Insect Biochem. Vol. 19, No. 8, pp. 775-779, 1989 0020-1790/89 $3.00 + 0.00 Printed in Great Britain. All rights reserved Copyright ~ 1989 Pergamon Press plc

SOLUBILIZATION OF ADENYLATE CYCLASE FROM CERATITIS CAPITA TA NEURAL MEMBRANES AND

INCORPORATION INTO LIPOSOMES

ALBERTO GUILLEN*, AMADOR HAROt, JOSE L. NIETO and ANGEL M. MUNICIO Department of Biochemistry, Faculty of Chemistry, Universidad Complutense, 28040-Madrid, Spain

(Received 12 December 1988; revised and accepted 28 July 1989)

Abstract--Solubilization of the adenylate cyclase from neural membranes of the dipterous Ceratitis capitata, by using several detergents, and regulatory characteristics of the solubilized enzyme were examined. Triton X-100 is the most effective detergent in solubilizing this enzyme activity. The adenylate cyclase in Triton X-100-solubilized preparations (105,000g supernatant) does not respond to either guanine nucleotides or fluoride and it apparently seems to be devoid of a functional regulatory component. When this preparation is centrifuged again at 300,000g for 30 min no enzyme activity is detectable in the supernatant, however only 8% of total activity is recovered in the pellet. The activation pattern for the enzyme in the 300,000g pellet is similar to that observed for the enzyme in the 105,000g supernatant. Incorporation of solubilized enzyme into dimyristoylphosphatidylcholine (DMPC), dipalmitoylphos- phatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC) or cholesterol-enriched DOPC liposomes increases the 300,000 g pellet adenylate cyclase activity in a similar extension; thus, this increase in enzyme activity appears to be independent not only on the phospholipid composition but also on the liposome fluidity.

Key Word Index: adenylate cyclase, solubilization, liposome fluidity

INTRODUCI'ION

Adenylate cyclasc is an enzyme localized within the plasma membrane which transduces an extracellular signal across the lipid bilayer generating cAMP as an intracellular messenger. This enzyme system consists, at least, of three components: the receptor (R) with a binding site for the extracellular messenger exposed at the cell surface, the guanine nucleotide-binding regulatory component (G) and the catalytic compo- nent (C), both having a ligand-binding site at the cytoplasmic surface (Clark, 1986; Caperson and Bourne, 1987). Although all three entities are integral proteins, G and C bind little detergent, indicating that only a relatively small fraction of their mass is actually interpolated into the bilayer, probably only the functional globular part of both G and C pene- trates into the cytosol-facing half of the bilayer (Neer, 1976; Ross and Gilman, 1980). On the other hand, R should be a transmembrane entity in order that it may interact appropriately with the G component localized at the cytosol surface (Houslay, 1981). Moreover, it has recently been reported that the C unit (Pfeuffer et al., 1985) and fl-adrenoceptors (Dohlman et al., 1987) are transmembrane proteins. For these reasons, adenylate cyclase solubilization requires the use of detergents and methods to solubi- lize peripheral membrane proteins are not useful.

Neural adenylate cyclase of the dipterous Ceratitis capitata is a plasma membrane-bound enzyme

*Present address: CCIPE, Rue de la Cardonille, 34094 Montpellier, France.

tAuthor to whom all correspondence should be addressed.

modulated by cations, fluoride, guanine-nucleotides, the diterpene forskolin and the neurotransmitter oc- topamine (Garcia and Haro, 1981; Garcia et al., 1981; Guillen et ai., 1987, 1989). In this paper we describe insect adenylate cyclase solubilization by using several anionic and non-ionic detergents and we compare the regulatory characteristics of solubilized enzyme with respect to those of membrane enzyme. Since reconstitution of membrane-bound enzymes and transport proteins with liposomes is one of the most useful techniques to study the functional aspects of these proteins (Banerjee and Datta, 1983), we also report the incorporation of insect solubilized adenylate cyclase into liposomes.

