Analysis of maple syrup urine disease in cell culture: use of substrates.
Branched-chain 2-oxo acid dehydrogenase activity in human skin fibroblasts against L-leucine, L-valine, L-isoleucine and derived 2-oxo acids was compared in incubations with 1 mmol/l of 1-14C-labelled substrate. The results suggested that the amino acids are the more suitable substrates for an estimation of decarboxylation activity in intact cells. In control cell lines (n = 12), 14CO2 release from amino acids was highest for valine and least for leucine. In a representative number of fibroblast strains of patients with different forms of MSUD (n = 11; residual decarboxylation activity 2-60% of the controls), 14CO2 release from the different amino acids was reduced to a similar degree. Additional measurement of 2-oxo[1-14C]acid release suggested that substrate supply to the branched-chain 2-oxo acid dehydrogenase complex was not rate limiting in the cell lines under investigation.
Schadewaldt
P
P
Diabetes-Forschungsinstitut, Universität Düsseldorf, FRG.
Beck
K
K
Wendel
U
U
D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D002478:Cells, Cultured; D005347:Fibroblasts / Q000201:enzymology*; D045782:Hemiterpenes; D006801:Humans; D007532:Isoleucine / Q000378:metabolism; D007651:Keto Acids / Q000378:metabolism; D007658:Ketone Oxidoreductases / Q000378:metabolism*; D007930:Leucine / Q000378:metabolism; D008375:Maple Syrup Urine Disease / Q000201:enzymology*; D009097:Multienzyme Complexes / Q000378:metabolism*; D013379:Substrate Specificity; D014633:Valine / Q000378:metabolism
D045782:Hemiterpenes; D007651:Keto Acids; D009097:Multienzyme Complexes; D007532:Isoleucine; C016211:alpha-keto-beta-methylvaleric acid; C001505:alpha-ketoisovalerate; C013082:alpha-ketoisocaproic acid; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D007930:Leucine; D014633:Valine