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Studies of acetylcholinesterase of the central nervous system of Galleria mellonella
Insect Biochem., 1973, 3, 231-242. [Scientechnica (Publishers) Ltd.]
231
STUDIES OF ACETYLCHOLINESTERASE OF THE CENTRAL NERVOUS SYSTEM OF G A L L E R I A M E L L O N E L L A M. HABIBULLA
ANn
R. W. NEWBURGH
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331, U.S.A.
(Received 22 January, 1972; revised 4 November, I972) ABSTRACT The central nervous system (CNS) of Galleria mellonella contains an active acetylcholinesterase, whose level changes during the development of the central nervous system. The enzyme occurs in five forms of different electrophoretic mobilities and the relative amounts of each form varies with the life state of Galleria. The molecular weight of the enzyme was estimated to be approximately 28o,ooo ± 15,ooo. It appeared to consist of four polypeptide subunits. Injection of the anticholinesterase, eserine, into the last larval stage inhibits the silk spinning and delays the formation of pupa by more than 2 weeks when compared with the controls. ACETYLCHOLINESTERASE [ACHE] (acetylcholine acetylhydrolase, E.C. 3.I.I.7) has been identified biochemically (Casida, I954; Chadwick, 1963) and cytochemically in electron micrographs of insect neural tissue (Smith and Treherne, 1965). Insect central nervous system neurons were shown to be relatively insensitive to applied acetylcholine [ACh] (Twarog and Roeder, 1957; Yamasaki and Narahashi, 1958; Calhoun, 1963; Kerkut, Pitman, and Walker, i969) , which led some investigators to suggest that the cholinergic system m a y not be present in insect nerves. H o w ever, from the studies of Smallman (1958), Smallman and Mansingh (1969) , and Shankland, Rose, and Donniger (1971) the presence in insects of the cholinergic system appears now to be well established. Since a direct relationship between acetylcholinesterase and neural development has been implicated in various organisms (Karczmar, i963) , this enzyme is a useful tool in studies on neurometarnorphosis. T h i s paper is a report of the changes occurring in acetylcholinesterase from Galleria mellonella during this process. MATERIALS AND M E T H O D S EXPERIMENTAL ANIMALS
The greater wax moth, Galleria mellonella L., was cultured by the method of Pipa (I963) with some modifications (Ishikawa and Newburgh, I97i ). The whole eggs, embryos, and certain isolated tissues from different life stages were used in this study. TISSUE PREPARATIONS
The insects were chilled in ice after CO2 anaesthetization. The various organs used in the experiments were dissected from chilled immobilized insects in a balanced salt solution (Martignoni and Scallion, i96i ) using a dissecting microscope. Whole insects or individual organs were homogenized in a solution containing o'o 5 M KPO,, p H 6"8; o'2 M NaC1; 0"5 per cent Triton X-ioo; and o.ooi M Na, EDTA. Two ml. of homogenizing solution were used for 0-6 g. wet weight of tissue. The homogenate was centrifuged at 8ooo g for io minutes. The pellet was
232
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again homogenized and centrifuged as previously described and the combined supernatants were used as the source of enzyme. It was determined that more than 95 per cent of the original activity was present in the supernatant. All operations were performed below 4 ° C. PURIFICATION OF THE ENZYME Since the specific activity of A C h E in the brain was high compared to other tissues, it was used as the source of the enzyme for purification. F o r this IOOO heads were homogenized with a mortar and pestle in 5 ml. of o ' o i M potassium phosphate buffer, p H 7"4, containing i35 rag. of a m m o n i u m sulphate. After centrifugation at 3ooo r.p.m, in a Servall centrifuge for 20 minutes, solid a m m o n i u m sulphate was added to the supernatant with stirring to bring it to 3 ° per cent saturation. T h i s solution was centrifuged for IO minutes at 5ooo r.p.m, and the supernatant was dialysed against o.oi M phosphate buffer, p H 7'4. T h e material in the dialysis bag was then concentrated using polyethelene glycol flakes (Carbowax). After withdrawing an aliquot for protein determination, the dialysate was subjected to polyacrylamide gel electrophoresis (Davis, 1964). T w e l v e tubes, 12'5 c m . × o ' 5 cm. diameter, each containing i ' S m l , of the small-pore gel and 0" 3 ml. of the large-pore gel were used. After the gels were prerun for 5 minutes at 15 mA., equal aliquots of the dialysate were applied in a 20 per cent sucrose solution and stacked for 5 minutes at 15 mA. Electrophoresis was conducted for I hour and 4 o minutes at z 5 mA., with ice-cold water circulating through the outer jacket of the apparatus. One of the gels was stained for activity with n-methylindoxyl acetate ( N M I A ) (Guilbault, Kuan, Tully, and Hackney, 197o). T o do this the gel was incubated in a test-tube containing N M I A (o'5 g. in zo ml. of o'I M KPOa, p H 7"5, diluted with an equal volume of glass-distilled water). R e values for the enzyme bands were calculated (Re mobility of enzyme band in cm./mobility of marker dye in cm.). T h e activity could be measured directly from gels washed in o'o5 M phosphate buffer, p H 7'5, by scanning at E~97 using a Gilford recording spectrophotometer with a scanning attachment. Immersion of gels in lO -5 ~ / e s e r i n e for 4 ° minutes prevented the appearance of activity bands. Regions of the gels corresponding to the R r values of the enzyme were isolated and again subjected to electrophoresis. T h e enzyme was again localized by staining one of the gels and the regions of unstained gels corresponding to the R v values of the enzyme were minced and eluted with o ' o i M phosphate buffer, p H 7"4, overnight in the cold. T h e eluates were pooled and centrifuged in a sucrose density gradient (5-20 per cent) at 39,00o r.p.m, for 5 hours at 4 ° C. in a preparative Spinco ultracentrifuge using the S W 39 head. F o u r ml. of 5 per cent sucrose containing o ' o i M phosphate buffer, p H 7"4, and o'5 ml. of the enzyme solution were layered on top of the gradient. After centrifugation, fractions containing 2o drops were collected. Protein in each fraction was measured by the Ezso/E~o m e t h o d of Warburg and Christian (Chaykin, I966 ). A C h E activity was measured using N M I A and measuring the extinction at E~97. A C h E of the electric organ of the electric eel was also centrifuged on a sucrose gradient and used as a reference standard. T h e isolated fractions were also subjected to gel electrophoresis and stained for protein and enzyme activity, and only a single band was found in either determination. MOLECULAR WEIGHT DETERMINATION A C h E molecular weight was estimated using the m e t h o d of W e b e r and Osborn (1969). Because of the high molecular weight of the enzyme, slight modifications were necessary. T h e concentration of methylene bisacrylamide was reduced to one-third, to facilitate migration of high molecular weight proteins, and the molarity of the buffer was reduced to one-half, to decrease the running time. Standard proteins of molecular weights in the range of 4 o , o o ° - 8 ° ° , ° ° ° (DNAse, 5"-globulin, catalase, and thyroglobulin) were utilized to construct a standard curve. For the electrophoresis of individual samples, the same procedure was used. T h e a m o u n t of protein applied to each gel varied from 4 ° to 4oo ~tg. DISSOCIATION OF THE ENZYME INTO SUBUNITS T h e isolated enzyme was dissolved in 6 M guanidine H C I containing o'I per cent 13-mercaptoethanol and dialysed against O'Ol per cent mercaptoethanol overnight. T h e dialysed protein was subjected to polyacrylamide gel electrophoresis to determine the presence of subunits. T h e staining was performed with methanolic Coomassie brilliant blue according to the m e t h o d of W e b e r and Osborn (1969).
