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Subcellular location of phosphoproteins in salivary glands of the lone star tick Amblyomma americanum (L.)

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Archives of Insect Biochemistry and Physiology 5:29-43 (1987)
Subcellular Location of Phosphoproteins in
Salivary Glands of the Lone Star Tick,
Amblyomma americanum (L.)
Janis L. McSwain, Stephen P. Schmidt, Deborah M. Claypool,
Richard C. Essenberg, and John R. Sauer
Departments of Entomology (J.L.M., D.M.C., J.R.S.) and Biochemistry (R.C.E.), Oklahoma
State University, Stillwater; Union Carbide Agricultural Products Co., Research Triangle Park,
North Carolina (S. P. S.)
Phosphoproteins were examined by electrophoresis and autoradiography in
fractions of tick salivary glands. When whole salivary glands were preincubated in 32Pi, then stimulated by 10 pM dopamine and subsequently
fractionated, substantial phosphate was incorporated into 45,000-, 47,000-,
and 62,000-dalton proteins of the plasma membrane-rich 11,500g pellet and
100,OOOg supernatant. When tissue homogenates were incubated in [-p3’P]
ATP prior to subcellular fractionation, the 62,000-, 47,000-, and 45,000-dalton
proteins were enhanced by cyclic AMP in all fractions and were most
prominent in the membrane-rich 11,500g fraction. Phosphoproteins of the
same molecular masses were also found in the 11,500g pellet and 100,OOOg
supernatant when labelled w i t h [y3’P] ATP in the presence of CAMP.
Key words: cyclic AMP, protein phosphorylation, subcellular fractionation, tick salivary
glands
INTRODUCTION
Reversible phosphorylation of specific proteins is recognized as a major
mechanism for the control of biological processes [l]. Phosphorylationldephosphorylation reactions initiated by hormones or neurotransmitters are
often mediated by cyclic adenosine 3‘,5’-monophosphate (cyclic AMP). Rodbell [2] has reviewed the literature showing that adenylate cyclase is activated
by many hormones and neurotransmitters, which cause an increase in intra-
Journal Article No. 4360 of the Agriculture Experiment Station, Oklahoma State University,
Stillwater. This research was supported in part by grant Al-13535 from the National Institutes
of Health and grant DCB-8415668 from the National Science Foundation.
Received April 28,1986; accepted November 26,1986.
Address reprint requests to Dr. J.R. Sauer, Dept. of Entomology, Oklahoma State University,
Stillwater, OK 74078.
0 1987 Alan R.
Liss, Inc.
30
McSwain et a1
cellular cyclic AMP. Cyclic AMP activates a cyclic AMP-dependent protein
kinase [3], which phosphorylates one or more proteins to effect in some way
the biological response.
Salivary glands are the primary organs of fluid excretion in ixodid female
ticks [4,5]. Fluid secretion by the salivary glands is likely controlled by nerves
and the neurotransmitter appears to be dopamine [6].
Sauer et al. [4] and Needham and Sauer [7l demonstrated that catecholamines such as dopamine stimulate fluid secretion and increase gland levels
of cyclic AMP. A dopamine-sensitive adenylate cyclase has also been identified in the glands [8]. At least 12 proteins whose states of phosphorylation
are increased by dopamine and CAMPhave been identified in whole-gland
and tissue homogenate experiments, respectively [9]. The following experiments were designed to determine the subcellular location of the phosphoproteins in tick salivary glands affected by cyclic AMP-dependent kinases.
MATERIALS AND METHODS
Materials
[32Pi]Orthophosphate (carrier free) and 10-20 Cilmmol [y-32P]ATP were
obtained from New England Nuclear, Boston, MA. Dopamine and cyclic
AMP were obtained from Sigma, St. Louis, MO.
