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The amino acid sequence of rat liver glucokinase deduced from cloned cDNA.
Andreone TL, Printz RL, Pilkis SJ, Magnuson MA, Granner DK
(1989) J Biol Chem 264: 363-9
MeSH Terms: Amino Acid Sequence, Animals, Base Sequence, Binding Sites, Cloning, Molecular, Codon, DNA, Glucokinase, Liver, Male, Molecular Sequence Data, Rats, Rats, Inbred Strains, Restriction Mapping
Show Abstract · Added February 23, 2011
Rat liver glucokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) was purified to homogeneity, cleaved, and subjected to amino acid sequence analysis. Forty-five percent of the protein sequence was obtained, and this information was used to design oligonucleotide probes to screen a rat liver cDNA library. A 1601-base pair cDNA (GK1) contained an open reading frame that encoded the amino acid sequences found in the peptides used to generate the oligonucleotide probes. A second cDNA was subsequently identified (GK.Z2), which is 2346 base pairs long and corresponds to nearly the entire glucokinase mRNA. Blot transfer analysis of hepatic RNA showed that glucokinase mRNA exists as a single species of about 2400 nucleotides. Four hours of insulin treatment of diabetic rats resulted in a 30-fold induction of this mRNA. GK.Z2 has a long open reading frame which, with the known partial peptide sequence, allowed us to deduce the primary structure of glucokinase. The enzyme is composed of 465 amino acids and has a mass of 51,924 daltons. Glucokinase has 53 and 33% amino acid sequence identities with the carboxyl-terminal domains of rat brain hexokinase I and yeast hexokinase, respectively. If conservative amino acid replacements are also considered, glucokinase is similar to these two enzymes at 75 and 63% of positions, respectively. The putative glucose- and ATP-binding domains of glucokinase were identified, and these regions appear to be highly conserved in the hexokinase family of enzymes.
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14 MeSH Terms
Rat glucokinase gene: structure and regulation by insulin.
Magnuson MA, Andreone TL, Printz RL, Koch S, Granner DK
(1989) Proc Natl Acad Sci U S A 86: 4838-42
MeSH Terms: Animals, Base Sequence, Cell Nucleus, Diabetes Mellitus, Experimental, Exons, Gene Expression Regulation, Genes, Glucokinase, Insulin, Introns, Liver, Male, Molecular Sequence Data, Rats, Rats, Inbred Strains, Restriction Mapping, Transcription, Genetic
Show Abstract · Added February 23, 2011
The glucokinase gene is 15.5-kilobases long, appears to be present as a single copy, and contains 10 exons that range in size from 96 to 977 base pairs. The transcription start site was located 127 nucleotides upstream from the translation initiation codon. The 5' flanking DNA contains several regions similar to defined promoter elements. These include a probable "TATA box," an Sp1 binding site, and several elements related to liver-specific gene expression. In addition, we determined that transcription of the glucokinase gene increased at least 20-fold when diabetic rats were treated with insulin for 2 hr.
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17 MeSH Terms
An alternate promoter in the glucokinase gene is active in the pancreatic beta cell.
Magnuson MA, Shelton KD
(1989) J Biol Chem 264: 15936-42
MeSH Terms: Adenoma, Islet Cell, Amino Acid Sequence, Animals, Base Sequence, Cloning, Molecular, Gene Amplification, Genes, Glucokinase, Insulinoma, Islets of Langerhans, Isoenzymes, Molecular Sequence Data, Pancreatic Neoplasms, Promoter Regions, Genetic, RNA Splicing, RNA, Messenger, Rats, Restriction Mapping, Sequence Homology, Nucleic Acid, Transcription, Genetic
Show Abstract · Added February 23, 2011
An alternate promoter in the glucokinase gene is active in the beta cell and produces a glucokinase mRNA which is longer and that has a different leader sequence and translation start site than the hepatic glucokinase mRNA. The glucokinase beta cell promoter is located at least 12 kilobases upstream from the glucokinase hepatic promoter. Transcription from the glucokinase beta cell promoter initiates over a region of 62 bases. The absence of a TATA box homology in the proximal promoter region may account for the diffuse transcriptional initiation. Translation of the beta cell glucokinase mRNA predicts a glucokinase isozyme that is different from the hepatic isozyme by 15 amino acids at the N terminus. The use of alternative promoters apparently enables the glucokinase gene to be regulated by insulin in the liver and by glucose in the beta cell, thus possibly constituting an important feedback control loop for maintaining glucose homeostasis. Alternate RNA splicing of the beta cell glucokinase mRNA predicts at least two beta cell glucokinase isoforms. An alternate splice acceptor site in the 4th exon of the glucokinase gene was identified in two glucokinase cDNAs from rat insulinoma tissue. Use of the alternate splice acceptor site results in a 51-nucleotide in frame deletion in the beta cell glucokinase mRNA and removal of 17 amino acids from a region of the protein situated between the putative glucose and ATP binding domains. Analysis of the pattern of RNA splicing in tissues containing beta cells indicates that the splice acceptor site utilized in producing hepatic glucokinase mRNA is also utilized in the beta cell.
