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PubMed1994

Choleragen catalyzes ADP-ribosylation of the stimulatory G protein heterotrimer but not its free alpha-subunit.

The heterotrimeric (alpha beta gamma) stimulatory G protein (Gs) mediates activation of adenylylcyclase. Gs is inactive when GDP is bound to the guanine nucleotide binding site of the alpha-subunit (Gs alpha). Gs can be activated by fluoroaluminate or by binding GTP or GTP analogues, (e.g., GTP gamma S) in place of GDP. Magnesium ion is also required for the activation of Gs, and Gs alpha is a substrate for ADP-ribosylation catalyzed by choleragen (CT). Gs activation can also be accompanied by dissociation of Gs alpha from the G beta gamma-subunit complex. When dissociated Gs subunits were separated by chromatography, isolated Gs alpha could not be ADP-ribosylated by CT unless G beta was added back. RM/1 antiserum against Gs alpha was used to immunoprecipitate Gs, and the subunit composition of the immunoprecipitate was determined. When Gs was incubated with 2 mM MgCl2, the Gs heterotrimer was immunoprecipitated, and Gs alpha could be ADP-ribosylated by CT. Activation of Gs with GTP gamma S or fluoroaluminate in the presence of 2 mM MgCl2 did not cause Gs subunit dissociation nor did it affect the ability of Gs alpha to be ADP-ribosylated by CT. MgCl2 caused a dose-dependent decrease in the amount of G beta that coprecipitated with Gs alpha in the absence as well as the presence of GTP gamma S or fluoroaluminate. Gs subunit dissociation was accompanied by a corresponding decrease in CT-catalyzed ADP-ribosylation of Gs alpha regardless of whether or not GTP gamma S or fluoroaluminate was bound to Gs alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate Ribose↗

PubMed2003

Three regulatory regions of the Aedes aegypti glutamine synthetase gene differentially regulate expression: identification of a crucial regulator in the first exon.

Aedes aegyptiglutamine synthetase (GS) is expressed constitutively at various developmental stages and its relative mRNA abundance increases in the midgut following blood feeding in support of the biosynthesis of chitin, a component of the peritrophic matrix. To understand the regulation of GS expression better, GS-luciferase reporter fusion genes were constructed and analysed in transiently transfected C6/36 cells. These studies have identified three GS regions: GS-A, -B and -C (C1, C2) that are required for efficient transcription. The crucial regulatory DNA sequence is located within 140 nucleotides of the GS-C region in the first exon. GS-B region between -209 and +4 contains a negative modulator that represses transcription of the GS-C promoter, but the 5'-GS-A region, between -476 and -282, can negate the transcription inhibition of GS-B and promote GS transcription of the GS-C promoter. Electrophoretic mobility shift assays showed that nuclear proteins for GS-A, GS-B and GS-C1 are present in the C6/36 cells, and therefore that GS-A, GS-B and GS-C1 indeed possess regulatory function. By contrast, nuclear proteins isolated from both cultured cells and midgut tissues bound to GS-C2, suggesting that GS-C2 plays an important role in GS transcription and that GS-C2 is regulated by several different and redundant transcription factors to achieve constitutive expression in a wide variety of tissues.

Aedes↗

PubMed1979

Glutamine synthetase regulation, adenylylation state, and strain specificity analyzed by polyacrylamide gel electrophoresis.

We used polyacrylamide gel electrophoresis to examine the regulation and adenylylation states of glutamine synthetases (GSs) from Escherichia coli (GS(E)) and Klebsiella aerogenes (GS(K)). In gels containing sodium dodecyl sulfate (SDS), we found that GS(K) had a mobility which differed significantly from that of GS(E). In addition, for both GS(K) and GS(E), adenylylated subunits (GS(K)-adenosine 5'-monophosphate [AMP] and GS(E)-AMP) had lesser mobilities in SDS gels than did the corresponding non-adenylylated subunits. The order of mobilities was GS(K)-AMP < GS(K) < GS(E)-AMP < GS(E). We were able to detect these mobility differences with purified and partially purified preparations of GS, crude cell extracts, and whole cell lysates. SDS gel electrophoresis thus provided a means of estimating the adenylylation state and the quantity of GS present independent of enzymatic activity measurements and of determining the strain origin. Using SDS gels, we showed that: (i) the constitutively produced GS in strains carrying the glnA4 allele was mostly adenylylated, (ii) the GS-like polypeptide produced by strains carrying the glnA51 allele was indistinguishable from wild-type GS(K), and (iii) strains carrying the glnA10 allele contained no polypeptide having the mobility of GS(K) or GS(K)-AMP. Using native polyacrylamide gels, we detected the increased amount of dodecameric GS present in cells grown under nitrogen limitation compared with cells grown under conditions of nitrogen excess. In native gels there was neither a significant difference in the mobilities of adenylylated and non-adenylylated GSs nor a GS-like protein in cells carrying the glnA10 allele.

