Inhibition of Recombinant Aldose-6-Phosphate Reductase from Peach Leaves by Hexose-Phosphates, Inorganic Phosphate and Oxidants.

Glucitol, also known as sorbitol, is a major photosynthetic product in plants from the Rosaceae family. This sugar alcohol is synthesized from glucose-6-phosphate by the combined activities of aldose-6-phosphate reductase (Ald6PRase) and glucitol-6-phosphatase. In this work we show the purification and characterization of recombinant Ald6PRase from peach leaves. The recombinant enzyme was inhibited by glucose-1-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate and orthophosphate. Oxidizing agents irreversibly inhibited the enzyme and produced protein precipitation. Enzyme thiolation with oxidized glutathione protected the enzyme from insolubilization caused by diamide, while incubation with NADP+ (one of the substrates) completely prevented enzyme precipitation. Our results suggest that Ald6PRase is finely regulated to control carbon partitioning in peach leaves.

Competitive inhibition of phosphoglucose isomerase of apple leaves by sorbitol 6-phosphate.

Apple leaf cytosolic phosphoglucose isomerase (PGI, EC 5.3.1.9) was purified to an apparent homogeneity with a specific activity of 2456 units/mg protein, and chloroplastic PGI was partially purified to a specific activity of 72 units/mg protein to characterize their biochemical properties. These two isoforms showed differential responses to heat treatment; incubation at 50 degrees C for 10 min resulted in a complete loss of the chloroplastic PGI activity, whereas the cytosolic PGI only lost 50% of its activity. Apple cytosolic PGI is a dimeric enzyme with a molecular mass of 66 kDa for each monomer. The activity of both isoforms was strongly inhibited by erythrose 4-phosphate (E4P) with a K(i) of 1.2 and 3.0 microM for the cytosolic PGI and chloroplastic PGI, respectively. Sorbitol 6-phosphate (Sor6P), an intermediate in sorbitol biosynthesis, was found to be a competitive inhibitor for both cytosolic and chloroplastic PGIs with a K(i) of 61 and 40 microM, respectively. PGIs from both spinach and tomato leaves were also inhibited by Sor6P in a similar manner. The possible physiological significance of this finding is discussed.

Preliminary studies on the inhibition of D-sorbitol-6-phosphate 2-dehydrogenase from Escherichia coli with substrate analogues.

D-Sorbitol-6-phosphate 2-dehydrogenase catalyzes the NADH-dependent conversion of D-fructose 6-phosphate to D-sorbitol 6-phosphate and improved production and purification of the enzyme from Escherichia coli is reported. Preliminary inhibition studies of the enzyme revealed 5-phospho-D-arabinonohydroxamic acid and 5-phospho-D-arabinonate as new substrate analogue inhibitors of the F6P catalyzed reduction with IC50 values of (40 +/- 1) microM and (48 +/- 3) microM and corresponding Km/IC50 ratio values of 14 and 12, respectively. Furthermore, we report here the phosphomannose isomerase substrate D-mannose 6-phosphate as the best inhibitor of E. coli D-sorbitol-6-phosphate 2-dehydrogenase yet reported with an IC50 = 7.5 +/- 0.4 microM and corresponding Km/IC50 ratio = about 76.

Effect of intracellular delivery of energy metabolites on intracellular Ca2+ in mouse islets of Langerhans.

Regulation of glucose-induced oscillations in intracellular Ca2+ concentration ([Ca2+]i) was investigated by using a novel technique, electroporation from an electrolyte-filled capillary, to deliver energy metabolites to the intracellular compartment of mouse islets. Intracellular application of ATP resulted in a nifedipine-sensitive increase in [Ca2+]i, consistent with a KATP-channel dependent mechanism of Ca2+ influx. [Ca2+]i in islets exposed to 10 mM glucose oscillated with a period of approximately 3 min, often superimposed with faster oscillations. Electroporation of ATP blocked all types of oscillations and elevated [Ca2+]i while delivery of ADP had no effect on oscillations. Intracellular delivery of glucose-6-phosphate or fructose-1,6-bisphosphate tended to transform slow oscillations to fast oscillations. These results demonstrate that modulation of ATP concentrations and glycolytic flux are important in development of slow oscillations.

