Horticultural availability and homeowner preferences drive plant diversity and composition in urban yards.

Understanding the factors that influence biodiversity in urban areas is important for informing management efforts aimed at enhancing the ecosystem services in urban settings and curbing the spread of invasive introduced species. We determined the ecological and socioeconomic factors that influence patterns of plant richness, phylogenetic diversity, and composition in 133 private household yards in the Minneapolis-Saint Paul Metropolitan area, Minnesota, USA. We compared the composition of spontaneously occurring plant species and those planted by homeowners with composition in natural areas (at the Cedar Creek Ecosystem Science Reserve) and in the horticulture pool of species available from commercial growers. Yard area and fertilizer frequency influenced species richness of the spontaneous species but expressed homeowner values did not. In contrast, the criteria that homeowners articulated as important in their management decisions, including aesthetics, wildlife, neatness and food provision, significantly predicted cultivated species richness. Strikingly, the composition of plant species that people cultivated in their yards resembled the taxonomic and phylogenetic composition of species available commercially. In contrast, the taxonomic and phylogenetic composition of spontaneous species showed high similarity to natural areas. The large fraction of introduced species that homeowners planted was a likely consequence of what was available for them to purchase. The study links the composition and diversity of yard flora to their natural and anthropogenic sources and sheds light on the human factors and values that influence the plant diversity in residential areas of a major urban system. Enhanced understanding of the influences of the sources of plants, both native and introduced, that enter urban systems and the human factors and values that influence their diversity is critical to identifying the levers to manage urban biodiversity and ecosystem services.

PFK-2/FBPase-2: maker and breaker of the essential biofactor fructose-2,6-bisphosphate.

Fructose-2,6-bisphosphate is responsible for mediating glucagon-stimulated gluconeogenesis in the liver. This discovery has led to the realization that this compound plays a significant role in directing carbohydrate fluxes in all eukaryotes. Biophysical studies of the enzyme that both synthesizes and degrades this biofactor have yielded insight into its molecular enzymology. Moreover, the metabolic role of fructose-2,6-bisphosphate has great potential in the treatment of diabetes.

Overexpression of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase in mouse liver lowers blood glucose by suppressing hepatic glucose production.

Hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is an important regulatory enzyme of glucose metabolism. By controlling the level of fructose-2,6-bisphosphate, an allosteric activator of the glycolytic enzyme 6-phosphofructo-1-kinase and an inhibitor of the gluconeogenic enzyme fructose-1,6-bisphosphatase, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase regulates hepatic glucose output. We studied the effects of adenovirus-mediated overexpression of this enzyme on hepatic glucose metabolism in normal or diabetic mice. These animals were treated with virus encoding either wild-type or bisphosphatase activity-deficient 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase. Seven days after virus injection, hepatic fructose-2,6-bisphosphate levels increased significantly in both normal and diabetic mice, with larger increases observed in animals with overexpression of the mutant enzyme. Blood glucose levels in normal mice overexpressing either enzyme were lowered, accompanied by increased plasma lactate, triglycerides, and FFAs. Blood glucose levels were markedly reduced in diabetic mice overexpressing the wild-type enzyme, and still more so in mice overexpressing the mutant form of the enzyme. The lower blood glucose levels in diabetic mice were accompanied by partially normalized plasma triglycerides and FFAs, increased plasma lactate, and increased liver glycogen levels, relative to diabetic mice treated with a control adenovirus. Our findings underscore the critical role played by hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in control of fuel homeostasis and suggest that this enzyme may be considered as a therapeutic target in diabetes.

cAMP-independent synergistic effects of insulin and dexamethasone on fructose 2,6-bisphosphate metabolism in H4IIE cells.

