Partial purification and characterization of rabbit-kidney brush-border (Ca2+ or Mg2+)-dependent adenosine triphosphatase.
The ATPase activity of rabbit-kidney brush border can be activated almost equally well by Ca2+ and Mg2+ and, therefore, should be called (Ca2+ or Mg2+)-ATPase. This enzyme was solubilized and enriched 14-fold by the following steps: pretreatment with papain removed 69% of alkaline phosphatase without attacking a significant portion of the ATPase activity. Addition of 1% cholate removed 65% of the protein but no ATPase activity. The combination of cholate (0.5%) and deoxycholate (0.4%) solubilized most of the ATPase activity and most of the remaining protein. A column chromatography of the extract on Sepharose CL-2B resulted in an 6.5-fold increase of specific ATPase activity. A precipitation by ammonium sulfate (40% saturation) produced an additional 1.9-fold increase. The yield of this partial purification was 16%. Towards the nucleotides UTP and GTP the enzyme showed an activity slightly higher, and towards ITP and CTP an activity slightly lower than that with ATP. ADP was split about half as fast as ATP. AMP was not accepted by the enzyme. Replacing MgCl2 by CaCl2 resulted in an ATPase activity of 92% of that with MgCl2. Using calcium- and magnesium-ATP as substrates, apparent Km values of 0.22 and 0.33 mM, respectively, were obtained. The gel electrophoresis revealed the enrichment of a protein with an apparent Mr of 95000 and also that of microvillus actin.
D000251:Adenosine Triphosphatases / Q000302:isolation & purification*; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases / Q000302:isolation & purification*; D002413:Cations, Divalent; D002845:Chromatography; D003902:Detergents; D007668:Kidney / Q000201:enzymology*; D008297:Male; D008871:Microvilli / Q000201:enzymology; D008970:Molecular Weight; D009711:Nucleotides / Q000378:metabolism; D010206:Papain; D011817:Rabbits; D012995:Solubility; D013379:Substrate Specificity
D002413:Cations, Divalent; D003902:Detergents; D009711:Nucleotides; D010206:Papain; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases