The Saccharomyces cerevisiae fermentation stress response protein Igd1p/Yfr017p regulates glycogen levels by inhibiting the glycogen debranching enzyme.

Wine fermentation imposes a number of stresses on Saccharomyces cerevisiae, and wine yeasts respond to this harsh environment by altering their transcriptional profile (Marks et al., 2008). We have labeled this change in gene expression patterns the fermentation stress response (FSR). An important component of the FSR is the increased expression of 62 genes for which no function has been identified for their protein products. We hypothesize that a function for these proteins may only be revealed late in grape must fermentation, when the yeast cells are facing conditions much more extreme than those normally encountered in laboratory media. We used affinity copurification to identify interaction partners for the FSR protein Yfr017p, and found that it interacts specifically with the glycogen debranching enzyme (Gdb1p). The expression of both of these proteins is strongly induced during wine fermentation. Therefore, we investigated the role of Yfr017p in glycogen metabolism by constructing wine yeast strains that lack this protein. These YFR017C null cells displayed a significant reduction in their ability to accumulate glycogen during aerobic growth and fermentation. Moreover, Yfr017p inhibits Gdb1p activity in vitro. These results suggest that Yfr017p functions as an inhibitor of Gdb1p, enhancing the ability of yeast cells to store glucose as glycogen. Therefore, we propose IGD1 (for inhibitor of glycogen debranching) as a gene name for the YFR017C ORF.

Crystal structure of Thermotoga maritima 4-alpha-glucanotransferase and its acarbose complex: implications for substrate specificity and catalysis.

4-alpha-Glucanotransferase (GTase) is an essential enzyme in alpha-1,4-glucan metabolism in bacteria and plants. It catalyses the transfer of maltooligosaccharides from an 1,4-alpha-D-glucan molecule to the 4-hydroxyl group of an acceptor sugar molecule. The crystal structures of Thermotoga maritima GTase and its complex with the inhibitor acarbose have been determined at 2.6A and 2.5A resolution, respectively. The GTase structure consists of three domains, an N-terminal domain with the (beta/alpha)(8) barrel topology (domain A), a 65 residue domain, domain B, inserted between strand beta3 and helix alpha6 of the barrel, and a C-terminal domain, domain C, which forms an antiparallel beta-structure. Analysis of the complex of GTase with acarbose has revealed the locations of five sugar-binding subsites (-2 to +3) in the active-site cleft lying between domain B and the C-terminal end of the (beta/alpha)(8) barrel. The structure of GTase closely resembles the family 13 glycoside hydrolases and conservation of key catalytic residues previously identified for this family is consistent with a double-displacement catalytic mechanism for this enzyme. A distinguishing feature of GTase is a pair of tryptophan residues, W131 and W218, which, upon the carbohydrate inhibitor binding, form a remarkable aromatic "clamp" that captures the sugar rings at the acceptor-binding sites +1 and +2. Analysis of the structure of the complex shows that sugar residues occupying subsites from -2 to +2 engage in extensive interactions with the protein, whereas the +3 glucosyl residue makes relatively few contacts with the enzyme. Thus, the structure suggests that four subsites, from -2 to +2, play the dominant role in enzyme-substrate recognition, consistent with the observation that the smallest donor for T.maritima GTase is maltotetraose, the smallest chain transferred is a maltosyl unit and that the smallest residual fragment after transfer is maltose. A close similarity between the structures of GTase and oligo-1,6-glucosidase has allowed the structural features that determine differences in substrate specificity of these two enzymes to be analysed.

[Effect of biogenic amines on glycogen degradation in isolated rat hepatocytes]. / Vliianie biogennykh aminov na degradatsiiu glikogena v izolirovannykh gepatotsitakh krysy.

