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Proteolytic enzymes in various embryonic stages of the eggs of Locusta migratoria migratorioides (R. and F.)
J. ins. Physiol.,
1957, Vol. 1, pp. 279 to 285.
Pergamon Press Ltd., London
PROTEOLYTIC ENZYMES IN VARIOUS EMBRYONIC STAGES OF THE EGGS OF LOCUSTR NITGRATOR12l MIGRATORIOIDlL!3 (R. AND F.) A. SHULOV,
I%. P. PENER,
S. KUK-~EIRI
and N’. ~ICHT~~STEIN
Departments of Zoology and Biological Chemistry, The Hebrew University, Jerusalem (Received
17 April
1957)
Abstract-The action of homogenates of eggs of Lac~st~ ~~~~to~ ~~~to~~es (R. and F.) in various stages of development on casein and on Ieucylglycylglycine at various pH values was investigated. The pH values of the homogenates of eggsin different stages were found to vary from 6.0 to 6.6. In no case was hydrolysis of casein or leucylglycylglycine obtained with freshly laid eggs. At pH 5.6 a distinct cleavage of casein was obtained with batches of eggs varying from stage VIII to stage XII (i.e. during the polypod stage and the beginning of the oligopod stage). This action increased towards the end of development. At pH 7.8 a distinct action on casein appeared occasionally with batches of eggs varying from stage X to stage XX (i.e. from the advanced polypod stage to the stage when the head of the embryo reaches the anterior end of the egg after blastokinesis). This action was always found in the batches with eggs from stages XVIII to XXIII (i.e. immediately after the end of blastokinesis to shortly before hatching) and markedly increased towards hatching. At pH 6.5 a slight action on casein appeared with the batches containing eggs of stages VIII-XII. The cleavage becsme distinct with stages X-XX, and increased further towards the end of development. A distinct action on leu~lglycylglyc~e, measured mainly at pH 7.8, occurred with stages III-XI and a greater one with eggs of all higher stages, It is concluded from the results that the homogenates of the eggs contain at least two kinds of endopeptidases.
IT HAS been shown that a glycerine extract of the eggs of the Desert Locust, Schistocemz gregmzk (Forskg), during the first few days of development attacks casein at pH 8 only to a very slight degree, if at all, while towards the end of the development, and especially shortly before hatching, hydrolysis is clearly pronounced (KuK-MEW, SHULOV and LICHTENSTEIN, 1954). It therefore seemed of interest to study the correlation between the different stages of development of Iocust eggs and the proteolytic activity. For this study, Locusta ~~~~~0~~ ~~g~at~~o~des (R. and F.) was chosen, and the action of homogenates of these eggs, at different embryonic stages (SHULOV and PENER, 1957), on casein and leucylglvcvlglvcine was investigated at various pH values. MATERIAL
AND
METHODS
The continuous breeding of L. m. ~~~~t~r~o~de~ was started with eggs which were kindly supplied in 1953 by the Anti-Locust Research Centre, London. The locusts were kept under crowded conditions and were of distinct gregayia phase. 279
280
A.
SHULOV,M. P. PENER,S. KUK-MEIRI ANDN. LICHTENSTEIN
The experiments described below were carried out with eggs laid between April 1955 and February 1956. The locusts laid their egg pods in tubes filled with moist sand (HUNTERThe tubes were checked and the egg pods removed JONES, 1956). daily. The eggs were considered to be 0 days old at the time of removal.* At fixed intervals, l-10 of the egg pods were opened. One out of each six eggs in each pod was killed in a fixing fluid (Bouin), and its embryonic stage was determined. The other five were used for enzymatic experiments. The number of eggs used in one series of experiments varied from 14 to 240. The eggs taken for the experiments were weighed, washed, and ground in a mortar with water to a fine suspension which was subsequently diluted in order to make its final volume (in ml) numerically equal to 10 times the weight of the eggs. An equal volume of 0.1 phosphate buffer at the appropriate pH was added. A casein solution (1 per cent) was prepared by dissolving the protein in excess of 0.1 in NaOH and by adding HCI drop by drop to the desired pH. The volume was made up by adding the appropriate phosphate buffer. DL-leucylglycylglycine was used in 2 per cent aqueous solution. For testing the proteinase activity by Anson’s method (NORTHROP, KUNITZ and HERRIOTT, 1948) 1 ml of each enzyme and of the casein solution were mixed. Controls run simultaneously did not contain substrate or enzyme. The reaction mixture was kept at 36°C for about 24 hr under toluene, 3 ml of 5 per cent trichloracetic acid was added, and the mixture was kept overnight in the cold (+ 5°C) and filtered. To 1 ml of the filtrate were added 8 ml of O-5 N NaOH and 3 ml of phenol reagent of Folin and Ciocalteou. The colour produced was measured in the KlettSommerson photoelectric calorimeter using filter 69. The colour values obtained with corresponding mixtures at the beginning of the experiments as well as the controls were subtracted. In some of the experiments a part of the filtrate obtained on addition of trichloracetic acid was also tested, after appropriate dilution with water, in the Beckman IJV Spectrophotometer at 273 rnp (HOLIDAY, 1936). The action of leucylglycylglycine was measured as follows: to 5 ml of leucylglycylglycine solution were added 5 ml of 0.1 M phosphate buffer and 2.5 ml of enzyme solution. Samples of 5 ml were tested by Sorensen form01 titration method at the beginning, and again after standing for 24 hr under toluene at 36°C. Controls were run with enzyme or substrate omitted. The pH values were measured with the aid of a Beckman glass-electrode pH meter. RESULTS In Table 1 are presented the results of experiments on the action of the egg homogenates at different stages of embryonic development on casein and on leucylglycylglycine at various pH values. The table shows that the morphological changes of a developing embryo are followed by qualitative as well as quantitative variations in the proteolytic activities. * The pods were kept in moist sand at 27 + 1°C.
