Analysis of the protein products encoded by variant glucokinase transcripts via expression in bacteria

Quaade, Christian; Hughes, Steven D.; Coats, Ward; Sestak, Andrea L.; Iynedjian, Patrick B.; Newgard, Christopher B.

doi:10.1016/0014-5793(91)80201-d

Cited by 15 publications

(10 citation statements)

References 21 publications

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“…of the rat liver GK was slightly greater than that of the human islet GK ( Table I). The Km values for GK expressed in the oocyte system are similar to those determined by in situ measurements of GK in hepatocytes and islets and to those obtained with recombinant GK produced by prokaryotic and eukaryotic expression (24,25). Our results differ from those reported previously in which the rat islet GK isoform had a greater V. than the rat hepatic GK isoform when expressed in either a eukaryotic or prokaryotic expression system (25).…”

Section: Methodssupporting

confidence: 74%

Coexpression of glucose transporters and glucokinase in Xenopus oocytes indicates that both glucose transport and phosphorylation determine glucose utilization.

Morita¹,

Yano²,

Niswender³

et al. 1994

J. Clin. Invest.

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A Xenopus oocyte expression system was used to examine how glucose transporters (GLUT 2 and GLUT 3) and glucokinase (GK) activity affect glucose utilization. Uninjected oocytes had low rates of both glucose transport and phosphorylation; expression of GLUT 2 or GLUT 3 increased glucose phosphorylation -20-fold by a low K., endogenous hexokinase at glucose concentrations -1 mM, but not at higher glucose concentrations. Coexpression of functional GK isoforms with GLUT 2 or 3 increased glucose utilization approximately an additional two-to threefold primarily at the physiologic glucose concentrations of 5-20 mM. The Km for glucose of both the hepatic and beta cell isoforms of GK, determined in situ, was -5-10 mM when coexpressed with either GLUT 2 or GLUT 3. The increase in glucose utilization by coexpression of GLUT 3 and GK was dependent upon glucose phosphorylation since two missense GK mutations linked with maturity-onset diabetes, 182:Val--Met and 228:Thr-+Met, did not increase glucose utilization despite accumulation of both a similar amount of immunoreactive GK protein and glucose inside the cell. Coexpression of a mutant GK and a normal GK isoform did not interfere with the function of the normal GK enzyme. Since the coexpression of GK and a glucose transporter in oocytes resembles conditions in the hepatocyte and pancreatic beta cell, these results indicate that increases in glucose utilization at glucose concentrations > 1 mM depend upon both a functional glucose transporter and GK. (J. Clin. Invest. 1994Invest. . 94:1373Invest. -1382

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Section: Methodssupporting

confidence: 74%

Coexpression of glucose transporters and glucokinase in Xenopus oocytes indicates that both glucose transport and phosphorylation determine glucose utilization.

Morita¹,

Yano²,

Niswender³

et al. 1994

J. Clin. Invest.

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“…However, this issue needs to be addressed directly. The higher efficiency ofbacterial expression ofliver glucokinase compared to the p-cell isoform has been reported by others (25) for the corresponding rat isoforms and suggests that the first 15 NH2-terminal amino acids affect stability and/or proper folding of glucokinase in E. coli cytosol.…”

Section: Resultsmentioning

confidence: 84%

Glucokinase mutations associated with non-insulin-dependent (type 2) diabetes mellitus have decreased enzymatic activity: implications for structure/function relationships.

