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Conversion of serine to glycine during the formation of egg-shells in the silkworm, Bombyx mori
J. InsectPhytiol.,1969, Vol. 15,pp, 25 to 32. Pergamon Press. Printed in Great Britain
CONVERSION OF SERINE TO GLYCINE DURING THE FORMATION OF EGG-SHELLS IN THE SILKWORM, BOMBYX MORI HIROYA
KAWASAKI,
HITOSHI SATO, MOTOR0 NOBUO OJIMA
Department of Biochemistry,
SUZUKI,
and
School of Dentistry, Iwate Medical College, Morioka, Japan
(Rectiwed 6 August 1968)
Abrtmct--The
egg-shell of the silkworm, Body% mm-i, contains waterinsoluble protein(s) of which the glycine wntent is particularly high. The female adults before emergence thus require considerable amounts of glycine for the synthesis of the protein(s). As was the case in the spinning larvae of B. nwri, serhe was found to serve as a potent precursor of glycine in the female pharate adult and the conversion of serine to glycine proceeded mainly with the loss of the &carbon of serine. INTRODUCTION
to the well-known reaction of serine-glycine interconversion, catalysed by serine hydroxymethyltransferase (EC 2.1 .2.1), a new reaction of glycine synthesis from serine was reported to occur in liver mitochondria of rat and other higher animals (KAWASAIU et al., 1966; SATOet al., 1967; KIKUCHIet al., 1967). In this new reaction the p-carbon ,of serine and CO8 were shown to be spec&ally converted to the u-carbon and carboxyl carbon of glycine, respectively, and the reaction required the presence of an ammonium salt. The reaction could be expressed by the following equation: serine+ CO,+NH, (+2H) + 2 glycine. In a previous study (KAWIISAKIet al., 1968), we investigated to what extent the above-mentioned new reaction could contribute in the conversion of serine to glycine in the spinning larvae of Bom&yx mti, taking advantage of the fact that serine had been shown to be an important precursor of glycine during’fibroin synthesis in this insect (F-A, 1960; M URAMATSU et al, 1961). The results indicated that the conversion of serine to glycine proceeded mainly with loss of the @zarbon of serine, probably being catalysed by serine hydroxymethyltransferase. Consequently, the contribution, if any, of the newly discovered reaction was of minor significance in the spinning larvae of B. mot-i. On the other hand, FUKUDAet al. (1961) revealed that an active synthesis of glycine took place in the female before emergence of B. mori in correlation to the formation of eggs of which glycine content was estimated to be high. This prompted us to examine whether and how serine could serve as the precursor of glycine in the pupal and pharate adult stage and to examine the status of glycine in the eggs. IN ADDITION
25
26
HIROYAKAWABAKI, HIT~SHISATO,MOTOKOSUZUKI,ANDNOBUOOJIMA
The experimental results showed that water-insoluble protein(s) in the egg-shell was responsible for the high glycine content of the egg and that glycine was supplied from serine mostly through the function of serine hydroxymethyltransferase in the female before emergence, the same as in the spinning larvae. These results are presented in this paper. MATERIALS
AND
METHODS
Silkworm and eggs The silkworms, B. mori strain K 203, at the end of the fifth instar were kindly supplied by Dr. K. Miya, Faculty of Agriculture, Iwate University, Morioka. The worms were kept at room temperature (26 _+2°C) throughout the development. The pupal plus pharate adult stage of this strain was of 8 to 9 days and the moths produced non-hibernating eggs. The female moths were allowed to lay eggs on filter paper. As soon as oviposition was finished, the eggs were collected after wetting the filter paper with water, washed with water, air-dried, and stored in a frozen state. FractionatGm of eggs Eggs were crushed in a mortar and transfered into a centrifuge tube with 5 vol. of cold water. After centrifugation at 800g for 10 min at 2”C, the precipitate was suspended in cold water and again homogenized in the mortar. This procedure was repeated several times until the supematant after centrifugation became transparent. The precipitate was washed with ethanol, methanol-chloroform (2 : 1, v/v)mixture at WC, ethanol, and ether, then dried in wacuo over PsO,. This fraction, consisting mainly of water-insoluble materials of the egg-shell, will be referred to as the insoluble fraction. The supematants were combined and cont. HCIO, was added to make the solution 0.6 M as to the acid. The resulting precipitate was collected by centrifugation, washed twice with 0.6 M HCIO,, defatted, and dried as described above to yield the soluble protein fraction. The deproteinized solution and washings with 0.6 M HClO, were combined, chilled in ice water, and neutralized with cont. KOH to pH 2. After removal of most of KCIO, by centrifugation, the solution was passed through a column of Dowex 50 x 8 (H+). Amino acids and peptides were then eluted from the column with 2 N ammonia, and the eluate was concentrated to dryness in vacua over cont. H,SO,. This fraction is referred to as the amino acid fraction. In an experiment, 116 mg (moisture 4.3 per cent) of the insoluble fraction and 7,9 mg (moisture 2.3 per cent) of the soluble protein fraction were obtained from 1 g of eggs. Analysis of the amino acid composition An automatic amino acid analyser, Hitachi KLA3B, was used for the assay of amino acids. Proteins were hydrolysed in a sealed tube with 6 N HCI for 24 hr in a boiling water-bath. The amino acid fractions which were found to contain
