Abstract
Gastric mucosal homogenates from hog were fractionated by differential and density gradient centrifugation and free-flow electrophoresis. The two major membrane fractions (FI and FII) thus obtained are distinct both enzymically and in terms of transport reactivity. This heterogenicity extends to their antigenic activity. Purified antibodies which were raised against the K+-ATPase-containing H+ transport fraction FI were of two types: inhibitory and non-inhibitory. Inhibitory antibodies reduced the K+-ATPase activity by approximately 80% and the K+-p-nitro-phenylphosphatase activity by approximately 40% in a concentration-dependent manner, while the small Mg++-dependent component of the enzyme activity was unaffected. Antibodies inhibiting the K+-ATPase also inhibited H+ transport. These antibodies did not cross-react with the other major membrane fraction isolated by free- flow electrophoresis, FII, and gave a single band on rocket immunoelectrophoresis. Antibodies against this FII fraction also did not react with the K+-ATPase and were heterogeneous, giving at least four bands with rocket immunoelectrophoresis and inhibiting both the 5'- nucleotidase and Mg++-ATPase of this fraction. Immunofluorescent staining of tissue sections showed that the FI was derived from the parietal cell of gastric tissue and was localized to the supranuclear area of the cell. Staining of isolated rat gastric cell suspensions by FI antibodies confirmed the selectivity of the antibody and showed a polar, plasma membrane localization. FII antibodies also largely stained the parietal cells in tissue sections. In the 16 hog tissues tested, FI antibodies cross-reacted only with gastric fundus, thyroid and weakly with thymus. Immunoelectronmicroscopy showed that FI antibodies reacted strongly with the secretory membrane at the apical cell surface of the parietal cells and at the secretory canaliculi, weakly with the apical surface of the zymogen cell, and not with the basal-lateral surface of the cells. Thus, the protontranslocating ATPase is localized in the parietal cells and in the region postulated to be the site of acid secretion.
Full Text
The Full Text of this article is available as a PDF (1.1 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Askari A. The effects of antibodies to Na+, K+-ATPase on the reactions catalyzed by the enzyme. Ann N Y Acad Sci. 1974;242(0):372–388. doi: 10.1111/j.1749-6632.1974.tb19104.x. [DOI] [PubMed] [Google Scholar]
- LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
- McCans J. L., Lane L. K., Lindenmayer G. E., Butler V. P., Jr, Schwartz A. Effects of an antibody to a highly purified Na+, K+-ATPase from canine renal medulla: separation of the "holoenzyme antibody" into catalytic and cardiac glycoside receptor-specific components. Proc Natl Acad Sci U S A. 1974 Jun;71(6):2449–2452. doi: 10.1073/pnas.71.6.2449. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nakane P. K. Recent progress in the peroxidase-labeled antibody method. Ann N Y Acad Sci. 1975 Jun 30;254:203–211. doi: 10.1111/j.1749-6632.1975.tb29170.x. [DOI] [PubMed] [Google Scholar]
- Rubin W., Ross L. L., Sleisenger M. H., Jefries G. H. The normal human gastric epithelia. A fine structural study. Lab Invest. 1968 Dec;19(6):598–626. [PubMed] [Google Scholar]
- Saccomani G., Chang H. H., Mihas A. A., Crago S., Sachs G. An acid transporting enzyme in human gastric mucosa. J Clin Invest. 1979 Aug;64(2):627–635. doi: 10.1172/JCI109503. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Saccomani G., Dailey D. W., Sachs G. The action of trypsin on the gastric (H+ + K+)-ATPase. J Biol Chem. 1979 Apr 25;254(8):2821–2827. [PubMed] [Google Scholar]
- Saccomani G., Stewart H. B., Shaw D., Lewin M., Sachs G. Characterization of gastric mucosal membranes. IX. Fractionation and purification of K+-ATPase-containing vesicles by zonal centrifugation and free-flow electrophoresis technique. Biochim Biophys Acta. 1977 Mar 1;465(2):311–330. doi: 10.1016/0005-2736(77)90081-5. [DOI] [PubMed] [Google Scholar]
- Sachs G., Chang H. H., Rabon E., Schackman R., Lewin M., Saccomani G. A nonelectrogenic H+ pump in plasma membranes of hog stomach. J Biol Chem. 1976 Dec 10;251(23):7690–7698. [PubMed] [Google Scholar]
- Schackmann R., Schwartz A., Saccomani G., Sachs G. Cation transport by gastric H+:K+ ATPase. J Membr Biol. 1977 Apr 22;32(3-4):361–381. doi: 10.1007/BF01905228. [DOI] [PubMed] [Google Scholar]
- Smith T. W., Wagner H., Jr Effects of (Na+ + K+)-ATPase-specific antibodies of enzymatic activity and monovalent cation transport. J Membr Biol. 1975;25(3-4):341–360. doi: 10.1007/BF01868583. [DOI] [PubMed] [Google Scholar]
- Soumarmon A., Cheret A. M., Lewin M. J. Localization of gastrin receptors in intact isolated and separated rat fundic cells. Gastroenterology. 1977 Oct;73(4 Pt 2):900–903. [PubMed] [Google Scholar]
- Sternberger L. A., Petrali J. P. Quantitative immunocytochemistry of pituitary receptors for luteinizing hormone-releasing hormone. Cell Tissue Res. 1975 Sep 17;162(2):141–176. doi: 10.1007/BF00209204. [DOI] [PubMed] [Google Scholar]
- Yoda A., Hokin L. E. On the reversibility of binding of cardiotonic steroids to a partially purified (Na+K)-activated adenosinetriphosphatase from beef brain. Biochem Biophys Res Commun. 1970 Aug 24;40(4):880–886. doi: 10.1016/0006-291x(70)90985-x. [DOI] [PubMed] [Google Scholar]