PROTEOLYTIC
ENZYMES
IN EGGS OF LOCUSTA
MIGRATORIA
MIGRATORIOIDES
281
Action on casein at pH 7.8. No significant action could be detectedin the batches of eggs in stages O-XIV (i.e. from the beginning of the development up to the last morphological stage before katatrepsis). However, in the batches of eggs ranging from stage X to stage XX (i.e. when the thoracic appendages of the embryo in a polypod stage begin turning medially until the head of the late oligopod embryo reaches the anterior end of the egg) proteolytic action was occasionally found. The batches of eggs of stages XVIII-XXIII (i.e. from the stage when half of the egg is occupied by the embryo after the end of blastokinesis until hatching) always showed proteolytic action. This action markedly increased towards the end of the development and at hatching. Action on casein at pH 5.6. No action was found at the beginning of development (stage 0), but a distinct action could be detected at stages VIII-XII (during the polypod and beginning of the late oligopod stages). This action increased in the subsequent stages, the rate of increase being higher towards the end of deveiopment and at hatching. Action on casein at pH 6.5. No action was found at stage 0 and only a very slight one, if any, at stages VIII-XII. This action increased and became distinct in the batches containing stages X-XX, increasing again towards the end of development and at hatching. Action on leucylglycylglycine at pH 7.8. No action was found at stages O-I (i.e. up to 2 days). A distinct one, however, was found with stages III-XI and there was an increase from XI until XIX, followed by a slight decrease towards the end of the development. As may be seen from Table 1, no endopeptidase activity (cleavage of casein) could be detected at the beginning of the development, under the conditions of At stages VIII-XII an action is present at pH 5.6 but no these experiments. action was found at pH 7.8. During stages X-XX there is evidence of action at pH 5.6 and occasionally at pH 7.8. From XVIII to XXIII action was found at both pH values, the cleavage at the lower value being predominant. Shortly before hatching and at hatching the cleavage increased at both pH values; in this case, however, the hydrolysis was not more pronounced in the acid medium. Several measurements carried out with the aid of the Beckman Spectrophotometer (wavelength 273 mk) gave results of the same order of magnitude as those obtained by the calorimetric method. Pre-incubation of the egg homogenates for 2 and 24 hr resulted in only a slight decrease, if any, in the cleavage of casein. These experiments were carried out at pH 7.8 with eggs of stages XX-XXIII. Cleavage of casein was also measured after an incubation of 72 hr at pH 7.8, with the eggs of stages XI-XVIII, XVII-XX, and XXI-XXIII. In all these cases the cleavage was 2 ‘to 3 times stronger than that obtained after 24 hr. The pH values of the egg homogenates in water varied from 6 to 6.6 (Table 2). Th ey were measured in 7 batches of eggs, each taken from several pods.
282
A.
SHULOV,
P. PENER,S.
M.
KLJK-MEIRI
AND
N.
LIWTENSTEIN
DISCUSSION
The results presented above suggest that in the developing egg there are at least two kinds of endopeptidases. One measured at pH 7.8 is probably trypsin-like, whereas the other, which is responsible for the cleavage of the casein at pH 5.6, TABLE
~-ACTION
OF HOMOGENATES OF Locusta
EGGS AT DIFFERENT STAGES
migratoria migratorioides
(The figures for the action on casein represent the increase in Klett-Sommerson calorimeter reading and
Serial number of expe~im~t
2 I____
!