SERINE TO CLYCINE CONVRRSION IN SILKWORM PHARATE ADULTS
27
several peptides were also hydrolysed with 2 N HCI for 6 hr in a boiling water-bath, since in the present study we were only interested in the total glycine content of the fractions. The methods of KRUEGER (1949) and of FRISELL et al. (1954) were also used to determine the amounts of glycine and serine, respectively. Measurement of radioactivity Badioactivities were measured with a gas-flow counter, which counted approximately 12O,OOO/minper 0.1 &i of W-compounds, and the measured radioactivities were expressed in counts/min at infinite thinness after appropriate corrections. Prior to the radioactivity assay of the insoluble fractions, aliquots were heated with 6 N HCl for 30 mm in a boiliig water-bath, which gave almost a clear solution and facilitated further procedures for the assay. Administration of 14C-compounds to the pupae and pharate adults, and preparation of radioactive products For the administration to the pupae or pharate adults, various W-compounds (specific radioactivity, 46-29 mCi/mM) were diluted with 0.14 M KC1 to give radioactivity of 15-45 x lo” counts/min per 0.05 ml of the solutions as indicated in Table 2. Each female pupa or pharate adult received an injection of 0.05 ml of the solution containing one of the W-compounds and was kept 4 hr at 25°C. At the end of the experimental period, the ova&a were taken out, washed in chilled 0.14 M KCl, and stored in a refrigerator at -20°C until used. The ovaries were fractionated by the same procedure described above for the eggs, and the fractions were examined for radioactivity. In some experiments, where the radioactivity of respiratory CO, was measured, the pharate adults were placed in a ventilating chamber immediately after the injection, and the respiratory CO, was trapped in 5% KOH. The CO, trapped was converted to BaCO, and assayed for radioactivity. Isolation of the radioactiveglycine and seriwfiom degradation of these amino at%3
the insoluble fraction of ovaries and
The insoluble fraction of ovaries, which was prepared from ovaries of at leastwenty pharate adults injected with the same W-compounds 1 day prior to emert gence, was hydrolysed with 6 N HCl for 8 hr under reflux. Separation of the neutral amino acid fraction from the hydrolysate, isolation and purification of glycine as the salt of nitronaphthalene &phonic acid, decomposition of the salt to yield free glycine, and further purification were carried out as described previously (KAWASAKI et al., 1968). After removal of glycine as the salt of sulphonic acid from the neutral amino acid fraction, the remaining sulphonic acid was also removed by the use of a column of Dowex 50 x 8 (H+). Serine was then isolated and purified by means of successive one-dimensional paper chromatography, first with phenol-waterammonia (77.5 : 21.5 : 1, v/v), then with tert-butanol-methyl ethyl ketone-waterdiethylamine (40 : 50 : 20 : 4, v/v).
28
Hnosxr SATO, M.~TOKOSumm,
HIROYAI&w-,
ANDNOBUO OJIMA
Radioactive glycine and serine were degraded by the methods described by and hONOFF (1950).
VERNON
RESULTS
AND DISCUSSION
Amino acid composition of eggs and ovaries An-ho acid compositions of the three fractions obtained from the laid eggs are shown in Table 1. The compositional patterns of these fractions differed &nificantly from one another, and one of the most remarkable differenceswas that in the glycine content. Glycine was the major constituent of the protein(s) in the insoluble fraction, and its amount in this fraction accounted for 91 per cent of the total amount of glycine in the egg. The high glycine content of the insoluble fraction agrees with an early analytical result given by TOMITA(1921) for B. mori egg-shell. T~LS
I--i&UN0
ACID COMPOSITION LAID
OF THE FRACTIONS OBTAINED FROM THE
EGGS AND FROM
OVARIPS OF 8
?fWTi
Laideggs Amino acida and ZUlXXlOnia
Lysine Hi&dine Ammonia A&nine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylakine
ovarie8*
Insoluble fraction
Soluble Amino protein acid fraction fraction WWg W)
7
5 44 26 4s 32 36 43 38 331 81 41 49 Trace 30 46 5s 18
26
14 60 18 58 2s 36 57 17 30 33 Trace 22 9 18 34
1.2 2.6 5.4 2.1 0.8 1.8 &2 0.6 2.6 5.1 0.2 0.9 0.5 0.8 1.1
21 19
0.7 0.3
Insoluble fraction
Soluble Amino protein acid fraction fraction (junolea/ pharate adults)
8 6 45 20 44 29 37 42 37 326 75 38 48 Trace 28 49 47 20
66 29 114 41 113 so 69 113 49 60 68 Trace 47 1s 40 67 38 43
1.3 4.5 6.7 1.7 1.0 3.2 18.8 06 4.9 5.5 0.4 2.0 1.2 0.9 1.1 ;::
Values arc uncorrected for destruction during hydrolysis. * Ovaries used were taken from the pharate adults 1 day before emergence.