Tissue Preparation
Male and female lone star ticks Arnblyornrnu arnericunurn (L.) were raised
according to the methods of Patrick and Hair [lo]. Salivary glands from adult
female ticks were used in all experiments. Feeding ticks that weighed 200 mg
or more were removed from the host (sheep), and glands were dissected at
4°C in a medium of modified, oxygenated TC-199 (Difco) at pH 7.0 containing
penicillin and streptomycin sulfate and buffered as described by Needham
and Sauer [V]. Approximately 30 pairs of glands were used in each experiment and were homogenized with a small hand tissue homogenizer with a
loose-fitting pestle in 1ml medium containing 0.25 M sucrose, 10 mM tricine
buffer (pH 7.2), 10 mM MgC12, and 0.05% p-amino-benzamidine, and 0.1
mM EDTA.*
Subcellular Fractionation
The crude homogenate was centrifuged at 9009 for 10 min, and the precipitate was washed twice. In some experiments the 9008 pellet was resuspended in buffer containing 67% sucrose and placed at the bottom of a 4-ml
(1/2” X 1-5/8”) centrifuge tube. Various concentrations of sucrose (Fig. 1)in
tricine buffer identical with the homogenization buffer were layered on the
top of the pellet (0.5-ml aliquots) beginning with the highest concentration.
*Abbreviations: EDTA = ethylenediarninetetraacetic acid; Hepes = 4-(2-hydroxylethyl)-lpiperazine ethane sulfonic acid; SDS = sodium dodecyl sulfate.
Protein Phosphorylation in Tick Salivary Gland
31
TISSUE H
o
P
m
Z
IE
D
IN I ML BUFFER
I
RESUSPENDED I N ,5PI..
67% SUCROSE
I N BUFFER
a
I
BUFFER
35 X SUCROSE
X
I
%
10 MIN,
g
I
SUCRoSE
45 X SUCROSE
M % SUCROSE
55 X SUCROSE
- PELLET
I
CENTRIFUGED I 1,500
+
6i2 SUCHUSE
SUPERNATANT
PELLET
CENTRIFUGED
SWINGING
BUCKET
1m,ooo
18 h
ROTOR
9
- LIGHT FRACTION
HEAVY FRACTION
Fig. 1. Subcellular fractionation of tick salivary glands.
The tube was centrifuged at 100,OOOgfor 16-18 h in a swinging bucket rotor.
The gradient was eluted by pumping 70% sucrose into the bottom of the
tube with an ISCO model D gradient fractionator, and ten-drop fractions
were collected. Absorbance at 254 nm was measured with an ISCO model
UA-2 ultraviolet analyzer.
The 9008 supernatant was centrifuged at 11,500g for 10 min, and the
precipitate was washed twice. The 11,5008 supernatant was centrifuged at
100,OOOg for 60 min to give the 100,OOOg precipitate and supernatant (soluble
fraction) (Fig. 1).
Enzyme Marker Assays
Na+,K+-ATPase [12] and adenylate cyclase [13] were used to identify
plasma membranes. Succinate dehydrogenase [14] activity was used to identify mitochondria, and glucose-6-phosphatase activity [15] was used to identify microsomes. Effectiveness of homogenization was determined by
32
McSwain et al
assaying for lactate dehydrogenase [16] in soluble and particulate fractions
(15,OOOg) of the crude homogenate.
Electron Microscopy
Tissue was collected, homogenized, and fractionated as illustrated in Figure 1. Light and heavy subfractions of the 900g pellet and total pellet preparations from 11,5008and 100,OOOg were processed for electron microscopy.
Fractions of salivary gland tissue were fixed in 2% cacodylate-buffered
glutaraldehyde for 1h (pH 7.4), rinsed in cacodylate buffer, then postfixed in
2% osmium tetroxide, all at room temperature. The fraction was en bloc
stained with 0.5% uranyl acetate at room temperature overnight. Tissue was
dehydrated in a graded series of ethyl alcohol before embedding in polybed
(Polysciences, Warrington, PA). Appropriate polymerized blocks were chosen for thin sections after viewing initial thick sections (1pm) stained with
Mallory's trichrome. Thin sections (70-90 nm) were obtained with a Sorvall
MT-2 ultramicrotome by using diamond knives. Sections were placed on 300mesh copper grids and stained with methanolic uranyl acetate and lead
citrate [lv.Sections were examined and photographed with a Phillips EM
200 electron microscope.
-
Electrophoresis and Autoradiography: Whole-Gland Method
Tissue was dissected as described above. Thirty pairs of salivary glands
were incubated for 1h at 37°C in 300 p1 buffered, oxygenated TC-199 (pH
7.0) containing 100 pCilml 32Pi.Following incubation, the glands were stimulated for 5 min in 300 pl TC-199 containing 10 pM dopamine. The glands
were then homogenized in 1ml tricine buffer as described above and fractionated according to the scheme outlined in Figure 1.
