6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase and small chemical activators affect enzyme activity of activating glucokinase mutants by distinct mechanisms.

Glucokinase (GK), a monomeric glucose-phosphorylating enzyme characterised by high structural flexibility, acts as a glucose sensor in pancreatic beta cells and liver. Pharmaceutical efforts to control the enzyme are hampered by an incomplete understanding of GK regulation. We investigated GK characteristics of wild-type and activating S64Y and G68V mutant proteins in the presence of various combinations of the synthetic activators RO-28-1675 and compound A, the endogenous activator fructose-2,6-bisphosphatase (FBPase-2), and the inhibitor mannoheptulose. S64Y impedes formation of a turn structure that is characteristic for the inactive enzyme conformation, and complex formation with compound A induces collision with the large domain. G68V evokes close contact of connecting region I and helix α13 with RO-28-1675 and compound A. Both mutants showed higher activity than the wild-type at low glucose and were susceptible to further activation by FBPase-2 and RO-28-1675, alone and additively. G68V was less active than S64Y, but was activatable by compound A. In contrast, compound A inhibited S64Y, and this effect was even more pronounced in combination with mannoheptulose. Mutant and wild-type GK showed comparable thermal stability and intracellular lifetimes. A GK-6-phosphofructo-2-kinase (PFK-2)/FBPase-2 complex predicted by in silico protein-protein docking demonstrated possible binding of the FBPase-2 domain near the active site of GK. In summary, activating mutations within the allosteric site of GK do not preclude binding of chemical activators (GKAs), but can alter their action into inhibition. Our postulated GK-PFK-2/FBPase-2 complex represents the endogenous principle of activation by substrate channelling which permits binding of other small molecules and proteins.

In vivo and ex vivo 19-fluorine magnetic resonance imaging and spectroscopy of beta-cells and pancreatic islets using GLUT-2 specific contrast agents.

The assessment of the ß-cell mass in experimental models of diabetes and ultimately in patients is a hallmark to understand the relationship between reduced ß-cell mass/function and the onset of diabetes. It has been shown before that the GLUT-2 transporter is highly expressed in both ß-cells and hepatocytes and that D-mannoheptulose (DMH) has high uptake specificity for the GLUT-2 transporter. As 19-fluorine MRI has emerged as a new alternative method for MRI cell tracking because it provides potential non-invasive localization and quantification of labeled cells, the purpose of this project is to validate ß-cell and pancreatic islet imaging by using fluorinated, GLUT-2 targeting mannoheptulose derivatives (19 FMH) both in vivo and ex vivo. In this study, we confirmed that, similar to DMH, 19 FMHs inhibit insulin secretion and increase the blood glucose level in mice temporarily (approximately two hours). We were able to assess the distribution of 19 FMHs in vivo with a temporal resolution of about 20 minutes, which showed a quick removal of 19 FMH from the circulation (within two hours). Ex vivo MR spectroscopy confirmed a preferential uptake of 19 FMH in tissue with high expression of the GLUT-2 transporter, such as liver, endocrine pancreas and kidney. No indication of further metabolism was found. In summary, 19 FMHs are potentially suitable for visualizing and tracking of GLUT-2 expressed cells. However, current bottlenecks of this technique related to the quick clearance of the compound and relative low sensitivity of 19 F MRI need to be overcome. Copyright © 2016 John Wiley & Sons, Ltd.

Dietary Mannoheptulose Does Not Significantly Alter Daily Energy Expenditure in Adult Labrador Retrievers.

