- Email: [email protected]
Distribution of calmodulin in insects as determined by radioimmunoassay
Camp. Biochem. Physiol. Vol. 8OC, No. 2, pp. 241-244. 1985 Printed in Great Britain
0306.4492185 $3.00 + 0.00 Pergamon Press Ltd
DISTRIBUTION OF CALMODULIN IN INSECTS DETERMINED BY RADIOIMMUNOASSAY
AS
MARK S. WRIGHT and BENJAMIN J. COOK Veterinary Toxicology and Entomology Research Laboratory, AgriculturaI Research Service, U.S. Department of Agriculture, P.O. Drawer GE, College Station, TX 77841, USA. Telephone: (409)260-9316
(Received 17 Jury 1984) Abstract-l.
The distribution of calmodulin in 11 separate tissues of the cockroach Leucophaea maderae The highest levels of this protein were found in Malpighian tubules, visceral muscle and the central nervous system. 2. Distribution of calmodulin was also determined for the following insect species: &is meliifera, Canthon imitator, Heiiothis zea (larvae) and Periplaneta americana. Abdominal tissues contained the highest levels of the calcium binding protein, while lesser amounts were found in the thorax and head. 3. The detected Levels of calmodulin suggest that this protein serves as an important regulator of bi~hemi~l functions in the viscera and nervous system of insects.
was determined by radioimmunoassay.
INTRODUCTION
The importance of calcium in muscular function has long been recognized (Ringer, 1886). Careful measurements of intracellular calcium in a variety of cells show that the concentration of this ion seldom exceeds 10s7 M in the resting state, while extracellular amounts are on the order of 10m3M. Such a large electrochemical gradient across cellular membranes provides an excellent basis for a signaling mechanism, because relatively minute transmembrane fluxes can radically change the intracellular level of the ion. This is particularly evident in the course of excitation~ontraction coupling in muscle, where the sudden influx of extracellular calcium can elevate the intracellular calcium levels to between 10e6 and IO-’ M and trigger contraction. Calcium is now recognized as an important intracellular messenger that regulates not only muscle but also glandular secretion and many enzymatic mechanisms such as adenylate cyclase (Brostrom et al., 1975) and glycogen synthase (Wang and Waisman, 1979). The recent discovery of the calcium binding protein calmodulin (CAM) has greatly expanded our understanding of the intracellular actions of calcium. This protein, for example, seems to regulate the phosphorylation of myosin light chain kinase, an important enzyme in the contraction process of vertebrate smooth muscle (Schaub Watterson, 1981; Conti and Anderson, 1980). In a series of preliminary experiments, we found that certain calmodulin inhibitors seemed to suppress both automyogenic contractions of the hindgut and the excitatory effects of the neuropeptide proctolin. Since CAM has a critical role in the contraction sequence of vertebrate smooth muscle, it seemed worthwhile to compare the presence and distribution of this protein in both visceral and nonvisceral tissues of a single insect species. ~A~RIA~
AND METHODS
Radioimmunoassay-grade bovine serum albumin (BSA), ether)-N,N,N’,N’-tetraet~ylene~lycol-his-(~-aminoethyl
acetic acid (EGTA), sodium chloride, boric acid and sodium borate were purchased from Sigma Chemical Company (St. Louis, MO). The CAM radioimmunoassay (RIA) kit was purchased from Amersham (Arlington Heights, IL). Leucophaea maderae, Periplaneta americana and Canton imitator larvae were taken from stock colonies maintained at VTERL. He&this zea and Apis mellifera were obtained from Dr R. Meola, Texas A & M University and B.J.C.. respectively. Tissues were excised and maintained at - 196°C until assayed. The freeze-dried reagents supplied in the RIA kit were rehydrated with distilled water, kept at 4°C and used within 4 weeks of receipt. Varying concentrations (5-1250 ng/ml) of the CAM standard samples were made by
