The Journal of Biochemistry, Vol. 52, No, 4, 1962• Studies on the Biosynthesis of Glycine in the Silkworm II. Conversion of Glyoxylic Acid to Glycine in the Intact Silkworm* By MICIIIKO MURAMATSU and KENSUKE SHIMURA (From the Laboratory of Biochemistry, Department of Agricultural. Chemistry, Tohoku University, Sendai) (Received for publication, June 11, 1962) In the previous paper**, it was demon- Silkworm-Silkworms (Bombvx mori L., a hybrid,. strated that serine was one of the important "Shi 122•~Nichi 122") at the beginning of the fifth precursors of glycine in the silkworm, Bombyx instar stage were kindly supplied from the Experi- mori (1). As another possible precursor of mental Sericultural Station of Miyagi Prefecture. Labeled Materials-Glyoxylic acid-I,2-C14 was pre glycine in the silkworm, glyoxylic acid seems pared from oxalate-C14 by the method of K r u p k a. to deserve a close investigation. et al, (6). Oxalate-C14, glycine-1-C14 and glycine-2- Previously F u k u d a et al. reported that C14 were purchased from the Radiochemical Centre, a relatively high level of glyoxylic acid was Amersham, England. Their specific activities were contained in the body fluid of the silkworm 3.3 x 105, 1.6 x 105 and 1.8 X 105 c.p.m. per ƒÊmole, res (2). K o i d e et al. (3, 4) and Fukuda et al. pectively. Experimental Procedure-Silkworms (10 worms in a. (5) found a transamination reaction between group) at the fifth instar stage were given injections- glyoxylic acid and alanine with the tissue ex- of C14 materials into the body cavity and immediate- tracts of silkworms and showed that the re- ly placed in a ventilating chamber. The expired action was much favoured for the glycine CO, was collected in hourly portions by absorption. formation. These facts suggest that glyoxy in NaOH and precipitated as BaCO3. After stated lic acid in the body fluid of the silkworm intervals, body fluid was collected in a test tube chil may be a possible precursor of glycine which led in ice and deproteinized by the addition of HCIO4 is a main component amino acid of silk fib to make a final concentration of 7.5 per cent. Silk-- roin. It has been uncertain, however, whether glands were quickly removed from the bleeded silk- the silkworm can actually utilize glyoxylic worms in the cold 0.15 M KCI solution, weighed, acid in the body fluid for the formation of homogenized with 3 volumes of water in a Waring- blendor and added with HCIO4 to make the final, glycine, in other words, the direction of concentration of 7.5 per cent. The protein precipitated. the reaction system participating in the was washed several times with 7.5 % HCIO4, then. transformation of glyoxylic acid into glycine with ethanol, ethanol-ether and ether. The radio- in vivo is still in doubt. activity of the protein was determined as described- In the present work, in order to clarify below. a role of glyoxylic acid in the biosynthesis of Isolation of Glyoxylic Acid in the Body Fluid-To- glycine in the silkworm, C14-labeled glyoxylic the deproteinized supernatant of the body fluid 0.5 acid and glycine were injected into silkworms 2,4-dinitrophenylhydrazine solution in 2NHC1 was added and after standing overnight at room tem and the extent of the interconversion of these perature the hydrazones of keto acids were separated compounds was investigated in vivo. according to the procedure of C a v a 11 i n i et al. (7). EXPERIMENTALS For paper chromatography, buthanol saturated with. 3 % NH4OH solution was used as the solvent system. * A part of this study was presented at the The bands of glyoxylic acid hydrazone corresponding Meeting of Agricultural Chemical Society of Japan, to the authentic sample were eluted with water from Tokyo, April 8, 1959. the paper, dried in vacuo and their specific activities-. ** Muramatsu, M., Nagayama,, H., arid Shimura, were determined. K., This journal, 49, 55 (1961) Isolation of Amino Acids-After extracting the by- 297 298 M. MURAMATSU and K. SHIMURA. .drazones of keto acids with ether, the , aqueous solu jection. This reduction in the specific activity tion was neutralized with 30 °% KOH and the resulting in the initial stage may be explained by a -potassium perchlorate was removed by centrifugation. dilution effect with the pooled glyoxylic acid, Amino acids in the supernatant were analyzed by which was estimated to be contained in an the same procedures as described in the previous amount of 5 to 10 ƒÊoles per worm (2). report (1). When glycine-l-C14 or glycine-2-C14 was Assay of Radioactivity-The radioactivity of the injected, little conversion of glycine into gly -sample was measured with a