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S L Bonting

Publications and source records attributed to S L Bonting.

At least 37 records · Page 2Linked to original sources
PubMed1984

Lack of immunological cross reactivity between the transport enzymes (Na+ + K+)-ATPase and (K+ + H+)-ATPase.

Goat antisera against (Na+ + K+)-ATPase and its isolated subunits and against (K+ + H+)-ATPase have been prepared in order to test for immune cross-reactivity between the two enzymes, whose catalytic subunits show great chemical similarity. None of the (Na+ + K+)-ATPase antisera cross-reacted with (K+ + H+)-ATPase or inhibited its enzyme activity. The same was true for the (K+ + H+)-ATPase antiserum with regard to (Na+ + K+)-ATPase and its subunits and its enzyme activity. So notwithstanding the chemical similarity of their subunits, there is no immunological cross-reactivity between these two plasma membrane ATPases.

Adenosine Triphosphatases↗
PubMed1984

Future ESA missions in biology.

A survey is given of the life sciences research program sponsored by the European Space Agency (ESA). This program rests on a number of facilities originated by ESA: Spacelab, Space sled, Biorack, Anthrorack, Eureca and its Botany - and Protein Crystallization facilities. They are all to be brought into space and returned by one of the NASA Space Shuttles. With these facilities a wide range of space biology research will be covered: cell biology, developmental biology, botany, human physiology, radiobiology, exobiology and biotechnology. Information is given on how to prepare, submit and execute an experiment proposal.

Europe↗
PubMed1984

Advanced biostack: experiment 1 ES 027 on Spacelab-1.

The radiobiological properties of the heavy ions of cosmic radiation were investigated on Spacelab 1 by use of biostacks, monolayers of biological test organisms sandwiched between thin foils of different types of nuclear track detectors. Biostacks were exposed to cosmic radiation at several locations with different shielding environments in the module and on the pallet. Evaluations of the physical and biological components of the experiment to date indicate that in general they survived the spaceflight in good condition. Dosimetric data are presented for the different shielding environments.

Animals↗
PubMed1983

Effects of dibutyryl cyclic GMP on rabbit pancreas secretion.

The isolated rabbit pancreas responds to the hormone cholecystokinin-pancreozymin and its C-terminal peptide with increases in protein secretion and in paracellular permeability. Dibutyryl cyclic GMP competitively inhibits these responses to the C-terminal octapeptide, but with different sensitivity. In low concentrations dibutyryl cyclic GMP lowers only the increase in the paracellular permeability, whereas in high concentrations it inhibits both the protein secretion and the permeability increase. The effect can be explained by assuming competition between dibutyryl cyclic GMP and the hormone at the level of the pancreozymin receptors.

Animals↗
PubMed1983

Hydrolysis of adenylyl imidodiphosphate in the presence of Na+ + Mg2+ by (Na+ + K+)-activated ATPase.

Contrary to what has usually been assumed, (Na+ + K+)-ATPase slowly hydrolyses AdoPP[NH]P in the presence of Na+ + Mg2+ to ADP-NH2 and Pi. The activity is ouabain-sensitive and is not detected in the absence of either Mg2+ or Na2+. The specific activity of the Na+ + Mg2+ dependent AdoPP[NH]P hydrolysis at 37 degrees C and pH 7.0 is 4% of that for ATP under identical conditions and only 0.07% of that for ATP in the presence of K+. The activity is not stimulated by K+, nor can K+ replace Na+ in its stimulatory action. This suggests that phosphorylation is rate-limiting. Stimulation by Na+ is positively cooperative with a Hill coefficient of 2.4; half-maximal stimulation occurs at 5-9 mM. The Km value for AdoPP[NH]P is 17 microM. At 0 degrees C and 21 degrees C the specific activity is 2 and 14%, respectively, of that at 37 degrees C. AMP, ADP and AdoPP[CH2]P are not detectably hydrolysed by (Na+ + K+)-ATPase in the presence of Na+ + Mg2+. In addition, AdoPP[NH]P undergoes spontaneous, non-enzymatic hydrolysis at pH 7.0 with rate constants at 0, 21 and 37 degrees C of 0.0006, 0.006 and 0.07 h-1, respectively. This effect is small compared to the effect of enzymatic hydrolysis under comparable conditions. Mg2+ present in excess of AdoPP[NH]P reduces the rate constant of the spontaneous hydrolysis to 0.005 h-1 at 37 degrees C, indicating that the MgAdoPP[NH]P complex is virtually stable to spontaneous hydrolysis, as is also the case for its enzymatic hydrolysis. A practical consequence of these findings is that AdoPP[NH]P binding studies in the presence of Na+ + Mg2+ with enzyme concentrations in the mg/ml range are not possible at temperatures above 0 degrees C. On the other hand, determination of affinity in the (Na+ + K+)-ATPase reaction by competition with ATP at low protein concentrations (microgram/ml range) remains possible without significant hydrolysis of AdoPP[NH]P even at 37 degrees C.

