Home About us Contact
|
Home About us Contact | ||
|
|
||
Min Incubation (min + incubation)
Selected AbstractsGlycogen content regulates insulin- but not contraction-mediated glycogen synthase activation in the rat slow-twitch soleus musclesACTA PHYSIOLOGICA, Issue 2 2009Y.-C. Lai Abstract Aim:, The aim of this study was to investigate the effect of glycogen content on glycogen synthase (GS) activation and phosphorylation in the slow-twitch soleus muscles after contraction, during insulin stimulation and when these two stimuli were combined. Methods:, Glycogen content was manipulated in vivo with 24 h fasting and fasting followed by 24 h refeeding. Soleus strips were electrically stimulated for 30 min in vitro, and GS activation and phosphorylation were investigated after an additional 30 min incubation with or without insulin. Results:, Fasting reduced glycogen content in soleus muscle by 40% and refeeding enhanced by 40%, compared to rats with free access to chow. Insulin-stimulated GS fractional activity was inversely correlated with glycogen content (R = ,0.95, P < 0.001, n = 24) and rate of glycogen synthesis was also inversely correlated with glycogen content (R = ,0.70, P < 0.001, n = 36). After contraction, GS fractional activity was increased to similar levels in muscles with low, normal and high glycogen content; rate of glycogen synthesis after contraction was also similar. After contraction, insulin additively increased GS activation at all glycogen contents. Group means of GS fractional activity was inversely correlated with GS Ser641 (R = ,0.93, P < 0.001) and Ser645,649,653,657 (R = ,0.85, P < 0.001) phosphorylation, but not with Ser7 phosphorylation. Conclusion:, Glycogen content regulates insulin- but not contraction-stimulated GS activation and glycogen synthesis in soleus muscles. Furthermore, phosphorylation of GS Ser641 and Ser645,649,653,657 seems to regulate GS activity in soleus. [source] Effect of Silicate-Substitution on Attachment and Early Development of Human Osteoblast-Like Cells Seeded on Microporous Hydroxyapatite Discs,ADVANCED ENGINEERING MATERIALS, Issue 1-2 2010Katharina Guth Hydroxyapatite (HA) is a well-established graft material used in bone repair. Silicon-substituted hydroxyapatite (SA; 0.8,wt% Si) has shown greater bone ingrowth and bone coverage than phase pure HA. To assess the effect of microporosity on sensitivity of cell attachment to surface physiochemistry, microporous SA and HA discs, and control Thermanox (TMX) discs were incubated with osteoblast-like cells (5,×,104 HOS-TE85 cells) under differing tissue culture conditions. To investigate early cellular attachment, organization, and differentiation, cells were also stained for integrin,,5,1, actin, and runt-related transcription factor (RUNX-2), respectively, after incubation on HA, SA, and TMX discs for 3 days. No significant differences emerged between HA, SA, and TMX discs in mean numbers of cells attached in serum free medium (SFM) over 90,min incubation. In contrast, significantly more cells were attached to SA than HA after 180,min incubation in complete medium (C-MEM) containing fetal calf serum (p,<,0.05). Cell attachment to SA and HA discs pre-conditioned in SFM supplemented with fibronectin (FN) was lower than discs pre-conditioned in C-MEM, suggesting sensitivity of an active FN conformation to the presence of co-adsorbates. Confocal microscopy demonstrated significantly more co-localization of integrin ,5,1 and actin on SA than HA. Translocalization of RUNX-2 to the nucleus was stronger in cells incubated on SA. Microporosity did not diminish the effect of surface physiochemistry on cell adhesion, and enhanced cell attachment for SA appears to be mediated by differences in the quality of adsorbed protein rather than via direct effects of substrate chemistry. [source] Hydrogen peroxide induces expression and activation of AMP-activated protein kinase in a dental pulp cell lineINTERNATIONAL ENDODONTIC JOURNAL, Issue 3 2008Y. Fukuyama Abstract Aim, To investigate the effects of hydrogen peroxide on cell viability and expression and activation of AMP-activated protein kinase (AMPK) in rat dental pulp cell line RPC-C2A. Methodology, RPC-C2A cells derived from rat dental pulp were maintained in MEM supplemented with 10% FBS at 37 °C, in a humidified atmosphere at 5% CO2. Cells were cultured in the presence or absence of H2O2 for up to 60 min at concentrations of from 0.1 to 3.0 mmol L,1. Cell viability was analysed by WST-1 reduction assay. Expression of AMPK subunit isoforms was analysed by Western