The charged milieu: a major player in fertilization reactions.

In previous studies, we have found that negatively charged, but not uncharged, amino acids and sugars block sea urchin fertilization. These studies were developed from modeling work in non-living systems using derivatized agarose beads that suggested that charge-charge bonding may control at least some adhesive interactions. In the present study, the effects of positively charged, negatively charged and uncharged molecules were examined in the sea urchin sperm-egg system in over 300 individual trials. The results indicate that depending on the specific molecules utilized, both sperm and egg are exquisitely sensitive to charged but not uncharged molecules and to pH changes in sea water caused by some of the charged molecules. It is shown that egg activation, as well as sperm motility and sperm-egg interactions, can be affected by charged molecules. One compound, fructose-1-phosphate blocked fertilization in S. purpuratus sea urchins but not in Lytechinus pictus sea urchins. These findings indicate that charge alone cannot explain all the results. In this case, the presence of a ketone instead of an aldehyde group indicates that species-specific components may control fertilization reactions. The present study is a comprehensive survey of the effects of charge, pH and molecular structure on the fertilization activation continuum in a model system of sea urchins.

Differences in regulatory properties between human and rat glucokinase regulatory protein.

The inhibition of glucokinase by rat and Xenopus GKRPs (glucokinase regulatory protein) is well documented. We report a comparison of the effects of human and rat GKRPs on glucokinase activity. Human GKRP is a more potent inhibitor of glucokinase than rat GKRP in the absence of fructose 6-phosphate or sorbitol 6-phosphate, and has a higher affinity for these ligands. However, human and rat GKRPs have similar affinities for fructose 1-phosphate and chloride. Residues that are not conserved between the rodent and human proteins affect both the affinity for fructose 6-phosphate and sorbitol 6-phosphate and the inhibitory potency of GKRP on glucokinase in the absence of these ligands.

The muscle fatty acid binding protein of spadefoot toad (Scaphiopus couchii).

Fatty acid binding protein was purified from skeletal muscle of the spadefoot toad (Scaphiopus couchii), an estivating species. While estivating, this animal relies on the fatty acid oxidation for energy. Hence we were interested in the behaviour of fatty acid binding protein under conditions of elevated urea (up to 200 mM) and potassium chloride such as exist during estivation. Also we examined whether there were interactions between glycolytic intermediates and the binding ability of the protein. The amount of bound fatty acid (a fluorescence assay using cis-parinarate) was not affected (P < 0.05) by glucose, fructose 6-phosphate or phosphoenolpyruvate at physiological concentrations. By contrast, glucose 6-phosphate increased the amount of bound cis-parinarate but the apparent dissociation constant was not different from the control. Fructose 1,6-bisphosphate but not fructose 2,6-phosphate decreased cis-parinarate binding by 40%, commensurate with doubling the apparent dissociation constant (1.15-2.62 microM). Urea, guanidinium and trimethylamine N-oxide at 200 mM increased cis-parinarate binding 60% over controls. Urea (1 M) and KCl (200 mM) did not affect cis-parinarate binding compared to controls. The interaction of this fatty acid transporter with fructose 1,6-bisphosphate is discussed in terms of reciprocal interaction with phosphofructokinase since fatty acid is also an inhibitor of phosphofructokinase.

Membrane potential-dependent conformational changes in mitochondrially bound hexokinase of brain.

Previously characterized monoclonal antibodies (Mabs) were used in a study of Type I hexokinase from rat brain. Based on the relative reactivity of these Mabs with soluble and mitochondrially bound forms, binding to mitochondria was shown to affect specific epitopic regions in both N- and C-terminal halves of the enzyme and to modulate conformational changes induced by binding of the ligands, Glc or ATP. Reactivities with Mabs recognizing epitopes in two defined regions of the N-terminal half and one defined region of the C-terminal half of the mitochondrially bound enzyme were selectively affected by mitochondrial membrane potential, or by addition of oligomycin, carboxyatractyloside, or bongkrekic acid. The Glc-6-P analog, 1 ,5-anhydroglucitol-6-P, was much more effective as a competitive inhibitor against extramitochondrial ATP than against intramitochondrial ATP generated by oxidative phosphorylation. These results provide further insight into the role of hexokinase-mitochondrial interactions in regulation of cerebral glucose metabolism.

