G. Saccomani

Characterization of gastric mucosal membranes: Composition of gastric cell membranes and polypeptide fractionation using ionic and nonionic detergents☆☆☆

Abstract Lipid, carbohydrate, amino acid, and polypeptide composition of 2 fractions of cell membranes obtained by sucrose-Ficoll zonal density gradient fractionation of gastric mucosal homogenates has been studied. The membranes with density = 1.04 contain 0.7 μmole lipid P and 0.54 μmole cholesterol/mg protein, while the membranes of density 1.10 contain only 0.25 μmole lipid P and 0.28 μmole cholesterol/mg protein. Phosphatidylcholine and phosphatidylethanolamine are the most abundant phospholipids. Free fatty acids were present. Carbohydrates were most abundant in the proteins of the Peak II membranes (density = 1.10). One polypeptide band was dominant on sodium dodecyl sulfate, Triton X-100, and Brij 36T gels, and the adenosine triphosphatase activity in Brij 36T gels was found to have a relative mobility of 0.14 and 0.19 in the two fractions.

ASPECTS OF PARIETAL CELL BIOLOGY: CELLS AND VESICLES

Many features of these gastric vesicles satisfy the requirements for the gastric H+ pump. For example, we have: (a) K+ requirement, (b) KA for K+ of about 30 mM; (c) identical cation sequence for tissue and vesicles, (d) similar anion sequence, (e) localization at the microvillus of the secretory canaliculus, (f) TI+ inhibiting H+ transport of both systems, and (g) the K+ gradient satisfying the osmotic gradient requirement for HCl-flow out of the parietal cell. Points that require explanation are lack of SCN- effects and regulation of KCl permeability.

Preparation and characterization of plasma membrane of cardiac tissue

A method of preparing highly purified plasma membrane from cardiac muscle is described using continuous ficoll-sucrose density gradient centrifugation in zonal rotors. 5′-Nucleotidase and ATPase (Mg2+ dependent, Na+ + K+ stimulated and ouabain inhibited) were found to be concentrated in the plasma membrane fraction. At 11% ficollsucrose Ca2+ and (Ca2+ + K+) stimulation of the ATPase was very small (13%) and very low level of succinic dehydrogenase activity was present. Electron microscopic studies of the fraction revealed the presence of membrane vesicles and sheets. Composition of these membranes was analyzed by 7% polyacrylamide gel electrophoresis in the presence of 1% SDS. One protein band of 68 000 Daltons accounted for 18% of protein on the gel. Lipid and carbohydrate stains revealed six and two bands respectively. Studies on the conformation of proteins within membranes were achieved using circular dichroism (CD) and optical rotatory dispersion (ORD) techniques. Corrections were applied using the pseudo reference state approach.

THE INTERACTION OF K+ WITH GASTRIC PARIETAL CELLS AND GASTRIC ATPASE*

The gastric H+ secretion in isolated cell requires K+ and is ATP dependent. There is also evidence in the cell system for Na+ inhibition of H+ secretion. The isolated gastric ATPase also shows K+ activation and inhibition by K+ or Na+ located on the ATP binding side of the enzyme, which corresponds to the cytoplasmic face of the enzyme. Gastric vesicles are activated in terms of transport activity by internal K+, and this site, inhibited by reagents that modify carboxyl groups, is required for enzyme turnover and transport.

Use of 1-anilino-8-naphthalene-sulfonate as a probe of gastric vesicle transport

SummaryThe interaction of 1-anilino-8-naphthalene-sulfonate (ANS) with vesicles derived from hog fundic mucosa was studied in the presence of valinomycin and with the addition of ATP. Evidence was found for two classes of sites, those rapidly accessible to ANS with aKD of 7.5 μm and those slowly accessible, but rapidly accessed in the presence of valinomycin with aKD of 2.5 μm. ATP transiently increases the quantum yield of the latter ANS binding sites only in the presence of valinomycin, but does not alter the number ofKD of those sites. The time course of this increase correlates with H+ uptake and Rb+ extrusion by those vesicles and H+ carriers such as tetrachlorsalicylanilide or nigericin abolish the ATP response. With ATP addition in the presence of SC14N and valinomycin there is transient uptake of SCN−. It is concluded that ANS is acting as a probe of a structural change dependent on a potential and H+ gradient.

Isolation and characterization of a liver-specific antigen (F-Ag) from human liver☆

Abstract A liver-specific antigen previously demonstrated in saline extracts of human liver has been purified by consecutive ammonium sulfate precipitation, ion-exchange chromatography and gel filtration. The purified antigen migrated as a single bank in gel electrophoresis. This antigen has a mol. wt of 44,000 daltons. Amino acid analysis showed the acidic amino acids to outnumber the basic amino acids while glycine is the most abundant neutral amino acid. No carbohydrate could be demonstrated in the purified preparation of this antigen.

Characterization of gastric mucosal membranes. VI. The presence of channel-forming substances

Abstract Highly purified gastric membranes were extracted by ionic or non-ionic detergents. Further fractionation was carried out on acrylamide gel electrophoresis. Some defined bands, incorporated into artificial bilayer membranes, produced discrete conductance changes characteristics of channel activity. A low molecular weight peak, purified through two cycles of electrophoresis was characterized as an anion-selective, positively voltage dependent channel, and a model to account for the action of the transport ATPase incoporating this channel concept is suggested.