blotting using antibodies to the catalytic ,1 and regulatory ,1 and ,1 subunit isoforms. The effect of silencing AMPK,1 on cell viability was determined using siRNA. Results, Exposure to H2O2 decreased cell viability in a time- and dose-dependent manner. The catalytic AMPK,1 subunit and its activated form, phospho-AMPK,, increased with exposure to H2O2 in a time- and dose-dependent manner, whereas the regulatory ,1 and ,1 subunits showed no change. Downregulation of AMPK,1 resulted in a reduction in cell viability in H2O2 -treated cells at a concentration of 0.1 mmol L,1 for 30 min incubation, indicating an increased sensitivity to H2O2. Conclusions, Reactive oxygen induced energy fuel gauge enzyme AMPK, expression and its activation by phosphorylation in RPC-C2A cells, suggesting that AMPK is essential for protection against H2O2 -induced nonapoptotic cell death. Therefore, AMPK may be a therapeutic modulation target for treatment of the dentine,pulp complex injured by reactive oxygen. [source] PARTIAL PURIFICATION AND CHARACTERIZATION OF A THERMOSTABLE TRYPSIN FROM PYLORIC CAECA OF TAMBAQUI (COLOSSOMA MACROPOMUM)JOURNAL OF FOOD BIOCHEMISTRY, Issue 3 2001RANILSON S. BEZERRA ABSTRACT A 38.5 kDa alkaline protease from pyloric caeca of tambaqui (Colossoma macropomumj, a tropical freshwater fish, was partially purified in three steps: thermal treatment (45Cfor 30 min), salting-out (ammonium sulfate at 40,80% of saturation) and gel filtration (Sephadex G-75), The purification and yield were 51.2-fold and 40%, respectively. The effects of pH, temperature, inhibitors, and substrates on proteolytic activities of partially purified enzyme were investigated. The optimum pH was 9.5, while the optimum temperature was 60C. This alkaline proteolytic activity remained unaltered after 30 min incubation at 55C. Active site inhibition provided additional evidence that this activity is attributed to a trypsin-like enzyme. [source] CHARACTERIZATION OF STOMACH AND PYLORIC CAECA PROTEINASES OF TAMBAQUI (COLOSSOMA MACROPOMUM)JOURNAL OF FOOD BIOCHEMISTRY, Issue 3 2000RANILSON DE SOUZA BEZERRA ABSTRACT The effects of pH, temperature, inhibitors, and substrates on proteolytic activities of crude extracts from the stomach, liver, pyloric caeca, and intestines of Colossoma macropomum were investigated. The highest acid and alkaline proteolytic activities were found in stomach and pyloric caeca, respectively. The optimum pH for the acid and alkaline proteases were 1.8 and 7.0,9.0, respectively, while the optimum temperatures were 35C and 65C. This alkaline protease thermal stability remained unaltered after 90 min incubation at 55C. A pepsin-like protease was responsible for most of the acidic proteolytic activity (Pepstatin A inhibited approximately 90%), whereas PMSF inhibited about 40% of the alkaline protease. The alkaline proteolytic activity has attractive thermal properties for industrial applications. [source] Toward a Microfluidic-Based Rapid Amylase Assay SystemJOURNAL OF FOOD SCIENCE, Issue 6 2009Richard J. Holmes ABSTRACT:, This article describes work into a prototype system for the assay of amylase, using microfludic technologies. The new system has a significantly shorter cycle time than the current laboratory methods, which generally use microtitre plates, yet is capable of generating significantly superior results. As such, we have shown that sensitivity is enhanced by a factor of 10 in the standard assay trials, and by a factor of 2 in the real-sample lab trials. In both assays, the use of a microreactor system reduced the reaction time by a factor of 6.2, from 20 min incubation to 3.2 min. Basing the conclusion on the Megazyme Cerealpha Standard Method, and using the Cerealpha units as a measure of assay efficiency, the typical response for the microfluidic assay was shown to be 1.0 × 10,3 CU/mL (standard deviation [SD] 2.5 × 10,4 CU/mL), compared to 2.56 × 10,4 CU/mL (SD 5.94 × 10,5 CU/mL) for the standard macroassay. It is believed that this improvement in the reaction schematics is due to the inherent advantages of microfluidic devices such as superior mixing, higher thermal efficiency, and enhanced reaction kinetics. [source] An algorithm for thorough background subtraction from high-resolution LC/MS data: application for detection of glutathione-trapped reactive metabolitesJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 9 2008Haiying Zhang Abstract A control sample background-subtraction algorithm was developed for thorough subtraction of background and matrix-related signals in high-resolution, accurate mass