Enzymatic synthesis of 5-azacytidine 5'-triphosphate from 5-azacytidine.

5-Azacytidine 5′-monophosphate (5-aza-CMP) was synthesized enzymatically from 5-azacytidine (5-aza-C) in a reaction catalyzed by uridine-cytidine kinase. In a second step, 5-azacytidine 5′-triphosphate (5-aza-CTP) was synthesized enzymatically from 5-aza-CMP using CMP kinase and nucleoside diphosphokinase. Due to the chemical instability of the triazide ring of 5-azacytosine at neutral and alkaline pH, the enzymatic synthesis and purification of the nucleotides by ion exchange chromatography were performed at acid pH. The enzymatically synthesized 5-aza-CTP had an ultraviolet absorbance spectrum at pH 5.5 similar to the spectrum of 5-aza-C. In the DNA-dependent RNA polymerase reaction, 5-aza-CTP inhibited the incorporation of [³H]CTP, but [³H]UTP, into RNA.     

Enzymatic activities for interconversion of purines in spirochetes.

Enzymatic activities that catalyze the interconversion of purines and purine derivatives were detected in cell extracts of Spirochaeta aurantia, Spirochaeta stenostrepta, Treponema succinifaciens, and Treponema denticola. Phosphoribosyltransferase activities present in cell extracts of each of the four spirochete species functioned in the conversion of adenine, hypoxanthine, and guanine to AMP, IMP, and GMP, respectively. Nucleotidase activities in the extracts mediated the formation of nucleosides from nucleotides. The conversion of adenosine, inosine, and guanosine to the respective purine bases was catalyzed by nucleoside phosphorylase and, in some instances, by nucleoside hydrolase activities. Guanine deaminase activity was found in both S. aurantia and S. stenostrepta, whereas adenosine deaminase activity was detected only in S. aurantia. Adenine deaminase activity in T. succinifaciens extracts was sensitive to O2 and was relatively resistant to heating. Our results indicate that the four species of spirochetes studied possess a broad spectrum of purine interconversion enzymes. It is suggested that these enzymes may function in metabolic processes important for the survival of spirochetes in nutrient-poor natural environments.     

Enzymatic synthesis of uniformly 32P-labeled polyribonucleotides and high-specific-activity ribonucleoside 5'-[alpha-32P]diphosphates

Uniformly 32P-labeled polyribonucleotides of high specific activity can be rapidly and easily synthesized from commercially available ribonucleoside 5'-[alpha-32P]triphosphates by using two enzymes in sequence. Myosin ATPase completely and irreversibly converted any triphosphates to diphosphates in 10 min. The product diphosphates, without purification, can be polymerized by polynucleotide phosphorylase (PNPase) in 1 h with an average yield of 60%. By choosing the desired molar ratio of radioactive and nonradioactive tri- or diphosphates, polymers of a wide range of specific activity can be obtained. Since myosin ATPase and PNPase both have little base specificity, the method can be used to synthesize a radiolabeled polymer of any desired base composition.