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P Owen

Publications and source records attributed to P Owen.

At least 19 recordsLinked to original sources
PubMed1979

Immunochemical analysis of membrane vesicles from Escherichia coli.

Membrane vesicles isolated from Escherichia coli ML 308--225 have been analyzed by crossed immunoelectrophoresis, and immunoprecipitates corresponding to the following cellular components have been identified: ATPase (EC 3.6.1,3), two or three NADH dehydrogenases (EC 1.6.99.3), D-lactate dehydrogenase (EC 1.1.1.27), glutamate dehydrogenase (EC 1.4.1.4), dihydro-orotate dehydrogenase (EC 1.3.3.1), 6-phosphogluconate dehydrogenase (EC 1.1.1.43), polynucleotide phosphorylase (EC 2.3.7.8), beta-galactosidase (EC 3.2.1.23), lipopolysaccharide, and Braun's lipoprotein. The cellular origin of many of the vesicle immunogens is determined, and Braun's lipoprotein is used as a marker to quantitate the extent of outer membrane contamination (less than 3%). Membrane antigens are also characterized with regard to their amphiphilic or hydrophilic properties by charge-shift crossed immunoelectrophoresis. Furthermore, the following immunogens cross-react with components in membrane vesicles prepared from Salmonella typhimurium: one of the three NADH dehydrogenases, ATPase, polynucleotide phosphorylase, 6-phosphogluconate dehydrogenase, Braun's lipoprotein, and three unidentified antigens. In the accompanying paper [Owen, P., & Kaback, H. R. (1979) Biochemistry 18 (following paper in this issue)] quantitative immunoadsorption is utilized to establish the topology of the vesicles with respect to the distribution of antigens on the inner and outer faces of the membrane.

Adenosine Triphosphatases↗
PubMed1979

Antigenic architecture of membrane vesicles from Escherichia coli.

The antigenic architecture of membrane vesicles prepared from Escherichia coli ML 308--225 has been studied using crossed immunoelectrophoresis. Progressive immunoadsorption experiments conducted with control vesicles and with physically disrupted vesicles were used to monitor and quantitate the expression of 14 different immunogens. Eleven immunogens, including NADH dehydrogenase (EC 1.6.33.3), D-lactate dehydrogenase (EC 1.1.1.27), dihydro-orotate dehydrogenase (EC 1.3.3.1), 6-phosphogluconate dehydrogenase (EC 1.1.1.43), polynucleotide phosphorylase (EC 2.3.7.8), and beta-galactosidase (EC 3.2.1.23), exhibit minimal expression (10% or less) unless the vesicles are disrupted. Three unidentified antigens are expressed to a similar extent in untreated and disrupted vesicles. Consideration of these and other results [Owen, P., & Kaback, H. R. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 3148] in terms of membrane polarity, dislocation of antigens, and possible transmembrane orientation of some immunogens reveals that over 95% of the membrane in the vesicle preparations is in the form of sealed sacculi with the same orientation as the intact cell. Furthermore, antigens are distributed across the membrane in a highly asymmetric manner, indicating that dislocation of components from the inner to the outer surface of the membrane during vesicle preparation does not occur to an extent exceeding 10%.

Antigen-Antibody Reactions↗
PubMed1979

Immunochemical analysis of triton X-100-insoluble residues from Micrococcus lysodeikticus membranes.

Triton X-100-insoluble residues from Micrococcus lysodeikticus membranes were analyzed by crossed immunoelectrophoresis after dispersal of the residues in sodium dodecyl sulfate (SDS). Conditions which produce no obvious distortion of the immunoprecipitate profile and which allow qualitative and quantitative analyses of the antigens present in the extracts are described. Two main antigens were detected; these were identified as succinate dehydrogenase (EC 1.3.99.1) and adenosine triphosphatase (EC 3.6.1.3). As determined by peak area estimations, the maximal release of succinate dehydrogenase and of adenosine triphosphatase from Triton X-100-insoluble membrane residues occurred at protein/SDS ratios of about 4.3:1 (0.2% SDS) and 6.8:1 (0.13% SDS), respectively. A comparison of enzyme activities of SDS extracts with those of untreated, control Triton X-100-insoluble membrane residues indicated that both the succinate dehydrogenase and the adenosine triphosphatase antigens were released with a full (or enhanced) catalytic potential at or below concentrations of SDS required to effect maximal solubilization of the enzyme in question. Evidence is also presented to suggest that the more acidic of the two components detected by crossed immunoelectrophoresis for the heterogeneous adenosine triphosphatase antigen is more sensitive to SDS than is the other. Both succinate dehydrogenase and adenosine triphosphatase lost catalytic activity and were denatured at protein/SDS ratios lower than 3.4:1.