Mannoheptulose (MH), a sugar found in avocados that inhibits glycolysis in vitro, has been preliminarily investigated as a novel food ingredient for dogs. This study aimed to determine the effects of dietary MH, delivered as an extract of un-ripened avocado, on energy expenditure (EE) in healthy adult Labrador Retriever dogs (total of 12 dogs, 26.99 ± 0.634 kg, 4.9 ± 0.2 y). The study was a double-blind, cross-over with each dog receiving both dietary treatments, control (CON) and MH (400 mg/kg of diet; 6 mg/kg BW), in random order. Resting and post-prandial (10 h) EE and respiratory quotient (RQ) were determined by indirect calorimetry (d 42). The following day, body composition was assessed using dual X-ray absorptiometry. Continuous activity monitoring was conducted using an Atical® accelerometer (d 43-47). A vastus lateralis muscle biopsy was obtained prior to the morning meal (d 49) and 4 h after consumption of their meal (d 56) to determine the protein content and phosphorylation of 5' adenosine monophosphate-activated protein kinase (AMPK). Diet did not affect body weight, resting EE or skeletal muscle AMPK phosphorylation. Dogs fed MH had significantly lower post-prandial RQ (p = 0.02) and ratio of fat to lean body mass (p = 0.02). Physical activity during light time periods (but not dark) was lower in dogs fed MH (p < 0.05) during weekends, but not on weekdays. These results suggest that MH affects energy balance of adult dogs, but that these effects are not dose dependent and not due to physical activity.

Physiological characterization of the human EndoC-ßH1 ß-cell line.

In the new human EndoC-ßH1 ß-cell line, a detailed analysis of the physiological characteristics was performed. This new human ß-cell line expressed all target structures on the gene and protein level, which are crucial for physiological function and insulin secretion induced by glucose and other secretagogues. Glucose influx measurements revealed an excellent uptake capacity of EndoC-ßH1 ß-cells by the Glut1 and Glut2 glucose transporters. A high expression level of glucokinase enabled efficient glucose phosphorylation, increasing the ATP/ADP ratio along with stimulation of insulin secretion in the physiological glucose concentration range. The EC50 value of glucose for insulin secretion was 10.3 mM. Mannoheptulose, a specific glucokinase inhibitor, blocked glucose-induced insulin secretion (GSIS). The nutrient insulin secretagogues l-leucine and 2-ketoisocaproate also stimulated insulin secretion, with a potentiating effect of l-glutamine. The Kir 6.2 potassium channel blocker glibenclamide and Bay K 8644, an opener of the voltage-sensitive Ca(2+) channel significantly potentiated GSIS. Potentiation of GSIS by IBMX and forskolin went along with a strong stimulation of cAMP generation. In conclusion, the new human EndoC-ßH1 ß-cell line fully mirrors the analogous physiological characteristics of primary mouse, rat and human ß-cells. Thus, this new human EndoC-ßH1 ß-cell line is very well suited for physiological ß-cell studies.

Non-invasive imaging of the endocrine pancreas (review).

The non-invasive imaging of the endocrine pancreas is currently considered by concerned institutions as a priority theme of research. Because the endocrine pancreas represents only about one percent of the pancreatic gland, highly specific tools are required for labelling the insulin-producing cells. The present review deals with the possible use of D-mannoheptulose for the non-invasive quantification of insulin-producing cells in the pancreas. This heptose is transported into hepatocytes and insulin-producing cells, but not other cell types, at the intervention of GLUT2. Its uptake coincides with the intracellular accumulation of acidic metabolites generated by phosphorylation of D-mannoheptulose. These metabolites remain in the islet cells after prolonged washing, suggesting that imaging of the pancreas at a relatively late time after intravenous administration of D-mannoheptulose could avoid significant extracellular contamination. Using tritiated D-mannoheptulose as tracer, a new method was designed for quantification of the total mass of insulin-producing cells in either isolated perfused rat pancreata or isolated pancreatic islets. Likewise, a preferential labelling of hepatocytes and insulin-producing transplanted cells can be achieved in vivo after administration of tritiated D-mannoheptulose. In the light of results obtained with 6-deoxy-6-iodo-D-glucose, it is proposed that 7-deoxy-7-iodo-D-mannoheptulose could be used for the non-invasive imaging of the endocrine pancreas.

Pancreatic fate of D-[3H] mannoheptulose.

