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Calmodulin activity in whole body and fat body tissue extracts of Heliothis virescens larvae
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS Pages 485-488
Vo1.158, No. 2,1989 Januaw 31,1989
CALMODULIN ACTIVITY IN WHOLE TISSUE EXTRACTS OF HELIOTHIS
Timothy
Memphis
BODY AND FAT BODY VIRESCENS LARVAE
D. Lockey and Donald D. Ourth
Department of Biology State University, Memphis,
TN
38152
Received November 16, 1988
Calmodulin is an activator of many enzymatic activities. Total calmodulin activity in tissue extracts of Heliothis virescens larvae (5th instar), assayed by cyclic phosphodiesterase activation, was 0.48 unit/gm for whole body and 22.2 units/gm for fat body. Specific calmodulin activity was 0.i unit/mg protein for whole body and 3.0 units/mg protein for fat body. The larval fat body is therefore the main site of calmodulin activity in this lepidopterous larva. ©1989AcademicPress,Inc.
Calmodulin purified
was first discovered
phosphodiesterase
by Cheung(1).
needed an activator
He found that
for enzymatic
activity to occur when compared with the enzymatic crude extract described
of phosphodiesterase(2).
as calmodulin
activity
of a
The activator was later
and found to be regulated
by the binding of
calcium(3). Calmodulin
is a member
calcium-binding myosin(4).
proteins
Calmodulin
of a group of structurally including
tropinin
is a heat-stable
C, parvalbumin
and is one of the most acidic proteins
having
an isoelectric
found in tissues
Calmodulin
amino acids and has a molecular weight of 16,700(5). acid sequence
of calmodulin
can be divided
contains
was first isolated
has been found in all eukaryotic acid sequence throughout
evolution(5).
vertebrates reptiles, isolated
of calmodulin
including
amphibians
into four structurally-
cells examined
It has been purified rabbit,
and fishes(4).
in protozoans,
fungi,
site(5).
from bovine brain and heart and (4,6,7).
has been conserved by various
the pig,
148
The amino
related domains with each domain having a calcium binding Calmodulin
and
protein of low molecular
weight
point of 3.9-4.3.
related
cat,
species
from other
chicken,
Calmodulin
The amino
turtle,
has also been
plants and many invertebrates(4)
485
0~6-291~89 $1.50 C o ~ r ~ h t © 1989 ~ Aca~mic Press, ~ c . All r ~ h ~ ~ repro~cfion in a T ~ r m rese~ed.
Vol. 158, No. 2, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
including three insect species(8,9,10). found in p r o k a r y o t e s
Calmodulin has not been
(4).
This paper describes the partial p u r i f i c a t i o n and i d e n t i f i c a t i o n of c a l m o d u l i n
from Heliothis virescens
(Lepidoptera:
Noctuidae)
preparations.
(tobacco budworm)
larvae
using whole body and fat body
An objective of the present study was to compare
c a l m o d u l i n activity between larval fat body and whole body tissues. C a l m o d u l i n has not before been described in a lepidopterous C a l m o d u l i n has, however, pupal
been purified from silkworm
larva.
(Bombyx mori)
fat body(10).