liquid chromatography/mass spectrometry (LC/MS) data to reveal ions of interest in an analyte sample. This algorithm checked all ions in the control scans within a specified time window around the analyte scan for potential subtraction of ions found in that analyte scan. Applying this method, chromatographic fluctuations between runs were dealt with and background and matrix-related signals in the sample could be thoroughly subtracted. The effectiveness of this algorithm was demonstrated using four test compounds, clozapine, diclofenac, imipramine, and tacrine, to reveal glutathione (GSH)-trapped reactive metabolites after incubation with human liver microsomes supplemented with GSH (30 µM compound, 45-min incubation). Using this algorithm with a ± 1.0 min control scan time window, a ± 5 ppm mass error tolerance, and appropriate control samples, the GSH-trapped metabolites were revealed as the major peaks in the processed LC/MS profiles. Such profiles allowed for comprehensive and reliable identification of these metabolites without the need for any presumptions regarding their behavior or properties with respect to mass spectrometric detection. The algorithm was shown to provide superior results when compared to several commercially available background-subtraction algorithms. Many of the metabolites detected were doubly charged species which would be difficult to detect with traditional GSH adduct screening techniques, and thus, some of the adducts have not previously been reported in the literature. Copyright © 2008 John Wiley & Sons, Ltd. [source] Regulation of glutamate carboxypeptidase II hydrolysis of N -acetylaspartylglutamate (NAAG) in crayfish nervous tissue is mediated by glial glutamate and acetylcholine receptorsJOURNAL OF NEUROCHEMISTRY, Issue 3 2005Albert K. Urazaev Abstract Glutamate carboxypeptidase II (GCPII), a glial ectoenzyme, is responsible for N -acetylaspartylglutamate (NAAG) hydrolysis. Its regulation in crayfish nervous tissue was investigated by examining uptake of [3H]glutamate derived from N -acetylaspartyl-[3H]glutamate ([3H]NAAG) to measure GCPII activity. Electrical stimulation (100 Hz, 10 min) during 30 min incubation with [3H]NAAG increased tissue [3H]glutamate tenfold. This was prevented by 2-(phosphonomethyl)-pentanedioic acid (2-PMPA), a GCPII inhibitor, suggesting that stimulation increased the hydrolysis of [3H]NAAG and metabolic recycling of [3H]glutamate. Antagonists of glial group II metabotropic glutamate receptors (mGLURII), NMDA receptors and acetylcholine (ACh) receptors that mediate axon,glia signaling in crayfish nerve fibers decreased the effect of stimulation by 58,83%, suggesting that glial receptor activation leads to stimulation of GCPII activity. In combination, they reduced [3H]NAAG hydrolysis during stimulation to unstimulated control levels. Agonist stimulation of mGLURII mimicked the effect of electrical stimulation, and was prevented by antagonists of GCPII or mGLURII. Raising extracellular K+ to three times the normal level stimulated [3H]NAAG release and GCPII activity. These effects were also blocked by antagonists of GCPII and mGLURII. No receptor antagonist or agonist tested or 2-PMPA affected uptake of [3H]glutamate. We conclude that NAAG released from stimulated nerve fibers activates its own hydrolysis via stimulation of GCPII activity mediated through glial mGLURII, NMDA and ACh receptors. [source] Melatonin protects against streptozotocin, but not interleukin-1,-induced damage of rodent pancreatic ,-cellsJOURNAL OF PINEAL RESEARCH, Issue 3 2001Annika K. Andersson In the present study, we examined whether melatonin can protect rodent pancreatic islets against streptozotocin (STZ) and interleukin-1, (IL-1,)-induced suppression of ,-cell function. Formation of free radicals, DNA damage and extensive DNA repair leading to depletion of intracellular nicotinamide adenine dinucleotide (NAD) may mediate STZ toxicity. Activation of inducible nitric oxide synthase and nitric oxide (NO) formation may cause IL-1,-induced ,-cell impairment. We also studied the effect of melatonin against STZ-induced hyperglycemia in C57BL/Ks mice. For in vitro studies, cultured rat islets were exposed to melatonin (100 ,M,1 mM) 30 min prior to STZ (0.5 mM) or IL-1, (25 U/mL) addition. After an additional 30 min incubation with STZ, islet function and NAD content were analyzed either acutely or after 18 hr of recovery in fresh culture medium. For IL-1, experiments, islets were incubated for 48 hr with the cytokine before evaluation of islet function. We found that melatonin