Chemical composition and rate of synthesis of the larval salivary secretion of the fly Rhynchosciara americana

J. Insect Physiol.,

1975, Vol. 21, pp. 643 to 657. Pevgamon Press. Printed in Great Britain

CHEMICAL COMPOSITION AND RATE OF SYNTHESIS OF THE LARVAL SALIVARY SECRETION OF THE FLY RHYNCHOSCIARA AMERICANA ANTONIO Departamento

G. DE BIANCHI

and WALTER

R. TERRA

de Bioquimica, Instituto de Quimica, Universidade de SPo Paulo, Caixa Postal 20.780, S”aoPaulo, Brasil (Received 26 July 1974)

Abstract-The salivary secretion of Rhynchosciara americana was chemically analysed. The secretion shows a yellow colour, with a pH of 7.5 and protein as its major component (94.5 per cent of the secretion dry weight). Carbohydrates are minor components of the secretion which amount to 3.4 per cent of the secretion dry weight, of which 2.3 per cent are neutral carbohydrates and l-l per cent are galactosamine. The major amino acids present in the secretion proteins are aspartic acid, glycine, serine, and glutamic acid. The salivary secretion proteins can be separated into eleven protein fractions by urea-acrylamide gel electrophoresis from which nine fractions are PAS positive. The salivary pigment moves together with the protein fraction No. 8, which is quantitatively the most important one, and has spectral characteristics identical to a haemolymph pigment. The higher rate of gland protein labelling by 14C-phenylalanine determined in viva and in ti’tro occurs around the middle of the spinning stage at the same time as the appearance of the large chromosomal puffs. The r6le of the salivary secretion in cocoon production is discussed. INTRODUCTION

THE COMMUNALcocoon of the larvae of Rhynchosciara americana is made up of calcium carbonate and of silk. The calcium carbonate seems to come from the Malpighian tubules, while the silk is derived from the salivary secretion of the larvae of these flies (TERRA and DE BIANCHI, 1974). The amino acid composition of the cocoon silk is very different from other known silks (TERRA and DE BIANCHI, 1974). Since the study of silk proteins is more easily accomplished with proteins in a soluble form, the best way to study them is in the secretion in which they are present. Furthermore, study of the salivary secretion proteins is a first step in the identification of which proteins are synthesized in the glands and which proteins are taken up from the haemolymph. The sequestration of proteins from the haemolymph by the salivary glands of R. americana was shown by BIANCHIet al. (1973). In this paper chemical analysis of the salivary secretion was carried out with emphasis on the proteins, which were studied by either SDS or urea-polyacrylamide We also measured the rate of salivary gland protein labelling gel electrophoresis. 643

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before and at different times during the spinning stage. This kind of experiment gives the times for the protein synthesis activity of the glands, which can be correlated to cocoon production and to the large gland chromosomal puffs which appear in the middle spinning stage. MATERIALS

AND METHODS

Animals The animals were raised’ in the laboratory according to LARA et al. (1965). Larvae of the same chronological age frequently differ in physiological responses. Due to this the fourth larval instar was divided into periods based on physiological markers. The division into periods adopted here was that introduced by TERRA et al. (1973). Salivary secretion collection Mature larvae near or at the beginning of the spinning stage (larvae from the second or third ,period of the fourth instar) were lightly compressed between the forefinger and the thumb producing little drops of secretion from the mouth which were then collected in a hollowed-out glass slide. After compression the amount of salivary gland protein amounts to 29 pg of protein per gland. When a sufficient amount of secretion had been collected its volume was measured and the secretion was used in the determinations, directly or after solubilization in suitable solvents. Isolation of the salivary glands The salivary glands were dissected from the animals under a stereoscopical microscope. The dissected glands were accumulated in Lara and Toledo’s physiological solution for Rhynchosciara (ARMELINet al., 1969) maintained at 0°C. Protein determinations The salivary glands were homogenized with a Potter-Elvehjem homogenizer in O-1 N NaOH and the proteins were determined in the homogenate, after centrifugation, using the method of LOWRY et al. (1951) according to OYAMA and EAGLE (1956). The salivary secretion proteins solubilized in 5% sodium desoxicolate in 0.01 M KOH were measured using the biuret reagent according to GORNALLet al. (1949). When 2 N NaOH were used to solubilize the salivary secretion proteins these were determined by the method of ELLMAN (1962) or by the method of LOWRY et al. (1951) according to OYAMAand EAGLE(1956). Serum albumin was used as standard in all cases. Carbohydrate determination The salivary glands were homogenized in 0.1 N NaOH, centrifuged, and the supernatant taken to 0.5 N HClO, and centrifuged again. The total gland acidsoluble carbohydrate was determined in the supernatant according to MOKRASCH (1954) using glucose as a standard.

