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M J Modak

Publications and source records attributed to M J Modak.

At least 73 records · Page 4Linked to original sources
PubMed1987

Inhibition of terminal deoxynucleotidyl transferase by adenine dinucleotides. Unique inhibitory action of Ap5A.

Terminal deoxynucleotidyltransferase (TdT) exhibits strong sensitivity to ATP and its dinucleotide analogues, Ap2A, Ap3A, Ap4A, Ap5A and Ap6A. Similar to ATP, all of the dinucleotides appear to be competitive inhibitors of TdT catalysis with respect to substrate deoxynucleoside triphosphates and effectively block the UV-mediated substrate cross-linking to TdT. Among the various dinucleotides, Ap5A and Ap6A (diadenosine 5'-5' penta- and hexaphosphate, respectively) are significantly more effective than dinucleotides containing 2, 3 or 4 phosphate backbones. Furthermore, Ap5A is found to be the only dinucleotide which has reactivity at both substrate- and primer-binding domains in TdT.

Adenine Nucleotides↗
PubMed1987

An affinity labeling of ras p21 protein and its use in the identification of ras p21 in cellular and tissue extracts.

We have carried out photoaffinity labeling of the ras p21 protein, a ras oncogene product, with [alpha-32P]GTP. Based on our studies, a sensitive, rapid, and specific assay for the detection of multiple forms of ras p21 has been developed. The specificity of this protocol is shown by (a) sensitivity of affinity labeling of ras p21 to known inhibitors of GTP binding and (b) immunoprecipitation of affinity labeled protein with anti-ras p21 serum. Detection and semiquantitation of ras p21 by this method is accomplished in less than 24 h and requires as little as 100,000 cells or about 5 mg of tissue sample from skin tumor, liver, and mammary tumor tissues. Furthermore, using this approach, we were able to detect the selective loss of one species of ras p21 in transplanted Morris hepatoma cells.

Affinity Labels↗
PubMed1987

Purification of high molecular mass species of calf thymus terminal deoxynucleotidyltransferase.

We have developed a simplified column chromatographic procedure for the simultaneous purification of two high molecular mass forms (58 kd and 45 kd) and a standard two subunit 44 kd from of terminal deoxynucleotidyltransferase (TdT) from calf thymus chromatin. The procedure involves high salt extraction of the chromatin fraction followed by successive chromatographies on phosphocellulose, DEAE sephadex, and hydroxylapatite matrices. While all 3 species of TdT comigrate throughout these steps, separation of individual species is achieved on a single stranded DNA agarose column. The combined yield of the 45 kd and 58 kd TdTs is quite high (approximately 8 mg/5000g tissue), 45 kd being the major species (approximately 60%) and the 58 kd constituting about 30%. The 44 kd species containing two subunits usually represents under 10% of the total. All the three forms of TdT showed similar specific activity and preference for purine deoxynucleoside triphosphates (dNTPs). The Km for individual dNTP with all three species of TdT is quite similar and decreases in the order dCTP greater than dTTP greater than dATP greater than dGTP. The Km for both synthetic primer and activated DNA with the 3 TdTs was, in increasing order, two subunit 44 kd less than 45 kd less than 58 kd TdT. Both 58 kd and 45 kd TdT displayed two optima for Mn++ (0.1 mM and 1 mM) and a single sharp optimum for Mg++ (2.5 mM). The two subunit 44 kd TdT exhibited a single but broad optimum for Mn++ (1 mM) and for Mg++ (10 mM).

Animals↗
PubMed1986

Solid phase synthesis of thymosin beta 9.

Thymosin beta 9, a 41 residue thymic polypeptide, has been synthesized by a solid phase method. A modification of the low HF method was used to deprotect and cleave the peptide from the resin. Thymosin beta 9 was then obtained in analytically pure form by a one-step purification procedure in 32% yield. The activity of thymosin beta 9 in the terminal deoxynucleotidyl transferase assay was greater than calf thymus fraction 5, but comparable to thymosin beta 4. In contrast to thymosin alpha 1, neither beta 4 nor beta 9 was active in the rosette inhibition assay.