In Table 1 are also listed the analytical results for the fractions obtained from ovaries of the pharate adult on the eighth day after pupation (1 day before emergence), with the intention to relate the results obtained with ovaries to the eggs since we used the pharate adult of this stage for tracer experiments with l*C-compounds
SERINE TO GLYCINE CONVERSION IN SILKWORM PHAFWTE ADULTS
29
(see below). The ovarian insoluble fraction was expected to contain water-insoluble
materialsoriginated from ovarian tissues other than eggs of various developmental stages. However, as can be seen from Table 1, the pattern of amino acid composition of the ovarian insoluble fraction resembled closely that of the insoluble fraction of the laid eggs, indicating that the insoluble fraction of ovaries also consisted of water-insoluble materials of eggs in ovarioles. The other two ovarian fractions differed significantly in the relative amounts of amino acids from those of the corresponding fractions of the laid eggs. The yield of the insoluble fraction relative to the other two fractions was slightly lower when compared to the case of the laid eggs. Thus, in the ovaries of pharate adults 1 day prior to emergence, 84 per cent of the total amount of glycine was found in the insoluble fraction. Incorporation of W from giycine-2-W into ovaries on different days afrer pupation and just after emergence The weight of the soluble protein fraction of ovaries began-to increase sign&cantly on the third day after pupation, but that of the insoluble fraction only on the sixth day (Fig. 1). The increase in weight of the insoluble fraction was due largely 35
3c
,-
25
,-
e
2c
E .E" g
I5
ICI-
5
0123456789
Days after pupation
t Emergence
FIG. 1. Change in weight of the insoluble fraction (- O-) and soluble protein fraction (-•-) of ovaries during development. The values given are for I pupa, or pharate adult, or for a moth just after emergence.
30
_
HIROYAKAWUAKI, HITOSHISATO,M~T~KO SUZUKI,ANDNOBUOOJIMA
to the egg-shell formation. On the fifth day after pupation only a few eggs at the proximal ends of the ovarioles had elastic hard shells, but next day the number of eggs with shell increased to about one-quarter of the total number of eggs when counted from the proximal ends of the ovarioles. The formation of shells spread to eggs at the distal parts of ovarioles in the following 2 days, and after emergence practically all of the eggs had a shell.
Days
oftrr
pupation
t Emergence
FIG. 2. incorporation of “C into the ovarian fractions during 4 hr after the injection of glycine-2-Y on the different days after pupation and just after emergence. - 0-, The insoluble fraction; -O--, the sohlble protein fraction; - x -, the amino acid fraction.
Fig. 2 shows that the incorporation of 14C of the injected glycine-2-14C into both the soluble and insoluble fractions increased markedly on the same days as when the increase in weight of these fractions was observable. Up to the fifth day the insoluble fraction did not incorporate appreciable amounts of 14C, but later on this fraction was the most active in incorporating 14C of glycine-2-14C. The maximum incorporation occurred on the seventh day when as much as about 30 per cent of the total radioactivity given was recovered in the insoluble fraction 4 hr after the injection. The incorporation decreased slightly on the eighth day and markedly after the emergence.
SERINETO GLYCINECOIWERSION IN SlLXWORMPHARATEADULTS
31
These data would indicate that the female pharate adults utilize glycine very effectively during the latter half of the pharate adult stage, particularly for the formation of egg-shells.