serial dilution of a 2SOOng/ml standard. The tissues were thawed in homogenization buffer (125 mM borate, 1 mM EGTA, 75 mM NaCl, pH 8.4) and homogenized using a Polytron”’ with a PT-IO generator. Nerve cords were homogenized with a glass micro tissue grinder using a five-fold excess of buffer (volume:weight). Next the tissues were sonicated four times using a BraunSonic 1510 sonicator at a setting of 100 W in 15 set pulses. The tissues were kept in an ice bath during this process. After homogenization, samples were removed for total protein assay by the method of Bradford (1976). The remaining homogenates were brought to a rapid boil in a microwave oven and quickly cooted in a dry ice-ethanol bath. The heat-treated samples were centrifuged at 15,000 # for 30 min and the supernatants were immunoassayed. ~mmunoassays were run in triplicate, using homogenization buffer that contained 2”~: BSA as a solvent. The standard curve was set up with known values
of CAM ranging from 0.5 to 125 ng per assay tube, along with controls to determine the total bound and background. Test samples were diluted to contain 50-100 ng/ml of CAM; dilutions were based on the assumption that CAM levels range from 0.1 to 1% of total protein present. The assay mixtures were incubated at 25°C for 18 hr. The S. aureus reagent was then added to each assay tube and incubated for 30 min with vortexing every 10min. The resulting immunoglobulin-S, aureu,y complex was centrifuged to a pellet at 1000 g for 10 min. The supernatant was decanted, the pellet rinsed twice and the mouth of the tubek blotted dry. The tubes were counted in a gamma spectrometer (Backman model 3000). The results obtained from the gamma spectrometer were plotted on semilog graph paper (Fig. 1). The resulting graph shows the percentage of CAM bound to the antibody vs the concentration of CAM in the standard samples. The con241
242
MARK S. WRIGHTand BENJAMIN J. COOK
0’ I
10
IO0
ng Calmodulin Fig. 1. ~lmodulirl RIA standard curve. A standard competition binding curve for calmodul~n binding was generated (see Materials and Methods). Each data point is an average of two assays which were done in triplicate. Bars represent SEM.
centration of CAM in the unknown samples was derived from the graph by calculating the percentage of CAM bound to the antibody, plotting the results on the ordinate and reading the sample CAM concentration off the abscissa. RESULTS AND
DISCUSSION
We used a radioimmunoassay (RIA) procedure for measuring calmodulin levels in insect tissues because attempts to use the phosphodiesterase (PDF) assay method (Cheung, 1970) proved quite unreliable. The RIA has great specificity and may be used to quantitate CAM when the PDE assay is unable to detect any measurable CAM. It is important to note that EGTA was included in all initial homogenization buffers, which gives the buffer calcium-chelating ability and yields calmodulin dissociated from various binding sites (Klee and Vanaman, 1982). A typical competition-binding curve for the CAM RIA is shown in Fig. I. The efficiency of the assay was monitored (1) by checking the recovery of internal standards of bovine brain CAM added to the tissue extracts and (2) by assay of serial dilutions of tissue extracts. This RIA was developed against rat testes CAM, and it has a limit of detection of 15 pg of CAM. The immunological response for CAM was the same regardless of whether the CAM was extracted from rat testes, bovine brain, sea pansey (Renilla reniformis) or peanut plant (ha&s hypoguea). Troponin C from rabbit skeletal muscle demonstrates only slight jmmunological cross-reactivity. This calcium binding protein would have to be increased 665-fold to achieve a 50% competition with CAM in the RIA (Chafouleas et ol., 1979). Calmodulin-like activity was detected in all tissues examined. The distribution in specific tissues of the cockroach L. maderae is illustrated by a series of histograms in Fig. 2. From the data recorded in this figure it is obvious that CAM is particularly abundant in the muscular organs of the viscera and the central nervous system. The large amount of immu-