Animals↗
PubMed1985

Observations on the suramin-mediated inhibition of cellular and viral DNA polymerases.

We have examined the sensitivity of various cellular and viral DNA polymerases to Suramin, an antitrypanosomal drug, which has been reported to exhibit antireverse transcriptase activity. We find that Suramin is a nonspecific inhibitor of all the viral and cellular DNA polymerases, including terminal deoxynucleotidyl transferase, and that the inhibition is most readily reversed by the addition of serum albumin. The drug appears to bind to all the enzyme proteins with no apparent selectivity. Binding of Suramin to enzyme has been found to result in the loss of both substrate and templateprimer binding abilities of various enzymes, confirming the nonspecific nature of protein-Suramin interaction.

DNA Nucleotidylexotransferase↗
PubMed1983

Mechanism of o-phenanthroline mediated inhibition of E. coli DNA polymerase I : formation of template-primer-metal-phenanthroline complexes with resultant loss of catalytic activity.

Inhibition of E. coli DNA polymerase I activity by 1,10 phenanthroline in the absence of reducing agents requires a high concentration of inhibitor (1-10 mM) depending upon the template primer used to direct the synthesis. We find that o-phenanthroline, unlike its non-chelating analogue, forms a divalent cation mediated complex with template-primers. Enzyme bound to such complexes is unable to catalyse either polymerization or nuclease functions.

Copper↗
PubMed1983

Biochemistry of terminal deoxynucleotidyltransferase: characterization and properties of photoaffinity labeling with 8-azidoadenosine 5'-triphosphate.

We have found that 8-azidoadenosine 5'-triphosphate (8-azido-ATP) and its photolyzed product are competitive inhibitors of terminal deoxynucleotidyltransferase with respect to substrate deoxynucleoside triphosphates. A detailed characterization of the inhibitory effect of 8-azido-ATP revealed that its mechanism of inhibition is identical with that reported for ATP [Modak, M. J. (1978) Biochemistry 17, 3116-3120]. Photoactivation of the azido-ATP-enzyme complex results in the covalent binding of azido-ATP to terminal deoxynucleotidyltransferase. No significant incorporation of prephotolyzed azido-ATP or unsubstituted ATP into enzyme protein is noted when complexes of these nucleotides with enzyme were exposed to identical photoactivation conditions. The majority of incorporated analogue was associated with the 26 000-dalton subunit of terminal deoxynucleotidyltransferase. Incorporation of azido-ATP was further found to be absolutely dependent on the presence of a divalent cation. All four deoxyribonucleoside triphosphates as well as ATP and guanosine 5'-triphosphate were able to compete with azido-ATP during the incorporation experiment as judged by the competitive reduction in the cross-linking of the photoaffinity analogue to terminal deoxynucleotidyltransferase (TDT). In addition, substrate binding site directed inhibitors, pyrophosphate and pyridoxal 5'-phosphate, effectively blocked the incorporation of azido-ATP into enzyme protein, while several other inhibitors of TDT catalysis, namely, ethylenediaminetetraacetic acid, alpha, alpha'-dipyridyl, 1,10-phenanthroline, p-(chloromercuri)benzoate, Rose Bengal, and the presence of 0.5 M KCl, influenced the cross-linking reaction to varying degrees. A tryptic peptide analysis of the azido-ATP-labeled 26K subunit of TDT revealed that the majority of the incorporated photoaffinity analogue was present in two peptides.

Adenosine Triphosphate↗
PubMed1983

The mechanism of inhibition of avian myeloblastosis virus reverse transcriptase by a dialdehyde derivative of ATP. Inactivation of essential sulfhydryl group function.

