Purification and some properties of 2', 3'-cyclic phosphodiesterase from the cell-free extract of Bacillus subtilis var. amyloliquefacus.

Abstract

A ribonucleoside 2',3'-cyclic phosphodiesterase (cyclic phosphodiesterase) having 3'-nucleotidase activity was purified from the cell-free extract of Bacillus subtilis var. amyloliquefacus, and its enzymatic properties were examined. The molecular weight was approximately 74,000. The enzyme was highly specific for ribonucleoside 2',3'-cyclic monophosphate (2',3'-cyclic mononucleotide), and ribonucleoside 3'-monophosphate (3'-mononucleotide), and also hydrolyzed bis p-nitrophenyl phosphate (bis p-NPP) but at a rate much slower than that of 3'-mononucleotide. Both cyclic phosphodiesterase and 3'-nucleotidase activities had an optimum pH of about 6.7, and phosphodiesterase activity against bis p-NPP indicated a relatively sharp pH optimum at 5.0. Only phosphodiesterase activity against bis p-NPP was greatly activated by Co2~, but both cyclic phosphodiesterase and 3'-nucleotidase activities were inhibited. However, Co2+ had an effect of protecting the enzyme against heat inactivation. 3'-Nucleotidase activity was not affected by EDTA, while phosphodiesterase activities against both 2',3'-cyclic mononucleotide and bis p-NPP were greatly inhibited. The enzyme hydrolyzed 3'-AMP with Km 0.046 mM and Vmax 1,585 µmol per hr per mg protein, bisp-NPP with Km 0.16 mM and Vn,a. 233 ,umol per hr per mg protein.In the course of studies on the physiological changes occurring after infection of Bacillus subtilis var. amyloliquefacus with phage, we became aware that a phosphodiesterase activity hydrolyzing bis p-nitrophenyl phosphate (bis p-NPP) under the acidic pH region (an optimal pH of about 5.0) exists in the phage uninfected cell-free extract, and that this activity did not change after infection of host cell with phage.In a preliminary experiment, attempts were made to characterize the substrate specificity using the enzyme partially purified by DEAF-cellulose chromatography. 487

Enzyme genomics: Application of general enzymatic screens to discover new enzymes

“…These small chromogenic substrates present convenient alternatives to complicated and laborious protocols that employ natural substrates. Both bis-pNPP and pNP-TMP have been applied successfully to characterize various phosphodiesterases and nucleases from both prokaryotes and eukaryotes [54][55][56][57]. Phosphodiesterases show high to significant affinity for both substrates (K m = 0.25-14.4 mM for bis-pNPP and 0.06-6 mM for pNP-TMP) within a broad pH range 7-10 (BRENDA database).…”

Enzyme genomics: Application of general enzymatic screens to discover new enzymes

“…These small chromogenic substrates present convenient alternatives to complicated and laborious protocols that employ natural substrates. Both bis‐ p NPP and p NP‐TMP have been applied successfully to characterize various phosphodiesterases and nucleases from both prokaryotes and eukaryotes [54–57]. Phosphodiesterases show high to significant affinity for both substrates ( K m = 0.25–14.4 mM for bis‐ p NPP and 0.06–6 mM for p NP‐TMP) within a broad pH range 7–10 (BRENDA database).…”