Evidence of nucleotidyl phosphatase activity associated with core protein sigma A of avian reovirus S1133.
Both avian reovirus core protein sigma A purified from virus-infected cell extracts and the purified bacterially expressed protein sigma A (e sigma A) were characterized for their nucleoside triphosphate (NTP) hydrolysis activity by thin-layer chromotography. Protein sigma A from both preparations has a nonspecific nucleotidyl phosphatase activity that hydrolyzes four types of NTP to their corresponding nucleoside di- and monophosphates and free phosphate. The divalent cation requirement for this activity of e sigma A was further examined by the addition of Mn(2+), Mg(2+), Ca(2+), and Zn(2+) ions. NTP hydrolysis by e sigma A was maximal when Mn(2+), Mg(2+), or Ca(2+) concentrations were 5, 4, or 1 mM, respectively. Addition of Mn(2+) or Mg(2+) stimulated the reactions up to 4- or 3-fold, respectively, higher than Ca(2+) (2.2-fold). However, Zn(2+) ion inhibited this activity of e sigma A. The results suggest that nucleotidyl phosphatase activity of e sigma A is absolutely dependent on the divalent cations Mn(2+), Mg(2+), or Ca(2+), but not Zn(2+). Similar results were obtained from the analysis of divalent cation requirements for the protein sigma A nucleotidyl phosphatase activity. Optimal pH for nucleotidyl phosphatase activity of protein sigma A from both preparations was determined using reaction mixtures buffered at different pH. The results show that the optimal activities of both proteins were similar and were achieved between pH 7.5 and 8.5.
Yin
Hsien Sheng
HS
Department of Veterinary Medicine, National Chung Hsing University, Taichung 403, Taiwan.
Su
Yu Pin
YP
Lee
Long Huw
LH
D017766:Acid Anhydride Hydrolases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002413:Cations, Divalent; D002478:Cells, Cultured; D002645:Chickens; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis / Q000187:drug effects; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D043583:Nucleoside-Triphosphatase; D030101:Orthoreovirus, Avian / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014758:Viral Core Proteins / Q000302:isolation & purification / Q000378:metabolism*; D015032:Zinc / Q000494:pharmacology
D002413:Cations, Divalent; D011994:Recombinant Proteins; D014758:Viral Core Proteins; D008345:Manganese; D017766:Acid Anhydride Hydrolases; D043583:Nucleoside-Triphosphatase; D008274:Magnesium; D015032:Zinc; D002118:Calcium