MATERIALS AND METHODS

Materials

[ct-32P]ATP (sp. act. 400 Ci/mmol) and [8-3 Hkyclic AMP (sp. act. 27.5 Ci/mmol) were obtained from The Radio- chemical Centre (Amersham, U.K.); nucleotides, octo- pamine, lipids, sucrose, Tris-HCl and detergents were purchased from Sigma Chemical Co. (St Louis, Mo.); 2-mercaptoethanol, tetrahydrofuran and inorganic salts were from Merck (Darmstadt, F.R.G.); forskolin was from Calbiochem (La Jolla, Calif.); 1,6-diphenyl-l,3,5-hexalriene was supplied by Aldrich Chemical Co. (Milwaukee, Wis.); Bio-Beads SM-2 was from Bio-Rad (Richmond, Calif.). All other reagents were of analytical grade.

Insect culture

C. capitata (Wiedemann) in the adult stage of develop- ment was ¢sed. Diet, temperature and humidity conditions of culturing were carefully controlled as previously described (Fernandez-Sousa et al., 1971).

775

776 ALBERTO GUILLEN et al.

Preparation o f solubilized adenylate cyclase

Plasma membranes from C. capitata heads were prepared as reported (Garcia and Haro, 1981). Membranes obtained from the 0.1 M sucrose interface after the gradient cen- trifugation step, used in this study, had the highest specific activity for adenylate cyclase and, since the enzyme was activated by the neurotransmitter octopamine (Guillen et al., 1989), the preparation is assumed to be highly rich in neural tissue. Membrane preparations were suspended in 50raM Tris-HC1 buffer, pH7.5 (25:C), containing 10raM 2-mercaptoethanol, 5 m M NaF, 5 raM MgSO.,, 100mM sucrose and 0.5% detergent (w/v) (solubilization buffer) to give a protein/detergent ratio of 1/5 (w/v). After standing for 30 rain on ice with stirring, the mixture was centrifuged at 105,000g for 60min in a Beckman L5-65B ultracentrifuge. The method of Holloway (1973) was used in order to remove excess detergent from the solubilized preparation. Bio-Beads SM-2 was added te the 105,000g supernatant fraction (0.25g/ml) and the mixture was left standing for 60rain on ice with stirring and then filtered through glass wool to remove the resin (preparation SI). In some experiments, the solubilized preparation was centrifuged at 300,000g for 30min and the pellet was resuspended in 5 0 m M Tris-HCl buffer, pH 7.5 (25~C), containing 10 mM 2-mercaptoethanol (incubation buffer), in the same volume as the supernatant (preparation $2).

Preparation q[" liposomes and incorporation t?/" solubilized enzyme into liposomes

Lipids in organic solvent were dried under a stream of nitrogen until the last trace of solvent was removed. The lipid was dispersed in incubation buffer by sonication on ice for 2-10 min, depending on the nature of the phospholipid acyl chain, at 12/am in a MSE sonicator (frequency 23 kHz) (Hebdon et al., 1979). The incorporation of solubilized enzyme into liposomes was carried out by incubating 0.375 ml of preparation S l with 0.125ml of liposomes for 12 h at 4~C with shaking (lipid concentration in tables and figures refers to the final incorporation volume) and 0.1 nd were added to adenylate cyclase assay mixture. In other experiments, samples were diluted after incorporation with 9 vol of incubation buffer and centrifuged at 300,000 g for 30 min. The pellet was resuspended in 0.5 ml of incubation buffer and 0.1 ml were used in the enzyme assay.