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Salivary secretion samples were hydrolysed in 3 N HCl, at lOO”C, for different lengths of time. The hydrolysates after passing through glass wool were divided into three aliquots. One was used for the determination of neutral carbohydrate according to DUBOISet al. (1956) and one to correct this result from the unspecific colour development resulting from the action of HaSO, upon the sample. Glucose was used as a standard. The third aliquot was used for the determination of hekosamines according to BOAS (1953). The standard used in this case was galactosamine-HCl. Salivary

secretion dry weight and pH

Samples of 50 ~1 of salivary secretion were weighed in weighing bottles after heating them at 100°C to constant weight. The pH of the salivary secretion was determined using the indicator paper ‘Neutralit’, pH 5-10, from Merck A. G., Darmstadt. The use of glass electrodes was not possible since the volume of secretion was too small and solidified soon after collection. Polyacrylamide

gel electrophmesis

Salivary secretion was solubilized in 0*05 M Tris-0.14 M glycine buffer, pH 8.3, containing 0.01 M methylamine and 3 M urea. The electrophoresis was performed in 3 M urea, 0.01 M methylamine 7% gels according to a modification of the system described by DAVIS (1964). Bromophenol blue was used for tracking and electrophoretic separation was obtained with a constant current of 5 mA per column. The gels were removed from the columns and the proteins stained with Amido black (PANYIMSand CHALKLEY,1969), while PAS staining was performed according to KORN and WRIGHT (1973) with lengthening of the time of treatment by Schiff reagent. The faint bands only appear after 24 to 48 hr. Controls not oxidized by periodic acid do not develop stained bands after those times. The Amido black stained gels were scanned at 650 nm in a microdensitometer (Spectrophotometer 240, Gilford Instrument). Peak areas were determined by planimetry. Determination

of the molecular weight of some salivary polypeptide

chains

Salivary secretion proteins solubilized in 1% SDS, 1% /3-mercaptoethanol, 0.01 M phosphate buffer, pH 7.0, were separated by electrophoresis in 10% polyacrylamide gels as described by WEBER and OSBORN(1969). The position of the tracking dye after the run was marked using a copper wire according to HEDRICKand SMITH (1968) and the staining and scanning performed as described above. The proteins used as standards were : serum albumin, egg albumin, chymotrypsinogen and myoglobin (all purchased from Sigma Chemical Company). Amino acid analysis Four aliquots of 50 ~1 of salivary secretion were sealed in evacuated glass ampoules containing 2.0 ml of 6 N HCl. Two ampoules were heated at 100°C for

