Protection of Vaccinia from Heat Inactivation by Nucleotide Triphosphates

The presence of adenosine triphosphate, guanosine triphosphate, cytosine triphosphate, or uridine triphosphate reduced the rate of inactivation of vaccinia when heated at 50 C. The virus-associated nucleoside triphosphate phosphohydrolases (adenosine triphosphatase, guanosine triphosphatase, cytosine triphosphatase, and uridine triphosphatase) and ribonucleic acid polymerase were also protected from heat inactivation by these compounds. These obervations are best explained by postulating that ribonucleoside triphosphates bind to enzymes in the virus particle, and that these enzyme-substrate complexes are more resistant to thermal denaturation than are the enzymes without their substrates. The kinetics of heat inactivation of the vaccinia ATP phosphohydrolase activity is biphasic, suggesting that there are two proteins in the vaccinia particle that have this enzyme activity but they have different kinetics of heat inactivation. Any of the vaccinia-associated nucleotide phosphohydrolase activities are protected from heat inactivation by the presence of any one of the respective nucleoside triphosphates. This observation suggests that there is a single enzymatic site in vaccinia that is able to react with any ribonucleoside triphosphate.     

Deoxyribonucleotide metabolism in Herpes simplex virus infected HeLa cells.

The effect of Rolly No. 11 strain herpes simplex virus infection of HeLa cells in culture on deoxynucleotide metabolism and the level of various enzymes concerned with the biosynthesis of DNA has been investigated. Of 18 enzyme activities studied, thymidine kinase, DNA polymerase and deoxyribonuclease were markedly augmented, a finding in agreement with previous reports. Deoxycytidine kinase, ribonucleotide reductase, thymidylate kinase and deoxycytidylate deaminase activities, in contrast with previous reports, did not increase; the activities of the other enzymes studied, also did not increase. Whereas most of the radioactivity derived from [14-C] thymidine in the acid-soluble fraction of the uninfected cells was present as deoxythymidine triphosphate, that present in the infected cells was primarily in the form of deoxythymidine monophosphate. Thus, in the infected cell deoxythymidylate kinase is a rate-limiting enzyme in the biosynthesis of deoxythymidine triphosphate. A marked increase in the pools of the four naturally occurring deoxynucleoside triphosphates (dTTP, dCTP, dATP, dGTP) was found. The rate of formation of the virus-induced enzymes was determined, as were the various nucleoside triphosphate pools and the other phosphorylated derivatives of thymidine; a maximum was reached for all these csmponents between 6 to 8 h post infection. Although an apparent greater synthesis of DNA occurred in the uninefected cells, when the specific activity of the radioactive deoxythymidine triphosphate was taken into account, there was actually a greater rate of DNA synthesis in the infected cells, with the peak at 8 h post infection.     

Effects of spin-labeled stearates on duck erythrocyte adenylate cyclase.

The effects of various spin-labeled stearates on duck erythrocyte adenylate cyclase were investigated. Only 2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl caused an increase in adenylate cyclase activity. It increased the basal rate by about 50%, and the activities stimulated by isoproterenol and isoproterenol plus guanosine 5'-[beta,gamma-imido]triphosphate by 35%. Upon analysis of the width parameter delta1 in the electron spin resonance spectra for both the basal enzyme activity and the stimulation obtained with effectors such as guanosine 5'-[beta,gamma-imido]triphosphate, isoproterenol, isoproterenol plus guanosine 5'-[beta,gamma-imido]triphosphate and NaF, a correlation of the changes of modification in adenylate cyclase activities was found. These findings suggest that the molecular environment of the enzyme has been modified.     

Adenosine triphosphate-linked control of Pseudomonas aeruginosa glucose-6-phosphate dehydrogenase

Extracts of Pseudomonas aeruginosa (ATCC 7700) cells grown on glucose, gluconate, or glycerol had enzyme activities related to the Entner-Doudoroff pathway. These activities were present in no more than trace amounts when the bacteria were grown on succinate. Fructose-1,6-diphosphate aldolase could not be detected in extracts of the bacteria grown on any of the above carbon sources. Therefore, it appears that P. aeruginosa degrades glucose via an inducible Entner-Doudoroff pathway. The apparent absence of fructose-1,6-diphosphate aldolase in cells growing on succinate suggests that the bacteria can form hexose and pentose phosphates from succinate by an alternate route. d-Glucose-6-phosphate dehydrogenase, a branch-point enzyme of the Entner-Doudoroff pathway, was purified 50-fold from glucose-grown cells. Its molecular weight, estimated by sucrose density gradient centrifugation, was found to be approximately 190,000. The enzyme was strongly inhibited by adenosine triphosphate, guanosine triphosphate, and deoxyguanosine triphosphate, which decreased the apparent binding of glucose-6-phosphate to the enzyme. It is suggested that adenine nucleotide-linked control of glucose-6-phosphate dehydrogenase may regulate the overall catabolism of hexose phosphates and prevent their wasteful degradation under certain conditions requiring gluconeogenesis.     