Following subcellular fractionation, an aliquot containing 60 p g protein
from each fraction and the supernatant was subjected to SDS polyacrylamide
gel electrophoresis and subsequent autoradiography as described by McSwain et al. [9].
Broken-Cell Method
Sixty pairs of salivary glands were dissected and homogenized in 1.0 ml
of buffer containing 50 mM Hepes buffer (pH 7.0) and 10 mM MgC12. The
tissue homogenate was separated into two equal aliquots of 500 pl each
containing approximately 9 mg of protein. Each aliquot was incubated in a
disposable glass tube containing 500 pl reaction medium prepared on ice and
consisting of 50 mM Hepes buffer (pH 7.0), 10 mM MgC12, and ,lo pM
cyclic AMP after a modification of the method of Rudolph and Krueger [B].
The phosphorylation reaction was initiated by adding 100 pl of 50 pM [y3'P]
ATP (specific activity 10-20 Cilmmol). The tubes were incubated in a shaking
water bath at 30°C for 5 min. Subcellular fractions were then collected as
described in Figure 1and analyzed by electrophoresis and autoradiography
as described above.
Separated Fraction Method
In another experiment, 30 pairs of salivary glands were dissected, homogenized, and subcellular fractionation was accomplished as described above.
Protein Phosphorylation in Tick Salivary Gland
33
Aliquots containing approximately 60 pg of protein for the 100,OOOg supernatant, 9008, 11,5OOg, and 100,OOOg pellets were incubated in disposable glass
test tubes containing 100 pl reaction medium prepared on ice and consisting
of 50 mM Hepes buffer (pH 7.0), 10 mM MgC12, and k10 pM cyclic AMP
after a modified method of Rudolph and Krueger [18]. The phosphorylation
reaction was initiated by adding 20 p1 of 48 pM [ Y - ~ ~ATP
P ] (specific activity
10-20 Cilmmol). The tubes were incubated in a shaking water bath at 30°C
for 5 min, then processed for electrophoresis and autoradiography as described.
RESULTS
Electron Microscopy
Significant compositional differences were observed in the 900g, 11,5OOg,
and lO0,OOOg pellets. The light subfraction following isopycnic flotation of
the 900g pellet (Fig. 2) contained numerous smooth and fewer rough membrane-bound vesicles. Differentiation between plasma membranes and
smooth endoplasmic reticulum was not possible; therefore, membrane-bound
vesicles lacking ribosomes were collectively labeled smooth membrane-bound
vesicles. Areas of cytoplasm were also present and contained numerous
small rough membrane-bound vesicles (Fig. 2).
The heavy subfraction of the 9008 pellet (Fig. 3) had a more heterogeneous
composition than the light subfraction. There were fewer smooth membranebound vesicles and more rough membrane-bound vesicles. Some intact mitochondria were also present in this subfraction. Numerous small, electrondense bodies, either attached to endoplasmic reticulum or free in the cytoplasm, produced large irregular-shaped structures. Areas of cytoplasm also
contained rough membrane-bound vesicles.
Fewer organelles, mostly fragmented, were observed in the 11,5008 pellet
(Fig. 4). Membrane-bound vesicles were few in number and relatively small.
The majority of membrane-bound vesicles present were smooth. Portions of
the cytoplasm contained few, if any, membrane-bound vesicles. Electrondense bodies were seen in the cytoplasm.
The lO0,OOOg pellet contained large amounts of small vesicles to which
were attached ribosomal-like structures (Fig. 5). Other electron-dense material appeared to be chromatin. Smooth membrane-bound vesicles were significantly reduced in size and number. No cytoplasmic fractions were
observed in this pellet.
Enzyme Activity in Subcellular Fractions
A cytosolic enzyme, lactate dehydrogenase, was used to determine efficiency of homogenization. Almost all of the enzyme activity appeared in the
supernatant following centrifugation for 3 min at 15,OOOg in an Eppendorf
microfuge (Table l), indicating good homogenization.
Relatively high total and specific adenylate cyclase and Na', K+ATPase
activities (plasma membrane markers) were consistently found in the 9008
pellet (Table 2), verifying the presence of plasma membranes as seen by
34
McSwain et al
Figs. 2-5. Electron micrographs of the 900,11,500, and 100,OOOg pellet from feeding lone star
tick salivary gland tissue. Magnification x 5,760.
