p p 1085 10 1091 Pergamon Press Lid 1979 Printed m Great Britain 0 International Socety for Neurochernistr! Ltd
Juiirnol qi .Vearorhurwsfri Vol 3:.
SHORT COMMUNICATION
Subcellular distribution of cyclic AMP phosphodiesterase in the ox neurohypophysis (Received 19 M a y 1978. Rrcrsrd 25 Augusr 1978. Accepted 9 October 1978)
CAMP AND,OR calcium ions play crucial roles in stimulussecretion coupling in a number of systems (RASMUSSEN & GOODMAN. 1977). In the neurohypophysis a definite role for C a 2 + has been established (for a recent survey. see THORNrt a/.. 1978). but the details of its action have not been clarified. The presence of CAMP in the neurohypophysis of rats and an increase of its concentration in rats that have been drinking 200 NaCl solution for 24 h has been demonstrated previously (RUOFF et a/., 1976). POIRIER et a/. (1977) and BONNErr a!. (1977) have published results of experiments on presence of adenylate cyclase in plasma membrane and secretory granule fractions from the ox neurohypophysis. The isolation from the neurohypophysis of both membrane-bound and soluble Ca' '-binding proteins (RUSSELL & THORN,1977) with similar properties as the modulator protein for cyclic nucleotide phosphodiesterase isolated et a / . , from whole brain and other tissues (WATTERSON 1976) stimulated us to look for presence of these enzymes and their distribution in the neurohypophysis. As far as we know. such studies have not been done previously. It was shown in the present work that cyclic AMP phosphodiesterase activity is present in plasma-membranes from a preparation of isolated nerve endings from bovine neurohypophjses. High activity was also found in high speed supernatants from bovine neurohypophysis homogenates and lysed neurosecretosomes (roughly 400, of the total activity). whereas only negligible activitj was associated with a purified secretory granule fraction.
Preparation of and fracrioriariori of isolatrd rierw eridiriqs (nrurosecrr?osomes). Bovine neurohypophyses. obtained from the Copenhagen Public Slaughterhouse. and transported on ice, were processed within 80 min of the animal's death. All operations were carried out at 4'C. One or two neurohypophyses at a time were homogenized in 4 m l of a medium containing 0.25 M-SUCTOSe and 20 x lo-' M-TES (pH 7.0) (sucrose-TES medium). A neurosecretosome fraction was obtained using the method of BINDLER et a / . (1967) with minor modifications as developed earlier in this laboratory (GRArzL ct a(.. 1977). The yield of protein in the neurosecretosome fraction u a h 3.9 & 0.8 (s.E., n = 6 ) mg per gland. The neurosecretosome pellets (see Fig. IA) from up to 12 neurohlpophyses were resuspended in 1 ml of sucrose TES medium. and 20ml of a lysis medium consisting of 5 x lo-' M-TES (pH 8.0) was added. After 1 h with gentle stirring 4 ml portions of this suspension were treated with a Branson Sonicator for 5 s at 0°C using 50 W. The suspension was subsequently centrifuged at 80,00Og,, for 45 min to obtain a crude membrane pellet. After resuspension, approx 1.5 mg of pellet protein in a volume of 1.5 ml of sucrose-TES medium a a s applied on the discontinuous sucrose gradient (buffered with 20 x lo-' M-TESto pH 7.0). composed of I.Oml lajers of 0.6 M-.0.95 M-. and 1.00 M-SUCrOse, and 0.5 ml each of I .2 M and 1.75 M-sucrose. Except for the last layer. this gradient was as described by VILHARDTrr a / . (1975). After centrifugation for 90 min at 60,0@3y,,. the following fractions were collected through the top of the tube using a Beckman gradient recovery system: fraction I . corresponding to the MATERIALS AND METHODS 0.25 M-sucrose layer, fraction 2 and 3 the upper and lower Matrriu1.c. &['HICAMP (spec. act. 26 Ci;10-3 M), halves, respectively, of the 0.6 M-sucrose lajer. fraction 4 2-['H]5'-AMP (spec. act. 19 Ci;IO-' M), adenosine 5'-ycomprising the 0.95 M- and the upper 3'4 of the 1.00 M-SUC[3'P]triphosphate (spec. act. 1.5 Ci/lO-' M), all as sodium rose and fraction 5 corresponding to the interface of the salts and '?'I for radioimmunoassay were purchased from 1.00/1.20 M-sucrose. the Radiochemical Centre, Amersham. U.K. Unlabelled The pellet in the bottom of the tube was discarded. nucleotides. adenosine and TES (A'-Tris(hydroxymethy1)Preparation of nrurosecrrtory grariules. Bovine neuroh) methyl-2-amino-ethane sulphonic acid) were from Sigma pophyses were fractionated by the differential centrifugaChemical Co.. St. Louis. MO. U.S.A. Lumagel was from tion method of DEAN& HOPE(1967) used and checked Lumac LSC Chemicals. Basel. and Poly(ethy1eneimine) by enzyme marker analyses earlier in this laborator! (Rcs(-)cellulose thin-layer sheets (PEI sheets) were Art. 5579 SELL & THORN.1975). Fraction I (30009 for 30 s. consisting from Merck. Darmstadt, F.R.G. All other chemicals were mainly of unhomogenized material) was discarded. Fracanalytical grade. tion 2. obtained after 14.000g for 15 min consists of crude mitochondria and plasma membrane. fraction 3, 26.000 y for 15 min, of crude neurosecretory granules. Fraction 4 Ahhreviariotis used: TES. N-Tris(hydroxymethy1)methyl- (105,000 y for 2 h) is a microsomal fraction and finally fraction S4 contains high speed supernatant. Fraction 3 was 2-amino-ethane sulphonic acid; PDE, 3':5'-cyclic AMP phosphodiesterase: 5'-Nuc, 5'-nucleotidase; AVP. arginine further loaded on a discontinuous sucrose gradient giving fractions A to D. Whereas fractions A and B contain some vasopressin.