Adenosine Monophosphate↗

PubMed2000

Control of gentamicin release from a calcium phosphate cement by admixed poly(acrylic acid).

The aim of this work was to develop a calcium phosphate cement (CPC) providing controlled release of the antibiotic gentamicin sulfate (GS) over at least 1 week. The CPC was made of beta-tricalcium phosphate [beta-TCP; beta-Ca(3)(PO(4))(2)], monocalcium phosphate monohydrate [MCPM; Ca(H(2)PO(4))(2). H(2)O] and water. Release of GS was controlled by admixture of poly(acrylic acid) (PAA). The effects on the GS release kinetics of the molecular weight of PAA, of the amount of admixed PAA, and of the pH of the release medium were investigated. A typical cement sample weighed 3.6 g and contained 100 mg of GS and between 0 and 150 mg of PAA. In the following, PAA content is expressed as the weight ratio, lambda, with respect to GS. At a low PAA content in the CPC (lambda < 0.7), GS was released over 1-2 days according to a square-root-of-time kinetics, but not all GS was released. The unreleased GS fraction increased from 0 to 58% with an increase of PAA content (up to lambda = 0.7). At high PAA content (lambda > 0.7), GS was released over a period of up to 8 days according to a combination of a square-root-of-time and a zero-order kinetics. The total GS fraction released increased again from 58 to 100% with an increase of the amount of PAA (up to lambda = 1.5). These observations were explained by molecular interaction between PAA and GS resulting in gel formation. The maximum fraction of GS released from the cement was indeed a function of the solubility of the PAA-GS (coacervate) complex in the release medium. Thus, GS release was controlled by two mechanisms: (1) diffusion of free GS molecules through the porous cement (square-root-of-time kinetics); and (2) dissociation of GS from the PAA-GS complex (zero-order kinetics). The first mechanism was predominant at low lambda, whereas the second mechanism became important at high lambda and later release times. As the solubility of the PAA-GS complex decreased with an increase in PAA molecular weight, the higher molecular weight PAA yielded more prolonged release periods of up to 8 days. Interestingly, the use of 450 kDa PAA at lambda = 1.00 provided an almost constant release profile over a period of 7 days. Gel formation between PAA and GS was explained in terms of hydrogen bonding of PAA carboxyl groups with GS amino groups. The molar ratio between carboxyl groups and amino groups in the gel was estimated to be approximately 1.9. In conclusion, admixture of PAA into calcium phosphate cement appeared to be a very elegant tool to control the release of the antibiotic over a period of 7 to 8 days.

Acrylic Resins↗

PubMed1990

Immunochemical analysis of the alpha-subunit of the stimulatory G-protein of adenylyl cyclase in patients with Albright's hereditary osteodystrophy.

Albright's hereditary osteodystrophy (AHO) is an autosomal dominant disorder characterized by an unusual phenotypic appearance and reduced biological activity of the alpha-subunit of the stimulatory G-protein of adenylyl cyclase (Gs alpha). In most AHO patients deficient Gs alpha activity is associated with generalized target organ resistance to hormones that act via stimulation of adenylyl cyclase. This form of the disorder is termed pseudohypoparathyroidism type Ia (PHP Ia). By contrast, other patients with Gs alpha deficiency fail to demonstrate clinical evidence of hormone resistance and are considered to have the related disorder pseudopseudohypoparathyroidism (pseudoPHP). Previous studies demonstrating deficient Gs alpha bioactivity in cell membranes from patients with AHO used functional assays that were unable to distinguish between reduced amounts of normal Gs alpha protein and normal amounts of defective Gs alpha protein. In the present study we used specific Gs alpha antisera to analyze immunoactive Gs alpha protein in erythrocyte and fibroblast membranes from 20 patients with AHO who had either normal or reduced levels of Gs alpha mRNA. Cell membranes were subjected to immunoblot analysis using Gs alpha antisera developed against synthetic peptides corresponding to amino acid sequences in the amino- or carboxyl-terminus of the Gs alpha molecule. Fibroblast membranes from patients with AHO who had reduced or normal levels of Gs alpha mRNA contained both the 45- and 52-kDa forms of the Gs alpha protein in quantities that were significantly less [mean +/- SE, 52 +/- 6%; (n = 8) for reduced mRNA and 35 +/- 19% (n = 2) for normal mRNA, percentage of control values] than those present in membranes from normal subjects. Similar reductions were found in the level of the 45-kDa form of Gs alpha in erythrocyte membranes from all AHO patients studied [40 +/- 4% (mean +/- SE) of control values]. No abnormal forms of Gs alpha protein were detected. Cell membranes from patients with PHP type Ia and from patients with pseudoPHP contained levels of immunoactive Gs alpha that were equivalently reduced (43 +/- 4% vs. 42 +/- 5%, respectively). By contrast, erythrocyte membranes from patients with PHP type Ib, who have normal Gs alpha activity, had normal levels of Gs alpha immunoactivity (101 +/- 7%). These results indicate that most patients with AHO have reduced levels of Gs alpha protein as the basis for deficient Gs alpha bioactivity.

