Epimer Of Glucose Research Articles

A mammalian protein homologous to fructosamine-3-kinase is a ketosamine-3-kinase acting on psicosamines and ribulosamines but not on fructosamines.

Fructosamine-3-kinase (FN3K) is an enzyme that appears to be responsible for the removal of fructosamines from proteins. In this study, we report the sequence of human and mouse cDNAs encoding proteins sharing 65% sequence identity with FN3K. The genes encoding FN3K and FN3K-related protein (FN3K-RP) are present next to each other on human chromosome 17q25, and they both have a similar 6-exon structure. Northern blots of mouse tissues RNAs indicate a high level of expression of both genes in bone marrow, brain, kidneys, and spleen. Human FN3K-RP was transfected in human embryonic kidney (HEK) cells, and the expressed protein was partially purified by chromatography on Blue Sepharose. Unlike FN3K, FN3K-RP did not phosphorylate fructoselysine, 1-deoxy-1-morpholino-fructose, or lysozyme glycated with glucose. In a more systematic screening for potential substrates for FN3K-RP, we found, however, that both enzymes phosphorylated ketosamines with a D-configuration in C3 (psicoselysine, 1-deoxy-1-morpholino-psicose, 1-deoxy-1-morpholino-ribulose, lysozyme glycated with allose-the C3 epimer of glucose, or with ribose). Tandem mass spectrometry and nuclear magnetic resonance analysis of the product of phosphorylation of 1-deoxy-1-morpholino-psicose by FN3K-RP indicated that this enzyme phosphorylates the third carbon of the sugar moiety. These results indicate that FN3K-RP is a ketosamine-3-kinase (ketosamine-3-kinase 2). This enzyme presumably plays a role in freeing proteins from ribulosamines or psicosamines, which might arise in a several step process, from the reaction of amines with glucose and/or glycolytic intermediates. This role is shared by fructosamine-3-kinase (ketosamine-3-kinase 1), which has, in addition, the unique capacity to phosphorylate fructosamines.

QLS motif in transmembrane helix VII of the glucose transporter family interacts with the C-1 position of D-glucose and is involved in substrate selection at the exofacial binding site.

The liver-type (GLUT2) and brain-type (GLUT3) human facilitative glucose transporters exhibit distinct kinetics (Km values for deoxyglucose transport of approximately 11 mM and approximately 1.5 mM, respectively) and patterns of substrate transport (GLUT2 is capable of D-fructose transport, while GLUT3 is not). Using a range of chimeric glucose transporters comprised of regions of GLUT2 and GLUT3 studied by expression in Xenopus oocytes after microinjection of cRNA, we have proposed that the seventh putative transmembrane helix is intimately involved in the selection of transported substrate and that this region plays an important role in determining the Km for 2-deoxyglucose [Arbuckle, M. I., Kane, S., Porter, L. M., Seatter, M. J., and Gould, G. W. (1996) Biochemistry 35, 16519-16527]. Inspection of the predicted amino acid sequence of this region reveals that GLUTs 1, 3, and 4 (high-affinity glucose transporters) contain a conserved QLS motif in this helix (residues 277-279 in human GLUT3). In the glucose/fructose transporter (GLUT2) this motif is replaced by HVA. To study the role of the QLS motif in substrate selection, we have engineered substitutions in this region between GLUT2 and GLUT3. GLUT3 (QLS > HVA) exhibits a Km for deoxyglucose transport identical to that of native GLUT3 but increased sensitivity for inhibition of deoxyglucose transport by D-fructose. However, unlike native GLUT3, this species is capable of transporting D-fructose. Compared to wild-type GLUT2, GLUT2 (HVA > QLS) exhibits a lower Km for deoxyglucose transport (approximately 3 mM vs approximately 11 mM), the ability to transport D-fructose is reduced, and D-fructose is a less efficient inhibitor of deoxyglucose transport. Analysis of the ability of a range of glucose epimers and analogues to inhibit transport by these species suggests that the QLS motif interacts with the incoming D-glucose at the C-1 position; this may be a key interaction in the high-affinity recognition of the transported substrate. We further argue that this interaction acts as a molecular filter that is involved in the selection of the transported substrate.

