Synthesis of deoxynucleoside-5'-tri-3'-diphosphates by Streptomyces adephospholyticus ATP:nucleotide pyrophosphokinase

ATP: nucleotide pyrophosphokinase (EC 2.7.6.4.) of Streptomyces adephospholyticus catalyzes an efficient transfer of the 5′-β,γ-pyrophosphoryl group of dATP to the four common deoxynucleoside-5′-triphosphates at their 3′-OH positions in the alkaline pH and in the presence of Co²⁺ ions, giving the respective 3′-pyrophosphoryl derivatives. Deoxyadenosine-5′-tri-3′-diphosphate was chromatographically prepared and structurally characterized.     

Interaction of guanosine cyclic 3',5'-phosphate dependent protein kinase with lin-benzoadenine nucleotides

Using the activated cGMP-dependent protein kinase in the presence of the phosphorylatable peptide [[Ala34]histone H2B-(29-35)], we found that lin-benzoadenosine 5'-diphosphate (lin-benzo-ADP) was a competitive inhibitor of the enzyme with respect to ATP with a Ki (22 microM) similar to the Kd (20 microM) determined by fluorescence polarization titrations. The Kd for lin-benzo-ADP determined in the absence of the phosphorylatable peptide, however, was only 12 microM. ADP bound with lower affinity (Ki = 169 microM; Kd = 114 microM). With [Ala34]histone H2B-(29-35) as phosphoryl acceptor, the Km for lin-benzo-ATP was 29 microM, and that for ATP was 32 microM. The Vmax with lin-benzo-ATP, however, was only 0.06% of that with ATP as substrate [0.00623 +/- 0.00035 vs. 11.1 +/- 0.17 mumol (min.mg)-1]. Binding of lin-benzo-ADP to the kinase was dependent upon a divalent cation. Fluorescence polarization revealed that Mg2+, Mn2+, Co2+, Ni2+, Ca2+, Sr2+, and Ba2+ supported nucleotide binding to the enzyme; Ca2+, Sr2+, and Ba2+, however, did not support any measurable phosphotransferase activity. The rank order of metal ion effectiveness in mediating phosphotransferase activity was Mg2+ greater than Ni2+ greater than Co2+ greater than Mn2+. Although these results were similar to those observed with the cAMP-dependent protein kinase [Hartl, F. T., Roskoski, R., Jr., Rosendahl, M. S., & Leonard, N. J. (1983) Biochemistry 22, 2347], major differences in the Vmax with lin-benzo-ATP as substrate and the effect of peptide substrates on nucleotide (both lin-benzo-ADP and ADP) binding were observed.     

Effect of nucleotides on translocation of sugar nucleotides and adenosine 3'-phosphate 5'-phosphosulfate into Golgi apparatus vesicles

Recent studies from this laboratory have suggested that rat-liver Golgi apparatus derived membranes contain different proteins which can translocate in vitro CMP-N-acetylneuraminic acid, GDP-fucose and adenosine 3'-phosphate 5'-phosphosulfate from an external compartment into a lumenal one. The aim of this study was to define the role of the nucleotide, sugar and sulfate moieties of sugar nucleotides and adenosine 3'-phosphate 5'-phosphosulfate in translocation of these latter compounds across Golgi vesicle membranes. Indirect evidence was obtained suggesting that the nucleotide (but not sugar or sulfate) is a necessary recognition feature for binding to the Golgi membrane (measured as inhibition of translocation) but is not sufficient for overall translocation; this latter event also depends on the type of sugar. Important recognition features for inhibition of translocation of the above sugar nucleotides and adenosine 3'-phosphate 5'-phosphosulfate were found to be the type of nucleotide base (purine or pyrimidine) and the position of the phosphate group in the ribose. Thus, UMP and CMP were found to be competitive inhibitors of translocation of CMP-N-acetylneuraminic acid, while AMP did not inhibit. Structural features of the nucleotides which were less important in inhibition of translocation (and thus presumably in binding) of the above sugar nucleotides and adenosine 3'-phosphate 5'-phosphosulfate were the number of phosphate groups in the nucleotide (CDP and CMP inhibited to a similar extent), the presence of ribose or deoxyribose in the nucleotide, a replacement of hydrogen in positions 5 of pyrimidines or 8 in purines by halogens or an azido group. The sugar or sulfate did not inhibit translocation of the above sugar nucleotides and adenosine 3'-phosphate 5'-phosphosulfate into Golgi vesicles and therefore appear not to be involved in their binding to the Golgi membrane.     

The specific, diverse effects of purine and pyrimidine nucleoside-5'-di(tri)-3'-diphosphates on the eucaryote translation system in vitro.

All the eight 5'-di(tri)-3'-diphosphates of four common ribonucleosides were prepared by the enzymic pyrophosphoryl transfer catalysed by Streptomyces adephospholyticus ATP:nucleotide pyrophosphokinase (E.C.2.7.6.4) form dATP to the respective 5'-phosphates, and their effects on the translation of mRNAs by a wheat germ system in vitro were studied. (p) ppPupp decreased the total 14C-leucine incorporation directed by a rat liver mRNA whereas (p) ppPypp did not. With a silkworm pupa ovary mRNA, distinctly reverase results were obtained. Gel electrophoretic profiles of the translation products disclosed the mRNA species-specific stimulatory or inhibitory effects for each of the polyphosphates tested.     

