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Changes of enzyme patterns in the integument of Locusta migratoria during formation of adult cuticle
Insect Biochem., 1976, VoL 6, pp. 79 to 83. Pergamon Press. Printed in Great Britain.
CHANGES OF ENZYME PATTERNS IN T H E I N T E G U M E N T OF LOCUSTA MIGRATORIA D U R I N G F O R M A T I O N OF A D U L T CUTICLE BERNHARDSURHOLT Zoologisches Institut der Universit/it Mfinster, Lehrstuhl ffir Tierphysiologie, D-44 Miinster, Hindenburgplatz 55, Deutschland (Received 12 March 1975; reused 6jTune 1975)
A b s t r a c t - - T h e activities of 'key enzymes' of the intermediate metabolism (HK, G-6-PDH, GAPDH, G D H , LDH, SDH, ME, GOT, GPT, GluDH) in the integument of the abdomen and of the wings of the migratory locust, Locusta raigratoria, were measured 24 hr before, 24 hr after, and 12 days after the last ecdysis. During this time, a significant correlation between these enzyme activity patterns and the formation of the adult cuticle could only be demonstrated in the integument of the wings. G D H , GluDH, and HK, however, were exceptions; their activities increased distinctly during the time after ecdysis.
INTRODUCTION
The activity of the enzymes dissolved in the supernatant and of the S D H in the suspension was assayed spectrophotometrically in glass cells (diameter I cm) in a constant volume of 2.0 ml at 25°C-/-1°C. According to BEaOMSY~ (1970) were determined: (1) H K - - hexokinase - - [ATP: v-hexose-6-phosphotransferase] EC 2.7.1.1 (2) G - 6 - P D H - glucose-6-phosphate dehydrogenase - - [D-glucose-6-phosphate: N A D oxidoreductase] EC 1.1.1.49 (3) G A P D H - - glyceraldehydephosphate dehydrog e n a s e - [D-glyceraldehyde-3-phosphate : NAD oxidoreductase (phosphorylating] EC 1.2.1.12 (4) G D H - - glycerol-3-phosphate dehydrogenase - [L-glycerol-3-phosphate: N A D oxidoreductase] EC 1.1.1.8 (5) L D H - - lactate dehydrogenase R [L-lactate: N A D oxidoreductase] EC 1.1.1.27 (6) G O T glutamate oxalacetate aminotransferase --[L-aspartate: 2-oxoglutarate aminotransferase] EC 2.6.1.1 (7) G P T - - glutamate pyruvate amlnotransferase - - [Lalanine: 2-oxoglutarate aminotransferase] EC 2.6.1.2 (8) G l u D H - - glutamate dehydrogenase - - [L-glutamate: NAD(P) oxidoreductase (deaminating] EC 1.4.1.3 The assay of succinate dehydrogenase was carried out according to BRUICZKAet al. (1968): (9) S D H - - succinate dehydrogenase - - [succinate: (aceeptor) oxidoreductase] EC 1.3.99.1 The 'malic-enzyme' was determined according to B u c ~ et al. (1964): (10) ME - - ' malic-enzyme'-- malate dehydrogenase (decarboxylating-- [L-malate: N A D P oxidoreductase (decarboxylating)] EC 1.1.1.40
DURINO the metamorphosis f r o m the last larval stage to the winged adult intense reactions of decomposition, resynthesis and synthesis take place in the integument. One of the m o s t important syntheses in this process is that of chitin. It was demonstrated that this synthesis--especially after ecdysis--resorts to glucose as precursor (see SURHOLT and ZEBE, 1972). U n t i l now, however, little has been known about the basal metabolism and the activities of the enzymes of the intermediate metabolism in the integument. T h e r e f o r e it seemed a necessary precondition to measure the activities of definite ' key e n z y m e s ' of the intermediate metabolism in this tissue of the insect body in order to detect correlations between the intermediate metabolism, the formation of a n e w cuticle, and chitin synthesis. M A T E R I A L S A N D METHODS Specimens of Locusta migratoria were taken from a synchronous culture (see SURHOLTand Z~E, 1972). The enzyme activities were measured 24 hr before (pharate adult), 24 hr after, and 12 days after the final ecdysis. For each measurement material, either from the wings or from the integumentary tissue from 10 individuals (five male, five female) of the same age was used. The integumentary tissue was prepared as described (I-IEINRICH et al., 1974). The tissue samples were homogenized using a Potter-Elvehjem homogenizer in 5 ml 0.1 M S6rensen-phosphate buffer (pH=7-5) and centrifuged at 20,000g for 20 min. The supernatant was taken for measurement of the soluble enzymes, the resuspended precipitation for measurement of S D H ; protein was determined in the supematant according to L o w a Y et aL (1951).
