Cellular distribution and some properties of 5'-nucleotidases in Bacillus subtilis K

• Published in: Journal of biochemistry (Tokyo) 1978-02, Vol.83 (2), p.409-421
• Main Authors: SHIIO, Isamu, OZAKI, Hachiro
• Format: Article
• Language: English
• Subjects: Bacillus subtilis - enzymology; Cations, Divalent - pharmacology; Hydrogen-Ion Concentration; Kinetics; Molecular Weight; Nucleotidases - isolation & purification; Nucleotidases - metabolism; Phosphates - metabolism; Spheroplasts - enzymology; Subcellular Fractions - enzymology; Substrate Specificity
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/oxfordjournals.jbchem.a131928

The distribution of 5′-nucleotidases of Bacillus subtilis K No. 231, an inosine-producing bacterium, was investigated. It was found that this organism has an extracellular 5′-nucleotidase as well as various cellular 5′-nucleotidases, which include a major one bound to the cell wall and minor ones in the cytoplasm and on the cytoplasmic membrane. The cell wallbound 5′-nucleotidase was specifically solubilized by treatment of the sonic cell debris with lysozyme, but not by treatment with various other hydrolyzing enzymes, detergents or high concentrations of inorganic salts. The extracellular enzyme was more repressible by inorganic phosphate than the cellular ones. Among the three cellular 5′-nucleotidase the cytoplasmic enzyme was the most repressible by inorganic phosphate. The crude preparations of the extracellular, cell wall-bound and cytoplasmic 5′-nucleotidase showed single peaks of activity on gel filtration, at the elution volumes corresponding to molecular weights of 67,000, 900,000 and 600,000, respectively. Upon DEAE-cellulose column chromatography of the peak fractions of the three preparations, two peaks (E1 and E2), three peaks (W1, W2, and W3) and two peaks (C1 and C2) were obtained, respectively. The molecular weights of E1, E2, W1, W2, W3, C1, and C2 were determined to be 27,000, 46,000, 37,000, 21,000, 640,000, 38,000, and 260,000, respectively. All seven enzymes showed optimum pH between 7.0 and 8.0, and specifically hydrolyzed the phosphomonoester bonds of purine nucleoside-5′-monophosphates but not those of 5′-pyrimidine nucleotides. The cellular enzymes showed slight activity toward UDP-glucose whereas the extracellular enzymes did not. None of the enzymes hydrolyzed 4-nitrophenylphosphate or bis(4-nitrophenyl)phosphate. All the enzymes showed the same Km values, 1.5–1.9 μM for AMP. High concentrations (0.1M and 1M) of NaCI, KCI, and (NH4)2SO4activated the cellular 5′-nucleotidases more strongly than the extracellular enzymes. Mg2+ and Mn2+ stimulated the activities of the cytoplasmic enzymes. C1 and C2, specifically. All the enzymes were inhibited by Zn2+, Cu2+, ATP, and PO42−. The PO42− inhibition was of mixed type with respect to AMP, and the inhibitor constant for PO42− was almost the same, 0.4–1.1 mM, for all the enzymes.