Fig. 2. Numerous smooth (SM) and fewer rough (RM) membrane-bound vesicles in the light
subfraction of the 9OOg pellet. Portions of the cytoplasm (C) also contained membrane-bound
vesicles.
Fig. 3. Rough (RM) and smooth (SM) membrane-bound vesicles, partially intact mitochondria
(M), and portions of cytoplasm ( C ) in the heavy subfraction of the 9OOg pellet.
Fig. 4. Smooth (SM) membrane-bound vesicles and portions of cytoplasm (C)were present
in the total 11,500g pellet.
Fig. 5. An abundance of free ribosomes (FR) (or possibly attached to endoplasmic reticulum)
and/or chromatin (CH) were present in the total 100,OOOg pellet.
Protein Phosphorylation in Tick Salivary Gland
35
TABLE 1. Lactate Dehydrogenase Activity in
Tick Salivary Gland Tissue*
Fraction
Crude
Supernatant
Pellet. 15.000~
Total enzyme activity
0.165
0.165
0.02
*Enzyme activity is expressed in international
units per ml. Nos. in table represent an average of
two experiments.
electron microscopy (Figs. 2, 3). Significantly lower total but higher specific
activities of these enzymes were measured in the 11,5008 pellet.
Succinate dehydrogenase (mitochondria1 marker) activity was low in the
11,500g pellet (Table 2); however, considerable activity was seen in the 900g
pellet, which agreed with the observation of mitochondria as seen with the
aid of electron microscopy (Fig. 3).
Glucose-6-phosphatase is often used as a marker for endoplasmic reticulum, but its activity is greatly reduced in some cells. In this tissue the activity
as well as the subcellular location of this enzyme was variable. In one
experiment, the greatest specific activity was found in the 100,OOOg pellet
(Table 2). In a different experiment, the greatest specific activity was found
in the lO0,OOOg supernatant.
In further attempts to see if membranes could be separated, the 900g pellet
was purified by isopycnic flotation using sucrose gradients (Fig. 1).Fractions
from the gradient that appeared highest in protein content were assayed for
Na+, K+-ATPase and adenylate cyclase (Fig. 6). In two experiments, greater
activity of both enzymes appeared in the light fractions. Specific activity of
the enzymes was also highest in the lightest fractions (Fig. 6). In another
experiment, total and specific activities of succinate dehydrogenase were
highest in the light fraction, but Na+, K+-ATPasewas highest in the heaviest
fractions, which indicated that the organelles are separable but they do not
necessarily separate the same way in different experiments. It is therefore
necessary to accompany each separation with appropriate enzyme assays.
Subcellular Phosphoproteins Labeled by the Whole-Gland Method
When whole salivary glands were preincubated in 32Pi to label ATP and
then stimulated with 10 pM dopamine and fractionated, radioactive phosphate was incorporated most conspicuously into 45,000-, 47,000-, and 62,000dalton proteins in the plasma membrane-rich 11,500g pellet (Fig. 7). These
same phosphoproteins were found in the 100,OOOg supernatant (cytoplasm),
but they either took up less radioactive phosphate or were at a lower concentration. The 45,000-dalton phosphoproteins were observed in all fractions
examined. The 47,000- and 62,000-dalton phosphoproteins were missing in
some fractions (9008; 100,OOOg; light isopycnic fraction).
Phosphorylation of Subcellular Proteins Labeled in Gland Homogenates and
Isolated Fractions
When tissue homogenates were incubated with [-p3*P] ATP with or without cyclic AMP and subsequently fractionated, the phosphorylation of
Total
protein
(mg)
5.66
3.68
0.09
Fraction
Crude
900g pellet
Light fraction'
Ouabain
inhibiteda
Ouabain
inhibiteda
Total
8.54
Ouabain
inhibiteda
Total
0.41
1.53
4.93
17.02
23.88
Condition
Total
4.61
17.04
1.34
4.62
1.51
4.22
11.5
5.9
Basal
Dopamineb
64.7
Dopamineb
29.6
45.6
Dopamineb
Basal
23.3
-
-
210.6 -
0.05
-
108.1 -
58.5
26.8 0.22
53.71
27.44
2.19
5.11
0.64
0.79
sp,
Succinate
Glucose-6
Total
(pmol
dehydrogenase
phosphatase
(pmol CAMP/
Sp. Act.
Sp. Act.