3
Time of experiment
Embryonic stage (SAULOV and PENER,
19%‘)
0
0
I
____I
Action on casein 7.8 rt 0.1 pE3. 6.5 $z 0.1. 5.6 rt 0.1
-2 -__
Action on Ioucylgtycylglycine
f7 +2 -9
__ d__ __ __ __ _. -I i I -
0.0
-
oG3
0.25
__-~-S
5
6
~~~____~ f10 +28 +81
l-2 -
-
+7 +23 +83
+2 -
-
-
-
probably belongs to the cathepsine-like type of enzymes. The cleavage of the casein found at pH 6.5 may be caused by the weak action of one of the enzymes or by both enzymes reacting together. As pointed out in our previous paper (KuK-MEIRI, Sautov and LXCHTENSTEIN, 1954), there is a possibility of the existence of a correlation in time between the TABLE
2-pH
VALUES OF HOMOGENATES OF EGGS OF Locusta VARIOUS EMBRYONIC STAGES
Embryonic
Serial number of Age of eggs experiments in Table 1 (days) 6
4and5
14
7
migratorioides
pII values
stages
7 and 8
16
migratoria
(zlz O-1)
III-XI
6.0
XI-XVIII
6-2
X-XIX
6.3
17
7
XI-XX
6.6
18
8
XVII-XVIII
6.4
26
13
28
XX-XXI
6.5
XXI-XXIII
1.5 I
I
I
6.5 I
AT
PROTEOLYI’IC
ENZYMES
IN EGGS OF LOCUSTA
MIGRATORLA
283
MIGWTORIOIDES
appearance of proteolytic enzymes and certain stages of the development of the eggs of Schistocercagregaria. The present paper shows that there is a correlation in time between the appearance of proteases and the different stages of egg development of Locusta migratoria migratorioides. OF EMBRYONIC DEVELOPMENT ON CASEIN AND LEUCYLGLYCYLGLYCINE AT VARIOUS pH VALUES those for the action on leucylglycylglycine represent the increase in titration value in ml of O-O.5N NaOH.)
17 ___--__-__
18
sz 1956 Jan. _~___________ 200 113 ____-~~___ 13.6 -~--__
19
20
Feb. 1956
Jan. 1956
July 1955
22 23 ______________ Jan. 1956
Dec. 1955
25 July 1955
78
77
38
110
85 47 ~____~______
15.6
17.9
16.1
19.4
17.8
16.1 _____
18.9
8
9
10
11,12
13
7 -___-___ XIxx __~___
XVIIXVIII
XVIIxx
+4 +40 -__
+39 +32 +114
f70 +53 +153 ___~__
1.0 0.1
21
-
-
XVIIIxx
11 11 __-_________ xx
XVIIIXXIII ~___
xxXXIII
+74 -
+45 +53 +142
+102 -
1.1
-
-
-__
+ 68. +55 f130
-
26 Jan. 1956 __~
27 Jan. 1956
28 Sept. 1955 ____
170
?
50
?
16.6
15
15
13
14
xxXXII ~_____~
xxXXII
XXIXXIII
XXIXXIII
XXIII
+44 ----__
t124 +79 +155
f138 +94 +149
+41 +48 -
+252 +215 +219
-
-
1.05 0.3
-
1.1
~~
1
31
132
Feb. 1956
17.05 17.05 __________
61
30
29
__ __ __ __ _.
XXIII __~___
~~ 1.0 -
/
XXIIIhatching
+170 -
I
XXIIIhatching
+ 183 +134 +218 ~__
I I--
33 JOY 1955 14 18.9 17
hatching
__ f300
__ -
-
_
The presence of the cathepsin-like endopeptidase in the eggs of Musca domestica was recently described by GREENBERGand PARETSKY(1955). Our experiments have shown that the proteolytic activity at pH 5.6 did not appear at the start of the development, but did so at quite early stages (VIII-XII). As found by FRUTON and his collaborators (FRUTON et al., 1951 ; JONES et al., 1952), intracellular animal and plant proteinases exhibit hydrolysing actions at a pH near 5, whereas at higher pH values transpeptidation reactions also occur. The catalysis of transpeptidation reactions is possibly a major function of the intracellular proteinases at physiological pH values (FRUTON and SIMMONDS, 1953). It is possible that the cathepsin-like enzyme found by us is instrumental in a similar way during the development of the locust egg. This suggestion should be considered on the basis of the fact that the pH value of the egg homogenates fluctuates between 6 and 6.6 at various stages of the development (Table 2). Further proof of the suggestion that this enzyme has cathepsin-like properties is furnished by the findings of NORMAN (1954). In his paper on the eggs of Melanoplus difleerentialis he states that the quantity of non-proteinic SH (probably glutathion) increases in the embryo during the development of the eggs. It is well known that glutathion is a natural activator of the cathepsins. The enzyme responsible for the cleavage of the casein, which has been found at pH 7.8, seems to belong to the trypsin group, well known from the digestive system of insects (DAY and WATERHOUSE,1953).
A.