Adenosine Triphosphate↗
PubMed1983

Properties of the Mg2+-induced low-affinity nucleotide binding site of (Na+ + K+)-activated ATPase.

The Mg2+-induced low-affinity nucleotide binding by (Na+ + K+)-ATPase has been further investigated. Both heat treatment (50-65 degrees C) and treatment with N-ethylmaleimide reduce the binding capacity irreversibly without altering the Kd value. The rate constant of inactivation is about one-third of that for the high-affinity site and for the (Na+ + K+)-ATPase activity. Thermodynamic parameters (delta H degree and delta S degree) for the apparent affinity in the ATPase reaction (Km ATP) and for the true affinity in the binding of AdoPP[NH]P (Kd and Ki) differ greatly in sign and magnitude, indicating that one or more reaction steps following binding significantly contribute to the Km value, which thus is smaller than the Kd value. Ouabain does not affect the capacity of low-affinity nucleotide binding, but only increases the Kd value to an extent depending on the nucleotide used. GTP and CTP appear to be most sensitive, ATP and ADP intermediately sensitive and AdoPP[NH]P and AMP least sensitive to ouabain. Ouabain reduces the high-affinity nucleotide binding capacity without affecting the Kd value. The nucleotide specificity of the low-affinity binding site is the same for binding (competition with AdoPP[NH]P) and for the ATPase activity (competition with ATP): AdoPP[NH]P greater than ATP greater than ADP greater than AMP. The low-affinity nucleotide binding capacity is preserved in the ouabain-stabilized phosphorylated state, and the Kd value is not increased more than by ouabain alone. It is inferred that the low-affinity site is located on the enzyme, more specifically its alpha-subunit, and not on the surrounding phospholipids. It is situated outside the phosphorylation centre. The possible functional role of the low-affinity binding is discussed.

Adenosine Triphosphate↗
PubMed1983

Studies on (K+ + H+)-ATPase. VI. Determination on the molecular size by radiation inactivation analysis.

(1) A (K+ + H+)-ATPase containing membrane fraction, isolated from pig gastric mucosa, has been further purified by means of zonal electrophoresis, leading to a 20% increase in specific activity and an increase in ratio of (K+ + H+)-ATPase to basal Mg2+-ATPase activity from 9 to 20. (2) The target size of (Na+ + K+)-ATPase, determined by radiation inactivation analysis, is 332 kDa, in excellent agreement with the earlier value of 327 kDa obtained from the subunit composition and subunit molecular weights. This shows that the Kepner-Macey factor of 6.4 X 10(11) is valid for membrane-bound ATPases. (3) The target size of (K+ + H+)-ATPase is 444 kDa, which, in connection with a subunit molecular weight of 110000, suggests a tetrameric assembly of the native enzyme. The ouabain-insensitive K+-stimulated p-nitrophenylphosphatase activity has a target size of 295 kDa. (4) In the presence of added Mg2+ the target sizes of the (K+ + H+)-ATPase and its phosphatase activity are decreased by about 15%, while that for the (Na+ + K+)-ATPase is not significantly changed. This observation is discussed in terms of a Mg2+-induced tightening of the subunits composing the (K+ + H+)-ATPase molecule.

