Trypsinization unmasks a Ca+2- stimulated ATPase activity from purified pig gastric microsomes

Trypsinization of gastric microsomal K⁺- stimulated ATPase in absence of ATP nearly abolished the K⁺- stimulated component of the enzyme activity without any significant effect on the basal (with Mg⁺² alone) activity. The K⁺- stimulated component, however, was completely restored by the ‘activator protein” partially purified form the soluble supernatant fraction of the pig gastric cells. On the other hand, trypsinization of the microsomes in presence of ATP significantly increased (2–3 fold) the basal rate with virtual elimination of the K⁺- stimulated component. Assay of the trypsinized microsomes in presence of the activator protein not only demonstrated complete restoration of the K⁺- stimulated ATPase but also revealed an additional activity which has been characterized as a Ca⁺²- stimulated ATPase.Tryptic digestion has recently been used as a tool to understand the mechanism of action of various transport enzymes such as Na⁺, K⁺- ATPase (1), Ca⁺²- ATPase (2,3) and gastric H⁺, K⁺- ATPase (4). Controlled tryptic digestion of purified enzymes under various conditions of ligand binding may provide us with many valuable informations regarding the molecular architecture of the enzyme protein. However, when dealing with a membrane system containing a host of many different intrinsic and extrinsic proteins one must be cautious about the interpretation of the trypsin effects. In the present paper we report the effects of trypsin digestion of the purified pig gastric microsomes on the microsomal K⁺- stimulated ATPase activity. Our studies demonstrated that digestion of the microsomes with trypsin in absence of ATP inactivated the K⁺- stimulated ATPase but the activity could be fully restored by the addition of partially purified activator protein (5). Microsomes treated with trypsin in presence of ATP responded to the activator protein to the same extent as that without ATP but in addition demonstrated the manifestation of another enzymatic activity which has been characterized as a Ca⁺²- stimulated ATPase. This is a preliminary report dealing primarily with the unmasking of a new ATPase after trypsin treatment. Detailed reports on the characterization and mechanism of action of the gastric Ca⁺²- stimulated ATPase will be published elsewhere.