284
SHULOV, M. P. PENER,S. KUK-MEIRIANDN. LICHTENSTEIN
ROONWAL (1937), d escribing the development of the midgut of Locusta migratoria migratorioides, pointed out that shortly after the blastokinesis definite groups of cells appear on the ends of both the proctodeal and the stomodeal invaginations which take part in the organization of the midgut. According to his observation (at 33”(Z), the blastokinesis occurs on the sixth day of the development and the provisional dorsal closure one day later. The definite midgut arises 5 days after the blastokinesis, i.e. 2 days before hatching, the whole development of the egg thus taking 13 days. Our experiments have been carried out at 27°C and the whole development takes 16-17 days (SHULOV and PENER, 1957). There is a slight difference as compared with the findings of HAMILTON(1950), who with the same insect at 26.7”C observed hatching after 15 days. The blastokinesis occurred in our experiments on the seventh-eighth day (at 27°C). The beginning of the activity of the trypsin-like enzyme thus coincides with the beginning of the organization of the midgut after blastokinesis (stage XVIII) in our experiments. The period of the prominent activity of this enzyme takes place simultaneously with the advancing organization of the midgut and reaches the maximum just before hatching and at hatching. Only a few papers deal with the occurrence of proteolytic enzymes in the eggs of insects. LICHTENSTEIN (1947) obtained cleavage of casein at pH values near 8 with glycerol extracts of the eggs of Bombyx mori only a few days before hatching. LICHTENSTEIN,BODENHEIMER and SHULOV(1949) described the action of glycerol extracts of developing eggs of Dociostaurus maroccanus kept in moist sand at 27°C. If one compares the results obtained by these authors with the study of BODENHEIMER and SHULOV(195 1) on the ecological embryology of this species, it will be seen that a distinct cleavage of casein at pH 8 failed to appear in extracts of eggs prepared before the end of blastokinesis. Here, too, the action on casein markedly increased in extracts made shortly before hatching. A considerable proteolytic action on casein, at pH 8, of the glycerol extracts of the eggs shortly before hatching was also found with Schistocerca gregaria (KuK-MEIRI, SHULOV and LICHTENSTEIN,1954). It appears from our results and from the preceding investigations that the appearance of a proteinase activity at alkaline pH values is connected with the development of the digestive system. REFERENCES BODENHEIMER F. S. and SHULOV A. (1951) Egg-development
and diapause in the Moroccan Locust. Bull. Res. Coun. Israel 1, 59-75. DAY M. F. and WATERHOUSE D. F. (1953) The mechanism of digestion (Ed. K. D. ROEDER). Insect PhysioZogy pp. 311-330. Wiley, New York; Chapman and Hall, London.
FRUTON J. S., JOHNSTON R. B. and FRIEDM. (1951) Elongation of peptide chains in enzymecatalysed transamidationreactions. J. biol. Chem. 190,39-53. FRUTONJ. S. and SIMMONDSS. (1953) General Biochemistry. Wiley, New York; Chapman
and Hall,
London.
GREENBERG B. and PARETSKY D. (1955) Proteolytic enzymes in the house fly, Musca domestica (L.).
Ann.
ent. Sot. Amer.
48, 46-50.
PROTEOLYTIC ENZYMES IN EGGSOFLOCUSTA
MIGRATORIA
MIGRATORIOIDES
285
HAMILTONA. G. (1950) Further studies on the relation of.humidity and temperature to the development of two species of African locusts-Locusta m&rat&a migratorioides (R. and F.) and Schistocerca gregaria (Forsk.). Trans. R. ent. Sot. Lond. 101, 1-58. HOLIDAY E. R. (1936) Spectrophotometry of proteins. I. Absorption spectra of tyrosine tryptophane and their derivatives. II. Estimation of tyrosine and tryptophane in proteins. Biochem. J. 30, 1795-1803. HUNTER-JONES P. (1956) Instructions for Rearing and Breeding Locusts in the Laboratory. Anti-Locust Research Centre, London. JONESM. E., HEARNW. R., FRIED M. and FRUTONJ. S. (1952) Transamidation reactions catalysed by cathepsin C. r. biol. Chem. 195, 645-656. KUK-MEIRI S., SHULOVA. and LICHTENSTEIN N. (1954) Proteases of the eggs of the Desert Locust (Schistocerca gregaria Forskal). Bull. Res. Corm. Israel 4, 66-68. LICHTENSTEINN. (1947) Proteolytic enzymes of insects. I. Proteases of the silkworm (Bombyx mom L.). Enzymologia 12. 156-165. LICHTENSTEIN N., BODENHEIMER F. S. and SHULOVA. (1949) Proteolytic enzymes of insects. II. Proteases of the eggs of the Moroccan Locust (Dociostaurns maroccanns Thnbg.). Enzymologia 13, 276-280. NORMANC. (1954) Quantitative study of distribution of sulfhydryl groups in the developing grasshopper (Melanoplus differentialis) embryo. Physiol. 2001. 27, 141-152. NORTHROPJ. H., KUNITZ M. and HERRIOTTR. M. (1948) Crystalline Enzymes. Columbia University Press, New York. ROOMVAL M. L. (1937) Studies on the embryology of the African Migratory Locust, Locusta migratoria migratorioides Reiche and Frm. (Orthoptera, Acrididae). II. Organogeny. Philos. Trans. B227, 175-244. SHULOVA. and PENERM. P. (1957) A contribution to knowledge of the development of the egg of Locusta migratoria migratorioides (R. and F.). Locnsta. In press.