Affinity labeling of Escherichia coli glucosamine-6-phosphate synthase with a fructose 6-phosphate analog--evidence for proximity between the N-terminal cysteine and the fructose-6-phosphate-binding site.

Glucosamine-6-phosphate synthase (GlcNP-synthase) catalyzes the formation of glucosamine 6-phosphate from fructose 6-phosphate using the gamma-amide functionality of glutamine as the nitrogen source. In the absence of glutamine, GlcNP-synthase was recently found to catalyze the formation of glucose 6-phosphate corresponding to a phosphoglucoisomerase-like activity. Here we report active-site directed, irreversible inhibition of Escherichia coli GlcNP-synthase (k(inact) = 0.60 +/- 0.05 min(-1), Kirr = 1.40 +/- 0.20 mM) by anhydro-1,2-hexitol 6-phosphates previously known as irreversible inhibitors of phosphoglucoisomerase. Enzyme inactivation with the tritiated affinity label, followed by tryptic digestion and purification of the radioactive fragments, allowed identification of three peptides. Two of them, accounting for 54% of the recovered radioactivity, are believed to result from the nucleophilic attack of side-chain carboxylates of Glu255 and Glu258 and thiol of Cys300 of the fructose-6-phosphate-binding site on the epoxide functionality of the inhibitor. The major peptide corresponds to derivatization of the N-terminal cysteine from the glutamine-binding site by the inhibitor. These results provide evidence for the close proximity of glutamine and fructose-6-phosphate-binding sites recently suggested by Bearne [Bearne, S. L. (1996) J. Biol. Chem. 271, 3052-3057].

Patterns of phosphoantigen stimulation of human Vgamma9/Vdelta2 T cell clones include Th0 cytokines.

This paper examines functional properties of human Vgamma9/Vdelta2 T cell lines and clones generated by in vitro culture with synthetic and natural (mycobacterial) phosphoantigenic molecules. It confirms the broad reactivity of Vgamma9/Vdelta2 T cell lines and clones toward phosphoantigens. Optimal recognition of phosphoantigens by Vgamma9/Vdelta2 T cells required accessory cells to occur, but did not require specialized antigen presenting cells. However, species origin of the APC was irrelevant as proliferation of Vgamma9/Vdelta2 T cells occurred in the presence of syngeneic, allogeneic or xenogeneic APC and was not restricted to APC of particular tissue origin. Moreover antigen uptake and processing was not required for recognition by Vgamma9/ Vdelta2 cells, as evidenced by the ability of fixed APCs to present phosphoantigens. Similarly, the expression of classical MHC class I and class II molecules was not required for phosphoantigen recognition by gammadelta T cells. However, gammadelta T cell clones responded to stimulation by several cytokines including IL-12, IFNgamma and TNFalpha. Finally, Vgamma9/Vdelta2 T cell clones preferentially produced both IFN-gamma and IL-4 in response to PHA or TUBAg stimulation, revealing that a Th0 pattern of cytokine production is frequent among these cells.

A new method for studying the binding of human IgE to CD23 and the inhibition of this binding.

CD23, a low-affinity receptor for IgE (Fc epsilonRII), is a type II membrane-bound glycoprotein expressed on many hematopoietic cells, particularly activated B-cells. CD23 binds to IgE at a domain homologous to Ca2+-dependent (C-type) animal lectin. This paper describes a binding assay by which only the specific binding of IgE to CD23 expressed on Epstein-Barr virus (EBV)-transformed B-cell line, L-KT9 cells, can be detected. This assay is useful in the search for CD23 ligands among many chemical compounds, because it is easily carried out and does not require the use of any radiolabeled reagents. Using the assay, we investigated the inhibition of IgE binding to CD23 by fucose-1-phosphate which has been reported to inhibit the binding of sCD23 to IgE [Delespesse, G., Sarfati, M., Wu, C.Y., Fournier, S., Letellier, M., 1992. The low affinity receptor for IgE. Immunol. Rev. 125, 77.] and the binding of CD23 to CD21 [Pochon, S., Graber, P., Yeager, M., Jansen, K., Bernard, A.R., Aubry, T.-P., Bonnefoy, J.-Y., 1992. Demonstration of second ligand for the low affinity receptor for immunoglobulin E (CD23) using recombinant CD23 reconstituted into flourescent liposomes. J. Exp. Med. 176, 389.]. Although both alpha- and beta-L-fucose-l-phosphate/di(cyclohexylammonium) salt decreased the extent of IgE binding to CD23, the inhibitory effects were not due to alpha- or beta-L-fucose-1-phosphate but to cyclohexylamine. The inhibitory effect of cyclohexylamine was dose dependent and the effect was decreased when inhibition tests were carried out in the presence of a 10-fold excess of IgE. These results suggest that cyclohexylamine specifically interacts with the binding of CD23 and IgE.