The dialdehyde derivative of ATP inhibits DNA synthesis by AMV reverse transcriptase, while the polymerase-associated ribonuclease H activity is significantly resistant to this reagent. Neither ATP nor its dialcohol form effectively block DNA synthesis, indicating that the aldehyde moiety is required for inhibition. The nature of the reactivity of dialdehyde-ATP with AMV reverse transcriptase has been examined and we find that: (a) inhibition is non-competitive with respect to substrate deoxynucleoside triphosphate concentration, suggesting that dialdehyde-ATP does not react at the substrate binding site; (b) pretreatment of enzyme with dialdehyde-ATP or sulfhydryl group binding reagents results in the complete loss of its template binding activity; however, treatment of preformed enzyme-template-primer complex with both inhibitors did not dissociate this complex; (c) the inhibitory effect of dialdehyde-ATP was completely reversed upon addition of reducing agents, such as dithiothreitol and sodium borohydride, indicating that dialdehyde-ATP reacts with the sulfhydryl groups present in AMV reverse transcriptase; (d) comparative studies carried out with the classical sulfhydryl reagent, dithiobisnitrobenzoic acid, revealed a remarkable similarity in its action to that of dialdehyde-ATP. We therefore conclude that the dialdehyde-ATP-mediated inhibition of AMV DNA polymerase is effected via blockage of essential sulfhydryl groups present in the enzyme protein.

Adenosine Triphosphate↗
PubMed1983

Rose Bengal mediated inhibition of DNA polymerases: mechanism of inhibition of avian myeloblastosis virus reverse transcriptase under photooxidative conditions.

DNA polymerases from prokaryotic, eukaryotic, and oncornaviral sources are irreversibly inactivated upon exposure to Rose Bengal in the presence of light (photooxidation conditions). Inactivation of these enzymes under dark (nonoxidative) conditions is totally reversible [Srivastava, S. K., & Modak, M. J. (1982) Biochemistry 21, 4633-4639]. The primary effect of photooxidation on the enzyme-Rose Bengal complex was found to be the loss of template-primer binding ability within 5 min of exposure to light. The presence of template-primer but not the substrate deoxynucleotides consistently provided partial protection against Rose Bengal mediated photooxidative inactivation. Preformed enzyme-template-primer complexes were not dissociated by Rose Bengal under these conditions. However, prolonged exposure (25-30 min) of this complex to light in the presence of Rose Bengal led to complete inactivation of catalytic activity without affecting the ability of enzyme to bind to template-primer. These results clearly indicate that oxidative inactivation of avian myeloblastosis virus reverse transcriptase in the presence of Rose Bengal involves a domain within the enzyme that contains the template-primer binding site as well as an additional site which is required for the expression of both the polymerization and nuclease activities of the reverse transcriptase.

Avian Leukosis Virus↗
PubMed1983

Synthesis of compositionally unique DNA by terminal deoxynucleotidyl transferase.

Studies on the composition and characterization of DNA product(s) synthesized by calf thymus terminal deoxynucleotidyl transferase were performed using homopolymeric single-stranded, calf thymus double-stranded, and native DNA resident in calf thymus chromatin preparations as priming DNA species. Synthesis was carried out using equimolar concentrations of all four deoxynucleoside triphosphates as substrates and Mg2+ or Mn2+ as an effective divalent cation. Irrespective of the nature of the priming DNA or the divalent cation, the DNA product contained 60-70% dGMP residues, 10-15% each of the two pyrimidine residues, and 5-10% dAMP residues. The product synthesized using chromatin DNA as initiator was predominantly single-stranded and its synthesis was resistant to actinomycin D. The predilection of terminal deoxynucleotidyl transferase to synthesize dGMP-rich products on natural or homopolymeric DNA primers suggests that such products may represent biologically important recognition signal sequences.

Animals↗
PubMed1983

Adenosine 5'-triphosphate (ATP)-mediated stimulation and suppression of DNA synthesis in lymphoid cells. II. Suppressive effect of ATP on murine T-cell functions.