Hormonal regulation of fructose 2,6-bisphosphate (Fru-2,6-P2) content was studied in H4IIE cells. These cells were found to be very sensitive to physiological concentrations of insulin. Addition of either insulin or dexamethasone alone increased Fru-2,6-P2 in a time- and dose-dependent manner, and the maximal effect of the hormones was seen at 1 h. Neither hormone had any measurable effect on cAMP levels. The effect of addition of both insulin and dexamethasone on Fru-2,6-P2 was synergistic. Insulin, but not dexamethasone, rapidly increased 6-phosphofructo-2-kinase (6PF-2-K) activity by causing dephosphorylation of the enzyme as judged by a decrease in the Km for fructose-6-phosphate. Addition of both hormones also resulted in a synergistic 10-fold increase in enzyme protein as measured by kinase activity and phosphoenzyme formation. Dexamethasone increased liver 6PF-2-K/Fru-2,6-P2 mRNA abundance by 10- to 12-fold as measured by a ribonuclease protection assay, and insulin increased it by only 4-fold. Effects were observed as early as 1 h after hormone addition, but addition of both hormones together showed no synergy. We conclude that the synergistic effects of insulin and dexamethasone on Fru-2,6-P2 content are mediated by a combination of stimulation of expression of the bifunctional enzyme gene by both hormones and insulin-induced modulation of the activation state of the bifunctional enzyme, both of which are mediated by cAMP-independent mechanisms.

Regulation of rat liver glucose-6-phosphatase gene expression in different nutritional and hormonal states: gene structure and 5'-flanking sequence.

The mRNA level of the catalytic subunit of rat liver glucose-6-phosphatase (Glu-6-Pase) was regulated by hormones commensurate with activity changes in vivo. Insulin exerts a dominant negative effect on the mRNA levels of Glu-6-Pase. Both mRNA levels and activities of the enzyme are low in the fed and refed state where insulin levels are elevated. Insulin administration to diabetic rats also decreases levels of mRNA and Glu-6-Pase activity. Insulin at a concentration of 1 nmol/l completely overcomes the stimulatory effect of glucocorticoids on Glu-6-Pase message levels in FAO hepatoma cells. The stimulatory response to glucocorticoid in FAO cells is biphasic, with maxima seen at 3 and 18 h after hormone addition (respectively 1.6- and 3.3-fold). 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) causes a fourfold increase in Glu-6-Pase mRNA at 3 h in FAO cells. The gene of rat liver Glu-6-Pase is 13 kilobases in length and comprised of 5 exons. The exon-intron structure is completely conserved when compared with the mouse and human genes. A 0.5-kb 3'-untranslated region, which is present in rat and mouse liver Glu-6-Pase cDNA, is absent in the Glu-6-Pase gene reported here, indicating the possible duplication of either the terminal fifth exon or the entire gene. The promoter region contains a consensus core CCAAT element at position -207 and a TATAAA at position -31. Several possible response elements have been identified in the 5'-flanking region (from a HindIII site at position -1641). A consensus glucocorticoid response element is located at base pair -1552, a 9/10 match of the insulin response sequence is located at position -1449, and a 7/8 match of the cAMP response element is located at position -164.

Characterization of glucokinase mutations associated with maturity-onset diabetes of the young type 2 (MODY-2): different glucokinase defects lead to a common phenotype.

Glucokinase (GK) is expressed in the pancreatic beta-cells and liver, and plays a key role in the regulation of glucose homeostasis. The enzymatic activity and thermal stability of wild-type (WT) GK and several mutant forms associated with maturity-onset diabetes of the young type 2 (MODY-2) were determined by a steady-state kinetic analysis of the purified expressed proteins. The eight MODY-2 mutations studied were Ala53Ser, Val367Met, Gly80Ala, Thr168Pro, Arg36Trp, Thr209Met, Cys213Arg, and Val226Met. These missense mutations were shown to have variable effects on GK kinetic activity. The Gly80Ala and Thr168Pro mutations resulted in a large decrease in Vmax and a complete loss of the cooperative behavior associated with glucose binding. In addition, the Gly80Ala mutation resulted in a sixfold increase in the half-saturating substrate concentration (S0.5) for ATP, and Thr168Pro resulted in eight- and sixfold increases in the S0.5 values for ATP and glucose, respectively. The Thr209Met and Val226Met mutations exhibited three- and fivefold increases, respectively, in the S0.5 for ATP, whereas the Cys213Arg mutation resulted in a fivefold increase in the S0.5 for glucose. These mutations also led to a small yet significant reduction in Vmax. Of all the mutations studied, only the Cys213Arg mutation had reduced enzymatic activity and decreased thermal stability. Two mutants, Ala53Ser and Val367Met, showed kinetic and thermal stability properties similar to those of WT. These mutants had increased sensitivities to the known negative effectors of GK activity, palmitoyl-CoA, and GK regulatory protein. Taken together, these results illustrate that the MODY-2 phenotype may be linked not only to kinetic alterations but also to the regulation of GK activity.

The cDNA sequence encoding the major intrinsic protein of frog lens.

