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Biomedical subjects

M J Modak

Publications and source records attributed to M J Modak.

At least 91 records · Page 5Linked to original sources
PubMed1980

Biochemistry of terminal deoxynucleotidyltransferase: identification, characterization, requirements, and active-site involvement in the catalysis of associated pyrophosphate exchange and pyrophosphorolytic activity.

Terminal deoxynucleotidyltransferase (TdT) has been found to catalyze both pyrophosphate exchange and pyrophosphorolysis reactions. Both reactions are strongly inhibited by antiserum to TdT. The reactions require the presence of a divalent cation, a single- or double-stranded oligomeric or polymeric DNA or RNA, and deoxyribonucleoside triphosphates (for PPi exchange only). Of the three divalent cations tested, Mg2+ and Co2+ are equally effective, while Mn2+ neither is used for catalysis nor inhibits the Mg2+-catalyzed reactions. Ribonucleoside triphosphates have been found to support the PPi exchange reaction to a minor extent and have no inhibitory effect on the catalysis mediated by dNTPs. Inhibition studies, using SH group inhibitors, Zn chelator, and a substrate binding site specific reagent, revealed that PPi exchange and pyrophosphorolysis reactions may be distinguished by differences in their sensitivity to inhibition by various reagents. While the PPi exchange reaction is strongly inhibited by sulfhydryl reagents, o-phenanthroline, and pyridoxal phosphate, the pyrophosphorolysis reaction is insensitive to these reagents. In addition, the pyrophosphorolysis reaction is also found not to require a free 3'-OH terminus of a primer. This difference in the susceptibility of the two reactions indicates that discrete active-site structures exist in TdT which catalyze PPi exchange and pyrophosphorolysis reactions.

Binding Sites↗
PubMed1980

Enzymatic activities associated with avian and murine retroviral DNA polymerases. Catalysis of and active site involvement in pyrophosphate exchange and pyrophosphorolysis reactions.

Reverse transcriptase isolated from avian myeloblastosis virus (AMV) and Rauscher murine leukemia virus (RLV) were examined for their ability to catalyze polymerization, ribonuclease H, pyrophosphate exchange, and pyrophosphorolysis reactions. A detailed characterization and a study of requirements for the expression of pyrophosphate exchange and pyrophosphorolysis reactions indicated that a variety of RNA and DNA template-primers supported these catalytic reactions. Furthermore, hydrogen bonding of template to primer was essential, although RNA:RNA template-primers, e.g. poly(rA) . (rU)9 or 70 S RNA . tRNA complex, were not utilized for these reactions. AMV enzyme required Mg2+, and RLV enzyme Mn2+, as the preferred divalent metal ion for the expression of these activities. Response of various catalytic reactions to site-specific inhibitors revealed that polymerization and pyrophosphate exchange reactions were susceptible to reagents that affected either the substrate or the template binding site, intrinsic zinc, or sulfhydryl groups. RNase H and pyrophosphorolysis activities, on the other hand, exhibited susceptibility only to the template site-specific reagent. We, therefore, conclude that RNase H and pyrophosphorolysis reactions are catalyzed through the template binding site while polymerization and pyrophosphate exchange reactions require additional participation of the substrate binding site, as well as that of intrinsic zinc and the presence of reactive sulfhydryl groups.

Avian Leukosis Virus↗
PubMed1980

Phenylglyoxal as a template site-specific reagent for DNA and RNA polymerases. Selective inhibition of initiation.

Phenylglyoxal, an arginine-specific reagent, strongly inhibits DNA polymerases isolated from eukaryotic, prokaryotic, and RNA tumor viral sources as well as Escherichia coli RNA polymerase. The inhibitory action of phenylglyoxal appears to be due to interference with the template binding function of these enzymes and implies the presence of an arginine residue at the template binding site of these enzymes from diverse sources and suggests that template dependent DNA, and perhaps RNA polymerases, may be mechanistically similar with respect to their template binding function. In contrast, the activity of terminal deoxynucleotidyl-transferase, a template-independent DNA polymerase isolated from calf thymus, is not inhibited by phenylglyoxal. A detailed analysis of the inhibitory process carried out using avian myeloblastosis virus (AMV) DNA polymerase as a test enzyme revealed that inclusion of template-primer during the preincubation with phenylglyoxal, but not substrate triphosphates or primer alone, protects the enzyme against phenylglyoxal inactivation. Furthermore, phenylglyoxal does not appear to inhibit the elongation of initiated DNA strands, but blocks the reinitiation of DNA synthesis.

Aldehydes↗
PubMed1980

A micromethod for determination of terminal deoxynucleotidyl transferase (TdT) in the diagnostic evaluation of acute leukemias.

