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G Saccomani

Publications and source records attributed to G Saccomani.

At least 37 records · Page 2Linked to original sources
PubMed1983

Purification of cells from livers of carcinogen-treated rats by free-flow electrophoresis.

The tumorigenic roles of specific types of cells that emerge during chemical hepatocarcinogenesis in rats could potentially be determined if the specific types of cells could be purified adequately. Type II cells, defined (J. M. Jacobs, T. P. Pretlow, N. Fausto, A. M. Pitts, and T. G. Pretlow, J. Natl. Cancer Inst., 66: 967-973, 1981) as small, slowly sedimenting cells with histochemically demonstrable gamma-glutamyl transpeptidase, have oval nuclei similar in size to those of lymphocytes. Adult male F344 rats were fed a choline-deficient diet containing 0.05% ethionine for 4 weeks. Liver cells were obtained in suspension by in situ perfusion of collagenase. A two-step process, i.e., sedimentation in an isokinetic gradient followed by free-flow electrophoresis, was used to purify type II cells. We obtained preparations of cells with 32.3 +/- 5.0% (S.D.) type II cells, 13.4 +/- 2.0% erythrocytes, 52.9 +/- 3.3% small nucleated cells, and only 1.4 +/- 0.3% hepatocytes. The small nucleated cells were Kupffer cells and cells smaller than Kupffer cells that included many lymphocytes and unidentified cells similar in size to lymphocytes. Because both the electrophoretic mobilities and the rates of sedimentation of type II cells and hepatocytes are different, there is some advantage to be obtained from the sequential use of these techniques that exploit independent differences in the physical properties of these cells.

Acyltransferases↗
PubMed1981

Reactivity of gastric (H+ + K+)-ATPase to N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline.

The K+-dependent ATPase and p-nitrophenyl phosphatase activity of, and formation of phosphoenzyme by, hog parietal cell membranes were inhibited in a time- and concentration-dependent manner by the carboxyl-activating reagent, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). The kinetics of inactivation was pseudo first order and was similar to the EEDQ-catalyzed incorporation of [14C]glycine ethyl ester. The most likely mechanism is the EEDQ-dependent formation of inter- and intramolecular amide bonds. Cross-linking between the subunits of the ATPase occurs with EEDQ treatment. The presence of K+ on the luminal face of the enzyme is able to prevent EEDQ inhibition of K+ ATPase activity (but not intermolecular cross-linking), whereas ATP enhanced the rate of inactivation. EEDQ reaction with the enzyme therefore allows investigation of K+- and ATP-dependent states of the enzyme.

Adenosine Triphosphatases↗
PubMed1981

Target molecular weight of the gastric (H+ + K+)-ATPase functional and structural molecular size.

The state of assembly of the (H+ + K+)-ATPase in purified hog gastric mucosa membranes was studied by target size analysis applied to radiation-induced enzyme inactivation and polypeptide degradation data. Radiation inactivated the Mg2+-ATPase, K+-stimulated ATPase, and p-nitrophenyl phosphatase activities of the membrane preparation with a dose dependence characteristic of a target size of 270,000-daltons. Radiation also bleached the major 100,000-dalton sodium dodecyl sulfate-gel electrophoresis band of this preparation, indicating a radiation-induced degradation. This apparent polypeptide degradation exhibited a dose dependency corresponding to a target size of 250,000 daltons in situ. It is suggested that the gastric ATPase is a trimeric assembly of the 100,000-dalton polypeptides.

Adenosine Triphosphatases↗
PubMed1980

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.

Adenosine Triphosphate↗
PubMed1980

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.

Adenosine Triphosphatases↗
PubMed1979

The action of trypsin on the gastric (H+ + K+)-ATPase.

The effect of trypsin on gastric (H+ + K+)-ATPase and K+-phosphatase was studied. Loss of both enzymic activities was biphasic, consisting of a fast and slow phase. Several peptides were produced from the original 105,000-dalton region of the sodium dodecyl sulfate electrophoretic separation, but only two, 87,000 and 47,000 daltons, were labeled following incubation with [gamma-33P]ATP. After a 30-min hydrolysis, 35% of the original peptide remained unaltered and appeared to be a glycoprotein. ATP and ADP abolished the second phase of tryptic inactivation of both activities and only two peptides, of 78,000 and 30,000 daltons, were found on the acrylamide gel in addition to the original 105,000-dalton region, neither of which was labeled by [gamma-33P]ATP. The protection was specific for these nucleotides, AMP, beta, gamma-methylene ATP, TTP, and pNPP being ineffective. Na+ and K+ at high concentrations reduced the rate of loss of activity but no change in the peptides produced was found. The level of phosphoenzyme was increased 2-fold by trypsin treatment, whereas the quantity of K+-sensitive phosphoenzyme remained relatively constant. Thus, the 105,000-dalton region is heterogeneous, consisting of a catalytic subunit (the active site is on a 47,000-dalton fragment), a glycoprotein, and another 105,000-dalton peptide. The action of trypsin is initially to prevent interconversion of a K+-insensitive to a K+-sensitive form of the phosphoenzyme, thus inhibiting hydrolysis.

