Direct Action of Insulin on Plasma Membrane ATPase Activity in Human Lymphocytes

LITTLE is known about the effects of insulin on lymphocytes. Helmreich and Eisen¹ concluded that it has insignificant effects, but others^2–5 have made a case for a role in inflammatory and immunological responses. We^6,7 have demonstrated that noradrenaline enhances the uptake of both glucose and potassium by lymphocytes, as does insulin in several tissues. We have associated this action of noradrenaline with a direct effect on membrane adenosine triphosphatase (ATPase) activity⁷. The observation⁸ that insulin bound to ‘Sepharose’ polymers enhances glucose transport while in contact only with the plasma membrane indicated that insulin might have a direct action similar to that of noradrenaline on membrane ATPase. The observations reported here show that insulin stimulates ATPase activity and glucose uptake in the lymphocyte and suggest a relationship between membrane ATPase activity and glucose transport.     

Change in Target Molecular Size of the Red Beet Plasma Membrane ATPase during Solubilization and Reconstitution

The plasma membrane ATPase from red beet (Beta vulgaris L.) storage tissue associated with either native plasma membrane vesicles, a detergent-solubilized enzyme preparation or reconstituted liposomes was subjected to radiation inactivation analysis to determine if changes in target molecular size occurred with modification of its amphipathic environment. For each preparation of the enzyme, the decline in ATP hydrolytic activity with increasing dose of γ-ray radiation demonstrated a simple exponential profile indicating the presence of a single target size. Analysis of the radiation inactivation profiles for the plasma membrane associated, solubilized, and reconstituted enzyme revealed target molecular sizes of 225 kilodaltons (kD), 129 kD, and 218 kD, respectively. These results suggest that the plasma membrane associated and reconstituted ATPase preparations consist of enzyme present as a dimer of 100 kD subunits while the solubilized enzyme is present in the monomeric form. These results also indicate that the 100 kD catalytic subunit most likely represents the minimal unit of ATP hydrolytic activity.     

Evidence for a beta-Aspartyl Phosphate Residue in the Phosphorylated Intermediate of the Red Beet Plasma Membrane ATPase

A borohydride reduction method was used to identify the phosphorylated amino acid in the phospho-enzyme of the red beet (Beta vulgaris L.) plasma membrane ATPase. Plasma membrane fractions were phosphorylated with unlabeled ATP in the presence of MgSO₄ at pH 6.5 and then treated with sodium [³H]borohydride. The borohydride-treated samples were subjected to hydrolysis in 6 normal HCl at 110°C for 22 hours and then analyzed by high voltage paper electrophoresis and thin layer chromatography. This analysis demonstrated the formation of labeled homoserine as the major reduction product when phosphorylated membrane samples were treated with sodium [³H]borohydride. This suggests that the phosphoryl group in the plasma membrane ATPase of red beet storage tissue is attached to the β-carboxyl side chain of an aspartic acid residue in the active site of the enzyme.     

Chemical Equivalence of Phosphoenzyme Reaction States in the Catalytic Mechanism of the Red Beet (Beta vulgaris L.) Plasma Membrane ATPase

A comparison of two phosphoryl enzyme reaction states associated with the plasma membrane ATPase of red beet (Beta vulgaris L.) storage tissue was carried out to determine if their differences in reactivity toward ADP and K⁺ was related to a structural difference in the site of phosphorylation. Using a pulse labeling method it was possible to produce preparations where either the ADP-sensitive and -insensitive phosphoenzyme forms or the ADP-insensitive phosphoenzyme form alone were trapped as trichloroacetic acid denatured protein. Following complete digestion with Pronase, both preparations yielded radioactive tripeptides with similar properties with respect to pH stability of the covalent bond linking the phosphate to the peptide, isoelectric point, and migration on cellulose thin layer plates. Since the preparation containing both intermediate reaction states behaved in a uniform manner during analysis and displayed properties similar to the preparation containing only the ADP-insensitive phosphoenzyme form, it was proposed that both phosphoenzyme forms were chemically equivalent and derived from the same region of the catalytic active site. The observation that ethyleneimine treatment of both preparations followed by trypsin digestion resulted in the production of tripeptides similar to the Pronase fragments would support this proposal since it suggests that the tripeptides from both phosphoenyzme states contain a lysine residue on the C terminal end and are adjacent to a cysteine residue on the N-terminal end. The chemical equivalence of these two phosphoenzyme reaction states suggests that their differences in reactivity toward ligands may be related to conformational changes associated with the catalytic and transport mechanism of this enzyme.     