1973, 3
AchE OF THE CENTRAL NERVOUS SYSTEM
233
PROTEIN DETERMINATION Gels were stained with Amidoschwartz and Coomassie brilliant blue by standard techniques to localize proteins. T h e excess dye was destained by diffusion with 7 per cent acetic acid. The protein in homogenates was determined by the method of Lowry, Rosebrough, Farr, and Randall (I95I). RADIOMETRIC ASSAY A microradiometric procedure was used for the assay of AChE (Rosenberg, Dalessio, Tremblay, and Woodman, 1971). T h e reaction mixture consisted of o'o5 M potassium phosphate (pH 6'8), o'2 M NaCI, o'ooi M Na2EDTA (pH 6"8), 15 X o ' I o a M acetylcholine iodide, and 2"7 × IO-a M [i-14C]acetylcholine iodide (specific activity 2"4 inc. per mmole total volume 5o lal., 37 ° C. for IO minutes). Th e reaction was stopped by adding i ml. of ice-cold glass-distilled water containing B.W.284C5I dibromide (Burroughs Wellcome), a specific inhibitor of ACHE. This mixture was then placed on an ion-exchange column (Biorad AG5oW-X8 lOO-2OO mesh, Na+). T h e column was prepared in disposable Pasteur pipettes and was 5 cm. in length. Th e eluate was collected in scintillation vials and included two I-ml. ice-cold glass-distilled water washes. Th e eluates were counted in a scintillation counter. Since the column retains acetylcholine and choline, the radioactivity obtained arises from the free [i-14C]acetate and the results are used to calculate the amount of acetylcholine hydrolysed.
ISOTOPE INCORPORATION A total of 50 ~1. of [4,5-~H]leucine was injected into 2o individuals of different stages. After 24 hours the isozymes of AChE were stained with N M I A after polyacrylamide gel electrophoresis. T h e activity bands were cut off and the radioactivity counted by standard methods after solubilizing the gel in hydrogen peroxide. ESERINE INJECTION Last larval stages collected over a period of i hour were anaesthetized with CO~ and 1-2 lal. of lO -3 M eserine were injected into each. Controls were injected with insect Ringer solution.
Table /.--INHIBITION OF A C H E ACTIVITY WITH ESERINE (PHYSOSTIGMINE) AND IS-BIS-(4-ALLYLDIMETHYLAMMONIUMPHENYL)PENTAN-3-ON E DIBROMIDE ~
INHIBITOR
PERCENTAGE INHIBITION OF ACHE
Eserine io -t5 M IO ~ M io-6M io -~ M
88"4 89"2 94"6 95"o
B.W.284C51 dibromide 2 × IO -~
M
2×io-6M 2XIO 8M 2 ×
io -z
M
96 96"2 96 '7 97"2
*B.W.284CsI dibromide (Burroughs Wellcome). T h e enzyme was from the head homogenate of the last larval stage. T h e radiometric assay is given in Methods.
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HABIBULLAAND NEWBURGH
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RESULTS Studies on the hydrolysis of ACh showed that at least 95 per cent of the activity was due to AChE on the basis of its inhibition by BW-284C 51 dibromide and eserine (Table I). Although these results are from the last larval stage head homogenates, similar results were obtained with other stages and tissue. In addition to the inhibition of the enzyme activity it was also found that injection of 2 gl. of lO -3 M eserine per larva inhibited Table / / . - - L o c A L I Z A T I O N OF A C H E IN DIFFERENT TISSUES AND CHANGES DURING DEVELOPMENT OF
TISSUE AND STAGE
Female reproductive system Developing egg Mature unfertilized egg Mature ovaries Fertilized egg Male reproductive system Testes Larvae Fat body Haemolymph Head, last larval instar Brain, last larval instar CNS, last larval instar Pupae Head Adult Head, pharate adult Adult (3 days old) Head Fat body Haemolymph Thoracic muscle
Galleria
ACHE ACTIVITY* (nmoles of ACh hydrolysed per lag. protein in i hour at 37 ° C.)
o o o
0'275
I .o8 0.067 o
o'83 4.88 5"29
i "39 4"60 2"I6 o o I "35
*Mean of 4 experiments. The deviation was less than 6 per cent. spinning of silk in 85 per cent of the cases, and delayed the transformation into pupae by more than i5 days. ACHE ACTIVITY AND CHANGES DURING METAMORPHOSIS T h e activity of A C h E was determined in different stages and tissues of Galleria and the results are shown in Table 11. A C h E could not be detected in the mature ovaries, developing egg, or the unfertilized egg. Activity was found in mature testes and the fertilized egg.
LOCALIZATION OF
1973, 3
AchE OF THE CENTRAL NERVOUS SYSTEM
235
The activity in the testes may be due to the presence of nerves in the preparation. It is reasonable to assume that the activity in the fertilized egg results from the initiation of neural development (Smallman and Mansingh, 1969). Except for the thoracic muscle, the activity in the larvae, pupae, and adult is localized in brain or the CNS. Since the thoracic muscle preparation would contain nerves, the activity in this organ may be the result of this and the presence of neuromuscular junctions. The level of
FIG. I . - - A c t i v i t y staining for acetylcholinesterase after polyacrylamide gel electrophoresis. (See text for details of methods.) T h e material applied to the column was the supernatant fraction isolated from larval heads. Left = stained for A C h E ; m i d d l e - - s t a i n e d for A C h E ; r i g h t - - s t a i n e d for A C h E after incubation in lO -5 M eserine for 3 ° minutes. FIG. 2 . - - F u r t h e r separation of the u p p e r band from Fig. i. T h e upper band from Fig. i was isolated by eluting with o.oi M phosphate buffer, p H 7"4. It was then re-electrophoresed on a 5 per cent polyacrylamide gel (see Methods) and stained with Coomassie brilliant blue.
AChE in the head of the last-instar larvae is lower than that in the pupae or adult head. A peak occurs in the head of the pharate adult and then declines as the insect ages. GEL-ELECTROPHORESIS OF A C H E FROM VARIOUS STAGES OF DEVELOPMENT
Under the conditions used, four activity bands were found in each stage. A typical electrophoretic pattern of AChE activity is shown in Fig. I. Although the reproduction
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Insect Biochem.
shown in Fig. i does not indicate it, the upper bands (RF=o.z3-o.25) consisted of two bands. T h e A C h E of the electric organ of the electric eel which was used as a reference had an RF value in this same region. T h e s e bands could be further resolved by isolating t h e m from the gel and doing a re-electrophoresis in a 5 per cent gel (Fig. z). T h e fast-moving A C h E activity bands had an R r value of between approximately o.58 and o'6i and, as seen in Fig. I, were well separated. F r o m these results it was concluded that four enzymatically active bands were present and represent isozymes of ACHE. It was then of interest to examine the possibility that
+ "
B
ol
."
.I
I.
. . .
I.
Oo
,4 B
G D
M
1234 Fro. 3.--Diagrammatic representation of AChE isozyme pattern of the head (except in the case of eggs) region of Galleria during different stages of development. I, Eggs (developing whole embryos); 2, larva; 3, pupa; 4, adult. A, B, C, and D designate four isozymes of ACHE. B1 denotes an isozyme which appears only in the adult stage. H is the position of the marker dye. The distance travelled by the marker dye in the separation gel was 7 cm. The dotted portion at the top of the figure represents the stacking gel. The procedures are given in the text. The widths of the bands are drawn to represent the relative amount of enzyme activity in each band at a particular stage of development, as determined by measuring AChE activity in the gel at E297.
at different stages of development different isozymes of A C h E are present. This was found to be true and the results are shown schematically in Fig. 3. Four forms of the enzyme were present in each of the four stages of development (embryo, larva, pupa, and adult). In the adult stage, one band (A) disappears and a new band (B1) appears. T h a t this result was not an artifact was shown by doing gel electrophoresis experiments with mixtures of the different stages (Fig. 4). When a mixture of larval and adult tissue was run, (F@ 4, 2) an additional band (B1) was apparent and this also was true when a mixture of pupa and adult material (Fig. 4, 5) was subjected to co-electrophoresis. T h e relative amounts of activity in the different bands also change during neurometamorphosis. T o determine this, the experiments consisted of adding the same
237
AchE OF THE CENTRAL NERVOUS SYSTEM
I973, 3
amount of protein (i20 lag.) to the gel, electrophoresing, and analysing the activity bands at E29v in a scanning attachment to a Gilford spectrophotometer. Typical results are shown in Table III, column 4. In the egg the predominant peak was B1, in the
+ •
~
I
n
2
5
.