Separation and partial characterization of two deoxyribonucleic acid polymerases from Spiroplasma citri.

The separation and partial characterization of two deoxyribonucleic acid polymerases from Spiroplasma citri have been achieved. The two enzymes had different elution properties on diethylaminoethyl (DEAE) cellulose and differed in their sensitivity to N-ethylmaleimide (NEM), preference for different template-primers, and sedimentation velocity in linear glycerol gradients. The first enzyme activity, ScA, was retained on DEAE-cellulose and was not inhibited by NEM. Activated deoxyribonucleic acid and poly(dA)-oligo(dT12) were the preferred template-primers. Arabinosyl-cytidine triphosphate had no effect. The sedimentation coefficient of ScA was 6.3s. The second activity, ScB, was not retained on DEAE-cellulose and was inhibited by NEM. Poly(dA)-oligo(dT12) was the preferred template-primer, whereas activated DNA was only poorly utilized. ScB was not affected by arabinosyl-cytidine triphosphate, and its sedimentation coefficient was 4.4s. The polymerization activities of the two enzymes were maximum at 37 to 40 degrees C.     

The role of nucleoside triphosphate pyrophosphohydrolase in in vitro nucleoside triphosphate-dependent matrix vesicle calcification

Nucleoside triphosphate pyrophosphohydrolase (EC 3.6.1.8) activity is associated with matrix vesicles purified from collagenase digests of fetal calf epiphyseal cartilage. This enzyme hydrolyzes nucleoside triphosphates to nucleotides and PPi, the latter inducing precipitation in the presence of Ca2+ and Pi. An assay for matrix vesicle nucleoside triphosphate pyrophosphohydrolase is developed using beta, gamma-methylene ATP as substrate. The assay is effective in the presence of matrix vesicle-associated ATPase, pyrophosphatase, and alkaline phosphatase activities. A soluble nucleoside triphosphate pyrophosphohydrolase is obtained from matrix vesicles by treatment with 5 mM sodium deoxycholate. The solubilized enzyme induced the precipitation of calcium phosphate in the presence of ATP, Ca2+, and Pi. Extraction of deoxycholate-solubilized enzymes from matrix vesicles with 1-butanol destroys nucleoside triphosphate pyrophosphohydrolase activity while enhancing the specific activities of ATPase, pyrophosphatase, and alkaline phosphatase. In solutions devoid of ATP and matrix vesicles, concentrations of PPi between 10 and 100 microM induce calcification in mixtures containing initial Ca2+ X P ion products of 3.5 to 7.9 mM2. This finding plus the discovery of nucleoside triphosphate pyrophosphohydrolase in matrix vesicles supports the view that these extracellular organelles induce calcium precipitation by the enzymatic production of PPi. Nucleoside triphosphate pyrophosphohydrolase is more active against pyrimidine nucleoside triphosphates than the corresponding purine derivatives. The pH optimum is 10.0 and the enzyme is neither activated nor inhibited by Mg2+ or Ca2+ ions or mixtures of the two. Vmax at pH 7.5 for beta, gamma-methylene ATP is 0.012 mumol of substrate hydrolyzed per min per mg of protein and Km is below 10 microM. The enzyme is irreversibly destroyed at pH 4 and is stable at pH 10.5.     

Polyploidy and aspartate-transcarbamylase activity in Hippocrepis comosa L.