]R]gSI~LTS I n order to show possible correlations between the intermediate metabolism and the chitin synthesis at 79
80
BERNHARD S U R H O L T
a) 1.000- U/rag Prot.(Iog) oIsO0"
-
~
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( ~)
-
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.
.
.
o.01o .
.
.
.
.
.
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b) % 250.
250
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(1)
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200.
150
150 130 113 ioo
100
100
S0-
rlnl la llN
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i
".
,.
H 21
1,1" nrl 76
"
SD
M H I[ 7 7 rtf~
34 Du H tO
I"1
T D
D
7
50
I0
0
Fig. 1. Activities of I0 'key enzymes' of the intermediate metabolism in the abdominal integument of Locusta migratoria 24 hr before (=pharate animal) (1), 24 hr after (2) and 12 days after (3) the adult ecdysis. (a) Data presented in a semi-logarithmic scale; (b) presentation of rates of activity as a percentage of the corresponding activity of HK ( = 100%).
different times before and after ecdysis, the activities of ten 'key enzymes' in the abdominal integument and in the tissue of the wings were measured 24 hr before (pharate animal) (1), 24 hr after (2), and 12 days after (3) the last ecdysis. The absolute values of the enzyme activities in relation to the soluble cell protein (~mole/min.mg protein) at these different times are represented on a semi-logarithmic scale; Fig. 1 is for the abdominal integument and Fig. 2 for the wing integument. I n addition the data in both diagrams are presented as a percentage in relation to the corresponding activities of the hexokinase ( -----100 %). Hence the following results were obtained for the abdominal integumentary tissue: GAPDH, HK, LDH, and G O T show the highest rates of activity both absolutely and relatively; the rate of G-6-PDH, G D H , G P T , and G I u D H and that of SDH and ME is below the activity of the first group of enzymes by at least one power of 10. These absolute values of the enzyme activities in relation to protein can be detected with small variations at the three different points of time.
Viewed relatively, i.e. as a percentage of the corresponding activity of HK, the rate of activity as a whole seems to decrease markedly during aging. But this is mainly due to a distinct increase in the absolute rate of activity of H K during the adult stage. The activities of the enzymes in the wing integument presents a somewhat different picture. The patterns of enzyme activity in grasshoppers 24 hr before (1) and 24 hr after (2) the final ecdysis differ significantly from those of 12 days adult locusts (3). Thus, contrary to the abdominal integument, the G P T attains at the first two points of measurement the proportion of HK, GAPDH, LDH, and G O T . Advancing in age, however, the activity of G P T decreases by about one power of 10. The G D H shows a change which is exact opposite. Its activity increases rapidly over the third point of measurement. Summing up, however, 12 days after shedding the larval cuticle a remarkable decrease of the activities of all enzymes occurs in the wings--much more distinctly than in the epidermis of the a b d o m e n - - w i t h the exception of that of H K as well
Changes of enzyme patterns in Locusta migratoria
a)
1.000 -
U/rag Prot. {log)
_
81
_
--I.000
0.500-
2)
(1)
(3)
-0.100
0:! 0 0 0.050-
lr
o.oi o 0.005-
0.001 -
-0.010
1I
-0001
m
b) % 250 -
25O (1)
(3)
(2)
;,P4
2OO
2oo 178
150
15o
132 tlg
100.