D-Mannoheptulose was recently postulated to be transported into cells by GLUT2. The validity of such an hypothesis was assessed by comparing the uptake of tritiated D-mannoheptulose by pancreatic islets versus pieces of pancreas and, in the latter case, by comparing results obtained in control rats versus animals injected with streptozotocin (STZ). The uptake of D-[3H] mannoheptulose by islets represents a time-related and temperature-sensitive process, inhibited by cytochalasin B and enhanced by D-glucose. The uptake of the tritiated heptose was much lower in pieces of pancreatic tissue and inhibited by D-glucose, at least in the STZ rats. Whether in pieces of pancreas exposed in vitro to D-[3H] mannoheptulose or after intravenous injection of the tritiated heptose, the radioactive content of the pancreatic tissue was lower in STZ rats than in control animals. This contrasted with an unaltered radioactive content of liver and muscle in the STZ rats, at least when treated with insulin. Suitably radiolabelled D-mannoheptulose or an analogue of the heptose could thus conceivably be used for quantification of the endocrine pancreatic mass.

Uptake of tritiated D-mannoheptulose by liver, pancreatic exocrine and endocrine cells.

Tritiated D-mannoheptulose, a ketoheptose known to inhibit D-glucose metabolism in hepatocytes and pancreatic islets, but not so in pancreatic acinar cells, was injected intravenously in streptozotocin-induced diabetic mice transplanted under the kidney capsule with islets from control mice of the same strain. One hour after the injection of the tritiated heptose, the radioactive content was 5-8 times higher in the liver and transplanted islets than in the pancreatic gland. It is proposed that suitably radiolabelled D-mannoheptulose could be used to label preferentially the endocrine moiety of the pancreatic gland, e.g., in the perspective of its non-invasive imaging.

Assessment by D-[(3)H]mannoheptulose uptake of B-cell density in isolated pancreatic islets from Goto-Kakizaki rats.

The uptake of D-[3H]mannoheptulose by isolated pancreatic islets was recently proposed as a tool to assess the relative contribution of insulin-producing cells to the total mass of the islets. In the present study, the uptake of the tritiated heptose over 60 min incubation at 37 degrees C was about 21% lower in islets from hereditarily diabetic rats (GK rats) than in islets from control animals, this decrease being virtually identical to that documented previously by morphometric analysis of islets from the same type of rats. The intracellular 3HOH space and extracellular [U-14C]sucrose space were not significantly different in control and diabetic rats, at least when the comparison was restricted to animals of the same sex. There was a trend, however, towards a somewhat lower D-[5-3H]-glucose intracellular distribution space in islets from GK rats, as compared to control animals. These findings provide further support to the validity of D-[3H]mannoheptulose uptake as a tool to assess the density of insulin-producing cells in isolated islets.

On the track to the beta-cell.

The imaging and quantification of the endocrine pancreas by a non-invasive procedure remains a challenge. In the prolongation of prior work on selected succinic acid esters and monosaccharide esters, it is proposed that D-mannoheptulose, which might be transported across the plasma membrane at the intervention of GLUT-2, could be used to label preferentially the endocrine moiety of the pancreatic gland. This heptose is taken up more efficiently by hepatocytes and islet cells, than by erythrocytes, parotid cells, acinar pancreatic cells or tumoural islet cells of either the RINm5F or INS-1 line. Likewise, D-mannoheptulose only inhibits D-glucose metabolism in hepatocytes and isolated islets. Its hexaacetate ester, however, inhibits the catabolism of the hexose in all cell types. The uptake of D-mannoheptulose represents a carrier-mediated process. Human islets behave like rat islets in terms of D-mannoheptulose uptake and inhibition by the heptose of both D-glucose metabolism and insulinotropic action. The use of radiolabelled analogs of D-mannoheptulose suitable for imaging of the endocrine pancreas is discussed. In the same perspective, it is proposed that advantage could be taken of the much greater accumulation of glycogen in insulin-producing cells, as compared to other pancreatic cell types, in situations of sustained hyperglycaemia.

Uptake of 1-deoxy-1-[125I]iodo-D-mannoheptulose by different cell types: in vitro and in vivo experiments.

