Localization and characterization of a parotid Ca(2+)-dependent ecto-ATPase

Area of Science:

• Biochemistry
• Cell Biology
• Physiology

Background:

• Parotid glands secrete saliva, a process involving ion transport.
• Ecto-ATPases are enzymes located on the cell surface, involved in extracellular ATP metabolism.
• Previous studies identified a Ca(2+)-ATPase in parotid membranes as an ecto-ATPase.

Purpose of the Study:

• To confirm the presence and characterize the ecto-ATPase activity in intact parotid acinar cells.
• To compare the inhibitor sensitivity and substrate specificity of the ecto-ATPase in intact cells with the previously identified membrane-bound Ca(2+)-ATPase.

Main Methods:

• Isolation of parotid acini.
• Enzymatic assays using various inhibitors (oligomycin, N-ethylmaleimide, ruthenium red, sodium azide, sodium orthovanadate, trifluoperazine) to probe ATPase activity.
• Assessment of nucleotide substrate specificity.

Main Results:

• The ecto-ATPase in intact parotid acinar cells showed insensitivity to oligomycin, N-ethylmaleimide, ruthenium red, and sodium azide.
• Approximately 22% inhibition was observed with sodium orthovanadate (Na3VO4) and 32% with trifluoperazine (TFP).
• The nucleotide substrate specificity pattern in intact cells closely mirrored that observed in isolated parotid membranes.

Conclusions:

• The ecto-ATPase activity in intact parotid acinar cells is consistent with a Ca(2+)-ATPase.
• The inhibitor profile and substrate specificity strongly suggest that the ecto-ATPase identified in intact cells is the same enzyme previously characterized in parotid membranes.
• This finding supports the role of this specific Ca(2+)-ATPase as an ecto-enzyme in parotid acinar cells.