nological activity in the superior longitudinal muscle isolated from the hindgut indicates that this calcium binding protein may mediate the contraction process in insects as it does in the smooth muscle of vertebrates (Adelstein and Klee, 1980). Such a prospect seems even more likely when the amount of CAM present in this isolated muscle is compared with that in the whole hindgut (VM and HG in Fig. 2). The muscle tissue probably accounts for more than 80% of the activity. Also, the low levels of CAM in the coxal muscle suggest that troponin C may be the principal calcium binding protein in insect skeletal muscle, just as it is in vertebrates (Berridge, 1980). In excretion, unlike the pressure driven system for blood filtration in most vertebrates, insects depend on an active secretory process across the epithelium of the Malpighian tubules (Maddrell, 1981). The surprisingly large amount of CAM present in Malpighian tubules probably reflects some kind of involvement with the active transport mechanisms of this secretion. Since CAM also has been implicated in neurotransmission and neurosecretion (Roufogalis, 1983), it was not unexpected to find high levels of the protein in the nervous tissue of insects. Calmodulin levels were determined in the head, thorax and abdomen of five insect species (Fig. 3). The abdominal tissues contained the largest amounts of the protein in most species, ranging from 4 to 8 ng of CAM per pg of protein. Smaller amounts usually were found in the head and thorax. However, in larvae of H. zea and in the adult dung beetle C. ~~~fu?~r substantial levels of CAM were detected in the thorax. Such anomalies might reflect the presence of a larger mass of visceral organs in the thorax of these insects than others. A comparison of calmodulin levels between the cockroach and the rat is shown in Table 1. Although the levels in the rat were determined by phosphodiesterase assay and those in the cockroach were not, it is still of interest to compare the interspecies ratios for CAM in the two animals. The brain to liver ratio in the rat is quite similar to the brain to fat body
Distribution
of calmodulin
in insects as determined
by radioimmunoassay
243
16 15 14
13
12
1 1
10
9
a
7
6
5
4
3
2
1 0 FG
OD
VM
HG
CM
B
TNC
ANC
MT
OV
FE
Fig. 2. Leucophueu mndercte calmodulin RIA activity. Representative tissues from L.. maderoe were assayed for calmoduiin. Tissues were assayed in triplicate using a calmodulin rddioimmunoassay. Determinations were done with 10 tissue samples from the foregut (FG). oviduct (OC), superior longitudinal muscle of the rectum (VM). hindgut (HG), coxal muscle (CM), brain (3). thoracic nerve cords (TNC), abdominal nerve cords @NC), Malpighian tubules (MT). ovaries (OV). fat body (FB). Bars represent SEM.
A.
mullifra
Ii.
zea
larva
C.
imitator
P.
amerlcana
L.
maderse
Fig. 3. Head (cross hatch), thoracic (black) and abdominal tissues (white) from A. melliferu, If.zeu, C. imiiafor, P. americuna and L. maderae were assayed in triplicate using a calmodulin radioimmunoassay. Determinations were done with four tissue samples from each insect. Each dete~ination was done in triplicate. Bars represent SEM.
244
MARK S. WRIGHT and BENJAMIN J. COOK Table 1. Comparison of mean tissue calmodulin levels L. maderae (ng/pg protein) 13.80 (brain) 14.04 (MT) 3.57 (FB)
Rat (ng/pg) protein) 16.47 (brain) 2.45 (kidney) 2.12 (liver)
Values for the rat were taken from Kakiuchl el al. (1975); a value of 40 units of calmodulin being equal to I pg of calmodulin (MT = Malpighian FB = fat tubules, body).
ratios in the insect. The CAM levels in the rat kidney are low compared to those in rat brain. However, the CAM detected in Malpighian tubules is almost the same as that in the insect brain. This undoubtedly reflects the difference in excretory mechanism between these two animals. Early experiments on insects indicated that CAM was not present in either flight muscle (Volmer, 1977) or the related nervous system of insects (Albin, 1975). However, more recent work has shown that this calcium-binding protein is present in flight muscle of locust (Cox et al., 1981), fat body of silk worm (Bodnaryk, 1984) and the head of the Mediterranean fruit fly (Dudoignon et al., 1983). These findings, along with those presented in this paper, strongly suggest that CAM serves as an important regulator of biochemical and physiological functions in insects.
