Drug-induced desensitization of insulinotropic actions of sulfonylureas

K(ATP)-channel-dependent and K(ATP)-channel-independent insulin-releasing actions of the sulfonylurea, tolbutamide, were examined in the clonal BRIN-BD11 cell line. Tolbutamide stimulated insulin release at both nonstimulatory (1.1 mM) and stimulatory (16. 7 mM) glucose. Under depolarizing conditions (16.7 mM glucose plus 30 mM KCl) tolbutamide evoked a stepwise K(ATP) channel-independent insulinotropic response. Culture (18 h) with tolbutamide or the guanidine derivative BTS 67 582 (100 microM) markedly reduced (P < 0. 001) subsequent responsiveness to acute challenge with tolbutamide, glibenclamide, and BTS 67 582 but not the imidazoline drug, efaroxan. Conversely, 18 h culture with efaroxan reduced (P < 0.001) subsequent insulinotropic effects of efaroxan but not that of tolbutamide, glibenclamide, or BTS 67 582. Culture (18 h) with tolbutamide reduced the K(ATP) channel-independent actions of both tolbutamide and glibenclamide. Whereas culture with efaroxan exerted no effect on the K(ATP) channel-independent actions of sulfonylureas, BTS 67 582 abolished the response of tolbutamide and inhibited that of glibenclamide. These data demonstrate that prolonged exposure to tolbutamide desensitizes both K(ATP)-channel-dependent and -independent insulin-secretory actions of sulfonylureas, indicating synergistic pathways mediated by common sulfonylurea binding site(s).     

Inhibition of nonenzymatic protein glycation and lipid peroxidation by drugs with antioxidant activity

We studied the effects of aminoguanidine (AG), beta-resorcylidene aminoguanidine (RAG), DL-penicillamine (PNCA) and captopril on early and advanced glycation of human serum albumin (HSA). We also assessed inhibition of lipid peroxidation by AG and RAG in erythrocytes. Incubation of HSA with D-glucose (20 mM, 37 degrees C for 21 days) led to the formation of Amadori products and fluorescent advanced glycation end-products (AGE). Only PNCA markedly reduced the formation of Amadori products, while all tested compounds markedly reduced the formation of AGE. AG and RAG also inhibited malondialdehyde formation in erythrocytes incubated with hydrogen peroxide. Addition of AG at concentrations from 1 microM to 1 mM caused a 10-80% inhibition of lipid peroxidation. Thus, AG and RAG inhibit toxic oxidative processes and may have therapeutic potential in a number of human diseases.     

Glucose Modification of Human Serum Albumin: A Structural Study

Structural changes associated with the exposure of human serum albumin (HSA) to glucose with or without the presence of Cu (II) have been characterized using a bank of methods for structural analysis including circular dichroism (CD), amino acid analysis (AAA), fluorescence measurements, SDS-PAGE, and boronate binding (which is a measure of Amadori product formation). We show that in the short-term (10 d) incubation mixtures, HSA is resistant to Cu (II)-mediated oxidative damage and that the early products of glycation of HSA had minimal effects on the folded structure. Amino acid analysis showed that there was no formation of advanced glycation endproducts (AGE), which can be measured by loss of lysine. This remained the case in longer term incubation of HSA (56 d) in the hyperglycemic concentration range (5–25 mM glucose) despite increased levels of Amadori product (60% boronate binding) and the formation of glycophore (Excitation 350, Emission 425). At high, nonphysiological concentrations (100 mM and 500 mM) of glucose, glycophore formation increased and 3 and 11 mol Lysine-glucose adduct/mol HSA were converted to AGE, respectively. This was accompanied by increased damage to tryptophan and protein-protein crosslinking but only minor tertiary structural change. In the presence of Cu (II), however, AGE formation was accompanied by extensive damage to histidine and tryptophan side chains, main chain fragmentation, and loss of both secondary and tertiary structure. Thus, changes in structure appear to be the result of oxidation as opposed to glycation, per se. © 1997 Elsevier Science Inc.     

