Large scale expression, purification and 2D crystallization of recombinant plant plasma membrane H+-ATPase.

P-type ATPases convert chemical energy into electrochemical gradients that are used to energize secondary active transport. Analysis of the structure and function of P-type ATPases has been limited by the lack of active recombinant ATPases in quantities suitable for crystallographic studies aiming at solving their three-dimensional structure. We have expressed Arabidopsis thaliana plasma membrane H+-ATPase isoform AHA2, equipped with a His(6)-tag, in the yeast Saccharomyces cerevisiae. The H+-ATPase could be purified both in the presence and in the absence of regulatory 14-3-3 protein depending on the presence of the diterpene fusicoccin which specifically induces formation of the H+-ATPase/14-3-3 protein complex. Amino acid analysis of the purified complex suggested a stoichiometry of two 14-3-3 proteins per H+-ATPase polypeptide. The purified H(+)-ATPase readily formed two-dimensional crystals following reconstitution into lipid vesicles. Electron cryo-microscopy of the crystals yielded a projection map at approximately 8 A resolution, the p22(1)2(1) symmetry of which suggests a dimeric protein complex. Three distinct regions of density of approximately equal size are apparent and may reflect different domains in individual molecules of AHA2.

The pharmacology of the gastric acid pump: the H+,K+ ATPase.

The gastric H+,K+ ATPase--the gastric acid pump--is the molecular target for the class of antisecretory drugs called the proton-pump inhibitors (PPIs). These compounds--omeprazole, lansoprazole, and pantoprazole--contain, as their core structure, 2-pyridyl methylsulfinyl benzimidazole. The H+,K+ ATPase is a heterodimer composed of a 1034-amino acid catalytic alpha peptide and a glycosylated 291-amino acid beta subunit. The alpha subunit probably contains 10 membrane-spanning sequences; the beta, a single transmembrane segment. The PPIs have a pKa of about 4.0; hence they accumulate only in the acidic secretory canaliculus of the stimulated parietal cell. Here they undergo conversion to a cationic sulfenamide, which then reacts with available cysteines on the extracytoplasmic face of the alpha subunit. Omeprazole reacts and forms disulfide bonds with cys813(822) and cys892; lansoprazole, with cys813(822), cys892, and cys321; and pantoprazole, with cys813 and -822. The antisecretory effect of the drugs reflects their short plasma half-life (approximately 60 min), the number of active pumps during that time, and the recovery of pumps following biosynthesis and reversal of inhibition. These drugs also show synergism with either amoxicillin or clari- thromycin in eradicating Helicobacter pylori, an organism shown to be important in duodenal and gastric ulcer disease. Their action is probably due to elevation of pH in the environment of the organism, rather than to any direct action.

Antiulcer agents. 5. Inhibition of gastric H+/K(+)-ATPase by substituted imidazo[1,2-a]pyridines and related analogues and its implication in modeling the high affinity potassium ion binding site of the gastric proton pump enzyme.

A number of substituted imidazo[1,2-a]pyridines and related analogues were selected for biochemical characterization in vitro against both the purified gastric proton pump enzyme, H+/K(+)-ATPase, and the intact gastric gland. The inhibitory activity in these two in vitro models was then examined for correlation with the gastric antisecretory potency determined for these compounds in vivo by using the histamine-stimulated Heidenhain pouch dog. Analysis of the biological data suggested that the inhibitory activity of the analogues determined in two in vitro models is predictive of their in vivo gastric antisecretory activity following intravenous, but not oral, administration. Furthermore, the good correlation observed between the in vitro and in vivo models suggests that these compounds are gastric proton pump inhibitors in vivo. A molecular modeling study of these compounds using the active analogue approach has defined the molecular volume which is shared by the active analogues, as well as the molecular volume which is common to the inactive analogues. Graphical representation of the difference between these molecular volumes can be interpreted in terms of a hypothetical description of the pharmacophore by means of which 3-(cyanomethyl)-2-methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine, Sch 28080 (1) and its analogues interact with the gastric proton pump enzyme, H+/K(+)-ATPase.

Inhibition of gastric H+/K+-ATPase by substituted imidazo[1,2-a]pyridines.