REFERENCES Adelstein R. L. and Klee C. B. (1980) Smooth muscle myosin light chain kinase. Cal. Ceil Function 1, 167-181. Abin E. E., Davidson S. K, and Newbrough R. W. (1975) Properties of cyclic nucleotide phosphodiesterase in the central nervous system of Manduca sexta. Biochim. biophys. Acta 377, 364380. Berridge M. J. (1980) Hormone action-a search for transducing mechanisms. In Insecf Biology in the Future (Edited by Locke M. and Smith D. S.), pp. 463478. Academic Press, London. Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, ^.^ ^_. L4&L34.
Bodnaryk R. P. (1984) Isolation and characterization of a calmodulin lacking trimethyllysine from the fat body of the silk worm, Bombyx mori. Insect Biochem. 14, 11-17. Brostrom C. O., Haung Y. C., Breckenridge B. M. and Wolff D. J. (1975) Identification of a calcium binding protein as a calcium dependent regulator of brain adenyl cyclase. Proc. natn. Acad. Sci. U.S.A. 72, 64-68. Chafouleas J. G., Dedman J. R., Munjaal R. P. and Means A. R. (1979) Calmodulin development and application of a sensitive radioimmunoassay. J. biol. Chem. 254, 10262-10267. Cheung W. Y. (1970) Cyclic 3’,5’-nucleotide phosphodiesterase. Demonstration of an activator. Biochem. kiophys. Res. Commun. 33, 533-538. Conti M. A. and Adelstein R. S. (1980) Phosohorvlation bv . cyclic adensine 3’-5’-monophosphate-dependent protein kinase regulates myosin light chain kinase. Fedn. Proc. Fedn. Am. Sots exp. Biol. 39, 1569-1573. Cox J. A., Kretsinger R. H. and Stein E. A. (1981) Sarcoplasmic calcium-binding proteins in insect muscle isolation and properties of locust calmodulin. Biochim. biophys. Acta 670, 441444. Dudoignon R. M., Gavilanes J. G., Henriquez R., Municio A. M. and Toro M. J. (1983) Calmodulin from the insect C. capita: isolation and molecular characterization. Comp. Biochem. Physiol. 76B, 643-647. Kakiuchi S., Yamazaki R., Teshima Y., Kinihiro U. and Mivamoto E. (1975) Multiple cyclic nucleotide phosphodiesterase activities from rat iissues and occurrence of a calcium-plus-magnesium-ion-dependent phosphodiesterase and its protein activator. Biochem. J. 146, 109-120. Klee C. B. and Vanaman T. C. (1982) Calmodulin. In Advances in Protein Chemistrv (Edited bv Anfinsen C. B., Edsall J. T. and Richard F.- I$, Vol. 35, pp. 215-321. Academic Press, New York. Maddrell S. H. P. (1981) The functional design of the insect excretory system. J. exp. Biol. 90, 1-15. Ringer S. (1886) Further experiments regarding the influence of small quantities of lime, potassium, and other salts on muscle tissue. J. Physiol. 7, 291-308. Roufogalis B. D. (1983) Calmodulin: its role in synaptic transmission. Trends Neurosci. 6, 238-241. Schaub M. C. and Watterson J. G. (1981) Control of the contractile process in muscle. Trends Pharmacol. Sci. 2, 279-282. Volmer H. (1977) Properties of cyclic nucleotide phosphodiesterase from the flight muscle of Locusta migratoria. Insect Biochem. 7, 411414. Wang J. H. and Waisman D. M. (1979) Calmodulin and its role in the second messenger system. In Current Topics in Cellular Regulation (Edited by Horecker B. L. and Stadtman E. R.), Vol. 15, pp. 47-107. Academic Press, New York. .