A cDNA clone encoding the frog lens major intrinsic protein (MIP) has been isolated and sequenced. The predicted protein of 28 kDa has high sequence identity and similarity to mammalian and avian lens MIP sequences. Frog lens MIP is encoded by a transcript of 4.4 kb.

Characterization of an intronic hormone response element of the rat liver/skeletal muscle 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene.

The glucocorticoid response element of the rat liver/skeletal muscle 6- phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene was characterized. The element is composed of two tandem hormone receptor binding sites separated by 12 base pairs. Addition of dexamethasone to HeLa cells transiently transfected with a chloramphenicol acetyl transferase (CAT) reporter plasmid containing the hormone response element and cotransfected with glucocorticoid receptor stimulated transcription 24-fold in an orientation- and position-independent manner. Deletion or mutation of essential G/C pairs of the distal binding site abolished hormone-stimulated CAT activity, whereas deletion or mutation of the proximal binding site decreased the hormone-stimulated response only slightly. Mutation of both distal and proximal binding sites resulted in complete loss of hormone-stimulated CAT activity. Experiments carried out using testosterone and progesterone with their respective receptors revealed qualitatively similar results to those seen with glucocorticoid. Binding of glucocorticoid receptor or androgen receptor DNA binding domains to the hormone response element, visualized by gel mobility shift, was unaffected in the proximal binding site mutant, markedly decreased in the distal binding site mutant, and abolished in the double mutant. In gel mobility shift analysis of separate distal and proximal binding sites, only the native distal site demonstrated high affinity binding to glucocorticoid and androgen receptor DNA binding domains. The results demonstrate that this element is responsible for glucocorticoid, androgen, and progesterone stimulation of transcription of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene and that the distal receptor binding site is dominant.

Fructose-2,6-bisphosphate and control of carbohydrate metabolism in eukaryotes.

Fructose-2,6-bisphosphate is an important intracellular biofactor in the control of carbohydrate metabolic fluxes in eukaryotes. It is generated from ATP and fructose-6-phosphate by 6-phosphofructo-2-kinase and degraded to fructose-6-phosphate and phosphate ion by fructose-2,6-bisphosphatase. In most organisms these enzymatic activities are contained in a single polypeptide. The reciprocal modulation of the kinase and bisphosphatase activities by post-translational modifications places the level of the biofactor under the control of extra-cellular signals. In general, these signals are generated in response to changing nutritional states, therefore, fructose-2,6-bisphosphate plays a role in the adaptation of organisms, and the tissues within them, to changes in environmental and metabolic states. Although the specific mechanism of fructose-2,6-bisphosphate action varies between species and between tissues, most involve the allosteric activation of 6-phosphofructo-1-kinase and inhibition of fructose-1,6-bisphosphatase. These highly conserved enzymes regulate the fructose-6-phosphate/fructose-1,6-bisphosphate cycle, and thereby, determine the carbon flux. It is by reciprocal modulation of these activities that fructose-2,6-bisphosphate plays a fundamental role in eukaryotic carbohydrate metabolism.

A retrospective evaluation of doxorubicin-based chemotherapy for dogs with right atrial masses and pericardial effusion.

OBJECTIVE: To report the outcome of doxorubicin-based chemotherapy as the sole treatment for dogs with echocardiographically identified right atrial masses and pericardial effusion. METHODS: A retrospective study of case records of dogs with right atrial masses treated with doxorubicin. Dogs were excluded from the study if they had any type of surgery performed such as pericardiectomy or right atrial mass resection, or if their chemotherapy protocol did not include doxorubicin. The data collected included signalment, history, physical examination findings, diagnostic test results and long-term survival. RESULTS: Dogs with right atrial masses and pericardial effusion that received doxorubicin-based chemotherapy alone had a median survival of 139 · 5 days (range 2 to 302 days). Chemotherapy side effects were frequent but mild. CLINICAL SIGNIFICANCE: Doxorubicin-based chemotherapy alone appears to be a viable treatment option for dogs with echocardiographically identified right atrial masses and pericardial effusion.

