Purification and characterization of a vanadate-sensitive nucleotide tri- and diphosphatase with acid pH optimum from rat bone.
Rat bone was extracted with KCl and Triton X-100, and a tartrate-resistant acid phosphatase activity was purified by protamine sulfate precipitation, ion-exchange chromatography (CM-cellulose), and gel filtration on Sephadex G-200 according to previously described procedures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining demonstrated a major band with an apparent monomer molecular size of approximately 14,000 Da. The enzyme is active with p-nitrophenylphosphate (p-NPP) but exhibits a 5- to 10-fold higher affinity towards several nucleotides of which ATP and ADP are the most readily hydrolyzed substrates based on kinetic studies. Based on sensitivity towards proteolytic treatment and detergent removal, as well as pH-optimum studies, a single enzyme was found to be responsible for activity towards nucleotide phosphates as well as p-NPP. This nucleotide tri- and diphosphatase constitutes around 15% of the total acid phosphatase activity in rat bone. The activity with ATP as substrate in contrast to that with p-NPP was inhibited in a noncompetitive fashion by MgCl2, sodium metavanadate, and p-chloromercuribenzoate. Enzyme activity with p-NPP and ATP is dependent on the presence of KCl and detergent and is activated by Fe3+ and ascorbate. The reported characteristics of the enzyme suggest that it functions as a unique membrane acid ATPase.
D000135:Acid Phosphatase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001842:Bone and Bones / Q000201:enzymology*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D009711:Nucleotides / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity; D014638:Vanadates; D014639:Vanadium / Q000494:pharmacology*
D009711:Nucleotides; D014639:Vanadium; D015636:Magnesium Chloride; D014638:Vanadates; D000255:Adenosine Triphosphate; D000135:Acid Phosphatase; D000251:Adenosine Triphosphatases; D008274:Magnesium