,
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.
m
n
I
m
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FIG. 4.--Diagrammatic representation of co-electrophoresis of AChEs from different stages of development. Other details as in Fig. 3. I, Larval ACHE; 2, larval+ adult ACHE. Note the additional band indicated by arrow corresponding to that of number 3; 3, adult ACHE; 4, pupal ACHE; 5, pupal~adult ACHE. Note the additional band indicated by arrow corresponding to that of number 6; 6, adult ACHE. Arrow=B1 band. larval stage C and D were the major peaks, in the pharate pupa band B again was the major peak, and in the pharate adult the major peaks were B and the new peak B 1. [aH]LEUClNE INTO A C H E [aH]Leucine was injected into insects at various stages of development. After z 4 hours the AChE isozymes were separated on gels, eluted, and analysed for incorporation of this amino-acid. The results of these studies are shown in Table III. In the larva bands C and D had the highest specific activity. The specific activity of band A in the pharate pupa was greater than in larva. The high specific activity of band B1 in the pharate adult is indicative of its rapid synthesis at this particular stage. INCORPORATION OF
PHYSICOCHEMICALSTUDIES The AChE from the larval heads was purified by a combination of ammonium sulphate precipitation, gel electrophoresis, and sucrose density gradient centrifugation techniques as described in the methods section (Table IV). Approximately a five-hundredfold purification was obtained for each of the four isozymic forms of the ACHE. Each isozyme was separated in a single peak on a sucrose gradient and, when the AChE of the electric eel was treated similarly, a single peak of active enzyme was found in the same position as the AChE band B of Galleria. The purity of each of the isolated isozymes was
238
Insect Biochem.
HABIBULLA AND NEWBURGH
established b y separation on polyacrylamide gel a n d d e t e r m i n a t i o n of the e n z y m e activity. Each of the isolated isozymes was dissociated in 6 M g u a n i d i n e HC1 cont a i n i n g o.i per cent [3-mercaptoethanol at r o o m t e m p e r a t u r e for 2 hours, a n d dialysed Table III.
INCORPORATION OF [4,5-aH]LEuCINE INTO THE DIFFERENT ISOZYMES OF ACETYLCHOLINESTERASE
ISOZYME BAND
STAGE
LABEL INCORPORATED
(c.p.m.)
0" 5 WIDTH X HEIGHT OF THE PEAK OF THE BAND SCANNED AT E297 (cm.)
A
Egg
B
2"31 8"1
C D
o'19
Pharate pupa
Pharate adult
2"1
B
30,000 47,00o
6'5
6,000 7,23 °
C D
168,ooo 152,79o
14'82 I4" 4
IO,6IO
A
82,000
B
6'2 16'8
13,23o 8,750
C D
147,ooo 84,6oo 56,568
6'6 5"4
12,820 10,480
A B B1 C D
I44,692 551,826 10,206 41,568
15"4 21 .o 2'4 3"3
9,4 °0 26,280 4,260 12,6oo
A
Last larval stage (after cessation of feeding)
SPECIFIC ACTIVITY
5'0
11,340
Fifty pl. of tritiated leucine (specific activity 20"8 c. per mmole) were injected into 20 individuals. The insects were incubated for 24 hours and the heads removed. These were then assayed for AChE by procedures as given under Methods.
Table/V.--PuRIFICATION OF ACHE FROM Galleria mellonella
FRACTION
SPECIFIC ACTIVITY (mmoles of ACh hydrolysed per mg. protein per hour at 37 ° C.)
I. Original homogenate
0"68
PERCENTAGE TOTAL ACTIVITY
IO0
(total mmoles of ACh hydrolysed in I hour at 37 ° C.--2'5) 2. 3° per cent saturated ammonium sulphate supernatant 3. Gel electrophoresis 4. Sucrose density gradient centrifuge (tubes 7-1o) The procedures are given under Methods.
3 "OO
20"4° 3o6'o
63.8 20
8.26
~973, 3
AChE OF THE CENTRAL NERVOUS SYSTEM
239
against o.ox M sodium phosphate buffer (pH 7"0) containing o.x per cent 13-mercaptoethanol overnight. The dialysate was then incubated at 37 ° C. for z hours in o.o1 M sodium phosphate buffer (pH 7"0) containing 1 per cent sodium dodecyl sulphate (SDS) and i per cent 13-mercaptoethanol. The protein concentration was 20 ~tg. per lOO Ill. The sample was then subjected to SDS polyacrylamide gel electrophoresis (Weber and Osborn, 1969). Under these conditions each of the isozymes dissociated into four subunits (Fig. 5). The molecular weight of each subunit was determined by using a standard
1000 =
o 2
2
3
×
100
4
10
1
,
,
,
,
I
,
0"5
,
~
I
I 1
MOBILITY
FIG. 5.--Diagrammatic representation of the electrophoresis of isozyme B of ACHE. The AChE (isozyme B) was treated with guanidine hydrocb_lorideand then separated by polyacrylamide gel electrophoresis. Details are provided in the text.
FIG. 6.--Graph showing relationship between molecular weight (MW) of the standard proteins and their mobilities in SDS polyaerylamide gel electrophoresis. The MW of the polypeptide subunits of AChE of Galleria are shown as small open circles and MW of AChE is represented as a big open circle. T=Thyroglobulin; C=catalase; G= -/-globulin; D=DNAse. Mobility=[distanee of protein migration)/(length after destaining)] × [(length before staining)/distance of dye migration)].
curve prepared from proteins of known molecular weight (Weber and Osborn, 1969)
(Fig. 6). The molecular weights of the polypeptide subunits ranged between 65,000 and 85,000, and the molecular weight of the protein containing four subunits was 28o,ooo :J: 15,000. DISCUSSION The absence of AChE in the mature ovary and developing and unfertilized egg, and the presence in the fertilized egg, is indicative of the initiation of neurogenesis. This is consistent with the results of Smallman and Mansingh (1969) and Shankland and others (1971). The increased AChE activity observed in the pharate pupal stage is likely to be the result of increased numbers of aeetylcholinesterase-rich neurons (Rockstein, 195o ,
240
HABIBULLAAND NEWBURGH
Insect Biochem.