MATERIALS
AND
METHODS
T w e n t y - f o u r H. v i r e s c e n s larvae were reared individually in plastic cups; each cup contained i0 ml of diet(ll,12). The cups were held at 24-25°C in a natural photoperiod. The larvae were 15 days old(5th instar) at the time of tissue preparation. The average weight of each larva was 0.371 gm. W h o l e body and fat body tissue p r e p a r a t i o n s were p r e p a r e d a c c o r d i n g to W a i s m a n et al.(13). Whole body tissues from 12 larvae were h o m o g e n i z e d for one min. in 5 volumes of 0.04 M Tris buffer, pH 7.5, and the h o m o g e n a t e was c e n t r i f u g e d for 30 min. at i0,000 rpm. The supernatant was removed and heated at 95°-i00°C for 6 min.(ll). The p r e c i p i t a t e d protein from h e a t i n g was removed by c e n t r i f u g a t i o n at 5,000 rpm for I0 min. The remaining supernatant was then e x t e n s i v e l y dialysized in 0.04 M Tris buffer, pH 7.5 at 4Oc. The fat body was surgically removed from 12 larvae and placed in a p r e w e i g h e d beaker to determine the total w e i g h t of fat body. The same p r o c e d u r e d e s c r i b e d above for p r e p a r a t i o n of whole body tissue was used to obtain an extract of larval fat body. Protein d e t e r m i n a t i o n of the whole body and fat body tissue extracts was by the Lowry method(14). Bovine serum albumin was used as the p r o t e i n standard. C a l m o d u l i n was q u a n t i t a t e d in the larval whole body and fat body p r e p a r a t i o n s by the method of Cheung(15). Calmodulin in the p r e s e n c e of calcium will activate cyclic nucleotide p h o s p h o d i e s t e r a s e which in turn e n z y m a t i c a l l y produces AMP from cAMP. A p r e - d e t e r m i n e d amount of enzyme extract (400 #i) was added to a reaction m i x t u r e that contained 40 mM Tris-HCL, pH 8.0, 5 mM MgS04, 50 #M CaCl2, 2 mM cAMP (Sigma, St. Louis, MO) and 5 m i l l i u n i t s of cAMP p h o s p h o d i e s t e r a s e (activator deficient)(Sigma, St. Louis, MO). The m i x t u r e was incubated at 30°C for i0 min. The enzyme r e a c t i o n was stopped by boiling for 1 min. The p h o s p h a t e was cleaved from AMP by addition of 5' n u c l e o t i d a s e (snake venom) (Sigma, St. Louis, M0) and incubated for i0 min. at 30°C. This reaction was stopped by the addition of 55% TCA. The p r e c i p a t e d protein was removed by c e n t r i f u g a t i o n at 3,000 rpm, and the s u p e r n a t a n t was used to determine c o l o r i m e t r i c a l l y the amount of inorganic p h o s p h a t e present in the whole body and fat body preparations(16).
486
Vol. 158, No. 2, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RESULTS
The total weight
content was 0.85 mg/ml calmodulin
virescens extracts
for whole body and 0.70 mg/ml
activity
in the tissue extracts
larvae was 0.48 unit/gm of total weight
calmodulin
The presence
for fat body
invertebrates
including
activity
calmodulin
two members
blue crab and meal worm(13). purified
calmodulin
found that silkworm m o l e c u l a r weight,
for fat body. of H.
for whole body
(Table i).
Specific
for whole body and 3.0
in whole body and fat body
larvae is consistent
They determined
Protein
(Table i).
of calmodulin
of H. virescens
a_!l.(13).
for fat body
activity was 0.i unit/mg protein
units/mg protein
procedure
fat body weight was 0.47 gm.
and 22.2 units/gm
extracts
DISCUSSION
of whole body used in the extraction
was 4.45 gm, and the total The total
AND
activity
with Waisman
e_tt
in different
phyla of
of the Phylum Arthropoda,
Bodnaryx and Morishima(10)
from the pupal calmodulin
the
have
fat body of the silkworm.
They
was similar to other calmodulins
high activity,
u.v.
in
spectrum and amino acid
composition(10). Calmodulin activitor
has previously
of many enzymatic
initial discovery indicating
activities(4).
of calmodulin
that calmodulin
to its importance Heliothis
been determined
The amount
of specific
results
function(4).
calmodulin
activity detected
found in the fat body extracts
calmodulin
for enzymatic
are calcium and calmodulin
larval
activity
dependent.
to be the main site for the enzymatic nutrients
Nearly all of the
Heliothis virescens
(Table i).
These
fat body likely
and has enzymes which
The insect conversion
and for the storage of nutritional
Table i.
in whole
is much less than the 3.0
indicate that the H. virescens
requires
due
(Table I).
(0.i unit/mg protein)
units/mg protein
larva,
activity was found in the fat body when
compared with whole body tissue body extracts
We report here the
in a lepidopterous
has been conserved by invertebrates
in enzymatic
calmodulin
to be an important
fat body is known of absorbed
reserves(17).
Calmodulin Activity in Larval Tissue Extracts Total Calmodulin Activity (unit/gm tissue)
Specific Calmodulin Activity (unit/mg protein)
Whole Body
0.48
0.i
Fat Body
22.2
3.0 487
Vol. 158, No. 2, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ACKNOWLEDGMENT
T h e a u t h o r s t h a n k Dr. W. Y. Cheung, St. J u d e C h i l d r e n ' s H o s p i t a l , M e m p h i s , TN for r e v i e w of the m a n u s c r i p t .
Research
REFERENCES
i.
2. 3. 4 5 6 7 8 9 i0 ii
12. 13. 14. 15. 16. 17.