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lysosomes and mitochondria, fraction D mainly consists of purified neurosecretory granules. All fractions were stored at -18°C until analysis for enzymes or arginine vasopressin. Assay of CAMP phosphodiesterase activity (EC 3.1.4.17 (PDE). In preliminary experiments we used the procedure of THOMPSON& APPLEMAN(1971). However, we had problems due to high blanks and low recovery as previously found by others (BOUDREAU & DRUMMOND, 1975; LYNCH & CHEUNG, 1975). We therefore developed the following procedure based on TLC:,samples containing l(t15 pg of protein were incubated for 30min in IOOpI 20 x w 3 M TES (pH7.5). 1 x 1 0 - 3 ~ - M g C I , and 0.5 x 1 0 - 3 ~ ~ ethanol having [jH]cAMP (spec. act. 2-3 ~ n C i / l O -M, been removed by lyophilization). The incubation temperature was 30°C to avoid loss of enzyme activity (EGRIEet al., 1977). At the end of the incubation period, lop1 of 10 x lo-’ M-CAMP,10 x 10-3 M-AMP and 10 x lo-’ Madenosine was added and the reaction was stopped by heating to 100°C for 5 min on an oil bath. After centrifugation for 5 min at 10,OOOy in a Beckman microfuge 1Opl of the supernatant were spotted on PEI sheets. The sheets were developed for 7 cm in redistilled water, dried under a stream of hot air, and developed in the same dimension for 7cm in 0.1 M-LICI. Three spots were visible under ultraviolet light: AMP (RF zed), CAMP (RF 0.5) and adenosine (RFc 0.8). The spots were cut out and placed in scintillation vials. They were then eluted for 15 min with 1.5 m14 M-NH,OH and 10 ml Lumagel was added. Tritium activity was measured on a Packard Tri-Carb liquid scintillation spectrometer (model 3375). Enzyme activity was calculated from the fraction of counts found in AMP + adenosine. The enzyme activity was proportional with time over the 30min period employed and with the amount of enzyme added in the assay. In preliminary experiments the concentration of cAMP was varied from lo-’ M to 1O-j M. N o difference in activity was found at M. A concentration of 0.5 mM concentrations above was chosen in order to measure total PDE and keep the sensitivity high. It was routinely tested that all applied tritium activity was recovered in the AMP, cAMP and adenosine spots. This was also the case when excess of enzyme was added to achieve 100% breakdown of CAMP, indicating that possible breakdown products of AMP (IMP) and adenosine (inosine and hypoxanthine) were either found together with these or were produced to a very limited extent. (The blank values varied somewhat with the age of the C3H]cAMP, but they were always below 1% of the counts applied.) In some articles the existence of cAMP phosphodiesterase with 2 difkrent K, values has been suggested. We have not tried to study the distribution of phosphodiesterase with different K, values since it is still under discussion whether there are 2 K,,, values or negative cooperativity (see e.g. RUSSELL et al., 1972). Consequently, only total activity was measured. Assay of 5‘-nucleotidase activity (EC 3 This was measured as described for PDE with the following differences: C3H]AMP instead of r3H]cAMP was added to a concentration of 0.1 x M and only 2-10 pg of sample was added. After spotting on PEI sheets the chromatograms were eluted with water only. Enzyme activity was proportional with time and with the amount of enzyme added. Recovery of counts was tested as for PDE. Mg’+-Na+-K+-dependent, ouabain sensitive ATPase (EC 3.6.1.4)(ATPase)was assayed as the difference between
the activities determined in the absence and presence of 1 x 1 0 - 3 ~ - o u a b a i nin the medium. The amount of ”P released from [y-”PIATP was measured as described by FORMBY et at. (1976), except that the reducing solution was omitted. The final concentrations of reactants in the incubationmedium were: 2 0 x ~O-’M-TES,pH7.0, 2 x I O - j M MgCI2, 100 x lo-’ M-NaCI, 10 x M-KCI, 0.01 x lo-’ M-[~-~’P]ATP,at a specific activity of 0.8-0.5 ci/ M, 36150 pg/ml protein, and, when present, ouabain 1 x lo-’ M,in the final volunie of 55 PI. Blank tubes contained no protein. Radioirnrnunoassay of arginine-vasopressin (AVP). Prior to assay samples were diluted at least 10-fold in 0.25 x 10-3 M-acetic acid and boiled for 5 min in a water bath. They were stable for several months at 4°C (NIELSEN, 1964). The radioimmunoassay (duplicate samples) was that described previously (GRATZLer al., 1977) modified mainly in the following way: the iodinated AVP was further purified on a Sephadex (3-25 superfine column (30 x 3 4 . The reaction mixture was incubated at 4°C for 2 days. The standard curve was performed in triplicate with 18 points included: from 5.9 to Z.SOOpg/ml. It was smoothed and interpolations were made with the ‘splining’ method, calculations being done on a Univac 1 I00 digital computer using an ascii Fortran program. It was tested that a sample dilution curve was parallel to thc standard curve. Protein was determined by the method of LOWRY et al. as modified by LOUSet al. (1956) with crystalline bovine serum albumin as standard. RESULTS AND DISCUSSION The distribution of protein, Mg2+-Na+-K +-dependent, ouabain sensitive ATPase, vasopressin, cAMP phosphodiesterase and 5‘-nucleotidase activities in the various subcellular fractions is shown in Table 1. From the EM of the neurosecretosome fraction (Fig. 1A) and from earlier freeze cleavage studies of the same fracet al., 1977) it appears that the neurosecretotion (GRATZL somes are only little contaminated by other tissue elements from the neurohypophysis. All fractions displayed some heterogeneity e.g. the RSA of 5‘-nucleotidase in most of the neurosecretosome membrane fractions were rather similar. Using the present fractionation procedure for plasma membranes, VILHARDTet al. (1975) found the fraction at the 0.6/0.95 M interface to be rich in plasma membranes, as measured by the activity of MgZ+-NaC-K+-dependent, ouabain-sensitive ATPase, with a low content of A V P and low contamination with microsomes and mitochondria as measured by glucose-6-phosphatase and monoamine oxidase activities, respectively. Consistent with these results, examination of our fractions showed that the highest ouabain sensitive specific activity of Mg”-Na+-K+, ATPase, and lowest specific activity of arginine-vasopressin was located to Fraction 2 (see Table 1). This was supported by electron microscopy (Fig. IB), this fraction displaying fairly uniformly sized, electron translucent vesicles, with little admixture of neurosecretory granules, or their dense core contents. Fraction 3 also possessed considerable ATPase activity. However, the relative specific activity of this marker was less than half of the value in Fraction 2. The AVP content was increased, and electron microscopy showed an enrichment in the incompletely lysed neurosecretory granules.
