An amino acid sequence in the active centre of phosphoglucomutase

Milstein, C.; Sanger, F.

doi:10.1042/bj0790456

Cited by 87 publications

(29 citation statements)

References 21 publications

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“…In addition, StcPGM exhibits considerable homology with StpPGM (Tauberger et al 2000), with which it has 76% similarity. Catalytic and metal-ion binding sites present in a wide range of PGM sequences from other sources could be identi®ed in the amino acid sequence of StcPGM (Milstein andSanger 1961 andDai et al 1992, respectively). Analysis of mRNA Northern blots using the StcPGM cDNA as a probe suggest a constitutive expression of this gene; the transcript was present at high levels in young and mature leaves, stems, roots, stolons and developing and mature tubers (Fig.…”

Section: Isolation and Molecular Characterisation Of Stcpgmmentioning

confidence: 98%

Antisense repression of cytosolic phosphoglucomutase in potato ( Solanum tuberosum ) results in severe growth retardation, reduction in tuber number and altered carbon metabolism

Fernie

Tauberger

Lytovchenko

et al. 2002

Planta

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The aim of this work was to investigate the role of cytosolic phosphoglucomutase (PGM; EC 5.4.2.2) in the regulation of carbohydrate metabolism. Many in vitro studies have indicated that PGM plays a central role in carbohydrate metabolism; however, until now the importance of this enzyme in plants has not been subject to reverse-genetics investigations. With this intention we cloned the cytosolic isoform of potato PGM (StcPGM) and expressed this in the antisense orientation under the control of the CaMV 35 S promoter in potato plants. We confirmed that these plants contained reduced total PGM activity and that loss in activity was due specifically to a reduction in cytosolic PGM activity. These plants were characterised by a severe phenotype: stunted aerial growth combined with limited root growth and a reduced tuber yield. Analysis of the metabolism of these lines revealed that leaves of these plants were inhibited in sucrose synthesis whereas the tubers exhibited decreased levels of sucrose and starch as well as decreased levels of glycolytic intermediates but possessed unaltered levels of adenylates. Furthermore, a broader metabolite screen utilising GC-MS profiling revealed that these lines contained altered levels of several intermediates of the TCA cycle and of amino acids. In summary, we conclude that cytosolic PGM plays a crucial role in the sucrose synthetic pathway within the leaf and in starch accumulation within the tuber, and as such is important in the maintenance of sink-source relationships.

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Section: Isolation and Molecular Characterisation Of Stcpgmmentioning

confidence: 98%

Antisense repression of cytosolic phosphoglucomutase in potato ( Solanum tuberosum ) results in severe growth retardation, reduction in tuber number and altered carbon metabolism

Fernie

Tauberger

Lytovchenko

et al. 2002

Planta

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“…The T-A-S-H-N motif that was identi®ed as the catalytic site of rat PGM (Milstein and Sanger 1961) is found at positions 179±183 of the Arabidopsis PGMp, except that the threonine residue is replaced by serine in PGMp. During the catalytic process, the hydroxyl group of the S-116 in the human PGM serves as the phosphate acceptor and donor (Ray and Peck 1972).…”

Section: Discussionmentioning

confidence: 99%

Molecular characterisation of a new mutant allele of the plastid phosphoglucomutase in Arabidopsis, and complementation of the mutant with the wild-type cDNA

Kofler¹,

Häusler²,

Schulz³

et al. 2000

Mol Gen Genet

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Screening of transposon-associated mutants of Arabidopsis thaliana for altered starch metabolism resulted in the isolation of a mutant that did not accumulate starch in any tissue or at any developmental stage (starch-free mutant, stf1). Allelism tests with known mutants showed that stf1 represents a new mutant allele of the plastid isoform of the enzyme phosphoglucomutase (PGMp). The mutation was mapped to chromosome 5. An Arabidopsis EST that showed significant homology to the cytosolic isoform of phosphoglucomutase (PGM) from maize was able to complement the mutant phenotype. The Arabidopsis EST was transcribed and translated in vitro and the protein product was efficiently imported into isolated chloroplasts and processed to its mature form. The lack of starch biosynthesis in stf1 is accompanied by the accumulation of soluble sugars. The rate of CO2 assimilation measured in individual leaves was substantially diminished only under conditions of high CO2 and low O2. Remarkably, stf1 exhibits an increase rather than a decrease in total leaf PGM activity, suggesting an induction of the cytosolic isoform(s) in the mutant. The substrate for PGM, glucose 6-phosphate, accumulated in stf1 during the day, resulting in 10-fold higher content than in the wild type at the end of the photoperiod.

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“…phosphate difEers from that in phosphorylated phosphoglucomutase (Milstein and Sanger, 1961) and phosphorylated alkaline phosphatase (Milstein, 1963) which are very resistant to acid, as much of the radioactivity of these P^^-enzymes is present as phosphoserine and phosphoserine-peptides after 30-90 minutes treatment at 100° C. in 5-7N-hydrochloric acid. In contrast to this, in our experiments, orthophosphate was the only radioactive product detected by electrophoresis and autoradiography (cf.…”

Section: Prelitninary Identification Of the Phosphorijhted Intermedimentioning

confidence: 99%

Studies of the Mechanism of Cation Transport

Charnock

1963

Aust J Exp Biol Med

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SUMMARY.Membrane-bound cation stimulated adenosine-triphosphatase is thought to be closely associated with active ion transport phenomena. As this enzyme system requires the combination of both sodium and potassium ions for full activation its mechanism of action has been investigated under these conditions. Using adenosine triphosphate labelled with P^ô nly in the terminal position as substrate for the reaction we have been able to show that ATP^2 is hydroiysed to adenosine diphosphate and radioactive orthophosphate via a phosphorylated intermediate. Sodium but not potassium ions are required for the formation of this intermediate and adenosine diphosphate; potassium ions are subsequently required for dephosphorylation of the intermediate complex with tlie release of P-''^ orthophosphate -that is, both sodium and potassium ions arc required for the turnover of the intermediate in a two step reaction.The cardiac glycoside ouabain (Strophanthin-G) is without effect upon the formation of this intermediate complex, but can completely inhibit the K+ -requiring dephosphorylation reaction. Preliminary experiments indicate that sodium ions are bound to the enzyme system during the phosphorylation reaction and are released when this complex is broken down. Solvent extraction procedures designed to remove phospholipid components did not decrease the specific activity of the phosphorylaled intenncdiate which suggests that it is phosphoprotein in nature.

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An amino acid sequence in the active centre of phosphoglucomutase

Cited by 87 publications

References 21 publications

Antisense repression of cytosolic phosphoglucomutase in potato ( Solanum tuberosum ) results in severe growth retardation, reduction in tuber number and altered carbon metabolism

Antisense repression of cytosolic phosphoglucomutase in potato ( Solanum tuberosum ) results in severe growth retardation, reduction in tuber number and altered carbon metabolism

Molecular characterisation of a new mutant allele of the plastid phosphoglucomutase in Arabidopsis, and complementation of the mutant with the wild-type cDNA

Studies of the Mechanism of Cation Transport

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