Partial purification and determination of some properties of an aminotransferase of pea (Pisum sativum L.) plants Public Deposited


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  • A transaminase (aminotransferase, EC 2. 6. 1) was extracted and purified approximately 148-fold from shoot tips of pea (Pisum sativum L. cv. Alaska) seedlings and some properties of the enzyme were determined. The purification included acetone precipitation followed by column chromatography on hydroxylapatite. With a-ketoglutarate as co-substrate, the acetone precipitated preparation of the enzyme transaminated the following aromatic amino acids: D, L-tryptophan, D, L-tyrosine and D, L-phenylalanine; as well as the following aliphatic amino acids: D, L-alanine, D, L-methionine and D, L-leucine. Of other keto acids tested, pyruvate and oxalacetate were more active than a-ketoglutarate, with D, L-tryptophan as amino acid. The pH optimum for the pea transaminase was 8.5. A partial dependence of the transaminase on pyridoxal phosphate was demonstrated, as approximately a 50% decrease in the activity of the acetone-precipitated preparation was observed when exogenous pyridoxal phosphate was omitted from reaction mixtures. The addition of pyridoxal phosphate inhibitors, semicarbazide or hydroxylamine, to the reaction mixtures further reduced the transaminase activity, providing additional evidence that pyridoxal phosphate is essential for the transaminase. Lineweaver-Burk double-reciprocal plots of the data from competition experiments suggested that D, L-tryptophan and D, L-tyrosine are noncompetitive substrates. These data suggested that more than one transaminase was present in the enzyme preparation from peas or that a single transaminase with more than one active site was present. Transaminase activity was routinely measured by assaying the amount of aromatic a-keto acid present in the reaction mixture after incubation. In addition, glutamate was identified as a product of the enzyme reaction by thin-layer chromatography. The presence of the aromatic a-keto acid and glutamate confirmed that the activity being assayed was that of an authentic transaminase. Further quantitative studies showed that equimolar amounts of indolepyruvic acid and glutamate were formed, which corroborated the conclusion that an authentic transaminase was being studied. The pea transaminase was found to be approximately three times as active with D-tryptophan as amino acid substrate than with L-tryptophan as substrate. It was speculated that the enzymic racemization of L-tryptophan to D-tryptophan prior to transamination might play a role in the control of free levels of tryptophan in pea seedlings. The evidence presented here indicated that an active transaminase is present in peas which catalyzes the conversion of tryptophan to indolepyruvic acid. Thus, the evidence presented here supports the conclusion that in cell-free extracts of shoot tips of green pea (Pisum sativum L.) seedlings, indolepyruvic acid is a potential intermediate in the biogenesis of the auxin-type hormone indoleacetic acid from tryptophan.
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