Serotonin and tyramine affected dissimilarly the enzyme activity involved in degradation of glycogen in isolated rat hepatocytes. Serotonin inhibited hydrolytic enzymes acid alpha-glucosidase, alpha-amylase and amylo-1,6-glucosidase but activated phosphorylase inducing a decrease of glycogen content in cells. Tyramine inhibited acid alpha-glucosidase and amylo-1,6-glucosidase, did not affect the alpha-amylase and phosphorylase activities and increased content of glycogen in cells. Tyramine may be used as stimulator of glycogen accumulation in liver cells.

Glycogen debranching enzyme 6 (AGL), enolase 1 (ENOSF1), ectonucleotide pyrophosphatase 2 (ENPP2_1), glutathione S-transferase 3 (GSTM3_3) and mannosidase (MAN2B2) metabolism computational network analysis between chimpanzee and human left cerebrum.

We identified significantly higher expression of the genes glycogen debranching enzyme 6 (AGL), enolase 1 (ENOSF1), ectonucleotide pyrophosphatase 2 (ENPP2_1), glutathione S-transferase 3 (GSTM3_3) and mannosidase (MAN2B2) from human left cerebrums versus chimpanzees. Yet the distinct low- and high-expression AGL, ENOSF1, ENPP2_1, GSTM3_3 and MAN2B2 metabolism networks between chimpanzee and human left cerebrum remain to be elucidated. Here, we constructed low- and high-expression activated and inhibited upstream and downstream AGL, ENOSF1, ENPP2_1, GSTM3_3 and MAN2B2 metabolism network between chimpanzee and human left cerebrum in GEO data set by gene regulatory network inference method based on linear programming and decomposition procedure, under covering AGL, ENOSF1, ENPP2_1, GSTM3_3 and MAN2B2 pathway and matching metabolism enrichment analysis by CapitalBio MAS 3.0 integration of public databases, including Gene Ontology, KEGG, BioCarta, GenMapp, Intact, UniGene, OMIM, etc. Our results show that the AGL, ENOSF1, ENPP2_1, GSTM3_3 and MAN2B2 metabolism network has more activated and less inhibited molecules in chimpanzee, but less activated and more inhibited in the human left cerebrum. We inferred stronger carbohydrate, glutathione and proteoglycan metabolism, ATPase activity, but weaker base excision repair, arachidonic acid and drug metabolism as a result of inducing cell growth in low-expression AGL, ENOSF1, ENPP2_1, GSTM3_3 and MAN2B2 metabolism network of chimpanzee left cerebrum; whereas stronger lipid metabolism, amino acid catabolism, DNA repair but weaker inflammatory response, cell proliferation, glutathione and carbohydrate metabolism as a result of inducing cell differentiation in high-expression AGL, ENOSF1, ENPP2_1, GSTM3_3 and MAN2B2 metabolism network of human left cerebrum. Our inferences are consistent with recent reports and computational activation and inhibition gene number patterns, respectively.

Identification of acceptor substrate binding subsites +2 and +3 in the amylomaltase from Thermus thermophilus HB8.

Glycoside hydrolase family 77 (GH77) belongs to the alpha-amylase superfamily (Clan H) together with GH13 and GH70. GH77 enzymes are amylomaltases or 4-alpha-glucanotransferases, involved in maltose metabolism in microorganisms and in starch biosynthesis in plants. Here we characterized the amylomaltase from the hyperthermophilic bacterium Thermus thermophilus HB8 (Tt AMase). Site-directed mutagenesis of the active site residues (Asp293, nucleophile; Glu340, general acid/base catalyst; Asp395, transition state stabilizer) shows that GH77 Tt AMase and GH13 enzymes share the same catalytic machinery. Quantification of the enzyme's transglycosylation and hydrolytic activities revealed that Tt AMase is among the most efficient 4-alpha-glucanotransferases in the alpha-amylase superfamily. The active site contains at least seven substrate binding sites, subsites -2 and +3 favoring substrate binding and subsites -3 and +2 not, in contrast to several GH13 enzymes in which subsite +2 contributes to oligosaccharide binding. A model of a maltoheptaose (G7) substrate bound to the enzyme was used to probe the details of the interactions of the substrate with the protein at acceptor subsites +2 and +3 by site-directed mutagenesis. Substitution of the fully conserved Asp249 with a Ser in subsite +2 reduced the activity 23-fold (for G7 as a substrate) to 385-fold (for maltotriose). Similar mutations reduced the activity of alpha-amylases only up to 10-fold. Thus, the characteristics of acceptor subsite +2 represent a main difference between GH13 amylases and GH77 amylomaltases.

