, but no NADH kinase activity. When polyphosphate replaced ATP, NADP+ production dropped to 2.5%. The addition of Ca2+ and/or bovine brain calmodulin did not significantly enhance the rate of NADP+ production.     

Purification of nuclear cAMP-independent protein kinases from rat ventral prostate

Two nuclear cAMP-independent protein kinases (designated PK-N1 and PK-N2) were purified from rat ventral-prostate and liver. The yield of enzyme units was 4-5% and 7-9% for each enzyme from the prostatic nuclei and liver nuclei, respectively. The average fold purification for prostatic nuclear protein kinase N1 and N2 was 1360 and 1833, respectively. The respective average specific activity of the two enzymes towards casein was 81,585 and 110,000 nmol 32P incorporated/hr/mg of enzyme. Protein kinase N1 comprised one polypeptide of Mr 35,000 which underwent phosphorylation in the presence of Mg2+ + ATP. Protein kinase N2 comprised two polypeptides Mr 40,000 and 30,000 of which only the Mr 30,000 polypeptide was autophosphorylated. Both enzymes were active towards casein, phosvitin, dephosphophosvitin, spermine-binding protein, and non-histone proteins in vitro. Little activity was detected towards histones. Both enzymes were stimulated by 150-200 mM NaCl. MgCl2 requirement varied with the protein substrate but was between 2-4 mM for both enzymes. With dephosphophosvitin as substrate, the apparent Km for ATP for N1 protein kinase was 0.01 mM. GTP did not replace ATP in this reaction. Protein kinase N2 was active in the presence of ATP or GTP. The apparent Km was 0.01 mM for ATP, but 0.1 mM for GTP.     

Chloroplast phosphoglycerate kinase from Euglena gracilis: endosymbiotic gene replacement going against the tide.

Two chloroplast phosphoglycerate kinase isoforms from the photosynthetic flagellate Euglena gracilis were purified to homogeneity, partially sequenced, and subsequently cDNAs encoding phosphoglycerate kinase isoenzymes from both the chloroplast and cytosol of E. gracilis were cloned and sequenced. Chloroplast phosphoglycerate kinase, a monomeric enzyme, was encoded as a polyprotein precursor of at least four mature subunits that were separated by conserved tetrapeptides. In a Neighbor-Net analysis of sequence similarity with homologues from numerous prokaryotes and eukaryotes, cytosolic phosphoglycerate kinase of E. gracilis showed the highest similarity to cytosolic and glycosomal homologues from the Kinetoplastida. The chloroplast isoenzyme of E. gracilis did not show a close relationship to sequences from other photosynthetic organisms but was most closely related to cytosolic homologues from animals and fungi.     

A comparative study of the purity, enzyme activity, and inactivation by hydrogen peroxide of commercially available horseradish peroxidase isoenzymes A and C

Horseradish peroxidase (HRP) is a commercially important enzyme that is available from a number of supply houses in a variety of grades of purity and isoenzymic combinations. The present article describes a comparative study made on nine HRP preparations. Six of these samples were predominantly composed of basic HRP, pl 8.5, and three of acidic HRP, pl 3.5. Two of the basic preparations were of lower purity than the others. The apparent molar catalytic activity of basic HRP with 0.5 mMABTS and 0.2 mM H(2)O(2) was around 950 s(-1) (about 770 s(-1) for the less pure samples) and with a 5 mM guaiacol and 0.6 mM H(2)O(2) was about 180 s(-1) for all the samples. A similar value (approximately 1000 s(-1)) was observed for acidic HRP but only at higher concentrations of ABTS (20 mM). With 20 mM guaiacol the molar catalytic activity of the acid isoenzyme was 65 s(-1). The apparent K(M) for ABTS of the acidic isoenzyme was 4 mM whereas for the basic isoenzyme it was 0.1 mM. All the enzymes were inactivated by H(2)O(2) when it was supplied as the only substrate. Under these conditions the partition ratio (r = number of catalytic cycles given by the enzyme before its inactivation), apparent dissociation constant (K(l)), and apparent rate constant of inactivation (k(inact)) were about twice as large for the acidic samples (1350, 2.6 mM, 9 . 10(-3) s(-1)) as for the basic (650, 1.3 mM, 5 . 10(-3) s(-1)). The apparent catalytic constant (k(cat)) was 3-4 times larger, and the efficiency of catalysis (k(cat)/K(l)) was double for the acidic isoenzyme, but the efficiency of inactivation (k(inact)/K(l)) was similar. The data obtained provide useful information for those using HRP isoenzymes for biotechnological applications (e.g., biosensors, bioreactors, or assays). (c) 1996 John Wiley & Sons, Inc.     

Characteristics of selective activation of cyclic AMP-dependent protein kinase isoenzymes by calcitonin and prostaglandin E2 in human breast cancer cells.

The characteristics of the cyclic AMP-dependent protein kinase isoenzyme response to calcitonin stimulation have been studied in two human breast cancer cell lines, T47D and MCF 7. Both cell lines possess calcitonin receptors, a calcitonin-responsive adenylate cyclase and the two isoenzymes of the cyclic AMP-dependent protein kinase, types I and II. The adenylate cyclase also responds to prostaglandin E2. Acute activation of the cyclic AMP-dependent protein kinase isoenzymes was determined by using a modification of a multiple small anion exchange column method [Livesey, Kemp, Re, Partridge & Martin (1982) J. Biol. Chem. 257, 14983-14987]. Control experiments showed that post-extraction activation did not influence the data. Calcitonin caused a rapid, selective activation of isoenzyme II in the T 47D cells with half-maximal response at 10(-10)M, and persisting for at least 24h. In MCF 7 cells calcitonin also caused a highly selective activation of isoenzyme II with half-maximal response at 5 X 10(-11) M, but the response was transient with a return to basal isoenzyme activity by 4-6 h. At this time further addition of calcitonin did not restimulate the cyclic AMP-dependent kinase activity. In neither cell line did calcitonin treatment result in activation of isoenzyme I. Prostaglandin E2, on the other hand, the only significant alternative agonist of adenylate cyclase in T 47D cells, activated isoenzymes I and II to an equal extent in these cells, illustrating that two hormones activating adenylate cyclase in the one cell type might exert different effects by their selective actions upon protein kinase isoenzymes.     

Mechanism of endogenous phosphorylation of microtubule proteins during GTP-induced microtubule assembly and implications for stability of the assembled structures

Cycle-purified microtubule protein from mammalian brain incorporated [32P]Pi upon incubation with [gamma-32P]GTP under the conditions used to promote assembly. This phosphorylation also occurred in the same proteins when phosphorylated with [gamma-32P]ATP and was only slightly stimulated by cAMP. GTP was a much less effective substrate than ATP. The transfer of phosphoryl groups from [gamma-32P]GTP to endogenous proteins followed a linear time-course and was stimulated by low concentrations of ATP and, more efficiently, by ADP. These data are in agreement with the predictions derived from a mechanism of phosphorylation by which [gamma-32P]GTP does not act as a phosphoryl donor for the protein kinase activity but, instead, only as a repository of high group transfer potential phosphoryl groups used to make [gamma-32P]ATP, from contaminating ADP, by means of the nucleoside diphosphate kinase activity. Using 100 mM fluoride, which suppressed protein phosphorylation without inhibiting the nucleoside diphosphate kinase activity, formation of [gamma-32P]ATP was detected. Fluoride was also able to protect microtubules from a slow depolymerization which was found to occur during long-term incubation of microtubules. This indicates that the phosphorylation observed in the presence of GTP is sufficient to destabilize microtubules.