(1/165) Factors affecting counteraction by methylamines of urea effects on aldose reductase.

The concentration of urea in renal medullary cells is high enough to affect enzymes seriously by reducing Vmax or raising Km, yet the cells survive and function. The usual explanation is that the methylamines found in the renal medulla, namely glycerophosphocholine and betaine, have actions opposite to those of urea and thus counteract its effects. However, urea and methylamines have the similar (not counteracting) effects of reducing both the Km and Vmax of aldose reductase (EC, an enzyme whose function is important in renal medullas. Therefore, we examined factors that might determine whether counteraction occurs, namely different combinations of assay conditions (pH and salt concentration), methylamines (glycerophosphocholine, betaine, and trimethylamine N-oxide), substrates (DL-glyceraldehyde and D-xylose), and a mutation in recombinant aldose reductase protein (C298A). We find that Vmax of both wild-type and C298A mutant generally is reduced by urea and/or the methylamines. However, the effects on Km are much more complex, varying widely with the combination of conditions. At one extreme, we find a reduction of Km of wild-type enzyme by urea and/or methylamines that is partially additive, whereas at the other extreme we find that urea raises Km for D-xylose of the C298A mutant, betaine lowers the Km, and the two counteract in a classical fashion so that at a 2:1 molar ratio of betaine to urea there is no net effect. We conclude that counteraction of urea effects on enzymes by methylamines can depend on ion concentration, pH, the specific methylamine and substrate, and identity of even a single amino acid in the enzyme.  (+info)

(2/165) Fingerprint patterns from laser-induced azido photochemistry of spin-labeled photoaffinity ATP analogs in matrix-assisted laser desorption/ionization mass spectrometry.

The photochemical reaction of azide derivatives induced by ultraviolet (UV) laser in matrix-assisted laser desorption/ionization mass spectrometry (MALDI) is reported. A novel synthesized class of azide aromatic derivatives, spin-labeled photoaffinity non-nucleoside adenosine triphosphate (ATP) analogs which are useful probes in study of muscle contraction mechanism, is used in this investigation. In the negative ion MALDI spectra of these ATP analogs, "fingerprint" peaks corresponding to [M - 10 - 1]-, [M - 12 - 1]-, [M - 16 - 1]-, [M - 26 - 1]-, [M - 28 - 1]-, [M - 41 - 1]-, and [M - 42 - 1]- were observed with relative intensities depending on the MALDI matrix. Only the [M - 16 - 1]- is present in the similar mass spectra of the analog in which the azido group is replaced by a hydrogen. A model is suggested for the photochemical reactions of azide derivatives under UV laser irradiation. The photoreaction fingerprint information is diagnostically useful in characterization of azido compounds, especially for spin-labeled photoaffinity non-nucleoside ATP analogs.  (+info)

(3/165) Difference in mechanism between glyceraldehyde- and glucose-induced insulin secretion from isolated rat pancreatic islets.

The effects of D-glyceraldehyde and glucose on islet function were compared in order to investigate the difference between them in the mechanism by which they induce insulin secretion. The stimulation of insulin secretion from isolated rat islets by 10 mM glyceraldehyde was not completely inhibited by either 150 microM diazoxide (an opener of ATP-sensitive K(+) channels) or 5 microM nitrendipine (an L-type Ca(2+)-channel blocker), whereas the stimulation of insulin secretion by 20 mM glucose was completely inhibited by either drug. The insulin secretion induced by glyceraldehyde was less augmented by 100 microM carbachol (a cholinergic agonist) than that induced by glucose. The stimulation of myo-inositol phosphate production by 100 microM carbachol was more marked in islets incubated with the hexose than with the triose. The content of glyceraldehyde 3-phosphate, a glycolytic intermediate, in islets incubated with glyceraldehyde was far higher than that in islets incubated with glucose, whereas the ATP content in islets incubated with the triose was significantly lower than that in islets incubated with the hexose. These results suggest that glyceraldehyde not only mimics the effect of glucose on insulin secretion but also has the ability to cause the secretion of insulin without the influx of Ca(2+ )through voltage-dependent Ca(2+) channels. The reason for the lower potency of the triose than the hexose in stimulating insulin secretion is also discussed.  (+info)

(4/165) Immunological evidence that non-carboxymethyllysine advanced glycation end-products are produced from short chain sugars and dicarbonyl compounds in vivo.