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2 Members
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20 MeSH Terms
Glucokinase gene structure. Functional implications of molecular genetic studies.
Magnuson MA
(1990) Diabetes 39: 523-7
MeSH Terms: Gene Expression Regulation, Enzymologic, Genes, Glucokinase, Humans, Islets of Langerhans, Liver
Show Abstract · Added May 27, 2010
Glucokinase is expressed in both the liver and the pancreatic beta-cell and plays a key role in the metabolism of glucose by both tissues. Expression of this enzyme is differentially regulated; hepatic glucokinase is stimulated by insulin and repressed by cAMP, whereas beta-cell glucokinase activity is increased by glucose. Recently, the glucokinase gene has been characterized and was found to contain two different transcription control regions. One region regulates transcription of the gene in the liver, whereas the other region, which lies at least 12 kilobases further upstream, controls transcription in the pancreatic beta-cell. The finding of two different transcription control regions in a single glucokinase gene provides a genetic basis for the tissue-specific differential regulation of glucokinase and will serve as the basis for further studies to identify and characterize the different regulatory elements and factors in the liver and beta-cell, which are presumably involved. Comparison of different glucokinase cDNAs isolated from hepatic, insulinoma, and islet cDNA libraries indicates that at least three glucokinase isoforms are generated by differential RNA processing of the glucokinase gene transcripts. Whether any of these glucokinase isoforms are functionally unique remains to be determined.
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6 MeSH Terms
Effects of alternate RNA splicing on glucokinase isoform activities in the pancreatic islet, liver, and pituitary.
Liang Y, Jetton TL, Zimmerman EC, Najafi H, Matschinsky FM, Magnuson MA
(1991) J Biol Chem 266: 6999-7007
MeSH Terms: Animals, Base Sequence, Cell Line, Exons, Glucokinase, Hexokinase, Islets of Langerhans, Isoenzymes, Liver, Mice, Molecular Sequence Data, Mutagenesis, Insertional, Mutagenesis, Site-Directed, Oligonucleotide Probes, Pituitary Gland, Polymerase Chain Reaction, RNA Splicing, RNA, Messenger, Rats, Restriction Mapping, Sequence Homology, Nucleic Acid, Transfection
Show Abstract · Added January 16, 2014
Different glucokinase isoforms are produced by tissue-specific alternative RNA splicing in the liver and pancreatic islet, the only tissues in which glucokinase activity has been detected. To determine whether differences in protein structure brought about by alternative RNA splicing have an effect on glucose phosphorylating activity, we expressed cDNAs encoding four different hepatic and islet glucokinase isoforms and determined the Km and Vmax of each. When the glucokinase B1 and L1 isoforms were expressed in eukaryotic cells, both high Km glucose phosphorylating activity and immunoreactive protein were detected. However, when the glucokinase B2 and L2 isoforms were expressed, both of which differ by deletion of 17 amino acids in a region between the putative glucose and ATP-binding domains, no high Km glucose phosphorylating activity and much less immunoreactive protein were detected. When the glucokinase B1 and B2 isoforms were expressed in Escherichia coli as fusion proteins with glutathione S-transferase, affinity-purified B1 fusion protein was able to phosphorylate glucose whereas the B2 fusion protein was not, thus indicating that the lack of glucose phosphorylating activity from both the B2 and L2 isoforms is due to lack of intrinsic activity in addition to accumulation of less protein. The Km values of the B1 and L1 isoforms, which differ from each other by 15 amino acids at the NH2 terminus, were similar, but the Vmax of the B1 isoform was 2.8-fold higher than that of the L1 isoform. Mutagenesis of the first two potential initiation codons in the glucokinase B1 cDNA from ATG to GTC (methionine to valine) indicated that the first ATG was crucial for activity and is, therefore, the likely translation initiation codon. Messenger RNAs encoding both the B2 and L2 isoforms of glucokinase were detected in islet and liver by polymerase chain reaction amplification of total cDNA, indicating that mRNAs utilizing this weak alternate splice acceptor site in the fourth exon are normally present in both the liver and islet but as minor components. A regulatory role for weak alternate splice acceptor and donor sites in the glucokinase gene was suggested by examining the expression of the gene in the pituitary and in AtT-20 cells. Interestingly, although glucokinase mRNAs of appropriate sizes were detected in both the AtT-20 cells and rat pituitaries, neither exhibited any detectable high Km glucose phosphorylating activity.(ABSTRACT TRUNCATED AT 400 WORDS)
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22 MeSH Terms
Tissue-specific regulation of glucokinase gene expression.
