Biochemical characterization of a V-ATPase of tracheal smooth muscle plasma membrane fraction.
A biochemical characterization of a Mg(2+)-ATPase activity associated with a plasma membrane fraction isolated from airway (tracheal) smooth muscle was performed. This enzyme is an integral part of the membrane remaining tightly bound after 0.6 M KCl extraction. This enzyme activity showed a cold inactivation in the presence of ATP and Mg2+. Also, this Mg(2+)-ATPase was stimulated by monovalent anions being Cl-, the best anion for such stimulation, even though Br- and I- were good substitutes and F- was ineffective. This Cl--stimulated activity showed a powerful nucleosidetriphosphatase activity having the following divalent cation specificity: Mg2+ > Mn2+ > Ca2+, where Zn2+ and Fe2+ were ineffective. This ATPase activity was not inhibited by ouabain oligomycin C and vanadate indicating that neither P- or F-ATPases were associated with this enzyme activity. However, the existence of a V-ATPase was shown by the significant inhibition causes by bafilomycin A1. Additionally, this V-ATPase seems to be coupled to Cl- conductor because duramycin inhibited this ATPase activity. The presence of a H+ pump associated to this V-ATPase was shown indirectly, through the stimulatory effect produced by uncouplers such as FCCP and 1799, which were able to produce significant stimulation of this V-ATPase indicating the existence of a H(+)-ATPase. Finally, the immunodetection of a 72 kDa polypeptide using a specific antibody against the A subunit (72 kDa) of V-ATPase from chromaffin granule demonstrated the presence of a V-ATPase in this plasma membrane fraction.
Pacheco
G
G
Catedras de Bioquímica y Patología General y Fisiopatología, Universidad Central de Venezuela, Caracas, Venezuela.
Lippo de Bécemberg
I
I
Gonzalez de Alfonzo
R
R
Alfonzo
M J
MJ
D000478:Alkylation; D000818:Animals; D000838:Anions; D015153:Blotting, Western; D017301:Ca(2+) Mg(2+)-ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002412:Cations; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D003080:Cold Temperature; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006219:Halogens / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D009130:Muscle, Smooth / Q000201:enzymology*; D009711:Nucleotides; D010042:Ouabain / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D013379:Substrate Specificity; D014132:Trachea / Q000201:enzymology* / Q000648:ultrastructure; D014475:Uncoupling Agents / Q000494:pharmacology; D014638:Vanadates / Q000494:pharmacology
D000838:Anions; D002412:Cations; D004791:Enzyme Inhibitors; D006219:Halogens; D009711:Nucleotides; D014475:Uncoupling Agents; D014638:Vanadates; D010042:Ouabain; D011189:Potassium Chloride; D017301:Ca(2+) Mg(2+)-ATPase