Crystal Structures of S120G Mutant and Wild Type of Human Nucleoside Diphosphate Kinase A in Complex with ADP

Nm23 was the first metastasis suppressor gene identified. This gene encodes a NDP kinase that also exhibits other properties like histidine protein kinase and interactions with proteins and DNA. The S120G mutant of NDPK-A has been identified in aggressive neuroblastomas and has been found to reduce the metastasis suppressor effect of Nm23. In order to understand the differences between the wild type and the S120G mutant, we have determined the structure of both mutant and wild type NDPK-A in complex with ADP. Our results reveal that there are no significant changes between the two enzyme versions even in the surroundings of the catalytic histidine that is required for NDP kinase activity. This suggests that the S120G mutation may affect an other protein property than NDP kinase activity.     

Plant Pyruvate Kinase

Pyruvate kinase is an important enzyme of glycolytic pathway that also functions in providing carbon skeleton for fatty acid biosynthesis. It has been purified to near homogeneity from Ricinus communis, Selenastrum minutum, Cynodon dactylon, Brassica campestris and B. napus, and characterised. Partially purified preparations are reported from several other sources. A phosphoenolpyruvate (PEP) phosphatase accompanies pyruvate kinase. In plants, two isozymes of pyruvate kinase are reported, namely cytosolic and plastidic. Isoforms of cytosolic pyruvate kinase have also been reported from spinach. In most cases pyruvate kinase is a tetrameric protein and the molecular mass lies between 200 to 250 kDa. The pH optimum is in the range of 6.2 to 7.5. It requires both Mg²⁺ and K⁺ for maximum activity. ATP, citrate, and oxalate inhibit pyruvate kinase in most cases. A sequential compulsory ordered mechanism of binding of substrates to the enzyme has been proposed.     

Purification and characterization of putrescine synthase from cucumber seedlings. A multifunctional enzyme involved in putrescine biosynthesis

The multifunctional enzyme, putrescine synthase has been purified fromCucumis sativus and characterized. This enzyme harbours agmatine iminohydrolase, ornithine transcarbamylase, putrescine transcarbamylase and carbamate kinase activities, whose concerted action results in agmatine → putrescine conversion. The enzyme resolved into two aggregation forms, enzyme aggregated and enzyme monomer upon electrophoresis at pH 8.3. Evidence has been provided by two-dimensional gel electrophoresis that both enzyme aggregated and enzyme monomer comprise of identical polypeptide chains. Under non-reducing conditions on sodium dodecyl sulphate-polyacrylamide gel electrophoresis, the protein moves as a single 150 KDa polypeptide; however, in the presence of 2-mercaptoethanol on sodium dodecyl sulphate-polyacrylamide gel elec trophoresis, it migrates as 3 polypeptides of molecular weight 48,000, 44,000 and 15,000. The enzyme undergoes age-dependentin vivo proteolytic degradation from a 66 KDa polypeptide (primary translational product), through 48 KDa polypeptide to 44 KDa species and finally to small molecular weight peptides. Preliminary results of this work were presented at Golden Jubilee and Annual General Body Meetings of Society of Biological Chemists (India) and the Second Congress of Asian and Ocean Biochemists (1980) held at Bangalore, 1981,Indian J. Biochem. Biophys.,18, 113.     

A phylogenetic analysis based on the gene encoding phosphoglycerate kinase

Summary We have determined the nucleotide sequence of both genomic and complementary DNA (cDNA) for the gene encoding the glycolytic enzyme phosphoglycerate kinase from the ciliated protozoan Tetrahymena thermophila. The amino acid sequence for the enzyme has also been derived from the cDNA sequence. The gene contains an open reading frame of 1260 nucleotides encoding 420 amino acids. Coding sequence in genomic DNA is interrupted by two introns at positions corresponding to introns 3 and 4 in mammalian phosphoglycerate kinase genes. The derived amino acid sequence was used to prepare a phylogeny by aligning the Tetrahymena sequence with 25 other phosphoglycerate kinase amino acid sequences. The Tetrahymena sequence is a typical eukaryotic sequence. There is recognizable and clear homology across species that cover nearly the complete range of life forms. The phylogenetic reconstruction of these sequences generally supports the conclusions that have been reached using rRNA sequences.Offprint requests to: R.E. Pearlman     

Regulation of lysine biosynthesis in Brevibacterium flavum

L-lysine synthesis pathway enzyme activities: β-aspartate kinase (EC.2.7.2.4), diaminopimelate decarboxylase (EC.4.1.1.20) for two L-lysine producing strains Brevibacterium flavum 22LD and RC-115 were studied. It has been found that β-aspartate kinase and diaminopimelate decarboxylase in the Br. flavum RC-115 are less sensitive to feed-back inhibition by lysine and threonine. It is supposed that desensitized β-aspartate kinase in the Br. flavum RC-115 can be determined by genetical changes of the regulatory properties of the β-aspartate kinase. Auxotrophity in the locus of homoserine dehydrogenase was tested and no homoserine dehydrogenase (EC.1.1.1.3) activity was found in either strain. The combination of these both types of mutation supplemented by the lack of catabolic repression in the RC-115 strain makes it an active lysine producer in the medium with high carbohydrates content.     