Adenosine Triphosphatases↗
PubMed1979

Transport characteristics of frog gastric membranes.

ATP-induced transport by fractions of frog gastric microsomes prepared either by density gradient centrifugation of by free flow electrophoresis were K+ dependent and hence considered due to a K+-activated ATPase. Significant activity of this enzyme was, however, only found in the anodic peak of the free flow electrophoretic separation, which in addition to separating transporting from non-transporting particles, also separated membranes containing a phosphorylatable peptide (Mr=105 000) region as the major peptide on SDS-polyacrylamide gel electrophoresis from those containing a peptide (Mr=44 000) on SDS-polyacrylamide gel electrophoresis. H+ uptake, measured either by acridine orange or 3,3'-diethyloxadicarbocyanine + tetrachlorosalicylanilide absorbance changes was dependent on K+ intravesicularly. Using 86Rb+, active extrusion of the cation followed ATP addition. SCN-, an inhibitor of acid secretion did not affect the latter, but blocked signals due to H+ uptake, in contrast to mammalian preparations.

Adenosine Triphosphatases↗
PubMed1979

Effect of phospholipase A2 on purified gastric vesicles.

The phospholipid and fatty acid composition and role of phospholipids in enzyme and transport function of gastric (H+ + K+)-ATPase vesicles was studied using phospholipase A2 (bee venom). The composition (%) was phosphatidyl-choline (PC) 33%; sphingomyelin (sph) 25%; phosphatidylethanolamine (PE) 22%; phosphatidylserine (PS) 11%; and phosphatidylinositol (PI) 8%. The fatty acid composition showed a high degree of unsaturation. In both fresh and lyophilized preparations, even with prolonged incubation, only 50% of phospholipids were hydrolyzed, but the amount of PE and PS disappearing was increased following lyophilization. There was a marked decrease in K+-ATPase activity (75%) but essentially no loss of the associated K+ p-nitrophenyl phosphatase was found. ATPase activity could be largely restored by various phospholipids (PE greater than PC greater than PS). There was also an increase in Mg2+-ATPase activity, partially reversed in fresh preparations by the addition of phospholipids (PE greater than PS greater than PC). Proton transport activity of the preparation was rapidly inhibited, initially due to a large increase in the HCl permeability of the preparation. Associated with these enzymatic and functional changes, the ATP-induced conformational changes, as indicated by circular dichroism spectra were inhibited.

Adenosine Triphosphatases↗
PubMed1979

Characterization of gastric mucosal membranes. X. Immunological studies of gastric (H+ + K+)-ATPase.

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.

Adenosine Triphosphatases↗
PubMed1979

An acid transporting enzyme in human gastric mucosa.

Isolation of a microsomal fraction from human gastric mucosa followed by density gradient centrifugation yielded a vesicular membrane preparation free of mitochondrial markers, containing a K+-activated, ouabain-insensitive ATPase with an activity of 20.7 mumol P1 released/mg protein per h. Sodium dodecyl sulfate gel electrophoresis showed that the human gastric membrane vesicles contained a major polypeptide of 110,000 daltons, which accounted for approximately or equal to 30% of the total protein stained and was phosphorylated by [gamma-32P]ATP and dephosphorylated in the presence of K+. Electron microscopy revealed the presence of vesicles with an average size of 0.13 micrometer in diameter. Addition of 0.65 microM ATP to this vesicular preparation resulted in the uptake of 17 nmol H+/mg protein which was dependent on the presence of K+. The gradient was dissipated by a combination of valinomycin and protonophore after consumption of the ATP. Incubation of fixed human fundic sections or human gastric biopsy with monospecific hog gastric membrane antibody followed by fluorescein-conjugated goat anti-rabbit gamma-globulin, showed fluorescent staining in the middle portion of the gastric glands. These data indicate that human stomach contains a H+ transport ATPase with characteristics similar to those established for lower species.

Adenosine Triphosphatases↗
PubMed1977

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

The 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 a KD of 7.5 micronM and those slowly accessible, but rapidly accessed in the presence of valinomycin with a KD of 2.5 micronM. ATP transiently increases the quantum yield of the latter ANS binding sites only in the presence of valinomycin, but does not alter the number of KD of those sites. The time course of this increase correlates with H+ uptake and Rb+ extrusion by those vesicles and H+ carries 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.

Anilino Naphthalenesulfonates↗
PubMed1977

Cation transport by gastric H+:K+ ATPase.