BACKGROUND: The Maillard reaction that leads to the formation of advanced glycation end-products (AGE) plays an important role in the pathogenesis of angiopathy in diabetic patients and in the aging process. Recently, it was proposed that AGE were not only created by glucose, but also by dicarbonyl compounds derived from the Maillard reaction, autoxidation of sugars and other metabolic pathways of glucose. In this study, we developed four types of non-carboxymethyllysine (CML) anti-AGE antibodies that recognized proteins modified by incubation with short chain sugars and dicarbonyl compounds. MATERIALS AND METHODS: AGE-modified serum albumins were prepared by incubation of rabbit serum albumin with glyceraldehyde, glycolaldehyde, methylglyoxal or glyoxal. After immunization of rabbits, four types of AGE-specific antisera were obtained that were specific for the AGE modification. To separate non-CML AGE antibodies (Ab) (non-CML AGE-Ab-2, -3, -4, and -5), these anti-AGE antisera were subjected to affinity chromatography on a matrix coupled with four kinds of AGE bovine serum albumin (BSA) or CML-BSA. These non-CML AGE antibodies were used to investigate the AGE content of serum obtained from diabetic patients on hemodialysis. RESULTS: Characterization of the four types of non-CML AGE antibodies obtained by immunoaffinity chromatography was performed by competitive ELISA and immunoblot analysis. Non-CML AGE-Ab-2 crossreacted with the protein modified by glyceraldehyde or glycolaldehyde. Non-CML AGE-Ab-3 and -Ab-4 specifically cross-reacted with protein modified by glycolaldehyde and methylglyoxal, respectively. NonCML AGE-Ab-5 cross-reacted with protein modified with glyoxal as well as methylglyoxal and glycolaldehyde. Three kinds of non-CML AGE (AGE-2, -4, and -5) were detected in diabetic serum as three peaks with apparent molecular weights of 200, 1.15, and 0.85 kD; whereas, AGE-3 was detected as two peaks with apparent molecular weights of 200 and 0.85 kD. CONCLUSION: We propose that various types of non-CML AGE are formed by the Maillard reaction, sugar autoxidation and sugar metabolism. These antibodies enable us to identify such compounds created by the Maillard reaction in vivo.  (+info)

(5/165) Glyceraldehyde preserves glucose concentrations in whole blood specimens.

BACKGROUND: Glucose concentrations decrease in blood specimens during transport/processing, primarily because of continuing metabolism (glycolysis) by erythrocytes. Several means to reduce the loss of glucose in blood specimens have been developed, but all have major drawbacks. Glyceraldehyde, which has antiglycolytic activity, was assessed for potential in preserving glucose in blood specimens. METHODS: Heparinized blood from volunteers was treated with glyceraldehyde and other agents. After incubation for various times, plasma concentrations of glucose and other common analytes were determined with prevalent commercial analyzers. RESULTS: The racemic mixture of glyceraldehyde (D,L-GA) preserved glucose concentrations for up to 8 h at room temperature. Half-maximal effect was attained with 0.9 mmol/L D,L-GA. Trials of the D and L stereoisomers individually indicated that the L isomer (L-GA) was responsible for all or most of the antiglycolytic activity of the racemic mixture. Other related compounds were ineffective. Measurements of most common clinical laboratory analytes were unaffected by the presence of D,L-GA or L-GA. CONCLUSIONS: Glyceraldehyde (D,L-GA or L-GA) effectively preserves glucose concentrations in whole blood specimens for up to 8 h. Specimens collected with D,L-GA or L-GA are suitable for analysis of many analytes commonly comeasured with glucose.  (+info)

(6/165) Pancreatic islet cells: effects of monosaccharides, glycolytic intermediates and metabolic inhibitors on membrane potential and electrical activity.

1. The effects of monosaccharides, glycolytic intermediates, metabolic inhibitors and anxia, have been studied on the membrane electrical activity of mouse pancreatic islet cells in vitro using a single intracellular micro-electrode for both voltage recording and current injection. 2. In addition to D-glucose (28mM), D-mannose (16-6mM), and L-leucin (10mM), the substances D-glyceraldehyde (11mM), and acetoacetate (20 mM), induced action potentials in islet cells but other glucos analogues and metabolic intermediates including L-glucose dod not. 3. Mannoheptulose 20 mM), but not D-galactose or 2-deoxy-D-glucose, antagonized the electrical activity induced in islet cells by D-glucose, 28mM. Prior treatment of the cells with mannoheptulose caused them to hyperpolarize and completely prevented the appearance of electrical activity on subsequent exposure to D-glucose. 4. Electrical activity induced by D0glucose 28mM, was progressively inhibited by phloridzin, 10mM, if the cells were exposed to D-glucose and inhibitor simultaneously, and abolished on pretreatment with inhibitor for 30-60 min. Phloridzin also caused depolarization of the islet cells which was independent of extracellular glucose. 5. Anoxia completely blocked the electrical activity induced by glucose but not that evoked by D-glyceraldehyde, L-leucine, tolbutamide or glibenclamide. 6. Iodoacetic acid, 5 mM, rapidly blocked glucose-induced electrical activity whilst that elicited by tolbutamide was relatively resistant to inhibition. 7. The nature and possible location of the glucoreceptor in pancreatic islet cells is discussed in relation to the origin and functional significance of glucose-induced electrical activity and insulin secretion.  (+info)

