Tissue culture provides a method for investigating inborn errors of metabolism at the cellular level and for studying the mechanisms by which enzymatic deficiencies give rise to abnormal phenotypes. Biochemical genetics has contributed important insights into the one cistron one polypeptide dictum. However genetic diseases affecting more than one enzyme or multienzyme complexes are poorly understood, because of difficulties in isolating and purifying these mitochondrial associated proteins and the need to develop sensitive assays for each component of the complex that can be reliably measured in normal and mutant cells. Our research emphasizes genetic diseases which affect multienzyme complexes or deficiency of sequential enzymes in a degradative pathway. Examples of the former are maple syrup urine disease (MSUD) and probably nonketotic hyperglycinemia and of the latter familial hyperlysinemia type I. Our approaches are (1) Enzymologic-including purification to homogeneity and characterization of the normal enzyme derived from bovine and human tissues. Kinetics, cofactor binding, and catalytic mechanisms are defined. An enzyme preparation from cultured normal human fibroblasts is compared to the highly purified enzyme. Similarity in kinetic parameters ensures the validity of our assays. An enzyme preparation from cultured mutant cells is then compared to that of normal cells and the defect in catalytic mechanism is characterized. Moreover, independent assays are developed for each component of the complex. Comparison of components of homogeneous enzyme with those in normal and mutant cells permits the localization and characterization of the enzyme defect. (2) Immunologic-purified enzyme and its components are used as antigen for preparation of specific polyclonal and monoclonal antibodies. Polyclonal antibody is used to measure enzyme levels, synthesis and stability in normal and mutant fibroblasts. Monoclonal antibodies recognize different antigenic sites of the enzyme and are used as probes for possible structural alterations in mutants. The availability of specific antisera to components of multienzyme complexes also will allow future studies of the mutation at the genetic level by recombinent DNA methods. (3) Complementation analysis by fusion of different mutant cells to form heterokaryons. Evidence for restoration of enzyme activity in heterokaryons suggests that the mutation affects different subunits of the multienzyme complex (interallelic) or different portions of the same subunit (intrallelic). When complementing pairs of mutant cells are detected, detailed enzymological characterization of the parent strains are carried out to define biochemical heterogeneity. Elucidation of structure-function relationships in multienzyme complexes by these studies provide insights for understanding the patients phenotype and perhaps for developing more effective therapy.