The DNA content of plants which were sampled in natural di-, tetra- and hexaploid populations of Hippocrepis comosa L. was estimated and the aspartate transcarbamylase activities of the corresponding cell-free extracts were compared. The amount of DNA is not exactly proportional to the number of genomes. The three kinds of populations do not differ in their aspartate transcarbamylase specific activity. While the enzyme properties are identical in the extracts derived from the diploid and hexaploid plants, the aspartate transcarbamylase present in the tetraploid cytotype shows a slightly lower affinity for one of its substrates and a significantly lower sensitivity to the feedback inhibitor UTP which is still observed after partial purification. These properties might be related to the previously reported greater ability of the tetraploid cytotype to adapt to a variety of biotopes.Abbreviations ATCase aspartate transcarbamylase - CAP carbamylphosphate - EDTA ethylenediaminetetracetic acid - Tris trihydroxymethylaminomethane - AMP adenosine monophosphate - ATP adenosine triphosphate - CMP cytidine monophosphate - CTP cytidine triphosphate - UMP uridine monophosphate - UTP uridine triphosphate     

Malic enzyme and fatty acid synthase in the uropygial gland and liver of embryonic and neonatal ducklings. Tissue-specific regulation of gene expression

Malic enzyme [L-malate-NADP oxidoreductase (decarboxylating), EC 1.1.1.40] and fatty acid synthase activities were barely detectable in the uropygial gland of duck embryos until 4 or 5 days before hatching, when they began to increase. These activities increased about 30- and 140-fold, respectively, by the day of hatching. Malic enzyme and fatty acid synthase activities were also very low in embryonic liver. However, hepatic malic enzyme activity did not increase until the newly hatched ducklings were fed. Hepatic fatty acid synthase began to increase the day before hatching and the rate of increase in enzyme activity accelerated markedly when the newly hatched ducklings were fed. Starvation of newly hatched or 12-day-old ducklings had no effect on the activities of malic enzyme and fatty acid synthase in the uropygial gland but markedly inhibited these activities in liver. Changes in the concentrations of both enzymes and in the relative synthesis rates of fatty acid synthase correlated with enzyme activities in both uropygial gland and liver. Developmental patterns for sequence abundance of malic enzyme and fatty acid synthase mRNAs in uropygial gland and liver were similar to those for their respective enzyme activities. Starvation of 4-day-old ducklings had no significant effect on the abundance of these mRNAs in uropygial gland but caused a pronounced decrease in their abundance in liver. It is concluded that developmental and nutritional regulation of these enzymes is tissue specific and occurs primarily at a pretranslational level in both uropygial gland and liver.     

Ectonucleotidase Activities Associated with Cholinergic Synaptosomes Isolated from Torpedo Electric Organ

Intact synaptosomes isolated from the electric organ of the electric ray Torpedo marmorata contain, at their surface, enzyme activities for the hydrolysis of externally applied nucleoside phosphates. The diazonium salt of sulfanilic acid, as a low-molecular-weight, slowly permeating, covalent inhibitory agent, selectively blocks these enzyme activities and leaves intracellular lactate dehydrogenase intact. The ectoenzymes comprise both a nucleoside triphosphate and diphosphate phosphohydrolase, as well as a 5'-nucleotidase. Activity of nonspecific ectophosphatases is absent. The nucleoside triphosphatase hydrolyzes almost equally well ATP, GTP, CTP, UTP, and ITP and is activated to a similar degree by Mg2+ or Ca2+. It has a high affinity for ATP (Km for ATP in the presence of Mg2+, 75 microM; in the presence of Ca2+, 66 microM). Maximal rates in the presence of Mg2+ and Ca2+ were very similar (34.8 and 32.5 nmol of Pi/min/mg of synaptosomal protein, respectively). Either Mg-ATP or Ca-ATP can act as a true substrate. ADP inhibits hydrolysis of ATP, but AMP is without effect. The nucleoside triphosphatase is not inhibited significantly by a number of inhibitors of mitochondrial Mg2+-ATPase or of Ca2+ + Mg2+-ATPases. It is, however, considerably inhibited by filipin and quercitin. The capacity of intact synaptosomes to hydrolyze also extracellular ADP, GDP, AMP, GMP, and IMP suggests that the nucleoside triphosphatase is part of an enzyme chain that causes complete hydrolysis of the respective nucleoside triphosphate to the nucleoside. We conclude that the cholinergic nerve terminals of the Torpedo electric organ can hydrolyze ATP released on coexocytosis with acetylcholine via an ectonucleoside triphosphatase activity that is different from known endogenous nerve terminal ATPases. The final product of the hydrolysis, adenosine, can then be salvaged by the nerve terminal for resynthesis of ATP. Other possible physiological functions of the ectonucleotidases are discussed.     