Adenylyl Cyclases↗

PubMed1992

Nucleotide sequence of a tobacco cDNA encoding plastidic glutamine synthetase and light inducibility, organ specificity and diurnal rhythmicity in the expression of the corresponding genes of tobacco and tomato.

A full-length cDNA encoding glutamine synthetase (GS) was cloned from a lambda gt10 library of tobacco leaf RNA, and the nucleotide sequence was determined. An open reading frame accounting for a primary translation product consisting of 432 amino acids has been localized on the cDNA. The calculated molecular mass of the encoded protein is 47.2 kDa. The predicted amino acid sequence of this precursor shows higher homology to GS-2 protein sequences from other species than to a leaf GS-1 polypeptide sequence, indicating that the cDNA isolated encodes the chloroplastic isoform (GS-2) of tobacco GS. The presence of C- and N-terminal extensions which are characteristic of GS-2 proteins supports this conclusion. Genomic Southern blot analysis indicated that GS-2 is encoded by a single gene in the diploid genomes of both tomato and Nicotiana sylvestris, while two GS-2 genes are very likely present in the amphidiploid tobacco genome. Western blot analysis indicated that in etiolated and in green tomato cotyledons GS-2 subunits are represented by polypeptides of similar size, while in green tomato leaves an additional GS-2 polypeptide of higher apparent molecular weight is detectable. In contrast, tobacco GS-2 is composed of subunits of identical size in all organs examined. GS-2 transcripts and GS-2 proteins could be detected at high levels in the leaves of both tobacco or tomato. Lower amounts of GS-2 mRNA were detected in stems, corolla, and roots of tomato, but not in non-green organs of tobacco. The GS-2 transcript abundance exhibited a diurnal fluctuation in tomato leaves but not in tobacco leaves. White or red light stimulated the accumulation of GS-2 transcripts and GS-2 protein in etiolated tomato cotyledons. Far-red light cancelled this stimulation. The red light response of the GS-2 gene was reduced in etiolated seedlings of the phytochrome-deficient aurea mutant of tomato. These results indicate a phytochrome-mediated light stimulation of GS-2 gene expression during greening in tomato.

Amino Acid Sequence↗

PubMed1998

Glutamine synthetase isoenzymes, oligomers and subunits from hairy roots of Beta vulgaris L. var. lutea.

A cytosolic and a plastidic isoenzyme of glutamine synthetase (GS; EC 6.3.1.2) were separated from hairy roots of Beta vulgaris L. var. lutea. The predominant activity was that of cytosolic GS 1; the relative proportion of plastidic GS 2 activity changed, however, depending on the growth conditions. Maximum activity of both isoenzymes was measured after growth with NO3- as the major N-source. Growth with NH4+ as the sole N-source or growth in constant darkness resulted in a significant decrease in GS 1 activity, whereas GS 2 activity was much less effected and thus contributed as much as 25% of total root GS activity. The isoenzymes GS 1 and GS 2 were active both in the octameric and tetrameric states. Both oligomers of GS 2 and octameric GS 1 were active under all growth conditions applied whereas tetrameric GS 1 was not active when the roots were grown under light-dark changes with NO3- as the major N-source. The molecular masses of the subunits were identical for both isoenzymes. Glutamine synthetase 1 was composed of up to four different 38-kDa subunits and two different 41-kDa subunits; GS 2 was assembled from one type of 38-kDa subunit and one type of 41-kDa subunit. The GS 2 subunits were most probably identical to two of the GS 1 subunits. The subunit composition of GS 1, but not of GS 2, changed depending on the growth conditions of the roots. Changes in GS 1 subunit composition were correlated with changes in GS 1 activity. The different growth conditions induced the specific assembly of different GS 1 isoenzymes which could, however, not be separated by anion-exchange chromatography but became evident only after two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Biopolymers↗

PubMed1983

Messenger RNA for glutamine synthetase. Review article.