.,° 50"
G
°
H
O.
S
.0 7
n~
39 lu
t2
n
-iL
I00 G S O
M E
,"3 ,s
& P ,DG
pHDH
L D
~
47
u
50
*"
nr ~
0
Fig. 2. Rates of activity of 10 'key enzymes' of the intermediate metabolism in the integument of the wings of Locusta migratoria 24 hr before (=pharate animal) (1), 24 hr after (2) and 12 days after (3) the last ecdysis. (a) and (b) see Fig. 1. as that of G D H and GIuDH. I n order to demonstrate fluctuations in the activity of each individual enzyme during metamorphosis and aging, the values measured 24 hr before the last ecdysis (pharate animal) were equated to 100% and the values of the two other points of measurement were related to these (see Fig. 3; a = a b d o m i n a l integument, b--wing integument). This manner of representation shows more clearly what was to a certain extent already apparent: In the integument of the abdomen the activities of the individual enzymes remain more or less at the same level, only the activity of G D H and H K increase rapidly in 12-day adult locusts; a remarkable decrease of enzyme activity occurs nowhere. I n the integument of the wings (Fig. 3b) a much more differentiated picture appears. For example, the G D H increases extraordinary from 24 hr before ecdysis to 12 days after ecdysis, just as the activity of GIuDH. The H K first decreases slightly in 24-hr-old adult locusts, in 12-day-old adults it rises too. The activities of a second group of enzymes at first demonstrate an obvious peak 24 hr after the ecdysis, but then, advancing in age, they decrease
more or less rapidly. Among these are the G O T , ME, G-6-PDH, and above all the G P T . I n a third group the enzyme activities remain at the same levels during the first two stages, while in 12-day adult locusts the activities quickly drop; these are the enzymes SDH, LDH, and G A P D H . Summing up, it can be said that the activity patterns of the measured enzymes of the intermediate metabolism demonstrate marked fluctuations in connection with the formation of the adult cuticle only in the wing integument, which means that their activities considerably diminished--with few exceptions (GDH, GluDH, and H K ) - - w i t h increasing distance from the final ecdysis. Some of these enzymes, for example G P T and G-6-PDH, dearly display their maximum activity at the peak of cuticle formation (i.e. 24 hr after ecdysis). DISCUSSION
Until now it has been unknown, which 'key enzymes' of distinct metabolic pathways of the intermediate metabolism are present in the integument of the insects and which activities.they have.
82
BERNHARD SURHOLT ~00, %
a)
b)
3GC
o GOH .HK
200
ooH
/
/
':'/
/
AOLuDH
+HK
100
200, %
o~
°~T
IOC G-6-POl
-
200
-
~,
°/o oSOI
lOC
~"-"---.~ o /
ALDH
+ GAPDH I
24 h before 24 h after
12 d aRer
24 h before 24 h after
12 d after
Fig. 3. The activities of the 10 'key enzymes' of the intermediate metabolism, in each case related separately to the values measured in pharate animals 24 hr before ecdysis (=100%). (a) in the integument of the abdomen; (b) in the integument of the wings. Therefore the analysis of the activities of defined ' key enzymes' during the period after apolysis was a precondition to detect possible correlations between the intermediate metabolism, the cuticle formation, and the chitin synthesis. Comparing the enzyme activities of the integument of the abdomen with that of the wings it becomes obvious that the pattern of the enzymes in the abdominal integument demonstrates no important fluctuations during metamorphosis and aging. This means that within the epidermal cells of this part of the integument of Locusts changes take place during the period of observation which are not so drastic as is probably the case in the wing integument. Similar observations were made for the glutamine-fructose-6-phosphate aminotransferase (SuRUOLT, 1975). I n the wing integument, however, the activities of most of the assayed enzymes show a marked decrease during the time after the final