CAMP minimg Total (Alminimg
Total
(mmoIimin/mg
formed) protein) (Aimin) protein) (mmolimin)
protein)
Basal
Sp. Act.
Total
(mmollmini
(mmolimin) mg protein) Condition
ATPase
Adenylate cyclase
TABLE 2. Enzyme Activity in Subcellular Fractions of Tick Salivary Gland Tissue
0.27
0.69
100,OOOg pellet
100,ooog
Ouabain
inhibiteda
Ouabain
inhibited”
Total
Ouabain
inhibited”
Total
Basal
Dopamineb
Basal
Dopamineb
Basal
Dopamineb
Basal
Dopamineb
7.31
1.87
6.10
1.87
4.42
1.73
1.60
0.28
5.26
1.34
2.13
1.34
1.17
0.45
1.10
0.19
31.7 42.1
74.4 0.012
123.0
29.3 N.D.d
25.6
23.9 N.D.
9.3
13.7
18.2
7.8
12.9
3.9
3.4
6.2
2.4
N.D.
N.D.
0.02
-
0.30
0.17
0.41
0.324
2.00
1.14
+
aNa , K + -ATPase activity.
b10 uM douamine.
CVaiuesreiresent activity in one ten-drop increment in each of the light and heavy fractions.
dN.D., none detected; results are representative. Results of adenylate cyclase and Na+, K + -ATPase were consistent in three separations except
isopycnic flotation of the 900g pellet. Results for succinate dehydrogenase and glucose-6-phosphatase were consistent in two other experiments except
glucose-6-phosphatase in the 100,OOOg pellets.
0.35
Ouabain
inhibited”
Total
0.72 Total
11,500g pellet
Heavy fraction‘
38
McSwain et a1
LIGHT
FRACTION
HEAVY
FRACTION
FRACTION NUMBER
5
7
t
LIGHT
FRACTION
-
HEAVY
FRACTION
FRACTION NUMBER
Fig. 6. lsopycnic flotation of 9OOg fraction. A Total Na+,K+-ATPase (mmol P, formedlmin in
total fraction), adenylate cyclase activity (pmol CAMP formed per fraction), and total protein
are plotted against fraction number from isopycnic gradient. Na+,K+-ATPase is 10 x actual
measured activity to facilitate graphics. B: Na+,K+-ATPase-specific activity i s expressed in
mmol Pi/rnin/mg protein, while adenylate cyclase specific activity is expressed in pmol CAMP/
minlmg protein after Stimulation by dopamine.
45,000-, 47,000-,and 62,000-dalton proteins was increased by cyclic AMP in
all fractions, although the phosphorylation is not as marked in the 9008
fraction (Fig. 8). As before, these phosphoproteins were most evident in the
11,5008fraction.
In another series of experiments, salivary glands were collected, fractionated, and the subcellular fractions were incubated with [y-32P]ATP with or
without cyclic AMP. In four replicates, results were inconsistent, and in some
Protein Phosphorylation in Tick Salivary Gland
39
3rigin
200.0
116.5
j4.0
i8.0
c
c
4-
13.0
Fig. 7. Autoradiograph of electrophoresed proteins showing incorporation of 32Pinto endogenous proteins of subcellular fractions of tick salivary gland tissue following incubation of
whole glands with 10 pM dopamine for 5 min. The locations of the 62,000-, 47,000-, and 45,000dalton proteins are indicated by arrows.
40
McSwain et a1
-Origin
- 200.0
- 116.5
e
X
-94.0
$
(3
s
a:
- 68.0
4-
43
Y
6
I
t
c
CAMP
-
+
+
-
+
-
900
11,500
100,000
X
X
9
9
X
9
+
-43.0
-
Supernatant
Fig. 8. Autoradiograph of electrophoresed proteins showing incorporation of 32P into endogenous proteins following incubation of tissue hornogenates in [y 32P]ATP f cyclic AMP
and subsequent subcellular fractionation. The locations of the 62,000-, 47,000-, and 45,000dalton proteins are indicated by arrows.
instances protein bands incorporated less 32P in the presence of cyclic AMP
as compared to the control paired experiment labeled without cyclic AMP.