Of the various eucaryotic tissues, where glutamine synthetase (GS) mRNA and its regulation have been investigated, the induction of GS by glucocorticoids in the embryonic chick retina represents one of the systems most extensively studied. GS mRNA was first identified at the polysomal level by immunochemical precipitation of fractionated polysomes containing nascent GS chains with anti-GS gamma-globulin. The mRNA has been shown to be polyadenylated at the 3' end; on this basis, it has been partially purified from embryonic chick retina as well as from N. Crassa by chromatography on oligo(dT)-cellulose or poly(U)-sepharose and translated in cell-free protein synthesizing systems derived from wheat germ. Hormonal regulation of GS activity studied in the embryonic retina, hepatoma tissue culture cells, or in other tissues is always shown to be mediated by GS mRNA. In the retina, hydrocortisone (HC) elicits an age-related and transcription-dependent induction of GS by enhancing the level of GS mRNA in the polysomes through an increased supply of this mRNA from the nucleus. Comparative studies of three inhibitors of transcription, viz. actinomycin D, leucanthone and proflavine on the induction of GS by HC indicate that the latter inhibits GS mRNA selectively and reversibly with minimal effects on other RNA synthesis. Since proflavine acts by competing with HC-receptor binding sites in the nuclei, further studies on its interaction with the retina genome are likely to help identify the DNA sequences involved in the GS induction. In bacteria, studies on the genetics and physiology of various mutants with lesions in the structural gene for GS show that the transcription of the GS gene (gln A) is regulated both positively and negatively by GS and the product of another gene gln F. Purification of GS mRNA to homogeneity cloning of its cDNA and development of assay systems for cell-free transcription of GS are other studies likely to advance our knowledge on GS mRNA and its regulation.

Animals↗

PubMed1998

Different effects of Gsalpha splice variants on beta2-adrenoreceptor-mediated signaling. The Beta2-adrenoreceptor coupled to the long splice variant of Gsalpha has properties of a constitutively active receptor.

The beta2-adrenoreceptor (beta2AR) couples to the G-protein Gs to mediate adenylyl cyclase activation. The splice variants of Gs alpha differ by a 15-amino acid insert between the Ras-like domain and the alpha-helical domain. The long splice variant of Gs alpha (Gs alphaL) binds GDP with lower affinity than the short splice variant (Gs alphaS), but the impact of this difference on the interaction of Gs alpha with the beta 2AR is not known. We studied the beta2 AR/Gs alpha interaction using receptor/G-protein fusion proteins (beta2 AR Gs alphaS and beta2 AR Gs alphaL) expressed in Sf9 cells. Fusion of the beta2 AR to Gs alpha promotes efficient coupling as shown by high-affinity agonist binding and GTPase and adenylyl cyclase activation and ensures fixed stoichiometry between receptor and G-protein. Importantly, fusion does not change the fundamental properties of the beta2 AR or Gs alpha. The beta2 AR in beta2 AR Gs alphaL showed hallmarks of constitutive activity (increased potency and intrinsic activity of partial agonists, increased efficacy of inverse agonists, and increased basal GTPase activity) compared with the beta2 AR in beta2 AR Gs alphaS. The apparent constitutive activity of the beta2 AR in beta2 AR Gs alphaL may be due to the lower GDP affinity of Gs alphaL compared with Gs alphaS, i.e. Gs alphaL is more often nucleotide-free than Gs alphaS and, therefore, more frequently available to stabilize the beta2 AR in the active (R*) state. This study demonstrates that subtle structural differences between closely related G-protein alpha-subunits can have important consequences for the functional properties of a G-protein-coupled receptor.

Adrenergic beta-Agonists↗

PubMed1984

Interaction of substrates with glutamine synthetase after limited proteolysis.