Lentiginosine, a dihydroxyindolizidine alkaloid that inhibits amyloglucosidase.

Lentiginosine, a dihydroxyindolizidine alkaloid, was extracted from the leaves of Astragalus lentiginosus with hot methanol and was purified to homogeneity by ion-exchange, thin-layer, and radial chromatography. A second dihydroxyindolizidine, the 2-epimer of lentiginosine, was also purified to apparent homogeneity from these extracts. Gas chromatography of the two isomers (as the TMS derivatives) showed that they were better than 95% pure; lentiginosine eluted at 8.65 min and the 2-epimer at 9.00 min. Both compounds had a molecular ion in their mass spectra of 157, and the NMR spectra demonstrated that both were dihydroxyindolizidines differing in the configuration of the hydroxyl group at carbon 2. Lentiginosine was found to be a reasonably good inhibitor of the fungal alpha-glucosidase, amyloglucosidase (Ki = 1 x 10(-5) M), but it did not inhibit other alpha-glucosidases (i.e., sucrase, maltase, yeast alpha-glucosidase, glucosidase I) nor any other glycosidases. The 2-epimer had no activity against any of the glycosidases tested.

Cystic fibrosis: enzymatic detection of a ciliostatic factor.

The ciliostatic cystic fibrosis (CF) factor has been investigated in order to develop an enzymatic assay for its detection. In saliva, this factor is associated with alpha-amylase. Removal or addition of the factor by dialysis does not affect the alpha-amylase catalytic activity of CF or control (normal) saliva. Crude preparations of the dialyzable factor from CF saliva are ciliostatic. Alpha-Amylase-catalyzed starch hydrolysis from both CF and control saliva was found to be insensitive to hydroxyalkylamine inhibitors so this method could not be used to detect differences in CF and control amylase. Since another carbohydrase, mammalian debranching enzyme, is much more sensitive to inhibition, the effect of the isolated crude factor on this enzyme was determined. The mean values for percentage of inhibition of debranching enzyme activity by fractions obtained from saliva of CF patients, obligate heterozygotes, and control subjects are: 32% (27 samples), 13% (21 samples), and 6% (11 samples), respectively. Fifteen of the CF values exceed the maximum 13% inhibition observed for controls. Indeed, five samples almost completely inhibited debranching enzyme. This inhibitory action provides the basis of an enzymatic assay for the detection of the ciliostatic CF factor.

Thiol/disulfide redox equilibrium between glutathione and glycogen debranching enzyme (amylo-1,6-glucosidase/4-alpha-glucanotransferase) from rabbit muscle.

Rabbit skeletal muscle glycogen debranching enzyme is inactivated in a kinetically biphasic manner by GSSG at pH 8.0. The rapid phase results in the loss of 30% activity, while the slower phase leads to total enzyme inactivation. Both the glucosidase and the transferase activities of the enzyme are inhibited by GSSG. The inactivation by disulfides is fully and rapidly reversed in a biphasic manner by reduction with excess reduced dithiothreitol or GSH. After a fast initial recovery of 70% of the initial activity, the remaining 30% of the activity is recovered more slowly. Equilibration of the enzyme with a redox buffer of GSH and GSSG shows a monophasic equilibration of the activity. The ratio of GSH/GSSG where the enzyme is 50% active (R0.5) is 0.06 +/- 0.03. The R0.5 does not vary significantly with the total concentration of glutathione species suggesting formation of protein-SSG mixed disulfides. The ratios of the observed second-order rate constants for GSSG inactivation and GSH reactivation do not lead to a correct value of the observed thiol/disulfide oxidation equilibrium constant. Although the enzyme has sulfhydryl groups, the oxidation of which leads to activity changes, the kinetic and thermodynamic resistance to oxidation suggests that the enzyme is not likely to be subject to regulation by thiol/disulfide exchange in vivo.