Chemical Properties of Syringomycin and Syringotoxin: Toxigenic Peptides Produced by Pseudomonas syringae

Syringomycin (SR) and syringotoxin (ST), wide spectrum antibiotics and phytotoxins isolated from ecotypic strains of Pseudomonas syringa , were purified to homogeneity and compared for their physicochemical properties. Acid hydrolysates of SR and ST were analysed for ninhydrin-reactive components by paper chromatography and the Durrum single-column method of amino acid analysis. Both active and base inactivated preparations of SR yielded substances tentatively identified as serine, phenylalanine, an unidentified basic amino acid, and arginine in a 2:1:2:1 mole ratio, respectively. Preparations of SR from ecotypic strains of P. syringae from pear, peach and millet hosts, had an identical amino acid composition which appeared to exclude a potential role of SR in the plant host specificity of P. syringae . ST isolated from a strain of P. syringae from a citrus host, contained substances tentatively identified as threonine, serine, glycine, ornithine, and the same unidentified basic amino acid found in SR in a 1:1:1:1:1 mole ratio. Although autoradiographs of paper chromatograms of acid hydrolysates of ¹⁴C-SR and various chromogenic reagents did not indicate the presence of substances other than amino acids, the nitrogen content of SR by combustion analyses was lower than expected which suggested the possible presence of another component.     

Modification of the Red Beet Plasma Membrane H-ATPase by Diethylpyrocarbonate

Incubation of the red beet (Beta vulgaris L.) plasma membrane H⁺-ATPase with micromolar concentrations of diethylpyrocarbonate (DEPC) resulted in inhibition of both ATP hydrolytic and proton pumping activity. Enzyme activity was restored when DEPC-modified protein was incubated with hydroxylamine, suggesting specific modification of histidine residues. Kinetic analyses of DEPC inhibition performed on both membrane-bound and solubilized enzyme preparations suggested the presence of at least one essential histidine moiety per active site. Inclusion of either ATP (substrate) or ADP (product and competitive inhibitor) in the modification medium reduced the amount of inhibition observed in the presence of DEPC. However, protection was not entirely effective in returning activity to noninhibited control values. These results suggest that the modified histidine does not reside directly in the ATP binding region of the enzyme, but is more likely involved in enzyme regulation through subtle conformational effects.     

Analysis of Sweet cherry (Prunus avium L.) Leaves for Plant Signal Molecules That Activate the syrB Gene Required for Synthesis of the Phytotoxin,Syringomycin, by Pseudomonas syringae pv syringae

An important aspect of the interaction of Pseudomonas syringae pv syringae with plant hosts is the perception of plant signal molecules that regulate expression of genes, such as syrB, required for synthesis of the phytotoxin, syringomycin. In this study, the leaves of sweet cherry (Prunus avium L.) were analyzed to determine the nature of the syrB-inducing activity associated with tissues of a susceptible host. Crude leaf extracts yielded high amounts of total signal activity of more than 12,000 units g-1 (fresh weight) based on activation of a syrB-lacZ fusion in strain B3AR132. The signal activity was fractionated by C18 reversed-phase high-performance liquid chromatography and found to be composed of phenolic glycosides, which were resolved in three regions of the high-performance liquid chromatography profile, and sugars, which eluted with the void volume. Two flavonol glycosides, quercetin 3-rutinosyl-4[prime]-glucoside and kaempferol 3-rutinosyl-4[prime]-glucoside, and a flavanone glucoside, dihydrowogonin 7-glucoside, were identified. The flavonoid glycosides displayed similar specific signal activities and were comparable in signal activity to arbutin, a phenyl [beta]-glucoside, giving rise to between 120 and 160 units of [beta]-galactosidase activity at 10 [mu]M. Although D-fructose exhibits intrinsic low level syrB-inducing signal activity, D-fructose enhanced by about 10-fold the signal activities of the flavonoid glycosides at low concentrations (e.g. 10 [mu]M). This demonstrates that flavonoid glycosides, which represent a new class of phenolic plant signals sensed by P. s. syringae, are in sufficient quantities in the leaves of P. avium to activate phytotoxin synthesis.     

