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The synthesis in vitro of the protein calliphorin by fat body from the larva of the blowfly, Calliphora erythrocephala
J. Insect Ph:kol., 1969, Vol. 15, pp. 1601 to 1605. Pergnmm Press. Printed in Great Britain
THE SYNTHESIS IN VITRO OF THE PROTEIN CALLIPHORIN BY FAT BODY FROM THE LARVA OF THE BLOWFLY, CALLIPHORA ER YTHROCEPHALA E. A. MUNN,
G. M. PRICE,
and
G. D. GREVILLE
Biochemistry Department, Agricultural Research Council Institute of Animal Physiology, Babraham, Cambridge, and Biochemistry Department, Agricultural Research Council Pest Infestation Laboratory, Slough, Bucks. (Received
20 February
1969)
Abstract-The soluble protein of fat bodies isolated from S-day-old larvae of Culliphora erythrocephala consists mainly of calliphorin, together with smaller amounts of a calliphorin-like protein designated Protein II. When the fat bodies were incubated with an amino acid mixture, calliphorin and Protein II were released into the suspending medium; label from [UJ4C]valine was incorporated into these proteins. It is concluded that calliphorin and Protein II are synthesized by the fat bodies and that net formation of calliphorin probably occurred in these experiments. The fat body is the likely source of the calliphorin found in the haemolymph.
INTRODUCTION
THE PRCITEIN calliphorin has been isolated in highly purified form from larvae and pupae of the blowfly Calliphora erythrocephala (MUNN et al., 1967). First detectable in extracts of 2-day larvae, calliphorin accounts for over 50 per cent of the scluble protein in the late larval stage (MUNN and GREVILLE, 1969). It is accompanied by relatively small amounts of another protein, designated Protein II, which has similar sedimentation properties but moves more slowly on electrophoresis. Fat bodies isolated from larval C. erythrocephala have been shown to synthesize protein when incubated, and to incorporate L-[UJ4C]valine into a number of proteins which were separated as zones by electrophoresis on acrylamide gel but were not otherwise characterized (PRICE, 1966; PRICE and BOSMAN, 1966). The present study establishes that the principal proteins synthesized by the fat body in vitro are calliphorin and Protein II. MATERIALS
AND
METHODS
Materials Water redistilled in glass and AnalaR chemicals were used throughout. L-[UJ4C]valine was purchased from the Radiochemical Centre, Amersham, Bucking’hamshire, Ionagar No. 2 from Oxoid Ltd., London, and hydrolysed starch from Connaught Medical Research Laboratories, Toronto, Canada. 1601
1602
E. A. MUNN,G. M. PRICE,AND
G. D. GREVILLE
Rearing method Blowflies were cultured by the procedure (1969).
described
in MUNN and GREVILLE
Isolation and incubation of fat bodies Fat bodies were isolated from 25 3-day larvae by the method of PRICE (1965) and incubated at 25°C with gentle shaking for 1 to l-5 hr in 2.5 ml of a medium containing 20 mM KCl, 30 mM MgCI, and 6 mM phosphate (final pH 7.0). The medium also contained nineteen unlabelled amino acids including L-valine, as used by PRICE (1966), and, where appropriate, L-[U-r4C]valine (3.75 PC). For the zero-time controls the fat bodies were put into the suspending medium at 0°C.
Extraction of fat bodies After the incubation, the fat bodies were separated from the suspending medium by centrifugation at lO,OOOg,, for 5 min at 2°C. The supernatant, which contained the proteins released by the fat bodies during the incubation, was decanted. The fat body tissue was thoroughly homogenized, by means of a small hand-operated Potter-Elvehjem-type homogenizer, in 2.5 ml of a solution containing 19 mM KCI, 29 mM MgCl,, and 6 mM phosphate (pH 7.0) and the homogenate was then centrifuged at lO,OOOg, for 15 min at 2°C. The supernatants from the two centrifugations were each diluted to 10 ml and centrifuged at 151,OOOg,, for 4 hr at about 4°C in an angle-head rotor, i.e. under conditions which sediment calliphorin. The pellets were taken up in known volumes (0*3O-5 ml) of a solution containing 0.15 M NaCl and 20 mM phosphate (pH 6.3). The zero-time controls were treated in exactly the same way.
