Purification and characterization of (Ca2+-Mg2+)-ATPase in rat liver lysosomal membranes.
A (Ca(2+)-Mg2+)-ATPase associated with rat liver lysosomal membranes was purified about 300-fold over the lysosomal membranes with a 7% recovery as determined from the pattern on polyacrylamide gel electrophoresis in the presence of SDS. The purification procedure included: preparation of lysosomal membranes, solubilization of the membrane with Triton X-100, WGA-Sepharose 6B, Con A-Sepharose, hydroxylapatite chromatography, and preparative polyacrylamide gel electrophoresis. The molecular mass, estimated by gel filtration with Sephacryl S-300 HR, was approximately 340 kDa, and SDS-polyacrylamide gel electrophoresis showed the enzyme to be composed of four identical subunits with an apparent molecular mass of 85 kDa. The isoelectric point of the purified enzyme was 3.6. The enzyme had a pH optimum of 4.5, a Km value for ATP of 0.17 mM and a Vmax of 71.4 mumol/min/mg protein at 37 degrees C. This enzyme hydrolyzed nucleotide triphosphates and ADP but did not act on p-nitrophenyl phosphate and AMP. The effects of Ca2+ and Mg2+ on the ATPase were not additive, thereby indicating that both Ca2+ and Mg(2+)-ATPase activities are manifested by the same enzyme. The (Ca(2+)-Mg2+)-ATPase differed from H(+)-ATPase in lysosomal membranes, since the enzyme was not inhibited by N-ethylmaleimide but was inhibited by vanadate. The effects of some other metal ions and compounds on this enzyme were also investigated. The N-terminal 18 residues of (Ca(2+)-Mg2+)-ATPase were determined.
Ezaki
J
J
Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka.
Himeno
M
M
Kato
K
K
D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000737:chemistry / Q000302:isolation & purification*; D002118:Calcium / Q000494:pharmacology; D002850:Chromatography, Gel; D003208:Concanavalin A; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D006882:Hydroxyapatites; D007425:Intracellular Membranes / Q000737:chemistry / Q000201:enzymology*; D007526:Isoelectric Point; D007700:Kinetics; D008099:Liver / Q000737:chemistry / Q000201:enzymology*; D008247:Lysosomes / Q000737:chemistry / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D011489:Protein Denaturation; D051381:Rats; D017208:Rats, Wistar; D012685:Sepharose / Q000031:analogs & derivatives; D013379:Substrate Specificity
D006882:Hydroxyapatites; D010446:Peptide Fragments; C042758:wheat germ agglutinin-sepharose 6MB; D003208:Concanavalin A; D000255:Adenosine Triphosphate; D012685:Sepharose; D017886:Durapatite; D017301:Ca(2+) Mg(2+)-ATPase; D008274:Magnesium; D002118:Calcium