I953) and may be associated with development of the optic lobe (Norlander, i967; Smallman and Mansingh, i969). Morphologically there is a considerable increase in the brain size during this stage and active cell division, as shown by a marked increase in the activity of thymidylate synthetase (Albin and Newburgh, I972 ). The acetylcholinesterase of the central nervous system of Galleria has a higher specific activity than is found in the cockroach (Lord, Gregory, and Burt, I967) or snail (Emson and Kerkut, I97I ). If this is generally true of the CNS insects it might explain why a relatively high concentration of acetylcholine (Io -~ M) is required to elicit excitation of insect neurons (Twarog and Roeder, I957; Yamasaki and Narahashi, x958; Calhoun, i963; Kerkut and others, ~969). From the studies reported here it appears likely that the acetylcholinesterase is associated only with neural tissue. This assumes, of course, that the activity found in non-neural tissues is due to contamination, and this seems reasonable since some degree of innervation occurs in these later tissues. Although it has been implied that the molecular weight of AChE in insects is in the order of 3-4 million (Dauterman, Talens, and van Aspen, i962), the molecular weight of this enzyme from Galleria is in the range of 280,000. This compares well with the (molecular weight of the AChE from the electric organ of the electric eel [260,0oo Leuzinger, Goldberg, and Cauvin, I969) ] and from the snail [24o,ooo (Emson and Kerkut, x97I)]. The AChE from developing chick tissue was shown to be as large as 420,000 (Wilson, Mettler, and Asmundson, i969). Dissociation of the Galleria acetylcholinesterase yielded four polypeptide subunits. This agrees with that found in snail brain (Emson and Kerkut, I97i ) but is different from the enzyme from the electric eel, where Leuzinger and others (i969) found the enzyme to be a tetramer of only two subunits. It appears that these subunits are combined in different ways during neurometamorphosis in Galleria to yield five different isozymes. At present, we do not have sufficient information to determine the subunit composition of each isozyme. Nevertheless, since isozymes are a refined expression of the enzymatic differentiation of a cell, certain mechanisms of neurometamorphosis in Galleria can be suggested. In Galleria, as in other holometabola, metamorphosis is maximal during egg to larval, larval to pupal, and pupal to adult development. Examination of the results of [3H]leucine incorporation are indicative of a genetic control of events that is manifested in a change in rate of synthesis of the different isozymes of ACHE. Of particular interest is the appearance of an additional isozymic form and disappearance of an earlier form in the adult stage. It would be of interest to determine whether these changes represent changes in the synthesis of the complete protein or particular subunits. Inhibition of AChE activity, by injecting eserine into larvae, appears seriously to interfere with normal metamorphosis. This suggests that the presence of an active acetylcholinesterase is required to initiate silk spinning and the larval to pupal transformation. One possible mechanism might be that these changes are under neuronal control and the normal process is altered owing to an increase in the amount of acetylcholine, which could result from an inhibition of AChE activity. REFERENCES ALBIN, E. A., and NEWBURGH,R. W. (I972), unpublished observations. CALHOUN, E. H. (x963), in Advances in Insect Physiology (ed. Beament, Treherne, and Wigglesworth), vol. I, pp. 1-46. New York: Academic Press.
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AChE OF THE CENTRAL NERVOUS SYSTEM
24I
CASlDA, J. E. (I954) , 'Comparative enzymology of certain insect acetylesterases in relation to poisoning by organophosphate insecticides', ft. Physiol., Lond., I27, zoP-~IP. CHADWICK, L. E. (I963), in Cholinesterases and Anticholinesterase Agents (ed. Koelle), pp. 741798. Berlin: Springer. CHAYKIN, S. (I966), Biochemistry Laboratory Techniques. New York: Wiley. DAUTERMAN,W. C., TALENS, A., and IVANASPEN (I962), 'Partial purification and properties of fly head cholinesterase ', J. Insect Physiol., 8, 1-I 4. DAvis, B. J. (I964), 'Disc electrophoresis. II. Methods and application to human serum proteins', Ann. N.Y. Acad. Sci., x2I, 4o4-4z7. EMSON, P. C., and KERKUT, G. A. (I97I), 'Acetylcholinesterase in snail brain', Comp. Biochem. Physiol., 39, 879-889. GUILBAULT,G. G., KUAN, S. S., TULLY, J., and tIACK.NEY,D. (I97O), 'New procedure for rapid and sensitive detection of cholinesterase separated by polyacrylamide gel electrophoresis', Analyt. Biochem., 36, 7z-77. ISHIKAWA, H., and NEWBURCH, R. W. (I97I) , 'Changes in RNA during metamorphosis of the central nervous tissue of Galleria', +7. Insect Physiol., I7, I I I3-I 124. KARCZMAR,A. G. (I 963), in Cholinesterases and AntichoIinesterase Agents (ed. KoeUe), pp. 129-186. Berlin: Springer. KERKUT, G. A., PITMAN, R. M., and WALXER, R. J. (~969), ' Sensitivity of neurons of the insect central nervous system to iontophoretically applied acetylcholine or GABA', Nature, Lond., 222, io75-io76. LEUZlNGER, W., GOLDBERC, M., and CAUVlN, E. (I969), 'Molecular properties of acetylcholinesterase',J, sol. Biol., 4o, 27-zz5. LORD, K. A., GREGORY, G. E., and BURT, P. E. (1967) , ' T h e penetration of acetylcholine into the central nervous system of the cockroach Periplaneta americana (L.)', ft. exp. Biol., 46, 153-159. LowRY, O. H., ROSEBROUGH,N. J., FARR,A. L., and RANDALL,R. J. (I 95 I), 'Protein measurement with the Folin phenol reagent', v~. biol. Chem., I93, 265-275. MARTIGNONI,M. E., and SCALLION,R. J. (I96I), 'Preparation and uses of hemocyte monolayers in vitro', Biol. Bull. mar. biol. Lab., Woods Hole, I2X, 5o7-52o. NORLANDER, R. (I967), in Invertebrate Nervous Systems (ed. Wiersma). University of Chicago Press.
PIPA, R. L. (1963) , ' Studies on the hexapod nervous system. VI. Ventral nerve cord shortening; a metamorphic process in GaUeria mellonella (L.)', Biol. Bull. mar. biol. Lab., Woods Hole, I24~ 203-302. ROCKSTEIN, M. (I95O), ' T h e relation of cholinesterase activity to change in cell number with age in the brain of adult worker honeybee', .7. cell. comp. Physiol., 55, 1I-z4. ROCKSTEIH,M. (1953), 'Physiology of aging in the adult worker honeybee', Biol. Bull. mar. biol. Lab., Woods Hole, IO5, I54-I59. ROSENBERG, R. N., DALESSIO,D. J., TREMBLAY,J., and WOODMAN, D. (I97I), 'Kinetics of acetylcholine synthesis and hydrolysis in myasthenia gravis', Science, N.Y., x73 , 644-645. SHANKLAHD,D. L., ROSE, J. A., and DONNIGER, C. (I97I), ' T h e cholinergic nature of the cercal nerve giant fiber synapse in the sixth abdominal ganglion of the American cockroach Periplaneta americana (L.)', ~t. Neurobiol., 2, 247-262. SMALLMAN, B. N. (I958), 'Physiological basis for the mode of action of organophosphorous insecticides', Proc. Xth Int. Cong. Ent., 2, 5-12. SMALLMAN, B. N., and MANSINOH, A. (x969), ' T h e cholinergic system in insect development', A. Rev. Ent., x4~ 387. SMITH, D. S., and TREHEENE,J. E. (I965), ' T h e electron microscopic localization of cholinesterase activity in the central nervous system of an insect, Periplaneta americana L.', J. Cell Biol., 26, 445-456. TWAROG, B. M., and ROEDER, K. D. (I957), 'Pharmacological observations of the desheathed last abdominal ganglion of the cockroach', Ann. Ent. Soc. Am., 5o, 231-237. WEBER, K., and OSBORN, M. (I969), ' T h e reliability of molecular weight determination by dodecyl sulphate polyacrylamide gel electrophoresis', ft. biol. Chem., 244, 44o6-4412.
24Z
HABIBULLAAND NEWBURGH
WILSON, B. W., METTLER, M. A., and ASMUNDSON,R. V. (1969), 'Acetylcholinesterases and nonspecific esterases in developing Avian tissues: Distribution and molecular weight of esterases in normal and dystrophic embryos and chicks', ft. exp. Zool., 172, 49-58. YAMASAKI, T., and NARAHASHI,T. (I958), 'Synaptic transmission in the cockroach', Nature, Lond., I82, 18o5-18o6.
Key Word Index: Galleria melloneUa, central nervous system, development, acetylcholine, acetylcholinesterase, enzyme, isozyme, molecular weight, protein subunit.