1957, Vol. 1, pp. 279 to 285.
Pergamon Press Ltd., London
PROTEOLYTIC ENZYMES IN VARIOUS EMBRYONIC STAGES OF THE EGGS OF LOCUSTR NITGRATOR12l MIGRATORIOIDlL!3 (R. AND F.) A. SHULOV,
I%. P. PENER,
S. KUK-~EIRI
and N’. ~ICHT~~STEIN
Departments of Zoology and Biological Chemistry, The Hebrew University, Jerusalem (Received
17 April
1957)
Abstract-The action of homogenates of eggs of Lac~st~ ~~~~to~ ~~~to~~es (R. and F.) in various stages of development on casein and on Ieucylglycylglycine at various pH values was investigated. The pH values of the homogenates of eggsin different stages were found to vary from 6.0 to 6.6. In no case was hydrolysis of casein or leucylglycylglycine obtained with freshly laid eggs. At pH 5.6 a distinct cleavage of casein was obtained with batches of eggs varying from stage VIII to stage XII (i.e. during the polypod stage and the beginning of the oligopod stage). This action increased towards the end of development. At pH 7.8 a distinct action on casein appeared occasionally with batches of eggs varying from stage X to stage XX (i.e. from the advanced polypod stage to the stage when the head of the embryo reaches the anterior end of the egg after blastokinesis). This action was always found in the batches with eggs from stages XVIII to XXIII (i.e. immediately after the end of blastokinesis to shortly before hatching) and markedly increased towards hatching. At pH 6.5 a slight action on casein appeared with the batches containing eggs of stages VIII-XII. The cleavage becsme distinct with stages X-XX, and increased further towards the end of development. A distinct action on leu~lglycylglyc~e, measured mainly at pH 7.8, occurred with stages III-XI and a greater one with eggs of all higher stages, It is concluded from the results that the homogenates of the eggs contain at least two kinds of endopeptidases.
IT HAS been shown that a glycerine extract of the eggs of the Desert Locust, Schistocemz gregmzk (Forskg), during the first few days of development attacks casein at pH 8 only to a very slight degree, if at all, while towards the end of the development, and especially shortly before hatching, hydrolysis is clearly pronounced (KuK-MEW, SHULOV and LICHTENSTEIN, 1954). It therefore seemed of interest to study the correlation between the different stages of development of Iocust eggs and the proteolytic activity. For this study, Locusta ~~~~~0~~ ~~g~at~~o~des (R. and F.) was chosen, and the action of homogenates of these eggs, at different embryonic stages (SHULOV and PENER, 1957), on casein and leucylglvcvlglvcine was investigated at various pH values. MATERIAL
AND
METHODS
The continuous breeding of L. m. ~~~~t~r~o~de~ was started with eggs which were kindly supplied in 1953 by the Anti-Locust Research Centre, London. The locusts were kept under crowded conditions and were of distinct gregayia phase. 279
280
A.
SHULOV,M. P. PENER,S. KUK-MEIRI ANDN. LICHTENSTEIN
The experiments described below were carried out with eggs laid between April 1955 and February 1956. The locusts laid their egg pods in tubes filled with moist sand (HUNTERThe tubes were checked and the egg pods removed JONES, 1956). daily. The eggs were considered to be 0 days old at the time of removal.* At fixed intervals, l-10 of the egg pods were opened. One out of each six eggs in each pod was killed in a fixing fluid (Bouin), and its embryonic stage was determined. The other five were used for enzymatic experiments. The number of eggs used in one series of experiments varied from 14 to 240. The eggs taken for the experiments were weighed, washed, and ground in a mortar with water to a fine suspension which was subsequently diluted in order to make its final volume (in ml) numerically equal to 10 times the weight of the eggs. An equal volume of 0.1 phosphate buffer at the appropriate pH was added. A casein solution (1 per cent) was prepared by dissolving the protein in excess of 0.1 in NaOH and by adding HCI drop by drop to the desired pH. The volume was made up by adding the appropriate phosphate buffer. DL-leucylglycylglycine was used in 2 per cent aqueous solution. For testing the proteinase activity by Anson’s method (NORTHROP, KUNITZ and HERRIOTT, 1948) 1 ml of each enzyme and of the casein solution were mixed. Controls run simultaneously did not contain substrate or enzyme. The reaction mixture was kept at 36°C for about 24 hr under toluene, 3 ml of 5 per cent trichloracetic acid was added, and the mixture was kept overnight in the cold (+ 5°C) and filtered. To 1 ml of the filtrate were added 8 ml of O-5 N NaOH and 3 ml of phenol reagent of Folin and Ciocalteou. The colour produced was measured in the KlettSommerson photoelectric calorimeter using filter 69. The colour values obtained with corresponding mixtures at the beginning of the experiments as well as the controls were subtracted. In some of the experiments a part of the filtrate obtained on addition of trichloracetic acid was also tested, after appropriate dilution with water, in the Beckman IJV Spectrophotometer at 273 rnp (HOLIDAY, 1936). The action of leucylglycylglycine was measured as follows: to 5 ml of leucylglycylglycine solution were added 5 ml of 0.1 M phosphate buffer and 2.5 ml of enzyme solution. Samples of 5 ml were tested by Sorensen form01 titration method at the beginning, and again after standing for 24 hr under toluene at 36°C. Controls were run with enzyme or substrate omitted. The pH values were measured with the aid of a Beckman glass-electrode pH meter. RESULTS In Table 1 are presented the results of experiments on the action of the egg homogenates at different stages of embryonic development on casein and on leucylglycylglycine at various pH values. The table shows that the morphological changes of a developing embryo are followed by qualitative as well as quantitative variations in the proteolytic activities. * The pods were kept in moist sand at 27 + 1°C.