Adenylate cyclase ctvsay

The adenylate cyclase assay mixture contained solubilized enzyme, 0 .05mM ATP (600,000cpm of [a-32P]ATP), 0.75 mM cAMP and modulators according to the experi- ment: 10mM MgCI 2, 1 mM MnCI 2, 15mM NaF, 0.05 mM forskolin, 0.1 mM GTP, 0.1 mM G p p N H p and 0.1 mM octopamine, in a final volume of 0.2 ml of incubation buffer. The reaction (10 min at 37~C with shaking) was stopped and the mixture was processed as reported (Garcia and Haro, 1981). [8 -~ HlCyclic AMP (20,000 cpm) was used as recovery marker. Forskolin was added to assay dispersed by sonica- tion on ice in incubation buffer. Adenylate cyclase specific activity is defined as pmol of cAMP per min per mg of protein. The experiments reported have been repeated at least twice and the results are the mean of duplicate determinations, the variability being less than 10%.

Fluorescence labeling and polarication measurements

1,6-Diphenyl-l,3,5-hexatriene (DPH) was incorporated into the hydrophobic portion of the liposomes by incubating for 30min at 3 7 C under stirring 1.1 ml of cholesterol- enriched DOPC liposomes (100/ag of DOPC) with 0.4 ml of a water-stable dispersion of the probe, previously dissolved in tetrahydrofuran (final concentration 9 # M). Fluorescence polarization (P) was measured as described (Guillen et al., 1984). Microviscosity (rT) was calculated from the P values through the relation 0 = 2 P / ( 0 . 4 6 - P) (Shinitzky and Barenholz, 1978). Assays were run in duplicate and the variations were less than 5%.

Other methods

Protein concentration was determined according to the method of Dulley and Grieve (1975) using bovine serum albumin as standard. Lipid phosphorous was measured in accordance with Rouser et al. (1966) in both membrane and solubilized preparations, water being first removed by calefaction at 110 C.

RESULTS AND DISCUSSION

Several d e t e r g e n t s have been a s s a y e d to solubi l ize m e m b r a n e - b o u n d a d e n y l a t e cyc lase f r o m prep- a r a t i o n s en r i ched in neu ra l t i ssue o f C. capitata: s o d i u m d o d e c y l s u l p h a t e a n d s o d i u m cho l a t e ( an ion ic de t e rgen t s ) a n d N o n i d e t P-40, Lub ro ! PX, T r i t o n

Table 1. Proteins, phospholipids (expressed as inorganic phosphate) and adenylate cyclase specific acitivity in 105,000g supernatants after solubilizing brain membranes with various

detergents

Adenylate cyclase specific activity ( p m o l × m i n ~ × m g ~)

Inorganic Without With Protein phosphate Bio-Beads Bio-Beads Yield

Detergent (%) (%) SM-2 SM-2 (%)

Cholate 16 + 0.5 I + 0.1 0.0 _+_ 0.0 0.0 +_ 0.0 0 SDS 59 + 0.9 19 + 1.6 0.0 _+ 0.0 0.0 _+ 0.0 0 Tween 20 18 + 1.3 3 ± 0.3 0.0 ~- 0.0 0.0 ~ 0.0 0 Brij 35 14 _+ 0.6 3 ~- 0.4 8.7 ~- 0.5 45.7 _- 2.6 15 Nonidet P-40 28_+2.0 26 t 2.1 14.3 + 1.1 1751 - 12.5 115 Lubrol PX 20 + 0.1 15 + 2.0 78.2 + 6.8 274.9 _- 10.0 130 Triton N-101 38+ 1.7 29+0.1 21.7+2.1 207.1 -_20.0 186 Triton X-100 38 + 2.1 25 -- 1.5 43.6 ± 4.0 867.8 _-+ 59.7 778

Proteins and inorganic phosphate are measured after treating 105,0O0g supernatants with Bio-Beads SM-2 to remove excess detergent and they are expressed as percentage of contents present in membranes. Adenylate cyclase activity is measured in the presence of 10raM MgCI:. Yield values correspond with enzyme activity values after adding Bio-Beads SM-2. In order to calculate solubilization yield, membrane adenylate cyclase activity (42.4 pmol × m i n i x mg -I ) is measured in the presence of 25 ,ul of membrane preparation (45 ,ug of protein), 10 mM MgCI: and 45#1 of solubilization buffer without detergent because NaF, which is included in the solubilization buffer, is a potent activator of membrane-bound adenylate eyclase. Data are the mean of duplicate samples of a rel~resentative experiment, the variability beit~g indicated.