FIG. 1A. Electron micrograph (10.000 x ) of the nciirosecretosome pellet (see Methods).
FIG 1B. Electron mlcrograph (54,600 x ) of purified plasma membrane (Frdctlon 2 ) from neurosecreto somes (see Methods).
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+
100 20.2 f 1.5 15.5 f 0.58 16.5 f 6.4 34.1 f 1.8 86.3 f 6.2 12.9 f 6.8 25.9 7.0 8.7 f 1.5 23.8 f 10.1 71.4 k 14.5
+
(5.6 1.2) 100 1 14.0 f 1.4 4.98 f 1.0 8.1 f 4.0 2.55 k 1.1 2.9 f 1.7 10.27 f 3.0 19.8 f 12.5 3.93 3.4 8.71 f 4.4 18.3 f 8.3 2.20 f 1.0 22.3 f 8.9 71.4 f 20
%
ATPase RSA
PROTEIN,
100 69.7 f 14.0 4.1 f 1.8 6.1 f 1.8 15.6 f 13.6 32.1 f 16.3 9.9 f 4.4 67.8 f 13.4
%
ATPase recovery
*
(23.3 f 2.4) 1 0.36 f 0.09 0.64 f 0.31 0.21 0.15 0.27 k 0.1 5 0.52 f 0.29 0.30 f 0.20
AVP RSA
ATPase, VASOPRESSIN, CAMP
100 5.0 f 1.3 21.3 f 7.6 5.1 f 2.9 16.2 f 5.9 23.6 f 9.5 19.5 f 11.1 85.7 f 8.0
%
AVP recovery
1.19 f 0.39 0.84 f 0.13 0.41 f 0.07 0.24 f 0.08
(0.34 f 0.07) I 0.75 f 0.08 0.49 f 0.13 0.66 f 0.10 1.23 f 0.11
(0.41 k 1.7) 1 2.08 f 0.48 2.97 f 2.25 4.05 k 0.43 3.37 f 0.56 2.01 f 0.31 1.08 f 0.19
+
I00 15.3 0.41 5.7 f 2.7 10.8 k 4.0 44.1 k 5.1 75.9 f 9.6 39.6 f 29.8 43.4 k 9.0 1.2 f 0.6 6.4 f 0.2 94.5 k 22.8
1.94 f 1.2 4.12 k 2.1 1.97 f 0.5 0.50 & 0.4
+
(0.34 f 0.10) I 2.97 k 2.2 1.21 f 1.2 10.6 4.8 1.34 f 1.0
(0.22 f 0.09) 1 1.56 f 0.73 1.67 f 0.61 2.55 f 1.11 2.63 k 1.18 2.10 &- 1.18 1.06 0.34
S’-Nuc R SA
100 9.0 f 0.5 7.5 k 6.2 40.9 f 16.7 14.8 f 6.7 72.0 f 3.3 11.8 5 9.5 34.8 5.1 4.1 f 0.4 3.5 f 0.8 54.1 f 12.8
100 21.8 f 10.2 8.2 f 1.2 5.9 f 0.2 20.0 2.3 20.7 f 7.1 19.2 f 5.8 73.0 f 14.4
%
5’-Nuc recovery
VARIOUS SUBCELLULAR FRACTIONS
100 29.1 f 6.1 11.6 f 12.3 5.7 f 0.6 20.2 & 6.0 19.4 f 7.7 13.0 k 0.9 69.9 5.3
%
PDE recovery
5’-NUCLEOTIDASE I N
PDE RSA
PHOSPHODIESTERASE A N D
Ref. fraction for recovery
The upper half of the table contains data from neurosecretosome preparations and further subfractions of them. The lower half contains data from secretory granule preparations. The absolute activities are given in brackets. (For PDE and 5‘Nuc nmol/pg prot. x h, for ATPase nmol/mg prot. x h, and for AVP ng/pg prot.) All figures are mean f S.E.M. of at least 3 individual experiments.
3 4 5 Gradient sum Homogenate of neurohypoph yses 2 3 4 s4 Dif. centrifug. sum A B C D Gradient sum
1
Crude membranes
Neurosecretosomes
Fraction
Protein recovery
TABLE1. DISTRIBUTTON OF
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Thus, Fraction 2 was considered t o represent the highcst concentration of neurosecretosomal plasma membranes.
Coinnients on the distribution of C A M P phosphodiesterase
(PDE) (1) Plasma menibrune isolafion. Roughly 1/3 of the phosphodiesterase activity was recovered in the crude membrane pellet (Table I). ATPase recovery was 70%. Since this enzyme is probably localized only on membranes. the missing 30% is likely to represent loss of protein. To get an estimate of membrane bound P D E activity, the recovery of about one third should be corrected for protein loss accordingly and thus should be approx 40%. The highest specific activity in subfractionation of the crude membranes was found in Fraction 2 (plasma membrane), but high specific activities were also found in Fractions 1 and 3. It would appear that P D E in Fraction 1 represents mainly soluble P D E and/or contamination from Fraction 2 and that P D E in Fractions 2 and 3 reprcsents plasma membrane bound PDE. The recovery of P D E was highest in Fraction 3, comparable to the recovery of ATPase. (2) Granule isolation. P D E activity was found in all fractions. The total recovery of P D E activity was always close t o protein recovery. This makes it unlikely that a different contribution of a possible modulator in the different experiments or fractions could explain the distribution found. The highest specific activity of P D E was found in the postmicrosomal fraction (S4). The recovery of P D E activity in this fraction was 44%, from which it can be deduced that roughly half of the P D E activity in the neurohypophysis is soluble. This is comparable to that found for neurosecretosomes. These results are in accordance with the results of CHEUNG(1967) for whole brain of rats. The crude granule preparation (Fraction 3) contained some P D E activity, but when this fraction was subfractionated o n a sucrose gradient the activity of P D E decreased with increasing density of the gradient, and was lowest in Fraction D. which contained the most purified granules. It should be noted that the very low activity of P D E in Fraction D could not be due to inhibition of PDE by sucrose since all fractions from the gradient were washed once in sucrose TES medium before analysis, which also involved a dilution 10 times.
Comments on the distrihution of 5'-nucleotidase and MgZ+-Na+-K +,ouabain-sensitive ATPase.