IdentiJication of proteins Immunodiffusion and immunoelectrophoresis were carried out in agar gel (1.5% w/v Ionagar No. 2 in 25 mM phosphate, pH 6-l), with antisera produced by injecting crude extracts of Calliphora pupae into rabbits and known to react with ten antigens present in extracts of Calliphora larvae (MUNN and GREVILLE, 1969). Antiserum to pure calliphorin was also used. Electrophoresis was carried out at 6 V/cm for 3 to 4 hr.
Estimation of amounts of protein Absolute quantities of proteins were not determined. The amounts present in the control and experimental samples extracted under identical conditions were compared on the basis of (a) the intensities of the precipitin lines obtained by immunodiffusion of twofold serial dilutions of the extracts and (b) of the intensity of staining by naphthalene black of zones separated by electrophoresis of the extracts on starch gel (9.6% w/v in 25 mM phosphate, pH 6.1).
Radioautography To obtain radioautographs, agar plates after immunodiffusion or immunoelectrophoresis were fixed in ethanol-glacial acetic acid-water (40 : 1 : 40 by vol.), dried, and then kept in close contact with X-ray film for 5 to 6 weeks.
CALLIPHORINSYNTHESISBY
LARVALFATBODY
1603
RESULTS
The immunodiffusion patterns show that the fat bodies contained calliphorin (arc 1) and small amounts of Protein II (arc 2) both at zero time (Fig. lb) and after the incubation (Fig. la). During the incubation calliphorin and Protein II were released into the suspending medium (Figs. 2a and 3a), no antigenic protein being found in the medium in the zero-time controls (Figs. 2b and 3b). In immunodiffusion tests (Fig. 3c, d) the precipitin lines obtained with serial dilutions of extracts of the fat bodies indicate that there was approximately as much calliphorin in the fat bodies after the incubation as there was at zero time, because the extract in each case gave a precipitin line when diluted 1 : 64 but not when diluted 1 : 128. Therefore, since calliphorin was released into the medium during the incubation, it seems likely that there was a net synthesis of this protein. Insufficient Protein II was present to form a precipitin line even when the extracts were diluted only 1 : 4. Arc 1 in the immunoelectrophoresis runs (Figs. 1 and 2) is ascribed to calliphorin because (a) it has the characteristic shape and position of the arc given by pure lcalliphorin (e.g. Fig. 2c, d of MUNN and GREVILLE, 1969); (b) Ouchterlony-type double diffusion tests carried out in agar gel with antiserum to pure calliphor:in proved that calliphorin was present in fat body extracts; and (c) the precipitin line given by fat body extract together with antiserum to crude pupal Calliphora extract joins up with the line given by fat body extract together with antiserum to pure calliphorin (Fig. 3i, j). Arc 2 in the immunoelectrophoresis is likewise ascribed to Protein II on the grounds of its characteristic shape and position, and this protein also has been identified in fat body extracts by Ouchterlony-type tests. Radioautographs of the patterns of arcs and precipitin lines show that label from L-[UJ4C] va 1ine was incorporated into calliphorin and into Protein II. Most of the labelled protein was released by the fat bodies during the incubation (Figs. 2c: and 3e). A small amount of labelled calliphorin and Protein II was, however, retained by them (Figs. lc and 3g) and, so far as can be judged from the intensities on the plates, at the end of the incubation the fat bodies contained calliphorin of low specific radioactivity. In the immunoelectrophoresis runs there was labelling of some material which formed a diffuse streak, or possibly arc (Figs. 1 and 2), and immunodiffusion tests indicated that, including calliphorin and Protein II, three, or possibly four, labelled proteins were released by the fat bodies during the incubation (Fig. 3k, 1). Further evidence that there was net synthesis of calliphorin during the incubation of the fat bodies was obtained by comparison of the intensity of staining by naphthalene black of zones obtained by starch-gel electrophoresis (Fig. 4). In this expseriment the fat bodies were homogenized in the suspending medium after incubation and the extract prepared as before; exactly the same procedure was app’lied to the zero-time control. The calliphorin zones, identified with a marker, thus represented the total amount of calliphorin present, and this is seen The experiment also confirms that to have increased during the incubation.
