Methods
Past work showed that both inhibition of H,K-ATPase activity and increased proteolytic susceptibility of HKa to proteolysis are closely associated with reduction of HKb disulfide bonds, as well as a high degree of a/b-subunit cooperativity. We now propose that HKb may protect the holoenzyme from conditions found in the extracellular environment. In one approach we compare the relative susceptibility of rabbit gastric HKb and renal NaKb to denaturation and proteolysis.

Results
The extracellular domain of HKb is highly resistant to tryptic digestion in the native state; resistance is much more profound than for the structurally related NaKb. Denaturation, or HK a/b subunit separation, is required to expose tryptic sites. The native HKb is also resistant to digestion by V8 protease which hydrolyzes proteins at anionic amino acids. Removal of terminal a-anomeric-linked galactose from b-subunit oligosaccharides does not appreciably alter the sensitivity of the b-subunit to tryptic digestion. However, complete removal of N-linked glycosylations make the HKb much more susceptible to pepsinolysis at pH <2.0.

Conclusions
We propose that (i) intrinsic folding of the b-subunit, (ii) cooperative interactive bonding forces between the a- and b-subunits , and (iii) oligosaccharides on the b-subunit provide an extracellular motif that is resistant to the lytic conditions of the gastric lumen. Further, that the b-subunit of all heterodimeric cation exchange pumps evolved as a protective element for the external surface. Protein folding, stabilized by disulfide bonding, with charged sites protected and hydrophobic sites exposed, would provide a stable structure in the face of large pH changes. N-linked oligosaccharides offer an effective aqueous interface. The H,K-ATPase is an obvious model, but analogous structural specializations must apply to all gastric luminally exposed proteins.