ecdysis (exceptions: HK, G D H , and GluDH) whereas the patterns of enzyme activities 24 hr before and 24 hr after imaginal ecdysis are very similar to those of the enzymes in the integument of the abdomen. These differences between the integument of the wings and that of the abdomen are due to a certain extent to the fact that the epidermal cells of the wings atrophy in adult insects of advanced age. I n detail it can be noticed, that the ' key enzyme' of the Embden-Meyerhof pathway, the GAPDH, with the one exception of the extracts from wings of 12 days adult locusts always has the highest measured rate of activity. The rate of activity of the enzymes L D H and H K is only a little lower. The relation between G A P D H and L D H distinctly suggests a capacity of the epidermal cells for anaerobic energy production. The very low activity of S D H as a ' key enzyme' of the citric acid cycle also leads one to this
Changes of enzyme patterns in Loeusta migratoria conclusion. I n a further comparison it is of interest that the activity of H K in the wings of 12-day-old adult locusts does not decrease, while that of G A P D H , and L D H at this age drops rapidly below that of H K ; consequently at this age the phosphorylation of hexoses is still possible to a relatively large extent. Also the activities of the two transaminases G O T and G P T reach values comparable to those of L D H and HK. This may be connected with the fact, that during the composition of a new cuticle great quantities of amino-acids are used to synthesize the scleroproteins. Thus, in the wing integument the activity of G P T is closely correlated with the formation of new cuticle, because its activity reaches a maximum 24 hr after ecdysis and drops extremely rapidly in 12-day adult grasshoppers (see Fig. 3 and SURHOLTand ZEBE, 1972). This is less extreme in G O T , ME, and G-6-PDH. T o the group of the less reactive enzymes belong the G-6-PDH, the G D H and the GIuDH. While the activity of G - 6 - P D H and the capacity of oxidative deamination remain almost on the same level during the time of measurement, the activity of G D H increases extremely rapidly in both types of the integumentary tissue with growing distance from the final ecdysis. SDH and ME, which show only very little activity, suggest a very low capacity of the citric acid cycle in the integument.
83
Acknowledgements---I am grateful to Prof. Dr. E. ZmB for valuable advice and criticism. I also wish to thank him for reading the manuscript. For correcting the English text I thank Mr. D. Thel3ien. REI~RF_~CES BEROMgYER H. U. (1970) Methoden der enzymatischen Analyse 1, 2. Auflage, Verlag Chemic, Weinheim. BRmCZKA D., P~rrE D., BRUNNERG., and MILLER F. (1968) Kompartimentierte Verteilung yon Enzymen in Rattenlebermitochondrien. Fur. jT. Biochem. 5, 294-304. BUCHERT., LUH W., and PETTED. (1964) Einfache und zusammengesetzte optische Tests mit Pyridinnucleotiden. Aus Hoppe-Seyler/Thierfelder Handbuch der physiologisch-chemischen Analyse, 10. Auflage, Bd. VI/A. Springer, Berlin. HEINRICHD., SURHOLTB., and Z~E E. (1974) Autoradiographische Untersuchungen zum Einbau von i'C-Glukose und SH-N-Acetylglukosamin bei der Wanderheuschrecke Locusta migratoria. Cytobiologie 9, 45-58. LowRY O. H., ROSEBnOUGHN. J., F~m~ A. L., and R~DALL R. J. (1951) Protein measurement with the Folin phenol reagent, ft. biol. Chem. 193, 265-275. SURHOLT B. (1975) Formation of glucesamine-6phosphate in chitin synthesis during ecdysis of the migratory locust, Loeusia migratoria. Insect Biochem. 6, In press. SUnHOLTB. and Z~E E. (1972) In vivo-Untersuchungen zur Chitin-Synthese an Wanderheuschrecken. a~. comp. Physiol. 78, 75-82.