C. capitata adenylate cyclase

Table 2. Adenylate cyclas¢ specific activity in the presence of several modulators for the Triton X-100-sol- ubilized preparation (preparation S~ ) in comparison with the enzyme activity for the membrane preparation

used as source of solubilized enzyme

777

Adenylate ~clase specific activity (pmol x min -I x m g - I )

Modulator Membrane-bound Triton X- 100-solubilired 10 mM MgC12 10 mM MgCI 2 + 0.05 mM forskolin 10mM MgCI2 + 15mM NaF 10mM MgCI2+0.1 mM GTP 10raM MgCI: + 0.1 mM GppNHp 10mM MgCI 2 + 0.1 mM GTP + 0.1 mM octopamine I mM MnCI 2

25.5 :t: 2.3 867.8 _+ 59.7 139.5+ 12.1 1622.8+ 110.2 416.6 _+ 30.7 826.0 + 24.3 42.0 -6 8.8 810.6 _ 53.1

120.3 + 10.0 932.8 _+ 70.5 67.6 + 5.4 848.9 +. 16.2 55.2 _+ 4.5 1739.0 + 55.5

Preparation s t is obtained as described under Materials and Methods. Values are the mean of duplicate samples of a representative experiment, the variability being indicated.

N-101, Tri ton X-100, Brij 35 and Tween 20 (non- ionic detergents). Solubilization buffers include the activator NaF which presents a protective effect against the enzyme thermal denaturat ion (Garcia et al., 1982). Results are shown in Table 1. Sodium dodecylsulphate is the most effective detergent in solubilizing insect plasma membrane proteins. For non-ionic detergents there is a correlation between the hydrophile-lipophile balance (HLB) and solubi- lizing power, as previously reported (Helenius and Simlons, 1975; Megias et al., 1986). In this way, Triton N-101 ( H L B = I 3 . 4 ) and Triton X-100 (HLB = 13.5) have minor HLB values, showing the greatest solubilizing powers. Moreover, detergents solubilize membrane inorganic phosphate, measured as a phospholipid index, this fact indicates the pres- ence of phospholipids in the solubilized preparations. So, it seems that solubilized proteins maintain, at least partially, their lipid environment.

Table i also shows the adenylate cyclase activity in 105,000g supernatants before and after adding Bit-Beads SM-2 to remove excess detergent. When anionic detergents or Tween 20 are used, the solubi- lized preparation has no detectable enzyme activity. It is in agreement with previous studies where alkyl anionic and cationic detergents had denaturing prop- erties at the concentrations used for the solubilization of membranes (Helenius and Simons, 1975). For the other detergents, adenylate cyclase activity is higher when supernatants are treated with Bit-Beads SM-2, Triton X-100-solubilized adenylate cyclase showing the maximal specific activity. On the other hand, it has been noticed that adenylate cyclase extraction from membranes by using non-ionic detergents in- creases the enzyme activity (Hebdon et al., 1981). In this respect, as shown in Table 1, the Triton X-100 solubilization yield is nearly 800%.

Table 2 shows the regulatory characteristics of both membrane and Tri ton Xo 100-solubilized adeny- late cyclase. In any case, Mn 2~ is more effective than Mg 2~ in promoting enzyme activity, however, fluoride, GTP, G p p N H p or octopamine, in the pres- ence of Mg 2+, do not increase Tri ton X-100-solubi- lized adenylate cyclase activity. This activation pattern for divalent ions, guanine nucleotides, fluoride or octopamine is identical when Lubrol PX is used (data not shown). S~nce non-ionic detergent- solubilized preparations do not respond to guanine nucleotides or fluoride, they appear to be apparently devoid of functional regulatory component.