Purification and characterization of an activated form of the protein tyrosine kinase Lck from an Escherichia coli expression system

The lymphocyte-specific, nonreceptor protein tyrosine kinase Lck has been purified from an Escherichia coli expression system using a monoclonal antibody column followed by dye-affinity chromatography. Polyacrylamide gel electrophoretic analysis of purified protein revealed a single 56 kDa band, indicating that recombinant Lck was purified to near-homogeneity. The purified enzyme displayed tyrosine kinase activity as measured by both autophosphorylation and phosphorylation of exogenous substrates. Biochemical properties including protein phosphorylation and kinetic characteristics of the enzyme have been assessed. Peptide map analysis revealed that bacterially expressed Lck is phosphorylated predominantly on the autophosphorylation site (tyrosine-394), which is characteristic for activated protein tyrosine kinases. Indeed, we found that the recombinant enzyme is approximately fivefold more active than Lck from resting T cells, which is extensively phosphorylated at the regulatory carboxy-terminal tyrosine residue (tyrosine-505). Thus, we have overproduced recombinant human Lck in E. coli and developed a simple two-step purification procedure which yields highly active enzyme. This will enable the identification and characterization of potential regulators and targets of Lck and thereby greatly facilitate studies which will clarify its role in T cell signal transduction. © 1994 Wiley-Liss, Inc.     

An allele (Pk-1 b ) from wild-caught mice that affects the activity and kinetics of erythrocyte and liver pyruvate kinase

A true breeding strain was made from a wild-caught mouse with low erythrocyte pyruvate kinase (E.C. 2.7.1.40) activity. This variation showed additive inheritance and segregated as an allele at a single locus (Pk-1 ^b). Mice homozygous for the reduced blood pyruvate kinase activity cosegregated for reduced liver activity. In both these tissues the variant enzyme had a lowered heat stability and reduced K _m values for ADP. An increased stimulation by FDP was also detected in the liver pyruvate kinase. No difference in the isoelectric point of the variant enzyme in either erythrocyte or liver was observed when compared with the enzyme from C57BL mice (Pk-1 ^a/Pk-1 ^a). It is concluded that Pk-1 is the structural gene for the erythrocyte and the major liver pyruvate kinase. No other tissue pyruvate kinase showed altered characteristics.This work was supported by a Medical Research Council grant.     

VARIATIONS IN AROMATIC AMINO ACID DECARBOXYLASE ACTIVITY TOWARDS DOPA AND 5-HYDROXYTRYPTOPHAN CAUSED BY pH CHANGES AND DENATURATION

—DOPA and 5-hydroxytryptophan (5-HTP) are generally supposed to be decarboxylated in mammalian tissues by a single enzyme, the two activities being present in constant ratio through a variety of purification procedures. It has now been shown that the ratio of activity of the liver enzyme towards the two substrates can be altered by mild treatments, such as might be used in solubilization of brain preparations. DOPA decarboxylase activity was preferentially inactivated by sodium dodecyl sulphate treatment, and 5-HTP decarboxylation by urea. Previous reports that the two substrates show different pH optima but are mutually competitive, have been confirmed. The K_m of the enzyme towards 5-HTP was lowest at pH 7.8 (the optimum pH for decarboxylation of this amino acid), but the variation with pH of the K_m towards DOPA was unrelated to the pH optimum for decarboxylation. There appeared to be no relation between the probable ionization state of the substrates and the pH dependence of the enzyme. Studies on the binding characteristics of the enzyme for the two products, dopamine and serotonin, did not show any specific saturable binding. It is proposed that the enzyme has a complex active site, with separate affinity sites for the two substrates, adjacent to a single catalytic site.     