Reduction of dental plaque deposition in humans by oolong tea extract.

The inhibitory effect of oolong tea extract (OTE) containing polymerized polyphenols on plaque deposition was examined in 35 human volunteers. Thirty-five human volunteers, aged 18-29 years, who received extensive oral prophylactic procedures were requested to refrain from all oral hygiene procedures for 4 days, and to rinse their mouth with 0.5 mg/ml OTE solution in 0.2% ethanol before and after every intake of food and before sleeping at night. No restriction regarding meals was given during the test period, except to refrain from teas or coffee. Plaque deposition was evaluated after disclosing the teeth with Erythrocin at the termination of this experiment. The study was repeated 1 week after the first trial, but only 0.2% ethanol without OTE was used for mouthrinsing in the second trial. OTE was found to significantly inhibit plaque deposition in volunteers, although mouthrinsing with OTE solution had no significant effect on the number of mutans streptococci in unstimulated whole saliva.

Reassessment of the catalytic mechanism of glycogen debranching enzyme.

The amylo-1,6-glucosidase catalytic activity of glycogen debranching enzyme allows it to hydrolyze alpha-D-glucosyl fluoride, in the absence or presence of glycogen or oligosaccharides, releasing equal amounts of fluoride and glucose at rates comparable to those seen with the natural substrates. 2-Deoxy-2-fluoro-alpha-D-glucosyl fluoride is found to be a poor substrate, rather than the covalent inhibitor that would be expected for a glucosidase which catalyzes hydrolysis of the glycosidic linkage with retention of anomeric configuration. In fact, analysis of the glucosidase reaction by NMR reveals that the debranching enzyme hydrolyzes the glycosidic linkage with inversion of configuration, releasing beta-D-glucose from both alpha-glucosyl fluoride and its natural substrate, the phosphorylase limit dextrin. In contrast, its transferase activity necessarily proceeds with retention of configuration. As has been seen with other "inverting" glycosidases, the debranching enzyme releases beta-D-glucose from beta-D-glucosyl fluoride in the presence of oligosaccharides such as maltohexaose and cyclomaltoheptaose but, unlike the others, not in their absence. An intermediate glucosyl-alpha-(1,6)-cyclomaltoheptaose has been detected by NMR analysis. In the presence of a water-soluble carbodiimide, a single mole of glycine ethyl ester is incorporated into each mole of the debranching enzyme, resulting in its inactivation when measured by the combined assay for both transferase and glucosidase activities. Measurement of the latter two activities independently indicates that it is the transferase activity which is inactivated, while the glucosidase activity, measured with alpha-D-glucosyl fluoride as substrate, is unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of enzymic digestion of amylose by free fatty acids in vitro contributes to resistant starch formation.

The effect of lipids on the enzymic breakdown of starch was investigated using an in vitro assay system. Mixtures of potato amylose, amylopectin and starch and various lipids were incubated at 37 degrees C for 10 min and subjected to digestion by alpha-amylase (EC 3.2.1.1) and amyloglucosidase (EC 3.2.1.33). Lauric, myristic, palmitic and oleic acids and lysolecithin inhibited enzymic hydrolysis of amylose by approximately 35% (P < 0.05). Stearic acid and cholesterol had no effect on the enzymic breakdown of amylose. Retrograded amylose was hydrolyzed less readily (P < 0.05) than solubilized amylose, but the breakdown was not further inhibited in the presence of lauric acid. Fatty acids had no effect on the enzymic hydrolysis of amylopectin, whereas inhibition by fatty acids of the breakdown of whole starch was consistent with only the amylose fraction being affected. The possibility that interactions between starch and fatty acids in the digestive tract could contribute to the formation of resistant starch is considered.