1604
E. A. MUNN,G. M. PRICE, AND
G. D. GREVILLE
calliphorin and Protein II are the major soluble protein components present in, and synthesized by, the fat bodies; since antiserum to pupal extracts was used, any antigens specific to larvae would escape detection in the immunological tests. DISCUSSION The principal protein found in fat bodies isolated from 3-day larvae of C. erythrocephala was calliphorin, and this was accompanied by relatively small amounts of Protein II and minor amounts of other soluble proteins (Fig. 4). These latter did not react appreciably with the antisera used in this work, which were formed against antigens extracted from pupae (Fig. 1). When the fat bodies were incubated in an amino acid mixture both calliphorin and Protein II were released into the incubation medium, and label from L-[U-14C]valine was incorporated into these two proteins. Most of the 14C incorporated was associated with the released protein, but a small amount was associated with the protein retained by the fat bodies. These observations are in accord with those of PRICE (1966), who found that when fat bodies from C. erythrocephala were incubated with amino acids there was a net synthesis of protein and incorporation of label from [U-i4C]valine; much of the labelled protein was released into the suspending medium during the incubation. PRICE (1966) also showed that protein released during the incubation of fat bodies in absence of [U-14C]valine did not become labelled on subsequent incubation with this compound, and so eliminated the possibility of isotopic exchange. The results in the present paper show that the protein which is synthesized consists almost entirely of calliphorin together with some Protein II. Price’s finding that there was a net synthesis of protein therefore supports the evidence presented here of a net synthesis of calliphorin. Using starch-gel electrophoresis, LAUFER (1960) found three proteins in fat bodies of female giant silk moths (Samiu Cynthia) with similar mobilities to those of three components of the haemolymph, and considered that the fat body was ‘strongly implicated’ as the source of these haemolymph proteins. LAUFER (1961) obtained further evidence for this in organ-culture experiments; two of the three proteins were detected in the medium in which fat body was cultured, and a malate dehydrogenase isoenzyme, corresponding in mobility to one in the haemolymph, also appeared in the culture medium. SHIGEMATSU(1958) found that, on incubation, fat bodies from the silkworm Bombyx mori synthesized proteins with the same electrophoretic mobilities on paper as those of the main globulin components of the haemolymph; the quantities were such that the fat body could be regarded as the sole source of these haemolymph globulins. Evidence that the larval fat body is the source of haemolymph proteins in the case of C. evythrocephala was obtained by PRICE and BOSMAN (1966); they found that when fat bodies of 4- to 7-day-larvae were incubated with amino acids the proteins released into the medium gave an electrophoretic pattern on acrylamide gel almost identical with that given by the haemolymph proteins. The principal protein of the haemolymph of Calliphora larvae is calliphorin (MUNN and
(ai
(b)
(¢)
(d)
FIC. 1. Immunoelectrophoresis patterns showing calliphorin (arc 1) and Protein II (arc 2) present in isolated larval fat bodies, (a) after incubation with an amino acid mixture containing [UJ4C]valine, (b) at zero time. Antiserum to crude pupal Calliphora extract was used. (c), (d) Radioautographs of the arcs in (a) and (b) respectively. For experimental details see Materials and Methods.
|
(a}
(bl
(c)
(d)
FIc. 2. Immunoelectrophoresis patterns given by antigens present in the m e d i u m in which the fat bodies were suspended; (a) after incubation, (b) zero-time controls. Calliphorin (arc 1) and Protein I I (arc 2) were released into the m e d i u m during the incubation, neither being detectable in the m e d i u m at zero time. A n t i s e r u m to crude pupal Calliphora extract was used. (c), (d) Radioautographs of (a) and (b) respectively. For experimental details see Materials and Methods.