Isolation of a cDNA for chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

A chicken liver cDNA for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was isolated from a Lambda ZAP2 phage library. The chicken liver cDNA codes for a protein that has 89.1, 88.4 and 88.0% amino acid identity with the human, rat and bovine liver isoforms, respectively. The kinetic properties of the rat and chicken liver enzymes, purified to homogeneity after expression in E. coli, were different including negative cooperativity for ATP binding and inhibition by Mg2+ for the chicken liver 6-phosphofructo-2-kinase but not for the rat liver kinase. Differences in the beta-loop ATP signature sequences in the chicken and rat liver kinase domains may explain the kinetic differences and represent the major divergence in the evolution of the enzyme from birds to mammals.

Photosynthetic carbon metabolism in leaves of transgenic tobacco (Nicotiana tabacum L.) containing decreased amounts of fructose 2,6-bisphosphate.

The aim of this work was to examine the role of fructose 2,6-bisphosphate (Fru-2,6-P2) in photosynthetic carbon partitioning. The amount of Fru-2,6-P2 in leaves of tobacco (Nicotiana tabacum L. cv. Samsun) was reduced by introduction of a modified mammalian gene encoding a functional fructose-2,6-bisphosphatase (EC 3.1.3.46). Expression of this gene in transgenic plants reduced the Fru-2,6-P2 content of darkened leaves to between 54% and 80% of that in untransformed plants. During the first 30 min of photosynthesis sucrose accumulated more rapidly in the transgenic lines than in the untransformed plants, whereas starch production was slower in the transgenic plants. On illumination, the proportion of 14CO2 converted to sucrose was greater in leaf disks of transgenic lines possessing reduced amounts of Fru-2,6-P2 than in those of the control plants, and there was a corresponding decrease in the proportion of carbon assimilated to starch in the transgenic lines. Furthermore, plants with smaller amounts of Fru-2,6-P2 had lower rates of net CO2 assimilation. In illuminated leaves, decreasing the amount of Fru-2,6-P2 resulted in greater amounts of hexose phosphates, but smaller amounts of 3-phosphoglycerate and dihydroxyacetone phosphate. These differences are interpreted in terms of decreased inhibition of cytosolic fructose-1,6-bisphosphatase resulting from the lowered Fru-2,6-P2 content. The data provide direct evidence for the importance of Fru-2,6-P2 in co-ordinating chloroplastic and cytosolic carbohydrate metabolism in leaves in the light.

Quantitative aspects of relationship between glucose 6-phosphate transport and hydrolysis for liver microsomal glucose-6-phosphatase system. Selective thermal inactivation of catalytic component in situ at acid pH.

Studies of the thermal stability of rat liver glucose-6-phosphatase (EC 3.1.3.9) were carried out to further elevate the proposal that the enzymic activity is the result of the coupling of a glucose-6-P-specific translocase and a nonspecific phosphohydrolase-phosphotransferase. Inactivation was observed when micorsomes were incubated at mild temperatures between pH 6.2 and 5.6. The rate of inactivation increased either with increasing hydrogen ion concentration or temperature. However, no inactivation was seen below 15 degrees in media as low as pH 5 or at neutral pH up to 37 degrees. The thermal stability of the enzyme may be controlled by the physical state of the membrane lipids and the degree of protonation of specific residues in the enzyme protein. Microsomes were exposed to inactivating conditions, and kinetic analyses were made of the glucose-6-P phosphohydrolase activities before and after supplementation to 0.4% sodium taurocholate. The results support the postulate and the kinetic characteristics of a given preparation of intact microsomes are determined by the relative capacities of the transport and catalytic components. Before detergent treatment, inactivation (i.e. a decrease in Vmax) was accompanied by a decrease in Km and a reduction in the fraction of latent activity, whereas only Vmax was depressed in disrupted preparations. The possibility that the inactivating treatments caused concurrent disruption of the microsomal membrane was ruled out. It is concluded that exposures to mild heat in acidic media selectively inactivate the catalytic component of the glucose-6-phosphatase system while preserving an intact permeability barrier and a functional glucose-6-P transport system. Analyses of kinetic data obtained in the present and earlier studies revealed several fundamental mathematical relationships among the kinetic constants describing the glucose-6-P phosphohydrolase activities of intact (i.e. the "system") and disrupted microsomes (i.e. the catalytic component). The quantitative relationships appear to provide a means to calculate a velocity constant (VT) and a half-saturation constant (KT) for glucose-6-P influx. The well documented, differential responses of the rat liver glucose-6-phosphatase system induced by starvation, experimental diabetes, or cortisol administration were analyzed in terms of these relationships. The possible influences of cisternal inorganic phosphate on the apparent kinetic constants of the intact system are discussed.

