guanosine-triphosphate and diazobenzenesulfonic-acid

Parotid acini were isolated and tested to further establish the presence of ecto-ATPase in the intact cells. Inhibitors were used to determine if the inhibitor profile of the ATPase was similar to that of a Ca(2+)-ATPase from parotid membranes identified previously as an ecto-ATPase. The Ca(2+)-ATPase of intact cells was insensitive to oligomycin (10 micrograms/ml), N-ethylmaleimide (NEM) (0.1 mM), ruthenium red (0.1 mM), sodium azide (1 mM), and was inhibited approximately 22% by sodium orthovanadate (Na3VO4) (1 mM). This profile was similar to the Ca(2+)-ATPase of intact cells. Trifluoperazine (TFP) (0.1 mM) inhibited the enzyme in intact cells by approximately 32%. The nucleotide substrate specificity of the enzyme also reflected very closely the pattern seen in isolated membranes.

Topics: Adenosine Triphosphatases; Animals; Azides; Calcium-Transporting ATPases; Cell Membrane; Cytidine Triphosphate; Diazonium Compounds; Ethylmaleimide; Guanosine Triphosphate; Inosine Triphosphate; Ouabain; Parotid Gland; Rats; Rats, Sprague-Dawley; Sodium Azide; Sulfanilic Acids; Trifluoperazine; Vanadates

An ATPase was isolated from synaptosomal plasma membranes derived from bovine cerebral cortex. The protein has an apparent molecular mass of 50 kDa and a pI of 5.3 to 5.9. It can be labelled by incubation of intact synaptosomes with azido-GTP or azido-ATP. The isolated ATPase can be activated to a similar extent in the presence of millimolar concentrations of Mg2+ or Ca2+. It does not hydrolyze ADP. Maximal activity is obtained between pH 7.5 and 8.5. Typical inhibitors of cytoplasmic ATPases do not affect enzyme activity. The enzyme is specifically inhibited after previous incubation of intact synaptosomes in the presence of the slowly membrane-permeable enzyme inhibitor diazotized sulfanilic acid. Incubation of intact synaptosomes with diazotized sulfanilic acid results in a small increase in the apparent molecular mass of the enzyme. Our results suggest that the active site of the membrane bound enzyme faces the extracellular medium. It thus would represent an ecto-ATPase.

Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Affinity Labels; Animals; Azides; Brain; Ca(2+) Mg(2+)-ATPase; Cattle; Diazonium Compounds; Guanosine Triphosphate; Hydrogen-Ion Concentration; Substrate Specificity; Sulfanilic Acids; Synaptic Membranes; Synaptosomes