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D W Martin

Publications and source records attributed to D W Martin.

At least 73 records · Page 4Linked to original sources
PubMed1984

Human purine nucleoside phosphorylase cDNA sequence and genomic clone characterization.

The isolation of a cDNA clone containing the complete coding region for human purine nucleoside phosphorylase (PNP) has been described previously. In this report we present the nucleotide sequence of this cDNA clone and compare the derived amino acid sequence, encoding a protein of 32 kilodaltons, with the published amino acid composition. Using a fragment of the cDNA clone as a probe, human PNP genomic clones from a bacteriophage lambda library have been isolated and the structural organization of the wild type PNP gene determined.

Base Sequence↗
PubMed1984

Cloning of human adenosine deaminase cDNA and expression in mouse cells.

A previously isolated partial cDNA sequence encoding human adenosine deaminase (ADA) was used to probe a cDNA library prepared from human cultured cell mRNA. Clones containing a combined overlapping length of 1462 bp were isolated and sequenced. One of these was found to include the entire ADA coding region. An open reading frame consisting of 363 codons was identified, predicting a polypeptide of Mr 40762. A mammalian expression plasmid was constructed, positioning the ADA coding sequence to be under transcriptional control of the mouse metallothionein promoter. Transfection of cultured mouse L-cells with this plasmid resulted in the acute expression of human ADA enzymatic activity, as assayed by isoelectric focusing.

Adenosine Deaminase↗
PubMed1984

Monomeric solubilized sarcoplasmic reticulum Ca pump protein: demonstration of Ca binding and dissociation coupled to ATP hydrolysis.

The sarcoplasmic reticulum Ca-pump protein can be solubilized in monomeric form in nonionic detergents with full retention of ATPase activity. It is impossible to prove directly that coupling between ATP hydrolysis and Ca2+ transport is maintained in the soluble state, because separate compartments for Ca2+ uptake and Ca2+ discharge are required to demonstrate this, but here we provide strong indirect evidence that coupling in fact persists, in both the forward and reverse directions of the normal pump cycle. Demonstration of coupling in the forward direction makes use of the fact that the solubilized protein is structurally labile in the absence of bound Ca2+. Loss of activity accompanying ATP hydrolysis in solution is quantitatively consistent with sequential Ca2+ binding and dissociation during the hydrolysis cycle. Coupling in the reverse direction is demonstrated by the dependence of ATP synthesis on Ca2+ concentration. Although substantial differences between solubilized (monomeric) and membrane-bound protein are shown to exist, as previously reported, they do not affect the main conclusion from this work, which is that the molecular machinery for free-energy coupling in active Ca2+ transport is an inherent property of each individual catalytic polypeptide chain of the pump protein.

Adenosine Triphosphate↗
PubMed1984

Specific cytotoxicity of arabinosylguanine toward cultured T lymphoblasts.

Purine nucleoside phosphorylase (PNP) deficiency in humans is associated with a severe T cell immunodeficiency. To understand further and exploit this T cell lymphospecificity, we have compared the cytotoxicities and metabolism of deoxyguanosine, the cytotoxic substrate of PNP and of arabinosylguanine, a deoxyguanosine analogue that is resistant to PNP cleavage, in T cell (8402) and B cell (8392) lines in continuous culture established from the same patient. In comparative growth rate experiments the T cells were 2.3-fold and 400-fold more sensitive to growth inhibition by deoxyguanosine and arabinosylguanine, respectively, than were the B cells. Only the T cells, but not the B cells, could phosphorylate in situ deoxyguanosine or arabinosylguanine to the corresponding triphosphate. Both the phosphorylation and cytotoxicity of arabinosylguanine in the T cell line could be prevented by deoxycytidine, which suggests that deoxycytidine-deoxyguanosine kinase initiated the intracellular metabolism and cytotoxicity of this nucleoside analogue. The sensitivity and selectivity of arabinosylguanine toward the T lymphoblastoid cells suggests a rational approach to the design of chemotherapeutic agents that are directed toward T cell malignancies and other T cell disorders.

Arabinonucleosides↗
PubMed1984

Injectable collagen in laryngeal rehabilitation.