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22 hr and the two others for 70 hr. After hydrolysis the ampoules were opened, and the materials were passed through glass wool and lyophilized. The residues were solubilized in a pH 2.20, O-2 N sodium citrate buffer and chromatographed in a Beckman Amino-acid Analyser (Model 120C) using a 2 hr schedule for protein hydrolysates based on SPACKMANet al. (1958). The identification of the peaks in the chromatograms was performed by comparing their elution time with those in a standard chromatogram and by measuring their 440/570 nm absorbance ratio (ZACHARIUSand TALLEY, 1962). The titres of acid-unstable amino acids were extrapolated to the zero time of hydrolysis according to HIRS et al. (1954). Determination of the rate of incorporation of labelled amino acids into salivary gland proteins In vivo: Four groups of 10 larvae were injected with a glass needle prepared from Microcaps of 15 ~1 (Drummond Scientific Co., Broomall, Pa.) with 2 ~1 of 14C-phenylalanine (1998 mC/mM; 0.1 mC/ml) and sacrificed after 30, 60, 90, and 120 min. The salivary glands were excised, homogenized in 1.0 ml of 0.1 N NaOH, and centrifuged. The supernatant was treated as described by the method of MANS and NOVELLI(1960). Proteins were measured as described by TERRAet al. (1974b). The radioactivity in the filters was determined in a liquid scintillation with an spectrometer (Beckman, Model LS-100) using PPO-POPOP-toluene efficiency of 74 per cent and a background of 56 counts/min. liz vitro: Four groups of 20 dissected salivary glands were incubated on 0.5 ml of R. americana incubation medium (TERRAet al., 1974a) for 60 min. At the end of this period, the glands were transferred to 0.5 ml of the medium containing 10 ~1 of 14C-phenylalanine (199.8 mC/mM; O-1 mC/ml) instead of the usual phenylalaThis incubation mixture was maintained with constant nine concentration. shaking at 28°C for 30, 60, 90, and 120 min. At the different time intervals the glands were picked from the medium and homogenized in O-5 ml of 0.1 N NaOH. The homogenate was treated as described for the in vivo experiments. RESULTS Protein and carbohydrate content in salivary glands during development The amount of protein and carbohydrate in the salivary glands rises continuously during the middle fourth instar (second period). When the larvae start to spin (third period), there is an abrupt fall in both protein and carbohydrate (Fig. 1). The protein content falls from 150 to 111 pg/gland. The mass of protein rises from the middle third period, becomes constant until the fifth period and then decreases continuously from the sixth period. The amount of carbohydrate decreases steadily from the beginning of the third period to the fifth period, when it becomes constant. Rate of protein labelling iz the salivary gland during development Part of the protein of the R. americana salivary secretion is sequestered from the haemolymph and part is synthesized in the salivary glands (BIANCHI et al., 1973).

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Periods 2

I

1 30

I

I

40 Larval

1

age,

I

50

I

I

60

I

days

FIG. 1. Total protein (Pr) and acid-soluble carbohydrate (Cb) in the salivary glands of R. a~y~ca~a. For each determination 10 freshly dissected salivary glands were homogenized in O-1 N NaOH. The homogenate was centrifuged at 12,100 g for 10 min. at 0°C. Protein and carbohydrate were determined in the supernatant as described in Materials and Methods. TABLE ~-BATE OF GLANDPROTEINLABELLING&z vivo BY r4C-PHENYLALANINE Bate of labelling

Period Second Third Fourth Fifth Pharate pupa

Counts/min per mg Counts/min per mg protein per hr * protein per hr$ 7110 10,000 52,000 47,000 27,000

6800 5840 20,200 47,000

Counts/min in gland Linear protein per larvae correlation per hr$ coefficient 5 1320 1755 4930 11,200

0.9740 OG3746 1*oooo 1*oooo 1.oooo

* The rates of protein IabeIling correspond to the slopes of straight lines drswn based on the rn~~~ square method from the specific activity data of labelled amino acid incorporation into gland protein, as a function of time. t The data of protein labelling were corrected to the same initial specific activity of the isotope used. In this calculation we used the phenylalanine titres present in the haemolymph in the different periods (TERRA et al., 1973) and the larval haemolymph volumes in the same periods (TERRA and BIGNCHI, unpublished titre in pharate pupa is unknown we could not results). Since the pheny~l~ne correct specific activity in this period. 1 The figures were calculated from the data of the second column in this table and from the protein masses present in the glands (Fig. 1). $ Calculated from the data of the specific activity as a function of time.

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The rate of gland protein labelling by 14C-phenylalanine, which is a function of protein synthesis, can be used therefore as an approximate measure of the rate of salivary secretion production. In &JO the amount of phenylalanine present in the haemolymph changes from one period to another (TERRAet al., 1973) and the protein masses of the glands also change (Fig. 1). For this reason corrections were applied to the data in order to permit a direct comparison of the results (Table 1). The in vitro data (Table 2) were corrected only for the variation of the protein in the gland since the specific activity of the isotope was constant in all experiments. In the in z&o and in oitro experiments, the higher rate of protein labelling occurs around the fifth period of the fourth instar. TABLE ~---RATE OF GLANDPROTEINLABELLINGin vitro

BY

14C-~~~~~~~~~

Rate of labelling

Period Third Fourth Fifth Pharate pupa

(Counts/min mg protein per hr *

Counts/min per mg protein per larvae per hr?