231
STUDIES OF ACETYLCHOLINESTERASE OF THE CENTRAL NERVOUS SYSTEM OF G A L L E R I A M E L L O N E L L A M. HABIBULLA
ANn
R. W. NEWBURGH
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331, U.S.A.
(Received 22 January, 1972; revised 4 November, I972) ABSTRACT The central nervous system (CNS) of Galleria mellonella contains an active acetylcholinesterase, whose level changes during the development of the central nervous system. The enzyme occurs in five forms of different electrophoretic mobilities and the relative amounts of each form varies with the life state of Galleria. The molecular weight of the enzyme was estimated to be approximately 28o,ooo ± 15,ooo. It appeared to consist of four polypeptide subunits. Injection of the anticholinesterase, eserine, into the last larval stage inhibits the silk spinning and delays the formation of pupa by more than 2 weeks when compared with the controls. ACETYLCHOLINESTERASE [ACHE] (acetylcholine acetylhydrolase, E.C. 3.I.I.7) has been identified biochemically (Casida, I954; Chadwick, 1963) and cytochemically in electron micrographs of insect neural tissue (Smith and Treherne, 1965). Insect central nervous system neurons were shown to be relatively insensitive to applied acetylcholine [ACh] (Twarog and Roeder, 1957; Yamasaki and Narahashi, 1958; Calhoun, 1963; Kerkut, Pitman, and Walker, i969) , which led some investigators to suggest that the cholinergic system m a y not be present in insect nerves. H o w ever, from the studies of Smallman (1958), Smallman and Mansingh (1969) , and Shankland, Rose, and Donniger (1971) the presence in insects of the cholinergic system appears now to be well established. Since a direct relationship between acetylcholinesterase and neural development has been implicated in various organisms (Karczmar, i963) , this enzyme is a useful tool in studies on neurometarnorphosis. T h i s paper is a report of the changes occurring in acetylcholinesterase from Galleria mellonella during this process. MATERIALS AND M E T H O D S EXPERIMENTAL ANIMALS
The greater wax moth, Galleria mellonella L., was cultured by the method of Pipa (I963) with some modifications (Ishikawa and Newburgh, I97i ). The whole eggs, embryos, and certain isolated tissues from different life stages were used in this study. TISSUE PREPARATIONS
The insects were chilled in ice after CO2 anaesthetization. The various organs used in the experiments were dissected from chilled immobilized insects in a balanced salt solution (Martignoni and Scallion, i96i ) using a dissecting microscope. Whole insects or individual organs were homogenized in a solution containing o'o 5 M KPO,, p H 6"8; o'2 M NaC1; 0"5 per cent Triton X-ioo; and o.ooi M Na, EDTA. Two ml. of homogenizing solution were used for 0-6 g. wet weight of tissue. The homogenate was centrifuged at 8ooo g for io minutes. The pellet was
232
HABIBULLA AND NEWBURGH
Insect Biochem.
again homogenized and centrifuged as previously described and the combined supernatants were used as the source of enzyme. It was determined that more than 95 per cent of the original activity was present in the supernatant. All operations were performed below 4 ° C. PURIFICATION OF THE ENZYME Since the specific activity of A C h E in the brain was high compared to other tissues, it was used as the source of the enzyme for purification. F o r this IOOO heads were homogenized with a mortar and pestle in 5 ml. of o ' o i M potassium phosphate buffer, p H 7"4, containing i35 rag. of a m m o n i u m sulphate. After centrifugation at 3ooo r.p.m, in a Servall centrifuge for 20 minutes, solid a m m o n i u m sulphate was added to the supernatant with stirring to bring it to 3 ° per cent saturation. T h i s solution was centrifuged for IO minutes at 5ooo r.p.m, and the supernatant was dialysed against o.oi M phosphate buffer, p H 7'4. T h e material in the dialysis bag was then concentrated using polyethelene glycol flakes (Carbowax). After withdrawing an aliquot for protein determination, the dialysate was subjected to polyacrylamide gel electrophoresis (Davis, 1964). T w e l v e tubes, 12'5 c m . × o ' 5 cm. diameter, each containing i ' S m l , of the small-pore gel and 0" 3 ml. of the large-pore gel were used. After the gels were prerun for 5 minutes at 15 mA., equal aliquots of the dialysate were applied in a 20 per cent sucrose solution and stacked for 5 minutes at 15 mA. Electrophoresis was conducted for I hour and 4 o minutes at z 5 mA., with ice-cold water circulating through the outer jacket of the apparatus. One of the gels was stained for activity with n-methylindoxyl acetate ( N M I A ) (Guilbault, Kuan, Tully, and Hackney, 197o). T o do this the gel was incubated in a test-tube containing N M I A (o'5 g. in zo ml. of o'I M KPOa, p H 7"5, diluted with an equal volume of glass-distilled water). R e values for the enzyme bands were calculated (Re mobility of enzyme band in cm./mobility of marker dye in cm.). T h e activity could be measured directly from gels washed in o'o5 M phosphate buffer, p H 7'5, by scanning at E~97 using a Gilford recording spectrophotometer with a scanning attachment. Immersion of gels in lO -5 ~ / e s e r i n e for 4 ° minutes prevented the appearance of activity bands. Regions of the gels corresponding to the R r values of the enzyme were isolated and again subjected to electrophoresis. T h e enzyme was again localized by staining one of the gels and the regions of unstained gels corresponding to the R v values of the enzyme were minced and eluted with o ' o i M phosphate buffer, p H 7"4, overnight in the cold. T h e eluates were pooled and centrifuged in a sucrose density gradient (5-20 per cent) at 39,00o r.p.m, for 5 hours at 4 ° C. in a preparative Spinco ultracentrifuge using the S W 39 head. F o u r ml. of 5 per cent sucrose containing o ' o i M phosphate buffer, p H 7"4, and o'5 ml. of the enzyme solution were layered on top of the gradient. After centrifugation, fractions containing 2o drops were collected. Protein in each fraction was measured by the Ezso/E~o m e t h o d of Warburg and Christian (Chaykin, I966 ). A C h E activity was measured using N M I A and measuring the extinction at E~97. A C h E of the electric organ of the electric eel was also centrifuged on a sucrose gradient and used as a reference standard. T h e isolated fractions were also subjected to gel electrophoresis and stained for protein and enzyme activity, and only a single band was found in either determination. MOLECULAR WEIGHT DETERMINATION A C h E molecular weight was estimated using the m e t h o d of W e b e r and Osborn (1969). Because of the high molecular weight of the enzyme, slight modifications were necessary. T h e concentration of methylene bisacrylamide was reduced to one-third, to facilitate migration of high molecular weight proteins, and the molarity of the buffer was reduced to one-half, to decrease the running time. Standard proteins of molecular weights in the range of 4 o , o o ° - 8 ° ° , ° ° ° (DNAse, 5"-globulin, catalase, and thyroglobulin) were utilized to construct a standard curve. For the electrophoresis of individual samples, the same procedure was used. T h e a m o u n t of protein applied to each gel varied from 4 ° to 4oo ~tg. DISSOCIATION OF THE ENZYME INTO SUBUNITS T h e isolated enzyme was dissolved in 6 M guanidine H C I containing o'I per cent 13-mercaptoethanol and dialysed against O'Ol per cent mercaptoethanol overnight. T h e dialysed protein was subjected to polyacrylamide gel electrophoresis to determine the presence of subunits. T h e staining was performed with methanolic Coomassie brilliant blue according to the m e t h o d of W e b e r and Osborn (1969).
