The objectives are: (1) to further characterize human 6-phosphofructokinase (PFK; EC. 2.7.1.11) isozymes in terms of their biochemical properties, cellular and subcellular localization, and the nature of the enzymatic lesions and their metabolic consequences in inherited PFK deficiency states; and (2) to undertake a detailed genetic dissection at the level of gene(s) localization, structure, organization, and mechanism(s) and regulation of expression with special reference to the mutant genes. These objectives will be pursued in the following specific studies: (1) Comparative structural and kinetic analyses of the three homotetramers; (2) Immunocytochemical localization of the three PFK subunits; (3) Elucidation of the biochemical and genetic bases of inherited PFK deficiency in patients with typical and atypical clinical syndromes; (4) Molecular cloning and characterization of the normal and mutant PFK genes; and (5) Elucidation of the control mechanisms involved in gene(s) expression in a number of pathophysiological states in humans; (6) Precise chromosomal localization of the PFK loci and investigation of the existence of complementation groups in glycogenosis type VII. These goals will be approached by: (1) Direct isolation and characterization of three homotetramers; (2) Immunocytochemical localization of the PFK subunits using subunit-specific antibodies; (3) Chromatographic, immunochemical and metabolic studies of the PFK-deficient humans; (4) Somatic cell and in situ hybridization methods; (5) Isolation of PFK-specific cDNAs and of natural and mutant PFK genes; and (6) Detailed structural and functional analyses of the cloned PFK genes in a number of pathophysiological states characterized by altered gene expression. These studies are expected to yield important clues as to the basic biochemical properties, genetic control mechanisms and physiological significance of PFK isozymes in human biology. These studies are expected to define the clinical syndromes, the nature of the genetic defects underlying them at the molecular level and their metabolic consequences.
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