( I ) Plasma menibranr i d a t i o n . There was a marked enrichment of 5'-nucleotidase in Fractions 2 and 3, but the recovery was highest in Fractions 3 and 4. Although the relative specific activities of ATPase and 5'-nucleotidase differ. the recoveries are comparable in all fractions. This suggests that part of the 5'-nucleotidase is localized t o plasma membrane in this system. ( 2 ) Granule isolation. The relative specific activity of 5'-nucleotidase was highest in Fraction 4. This may indicate that this fraction contains high amounts of plasma membrane. High activity was also found in Fraction B. This fraction has a considerable content of nongranular membrane (DEAN& HOPE, 1967).
'
With the technical assistance of E. ENGDERG,I. KlELDSEN and B. LYNDERUP. 'Author t o whom all correspondence should be sent.
Coniments on possible Jirnction of c A M P phosphodiesterase in the neurohypophysis The specific activity found in the neurosecretosomal membrane fraction in the present experiments is comparable to that found in synaptic membranes from porcine brain (EGRIEet al., 1977). Common t o these membranes is a function in release of signal molecules. cAMP metabolism could play an important role in these functions in several ways. In our laboratory TREIMAN et al. (in press) have recently demonstrated a CAMP-dependent phosphorylation of several protein bands in the neurosecretosomal membranes. One of these hands was specifically influenced by Cazi in 1 0 - 4 ~ concentration.
Acknowledgements-We are greatly indebted t o Professor 0. BEHNKE,Institute of Medical Anatomy C and to Dr. MAGNUSBUNDGAAKD. institute of Medical Physiology A of the University of Copenhagen for carrying out the electron microscopy. The work was supported in part by the N O V O Foundation and the Danish Medical Research Council. Institute of Medical Physiology C , Uniuersity of Copenhagen, Panum Institute, Blegdanisvej 3C, 2200 Copenhagen N , Denmark
C. TORP-PEDERSEN M. TREIMAN N. A. THORN"^
REFERENCES
M. (1967) Isolated BINDLERE., LABELLAF. S. & SANWAL nerve endings (neurosecretosomes) from the posterior pituitary. J . Cell. Biol. 34, 185-205. BONNED., NICOLASP., LAURERM., CAMER M., TIXIERVIDALA. & COHENP. (1977) Evidence for an adenylatecyclase activity in neurosecretory granule membranes from bovinc neurohypophysis. Eur. J . Biochem. 78, 337-342. BOUDREAU R. J. & DRUMMOND G. J. (1975) A modified assay of 3',5'-cyclic-AMP phosphodiestcrase. Analyt. Biochem. 63, 388-399. CHEUNCW. Y . (1967) Properties of cyclic 3',5'-nucleotide phosphodiesterase from rat brain. Biochemistry, Easton 6, 1079-1087. DEANC. R. & HOPED. B. (1967) The isolation of purified neurosecretory granules from bovine pituitary posterior lobes. Biochem. J . 104, 1082-1088. J. A,, FLANGAS A. L. & SIEGEL EGRIEJ. C . , CAMPBELL F. L. (1977) Regional, cellular and subcellular distribution of calcium-activated cyclic nucleotidc phosphodiesterase and calcium-dependent regulator in porcine brain. J . Neurochem. 28, 1207-1213. FORMBY B., CAPITOK., EGEBERGJ. & HEDESKOVC. J. (1976) Ca-activated ATPase activity in subcellular fractions of mouse pancreatic islets. Am. J . Physiol. 230, 441448. GRATZL M., DAHLG . , RUSSELL J. T. & THORN N. A. (1977) Fusion of neurohypophyseal membranes in vitro. Biochim. biophys. acta 470, 45-51. C . M. & SCHOUM. (1956) Kolorimetrisk Lows P., PLENN bestemmelse af totalprotein og globulin i spinalvreske. Afprmvning af Lowry-metoden. Nord Med. 55, 693-695. LYNCHT. J . & CHEUNG W. Y. (1975) Underestimation of cyclic 3',5'-nucleotide phosphodiesterase activity by a radioisotopic assay using an anionic-exchange resin. Analyt. Biochem. 67, 130-138.
Short communication AA. T. (1964) Posterior pituitary hormones. Thesis NICLSCN Pharmaceutical High School. Copenhagen. POIRIFRG., LABRIE F., LEMAYH., DUPORCTA., SAVARY M. & PELLLTICR G. (1977) Purification of plasma membrane fractions from the bovine pars intermedia and neurohypophyseal lobe and properties of associated adenylate cyclase. Can. J . Eiochern. 55, 555 -566. RASMUSSEN H. & GOODMAN D. B. P. (1977) Relationship between calcium and cyclic nucleotides in cell activation. Physiol. Rev. 57, 421-509. RUOFFH. J.. MATHISONR. & LEDERISK. (1976) Cyclic 3',5'-adenosine monophosphate in the hypothalamoneurohypophysial system of normal, NaCI-treated and lactating rats. Neuroetidocrinolo~~~ 22, 18-29. RUSSELL J. T. & THORNN. A. (1975) Adenosine triphosphate dependent calcium uptake by subcellular fractions from bovine neurohypophyses. Acta physiof. scand. 93, 3 64- 377. RUSSELL J. T. & THORNN. A. (1977) Isolation and purification of calcium-binding proteins from bovine neurohypophyses. Biochim. hiophys. .4rta 491, 398-408.
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RUSSELL T. k., THOMPSONw. J., SCHNEIDER F. w. & APPLEMAN M. M. (1972) 3',5'-Cyclic adenosine monophosphate phosphodiesterase: negative cooperativity. Proc. nurn. Acad. Sri.. U S A . 69, 17911795. THOMPSON W. J. & APPLEMANM. N. (1971) Characterization of cyclic nucleotidc phosphodiesterases of rat tissues. J . h i d . Chern. 246, 3145-3150. THORN N. A., R U S S ~ LJ.LT., TORP-PEDERSEN C. & TREIMAN M. (1978) Calcium and neurosecretion. Ann. N . Y. Arad. Sci. 307. 6 18-639. VILHARVTH., BAKERR. & HOPED. B. (1975) Isolation and protein composition of membranes of neurosecretory vesicles and plasma membranes from the neural lobe of the bovine pituitary gland. Biockem J . 148, 5765. WATTERSON D. M., HARRELSON W. G., JR.. KLLLERP. M.. SHARIEF F. & VANAMANT. C. (1976) Structural similarities between the CaZ'dependent regulatory proteins of 3',5'-cyclic nucleotide phosphodiesterase and actomyosin ATPase. J . hioL Chrni. 251, 4501-4513.
