Abstract:
Attempts were made to form indoleacetic acid in cellfree extracts of mung bean (Vigna radiata) shoots. The extracts were incubated with radiolabelled tryptophan and other substrates and cofactors thought to be involved in indoleacetic acid biosynthesis. After incubation indolepyruvate and indoleacetic acid were separated and quantified by HPLC. There was no significant difference in the conversion of tryptophan to indolepyruvate and indoleacetic acid between the incubations and control incubations using boiled extract. The concentrations of indolepyruvate and indoleacetic acid in mung bean hypocotyl suspension cultures were measured using GC-MS SIM over the growth of the culture, a period of 29 days. Indoleacetic acid concentrations, although scattered, mostly remained at constant low levels in the range of 6 to 9ng/g fwt of culture. The indolepyruvate levels steadily increased to a maximum level after 14 days, then remained at this level, 10 to 12 ng/g fwt, for the remainder of the culture period. This plateau in indolepyruvate concentration matched the period that the suspension culture was in the logarithmic phase of growth. An aromatic amino acid aminotransferase was purified over 33,000 fold from the shoots and primary leaves of mung beans, as determined using a tryptophan aminotransferase activity assay. The enzyme was a monomer, with a molecular weight of about 58kDa. The pH optimum was broad, with a maximum at about 8.6. The relative activities of the aromatic amino acids were: tryptophan 100, tyrosine 83 and phenylalanine 75, and the Kms were 0.095, 0.08 and 0.07mM respectively. The enzyme was able to use 2-oxoglutarate, oxaloacetate and pyruvate as the oxo acid substrate at relative activities 100, 128 and 116 and Kms 0.65, 0.25 and 0.24mM respectively In addition to the aromatic amino acids the enzyme was able to transaminate alanine, arginine, leucine and lysine to a lesser extent, and showed slight activity with asparagine, aspartate, histidine, valine and D-tryptophan and tyrosine. Inhibition studies showed that the alanine, aspartate and histidine activities were part of the aromatic amino acid aminotransferase activity. The enzyme was not inhibited by indoleacetic acid, although naphthaleneacetic acid did inhibit slightly. There was evidence of substrate inhibition by hydroxyphenylpyruvate at high concentrations. Addition of the cofactor pyridoxal phosphate only slightly increased the activity of the enzyme. The enzyme was blotted onto a PVDF membrane cleaved by in situ trypsin digest. Three of the tryptic fragments were sequenced. These fragments had approximately 60% sequence similarity with plant aspartate aminotransferases and tyrosine aminotransferases.