counteracted STZ-induced inhibition of glucose metabolism and insulin release in cultured rat islets after 18 hr of recovery. Moreover, NAD levels were higher in the melatonin-treated group at this time point. Melatonin had no effect on IL-1,-induced islet inhibition of glucose oxidation or NO formation. Diabetes induced by STZ (140 mg/kg body weight; i.v.) was effectively prevented by administration of melatonin (100 mg/kg body weight; i.p.) 30 min before STZ injection. We conclude that the protective effects of melatonin against ,-cell damage may be related to interference with DNA damage and poly(ADP-ribose) polymerase (PARP) activation rather than through effects on NO generation pathways. [source] MYELINATION DEFICIT IN NERVE OF SUCKLING RATS DUE TO CYCLOLEUCINE -INDUCED DEFICIENCY OF METHYL DONORSJOURNAL OF THE PERIPHERAL NERVOUS SYSTEM, Issue 1 2000R. Bianchi We used cycloleucine (CL) , which prevents methionine conversion to S-adenosyl-methionine (SAMe) by inhibiting ATP-L-methionine-adenosyl-transferase (MAT) , to characterize the lipid and protein changes induced by methyl donors deficit in peripheral nerve and brain myelin in rats during development. We have previously shown that CL (400 mg/kg ip) given to suckling rats at days 7, 8, 12, and 13 after birth reduced brain and sciatic nerve weight gain, brain myelin content, protein, phospholipid (PL), and galactolipid concentration in comparison to control. Among PLs, only sphingomyelin (SPH) significantly increased by 35,50%. SAMe p-toluensulphonate (SAMe-SD4) (100 mg/kg, ip) given daily from day 7, as with exogenous SAMe, partially prevented some lipid alterations induced by CL, particularly galactolipid and SPH. To test the ability of CL to affect PL metabolism we have measured de novo PL biosynthesis, ex vivo in nerve homogenates (in comparison with brain homogenates) from control and CL-treated animals killed at day 18 after birth, starting from labelled substrates ([3H]-choline, specific activity 20 mCi/mmol) in a Tris/HCl buffer, containing 5 mM MgCl2, 0.2 mM EDTA, 0.1 mM ATP, and 0.5 mM of the labelled substrates. After 60 min incubation, lipids were extracted, PL separated by TLC, and corresponding silica gel fractions scraped and counted in a liquid scintillator. Phosphatidylcholine enrichment in labelled choline resulted in slight increases in brain and sciatic nerve of CL-treated rats, suggesting an increased synthesis rate via the Kennedy pathway, possibly due to the reduced availability of methyl donors. Interestingly, choline incorporation into SPH in brain and nerve myelin resulted in significant increases of 30,40%. In agreement with the observed decrease of galactolipid content and the relative increase in SPH, these data suggest an alteration in sphingolipid metabolism after CL. Among proteins, in sciatic nerves of CL-treated pups the relative content of a number of polypeptides, namely the 116, 90, 66, 58, and 56 kDa bands, decreased, whereas others increased; the most abundant PNS protein, protein zero, remained unchanged. The analyses of myelin basic protein isoforms revealed a dramatic increase in the 14.0 and 18.5 forms, indicating early active myelination. SAMe-SD4 treatment counteracted, and in some cases normalized, these changes. In summary, methyl donor deficiency induced by MAT inhibition produces myelin lipid and protein alterations, partly counteracted by SAMe-SD4 administration. The financial support of Telethon-Italy (grant No. D 51) is gratefully acknowledged. [source] Purification and some properties of a cysteine proteinase from sorghum malt variety SK5912JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 2 2004Augustine C Ogbonna Abstract A cysteine proteinase from sorghum malt variety SK5912 was purified by a combination of 4 M sucrose fractionation, ion-exchange chromatography on Q- and S-Sepharose (fast flow), gel filtration chromatography on Sephadex G-100 and hydrophobic interaction chromatography on Phenyl Sepharose CL-4B. The enzyme was purified 8.4-fold to give a 13.4% yield relative to the total activity in the crude extract and a final specific activity of 2057.1 U mg,1 protein. SDS,PAGE revealed two migrating protein bands corresponding to apparent relative molecular masses of 55 and 62 kDa, respectively. The enzyme was optimally active at pH 6.0 and 50 °C, not influenced across a relatively broad pH range of 5.0,8.0 and retained over 60% activity at 70 °C after 30-min incubation. It was highly significantly (P < 0.001) inhibited by Hg2+, appreciably (P < 0.01) inhibited by Ag+, Ba2+ and Pb2+ but highly