I
0306.4492185 $3.00 + 0.00 Pergamon Press Ltd
DISTRIBUTION OF CALMODULIN IN INSECTS DETERMINED BY RADIOIMMUNOASSAY
AS
MARK S. WRIGHT and BENJAMIN J. COOK Veterinary Toxicology and Entomology Research Laboratory, AgriculturaI Research Service, U.S. Department of Agriculture, P.O. Drawer GE, College Station, TX 77841, USA. Telephone: (409)260-9316
(Received 17 Jury 1984) Abstract-l.
The distribution of calmodulin in 11 separate tissues of the cockroach Leucophaea maderae The highest levels of this protein were found in Malpighian tubules, visceral muscle and the central nervous system. 2. Distribution of calmodulin was also determined for the following insect species: &is meliifera, Canthon imitator, Heiiothis zea (larvae) and Periplaneta americana. Abdominal tissues contained the highest levels of the calcium binding protein, while lesser amounts were found in the thorax and head. 3. The detected Levels of calmodulin suggest that this protein serves as an important regulator of bi~hemi~l functions in the viscera and nervous system of insects.
was determined by radioimmunoassay.
INTRODUCTION
The importance of calcium in muscular function has long been recognized (Ringer, 1886). Careful measurements of intracellular calcium in a variety of cells show that the concentration of this ion seldom exceeds 10s7 M in the resting state, while extracellular amounts are on the order of 10m3M. Such a large electrochemical gradient across cellular membranes provides an excellent basis for a signaling mechanism, because relatively minute transmembrane fluxes can radically change the intracellular level of the ion. This is particularly evident in the course of excitation~ontraction coupling in muscle, where the sudden influx of extracellular calcium can elevate the intracellular calcium levels to between 10e6 and IO-’ M and trigger contraction. Calcium is now recognized as an important intracellular messenger that regulates not only muscle but also glandular secretion and many enzymatic mechanisms such as adenylate cyclase (Brostrom et al., 1975) and glycogen synthase (Wang and Waisman, 1979). The recent discovery of the calcium binding protein calmodulin (CAM) has greatly expanded our understanding of the intracellular actions of calcium. This protein, for example, seems to regulate the phosphorylation of myosin light chain kinase, an important enzyme in the contraction process of vertebrate smooth muscle (Schaub Watterson, 1981; Conti and Anderson, 1980). In a series of preliminary experiments, we found that certain calmodulin inhibitors seemed to suppress both automyogenic contractions of the hindgut and the excitatory effects of the neuropeptide proctolin. Since CAM has a critical role in the contraction sequence of vertebrate smooth muscle, it seemed worthwhile to compare the presence and distribution of this protein in both visceral and nonvisceral tissues of a single insect species. ~A~RIA~
AND METHODS
Radioimmunoassay-grade bovine serum albumin (BSA), ether)-N,N,N’,N’-tetraet~ylene~lycol-his-(~-aminoethyl
acetic acid (EGTA), sodium chloride, boric acid and sodium borate were purchased from Sigma Chemical Company (St. Louis, MO). The CAM radioimmunoassay (RIA) kit was purchased from Amersham (Arlington Heights, IL). Leucophaea maderae, Periplaneta americana and Canton imitator larvae were taken from stock colonies maintained at VTERL. He&this zea and Apis mellifera were obtained from Dr R. Meola, Texas A & M University and B.J.C.. respectively. Tissues were excised and maintained at - 196°C until assayed. The freeze-dried reagents supplied in the RIA kit were rehydrated with distilled water, kept at 4°C and used within 4 weeks of receipt. Varying concentrations (5-1250 ng/ml) of the CAM standard samples were made by
