Acid Adenosine Triphosphatase from Chicken Liver Lysosomes

I. Partial Purification and Some Properties

Abstract

Acid ATPase [ATP phosphohydrolase, EC 3. 6. 1. 3] of chicken liver lysosomes was purified from the 100, 000×g supernatant of lysosomal extract, by precipitation with acetone, column chromatographies on DEAE-cellulose and Sephadex G-150, and isoelectric focusing, with an overall recovery of 4%. and 70-fold increase in specific activity. The purified enzyme preparation showed a major band and two additional minor components on sodium dodecyl sulphate (SDS)-polyacrylamide disc gel electrophoresis.
The enzyme thus purified had a molecular weight of about 55, 000 according to gel filtration on Sephadex G-150, or about 60, 000 according to SDS-polyacrylamide gel electrophoresis. On isoelectric focusing, two enzyme forms having isoelectric point (pI) of 5.08 and 5.18 appeared. The purified enzyme hydrolyzed not only ATP but also bis (p-nitrophenyl) phosphate. According to the distribution patterns of DEAE-cellulose and Sephadex G-150, the same enzyme hydrolyzed both ATP and bis (p-nitrophenyl) phosphate. The optimal pH of ATP hydrolysis was 5.4, whereas that of bis(p-nitrophenyl) phosphate was 7.0.
The enzyme hydrolyzed nucleoside triphosphates, inorganic pyrophosphate and bis(p-nitrophenyl) phosphate, but did not act on nucleoside monophosphates, phosphomonoesters and sulphate monoesters. ATP was the best substrate of all compounds tested, although the value of K_m was comparable to those for other nucleoside triphosphates. The products of ATP hydrolysis were ADP and inorganic phosphate. Subsequent hydrolysis of ADP to AMP was not detected by Avicel SF-cellulose thin-layer chromatography.
ATPase activity was activated by reducing agents such as ascorbic acid, but SH blocking agents, Hg²⁺, p-chloromercuriphenyl sulphonic acid and PCMB were inhibitory to enzyme activity. Na⁺, K⁺, ouabain and oligomycin, which is known to affect the activities of ATPases in plasma membrane and mitochondria, were without effect.