Evaluation of the Role of Syringomycin in Plant Pathogenesis by Using Tn5 Mutants of Pseudomonas syringae pv. syringae Defective in Syringomycin Production

Syringomycin is a necrosis-inducing phytotoxin produced by Pseudomonas syringae pv. syringae. To determine whether syringomycin production is a determinant in virulence or pathogenicity, we isolated nontoxigenic (Tox⁻) Tn5-containing mutants and then quantitatively evaluated them for the ability to multiply and cause disease in immature sweet-cherry fruits. Transposon Tn5 was delivered to Tox⁺ strain B301D-R by using the suicide vector, pGS9, and the resultant kanamycin-resistant (Km^r) colonies were screened for changes in syringomycin production by testing for antibiosis against Geotrichum candidum. Southern blot analysis of KpnI-and EcoRI-digested DNA showed that 15 (0.3%) Tox⁻ mutants were isolated which had Tn5 inserted into 1 of 14 distinct loci. Phenotypic characterization of the Tox⁻ mutants identified three major groups, which were differentiated by pathogenicity and ability to cause a tobacco hypersensitive reaction (HR). The eight strains in group A were pathogenic (Path⁺) in cherry fruit assays, but the disease index was 17 to 66% lower (significant at P = 0.01) than for the parental Tox⁺ strain, B301D-R. The population dynamics of group A strains W4S770 and W4S116 in cherry fruits were, however, indistinguishable from that of strain B301D-R. The remaining seven Tox⁻ strains were nonpathogenic; group B strain W4S2545 (Path⁻ HR⁺) and group C strain W4S468 (Path⁻ HR⁻) developed significantly lower populations (10⁵ to 10⁷ CFU per cherry fruit) 3 days after inoculation than strain B301D-R did (nearly 10⁹ CFU per fruit). The data indicate that syringomycin is not essential for pathogenicity, but contributes significantly to virulence.     

Photoinactivation of Detergent-Solubilized Plasma Membrane ATPase from Rosa damascena: Action Spectra

The photochemistry of vesicular and detergent-solubilized preparations of plasma membrane-associated ATPase was investigated in Rosa damascena. The cholate-solubilized ATPase activity fractionated into two peaks on a Sephadex G-150 column with simple, but different ultraviolet (UV) sensitivities. The larger enzyme was UV sensitive; the smaller enzyme was relatively insensitive. The activity of both ATPase fractions depended on environment: both were inactive in cholate, relatively inactive in phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, and active in phosphatidylglycerol and phosphatidylserine. The UV sensitivities of both fractions also depended on their environment. For the UV sensitive fraction, the action spectrum differed in the 300 to 400 nanometers range when the fraction was irradiated with and without lipids. For the resistant fraction, UV sensitivity at 290 nanometers differed (up to 6-fold) in different lipids. The resistant fraction solubilized in octylglucoside had an action spectrum very different from that in cholate or in lipid vesicles. The absorption spectra of the different preparations reflected the action spectra. For both UV sensitive and insensitive fractions, the action spectra for photoinactivation had peaks at 290 nanometers, suggesting that the chromophores were tryptophanyl residues. The loss of ATPase activity was strictly correlated with the loss of fluorescence from tryptophan in the partially purified enzymes. Cs⁺ protected the UV sensitive activity but not the insensitive one. We propose a model which explains the difference in UV sensitivities based on the positions of the tryptophan residues in the two proteins.     

Syringomycin-Stimulated Phosphorylation of the Plasma Membrane H-ATPase from Red Beet Storage Tissue