D-mannoheptulose was recently proposed as a tool to label preferentially insulin-producing cells in the pancreatic gland in the perspective of the non-invasive imaging of the endocrine pancreas. In such a perspective, we have now synthesized 1-deoxy-1-[125I]iodo-D-mannoheptulose ([125I]MH) and examined its uptake by different rat cell types. No phosphorylation of [125I]MH by bovine heart hexokinase could be detected. The apparent distribution space of [125I]MH largely exceeded that of [U-14C]sucrose, considered as an extracellular marker, in erythrocytes, parotid cells, hepatocytes, pancreatic pieces and isolated pancreatic islets. Relative to the mean intracellular distribution space of 3HOH, that of [125I]MH was not significantly different in pancreatic pieces from either normal rats or streptozotocin-induced diabetic animals (STZ rats). In pancreatic islets, the uptake of [125I]MH was decreased at low temperature, but failed to be significantly affected by cytochalasin B. Sixty min after the intravenous injection of [125I]MH, the radioactive content of selected organs displayed the following hierarchy: muscle

D-mannoheptulose phosphorylation by hexokinase isoenzymes.

D-mannoheptulose is a specific inhibitor of D-glucose phosphorylation by hexokinase isoenzymes. In the present study, the phosphorylation of this heptose was investigated by either a spectrophotometric or radioisotopic procedure. Using yeast hexokinase, the phosphorylation of 25 mM D-mannoheptulose only represented 0.02% of that of 5 mM D-glucose. Such a percentage was increased to 3.93% in the case of bovine heart hexokinase. In the latter case, the Km for D-mannoheptulose was close to 0.2 mM and both D-glucose (0.1-1.0 mM) and D-glucose 6-phosphate (also 0.1-1.0 mM) inhibited the phosphorylation of the heptose (0.03-0.60 mM). Human B-cell glucokinase also catalyzed the phosphorylation of D-mannoheptulose (0.1 mM), which was now increased in a bell-shaped manner by D-glucose (1.0-20 mM). Likewise, rat parotid gland, liver and pancreatic islet homogenates catalyzed the phosphorylation of D-[3H]mannoheptulose. The results obtained in these three tissues differed from one another by their absolute values (per mg wet wt.), relative values (by reference to the phosphorylation rate of 10 mM D-glucose), and sensitivity to inhibition by D-glucose (10 mM).

Assessment of B-cell mass in isolated islets exposed to D-[3H]mannoheptulose.

D-mannoheptulose was recently proposed to be transported into cells mainly at the intervention of GLUT2. In the present study, it was investigated whether advantage could be taken from such a situation to assess the contribution of insulin-producing B-cells to the total mass of isolated rat pancreatic islets. After 60 min incubation at 37 degrees C in the presence of 8.3 mM D-glucose, the intracellular distribution space of D-[3H]mannoheptulose (0.1 mM) averaged, in islets from control and streptozotocin-induced diabetic rats, respectively 49.0+/-2.3 and 6.2+/-1.5% of the corresponding intracellular 3HOH space, all values being corrected for extracellular contamination as judged from the distribution space of [U-14C]sucrose (1.0 mM). These findings indicate that the present approach indeed allows to assess the relative contribution of B-cells to total islet mass for purpose of comparison between animals with different metabolic and/or hormonal status.

D-mannoheptulose uptake and its metabolic and secretory effects in human pancreatic islets.

D-mannoheptulose was recently proposed to be transported into cells at the intervention of GLUT2. Since GLUT1, rather than GLUT2, represents the major carrier system for the transport of monosaccharides across the islet B-cell plasma membrane in human subjects, the uptake of D-mannoheptulose and its metabolic and secretory effects were investigated in human islets. The uptake of D-glucose reached much more rapidly a close-to-equilibrium value in isolated islets than in pieces of pancreas obtained from the same donor. The distribution space of D-[3H]mannoheptulose in the human islets largely exceeded that of [U-14C]sucrose, considered as an extracellular marker, and did not differ significantly from that of 3HOH. In the human islets, the heptose (10.0 mM) inhibited both D-[5-3H]glucose utilization and D-[U-14C] glucose oxidation, and decreased glucose-stimulated insulin release to the same extent as D-mannoheptulose hexaacetate. These findings indicate that a suitable radioactive analog of D-mannoheptulose could be used, in human like in rat islets, for preferential labelling of the endocrine moiety of the pancreatic gland.

Effects of D-mannoheptose and D-glycero-D-gulo-heptose upon D-glucose metabolism and insulinotropic action in rat pancreatic islets and D-glucose phosphorylation by hexokinase isoenzymes: comparison with D-mannoheptulose.