A vesicular microsomal fraction isolated from hog fundic mucosa demonstrates the capacity to take up equal amounts of RB+ and Cl-. The amount of the Rb+ uptake is sensitive to the extravesicular osmolarity, and rate of uptake is sensitive to temperature. 86Rb+ efflux is dependent upon the cation composition of the diluting solution. ATP, but not beta-gamma methylene ATP, induces a reversible efflux of 86Rb+ from loaded vesicles, and this is dependent upon a functional K+-ATPase. The ATP induced efflux is not affected by CCCP (carbonyl cyanide m-chlorophenylhydrazone) or TCS (tetrachlorosalicylanilide) nor by lipid soluble ions or valinomycin. Nigericin inhibits the efflux by 40%. Uptake of the lipid soluble ion 14C-SCN- has been demonstrated and is enhanced by ATP only in the presence of valinomycin. The results are consistent with a neutral or isopotential exchange of H+ for Rb+ mediated by K+-ATPase.

Adenosine Triphosphatases↗
PubMed1977

Characterization of gastric mucosal membranes. IX. Fractionation and purification of K+-ATPase-containing vesicles by zonal centrifugation and free-flow electrophoresis technique.

Methods are described for purification of a vesicular membrane fraction of hog gastric mucosa using differential centrifugation, density gradient separation on zonal rotors and free-flow electrophoresis. As a result a fraction is obtained enriched 40-fold in terms of K(+)-ATPase and free of any other enzyme marker other than K(+)-activated p-nitrophenyl phosphatase. The 5'-nucleotidase and basal Mg(2+)-ATPase are clearly separated from the latter enzymes. Osmotic shock, Triton X-100 treatment or K+ ionophores increased the K(+)-ATPase activity in isotonic conditions, but K(+)-p-nitrophenyl phosphatase is not affected by these treatments, nor is the ATPase activity in the presence of NH4+. The results suggest that the electrophoretic fraction contains a major population of tight vesicles, whose permeability to K+ is rate limiting for the ATPase activity but not for the p-nitrophenyl phosphatase activity. It is concluded that K+ site for the ATPase is internal whereas the K+ site for the p-nitrophenyl phosphatase is external, hence, the K+ site must be mobile across the membrane.

4-Nitrophenylphosphatase↗
PubMed1977

Proton transport by gastric membrane vesicles.

A highly purified membrane fraction was derived from hog gastric mucosa by a combination of differential and density gradient centrifugation and free flow electrophoresis. This final fraction was 35-fold enriched with respect to cation activated ouabain-insensitive ATPase. Antibody against this fraction was shown to be bound to the luminal surface of the gastric glands. The addition of ATP to this fraction or the density gradient fraction resulted in H+ uptake into an osmotically sensitive space. The apparent Km for ATP was 1.7-10(-4) M in the absence of a K+ gradient similar to that found for ATPase activity. The reaction is specific for ATP and requires cation in the sequence K+ greater than Rb+ greater than Cs+ greater than Na+ greater than Li+ and inhibited by ATPase inhibitors such as N,N'-dicylclohexyl-carbodiimide. Maximal H+ uptake occurs with an outward K+ gradient but the minimal apparent KA is found in the absence of a K+ gradient. The pH optimum for H+ uptake is between 5.8 and 6.2 which corresponds to the pH range for phosphroylation of the enzyme, but is considerably less than the pH maximum of the K+ dependent dephosphorylation. In the presence of an inward K+ gradient, protonophores such as tetrachlorsalicylanilide only partially abolish the H+ gradient but valinomycin dissipates 75% of the gradient, and nigericin abolishes the gradient. The vesicles therefore have a low K+ conductance but a measurable H+ conductance, hence a K+ gradient can produce an H+ gradient in the presence of valinomycin. The uptake and spontaneous leak of H+ are temperature sensitive with a similar transition temperature. Ultraviolet irradiation inactivates ATPase and proton transport at the same rate, approximately at twice the rate of p-nitrophenylphosphatase inactivation. It is concluded that H+ uptake by these vesicles is probably due to a dimeric (H+ + K+)-ATPase and is probably non-electrogenic.

Adenosine Triphosphatases↗
PubMed1977

Metabolic and membrane aspects of gastric H+ transport.

Metabolic properties of dog gastric mucosa, investigated by substrate level measurements, implicate the Krebs cycle as the major energy-yielding metabolic pathway but are equivocal in terms of an ATP-based H+ secretion. Purification of gastric membranes by centrifugation and free flow electrophoresis results in a class of membrane vesicles enriched in K+-ATPase and capable of ATP-energized H+ uptake. Immunohistochemistry shows these to be derived from the parietal cell. H+ uptake by the vesicles is accompanied by K+ efflux, and movement of either ion is not potential-coupled. The simplest interpretation of these transport studies is uptake of KCl by the vesicles by passive diffusion followed by active H+:K+ exchange. In some respects, however, this model fails to conform to the expectations from in vitro studies. It may be, therefore, that another pump (i.e., redox) or another membrane component (i.e., Cl- conductance) is lost during purification. The properties of the vesicles are such, however, as to establish their role in H+ secretion by the stomach.

Adenosine Diphosphate↗