Previous studies [Dautry-Varsat, A., Cohen, G. N., & Stadtman, E.R. (1979) J. Biol. Chem. 254, 3124-3128; Lei, M., Aebi, U., Heidner, E. G., & Eisenberg, D. (1979) J. Biol. Chem. 254, 3129-3134] have shown that Escherichia coli glutamine synthetase (GS) can be cleaved by proteases to form a limited digestion species called nicked glutamine synthetase (GS). The present study gives the amino acid sequence of the protease-sensitive region of glutamine synthetase. The present study also shows that GS is enzymatically active, but this activity is low compared to the activity of GS. The apparent Michaelis constant value for glutamate was 90 mM for GS as compared to 3 mM for GS, while the Michaelis constant values for ATP were similar for GS and GS*. The dissociation constant values for ATP, as determined by intrinsic fluorescence measurements, were similar for GS and GS*. Glutamate decreased the dissociation constant value of ATP for GS because of synergism between the two binding sites; glutamate did not decrease the dissociation constant value of ATP for GS*. The glutamate analogue methionine sulfoximine bound very tightly to GS and inactivated the enzyme in the presence of ATP. Methionine sulfoximine did not appear to bind to GS* and did not inactivate GS* in the presence of ATP. The ATP analogue 5'-[p-(fluorosulfonyl)benzoyl]adenosine bound to GS and inactivated the enzyme by forming a covalent bond with it. Glutamate accelerated this inactivation because of the synergism between the ATP and glutamate binding sites of GS.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗

PubMed2000

Good outcome of children with acute myeloid leukemia and t(8;21)(q22;q22), even when associated with granulocytic sarcoma: a report from a single institution in Argentina.

BACKGROUND: The association between t(8;21) and granulocytic sarcoma (GS) is well known, but to the authors' knowledge the prognostic significance of GS in these patients has not been defined clearly. METHODS: Between January 1990 and July 1999 174 children with acute myeloid leukemia were admitted to the study institution. Translocation (8;21) was identified in 20 patients (11.5%). Eighteen patients were evaluable for the current study and 8 presented with GS at the time of diagnosis (GS+). RESULTS: The authors defined two groups of patients: those who were GS+ and those who were GS-. One patient in the GS+ group and two patients in the GS- group died during the induction phase of the study. Complete remission was achieved in seven patients in the GS+ group and eight patients in the GS- group. Two patients developed a recurrence in the GS+ group as did one patient in the GS- group. The event free-survival probability (the standard error) was 58% (18%) in the GS+ group and 70% (14%) in the GS- group. Localization of GS was in only one site in seven patients and at multiple sites in one patient. Patients with an epidural mass received local radiotherapy (one patient) or surgery (two patients). Two of these patients developed paraplegia as sequelae: one patient after surgery and one patient after radiotherapy. One patient with orbital GS received local radiotherapy because of progressive proptosis. The remaining four patients had a complete resolution of the GS with chemotherapy only. CONCLUSIONS: In the current study of patients with t(8;21)(q22;q22), the presence of granulocytic sarcoma was not found to be an adverse prognostic factor. However, careful attention should be paid, especially to patients with an epidural site, to avoid sequelae. Chemotherapy appears to be the optimum treatment for these children.

Adolescent↗

PubMed2003

Altered kinetic properties of tyrosine-183 to cysteine mutation in glutamine synthetase of anabaena variabilis strain SA1 is responsible for excretion of ammonium ion produced by nitrogenase.

A L-methionine- D, L-sulfoximine-resistant mutant of the cyanobacterium Anabaena variabilis, strain SA1, excreted the ammonium ion generated from N(2) reduction. In order to determine the biochemical basis for the NH(4)(+)-excretion phenotype, glutamine synthetase (GS) was purified from both the parent strain SA0 and from the mutant. GS from strain SA0 (SA0-GS) had a pH optimum of 7.5, while the pH optimum for GS from strain SA1 (SA1-GS) was 6.8. SA1-GS required Mn(+2) for optimum activity, while SA0-GS was Mg(+2) dependent. SA0-GS had the following apparent K(m) values at pH 7.5: glutamate, 1.7 m M; NH(4)(+), 0.015 m M; ATP, 0.13 m M. The apparent K(m) for substrates was significantly higher for SA1-GS at its optimum pH (glutamate, 9.2 m M; NH(4)(+), 12.4 m M; ATP, 0.17 m M). The amino acids alanine, aspartate, cystine, glycine, and serine inhibited SA1-GS less severely than the SA0-GS. The nucleotide sequences of glnA (encoding glutamine synthetase) from strains SA0 and SA1 were identical except for a single nucleotide substitution that resulted in a Y183C mutation in SA1-GS. The kinetic properties of SA1-GS isolated from E. coli or Klebsiella oxytoca glnA mutants carrying the A. variabilis SA1 glnA gene were also similar to SA1-GS isolated from A. variabilis strain SA1. These results show that the NH(4)(+)-excretion phenotype of A. variabilis strain SA1 is a direct consequence of structural changes in SA1-GS induced by the Y183C mutation, which elevated the K(m) values for NH(4)(+) and glutamate, and thus limited the assimilation of NH(4)(+) generated by N(2) reduction. These properties and the altered divalent cation-mediated stability of A. variabilis SA1-GS demonstrate the importance of Y183 for NH(4)(+) binding and metal ion coordination.

