Addition Of L-leucine Research Articles

L-Tryptophan-.ALPHA.-ketoisocaproate Aminotransferase from Pseudomonas sp.

l-Tryptophan-α-ketoisocaproate aminotransferase (tentative term) was purified from Pseudomonas sp., utilizing α-aminoisobutyrate. The purified enzyme was shown to be homogeneous by polyacrylamide gel disc electrophoresis and ultracentrifugation. The molecular weight of this enzyme was determined to be 90,000±5,000 by sedimentation equilibrium and 1l0,000±5,000 by Sephadex G-150 gel filtration. The holoenzyme exhibited absorption maxima at 330 nm and 415 nm. The addition of l-leucine shifted the 415 nm peak to 330 nm.This enzyme showed a very broad substrate specificity, that is, high activity toward the branched chain, straight chain and aromatic amino acids and the corresponding α-keto acids, but it was not active to l-glutamate and α-ketoglutarate. The apparent Michaelis constants were determined as follows: l-leucine 0.4 mm, l-isoleucine 0.5 mm, l-valine 4.5 mm, l-phenylalanine 0.8 mm, l-tryptophan 1.4 mm, l-tyrosine 1.0 mm, α-ketoisocaproate 0.03 mm, α-ketoisovalerate 0.11 mm and phenylpyruvate 0.08 mm.

Studies on amino acid inhibition of monosaccharide exit from anuran small intestinal epithelium.

1. The effect of the addition of amino acids to the intestinal lumen upon the movement of the monosaccharide alpha-methyl-D-glucopyranoside (alpha MG) from the preloaded epithelium into the blood and into the lumen of the vascularly perfused frog small intestine has been studied. 2. The neutral hydrophobic amino acids tryptophan, leucine, phenylalanine, tyrosine, isoleucine, valine, norleucine and cycloleucine all rapidly inhibit the exit of alpha MG out of the epithelium into the vascular bed. They stimulate backflux of the sugar from the epithelium into the lumen to a very much smaller extent. 3. L-Leucine is a more effective inhibitor of alpha MG exit into the blood than is D-leucine. Near-maximal inhibition of alpha MG exit is seen with 10 mM-L-leucine in the intestinal lumen. 4. The addition of leucine (10 mM) to the lumen of the intestine preloaded with alpha MG approximately halves the rate constant for alpha MG washout into the blood from 12.6 +/- 1.7 (4) x 10(-3) to 5.5 +/- 1.4 (4) x 10(-3) min-1, without appreciably altering the pool of monosaccharide in the tissue. The inhibitory effect of L-leucine upon alpha MG exit into the blood is not abolished by the presence of phlorizin (5 x 10(-5) M) in the intestinal lumen. 5. The complex pattern of inhibition of alpha MG transfer from the lumen to the blood observed upon the addition of L-leucine to the lumen is consistent with the finding that the amino acid inhibits the exit of the monosaccharide out of the epithelium into the blood in addition to any inhibitory effect upon sugar entry across the brush border. 6. It is suggested that alpha MG may be a substrate for a proposed transport system for neutral hydrophobic amino acids which, it is suggested, is present in the basolateral membrane of the epithelial cell.

The effects L-leucine on the synthesis of urea, glutamate and glutamine by isolated rat liver cells.

With either alanine or a mixture of 15 different amino acids as nitrogen source, the addition of L-leucine inhibited the synthesis of urea by isolated rat liver cells. With alanine present leucine promoted the production of glutamate and glutamine. Comparison of effects of leucine on soluble glutamate dehydrogenase, mitochondria and isolated cells supports the postulate that leucine exerts its effect through activation of glutamate dehydrogenase. It is suggested that this latter enzyme may not be as important for the production of NH3 for carbamoyl phosphate synthesis as has been considered hitherto.

THE AMINO ACID REQUIREMENTS FOR PYOCYANIN PRODUCTION

A medium consisting of acid-hydrolysed casein, glycerol, and a salt mixture has been shown to yield pyocyanin by Pseudomonas aeruginosa equal in amount to that obtained from glycerol peptone agar. The monoaminomonocarboxylic acids fraction obtained from casamino acids (Bacto) has been shown to be the source of nitrogen essential to pigment formation. Glycine, dl-alanine, dl-valine, or l-tyrosine have been shown to produce pyocyanin when employed as sole sources of nitrogen. The addition of l-leucine to media containing glycine or dl-alanine markedly increased pyocyanin formation. The addition of dl-phenylalanine to a synthetic medium inhibited pigment production. A synthetic medium consisting of dl-alanine or glycine at 0.4% concentration combined with 0.8% l-leucine, 1.0% glycerol, and a salt mixture has been shown to be the most suitable medium for pyocyanin production by five representative strains of Pseudomonas aeruginosa.