Abstract
A metal ion-activated acid adenosine triphosphatase (ATPase) of chicken liver lysosomes was purified from the 100000×g supernatant of lysosomal extract, by fractionation with (NH4)2SO4, column chromatographies on Phenyl-Separose CL-4B, Sephacryl S-300 and Affi-Gel 501, isoelectric focusing, and gel filtration on Sepharose 6B, with a recovery of 0.38% and a 19-fold increase in specific activity.Although the purified enzyme had a molecular weight ranging from 700000 to 800000 according to Sephacryl S-300 gel filtration, the enzyme was dissociated into several subunits having molecular weights ranging from 26000 to 93000 on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. By isoelectric focusing, the pI of the enzyme was found to be 4.12. The percentage activity rations of acid phosphatase and phosphodiesterase to ATPase in the enzyme preparations became lower with the progress of purification, those in the finally purified preparation being much lower than reported previously.The enzyme preparation mainly hydrolyzed nucleoside triphosphates, but also acted weakly on adenosine diphosphate (ADP), adenosine monophosphate (AMP), p-nitrophenylphosphate or bis(p-nitophenyl)phosphate. In terms of Km values, adenosine triphoshate (ATP) was the most accessible to the enzyme of all the nucleoside triphosphates tested. At 0.1 mM, nucleoside triphosphates were hydrolyzed in the absence of metal ions in the following order : ATP>GTP>CTP, UTP>dTTP. IN the presence of metal ions, the order of nucleotide preference was only slightly affected.The ATPase activity was markedly inhibited by HgCl2, but other reagents including several SH-blockers and ATPase inhibitors were without effect.