Amino Acid Sequence↗

PubMed2003

The Gleason score shift: score four and seven years ago.

PURPOSE: There is some evidence that Gleason scores (GS) have shifted over time, although documentation of the extent to which this has occurred and its clinical significance is sparse. METHODS AND MATERIALS: Three types of analyses were performed to characterize the GS shift. First, the time-related changes in GS, T-stage category, and PSA for all 983 patients treated with conformal radiotherapy at our institution were examined between 1992 and 1997. Then a matched-pair analysis of patients treated without androgen deprivation was implemented. Patients were matched for grouped GS, grouped PSA, T category, and grouped radiation dose; 242 patients (Cohort 1) treated between 1992 and 1994 were compared to 242 patients treated between 1995 and 1997 (Cohort 2). Follow-up for each patient in Cohort 1 was truncated to match the length of follow-up for his counterpart in Cohort 2 (median follow-up 42 vs. 47 months). The ASTRO consensus definition of a rising PSA was used. Finally, the pathology slides of 106 patients treated with radiotherapy at another institution between 1987 and 1993 underwent a blind second review by one of the study pathologists (P.T.) in 1998. RESULTS: The percentages of prostate cancer patients treated with radiotherapy at our institution from 1992 to 1994 vs. 1995 to 1997 were GS 2-5: 49% vs. 16%, GS 6: 27% vs. 59%, GS 7: 20% vs. 19%, and GS > or =8: 8.3% vs. 6%. There was no clear difference in T category over time with 92-96% having T1-T2 disease. The percentage of patients with a PSA < or = 10 ng/mL rose rapidly from 35% in 1992 to 63% in 1994. In corroboration of these findings, the pathologic review of specimens from 106 patients at another institution yielded changes between the initial reading to the recent reading of 50% to 6% for GS 2-5, 22% to 31% for GS 6, 16% to 25% for GS 7, and 12% to 25% for GS 8-10 (p < 0.0001). From the matched-pair analysis, 5-year Kaplan-Meier bNED rates were 68% and 82% (p = 0.03) favoring Cohort 2. CONCLUSIONS: During the study period, pretreatment PSA values declined and T category remained stable; yet, GS increased. Upgrading of GS would be expected to lead to an improvement in outcome for all GS groups because of the Will Rogers effect, which explains in part the results of the matched-pair analysis. The GS shift confounds retrospective series spanning the 1990s.

Humans↗

PubMed1998

Directly energized uptake of beta-estradiol 17-(beta-D-glucuronide) in plant vacuoles is strongly stimulated by glutathione conjugates.

