Uptake of glucose in mammalian cells occurs by facilitated diffusion by glucose transporters. Conversion of glucose to glucose-6-phosphate (G-6-P) catalyzed by hexokinases plays a critical role by maintaining downhill gradient necessary for glucose entry into cells. The major focus of the proposal is on hexoskinase II (HKII) which, like HKI and HKIII is a 100 kD isoform that is inhibited by physiologic concentrations of G-6-P. HKII is selectively found in adipose tissue, skeletal muscle and heart, tissues in which insulin stimulates glucose uptake and utilization. Studies have shown that insulin increase HKII activity in these tissues. The recent cloning of the HKII cDNA and gene make it possible to study structure and regulation of the gene. The ratio of glucose transporters and HKs in a cell can be manipulated so that relative contributions of each glucose uptake and metabolism can be studied.
Aim 1 will establish whether glucagon (cAMP), glucocorticoids and insulin modulate HKII gene transcription. If so, the respective hormone-response elements will be mapped. Transgenic mice will be used to determine the promoter elements responsible for the unique tissue distribution of HKII.
Aim 2 will use specific mutations to locate the glucose, ATP and G-6-P binding domains. The HKII, the N- and C-halves will be expressed in E. coli and compared for function with HKI and gulucokinae (GK). Whether catalytic and regulatory domains co-exist in each half of HKII or separate halves will be determined. The ultimate goal is to provide a crystallographic analysis.
Aim 3 is to define the kinetics of expressed HKs, and their interactions with selected transporter isoforms in frog oocytes. This approach will determine how HKs control metabolic flux and whether compartmentation is involved in the process.
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