Kinetin inhibits protein oxidation and glycoxidation in vitro

We tested the ability of N(6)-furfuryladenine (kinetin) to protect against oxidative and glycoxidative protein damage generated in vitro by sugars and by an iron/ascorbate system. At 50 microM, kinetin was more efficient (82% inhibition) than adenine (49% inhibition) to inhibit the bovine serum albumin (BSA)-pentosidine formation in slow and fast glycation/glycoxidation models. Kinetin also inhibited the formation of BSA-carbonyls after oxidation significantly more than adenine did. However both compounds inhibited the advanced glycation end product (AGE) formation to the same extent (59-68% inhibition). At 200 microM, kinetin but not adenine, limited the aggregation of BSA during glycation. These data suggest that kinetin is a strong inhibitor of oxidative and glycoxidative protein-damage generated in vitro.     

Amadorins: novel post-Amadori inhibitors of advanced glycation reactions

The present review focuses on the background and progress that led to discovery of specific inhibition of post-Amadori formation of advanced glycation end products, or AGEs. The "classic" or Hodge pathway begins with glucose condensation with amino groups to form a Schiff base aldimine adduct that undergoes rearrangement to a ketoamine Amadori product. This pathway is considered an important route to AGE formation that has been implicated in glucose-mediated damage in vivo (3-5). We recently described a facile procedure for isolation of proteins rich in Amadori adducts but free of AGEs, thus permitting study of pathways of conversion of Amadori compounds to AGEs. This in turn led to a unique and rapid post-Amadori screening assay for putative "Amadorins," which we define here as inhibitors of the conversion of Amadori intermediates to AGEs in the absence of excess free or reversibly bound (Schiff base) sugar. Our screening assay then led to the identification of pyridoxamine (Pyridorin) as the first member of this class of Amadorin compounds. Rather unexpectedly, the assay also led to the clear demonstration that the well-known AGE inhibitor aminoguanidine, currently in Phase 3 clinical trials for treatment of diabetic nephropathy, has negligible Amadorin activity. In view of the importance of Amadori compounds as intermediates in AGE formation in vivo, the therapeutic potential of Pyridorin is currently being investigated and is now showing highly promising results in different animal models.     

Kinetic properties of human placental glucose-6-phosphate dehydrogenase

The kinetic properties of placental glucose-6-phosphate dehydrogenase were studied, since this enzyme is expected to be an important component of the placental protection system. In this capacity it is also very important for the health of the fetus. The placental enzyme obeyed "Rapid Equilibrium Ordered Bi Bi" sequential kinetics with K(m) values of 40+/-8 microM for glucose-6-phosphate and 20+/-10 microM for NADP. Glucose-6-phosphate, 2-deoxyglucose-6-phosphate and galactose-6-phosphate were used with catalytic efficiencies (k(cat)/K(m)) of 7.4 x 10(6), 4.89 x 10(4) and 1.57 x 10(4) M(-1).s(-1), respectively. The K(m)app values for galactose-6-phosphate and for 2-deoxyglucose-6-phosphate were 10+/-2 and 0.87+/-0.06 mM. With galactose-6-phosphate as substrate, the same K(m) value for NADP as glucose-6-phosphate was obtained and it was independent of galactose-6-phosphate concentration. On the other hand, when 2-deoxyglucose-6-phosphate used as substrate, the K(m) for NADP decreased from 30+/-6 to 10+/-2 microM as the substrate concentration was increased from 0.3 to 1.5 mM. Deamino-NADP, but not NAD, was a coenzyme for placental glucose-6-phosphate dehydrogenase. The catalytic efficiencies of NADP and deamino-NADP (glucose-6-phosphate as substrate) were 1.48 x 10(7) and 4.80 x 10(6) M(-1)s(-1), respectively. With both coenzymes, a hyperbolic saturation and an inhibition above 300 microM coenzyme concentration, was observed. Human placental glucose-6-phosphate dehydrogenase was inhibited competitively by 2,3-diphosphoglycerate (K(i)=15+/-3 mM) and NADPH (K(i)=17.1+/-3.2 microM). The small dissociation constant for the G6PD:NADPH complex pointed to tight enzyme:NADPH binding and the important role of NADPH in the regulation of the pentose phosphate pathway.     