Cheung, W. Y. (1970) Biochem. Biophys. Res. Commun. 38, 533538. Cheung, W. Y. (1971) J. Biol. Chem. 246, 2 8 5 9 - 2 8 6 9 . Teo, T.S. a n d Wang, J. H. (1973) J. Biol. Chem. 248, 59505955. Klee, C. B. and V a n a m a n , T. C. (1982) A d v a n c e s in P r o t e i n C h e m i s t r y , p. 213-321. A c a d e m i c Press, N e w York. Klee, C. B., Crouch, T. H. and Richman, P. G. (1980) Ann. Rev. Biochem. 49, 489-515. Teo, T. S., Wang, T. H. and Wang, J. H. (1973) J. Biol. Chem. 248, 588-595. Lin, Y. M., Liu, Y. P. a n d Cheung, W. Y. (1975) FEBS Lett. 49, 356-360. Y a m a n a k a , M. K. and Kelly, L. E. (1981) Biochem. Biophys. Acta 674, 277-286. Cox, J. A., K r e t s i n g e r , R. H. a n d Stein, E. A. (1981) Biochem. Biophys. A c t a 670, 441-444. B o d n a r y k , R. P. and M o r i s h i m a , I. (1984) I n s e c t Biochem. 14, 11-17. R a u l s t o n , J. R. and King, E. G. (1984) A d v a n c e s a n d c h a l l e n g e s in i n s e c t rearing. A g r i c u l t u r a l R e s e a r c h Service, p. 167-175. U.S. D e p a r t m e n t of A g r i c u l t u r e , N e w Orleans. Ourth, D. D. (1988) J. Econ. Entomol. 81, 148-151. W a i s m a n , D., Stevens, F. C. and Wang, J. H. (1975) Biochem. Biophys. Res. Commun. 65, 975-982. Lowry, O. H., R o s e b r o u g h , N. J., Farr, A. L. and R a n d a l l , R. J. (1951) J. Biol. Chem. 193, 265-275. Cheung, W. Y. (1980) C a l c i u m and Cell F u n c t i o n , Vol. I, pp. 1-40. A c a d e m i c Press, N e w York. Ames, B. (1966) M e t h o d s in E n z y m o l o g y , 8, 115-118. G a r d n e r , M. (1972) T h e B i o l o g y of I n v e r t e b r a t e s , p. 366-367. M c G r a w a n d Hill, N e w York.
488
Vo1.158, No. 2,1989 Januaw 31,1989
CALMODULIN ACTIVITY IN WHOLE TISSUE EXTRACTS OF HELIOTHIS
Timothy
Memphis
BODY AND FAT BODY VIRESCENS LARVAE
D. Lockey and Donald D. Ourth
Department of Biology State University, Memphis,
TN
38152
Received November 16, 1988
Calmodulin is an activator of many enzymatic activities. Total calmodulin activity in tissue extracts of Heliothis virescens larvae (5th instar), assayed by cyclic phosphodiesterase activation, was 0.48 unit/gm for whole body and 22.2 units/gm for fat body. Specific calmodulin activity was 0.i unit/mg protein for whole body and 3.0 units/mg protein for fat body. The larval fat body is therefore the main site of calmodulin activity in this lepidopterous larva. ©1989AcademicPress,Inc.
Calmodulin purified
was first discovered
phosphodiesterase
by Cheung(1).
needed an activator
He found that
for enzymatic
activity to occur when compared with the enzymatic crude extract described
of phosphodiesterase(2).
as calmodulin
activity
of a
The activator was later
and found to be regulated
by the binding of
calcium(3). Calmodulin
is a member
calcium-binding myosin(4).
proteins
Calmodulin
of a group of structurally including
tropinin
is a heat-stable
C, parvalbumin
and is one of the most acidic proteins
having
an isoelectric
found in tissues
Calmodulin
amino acids and has a molecular weight of 16,700(5). acid sequence
of calmodulin
can be divided
contains
was first isolated
has been found in all eukaryotic acid sequence throughout
evolution(5).
vertebrates reptiles, isolated
of calmodulin
including
amphibians
into four structurally-
cells examined
It has been purified rabbit,
and fishes(4).
in protozoans,
fungi,
site(5).
from bovine brain and heart and (4,6,7).
has been conserved by various
the pig,
148
The amino
related domains with each domain having a calcium binding Calmodulin
and
protein of low molecular
weight
point of 3.9-4.3.
related
cat,
species
from other
chicken,
Calmodulin
The amino
turtle,
has also been
plants and many invertebrates(4)
485
0~6-291~89 $1.50 C o ~ r ~ h t © 1989 ~ Aca~mic Press, ~ c . All r ~ h ~ ~ repro~cfion in a T ~ r m rese~ed.