ELISA detection of restriction site polymorphisms in the pig ryanodine receptor locus

We compare two strategies for ELISA detection of restriction site polymorphisms (EDRSP) that are suitable for high-throughput genotyping of the pig ryanodine receptor point mutation (RYR1 ^hal ). In both procedures, target DNA is amplified by PCR with one primer that is 5′ biotinylated and a second primer that is 5′ fluoresceinylated. PCR products are captured in duplicate wells on a streptavidin-coated, 96-well plate. The duplicates may be treated in two ways. In a single restriction enzyme assay, one duplicate is exposed to a restriction enzyme that cuts one allele specifically, and the second duplicate is exposed to no restriction enzyme. In a dual restriction enzyme assay, the second replicate is exposed to a second restriction enzyme that cuts the alternate allele specifically. Thereafter, the two procedures are similar; anti-fluorescein antibodies conjugated to peroxidase are allowed to bind to the fluoresceinylated ends, the plate is washed, and a substrate is converted to a colored end product. The ratio of the absorbances in the two wells is used to classify subjects by genotype. When the dual restriction enzyme assay is run, three genotype groups are easily distinguishable. When the single restriction enzyme assay is run, heterozygotes generate values that may overlap with those of the homozygotes that are not cut by the restriction enzyme. Dual restriction enzyme assays are more accurate than single restriction enzyme assays; however, single restriction enzyme assays are sufficient for identifying pigs that carry RYR1 ^hal . Received: 30 December 1997 / Accepted: 20 April 1998     

Tyrosine Hydroxylase Inactivation Following cAMP-Dependent Phosphorylation Activation

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, is activated following phosphorylation by the cAMP-dependent protein kinase (largely by decreasing the K^m of the enzyme for its pterin co-substrate). Following its phosphorylation activation in rat striatal homogenates, we find that tyrosine hydroxylase is inactivated by two distinct processes. Because cAMP is hydrolyzed in crude extracts by a phospho-diesterase, cAMP-dependent protein kinase activity declines following a single addition of cAMP. When tyrosine hydroxylase is activated under these transient phosphorylation conditions, inactivation is accompanied by a reversion of the activated kinetic form (low apparent K^m for pterin co-substrate, ≤0.2 mM) to the kinetic form characteristic of the untreated enzyme (high apparent K^m, ≥1.0 mM). This inactivation is readily reversed by the subsequent addition of cAMP. When striatal tyrosine hydroxylase is activated under constant phosphorylation conditions (incubated with purified cAMP-dependent protein kinase catalytic subunit), however, it is also inactivated. This second inactivation process is irreversible and is characterized kinetically by a decreasing apparent V^max with no change in the low apparent K^m for pterin co-substrate (0.2 mM). The latter inactivation process is greatly attenuated by gel filtration which resolves a low-molecular-weight inactivating factor(s) from the tyrosine hydroxylase. These results are consistent with a regulatory mechanism for tyrosine hydroxylase involving two processes: in the first case, reversible phosphorylaton and dephos-phorylation and, in the second case, an irreversible loss of activity of the phosphorylated form of tyrosine hydroxylase.     

Thymidine phosphorylation in synchronous cultures of Tetrahymena pyriformis GL

Recently it has been established that thymidine can be phosphorylated in two ways in Tetrahymena pyriformis

1. 1. by action of thymidine kinase

2. 2. by action of nucleoside phosphotransferase.

The present report confirms that thymidine kinase is a peak enzyme during S phase. It is suggested that a different thymidine concentration in the thymidine kinase assay might explain why previous workers have been unable to find thymidine kinase in Tetrahymena.     

Expression of tomato thymidine kinase 1 by means of the baculovirus expression vector system

ABSTRACT

Tomato thymidine kinase 1 (ToTK1) is a deoxyribonucleoside kinase (dNK) that has been subject to study because of its potential to phosphorylate the nucleoside analogue 3-azido-2,3-dideoxythymidine (azidothymidine, AZT) equally well as its natural substrate thymidine (dThd). The combination of ToTK1 and AZT has been tested in two animal studies for its efficiency and use in suicide gene therapy for malignant glioma. The determination of the 3D structure of ToTK1 might shed light on the structure–function relationships of nucleoside activation by this enzyme and thereby show routes toward further improvement of ToTK1 and other TK1-like dNKs for suicide gene therapy. Here we report the successful expression of both full-length ToTK1 and a C-terminal truncated ToTK1 in Spodoptera frugiperda and Trichoplusia ni insect cells using the baculovirus expression vector system. This constitutes a further step on the road to determine the 3D structure of the first TK1 of plant origin, but also an enzyme with great potential for dNK-mediated suicide gene therapy.     