A hydrophobic imidazopyridine, SCH 28080 (3-cyanomethyl-2-methyl-8-phenylmethoxy)imidazo[1,2-a]pyridine) has previously been shown to inhibit gastric acid secretion in vivo and in vitro. Studies of isolated gastric H+/K+-ATPase have demonstrated that SCH 28080 reversibly inhibited the enzyme and competitively interacted with the K+-stimulated ATPase and p-nitrophenylphosphatase activities of the H+/K+-ATPase. To elucidate the mechanism of inhibition further, for example to establish whether the inhibitor interaction occurs on the luminal or the cytosolic side of the enzyme or if compound pKa influences inhibition, SCH 28080 and three analogues have been studied. We have examined the effects on K+-stimulated ATPase activity in isolated ion-permeable membrane vesicles at different pH values and KCl concentrations. In ion-tight membrane fractions the effect on acid formation was estimated. The results are in agreement with the hypothesis that the protonated, and thus positively charged, form of SCH 28080 is the active species, and that the inhibitory effect is exerted by binding of the compound to the luminal side of the H+/K+-ATPase.

Inhibition of gastric H+,K+-ATPase and acid secretion by SCH 28080, a substituted pyridyl(1,2a)imidazole.

A hydrophobic amine, SCH 28080, 2-methyl-8-(phenylmethoxy)imidazo(1,2a)pyridine-3-acetonitrile, previously shown to inhibit gastric acid secretion in vivo and in vitro, was also shown to inhibit basal and stimulated aminopyrine accumulation in isolated gastric glands when histamine, high K+ concentrations, or dibutyryl cAMP were used as secretagogues. Stimulated, but not basal, oxygen consumption was also inhibited. Neutralization of the acid space of the parietal cell by high concentrations of the weak base, imidazole, reduced the potency of the drug, suggesting that SCH 28080 was active when protonated. Studies on the isolated H+,K+-ATPase showed that the compound inhibited the enzyme competitively with K+, whether ATP or p-nitrophenyl phosphate were used as substrates. In contrast, the inhibition was mixed with respect to p-nitrophenyl phosphate and uncompetitive with respect to ATP. The drug reduced the steady state level of the phosphoenzyme but not the observed rate constant for phosphoenzyme formation in the absence of K+ nor the quantity of phosphoenzyme reacting with K+. The drug quenched the fluorescence of fluorescein isothiocyanate-modified enzyme and also inhibited the ATP-independent K+ exchange reaction of the H+,K+-ATPase. Its action on gastric acid secretion can be explained by inhibition of the H+,K+-ATPase by reversible complexation of the enzyme. This class of compound, therefore, acts as a reversible inhibitor of gastric acid secretion.

Chronic effects of dichloromethane on amino acids, glutathione and phosphoethanolamine in gerbil brain.

Mongolian gerbils were exposed to dichloromethane for three months by continuous inhalation at 210 ppm. Total free tissue amino acids, glutathione, and phosphoethanolamine were determined in the vermis posterior of the cerebellum and the frontal cerebral cortex. These two brain areas were chosen because humans occupationally exposed to dichloromethane have shown abnormalities in the electroencephalogram of the frontal part of the cerebral cortex. This study showed that long-term exposure of gerbils to dichloromethane (210 ppm) for three months leads to decreased levels of glutamate, gamma-aminobutyric acid, and phosphoethanolamine in the frontal cerebral cortex, while glutamine and gamma-aminobutyric acid are elevated in the posterior cerebellar vermis.

Chronic effects of perchloroethylene and trichloroethylene on the gerbil brain amino acids and glutathione.

Mongolian gerbils were exposed for 12 months to trichloroethylene (TCE) 50 or 150 ppm or perchloroethylene (PCE) 120 ppm. Vermis posterior of cerebellum and hippocampus were used for measurement of high-affinity uptake and release of 3H-gamma-aminobutyric acid (GABA) and 14C-glutamate, as well as for determination of total free tissue amino acids and glutathione. Glutathione was significantly elevated in the hippocampus of animals exposed to 150 ppm or TCE. Levels of amino acids were not appreciably affected. After PCE exposure, 120 ppm for 12 months, taurine significantly decreased in the hippocampus and even more in the posterior part of the cerebellar vermis. Glutamine was elevated in the hippocampus. No other significant changes in amino acids or glutathione were observed. After exposing the animals to TCE (50 and 150 ppm) for 12 months, the accumulation of glutamate by the posterior part of the cerebellar vermis increased in a dose-dependent manner, but no significant changes were observed in the hippocampus. The uptake of glutamate and GABA in cerebellum and hippocampus were unaffected after PCE exposure to 120 ppm for 12 months. The potassium-stimulated release of glutamate and GABA was unaffected in hippocampal tissue slices from gerbils exposed to 50 and 150 ppm TCE.