Synthesis of pyrimidine nucleotides in the heart: uridine and cytidine kinase activity

These experiments were designed to determine through the study of uridine and cytidine kinase activity, the precise mechanisms of plasma nucleoside salvage leading to pyrimidine nucleotide synthesis in the rat heart. The kinetic parameters were: Km = 10 microM, V = 4 nmol g-1 min-1 for cytidine kinase activity and Km = 43 microM and V = 18 nmol g-1 min-1 for uridine kinase activity. Competing activity as concerns the two nucleosides was shown to occur, suggesting that in the rat myocardium as in other cells, one and the same enzyme phosphorylates both uridine and cytidine. UTP and CTP were shown to exert a potent inhibitory action on nucleoside phosphorylation; two factors thus exert a joint influence on the control of pyrimidine nucleotide synthesis in the rat heart: the extracellular concentration of precursor and the intracellular level of UTP and CTP. The kinetic parameters for kinase activities are discussed, taking into account the actual concentration of plasmatic nucleosides. Comparison of these data with respectively those for incorporation of nucleosides into the pyrimidine nucleotides of isolated rat heart and with nucleotide turnover rates in vivo suggests that, under physiological conditions, the utilization of plasma cytidine is crucial to the synthesis of myocardial pyrimidine synthesis.     

Modified 5'-nucleotides resistant to 5'-nucleotidase: isolation of 3-(3-amino-3-carboxypropyl) uridine 5'-phosphate and N2, N2-dimethylguanosine 5'-phosphate from snake venom hydrolysates of transfer RNA.

A procedure for the quantitative measurement of the O2'-methylnucleoside constitutents of RNA has recently been developed in this laboratory (Gray, M.W. Can. J. Biochem. 53, 735-746 (1975)). This assay method is based on the resistance of O2'-methylnucleoside 5'-phosphates (pNm) (generated by phosphodiesterase hydrolysis of RNA) to subsequent dephosphorylation by venom 5'-nucleotidase (EC 3.1.3.5). In the present investigation, two base-modified 5'-nucleotides, each displaying an unusual resistance to 5'-nucleotidase, have been identified. These compounds have been characterized by a variety of techniques as N2, N2-dimethylguanosine 5'-phosphate (pm2/2G) and 3-(3-amino-3-carboxypropyl)uridine 5'-phosphate (p4abu3U). Because of their resistance to 5'-nucleotidase, pm2/2G and p4abu3U are isolated along with the pNm in the mononucleotide fraction of venom hydrolysates of transfer RNA. Under hydrolysis conditions, the stability of p4abu3U is comparable to that of a pNm, allowing quantitative assay of the nucleotide. The proportion (mean +/- SD) of p4abu3U in venom hydrolysates of wheat embryo and Escherichia coli tRNA has been determined to be 0.35 +/- 0.03 (n=5) and 0.14 +/- 0.02 (n=4) mol%, respectively. The absence of p4abu3U in venom hydrolysates of yeast tRNA implies the absence of the corresponding nucleoside in yeast tRNA, in agreement with existing data. The variable recovery of pm2/2G from venom hydrolysates of wheat embryo and yeast tRNA indicates that under hydrolysis conditions, this base-modified nucleotide is only partially resistent to 5'-nucleotidase. The complete absence of pm2/2G in venom hydrolysates of E. coli tRNA is consistent with the known absence of N2, N2-dimethylguanosine in this RNA. These observations demonstrate that resistance to 5'-nucleotidase is a necessary but not sufficient criterion for concluding that a 5'-nucleotide is O2'-methylated. When applied to wheat embryo ribosomal RNA, the analytical methods described in this report failed to reveal any compound having the distinctive charge properties of p4abu3U. It therefore appears that 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, recently characterized as a constituent of the 18 S rRNA of Chinese hamster cells (Saponara, A.G. & Enger, M.D. Biochim. Biophys. Acta 349, 61-77 (1974)), may not be present in wheat embryo ribosomal RNA.     

Synthesis and template-directed polymerization of adenylyl(3'-5')adenosine cyclic 2', 3'-phosphate.

Adenylyl(3'-5')adenosine cyclic 2',3'-phosphate (A-A greater than p) was synthesized and its polymerization was attempted under various conditions inthe presence of poly(uridylic acid) and1,3-propanediamine. Reaction at -20 degrees C for 16 days gave polymerized products (up to the 8-mer) in 15% yield and was proved to be dependent on the template. Reaction at 0 degrees C for 16 days gave more extensive (up to the 10-mer) and more efficient (35%) polymerization. The newly formed phosphodiester linkage was exclusively 2'-5'. These results are discussed in comparison with the monomer-condensation reaction.