A further characterization of the cinnamon gene in Drosophila melanogaster

Summary Two mutants of Saccharomyces cerevisiae lacking pyruvate kinase (EC.2.7.1.40) are described. The mutations are recessive, segregate 2⁺:2^- in tetrads and do not complement each other. Single-step spontaneous revertants, isolated on glucose plates, get back pyruvate kinase activity. The enzymes from various revertants display a wide spectrum of specific activity, thermolability and altered affinity for ligands such as P-enol pyruvate, ADP and fructose 1,6-diphosphate. The mutants produce materials crossreacting to the rabbit antibody raised against purified pyruvate kinase from the wild type yeast. These mutations thus define the structural gene of pyruvate kinase.The mutations map on the leaft arm of chromosome I and form a single complementation group with five other pyruvate kinase mutations in the pyk1 gene that was earlier suggested to be a regulatory locus controlling the synthesis of this enzyme. A comparative study of these mutants has been made with the structural mutants described here.     

Sphingosine kinase A is a pleiotropic and essential enzyme for Leishmania survival and virulence

Sphingosine kinase is a key enzyme in sphingolipid metabolism, catalysing the conversion of sphingosine or dihydrosphingosine into sphingosine‐1‐phosphate or dihydrosphingosine‐1‐phosphate respectively. In mammals, sphingosine‐1‐phosphate is a powerful signalling molecule regulating cell growth, differentiation, apoptosis and immunity. Functions of sphingosine kinase or sphingosine‐1‐phosphate in pathogenic protozoans are virtually unknown. While most organisms possess two closely related sphingosine kinases, only one sphingosine kinase homologue (SKa) can be identified in Leishmania, which are vector‐borne protozoan parasites responsible for leishmaniasis. Leishmania SKa is a large, cytoplasmic enzyme capable of phosphorylating both sphingosine and dihydrosphingosine. Remarkably, deletion of SKa leads to catastrophic defects in both the insect stage and mammalian stage of Leishmania parasites. Genetic and biochemical analyses demonstrate that proper expression of SKa is essential for Leishmania parasites to remove toxic metabolites, to survive stressful conditions, and to cause disease in mice. Therefore, SKa is a pleiotropic enzyme with vital roles throughout the life cycle of Leishmania. The essentiality of SKa and its apparent divergence from mammalian counterparts suggests that this enzyme can be selectively targeted to reduce Leishmania infection.     

Purification of leucoplast pyruvate kinase from developing castor bean endosperm

Leucoplast pyruvate kinase from endosperm of developing castor oil seeds (Ricinus communis L.; cv Baker) has been purified 1370-fold to a specific activity of 41.1 micromoles pyruvate produced per minute per milligram protein. Nondenaturing polyacrylamide gel electrophoresis of the purified enzyme resulted in a single protein staining band that co-migrated with pyruvate kinase activity. However, following sodium dodecyl sulfate polyacrylamide electrophoresis, two major protein staining bands of 57.5 and 44 kilodaltons, which occurred in an approximate 2:1 ratio, respectively, were observed. The native molecular mass was approximately 305 kilodaltons. Rabbit antiserum raised against the final enzyme preparation effectively immunoprecipitated leucoplast pyruvate kinase. The 57.5- and 44-kilodalton polypeptides are immunologically related as both proteins cross-reacted strongly on Western blots probed with the rabbit anti-(developing castor seed endosperm leucoplast pyruvate kinase) immunoglobulin that had been affinity-purified against the 57.5-kilodalton polypeptide. In contrast, pyruvate kinases from the following sources showed no immunological cross-reactivity with the same immunoglobulin: the cytosolic enzyme from developing or germinating castor bean endosperm; chloroplastic pyruvate kinase from expanding leaves of the castor oil plant; chloroplastic or cytosolic pyruvate kinase from the green alga, Selenastrum minutum; and mammalian or bacterial pyruvate kinases.     

Purification and characterization of X-prolyl dipeptidyl peptidase fromLactobacillus casei subsp.casei LLG

X-Prolyl dipeptidyl peptidase, which hydrolysed X-Pro-Y almost specifically, has been purified to homogeneity from crude cell-free extracts ofLactobacillus casei subsp.casei LLG using fast protein liquid chromatography equipped with preparative and analytical anion exchange columns. The enzyme was purified to 274-fold by ammonium sulphate fractionation, and by two successive ion-exchange chromatographies with a recovery of 34%. The purified enzyme appeared as a single band on both native-polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulphate (SDS)-PAGE and had a molecular mass of 79 kDa. The pH and the temperature optima by the purified enzyme were 7.0 and 50°C, respectively. X-PDP was a serine-dependent enzyme, as both diisopropylfluorophosphate and phenylmethylsulphonylfluoride caused complete inhibition of the enzyme activity. The Michaelis constant (K _m ) and maximum reaction velocity (V _max ) values were 0.2 mm and 43 mm per milligram, respectively.     