Ditution
(a)
(e)
(b)
(f)
(¢)
(gl
(d)
(h)
1:
4
8
16
32
64
128
256
1
2
•
8
16
32
64
(il
(j) Ditution I :
(k) DItution 1:
Fxc. 3 (a)-(d). Immunodiffusion precipitin lines due to calliphorin before and after the incubation of the fat bodies, (a) in suspending m e d i u m after the incubation, (b) in suspending m e d i u m at zero time, (c) in fat bodies after the incubation, (d) in fat bodies at zero time. T w o f o l d serial dilutions of extracts prepared as described in Materials and Methods were placed in the wells, ranging from 1 : 4 on the left to 1 : 256 on the right; the slots contained antiserum to crude pupal Calliphora extract. (e), (f), (g), (h) Radioautographs of (a), (b), (c), (d) respectively. (i), (j) Immunodiffusion patterns obtained with extracts of fat bodies, (i) after incubation and (j) at zero time; *, slots containing antiserum to crude pupal extract; t, slot containing antiserum to pure calliphorin; in this experiment the proteins were not concentrated by high-speed centrifugation (see Materials and Methods) and the serial twofold dilutions began with undiluted extract on the left. (k) I m m u n o diffusion pattern and (I) corresponding radioautograph obtained from suspending m e d i u m after the incubation of the fat bodies, showing lines due to three labelled proteins ; the proteins in the m e d i u m were concentrated by high-speed centrifugation as in (a)-(d).
P
C
(a)
(b)
(c)
i iii
i ii : iii!i~i
(d)
(e)
Fro. 4. Patterns obtained by staining protein with naphthalene black after sta:rch-gel electrophoresis of extracts prepared from fat bodies together with the suspending medium, (a) before incubation and (b) after incubation, equivalent amounts of the two extracts being applied to the gel. (c) Repeat of (b) in a different experiment, run together with (d) a pure calliphorin marker. (e) Traces obtained from (a) and (b) by means of a Double Beam Recording Microdensitometer (Joyce, Loebl and Co. Ltd., Gateshead-on-Tyne) : Z, trace from (a) (zero time) and I, 1:race from (b) (incubated). The peak height of I is 1.26 times that of Z. C, calliphorin; P, Protein II.
CALLIPHORIN SYNTHESISBY LARVALFAT BODY
GREVILLE, 1969). From the observations the fat body is its most likely source.
in the present
1605
paper it appears that
Aciinowledgements-We are grateful to Dr. A. FEINSTEIN for advice and criticism and to Dr. R. W. Cox for help with the use of the microdensitometer. REFERENCES LAUFERH. (1960) Blood proteins in insect development. Ann. N. Y. Acad. Sci. 89,490-515. LAUFER H. (1961) Forms of enzymes in insect development. Ann. N. Y. Acad. Sci. 94, 825-8X. MUNN E. A., FEINSTEIN A., and GREVILLE G. D. (1967) A major protein constituent of pupae of the blowfly Calliphora erythrocephala. Biochem. J. 102, SP-6P. MUNN E. A. and GREVILLE G. D. (1969) The soluble proteins of developing Calliphora erythrocephala, particularly calliphorin, and similar proteins in other insects. J. Insect Physiol. In press. PRICE G. M. (1965) Nucleic acids in the larva of the blowfly Calliphora erythrocephala. J. Insect Physiol. 11,869478. PRICE G. M. (1966) The in vitro incorporation of [U-*4C]valine into fat body protein of the larva of the blowfly, Calliphora erythrocephala. J. Insect Physiol. 12, 731-740. PRICE G. M. and BOSMANT. (1966) The electrophoretic separation of proteins isolated from the larva of the blowfly, Calliphora erythrocephala. ‘J. Insect Physiol. 12, 741-745. SHICEMATSU H. (1958) Synthesis of blood protein by the fat body in the silkworm, Bombyx mori L. Nature, Lond. 182, 880-882.