The possible use of D-mannoheptose or D-glycero-D-gulo-heptose as substitute of D-mannoheptulose for specific inhibition of D-glucose phosphorylation, metabolism and insulinotropic action was investigated in the present study. The two aldoheptoses failed to duplicate the effect of D-mannoheptulose upon the phosphorylation of D-glucose by yeast hexokinase, bovine heart hexokinase or human B-cell glucokinase. They were poorly phosphorylated by the low-Km hexokinase isoenzymes or liver B-cell glucokinase. D-mannoheptose failed to reproduce the inhibitory action of D-mannoheptulose upon D-glucose metabolism by isolated rat pancreatic islets. Whilst D-glycero-D-gulo-heptose failed to affect glucose-induced insulin release, D-mannoheptose slightly enhanced glucose-induced insulin release when tested at low concentrations (0.75-1.5 mM) and progressively decreased insulin output at higher concentration (3. 0-20.0 mM) in islets exposed to a high (16.7 mM), but not physiological (8.3 mM), concentration of D-glucose. D-mannoheptose, however, also caused a modest inhibition of insulin release evoked by 2-ketoisocaproate. It is concluded, therefore, that neither D-mannoheptose nor D-glycero-D-guloheptose can be considered as suitable substitutes of D-mannoheptulose.

Glucose-mediated Ca(2+) signalling in single clonal insulin-secreting cells: evidence for a mixed model of cellular activation.

Using clonal insulin-secreting BRIN-BD11 cells, we have assessed whether the graded response of the whole cell population to glucose can be accounted for by a dose-dependent recruitment of individual cells, an amplification of the response of the recruited cells or both. Cytosolic free Ca(2+) concentration ([Ca(2+)](i)) is an established index of beta-cell function. We used fura-2 microfluorescence techniques to assess the [Ca(2+)](i) responsiveness of single BRIN-BD11 cells to glucose and other secretagogues. Glucose (1-16.7 mM) evoked oscillatory [Ca(2+)](i) rises in these cells resembling those found in parental rat pancreatic beta-cells. The percentage of glucose-responsive cells was 11% at 1 mM and increased to 40-70% at 3-16.7 mM glucose, as assessed by a single-stimulation protocol. This profile was unrelated to possible differences in the cell cycle, as inferred from experiments where the cultured cells were synchronized by a double thymidine block protocol. Individual cells exhibited variable sensitivities to glucose (threshold range: 1-5 mM) and a variable dose-dependent amplification of the [Ca(2+)](i) responses (EC(50) range: 2-10 mM), as assessed by a multiple-stimulation protocol. Glyceraldehyde and alpha-ketoisocaproic acid had glucose-like effects on [Ca(2+)](i). The data support a mixed model for the activation of insulin-secreting cells. Specifically, the graded secretory response of the whole cell population is likely to reflect both a recruitment of individual cells with different sensitivities to glucose and a dose-dependent amplification of the response of the recruited cells.

Hexokinase activity alters sugar-nucleotide formation in maize root homogenates.

Two pools of hexokinase activities differing in sensitivity to ADP inhibition were characterised in maize roots. In order to evaluate how glucose utilisation could be affected by these hexokinases, glucose-6-P and NDP-5'-sugar levels were measured after a D-[U-14C]glucose pulse in root extracts in the presence of 0 or 1 mM ADP. Analysis of radio-labelled activated sugars by paper chromatography revealed that: (1) without ADP, nearly 20% of the 14C appeared in NDP-5'-sugars; (2) 0.1 mM ADP inhibited 14C-NDP-5'-sugar formation by 85%; and (3) with 1 mM ADP, 14C-NDP-5'-sugars were undetectable, but substantial (14%) 14C accumulated as glucose-6-P. Mannoheptulose, a hexokinase inhibitor, blocked the NDP-5'-sugar formation, but did not modify the amount of 14C-glucose-6-P in root extracts either with or without ADP. The analysis of the hexokinase activities with 0.8 mM glucose in maize root extracts showed that: (1) mitochondrial hexokinase activity was totally inhibited by 30 mM mannoheptulose; and (2) the cytosolic hexokinase was inhibited by only 30%. These data suggest that NDP-5'-sugar synthesis is sensitive to ADP fluctuations and that mannoheptulose affects preferentially the mitochondrial-bound hexokinase, but the cytosolic form is less sensitive. We propose that the mitochondrial hexokinase is the main energy charge sensor in this pathway in maize.