significantly (P < 0.001) activated by Co2+, Mn2+ and Sr2+. The proteinase was equally highly significantly (P < 0.001) inhibited by both iodoacetate and p -chloromercuribenzoate and hydrolysed casein to give the following kinetic constants: Km = 0.33 mg ml,1; Vmax = 0.08 µmol ml,1 min,1. Copyright © 2004 Society of Chemical Industry [source] Eradication of Multiple Myeloma and Breast Cancer Cells by TH9402-mediated Photodynamic Therapy: Implication for Clinical Ex Vivo Purging of Autologous Stem Cell Transplants,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 6 2000N. Brasseur ABSTRACT High-dose chemotherapy combined with autologous transplantation using bone marrow or peripheral blood-derived stem cells (PBSC) is now widely used in the treatment of hematologic malignancies as well as some solid tumors like breast cancer (BC). However, some controversial results were recently obtained in the latter case. The presence of malignant cells in the autograft has been associated with the recurrence of the disease, and purging procedures are needed to eliminate this risk. The aim of this study was to evaluate the potential of the photosensitizer 4,5-dibromorhodamine methyl ester (TH9402), a dibrominated rhodamine derivative, to eradicate multiple myeloma (MM) and BC cell lines, while sparing more than 50% of normal pluripotential blood stem cells from healthy volunteers. The human BC MCF-7 and T-47D and MM RPMI 8226 and NCI-H929 cell lines were used to optimize the photodynamic purging process. Cell concentration and the cell suspension thickness as well as the dye and light doses were varied in order to eventually treat 1,2 L of apheresis. The light source consisted of two fluorescent scanning tubes emitting green light centered about 515 nm. The cellular uptake of TH9402 was measured during the incubation and washout periods and after photodynamic treatment (PDT) using spectrofluorometric analysis. The limiting dilution assay showed that an eradication rate of more than 5 logs is obtained when using a 40 min incubation with 5,10 ,M dye followed by a 90 min washout period and a light dose of 5,10 J/cm2 (2.8 mW/cm2) in all cell lines. Agitating the 2 cm thick cell suspension containing 20 × 106 cells/mL during PDT was essential for maximal photoinactivation. Experiments on mobilized PBSC obtained from healthy volunteers showed that even more drastic purging conditions than those found optimal for maximal eradication of the malignant cell lines were compatible with a good recovery of hematopoietic progenitors cells. The absence of significant toxicity towards normal hematopoietic stem cells, combined with the 5 logs eradication of cancer cell lines induced by this procedure suggests that TH9402 offers an excellent potential as an ex vivo photodynamic purging agent for autologous transplantation in MM and BC treatment. [source] Oligocarrageenans and tissue-dependant oxidative burst in Solieria chordalis (Rhodophyceae, Gigartinales)PHYCOLOGICAL RESEARCH, Issue 1 2008Erwan Ar Gall SUMMARY The release of hydrogen peroxide by thallus fragments of the rhodophycean Solieria chordalis (C. Agardh) J. Agardh has been documented both in the presence and in the absence of oligosaccharides. Within 1 h, ramuli were able to release large amounts of peroxide in the absence of any chemical stress. Among potential elicitors tested, only degree of polymerization 1 (DP1) and DP7-8 oligo-iota-carrageenans stimulated defense mechanisms in both axes and ramuli as shown by the occurrence of an oxidative burst. Chopping of the tissues had no effect on the intensity of the burst, therefore suggesting that mainly cortical cell layers were involved in the process. After 5 min incubation, a dose of 125 ,g mL,1 of an oligomeric mixture containing a large proportion of DP1 units proved to be sufficient to obtain a maximal response. The intensity of the burst was significantly higher with isolated ramuli than with pieces of the axis, with outer peroxide accumulations reaching 200 nmol g,1 fresh weight of treated tissue. Altogether, our results show that S. chordalis is able to react to a simulated pathogen attack by an oxidative burst and that the capacity to carry out an oxidative burst is stronger in ramuli than in axes. [source] Hg2+ Reacts with Different Components of the NADPH: Protochlorophyllide Oxidoreductase MacrodomainsPLANT BIOLOGY, Issue 3 2004K. Solymosi Abstract: The molecular background of Hg2+ -induced inhibition of protochlorophyllide (Pchlide) photoreduction was investigated in homogenates of dark-grown wheat leaves. Our