serial dilution of a 2SOOng/ml standard. The tissues were thawed in homogenization buffer (125 mM borate, 1 mM EGTA, 75 mM NaCl, pH 8.4) and homogenized using a Polytron”’ with a PT-IO generator. Nerve cords were homogenized with a glass micro tissue grinder using a five-fold excess of buffer (volume:weight). Next the tissues were sonicated four times using a BraunSonic 1510 sonicator at a setting of 100 W in 15 set pulses. The tissues were kept in an ice bath during this process. After homogenization, samples were removed for total protein assay by the method of Bradford (1976). The remaining homogenates were brought to a rapid boil in a microwave oven and quickly cooted in a dry ice-ethanol bath. The heat-treated samples were centrifuged at 15,000 # for 30 min and the supernatants were immunoassayed. ~mmunoassays were run in triplicate, using homogenization buffer that contained 2”~: BSA as a solvent. The standard curve was set up with known values
of CAM ranging from 0.5 to 125 ng per assay tube, along with controls to determine the total bound and background. Test samples were diluted to contain 50-100 ng/ml of CAM; dilutions were based on the assumption that CAM levels range from 0.1 to 1% of total protein present. The assay mixtures were incubated at 25°C for 18 hr. The S. aureus reagent was then added to each assay tube and incubated for 30 min with vortexing every 10min. The resulting immunoglobulin-S, aureu,y complex was centrifuged to a pellet at 1000 g for 10 min. The supernatant was decanted, the pellet rinsed twice and the mouth of the tubek blotted dry. The tubes were counted in a gamma spectrometer (Backman model 3000). The results obtained from the gamma spectrometer were plotted on semilog graph paper (Fig. 1). The resulting graph shows the percentage of CAM bound to the antibody vs the concentration of CAM in the standard samples. The con241
242
MARK S. WRIGHTand BENJAMIN J. COOK
0’ I
10
IO0
ng Calmodulin Fig. 1. ~lmodulirl RIA standard curve. A standard competition binding curve for calmodul~n binding was generated (see Materials and Methods). Each data point is an average of two assays which were done in triplicate. Bars represent SEM.
centration of CAM in the unknown samples was derived from the graph by calculating the percentage of CAM bound to the antibody, plotting the results on the ordinate and reading the sample CAM concentration off the abscissa. RESULTS AND
DISCUSSION
We used a radioimmunoassay (RIA) procedure for measuring calmodulin levels in insect tissues because attempts to use the phosphodiesterase (PDF) assay method (Cheung, 1970) proved quite unreliable. The RIA has great specificity and may be used to quantitate CAM when the PDE assay is unable to detect any measurable CAM. It is important to note that EGTA was included in all initial homogenization buffers, which gives the buffer calcium-chelating ability and yields calmodulin dissociated from various binding sites (Klee and Vanaman, 1982). A typical competition-binding curve for the CAM RIA is shown in Fig. I. The efficiency of the assay was monitored (1) by checking the recovery of internal standards of bovine brain CAM added to the tissue extracts and (2) by assay of serial dilutions of tissue extracts. This RIA was developed against rat testes CAM, and it has a limit of detection of 15 pg of CAM. The immunological response for CAM was the same regardless of whether the CAM was extracted from rat testes, bovine brain, sea pansey (Renilla reniformis) or peanut plant (ha&s hypoguea). Troponin C from rabbit skeletal muscle demonstrates only slight jmmunological cross-reactivity. This calcium binding protein would have to be increased 665-fold to achieve a 50% competition with CAM in the RIA (Chafouleas et ol., 1979). Calmodulin-like activity was detected in all tissues examined. The distribution in specific tissues of the cockroach L. maderae is illustrated by a series of histograms in Fig. 2. From the data recorded in this figure it is obvious that CAM is particularly abundant in the muscular organs of the viscera and the central nervous system. The large amount of immu-