PROTEOLYTIC
ENZYMES
IN EGGS OF LOCUSTA
MIGRATORIA
MIGRATORIOIDES
281
Action on casein at pH 7.8. No significant action could be detectedin the batches of eggs in stages O-XIV (i.e. from the beginning of the development up to the last morphological stage before katatrepsis). However, in the batches of eggs ranging from stage X to stage XX (i.e. when the thoracic appendages of the embryo in a polypod stage begin turning medially until the head of the late oligopod embryo reaches the anterior end of the egg) proteolytic action was occasionally found. The batches of eggs of stages XVIII-XXIII (i.e. from the stage when half of the egg is occupied by the embryo after the end of blastokinesis until hatching) always showed proteolytic action. This action markedly increased towards the end of the development and at hatching. Action on casein at pH 5.6. No action was found at the beginning of development (stage 0), but a distinct action could be detected at stages VIII-XII (during the polypod and beginning of the late oligopod stages). This action increased in the subsequent stages, the rate of increase being higher towards the end of deveiopment and at hatching. Action on casein at pH 6.5. No action was found at stage 0 and only a very slight one, if any, at stages VIII-XII. This action increased and became distinct in the batches containing stages X-XX, increasing again towards the end of development and at hatching. Action on leucylglycylglycine at pH 7.8. No action was found at stages O-I (i.e. up to 2 days). A distinct one, however, was found with stages III-XI and there was an increase from XI until XIX, followed by a slight decrease towards the end of the development. As may be seen from Table 1, no endopeptidase activity (cleavage of casein) could be detected at the beginning of the development, under the conditions of At stages VIII-XII an action is present at pH 5.6 but no these experiments. action was found at pH 7.8. During stages X-XX there is evidence of action at pH 5.6 and occasionally at pH 7.8. From XVIII to XXIII action was found at both pH values, the cleavage at the lower value being predominant. Shortly before hatching and at hatching the cleavage increased at both pH values; in this case, however, the hydrolysis was not more pronounced in the acid medium. Several measurements carried out with the aid of the Beckman Spectrophotometer (wavelength 273 mk) gave results of the same order of magnitude as those obtained by the calorimetric method. Pre-incubation of the egg homogenates for 2 and 24 hr resulted in only a slight decrease, if any, in the cleavage of casein. These experiments were carried out at pH 7.8 with eggs of stages XX-XXIII. Cleavage of casein was also measured after an incubation of 72 hr at pH 7.8, with the eggs of stages XI-XVIII, XVII-XX, and XXI-XXIII. In all these cases the cleavage was 2 ‘to 3 times stronger than that obtained after 24 hr. The pH values of the egg homogenates in water varied from 6 to 6.6 (Table 2). Th ey were measured in 7 batches of eggs, each taken from several pods.
282
A.
SHULOV,
P. PENER,S.
M.
KLJK-MEIRI
AND
N.
LIWTENSTEIN
DISCUSSION
The results presented above suggest that in the developing egg there are at least two kinds of endopeptidases. One measured at pH 7.8 is probably trypsin-like, whereas the other, which is responsible for the cleavage of the casein at pH 5.6, TABLE
~-ACTION
OF HOMOGENATES OF Locusta
EGGS AT DIFFERENT STAGES
migratoria migratorioides
(The figures for the action on casein represent the increase in Klett-Sommerson calorimeter reading and
Serial number of expe~im~t
2 I____
!