Control of glycolysis in vertebrate skeletal muscle during exercise.

The gastrocnemius muscle of the frog (Rana temporaria) has a high capacity for anaerobic glycolysis from glycogen. Glycolytic metabolites and effectors of phosphofructokinase, particularly the hexose bisphosphates, were followed in muscle during exercise (swimming between 5 s and 5 min), recovery (rest for up to 2 h after 5 min of swimming), and repeated exercise (swimming for up to 60 s after 2 h of recovery). Glycogen phosphorylase and phosphofructokinase were swiftly activated with exercise. The hexose bisphosphates followed markedly different time courses. Fructose 1,6-bisphosphate was transiently increased in both exercise and repeated exercise. This appears to be an effect rather than a cause of phosphofructokinase activation. Glucose 1,6-biphosphate was accumulated only while phosphofructokinase was active and was unchanged at other times. Fructose 2,6-biphosphate showed a 10-fold transient increase on exercise in rested frogs, almost disappeared from the muscle during recovery, and did not change during repeated exercise. Fructose 2,6-biphosphate is a potent activator of phosphofructokinase in vitro under near physiological assay conditions, and it may serve this function also in vivo during exercise. Glucose 1,6-biphosphate could be an activator of phosphofructokinase in repeated exercise when fructose 2,6-biphosphate is not available.

Functional organization of mammalian hexokinases: characterization of chimeric hexokinases constructed from the N- and C-terminal domains of the rat type I and type II isozymes.

Chimeric hexokinases consisting of either the N-terminal half of Type I hexokinase fused with the C-terminal half of the Type II isozyme (NICII) or the inverse pair (NIICI), along with the parental isozymes, were expressed in COS-1 cells. The thermal stability of the chimeras was intermediate between that of the highly labile Type II isozyme and the relatively stable Type I hexokinase. In their Kms for substrates, Glc and ATP, the chimeric enzymes were similar to the parental isozyme from which the C-terminal half was derived. Although the Type I and Type II isozymes were similar in their sensitivity to inhibition (competitive vs ATP) by the Glc-6-P analogs, 1,5-anhydroglucitol 6-phosphate (AnGlc-6-P), and Glc-1,6-bisphosphate, the chimeric enzymes differed markedly, with the NIICI chimera being much more sensitive and the NICII chimera much less sensitive than either parental form to these inhibitors. In contrast, the response of the chimeras to Pi, either as an antagonist of inhibition by AnGlc-6-P or, at higher concentrations, as an inhibitor, was correlated with the origin of the N-terminal domain. The results are consistent with the view that catalytic function is associated with the C-terminal domain of the Type I isozyme, with regulatory function--inhibition by Glc-6-P and its analogs and antagonism of this inhibition by Pi--being mediated by the N-terminal domain.

Mechanism of hemolysis of canine erythrocytes induced by L-sorbose.

The cause of species difference in the susceptibility of erythrocytes to L-sorbose, and the difference in the hemolytic effect of sorbose on high potassium-containing (HK) and low potassium-containing (LK) canine erythrocytes were examined. L-Sorbose was phosphorylated in canine erythrocytes, but not in human erythrocytes. Furthermore, sorbose-1-phosphate, a metabolite of L-sorbose, strongly inhibited the hexokinase of LK canine erythrocytes, but not that of HK canine erythrocytes. These results strongly indicated that inhibition of hexokinase by sorbose-1-phosphate in LK erythrocytes induced severe glycolytic limitation in these cells, resulting in hemolysis, and that HK erythrocytes are resistant to sorbose-induced hemolysis because these cells have a high hexokinase activity.