TABLE 2-RADIOA~TWE PRODUCTS FORMED DURING4 hr AFTW THE INJECTIONOF VARIOUS “C-COMPOuNDSTO THE PHNUTE ADULT1 day BEFOREEMERGENCE Radioactivity recovered “C-Compound
injected
In ovarial fractions In CO, Per pharate expired adult Insol.* Sol.* A.A.* (10’ counts/ (% of the total radioactivity injected) h) DL-Serb-1-“C DL-seri,ne-3-“C
L-Serine-3-W H%OONa D-Glucose-1-l*C NaHWO Glycine-1:14C Glycine-2-W
30 30 15 45 45 45 15 15
6.5 19.1 37.8 49.2 15.6 84.9 7.6 2.0
17.8 3.4 6.8 2.2 2.0 25:* 26.8
5.6 2.6 5.1 l-6 1.9 0.2 5.3 5.5
5.2 5.3 3.7 2.7 l-5 0.5 2.5 2.6
Specific radioactivities of amino acids in the insoluble fraction of ovaries Glycine Serine (C-l : C-2) (C-l : C-2 : C-3) (counts/min per pole) 526 9 8 9 16 15 450 436
(100 : 0) (60:40) (59 : 41) (70 : 30) (61 : 39) (100 : 0) (100 : 0) (0 : 100)
732 729 710 684 125 20 168 192
(100 : 0 : 0) (1 : 1 : 98) (1 : 1 : 98) (2 : 1 : 97) (14 : 9 : 77) (100 : 0 : 0) (100 : 0 : 0) (1 : 85 : 14)
* InsoL, the insoluble fraction; Sol., the soluble protein fraction ; A.A., the amino acid fraction.
Serine as the precursor of glycine Various I%-compounds including serine- 14C were administered to the female pharate adult 1 day before emergence, and radioactive products formed during 4 hr after the injection were analysed. The results are listed in Table 2. The general features of the results obtained are essentially similar to those observed from the spinning larvae (KAWASAKIet al., 1968), and as was discussed in detail in the previous paper with respekt to the interpretation of the labelling patterns of the l*C:products in the spinning larvae, the data in Table 2 are consistent with the view that serine served as a potent precursor of glycine in the female pharate adult and that glycine was formed from serine mainly with the loss of the p-carbon of serine, probably being catalysed by serine hydroxymethyltransferase; and the p-carbon thus liberated was mostly oxidized to CO,; the contribution, if any, of the newly discovered reaction for glycine biosynthesis (KAWASAKIet al., 1966) appears to be of minor significance in the female pharate adult the same as in the spinning larvae. With the spinning larvae, evidence was obtained suggesting that D-serine was also utilized to some extent (KAWASAKIet al., 1968), but in the female pharate adult D-serine does not seem to be utilized (see Table 2).
32
HIROYA KAWABAKI,HITOSHI SATO, MOTOKO SUZUKI,AND NOBUOOJIMA
Ackmwh&maent-The authors are grateful to Dr. K. MIYA, Iwate University Faculty of Agriculture, for his invaluable advice and for his kindness in providing the silkworms. REFERENCES FRISBLLW. R., MEECHL. A., and MACKENZIE C. G. (1954) A simplified photometric analysis for serine and formaldehyde. J. biol. Chew. 207, 709-716. FUKUDAT. (1960) Biochemical studies on the formation of the silkprotein-VI. Conversion of serine to glycine in the silkworm larva. J. Biochem., Tokyo 47, 581-583. FIJKU~AT., DUCI&TBAV-BO~SON GH., and FLORKINM. (1961) Contributions to silkworm biochemistry-XXIV. Breakdown and biosynthesis of amino acids during the development of Bombyx mori L. Archs int. Physiol. Biochim. 69, 701-719. KAWASAKIH., OJIMA N., TADA H., and SATO H. (1968) Conversion of serine to glycine in the spinning larvae of silkworm, Bombyx mori. J. Biochew., Tokyo 64,247-250. KAWASAKIH., SATOT., and KIKUCHI G. (1966) A new reaction for glycine biosynthesis.
B&hem. biophys. Rts. Commun. 23,227-233. KIKVCHI G., SATOT., KAWASAKI H., KOCXI H., and MOTOKAWAY. (1967) On the mechanism of new reaction for glycine synthesis in liver mitochondria. Abstr. 7th int. Congr. B&hem. Tokyo, p. 763. The Science Council of Japan, Tokyo. KRUEG~ R. (1949) &x die quantitative Bestimm~mg kleiner Mengen Glykokoll in Blut, Ham und Proteinhydrolysaten. Hdv. chim. Actu 32,238-251. M~xsu M., NAGAYAMA H., and SH~MUIUK. (1961) Studies on the biosynthesis of glycine in the silkworm-I. Formation of glycine from serine. y. Biocha., To&y0
49, 55-58. SATO T., M~T~KAWA Y., KOCHI H., and KIKUCZEII G. (1967) Glycine synthesis by extracts of acetone powder of rat-liver mitochondria. B&hem. biophys. Ru. Commwt. 28, 495-501. TOMITAM. (1921) ober die chemische 2 usammensetzung der Eischale des Seidenspinners. Biochem. 2. 116,40-47. VERNON L. P. and ARONOFFS. (1950) Metabolism of soybean leaves-II. Amino acids formed during short-term photosynthesis. At&s Biochem. Biophyr. 29, 179-186.