Glottic insufficiency has been successfully managed by Teflon augmentation of paretic vocal cords over the past two decades. Collagen fulfills the criteria for a suitable bioimplant and is an attractive alternative as it is a normal constituent of the extracellular protein matrix. This study explores the use of modified bovine collagen injected into the canine larynx. Clinical situations are simulated by creating lesions and injecting collagen at different sites. The technique permits precise placement and quantification. Histologic study of the collagen implant fails to show evidence of significant foreign body reaction. The injected collagen appears similar to viable connective tissue and is colonized by host fibroblasts and nourishing capillaries.

Animals↗
PubMed1983

Active unit of solubilized sarcoplasmic reticulum calcium adenosinetriphosphatase: an active enzyme centrifugation analysis.

Sarcoplasmic reticulum calcium adenosinetriphosphatase (Ca2+-ATPase) was solubilized to monomeric form with the nonionic detergent n-dodecyl octaethylene glycol monoether (C12E8). Equilibrium ultracentrifugation analysis indicated that this preparation is initially greater than 75% monomer, the remainder being best described as a tetramer. In the presence of substrates, this preparation has ATPase activity comparable to that of leaky sarcoplasmic reticulum vesicles. The possibility of substrate-induced oligomerization of the monomer under ATPase activity assay conditions was tested. Active enzyme centrifugation analysis demonstrated that ATPase activity sedimented with a rate which can only be attributed to a monomeric particle. The sedimentation rate was invariant over a 6-fold concentration range comparable to that used in activity assays. The portion of the protein that sediments as an oligomer when measurements are based on the movement of protein (A280) is not seen when measurements are based on the movement of activity. The data demonstrate that the monomer represents the minimal ATPase active unit of Ca2+-ATPase.

Animals↗
PubMed1983

Direct photoaffinity labeling of the catalytic site of mouse ribonucleotide reductase by CDP.

Ribonucleotide reductase reduces all four ribonucleoside diphosphates to the deoxyribonucleotides required for DNA synthesis. The enzyme is composed of two nonidentical subunits, M1 and M2. The 89-kilodalton M1 subunit contains at least two allosteric sites which, by binding nucleotide effectors, regulate the catalytic activity and substrate specificity of the enzyme. We now show that in addition, protein M1 contains a substrate-binding (catalytic) site which is specifically photolabeled after UV irradiation in the presence of the natural substrate, [32P]CDP. The photolabeling of protein M1 by [32P]CDP required the presence of the second subunit, protein M2, and ATP, the positive allosteric effector for CDP reduction. The negative effectors, dATP, dGTP, and dTTP, inhibited the photolabeling of wild type protein M1. Deoxy-ATP did not inhibit the labeling of a mutant protein M1 that is resistant to feedback inhibition by dATP. In addition, hydroxyurea and 4-methyl-5-aminoisoquinoline thiosemicarbazone, two inhibitors of ribonucleotide reductase which affect protein M2, also inhibited the [32P]CDP labeling of protein M1. These data provide new insights into the role and interaction of the two ribonucleotide reductase subunits, proteins M1 and M2, and the mechanism of action of the allosteric effectors.

Affinity Labels↗
PubMed1983

Cloning of human purine-nucleoside phosphorylase cDNA sequences by complementation in Escherichia coli.

We have obtained cDNA clones that contain the entire coding region of the human purine-nucleoside phosphorylase (PNP; EC 2.4.2.1) mRNA. The cDNA sequences were generated by reverse transcription of PNP-enriched mRNA obtained by immunoadsorption of HeLa cell polyribosomes with monospecific antibody to human PNP. cDNA molecules that were close in length to PNP mRNA were separated by agarose gel electrophoresis and inserted into the Pst I site of the plasmid pBR322. Plasmid DNA from the pooled clones was used to transform PNP-deficient Escherichia coli cells, and those transformants that phenotypically expressed PNP were isolated on selective media. The presence of human PNP in the selected bacterial cells was detected by immunoprecipitation with human PNP antibody.

Base Sequence↗
PubMed1982

Direct photoaffinity labeling of an allosteric site of subunit protein M1 of mouse ribonucleotide reductase by dATP. Evidence for two independent binding interactions within the allosteric specificity site.