The syringomycin-stimulated in vitro protein phosphorylation of the plasma membrane H⁺-ATPase of red beet (Beta vulgaris L.) storage tissue was investigated. Peptides representing the H⁺-ATPase N and C termini and nucleotide binding site (P-2, P-3, and P-1, respectively) were synthesized, and rabbit antisera against each were produced. In western immunoblots of purified plasma membranes, these antisera immunoreacted with the 100-kilodalton polypeptide of the H⁺-ATPase and with other smaller polypeptides. The smaller polypeptides appeared to be degraded forms of the intact 100-kilodalton polypeptide. Immunoprecipitation experiments showed that plasma membranes treated with syringomycin had increased protein phosphorylation rates of the 100-kilodalton polypeptide. Optimal phosphorylation levels were achieved with 25 micromolar free Ca²⁺. Phosphoserine and phosphothreonine were detected in the immunoprecipitates. Washed immunoprecipitates generated with anti-P-1 possessed protein phosphorylation activity. This immunoprecipitate activity was not stimulated by syringomycin, but it was inhibited when plasma membranes were treated with sodium deoxycholate before immunoprecipitation. The findings show that syringomycin stimulates the phosphorylation of the plasma membrane H⁺-ATPase and that specific protein kinase(s) are probably associated with the enzyme.     

Characterization of the argA Gene Required for Arginine Biosynthesis and Syringomycin Production by Pseudomonas syringae pv. syringae

Two types of necrosis-inducing lipodepsipeptide toxins, called syringomycin and syringopeptin, are major virulence factors of Pseudomonas syringae pv. syringae strain B301D. A previous study showed that a locus, called syrA, was required for both syringomycin production and plant pathogenicity, and the syrA locus was speculated to encode a regulator of toxin production. In this study, sequence analysis of the 8-kb genomic DNA fragment that complements the syrA phenotype revealed high conservation among a broad spectrum of fluorescent pseudomonads. The putative protein encoded by open reading frame 4 (ORF4) (1,299 bp) in the syrA locus region exhibited 85% identity to ArgA, which is involved in arginine biosynthesis in Pseudomonas aeruginosa. Growth of strain W4S2545, the syrA mutant, required supplementation of N minimal medium with arginine. Similarly, syringomycin production of syrA mutant W4S2545 was restored by the addition of arginine to culture media. Furthermore, the insertion of Tn5 in the genome of the syrA mutant W4S2545 was localized between nucleotides 146 and 147 in ORF4, and syringomycin production was complemented in trans with the wild-type DNA fragment containing intact ORF4. These results demonstrate that the syrA locus is the argA gene of P. syringae pv. syringae and that argA is directly involved in arginine biosynthesis and therefore indirectly affects syringomycin production because of arginine deficiency.     

Plasma Membrane ATPase Activity following Reversible and Irreversible Freezing Injury

Plasma membrane ATPase has been proposed as a site of functional alteration during early stages of freezing injury. To test this, plasma membrane was purified from Solanum leaflets by a single step partitioning of microsomes in a dextran-polyethylene glycol two phase system. Addition of lysolecithin in the ATPase assay produced up to 10-fold increase in ATPase activity. ATPase activity was specific for ATP with a K_m around 0.4 millimolar. Presence of the ATPase enzyme was identified by immunoblotting with oat ATPase antibodies. Using the phase partitioning method, plasma membrane was isolated from Solanum commersonii leaflets which had four different degrees of freezing damage, namely, slight (reversible), partial (partially reversible), substantial and total (irreversible). With slight (reversible) damage the plasma membrane ATPase specific activity increased 1.5- to 2-fold and its K_m was decreased by about 3-fold, whereas the specific activity of cytochrome c reductase and cytochrome c oxidase in the microsomes were not different from the control. However, with substantial (lethal, irreversible) damage, there was a loss of membrane protein, decrease in plasma membrane ATPase specific activity and decrease in K_m, while cytochrome c oxidase and cytochrome c reductase were unaffected. These results support the hypothesis that plasma membrane ATPase is altered by slight freeze-thaw stress.     

Characterization of ATPase Activity Associated with Plasma Membrane from Vigna Hypocotyls