Juiirnol qi .Vearorhurwsfri Vol 3:.
SHORT COMMUNICATION
Subcellular distribution of cyclic AMP phosphodiesterase in the ox neurohypophysis (Received 19 M a y 1978. Rrcrsrd 25 Augusr 1978. Accepted 9 October 1978)
CAMP AND,OR calcium ions play crucial roles in stimulussecretion coupling in a number of systems (RASMUSSEN & GOODMAN. 1977). In the neurohypophysis a definite role for C a 2 + has been established (for a recent survey. see THORNrt a/.. 1978). but the details of its action have not been clarified. The presence of CAMP in the neurohypophysis of rats and an increase of its concentration in rats that have been drinking 200 NaCl solution for 24 h has been demonstrated previously (RUOFF et a/., 1976). POIRIER et a/. (1977) and BONNErr a!. (1977) have published results of experiments on presence of adenylate cyclase in plasma membrane and secretory granule fractions from the ox neurohypophysis. The isolation from the neurohypophysis of both membrane-bound and soluble Ca' '-binding proteins (RUSSELL & THORN,1977) with similar properties as the modulator protein for cyclic nucleotide phosphodiesterase isolated et a / . , from whole brain and other tissues (WATTERSON 1976) stimulated us to look for presence of these enzymes and their distribution in the neurohypophysis. As far as we know. such studies have not been done previously. It was shown in the present work that cyclic AMP phosphodiesterase activity is present in plasma-membranes from a preparation of isolated nerve endings from bovine neurohypophjses. High activity was also found in high speed supernatants from bovine neurohypophysis homogenates and lysed neurosecretosomes (roughly 400, of the total activity). whereas only negligible activitj was associated with a purified secretory granule fraction.
Preparation of and fracrioriariori of isolatrd rierw eridiriqs (nrurosecrr?osomes). Bovine neurohypophyses. obtained from the Copenhagen Public Slaughterhouse. and transported on ice, were processed within 80 min of the animal's death. All operations were carried out at 4'C. One or two neurohypophyses at a time were homogenized in 4 m l of a medium containing 0.25 M-SUCTOSe and 20 x lo-' M-TES (pH 7.0) (sucrose-TES medium). A neurosecretosome fraction was obtained using the method of BINDLER et a / . (1967) with minor modifications as developed earlier in this laboratory (GRArzL ct a(.. 1977). The yield of protein in the neurosecretosome fraction u a h 3.9 & 0.8 (s.E., n = 6 ) mg per gland. The neurosecretosome pellets (see Fig. IA) from up to 12 neurohlpophyses were resuspended in 1 ml of sucrose TES medium. and 20ml of a lysis medium consisting of 5 x lo-' M-TES (pH 8.0) was added. After 1 h with gentle stirring 4 ml portions of this suspension were treated with a Branson Sonicator for 5 s at 0°C using 50 W. The suspension was subsequently centrifuged at 80,00Og,, for 45 min to obtain a crude membrane pellet. After resuspension, approx 1.5 mg of pellet protein in a volume of 1.5 ml of sucrose-TES medium a a s applied on the discontinuous sucrose gradient (buffered with 20 x lo-' M-TESto pH 7.0). composed of I.Oml lajers of 0.6 M-.0.95 M-. and 1.00 M-SUCrOse, and 0.5 ml each of I .2 M and 1.75 M-sucrose. Except for the last layer. this gradient was as described by VILHARDTrr a / . (1975). After centrifugation for 90 min at 60,0@3y,,. the following fractions were collected through the top of the tube using a Beckman gradient recovery system: fraction I . corresponding to the MATERIALS AND METHODS 0.25 M-sucrose layer, fraction 2 and 3 the upper and lower Matrriu1.c. &['HICAMP (spec. act. 26 Ci;10-3 M), halves, respectively, of the 0.6 M-sucrose lajer. fraction 4 2-['H]5'-AMP (spec. act. 19 Ci;IO-' M), adenosine 5'-ycomprising the 0.95 M- and the upper 3'4 of the 1.00 M-SUC[3'P]triphosphate (spec. act. 1.5 Ci/lO-' M), all as sodium rose and fraction 5 corresponding to the interface of the salts and '?'I for radioimmunoassay were purchased from 1.00/1.20 M-sucrose. the Radiochemical Centre, Amersham. U.K. Unlabelled The pellet in the bottom of the tube was discarded. nucleotides. adenosine and TES (A'-Tris(hydroxymethy1)Preparation of nrurosecrrtory grariules. Bovine neuroh) methyl-2-amino-ethane sulphonic acid) were from Sigma pophyses were fractionated by the differential centrifugaChemical Co.. St. Louis. MO. U.S.A. Lumagel was from tion method of DEAN& HOPE(1967) used and checked Lumac LSC Chemicals. Basel. and Poly(ethy1eneimine) by enzyme marker analyses earlier in this laborator! (Rcs(-)cellulose thin-layer sheets (PEI sheets) were Art. 5579 SELL & THORN.1975). Fraction I (30009 for 30 s. consisting from Merck. Darmstadt, F.R.G. All other chemicals were mainly of unhomogenized material) was discarded. Fracanalytical grade. tion 2. obtained after 14.000g for 15 min consists of crude mitochondria and plasma membrane. fraction 3, 26.000 y for 15 min, of crude neurosecretory granules. Fraction 4 Ahhreviariotis used: TES. N-Tris(hydroxymethy1)methyl- (105,000 y for 2 h) is a microsomal fraction and finally fraction S4 contains high speed supernatant. Fraction 3 was 2-amino-ethane sulphonic acid; PDE, 3':5'-cyclic AMP phosphodiesterase: 5'-Nuc, 5'-nucleotidase; AVP. arginine further loaded on a discontinuous sucrose gradient giving fractions A to D. Whereas fractions A and B contain some vasopressin.