Gidh‐Jain

Takeda

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

195

106

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The glycolytic enzyme glucokinase plays an important role in the regulation of insuln secretion and recent studies have shown that mutations in the human glucokinas gene are a common cause of an autosomal domint form of non-inssulin-dependent (type 2) diabetes meilitus (NIIDDM) that has an onset often during childhood. The majority of the mutations that have been identified are missense mutations that result in the syntheis of a glucokinas molecule with an altered amino acid sequence. To characterize the effect of these mutations on the catalytic properties of human 3-cell glucokinase, we have expressed native and mutant forms of this protein in Eschenchia coli. AU of the missense mutations show chnges in enzyme activity including a decrease in V.. and/or increase in K. for glucose. Using a model for the threedimensional structure of human glucokinas based on the crystal structure of the related enzyme yeast hexokinae B, the mutations map primarily to two regions of the protein. One group of mutations is lcated in the active site cleft separating the two domains of the enzyme as wefl as in surface loops leading into this cleft. These mutations usually result in large reductions in enzyme activity. The second group of mutations is located far from the active site in a region that is predicted to undergo a substrate-induced conformational change that results in closure of the active site cleft. These mutations show a smail -2-fold reduction in V., and a 5-to 10-fold increase in K. for glucose. The characterization of mutations in glucokinase that are asted with a distinct and readily recognizable form of NIDDM has led to the identification of key amino acids involved in glucokinas catalysis and locaized functionaUly important regions of the glucokinas molecule.

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“…3A); In the face of increased rates of glucose transport, even at low glucose, glucose phosphorylation likely becomes the rate-limiting step for generation of glucose-related signals. Although AtT-20ins cells express the mature islet glucokinase transcript (23), which when prepared as cDNA and expressed in bacteria clearly encodes glucokinase activity (38), their predominant glucose phosphorylating enzyme is hexokinase [Km for glucose of ='10 ILM (39)]. Support for the notion that hexokinase can exert a dominant effect is provided by a recent study showing that overexpression of hexokinase I in normal islets results in a shift in the glucose dose-response curve such that the transfected cells are maximally responsive to glucose at subphysiological levels (40).…”

Section: Discussionmentioning

confidence: 99%

Engineering of glucose-stimulated insulin secretion and biosynthesis in non-islet cells.

Hughes

Johnson

Quaade

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

118

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The high-capacity glucose transporter known as GLUT-2 and the glucose phosphorylating enzyme glucokinase are thought to be key components of the "glucose-sensing apparatus" that regulates insulin release from the 13 cells of the islets of Langerhans in response to changes in external glucose concentration. AtT-20ins cells are derived from anterior pituitary cells and are like 13 cells in that they express glucokinase and have been engineered to secrete correctly processed insulin in response to analogs of cAMP, but, unlike 13 cells, they fail to respond to glucose and lack GLUT-2 expression. Herein we demonstrate that stable transfection of AtT-20ins cells with the GLUT-2 cDNA confers glucose-stimulated insulin secretion and glucose regulation of insulin biosynthesis and also results in glucose potentiation of the secretory response to non-glucose secretagogues. This work represents a first step toward creation of a genetically engineered "artificial 13 cell."The pancreatic islets of Langerhans secrete glucoregulatory hormones in response to changes in circulating levels of key metabolic fuels. In the case of glucose, transport into the 1 cell and metabolism of this sugar are absolute requirements for secretion, leading to the hypothesis that its specific stimulatory effect is mediated by and proportional to its flux rate through glycolysis and related pathways (1-6). In addition to its acute effects on the release of prestored insulin, glucose stimulates de novo insulin biosynthesis by increasing insulin gene transcription, stabilizing insulin mRNA, enhancing translation of the insulin transcript into protein, and stimulating the rate of conversion of proinsulin to insulin (7)(8)(9)(10)(11).A substantial body of evidence has accumulated implicating a specific facilitated-diffusion-type glucose transporter known as GLUT-2 and the glucose phosphorylating enzyme glucokinase in the control of glucose metabolism in islet pB cells. Both proteins are members of gene families; GLUT-2 is unique among the five-member family of glucose transporter proteins (GLUTs 1-5; see refs. 12 and 13 for review)

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Analysis of the protein products encoded by variant glucokinase transcripts via expression in bacteria

Cited by 15 publications

References 21 publications

Coexpression of glucose transporters and glucokinase in Xenopus oocytes indicates that both glucose transport and phosphorylation determine glucose utilization.

Coexpression of glucose transporters and glucokinase in Xenopus oocytes indicates that both glucose transport and phosphorylation determine glucose utilization.

Glucokinase mutations associated with non-insulin-dependent (type 2) diabetes mellitus have decreased enzymatic activity: implications for structure/function relationships.

Engineering of glucose-stimulated insulin secretion and biosynthesis in non-islet cells.

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