1973, 3
AchE OF THE CENTRAL NERVOUS SYSTEM
233
PROTEIN DETERMINATION Gels were stained with Amidoschwartz and Coomassie brilliant blue by standard techniques to localize proteins. T h e excess dye was destained by diffusion with 7 per cent acetic acid. The protein in homogenates was determined by the method of Lowry, Rosebrough, Farr, and Randall (I95I). RADIOMETRIC ASSAY A microradiometric procedure was used for the assay of AChE (Rosenberg, Dalessio, Tremblay, and Woodman, 1971). T h e reaction mixture consisted of o'o5 M potassium phosphate (pH 6'8), o'2 M NaCI, o'ooi M Na2EDTA (pH 6"8), 15 X o ' I o a M acetylcholine iodide, and 2"7 × IO-a M [i-14C]acetylcholine iodide (specific activity 2"4 inc. per mmole total volume 5o lal., 37 ° C. for IO minutes). Th e reaction was stopped by adding i ml. of ice-cold glass-distilled water containing B.W.284C5I dibromide (Burroughs Wellcome), a specific inhibitor of ACHE. This mixture was then placed on an ion-exchange column (Biorad AG5oW-X8 lOO-2OO mesh, Na+). T h e column was prepared in disposable Pasteur pipettes and was 5 cm. in length. Th e eluate was collected in scintillation vials and included two I-ml. ice-cold glass-distilled water washes. Th e eluates were counted in a scintillation counter. Since the column retains acetylcholine and choline, the radioactivity obtained arises from the free [i-14C]acetate and the results are used to calculate the amount of acetylcholine hydrolysed.
ISOTOPE INCORPORATION A total of 50 ~1. of [4,5-~H]leucine was injected into 2o individuals of different stages. After 24 hours the isozymes of AChE were stained with N M I A after polyacrylamide gel electrophoresis. T h e activity bands were cut off and the radioactivity counted by standard methods after solubilizing the gel in hydrogen peroxide. ESERINE INJECTION Last larval stages collected over a period of i hour were anaesthetized with CO~ and 1-2 lal. of lO -3 M eserine were injected into each. Controls were injected with insect Ringer solution.
Table /.--INHIBITION OF A C H E ACTIVITY WITH ESERINE (PHYSOSTIGMINE) AND IS-BIS-(4-ALLYLDIMETHYLAMMONIUMPHENYL)PENTAN-3-ON E DIBROMIDE ~
INHIBITOR
PERCENTAGE INHIBITION OF ACHE
Eserine io -t5 M IO ~ M io-6M io -~ M
88"4 89"2 94"6 95"o
B.W.284C51 dibromide 2 × IO -~
M
2×io-6M 2XIO 8M 2 ×
io -z
M
96 96"2 96 '7 97"2
*B.W.284CsI dibromide (Burroughs Wellcome). T h e enzyme was from the head homogenate of the last larval stage. T h e radiometric assay is given in Methods.
234
HABIBULLAAND NEWBURGH
Insect Biochem.
RESULTS Studies on the hydrolysis of ACh showed that at least 95 per cent of the activity was due to AChE on the basis of its inhibition by BW-284C 51 dibromide and eserine (Table I). Although these results are from the last larval stage head homogenates, similar results were obtained with other stages and tissue. In addition to the inhibition of the enzyme activity it was also found that injection of 2 gl. of lO -3 M eserine per larva inhibited Table / / . - - L o c A L I Z A T I O N OF A C H E IN DIFFERENT TISSUES AND CHANGES DURING DEVELOPMENT OF
TISSUE AND STAGE
Female reproductive system Developing egg Mature unfertilized egg Mature ovaries Fertilized egg Male reproductive system Testes Larvae Fat body Haemolymph Head, last larval instar Brain, last larval instar CNS, last larval instar Pupae Head Adult Head, pharate adult Adult (3 days old) Head Fat body Haemolymph Thoracic muscle
Galleria
ACHE ACTIVITY* (nmoles of ACh hydrolysed per lag. protein in i hour at 37 ° C.)
o o o
0'275
I .o8 0.067 o
o'83 4.88 5"29
i "39 4"60 2"I6 o o I "35
*Mean of 4 experiments. The deviation was less than 6 per cent. spinning of silk in 85 per cent of the cases, and delayed the transformation into pupae by more than i5 days. ACHE ACTIVITY AND CHANGES DURING METAMORPHOSIS T h e activity of A C h E was determined in different stages and tissues of Galleria and the results are shown in Table 11. A C h E could not be detected in the mature ovaries, developing egg, or the unfertilized egg. Activity was found in mature testes and the fertilized egg.
LOCALIZATION OF
1973, 3
AchE OF THE CENTRAL NERVOUS SYSTEM
235
The activity in the testes may be due to the presence of nerves in the preparation. It is reasonable to assume that the activity in the fertilized egg results from the initiation of neural development (Smallman and Mansingh, 1969). Except for the thoracic muscle, the activity in the larvae, pupae, and adult is localized in brain or the CNS. Since the thoracic muscle preparation would contain nerves, the activity in this organ may be the result of this and the presence of neuromuscular junctions. The level of
FIG. I . - - A c t i v i t y staining for acetylcholinesterase after polyacrylamide gel electrophoresis. (See text for details of methods.) T h e material applied to the column was the supernatant fraction isolated from larval heads. Left = stained for A C h E ; m i d d l e - - s t a i n e d for A C h E ; r i g h t - - s t a i n e d for A C h E after incubation in lO -5 M eserine for 3 ° minutes. FIG. 2 . - - F u r t h e r separation of the u p p e r band from Fig. i. T h e upper band from Fig. i was isolated by eluting with o.oi M phosphate buffer, p H 7"4. It was then re-electrophoresed on a 5 per cent polyacrylamide gel (see Methods) and stained with Coomassie brilliant blue.
AChE in the head of the last-instar larvae is lower than that in the pupae or adult head. A peak occurs in the head of the pharate adult and then declines as the insect ages. GEL-ELECTROPHORESIS OF A C H E FROM VARIOUS STAGES OF DEVELOPMENT
Under the conditions used, four activity bands were found in each stage. A typical electrophoretic pattern of AChE activity is shown in Fig. I. Although the reproduction
236
HABIBULLAAND NEWBURGH
Insect Biochem.
shown in Fig. i does not indicate it, the upper bands (RF=o.z3-o.25) consisted of two bands. T h e A C h E of the electric organ of the electric eel which was used as a reference had an RF value in this same region. T h e s e bands could be further resolved by isolating t h e m from the gel and doing a re-electrophoresis in a 5 per cent gel (Fig. z). T h e fast-moving A C h E activity bands had an R r value of between approximately o.58 and o'6i and, as seen in Fig. I, were well separated. F r o m these results it was concluded that four enzymatically active bands were present and represent isozymes of ACHE. It was then of interest to examine the possibility that
+ "
B
ol
."
.I
I.
. . .
I.
Oo
,4 B
G D
M
1234 Fro. 3.--Diagrammatic representation of AChE isozyme pattern of the head (except in the case of eggs) region of Galleria during different stages of development. I, Eggs (developing whole embryos); 2, larva; 3, pupa; 4, adult. A, B, C, and D designate four isozymes of ACHE. B1 denotes an isozyme which appears only in the adult stage. H is the position of the marker dye. The distance travelled by the marker dye in the separation gel was 7 cm. The dotted portion at the top of the figure represents the stacking gel. The procedures are given in the text. The widths of the bands are drawn to represent the relative amount of enzyme activity in each band at a particular stage of development, as determined by measuring AChE activity in the gel at E297.
at different stages of development different isozymes of A C h E are present. This was found to be true and the results are shown schematically in Fig. 3. Four forms of the enzyme were present in each of the four stages of development (embryo, larva, pupa, and adult). In the adult stage, one band (A) disappears and a new band (B1) appears. T h a t this result was not an artifact was shown by doing gel electrophoresis experiments with mixtures of the different stages (Fig. 4). When a mixture of larval and adult tissue was run, (F@ 4, 2) an additional band (B1) was apparent and this also was true when a mixture of pupa and adult material (Fig. 4, 5) was subjected to co-electrophoresis. T h e relative amounts of activity in the different bands also change during neurometamorphosis. T o determine this, the experiments consisted of adding the same
237
AchE OF THE CENTRAL NERVOUS SYSTEM
I973, 3
amount of protein (i20 lag.) to the gel, electrophoresing, and analysing the activity bands at E29v in a scanning attachment to a Gilford spectrophotometer. Typical results are shown in Table III, column 4. In the egg the predominant peak was B1, in the
+ •
~
I
n
2
5
.