THE SYNTHESIS IN VITRO OF THE PROTEIN CALLIPHORIN BY FAT BODY FROM THE LARVA OF THE BLOWFLY, CALLIPHORA ER YTHROCEPHALA E. A. MUNN,
G. M. PRICE,
and
G. D. GREVILLE
Biochemistry Department, Agricultural Research Council Institute of Animal Physiology, Babraham, Cambridge, and Biochemistry Department, Agricultural Research Council Pest Infestation Laboratory, Slough, Bucks. (Received
20 February
1969)
Abstract-The soluble protein of fat bodies isolated from S-day-old larvae of Culliphora erythrocephala consists mainly of calliphorin, together with smaller amounts of a calliphorin-like protein designated Protein II. When the fat bodies were incubated with an amino acid mixture, calliphorin and Protein II were released into the suspending medium; label from [UJ4C]valine was incorporated into these proteins. It is concluded that calliphorin and Protein II are synthesized by the fat bodies and that net formation of calliphorin probably occurred in these experiments. The fat body is the likely source of the calliphorin found in the haemolymph.
INTRODUCTION
THE PRCITEIN calliphorin has been isolated in highly purified form from larvae and pupae of the blowfly Calliphora erythrocephala (MUNN et al., 1967). First detectable in extracts of 2-day larvae, calliphorin accounts for over 50 per cent of the scluble protein in the late larval stage (MUNN and GREVILLE, 1969). It is accompanied by relatively small amounts of another protein, designated Protein II, which has similar sedimentation properties but moves more slowly on electrophoresis. Fat bodies isolated from larval C. erythrocephala have been shown to synthesize protein when incubated, and to incorporate L-[UJ4C]valine into a number of proteins which were separated as zones by electrophoresis on acrylamide gel but were not otherwise characterized (PRICE, 1966; PRICE and BOSMAN, 1966). The present study establishes that the principal proteins synthesized by the fat body in vitro are calliphorin and Protein II. MATERIALS
AND
METHODS
Materials Water redistilled in glass and AnalaR chemicals were used throughout. L-[UJ4C]valine was purchased from the Radiochemical Centre, Amersham, Bucking’hamshire, Ionagar No. 2 from Oxoid Ltd., London, and hydrolysed starch from Connaught Medical Research Laboratories, Toronto, Canada. 1601
1602
E. A. MUNN,G. M. PRICE,AND
G. D. GREVILLE
Rearing method Blowflies were cultured by the procedure (1969).
described
in MUNN and GREVILLE
Isolation and incubation of fat bodies Fat bodies were isolated from 25 3-day larvae by the method of PRICE (1965) and incubated at 25°C with gentle shaking for 1 to l-5 hr in 2.5 ml of a medium containing 20 mM KCl, 30 mM MgCI, and 6 mM phosphate (final pH 7.0). The medium also contained nineteen unlabelled amino acids including L-valine, as used by PRICE (1966), and, where appropriate, L-[U-r4C]valine (3.75 PC). For the zero-time controls the fat bodies were put into the suspending medium at 0°C.
Extraction of fat bodies After the incubation, the fat bodies were separated from the suspending medium by centrifugation at lO,OOOg,, for 5 min at 2°C. The supernatant, which contained the proteins released by the fat bodies during the incubation, was decanted. The fat body tissue was thoroughly homogenized, by means of a small hand-operated Potter-Elvehjem-type homogenizer, in 2.5 ml of a solution containing 19 mM KCI, 29 mM MgCl,, and 6 mM phosphate (pH 7.0) and the homogenate was then centrifuged at lO,OOOg, for 15 min at 2°C. The supernatants from the two centrifugations were each diluted to 10 ml and centrifuged at 151,OOOg,, for 4 hr at about 4°C in an angle-head rotor, i.e. under conditions which sediment calliphorin. The pellets were taken up in known volumes (0*3O-5 ml) of a solution containing 0.15 M NaCl and 20 mM phosphate (pH 6.3). The zero-time controls were treated in exactly the same way.