Adenosine Triphosphatases↗
PubMed1983

Detergent-induced specificity of an antirhodopsin serum for opsin. Micro-complement fixation studies.

An antiserum elicited in rabbit against dark-adapted rod outer segment membranes has been characterized by means of the micro-complement fixation technique. Both particulate rhodopsin and opsin, either biochemically intact or denatured and either membrane-bound or in the absence of lipids, are able to interact with the antiserum. Solubilization of the antigens in increasing concentrations of Emulphogene BC-720 leads to complete loss of complement fixation with both rhodopsin and opsin, but in the case of opsin this requires almost 10-times more detergent. In the case of opsin this masking phenomenon is preceded by a drastic exposure of antigenic sites which in the membrane vesicles are not accessible to the antibodies. Absorption experiments show that the antigenic sites on membrane-bound rhodopsin and opsin, as well as on Emulphogene BC-720-solubilized opsin, are of the same nature. Competition experiments show that the masking effect of the detergent is due to an inhibition of the primary antigen-antibody interaction and not to the inhibition of lattice formation. The use of detergents other than Emulphogene BC-720 further demonstrates that detergents more efficiently mask the antigenicity of conformationally intact than of denatured rhodopsinoids. The balance between the masking and the denaturing efficiency of a particular detergent determines whether a detergent-induced immunological discrimination can be observed between rhodopsin and opsin. The detergent-induced masking effects described are typical for the present antiserum and are probably dependent on methodological details of the immunization procedure.

Animals↗
PubMed1982

Studies on (K+ + H+)-ATPase V. Chemical composition and molecular weight of the catalytic subunit.

(1) A (K+ + H+)-ATPase preparation from porcine gastric mucosa is solubilized in sodium dodecyl sulfate, and is subjected to gel filtration. (2) A main subunit fraction is obtained, which is a protein carbohydrate lipid complex, containing 88% protein, 7% carbohydrate and 5% phospholipid. The Detailed composition of the protein and carbohydrate moieties are reported. (3) Sedimentation analysis of the subunit preparation, after detergent removal, reveals no heterogeneity, but the subunits readily undergo aggregation. (4) Acylation of the subunit preparation with citraconic anhydride causes a clear shift of the band obtained after SDS gel electrophoresis, but the absence of broadening and splitting of the band pleads against subunit heterogeneity. (5) Treatment of the subunit preparation with dansyl chloride indicates that the NH2 terminus is blocked, which favors the assumption of homogeneity of the protein. (6) Binding studies with concanavalin A indicate that at least 86% of the subunit preparation is composed of glycoprotein. (7) These findings, taken together, strongly suggest that there is a single subunit which is a glycoprotein and which represents the catalytic subunit of the enzyme. From sedimentation equilibrium analysis a molecular mass value of 119 kDa (S.E. 3, n = 6) is calculated for protein + carbohydrate and of 110 kDa (S.E. 3, N = 6) for protein only. (8) In combination with the molecular mass of 444 kDa (S. E. 10, n = 4) obtained for the intact enzyme by radiation inactivation we conclude that the enzyme appears to be composed of a homo-tetramer of catalytic subunits.

Adenosine Triphosphatases↗
PubMed1982

Studies on the catalytic hydrogenation of biomembranes. Hydrogenation of phosphatidylcholine liposomes by two water-soluble rhodium-phosphine catalysts.

The reactions of two water-soluble amphiphiolic rhodium-phosphine hydrogenation catalysts, chlorotris(sodium diphenylphosphinobenzene-m-sulfonate)rhodium(I) and chlorotris(bissodium diphenylphosphinoundecylphosphate)rhodium(I), with aqueous dispersions of unsaturated phosphatidylcholines have been studied. Under mild reaction conditions (pH2 = 1.2 atm, 37 degrees C, pH 6.9) hydrogenation of aqueous dioleoylphosphatidylcholine dispersions, prepared by sonication, was achieved. This reaction was not affected by the presence of high salt concentrations. The rate of hydrogenation was independent of catalyst concentration. The reaction with dioleoylphosphatidylcholine dispersions, prepared by the ethanolic injection method, was preceded by a lag period of about 5 h. The reaction with dioleoylphosphatidylcholine dispersions, obtained by vigorous shaking, was rather slow, suggesting the presence of a penetration barrier. The reaction with dioleoylphosphatidylcholine proceeds via the isomerisation of the oleoyl to the elaidoyl moiety, followed by hydrogenation of the elaidoyl moiety. The possible interactions of the catalysts with the bilayer are considered and the implications of these findings are discussed.