earlier work showed that 15 min incubation with 10 -2 M Hg2+ completely inhibits the activity of NADPH: Pchlide oxidoreductase. Detailed analysis of spectra recorded at 10 K indicated the appearance of emission bands at 638 and 650 nm, which are characteristic for NADP+ -Pchlide complexes. Fluorescence emission spectra recorded with different excitation wavelengths, fluorescence lifetime measurements and the analysis of acetone extractions revealed that Hg2+ can also react directly with Pchlide, resulting in protopheophorbide formation. At 10 -3 M Hg2+, the phototransformation was complete but the blue shift of the chlorophyllide emission band speeded up remarkably. This indicates oxidation of the NADPH molecules that have a structural role in keeping together the etioplast inner membrane components. We suggest a complex model for the Hg2+ effect: depending on concentration it can react with any components of the NADPH: Pchlide oxidoreductase macrodomains. [source] Functional importance of the actin cytoskeleton in contraction of bovine iris sphincter muscleAUTONOMIC & AUTACOID PHARMACOLOGY, Issue 3 2002J. A. C. Filipe Summary 1 The contractile capacity of smooth muscle cells depends on the cytoskeletal framework of the cell. The aim of this study was to determine the functional importance of both the actin and the tubulin components of the cytoskeleton in contractile responses of the bovine isolated iris sphincter muscle. 2 In each preparation, two contractions to the muscarinic agonist carbachol were obtained. The maximum responses of the first contractions were taken as 100%. The second contractions to carbachol were elicited in the presence of either cytochalasin B (50 and 5 ,m), an inhibitor of the actin cytoskeleton, or colchicine (100 ,m), an inhibitor of the tubulin cytoskeleton (30 min incubation). 3 Cytochalasin B, at a concentration of 50 ,m, significantly decreased the contractions induced by carbachol, with the maximum response reduced to 21.8 ± 6.6% (n = 12) of the initial maximum. The maximal contractions to carbachol in the presence of colchicine reached 96.2 ± 7.9% (n = 9) of the initial contraction, which was not significantly different from control second responses to carbachol with neither drug present, which reached 113.3 ± 7.6% (n = 7). 4 The effect of cytochalasin B was dose-dependent, since at a lower concentration of 5 ,m, the drug decreased the maximum contraction to carbachol to 60.3 ± 8.8% (n = 6). The effect of cytochalasin B was at least partially reversible, since after the use of the higher concentration of 50 ,m, contractions to carbachol increased to 62.3 ± 15.5% (n = 4) of the maximal response, after 1 h repeated washing of the preparations. 5 Cytochalasin D, at a concentration of 50 ,m, completely abolished the contractions induced by carbachol (n = 4). 6 These findings suggest that in bovine iris sphincter muscle, contractions to carbachol are highly dependent, from a functional point of view, on actin polymerization, and not, to any important degree, on the polymerization of tubulin. [source] Impaired enzyme-to-enzyme channelling between hexokinase isoenzyme(s) and phosphoglucoisomerase in rat pancreatic islets incubated at a low concentration of D -glucoseCELL BIOCHEMISTRY AND FUNCTION, Issue 1 2005Ying Zhang Abstract It was recently proposed that in rat pancreatic islets exposed to 8.3,mMD -glucose, ,- D -glucose-6-phosphate undergoes enzyme-to-enzyme channelling between hexokinase isoenzyme(s) and phosphoglucoisomerase. To explore the identity of the hexokinase isoenzyme(s) involved in such a tunnelling process, the generation of 3HOH from the ,- and ,-anomers of either D -[2- 3H]glucose or D -[5- 3H]glucose was now measured over 60,min incubation at 4°C in pancreatic islets exposed only to 2.8,mMD -glucose, in order to decrease the relative contribution of glucokinase to the phosphorylation of the hexose. Under these experimental conditions, the ratio for 3HOH production from D -[2- 3H]glucose/D -[5- 3H]glucose at anomeric equilibrium (39.7,±,11.6%) and the ,/, ratios for the generation of 3HOH from either the D -[2- 3H]glucose anomers (70.9,±,12.6%) or the D -[5- 3H]glucose anomers (59.6,±,12.4%) indicated that a much greater fraction of ,- D -glucose-6-phosphate escapes from the process of enzyme-to-enzyme channelling in the islets exposed to 2.8,mM, rather than 8.3,mMD -glucose. These findings suggest, therefore, that the postulated process of enzyme-to-enzyme channelling involves mainly glucokinase. Copyright © 2004 John Wiley & Sons, Ltd. [source] | ||||||||||||||||