Determination of Galactose in Human Blood by High‐Performance Liquid Chromatography: Comparison with an Enzymatic Method and Application to the Pharmacokinetic Study of Galactose in Patients with Liver Dysfunction

Galactose, the C‐4 epimer of glucose, is an agent of choice for the quantitation of liver function. A simple, precise, and accurate high‐performance liquid chromatographic (HPLC) assay with refractive index detection was developed for the determination of galactose in human whole blood. The method consists of organic solvent‐heavy metal deproteinization procedures and reversed‐phase chromatography on a cation‐exchange column in the calcium form. Calibration graphs were linear over the concentration range 100–2500 µg/mL, with correlation coefficients >0.999. The within‐day coefficient of variation (CV) ranged from 2.08 to 8.94%, and the between‐day CV ranged from 1.61 to 10.9%. The limit of quantitation was 100 µg/mL in whole blood. However, the limit of detection was 75 µg/mL based on a signal‐to‐noise ratio of ⩾3. Eight structurally related sugars and polyols were investigated to check for potential interferences using the analytical condition of the assay. The possible metabolites of galactose present in the body were also checked to determine the specificity of this assay. The proposed HPLC assay was compared with an enzymatic assay and an excellent correlation was observed (HPLC = 1.0299Enz. – 12.907, r = 0.952, p < 0.001). This HPLC method has been successfully applied to the pharmacokinetic study of galactose in six patients with liver dysfunction. Following the intravenous administration of a dose of 0.5 g/kg body weight, galactose pharmacokinetics followed a nonlinear two‐compartment model with Michaelis–Menten elimination from the central compartment. The maximum elimination rate and the Michaelis–Menten constant of galactose in these patients were 44.0 ± 14.9 µg/mL/min and 478 ± 250 µg/mL, respectively. The distribution parametersK12andK21were 0.0749 ± 0.0365 and 0.147 ± 0.081 min−1, respectively. The volume of the central compartment was 0.204 ± 0.045 L/kg body weight.

Subsite specificity of the active site of glucosyltransferases from Streptococcus sobrinus.

Oral bacterial glucosyltransferases use sucrose as a substrate in synthesis of either alpha-1,3 water-insoluble glucans (GTF-I) or alpha-1,6 water-soluble glucans (GTF-S). The binding specificity of the glucosyl and fructosyl subsites of the sucrose-binding site was examined to identify ligands that bind exclusively to each subsite. Such compounds can be used as reporter ligands to localize the subsite binding of any reversible or irreversible active site inhibitor. In examining potential subsite-specific ligands, binding affinity to GTF-I was consistently stronger than binding to GTF-S. Fructose was found to be a moderate GTF inhibitor, but free glucose, alpha-methylglucoside and glucose epimers were very weak inhibitors. In contrast, glucose transition-state analogues, D-glucano-1,5-lactone, 1-deoxynojirimycin (dNJ), and most N-alkyl derivatives of dNJ were moderate to strong inhibitors; in particular N-methyl-dNJ was found to be the strongest GTF inhibitor identified to date. Multiple inhibitor kinetic analysis established nonexclusive binding of fructose and dNJ at the respective subsites. Binding of fructose and N-alkyl-dNJ derivatives was, to a small degree, partially exclusive. Fructose and dNJ were used as reporter ligands to localize the subsite specificity of two test inhibitors: a reversible inhibitor, Zn2+, and an irreversible inhibitor, diethyl pyrocarbonate (DEP). Zn2+ paired with dNJ in multiple inhibitor kinetic analysis showed no competition between the inhibitors, while Zn2+ paired with fructose decreased ligand affinity 7-fold, establishing Zn2+ binding exclusively at the fructose subsite. Analogous experiments adapted to the irreversible inhibitor DEP indicated that it reacts at both subsites or induces a protein conformational change at one subsite that alters ligand binding at the adjacent subsite.

