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Expression and Characterization of the β 1 Isoform of Glucokinase Regulatory Protein (GKRP) from Homo sapiens β‐Islet Cells
Author(s) -
Carr Kevin Ricardo,
Taylor Brandon Christopher,
Shipman Lance Winston
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a955
Subject(s) - glucokinase , biochemistry , gene isoform , intein , biology , isozyme , chemistry , microbiology and biotechnology , enzyme , rna splicing , gene , rna
Glucokinase (GK) is a member of a superfamily of hexokinases that phosphorylates glucose to glucose‐6‐phosphate and is modulated by a regulatory protein (GKRP), with association of the inhibitory complex occurring in the presence of fructose‐6‐phosphate (F6P) and dissociation promoted in the presence of fructose‐1‐phosphate (F1P). Using complementary single‐stranded synthetic oligonucleotides to construct duplex DNA corresponding to the unique 3′‐terminus of β‐islet mRNA splicing variant, the chimeric gene was subcloned and overexpressed in E. coli yielding the putative β 1‐GKRP isoform produced in pancreatic β‐islet cells as described by Alvarez, et al. After purification of β 1‐GKRP by metal chelation affinity chromatography, the variant protein was tested for its ability to bind and inhibit human liver glucokinase. A qualitative differential binding assay was developed using a chitin binding domain/glucokinase (CBD/GK) fusion protein immobilized on a chitin column as bait against β 1‐GKRP as the mobile phase. Also, the inhibition of GK by B1‐GKRP in the presence of either F6P or F1P was observed and compared to that of rat liver GKRP by monitoring glucokinase activity as coupled to either pyruvate kinase/ lactate dehydrogenase or glucose‐6‐phosphate dehydrogenase activity. Andrew W. Mellon Faculty Career Enhancement