Forskolin also stimulates solubilized adenylate cyclase, therefore the diterpene does not require a functional G component to act. At a concentration of 0.05 mM, the diterpene activates membrane adeny- late cyclase from 5 to 6-fold, whereas solubilized activity is only stimulated by two to three times the basal enzyme level. This observation may suggest that in the course of solubilization an unknown protein or a specific lipid necessary to lead maximal activation could be removed, in agreement with previous studies (Pfeuffer et al., 1985).

When Triton X-100-solubilized preparations are centrifuged again at 300,000 g for 30 min no enzyme activity is detectable in the supernatant, however only an 8% of total activity is recovered in the pellet (Fig. !). This fact could be attributed to a loss of a component necessary for a fully active enzyme during the centrifugation. Thus, when the 300,000 g pellet is resuspended in the centrifugation supernatant, the enzyme activity is partially restored. Otherwise, the activation pattern for the enzyme in the 300,000g pellet (preparation $2) is similar (data not shown) to that described above for the enzyme in the 105,000g supernatant (preparation S~). On the other hand, we

I

f 2 0 0 I: N

i . m

.> 100

m m Fig. 1. Total activity of Triton X-100-solubilized adenylate cyclase in the preparation St (white bar) and in the prepara- tion S 2 when the 300,000 8 centrifugation pellet is resus- pended in incubation buffer (solid bar), in solubilization buffer without Triton X-100 (dotted bar) or in the centrifu- gation supernatant (striped bar). (Preparations S~ and $2 are obtained as described under Materials and Methods; en- zyme activity is measured in the presence of I0 mM MgCI2; bars represent means of duplicate samples of a represen-

tative experiment and deviations are indicated.)

778 ALBERTO GUILLEN et al.

Table 3. Triton X-100-solubilized adenylate cyclase activity in the preparation S 2 incorporated into dipalmitoylphosphatidylcholine

liposomes (DPPC) by using two incorporation procedures Adenylate cyclas¢ activity (pmol x rain ~ x I00 / t l i)

Conditions of incorporation 10mM MgCI 2 10mM MgCI2+0.1 mM GTP

Control 29.1 + 1.4 30.0 + 2.3 Incubation

0.TraM DPPC 40.8_+4.0 39.4_+ 1.1 7raM DPPC 154.8_+9.5 141.3_+3.2

Sonication 0.7 mM DPPC 43.0 _+ 0.5 38.9 + 3.6 7 mM DPPC 77.8 + 6.6 68.8 _+ 5.4

Preparation S 2 is obtained as described under Materials and Meth- ods.

Controls correspond to preparation $2 without incorporating into liposomes. Values are the mean of duplicate samples of a representative experiment, the variability being indicated.

have observed in C. capitata neural adenylate cyclase, either membrane-bound or solubilized, that an in- crease in fluidity of the catalytic unit lipid environ- ment involves a decrease in enzyme activity (unpublished data). In this way, we have studied the incorporation of Triton X-100-solubilized enzyme into liposomes of defined phospholipid composition as a membrane model.

Optimal conditions for the incorporation of Triton X-100-solubilized adenylate cyclase into liposomes have been assayed. Table 3 shows adenylate cyclase activity in the 300,000 g pellet when the preparation S~ is incubated for 12h at 4°C with shaking or sonicated on ice for 5 min with DPPC iiposomes. The first method's results are more advantageous than the second one's, so it is used in the following experi- ments. The preparation S~ incorporated into DPPC liposomes do not exhibit activation by GTP neither before (data not shown) nor after 300,000g centri- fugation. Since activation by guanine nucleotides cannot be restored in any case by reconstitution in liposomes, Triton X-100 could only solubilize the

catalytic unit, the G component remaining in the 105,000g pellet; although it is also possible that the G component is found in the supernatant but in a non-functionai form.