DISCUSSION
Fractionation of Tick Salivary Glands
Measurement of lactate dehydrogenase activity indicated that glands were
homogenized uniformly prior to fractionation. The activity of the cytosolic
enzyme was found almost entirely in the supernatant following centrifugation. Typically mitochondria require 10,000-12,OOOg to be sedimented into
Protein Phosphorylation in lick Salivary Gland
41
the pellet fraction. However, in tick salivary gland homogenates most mitochondria were pelleted with much less centrifugal force (900g) as suggested
by high succinate dehydrogenase activity in the 900g fraction and evidence
from electron microscopy. The 9008 fraction also contained high amounts of
plasma membrane marker enzymes Na +,K+-ATPase and adenylate cyclase.
Plasma membranes were also observed by electron microscopy in this fraction. When the 9008 fraction was resuspended and separated further by
isopycnic flotation on a sucrose density gradient, plasma membranes were
clearly separated on the gradient as indicated by electron microscopy and by
Na', K+-ATPase and adenylate cyclase activities. However, despite good
separations in two experiments (these values are listed in Table 2), organelles
separated differently in another experiment, with plasma membranes concentrated in the heavy fraction in one experiment and in the light fraction in
another. Reasons for this variation are not clear, but possibly variations in
physiological stage of tick feeding from which the glands were obtained or
slight variations in hand homogenization of tissue affected size of vesicles
formed and thereby location on the gradient. There is substantial rapid
proliferation of plasma membranes in the salivary glands during the latter
stages of tick feeding [19].
However, consistent collection of plasma membranes was accomplished
by pelleting the 9008 supernatant at 11,500g. This fraction exhibited high
specific adenylate cyclase and Na', K+-ATPase activities, little succinate
dehydrogenase activity, and some glucose-6-phosphatase activities. Observation of this fraction by electron microscopy also indicated good collection
of plasma membranes.
The highest specific glucose-6-phosphatase activity was observed in the
100,OOOg pellet, which indicated that most microsomes can be collected in
this manner. Only small amounts of adenylate cyclase and Na+, K+-ATPase
activities were measured in this fraction. The 100,OOOg supernatant had relatively low Naf, K+-ATPaseand adenylate cyclase activities and no succinate
dehydrogenase qctivity but some glucose-6-phosphatase activity.
The combined results indicate that plasma membranes from tick salivary
glands can be collected by centrifuging the 9008 supernatant at 11,500g.
Reasonably clean microsomes can be collected by centrifugation of the 11,500g
supernatant at 100,OOOg.
Phosphorylation of Proteins in Subcellular Fractions
In previous studies [9], we demonstrated that various proteins in whole
tick salivary glands incorporated phosphate in response to dopamine and
cAMP and that cAMP stimulated phosphorylation of some of these in gland
homogenates. Proteins with molecular masses of 148,000, 102,000, 62,000,
55,000, 47,000, 45,000, and 37,000 daltons were of particular interest because
they were consistently labeled in all three types of experiments. Proteins
having molecular masses of 45,000, 47,000, and 62,000 were the most substantially phosphorylated in these experiments. The purpose of the present
study was to determine their subcellular location. In whole-gland experiments, when glands were preincubated in 32Piand then stimulated by 10 pM
dopamine and fractionated, phosphoproteins having molecular masses of
42
McSwain et a1
62,000, 47,000, and 45,000 daltons were found to an appreciable extent in the
plasma membrane-rich 11,500g pellet, the heavy fraction of the 9008 pellet,
and the supernatant. In experiments in which tissue homogenates were
incubated with [ Y - ~ ~ATP
P ] with or without cyclic AMP prior to fractionation,
three proteins with the same apparent molecular masses appeared in all
fractions but were again most prominent in the 900g and 11,500g fractions.
Phosphorylation of these three proteins was also enhanced by cyclic AMP in
this procedure. When isolated fractions were incubated with CAMP,45,000and 62,000-dalton phosphoproteins were present in the 11,500g fraction folP ] but the 47,000-dalton
lowing incubation of tissue with cAMP and [ Y - ~ ~ATP,
protein was not, suggesting that factors in other organelles may be responsible for phosphorylating this protein in response to cAMP in whole glands.
The 47,000-dalton protein was found only in the supernatant. In two replications of this experiment, the phosphorylation of this protein was not
enhanced by cyclic AMP, while in two additional replications the phosphorylation was enhanced by cyclic AMP (data not shown). The dissimilar results
may result from slight contamination by other organelles during some separations. In labeling isolated fractions, it is possible that factors necessary for
the reaction were missing.
