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Recently, we provided preliminary evidence for calcium (Ca2+)/ calmodulin (CaM) stimulation of plant glutamate decarboxylase (GAD; EC 4.1.1.15). In the present study, a detailed characterization of the phenomenon is described. GAD was partially purified from various soybean (Glycine max L. Merr.) tissues (developing seed coat and cotyledons, leaf, and root) in the presence of EDTA by a combination of ammonium sulfate precipitation and anion-exchange fast protein liquid chromatography. GAD activity showed a sharp optimum at pH 5.8, with about 12% of maximal activity at pH 7. It was stimulated 2- to 8-fold (depending on the tissue source) in the presence of Ca2+/CaM at pH 7 but not at pH 5.8. Furthermore, when the protease inhibitor phenylmethylsulfonyl fluoride was omitted from the purification procedure, GAD activity was insensitive to Ca2+/CaM but was similar in magnitude to CaM-stimulated activity. The stimulation by Ca2+/CaM was fully inhibited by the CaM antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and trifluoperazine. With saturating CaM or Ca2+, the concentrations of Ca2+ and CaM required for half-maximal stimulation were about 7 to 11 micromolars and 25 nM, respectively. The effect of Ca2+ and CaM appeared to be through a 2.4-fold stimulation of Vmax and a 55% reduction in Km. The results suggested that GAD is activated via Ca2+ signal transduction.

cDNAs encoding the large subunit and a possibly truncated small subunit of the potato tuber (Solanum tuberosum L.) adenosine 5'-diphosphate-glucose pyrophosphorylase have been expressed in Escherichia coli (A.A. Iglesias, G.F. Barry, C. Meyer, L. Bloksberg, P.A. Nakata, T. Greene, M.J. Laughlin, T.W. Okita, G.M. Kishore, J. Preiss, J Biol Chem [1993] 268: 1081-1086). However, some properties of the transgenic enzyme were different from those reported for the enzyme from potato tuber. In this work, extension of the cDNA was performed to elongate the N terminus of the truncated small subunit by 10 amino acids. This extension is based on the almost complete conservation seen at the N-terminal sequence for the potato tuber and the spinach leaf small subunits. Expressing the extended cDNA in E. coli along with the large subunit cDNA yielded a transgenic heterotetrameric enzyme with similar properties to the purified potato tuber enzyme. It was also found that the extended small subunit expressed by itself exhibited high enzyme activity, with lower affinity for activator 3-phosphoglycerate and higher sensitivity toward inorganic phosphate inhibition. It is proposed that a major function of the large subunit of adenosine 5'-diphosphate-glucose pyrophosphorylases from higher plants is to modulate the regulatory properties of the native heterotetrameric enzyme, and the small subunit's major function is catalysis.

The ras GTPase-activating protein (GAP), identified and characterized in mammalian cells, stimulates the intrinsic GTPase activity of ras proteins. We have previously proposed that the IRA genes, negative regulators of RAS genes in Saccharomyces cerevisiae, encode yeast homologs of the mammalian GAP. In this paper, we present the following evidence that a product of the IRA2 gene exhibits GAP activity similar to that of the mammalian GAP protein. (i) Extracts of yeast cells overexpressing IRA2 stimulated the GTPase activity of the yeast RAS2 protein. (ii) An epitope for a monoclonal antibody (12CA5) was added to the N terminus of the IRA2 protein. The GAP activity of extracts prepared from cells expressing this fusion protein was shown to be immunoprecipitable by 12CA5. (iii) An IRA2 protein fused to glutathione S-transferase (GST) was produced and partially purified from Escherichia coli cells. GAP activity was detected with this purified GST-IRA2 fusion protein. (iv) The GAP activity of IRA2 proteins described above did not stimulate the GTPase activity of the RAS2Val19 protein, a protein having an amino acid alteration analogous to that found in mammalian oncogenic ras proteins. This result parallels studies showing that mammalian GAP is incapable of stimulating the GTPase activity of mammalian oncogenic proteins. The remarkable conservation between the GAP activity in mammalian and yeast cells supports the idea that the function of GAP is to negatively regulate ras proteins in mammalian cells.