Incorporation of solubilized enzyme (preparation S~) into DMPC, DSPC and DOPC liposomes in- creases the 300,000 g pellet adenylate cyclase activity by the same extent that incorporation into DPPC liposomes does [Fig. 2(A)]. Thus, the increase in enzyme activity appears to be independent not only on the phospholipid composition but also on the liposome fluidity. In order to corroborate this obser- vation, incorporation of solubilized enzyme into cholesterol-enriched DOPC liposomes has been carried out [Fig. 2(B)]. Cholesterol increases the microviscosity of DOPC liposomes, as shown by DPH-fluorescence polarization measurements, but reconstitution of the enzyme activity does not correlate with liposome fluidity.

Figure 3 shows the different temperature depen- dence of Triton X-100-solubilized adenylate cyclase activity before (preparation S~) or after (preparation $2) 300,000g centrifugation, the preparation $2 being more sensitive to thermal inactivation. Likewise, the protective effect against thermal denaturation of in- corporating the preparation S~ into DOPC liposomes is shown. The protective effect is higher for the enzyme in the 300,000g pellet.

Therefore, incorporation of solubilized adenylate cyclase into liposomes, whatever their fluidity, ap- pears to provide a supportive milieu for the enzyme, which would partially prevent the loss of the com- ponent necessary for a fully enzyme activity in the course of the 300,000g centrifugation. Since we have previously observed a correlation between the order degree of the catalytic unit annular lipids and adenylate cyclase activity, the catalytic unit lipid environment could be partially removed during the 300,O00g centrifugation, distorting the annulus of the phospholipids that are in residence at the

~ 250

'T =1.

• r; 15o T=

l >

A

!

° CONTROL DMP¢ DPP¢ I ~ P c

phospholipids DOPC

B

l COm~OL 011 O.Sll I11

c_J-~esterol/dopc

2.5

1.5

0.5

1.S11

| G. >~

O

> o U

E

Fig. 2. (A) Triton X-100-solubilized adenylate cyclase activity in the preparation $2 incorporated into dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphos- phatidylcholine (DSPC) or dioleoylphosphatidylcholine (DOPC) liposomes. (B) Triton X-100-solubilized adenylate cyclase activity in the preparation $2 incorporated into cholesterol-enriched DOPC liposomes and microviscosity values for these liposomes. (Preparation $2 is obtained as described under Materials and Methods; enzyme activity is measured in the presence of 10 mM MgCI2; controls correspond to preparation $2 without incorporation into liposomes; phospholipid concentration is 7 mM; bars represent

means of duplicate samples of a representative experiment and deviations are indicated.)

C. capitata adenylate cyclase 779

150

100

50

' ' 5 5 25 35 45 temperature, °C

Fig. 3. Temperature dependence of Triton X- 100-solubilized adenylate cyclase in the preparation S~ (circles) or in the preparation $2 (squares), before (open symbols) or after (solid symbols) incorporation into 7mM dioleoylphos- phatidylcholine liposomes. (Preparations S~ and $2 are obtained as described under Materials and Methods; en- zyme activity is measured in the presence of 10 mM MgCI2; the values given are means from duplicate samples of a representative experiment, the variability being less than

10%.)

l ipid-protein interface. This distortion thus would be responsible for the lesser enzyme activity and the higher sensitivity to thermal denaturation found in the preparation $2.

REFERENCES

Banerjee R. K. and Datta A. G. (1983) Proteoliposome as the model for the study of membrane-bound enzymes and transport proteins. Molec. cell. Biochem. 50, 3-15.

Casperson G. F. and Bourne H. R. (1987) Biochemical and molecular genetic analysis of hormone-sensitive adenylate cyclase. A. Rev. Pharmac. Toxic. 27, 371-384.

Clark R. B. (1986) Desensitization of hormonal stimuli coupled to regulation of cyclic AMP levels. In Advances in Cyclic Nucleotide and Protein Phosphorylation Research (Edited by Greengard P. and Robison G. A.), Vol. 20, pp. 151-209. Raven Press, New York.

Dohlman H. G., Caron M. G. and Lefkowitz R. J. (1987) A family of receptors coupled to guanine nucleotide regulatory proteins. Biochemistry 26, 2657-2664.