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lysosomes and mitochondria, fraction D mainly consists of purified neurosecretory granules. All fractions were stored at -18°C until analysis for enzymes or arginine vasopressin. Assay of CAMP phosphodiesterase activity (EC 3.1.4.17 (PDE). In preliminary experiments we used the procedure of THOMPSON& APPLEMAN(1971). However, we had problems due to high blanks and low recovery as previously found by others (BOUDREAU & DRUMMOND, 1975; LYNCH & CHEUNG, 1975). We therefore developed the following procedure based on TLC:,samples containing l(t15 pg of protein were incubated for 30min in IOOpI 20 x w 3 M TES (pH7.5). 1 x 1 0 - 3 ~ - M g C I , and 0.5 x 1 0 - 3 ~ ~ ethanol having [jH]cAMP (spec. act. 2-3 ~ n C i / l O -M, been removed by lyophilization). The incubation temperature was 30°C to avoid loss of enzyme activity (EGRIEet al., 1977). At the end of the incubation period, lop1 of 10 x lo-’ M-CAMP,10 x 10-3 M-AMP and 10 x lo-’ Madenosine was added and the reaction was stopped by heating to 100°C for 5 min on an oil bath. After centrifugation for 5 min at 10,OOOy in a Beckman microfuge 1Opl of the supernatant were spotted on PEI sheets. The sheets were developed for 7 cm in redistilled water, dried under a stream of hot air, and developed in the same dimension for 7cm in 0.1 M-LICI. Three spots were visible under ultraviolet light: AMP (RF zed), CAMP (RF 0.5) and adenosine (RFc 0.8). The spots were cut out and placed in scintillation vials. They were then eluted for 15 min with 1.5 m14 M-NH,OH and 10 ml Lumagel was added. Tritium activity was measured on a Packard Tri-Carb liquid scintillation spectrometer (model 3375). Enzyme activity was calculated from the fraction of counts found in AMP + adenosine. The enzyme activity was proportional with time over the 30min period employed and with the amount of enzyme added in the assay. In preliminary experiments the concentration of cAMP was varied from lo-’ M to 1O-j M. N o difference in activity was found at M. A concentration of 0.5 mM concentrations above was chosen in order to measure total PDE and keep the sensitivity high. It was routinely tested that all applied tritium activity was recovered in the AMP, cAMP and adenosine spots. This was also the case when excess of enzyme was added to achieve 100% breakdown of CAMP, indicating that possible breakdown products of AMP (IMP) and adenosine (inosine and hypoxanthine) were either found together with these or were produced to a very limited extent. (The blank values varied somewhat with the age of the C3H]cAMP, but they were always below 1% of the counts applied.) In some articles the existence of cAMP phosphodiesterase with 2 difkrent K, values has been suggested. We have not tried to study the distribution of phosphodiesterase with different K, values since it is still under discussion whether there are 2 K,,, values or negative cooperativity (see e.g. RUSSELL et al., 1972). Consequently, only total activity was measured. Assay of 5‘-nucleotidase activity (EC 3 This was measured as described for PDE with the following differences: C3H]AMP instead of r3H]cAMP was added to a concentration of 0.1 x M and only 2-10 pg of sample was added. After spotting on PEI sheets the chromatograms were eluted with water only. Enzyme activity was proportional with time and with the amount of enzyme added. Recovery of counts was tested as for PDE. Mg’+-Na+-K+-dependent, ouabain sensitive ATPase (EC 3.6.1.4)(ATPase)was assayed as the difference between
the activities determined in the absence and presence of 1 x 1 0 - 3 ~ - o u a b a i nin the medium. The amount of ”P released from [y-”PIATP was measured as described by FORMBY et at. (1976), except that the reducing solution was omitted. The final concentrations of reactants in the incubationmedium were: 2 0 x ~O-’M-TES,pH7.0, 2 x I O - j M MgCI2, 100 x lo-’ M-NaCI, 10 x M-KCI, 0.01 x lo-’ M-[~-~’P]ATP,at a specific activity of 0.8-0.5 ci/ M, 36150 pg/ml protein, and, when present, ouabain 1 x lo-’ M,in the final volunie of 55 PI. Blank tubes contained no protein. Radioirnrnunoassay of arginine-vasopressin (AVP). Prior to assay samples were diluted at least 10-fold in 0.25 x 10-3 M-acetic acid and boiled for 5 min in a water bath. They were stable for several months at 4°C (NIELSEN, 1964). The radioimmunoassay (duplicate samples) was that described previously (GRATZLer al., 1977) modified mainly in the following way: the iodinated AVP was further purified on a Sephadex (3-25 superfine column (30 x 3 4 . The reaction mixture was incubated at 4°C for 2 days. The standard curve was performed in triplicate with 18 points included: from 5.9 to Z.SOOpg/ml. It was smoothed and interpolations were made with the ‘splining’ method, calculations being done on a Univac 1 I00 digital computer using an ascii Fortran program. It was tested that a sample dilution curve was parallel to thc standard curve. Protein was determined by the method of LOWRY et al. as modified by LOUSet al. (1956) with crystalline bovine serum albumin as standard. RESULTS AND DISCUSSION The distribution of protein, Mg2+-Na+-K +-dependent, ouabain sensitive ATPase, vasopressin, cAMP phosphodiesterase and 5‘-nucleotidase activities in the various subcellular fractions is shown in Table 1. From the EM of the neurosecretosome fraction (Fig. 1A) and from earlier freeze cleavage studies of the same fracet al., 1977) it appears that the neurosecretotion (GRATZL somes are only little contaminated by other tissue elements from the neurohypophysis. All fractions displayed some heterogeneity e.g. the RSA of 5‘-nucleotidase in most of the neurosecretosome membrane fractions were rather similar. Using the present fractionation procedure for plasma membranes, VILHARDTet al. (1975) found the fraction at the 0.6/0.95 M interface to be rich in plasma membranes, as measured by the activity of MgZ+-NaC-K+-dependent, ouabain-sensitive ATPase, with a low content of A V P and low contamination with microsomes and mitochondria as measured by glucose-6-phosphatase and monoamine oxidase activities, respectively. Consistent with these results, examination of our fractions showed that the highest ouabain sensitive specific activity of Mg”-Na+-K+, ATPase, and lowest specific activity of arginine-vasopressin was located to Fraction 2 (see Table 1). This was supported by electron microscopy (Fig. IB), this fraction displaying fairly uniformly sized, electron translucent vesicles, with little admixture of neurosecretory granules, or their dense core contents. Fraction 3 also possessed considerable ATPase activity. However, the relative specific activity of this marker was less than half of the value in Fraction 2. The AVP content was increased, and electron microscopy showed an enrichment in the incompletely lysed neurosecretory granules.
FIG. 1A. Electron micrograph (10.000 x ) of the nciirosecretosome pellet (see Methods).