CONVERSION OF SERINE TO GLYCINE DURING THE FORMATION OF EGG-SHELLS IN THE SILKWORM, BOMBYX MORI HIROYA
KAWASAKI,
HITOSHI SATO, MOTOR0 NOBUO OJIMA
Department of Biochemistry,
SUZUKI,
and
School of Dentistry, Iwate Medical College, Morioka, Japan
(Rectiwed 6 August 1968)
Abrtmct--The
egg-shell of the silkworm, Body% mm-i, contains waterinsoluble protein(s) of which the glycine wntent is particularly high. The female adults before emergence thus require considerable amounts of glycine for the synthesis of the protein(s). As was the case in the spinning larvae of B. nwri, serhe was found to serve as a potent precursor of glycine in the female pharate adult and the conversion of serine to glycine proceeded mainly with the loss of the &carbon of serine. INTRODUCTION
to the well-known reaction of serine-glycine interconversion, catalysed by serine hydroxymethyltransferase (EC 2.1 .2.1), a new reaction of glycine synthesis from serine was reported to occur in liver mitochondria of rat and other higher animals (KAWASAIU et al., 1966; SATOet al., 1967; KIKUCHIet al., 1967). In this new reaction the p-carbon ,of serine and CO8 were shown to be spec&ally converted to the u-carbon and carboxyl carbon of glycine, respectively, and the reaction required the presence of an ammonium salt. The reaction could be expressed by the following equation: serine+ CO,+NH, (+2H) + 2 glycine. In a previous study (KAWIISAKIet al., 1968), we investigated to what extent the above-mentioned new reaction could contribute in the conversion of serine to glycine in the spinning larvae of Bom&yx mti, taking advantage of the fact that serine had been shown to be an important precursor of glycine during’fibroin synthesis in this insect (F-A, 1960; M URAMATSU et al, 1961). The results indicated that the conversion of serine to glycine proceeded mainly with loss of the @zarbon of serine, probably being catalysed by serine hydroxymethyltransferase. Consequently, the contribution, if any, of the newly discovered reaction was of minor significance in the spinning larvae of B. mot-i. On the other hand, FUKUDAet al. (1961) revealed that an active synthesis of glycine took place in the female before emergence of B. mori in correlation to the formation of eggs of which glycine content was estimated to be high. This prompted us to examine whether and how serine could serve as the precursor of glycine in the pupal and pharate adult stage and to examine the status of glycine in the eggs. IN ADDITION
25
26
HIROYAKAWABAKI, HIT~SHISATO,MOTOKOSUZUKI,ANDNOBUOOJIMA
The experimental results showed that water-insoluble protein(s) in the egg-shell was responsible for the high glycine content of the egg and that glycine was supplied from serine mostly through the function of serine hydroxymethyltransferase in the female before emergence, the same as in the spinning larvae. These results are presented in this paper. MATERIALS
AND
METHODS
Silkworm and eggs The silkworms, B. mori strain K 203, at the end of the fifth instar were kindly supplied by Dr. K. Miya, Faculty of Agriculture, Iwate University, Morioka. The worms were kept at room temperature (26 _+2°C) throughout the development. The pupal plus pharate adult stage of this strain was of 8 to 9 days and the moths produced non-hibernating eggs. The female moths were allowed to lay eggs on filter paper. As soon as oviposition was finished, the eggs were collected after wetting the filter paper with water, washed with water, air-dried, and stored in a frozen state. FractionatGm of eggs Eggs were crushed in a mortar and transfered into a centrifuge tube with 5 vol. of cold water. After centrifugation at 800g for 10 min at 2”C, the precipitate was suspended in cold water and again homogenized in the mortar. This procedure was repeated several times until the supematant after centrifugation became transparent. The precipitate was washed with ethanol, methanol-chloroform (2 : 1, v/v)mixture at WC, ethanol, and ether, then dried in wacuo over PsO,. This fraction, consisting mainly of water-insoluble materials of the egg-shell, will be referred to as the insoluble fraction. The supematants were combined and cont. HCIO, was added to make the solution 0.6 M as to the acid. The resulting precipitate was collected by centrifugation, washed twice with 0.6 M HCIO,, defatted, and dried as described above to yield the soluble protein fraction. The deproteinized solution and washings with 0.6 M HClO, were combined, chilled in ice water, and neutralized with cont. KOH to pH 2. After removal of most of KCIO, by centrifugation, the solution was passed through a column of Dowex 50 x 8 (H+). Amino acids and peptides were then eluted from the column with 2 N ammonia, and the eluate was concentrated to dryness in vacua over cont. H,SO,. This fraction is referred to as the amino acid fraction. In an experiment, 116 mg (moisture 4.3 per cent) of the insoluble fraction and 7,9 mg (moisture 2.3 per cent) of the soluble protein fraction were obtained from 1 g of eggs. Analysis of the amino acid composition An automatic amino acid analyser, Hitachi KLA3B, was used for the assay of amino acids. Proteins were hydrolysed in a sealed tube with 6 N HCI for 24 hr in a boiling water-bath. The amino acid fractions which were found to contain