ITPase Activity in Dry Blood Spots is Comparable with That in Fresh Erythrocytes

Inosine triphosphate pyrophosphohydrolase (ITPase) catalyzing the pyrophosphohydrolysis of inosine triphosphate, deoxyinosine triphosphate and xanthosine triphosphate is involved in the metabolism and tolerance of thiopurine drugs. ITPase activity plays an important role in the prediction of toxicity to thiopurine therapy. Activities in dry blood spots were compared with fresh erythrocytes. Samples were incubated with inosine triphosphate, then inosine monophosphate was determined by a capillary electrophoresis method. Calculated enzyme activities obtained from dry blood spots were in good accordance with activity in fresh erythrocytes.     

THYMIDINE TRIPHOSPHATE SYNTHESIS IN TETRAHYMENA : I. Studies on Thymidine Kinase

The amount of thymidine-H³ converted to thymidine-H³ monophosphate in 30 min formed the basis for assays of thymidine kinase in cell extracts from Tetrahymena pyriformis. The optimal concentration of adenosine triphosphate is lower than that required by other cell types. Thymidine triphosphate does not exercise any feedback control of the enzyme. Other deoxyprimidine nucleotides were tested, but these also failed to exhibit any feedback inhibition. At suboptimal adenosine triphosphate levels, thymidine triphosphate and other deoxypyrimidine nucleotides stimulate the reaction, suggesting that these nucleotides may act either directly or indirectly as phosphate donors in the crude enzyme preparations. This possibility was affirmed when thymidine triphosphate and deoxycytidine triphosphate were shown to be capable of limited phosphorylation of thymidine. Comparison of enzymatic activities in logarithmically growing culture and stationary phase culture, in which nuclear DNA synthesis has virtually ceased, reveals no change in enzymatic activity. The results suggest that thymidine kinase is a constitutive enzyme in Tetrahymena.     

ITPase activity in dry blood spots is comparable with that in fresh erythrocytes

Inosine triphosphate pyrophosphohydrolase (ITPase) catalyzing the pyrophosphohydrolysis of inosine triphosphate, deoxyinosine triphosphate and xanthosine triphosphate is involved in the metabolism and tolerance of thiopurine drugs. ITPase activity plays an important role in the prediction of toxicity to thiopurine therapy. Activities in dry blood spots were compared with fresh erythrocytes. Samples were incubated with inosine triphosphate, then inosine monophosphate was determined by a capillary electrophoresis method. Calculated enzyme activities obtained from dry blood spots were in good accordance with activity in fresh erythrocytes.     

Biopterin

An enzyme that catalyzes the conversion of 2-amino-6-(5'-triphosphoribosyl)amino-5- or 6-formamido-6-hydroxypyrimidine, but not of guanosine triphosphate, to quinonoid 6-(D-erythro-1'-2'-3'-trihydroxypropyl)dihydropterin triphosphate and formic acid has been purified to homogeneity from some mammalian brain and liver. The enzyme of a single strand is a basic protein of 9177 daltons consisting of 68 amino acid residues--except the enzyme from rat brain, which has one additional aspartic acid as residue 7. The enzyme possesses three free SH groups and, in its most active form, 1 mol of phosphate per mole of enzyme. Peptides isolated after hydrolysis with trypsin, chymotrypsin, or weak acid were separated by thin-layer chromatography and sequenced manually by Edman degradation. The complete sequence of the molecule was established as follows: (formula: see text)     

Biochemical and Structural Studies of 6-Carboxy-5,6,7,8-tetrahydropterin Synthase Reveal the Molecular Basis of Catalytic Promiscuity within the Tunnel-fold Superfamily

6-Pyruvoyltetrahydropterin synthase (PTPS) homologs in both mammals and bacteria catalyze distinct reactions using the same 7,8-dihydroneopterin triphosphate substrate. The mammalian enzyme converts 7,8-dihydroneopterin triphosphate to 6-pyruvoyltetrahydropterin, whereas the bacterial enzyme catalyzes the formation of 6-carboxy-5,6,7,8-tetrahydropterin. To understand the basis for the differential activities we determined the crystal structure of a bacterial PTPS homolog in the presence and absence of various ligands. Comparison to mammalian structures revealed that although the active sites are nearly structurally identical, the bacterial enzyme houses a His/Asp dyad that is absent from the mammalian protein. Steady state and time-resolved kinetic analysis of the reaction catalyzed by the bacterial homolog revealed that these residues are responsible for the catalytic divergence. This study demonstrates how small variations in the active site can lead to the emergence of new functions in existing protein folds.     