A micromethod for the determination of TdT in peripheral leukocytes and bone marrow cells has been developed that allows unequivocal identification and quantitation of TdT in less than 1 X 10(6) leukocytes from ALL patients, i.e., in 1 ml of peripheral blood and/or 0.5 ml of bone marrow obtained during routine clinical sampling. The method involves disruption of cell pellet with high salt and detergent followed by centrifugation of extracts at 12,000 X g and partial purification on phosphocellulose matrix by a batch elution technique using a standard laboratory microcentrifuge. Using this microassay, TdT activities have been determined in 500 samples of peripheral blood and bone marrow of 240 adult patients with acute leukemias (86 ALL, 108 ANLL, 44 blastic CML, two acute leukemias following P. vera). From an analysis of our data based on TdT activity, cell surface markers and growth patterns in soft agar and observations published in the literature, it can be concluded that the frequencies of TdT + phenotypes in the various clinical-morphological diagnostic groups are approximately 95% in ALL, 10% in ANLL, 50% in AUL, and 35% in blastic CML. Since the presence of high TdT activity is clearly associated with clinical response to specific forms of chemotherapy in blastic CML and most probably, also in ANLL, the determination of TdT should be considered in all cases of acute leukemias to objectively define prognostically important subgroups which can not be diagnosed by conventional means.

Acute Disease↗
PubMed1979

Reverse transcriptase-associated ribonuclease H does not require zinc for catalysis.

omicron-Phenanthroline, a zinc chelating agent, is known to inhibit the DNA polymerase activity of cellular DNA-dependent and viral RNA-dependent DNA polymerases. We find that omicron-phenanthroline does not inhibit the reverse transcriptase-associated RNase H activity of retroviruses. Kinetic studies, using DNA template-primers as an inhibitor of RNase H, suggest that zinc does not play any role in template-primer binding by reverse transcriptase. These results also indicate a distinct binding site for the template and triphosphate substrates. Cellular RNase H from calf thymus and RNase H-II from Rauscher leukemia virus are likewise resistant to omicron-phenanthroline inhibition, implying non-involvement of zinc in the nucleic acid hydrolysis by these enzymes.

Avian Myeloblastosis Virus↗
PubMed1978

Biochemistry of terminal deoxynucleotidyltransferase: mechanism of inhibition by adenosine 5'-triphosphate.

The polymerization of deoxyribunucleoside triphosphate catalyzed by terminal deoxyribonucleotidyltransferase (TdT, EC 2.7.7.31) is severely inhibited by the addition of ribonucleoside triphosphates, ATP being the most potent inhibitor. Examination of the inhibitory effect of ATP using oligo(dA)12-18 as well as activated DNA as primers revealed that (a) ATP inhibition is not due to its addition onto a 3'-OH primer terminus ad judged by the lack of incorporation of labeled ATP, although under similar conditions incorporation of GTP can be demonstrated, (b) a consistent degree of inhibition was noted independent of primer or enzyme concentration; (c) addition of ATP to an ongoing reaction promptly reduces the rate of polymerization; (d) kinetic studies indicate a competitive (with respect to substrate deoxy triphosphate) pattern of inhibition; (e) addition of excess deoxyribotriphosphate promptly relieves the inhibition. Unlike ATP, other ribotriphosphates yield a mixed pattern of inhibition partly mediated by competitive mechanisms. GTP and CTP and to a minor extent UTP are incorporated into DNA in the presence or absence of deoxy triphosphate. Furthermore, addition of ATP also inhibits incorporation of GTP and CTP.

Adenosine Triphosphate↗
PubMed1978

Template-specific requirements for DNA synthesis by the Mason-Pfizer monkey virus DNA polymerase: unique aspects.

The biochemical properties of DNA polymerase purified from Mason-Pfizer monkey virus were studied, with respect to synthetic and natural template-primer utilization. Thes studies revealed the following new information about the Mason-Pfizer monkey virus enzyme: (a) Mason-Pfizer monkey virus polymerase was found to prefer template: primer molar nucleotide ratios of 2.5-5: 1 for optimal rates of synthesis with poly(C) .(dG)12-18 as template-primer. (b) Poly(A)-directed synthesis was stimulated by the addition of low concentrations of inorganic phosphate to the reaction mixture. (c) Poly(2' -O-methyl-cytidylate), poly(rCm), was the only template studied for which Mn2+ proved the preferred divalent cation. Combinations of divalent cations stimulated rather than inhibited poly(rCm)-directed poly(dG) synthesis by the Mason-Pfizer monkey virus enzyme. (d) Heteropolymeric regions of rabbit globin mRNA and avian myeloblastosis virus 70 S RNA could be copied by the Mason-Pfizer monkey virus polymerase with oligo(dT), oligo(U) or in the case of avian myeloblastosis virus RNA, endogenous primers. In all such studies, Mg2+ was the preferred divalent cation and a distinct preference for the DNA primer in the reverse transcription of natural RNAs was observed. These new findings necessitated comparative studies with the DNA polymerases from Rauscher murine leukemia virus and murine mammary tumor virus, as representative type C and type B retroviruses. Although the Mason-Pfizer monkey virus enzyme was found to share some properties in common with both type C and type B mammalian viral enzymes, certain of the above properties rendered it unique among the polymerases examined.