3
Time of experiment
Embryonic stage (SAULOV and PENER,
19%‘)
0
0
I
____I
Action on casein 7.8 rt 0.1 pE3. 6.5 $z 0.1. 5.6 rt 0.1
-2 -__
Action on Ioucylgtycylglycine
f7 +2 -9
__ d__ __ __ __ _. -I i I -
0.0
-
oG3
0.25
__-~-S
5
6
~~~____~ f10 +28 +81
l-2 -
-
+7 +23 +83
+2 -
-
-
-
probably belongs to the cathepsine-like type of enzymes. The cleavage of the casein found at pH 6.5 may be caused by the weak action of one of the enzymes or by both enzymes reacting together. As pointed out in our previous paper (KuK-MEIRI, Sautov and LXCHTENSTEIN, 1954), there is a possibility of the existence of a correlation in time between the TABLE
2-pH
VALUES OF HOMOGENATES OF EGGS OF Locusta VARIOUS EMBRYONIC STAGES
Embryonic
Serial number of Age of eggs experiments in Table 1 (days) 6
4and5
14
7
migratorioides
pII values
stages
7 and 8
16
migratoria
(zlz O-1)
III-XI
6.0
XI-XVIII
6-2
X-XIX
6.3
17
7
XI-XX
6.6
18
8
XVII-XVIII
6.4
26
13
28
XX-XXI
6.5
XXI-XXIII
1.5 I
I
I
6.5 I
AT
PROTEOLYI’IC
ENZYMES
IN EGGS OF LOCUSTA
MIGRATORLA
283
MIGWTORIOIDES
appearance of proteolytic enzymes and certain stages of the development of the eggs of Schistocercagregaria. The present paper shows that there is a correlation in time between the appearance of proteases and the different stages of egg development of Locusta migratoria migratorioides. OF EMBRYONIC DEVELOPMENT ON CASEIN AND LEUCYLGLYCYLGLYCINE AT VARIOUS pH VALUES those for the action on leucylglycylglycine represent the increase in titration value in ml of O-O.5N NaOH.)
17 ___--__-__
18
sz 1956 Jan. _~___________ 200 113 ____-~~___ 13.6 -~--__
19
20
Feb. 1956
Jan. 1956
July 1955
22 23 ______________ Jan. 1956
Dec. 1955
25 July 1955
78
77
38
110
85 47 ~____~______
15.6
17.9
16.1
19.4
17.8
16.1 _____
18.9
8
9
10
11,12
13
7 -___-___ XIxx __~___
XVIIXVIII
XVIIxx
+4 +40 -__
+39 +32 +114
f70 +53 +153 ___~__
1.0 0.1
21
-
-
XVIIIxx
11 11 __-_________ xx
XVIIIXXIII ~___
xxXXIII
+74 -
+45 +53 +142
+102 -
1.1
-
-
-__
+ 68. +55 f130
-
26 Jan. 1956 __~
27 Jan. 1956
28 Sept. 1955 ____
170
?
50
?
16.6
15
15
13
14
xxXXII ~_____~
xxXXII
XXIXXIII
XXIXXIII
XXIII
+44 ----__
t124 +79 +155
f138 +94 +149
+41 +48 -
+252 +215 +219
-
-
1.05 0.3
-
1.1
~~
1
31
132
Feb. 1956
17.05 17.05 __________
61
30
29
__ __ __ __ _.
XXIII __~___
~~ 1.0 -
/
XXIIIhatching
+170 -
I
XXIIIhatching
+ 183 +134 +218 ~__
I I--
33 JOY 1955 14 18.9 17
hatching
__ f300
__ -
-
_
The presence of the cathepsin-like endopeptidase in the eggs of Musca domestica was recently described by GREENBERGand PARETSKY(1955). Our experiments have shown that the proteolytic activity at pH 5.6 did not appear at the start of the development, but did so at quite early stages (VIII-XII). As found by FRUTON and his collaborators (FRUTON et al., 1951 ; JONES et al., 1952), intracellular animal and plant proteinases exhibit hydrolysing actions at a pH near 5, whereas at higher pH values transpeptidation reactions also occur. The catalysis of transpeptidation reactions is possibly a major function of the intracellular proteinases at physiological pH values (FRUTON and SIMMONDS, 1953). It is possible that the cathepsin-like enzyme found by us is instrumental in a similar way during the development of the locust egg. This suggestion should be considered on the basis of the fact that the pH value of the egg homogenates fluctuates between 6 and 6.6 at various stages of the development (Table 2). Further proof of the suggestion that this enzyme has cathepsin-like properties is furnished by the findings of NORMAN (1954). In his paper on the eggs of Melanoplus difleerentialis he states that the quantity of non-proteinic SH (probably glutathion) increases in the embryo during the development of the eggs. It is well known that glutathion is a natural activator of the cathepsins. The enzyme responsible for the cleavage of the casein, which has been found at pH 7.8, seems to belong to the trypsin group, well known from the digestive system of insects (DAY and WATERHOUSE,1953).
A.
