Biosynthesis of L-alanine, a major amino acid of fibroin in Samia cynthia ricini

The derivation of alanine in fibroin was investigated using NMR and selecti ve isotopic labelling. (H2O)-H-2 infused orally into 5th instar larvae was incorporated into the proton of the methyl group of alanine in fibroin. Pro ton exchange among alanine, glycine and serine was also found. Incorporatio n of C-13 from [2-C-13]acetate into alanine C2 and C3 and glycine C2 in fib roin, and also C4 of free glutamine plus glutamate was observed in vivo. He molymph contained a peak for C4 of glutamate plus glutamine, and an alanine C3 peak appeared transiently. Thus, it is suggested that the C-skeleton of alanine formed was derived from L-malate via the TCA-cycle, and that this alanine is utilized in part for fibroin synthesis. Spectra of the hemolymph extract of larvae infused orally with [N-15(2)]urea showed no 1(5N)-compou nds, whereas those of larvae injected subcutaneously showed only one peak o f urea, whose intensity decreased with time, as shown in the in vivo spectr a of a living larva infused with [N-15(2)]urea. The solution NMR spectrum o f fibroin showed no N-15-labelled compounds. Temporal changes in the peak i ntensities of six compounds in the spectra of a Living larva infused with [ N-15]ammonium demonstrated a process in which N-15 was incorporated into fi broin containing N-15-alanine through the amide group of glutamine and the amino group of glutamate. Thus, alanine biosynthesis from the. TCA-cycle or iginates mainly from water, L-malate and ammonium. The fact that no N-15-ur ea was detected in the hemolymph extract of larvae infused with [N-15]ammon ium suggests that N-15-urea found in the above in vivo spectra may be that accumulated in the hindgut. Thus, excess ammonium in the body causes the pr oduction of urea by the urea-cycle. In Samia larvae, urea was not reutilize d but excreted. The metabolic relationships between the assimilation of amm onium and the function of the urea-cycle are discussed. (C) 2000 Elsevier S cience Ltd. All rights reserved.