Purification and characterization of a cytoplasmic apyrase plumules of Pisum sativum Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/z029p849z

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  • The 105,000 x g supernatant fluid (S[subscript HS]) from extracts of pea seedlings (Pisum sativum, var. Alaska) etiolated for 60 hours yielded a very potent adenosine triphosphatase activity. This activity was designated as an apyrase when the reaction stoichiometry indicated that adenosine triphosphate (ATP) was hydrolyzed to adenosine monophosphate (AMP) and two moles of inorganic phosphate (P[subscript i]). The apyrase has been purified 88-fold with 40% recovery from acetone powders of the S[subscript HS]. This 6-day procedure involved filtration of the acetone powder solutions through Sephadex G-200 and G-75 followed by protamine sulfate treatment to remove polyanionic material. After film dialysis the enzyme activity was chromatographed on carboxymethyl cellulose (CMC) with a gradient NaCl elution. A second more rapid purification scheme was used to purify extracts of acetone powders to 79- to 100-fold with overall yield of 67% to >100%. This 15-hour scheme involved protamine treatment of the acetone powder extracts, followed by ammonium sulfate fractionation in the presence of 0.01 M ethylenediaminetetraacetic acid (EDTA) with pH being continuously maintained at 7.8 during salt dissolution. The precipitate obtained between 2.7 to 4.0 molal ammonium sulfate was film dialyzed and then chromatographed on CMC with NaCl gradient elution. With ATP as substrate the apyrase (ATPase activity) showed a temperature optimum at 30°C and an optimum of the apparent V[subscript m] at pH 6.1. With ADP as the substrate the enzyme (ADPase activity) showed an optimum of the apparent V[subscript m] at pH 7.1. The double-reciprocal plots of the ATPase activity showed an upward deviation at high substrate concentration. However, the ADPase reaction showed an upward deviation in these plots at low substrate concentration. The ATPase reaction differed from the ADPase by showing diminished activity in maleate and imidazole buffers. Divalent cations were required for both reactions; the most effective were Mn⁺⁺>Ca⁺⁺>Mg⁺⁺. No phosphoesterase or inorganic pyrophosphatase activity were detected. Triphosphates of guanosine, cytidine, inosine, and uridine were hydrolyzed faster than ATP. In contrast to other apyrases in the literature, the pea seedling enzyme was seen to hydrolyze ADP faster than ATP. Co-purification and non-additivity of the two activities indicated that they were due to one enzyme.
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