Animals↗
PubMed1978

Reverse transcriptase-associated RNase H activity. II. Inhibition by natural and synthetic RNA.

The RNase H activity associated with purified avian myeloblastosis virus and Rauscher murine leukemia virus DNA polymerases is inhibited by homopolymeric RNA molecules, although the efficiency of inhibition by each homopolymer appears enzyme specific. Formation of duplex RNA molecules abolished the inhibitory activity. In contrast to these results, DNA polymerase-independent RNase H activities, such as the RNase H-II from Rauscher murine leukemia virus and calf thymus RNase H, were unaffected by the addition of exogenous RNA molecules to reaction mixtures. These results support the concept (M. J. Modak and S. L. Marcus, J. Virol. 22:253--256, 1977) that the catalytic site of DNA polymerase-associated RNase H activity may be that which is also involved in template binding. Naturally occurring RNA molecules of oncornaviral, procaryotic, or eucaryotic origin also proved to be efficient inhibitors of avian myeloblastosis virus DNA polymerase-associated RNase H activity. In contrast to this result, naturally occurring RNA molecules, at concentrations which inhibited the avian myeloblastosis virus enzyme, did not inhibit Rauscher murine leukemia virus DNA polymerase-catalyzed RNase H activity. This finding represents a new biochemical distinction between the two reverse transcriptases, and may be indicative of differences in the relative affinities of these enzymes for natural RNA molecules.

Avian Leukosis Virus↗
PubMed1977

Purification and properties of Rauscher leukemia virus DNA polymerase and selective inhibition of mammalian viral reverse transcriptase by inorganic phosphate.

Rauscher leukemia virus RNA-directed DNA polymerase has been purified to near homogeneity (greater than 90% pure) using affinity chromatography on polycytidylate-agarose with over 85% recovery of input enzymatic activity. The purified enzyme has a molecular weight of approximately 70,000 and appears to consist of a single polypeptide chain. The enzyme is free of DNase, but has RNase H activity. Analysis of the requirements for optimal rates of DNA synthesis by this enzyme using synthetic and natural template-primers has revealed template-specific variations in such requirements. During these studies it was observed that DNA synthesis catalyzed by Rauscher leukemia virus DNA polymerase is inhibited by the addition of inorganic phosphate. An analysis of the mechanism of phosphate inhibition was carried out using the synthetic template-primer poly(A)-(dT)10. It appears that by some mechanism, possibly involving the substrate binding site of the enzyme, phosphate ions inhibit DNA synthesis with a more acute effect on the rate of chain growth than on that of initiation. The extension of these studies to DNA synthesis catalyzed by a variety of mammalian type C viral reverse transcriptases revealed that low levels ( less than or equal to 2 mM) of inorganic phosphate strongly inhibited DNA synthesis. The susceptibility to phosphate inhibition appears unique to mammalian type C viral enzymes since the type B viral enzyme, Escherichia coli DNA polymerase I, avian myeloblastosis virus and Mason Pfizer monkey tumor virus reverse transcriptase and cellular DNA polymerases alpha and gamma are not inhibited by inorganic phosphate. This phenomenon of phosphate inhibition of various DNA polymerases, therefore, provides a new basis for the differentiation of the sources and nature of these enzymes.

Chromatography, Affinity↗
PubMed1977

Specific inhibition of DNA polymerase-associated RNase H by DNA.

The RNase H activity associated with several RNA-directed DNA polymerases is inhibited by the addition of DNA, in contrast to RNase H activity from enzymes devoid of polymerizing activity. Kinetic investigations of the inhibitory effect of DNA, using purified Rauscher leukemia virus DNA polymerase as a test enzyme, revealed that the addition of DNA to an ongoing RNase H reaction causes an immediate cessation of RNase H activity. Concomitant initiation of DNA synthesis by inhibitory DNA can occur, provided that appropriate substrate and primer is available. Thus, in addition to providing a simple test for the distinction between polymerase-associated and polymerase-independent RNase H activity, this study strongly supports the concepts that (i) RNase H activity expressed by several mammalian oncoviral reverse transcriptases is an integral part of that molecule, and (ii) that the catalytic site of RNase H activity is also involuved in template-primer binding.

DNA↗