Adenosine Triphosphatases↗
PubMed1978

Evaluation of weighted discriminating power calculations as an aid to the selection of chromatographic systems for the analyses of drugs.

In toxicological analyses some chromatographic separations are more important than others. Two weighting methods for chromatographic data are described which incorporate the importance of particular separations into discriminating power calculations. The data were abstracted from a publication on the separations of acidic drugs on thin-layer chromatographic systems. When compared with non-weighted discriminating power calculations, those obtained with the weighting procedure did not give any advantage.

Chromatography, Thin Layer↗
PubMed1978

Choice of thin-layer chromatographic systems for the routine screening for neutral drugs during toxicological analyses.

The efficiencies of fifteen thin-layer silica gel chromatographic systems for separating commonly encountered neutral drugs are compared. The discriminating powers of the systems are measured both individually and in combination. Chloroform-acetone (4:1) is found to be the best system. The combination of the ethyl acetate-methanol-ammonia (85:10:5) system with this gives the best pair of systems. A suitable sequence of spray reagents is also suggested. The chloroform-acetone (4:1) system is recommended as the best system to use when screening for both acidic and neutral drugs.

Chromatography, Gel↗
PubMed1978

Choice of thin-layer chromatographic systems for the routine screening for acidic drugs during toxicological analyses.

The efficiencies of fifteen thin-layer silica gel chromatographic systems in separating commonly encountered acidic drugs are compared. The discriminating powers of the systems are measured both individually and in combination. Ethyl acetate and chloroform-methanol (9:1) are found to be the two best systems. The combination of the ethyl acetate-methanol-ammonia (85:10:5) system with either of these gives the best pair of systems. Various sequences of spray reagents are also examined.

Chromatography, Thin Layer↗
PubMed1978

Molecular structure of membrane vesicles from Escherichia coli.

The molecular architecture of membrane vesicles prepared from Escherichia coli ML 308-225 has been studied by using crossed immunoelectrophoresis, and a reference pattern of 52 discrete immunoprecipitates has been established. Progressive immunoadsorption experiments conducted with untreated control vesicles and with physically disrupted vesicles demonstrate that the membrane-associated immunogens fall into two categories: (i) those immunogens typified by ATPase (ATP phosphohydrolase, EC 3.6.1.3) and NADH dehydrogenase [NADH: (acceptor) oxidoreductase, EC 1.6.99.3] whose expression is minimal unless the vesicles are disrupted; and (ii) immunogens such as Braun's lipoprotein that are expressed to similar extents in untreated and in disrupted vesicles. A mathematical relationship between the peak area subtended by an immunoprecipitate in the crossed immuno-electrophoresis system and the quantity of vesicles used in the adsorption process has been derived. This relationship allows quantitation of the degree to which specific membrane immunogens partition between exposed and unexposed surfaces of the vesicle membrane. The results demonstrate conclusively that >95% of the membrane in the vesicle preparations is in the form of sealed sacculi with the same polarity as the intact cell. Moreover, the findings provide a strong indication that dislocation of immunogens from the inner to the outer surface of the membrane during vesicle preparation does not occur to an extent exceeding 11%.

Adenosine Triphosphatases↗
PubMed1978

Immunochemical analysis of inner and outer membranes of Escherichia coli by crossed immunoelectrophoresis.

Isolated membrane fractions of Escherichia coli K-12 yielded complex immunoprecipitate patterns when Triton X-100 and sodium dodecyl sulfate extracts were examined by crossed immunoelectrophoresis with antienvelope immunoglobulins. Twelve of the 46 antigens in the immunoprecipitate patterns of inner (plasma) membranes were identified by zymograms and/or by the use of specific antisera. The following enzyme activities were detected in immunoprecipitates: 6-phosphogluconate dehydrogenase (EC 1.1.1.43); adenosine triphosphatase (EC 3.6.1.3); glutamate dehydrogenase (EC 1.4.1.4), two separate components; malate dehydrogenase (EC 1.1.1.37); dihydroorotate dehydrogenase (EC 1.3.3.1); succinate dehydrogenase (EC 1.3.99.1); lactate dehydrogeanse (EC 1.1.1.27); reduced nicotinamide adenine dinucleotide dehydrogenase (EC 1.6.99.3); protease (EC 3.4.21.1); and glycerol 3-phosphate dehydrogenase (EC 1.1.99.5). The corresponding immunoprecipitate pattern for isolated outer membranes consisted of at least 25 discrete antigens and differed strikingly from that obtained with inner membranes. Two major immunogens were identified as lipopolysaccharide and Braun lipoprotein. A protease-active immunoprecipitate was also detected in this fraction, but attempts to identify the Rosenbusch matrix protein in the crossed immunoelectrophoretic profile were unsuccessful.