,
°-.,. .
.
m
n
I
m
~
r a i n
4
. .
.
.
. ,
.
.
.
n
5
6
FIG. 4.--Diagrammatic representation of co-electrophoresis of AChEs from different stages of development. Other details as in Fig. 3. I, Larval ACHE; 2, larval+ adult ACHE. Note the additional band indicated by arrow corresponding to that of number 3; 3, adult ACHE; 4, pupal ACHE; 5, pupal~adult ACHE. Note the additional band indicated by arrow corresponding to that of number 6; 6, adult ACHE. Arrow=B1 band. larval stage C and D were the major peaks, in the pharate pupa band B again was the major peak, and in the pharate adult the major peaks were B and the new peak B 1. [aH]LEUClNE INTO A C H E [aH]Leucine was injected into insects at various stages of development. After z 4 hours the AChE isozymes were separated on gels, eluted, and analysed for incorporation of this amino-acid. The results of these studies are shown in Table III. In the larva bands C and D had the highest specific activity. The specific activity of band A in the pharate pupa was greater than in larva. The high specific activity of band B1 in the pharate adult is indicative of its rapid synthesis at this particular stage. INCORPORATION OF
PHYSICOCHEMICALSTUDIES The AChE from the larval heads was purified by a combination of ammonium sulphate precipitation, gel electrophoresis, and sucrose density gradient centrifugation techniques as described in the methods section (Table IV). Approximately a five-hundredfold purification was obtained for each of the four isozymic forms of the ACHE. Each isozyme was separated in a single peak on a sucrose gradient and, when the AChE of the electric eel was treated similarly, a single peak of active enzyme was found in the same position as the AChE band B of Galleria. The purity of each of the isolated isozymes was
238
Insect Biochem.
HABIBULLA AND NEWBURGH
established b y separation on polyacrylamide gel a n d d e t e r m i n a t i o n of the e n z y m e activity. Each of the isolated isozymes was dissociated in 6 M g u a n i d i n e HC1 cont a i n i n g o.i per cent [3-mercaptoethanol at r o o m t e m p e r a t u r e for 2 hours, a n d dialysed Table III.
INCORPORATION OF [4,5-aH]LEuCINE INTO THE DIFFERENT ISOZYMES OF ACETYLCHOLINESTERASE
ISOZYME BAND
STAGE
LABEL INCORPORATED
(c.p.m.)
0" 5 WIDTH X HEIGHT OF THE PEAK OF THE BAND SCANNED AT E297 (cm.)
A
Egg
B
2"31 8"1
C D
o'19
Pharate pupa
Pharate adult
2"1
B
30,000 47,00o
6'5
6,000 7,23 °
C D
168,ooo 152,79o
14'82 I4" 4
IO,6IO
A
82,000
B
6'2 16'8
13,23o 8,750
C D
147,ooo 84,6oo 56,568
6'6 5"4
12,820 10,480
A B B1 C D
I44,692 551,826 10,206 41,568
15"4 21 .o 2'4 3"3
9,4 °0 26,280 4,260 12,6oo
A
Last larval stage (after cessation of feeding)
SPECIFIC ACTIVITY
5'0
11,340
Fifty pl. of tritiated leucine (specific activity 20"8 c. per mmole) were injected into 20 individuals. The insects were incubated for 24 hours and the heads removed. These were then assayed for AChE by procedures as given under Methods.
Table/V.--PuRIFICATION OF ACHE FROM Galleria mellonella
FRACTION
SPECIFIC ACTIVITY (mmoles of ACh hydrolysed per mg. protein per hour at 37 ° C.)
I. Original homogenate
0"68
PERCENTAGE TOTAL ACTIVITY
IO0
(total mmoles of ACh hydrolysed in I hour at 37 ° C.--2'5) 2. 3° per cent saturated ammonium sulphate supernatant 3. Gel electrophoresis 4. Sucrose density gradient centrifuge (tubes 7-1o) The procedures are given under Methods.
3 "OO
20"4° 3o6'o
63.8 20
8.26
~973, 3
AChE OF THE CENTRAL NERVOUS SYSTEM
239
against o.ox M sodium phosphate buffer (pH 7"0) containing o.x per cent 13-mercaptoethanol overnight. The dialysate was then incubated at 37 ° C. for z hours in o.o1 M sodium phosphate buffer (pH 7"0) containing 1 per cent sodium dodecyl sulphate (SDS) and i per cent 13-mercaptoethanol. The protein concentration was 20 ~tg. per lOO Ill. The sample was then subjected to SDS polyacrylamide gel electrophoresis (Weber and Osborn, 1969). Under these conditions each of the isozymes dissociated into four subunits (Fig. 5). The molecular weight of each subunit was determined by using a standard
1000 =
o 2
2
3
×
100
4
10
1
,
,
,
,
I
,
0"5
,
~
I
I 1
MOBILITY
FIG. 5.--Diagrammatic representation of the electrophoresis of isozyme B of ACHE. The AChE (isozyme B) was treated with guanidine hydrocb_lorideand then separated by polyacrylamide gel electrophoresis. Details are provided in the text.
FIG. 6.--Graph showing relationship between molecular weight (MW) of the standard proteins and their mobilities in SDS polyaerylamide gel electrophoresis. The MW of the polypeptide subunits of AChE of Galleria are shown as small open circles and MW of AChE is represented as a big open circle. T=Thyroglobulin; C=catalase; G= -/-globulin; D=DNAse. Mobility=[distanee of protein migration)/(length after destaining)] × [(length before staining)/distance of dye migration)].
curve prepared from proteins of known molecular weight (Weber and Osborn, 1969)
(Fig. 6). The molecular weights of the polypeptide subunits ranged between 65,000 and 85,000, and the molecular weight of the protein containing four subunits was 28o,ooo :J: 15,000. DISCUSSION The absence of AChE in the mature ovary and developing and unfertilized egg, and the presence in the fertilized egg, is indicative of the initiation of neurogenesis. This is consistent with the results of Smallman and Mansingh (1969) and Shankland and others (1971). The increased AChE activity observed in the pharate pupal stage is likely to be the result of increased numbers of aeetylcholinesterase-rich neurons (Rockstein, 195o ,
240
HABIBULLAAND NEWBURGH
Insect Biochem.
