Scope and mechanism of carbohydrase action. Stereocomplementary hydrolytic and glucosyl-transferring actions of glucoamylase and glucodextranase with alpha- and beta-D-glucosyl fluoride

Rhizopus niveus glucoamylase and Arthrobacter globiformis glucodextranase, which catalyze the hydrolysis of starch and dextrans, respectively, to form D-glucose of inverted (beta) configuration, were found to convert both alpha- and beta-D-glucosyl fluoride to beta-D-glucose and hydrogen fluoride. Each enzyme directly hydrolyzes alpha-D-glucosyl fluoride but utilizes th beta-anomer in reactions that require 2 molecules of substrate and yield glucosyl transfer products which are then rapidly hydrolyzed to form beta-D-glucose. Various D-glucopyranosyl compounds serve as acceptors for such reactions. Mixtures of beta-D-glucosyl fluoride and methyl-alpha-D-glucopyranoside[14C], incubated with either enzyme, yielded both methyl-alpha-D-glucopyranosyl-(1 leads to 4)-alpha-D-[14C]glucopyranoside and methyl-alpha-D-glucopyranosyl-(1 leads to 6)-alpha-D-[14C]glucopyranoside. Glucoamylase produced more of the alpha-maltoside; glucodextranase produced more of the alpha-isomaltoside. Thus, both "exo-alpha-glucan hydrolases" emerge as glucosylases that catalyze stereospecifically complementary hydrolytic and transglucosylative reactions with glucosyl donors of opposite configuration. These reactions not only provide a new view of the catalytic capabilities of these supposedly strict hydrolases; they also furnish a basis for defining a detailed mechanism for catalysis. Present results, together with those of several recent studies from this laboratory (especially similar findings obtained with beta-amylase acting on alpha- and beta-maltosyl fluoride (Hehre, E. J., Brewer, C. F., and Genghof, D. S. (1979) J. Biol. Chem. 254, 5942-5950), provide strong new evidence for the functional flexibility of the catalytic groups of carbohydrases.     

Differentiation of human non-salivary, non-pancreatic alpha-amylase from salivary and pancreatic alpha-amylases by use of FG5P

Human non-salivary, non-pancreatic alpha-amylase (yHXA) is the gene product of a newly found human alpha-amylase gene expressed in yeast. Its mode of action on a fluorogenic derivative of p-nitrophenyl alpha-maltopentaoside, FG5P (FG-G-G-G-G-P), was examined at various pH values to elucidate the difference between yHXA and pancreatic or salivary alpha-amylase. The product analysis of the digests by HPLC showed that the enzyme hydrolyzed FG5P to FG3 (FG-G-G) and p-nitrophenyl alpha-maltoside (G-G-P) and to FG4(FG-G-G-G) and p-nitrophenyl alpha-glucoside (G-P), and the ratio of the two reactions changed with pH. The three enzymes differed from each other in the mode of action at pH 5.5. The molar ratio of FG4 to FG3 in the digest with yHXA was the largest. This suggested that the expression of the new gene in human can be detected by the use of FG5P as the substrate in the alpha-amylase assay.     

Regulation of carbohydrate transport activities in Salmonella typhimurium: use of the phosphoglycerate transport system to energize solute uptake.

The phosphoglycerate transport system was employed to supply energy-depleted, lysozyme-treated Salmonella typhimurium cells with a continuous intracellular source of phosphoenolpyruvate. When the cells had been induced to high levels of the phosphoglycerate transport system, a low extracellular concentration of phosphoenolpyruvate (0.1 mM) half maximally stimulated uptake of methyl alpha-glucoside via the phosphoenolpyruvate:sugar phosphotransferase system. If the phosphoglycerate transport system was not induced before energy depletion, 100 times this concentration of phosphoenolpyruvate was required for half-maximal stimulation. Phosphoenolpyruvate could not be replaced by other energy sources if potassium fluoride (an inhibitor of enolase) was present. Inhibition of [14C]-glycerol uptake into energy-depleted cells by methyl alpha-glucoside was demonstrated. A concentration of phosphoenolpyruvate which stimulated methyl alpha-glucoside accumulation counteracted the inhibitory effect of the glucoside. In the presence of potassium fluoride, phosphoenolpyruvate could not be replaced by other energy sources. Inhibition of glycerol uptake by methyl alpha-glucoside in intact untreated cells was also counteracted by phosphoenolpyruvate, but several energy sources were equally effective; potassium fluoride was without effect. These and other results were interpreted in terms of a mechanism in which the relative proportions of the phosphorylated and nonphosphorylated forms of a cell constituent influence the activity of the glycerol transport system.     