284
SHULOV, M. P. PENER,S. KUK-MEIRIANDN. LICHTENSTEIN
ROONWAL (1937), d escribing the development of the midgut of Locusta migratoria migratorioides, pointed out that shortly after the blastokinesis definite groups of cells appear on the ends of both the proctodeal and the stomodeal invaginations which take part in the organization of the midgut. According to his observation (at 33”(Z), the blastokinesis occurs on the sixth day of the development and the provisional dorsal closure one day later. The definite midgut arises 5 days after the blastokinesis, i.e. 2 days before hatching, the whole development of the egg thus taking 13 days. Our experiments have been carried out at 27°C and the whole development takes 16-17 days (SHULOV and PENER, 1957). There is a slight difference as compared with the findings of HAMILTON(1950), who with the same insect at 26.7”C observed hatching after 15 days. The blastokinesis occurred in our experiments on the seventh-eighth day (at 27°C). The beginning of the activity of the trypsin-like enzyme thus coincides with the beginning of the organization of the midgut after blastokinesis (stage XVIII) in our experiments. The period of the prominent activity of this enzyme takes place simultaneously with the advancing organization of the midgut and reaches the maximum just before hatching and at hatching. Only a few papers deal with the occurrence of proteolytic enzymes in the eggs of insects. LICHTENSTEIN (1947) obtained cleavage of casein at pH values near 8 with glycerol extracts of the eggs of Bombyx mori only a few days before hatching. LICHTENSTEIN,BODENHEIMER and SHULOV(1949) described the action of glycerol extracts of developing eggs of Dociostaurus maroccanus kept in moist sand at 27°C. If one compares the results obtained by these authors with the study of BODENHEIMER and SHULOV(195 1) on the ecological embryology of this species, it will be seen that a distinct cleavage of casein at pH 8 failed to appear in extracts of eggs prepared before the end of blastokinesis. Here, too, the action on casein markedly increased in extracts made shortly before hatching. A considerable proteolytic action on casein, at pH 8, of the glycerol extracts of the eggs shortly before hatching was also found with Schistocerca gregaria (KuK-MEIRI, SHULOV and LICHTENSTEIN,1954). It appears from our results and from the preceding investigations that the appearance of a proteinase activity at alkaline pH values is connected with the development of the digestive system. REFERENCES BODENHEIMER F. S. and SHULOV A. (1951) Egg-development
and diapause in the Moroccan Locust. Bull. Res. Coun. Israel 1, 59-75. DAY M. F. and WATERHOUSE D. F. (1953) The mechanism of digestion (Ed. K. D. ROEDER). Insect PhysioZogy pp. 311-330. Wiley, New York; Chapman and Hall, London.
FRUTON J. S., JOHNSTON R. B. and FRIEDM. (1951) Elongation of peptide chains in enzymecatalysed transamidationreactions. J. biol. Chem. 190,39-53. FRUTONJ. S. and SIMMONDSS. (1953) General Biochemistry. Wiley, New York; Chapman
and Hall,
London.
GREENBERG B. and PARETSKY D. (1955) Proteolytic enzymes in the house fly, Musca domestica (L.).
Ann.
ent. Sot. Amer.
48, 46-50.
PROTEOLYTIC ENZYMES IN EGGSOFLOCUSTA
MIGRATORIA
MIGRATORIOIDES
285
HAMILTONA. G. (1950) Further studies on the relation of.humidity and temperature to the development of two species of African locusts-Locusta m&rat&a migratorioides (R. and F.) and Schistocerca gregaria (Forsk.). Trans. R. ent. Sot. Lond. 101, 1-58. HOLIDAY E. R. (1936) Spectrophotometry of proteins. I. Absorption spectra of tyrosine tryptophane and their derivatives. II. Estimation of tyrosine and tryptophane in proteins. Biochem. J. 30, 1795-1803. HUNTER-JONES P. (1956) Instructions for Rearing and Breeding Locusts in the Laboratory. Anti-Locust Research Centre, London. JONESM. E., HEARNW. R., FRIED M. and FRUTONJ. S. (1952) Transamidation reactions catalysed by cathepsin C. r. biol. Chem. 195, 645-656. KUK-MEIRI S., SHULOVA. and LICHTENSTEIN N. (1954) Proteases of the eggs of the Desert Locust (Schistocerca gregaria Forskal). Bull. Res. Corm. Israel 4, 66-68. LICHTENSTEINN. (1947) Proteolytic enzymes of insects. I. Proteases of the silkworm (Bombyx mom L.). Enzymologia 12. 156-165. LICHTENSTEIN N., BODENHEIMER F. S. and SHULOVA. (1949) Proteolytic enzymes of insects. II. Proteases of the eggs of the Moroccan Locust (Dociostaurns maroccanns Thnbg.). Enzymologia 13, 276-280. NORMANC. (1954) Quantitative study of distribution of sulfhydryl groups in the developing grasshopper (Melanoplus differentialis) embryo. Physiol. 2001. 27, 141-152. NORTHROPJ. H., KUNITZ M. and HERRIOTTR. M. (1948) Crystalline Enzymes. Columbia University Press, New York. ROOMVAL M. L. (1937) Studies on the embryology of the African Migratory Locust, Locusta migratoria migratorioides Reiche and Frm. (Orthoptera, Acrididae). II. Organogeny. Philos. Trans. B227, 175-244. SHULOVA. and PENERM. P. (1957) A contribution to knowledge of the development of the egg of Locusta migratoria migratorioides (R. and F.). Locnsta. In press.