Antigens, Bacterial↗
PubMed1977

Membrane asymmetry and expression of cell surface antigens of Micrococcus lysodeikticus established by crossed immunoelectrophoresis.

Crossed immunoelectrophoresis of Triton X-100-solubilized plasma membranes of Micrococcus lysodeikticus established the presence of 27 discrete antigens. Individual antigens were identified as membrane components possessing enzyme activity by zymogram staining procedures and by reactivity of certain antigens with a selection of four lectins in the crossed-immunoelectrophoresis (immunoaffinoelectrophoresis) system. Absorption experiments with intact, stable protoplasts and isolated membranes established the asymmetric nature of the M. lysodeikticus plasma membranes. Of the 14 antigens with determinants accessible solely on the cytoplasmic face of the membrane, four possessed individual dehydrogenase activities, and a fifth was identifiable as a component possessing adenosine triphosphatase (EC 3.6.1.3) activity. Evidence from absorption studies with isolated membranes suggested that antigens such as the adenosine triphosphatase complex were more readily accessible to reaction with antibodies than was succinate dehydrogenase (EC 1.3.99.1), for example. Twelve antigens were located on the protoplast surface as determined by antibody absorption, and the succinylated lipomannan was identified as a major antigen. At least five other antigens possessed sugar residues that interacted with concanavalin A. With the antisera generated to isolated membranes, there was no evidence suggesting that any of these antigens was not detectable on either surface of the plasma membrane. From absorption experiments with washed, whole cells of M. lysodeikticus, it was concluded that the immunogens on the protoplast surface were also detectable on the surface of the intact cell. However, some of the components such as the succinylated lipomannan appeared to be exposed to a greater extent than others. The cytoplasmic fraction from M. lysodeikticus was used as an antigen source to generate antibodies, and 97 immunoprecipitates were resolvable by crossed immunoelectrophoresis. In the cytoplasm-anticytoplasm reference immunoelectrophoresis pattern of precipitates, three of the immunoprecipitates unique to the cytoplasmic fraction were identifiable by zymogram staining procedures as catalase (EC 1.11.1.6), isocitrate dehydrogenase (EC 1.1.1.42), and polynucleotide phosphorylase (EC 2.3.7.8). The identification of membrane and cytoplasmic antigens (including the above-mentioned enzymes) provides a sensitive analytical system for monitoring cross-contamination and antigen distribution in cellular fractions.

Adenosine Triphosphatases↗
PubMed1977

Identification and quantitation of solubilized I blood group substance by wheat germ agglutinin using quantitative immunoelectrophoresis.

Wheat germ agglutinin (WGA) has been shown to react specifically with solubilized I blood group substance, purified from papain treated human erythrocyte membranes. WGA and I react to form an affinity precipitate in immunodiffusion gels, a reaction which can be blocked by the incorporation of N-acetyl glucosamine into the gel. The I material was a strong inhibitor of both anti-I cold hemagglutination and WGA hemagglutination reactions. Utilizing the techniques of crossed immunoelectrophoresis we have clearly established that WGA and anti-I IgM cold antibody are reacting with the same membrane macromolecule (I antigen). WGA was then used in a rocket affinoelectrophoretic assay system to quantitate I substance. The limits of detection in this system was 25 ng.

Acetylglucosamine↗
PubMed1975

Effects of increased mechanical work by isolated perfused rat heart during production or uptake of ketone bodies. Assessment of mitochondrial oxidized to reduced free nicotinamide-adenine dinucleotide ratios and oxaloacetate concentrations.