Magnuson MA
(1992) J Cell Biochem 48: 115-21
MeSH Terms: Animals, DNA, Exons, Gene Expression Regulation, Enzymologic, Glucokinase, Organ Specificity, Promoter Regions, Genetic, RNA Splicing
Show Abstract · Added May 27, 2010
Glucokinase contributes to the maintenance of blood glucose homeostasis by catalyzing the high Km phosphorylation of glucose in the liver and the pancreatic beta cell, the only two tissues known to express this enzyme. Molecular biological studies of the glucokinase gene and its products have advanced our understanding of how this gene is differentially regulated in the liver and beta cell. The production of an active glucokinase isoform is determined by both transcriptional and post-transcriptional events. Two different promoter regions that are widely separated in a single glucokinase gene are used to produce glucokinase mRNAs in the liver, pancreatic beta cell, and pituitary. The different transcription control regions are tissue-specific in their expression and are differentially regulated. In liver, glucokinase gene expression is regulated by insulin and cAMP, whereas in the beta cell it is regulated by glucose. The upstream glucokinase promoter region, which gives rise to the glucokinase mRNA in pituitary and pancreas, is structurally and functionally different from the downstream promoter region, which gives rise to the glucokinase mRNA in liver. The use of distinct promoter regions in a single glucokinase gene enables a different set of transcription factors to be utilized in the liver and islet, thus allowing a functionally similar gene product to be regulated in a manner consistent with the different functions of these two tissues. In addition, the splicing of the glucokinase pre-mRNA is regulated in a tissue-specific manner and can affect the activity of the gene product.(ABSTRACT TRUNCATED AT 250 WORDS)
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8 MeSH Terms
Concordant glucose induction of glucokinase, glucose usage, and glucose-stimulated insulin release in pancreatic islets maintained in organ culture.
Liang Y, Najafi H, Smith RM, Zimmerman EC, Magnuson MA, Tal M, Matschinsky FM
(1992) Diabetes 41: 792-806
MeSH Terms: Animals, Cells, Cultured, DNA, Dose-Response Relationship, Drug, Enzyme Induction, Glucokinase, Glucose, Hexokinase, Insulin, Insulin Secretion, Islets of Langerhans, Kinetics, Male, Microscopy, Electron, Models, Biological, RNA, Messenger, Rats, Rats, Inbred Strains, Time Factors
Show Abstract · Added May 27, 2010
Using cultured islets as the experimental system, this study established dosage-response and time-dependency curves of the inductive glucose effect on glucose-stimulated insulin release, glucose usage, and glucokinase activity. Glucose-stimulated insulin release in islets cultured for 1, 2, or 7 days was increased as a function of glucose concentration in the culture medium and as a function of time. Glucose usage in the cultured islets showed a close relationship with glucose concentration in the culture medium at both 2 and 7 days of culture. Glucokinase activity increased in islets cultured for 1, 2, or 7 days as a function of increasing glucose concentrations in the culture medium and as a function of time. The V(max) of glucokinase in islets cultured for 7 days in medium containing 30 mM glucose was twice the value of freshly isolated islets and was almost fivefold higher than that in islets cultured for 7 days in 3 mM glucose. The glucose induction of glucose-stimulated insulin release, of glucose usage, and of glucokinase activity were tightly correlated. The biochemical mechanisms of glucose induction of islet glucokinase were further studied. Immunoblotting with an antibody against C-terminal peptide of glucokinase showed that densities of a 52,000-kD protein band from tissue extracts of islets cultured for 7 days in 3, 12, and 30 mM glucose were 25, 44, and 270% compared with that of extract from freshly isolated islets (100%). RNA blot analysis of glucokinase mRNA demonstrated virtually the same levels in fresh islets and islets after 7 days of culture in 3 or 30 mM glucose. The adaptive response of glucokinase to glucose appears therefore to be occurring at a translational or posttranslational site in cultured islets. These data greatly strengthen the concept that glucose is the regulator that induces the activity of glucokinase, which in turn determines the rate change of glucose usage as well as glucose-stimulated insulin release from beta-cells. Thus, the hypothesis that glucokinase is the glucose sensor of beta-cells is strengthened further.