Long lasting changes in gerbil brain after chronic ethanol exposure: a quantitative study of the glial cell marker S-100 and DNA.

Glial S-100 protein, soluble protein, and DNA were quantitatively studied in brains of gerbils chronically exposed to ethanol in a nutritionally complete fluid diet. Eight different brain areas were studied. After exposure to ethanol for 3 months followed by a 4-month post-treatment ethanol-free period, increased amounts of S-100 protein per wet weight were found in the frontal cerebral cortex, the sensory-motor cerebral cortex, the posterior cerebellar vermis, and the brainstem. The increase of S-100 in the posterior cerebellar vermis was paralleled by an increase in DNA per wet weight, which was also increased in the anterior cerebellar vermis. However, a decreased content of DNA was observed in the frontal cerebral cortex, despite the increase of S-100 protein, suggesting a cell loss affecting cells other than astroglial in this area. In the cerebellar vermis, elevated concentrations of soluble proteins per wet weight were found, whereas a decreased amount was found in the anterior cerebellar hemispheres. It is suggested that the S-100 protein acts as a marker for astroglial cell volume and that a concomitant increase of S-100 protein and DNA might indicate an increase in the number of astroglial cells. Thus, our results obtained after ethanol exposure and subsequent ethanol abstinence are compatible with changes consisting of astroglial hypertrophy in the cortex areas and brainstem, as well as astroglial hypertrophy and/or proliferation in the posterior cerebellar vermis, a clear sign of the preceding noxae.

The effect of S-100a and S-100b proteins and Zn2+ on the assembly of brain microtubule proteins in vitro.

The homologous proteins S-100a and S-100b affect the microtubule system in a distinctly different way in the presence of low molar ratios of Zn2+. Assembly of brain microtubule proteins can be almost completely inhibited and rapid disassembly can be induced by low molar amounts of S-100b in the presence of low molar ratios [2-4] of Zn2+. Higher molar ratios per S-100b (greater than 4) potentiate the general Zn2+ effect, promoting the formation of sheets of microtubules. However, the effect of S-100a is quite different, no inhibition of assembly can be observed and the presence of S-100a seems to protect the microtubule proteins against the effect of Zn2+ by chelating the Zn2+ and decreasing the free metal-ion concentration. S-100a or S-100b cannot bind to the microtubule polymer-form, either in the absence or in the presence of Zn2+.

Trichloroethylene: long-lasting changes in the brain after rehabilitation.

The exposure of adult Mongolian gerbils to 60 or 320 ppm trichloroethylene (TCE) by continuous inhalation during 3 months, followed by a period of 4 months free of exposure, causes biochemical changes in the hippocampus, the posterior part of cerebellar vermis and in the brain stem, compatible with alterations comprising astroglial hypertrophy and/or proliferation. General and experimental neuropathology have been able to raise our knowledge of the glial cells, and a number of distinct tissue syndromes are characterized and pathogenetically determined by changes in the astroglial component of the nervous system. An astroglial reaction, comprising hypertrophy or proliferation, is known to be caused by exogenous and endogenous noxae. Ultrastructural evidences on neuronal cell alterations, such as decreased amounts of microtubules and increased content of lysosomes and myelin bodies, were observed. The biochemical and morphological data presented indicate that TCE is neurotoxic after moderate exposure levels and periods. Some brain areas seem to be more sensitive than others.

Effects of trichloroethylene inhalation on proteins of the gerbil brain.

Inhalation exposure of adult Mongolian gerbils to 320 ppm of trichloroethylene (TCE) during 8 weeks causes a decrease of soluble proteins per wet weight in frontal cerebral cortex, cerebellar anterior part of the hemispheres and in the posterior part of vermis, as well as in hippocampus, although the levels of S 100, a glial cytoplasmic protein, showed an overgoing increase back to control levels, or a significant increase. In the sensory-motor cortex, an overgoing increase of soluble proteins, as well as of the S 100, were observed during the exposure period. One of the major soluble polypeptides (m.w. 50,000--52,000) of cerebral cortex, the cerebellar hemispheres and the brain stem, decreased at the end of the exposure period. Possible candidates for such a polypeptide are among others the subunit of microtubular protein or a subunit of (Na+K+)-ATPase. The results show that inhalation of TCE effect various brain areas differently. The observed biochemical changes could be interpreted as an adaptation and in some brain areas neuronal cells seem to be more sensitive than glial cells to TCE.