Pancreatic fate of 14C-labelled hexoses.

In order to assess the respective contribution of the exocrine and endocrine moieties of the pancreas to the overall net uptake of selected monosaccharides by the pancreatic gland, the apparent distribution space of L-[1-14C]glucose, 3-O-[14C-methyl]-D-glucose, D-[U-14C]glucose, D-[U-14C]mannose and D-[U-14C]fructose was measured in pieces of pancreas obtained from either control rats or animals injected with streptozotocin. Although the time course for the uptake of 3-O-[14C-methyl]-D-glucose, D-[U-14C]glucose, D-[U-14C]mannose and D-[U-14C]fructose was much slower in the pieces of pancreas than that previously documented in isolated pancreatic islets, no significant difference could, as a rule, be detected between the results obtained in pancreatic pieces of control and streptozotocin rats. A comparable situation prevailed in the pancreas of animals examined 3 min after the intravenous injection of 3-O-[14C-methyl]-D-glucose. D-Glucose inhibited the uptake of 3-O-[14C-methyl]-D-glucose and that of D-[U-14C]fructose. Likewise, 3-O-methyl-D-glucose inhibited the uptake of D-[U-14C]glucose. Cytochalasin B (20 microm) also inhibited the uptake of 3-O-[14C-methyl]-D-glucose and D-[U-14C]glucose, but not that of D-[U-14C]fructose. D-Mannoheptulose hexaacetate, but not the unesterified heptose, inhibited the metabolism of tritiated and 14C-labelled D-glucose, as well as the net uptake of D-[U-14C]glucose and D-[U-14C]mannose and, to a lesser extent, that of D-[U-14C]fructose. These findings indicate that despite marked differences between endocrine and exocrine pancreatic cells in terms of both the time course for the uptake of several hexoses and the inhibition of their phosphorylation by D-mannoheptulose, little or no preferential labelling of the endocrine moiety of the pancreas by the 14C-labelled hexoses is observed, at least when judged from their distribution space in pancreatic pieces or the whole pancreatic gland. Nevertheless, the findings made with D-mannoheptulose and its hexaacetate ester raise the view that this heptose could conceivably be used to achieve a sizeable preferential labelling of the endocrine pancreas under the present experimental conditions.

KATP channel-dependent and -independent pathways of insulin secretion in isolated islets from fa/fa Zucker rats.

We hypothesized that altered insulin secretory patterns in obese (fa/fa) Zucker rats might be caused by changes in downstream stimulus-secretion coupling events, such as ATP-dependent potassium (KATP) channel activity. The functions of KATP-dependent and -independent pathways of insulin secretion were therefore compared in lean and fa/fa Zucker rat isolated islets. KATP channel function was normal in fa/fa rat islets, as assessed by responsiveness to direct channel inactivators glybenclamide and quinine and by the receptor-mediated response to epinephrine and somatostatin. Altered sensitivity to glucose and mannoheptulose were explained by upstream alterations in glucose metabolism documented earlier. Despite normal inactivation of KATP channels by ATP depletion of fa/fa rat islets, glucose-stimulated insulin secretion was not inhibited, leading to studies of a putative KATP-independent pathway. When islets were depolarized by incubating with 30 mM potassium and 0.25 mM diazoxide to bypass KATP channels, glucose elicited a concentration-dependent response in both phenotypes. This response required glucose metabolism and Ca2+, as proven by experiments with nonmetabolizable glucose analogs and calcium chelation, but was only partially inhibited by a glycolytic inhibitor. Intermediates or products of oxidative metabolism are likely involved because alpha-ketoisocaproate also elicited a KATP-independent insulin response. The pattern of responses was similar in lean and fa/fa rat islets, indicating that neither of these pathways explains the insulin secretion by fa/fa rat islets depleted of ATP. In conclusion, phenotype-related differences in KATP channel function were consistent with upstream changes in glucose metabolism in fa/fa rat islets. Further studies are required to understand the basis of insulin secretion in ATP-depleted islets from fa/fa rats.

