Subcellular location of phosphoproteins in salivary glands of the lone star tick Amblyomma americanum (L.)код для вставкиСкачать
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  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 , 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 . Sauer et al.  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 . At least 12 proteins whose states of phosphorylation are increased by dopamine and CAMPhave been identified in whole-gland and tissue homogenate experiments, respectively . 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  and adenylate cyclase  were used to identify plasma membranes. Succinate dehydrogenase  activity was used to identify mitochondria, and glucose-6-phosphatase activity  was used to identify microsomes. Effectiveness of homogenization was determined by 32 McSwain et al assaying for lactate dehydrogenase  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. . 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 . 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 . 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 , 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).