CHANGES OF ENZYME PATTERNS IN T H E I N T E G U M E N T OF LOCUSTA MIGRATORIA D U R I N G F O R M A T I O N OF A D U L T CUTICLE BERNHARDSURHOLT Zoologisches Institut der Universit/it Mfinster, Lehrstuhl ffir Tierphysiologie, D-44 Miinster, Hindenburgplatz 55, Deutschland (Received 12 March 1975; reused 6jTune 1975)
A b s t r a c t - - T h e activities of 'key enzymes' of the intermediate metabolism (HK, G-6-PDH, GAPDH, G D H , LDH, SDH, ME, GOT, GPT, GluDH) in the integument of the abdomen and of the wings of the migratory locust, Locusta raigratoria, were measured 24 hr before, 24 hr after, and 12 days after the last ecdysis. During this time, a significant correlation between these enzyme activity patterns and the formation of the adult cuticle could only be demonstrated in the integument of the wings. G D H , GluDH, and HK, however, were exceptions; their activities increased distinctly during the time after ecdysis.
INTRODUCTION
The activity of the enzymes dissolved in the supernatant and of the S D H in the suspension was assayed spectrophotometrically in glass cells (diameter I cm) in a constant volume of 2.0 ml at 25°C-/-1°C. According to BEaOMSY~ (1970) were determined: (1) H K - - hexokinase - - [ATP: v-hexose-6-phosphotransferase] EC 2.7.1.1 (2) G - 6 - P D H - glucose-6-phosphate dehydrogenase - - [D-glucose-6-phosphate: N A D oxidoreductase] EC 1.1.1.49 (3) G A P D H - - glyceraldehydephosphate dehydrog e n a s e - [D-glyceraldehyde-3-phosphate : NAD oxidoreductase (phosphorylating] EC 1.2.1.12 (4) G D H - - glycerol-3-phosphate dehydrogenase - [L-glycerol-3-phosphate: N A D oxidoreductase] EC 1.1.1.8 (5) L D H - - lactate dehydrogenase R [L-lactate: N A D oxidoreductase] EC 1.1.1.27 (6) G O T glutamate oxalacetate aminotransferase --[L-aspartate: 2-oxoglutarate aminotransferase] EC 2.6.1.1 (7) G P T - - glutamate pyruvate amlnotransferase - - [Lalanine: 2-oxoglutarate aminotransferase] EC 2.6.1.2 (8) G l u D H - - glutamate dehydrogenase - - [L-glutamate: NAD(P) oxidoreductase (deaminating] EC 1.4.1.3 The assay of succinate dehydrogenase was carried out according to BRUICZKAet al. (1968): (9) S D H - - succinate dehydrogenase - - [succinate: (aceeptor) oxidoreductase] EC 1.3.99.1 The 'malic-enzyme' was determined according to B u c ~ et al. (1964): (10) ME - - ' malic-enzyme'-- malate dehydrogenase (decarboxylating-- [L-malate: N A D P oxidoreductase (decarboxylating)] EC 1.1.1.40
DURINO the metamorphosis f r o m the last larval stage to the winged adult intense reactions of decomposition, resynthesis and synthesis take place in the integument. One of the m o s t important syntheses in this process is that of chitin. It was demonstrated that this synthesis--especially after ecdysis--resorts to glucose as precursor (see SURHOLT and ZEBE, 1972). U n t i l now, however, little has been known about the basal metabolism and the activities of the enzymes of the intermediate metabolism in the integument. T h e r e f o r e it seemed a necessary precondition to measure the activities of definite ' key e n z y m e s ' of the intermediate metabolism in this tissue of the insect body in order to detect correlations between the intermediate metabolism, the formation of a n e w cuticle, and chitin synthesis. M A T E R I A L S A N D METHODS Specimens of Locusta migratoria were taken from a synchronous culture (see SURHOLTand Z~E, 1972). The enzyme activities were measured 24 hr before (pharate adult), 24 hr after, and 12 days after the final ecdysis. For each measurement material, either from the wings or from the integumentary tissue from 10 individuals (five male, five female) of the same age was used. The integumentary tissue was prepared as described (I-IEINRICH et al., 1974). The tissue samples were homogenized using a Potter-Elvehjem homogenizer in 5 ml 0.1 M S6rensen-phosphate buffer (pH=7-5) and centrifuged at 20,000g for 20 min. The supernatant was taken for measurement of the soluble enzymes, the resuspended precipitation for measurement of S D H ; protein was determined in the supematant according to L o w a Y et aL (1951).