Preparation of non-reducing-end substituted p-nitrophenyl alpha-maltopentaoside (FG5P) as a substrate for a coupled enzymatic assay for alpha-amylases

p-Nitrophenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)-O-alpha-D - glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-alpha-D-glucopyranoside, FG5P, was prepared, taking advantage of the action of Bacillus macerans cyclodextrin glucanotransferase on a mixture of O-6-deoxy-6-[(2-pyridyl)-amino]-alpha-D-glucopyranosyl-(1----4)-O-alpha- D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-D-glucose and p-nitrophenyl alpha-glucoside. The maltopentaose derivative is resistant to alpha-glucosidase and is suitable as a substrate for the alpha-amylase assay coupled with alpha-glucosidase in which the activity of alpha-amylase is determined by measuring the amount of p-nitrophenol liberated by alpha-glucosidase from p-nitrophenyl alpha-glucoside and p-nitrophenyl alpha-maltoside produced by the action of alpha-amylase. This alpha-amylase assay method was applied for determination of alpha-amylases in human serum.     

Actions of three human alpha-amylases expressed in yeast on modified substrates and the amino acid residues causing their different actions

The mode of action of an alpha-amylase (yHXA) which was the gene product of a newly found human alpha-amylase gene expressed in yeast on synthetic substrates was compared with those of the gene products (yHSA and yHPA) of human salivary and pancreatic alpha-amylase gene in yeast. The substrates used were phenyl alpha-maltopentaoside (G5 phi) and its derivatives in which the CH2OH groups of the non-reducing-end glucose residues were converted to CH2NH2 (AG5 phi), COOH (CG5 phi), or CH2I (IG5 phi). The digests were subjected to HPLC to determine the amounts of products. The HPLC analysis revealed that yHXA and yHSA bound G5 phi to their active sites in similar manners to give the same products, while yHPA hydrolyzed it in a different way. Modifications of the non-reducing-end glucose of G5 phi caused change of the binding mode to the active sites of the enzymes. AG5 phi and CG5 phi were hydrolyzed by the enzymes to give more phenyl alpha-glucoside (G phi) and less phenyl alpha-maltoside (G2 phi), while IG5 phi gave more G2 phi and less G phi, compared with G5 phi. The substrate binding mode of yHXA changed more extensively than that of yHSA. The results suggested that there exists an amino acid replacement between yHXA and yHSA. The amino acid residues replaced are neither acidic nor basic, are located in subsite S3, and interact with the CH2OH residue of the non-reducing-end glucose residue of G5 phi.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of Acremonium implicatum alpha-glucosidase having regioselectivity for alpha-1,3-glucosidic linkage

alpha-Glucosidase with a high regioselectivity for alpha-1,3-glucosidic linkages for hydrolysis and transglucosylation was purified from culture broth of Acremonium implicatum. The enzyme was a tetrameric protein (M.W. 440,000), of which the monomer (M.W. 103,000; monomeric structure was expected from cDNA sequence) was composed of two polypeptides (M.W. 51,000 and 60,000) formed possibly by posttranslational proteolysis. Nigerose and maltose were hydrolyzed by the enzyme rapidly, but slowly for kojibiose. The k(0)/K(m) value for nigerose was 2.5-fold higher than that of maltose. Isomaltose was cleaved slightly, and sucrose was not. Maltotriose, maltotetraose, p-nitrophenyl alpha-maltoside and soluble starch were good substrates. The enzyme showed high affinity for maltooligosaccharides and p-nitrophenyl alpha-maltoside. The enzyme had the alpha-1,3- and alpha-1,4-glucosyl transfer activities to synthesize oligosaccharides, but no ability to form alpha-1,2- and alpha-1,6-glucosidic linkages. Ability for the formation of alpha-1,3-glucosidic linkage was two to three times higher than that for alpha-1,4-glucosidic linkage. Eight kinds of transglucosylation products were synthesized from maltose, in which 3(2)-O-alpha-nigerosyl-maltose and 3(2)-O-alpha-maltosyl-maltose were novel saccharides.     

Quantitative study on anomeric forms of glucose produced by alpha-glucosidases.