Oxidative stress-inducing carbonyl compounds from common foods: novel mediators of cellular dysfunction

BACKGROUND: The general increase in reactive oxygen species generated from glucose-derived advanced glycation endproducts (AGEs) is among the key mechanisms implicated in tissue injury due to diabetes. AGE-rich foods could exacerbate diabetic injury, at least by raising the endogenous AGE. MATERIALS AND METHODS: Herein, we tested whether, prior to ingestion, diet-derived AGEs contain species with cell activating (TNFalpha), chemical (cross-linking) or cell oxidative properties, similar to native AGEs. Glutathione (GSH) and GSH peroxidase (GPx) were assessed after exposure of human umbilical vein endothelial cell (HUVECs) to affinity-purified food-AGE extracts, each exposed to 250 degrees C, for 10 min, along with synthetic AGEs. RESULTS: Animal product-derived AGE, like synthetic methylglyoxal-bovine serum albumin (MG-BSA), AGE-BSA, and AGE-low density lipoprotein (AGE-LDL), induced a dose- and time-dependent depletion of GSH (()60-75%, p, 0.01) and an increase in GPx activity (()500-600%, p < 0.01), consistent with marked TNFalpha and cross-link formation (p < 0.05); this contrasted with the low bioreactivity of starch/vegetable AGE-extracts, which was similar to that of control BSA and CML- BSA and BSA (p:NS). Anti-AGE-R1,2,3 and -RAGE IgG each inhibited cell-associated (125) I-dAGE by approximately 30-55%; GSH/GPx were effectively blocked by N-acetyl-cysteine (NAC, 800 uM, p < 0.01) and aminoguanidine-HCl (AG, 100 uM, p < 0.01). CONCLUSION: Thus, food-derived AGE, prior to absorption, contain potent carbonyl species, that can induce oxidative stress and promote inflammatory signals.     

Modification of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides with the 2',3'-dialdehyde derivative of NADP+ (oNADP+)

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides is irreversibly inactivated by the 2,3'-dialdehyde of NADP+ (oNADP+) in the absence of substrate. The inactivation is first order with respect to NADP+ concentration and follows saturation kinetics, indicating that the enzyme initially forms a reversible complex with the inhibitor followed by covalent modification (KI = 1.8 mM). NADP+ and NAD+ protect the enzyme from inactivation by oNADP+. The pK of inactivation is 8.1. oNADP+ is an effective coenzyme in assays of glucose-6-phosphate dehydrogenase (Km = 200 microM). Kinetic evidence and binding studies with [14C] oNADP+ indicate that one molecule of oNADP+ binds per subunit of glucose-6-phosphate dehydrogenase when the enzyme is completely inactivated. The interaction between oNADP+ and the enzyme does not generate a Schiff's base, or a conjugated Schiff's base, but the data are consistent with the formation of a dihydroxymorpholino derivative.     

Antiglycation effect of gliclazide on in vitro AGE formation from glucose and methylglyoxal

Gliclazide, a sulfonylurea widely used for treatment of diabetes mellitus, is known to scavenge reactive oxygen species. To clarify whether its antioxidative ability interferes with the glycation processes, we incubated bovine serum albumin (BSA) with 1 M glucose or 1 mM methylglyoxal, in the presence or absence of gliclazide, and observed the formation of advanced glycation end products (AGEs). AGE production was assessed by AGE-specific fluorescence, an enzyme-linked immunosorbent assay (ELISA), and Western blotting. The fluorescence at excitation/emission wavelengths of 320/383 nm and 335/385 nm was definitely increased by incubating BSA with 1 M glucose or 1 mM methylglyoxal, and 1 mM gliclazide significantly blunted the fluorescent augmentation, in both wavelengths, in a dose-dependent fashion. Gliclazide almost equaled to aminoguanidine, a putative antiglycation agent, in the inhibitory effect on the glucose-induced fluorescence, while the methylglyoxal-derived fluorescent formation was less suppressed by gliclazide than by aminoguanidine. The AGE concentrations determined by ELISA showed similar results. Incubation of BSA with 1 M glucose or 1 mM methylglyoxal yielded an apparent increase in carboxymethyllysine or argpyrimidine. Both AGEs were significantly lowered by 1 mM gliclazide and a reduction of glucose-derived carboxymethyllysine was comparable to that caused by aminoguanidine. The results of Western blotting supported the findings in ELISA. To our knowledge, the present study provides the first evidence of the antiglycation effect of gliclazide on in vitro AGE formation from glucose and methylglyoxal.     