A directly energized vacuolar pump for glutathione (GS) conjugates has been described for several plant species. Since glucuronate conjugates also occur in plants, we addressed the question whether plant vacuoles take up the abiotic glucuronate conjugate estradiol 17-(beta-glucuronide) (E217G) via a GS conjugate pump, which in some cases has been reported to accept various organic anions as substrates, or via a distinct glucuronate transporter. Uptake studies into vacuoles from rye and barley were performed with E217G and metolachlor-GS (MOC-GS), a substrate of the GS conjugate ATPase, to compare glucuronate conjugate transport into vacuoles containing endogenous flavone glucuronides with those lacking specific glucuronate conjugates, respectively. Our results indicate that E217G and MOC-GS are taken up into vacuoles of both plants via a directly energized mechanism since transport was (i) strictly ATP-dependent; (ii) inhibited by vanadate but not by bafilomycin A1, azide, verapamil, nor by dissipation of the vacuolar DeltapH or DeltaPsi; (iii) E217G uptake into rye vacuoles was partially driven by other nucleotides in the following order of efficiency: ATP > GTP > UTP congruent with CTP, whereas the non-hydrolyzable ATP analogue 5'-adenylyl-beta,gamma-imidodiphosphate, ADP, or PPi did not energize uptake. E217G transport into rye vacuoles was saturable (Km approximately 0.2 mM). The rye-specific luteolin glucuronides decreased uptake rates of E217G and MOC-GS into rye and barley vacuoles to comparable degrees with the mono- and diglucuronidated derivatives (40-60% inhibition) being more effective than the triglucuronide. Inhibition of E217G uptake by luteolin 7-O-diglucuronide was competitive (Ki = 120 microM). Taurocholate had no effect on E217G transport, and uptake of MOC-GS was not inhibited by E217G. Although GS conjugates and oxidized GS decreased MOC-GS transport, E217G uptake into rye and barley vacuoles was stimulated up to 7-fold in a concentration-dependent manner by these substances, with dinitrobenzene-GS being most effective. The stimulation of the GS conjugates was not due to detergent or redox effects and was specific for the E217G pump. GS conjugate stimulation of glucuronate uptake was unique for plants as E217G uptake into yeast microsomal vesicles was not affected. By comparison with a DeltaYCF1 yeast mutant, defective in vacuolar transport of GS conjugates mediated by YCF1, it was shown that E217G was taken up into yeast vesicles via a YCF1-independent directly energized pump. These results indicate that E217G as a glucuronate conjugate is transported across the vacuolar membranes of plants and yeast by a carrier distinct from the GS conjugate ATPase.

Adenosine Triphosphate↗

PubMed1989

Glutamine synthetase heterogeneous expression as a marker for the cellular lineage of preneoplastic and neoplastic liver populations.

The distribution of glutamine synthetase (GS) in rat liver and in putative preneoplastic and neoplastic lesions was studied at stages of hepatocarcinogenesis induced by diethylnitrosamine (DEN), 2-acetylaminofluorene (AAF) and aflatoxin B1 (AFB1) using immunohistochemistry. In control rats, GS was localized entirely to rings 1-3 cells deep surrounding the central veins. Exposure to DEN or AAF resulted in a reduction of these GS-positive (GS+) zones by 96 and 61% respectively, due to necrosis of the cells in this compartment, while AFB1 had little effect (less than 14%). At later times GS+ foci, adenomas and carcinomas developed in animals exposed to DEN or AAF, but not AFB1, corresponding to the acute effects. Small GS+ foci also identified by other phenotypic abnormalities (e.g. gamma-glutamyltransferase) were exclusively associated with the regenerating GS+ zone as were a few collections of GS+ hepatocytes, the nature of which was uncertain. Although accounting for a very small fraction (less than 3%) of total foci, these GS+ foci or GS+ hepatocytes apparently gave rise to a substantial fraction of adenomas that were GS+ (DEN: 43% and AAF: 33%) and an even higher percentage of GS+ carcinomas (DEN: 59% and AAF: 39%). No GS+ neoplasms were induced by AFB1. Statistical evaluation of these data strongly suggests that GS+ neoplasms originate through initiation of hepatocytes which possess this particular phenotype. Thus, GS might serve as a specific marker for tracing cell lineage relationships during hepatocarcinogensis. The GS+ phenotype which confers glutamine independence may also provide a growth advantage.

2-Acetylaminofluorene↗

PubMed1986

Mode of antibacterial action by gramicidin S.

To elucidate the mode of antibacterial action by gramicidin S (GS), a detailed experiment on GS distribution on bacteria cells was carried out. 14C-Labeled gramicidin S ([14C]GS) was incubated with cells of Gram-positive Bacillus subtilis and Gram-negative Escherichia coli, and the amount of [14C]GS adsorbed on the cells was measured. Adsorption on B. subtilis cells was observed from 1 microgram/ml of [14C]GS. As the concentration of [14C]GS increased, the amount adsorbed on B. subtilis increased discontinuously, producing a curve which had three plateaus. On the other hand, [14C]GS was not easily adsorbed on E. coli cells at lower concentrations, but the amount adsorbed increased above 6 micrograms/ml, and the cells were temporarily saturated with GS at 10 micrograms/ml, which is the minimum inhibitory concentration for E. coli. The amount of [14C]GS adsorbed on the protoplast membrane of B. subtilis was the same as that of natural cells. However, the amount of [14C]GS adsorbed on the cell wall dropped to about 20% of that of natural bacteria. These facts indicate that GS is adsorbed on the cell membrane of bacteria particularly. The uptake of amino acid or glucose in B. subtilis was inhibited by GS. Therefore, it is concluded that GS damages the phospholipid bilayer of the cell membrane by adsorption, and prevents the functioning of the cell membrane. The amount of [14C]GS adsorbed on the spheroplast membrane of E. coli increased remarkably as compared with natural cells, even at a lower concentration of GS. The poor GS adsorption on E. coli cells may be due to the permeability barrier of the E. coli cell wall.