The suppressive effects of ATP on murine T-cell functions were studied. The suppressive effects of ATP as well as adenosine on the DNA synthesis of spleen cells are due to the presence of mature T-cells, because ATP has no suppressive effect on athymic nu/nu spleen cells. Further characterization of the cells which are responsible for ATP-mediated suppression of DNA synthesis revealed that the cells are nylon wool-adherent T-cells and PHA-reactive T-cells. In addition, the suppressive effects of ATP on both spontaneous and mitogen-induced proliferative responses are stronger than that of adenosine, and T-cells are more sensitive to ATP than B-cells. The observation that both ATP and adenosine have unique effects on T-cells compared to B-cells may contribute toward explaining why patients with severe combined immunodeficiency (SCID) associated with adenosine deaminase (ADA) deficiency have greater T-cell than B-cell abnormalities.

Adenosine↗
PubMed1982

Biochemistry of terminal deoxynucleotidyl transferase. Conditions for and characterization of ultraviolet light mediated substrate cross-linking to terminal deoxynucleotidyl transferase.

Calf thymus terminal deoxynucleotidyl transferase may be cross-linked to substrate deoxynucleoside triphosphate as well as ATP in a reaction mediated by ultraviolet irradiation. The cross-linking requires the presence of divalent cation but is independent of primer. The cross-linking of substrate to enzyme is extremely sensitive to substrate binding site directed reagents, e.g. pyrophosphate and pyridoxal 5'-phosphate, and to o-phenanthroline, a zinc-directed chelator. The cross-linking reaction is competitively inhibited by all four deoxynucleoside triphosphates, ATP, and GTP, but is resistant to sulfhydryl reagents. When primer is provided in the reaction mixture, cross-linking of both substrate and primer to 26K subunit of terminal deoxynucleotidyl transferase may be demonstrated.

Animals↗
PubMed1981

Divalent cation-dependent pyridoxal 5'-phosphate inhibition of Rauscher leukemia virus DNA polymerase: characterization and mechanism of action.

We have shown that pyridoxal 5'-phosphate is an effective inhibitor of Rauscher leukemia virus DNA polymerase (Biochemistry 15 (1976) 3620). Detailed studies of this inhibition revealed that, in addition to the phosphate and aldehyde groups of pyridoxal phosphate, the presence of a divalent cation is essential for the inhibitory action. The synthesis directed by template primers containing GC base-pairs exhibited more resistance to pyridoxal phosphate inhibition than did that directed by AT base-paired templates. Maximal inhibitory activity of pyridoxal phosphate, however, is noted in the presence of Mn2+, irrespective of which template-primer is used to direct the DNA synthesis. The action of pyridoxal phosphate on the substrate binding site may be deduced from the observations that: (a) only the substrate triphosphate is able to reverse the pyridoxal phosphate-mediated inhibition; (b) the inhibition kinetics exhibit a classical competitive pattern with the substrate; (c) analogous to substrate deoxynucleoside triphosphates the inhibitor is also accepted only in the form of its divalent metal ion complex; and (d) substrate site-specific labeling of RLV DNA polymerase has been shown to occur by linking covalently the pyridoxal phosphate bound to a lysine residue at the substrate binding site.

Binding Sites↗
PubMed1981

Adenosine-5'-triphosphate-(ATP) mediated stimulation and suppression of DNA synthesis in lymphoid cells. I. Characterization of ATP responsive cells in mouse lymphoid organs.

The effects of various nucleotides and nucleosides on DNA synthesis of mouse lymphocyte populations were studied. Significant stimulation of DNA synthesis was observed in the cells from bone marrow or the thymus in the presence of ATP. In contrast, the DNA synthesis of the cells from spleen, lymph nodes, and peripheral blood was markedly inhibited by ATP. Guanosine 5'-triphosphate had nonspecific stimulatory effects on the DNA synthesis of various lymphoid cells, whereas cytidine triphosphate had no effect. When thymocytes or bone marrow cells were separated by 1 x g velocity sedimentation, a distinct cell population was identified as being responsible for the ATP-mediated stimulation of DNA synthesis. Further characterization of ATP-responsive cells revealed that the highest concentration of terminal deoxynucleotidyl transferase, a marker enzyme for precursor T cells, was present in these cells. ATP-mediated stimulation of DNA synthesis may, therefore, serve as a specific marker restricted to a certain population of differentiating T cells.

Adenine↗