The combined results of these and previous protein phosphorylation studies strongly suggest that the plasma-membrane and supernatant of tick
salivary glands contain the major phosphoproteins (45,000,47,000, and 62,000
daltons) whose level of phosphate is increased by factors that affect fluid
secretion. Other proteins in other organelles are phosphorylated in the presence of CAMP, but the importance of these to fluid secretion is unclear
because they are not identifiable in isolated fractions following stimulation of
whole glands with dopamine.
Tick salivary glands are muticellular and multifunctional, and tissue differentiation occurs rapidly following attachment and feeding. The glands are
involved in various synthetic and secretory functions. It seems probable that
many of these are modulated either directly or indirectly through specific
protein phosphorylationldephosphorylation.
LITERATURE CITED
1. Krebs EG: The phosphorylation of proteins: A major mechanism for biological regulation.
Biochem SOCTrans 23, 813 (1985).
2. Rodbell J: The role of hormone response and GTP-regulatory proteins in membrane
transduction. Nature 284, 17 (1980).
3. Rubin CS, Rosen OM: Protein phosphorylation. Annu Rev Biochem 44, 831 (1975).
4. Sauer JR, Mincolla I'M, Needham GR: Adrenaline and cyclic AMP stimulated uptake of
chloride and fluid secretion by isolated salivary glands of the lone star tick. Comp Biochem
Physiol53C, 63 (1976).
5. Sauer JR: Acarine salivary glands-physiological relationships. J Med Entomoll4, 1(1977).
6. Kaufman WR, Phillips JE: Ion and water balance in the ixodid tick Dermacenfor andersoni.
11. Mechanism and control of salivary secretion. J Exp Biol58, 537 (1973).
7. Needham GR, Sauer JR: Control of fluid secretion by isolated salivary glands of the lone
star tick. J Insect Physiol22, 1893 (1975).
8. Schmidt SP, Essenberg RC, Sauer JR: A dopamine sensitive adenylate cyclase in the
salivary glands of Amblyomma americanum (L.). Comp Biochem Physiol 72C, 9 (1982).
Protein Phosphorylation in lick Salivary Gland
43
9. McSwain JL, Essenberg RC, Sauer JR: Cyclic AMP mediated phosphorylation of endogenous proteins in the salivary glands of the lone star tick, Amblyomma americanum (L.).
Insect Biochem 15, 789 (1985).
10. Patrick CD, Hair JA: Laboratory rearing procedures and equipment for multi-host ticks
(Acarina: Ixodidae). J Med Entomol12, 389 (1975).
11. Needham GR, Sauer JR: Involvement of calcium and cyclic AMP in controlling ixodid tick
salivary fluid secretion. J Parasitol65, 531 (1979).
12. Adolfsen R, Moudrianakis EN: Purification and properties of two soluble coupling factors
of oxidative phosphorylation from Alcaligenes faecalis. Biochemistry 20, 2247 (1971).
13. Schmidt SP, Essenberg RC, Sauer JR: Evidence for a D1 dopamine receptor in the salivary
glands of Amblyomma americanum (L.). J Cyclic Nucleotide Res 7, 275 (1981).
14. Harmon HJ, Crane FL: Inhibition of mitochondria1 electron transport by hydrophilic metal
chelators. Determination of dehydrogenase topography. Biochim Biophys Acta 440, 45
(1976).
15. Moores DJ: Isolation of Golgi apparatus. Methods Enzymol22, 130 (1971).
16. Bernstein LH, Everse J: Determination of the isoenzyme levels of lactate dehydrogenase.
Methods Enzymol41, 47 (1975).
17. Venable JH, Coggeshall R: A simplified lead citrate stain for use in electron microscopy. J
Cell Biol25, 407 (1965).
18. Rudolph SA, Krueger BK: Endogenous protein phosphorylation and dephosphorylation.
In: Adv Cyclic Nucleotide Research. Brooker G, Greengard P, Robinson GA, eds. Raven
Press, New York, Vol10, pp 107-133 (1979).
19. Fawcett DW, Binnington K, Voigt WP: The cell biology of the ixodid tick salivary gland.
In: Morphology, Physiology and Behavioral Biology of Ticks. Sauer JR, Hair JA, eds. Ellis
Honvood Limited, Chichester, pp 22-45 (1986).
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subcellular, amblyomma, tick, loner, star, gland, salivary, american, phosphoprotein, location
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