Vol. 158, No. 2, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
including three insect species(8,9,10). found in p r o k a r y o t e s
Calmodulin has not been
(4).
This paper describes the partial p u r i f i c a t i o n and i d e n t i f i c a t i o n of c a l m o d u l i n
from Heliothis virescens
(Lepidoptera:
Noctuidae)
preparations.
(tobacco budworm)
larvae
using whole body and fat body
An objective of the present study was to compare
c a l m o d u l i n activity between larval fat body and whole body tissues. C a l m o d u l i n has not before been described in a lepidopterous C a l m o d u l i n has, however, pupal
been purified from silkworm
larva.
(Bombyx mori)
fat body(10).
MATERIALS
AND
METHODS
T w e n t y - f o u r H. v i r e s c e n s larvae were reared individually in plastic cups; each cup contained i0 ml of diet(ll,12). The cups were held at 24-25°C in a natural photoperiod. The larvae were 15 days old(5th instar) at the time of tissue preparation. The average weight of each larva was 0.371 gm. W h o l e body and fat body tissue p r e p a r a t i o n s were p r e p a r e d a c c o r d i n g to W a i s m a n et al.(13). Whole body tissues from 12 larvae were h o m o g e n i z e d for one min. in 5 volumes of 0.04 M Tris buffer, pH 7.5, and the h o m o g e n a t e was c e n t r i f u g e d for 30 min. at i0,000 rpm. The supernatant was removed and heated at 95°-i00°C for 6 min.(ll). The p r e c i p i t a t e d protein from h e a t i n g was removed by c e n t r i f u g a t i o n at 5,000 rpm for I0 min. The remaining supernatant was then e x t e n s i v e l y dialysized in 0.04 M Tris buffer, pH 7.5 at 4Oc. The fat body was surgically removed from 12 larvae and placed in a p r e w e i g h e d beaker to determine the total w e i g h t of fat body. The same p r o c e d u r e d e s c r i b e d above for p r e p a r a t i o n of whole body tissue was used to obtain an extract of larval fat body. Protein d e t e r m i n a t i o n of the whole body and fat body tissue extracts was by the Lowry method(14). Bovine serum albumin was used as the p r o t e i n standard. C a l m o d u l i n was q u a n t i t a t e d in the larval whole body and fat body p r e p a r a t i o n s by the method of Cheung(15). Calmodulin in the p r e s e n c e of calcium will activate cyclic nucleotide p h o s p h o d i e s t e r a s e which in turn e n z y m a t i c a l l y produces AMP from cAMP. A p r e - d e t e r m i n e d amount of enzyme extract (400 #i) was added to a reaction m i x t u r e that contained 40 mM Tris-HCL, pH 8.0, 5 mM MgS04, 50 #M CaCl2, 2 mM cAMP (Sigma, St. Louis, MO) and 5 m i l l i u n i t s of cAMP p h o s p h o d i e s t e r a s e (activator deficient)(Sigma, St. Louis, MO). The m i x t u r e was incubated at 30°C for i0 min. The enzyme r e a c t i o n was stopped by boiling for 1 min. The p h o s p h a t e was cleaved from AMP by addition of 5' n u c l e o t i d a s e (snake venom) (Sigma, St. Louis, M0) and incubated for i0 min. at 30°C. This reaction was stopped by the addition of 55% TCA. The p r e c i p a t e d protein was removed by c e n t r i f u g a t i o n at 3,000 rpm, and the s u p e r n a t a n t was used to determine c o l o r i m e t r i c a l l y the amount of inorganic p h o s p h a t e present in the whole body and fat body preparations(16).
486
Vol. 158, No. 2, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RESULTS
The total weight
content was 0.85 mg/ml calmodulin
virescens extracts
for whole body and 0.70 mg/ml
activity
in the tissue extracts
larvae was 0.48 unit/gm of total weight
calmodulin
The presence
for fat body
invertebrates
including
activity
calmodulin
two members
blue crab and meal worm(13). purified
calmodulin
found that silkworm m o l e c u l a r weight,
for fat body. of H.
for whole body
(Table i).