Adsorption↗

PubMed2002

The membrane-proximal domain of vesicular stomatitis virus G protein functions as a membrane fusion potentiator and can induce hemifusion.

Recently we showed that the membrane-proximal stem region of the vesicular stomatitis virus (VSV) G protein ectodomain (G stem [GS]), together with the transmembrane and cytoplasmic domains, was sufficient to mediate efficient VSV budding (C. S. Robison and M. A. Whitt, J. Virol. 74:2239-2246, 2000). Here, we show that GS can also potentiate the membrane fusion activity of heterologous viral fusion proteins when GS is coexpressed with those proteins. For some fusion proteins, there was as much as a 40-fold increase in syncytium formation when GS was coexpressed compared to that seen when the fusion protein was expressed alone. Fusion potentiation by GS was not protein specific, since it occurred with both pH-dependent as well as pH-independent fusion proteins. Using a recombinant vesicular stomatitis virus encoding GS that contained an N-terminal hemagglutinin (HA) tag (GS(HA) virus), we found that the GS(HA) virus bound to cells as well as the wild-type virus did at pH 7.0; however, the GS(HA) virus was noninfectious. Analysis of cells expressing GS(HA) in a three-color membrane fusion assay revealed that GS(HA) could induce lipid mixing but not cytoplasmic mixing, indicating that GS can induce hemifusion. Treatment of GS(HA) virus-bound cells with the membrane-destabilizing drug chlorpromazine rescued the hemifusion block and allowed entry and subsequent replication of GS(HA) virus, demonstrating that GS-mediated hemifusion was a functional intermediate in the membrane fusion pathway. Using a series of truncation mutants, we also determined that only 14 residues of GS, together with the VSV G transmembrane and cytoplasmic tail, were sufficient for fusion potentiation. To our knowledge, this is the first report which shows that a small domain of one viral glycoprotein can promote the fusion activity of other, unrelated viral glycoproteins.

Amino Acid Sequence↗

PubMed2005

Impact of genetic versus environmental factors on the control of muscle glycogen synthase activation in twins.

Storage of glucose as glycogen accounts for the largest proportion of muscle glucose metabolism during insulin infusion in normal and insulin-resistant subjects. Studies in first-degree relatives have indicated a genetic origin of the defective insulin activation of muscle glycogen synthase (GS) in type 2 diabetes. The aim of this study was to evaluate the relative impact of genetic versus nongenetic factors on muscle GS activation and regulation in young and elderly twins examined with a 2-h euglycemic-hyperinsulinemic (40 mU x m(-2) x min(-1)) clamp combined with indirect calorimetry and excision of muscle biopsies. The etiological components were determined using structural equation modeling. Fractional GS activity; GS phosphorylation at sites 2, 2 + 2a, and 3a + 3b corrected for total GS protein; and GS kinase 3 (GSK3) activity were similar in both age groups, whereas total GS activity and protein were lower in elderly compared with younger twins. GS fractional activity increased significantly during insulin stimulation in both young and elderly twins. Conversely, there was a significant decrease in GS phosphorylation at site 3a + 3b and GSK3 activity during insulin stimulation in both age groups, whereas GS phosphorylation at site 2 and 2 + 2a only decreased on insulin stimulation in the younger twins. The increment in whole-body glucose disposal (Rd) and nonoxidative glucose metabolism (insulin - basal) correlated significantly with the increment in GS fractional activity. Fractional GS activity had a major environmental component in both age groups. GSK3 activity exhibited a genetic component in young (basal: a2 = 0.42; insulin: a2 = 0.58) and elderly (insulin: a2 = 0.56) twins. Furthermore, GS phosphorylation at site 2 (insulin: a2 = 0.69) in the elderly and at site 3a + 3b (insulin: a2 = 0.50) in the young twins had a genetic component. In conclusion, GSK3 activity and GS phosphorylation, particularly at sites 2 and 3a + 3b, had major genetic components. Total and fractional GS activities per se were, on the other hand, predominantly controlled by environmental factors. Moreover, GS activity was intact with increasing age, despite a significant reduction in nonoxidative glucose metabolism.

Adult↗