Arginine decarboxylase (polyamine synthesis) mutants of Arabidopsis thaliana exhibit altered root growth

Putrescine and polyamines are produced by two alternative pathways in plants. One pathway starts with the enzyme arginine decarboxylase; the other with ornithine decarboxylase. The authors developed an in vivo screening strategy to identify mutants with low levels of arginine decarboxylase activity. The screen requires both a primary screen of the M2 generation and a secondary screen of the M3 generation. The method used was to screen 15 000 EMS-mutagenized M2 seedlings for low levels of arginine decarboxylase (ADC) activity and identified seven mutants that fall into two complementation groups. These mutants have from 20% to 50% of wild-type enzyme activity. Morphological alterations common among the mutants include increased levels of lateral root branching. The authors obtained a double mutant combining the alleles with the lowest activities from the two complementation groups; this has lower ADC enzyme activity and putrescine levels than either of the single mutants. The double mutant has highly kinked roots that form a tight cluster; it also has narrower leaves, sepals, and petals than either single mutant or wild-type, and delayed flowering. These results suggest there may be more than one ADC gene in Arabidopsis, and that ADC and polyamine levels play roles in root meristem function and in lateral growth of leaf-homolog organs.     

Characterization of thymidine kinase and phosphorylation of deoxyribonucleosides in Chlamydomonas reinhardti.

Summary Using gel filtration chromatography, we find a single peak of deoxythymidine phosphorylating activity in Chlamydomonas reinhardti. This activity has characteristics of a thymidine kinase, in that (1) it will utilize ATP (or dATP) or CTP (or dCTP) as phosphoryl donor, but not AMP or phenyl phosphate, and (2) it is inhibited by dTTP (and less so by dTDP, dUTP, and dUDP) but is unaffected by 3–5 cyclic AMP.Partially purified Chlamydomonas thymidine kinase has a pH optimum near 8.5, and a molecular weight of 80,000 to 85,000 daltons. Kinetic studies indicate a ping-pong mechanism with a Km for thymidine of 1.5x10^-7 moles per liter. 5-Bromo-and 5-fluorodeoxyuridine, and to a lesser degree deoxyuridine, are competitive inhibitors, but significant phosphorylation of these nucleosides could not be demonstrated in vitro by thymidine kinase.While thymidine is phosphorylated to dTMP by crude Chlamydomonas extracts, greater than 80% of the product formed by the partially purified enzyme is dTTP. Further, the gel filtration elution position of the single deoxythymidylate kinase activity present in cell extracts coincides with that of thymidine kinase. These results suggest that a multifunctional enzyme, rather than three separate phosphorylating activities, may be responsible for dTTP formation.Abbreviations MES 2(N-morpholino) ethanesulfonic acid - TES N-tris (hydroxymethyl) methyl-2-amino ethanesulfonic acid - tris tris-hydroxyamino methane - NEM N-ethyl maleimide - PEI polyethyleneimine - TLC thin-layer chromatography; nucleotides abbreviated by CBN rules     

A Nucleoside Diphosphate Kinase from Paramecium tetraurelia with Protein Kinase Activity

Nucleoside diphosphate kinase (NDP kinase) from Paramecium was purified to homogeneity. The native enzyme was 80 kDa (by gel filtration), with subunits of 18 and 20 kDa. Near the amino terminus, 15 of 20 residues were identical with those in human NDP kinase, and 17 of 20 with the awd gene product from Drosophila. NDP kinase bound α-labeled ATP and GTP, and a photoreactive GTP analog labeled both subunits. Purified NDP kinase underwent autophosphorylation on a histidine and a serine residue using either ATP or GTP as a substrate. The enzyme also catalyzed acid-stable phosphorylation of casein and phosvitin. This protein kinase activity is distinct from the histidine phosphorylation that is part of the NDP kinase catalytic cycle. Antiserum against the purified protein from Paramecium cross-reacted with 16- to 20-kDa proteins in most species tested, and with a larger protein (44 kDa) in Paramecium, Xenopus, and two human lines. The multiple forms (20 and 44 kDa) of the NDP kinase in Paramecium and its protein kinase activity, suggest that the protein is more than a housekeeping enzyme; it may have regulatory roles such as those of the NDP kinase-like awd protein of Drosophila and Nm23 protein of humans.