Lysines 72, 80 and 213 and aspartic acid 210 of the Lactococcus lactis LacR repressor are involved in the response to the inducer tagatose-6-phosphate leading to induction of lac operon expression.

Site-directed mutagenesis of the Lactococcus lactis lacR gene was performed to identify residues in the LacR repressor that are involved in the induction of lacABCDFEGX operon expression by tagatose-6-phosphate. A putative inducer binding domain located near the C-terminus was previously postulated based on homology studies with the Escherichia coli DeoR family of repressors, which all have a phosphorylated sugar as inducer. Residues within this domain and lysine residues that are charge conserved in the DeoR family were changed into alanine or arginine. The production of the LacR mutants K72A, K80A, K80R, D210A, K213A and K213R in the LacR-deficient L.lactis strain NZ3015 resulted in repressed phospho-beta-galactosidase (LacG) activities and decreased growth rates on lactose. Gel mobility shift assays showed that the complex between a DNA fragment carrying the lac operators and LacR mutants K72A, K80A, K213A and D210A did not dissociate in the presence of tagatose-6-phosphate, in contrast to wild type LacR. Other mutations (K62A/K63A, K72R, K73A, K73R, T212A, F214R, R216R and R216K) exhibited no gross effects on inducer response. The results strongly suggest that the lysines at positions 72, 80 and 213 and aspartic acid at position 210 are involved in the induction of lac operon expression by tagatose-6-phosphate.

The regulatory protein of liver glucokinase.

Fructose, sorbitol and D-glyceraldehyde stimulate the rate of glucose phosphorylation in isolated hepatocytes. This effect is mediated by fructose 1-phosphate, which releases the inhibition exerted by a regulatory protein on liver glucokinase. In the presence of fructose 6-phosphate, the regulatory protein binds to, and inhibits, liver glucokinase. Fructose 1-phosphate antagonizes this inhibition by causing dissociation of the glucokinase-regulatory protein complex. Both phosphate esters act by binding to the regulatory protein, and by presumably causing changes in its conformation. The regulatory protein behaves as a fully competitive inhibitor. It inhibits liver glucokinase from various species, and rat islet glucokinase, but has no effect on hexokinases from mammalian tissues or from yeast, or on glucokinase from microorganisms. Kinetic studies indicate that the regulatory protein binds to glucokinase at a site distinct from the catalytic site. Several phosphate esters, mainly polyol-phosphates, were found to mimick the effect of fructose 6-phosphate. The most potent is sorbitol 6-phosphate, suggesting that fructose 6-phosphate is recognized by the regulatory protein in its open-chain configuration. Other phosphate esters and Pi have a fructose 1-phosphate-like effect. The stimulatory effect of fructose on glucose phosphorylation is observed not only in isolated hepatocytes but also in the livers of anesthetized rats. This suggests that fructose could be a nutritional signal causing an increase in the hepatic glucose uptake.

Effects of mannose-6-phosphate on receptor-mediated endocytosis of insulin-like growth factor-II.

The insulin-like growth factor-II (IGF-II) and lysosomal enzymes containing a mannose-6-phosphate (M6P) recognition site bind to different sites of the same receptor molecule. We have observed that M6P increases the receptor-mediated uptake of IGF-II into IM-9 cells. We now confirm this phenomenon in a different line, the H-35 rat hepatoma cells, and present additional characterization of the underlying mechanisms. When incubated in the presence of radiolabeled IGF-II, H-35 cells accumulated, in a time-dependent fashion, radioactivity that was resistant to removal by trypsin digestion at 15 C, indicating that it was endocytosed. In the presence of 3 mM M6P, endocytosed counts were approximately 2-fold higher after 5 min of incubation, an enhancement that peaked at 10 min, then declined, but was still evident after 40 min (1.5-fold). The rate of release of cell-associated IGF-II, degraded or intact, as measured in a chase experiment, was not affected by M6P. These observations indicate that M6P increased accumulation of IGF-II by accelerating its rate of endocytosis rather than by interfering with IGF-II degradation or with the recycling of intact hormone-receptor complexes to the cell surface. Electrophoresis after affinity cross-linking of labeled cells demonstrated that the enhancement in radioactivity could be located at a molecular size of approximately 250 kDa, corresponding to IGF-II-receptor complexes. Preincubation with M6P did not significantly alter the specific binding of IGF-II to the cell surface of H-35 cells, as measured by a binding assay at 4 C. Finally, pretreatment with cycloheximide for up to 8 h, to remove all newly synthesized lysosomal enzymes bound to the M6P/IGF-II receptor, did not affect IGF-II endocytosis beyond what could be accounted for by some loss of receptor, suggesting that the observed effect of M6P is due to the binding of M6P itself to the receptor and not to displacement of lysosomal enzymes. We conclude that simultaneous occupancy of the M6P/IGF-II receptor by ligands on both binding sites enhances its rate of endocytosis.

