Mutations in the glucokinase (GK) gene cause type-2 maturity-onset diabetes of the young type 2 (MODY-2) and GK-linked hyperinsulinaemia (GK-HI). Recombinant adenoviruses expressing the human wild-type islet GK or one of four mutant forms of GK, (the MODY-2 mutants E70K, E300K and V203A and the GK-HI mutant V455M) were transduced into glucose-responsive insulin-secreting β-HC9 cells and tested functionally in order to initiate the first analysis in vivoof recombinant wild-type and mutant human islet GK. Kinetic analysis of wild-type human GK showed that the glucose S0.5 and Hill coefficient were similar to previously published data in vitro (S0.5 is the glucose level at the half-maximal rate). E70K had half the glucose affinity of wild-type, but similar enzyme activity. V203A demonstrated decreased catalytic activity and an 8-fold increase in glucose S0.5 when compared with wild-type human islet GK. E300K had a glucose S0.5 similar to wild-type but a 10-fold reduction in enzyme activity. E300K mRNA levels were comparable with wild-type GK mRNA levels, but Western-blot analyses demonstrated markedly reduced levels of immunologically detectable protein, consistent with an instability mutation. V455M was just as active as wild-type GK, but with a markedly reduced S0.5. The effects of the different GK mutants on glucose-stimulated insulin release support the kinetic and expression data. These experiments show the utility of a combined genetic, biochemical and cell-biological approach to the quantification of functional and structural changes of human GK that result from MODY-2 and GK-HI mutations.
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September 1999
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Research Article|
August 24 1999
Cell-biological assessment of human glucokinase mutants causing maturity-onset diabetes of the young type 2 (MODY-2) or glucokinase-linked hyperinsulinaemia (GK-HI)
Charlotte V. BURKE;
Charlotte V. BURKE
*Harrison Department of Surgical Research, University of Pennsylvania Medical Center, 313 Stemmler Hall, 36th and Hamilton Walk, Philadelphia, PA 19104, U.S.A.
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Carol W. BUETTGER;
Carol W. BUETTGER
†Department of Biochemistry and Biophysics and the Diabetes Research Center, University of Pennsylvania Medical Center, 501 Stemmler Hall, 36th and Hamilton Walk, Philadelphia, PA 19104, U.S.A.
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Elizabeth A. DAVIS;
Elizabeth A. DAVIS
†Department of Biochemistry and Biophysics and the Diabetes Research Center, University of Pennsylvania Medical Center, 501 Stemmler Hall, 36th and Hamilton Walk, Philadelphia, PA 19104, U.S.A.
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Steven J. MCCLANE;
Steven J. MCCLANE
*Harrison Department of Surgical Research, University of Pennsylvania Medical Center, 313 Stemmler Hall, 36th and Hamilton Walk, Philadelphia, PA 19104, U.S.A.
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Franz M. MATSCHINSKY;
Franz M. MATSCHINSKY
†Department of Biochemistry and Biophysics and the Diabetes Research Center, University of Pennsylvania Medical Center, 501 Stemmler Hall, 36th and Hamilton Walk, Philadelphia, PA 19104, U.S.A.
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Steven E. RAPER
Steven E. RAPER
1
*Harrison Department of Surgical Research, University of Pennsylvania Medical Center, 313 Stemmler Hall, 36th and Hamilton Walk, Philadelphia, PA 19104, U.S.A.
‡Institute for Human Gene Therapy, University of Pennsylvania Medical Center, 607 BRB II/III, 421 Curie Boulevard, Philadelphia, PA 19104, U.S.A.
1To whom correspondence should be sent, at the Institute for Human Gene Therapy address (e-mail seraper!mail.med.upenn.edu).
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Publisher: Portland Press Ltd
Received:
January 21 1999
Revision Received:
May 06 1999
Accepted:
June 14 1999
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1999
1999
Biochem J (1999) 342 (2): 345–352.
Article history
Received:
January 21 1999
Revision Received:
May 06 1999
Accepted:
June 14 1999
Citation
Charlotte V. BURKE, Carol W. BUETTGER, Elizabeth A. DAVIS, Steven J. MCCLANE, Franz M. MATSCHINSKY, Steven E. RAPER; Cell-biological assessment of human glucokinase mutants causing maturity-onset diabetes of the young type 2 (MODY-2) or glucokinase-linked hyperinsulinaemia (GK-HI). Biochem J 1 September 1999; 342 (2): 345–352. doi: https://doi.org/10.1042/bj3420345
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