Specific
for whole body and 3.0
in whole body and fat body
larvae is consistent
They determined
Protein
(Table i).
of calmodulin
of H. virescens
a_!l.(13).
for fat body
activity was 0.i unit/mg protein
units/mg protein
procedure
fat body weight was 0.47 gm.
and 22.2 units/gm
extracts
DISCUSSION
of whole body used in the extraction
was 4.45 gm, and the total The total
AND
activity
with Waisman
e_tt
in different
phyla of
of the Phylum Arthropoda,
Bodnaryx and Morishima(10)
from the pupal calmodulin
the
have
fat body of the silkworm.
They
was similar to other calmodulins
high activity,
u.v.
in
spectrum and amino acid
composition(10). Calmodulin activitor
has previously
of many enzymatic
initial discovery indicating
activities(4).
of calmodulin
that calmodulin
to its importance Heliothis
been determined
The amount
of specific
results
function(4).
calmodulin
activity detected
found in the fat body extracts
calmodulin
for enzymatic
are calcium and calmodulin
larval
activity
dependent.
to be the main site for the enzymatic nutrients
Nearly all of the
Heliothis virescens
(Table i).
These
fat body likely
and has enzymes which
The insect conversion
and for the storage of nutritional
Table i.
in whole
is much less than the 3.0
indicate that the H. virescens
requires
due
(Table I).
(0.i unit/mg protein)
units/mg protein
larva,
activity was found in the fat body when
compared with whole body tissue body extracts
We report here the
in a lepidopterous
has been conserved by invertebrates
in enzymatic
calmodulin
to be an important
fat body is known of absorbed
reserves(17).
Calmodulin Activity in Larval Tissue Extracts Total Calmodulin Activity (unit/gm tissue)
Specific Calmodulin Activity (unit/mg protein)
Whole Body
0.48
0.i
Fat Body
22.2
3.0 487
Vol. 158, No. 2, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ACKNOWLEDGMENT
T h e a u t h o r s t h a n k Dr. W. Y. Cheung, St. J u d e C h i l d r e n ' s H o s p i t a l , M e m p h i s , TN for r e v i e w of the m a n u s c r i p t .
Research
REFERENCES
i.
2. 3. 4 5 6 7 8 9 i0 ii
12. 13. 14. 15. 16. 17.
Cheung, W. Y. (1970) Biochem. Biophys. Res. Commun. 38, 533538. Cheung, W. Y. (1971) J. Biol. Chem. 246, 2 8 5 9 - 2 8 6 9 . Teo, T.S. a n d Wang, J. H. (1973) J. Biol. Chem. 248, 59505955. Klee, C. B. and V a n a m a n , T. C. (1982) A d v a n c e s in P r o t e i n C h e m i s t r y , p. 213-321. A c a d e m i c Press, N e w York. Klee, C. B., Crouch, T. H. and Richman, P. G. (1980) Ann. Rev. Biochem. 49, 489-515. Teo, T. S., Wang, T. H. and Wang, J. H. (1973) J. Biol. Chem. 248, 588-595. Lin, Y. M., Liu, Y. P. a n d Cheung, W. Y. (1975) FEBS Lett. 49, 356-360. Y a m a n a k a , M. K. and Kelly, L. E. (1981) Biochem. Biophys. Acta 674, 277-286. Cox, J. A., K r e t s i n g e r , R. H. a n d Stein, E. A. (1981) Biochem. Biophys. A c t a 670, 441-444. B o d n a r y k , R. P. and M o r i s h i m a , I. (1984) I n s e c t Biochem. 14, 11-17. R a u l s t o n , J. R. and King, E. G. (1984) A d v a n c e s a n d c h a l l e n g e s in i n s e c t rearing. A g r i c u l t u r a l R e s e a r c h Service, p. 167-175. U.S. D e p a r t m e n t of A g r i c u l t u r e , N e w Orleans. Ourth, D. D. (1988) J. Econ. Entomol. 81, 148-151. W a i s m a n , D., Stevens, F. C. and Wang, J. H. (1975) Biochem. Biophys. Res. Commun. 65, 975-982. Lowry, O. H., R o s e b r o u g h , N. J., Farr, A. L. and R a n d a l l , R. J. (1951) J. Biol. Chem. 193, 265-275. Cheung, W. Y. (1980) C a l c i u m and Cell F u n c t i o n , Vol. I, pp. 1-40. A c a d e m i c Press, N e w York. Ames, B. (1966) M e t h o d s in E n z y m o l o g y , 8, 115-118. G a r d n e r , M. (1972) T h e B i o l o g y of I n v e r t e b r a t e s , p. 366-367. M c G r a w a n d Hill, N e w York.
488