Structural requirements of alloxan and ninhydrin for glucokinase inhibition and of glucose for protection against inhibition.

1. In order to elucidate the mechanism underlying the interactions between glucose and alloxan when competing for the sugar binding site of glucokinase from pancreatic B-cells or liver, the structural requirements of the enzyme for inhibition by alloxan and for protection by glucose were determined. 2. With a half-maximal inhibitory concentration of 5 microM, alloxan was the most potent pyrimidine derivative inhibitor of glucokinase. Uramil was a less potent enzyme inhibitor. A variety of other pyrimidine derivatives and related substances were ineffective. 3. Ninhydrin also inhibited glucokinase with a half-maximal inhibitory concentration of 5 microM. Isatin was a slightly less potent enzyme inhibitor. Several other indoline derivatives were ineffective. 4. Only glucose derivatives with a sufficiently bulky substituent in position C-2, such as the glucokinase substrates glucose and mannose and the inhibitors mannoheptulose, glucosamine, and N-acetylglucosamine, protected glucokinase against inhibition by alloxan by binding to the active site of the enzyme. Glucose epimers which differed in other positions did not protect the enzyme against alloxan inhibition. 5. DTT (dithiothreitol) protected glucokinase against inhibition by alloxan and reversed the inhibition of the enzyme induced by alloxan. Thus the mechanism of glucokinase inhibition by alloxan and other inhibitors, such as uramil and ninhydrin, is an oxidation of functionally essential SH groups of the enzyme, where the most reactive keto group of the inhibitor acts as the hydrogen acceptor. The protective action of glucose and several C-2 epimers demonstrates that these functionally essential SH groups are situated in the sugar binding site of the glucokinase. 6. The present results support our contention, that the pancreatic B-cell glucokinase is the major target mediating the inhibition of insulin secretion by alloxan.

Phosphoglucomutase: its role in the response of pancreatic islets to glucose epimers and anomers

Rat pancreatic islets display phosphoglucomutase activity. The velocity of glucose-1-phosphate conversion to glucose-6-phosphate is increased in a dose-related fashion by glucose-1,6-bisphosphate. The islet homogenate, like purified muscle phosphoglucomutase, also catalyzes the synthesis of glucose-1,6-bisphosphate from glucose-6-phosphate and fructose-1,6-bisphosphate. The rate of the latter reaction is about 10,000 times lower than that of glucose-1-phosphate conversion to glucose-6-phosphate in the presence of glucose-1,6-bisphosphate. D-glucose and D-mannose, but not D-galactose nor D-fructose, markedly increase the islet content in glucose-1,6-bisphosphate. Such a content is twice higher in islets exposed for 5 minutes to alpha-D-glucose than in islets exposed to beta-D-glucose. The process of glucose-1,6-bisphosphate synthesis, as catalyzed by the alpha-stereospecific phosphoglucomutase, may play a role in the metabolic and, hence, secretory responses of the islets to glucose epimers and anomers.

Further identification of the nature and linkage of the carbohydrate in hemoglobin A 1c

We have found that Hb A 1c contains neutral sugars which are only partially hydrolyzed from the N-termini of β chains. In both normal and diabetic Hb A 1c, 0.2–0.3 equivalents of hexose were released, composed primarily of glucose and mannose in a 3:1 ratio. When Hb A 1c was reduced with 3HNa BH 4 and then treated with periodic acid, most of the radioactivity was recovered as 3H-formic acid with much lesser amounts of 3H-formaldehyde. From these results, we propose that in the red cell, glucose binds to the α-amino position of hemoglobin β-chains (valine) in an aldimine (Schiff base) linkage. This aldimine can then partially rearrange in a reversible manner to form a ketoamine linkage which is stable to acid hydrolysis. This Amadori-type rearrangement accounts for the formation of mannose, the C-2 epimer of glucose, as well as the inability to demonstrate 3HNa BH 4 reduction at the C-1 position.