Linear correlation coefficient1

13,000 40,000 57,000 44,000

2520 9760 13,570 9420

1~0000 1~0000 0.8702 0.9171

* The rates of protein labelling correspond to the slopes of straight lines drawn based on the minimum square method from the specific activity data of labelled amino acid incorporation into gland protein, as a function of time, as described in Materials and Methods. + The figures were calculated from the data of the first column in this table and from the protein masses present in the glands (Fig. 1). 1 Calculated from the data of the specific activity as a function of time.

General aspects of the salivary secretion The salivary secretion obtained as described in Materials and Methods is yellow, has a pH of 7.5, and protein is its major component (Table 3). The secretion after drying becomes an insoluble glossy film, fitting the silk definition proposed by RUDALL and KENCHINGTON (1971). The salivary secretion displays a yellow-greenish fluorescence when illuminated with long-wave U.V. light (315-380 nm). Its absorption spectrum, recorded at pH 8.3, 1% SDS, O-1 M Tris-HCl buffer, shows absorption peaks at 344, 362, and 383 nm (Fig. 2). When the absorption spectrum of the salivary secretion is recorded in pH 9.5, O-1 M carbonate-bicarbonate buffer its spectrum shows just one peak at 310 nm (Fig. 2). The salivary secretion is insoluble in water or physiological solution. Fresh salivary secretion solubilizes easily in any of the following solutions : pH 9.5,O.l M carbonattibicarbonate buffer, 0.1 N NaOH, 1% SDS, or in 3 M urea.

LARVAL SALIVARYSECRETIONOF

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TABLE 3-R. americana SALIVARYSECRRTIONCOMPOSITION

Determination Dry weight * Protein* Neutral carbohydrates-t Galactosaminet

g/100 ml 7.2 f 2-4 6.80 * 0.07 0.163 0.080

o/0 of secretion dry weight 100.0 94;5 2.3 1.1

* Figures represent mean of 10 determinations and standard deviation. t Extrapolated values for zero time of hydrolysis as shown in Table 4.

l.O(

(A) 0.5c

A,

nm

FIG. 2. Absorption spectrum of the salivary secretion of R. americana in pH 8.3, 0.1 M NaCl, 1 y0 SDS, O-1 M Tris-HCl buffer (open circles) and in pH 9.5,O.l M carbonatebicarbonate buffer (solid circles). The two solutions had different concentrations of salivary secretion.

Carbohydrates A kinetic study of the yield of free neutral sugars during mild acid hydrolysis of the salivary secretion shows that after 1 hr of hydrolysis maximal carbohydrate recovery is obtained (Table 4). After this, there is a decrease in the recovery of the

‘6.50

ANTONIO G. DE BIANCHI AND WALTER R.TERRA TABLE~-~CUPERATIONOFNEUTRALSUGARSANDGALACTOSAMINEAFTERHYDROLYSIS OF R. americana SALIVARYSECRETION Recuperation(g/lOOml) Time of hydrolysis (hr)

Galactosamine

Neutral sugars

-

o-097 0.142 0.057 0.045 0.012 -

0 1 2 4

0.076 0.082 0.069 0.069 0.060 0.080

14 Value of extrapolation

0.163

neutral sugars which follows an apparent first-order

kinetics. The best figure for the amount of neutral sugars present in the salivary secretion was considered to be the extrapolated value corresponding to the zero time of hydrolysis, using the points with first-order decay. This type of approach is currently used in the determination of acid-unstable amino acids (HIRS et al., 1954) and has also been used in the quantification of carbohydrates (TERRA and DE BIANCHI, 1974). The same reasoning as above can be used to justify the use of the extrapolated value as the best figure for galactosamine (Table 4), which is the only hexosamine present in the salivary secretion. This conclusion is drawn from the results obtained by ion exchange chromatography, ‘where the only ninhydrin-positive peak that does not correspond to any amino-acid standard has an elution time identical to that of galactosamine. TABLE S-AMOUNTANDMIGRATIONOFTHEPROTEINFRACTIONSFROMTHESALIVARY SECRETION AFTER SEPARATION BY 3 M UREA-ACRYLAMIDE GEL ELECTROPHORESIS Amount ( y0 total area)t Fraction No.