Modification of phospholipase C and phospholipase A2 activities during poliovirus infection

The infection of HeLa cells by poliovirus leads to profound alterations in the activities of both phospholipase C and the A23187-stimulated phospholipase A2. As early as the third hour after poliovirus infection, the activity of phospholipase C is enhanced, as measured by the increase in inositol triphosphate (IP3) in the cells. By the fifth hour post-infection there is a 5-fold increase in IP3 in the infected cells. Therefore, the synthesis of the bulk of poliovirus proteins and poliovirus genomes takes place in cells containing a high and sustained increase in IP3. This augmentation in IP3 is dependent on the multiplicity of infection used. Poliovirus gene expression is required to induce the increase in phospholipase C activity, since the presence of cycloheximide or guanidine blocked it. In contrast to the activation of phospholipase C induced by poliovirus, there is a drastic blockade of the A23187-induced phospholipase A2 activity, measured as the release of [3H]arachidonic acid to the medium. This action on phospholipase A2 is dependent on poliovirus gene expression because it was prevented by cycloheximide or 3-methylquercetin. To our knowledge this is the first report analyzing these two activities in animal virus-infected cells. The findings described may help to explain the profound modifications of both membrane permeability and lipid metabolism undergone by poliovirus-infected cells.     

Regulation fo protein kinase C activity by various lipids

Protein kinase C has recently attracted considerable attention because of its importance in the control of cell division, cell differentiation, and signal transduction across the cell membrane. The activity of this enzyme is altered by several lipids such as diacylglycerol, free fatty acids, lipoxins, gangliosides, and sulfatides. These lipids may interact with protein kinase C either directly or through calcium ions and produce their regulatory effect (activation or inhibition) on the activities of the enzymes phosphorylated by this kinase. These processes widen our perspective of the regulation of intercellular and intracelluular communication.Abbreviations used (PK-C) Protein kinase C - (cAMP-PK) cAMP dependent protein kinase - (DAG) diacylglycerol - (PtdSer) phosphatidylserine - (InsP ₃) inositol 1,4,5-trisphosphate - (PtdIns 4,5-P₂) inositol 4,5 bisphosphate - (FFA) free fatty acid - (MBP) myelin basic protein - (ATP) adenosine triphosphate - (GTP) guanine triphosphate - (TPA) 12-tetradecanoylphorbol-13-acetate - (EGF) epidermal growth factor - (PDGF) platelet derived growth factor - (NeuNAc) and N-acetylneuraminic acid     

Nucleotide Phosphohydrolase in Purified Vaccinia Virus

Purified infectious vaccinia virus has been shown to contain an enzyme or enzymes that remove the terminal phosphate group from adenosine triphosphate (ATP), guanosine triphosphate (GTP), uridine triphosphate (UTP), and cytidine triphosphate (CTP). The K(m) for ATP of this enzyme is 5.5 x 10(-4)m, and the relative rates of the reaction with ATP, GTP, UTP, and CTP are 1.00, 0.34, 0.15, and 0.29, respectively. The virus enzyme does not react with any of the diphosphates. The rate of the reaction is proportional to the amount of virus added and is linear for 130 min. The virus nucleotide phosphohydrolase activity is greatly stimulated by Mg(++) and very slightly stimulated by Ca(++). The small residual activity observed in the absence of divalent cations is completely inhibited by ethylenediaminetetraacetic acid. Neither Na(+) nor K(+) ions, nor any mixture of these, was found to stimulate the reaction significantly, and ouabain, at 10(-4)m, inhibited the reaction by only 27%. The response of the vaccinia enzyme to mono- and divalent cations and to ouabain indicates that the vaccinia enzyme has different properties from those associated with microsomes and mitochondria.     

Difference in thiamine metabolism between extensor digitorum longus and soleus muscles

1. Thiamine diphosphate level was higher in soleus muscle than in extensor digitorum longus muscle in various animals, whereas thiamine triphosphate level was less in the former muscle than in the latter except for mouse. 2. 2-Oxoglutarate dehydrogenase, transketolase and thiamine pyrophosphokinase activities were higher in soleus muscle than in extensor digitorum longus in rat and guinea pig. 3. The differences between rat two muscle phenotypes in thiamine diphosphate, but not thiamine triphosphate, level and the thiamine-related enzyme activities disappeared after denervation. 4. Tenotomy had little effect on thiamine phosphate levels and the thiamine-related enzyme activities in rat skeletal muscles.