Catalysis↗
PubMed1982

Nucleotide content of isolated bovine rod outer segments.

Endogenous nucleotide levels of isolated intact ROS were analyzed by high pressure liquid chromatography. Intact bovine ROS showed a total nucleotide concentration of 1.0 mM, ATP and GTP being the major components (0.1-0.2 mol per mol of rhodopsin). When intact ROS resuspended in sucrose-ficoll medium were diluted in a Ringer-Krebs bicarbonate, the nucleotide ratios were markedly changed, but the total concentration remained unchanged. The concentration of cyclic nucleotides (cAMP and cGMP) was markedly enhanced when ROS were placed in Ringer buffer, whereas ATP and GTP concentration was reduced. Total nucleotide concentration is 100% higher in intact than in leaky plasma membrane ROS. Upon illumination, no change was observed in the nucleotide levels of ROS in sucrose-ficoll medium. However ATP, GTP and cAMP levels were reduced when ROS in Ringer medium were exposed to light while cGMP concentration showed no change. Relevance of relative nucleotide content and ionic concentration to the transduction phenomenon in photoreceptor is discussed.

Adenosine Triphosphate↗
PubMed1981

Studies on (Na+ + K+)-activated ATPase. XLIX. Content and role of cholesterol and other neutral lipids in highly purified rabbit kidney enzyme preparation.

(1) The neutral lipids and the free and bound fatty acids of a highly purified (Na+ + K+)-ATPase preparation from rabbit kidney outer medulla have been analysed. (2) On a dry weight basis, the total lipid content is nearly the same as the total protein content, and consists for 66% of phospholipids and for 34% of neutral lipids and free fatty acids. In the latter category cholesterol is the main component (71%). (3) On a molar basis the enzyme preparation contains 382 mol phospholipids, 67 mol free fatty acids, 9, 16 and 12 mol mono-, di- and triacylglycerols, 249 and 19 mol free and esterified cholesterol per mol enzyme. (4) The fatty acid composition of each lipid and of the free fatty acid fraction, present in the enzyme preparation, is reported. (5) All cholesterol and part of the phospholipids can be removed by hexane extraction, leaving 66% of the (Na+ + K+)-ATPase activity. Oxidation of all cholesterol to cholest-4-en-3-one by cholesterol oxidase leaves 85% of the (Na+ + K+)-ATPase activity. These results indicate that cholesterol is not essential for (Na+ + K+)-ATPase activity.

Animals↗
PubMed1981

Studies on (K+ + H+)-ATPase. IV. Effects of phospholipase C treatment.

(1) The total phospholipid content of a gradient purified (K+ + H+)-ATPase preparation from pig gastric mucosa is 105 mumol per 100 mg protein, and consists of 29% sphingomyelin, 29% phosphatidylcholine, 28% phosphatidylethanolamine, 10% phosphatidylserine and 4% phosphatidylinositol. The cholesterol content corresponds to 50 mumol per 100 mg protein. (2) Treatment with phospholipase C (from Clostridium welchii and Bacillus cereus) results in an immediate decrease of the phosphate content. Up to 50% of the phospholipids are hydrolyzed by each phospholipase C preparation alone, without further hydrolysis by increased phospholipase concentration or prolonged incubation time. Combined treatment with the two phospholipase C preparations, sequentially or simultaneously, hydrolyzes up to 65% of the phospholipids. (3) The (K+ + H+)-ATPase and K+ stimulated p-nitrophenylphosphatase activities are decreased proportionally with the total phospholipid content, indicating that these enzyme activities are dependent on phospholipids. (4) Phospholipase C treatment does not change optimal pH, Km value for ATP and temperature dependence of the gastric (K+ + H+)-ATPase, but slightly decreases the Ka value for K+. (5) Phospholipase C treatment lowers the AdoPP[NH]P binding and phosphorylation capacities, suggesting that inactivation occurs primarily on the substrate binding level. (6) Most of the results can be understood by assuming that hydrolysis of the phospholipids by phospholipase C leads to aggregation of the membrane protein molecules and complete inactivation of the aggregated ATPase molecules.