IdentiJication of proteins Immunodiffusion and immunoelectrophoresis were carried out in agar gel (1.5% w/v Ionagar No. 2 in 25 mM phosphate, pH 6-l), with antisera produced by injecting crude extracts of Calliphora pupae into rabbits and known to react with ten antigens present in extracts of Calliphora larvae (MUNN and GREVILLE, 1969). Antiserum to pure calliphorin was also used. Electrophoresis was carried out at 6 V/cm for 3 to 4 hr.
Estimation of amounts of protein Absolute quantities of proteins were not determined. The amounts present in the control and experimental samples extracted under identical conditions were compared on the basis of (a) the intensities of the precipitin lines obtained by immunodiffusion of twofold serial dilutions of the extracts and (b) of the intensity of staining by naphthalene black of zones separated by electrophoresis of the extracts on starch gel (9.6% w/v in 25 mM phosphate, pH 6.1).
Radioautography To obtain radioautographs, agar plates after immunodiffusion or immunoelectrophoresis were fixed in ethanol-glacial acetic acid-water (40 : 1 : 40 by vol.), dried, and then kept in close contact with X-ray film for 5 to 6 weeks.
CALLIPHORINSYNTHESISBY
LARVALFATBODY
1603
RESULTS
The immunodiffusion patterns show that the fat bodies contained calliphorin (arc 1) and small amounts of Protein II (arc 2) both at zero time (Fig. lb) and after the incubation (Fig. la). During the incubation calliphorin and Protein II were released into the suspending medium (Figs. 2a and 3a), no antigenic protein being found in the medium in the zero-time controls (Figs. 2b and 3b). In immunodiffusion tests (Fig. 3c, d) the precipitin lines obtained with serial dilutions of extracts of the fat bodies indicate that there was approximately as much calliphorin in the fat bodies after the incubation as there was at zero time, because the extract in each case gave a precipitin line when diluted 1 : 64 but not when diluted 1 : 128. Therefore, since calliphorin was released into the medium during the incubation, it seems likely that there was a net synthesis of this protein. Insufficient Protein II was present to form a precipitin line even when the extracts were diluted only 1 : 4. Arc 1 in the immunoelectrophoresis runs (Figs. 1 and 2) is ascribed to calliphorin because (a) it has the characteristic shape and position of the arc given by pure lcalliphorin (e.g. Fig. 2c, d of MUNN and GREVILLE, 1969); (b) Ouchterlony-type double diffusion tests carried out in agar gel with antiserum to pure calliphor:in proved that calliphorin was present in fat body extracts; and (c) the precipitin line given by fat body extract together with antiserum to crude pupal Calliphora extract joins up with the line given by fat body extract together with antiserum to pure calliphorin (Fig. 3i, j). Arc 2 in the immunoelectrophoresis is likewise ascribed to Protein II on the grounds of its characteristic shape and position, and this protein also has been identified in fat body extracts by Ouchterlony-type tests. Radioautographs of the patterns of arcs and precipitin lines show that label from L-[UJ4C] va 1ine was incorporated into calliphorin and into Protein II. Most of the labelled protein was released by the fat bodies during the incubation (Figs. 2c: and 3e). A small amount of labelled calliphorin and Protein II was, however, retained by them (Figs. lc and 3g) and, so far as can be judged from the intensities on the plates, at the end of the incubation the fat bodies contained calliphorin of low specific radioactivity. In the immunoelectrophoresis runs there was labelling of some material which formed a diffuse streak, or possibly arc (Figs. 1 and 2), and immunodiffusion tests indicated that, including calliphorin and Protein II, three, or possibly four, labelled proteins were released by the fat bodies during the incubation (Fig. 3k, 1). Further evidence that there was net synthesis of calliphorin during the incubation of the fat bodies was obtained by comparison of the intensity of staining by naphthalene black of zones obtained by starch-gel electrophoresis (Fig. 4). In this expseriment the fat bodies were homogenized in the suspending medium after incubation and the extract prepared as before; exactly the same procedure was app’lied to the zero-time control. The calliphorin zones, identified with a marker, thus represented the total amount of calliphorin present, and this is seen The experiment also confirms that to have increased during the incubation.