]R]gSI~LTS I n order to show possible correlations between the intermediate metabolism and the chitin synthesis at 79
80
BERNHARD S U R H O L T
a) 1.000- U/rag Prot.(Iog) oIsO0"
-
~
"
~
( ~)
-
~I
(2)
---
~1
-1.000
~"~ ':'
- - ( 3)
.lliill111111 o.o I1 O.lOO .
.
.
.
o.01o .
.
.
.
.
.
o. oo
.
0.005"
0.001-
-0.001
b) % 250.
250
(2)
(1)
(3) ZOO
200.
150
150 130 113 ioo
100
100
S0-
rlnl la llN
g Io. m~ S 37 D
i
".
,.
H 21
1,1" nrl 76
"
SD
M H I[ 7 7 rtf~
34 Du H tO
I"1
T D
D
7
50
I0
0
Fig. 1. Activities of I0 'key enzymes' of the intermediate metabolism in the abdominal integument of Locusta migratoria 24 hr before (=pharate animal) (1), 24 hr after (2) and 12 days after (3) the adult ecdysis. (a) Data presented in a semi-logarithmic scale; (b) presentation of rates of activity as a percentage of the corresponding activity of HK ( = 100%).
different times before and after ecdysis, the activities of ten 'key enzymes' in the abdominal integument and in the tissue of the wings were measured 24 hr before (pharate animal) (1), 24 hr after (2), and 12 days after (3) the last ecdysis. The absolute values of the enzyme activities in relation to the soluble cell protein (~mole/min.mg protein) at these different times are represented on a semi-logarithmic scale; Fig. 1 is for the abdominal integument and Fig. 2 for the wing integument. I n addition the data in both diagrams are presented as a percentage in relation to the corresponding activities of the hexokinase ( -----100 %). Hence the following results were obtained for the abdominal integumentary tissue: GAPDH, HK, LDH, and G O T show the highest rates of activity both absolutely and relatively; the rate of G-6-PDH, G D H , G P T , and G I u D H and that of SDH and ME is below the activity of the first group of enzymes by at least one power of 10. These absolute values of the enzyme activities in relation to protein can be detected with small variations at the three different points of time.
Viewed relatively, i.e. as a percentage of the corresponding activity of HK, the rate of activity as a whole seems to decrease markedly during aging. But this is mainly due to a distinct increase in the absolute rate of activity of H K during the adult stage. The activities of the enzymes in the wing integument presents a somewhat different picture. The patterns of enzyme activity in grasshoppers 24 hr before (1) and 24 hr after (2) the final ecdysis differ significantly from those of 12 days adult locusts (3). Thus, contrary to the abdominal integument, the G P T attains at the first two points of measurement the proportion of HK, GAPDH, LDH, and G O T . Advancing in age, however, the activity of G P T decreases by about one power of 10. The G D H shows a change which is exact opposite. Its activity increases rapidly over the third point of measurement. Summing up, however, 12 days after shedding the larval cuticle a remarkable decrease of the activities of all enzymes occurs in the wings--much more distinctly than in the epidermis of the a b d o m e n - - w i t h the exception of that of H K as well
Changes of enzyme patterns in Locusta migratoria
a)
1.000 -
U/rag Prot. {log)
_
81
_
--I.000
0.500-
2)
(1)
(3)
-0.100
0:! 0 0 0.050-
lr
o.oi o 0.005-
0.001 -
-0.010
1I
-0001
m
b) % 250 -
25O (1)
(3)
(2)
;,P4
2OO
2oo 178
150
15o
132 tlg
100.