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19 MeSH Terms
Heterogeneous expression of glucokinase among pancreatic beta cells.
Jetton TL, Magnuson MA
(1992) Proc Natl Acad Sci U S A 89: 2619-23
MeSH Terms: Amino Acid Sequence, Animals, Fluorescent Antibody Technique, Gene Expression, Glucokinase, Insulin, Islets of Langerhans, Male, Molecular Sequence Data, Monosaccharide Transport Proteins, RNA, Messenger, Rats, Rats, Inbred Strains
Show Abstract · Added May 27, 2010
The cellular location of glucokinase (GK), a key component of the glucose-sensing mechanism of the pancreatic islet, was determined using immunocytochemical techniques. In rat islets, GK immunoreactivity was detected only in beta cells with no immunoreactivity detected in alpha, delta, or pancreatic polypeptide-containing (PP) cells. However, within various beta cells, GK immunoreactivity varied considerably. Most beta cells displayed relatively low levels of cytoplasmic immunoreactivity whereas other beta cells stained intensely for this enzyme. Colocalization studies of GK and GLUT2, the high Km glucose transporter of beta cells, confirmed that these proteins are located in different subcellular domains of beta cells. The lack of GK immunoreactivity in glucagon- and somatostatin-secreting cells in islets suggests that these cells are not directly responsive to glucose or utilize a fundamentally different mechanism for sensing glucose fluctuations. Moreover, the differential expression of GK among pancreatic beta cells suggests that glucose phosphorylation is the probable enzymatic control point for the functional diversity of these cells.
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13 MeSH Terms
Multiple elements in the upstream glucokinase promoter contribute to transcription in insulinoma cells.
Shelton KD, Franklin AJ, Khoor A, Beechem J, Magnuson MA
(1992) Mol Cell Biol 12: 4578-89
MeSH Terms: 3T3 Cells, Animals, Base Sequence, Cloning, Molecular, Cricetinae, DNA, Gene Expression Regulation, Enzymologic, Glucokinase, Insulin, Insulinoma, Islets of Langerhans, Mice, Molecular Sequence Data, Mutagenesis, Organ Specificity, Promoter Regions, Genetic, Transcription Factors, Transcription, Genetic, Transfection, Tumor Cells, Cultured
Show Abstract · Added February 23, 2011
beta-cell type-specific expression of the upstream glucokinase promoter was studied by transfection of fusion genes and analysis of DNA-protein interactions. A construct containing 1,000 bp of 5'-flanking DNA was efficiently expressed in HIT M2.2.2 cells, a beta-cell-derived line that makes both insulin and glucokinase, but not in NIH 3T3 cells, a heterologous cell line. In a series of 5' deletion mutations between bases -1000 and -100 (relative to a base previously designated +1), efficient expression in HIT cells was maintained until -280 bp, after which transcription decreased in a stepwise manner. The sequences between -180 and -1 bp contributing to transcriptional activity in HIT cells were identified by studying 28 block transversion mutants that spanned this region in 10-bp steps. Two mutations reduced transcription 10-fold or more, while six reduced transcription between 3- and 10-fold. Three mutationally sensitive regions of this promoter were found to bind to a factor that was expressed preferentially in pancreatic islet beta cells. The binding sites, designated upstream promoter elements (UPEs), shared a consensus sequence of CAT(T/C)A(C/G). Methylation of adenine and guanine residues within this sequence prevented binding of the beta-cell factor, as did mutations at positions 2, 3, and 5. Analysis of nuclear extracts from different cell lines identified UPE-binding activity in HIT M2.2.2 and beta-TC-3 cells but not in AtT-20, NIH 3T3, or HeLa cells; the possibility of a greatly reduced amount in alpha-TC-6 cells could not be excluded. UV laser cross-linking experiments supported the beta-cell type expression of this factor and showed it to be approximately 50 kDa in size. Gel mobility shift competition experiments showed that this beta-cell factor is the same that binds to similar elements, termed CT boxes, in the insulin promoter. Thus, a role for these elements (UPEs or CT boxes), and the beta-cell factor that binds to them, in determining the expression of genes in the beta cells of pancreatic islets is suggested.
1 Communities
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20 MeSH Terms