Regulation of glucose production by the liver.

Glucose is an essential nutrient for the human body. It is the major energy source for many cells, which depend on the bloodstream for a steady supply. Blood glucose levels, therefore, are carefully maintained. The liver plays a central role in this process by balancing the uptake and storage of glucose via glycogenesis and the release of glucose via glycogenolysis and gluconeogenesis. The several substrate cycles in the major metabolic pathways of the liver play key roles in the regulation of glucose production. In this review, we focus on the short- and long-term regulation glucose-6-phosphatase and its substrate cycle counter-part, glucokinase. The substrate cycle enzyme glucose-6-phosphatase catalyzes the terminal step in both the gluconeogenic and glycogenolytic pathways and is opposed by the glycolytic enzyme glucokinase. In addition, we include the regulation of GLUT 2, which facilitates the final step in the transport of glucose out of the liver and into the bloodstream.

Evidence for the participation of independent translocation for phosphate and glucose 6-phosphate in the microsomal glucose-6-phosphatase system. Interactions of the system with orthophosphate, inorganic pyrophosphate, and carbamyl phosphate.

The interactions of Pi, PPi, and carbamyl-P with the hepatic glucose-6-phosphatase system were studied in intact and detergent-disrupted microsomes. Penetration of PPi and carbamyl-P into intact microsomes was evidenced by their reactions with the enzyme located exclusively on the luminal surface. Lack of effects of carbonyl cyanide m-chlorophenylhydrazone and valinomycin + KCl indicated that pH gradients and/or membrane potentials that could influence the kinetics of the system are not generated during metabolism of PPi and glucose-6-P by intact microsomes. With disrupted microsomes, only competitive interactions were seen among glucose-6-P, Pi, PPi, and carbamyl-P. With intact microsomes, Pi, PPi, and carbamyl-P were relatively weak, noncompetitive inhibitors of glucose-6-phosphatase, and PPi hydrolysis was inhibited competitively by Pi and carbamyl-P but noncompetitively by glucose-6-P. Analysis of the kinetic data in combination with findings from other studies that a variety of inhibitors of the glucose-6-P translocase (T1) does not affect PPi hydrolysis provide compelling evidence that permeability of microsomes to Pi, PPi, and carbamyl-P is mediated by a second translocase (T2). Some properties of the microsomal anion transporters are described. If the characteristics of the glucose-6-phosphatase system as presently defined in intact microsomes apply in vivo, glucose-6-P hydrolysis appears to be the predominant, if not the exclusive, physiologic function of the system. Both the "noncompetitive character" and the relative ineffectiveness of Pi as an inhibitor of glucose-6-phosphatase of intact microsomes result from the rate limitation imposed by T1 that prevents equilibration of glucose-6-P across the membrane. In microsomes from fed rats, where T1 is less rate restricting, about one-half as much Pi was required to give 50% inhibition compared with microsomes from fasted or diabetic rats. Thus, any treatment or agent that alters the kinetic relationship between transport and hydrolysis of glucose-6-P (e.g. endocrine or nutritional status) is an essential consideration in analyses of kinetic data for the glucose-6-phosphatase system.

Isolation of bovine liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase cDNA: bovine liver and heart forms of the enzyme are separate gene products.

In order to ascertain whether the heart and liver forms of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase were products of two different genes or arose via alternative splicing of a single gene, the bovine liver cDNA of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was isolated from a lambda gt10 phage library and its sequence compared with that of bovine heart cDNA. The deduced amino acid sequence of the bovine liver cDNA was also compared with the amino acid sequence of the human and rat liver phosphofructo-2-kinase/fructose-2,6-bisphosphatase enzyme. The bovine liver cDNA codes for a protein that has 81.6% amino acid identity with the bovine heart form and 97.0 and 98.3% identity with the rat and human liver forms of the enzyme, respectively. Comparison of the nucleotide sequences of the two bovine cDNAs and their deduced amino acid sequences demonstrates that while there is conservation of the active sites of liver/muscle and heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases they are encoded by different genes.

Aluminum ions are required for stabilization and inhibition of hepatic microsomal glucose-6-phosphatase by sodium fluoride.