(7/165) Glyceraldehyde phosphate at the reducing terminus of Salmonella Q haptens. Salmonella montevideo.

The O antigen polysaccharide of Salmonella montevideo was isolated from a core-defective mutant by the phenol/water procedure, and was suspected to contain phosphomonester and cyclic phosphodiester at its reducing end in anology to the O hapten from Salmonella typhimurium (Kent and Obsborn, 1968. Therefore, it was chromatographed on a DEAE-cellulose column. Whereas one part eluted with water the other part of the polysaccharide could only be eluted with buffer. Both fractions were further purified on Sephadex G100 and contained mannose, glucose, N-acetylglucosamine and phosphate in a molar ratio of 4:1:1: less than 0.1. In order to specifically label the reducing end phosphate was removed enzymatically, or the presumed cyclic diester was cleaved by mild hydrolysis, and the fractions were reduced with sodium horo[3H]hydride. Both fractions yield mainly [3H]glycerol after hydrolysis and paper chromatogaphy. In addition, [3H]mannitol and [H]monohydroxyacetone could be identified by paper chromatography and were concluded to be the result of phosphate migration and beta-elimination reactions taking place during the isolation procedure and the various treatments prior to sodium boro[3H]hydride reduction. These findings in addition to periodate oxidation studies indicated that the O antigen polysaccharide of Salmonella montevideo had glyceraldehyde phosphate at its reducing end. From the incorporation of 3H into the polysaccharide the O antigen was calculated to consist of about 19 repeating units of 6 sugar residues each.  (+info)

(8/165) Differential regulation of [Ca2+]i oscillations in mouse pancreatic islets by glucose, alpha-ketoisocaproic acid, glyceraldehyde and glycolytic intermediates.

Glucose induces slow oscillations of the cytoplasmic Ca2+ concentration in pancreatic beta-cells. In order to elucidate the mechanisms responsible for the slow [Ca2+]i oscillations the effects of various nutrient insulin secretagogues on glucose-induced [Ca2+]i oscillations in intact mouse pancreatic islets and single beta-cells were studied. These were the glycolytic intermediates, glyceraldehyde and pyruvate, and the mitochondrial substrate, alpha-ketoisocaproic acid (KIC). Glucose, at a 10 or 15 mM concentration, induced the typical slow oscillations of [Ca2+]i (0.4 min(-1)). At higher glucose concentrations the frequency of these oscillations decreased further (0.2 min(-1)). Glyceraldehyde, an insulin secretagogue like glucose, did not cause slow oscillations of [Ca2+]i in the absence of glucose. However, it exhibited a synergistic action with glucose. Glyceraldehyde, at 3 or 10 mM concentration, induced slow [Ca2+]i oscillations at a substimulatory concentration of 5 mM glucose (0.3-0.4 min(-1)) and reduced the frequency of the glucose-induced [Ca2+]i oscillations at stimulatory concentrations of 10 or 15 mM glucose (0.2 min(-1)). KIC (5 or 10 mM) as well as pyruvate (10 mM), the end product of glycolysis, and its ester methyl pyruvate (10 mM), did not cause slow oscillations of [Ca2+]i in the absence of glucose. In contrast to glyceraldehyde, however, all three compounds were capable of preventing the slow [Ca2+]i oscillations induced by glucose. Mannoheptulose (2 mM), an inhibitor of glucokinase and glucose-induced insulin secretion, reversibly blocked any kind of [Ca2+]i oscillation and returned the [Ca2+]i to a basal level through its ability to inhibit glycolytic flux. It can be concluded therefore that only substrates which generate a glucokinase-mediated metabolic flux through glycolysis and produce glycolytic ATP can induce slow [Ca2+]i oscillations in pancreatic beta-cells.  (+info)