Influence of vitamin D metabolites on proteoglycan synthesis by chick growth cartilage in organ culture

butunoate. glutamate, leucine, isoleucine, phenylalanine. threonine and valine; the organic acids glyoxylate, glycollate, oxalate, succinate, malonate, malate, citrate and glycerate; the fatty acids palmitate and stearate; the sterols campesterol, stigmasterol and sitosterol; the nitrogenous compounds urea, cytosine and uridine; and the poly-hydroxy compounds glycerol, a-glycerophosphatc. /I-glycerophosphate, 4-deoxy-arabinitol and quinate. "C-labelling was however detected in compounds whose biosynthesis includes either the incorporation o f glycine or the methyl group from NS. '" methylene-THF into the molecule. An increase in mass by 1 observed in adenine derived from cells incubated with [ 1 -"C]glycine, is consistent with the known mechanism for purine synthesis that involves the incorporation of the whole molecule of glycine ([ 1 -"C]glycine) into 5'-phosphoribosyl-glycineamide and the subsequent addition of two carbons from N',"'-methylene-THF. An increase in mass by 3 was observed in adenine derived from cells incubated on [2-"C]~lycine. This is consistent with the incorporation of [23C]glycine into 5-phosphoribosyl-glycineamide and the utilization of N'."'-methylene-THF formed from [2-'3C]glycine. to form 5-phosphoribosyl-N-formylglycineamide and 5-phosphoribosyl-5-formamido-4-imidazole carboxamide during adenine biosynthesis. The "C-label was also detected in methionine from cells incubated with [2-'3C]glycine, and the methionine mass spectrum indicated that the label was in the methyl group derived from N'."'-methylene-THF. I3C-labelling was also detected in each of three unknown compounds A, B and C (Table I ) . The labelling pattern of A was similar to that of glycine, of B to serine, and of C to ethanolamine. A, B and C were only detected when excess glycine was added (producing excess serine and ethanolamine) and probably result from a detoxification reaction involving the substitution of a group of mass 45 for a hydrogen of the amino group. These experiments indicated that the conversion of [2-I3C]glycine to [2,3-"C]serine occurred in carrot cells as demonstrated previously by n.m.r. in tobacco cells. GC-MS techniques were in addition able to show the exchange of '3C-labelled glycine and serine from the 'free pool' into proteins, and that the "C-labelled glycine and the N'.'"-methylene-THF formed from it were utilized in biosynthetic reactions.