A kinetic investigation of the aps-kinase from Chlamydomonas reinhardii CW 15

The reaction kinetics of APS-kinase from Chlamydomonas reinhardii showed that the enzyme formed PAPS from APS upon the addition of ATP. Evidence for a ³⁵S-labelled protein intermediate between APS and PAPS has been obtained. The APS-kinase activity could only be measured in the presence of low concentrations of APS (20 ± 10 μM) and of ATP (0.2 ± 0.05 mM) due to substrate inhibition. The inhibition was partially overcome by low concentrations of 3′,5′-PAP (10,μM). The rates of PAPS formation obtained with cell extracts from the alga varied from 2 to 6 nM PAPS/mg protein/min (33–100 × 10^?12 kat/mg).     

Inhibition of the Bacterial Cell Wall Synthesis in vitro by Enduracidin,a New Polypeptide Antibiotic

Adenosine 5′-phosphosulfate (APS) kinase from a thermophilic bacterium, Bacillus st ear other mophilus, was purified to apparent homogeneity. The apparent molecular weight was 50 kDa, consisting of two 26-kDa subunits. The enzyme was very thermostable and lacked cysteine and methionine residues. Enzyme activity was more stimulated with Mn^{2 +} , Zn^{2 +}, or Co^{2 +} than with Mg^{2 +} and the K_m for ATP and APS were 220 µM and 42 µM, respectively.     

A comparison of the carcinogen-DNA adducts formed in rat liver in vivo after administration of single or multiple doses of N-methyl-4-aminoazobenzene

N-Methyl-4-aminoazobenzene (MAB) is believed to be metabolized in the liver to an electrophilic N-sulfonyloxy ester which binds covalently to cellular macromolecules, resulting in the induction of hepatic neoplasia. Previous in vivo studies in the rat detected only two hepatic MAB-DNA adducts, 3-(deoxyguanosin-N²-yl)-MAB(N²-dG) and N-(deoxyguanosin-8-yl)-MAB(C8-dG), which respectively accounted for 25% and 70% of the total MAB bound to DNA at 8 h after a single dose of the carcinogen. Subsequently, the C8-dG adduct was shown to be rapidly lost from the DNA while the N²-dG adduct was a persistent lesion. Since a single dose of MAB is not sufficient for complete carcinogenic activity, we sought to identify the MAB-DNA adducts present in rat liver after multiple oral doses of [³H]MAB. The MAB was administered by intubation at a level of 0.2 mmol/kg for 1, 3 or 4 doses and animals were sacrificed at 8 h after the last dose. Hepatic DNA was isolated by extraction and hydroxylapatite chromatography and was enzymatically hydrolyzed to MAB-mononucleoside adducts, which were quantitated by high pressure liquid chromatography (HPLC). After 3 doses, N²-dG, C8-dG, and an unknown adduct were detected. By 4 doses, these accounted for 51%, 25% and 23% of the total adducts. This data is consistent with rapid removal of the C8-dG derivative and the relative persistence of the N²-dG and the unknown adduct. The latter was shown to exhibit chromatographic and pH-dependent solvent partitioning properties that were identical to a product also present in DNA treated with the synthetic ultimate carcinogen, N-benzoyloxy-MAB. Analysis of this adduct by field desorption mass spectrometry (M⁺ = 460) and, after perdeuteromethylation, by electron impact mass spectrometry (M⁺ = 528; M-N(CH₃)(CD₃) = 481) indicated the structure to be a deoxyadenosin-N⁶-yl derivative substituted through an aromatic ring of MAB. Further analysis by 270 MHz ¹H-NMR spectroscopy allowed complete assignment of the MAB and adenyl resonances and was uniquely consistent with a 3-(deoxyadenosin-N⁶-yl)-MAB structure. Since this persistent adduct is potentially mutagenic due to possible tautomeric equilibria between the N⁶-amino and N⁶-imino structures, it may represent an initiating lesion in MAB hepatocarcinogenesis.     