SERINE TO CLYCINE CONVRRSION IN SILKWORM PHARATE ADULTS
27
several peptides were also hydrolysed with 2 N HCI for 6 hr in a boiling water-bath, since in the present study we were only interested in the total glycine content of the fractions. The methods of KRUEGER (1949) and of FRISELL et al. (1954) were also used to determine the amounts of glycine and serine, respectively. Measurement of radioactivity Badioactivities were measured with a gas-flow counter, which counted approximately 12O,OOO/minper 0.1 &i of W-compounds, and the measured radioactivities were expressed in counts/min at infinite thinness after appropriate corrections. Prior to the radioactivity assay of the insoluble fractions, aliquots were heated with 6 N HCl for 30 mm in a boiliig water-bath, which gave almost a clear solution and facilitated further procedures for the assay. Administration of 14C-compounds to the pupae and pharate adults, and preparation of radioactive products For the administration to the pupae or pharate adults, various W-compounds (specific radioactivity, 46-29 mCi/mM) were diluted with 0.14 M KC1 to give radioactivity of 15-45 x lo” counts/min per 0.05 ml of the solutions as indicated in Table 2. Each female pupa or pharate adult received an injection of 0.05 ml of the solution containing one of the W-compounds and was kept 4 hr at 25°C. At the end of the experimental period, the ova&a were taken out, washed in chilled 0.14 M KCl, and stored in a refrigerator at -20°C until used. The ovaries were fractionated by the same procedure described above for the eggs, and the fractions were examined for radioactivity. In some experiments, where the radioactivity of respiratory CO, was measured, the pharate adults were placed in a ventilating chamber immediately after the injection, and the respiratory CO, was trapped in 5% KOH. The CO, trapped was converted to BaCO, and assayed for radioactivity. Isolation of the radioactiveglycine and seriwfiom degradation of these amino at%3
the insoluble fraction of ovaries and
The insoluble fraction of ovaries, which was prepared from ovaries of at leastwenty pharate adults injected with the same W-compounds 1 day prior to emert gence, was hydrolysed with 6 N HCl for 8 hr under reflux. Separation of the neutral amino acid fraction from the hydrolysate, isolation and purification of glycine as the salt of nitronaphthalene &phonic acid, decomposition of the salt to yield free glycine, and further purification were carried out as described previously (KAWASAKI et al., 1968). After removal of glycine as the salt of sulphonic acid from the neutral amino acid fraction, the remaining sulphonic acid was also removed by the use of a column of Dowex 50 x 8 (H+). Serine was then isolated and purified by means of successive one-dimensional paper chromatography, first with phenol-waterammonia (77.5 : 21.5 : 1, v/v), then with tert-butanol-methyl ethyl ketone-waterdiethylamine (40 : 50 : 20 : 4, v/v).
28
Hnosxr SATO, M.~TOKOSumm,
HIROYAI&w-,
ANDNOBUO OJIMA
Radioactive glycine and serine were degraded by the methods described by and hONOFF (1950).
VERNON
RESULTS
AND DISCUSSION
Amino acid composition of eggs and ovaries An-ho acid compositions of the three fractions obtained from the laid eggs are shown in Table 1. The compositional patterns of these fractions differed &nificantly from one another, and one of the most remarkable differenceswas that in the glycine content. Glycine was the major constituent of the protein(s) in the insoluble fraction, and its amount in this fraction accounted for 91 per cent of the total amount of glycine in the egg. The high glycine content of the insoluble fraction agrees with an early analytical result given by TOMITA(1921) for B. mori egg-shell. T~LS
I--i&UN0
ACID COMPOSITION LAID
OF THE FRACTIONS OBTAINED FROM THE
EGGS AND FROM
OVARIPS OF 8
?fWTi
Laideggs Amino acida and ZUlXXlOnia
Lysine Hi&dine Ammonia A&nine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylakine
ovarie8*
Insoluble fraction
Soluble Amino protein acid fraction fraction WWg W)
7
5 44 26 4s 32 36 43 38 331 81 41 49 Trace 30 46 5s 18
26
14 60 18 58 2s 36 57 17 30 33 Trace 22 9 18 34
1.2 2.6 5.4 2.1 0.8 1.8 &2 0.6 2.6 5.1 0.2 0.9 0.5 0.8 1.1
21 19
0.7 0.3
Insoluble fraction
Soluble Amino protein acid fraction fraction (junolea/ pharate adults)
8 6 45 20 44 29 37 42 37 326 75 38 48 Trace 28 49 47 20
66 29 114 41 113 so 69 113 49 60 68 Trace 47 1s 40 67 38 43
1.3 4.5 6.7 1.7 1.0 3.2 18.8 06 4.9 5.5 0.4 2.0 1.2 0.9 1.1 ;::
Values arc uncorrected for destruction during hydrolysis. * Ovaries used were taken from the pharate adults 1 day before emergence.