Site-directed mutagenesis studies on the determinants of sugar specificity and cooperative behavior of human beta-cell glucokinase.

The determinants of sugar specificity and cooperative behavior of human beta-cell glucokinase were studied by mutating several active site residues and performing a steady-state kinetic analysis of the purified mutant and wild-type enzymes after their expression in Escherichia coli. Asn-204, Glu-256, and Glu-290 were predicted from molecular modeling to interact with the 3-OH, 4-OH, 2-OH, and 1-OH groups of glucose. Mutation of these residues resulted in enzymes with decreased values of kcat and increased values of Km for glucose, mannose, and 2-deoxyglucose. Lys-56 is also predicted to make an interaction with the side chain of Glu-256 and its mutation increased the Km for glucose, deoxyglucose, mannose, and fructose by 4-, 4-, 3-, and 10-fold, respectively, and also increased the kcat for fructose by 5-fold. The Ki values for N-acetylglucosamine and mannoheptulose for the wild-type enzyme were 0.2 and 0.8 mM, respectively, and mutation of glucose binding residues to alanine resulted in an increase of about 3 orders of magnitude in these Ki values. Mutation of residues that directly hydrogen bond glucose hydroxyls (Asn-204, Glu-256, and Glu-290) to alanine resulted in enzymes that did not exhibit cooperative behavior, but mutation of Lys-56 or other residues that do not directly contact glucose had no effect on the Hill coefficient. Only glucose and deoxyglucose exhibited cooperative behavior. The results 1) confirm the predictions of the model that Asn-204, Glu-256, and Glu-290 are important residues involved in catalysis and hydrogen bonding glucose hydroxyl groups, 2) provide evidence for a role of Lys-56 in hexose binding, and 3) are consistent with the cooperative behavior of glucokinase being mediated by interactions of other regions of the protein with the highly conserved active site glucose binding residues.

Kinetic characterization of Na+/D-mannose cotransport in dog kidney: comparison with Na+/D-glucose cotransport.

Brush-border membrane vesicles (BBMV) prepared from whole dog kidney cortex, or separately from outer cortex (OC) and outer medulla (OM), were used to study the kinetics and inhibition specificity of Na(+)-dependent D-mannose cotransport. In BBMV from whole cortex the measured parameters for Na+/D-mannose uptake were Km = 0.07 +/- 0.01 mM and Vmax = 4.19 +/- 0.24 nmol/mg protein per min (n = 36). In OC BBMV the Km for Na+/D-mannose was 0.04 mM, Vmax = 3.41 nmol/mg per min. In OM the Km was 0.06 +/- 0.02 mM Vmax = 0.18 nmol/mg per min. Thus only about 5% of Na+/D-mannose activity occurs in OM. Both mannoheptulose (Ki = 5.6 mM) and methyl alpha-D-mannoside (Ki = 0.05 mM) are competitive inhibitors of Na+/D-mannose uptake, but at comparable concentrations have little effect on Na+/D-glucose uptake. Phlorizin is a noncompetitive inhibitor of Na+/D-mannose uptake (Ki = 4.45 microM) but a more potent and competitive inhibitor (Ki = 0.58 microM) of Na+/D-glucose uptake. Phloretin (Ki = 104 microM) is a noncompetitive inhibitor of Na+/D-mannose uptake in BBMV. We conclude that Na+/D-mannose uptake is mediated by a unique high-affinity carrier located in the OC presumably at the luminal surface of the proximal convoluted tubule, with strong specificity requirements for sugars with mannose-like structures (i.e., axial C-2 hydroxyl group). Phlorizin is an inhibitor of both Na+/D-mannose and Na+/D-glucose cotransporters but is approx. 10 times less potent for the Na+/D-mannose system and also has a different mode of inhibition (i.e., noncompetitive vs. competitive). The different phlorizin inhibitory mechanisms on the Na+/D-glucose and Na+/D-mannose cotransporters may be mediated by distinct hydrophobic and sugar binding sites that characterize phlorizin-carrier interaction.