Production of a monoclonal antibody against the hRRM2 subunit of ribonucleotide reductase and immunohistochemistry study of human cancer tissues.

We report production of a monoclonal antibody against the hRRM2 subunit of ribonucleotide reductase and immunohistochemistry (IHC) staining of human cancer tissues available in paraffin block. BALB/c mice were immunized with purified hRRM2 protein, and splenocytes from these mice were fused with mice myeloma cell lines by using standard hybridoma production techniques. Resulting hybridomas producing anti-hRRM2 antibodies were screened by enzyme-linked immunosorbent assay (ELISA). The specificity was determined by limiting serial dilutions. Clones were chosen for antibody production based on their activities on paraffin-embedded human tissues. They were then isotyped and shown to produce immunoglobulin M (IgM) antibodies against hRRM2. Using these antibodies, we performed Western blot on oropharyngeal KB cancer cell lines and immunohistochemistry staining of available paraffin-embedded cancer tissues. Interestingly, cancer tissues stained positive with the anti-hRRM2 antibody but not normal tissues. Colon, stomach, liver, lung, pancreatic, and breast cancer had the strongest staining. No staining was identified on astrocytoma, mesothelioma, or myeloma. Our findings were validated with data from reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrating overexpression of hRRM2 in breast cancer tissues compared to matched noncancer tissues. We propose that IHC with this monoclonal anti-hRRM2 antibody may be useful for ribonucleotide reductase research and as a biomarker for tumorgenesis.

An improved method for detection of Leishmania amastigotes by an antibody probe against the small subunit of leishmanial ribonucleotide reductase.

By taking advantage of an antibody raised against the small M2 subunit of ribonucleotide reductase of Leishmania that reacts with the enzyme in the nucleus of the parasite but does not cross-react with the same enzyme of the host macrophage, an improved fluorescence-staining method is developed for enumeration of leishmanial amastigotes inside the macrophage. The method offers an accurate and easy way of counting, compared with Giemsa staining.

Glutathione-dependent hydrogen donor system for calf thymus ribonucleoside-diphosphate reductase.

Purified calf thymus ribonucleoside-diphosphate reductase (2'-deoxyribonucleoside-diphosphate:oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1), showed an absolute requirement for a dithiol as hydrogen donor, whereas the natural monothiol glutathione (GSH) was inactive per se. However, a protein partially purified from thymus coupled the oxidation of GSH to the formation of deoxyribonucleotides by ribonucleotide reductase. In analogy with the ribonucleotide reductase system of Escherichia coli this protein was called glutaredoxin [Holmgren, A. (1976) Proc. Natl. Acad. Sci. USA 73, 2275-2279]. Thymus glutaredoxin had the following properties: (i) its molecular weight determined by gel chromatography was about 12,000; (ii) it was active iwth ribonucleotide reductase in the presence of GSH, NADPH, and glutathione reductase but had no activity with NADPH and thioredoxin reductase; and (iii) it was immunologically different from thioredoxin because it did not bind to antithioredoxin immunoadsorbents. Experiments on the crossreactivity of thymus and E. coli ribonucleotide reductases and the corresponding thioredoxin and glutaredoxin systems showed essentially no specificity for the homologous thioredoxin but a high species specificity for the homologous glutaredoxin.

Ribonucleoside diphosphate reductase induced by bacteriophage T4. III. Isolation and characterization of proteins B1 and B2.

Ribonucleoside diphosphate reductase determined by bacteriophage T4 consists of a tight complex (alpha2beta2) of the polypeptide chains alpha (Mr = 80,000 to 85,000) and beta (Mr = 35,000). The alpha2 dimer (= protein B1) was purified from Escherichia coli B infected with T4 mutant nrdB55 (Yeh, Y.C., and Tessman, I. (1972) Virology 47, 767-772) which carries an amber mutation in the gene coding for the beta polypeptide chain. Protein B1 contained binding sites for dATP, an allosteric effector of the reductase. The beta2 dimer (= protein B2) was purified by selective desorption with 1 M guanidine HCl from a dATP-Sepharose affinity column containing adsorbed native T4 ribonucleotide reductase. Protein B2, isolated this way, was enzymatically inactive due to partial loss of its iron but it could be reactivated by treatment with ferrous iron. Active protein B2 contained two atoms of non-heme iron per molecule and exhibited the optical and electron spin resonance spectra previously demonstrated in the native enzyme. The T4-induced proteins B1 and B2 were unable to reduce ribonucleotides when assayed separately but were active in combination. The proteins did not form catalytically functional hybrids with proteins B1 and B2 of Escherichia coli ribonucleotide reductase, neither did they cross-react immunologically with the latter. 5-Hydroxymethyl-dCTP, at concentrations above 10 muM, was a positive allosteric effector of T4 ribonucleotide reductase promoting the reduction of the pyrimidine ribonucleotides CDP and UDP. The nucleotide had little effect on E. coli ribonucleotide reductase.