nological activity in the superior longitudinal muscle isolated from the hindgut indicates that this calcium binding protein may mediate the contraction process in insects as it does in the smooth muscle of vertebrates (Adelstein and Klee, 1980). Such a prospect seems even more likely when the amount of CAM present in this isolated muscle is compared with that in the whole hindgut (VM and HG in Fig. 2). The muscle tissue probably accounts for more than 80% of the activity. Also, the low levels of CAM in the coxal muscle suggest that troponin C may be the principal calcium binding protein in insect skeletal muscle, just as it is in vertebrates (Berridge, 1980). In excretion, unlike the pressure driven system for blood filtration in most vertebrates, insects depend on an active secretory process across the epithelium of the Malpighian tubules (Maddrell, 1981). The surprisingly large amount of CAM present in Malpighian tubules probably reflects some kind of involvement with the active transport mechanisms of this secretion. Since CAM also has been implicated in neurotransmission and neurosecretion (Roufogalis, 1983), it was not unexpected to find high levels of the protein in the nervous tissue of insects. Calmodulin levels were determined in the head, thorax and abdomen of five insect species (Fig. 3). The abdominal tissues contained the largest amounts of the protein in most species, ranging from 4 to 8 ng of CAM per pg of protein. Smaller amounts usually were found in the head and thorax. However, in larvae of H. zea and in the adult dung beetle C. ~~~fu?~r substantial levels of CAM were detected in the thorax. Such anomalies might reflect the presence of a larger mass of visceral organs in the thorax of these insects than others. A comparison of calmodulin levels between the cockroach and the rat is shown in Table 1. Although the levels in the rat were determined by phosphodiesterase assay and those in the cockroach were not, it is still of interest to compare the interspecies ratios for CAM in the two animals. The brain to liver ratio in the rat is quite similar to the brain to fat body
Distribution
of calmodulin
in insects as determined
by radioimmunoassay
243
16 15 14
13
12
1 1
10
9
a
7
6
5
4
3
2
1 0 FG
OD
VM
HG
CM
B
TNC
ANC
MT
OV
FE
Fig. 2. Leucophueu mndercte calmodulin RIA activity. Representative tissues from L.. maderoe were assayed for calmoduiin. Tissues were assayed in triplicate using a calmodulin rddioimmunoassay. Determinations were done with 10 tissue samples from the foregut (FG). oviduct (OC), superior longitudinal muscle of the rectum (VM). hindgut (HG), coxal muscle (CM), brain (3). thoracic nerve cords (TNC), abdominal nerve cords @NC), Malpighian tubules (MT). ovaries (OV). fat body (FB). Bars represent SEM.
A.
mullifra
Ii.
zea
larva
C.
imitator
P.
amerlcana
L.
maderse
Fig. 3. Head (cross hatch), thoracic (black) and abdominal tissues (white) from A. melliferu, If.zeu, C. imiiafor, P. americuna and L. maderae were assayed in triplicate using a calmodulin radioimmunoassay. Determinations were done with four tissue samples from each insect. Each dete~ination was done in triplicate. Bars represent SEM.
244
MARK S. WRIGHT and BENJAMIN J. COOK Table 1. Comparison of mean tissue calmodulin levels L. maderae (ng/pg protein) 13.80 (brain) 14.04 (MT) 3.57 (FB)
Rat (ng/pg) protein) 16.47 (brain) 2.45 (kidney) 2.12 (liver)
Values for the rat were taken from Kakiuchl el al. (1975); a value of 40 units of calmodulin being equal to I pg of calmodulin (MT = Malpighian FB = fat tubules, body).
ratios in the insect. The CAM levels in the rat kidney are low compared to those in rat brain. However, the CAM detected in Malpighian tubules is almost the same as that in the insect brain. This undoubtedly reflects the difference in excretory mechanism between these two animals. Early experiments on insects indicated that CAM was not present in either flight muscle (Volmer, 1977) or the related nervous system of insects (Albin, 1975). However, more recent work has shown that this calcium-binding protein is present in flight muscle of locust (Cox et al., 1981), fat body of silk worm (Bodnaryk, 1984) and the head of the Mediterranean fruit fly (Dudoignon et al., 1983). These findings, along with those presented in this paper, strongly suggest that CAM serves as an important regulator of biochemical and physiological functions in insects.