Mechanisms of action of cyclosporin A on islet alpha- and beta-cells. Effects on cAMP- and calcium-dependent pathways.

The involvement of cAMP- and calcium-dependent pathways on the inhibitory effect of CsA (0.5 micrograms/ml) on insulin and glucagon release was studied in collagenase-isolated islets. CsA suppressed by 50% the release of insulin in pertussis toxin treated islets stimulated by 20 mM D-glucose. CsA blocked glucagon and insulin release induced by 0.2 mM IBMX (80% and 50% respectively). Similarly it inhibited glucagon and insulin release induced by 1 microM A23187 (53% and 40% respectively). CsA also abolished 0.1 microM glucagon-induced insulin release and 10 ng/ml VIP-induced glucagon release (70% and 38% respectively). The glucagon response to 2 mM D-glucose and to 10 mM arginine was decreased 25% and 45% respectively by CsA. The inhibitory effect of 0.1 microM somatostatin on insulin release was significantly abolished by CsA (p less than 0.001 vs control). On the other hand 1 microM forskolin induced insulin and glucagon release was not modified by CsA. Rats treated with CsA (10 mg/kg body wt) during 10 days showed hyperglycaemia, hypoglucagonemia and higher contents of pancreatic glucagon. It is concluded that CsA affects alpha- and beta-cell function, in vivo and in vitro, acting through calcium and cAMP-dependent pathways. This latter pathway involves the Ca(2+)-calmodulin dependent phosphodiesterase and the regulatory proteins Gs and Gi.     

Uptake of advanced glycation end products by proximal tubule epithelial cells via macropinocytosis

Chronic hyperglycaemia during diabetes leads to non-enzymatic glycation of proteins to form advanced glycation end products (AGEs) that contribute to nephropathy. We describe AGE uptake in LLC-PK1 and HK2 proximal tubule cell lines by macropinocytosis, a non-specific, endocytic mechanism. AGE–BSA induced dorsal circular actin ruffles and amiloride-sensitive dextran–TRITC uptake, significantly increased AGE–BSA–FITC uptake (167 ± 20% vs BSA control, p < 0.01) and was ezrin-dependent. AGE–BSA–FITC uptake was significantly inhibited by amiloride and inhibitors of Arf6, Rac1, racGEF Tiam1, PAK1 and actin polymerisation. AGE–BSA–FITC, Arf6 and PIP2 co-localised within dorsal circular actin ruffles. AGE–BSA increased PAK1 kinase activity (212 ± 41% vs control, p < 0.05) and protein levels of Tiam1, a Rac1 activator. AGE–BSA significantly increased TGF-β₁ protein levels (160 ± 6%, p < 0.001 vs BSA), which were significantly inhibited by inhibitors of Arf6 (82 ± 19%, p < 0.001 vs AGE) and actin polymerisation (107 ± 11%, p < 0.001 vs AGE), suggesting AGEs partially exert their profibrotic effects via macropinocytosis. PAK1 and PIP5Kγ siRNA significantly decreased AGE–BSA–FITC uptake (81 ± 6% and 64 ± 7%, respectively, p < 0.05 vs control for both), and AGE-stimulated TGF-β₁ protein release (99 ± 15% and 49 ± 8% of control, p < 0.05 and p < 0.001, respectively). Inhibition of AGE uptake by macropinocytosis inhibitors and a neutralising TGF-β antibody, reversed the AGE-induced decrease in surface Na⁺K⁺ATPase, suggesting AGE uptake by macropinocytosis may contribute to diabetic kidney fibrosis and/or EMT by modulating this pump. Understanding methods of cellular uptake and signalling by AGEs may lead to novel therapies for diabetic nephropathy.     

