Shigeo Nishi

Human hexokinase: sequences of amino- and carboxyl-terminal halves are homologous.

cDNA clones encoding human hexokinase have been isolated from an adult kidney library. Analysis of this 917 amino acid protein (Mr = 102,519) indicates that the sequences of the NH2- and COOH-terminal halves, corresponding to the regulatory and catalytic domains, respectively, are homologous; and that eukaryotic hexokinases evolved by duplication of a gene encoding a protein of 450 amino acids. The COOH-terminal half of the protein created by this gene duplication retained the glucose binding site and glucose phosphorylating activity while the substrate binding sites of the NH2-terminal half evolved into a new allosteric effector site.

Human pancreatic beta-cell glucokinase:cDNA sequence and localization of the polymorphic gene to chromosome 7, band p 13

SummaryThe glucose phosphorylating enzyme glucokinase plays an important role in the regulation of glucose homeostasis. Studies in rodents indicate that pancreatic Beta cells and hepatocytes express different isoforms of this protein as a consequence of the presence of tissue-specific promoters and exon 1 sequences which are spliced to a shared group of nine exons which encode most of the mRNA and protein. Here, we report the isolation and characterization of cDNA clones encoding human Beta-cell glucokinase. The sequence of human Beta-cell glucokinase shows 97% amino acid identity with that of the cognate rat protein. We also mapped the human glucokinase gene to the short arm of chromosome 7 by analysing its segregation in a panel of reduced human mouse somatic cell hybrids. In situ hybridization to metaphase chromosomes confirmed the localization of the human glucokinase gene to chromosome 7 and indicated that it was in band p 13. A microsatellite DNA polymorphism that can be typed using the polymerase chain reaction was identified upstream of exon 1 a, the Beta-cell specific first exon. The glucokinase cDNA clone and highly informative DNA polymorphism will be useful for examining the role of this gene in the pathogenesis of diabetes mellitus.

Mutations of the human glucokinase gene and diabetes mellitus.

The enzyme glucokinase catalyzes the phosphorylation of glucose and plays a key role in the regulation o f insulin secretion by pancreatic beta cells and glucose disposal in hepatocytes. Recent studies have shown that mutations in the gene encoding this key regulatory enzyme of glycolysis are a common cause of an autosomal dominant form of non-insulin-dependent (type 2) diabetes mellitus that has an onset often during childhood. The association of mutations in the glucokinase gene with impaired pancreatic cell function underscores the importance of glycolysis in the regulation of insulin secretion and suggests that mutations in other genes expressed in the beta-cell that also control rate-limiting steps in glucose metabolism may lead to diabetes.

Mutations in the glucokinase gene are not a major cause of late-onset type 2 (non-insulin-dependent) diabetes mellitus in Japanese subjects

The role of the glucokinase gene in the development of diabetes in a group of 349 Japanese subjects with late‐onset Type 2 diabetes was examined. These diabetic subjects and 197 non‐diabetic controls were typed at two simple tandem repeat DNA polymorphisms in the glucokinase gene termed GCK2 and GCK3. Six and five alleles were evident in Japanese subjects at GCK2 and GCK3, respectively. There were no significant differences in allele, genotype or haplotype frequencies between diabetic and normal groups. In addition, the glucokinase gene of 340 diabetic and 170 non‐diabetic Japanese subjects was screened for mutations using single strand conformation polymorphism analysis. Four nucleotide substitutions were identified: a silent substitution in exon 4 in the codon for proline 145 (CCC→CCG), and A→T, C→G, and C→A substitutions in introns 1b, 3, and 5, respectively. There were no significant differences in the frequencies of these nucleotide substitutions between diabetic and non‐diabetic groups. These results suggest that glucokinase gene defects are not a major cause of late‐onset Type 2 diabetes in Japanese subjects.