Stabilization and inhibition of hepatic microsomal glucose-6-P phosphohydrolase (EC 3.1.3.9) by F- requires the presence of Al3+ ions. At millimolar concentrations, reagent grade NaF inhibited glucose-6-P hydrolysis and protected the enzyme against inactivation induced by heat in the presence of 0.025% (w/v) Triton X-100 or by reaction of the catalytic site with the histidine-specific reagent, diethyl pyrocarbonate. The presence of millimolar EDTA in all test systems abolished the effectiveness of NaF, yet EDTA by itself was without significant influence on the kinetics of phosphohydrolase reaction, the thermal stability of the enzyme or its reactivity with diethyl pyrocarbonate. Although ultrapure NaF was ineffectual in all test systems, its potency as a competitive inhibitor or protective agent was markedly increased by micromolar AlCl3 or when assays were carried out in flint glass test tubes. The latter response is explained by the well documented ability of fluoride solutions to extract Al3+ from glass at neutral pH. Our analysis indicates that the effectiveness of fluoride in all test systems derives from the formation of a specific complex with Al3+, most likely Al(F)4-. The apparent dissociation constant for interaction of the enzyme and Al(F)4- is 0.1 microM. The combination of NaF and AlCl3 holds promise as an unusually effective and versatile means to stabilize this notoriously labile enzyme during efforts to purify it.

Expression of human liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in Escherichia coli. Role of N-2 proline in degradation of the protein.

Human liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is 96% identical to the rat and bovine liver enzymes, and all of the critical catalytic and substrate binding residues in both the kinase and bisphosphatase domains are conserved in the three enzymes. However, in contrast to rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, which is readily expressed in an Escherichia coli T-7 RNA polymerase-based expression system, the human liver bifunctional enzyme could not be expressed in this system. Western blot and slot blot analysis revealed that although both the bifunctional enzyme protein and its mRNA were rapidly induced by the addition of isopropyl-1-thio-beta-D-galactopyranoside, the protein underwent rapid degradation. Deletion of the N-2 proline residue or its mutation to arginine, the corresponding residue in the rat liver enzyme, revealed that this proline residue was responsible for its rapid degradation. The Pro-2-->Arg mutant could be expressed with a high yield (20 mg/liter) in E. coli. The results support the hypothesis that a proline residue at N-2 facilitates bifunctional enzyme degradation in E. coli. The E. coli expressed mutant form was purified to homogeneity by phosphocellulose chromatography, and its kinetic properties were compared with those of the rat liver enzyme. The kinetic properties of the two enzymes were identical except for the presence of substrate (fructose 6-phosphate) inhibition of the human liver enzyme but not of the rat liver enzyme. The ability to express and purify large amounts of human liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase will permit structure/function and x-ray crystal structure studies of the enzyme and ultimately its targeting for drug therapy.

Characterization of glucokinase-binding protein epitopes by a phage-displayed peptide library. Identification of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as a novel interaction partner.

The low affinity glucose-phosphorylating enzyme glucokinase shows the phenomenon of intracellular translocation in beta cells of the pancreas and the liver. To identify potential binding partners of glucokinase by a systematic strategy, human beta cell glucokinase was screened by a 12-mer random peptide library displayed by the M13 phage. This panning procedure revealed two consensus motifs with a high binding affinity for glucokinase. The first consensus motif, LSAXXVAG, corresponded to the glucokinase regulatory protein of the liver. The second consensus motif, SLKVWT, showed a complete homology to the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2), which acts as a key regulator of glucose metabolism. Through yeast two-hybrid analysis it became evident that the binding of glucokinase to PFK-2/FBPase-2 is conferred by the bisphosphatase domain, whereas the kinase domain is responsible for dimerization. 5'-Rapid amplification of cDNA ends analysis and Northern blot analysis revealed that rat pancreatic islets express the brain isoform of PFK-2/FBPase-2. A minor portion of the islet PFK-2/FBPase-2 cDNA clones comprised a novel splice variant with 8 additional amino acids in the kinase domain. The binding of the islet/brain PFK-2/ FBPase-2 isoform to glucokinase was comparable with that of the liver isoform. The interaction between glucokinase and PFK-2/FBPase-2 may provide the rationale for recent observations of a fructose-2,6-bisphosphate level-dependent partial channeling of glycolytic intermediates between glucokinase and glycolytic enzymes. In pancreatic beta cells this interaction may have a regulatory function for the metabolic stimulus-secretion coupling. Changes in fructose-2,6-bisphosphate levels and modulation of PFK-2/FBPase-2 activities may participate in the physiological regulation of glucokinase-mediated glucose-induced insulin secretion.