Masanao Miwa

Enzyme cleaving the 5'-terminal methylated blocked structure of messenger RNA.

Recently we purified a novel phosphodiesterase from cultured tobacco cells to an apparently homogeneous state by polyacrylamide gel electrophoresis [l] . The enzyme has a pH optimum at around 6, and is fully active in the presence of EDTA. This tobacco acid phosphodiesterase also shows pyrophosphatase activity, and hydrolyzes various phosphodiesters and pyrophosphate bonds, including those in cyclic AMP, ATP, ADP and NAD: However, it does not hydrolyze polynucleotides. The recent discovery of the 5’-terminal methylated blocked structure of various viral and mammalian mRNA [2131, prompted us to determine the susceptibility of the pyrophosphate bond in this structure to this novel phosphodiesterase. This paper reports that the tobacco phosphodiesterase preferentially cleaves the 5’-terminal blocked structure of cytoplasmic polyhedrosis (CP) virus mRNA releasing pm’G from it, but does not degrade the polynucleotide chain of the mRNA.

Studies on anti-poly(adenosine diphosphate ribose) antibody.

Summary Specific antibody against poly(ADP-Rib) was produced in a rabbit by injecting poly(ADP-Rib) mixed with methylated bovine serum albumin. Under standardized conditions, 1 mg of purified anti-poly(ADP-Rib) antibody combined with 400 pmoles (4 μg) of poly(ADP-Rib) and was retained on a millipore filter. The binding of [ 14 C]poly-(ADP-Rib) was not inhibited by poly(A) or other related nucleotides.

Studies on poly(adenosine diphosphate-ribose): XI. Purification of poly(adenosine diphosphate-ribose) on a hydroxylapatite column

Abstract Hydroxylapatite column chromatography was used for purification of poly (ADP-Rib). With an increasing concentration gradient of phosphate buffer (pH 6.8) RNA, DNA, and poly (ADP-Rib) with a chain of 17–18 ADP-ribose units were eluted successively. RNase and DNase digestion of a crude extract containing RNA, DNA, and poly(ADP-Rib) before chromatography resulted in a better separation of poly (ADP-Rib). Poly (ADP-Rib) with a shorter chain length were eluted with more dilute phosphate buffer and a linear relationship was obtained between the chain length of the material eluted and the phosphate concentration of the elution buffer.

Degradation of poly(adenosine diphosphate ribose) by homogenates of various normal tissues and tumors of rats.

Abstract Poly(ADP-ribose) glycohydrolase, which was first identified in calf thymus and rat liver, was found to be present in all normal rat tissues and tumors. Of the tissues examined, the testis had the highest activity. The major pathway of degradation of poly(ADP-ribose) in all tissues was through poly(ADP-ribose) glycohydrolase. Phosphodiesterase, which was first found in rat liver, was only of minor importance in hydrolysis of poly(ADP-ribose). These findings suggest the importance of poly(ADP-ribose) glycohydrolase both in normal and tumor tissues for the rapid turnover of poly(ADP-ribose).

Depression of DNA polymerase activity in chromatin by incubation with NAD

Abstract DNA polymerase activity of a disrupted nuclear preparation of rat liver was depressed by preincubation with NAD. This depression was observed either using endogenous DNA or poly d(A-T) as template. DNA polymerase activity in a solubilized fraction from the disrupted nuclear preparation, was also reduced when obtained from a preparation which had been preincubated with NAD. Exogenously added E. coli DNA polymerase could be used equally well by the disrupted nuclear preparation, preincubated either with or without NAD. Addition of purified poly (ADP-Rib) did not inhibited the activity of DNA polymerase using poly d(A-T) or endogenous DNA as template.

NMR analyses of conformation of ribosyl adenosine 5′,5″-bis(phosphate) in aqueous solution

Summary NMR analyses of spin coupling constants, nuclear Overhauser effects and Gd(III)-induced relaxation rates were made of ribosyl adenosine 5′,5″-bis(phosphate), [Ado(P)-Rib-p], in aqueous solution. The α-configuration of the Cl″ atom of the Rib-P moiety was confirmed. The Ado(P) moiety takes the anti form about the glycosidic bond, the gg form about the exocyclic bond, and the 2′-endo and 3′-endo forms of the ribose ring with nearly equal fractional populations. The ribose ring of the Rib-P moiety preferentially takes the gt/tg-2″-endo form. The Ado(P)-Rib-P molecule has a fairly extended overall molecular conformation.

Poly(adenosine diphosphate ribose) glycohydrolase in Physarum polycephalum.

Abstract Poly(adenosine diphosphate ribose) glycohydrolase, which has thus far only been found in mammalian tissues, was found for the first time in the primitive eukaryotic slime mold Physarum polycephalum. The hydrolytic product of poly(adenosine diphosphate ribose) with this enzyme was identified as adenosine diphosphate ribose by paper and thin-layer chromatography. It is likely that the enzyme caused exoglycosidic hydrolysis. The optimal pH of this enzyme was 6.0, and the Km value was 4.3 μ m , as adenosine diphosphate ribose residues of polymer. Adenosine diphosphate ribose, ADP and ATP at a concentration of 0.1m m strongly inhibited the enzyme activity. 3′,5′-Cyclic AMP was inhibitory at a concentration of 1m m . The molecular weight of this enzyme was estimated to be 57,000.

Inhibition of deoxyribonuclease in an extract of rat liver nuclei by poly(ADP-ribose).

Summary A deoxyribonuclease inhibited by poly(ADP-Rib) was found in an extract of rat liver nuclei. Of the compounds tested, (i.e. poly(ADP-Rib), poly(A), NAD, ADP-Rib, rRNA, and tRNA) only poly(ADP-Rib) and poly(A) inhibited deoxyribonuclease activity. The deoxyribonuclease activity was separated by DEAE-cellulose column chromatography into two distinct fractions, only one of which was inhibited by poly(ADP-Rib) and poly(A). Both fractions degraded poly[d(A-T)] exonucleolitically.

Phosphodiesterase from cultured tobacco cells. Physical and chemical properties.

Phosphodiesterase isolated from suspension cultures of tobacco cells showed high affinity for concanavalin A-Sepharose and gave single superimposed bands of protein and carbohydrates on disc gel electrophoresis, suggesting that it is a glycoprotein. It contains 14% carbohydrate by weight, and has relatively high contents of basic and aromatic amino acids. Its isoelectric point is at pH 8.8, and the molecular weight of its subunits was estimated as 72 000 from a plot of the retardation coefficient on sodium dodecyl sulfate gel electrophoresis versus the molecular weight. The enzyme was catalytically active in an immobilized state on a concanavalin A-Sepharose column.

Enhancement by m-Aminobenzamide of Methylazoxymethanol Acetate-Induced Hepatoma of the Small Fish “Medaka” (Oryzias latipes)

A poly(ADP-ribosyl)ation reaction is suggested to be involved in DNA repair, mutagenesis, cell transformation, and cell differentiation [1–3].