Dulley J. R. and Grieve P. A. (1975) A simple technique for eliminating interference by detergents in the Lowry method of protein determination. Analyt. Biochem. 64, 136-141.

Fernandez-Sousa J. M., Municio A. M. and Ribera A. (1971) Biochemistry of the development of the insect C. capitata. Changes in the positional distribution of fatty acids in diacylethanolamine phosphoglycerides. Biochim.

biophys. Acta 231, 527-534. Garcia J. L. and Haro A. (1981) Activation of adenylat¢

cyclase activity in C. capitata brain membranes by cations and anions. Comp. Biochem. Physiol. 68C, 109-113.

Garcia J. L., Haro A. and Municio A. M. (1981) Regulation of adenylate cyclase from brain membranes of the insect C. capitata. Comp. Biochem. Physiol. 70B, 57-62.

Garcia J. L., Haro A. and Municio A. M. (1982) Thermal stability of the membrane-bound adenylate cyclas¢ from C. capitata brain. Comp. Biochem. Physiol. 71B, 19-22.

Guillen A., Haro A. and Municio A. M. (1984) Regulatory mechanisms of fatty acid isomers on adenylate cyclase activity from C. capitata brain. Molec. cell. Biochem. 65, 83-88.

Guillen A., Haro A. and Municio A. M. (1987) Regulation of forskolin of octopamine-stimulated adenylate cyclase from brain of the dipterous C. capitata. Archs Biochem. Biophys. 254, 234-240.

Guillen A., Haro A. and Municio A. M. (1989) A possible new class of octopamine receptors coupled to adenylate cyclase in the brain of the dipterous C. capitata. Pharma- cological characterization and regulation of 3H-octo- pamine binding. Life Sci. 45, 655-662.

Hebdon G. M., Le Vine H., Sahyoun N. E., Schmitges C. J. and Cuatrecasas P. (1981) Specific phospholipids are required to reconstitute adenylate cyclase solubilized from rat brain. Proc. natn. Acad. Sci. U.S.A. 78, 120-123.

Hebdon G. M., Le Vine H., Minard R. B., Sahyoun N. E., Schmitges C. J. and Cuatrecasas P. (1979) Incorporation of rat brain adenylate cyclas¢ into artificial phospholipid vesicles. J. biol. Chem. 254, 10459-10465.

Helenius A. and Simons K. (1975) Solubilization of mem- branes by detergents. Biochim. biophys. Acta 415, 29-79.

Holloway P. W. (1973) A simple procedure for removal of Triton X-100 from protein samples. Analyt. Biochem. 53, 304-308.

Houslay M. D. (1981) Mobile receptor and collision cou- pling mechanisms for the activation of adenylate cyclase by glucagon. In Advances in Cyclic Nucleotide Research (Edited by Greengard P. and Robison G. A.), Vol. 14, pp. 111-119. Raven Press, New York.

Megias A., Saborido A. and Municio A. M. (1986) Solubi- lization of microsomal membrane proteins from C. capi- tara. A comparative study of non-hydrolytic methods. Comp. Biochem. Physiol. g3B, 287-293.

Neer E. J. (1976) The size of adenylate cyclase and guanylate cyclase from the rat renal medulla. J. supramolec. Struct. 4, 51-61.

Pfeuffer E., Mollner S. and Pfeuffer T. (1985) Adenylate cyclase from bovine brain cortex: purification and charac- terization of the catalytic unit. EMBO J. 4, 3675-3679.

Ross E. M. and Gilman A. G. (1980) Biochemical properties of hormone sensitive adenylate cyclase. A. Rer. Biochem. 49, 553-564.

Rouser G., Siakotos A. N. and Fleischer S. (1966) Analysis of phospholipids by thin layer chromatography and anal- ysis of spots. Lipids 1, 8546.

Shinitzky M. and Barenholz Y. (1978) Fluidity parameters of lipid regions determined by fluorescence polarization. Biochim. biophys. Acta 515, 367-394.