Studies on the Presence and Role of Tryptophan in Pig Heart Mitochondrial Malate Dehydrogenase

• Published in: The Journal of biological chemistry 1968-12, Vol.243 (23), p.6284-6290
• Main Authors: Chan, T L, Schellenberg, K A
• Format: Article
• Language: English
• Subjects: Alcohol Oxidoreductases; Animals; Bromine; Chromatography, Gas; Chromatography, Ion Exchange; Chromatography, Paper; Fluorescence; Indoles; L-Lactate Dehydrogenase; Malate Dehydrogenase; Mitochondria, Muscle - enzymology; Models, Chemical; Myocardium - enzymology; Nitrobenzenes; Spectrum Analysis; Swine; Tritium; Tryptophan; Ultraviolet Rays
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)94489-3

Several different experimental approaches have shown the presence of tryptophan in malate dehydrogenase from pig heart mitochondria. The methods used were ( a ) fluorescence measurement of tryptophan content after acid hydrolysis and separation of amino acids by paper chromatography, ( b ) recovery of tryptophan labeled with tritium after incubation of the enzyme with tritiated substrates, ( c ) determination of tryptophan content of acid and alkaline hydrolysates with the amino acid analyzer and (after silylation) by gas chromatography, ( d ) the ultraviolet spectral method of Edelhoch ( Biochemistry , 6, 1948 (1967)), and ( e ) determination of tryptophan by reaction with 2-hydroxy-5-nitrobenzyl bromide. The estimation of tryptophan by fluorescence, tritium-labeling, the amino acid analyzer, or gas chromatography of acid or alkaline hydrolysates usually indicated 0.3 to 1.1 residues per molecule of enzyme, whereas the two nonhydrolytic methods (ultraviolet absorption and reaction with 2-hydroxy-5-nitrobenzyl bromide) indicated about 2 tryptophanyl residues per molecule of enzyme. The protein acquires about 0.5 atom of tritium per molecule of enzyme from labeled substrate under conditions similar to the previously reported labeling of yeast alcohol dehydrogenase and rabbit muscle lactate dehydrogenase. The positive results in three different DPN-linked enzymes suggest that in some members, at least, of this class of enzymes dehydrogenation of an enzyme tryptophanyl residue takes place through the intermediacy of an indolenine or equivalent structure. The structure of the adduct of 2-hydroxy-5-nitrobenzyl bromide with 3-methylindole was determined as a model for the primary adduct of the reagent with protein tryptophanyl residues.