.,° 50"
G
°
H
O.
S
.0 7
n~
39 lu
t2
n
-iL
I00 G S O
M E
,"3 ,s
& P ,DG
pHDH
L D
~
47
u
50
*"
nr ~
0
Fig. 2. Rates of activity of 10 'key enzymes' of the intermediate metabolism in the integument of the wings of Locusta migratoria 24 hr before (=pharate animal) (1), 24 hr after (2) and 12 days after (3) the last ecdysis. (a) and (b) see Fig. 1. as that of G D H and GIuDH. I n order to demonstrate fluctuations in the activity of each individual enzyme during metamorphosis and aging, the values measured 24 hr before the last ecdysis (pharate animal) were equated to 100% and the values of the two other points of measurement were related to these (see Fig. 3; a = a b d o m i n a l integument, b--wing integument). This manner of representation shows more clearly what was to a certain extent already apparent: In the integument of the abdomen the activities of the individual enzymes remain more or less at the same level, only the activity of G D H and H K increase rapidly in 12-day adult locusts; a remarkable decrease of enzyme activity occurs nowhere. I n the integument of the wings (Fig. 3b) a much more differentiated picture appears. For example, the G D H increases extraordinary from 24 hr before ecdysis to 12 days after ecdysis, just as the activity of GIuDH. The H K first decreases slightly in 24-hr-old adult locusts, in 12-day-old adults it rises too. The activities of a second group of enzymes at first demonstrate an obvious peak 24 hr after the ecdysis, but then, advancing in age, they decrease
more or less rapidly. Among these are the G O T , ME, G-6-PDH, and above all the G P T . I n a third group the enzyme activities remain at the same levels during the first two stages, while in 12-day adult locusts the activities quickly drop; these are the enzymes SDH, LDH, and G A P D H . Summing up, it can be said that the activity patterns of the measured enzymes of the intermediate metabolism demonstrate marked fluctuations in connection with the formation of the adult cuticle only in the wing integument, which means that their activities considerably diminished--with few exceptions (GDH, GluDH, and H K ) - - w i t h increasing distance from the final ecdysis. Some of these enzymes, for example G P T and G-6-PDH, dearly display their maximum activity at the peak of cuticle formation (i.e. 24 hr after ecdysis). DISCUSSION
Until now it has been unknown, which 'key enzymes' of distinct metabolic pathways of the intermediate metabolism are present in the integument of the insects and which activities.they have.
82
BERNHARD SURHOLT ~00, %
a)
b)
3GC
o GOH .HK
200
ooH
/
/
':'/
/
AOLuDH
+HK
100
200, %
o~
°~T
IOC G-6-POl
-
200
-
~,
°/o oSOI
lOC
~"-"---.~ o /
ALDH
+ GAPDH I
24 h before 24 h after
12 d aRer
24 h before 24 h after
12 d after
Fig. 3. The activities of the 10 'key enzymes' of the intermediate metabolism, in each case related separately to the values measured in pharate animals 24 hr before ecdysis (=100%). (a) in the integument of the abdomen; (b) in the integument of the wings. Therefore the analysis of the activities of defined ' key enzymes' during the period after apolysis was a precondition to detect possible correlations between the intermediate metabolism, the cuticle formation, and the chitin synthesis. Comparing the enzyme activities of the integument of the abdomen with that of the wings it becomes obvious that the pattern of the enzymes in the abdominal integument demonstrates no important fluctuations during metamorphosis and aging. This means that within the epidermal cells of this part of the integument of Locusts changes take place during the period of observation which are not so drastic as is probably the case in the wing integument. Similar observations were made for the glutamine-fructose-6-phosphate aminotransferase (SuRUOLT, 1975). I n the wing integument, however, the activities of most of the assayed enzymes show a marked decrease during the time after the final