1604
E. A. MUNN,G. M. PRICE, AND
G. D. GREVILLE
calliphorin and Protein II are the major soluble protein components present in, and synthesized by, the fat bodies; since antiserum to pupal extracts was used, any antigens specific to larvae would escape detection in the immunological tests. DISCUSSION The principal protein found in fat bodies isolated from 3-day larvae of C. erythrocephala was calliphorin, and this was accompanied by relatively small amounts of Protein II and minor amounts of other soluble proteins (Fig. 4). These latter did not react appreciably with the antisera used in this work, which were formed against antigens extracted from pupae (Fig. 1). When the fat bodies were incubated in an amino acid mixture both calliphorin and Protein II were released into the incubation medium, and label from L-[U-14C]valine was incorporated into these two proteins. Most of the 14C incorporated was associated with the released protein, but a small amount was associated with the protein retained by the fat bodies. These observations are in accord with those of PRICE (1966), who found that when fat bodies from C. erythrocephala were incubated with amino acids there was a net synthesis of protein and incorporation of label from [U-i4C]valine; much of the labelled protein was released into the suspending medium during the incubation. PRICE (1966) also showed that protein released during the incubation of fat bodies in absence of [U-14C]valine did not become labelled on subsequent incubation with this compound, and so eliminated the possibility of isotopic exchange. The results in the present paper show that the protein which is synthesized consists almost entirely of calliphorin together with some Protein II. Price’s finding that there was a net synthesis of protein therefore supports the evidence presented here of a net synthesis of calliphorin. Using starch-gel electrophoresis, LAUFER (1960) found three proteins in fat bodies of female giant silk moths (Samiu Cynthia) with similar mobilities to those of three components of the haemolymph, and considered that the fat body was ‘strongly implicated’ as the source of these haemolymph proteins. LAUFER (1961) obtained further evidence for this in organ-culture experiments; two of the three proteins were detected in the medium in which fat body was cultured, and a malate dehydrogenase isoenzyme, corresponding in mobility to one in the haemolymph, also appeared in the culture medium. SHIGEMATSU(1958) found that, on incubation, fat bodies from the silkworm Bombyx mori synthesized proteins with the same electrophoretic mobilities on paper as those of the main globulin components of the haemolymph; the quantities were such that the fat body could be regarded as the sole source of these haemolymph globulins. Evidence that the larval fat body is the source of haemolymph proteins in the case of C. evythrocephala was obtained by PRICE and BOSMAN (1966); they found that when fat bodies of 4- to 7-day-larvae were incubated with amino acids the proteins released into the medium gave an electrophoretic pattern on acrylamide gel almost identical with that given by the haemolymph proteins. The principal protein of the haemolymph of Calliphora larvae is calliphorin (MUNN and
(ai
(b)
(¢)
(d)
FIC. 1. Immunoelectrophoresis patterns showing calliphorin (arc 1) and Protein II (arc 2) present in isolated larval fat bodies, (a) after incubation with an amino acid mixture containing [UJ4C]valine, (b) at zero time. Antiserum to crude pupal Calliphora extract was used. (c), (d) Radioautographs of the arcs in (a) and (b) respectively. For experimental details see Materials and Methods.
|
(a}
(bl
(c)
(d)
FIc. 2. Immunoelectrophoresis patterns given by antigens present in the m e d i u m in which the fat bodies were suspended; (a) after incubation, (b) zero-time controls. Calliphorin (arc 1) and Protein I I (arc 2) were released into the m e d i u m during the incubation, neither being detectable in the m e d i u m at zero time. A n t i s e r u m to crude pupal Calliphora extract was used. (c), (d) Radioautographs of (a) and (b) respectively. For experimental details see Materials and Methods.