Anomeric forms of glucose produced from phenyl alpha-maltoside, maltose, or phenyl alpha-glucoside have been determined quantitatively by simultaneous measurements of optical rotation and reducing power, for eight kinds of glucose-producing 1,4-alpha-glucosyl hydrolases, including glucose-forming amylase from human urine, and alpha-glucosidases from pig serum, honey bee, buckwheat seed, rice seed, sugar beet seed, flint corn seed, and brewer's yeast. All the eight enzymes studied were found to produce alpha-glucose exclusively.     

Examination of aglycone-binding site of human salivary alpha-amylase by means of transglycosylation reactions

The active site of human salivary alpha-amylase is composed of tandem subsites (S3, S2, S1, S1',S2', etc.) geometrically complementary to several glucose residues, and the glycosidic linkage of the substrate is split between S1 and S1'. As a matter of convenience, the subsites to which the non-reducing-end part (glycone) and the reducing-end part (aglycone) of the substrate being hydrolyzed are bound are named the glycone-binding site (S3, S2, S1) and the aglycone-binding site (S1', S2'), respectively. The features of the aglycone-binding site of human salivary alpha-amylase were examined by means of transglycosylation reaction using phenyl alpha-maltoside (GG phi: G-G-phi) and its derivatives (GAG phi: G-AG-phi, GCG phi: G-CG-phi, AGG phi: AG-G-phi, and CGG phi: CG-G-phi) in which one of the glucose residues (G) has been converted to 6-amino-6-deoxy-glucose (AG) or glucuronic acid (CG) residue as the acceptor. A fluorogenic derivative of maltotetraose, p-nitrophenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)-O-alpha-D -glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-alpha-D- glucopyranosyl-(1----4)-alpha-D-glucopyranoside (FG4P, FG-G-G-G-P), was used as the substrate. HSA catalyzed both hydrolysis of FG4P to FG3 (FG-G-G) and p-nitrophenyl alpha-glucoside (G-P) and transfer of the FG3 residue of FG4P to the acceptors. Transfer to GAG phi occurred more effectively than to GG phi. Transfers to GCG phi and CGG phi were less than to GG phi and very little transfer to AGG phi occurred.(ABSTRACT TRUNCATED AT 250 WORDS)     

1,2-alpha-l-Fucosynthase: a glycosynthase derived from an inverting alpha-glycosidase with an unusual reaction mechanism

Fucosyloligosaccharides have great therapeutic potential. Here we present a new route for synthesizing a Fucalpha1,2Gal linkage by introducing glycosynthase technology into 1,2-alpha-l-fucosidase. The enzyme adopts a unique reaction mechanism, in which asparagine-423 activated by aspartic acid-766 acts as a base while asparagine-421 fixes both a catalytic water and glutamic acid-566 (an acid) in the proper orientations. Glycosynthase activity of N421G, N423G, and D766G mutants was examined using beta-fucosyl fluoride and lactose, and among them, the D766G mutant most effectively synthesized 2'-fucosyllactose. 1,2-alpha-l-Fucosynthase is the first glycosynthase derived from an inverting alpha-glycosidase and from a glycosidase with an unusual reaction mechanism.     

Detection of human urinary alpha-amylase encoded by the AMY2B gene using a fluorogenic substrate, FG5P.

The existence of alpha-amylase (HXA) encoded by alpha-amylase gene AMY2B in healthy humans was examined using a fluorogenic substrate, FG5P (FG-G-G-G-G-P: FG, 6-deoxy-6-[(2-pyridyl)amino]-D-glucose residue; G, glucose residue; P, p-nitrophenyl residue; -, alpha-1,4-glycosidic bond). Chromatofocusing of urine from a healthy human was carried out. FG5P was digested with the fractions exhibiting alpha-amylase activity and each digest at an early stage was analyzed by HPLC. FG5P was hydrolyzed to FG3 (FG-G-G) and p-nitrophenyl alpha-maltoside (G-G-P), and to FG4 (FG-G-G-G) and p-nitrophenyl alpha-glucoside (G-P). The molar ratios of FG4 to FG3 (FG4/FG3) in the digests with basic fractions were larger than those in the digests of human pancreatic alpha-amylase (HPA, 1.11) and human salivary alpha-amylase (HSA, 0.51). Considering that the value for the AMY2B gene product with yeast (yHXA) is 1.88, a value of more than 1.11 implies that HXA exists. The amount of HXA was determined after removal of HSA on an anti-human salivary alpha-amylase antibody bound column. The FG4/FG3 values for six urine samples free from HSA were 1.23-1.26. Assuming that the FG4/FG3 value for HXA is the same as that for yHXA, the ratios of HXA and HPA were estimated to be 1:5.4-4.1. The results obtained showed that the AMY2B gene is usually expressed as HXA in healthy humans.     