4-Nitrophenylphosphatase↗
PubMed1981

Effects of clanobutin on pancreatic secretion in vitro.

The effects of clanobutin (4-[4-chloro-N-(4-methoxyphenyl)-benzamido]butyric acid) were investigated on two in vitro pancreatic preparations. In rat pancreatic lobules clanobutin (7.2 mM) stimulated the secretion of amylase and radiolabeled proteins to the same or higher extent as did CCK-PZ and carbachol in maximally active doses, but with a delay of 1-2 h. In the isolated rabbit pancreas clanobutin (3.3 mM) stimulated protein secretion about half as much as did carbachol, but without significant delay. The effect of clanobutin on protein secretion was prevented by prior application of carbachol, was not affected by the omission of Ca2+ from the medium and was only partly inhibited by the presence of atropine (0.1 mM). Prior addition of clanobutin did not prevent the effects of carbachol on enzyme secretion. In addition, clanobutin (3.3 mM) inhibited fluid secretion in the rabbit pancreas by about 50 percent; the inhibition was reversible. No effects on paracellular permeability were found with sucrose used as test substance. The findings indicate that clanobutin has opposite effects on enzyme and fluid secretion by rat and rabbit pancreas, which are not due to circulatory effects.

Amylases↗
PubMed1981

Role of calcium in exocrine pancreas secretion. VII. Effect of sodium on enzyme secretion and calcium metabolism in rabbit pancreatic fragments and acinar cells.

1. The role of sodium in the pancreatic stimulus-secretion coupling has been studied. 2. Reduction of the extracellular sodium concentration or addition of ouabain to the medium inhibits the stimulation of enzyme secretion by carbachol. 3. Incubation in low sodium medium or in the presence of ouabain increases the exchangeable calcium content, but not the total calcium content of acinar cells. 4. Depending on preincubation time and the substrate replacing sodium in the low sodium medium, the carbachol-induced Ca45(2)+ efflux may be blocked, but it is not blocked by addition of ouabain to a normal Na+ medium. 5. Stimulation of enzyme secretion by the calcium ionophore A23187 in the presence of external calcium is inhibited in low sodium as well as in ouabain containing media. 6. These findings suggest that reduction of the Na+ gradient across the plasma membrane blocks the coupling between intracellular calcium release and exocytosis, while under certain conditions it also blocks the coupling between hormone-receptor interactions and intracellular calcium release.

Animals↗
PubMed1981

(Na/ + K+)ATPase has one functioning phosphorylation site per alpha subunit.

(Na+ + K+)ATPase contains two different subunits, a catalytic subunit (alpha) and a subunit with uncertain function (beta). The enzyme binds ATP, ouabain and vanadate, and can be phosphorylated by ATP as well as by inorganic phosphate. From the previously reported maximal binding and phosphorylation capacities of 3.5--4.3 nmol P per mg protein (based on Lowry protein determination) and the earlier molecular weight value of approximately 250,000, a molar binding and phosphorylation capacity of 0.87--1.07 mol per mol enzyme was derived. As it is generally agreed that the enzyme molecule contains two alpha subunits or even a multiple of two, it has been suggested that the enzyme operates by means of a so-called "half-of-the-sites mechanism" whereby only of the two alpha subunits can be phosphorylated at any one time. We now present evidence that every alpha subunit can be phosphorylated simultaneously, which rules out the operation of such a mechanism.

Adenosine Triphosphate↗