&lI*

650 nm

310 nm

1 2

0.023 0.046 >

18

5

3 4

0.24 0.27

2 6

1 5

6 5 7 8 9 10 11

0.35 0.31 > 0.44 0.59 0.73 0.76 > 0.80

5

4

5 58

6 75

6

5

* Ratio between the migration of protein fractions and the migration of bromophenol blue. -t Percentages are referred to the total amount of protein which entered the separating gel.

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Electrophoresis of the secreted proteins Since ‘the salivary secretion is insoluble in pH 8.3 Tris-glycine buffer, the samples were solubilized in this buffer after the addition of 3 M urea and methylamine 0.01 M. Electrophoresis carried out in this buffer results in good resolution of the protein fractions and the salivary pigment migrates together with the protein fraction No. 8, which is quantitatively the most important one (Fig. 3 and Table 5). Since the salivary pigment has an absorption peak at 310 nm (Fig. 2) a densitometric scanning was recorded at this wavelength after a recording at 650 run. Table 5 shows that fraction No. 8 is the only fraction whose absorbance increases at 310 nm in comparison to the absorbance at 650 nm. This result confirms that the salivary pigment is present in fraction No. 8. The majority of the salivary protein fraction is PAS positive, although only the fraction Nos. 1,2,9, and 10 stain heavily (Fig. 3). It is important to note that the most faint bands both in the Amido black and in the PAS staining are sometimes missing in some electrophoretograms.

t

FIG. 3. Densitometric scan of a 3 M urea-acrylamide gel electrophoretogram of proteins from the salivary secretion of R. americana. AB, Amido black staining; PAS, PAS staining. In the diagram of the PAS-stained gel the shading is proportional to the staining.

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There was a retention of proteins on the top of the stacking gel which could be a consequence of an unspecific aggregation of the secreted protein in a similar way as seems to occur in the salivary secretion of Chironomus (Wosus et al., 1970). Determination of the molecular weight of some polypeptide fractions The results of an SDS-10% polya~~l~de gel electrophoresis of the salivary secretion stained with Amido black is presented in Fig. 4. Table 6 shows the molecular weights of fractions Nos. 8 to 13.

12

3 456769

IO

II

12 13

14 f

FIG. 4. Densitometric scan of an proteins from the salivary’secretion PAS, PAS staining. In the diagram tional

SDS-acrylamide gel electrophoretogram of of R. ~rn~~c~. AB, Amid0 black staining; of the PAS-stained gel the shading is proporto the staining.

In these conditions the lemon-coloured salivary pigment migrates close to the tracking dye, which may indicate a denaturation followed by dissociation of the c~omophore from the protein. A comparison of densitometric scannings at 310 and 650 nm was impossible in SDS gels since in these conditions the pigment is removed from the gels during Amido black staining.

LARVAL SALIVARY SECRETIONOF RHYNCHOSCIARA

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TABLE ~-AMOUNT, MIGRATION AND MOLECULAR WRIGHT OF THE POLYPEPTIDE FRACTIONS FROM THE SALIVARY SECRETIONAFTER SEPARATIONBY SDS-ACRYLAMIDE GEL ELECTROPHORESIS

Fraction No.

Rlll

2

0.048

3 4 5 6

Molecular weight (daltons x 10e3)

Amount (% of total area at 650 nm)

-

21

0.13 0.18

106 90

38

0.21 0.24

86 76

10

7 8 1 9 10 11

0.26 0.31 0.024 0.35 0.43 0.55

69 64 - 1 53 45 31

13 12 14

0.70 0.65 0.84

20 23 > -

2 3 5 2 10 10

The majority of the polypeptide fractions in the SDS gels are lightly PAS stained with the exception of fraction Nos. 13 and 8 that are more heavily stained (Fig. 4). Amino acid composition of the salivary secretion The amino acid composition of the salivary secretion is shown in Table 7. The major amino acids present are: aspartic acid, glycine, serine, glutamic acid and threonine. DISCUSSION