Bacterial phosphotransferase system: regulation of the glucose and mannose enzymes II by sulfhydryl oxidation

We have investigated the effect of oxidizing agents on methyl alpha-glucoside phosphorylation by the Escherichia coli phosphotransferase system (PTS). Oxidizing agents inhibited methyl alpha-glucoside phosphorylation at low methyl alpha-glucoside concentrations, and the degree of inhibition was shown to decrease with increasing concentrations of methyl alpha-glucoside. Results of studies with mutant bacteria and substrate analogues of the glucose and mannose enzymes II showed that contrary to the interpretation of Robillard and Konings [Robillard, G. T., & Konings, W. N. (1981) Biochemistry 20, 5025-5032] the apparent change in the Km value for methyl alpha-glucoside phosphorylation induced by sulfhydryl oxidation is not due to the formation of a low-affinity, oxidized form of the glucose enzyme II. Rather, the results are explained by the presence of two phosphotransferase systems that phosphorylate methyl alpha-glucoside with different affinities and that are differentially sensitive to oxidizing agents. The low Km system corresponds to the glucose enzyme II, which is strongly inhibited by potassium ferricyanide, phenazine methosulfate, and plumbagin. The high Km system corresponds to the mannose enzyme II, which is less sensitive to inhibition by these oxidizing agents. This differential sensitivity to inhibition by oxidizing agents can account for the apparent Km change for methyl alpha-glucoside phosphorylation reported by Robillard and Konings. The physiological significance of sulfhydryl oxidation in the enzymes II of the PTS has yet to be ascertained.     

Inhibition and binding modes of low-molecular-weight inhibitors of porcine pancreatic alpha-amylase.

Inhibition of porcine pancreatic alpha-amylase (1,4-alpha-D-glucan glucanohydrase) [EC 3.2.1.1] with maltotriitol (G3OH) and 4-phenylimidazole was investigated by using maltohexaitol (G6OH) and p-nitrophenyl-alpha-D-maltoside (G2PNP) as substrates. When G6OH was the substrate, both G3OH and 4-phenylimidazole behaved as competitive inhibitors. On the other hand, when G2PNP was the substrate, G3OH behaved as a competitive inhibitor, whereas 4-phenylimidazole behaved as a non-competitive inhibitor. Further inhibition study in the presence of both G3OH and 4-phenylimidazole, with G6OH as the substrate, showed that the two inhibitors compete with each other for the active site of the enzyme. Based on a consideration of the productive (reactive) binding modes of G2PNP and G6OH, and a nonproductive (nonreactive) binding mode of G2PNP, it is suggested that the binding sites of the two inhibitors may be partially overlapping around the catalytic site of the enzyme and that the rest of the binding site of each inhibitor lies along the substrate binding cleft of the enzyme.     

Studies on the active site of Taka-amylase A: its action on phenyl maltooligosides with a charge at their non-reducing-ends

Five modified moltooligosaccharides, phenyl O-6-amino-6-deoxy-alpha-D- glucopyranosyl- (1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1-- --4)- alpha-D-glucopyransoide (AG4P), phenyl O-(alpha-D-glucopyranosyluronic acid)-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-d-glucopyran osy l- (1----4)-alpha-D-glucopyranoside (CG4P), phenyl O-6-amino-6-deoxy-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyra nos yl- (1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1-- --4)- alpha-D-glucopyranoside (AG5P), phenyl O-(alpha-D-glucopyranosyluronic acid)-(1----4)-O-alpha-D-glucopyranosyl- (1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1-- --4)- alpha-D-glucopyranoside (CG5P), and phenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)- O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-a lph a-D- glucopyranoside (FG4P), were prepared to examine the active site of Taka-amylase A (TAA) [EC 3.2.1.1, Aspergillus oryzae]. Phenyl alpha-maltotetraoside (G4P) was predominantly hydrolyzed by TAA to maltose and phenyl alpha-maltoside (G2P). While G2P, phenyl alpha-glucoside (GP), and phenol were liberated from AG4P in the ratio of 7:63:30. G4P, phenyl alpha-maltotrioside (G3P), G2P, and GP were liberated from G5P in the ratio of 1:20:73:6, but AG5P was almost completely hydrolyzed to modified maltotriose and G2P. On the hydrolysis of CG4P and CG5P, no remarkable change was observed except for a decrease in the relative reaction rates compared with G4P and G5P, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and structural characterization of O-beta-ribosyl-(1"----2'')-adenosine-5"-phosphate in yeast methionine initiator tRNA.

