Further studies of L-arginine biosynthesis in germinating pea seeds. Evidence for the presence of argininosuccinate synthetase in cotyledon extracts.

Abstract

The synthesis and metabolism of arginine in germinating peas was examined by supplying micromolar quantities of L-citruiline-carbamyl-14C, DL-arginine-carbamyl-14C, and DL-arginine-5-14C to imbibing seeds. Citrulline was readily incorporated into arginine, but the labelled arginine solutions were not extensively metabolized.Extracts of 1-day-old cotyledons were found to catalyze the synthesis of arginine from citrulline in a reaction having absolute requirements for ATP, L-aspartate, and magnesium ions. The extracts were fractionated by (NH4)2SO4 precipitation followed by gel filtration on columns of Sephadex G-50 and G-200. These treatments increased the specific enzyme activity by approximately 36 times. After such treatments the preparations still contained appreciable amounts of argininosuccinate lyase (L-argininosuccinate arginine-lyase, EC 4.3.2.1) activity. The rate of arginine synthesis was altered by increasing the concentrations of L-citrulline, L-aspartate, and ATP. The latter compounds were found to be inhibitory at concentrations of 1 μmole/ml and 4 μmoles/ml, respectively. Arginine synthesis was markedly affected by pH and by additions of arginine and argininosuccinate. It is concluded that germinating pea cotyledons contain appreciable levels of argirrinosuccmate synthetase (L-citrulline:L-aspartate ligase (AMP), EC 6.3.4.5), and furthermore, that this enzyme has importance in arginine biosynthesis during germination.