FIG 1B. Electron mlcrograph (54,600 x ) of purified plasma membrane (Frdctlon 2 ) from neurosecreto somes (see Methods).
1087
+
100 20.2 f 1.5 15.5 f 0.58 16.5 f 6.4 34.1 f 1.8 86.3 f 6.2 12.9 f 6.8 25.9 7.0 8.7 f 1.5 23.8 f 10.1 71.4 k 14.5
+
(5.6 1.2) 100 1 14.0 f 1.4 4.98 f 1.0 8.1 f 4.0 2.55 k 1.1 2.9 f 1.7 10.27 f 3.0 19.8 f 12.5 3.93 3.4 8.71 f 4.4 18.3 f 8.3 2.20 f 1.0 22.3 f 8.9 71.4 f 20
%
ATPase RSA
PROTEIN,
100 69.7 f 14.0 4.1 f 1.8 6.1 f 1.8 15.6 f 13.6 32.1 f 16.3 9.9 f 4.4 67.8 f 13.4
%
ATPase recovery
*
(23.3 f 2.4) 1 0.36 f 0.09 0.64 f 0.31 0.21 0.15 0.27 k 0.1 5 0.52 f 0.29 0.30 f 0.20
AVP RSA
ATPase, VASOPRESSIN, CAMP
100 5.0 f 1.3 21.3 f 7.6 5.1 f 2.9 16.2 f 5.9 23.6 f 9.5 19.5 f 11.1 85.7 f 8.0
%
AVP recovery
1.19 f 0.39 0.84 f 0.13 0.41 f 0.07 0.24 f 0.08
(0.34 f 0.07) I 0.75 f 0.08 0.49 f 0.13 0.66 f 0.10 1.23 f 0.11
(0.41 k 1.7) 1 2.08 f 0.48 2.97 f 2.25 4.05 k 0.43 3.37 f 0.56 2.01 f 0.31 1.08 f 0.19
+
I00 15.3 0.41 5.7 f 2.7 10.8 k 4.0 44.1 k 5.1 75.9 f 9.6 39.6 f 29.8 43.4 k 9.0 1.2 f 0.6 6.4 f 0.2 94.5 k 22.8
1.94 f 1.2 4.12 k 2.1 1.97 f 0.5 0.50 & 0.4
+
(0.34 f 0.10) I 2.97 k 2.2 1.21 f 1.2 10.6 4.8 1.34 f 1.0
(0.22 f 0.09) 1 1.56 f 0.73 1.67 f 0.61 2.55 f 1.11 2.63 k 1.18 2.10 &- 1.18 1.06 0.34
S’-Nuc R SA
100 9.0 f 0.5 7.5 k 6.2 40.9 f 16.7 14.8 f 6.7 72.0 f 3.3 11.8 5 9.5 34.8 5.1 4.1 f 0.4 3.5 f 0.8 54.1 f 12.8
100 21.8 f 10.2 8.2 f 1.2 5.9 f 0.2 20.0 2.3 20.7 f 7.1 19.2 f 5.8 73.0 f 14.4
%
5’-Nuc recovery
VARIOUS SUBCELLULAR FRACTIONS
100 29.1 f 6.1 11.6 f 12.3 5.7 f 0.6 20.2 & 6.0 19.4 f 7.7 13.0 k 0.9 69.9 5.3
%
PDE recovery
5’-NUCLEOTIDASE I N
PDE RSA
PHOSPHODIESTERASE A N D
Ref. fraction for recovery
The upper half of the table contains data from neurosecretosome preparations and further subfractions of them. The lower half contains data from secretory granule preparations. The absolute activities are given in brackets. (For PDE and 5‘Nuc nmol/pg prot. x h, for ATPase nmol/mg prot. x h, and for AVP ng/pg prot.) All figures are mean f S.E.M. of at least 3 individual experiments.
3 4 5 Gradient sum Homogenate of neurohypoph yses 2 3 4 s4 Dif. centrifug. sum A B C D Gradient sum
1
Crude membranes
Neurosecretosomes
Fraction
Protein recovery
TABLE1. DISTRIBUTTON OF
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Thus, Fraction 2 was considered t o represent the highcst concentration of neurosecretosomal plasma membranes.
Coinnients on the distribution of C A M P phosphodiesterase
(PDE) (1) Plasma menibrune isolafion. Roughly 1/3 of the phosphodiesterase activity was recovered in the crude membrane pellet (Table I). ATPase recovery was 70%. Since this enzyme is probably localized only on membranes. the missing 30% is likely to represent loss of protein. To get an estimate of membrane bound P D E activity, the recovery of about one third should be corrected for protein loss accordingly and thus should be approx 40%. The highest specific activity in subfractionation of the crude membranes was found in Fraction 2 (plasma membrane), but high specific activities were also found in Fractions 1 and 3. It would appear that P D E in Fraction 1 represents mainly soluble P D E and/or contamination from Fraction 2 and that P D E in Fractions 2 and 3 reprcsents plasma membrane bound PDE. The recovery of P D E was highest in Fraction 3, comparable to the recovery of ATPase. (2) Granule isolation. P D E activity was found in all fractions. The total recovery of P D E activity was always close t o protein recovery. This makes it unlikely that a different contribution of a possible modulator in the different experiments or fractions could explain the distribution found. The highest specific activity of P D E was found in the postmicrosomal fraction (S4). The recovery of P D E activity in this fraction was 44%, from which it can be deduced that roughly half of the P D E activity in the neurohypophysis is soluble. This is comparable to that found for neurosecretosomes. These results are in accordance with the results of CHEUNG(1967) for whole brain of rats. The crude granule preparation (Fraction 3) contained some P D E activity, but when this fraction was subfractionated o n a sucrose gradient the activity of P D E decreased with increasing density of the gradient, and was lowest in Fraction D. which contained the most purified granules. It should be noted that the very low activity of P D E in Fraction D could not be due to inhibition of PDE by sucrose since all fractions from the gradient were washed once in sucrose TES medium before analysis, which also involved a dilution 10 times.
Comments on the distrihution of 5'-nucleotidase and MgZ+-Na+-K +,ouabain-sensitive ATPase.