Advanced glycation endproduct induces ROS accumulation, apoptosis, MAP kinase activation and nuclear O-GlcNAcylation in human cardiac myocytes

Accumulation of advanced glycation endproduct (AGE) has been implicated in the pathogenesis of diabetic complications. However, the precise role and mechanism behind AGE-associated diabetic heart injury are not fully clear. This study was designed to evaluate the effect of AGE on accumulation of reactive oxygen species (ROS), apoptosis, mitogen-activated protein kinase (MAPK) activation and nuclear O-GlcNAcylation in fetal human cardiac myocytes. Myocytes were maintained for 24-72 h in a defined culture medium containing high glucose, the AGE carbon precursor methylglyoxal (MG), and MG-AGE derived from MG and bovine serum albumin (BSA). Generation of ROS was detected by 5-(6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate. Apoptosis was evaluated by caspase-3 activity and quantitative DNA fragmentation. Both high glucose (25.5 mM) and MG (200 microM) significantly enhanced ROS and AGE formation with greater effects elicited by MG. Both high glucose and MG-AGE significantly facilitated apoptosis with a more predominant effect from MG-AGE. In addition, phosphorylation of MAPK cascade [extracellular signal-regulated kinase-1/2 (ERK1/2) and p38] and nuclear O-GlcNAcylation were enhanced in MG-AGE-treated myocytes, similar to those elicited by high glucose. MG-AGE-induced phosphorylation of ERK1/2 and p38 was nullified by neutralizing AGE with specific anti-AGE antibody but not nonspecific antiserum. Collectively, these results indicated that AGE or its precursor MG may trigger ROS generation, apoptosis, MAPK activation and nuclear O-GlcNAcylation in human cardiac myocytes, in a manner reminiscent of high extracellular glucose.     

1L-myo-inositol 1-phosphate synthase from pollen of Lilium longiflorum. An ordered sequential mechanism

1L-Inositol 1-phosphate synthase (EC 5.5.1.4) devoid of bound NAD+ was isolated from mature pollen of Lilium longiflorum ( Easter lily ). The enzyme has a molecular weight of 157,000 +/- 15,000 and a subunit weight of 61,000 +/- 5,000. Kinetic studies of the uninhibited reaction and of inhibition by 2-deoxy-D-glucose 6-phosphate and NADH show the reaction to be ordered sequential with NAD+ adding first. The Michaelis constants for NAD+ and D-glucose 6-phosphate are 2.4 and 65 microM, respectively. The Ki for 2-deoxy-D-glucose 6-phosphate was 8.7 and 2.0 microM, respectively, when D-glucose 6-phosphate or NAD+ was varied. The Ki for NADH and variable NAD+ was 4.7 microM and, for NADH and variable D-glucose 6-phosphate, 3.9 microM.     

Synthesis and evaluation of novel 2,3,5-triaryl-4H,2,3,3a,5,6,6a-hexahydropyrrolo[3,4-d]isoxazole-4,6-diones for advanced glycation end product formation inhibitory activity

The synthesis of some biologically interesting pyrrolo-isoxazolidine derivatives was accomplished by the 1,3-dipolar cycloaddition reaction of substituted azomethine N-oxides 1 with substituted N-aryl maleimides 2 leading to the formation of new stereoisomeric 2,3,5-triaryl-4H,2,3,3a,5,6,6a-hexahydropyrrolo[3,4-d]isoxazole-4,6-dione derivatives 3 in excellent yields. The synthesized compounds have been screened for their advanced glycation end (AGE) product formation inhibitory activity on the basis of their ability to inhibit the formation of AGEs in the bovine serum albumin (BSA)-glucose assay. All the synthesized compounds have been found to exhibit significant activity against AGE formation.     

Proteomic analysis of the site specificity of glycation and carboxymethylation of ribonuclease

Proteomic analysis using electrospray liquid chromatography-mass spectrometry (ESI-LC-MS) has been used to compare the sites of glycation (Amadori adduct formation) and carboxymethylation of RNase and to assess the role of the Amadori adduct in the formation of the advanced glycation end-product (AGE), N(epsilon)-(carboxymethyl)lysine (CML). RNase (13.7 mg/mL, 1 mM) was incubated with glucose (0.4 M) at 37 degrees C for 14 days in phosphate buffer (0.2 M, pH 7.4) under air. On the basis of ESI-LC-MS of tryptic peptides, the major sites of glycation of RNase were, in order, K41, K7, K1, and K37. Three of these, in order, K41, K7, and K37 were also the major sites of CML formation. In other experiments, RNase was incubated under anaerobic conditions (1 mM DTPA, N2 purged) to form Amadori-modified protein, which was then incubated under aerobic conditions to allow AGE formation. Again, the major sites of glycation were, in order, K41, K7, K1, and K37 and the major sites of carboxymethylation were K41, K7, and K37. RNase was also incubated with 1-5 mM glyoxal, substantially more than is formed by autoxidation of glucose under experimental conditions, but there was only trace modification of lysine residues, primarily at K41. We conclude the following: (1) that the primary route to formation of CML is by autoxidation of Amadori adducts on protein, rather than by glyoxal generated on autoxidation of glucose; and (2) that carboxymethylation, like glycation, is a site-specific modification of protein affected by neighboring amino acids and bound ligands, such as phosphate or phosphorylated compounds. Even when the overall extent of protein modification is low, localization of a high proportion of the modifications at a few reactive sites might have important implications for understanding losses in protein functionality in aging and diabetes and also for the design of AGE inhibitors.     