A quench-flow kinetic investigation of calcium ion accumulation by isolated cardiac sarcoplasmic reticulum. Dependence of initial velocity on free calcium ion concentration and influence of preincubation with a protein kinase,MgATP, and cyclic AMP

Ca²⁺ accumulation at pH 6.8 by isolated rabbit heart microsomes derived chiefly from sarcoplasmic reticulum was investigated by a quench-flow technique. The reaction was terminated at preset times by addition to the reaction mixture of an equal volume of 10 to 50 mM ethyleneglycol-bis-(β-aminoethyl ether)-N,N′-tetraacetic acid buffered at pH 6.0. The initial velocity of Ca²⁺ accumulation by microsomal preparations exhibiting a steady state Ca²⁺ accumulation of 25.6 nmol Ca²⁺/mg increased from 3.67 to 33.4 nmol Ca²⁺/mg · s as the free Ca²⁺ concentration was raised from 0.2 to 18.9 μM. Preincubation of the cardiac microsomes with a partly purified soluble cardiac cyclic AMP-dependent protein kinase, MgATP, and cyclic AMP lead to a significant increase in the initial Ca²⁺ accumulation rate. The amounts of Ca²⁺ that were found to accumulate in the first 200 ms of the reaction are comparable to the quantities of the ion that according to literature data need to be removed from the myofilaments and the myoplasm for induction of relaxation of the myocardial fibers.     

Crystal Structure of Yeast FAD Synthetase (Fad1) in Complex with FAD

Flavin adenine dinucleotide (FAD) synthetase is an essential enzyme responsible for the synthesis of FAD by adenylation of riboflavin monophosphate (FMN). We have solved the 1.9 Å resolution structure of Fad1, the yeast FAD synthetase, in complex with the FAD product in the active site. The structure of Fad1 shows it to be a member of the PP-ATPase superfamily. Important conformational differences in the two motifs involved in binding the phosphate moieties of FAD compared to the Candida glabrata FMNT ortholog suggests that this loop is dynamic and undergoes substantial conformational changes during its catalytic cycle.     

Variations du taux d'amp cyclique et des activités spécifiques de l'adénylate cyclase et de l'amp cyclique-phosphodiestérase pendant le cycle cellulaire d'un actinomycète

The variations in the concentrations of intra- and extracellular cyclic AMP and in the specific activities of adenylate cyclase (EC 4.6.1.1) and cyclic AMP phosphodiesterase (EC 3.1.4.17) have been monitored in synchronized culture of Nocardia restricta, a prokaryote belonging to the group of Actinomycetes. At the beginning of the cell cycel, during a first period of RNA and protein synthesis, there is an increasing synthesis of adenylate cyclase which can be suppressed in the presence of chloramphenicol or rifampicin. Simultaneously, the specific activity of cyclic AMP phosphodiesterase decreases and the concentrations of intra- and extracellular cyclic AMP rise. After the end of DNA replication, during a second period of RNA and protein synthesis, the specific activity of cyclic AMP phosphodiesterase increases; during the same time, the specific activity of adenylate cyclase and the level of intracellular cyclic AMP drop. It appears that the overall metabolism of cyclic AMP is coordinated so that the cyclic AMP level will be high at the beginning of DNA replication and will fall thereafter. The results are discussed in comparison with known data about the variations of cyclic AMP during the cell cycle of mammalian cells in cultures.     

Reactions of adenosine 5′-phosphorimidazolide with adenosine analogs on a polyuridylic acid template: The uniqueness of the 2′-3′-unsubstituted β-ribosyl system

We have studied the reactions between adenosine 5′-phosphorimidazolide and various adenosine analogs on a poly(U) template. The nucleosides were adenosine (I), 2′-deoxyadenosine (II), 3′-deoxyadenosine (III), 2′-O-methyladenosine (IV), 3′-O-methyladenosine (V), 9-β-d-xylofuranosyladenine (VI), and 9-β-d-arabinofuranosyladenine (VII). We find that the various analogs form triple helices with poly(U) which are of comparable stability, but that only the β-riboside takes part in an efficient template-directed condensation.     

A heterodimer of human 3′-phospho-adenosine-5′-phosphosulphate (PAPS) synthases is a new sulphate activating complex

3′-Phospho-adenosine-5′-phosphosulphate (PAPS) synthases are fundamental to mammalian sulphate metabolism. These enzymes have recently been linked to a rising number of human diseases. Despite many studies, it is not yet understood how the mammalian PAPS synthases 1 and 2 interact with each other. We provide first evidence for heterodimerisation of these two enzymes by pull-down assays and Förster resonance energy transfer (FRET) measurements. Kinetics of dimer dissociation/association indicates that these heterodimers form as soon as PAPSS1 and -S2 encounter each other in solution. Affinity of the homo- and heterodimers were found to be in the low nanomolar range using anisotropy measurements employing proteins labelled with the fluorescent dye IAEDANS that - in spite of its low quantum yield - is well suited for anisotropy due to its large Stokes shift. Within its kinase domain, the PAPS synthase heterodimer displays similar substrate inhibition by adenosine-5′-phosphosulphate (APS) as the homodimers. Due to divergent catalytic efficacies of PAPSS1 and -S2, the heterodimer might be a way of regulating PAPS synthase function within mammalian cells.     