REFERENCES Adelstein R. L. and Klee C. B. (1980) Smooth muscle myosin light chain kinase. Cal. Ceil Function 1, 167-181. Abin E. E., Davidson S. K, and Newbrough R. W. (1975) Properties of cyclic nucleotide phosphodiesterase in the central nervous system of Manduca sexta. Biochim. biophys. Acta 377, 364380. Berridge M. J. (1980) Hormone action-a search for transducing mechanisms. In Insecf Biology in the Future (Edited by Locke M. and Smith D. S.), pp. 463478. Academic Press, London. Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, ^.^ ^_. L4&L34.
Bodnaryk R. P. (1984) Isolation and characterization of a calmodulin lacking trimethyllysine from the fat body of the silk worm, Bombyx mori. Insect Biochem. 14, 11-17. Brostrom C. O., Haung Y. C., Breckenridge B. M. and Wolff D. J. (1975) Identification of a calcium binding protein as a calcium dependent regulator of brain adenyl cyclase. Proc. natn. Acad. Sci. U.S.A. 72, 64-68. Chafouleas J. G., Dedman J. R., Munjaal R. P. and Means A. R. (1979) Calmodulin development and application of a sensitive radioimmunoassay. J. biol. Chem. 254, 10262-10267. Cheung W. Y. (1970) Cyclic 3’,5’-nucleotide phosphodiesterase. Demonstration of an activator. Biochem. kiophys. Res. Commun. 33, 533-538. Conti M. A. and Adelstein R. S. (1980) Phosohorvlation bv . cyclic adensine 3’-5’-monophosphate-dependent protein kinase regulates myosin light chain kinase. Fedn. Proc. Fedn. Am. Sots exp. Biol. 39, 1569-1573. Cox J. A., Kretsinger R. H. and Stein E. A. (1981) Sarcoplasmic calcium-binding proteins in insect muscle isolation and properties of locust calmodulin. Biochim. biophys. Acta 670, 441444. Dudoignon R. M., Gavilanes J. G., Henriquez R., Municio A. M. and Toro M. J. (1983) Calmodulin from the insect C. capita: isolation and molecular characterization. Comp. Biochem. Physiol. 76B, 643-647. Kakiuchi S., Yamazaki R., Teshima Y., Kinihiro U. and Mivamoto E. (1975) Multiple cyclic nucleotide phosphodiesterase activities from rat iissues and occurrence of a calcium-plus-magnesium-ion-dependent phosphodiesterase and its protein activator. Biochem. J. 146, 109-120. Klee C. B. and Vanaman T. C. (1982) Calmodulin. In Advances in Protein Chemistrv (Edited bv Anfinsen C. B., Edsall J. T. and Richard F.- I$, Vol. 35, pp. 215-321. Academic Press, New York. Maddrell S. H. P. (1981) The functional design of the insect excretory system. J. exp. Biol. 90, 1-15. Ringer S. (1886) Further experiments regarding the influence of small quantities of lime, potassium, and other salts on muscle tissue. J. Physiol. 7, 291-308. Roufogalis B. D. (1983) Calmodulin: its role in synaptic transmission. Trends Neurosci. 6, 238-241. Schaub M. C. and Watterson J. G. (1981) Control of the contractile process in muscle. Trends Pharmacol. Sci. 2, 279-282. Volmer H. (1977) Properties of cyclic nucleotide phosphodiesterase from the flight muscle of Locusta migratoria. Insect Biochem. 7, 411414. Wang J. H. and Waisman D. M. (1979) Calmodulin and its role in the second messenger system. In Current Topics in Cellular Regulation (Edited by Horecker B. L. and Stadtman E. R.), Vol. 15, pp. 47-107. Academic Press, New York. .
I