Ditution
(a)
(e)
(b)
(f)
(¢)
(gl
(d)
(h)
1:
4
8
16
32
64
128
256
1
2
•
8
16
32
64
(il
(j) Ditution I :
(k) DItution 1:
Fxc. 3 (a)-(d). Immunodiffusion precipitin lines due to calliphorin before and after the incubation of the fat bodies, (a) in suspending m e d i u m after the incubation, (b) in suspending m e d i u m at zero time, (c) in fat bodies after the incubation, (d) in fat bodies at zero time. T w o f o l d serial dilutions of extracts prepared as described in Materials and Methods were placed in the wells, ranging from 1 : 4 on the left to 1 : 256 on the right; the slots contained antiserum to crude pupal Calliphora extract. (e), (f), (g), (h) Radioautographs of (a), (b), (c), (d) respectively. (i), (j) Immunodiffusion patterns obtained with extracts of fat bodies, (i) after incubation and (j) at zero time; *, slots containing antiserum to crude pupal extract; t, slot containing antiserum to pure calliphorin; in this experiment the proteins were not concentrated by high-speed centrifugation (see Materials and Methods) and the serial twofold dilutions began with undiluted extract on the left. (k) I m m u n o diffusion pattern and (I) corresponding radioautograph obtained from suspending m e d i u m after the incubation of the fat bodies, showing lines due to three labelled proteins ; the proteins in the m e d i u m were concentrated by high-speed centrifugation as in (a)-(d).
P
C
(a)
(b)
(c)
i iii
i ii : iii!i~i
(d)
(e)
Fro. 4. Patterns obtained by staining protein with naphthalene black after sta:rch-gel electrophoresis of extracts prepared from fat bodies together with the suspending medium, (a) before incubation and (b) after incubation, equivalent amounts of the two extracts being applied to the gel. (c) Repeat of (b) in a different experiment, run together with (d) a pure calliphorin marker. (e) Traces obtained from (a) and (b) by means of a Double Beam Recording Microdensitometer (Joyce, Loebl and Co. Ltd., Gateshead-on-Tyne) : Z, trace from (a) (zero time) and I, 1:race from (b) (incubated). The peak height of I is 1.26 times that of Z. C, calliphorin; P, Protein II.
CALLIPHORIN SYNTHESISBY LARVALFAT BODY
GREVILLE, 1969). From the observations the fat body is its most likely source.
in the present
1605
paper it appears that
Aciinowledgements-We are grateful to Dr. A. FEINSTEIN for advice and criticism and to Dr. R. W. Cox for help with the use of the microdensitometer. REFERENCES LAUFERH. (1960) Blood proteins in insect development. Ann. N. Y. Acad. Sci. 89,490-515. LAUFER H. (1961) Forms of enzymes in insect development. Ann. N. Y. Acad. Sci. 94, 825-8X. MUNN E. A., FEINSTEIN A., and GREVILLE G. D. (1967) A major protein constituent of pupae of the blowfly Calliphora erythrocephala. Biochem. J. 102, SP-6P. MUNN E. A. and GREVILLE G. D. (1969) The soluble proteins of developing Calliphora erythrocephala, particularly calliphorin, and similar proteins in other insects. J. Insect Physiol. In press. PRICE G. M. (1965) Nucleic acids in the larva of the blowfly Calliphora erythrocephala. J. Insect Physiol. 11,869478. PRICE G. M. (1966) The in vitro incorporation of [U-*4C]valine into fat body protein of the larva of the blowfly, Calliphora erythrocephala. J. Insect Physiol. 12, 731-740. PRICE G. M. and BOSMANT. (1966) The electrophoretic separation of proteins isolated from the larva of the blowfly, Calliphora erythrocephala. ‘J. Insect Physiol. 12, 741-745. SHICEMATSU H. (1958) Synthesis of blood protein by the fat body in the silkworm, Bombyx mori L. Nature, Lond. 182, 880-882.