Purification and partial characterization of Ca(2+)-dependent ribonucleotide reductase from Streptomyces aureofaciens.
Ribonucleotide reductase (EC 1. 17. 4. 1.) is an essential enzyme providing 2'-deoxy-ribonucleotides for DNA replication. Ribonucleotide reductase from Streptomyces aureofaciens was purified 3365-fold with a yield of 6.5%. After homogenization of cells by ultrasonic homogenizer and DNA removing by 7% (w/v) solution of streptomycin sulphate, the sample was chromatographed on a DEAE-Sepharose CL 6 B, Phenyl-Sepharose CL 4 B, Heparin-Sepharose CL 6 B and a Sephacryl S-200. The specific activity of the purified protein was 1740 pmol per s per mg. Sephacryl S-200 chromatography and sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed that in the presence of calcium ions the enzyme appears to be a dimer with an apparent molecular weight of 125.9 kDa. In the absence of calcium dimer dissociates into a monomer with the apparent molecular weight of 64.3 kDa. On the basis of these results, we suggest that calcium plays a role in the formation of the dimer, which is the biologically active form of ribonucleotide reductase.
Racay
P
P
Department of Biochemistry, Faculty of Natural Sciences, Comenius University, Bratislava, Slovak Republic.
Kollárová
M
M
D002118:Calcium / Q000494:pharmacology*; D004800:Enzymes, Immobilized; D008970:Molecular Weight; D012264:Ribonucleotide Reductases / Q000302:isolation & purification*; D013304:Streptomyces aureofaciens / Q000201:enzymology*
D004800:Enzymes, Immobilized; D012264:Ribonucleotide Reductases; D002118:Calcium