Regulation of glucokinase by a fructose-1-phosphate-sensitive protein in pancreatic islets.

In the post-microsomal supernatant of pancreatic islets, prepared from fasted or fed rats, D-fructose 1-phosphate increased the activity of glucokinase by 20-30% as measured in the presence of D-glucose 6-phosphate and D-fructose 6-phosphate. Such an activation was less marked than that found in liver extracts. The islet cytosol was also found to inhibit purified liver glucokinase, and this effect was antagonized by D-fructose 1-phosphate. In the presence of hexose 6-phosphates, partially purified islet glucokinase was inhibited by the hepatic glucokinase regulatory protein in a D-fructose-1-phosphate-sensitive manner. In intact islets, D-glyceraldehyde stimulated the generation of 14C-labelled D-fructose 1-phosphate from D-[U-14C]glucose and increased the production of 3H2O from D-[5-3H]glucose. These findings suggest that the activity of glucokinase in islet cells may be regulated by a protein mediating the antagonistic effects of D-fructose 6-phosphate and D-fructose 1-phosphate in a manner qualitatively similar to that operating in hepatocytes, but with lower efficiency.

Mannose-6-phosphate stimulates proliferation of neuronal precursor cells.

The mitogenic signal function of mannose-6-phosphate (Man-6-P)/insulin-like growth factor II (IGF-II) receptors was studied in neuronal precursor cells from developing rat brain (E15). About 30% of the cellular Man-6-P/IGF-II receptors were present on the cell surface. Man-6-P and IGF-II stimulated DNA synthesis twofold and their effects were additive. Antibody 3637 to the Man-6-P/IGF-II receptor blocked the response to Man-6-P but not that to IGF-II. Other phosphorylated hexoses were also active. Fructose-1-phosphate was equally potent with Man-6-P, whereas glucose-6-phosphate was 5 times less potent. We conclude that Man-6-P-containing proteins and IGF-II act as mitogens in developing brain by interaction with the Man-6-P/IGF-II receptor and the IGF-I receptor, respectively.

A bisubstrate analog inhibitor for alpha(1----2)-fucosyltransferase.

Porcine submaxillary beta-galactoside alpha(1----2)-fucosyltransferase is known to transfer a fucosyl residue from guanosine 5'-diphosphofucose (GDP-fucose) to the 2-OH group of beta-D-galactopyranosides with inversion of configuration at the fucopyranosyl anomeric carbon. A bisubstrate analog (1) of the postulated transition-state for this reaction, which has O-2 of phenyl beta-D-galactopyranoside attached to the terminal phosphorous of GDP through a flexible ethylene bridge, has been chemically synthesized and evaluated as an inhibitor of this enzyme. Compound 1 was found to be a competitive inhibitor with respect to both GDP-fucose and phenyl beta-D-galactopyranoside for both the membrane-bound and soluble forms of the fucosyltransferase. It was also a competitive inhibitor with respect to the alternate acceptor beta DGal(1----3)beta DGlcNAcO(CH2)8-COOMe. The Ki values were in the range 2.3-16 microM. Compound 1 is the first example of a bisubstrate analog inhibitor for a glycosyltransferase which binds to both the acceptor and donor recognition sites of the enzyme. The potential of a bisubstrate analog strategy for the production of specific glycosyltransferase inhibitors is discussed.