In Table 1 are also listed the analytical results for the fractions obtained from ovaries of the pharate adult on the eighth day after pupation (1 day before emergence), with the intention to relate the results obtained with ovaries to the eggs since we used the pharate adult of this stage for tracer experiments with l*C-compounds
SERINE TO GLYCINE CONVERSION IN SILKWORM PHAFWTE ADULTS
29
(see below). The ovarian insoluble fraction was expected to contain water-insoluble
materialsoriginated from ovarian tissues other than eggs of various developmental stages. However, as can be seen from Table 1, the pattern of amino acid composition of the ovarian insoluble fraction resembled closely that of the insoluble fraction of the laid eggs, indicating that the insoluble fraction of ovaries also consisted of water-insoluble materials of eggs in ovarioles. The other two ovarian fractions differed significantly in the relative amounts of amino acids from those of the corresponding fractions of the laid eggs. The yield of the insoluble fraction relative to the other two fractions was slightly lower when compared to the case of the laid eggs. Thus, in the ovaries of pharate adults 1 day prior to emergence, 84 per cent of the total amount of glycine was found in the insoluble fraction. Incorporation of W from giycine-2-W into ovaries on different days afrer pupation and just after emergence The weight of the soluble protein fraction of ovaries began-to increase sign&cantly on the third day after pupation, but that of the insoluble fraction only on the sixth day (Fig. 1). The increase in weight of the insoluble fraction was due largely 35
3c
,-
25
,-
e
2c
E .E" g
I5
ICI-
5
0123456789
Days after pupation
t Emergence
FIG. 1. Change in weight of the insoluble fraction (- O-) and soluble protein fraction (-•-) of ovaries during development. The values given are for I pupa, or pharate adult, or for a moth just after emergence.
30
_
HIROYAKAWUAKI, HITOSHISATO,M~T~KO SUZUKI,ANDNOBUOOJIMA
to the egg-shell formation. On the fifth day after pupation only a few eggs at the proximal ends of the ovarioles had elastic hard shells, but next day the number of eggs with shell increased to about one-quarter of the total number of eggs when counted from the proximal ends of the ovarioles. The formation of shells spread to eggs at the distal parts of ovarioles in the following 2 days, and after emergence practically all of the eggs had a shell.
Days
oftrr
pupation
t Emergence
FIG. 2. incorporation of “C into the ovarian fractions during 4 hr after the injection of glycine-2-Y on the different days after pupation and just after emergence. - 0-, The insoluble fraction; -O--, the sohlble protein fraction; - x -, the amino acid fraction.
Fig. 2 shows that the incorporation of 14C of the injected glycine-2-14C into both the soluble and insoluble fractions increased markedly on the same days as when the increase in weight of these fractions was observable. Up to the fifth day the insoluble fraction did not incorporate appreciable amounts of 14C, but later on this fraction was the most active in incorporating 14C of glycine-2-14C. The maximum incorporation occurred on the seventh day when as much as about 30 per cent of the total radioactivity given was recovered in the insoluble fraction 4 hr after the injection. The incorporation decreased slightly on the eighth day and markedly after the emergence.
SERINETO GLYCINECOIWERSION IN SlLXWORMPHARATEADULTS
31
These data would indicate that the female pharate adults utilize glycine very effectively during the latter half of the pharate adult stage, particularly for the formation of egg-shells.