Chalcone synthase from cell suspension cultures of Daucus carota L

Chalcone synthase (CHS) has been partially purified about 35-fold. Withdrawal of 2-mercaptoethanol after precipitation with ammonium sulfate led to higher stability during further purification steps. In order to determine CHS activity, two procedures [according to Schr?der et al. (1979) Plant Sci. Lett. 14, 281-286] were applied. The radioactivity extracted with ethyl acetate from the assay mixture (total products) was compared to 14C-labeled flavanone purified by TLC. The activity of CHS increased with bovine serum albumin (BSA) or 2-mercaptoethanol in the assay. Both effects were synergistic, but BSA did not promote "side products" as 2-mercaptoethanol did. BSA (10 mg ml-1) and 2-mercaptoethanol (1.4 mM) were components of the standard assay. Under these conditions, the CHS from Daucus carota had different pH optima for naringenin formation (7.9) and eriodictyol formation (6.8). The apparent Km values were 0.6 microM for 4-coumaroyl-CoA (pH 7.9), 7.7 microM for caffeoyl-CoA (pH 6.8), and 3.0 microM for malonyl-CoA (pH 7.9). Substrate inhibition was observed with 4-coumaroyl-CoA (greater than 10 microM) and malonyl-CoA (greater than 50 microM). The inhibitory activity of various flavonoids and related compounds (100 microM) was investigated. Naringenin and naringenin-chalcone inhibited eriodictyol formation totally and naringenin formation by 50%. In contrast, eriodictyol and eriodictyol-chalcone inhibited only eriodictyol formation by 40%. It was shown that the inhibition with naringenin was fully uncompetitive. These in vitro data support the view that the true substrate of CHS in D. carota is 4-coumaroyl-CoA.     

Effects of sperm treatments on the in vitro development of bovine oocytes in semidefined and defined media

Bovine in vitro matured oocytes were inseminated with frozen-thawed spermatozoa prepared by A) swim-up through Fert-TALP supplemented with hyaluronic acid (HYA, 1 mg/ml), heparin (5.0 microg/ml) and bovine serum albumin (BSA, 6 mg/ml) or B) washing by centrifugation in modified Brackett-Oliphant medium (mBO) supplemented with 10 mM caffeine-sodium benzoate. For Method A, in vitro fertilization (IVF) was performed in Fert-TALP supplemented with 6 mg/ml BSA, 5.0 microg/ml heparin, 20 microM D-penicillamine, 10 microM hypotaurine and 1 microM epinephrine. For Method B it was performed in mBO medium supplemented with 10 mg globulin-free BSA/ml and 10 microg heparin/ml. Presumptive zygotes were cultured in 1 of 3 culture media: 1) BSAITS - TCM 199 supplemented with 10 mg/ml BSA and ITS (5 microg/ml insulin, 5 microg/ml transferrin, and 5 ng/ml sodium selenite); 2) BECM - bovine embryo culture medium; and 3) BECM supplemented with ITS. Altogether, a significantly higher proportion of oocytes developed to the blastocyst stage after insemination with spermatozoa prepared by Method A than by Method B (17.9 vs 7.1%, respectively; P < 0.001). For Method A, the cleavage rate and the proportion of zygotes with >2 cells 48 h after insemination did not differ significantly between any of the 3 culture media assayed, but blastocyst formation was significantly stimulated in BSAITS and BECMITS compared with that in BECM (20.7 and 22.1% vs 10.7%, respectively; P < 0.05). For Method B, the cleavage rate and the proportion of zygotes with >2 cells were significantly lower in BSAITS than in BECM and BECMITS (56.4 and 28.7% vs 71.6 and 42.1%; and 70.2 and 51.1%, respectively; P < 0.05). However, no significant differences were recorded in blastocyst development rates between any of the culture media assayed (6.4 to 7.4%; P > 0.05).     