Metabolic effects of increased mechanical work were studied by comparing isolated pumping rat hearts perfused by the atrial-filling technique with aortic-perfused non-pumping hearts perfused by the technique of Langendorff. The initial medium usually contained glucose (11 mm) and palmitate (0.6 mm bound to 0.1 mm albumin). During increased heart work (comparing pumping with non-pumping hearts) the uptake of oxygen and glucose increased threefold, but that of free fatty acids was unchanged. Tissue contents of alpha-oxoglutarate, NH4+, malate, lactate, pyruvate and Pi rose with increased heart work, but contents of ATP, phosphocreatine and citrate fell. Ketone bodies were produced with a ratio of beta-hydroxybutyrate/acetoacetate of about 3:1 in both pumping and non-pumping hearts but with higher net production rates in non-pumping hearts. When ketone bodies were added in relatively high concentrations (total 4 mm) to a glucose (11 mm) medium the medium, ratios of beta-hydroxybutyrate/acetoacetate were not steady even after 60 min of perfusion. The validity of calculating mitochondrial free NAD+/NADH ratios from the tissue contents of the reactants of the glutamate dehydrogenase system or the beta-hydroxybutyrate dehydrogenase system is assessed. The activities of these enzymes are considerably less in the rat heart than in the rat liver, introducing reservations into the application to the heart of the principles used by Williamson et al. (1967) for calculation of mitochondrial free NAD+/NADH ratios of liver mitochondria...

Acetoacetates↗
PubMed1975

Antigenic and enzymatic architecture of Micrococcus lysodeikticus membranes established by crossed immunoelectrophoresis.

By crossed immunoelectrophoresis with membrane antiserum, 17 antigens have been detected in fractions from plasma membranes of M. lysodeikticus solubilized with Triton X-100. Absorption tests with protoplasts have demonstrated that eight of the antigens are expressed on the surface. Of these antigens the major one has been identified as a succinylated mannan. Five of the principal immunoprecipitates unaffected by absorption with protoplasts were shown by zymograms to possess the following enzymic activites: succinate dehydrogenase (EC 1.3.99.1), ATPase (EC 3.6.1.3), NADH dehyrogenase (EC 1.6.99.3)(two separate components), and malate dehydrogenase (EC 1.1.1.37). These enzymes or enzyme-complexes are, therefore, not expressed on the outer surface of the protoplast membrane.

Adenosine Triphosphatases↗
PubMed1975

Effects of glucose, insulin and potassium infusion on tissue metabolic changes within first hour of myocardial infarction in the baboon.

The effects of infusions of glucose, insulin and potassium (GIK) on the heart tissue metabolic changes found in adult baboons 60 min after coronary artery ligation were studied. Biopsies taken from 11 baboons without coronary artery ligation gave control values. A second group of 46 baboons had coronary artery ligation. A third group of 17 baboons received an infusion of KCl after coronary artery ligation. A fourth group of 26 baboons received infusion of GIK. Coronary artery ligation resulted in the expected fall of ATP, creatine phosphate, glycogen, tissue (K+/Na+) ratio, and tissue pH, and rise of inorganic phosphate, lactare, lactate/pyruvate ratio and alpha-glycerophosphate in the infarction zones. Compared with ligation, additional infusions of GIK approximately doubled the contents of creatine phosphate and glycogen in the infarct zones, increased the content of ATP in the central infarct zone, and decreased the content of inorganic phosphate in the peripheral infarct zone. Other GIK effects were that the tissue (K+/Na+) ratio rose in the peripheral infarct zone, and the content of both glycogen and lactate rose in the peri-infarct and non-ischemic zones; the pH of tissue homogenates did not decrease. KCl infusions had few effects compared with the ligation group. GIK infusions exerted a beneficial effect when compared with infusions of KCl in that tissue creatine phosphate rose in the peripheral infarct and nonischemic zones; the tissue K+/Na+ ratio rose in the peripheral infarct, peri-infarct, and nonischemic zones; and the lactate/pyruvate ratio fell in the infarct zone. It is proposed that GIK counteracted early tissue metabolic deterioration in the infarcting baboon heart.

Adenosine Triphosphate↗
PubMed1975

Estimated glycolytic flux in infarcting heart.

Regional ischemia results in infarction even in the presence of residual oxidative metabolism. Although glycolytic flux is relatively inhibited at the level of phosphofructokinase, glucose competes more effectively than does free fatty acid for the residual oxygen supply. Glycogen is not the major energy source until effective collateral flow is virtually zero.

Adenosine Triphosphate↗