thin mica end-window oxylic acid was observed over a period of 10 -Geiger-Muller counter and corrected for the back- minutes to 18 hours after injection. Almost ground and self-absorption. the same results were obtained from three different experiments, therefore this reverse RESULTS reaction seems to occur to very small, if any, Radioactivity of Glyoxylic Acid in the Body extent in the silkworm. Fluid-Changes in the specific activity of gly Distribution of Radioactivity in the Amino Acid oxylic acid in the body fluid after the injec Fraction-The conversion of labeled glyoxylic tion of glyoxylate-l,2-C14 were presented in acid , to amino acids in the body fluid was Table I. A gradual decrease in the specific shown in Table II. Most of the radioactivity activity after 20 minutes after injection was of total amino acid fraction was found in the TABLE I neutral and basic amino acids fraction, in Radioactivity of Glyoxylic Acid in the Body Fluid TABLE II after Injection of Glyoxylate-1,2-C14, Distribution of Radioactivity among the Amino Glycine-1-C14and Glycine-2-014 Acids in the Body Fluid of the Silkworm Injected with Glyoxylate-l,2-C14 The analysis was carried out with the same samples as Table I. Amino acids were fractionat 0.05m1. of glyoxylate-l,2-C14 (4.0•~ 104c.p.m.), ed by Dowex-50 column chromatography (1). glycine-l-C14 (2.7•~ 104 c.p.m,) and glycine-2-C74 which 80 to 90 percent of radioactivity was (5.0•~ 104c.p.m.) in 0.15 MKCI solution was inject- recovered in glycine and serine*. Changes ed,per, worm,. respectively. The specific activities in the specific activity of glycine were shown ,of these labeled compounds were 3.3•~105, 1.6•~ in Table III. No appreciable change in the 105, and 1.8•~ 105 c.p.m. per ,ƒÊmole in order. specific activity of glycine was observed dur 10 worms^w: e" used as a group. Radioactivity of glyoxylate was determined as its 2,4-dinitro- ing the experimental period. The specific phenylhydrazone. * A lower recovery in the radioactivity of gly observed. It was noted that glyoxylic acid cine and serine of the sample at 50 minutes after injected (0.12 ƒÊmole per worm) was diluted injection is due to unexpected loss in the analytical about one-hundredth by 20 minutes after in- procedure. Glycine Formation from C14-Glyoxylate in Silkworms TABLE III ly incorporated into the posterior silkgland, SpecificActiuity of Glycine and Serine in the Body in which silk fibroin is synthesized, is the same Fluid after Injection of Glyoxylate-1,2-C14 phenomenon as observed when C14-labeled glycine was injected (10), although the rate of incorporation of glyoxylic-C14 into protein was slower than that of glycine. Radioactivity of Respiratory CO2-The total radioactivity of CO, evolved after the injec tion of labeled glyoxylic acid was shown in Fig. 2. It was observed that a small but. The amount of glycine and serine was deter- significant portion of glyoxylic acid was ex mined by the methods of K r u e g e r (8) and pired as CO2.The total radioactivity increa F r i s e I 1 (9), respectively. activity of serine was lower than that of gly cine, being about 30 to 60 per cent of that of glycine. This fact indicates that glycine was directly formed from, glyoxylic acid, but not via serine. Radioactivity of the Silkgland and BodyFluid Proteins-The specific activities of the silk- gland and body fluid proteins after the in jection of glyoxylate-l,2-CL4 were persented in Fig. 1. A gradual increase in the radio- activity of the protein of posterior silkgland FIG. 2. Radioactivity of respiratory COT after injection of glyoxylate-1,2-C14, glycine-1-C14' and glycine-2-C14. Radioactivities are expressed in total c.p.m. of CO2 expired by ten worms, -0- after injection of glyoxylate-l,2- C14,-• - after injection of glycine-l C14, -•~- after injection of glycine-2- C14. sed approximately as a linear function of time- and the specific activity was constant during. the experimental period. In the case of the injection of glycine, however, little radioactiv FIG. 1. Incorporation of C14 into proteins ity was found in respiratory CO2. of silkgland and 'body fluid after injection of glyoxylate-l,2-C14. DISCUSSION was observed, while little activity was found Using C14-labeled glyoxylate, it was de in the proteins of the middle silkgland and monstrated that glyoxylic acid in the body the body fluid. The fact that the isotope of fluid was actually utilized for the formation, of glycine in the intact silkworm (Table I) glyoxylate was actively and almost exclusive 300 M. MURAMATSU and K. SHIMURA Further, it was also demonstrated that the appeared possible to occur in the silkworm.