Storage of the salivary secretion R. americana salivary glands grow by cell enlargement without cell division. These glands grow and accumulate salivary secretion until the third period (Fig. 1). At this time the larvae begin to spin a communal cocoon and there occurs a significant decrease in the amount of protein present in the glands (Fig. 1). This decrease is chemical evidence of the participation of the salivary secretion in cocoon production. If we assume that the protein present in the pupal gland, at a stage when histolysis is just starting, is mostly structural protein (82 pg/gland) and subtract this figure from the maximum protein value determined (150 pg/gland), we can estimate that the highest amount of protein that the gland can store is 68 pg/gland. The amount of secretion protein stored by larvae is 68 x 2 = 136 ,ug. Since the total protein incorporated into the finished cocoon of R. americana is 1170 pg/larva (TERRA and DE BIANCHI, 1975) we can conclude that the amount of stored protein in

654

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TABLE ~-AMINO ACID COMPOSITION OF R. amevicana SALIVARYSECRETIONAND COCOON

Amino acid Aspartic acid Glycine Serine Glutamic acid Threonine Leucine Lysine Alanine Isoleucine Q-Cystine VaIine Arginine Tyrosine Phenylalanine Proline Methionine Histidine

Cocoon * 10.9 8.1 IO.2 11.6 9.3 7.3 4.2 6.4 5.5 4.5 4.4 3.1 3.0 2.6 1.3 1.0 0.8

Secretion

’

16.1 13.0 9.6 9.6 8.6 7.0 5.7 5.2 4.7 4.5 6.0 2.5 4.0 3.4 3.1 1.0 2.0

* Cocoon-insoluble proteins, according to TERRA and DE BIANCHI(1974).

the salivary glands at the time when the larvae begin to spin is only 10 per cent of the total protein that is extruded during the spinning. As a consequence 90 per cent of the secretion proteins are synthesized after the beginning of spinning, when the larvae do not eat any more. From the second day of the third period until the fifth period, the weight in lug of protein/gland remains constant (Fig. l), while protein synthesis increases (Tables 1 and 2). These results suggest that the glands are synthesizing mainly proteins which have been secreted. Protein synthesis in the salivary glands The highest rate of gland protein labelling by 14C-phenylalanine occurs around the middle spinning stage (fifth period of the fourth instar, Tables 1 and 2), although a high rate also occurs in the pharate pupa (Table 2). It is not possible to draw direct conclusions on the rate of protein synthesis only from proteinlabelling rate data (DINA~MARCAand LEVENBOOK, 1966). However, our data can be a real estimate of the rates of protein synthesis, since the highest increment of proteins in the communal cocoon occurs at the end of the spinning stage (between the fifth and the pharate pupal period) according to TERRA and DE BIANCHI (1975) as expected by the protein labelling rate data which must represent instantaneous rates.

LARVAL SALIVARY

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65.5

The enlargement of the B-2 salivary gland DNA puff (BREUER and PAVAN,1955) is coincident with the phase of the highest protein synthesis rate. The appearance of chromosomal puffs correlated with a high rate of amino acid incorporation into proteins was also found in Sarcophaga bullata (GOLDBERG et al., 1969). Salivary secretion composition The chemical analysis of the salivary secretion accounted by 98 per cent of the dry weight. The amounts of protein and carbohydrates present in R. americana salivary secretion (Table 3) are of the same order as in the salivary secretion of Camptochironomus (GROSSBACH, 1969). The ammo acid compositions of both secretions are, however, very different. Camptochironomus secretion is mainly composed of basic amino acids (GROSSBACH, 1969) while in R. americana secretion the acid amino acids are the major ones (Table 7). A lemon-coloured, fluorescent chromoprotein is found in the haemolymph of R. americana larvae. This pigment has an absorption peak at 310 nm in a neutral saline medium (TERRA et al., 1974b), absorption peaks at 342, 358, and 379 nm in chloroform-methanol-saturated water and a similar three-peaked absorption spectra in other denaturating conditions such as when submitted to heat or extreme pHs (TERRA and DE BIANCHI, unpublished results). The absorption spectrum of the salivary secretion in carbonate-bicarbonate buffer (Fig. 2) is identical to the spectrum of the haemolymph lemon-coloured chromoprotein in neutral saline, while the spectrum of the secretion in 1% SDS (Fig. 2) is similar to that of haemolymph chromoprotein in denaturating conditions. The data shown here indicate that both the haemolymph lemon-coloured chromoprotein and the one from the salivary secretion could be the same. Since the salivary glands sequester proteins from the haemolymph (BIANCHI et al., 1973), the lemon-coloured chromoprotein present in the salivary secretion could come from the haemolymph. The Tris-glycine-urea gel electrophoretogram is the best approximation we have about the migration of the secretion proteins since in the absence of urea they do not solubilize and in the SDS-gel we do not obtain any information about the chromoprotein which seems to dissociate in these conditions. Although in Fig. 3 we report the existence of eleven protein fractions in urea gels, we must bear in mind that in these conditions some variation in the number of the faint bands occurs. This variation should be a consequence of the low concentration of urea used which is sufficient to solubilize the proteins but is not sufficiently high to ensure complete dissociation of the proteins, resulting in unequal retention in the top of the gels among different runs. The fractionation .of the secretion polypeptides in SDS gels (Fig. 4) gave very reproducible results and we did not detect any retention of material at the top of the gels. Thus the finding that about 80 per cent of the secretory polypeptide chains has a molecular weight lower than 1 x 105 daltons (Table 6) is significant.