I953) and may be associated with development of the optic lobe (Norlander, i967; Smallman and Mansingh, i969). Morphologically there is a considerable increase in the brain size during this stage and active cell division, as shown by a marked increase in the activity of thymidylate synthetase (Albin and Newburgh, I972 ). The acetylcholinesterase of the central nervous system of Galleria has a higher specific activity than is found in the cockroach (Lord, Gregory, and Burt, I967) or snail (Emson and Kerkut, I97I ). If this is generally true of the CNS insects it might explain why a relatively high concentration of acetylcholine (Io -~ M) is required to elicit excitation of insect neurons (Twarog and Roeder, I957; Yamasaki and Narahashi, x958; Calhoun, i963; Kerkut and others, ~969). From the studies reported here it appears likely that the acetylcholinesterase is associated only with neural tissue. This assumes, of course, that the activity found in non-neural tissues is due to contamination, and this seems reasonable since some degree of innervation occurs in these later tissues. Although it has been implied that the molecular weight of AChE in insects is in the order of 3-4 million (Dauterman, Talens, and van Aspen, i962), the molecular weight of this enzyme from Galleria is in the range of 280,000. This compares well with the (molecular weight of the AChE from the electric organ of the electric eel [260,0oo Leuzinger, Goldberg, and Cauvin, I969) ] and from the snail [24o,ooo (Emson and Kerkut, x97I)]. The AChE from developing chick tissue was shown to be as large as 420,000 (Wilson, Mettler, and Asmundson, i969). Dissociation of the Galleria acetylcholinesterase yielded four polypeptide subunits. This agrees with that found in snail brain (Emson and Kerkut, I97i ) but is different from the enzyme from the electric eel, where Leuzinger and others (i969) found the enzyme to be a tetramer of only two subunits. It appears that these subunits are combined in different ways during neurometamorphosis in Galleria to yield five different isozymes. At present, we do not have sufficient information to determine the subunit composition of each isozyme. Nevertheless, since isozymes are a refined expression of the enzymatic differentiation of a cell, certain mechanisms of neurometamorphosis in Galleria can be suggested. In Galleria, as in other holometabola, metamorphosis is maximal during egg to larval, larval to pupal, and pupal to adult development. Examination of the results of [3H]leucine incorporation are indicative of a genetic control of events that is manifested in a change in rate of synthesis of the different isozymes of ACHE. Of particular interest is the appearance of an additional isozymic form and disappearance of an earlier form in the adult stage. It would be of interest to determine whether these changes represent changes in the synthesis of the complete protein or particular subunits. Inhibition of AChE activity, by injecting eserine into larvae, appears seriously to interfere with normal metamorphosis. This suggests that the presence of an active acetylcholinesterase is required to initiate silk spinning and the larval to pupal transformation. One possible mechanism might be that these changes are under neuronal control and the normal process is altered owing to an increase in the amount of acetylcholine, which could result from an inhibition of AChE activity. REFERENCES ALBIN, E. A., and NEWBURGH,R. W. (I972), unpublished observations. CALHOUN, E. H. (x963), in Advances in Insect Physiology (ed. Beament, Treherne, and Wigglesworth), vol. I, pp. 1-46. New York: Academic Press.
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AChE OF THE CENTRAL NERVOUS SYSTEM
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CASlDA, J. E. (I954) , 'Comparative enzymology of certain insect acetylesterases in relation to poisoning by organophosphate insecticides', ft. Physiol., Lond., I27, zoP-~IP. CHADWICK, L. E. (I963), in Cholinesterases and Anticholinesterase Agents (ed. Koelle), pp. 741798. Berlin: Springer. CHAYKIN, S. (I966), Biochemistry Laboratory Techniques. New York: Wiley. DAUTERMAN,W. C., TALENS, A., and IVANASPEN (I962), 'Partial purification and properties of fly head cholinesterase ', J. Insect Physiol., 8, 1-I 4. DAvis, B. J. (I964), 'Disc electrophoresis. II. Methods and application to human serum proteins', Ann. N.Y. Acad. Sci., x2I, 4o4-4z7. EMSON, P. C., and KERKUT, G. A. (I97I), 'Acetylcholinesterase in snail brain', Comp. Biochem. Physiol., 39, 879-889. GUILBAULT,G. G., KUAN, S. S., TULLY, J., and tIACK.NEY,D. (I97O), 'New procedure for rapid and sensitive detection of cholinesterase separated by polyacrylamide gel electrophoresis', Analyt. Biochem., 36, 7z-77. ISHIKAWA, H., and NEWBURCH, R. W. (I97I) , 'Changes in RNA during metamorphosis of the central nervous tissue of Galleria', +7. Insect Physiol., I7, I I I3-I 124. KARCZMAR,A. G. (I 963), in Cholinesterases and AntichoIinesterase Agents (ed. KoeUe), pp. 129-186. Berlin: Springer. KERKUT, G. A., PITMAN, R. M., and WALXER, R. J. (~969), ' Sensitivity of neurons of the insect central nervous system to iontophoretically applied acetylcholine or GABA', Nature, Lond., 222, io75-io76. LEUZlNGER, W., GOLDBERC, M., and CAUVlN, E. (I969), 'Molecular properties of acetylcholinesterase',J, sol. Biol., 4o, 27-zz5. LORD, K. A., GREGORY, G. E., and BURT, P. E. (1967) , ' T h e penetration of acetylcholine into the central nervous system of the cockroach Periplaneta americana (L.)', ft. exp. Biol., 46, 153-159. LowRY, O. H., ROSEBROUGH,N. J., FARR,A. L., and RANDALL,R. J. (I 95 I), 'Protein measurement with the Folin phenol reagent', v~. biol. Chem., I93, 265-275. MARTIGNONI,M. E., and SCALLION,R. J. (I96I), 'Preparation and uses of hemocyte monolayers in vitro', Biol. Bull. mar. biol. Lab., Woods Hole, I2X, 5o7-52o. NORLANDER, R. (I967), in Invertebrate Nervous Systems (ed. Wiersma). University of Chicago Press.
PIPA, R. L. (1963) , ' Studies on the hexapod nervous system. VI. Ventral nerve cord shortening; a metamorphic process in GaUeria mellonella (L.)', Biol. Bull. mar. biol. Lab., Woods Hole, I24~ 203-302. ROCKSTEIN, M. (I95O), ' T h e relation of cholinesterase activity to change in cell number with age in the brain of adult worker honeybee', .7. cell. comp. Physiol., 55, 1I-z4. ROCKSTEIH,M. (1953), 'Physiology of aging in the adult worker honeybee', Biol. Bull. mar. biol. Lab., Woods Hole, IO5, I54-I59. ROSENBERG, R. N., DALESSIO,D. J., TREMBLAY,J., and WOODMAN, D. (I97I), 'Kinetics of acetylcholine synthesis and hydrolysis in myasthenia gravis', Science, N.Y., x73 , 644-645. SHANKLAHD,D. L., ROSE, J. A., and DONNIGER, C. (I97I), ' T h e cholinergic nature of the cercal nerve giant fiber synapse in the sixth abdominal ganglion of the American cockroach Periplaneta americana (L.)', ~t. Neurobiol., 2, 247-262. SMALLMAN, B. N. (I958), 'Physiological basis for the mode of action of organophosphorous insecticides', Proc. Xth Int. Cong. Ent., 2, 5-12. SMALLMAN, B. N., and MANSINOH, A. (x969), ' T h e cholinergic system in insect development', A. Rev. Ent., x4~ 387. SMITH, D. S., and TREHEENE,J. E. (I965), ' T h e electron microscopic localization of cholinesterase activity in the central nervous system of an insect, Periplaneta americana L.', J. Cell Biol., 26, 445-456. TWAROG, B. M., and ROEDER, K. D. (I957), 'Pharmacological observations of the desheathed last abdominal ganglion of the cockroach', Ann. Ent. Soc. Am., 5o, 231-237. WEBER, K., and OSBORN, M. (I969), ' T h e reliability of molecular weight determination by dodecyl sulphate polyacrylamide gel electrophoresis', ft. biol. Chem., 244, 44o6-4412.
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WILSON, B. W., METTLER, M. A., and ASMUNDSON,R. V. (1969), 'Acetylcholinesterases and nonspecific esterases in developing Avian tissues: Distribution and molecular weight of esterases in normal and dystrophic embryos and chicks', ft. exp. Zool., 172, 49-58. YAMASAKI, T., and NARAHASHI,T. (I958), 'Synaptic transmission in the cockroach', Nature, Lond., I82, 18o5-18o6.
Key Word Index: Galleria melloneUa, central nervous system, development, acetylcholine, acetylcholinesterase, enzyme, isozyme, molecular weight, protein subunit.