Specificity of an isolated salivary factor material to cystic fibrosis.

An oyster gill ciliostatic factor material has been isolated from the saliva of patients with cystic fibrosis (CF) by utilizing its ability to bind to alpha-amylase. It was quantitatively assayed by its ability to reversibly inhibit rabbit muscle glycogen debranching enzyme. The specificity of this CF factor material was investigated by comparing activities from the saliva of CF homozygotes (patients) varying in age, sex, and the severity of the disease; CF obligate heterozygotes (carriers); normal control subjects who had no family history of CF; non-CF asthmatic and allergic bronchitis patients; non-CF immunologically deficient patients with chronic respiratory problems; non-CF juvenile diabetic patients; non-CF pancreatic insufficiency patients; non-CF patients with obstructive liver cirrhosis; and non-CF patients with ectodermal dysplasia. The results show that the CF factor material isolated from CF saliva is specific to subjects with cystic fibrosis and is not associated with similar non-CE chronic disease states, nor is it produced as a result of an organ pathology associated with CF. There was no correlation between the amount of factor present in an individual CF homozygote sample and the severity of the disease. In the case of both the CF homozygote and heterozygote samples, there was also no correlation in either age or sex and the amount of factor present. The degree of inhibition produced by CF homozygotes compared to CF heterozygotes is characteristic of the autosomal recessive mode of inheritance of CF. This finding appears to associate the isolated CF factor material with the affected CF gene and suggests that the factor material is related in some way to the genetic lesion in CF.

Cystic fibrosis: isolation and physical properties of a salivary cystic fibrosis factor.

A ciliostatic factor has been isolated from cystic fibrosis (CF) saliva by dialyzing it from purified alpha-amylase prepared by a glycogen-complex method. This method of isolating the CF factor is an improvement over the previously employed heparin procedure. The activity of the isolated factor is proportional with concentration using the oyster gill ciliostatic assay and in its inhibition of mammalian glycogen debranching enzyme. The ciliostatic action of the factor can be reversed by heparin under certain conditions. The type of inhibition of the debranching enzyme by the isolated CF factor indicates that its chemical structure is similar to that observed with hydroxyalkylamines and polyamine metabolites. Physical properties of the isolated factor indicate that it is of low molecular weight and is labile as a function of pH and temperature. At neutral pH the conditions under which it is maintained have a direct effect on the length of time that it is stable.

Inhibition of glycogenolysis by a glucose analogue in the working rat heart.

The effects of BAY o 1248, an inhibitor of alpha-amylo-1, 6-glucosidase, on glycogenolysis and post-ischemic functional recovery were investigated in isolated perfused rat hearts. Working rat hearts were perfused during 30 min with 11 mm glucose (controls) and, in some hearts, with 1 microM insulin or 5 mM lactate to increase their glycogen concentration. The hearts were then submitted to 10 min of no-flow ischemia and reperfused during 15 min with 11 mM glucose alone. Glycogen content was increased by 50% in hearts perfused with insulin or lactate. During ischemia, glycogen breakdown was similar in the control and lactate groups, but was abolished in the insulin-group. At reperfusion, functional recovery was improved in glycogen-loaded hearts compared to controls. When hearts were perfused with 1 mM BAY o 1248, added before ischemia, glycogenolysis was inhibited in the three groups and functional recovery was hampered in both the control and lactate groups. In the insulin group, however, the functional recovery was barely affected by BAY o 1248. We conclude that: (i) BAY o 1248 is an inhibitor of heart glycogen breakdown; (ii) the consequences of inhibition of ischemic glycogenolysis on post-ischemic functional recovery depend on the conditions; and (iii) the protective effect of insulin does not result from ischemic glycogenolysis.