656

ANTONIO G. DE BIANCHIANDWALTER R. Tnnn~

.&&vary secretion and the COCOOG The ammo acid composition of the salivary secretion and of the insoluble organic material of the cocoon of R. americana shows some differences (Table 7). The discrepancy found is not great, however, since the five major amino acids present in both materials are the same and the amount of acid amino acids and the summation of the low molecular weight ammo acids glycine, alanine and serine are very similar. The differences seen in Table 7 could be a consequence of the fact that the salivary secretion accounts for the whole organic material of the cocoon, not only for the insoluble organic fraction. In this connexion it is interesting to note the occurrence of soluble proteins in the cocoon which could be present in part in the silk extruded by the larvae and are not solely derived from the faeces and/or haemolymph contaminants produced during remotion of the larvae from the communal cocoon (TERRA and DE BIANCHI, 1974). The differences’ found between the amount of carbohydrates present in the salivary secretion and in the insoluble fraction of the cocoon are so large that the explanation given for the amino acid discrepancies does not hold here. The insoluble fraction of the cocoon contains 18 per cent (w/w) of neutral carbohydrates and 4-l per cent (w/w) of galactosamine (TERRA and DE BIANCHI, 1974), while the salivary secretion contains 2.3 per cent (w/w) of neutral carbohydrates and 1-l per cent (w/w) of galactosamine (Table 3). The reason for these differences is not yet clear. One possibility is that there is an unequal distribution of the carbohydrate components of the secretion inside the lumen of the gland so that when the animal is compressed only some components are collected. Ack~o~~e~~ts-We are much indebted to Professor F. J. S. LARA for helpful discussions and for laboratory facilities. This work was supported by grants from the Funda@io de Amparo 1 Pesquisa do Estado de Sgo Paul0 (FAPESP).

REFERENCES ARMELINII. A., -CHINI R., and LARA F. J. S. (1969) Extraction and characterization of newly synthesized RNA from whole cells and cellular fractions of R. angelue. Biochim. biophys. Actu 190,358-367. DE BIANCEX~ A. G., TERRAW. R., and LARA F. J. S. (1973) Formation of salivary secretion in Rhynchosciara americana-I. Kinetics of labelled amino acid incorporation. r. Cell biol. 58,470476. BOAS N. F. (1953) Method for determination of hexosamines in tissues. r. biol. Chem. 204, 553-563. BREUER M. E. and PAVANC. (1955) Behavior of polytene chromosomes of lrl. angelue at different stages of larval development. Ckromosoma 7, 371-386. DAVIS B. J. (1964) Disc electrophoresis-II, Methods and application to human serum proteins. Ann. N. Y. Acad. Sci. 121,404-427. L. (1966) Oxidation, utilization and incorporation into DIN-RCA M. L. and L moox proteins of alanine and lysine during metamorphosis of the blowfly Phormia regina (Meigen). Arch. Biochem. 117, 110-119. DUBOISM., GILLES K. A., HAMILTONJ. K., REBERSP. A., and SMITH, F. (1956) Calorimetric methods for determination of sugars and related substances. A&y& Chem. 28, 350-356.

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