Monkey liver microsomal glucose-6-phosphatase]

Kinetic studies indicate that glucose-6-phosphatase is a multifunctional enzyme. a) Phosphohydrolase activities. The mannose-6-phosphatase activity is low (Km = 8 mM, VM = 90 nmoles. min-1mg-1). The enzyme shows a strong affinity for glucose-6-phosphate (Km = 2.5 mM, VM = 220 nmoles.min-1mg-1). beta-glycerophosphate (K1 = 30 mM), D-glucose (Ki = 120 mM) are mixed type inhibitors; pyrophosphate (Ki = 2 mM) is a non competitive one. b) Phosphotransferase activities. Di and triphosphate adenylic nucleosides or phosphoenol pyruvate are not substrates. Carbamylphosphate serves as a phosphoryl donor with D-glucose as acceptor. The phosphate transfer is consisstent with a random mechanism in which the binding of one substrate increases the enzymes affinity for the second substrate. Apparent Km values for carbamyl-phosphate range from 5.2 mM (D-glucose concentration leads to infinity) to 8 mM (D-glucose concentration leads to 0). The corresponding apparent Km values for D-glucose are 59 mM (carbamyl-phosphate concentration leads to infinity) to 119 mM (carbamyl-phosphate concentration leads to 0). Maximal reaction velocity with infinite levels of both substrates is 270 nmoles.min-1.mg-1. Pyrophosphate is a poor phosphoryl donnor (Km = 55 mM with D-glucose concentration 250 mM). In addition we do not find any latency; detergents, namely sodium deoxycholate, Triton X 100 do not affect or inhibit glucose-6-phosphatase activity.     

Favored and disfavored pathways of protein crosslinking by glucose: glucose lysine dimer (GLUCOLD) and crossline versus glucosepane

We describe the isolation and molecular characterization of a novel glucose-lysine dimer crosslink 1,3-bis-(5-amino-5-carboxypentyl)-4-(1′,2′,3′,4′-tetrahydroxybutyl)-3H-imidazolium salt, named GLUCOLD. GLUCOLD was easily formed from the Amadori product (fructose–lysine). However, when BSA was incubated with 100 mM glucose for 25 days, the levels of the lysine-lysine glucose crosslinks GLUCOLD and CROSSLINE were only 21 and <1 pmol/mg, respectively, compared to 611 pmol/mg protein for the lysine-arginine GLUCOSEPANE crosslink, in spite of more than 20 potential lysine-lysine crosslinking sites in the protein. Mechanistic investigation revealed that metal-free phosphate ions catalyzed formation of fructose–lysine and all three crosslinks from amino acids, while cationic MOPS buffer had an opposite effect. This together with the rapid formation of N ⁶-1,4-dideoxy-5,6-dioxoglucosone derivatives by dicarbonyl trapping agents, such as 1,2-diaminobenzene or γ-guanidinobutyric acid, strongly suggests that enolization of the Amadori product and trapping of the 5,6-dioxo derivative by arginine residues constitutes the major pathway for glucose-mediated crosslinking in proteins.     

Enzyme-catalyzed synthesis of optically pure R(+)-phenylethanol

Summary A dehydrogenase catalyzing the NADPH-dependent reduction of acetophenone to R(+)-phenylethanol was found inLactobacillus kefir. A continuous conversion of 10 mM acetophenone with a yield of up to 90% was achieved in an enzyme-membrane-reactor with simultaneous NADPH-regeneration using glucose-6-phosphate and glucose-6-phosphate dehydrogenase. Enzyme-catalyzed oxidation of phenylethanol occurred with the R(+)-enantiomer only, whereas S(–) was not converted by the enzyme. The formation of R(+)-phenylethanol with an enantiomeric excess of 100% was confirmed by chiral HPLC.