As part of a structure-function analysis of the higher-plant ADP-glucose pyrophosphorylase (AGP), we used a random mutagenesis approach in combination with a novel bacterial complementation system to isolate over 100 mutants that were defective in glycogen production (T.W. Greene, S.E. Chantler, M.L. Khan, G.F. Barry, I. Preiss, T.W. Okita [1996] Proc Natl Acad Sci USA 93: 1509-1513). One mutant of the large subunit M27 was identified by its capacity to only partially complement a mutation in the structural gene for the bacterial AGP (glg C), as determined by its light-staining phenotype when cells were exposed to I2 vapors. Enzyme-linked immunosorbent assay and enzymatic pyrophosphorylysis assays of M27 cell extracts showed that the level of expression and AGP activity was comparable to those of cells that expressed the wild-type recombinant enzyme. Kinetic analysis indicated that the M27 AGP displays normal Michaelis constant values for the substrates glucose-1-phosphate and ATP but requires 6- to 10-fold greater levels of 3-phosphoglycerate (3-PGA) than the wild-type recombinant enzyme for maximum activation. DNA sequence analysis showed that M27 contains a single point mutation that resulted in the replacement of aspartic acid 413 to alanine. Substitution of a lysine residue at this site almost completely abolished activation by 3-PGA. Aspartic acid 413 is adjacent to a lysine residue that was previously identified by chemical modification studies to be important in the binding of 3-PGA (K. Ball, J. Preiss [1994] J Biol Chem 269: 24706-24711). The kinetic properties of M27 corroborate the importance of this region in the allosteric regulation of a higher-plant AGP.

The activity of highly purified pyrophosphate:fructose-6-phosphate 1-phosphotransferase (PFP) from barley (Hordeum vulgare) leaves was studied under conditions where the catalyzed reaction was allowed to approach equilibrium. The activity of PFP was monitored by determining the changes in the levels of fructose-6-phosphate, orthophosphate, and fructose-1,6-bisphosphate (Fru-1,6-bisP). Under these conditions PFP activity was not dependent on activation by fructose-2,6-bisphosphate (Fru-2,6-bisP). Inclusion of aldolase in the reaction mixture temporarily restored the dependence of PFP on Fru-2,6-bisP. Alternatively, PFP was activated by Fru-1,6-bisP in the presence of aldolase. It is concluded that Fru-1,6-bisP is an allosteric activator of barley PFP, which can substitute for Fru-2,6-bisP as an activator. A significant activation was observed at a concentration of 5 to 25 micromolar Fru-1,6-bisP, which demonstrates that the allosteric site of barley PFP has a very high affinity for Fru-1,6-bisP. The high affinity for Fru-1,6-bisP at the allosteric site suggests that the observed activation of PFP by Fru-1,6-bisP constitutes a previously unrecognized in vivo regulation mechanism.

Incarnant une histoire séculaire riche et mature, les médinas marocaines regorgent d'édifices et d'espaces de haute qualité patrimoniale. Cependant, ces tissus anciens, caractérisés par une haute qualité patrimoniale, sont aujourd'hui soumis à de multiples formes de dégradation dues, en partie, à la forte pression et aux différents types d'usages auxquels ils sont confrontés, outre le manque d'entretien et la limite des actions de restauration qui sont assurées par les acteurs en charge de ces tissus patrimoniaux. En effet, la restauration de certains édifices (fontaines, fondouks, medersas, portes, etc.) et espaces publics (places, jardins, etc.), qui présentent une qualité patrimoniale particulière, sont confinés aux musées en raison d'une restauration sans utilisation active de ces derniers tissus historiques. Une fois restauré, le bâtiment devient un objet ornemental et décoratif, privant la population d'un nombre important d'équipements et de lieux urbains, caractérisés par une haute valeur d'usage et des messages sociaux et culturels forts. Ces bâtiments et espaces étaient accessibles et fonctionnels depuis longtemps. Cependant, une fois qu'ils ont retrouvé leur aspect d'origine et qu'ils ne sont plus utilisables, ils sont privés de leur vocation, perdant leur vie et leur âme, puis leur raison d'être. Cela contribue profondément à la dégradation et à la mise en danger de ces bâtiments et espaces patrimoniaux. Ainsi, une réhabilitation active du bâtiment et de son usage pourrait le sauver du déclin, permettre un renouvellement de son rôle et de sa fonction et l'adapter aux besoins réels. Ainsi, le présent article vise à présenter une analyse de quelques actions de restauration réalisées dans les médinas marocaines, afin de mettre en évidence leurs limites et de découvrir, à travers des études de cas, la pertinence de l'approche basée sur « l'utilisation active » dans la préservation et la valorisation des tissus anciens. Convaincu que « les pierres qui ne sont pas habitées sont des pierres mortes », l'énoncé mérite réflexion et débat.