( I ) Plasma menibranr i d a t i o n . There was a marked enrichment of 5'-nucleotidase in Fractions 2 and 3, but the recovery was highest in Fractions 3 and 4. Although the relative specific activities of ATPase and 5'-nucleotidase differ. the recoveries are comparable in all fractions. This suggests that part of the 5'-nucleotidase is localized t o plasma membrane in this system. ( 2 ) Granule isolation. The relative specific activity of 5'-nucleotidase was highest in Fraction 4. This may indicate that this fraction contains high amounts of plasma membrane. High activity was also found in Fraction B. This fraction has a considerable content of nongranular membrane (DEAN& HOPE, 1967).
'
With the technical assistance of E. ENGDERG,I. KlELDSEN and B. LYNDERUP. 'Author t o whom all correspondence should be sent.
Coniments on possible Jirnction of c A M P phosphodiesterase in the neurohypophysis The specific activity found in the neurosecretosomal membrane fraction in the present experiments is comparable to that found in synaptic membranes from porcine brain (EGRIEet al., 1977). Common t o these membranes is a function in release of signal molecules. cAMP metabolism could play an important role in these functions in several ways. In our laboratory TREIMAN et al. (in press) have recently demonstrated a CAMP-dependent phosphorylation of several protein bands in the neurosecretosomal membranes. One of these hands was specifically influenced by Cazi in 1 0 - 4 ~ concentration.
Acknowledgements-We are greatly indebted t o Professor 0. BEHNKE,Institute of Medical Anatomy C and to Dr. MAGNUSBUNDGAAKD. institute of Medical Physiology A of the University of Copenhagen for carrying out the electron microscopy. The work was supported in part by the N O V O Foundation and the Danish Medical Research Council. Institute of Medical Physiology C , Uniuersity of Copenhagen, Panum Institute, Blegdanisvej 3C, 2200 Copenhagen N , Denmark
C. TORP-PEDERSEN M. TREIMAN N. A. THORN"^
REFERENCES
M. (1967) Isolated BINDLERE., LABELLAF. S. & SANWAL nerve endings (neurosecretosomes) from the posterior pituitary. J . Cell. Biol. 34, 185-205. BONNED., NICOLASP., LAURERM., CAMER M., TIXIERVIDALA. & COHENP. (1977) Evidence for an adenylatecyclase activity in neurosecretory granule membranes from bovinc neurohypophysis. Eur. J . Biochem. 78, 337-342. BOUDREAU R. J. & DRUMMOND G. J. (1975) A modified assay of 3',5'-cyclic-AMP phosphodiestcrase. Analyt. Biochem. 63, 388-399. CHEUNCW. Y . (1967) Properties of cyclic 3',5'-nucleotide phosphodiesterase from rat brain. Biochemistry, Easton 6, 1079-1087. DEANC. R. & HOPED. B. (1967) The isolation of purified neurosecretory granules from bovine pituitary posterior lobes. Biochem. J . 104, 1082-1088. J. A,, FLANGAS A. L. & SIEGEL EGRIEJ. C . , CAMPBELL F. L. (1977) Regional, cellular and subcellular distribution of calcium-activated cyclic nucleotidc phosphodiesterase and calcium-dependent regulator in porcine brain. J . Neurochem. 28, 1207-1213. FORMBY B., CAPITOK., EGEBERGJ. & HEDESKOVC. J. (1976) Ca-activated ATPase activity in subcellular fractions of mouse pancreatic islets. Am. J . Physiol. 230, 441448. GRATZL M., DAHLG . , RUSSELL J. T. & THORN N. A. (1977) Fusion of neurohypophyseal membranes in vitro. Biochim. biophys. acta 470, 45-51. C . M. & SCHOUM. (1956) Kolorimetrisk Lows P., PLENN bestemmelse af totalprotein og globulin i spinalvreske. Afprmvning af Lowry-metoden. Nord Med. 55, 693-695. LYNCHT. J . & CHEUNG W. Y. (1975) Underestimation of cyclic 3',5'-nucleotide phosphodiesterase activity by a radioisotopic assay using an anionic-exchange resin. Analyt. Biochem. 67, 130-138.
Short communication AA. T. (1964) Posterior pituitary hormones. Thesis NICLSCN Pharmaceutical High School. Copenhagen. POIRIFRG., LABRIE F., LEMAYH., DUPORCTA., SAVARY M. & PELLLTICR G. (1977) Purification of plasma membrane fractions from the bovine pars intermedia and neurohypophyseal lobe and properties of associated adenylate cyclase. Can. J . Eiochern. 55, 555 -566. RASMUSSEN H. & GOODMAN D. B. P. (1977) Relationship between calcium and cyclic nucleotides in cell activation. Physiol. Rev. 57, 421-509. RUOFFH. J.. MATHISONR. & LEDERISK. (1976) Cyclic 3',5'-adenosine monophosphate in the hypothalamoneurohypophysial system of normal, NaCI-treated and lactating rats. Neuroetidocrinolo~~~ 22, 18-29. RUSSELL J. T. & THORNN. A. (1975) Adenosine triphosphate dependent calcium uptake by subcellular fractions from bovine neurohypophyses. Acta physiof. scand. 93, 3 64- 377. RUSSELL J. T. & THORNN. A. (1977) Isolation and purification of calcium-binding proteins from bovine neurohypophyses. Biochim. hiophys. .4rta 491, 398-408.
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RUSSELL T. k., THOMPSONw. J., SCHNEIDER F. w. & APPLEMAN M. M. (1972) 3',5'-Cyclic adenosine monophosphate phosphodiesterase: negative cooperativity. Proc. nurn. Acad. Sri.. U S A . 69, 17911795. THOMPSON W. J. & APPLEMANM. N. (1971) Characterization of cyclic nucleotidc phosphodiesterases of rat tissues. J . h i d . Chern. 246, 3145-3150. THORN N. A., R U S S ~ LJ.LT., TORP-PEDERSEN C. & TREIMAN M. (1978) Calcium and neurosecretion. Ann. N . Y. Arad. Sci. 307. 6 18-639. VILHARVTH., BAKERR. & HOPED. B. (1975) Isolation and protein composition of membranes of neurosecretory vesicles and plasma membranes from the neural lobe of the bovine pituitary gland. Biockem J . 148, 5765. WATTERSON D. M., HARRELSON W. G., JR.. KLLLERP. M.. SHARIEF F. & VANAMANT. C. (1976) Structural similarities between the CaZ'dependent regulatory proteins of 3',5'-cyclic nucleotide phosphodiesterase and actomyosin ATPase. J . hioL Chrni. 251, 4501-4513.