The M1 subunit of ribonucleotide reductase contains two kinds of allosteric sites, the activity site and the specificity site, which regulate the overall catalytic activity and the substrate specificity of the enzyme, respectively. The effector nucleotides, dGTP and dTTP, bind only to the specificity site; dATP and ATP bind to both sites. Partially purified protein M1 was photolabeled specifically after UV irradiation in the presence of [32P]dATP. The labeling occurred exclusively at the allosteric specificity site as evidenced by 1) total inhibition of the labeling by dGTP and dTTP, 2) normal photoincorporation of [32P]dATP by mutant protein M1 molecules that lack a functional activity site, and 3) coidentity of one-dimensional peptide maps of protein M1 labeled with either [32P]dATP or [32P]dTTP. A mutant protein M1 that is resistant to normal regulation by dGTP and dTTP (indicating an alteration in the allosteric specificity site) showed normal photoincorporation of [32P]dATP (but not [32P]dTTP). This labeling was not inhibited by dGTP or dTTP. Our data suggest that this mutation has altered the binding of dGTP and dTTP but not dATP (or ATP) at the specificity site. Thus, by the combination of genetic and photolabeling techniques, two independent nucleotide binding interactions occurring within this one complex regulatory domain can be distinguished.

Affinity Labels↗
PubMed1982

Presence of a DNA demethylating activity in the nucleus of murine erythroleukemic cells.

Nucleoplasm prepared from murine erythroleukemia cells was assayed from enzymatic activity which removes methyl groups from DNA methylated at the internal C of the sequence 5'-CCGG-3' (Hpa II sites). Under the conditions of assay, the release of methyl groups was linear with time for 1 h, and up to 2 pmol could be released per microgram of extract protein. The activity was proportional to the amount of added extract and was sensitive to heat ant to proteinase K exposure. The initial velocity was hyperbolic with increasing amounts of substrate, and the Km for methyl groups was estimated to be between 1 and 1.5 microM. These results show that DNA demethylation catalyzed by extracts from cell nuclei can occur in vitro where no DNA synthesis is occurring and suggest that such a process might occur in vivo to alter DNA methylation during differentiation.

Animals↗
PubMed1982

Phosphorylation of calcium adenosinetriphosphatase by inorganic phosphate: reversible inhibition at high magnesium ion concentrations.

Magnesium stimulates phosphorylation of the calcium pump protein of the sarcoplasmic reticulum by inorganic phosphate, but the effect is reversed by high [Mg2+]. This reversal is readily explained in terms of the generally accepted existence of two conformational states of the enzyme, E1 and E2. E2 is the form of the enzyme that can be phosphorylated by Pi, and it has one binding site for Mg2+. E1 is the form of the enzyme that has two high-affinity Ca2+ binding sites, and it is phosphorylated by ATP when Ca2+ is bound. Mg2+ can bind weakly to the two Ca2+ sites and to a third site known to be present on E1; this stabilizes E1 at the expense of E2 when [Mg2+] is large. Stabilization of E1 at pH 6.2 and 25 degrees C was found to be a highly cooperative function of [Mg2+] and was not prevented by increasing [Pi]. The latter result requires the existence of a binding site for Pi on E1, with an affinity for Pi comparable to that of E2. Cooperativity with respect to [Mg2+] requires that E2 is the stable state of the enzyme in the absence of ligands, with an equilibrium constant [E2]/[E1] on the order of 10(3) or higher at pH 6.2 and 25 degrees C.

Binding Sites↗
PubMed1982

Direct photoaffinity labeling of an allosteric site on subunit protein M1 of mouse ribonucleotide reductase by dTTP.

The protein M1 subunit of ribonucleotide reductase contains at least two allosteric nucleotide binding sites that control the capacity of the enzyme to reduce ribonucleotides to the deoxyribonucleotides required for DNA synthesis. Direct photoaffinity labeling of partially purified protein M1 from mouse T-lymphoma (S49) cells was observed after UV irradiation in the presence of dTTP at 0 degrees C. The relative molar incorporation of nucleotide per subunit was 4-8%. Competition experiments showed that the dTTP was bound to an allosteric domain genetically and kinetically defined as the substrate specificity site of the enzyme. An altered protein M1 isolated from a thymidine-resistant mutant cell line showed significantly decreased photoincorporation of dTTP, consistent with the fact that its CDP reductase activity is resistant to feedback inhibition by dTTP. Specific photolabeling of several other proteins with pyrimidine and purine nucleotides was also found, indicating the general usefulness of direct photoaffinity labeling in the study of enzymes involved in nucleotide and nucleic acid metabolism.

Affinity Labels↗