Phosphorylation of an actin · tropomyosin · troponin complex from human skeletal muscle

A human skeletal actin · tropomyosin · troponin complex was phosphorylated in the presence of [γ-³²P]ATP, Mg²⁺, adenosine 3′:5′-monophosphate (cyclic AMP) and cyclic AMP-dependent protein kinase (protein kinase). Phosphorylation was not observed when the actin complex was incubated in the absence of protein kinase or 1 μM cyclic AMP. In the presence of 10⁻⁷ M Ca²⁺ and protein kinase 0.1 mole of [³²P]phosphate per 196 000 g of protein was incorporated. This was two-fold higher than the [³²P]phosphate content of a rabbit skeletal actin complex but two-fold lower than that of a bovine cardiac actin complex. At high Ca²⁺, 5 · 10⁻⁵ M, little change in the phosphorylation of a human skeletal actin complex occurred. Phosphoserine and phosphothreonine were identified in the [³²P]phosphorylated actin complex. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that 60% of the label was associated with the tropomyosin binding component of troponin. The inhibitory component of troponin contained 16% of the bound [³²P]phosphate. Increasing the Ca²⁺ concentration did not significantly decrease the [³²P]phosphate content of the phosphorylated proteins in the actin complex. No change in the distribution of phosphoserine or phosphothreonine was observed. Half maximal calcium activation of the ATPase activity of reconstituted human skeletal actomyosin made with the [³²P]phosphorylated human skeletal actin complex was the same as a reconstituted actomyosin made with an actin complex incubated in the absence of protein kinase at low or high Ca²⁺.     

Phosphorylation of Escherichia coli RNA polymerase by rabbit skeletal muscle protein kinase

Rabbit skeletal muscle protein kinase catalyzes the phosphorylation of DNA-dependent RNA polymerase of Escherichia coli in the presence of adenosine 3′,5′-monophosphate and ATP. The phosphorylation occurs on one (or more) serine residue(s) in the σ-factor under reaction conditions similar to those employed for RNA synthesis. The phosphorylation of RNA polymerase and its stimulation by protein kinase are inhibited by a specific heat-stable inhibitor from rabbit skeletal muscle. With conditions more favorable for the protein kinase reaction, phosphorylation of RNA polymerase also occurs on the β subunit of the core enzyme, but this reaction occurs at a much slower rate than the phosphorylation of the σ-factor.     

Studies on the synthesis of CDP-diacylglycerol: Stimulation by GTP and inhibition by ATP and fluoride

The rat liver microsomal enzyme CTP: phosphatidate cytidylyltransferase (EC 2.7.7.41) which catalyzes the formation of CDP-diacylglycerol has been found to be markedly stimulated by GTP. The requirement for GTP is absolute, the novel GTP analogues such as guanosine 5′-[β,γ-methylene]-triphosphate, guanosine 5′-[α,β-methylene]-triphosphate, guanosine 5′-[β,γ-imido]-triphosphate and guanosine 3′-diphosphate 5′-diphosphate are without significant effect. Maximal stimulation occurs at 1 mM GTP. ATP at a concentration of 5 mM totally inhibits the formation of CDP-diacylglycerol even in the presence of optimal GTP concentration. Analogues of ATP such as adenosine 5′-[α,β-methylene]-triphosphate, adenosine 5′-[β,γ-methylene]-triphosphate and adenosine 5′-[β,γ-imido]-triphosphate are without effect on the reaction. The addition of fluoride (8 mM) likewise abolishes the stimulatory effect of GTP.     

Stimulation of guanylate cyclase activity by several fatty acids.

Guanylate cyclase of plasma membrane of isolated rat fat cells was activated 7 to 11 fold by oleic acid, linoleic acid, linolenic acid or arachidonic acid. The activation of the enzyme by linoleic acid or oleic acid was influenced by the concentration of enzyme protein and that of the fatty acid. At 158 μg/ml of enzyme protein, 0.6 mM linoleic acid produced maximal activation of 12 fold which was partially reversed by washing. Particulate guanylate cyclase of cerebral cortex and liver was also activated by linoleic acid.