ecdysis (exceptions: HK, G D H , and GluDH) whereas the patterns of enzyme activities 24 hr before and 24 hr after imaginal ecdysis are very similar to those of the enzymes in the integument of the abdomen. These differences between the integument of the wings and that of the abdomen are due to a certain extent to the fact that the epidermal cells of the wings atrophy in adult insects of advanced age. I n detail it can be noticed, that the ' key enzyme' of the Embden-Meyerhof pathway, the GAPDH, with the one exception of the extracts from wings of 12 days adult locusts always has the highest measured rate of activity. The rate of activity of the enzymes L D H and H K is only a little lower. The relation between G A P D H and L D H distinctly suggests a capacity of the epidermal cells for anaerobic energy production. The very low activity of S D H as a ' key enzyme' of the citric acid cycle also leads one to this
Changes of enzyme patterns in Loeusta migratoria conclusion. I n a further comparison it is of interest that the activity of H K in the wings of 12-day-old adult locusts does not decrease, while that of G A P D H , and L D H at this age drops rapidly below that of H K ; consequently at this age the phosphorylation of hexoses is still possible to a relatively large extent. Also the activities of the two transaminases G O T and G P T reach values comparable to those of L D H and HK. This may be connected with the fact, that during the composition of a new cuticle great quantities of amino-acids are used to synthesize the scleroproteins. Thus, in the wing integument the activity of G P T is closely correlated with the formation of new cuticle, because its activity reaches a maximum 24 hr after ecdysis and drops extremely rapidly in 12-day adult grasshoppers (see Fig. 3 and SURHOLTand ZEBE, 1972). This is less extreme in G O T , ME, and G-6-PDH. T o the group of the less reactive enzymes belong the G-6-PDH, the G D H and the GIuDH. While the activity of G - 6 - P D H and the capacity of oxidative deamination remain almost on the same level during the time of measurement, the activity of G D H increases extremely rapidly in both types of the integumentary tissue with growing distance from the final ecdysis. SDH and ME, which show only very little activity, suggest a very low capacity of the citric acid cycle in the integument.
83
Acknowledgements---I am grateful to Prof. Dr. E. ZmB for valuable advice and criticism. I also wish to thank him for reading the manuscript. For correcting the English text I thank Mr. D. Thel3ien. REI~RF_~CES BEROMgYER H. U. (1970) Methoden der enzymatischen Analyse 1, 2. Auflage, Verlag Chemic, Weinheim. BRmCZKA D., P~rrE D., BRUNNERG., and MILLER F. (1968) Kompartimentierte Verteilung yon Enzymen in Rattenlebermitochondrien. Fur. jT. Biochem. 5, 294-304. BUCHERT., LUH W., and PETTED. (1964) Einfache und zusammengesetzte optische Tests mit Pyridinnucleotiden. Aus Hoppe-Seyler/Thierfelder Handbuch der physiologisch-chemischen Analyse, 10. Auflage, Bd. VI/A. Springer, Berlin. HEINRICHD., SURHOLTB., and Z~E E. (1974) Autoradiographische Untersuchungen zum Einbau von i'C-Glukose und SH-N-Acetylglukosamin bei der Wanderheuschrecke Locusta migratoria. Cytobiologie 9, 45-58. LowRY O. H., ROSEBnOUGHN. J., F~m~ A. L., and R~DALL R. J. (1951) Protein measurement with the Folin phenol reagent, ft. biol. Chem. 193, 265-275. SURHOLT B. (1975) Formation of glucesamine-6phosphate in chitin synthesis during ecdysis of the migratory locust, Loeusia migratoria. Insect Biochem. 6, In press. SUnHOLTB. and Z~E E. (1972) In vivo-Untersuchungen zur Chitin-Synthese an Wanderheuschrecken. a~. comp. Physiol. 78, 75-82.