We report in this paper on the complete structure determination of the modified nucleotide A*, now called Ar(p), that was previously identified in yeast methionine initiator tRNA as an isomeric form of O-ribosyl-adenosine bearing an additional phosphoryl-monoester group on its ribose2 moiety. By using the chemical procedure of periodate oxidation and subsequent beta-elimination with cyclohexylamine on mono- and dinucleotides containing Ar(p), we characterized the location of the phosphate group on the C-5" of the ribose2 moiety, and the linkage between the two riboses as a (1"----2')-glycosidic bond. Since the structural difference between phosphatase treated Ar(p) and authentic O-alpha-ribosyl-(1"----2')-adenosine from poly(ADP-Ribose) was previously assigned to an isomeric difference in the ribose2-ribose1 linkage, the (1"----2')-glycosidic bond of Ar(p) was deduced to have a beta-spatial configuration. Thus, final chemical structure for Ar(p) at the position 64 in yeast initiator tRNA(Met) has been established as O-beta-ribosyl-(1"----2')-adenosine-5"-phosphate. This nucleotide is linked by a 3',5'-phosphodiester bond to G at the position 65.     

Osteoarthritis-susceptibility locus on chromosome 11q, detected by linkage.

We present a two-stage genomewide scan for osteoarthritis-susceptibility loci, using 481 families that each contain at least one affected sibling pair. The first stage, with 272 microsatellite markers and 297 families, involved a sparse map covering 23 chromosomes at intervals of approximately 15 cM. Sixteen markers that showed evidence of linkage at nominal P相似文献   

Multiple amino acid substitutions in hemagglutinin are necessary for wild-type measles virus to acquire the ability to use receptor CD46 efficiently

Measles virus (MV) possesses two envelope glycoproteins, namely, the receptor-binding hemagglutinin (H) and fusion proteins. Wild-type MV strains isolated in B-lymphoid cell lines use signaling lymphocyte activation molecule (SLAM), but not CD46, as a cellular receptor, whereas MV vaccine strains of the Edmonston lineage use both SLAM and CD46 as receptors. Studies have shown that the residue at position 481 of the H protein is critical in determining the use of CD46 as a receptor. However, the wild-type IC-B strain with a single N481Y substitution in the H protein utilizes CD46 rather inefficiently. In this study, a number of chimeric and mutant H proteins, and recombinant viruses harboring them, were generated to determine which residues of the Edmonston H protein are responsible for its efficient use of CD46. Our results show that three substitutions (N390I and E492G plus N416D or T446S), in addition to N481Y, are necessary for the IC-B H protein to use CD46 efficiently as a receptor. The N390I, N416D, and T446S substitutions are present in the H proteins of all strains of the Edmonston lineage, whereas the E492G substitution is found only in the H protein of the Edmonston tag strain generated from cDNAs. The T484N substitution, found in some of the Edmonston-lineage strains, resulted in a similar effect on the use of CD46 to that caused by the E492G substitution. Thus, multiple residues in the H protein that have not previously been implicated have important roles in the interaction with CD46.     

Endocytosis of gentamicin in a proximal tubular renal cell line.

