[Aminoglycoside-3'-phosphotransferase from Actinomyces fradiae. Its isolation, purification and properties]. / Aminoglikozid-3'-fosfotransferaza iz Actinomyces fradiae. Vydelenie, ochistka i svoistva.

Aminoglycoside phosphotransferase was isolated from the mycelium of Act. fradiae, the neomycin-producing organism, with paromomycin, neomycin and to a less extent ribostamycin being substrates of aminoglycoside-phosphotransferase. It was purified to homogenous state. The maximum activity of the enzyme preparations was observed at pH 7.7--7.8;KM for neomycin and paromomycin was about 20 micron and KM for ATP was 150 micron. Mg2+ ions were necessary for the enzyme activity. None of the divalent cations tested could replace the magnesium ions in the reaction of phosphorylation catalyzed by the enzyme. High sensitivity to the ionic strength of the buffer was characteristic of the enzyme. It lost about 80 per cent of the initial activity at a concentration of KC1 equal to 1.0 M. The molecular mass of the enzyme from the mycelium of Act. fradiae was determined by the method of gel-filtration through sefadex G-100. It was about 22,000. High stability was characteristic of the enzyme. The fingings indicate that aminoglycoside phosphotransferase from Act. fradiae differs from the described aminoglycoside-3'-phosphotransferases isolated from antibiotic resistant bacteria.

[Aminoglycoside-3'-phosphotransferase from Actinomyces fradiae. The identification of its inactivation product]. / Aminoglikozid-3'-fosfotransferaza iz Actinomyces fradiae. Identifikatsiia produkta inaktivatsii.

Neomycin phosphate was obtained as a result of neomycin phosphorylation with aminoglycoside-phosphotransferase from Act. fradiae. It was isolated from the reaction mixture and purified. Successive ion exchange chromatography on columns with Amberlite IRC-50 (NH+4 form), Dowex 1 X 10 (OH- form) and Amberlite CG-50 (NH+4 form) was used for purification of the inactivation product. The findings of the elementary analysis of neomycin phosphate showed the presence of 1 mole of phosphorus per 1 mole of the antibiotic. From the results of the chemical analysis, IR- and NMR-spectrometry neomycin phosphate and neamine phosphate obtained from it by methanolysis were identified as neomycin-3'-phosphate and neamine-3'-phosphate, respectively. The data indicate that the enzyme isolated from Act. fradiae is aminoglycoside-3'-phosphotransferase.

[Isolation and substrate specificity of neomycin (paromomycin)--phosphotransferase from Actinomyces fradiae, a producer of neomycin]. / Vydelenie i substratnaia spetsifichnost' neomitsin (paromomitsin)--fosfotransferazy iz Actinomyces fradiae--produtsenta neomitsina.

Neomycin (paromomycin) phosphotransferase was isolated from the mycelium and fermentation broth filtrates of Act. fradiae. The substance was partially purified by means of fractionation with ammonium sulphate followed by gel-filtration through Sefadex G-100. The extracellular and intracellular forms of the enzyme had the same substrate specificity and used only neomycin and paromomycin as substrates. The other aminoglycosides, including kanamycins A and B, lividomycin and ribostamycin were not used. The both forms had the same thermolability. The intracellular form of the enzyme was detected in the mycelium at the early stages of the organism development, while the extracellular form was found in detectable amounts in the culture medium only at the late stages of the actinomycete development. Therefore, the neomycin-producing organism, i.e. Act. fradiae had one enzyme which phosphorilated neomycin and paromomycin and was excreted from the mycellium into the culture medium during the fermentation process.