A plasma membrane fraction was isolated from the hypocotylsof cowpea {Vigna unguiculata) by a combination of differentialcentrifugation and sucrose density gradient centrifugation.The ATPase activity of this fraction was dependent on divalentcations (Mn²⁺>Mg²⁺>Co²⁺>Ca²⁺>Fe²⁺>Zn²⁺>Ni²⁺)but was not further stimulated by monovalent cations (K⁺ and/orNa⁺). The pH optimum for the activation of ATPase by Mg²⁺ was7.0. This fraction hydrolyzed ATP or UTP as a substrate andthe ATPase activity obeyed a Michaelis-Menten type of kinetics.The K_m for MgATP ranged from 0.65 to 1.1 mM. The ATPase activitywas inhibited by inhibitors such as N, N'- dicyclohexylcarbodiimide,diethylstilbestrol and triphenyltin chloride, all of which arereported to block proton (H⁺) transport in plant cells, butwas insensitive to those of mitochondrial ATPase such as oligomycinand sodium azide. The ATPase activity was not stimulated bytreatment with ionophores (e.g., carbonyl cyanide p-trifluoromethoxyphenylhydrazone,3,5-di-ter-butyl-4-hydroxybenzilidenemalononitrile and valinomycin⁺KCl)which would be expected to dissipate the electrochemical potentialdifference of H⁺ or the membrane potential difference. The characteristics of the ATPase are compared with those ofplasma membrane ATPases of other plants and its possible rolein H⁺-transport is discussed. ¹ Present address: Institute of Applied Biochemistry, Yagi MemorialPark, Mitake, Gifu 505-01, Japan or Laboratory for Plant EcologicalStudies, Faculty of Science, Kyoto University, Kyoto 606, Japan. (Received April 20, 1984; Accepted August 14, 1984)     

Effect of Arsenic on Bacterial Growth and Plasma Membrane ATPase Activity

The effects of arsenic in the forms of arsenite and arsenate on bacterial growth and plasma membranes' ATPase activity was studied. Correlation of the rate of ATP hydrolysis was found to be correlated with bacterial resistance to toxic arsenic ions. Detoxification of arsenate by resistant cultures of bacteria was suggested to be related to an increase in bacterial ATPase activity and the degree of ATPase mobilization.     

Molecular and Physiological Characterization of Pseudomonas syringae pv. tomato and Pseudomonas syringae pv. maculicola Strains That Produce the Phytotoxin Coronatine

The chlorosis-inducing phytotoxin coronatine is produced by several Pseudomonas syringae pathovars, including glycinea, morsprunorum, atropurpurea, and the closely related tomato and maculicola. To date, all coronatine-producing pv. glycinea, morsprunorum, and atropurpurea strains that have been examined carry the gene cluster that controls toxin production on a large plasmid. In the present study the genomic location of the coronatine gene cluster was determined for coronatine-producing strains of the pv. tomato-maculicola group by subjecting their genomic DNA to pulsed-field electrophoresis and Southern blot analysis with a hybridization probe from the coronatine gene cluster. The cluster was chromosomally borne in 10 of the 22 strains screened. These 10 strains infected both crucifers and tomatoes but could not use sorbitol as a sole source of carbon. The remaining 12 coronatine-producing strains had plasmid-borne toxin gene clusters and used sorbitol as a carbon source. Only one of these strains was pathogenic on both crucifers and tomatoes; the remainder infected just tomatoes. Restriction fragment length polymorphism analysis of the pv. tomato-maculicola coronatine gene clusters was performed with probes from P. syringae pv. tomato DC3000, a tomato and crucifer pathogen. Although the coronatine cluster appeared, in general, to be highly conserved across the pv. tomato-maculicola group, there were significant differences between plasmid-borne and chromosomally borne genes. The extensively studied coronatine cluster of pv. glycinea 4180 closely resembled the plasmid-borne clusters of the pv. tomato-maculicola group.     

Phytotoxin production by Pseudomonas syringae pv. syringae: Syringopeptin production by syr mutants defective in biosynthesis or secretion of syringomycin

Abstract Syringomycin and syringopeptin are lipodepsipeptide phytotoxins produced by Pseudomonas syringae pv. syringae . Four syr genes were identified previously and hypothesized to be involved in the regulation ( syrA ), biosynthesis ( syrB and syrC ), or export ( syrD ) of syringomycin. This study determines the influence of syr mutations on the composition of phytotoxic metabolites produced by P. syringae pv. syringae strain B301D-R. Levels of syringomycin and syringopeptin produced in liquid cultures were estimated by reverse phase HPLC analyses and differential antimicrobial assays. Significant quantities of syringopeptin were produced by both syrB and syrC mutants despite their inability to produce syringomycin. Only trace quantities of both lipodepsipeptides were produced by syrA and syrD mutants of P. syringae pv. syringae . These results indicate that syringomycin and syringopeptin are synthesized by separate pathways, but may share common mechanisms for secretion and regulation.