The mechanisms by which aminoglycosides are accumulated in renal proximal tubular cells remain unclear. Adsorptive mediated endocytosis, via a common pathway for cationic proteins, or receptor endocytosis, mediated by the glycoprotein 330/megalin, have been proposed to be involved in gentamicin transport in renal cells. We used the LLC-PK1 cell line, derived from the pig proximal tubule, to explore further the regulation of gentamicin endocytosis in these cells and to determine the role of clathrin mediated endocytosis and G proteins in this function. Gentamicin endocytosis was strictly temperature dependent, whereas total uptake (endocytosis plus binding) did not significantly differ at 4 or 37 degrees C. Substances that suppress receptor mediated, clathrin dependent endocytosis, such as monensin, phenylarsine oxide and dansylcadaverine, or inhibit caveolae mediated endocytosis, such as nystatin, did not affect gentamicin entrance in LLC-PK1 cells. Among substances that disrupt the actin cytoskeleton, only cytochalasin D, that is active also on fluid phase endocytosis, significantly reduced the intracellular concentrations of the aminoglycoside. Other maneuvers that perturb clathrin dependent endocytosis without affecting clathrin independent pathway, such as acidification of cytosol or incubation in hypertonic medium, were also without effect. Mastoparan, a well known stimulator of heterotrimeric G proteins, strongly increased endocytosis of gentamicin, and the same effect was evident with two other G protein stimulators, aluminum fluoride and fluoride alone; however the effect seems not to be mediated by an activation of adenylyl cyclase. In conclusion, gentamicin endocytosis in LLC-PK1 cells is probably clathrin independent, limited by cytochalasin D, which interacts with cytoskeleton, and increased by substances like mastoparan and aluminum fluoride, which activate heterotrimeric G proteins.     

Genetic loss of D-amino acid oxidase activity reverses schizophrenia-like phenotypes in mice

Reduced function of the N -methyl- d -aspartate receptor (NMDAR) has been implicated in the pathophysiology of schizophrenia. The NMDAR contains a glycine binding site in its NR1 subunit that may be a useful target for the treatment of schizophrenia. In this study, we assessed the therapeutic potential of long-term increases in the brain levels of the endogenous NMDAR glycine site agonist D-serine, through the genetic inactivation of its catabolic enzyme D-amino acid oxidase (DAO) in mice. The effects of eliminating DAO function were investigated in mice that display schizophrenia-related behavioral deficits due to a mutation ( Grin 1 ^D481N ) in the NR1 subunit that results in a reduction in NMDAR glycine affinity. Grin 1 ^D481N mice show deficits in sociability, prolonged latent inhibition, enhanced startle reactivity and impaired spatial memory. The hypofunctional Dao 1 ^G181R mutation elevated brain levels of D-serine, but alone it did not affect performance in the behavioral measures. Compared to animals with only the Grin 1 ^D481N mutation, mice with both the Dao1 ^G181R and Grin 1 ^D481N mutations displayed an improvement in social approach and spatial memory retention, as well as a reversal of abnormally persistent latent inhibition and a partial normalization of startle responses. Thus, an increased level of D-serine resulting from decreased catalysis corrected the performance of mice with deficient NMDAR glycine site activation in behavioral tasks relevant to the negative and cognitive symptoms of schizophrenia. Diminished DAO activity and elevations in D-serine may serve as an effective therapeutic intervention for the treatment of psychiatric symptoms.     

Association of <Emphasis Type="Italic">NAT2</Emphasis> polymorphisms with susceptibility to psoriasis in the Moscow population

N-acetyltransferase 2 (NAT2) is a key enzyme of biotransformation phase II that metabolizes genotoxic compounds such as carcinogens and mutagens in different types of cells. A decreased NAT2 activity may correlate with sensitivity to harmful environmental factors, thus increasing susceptibility to different multifactorial diseases, including dermatologic conditions like psoriasis. A biochip developed in our lab to detect 17 NAT2 SNPs was tested on 279 clinical DNA samples from 180 patients with psoriasis and 99 healthy individuals, all residents of Moscow. Six polymorphisms that are most common in European populations (282C > T, 341T > C, 481C > T, 590G > A, 803A > G, and 857G > A) were detected. The NAT2 allele and genotype frequencies for individual SNPs did not differ between patients and healthy individuals. The frequency of the slow acetylation phenotype was increased in patients with type II psoriasis and in normosthenic patients as compared to controls (OR = 1.76, P = 0.177 and OR = 2.07, P = 0.050, respectively). Genotype 341C/C,481T/T,803G/G was significantly more frequent in patients who smoked at least one pack of cigarettes per day and in those who regularly consumed alcohol than in controls (OR = 7.42, P = 0.008 and OR = 106.11, P = 0.003, respectively). The frequency of genotype 341T/T, 481C/C, 590A/-, 803A/A was increased in patients with adverse reactions to medications (OR = 2.05, P = 0.099). Thus, our data suggest that some NAT2 genotypes in combination with certain lifestyles can be considered risk factors of psoriasis in the Moscow population.     

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)