TABLE 2-RADIOA~TWE PRODUCTS FORMED DURING4 hr AFTW THE INJECTIONOF VARIOUS “C-COMPOuNDSTO THE PHNUTE ADULT1 day BEFOREEMERGENCE Radioactivity recovered “C-Compound
injected
In ovarial fractions In CO, Per pharate expired adult Insol.* Sol.* A.A.* (10’ counts/ (% of the total radioactivity injected) h) DL-Serb-1-“C DL-seri,ne-3-“C
L-Serine-3-W H%OONa D-Glucose-1-l*C NaHWO Glycine-1:14C Glycine-2-W
30 30 15 45 45 45 15 15
6.5 19.1 37.8 49.2 15.6 84.9 7.6 2.0
17.8 3.4 6.8 2.2 2.0 25:* 26.8
5.6 2.6 5.1 l-6 1.9 0.2 5.3 5.5
5.2 5.3 3.7 2.7 l-5 0.5 2.5 2.6
Specific radioactivities of amino acids in the insoluble fraction of ovaries Glycine Serine (C-l : C-2) (C-l : C-2 : C-3) (counts/min per pole) 526 9 8 9 16 15 450 436
(100 : 0) (60:40) (59 : 41) (70 : 30) (61 : 39) (100 : 0) (100 : 0) (0 : 100)
732 729 710 684 125 20 168 192
(100 : 0 : 0) (1 : 1 : 98) (1 : 1 : 98) (2 : 1 : 97) (14 : 9 : 77) (100 : 0 : 0) (100 : 0 : 0) (1 : 85 : 14)
* InsoL, the insoluble fraction; Sol., the soluble protein fraction ; A.A., the amino acid fraction.
Serine as the precursor of glycine Various I%-compounds including serine- 14C were administered to the female pharate adult 1 day before emergence, and radioactive products formed during 4 hr after the injection were analysed. The results are listed in Table 2. The general features of the results obtained are essentially similar to those observed from the spinning larvae (KAWASAKIet al., 1968), and as was discussed in detail in the previous paper with respekt to the interpretation of the labelling patterns of the l*C:products in the spinning larvae, the data in Table 2 are consistent with the view that serine served as a potent precursor of glycine in the female pharate adult and that glycine was formed from serine mainly with the loss of the p-carbon of serine, probably being catalysed by serine hydroxymethyltransferase; and the p-carbon thus liberated was mostly oxidized to CO,; the contribution, if any, of the newly discovered reaction for glycine biosynthesis (KAWASAKIet al., 1966) appears to be of minor significance in the female pharate adult the same as in the spinning larvae. With the spinning larvae, evidence was obtained suggesting that D-serine was also utilized to some extent (KAWASAKIet al., 1968), but in the female pharate adult D-serine does not seem to be utilized (see Table 2).
32
HIROYA KAWABAKI,HITOSHI SATO, MOTOKO SUZUKI,AND NOBUOOJIMA
Ackmwh&maent-The authors are grateful to Dr. K. MIYA, Iwate University Faculty of Agriculture, for his invaluable advice and for his kindness in providing the silkworms. REFERENCES FRISBLLW. R., MEECHL. A., and MACKENZIE C. G. (1954) A simplified photometric analysis for serine and formaldehyde. J. biol. Chew. 207, 709-716. FUKUDAT. (1960) Biochemical studies on the formation of the silkprotein-VI. Conversion of serine to glycine in the silkworm larva. J. Biochem., Tokyo 47, 581-583. FIJKU~AT., DUCI&TBAV-BO~SON GH., and FLORKINM. (1961) Contributions to silkworm biochemistry-XXIV. Breakdown and biosynthesis of amino acids during the development of Bombyx mori L. Archs int. Physiol. Biochim. 69, 701-719. KAWASAKIH., OJIMA N., TADA H., and SATO H. (1968) Conversion of serine to glycine in the spinning larvae of silkworm, Bombyx mori. J. Biochew., Tokyo 64,247-250. KAWASAKIH., SATOT., and KIKUCHI G. (1966) A new reaction for glycine biosynthesis.
B&hem. biophys. Rts. Commun. 23,227-233. KIKVCHI G., SATOT., KAWASAKI H., KOCXI H., and MOTOKAWAY. (1967) On the mechanism of new reaction for glycine synthesis in liver mitochondria. Abstr. 7th int. Congr. B&hem. Tokyo, p. 763. The Science Council of Japan, Tokyo. KRUEG~ R. (1949) &x die quantitative Bestimm~mg kleiner Mengen Glykokoll in Blut, Ham und Proteinhydrolysaten. Hdv. chim. Actu 32,238-251. M~xsu M., NAGAYAMA H., and SH~MUIUK. (1961) Studies on the biosynthesis of glycine in the silkworm-I. Formation of glycine from serine. y. Biocha., To&y0
49, 55-58. SATO T., M~T~KAWA Y., KOCHI H., and KIKUCZEII G. (1967) Glycine synthesis by extracts of acetone powder of rat-liver mitochondria. B&hem. biophys. Ru. Commwt. 28, 495-501. TOMITAM. (1921) ober die chemische 2 usammensetzung der Eischale des Seidenspinners. Biochem. 2. 116,40-47. VERNON L. P. and ARONOFFS. (1950) Metabolism of soybean leaves-II. Amino acids formed during short-term photosynthesis. At&s Biochem. Biophyr. 29, 179-186.