A comparative study of (Ca(2+)-Mg2+)-ATPase on the lysosomal membrane and ecto-ATPase on the plasma membrane from rat liver.
We showed that rat liver lysosomes possess at least two types of ATPase besides H(+)-translocating ATPase (H(+)-ATPase); namely, N-ethylmaleimide (NEM)-sensitive and bafilomycin A1-insensitive Mg(2+)-ATPase [ATPase I] and NEM- and bafilomycin A1-insensitive (Ca(2+)-Mg2+)-ATPase [ATPase II] [Hayashi H., et al. Chem. Pharm. Bull., 37, 2783-2786 (1992)]. Subcellular fractionation showed the presence of similar activity of (Ca(2+)-Mg2+)-ATPase not only in the Golgi but also in the plasma membrane fractions, of which plasma membrane had the highest activity, most probably due to the ecto-ATPase. In this study, we partially purified the (Ca(2+)-Mg2+)-ATPases from both lysosomal and plasma membranes and compared their properties. Both enzymes had quite similar characteristics including: (1) broad pH-activity profiles with two pH optima at 4.5 and 7.0, (2) K(m) values for ATP being 21-27 microM (at pH 4.5) and 18-14 microM (at pH 7.0) (in the presence of CaCl2), (3) hydrolysis of both nucleoside triphosphates and diphosphate (ADP), (4) inhibition only by vanadate and 4,4'-diisothiocyanatostilbene 2,2'-disulfonic acid (DIDS) at pH 4.0 (but not at pH 7.0), (5) acidic pI values that were shifted by neuraminidase digestion, and (6) a positive reaction against an antibody to the N-terminal peptide of ecto-ATPase.
Adachi
T
T
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Kanazawa University, Ishikawa, Japan.
Arai
K
K
Ohkuma
S
S
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology*; D003300:Copper / Q000378:metabolism; D005459:Fluorides / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D008099:Liver / Q000201:enzymology / Q000648:ultrastructure*; D008247:Lysosomes / Q000201:enzymology*; D008274:Magnesium / Q000378:metabolism; D051381:Rats; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity; D013431:Sulfates / Q000378:metabolism; D013447:Sulfites / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
D013431:Sulfates; D013447:Sulfites; D003300:Copper; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase; C027201:ectoATPase; D008274:Magnesium; D015032:Zinc; D005459:Fluorides; D002118:Calcium