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1.  Molybdenum cofactor biosynthesis in humans. Identification of two complementation groups of cofactor-deficient patients and preliminary characterization of a diffusible molybdopterin precursor. 
Journal of Clinical Investigation  1989;83(3):897-903.
Molybdenum cofactor deficiency is a devastating disease with affected patients displaying the symptoms of a combined deficiency of sulfite oxidase and xanthine dehydrogenase. Because of the extreme lability of the isolated, functional molybdenum cofactor, direct cofactor replacement therapy is not feasible, and a search for stable biosynthetic intermediates was undertaken. From studies of cocultured fibroblasts from affected individuals, two complementation groups were identified. Coculture of group A and group B cells, without heterokaryon formation, led to the appearance of active sulfite oxidase. Use of conditioned media indicated that a relatively stable, diffusible precursor produced by group B cells could be used to repair sulfite oxidase in group A recipient cells. Although the extremely low levels of precursor produced by group B cells preclude its direct characterization, studies with a heterologous, in vitro reconstitution system suggest that the precursor that accumulates in group B cells is the same as a molybdopterin precursor identified in the Neurospora crassa molybdopterin mutant nit-1, and that a converting enzyme is present in group A cells which catalyzes an activation reaction analogous to that of a converting enzyme identified in the Escherichia coli molybdopterin mutant ChlA1.
PMCID: PMC303764  PMID: 2522104
2.  Type II hyperprolinemia. Delta1-pyrroline-5-carboxylic acid dehydrogenase deficiency in cultured skin fibroblasts and circulating lymphocytes. 
Journal of Clinical Investigation  1976;58(3):598-603.
Type II hyperprolinemia is an inherited abnormality in amino acid metabolism characterized by elevated plasma proline concentrations, iminoglycinuria, and the urinary excretion of delta1-pyrroline compounds. To define the enzymologic defect of this biochemical disorder, we developed a specific, sensitive radioisotopic assay for the proline degradative enzyme delta1-pyrroline-5-carboxylic acid dehydrogenase. Using this assay, we have shown an absence of delta1-pyrroline-5-carboxylic acid dehydrogenase activity in the cultured fibroblasts from three patients with type II hyperprolinemia. We confirmed this result on cultured cells by demonstrating a similar absence of delta1-pyrroline-5-carboxylic acid dehydrogenase activity in extracts prepared from the peripheral leukocytes of these patients. Additionally, we found significantly decreased levels of delta1-pyrroline-5-carboxylic acid dehydrogenase activity in the leukocyte extracts from five obligate heterozygotes for type II hyperprolinemia. We also demonstrated a reduction in leukocyte delta1-pyrroline-5-carboxylic acid dehydrogenase activity in three successive generations of a family. These results prove that an absence of delta1-pyrroline-5-carboxylic acid dehydrogenase is the enzymologic defect in type II hyperprolinemia and that this defect is inherited in an autosomal recessive fashion.
PMCID: PMC333218  PMID: 956388
3.  Metabolism of [1-(14)C] and [2-(14)C] leucine in cultured skin fibroblasts from patients with isovaleric acidemia. Characterization of metabolic defects. 
Journal of Clinical Investigation  1976;58(1):164-172.
Leucine metabolism in cultured skin fibroblasts from patients with isovaleric acidemia was compared with that in normal fibroblasts and in cells from patients with maple syrup urine disease using [1-(14)C] and [2-(14)C] leucine as substrates. Inhibitory effects of methylenecyclopropylacetic acid on leucine metabolism in normal cells were also investigated. Production of 14CO2 from [2-(14)C] leucine was very reduced (96-99%) in both types of mutant cells. Radioactive isovaleric acid accumulated in assay media with isovaleric acidemia cells but not in those with maple syrup urine disease cells. Unexpectedly, 14CO2 production from [1-(14)C] leucine was partially depressed (80%) in isovaleric acidemia cells whereas in maple syrup urine disease cells it was strongly depressed (99%) as expected. These two mutant cells were clearly distinguished by detection of 14C-isovaleric acid accumulation after incubation with [2-(14)C] leucine. A pattern of inhibition of leucine oxidation similar to that seen in isovaleric acidemia cells was induced in normal cells by the addition of 0.7 mM methylenecyclopropylacetic acid to the assay medium. The partial inhibition of [1-(14)C] leucine oxidation seen in isovaleric acidemia cells and also in normal cells in the presence of the inhibitor appears to be, at least in part, due to an accumulation of isovalerate in the cells. Isovaleric acid (5-10) mM) inhibited [1-(14)C] leucine oxidation 32-68% when added to the assay medium with normal cells. Addition of flavin adenine dinucleoside to culture medium or assay medium or both did not restore oxidation of either leucine substrate in isovaleric acidemia cells.
PMCID: PMC333167  PMID: 932204

Results 1-3 (3)