Oxidation of leucine in human lymphocytes.
Appropriate conditions for analysis of leucine oxidation in peripheral human lymphocytes were determined. Lymphocytes, isolated by Ficoll centrifugation, were washed in phosphate-buffered saline without additions, with 5 mmol/l ADP or with 5 mmol/l ADP plus 25 mmol/l NaF for determination of transaminase activity, and total and basal activity of branched chain keto acid dehydrogenase (BCKA-D), respectively. Cells were incubated for 20-60 min with [1-14C]-leucine, or [1-14C]-alpha-ketoisocaproic acid (KIC) in the presence of various concentrations of the respective unlabelled substrates. Pyridoxal phosphate (0.1 mmol/l) augmented transaminase activity, coenzyme A, NAD and thiaminepyrophosphate (0.5 mmol/l each) enhanced BCKA-D activity. Apparent Km values for transamination and for BCKA-D were 40 and 17 microM, respectively. Total capacity for leucine transamination was about five times greater than for oxidation of KIC. The mean activity state of BCKA-D was 30%. Oxidation of KIC declined when 5.6 mmol/l glucose was added to the medium. It is concluded that: (1) BCKA-D is rate-limiting for leucine catabolism in peripheral human lymphocytes, (2) the BCKA-D complex is normally only partially active, and (3) flux of leucine through BCKA-D is inhibited by physiological glucose concentrations.
Schauder
P
P
Division of Gastroenterology and Metabolism, University of Göttingen, FRG.
Schäfer
G
G
D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D000328:Adult; D005260:Female; D006801:Humans; D007658:Ketone Oxidoreductases / Q000097:blood; D007930:Leucine / Q000097:blood*; D051276:Leucine Transaminase; D008214:Lymphocytes / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D009097:Multienzyme Complexes / Q000097:blood; D010084:Oxidation-Reduction; D000637:Transaminases / Q000097:blood
D009097:Multienzyme Complexes; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D000637:Transaminases; D051276:Leucine Transaminase; D007930:Leucine