Biochimica et BiophysicaActa, 1091 (1991) 213-221 © 1991 ElsevierScience Publishers B.V. (BiomedicalDivision)0167-4889/91/$03.50 ADONIS 0167488991000790

213

BBAMCR 12844

Apparent activation of rabbit lung membrane adenylate cyclase by cytosolic proteins possessing adenylate kinase activity Eugenia V. Nupenko, Michail P. Panchenko, Marina G. Starikova, Alexander V. Grishin and Vsevolod A. Tkachuk Laboratory of MolecularEndocrinology, CardiologyResearch Center of the U.S.S.R,, Moscow(U.S.S.R.)

(Received 27 April 1990) (Revised manuscriptreceived25 July 1990)

Key words: Adenylatecyclase: Adenylatekinase: Lung cytosolic activator Lung cytosolic fraction (235 000 × g su0ernatant) activates cAMP synthesis by lung membrane adenylate cyclase (AC). 23 kDa and 29 kDa proteins were isolated from rabbit lung cytosolic fraction in a homogeneous state, as 'activators' of lung membrane AC. Both of these proteins possess high adenylate kinase (AK) activity and are able to mimic the 'activating' effect of lung cytosol on the lung membrane AC in the standard incubation mixture devoid of adenylate kinase. The activating effect is abolished in the presence of adenylate kinase inhibitor DAPP and after heat- or trypsin-treatment of the cytosolic fraction. Commercial adenylate kinase or nonionic detergent Lubrol PX activate cAMP synthesis by lung membrane AC in a similar manner to that of cytosolic fraction, in the presence of commercial adenylate kinase or Lubrol PX no activating effect of the cytosolic fraction on lung membrane AC is revealed. The ability of cytosolic fraction, commercial adenylate kinase, Lubrol PX or purified 23 kDa and 29 kDa proteins to activate cAMP synthesis by lung mtmbrane AC correlates with their ability to support the constant ATP (AC substrate) concentration in the AC assay mixture. Our data indicate that 'activation' of lung membrane AC in the presence of cytosolic fraction may be produced by cytosolic adenylate kinase activity which regenerates ATP from AMP in the presence of creatine kinase and creatine phosphate providing the substrate for cAMP synthesis by AC.

Introduction Hormone-sensitive adenylate cyclase (AC) is a membrane-bound oligomeric protein complex consisting of at least the catalytic component, the stimulatory and the inhibitory guanyl nucleotide-binding regulatory proteins (designated as Gs and Gi) and the stimulatory and the inhibitory hormonal receptors (see Refs. 1-6 for a review).

Abbreviations: AC, adenylate cyclase (ATPopyrophosphatelyase, cycling; EC 4.6.1.1); AK, adenylate kinase (ATP:AMP phosphotransferase; EC 2.7.4.3); DAPP, pl,p5 di(adenosine.5,)penta. phosphate; DTT, dithiothreitol; GTP~,S, guanosine-5'-O-(3thiotriphosphate); Gpp[NH]p, guanosine 5'-(/Ly-imido)triphosphate; GDPflS, guanosine-5',.O-(2-thiodiphosphate);PEl-cellulose, polyethylenemine cellulose; PMSF, phenylmethylsulfonyl fluoride; SDSPAGE, sodium dodecyl sulfate polyacrylamidegel electrophoresis. Correspondence: V.A Tkachuk, Laboratoryof MolecularEndocrinology, Insti~a~t~of ExpermentalCardi"flogy,CardiologyResearchCenter of the U.S.S.R., Acaf:!emyof Medical Sciences, 3rd Cherepkovskaya Str. 15A, Moscow 12~552, U.S.S.R.

Several cytosolic protein factors have been described which modulate membrane-bound AC activity [7-19]. Some of these modulators (for instance, calmodulin [20-26]) have been identified already; however, the identity and mode of action of other cytosolic AC modulators remain unclear. Lung membrane AC activity is strongly dependent on the presence of lung cytosolic fraction [27-31]. Lung cytosolic factor(sL which activate AC have been separated into two components by gel filtration. The low-molecular-weight component was identified as GTP, but the second component (presumably protein) was not identified [27-31]. The aim of the present work was to purify the protein component o f the cvtosolic 'activator' of lung membrane AC and to establish the mechanism of its stimulatory action on lung membrane AC. Materials and Methods Isolation of plasma membrane and cytosolic fractions from rabbit lung Male rabbits weighing 2.5-3.0 kg were killed by decapitation and lungs were rapidly removed (all the

214 subsequent manipulations were carried out at 2-4 ° C). The lungs were dissected free of bronchi and vasculature, rinsed with chilled 0.9~ NaCI, placed in 5 vol. (w/v) of buffer A (20 mM Tris-HCl (pH 8.0 at 4 o C), 1 mM EDTA and 0.1 mM PMSF) and homogenized with a Polytron homogenizer for 1 n-fin at high speed. The homogenate was filtered through two layers of gauze and combined with 5 vol. of buffer A. The suspension was centrifuged at 235 000 × g for 90 rain in a type 45Ti rotor (Beckman). The clear cytosolic fraction was carefully collected with a syringe, divided into small aliquots (100-500 #1), and stored in liquid nitrogen or used for isolation of soluble 'activator' of membrane-bound lung AC. The membrane pellet was suspended in 5-7 vol. of buffer B (20 mM Tris-HCl (pH 8.0 at 4°C), 1 mM EDTA, 250 mM sucrose) to a final membrane prtoein concentration 12-15 mg/ml, layerd on a 0.8 M sucrose solution in 20 mM Tris-HC! (pH 8.0 at 4°C), 1 mM EDTA (15 ml membrane suspension to 20 ml of 0.8 M sucrose solution) and ultracentrifuged at 132 000 × g for 90 rain in a type SW 27 bucket rotor (Beckman). The plasma membrane fraction, floating between 0.25 and 0.8 M sucrose solutions was collected with a syringe, diluted 3-fold with buffer A without PMSF and centrifuged at 132000 × g for 60 min. The membrane pellet was suspended in buffer A without PMSF to a final membrane protein concentration of 3-4 mg/ml, divided into small aliquots (50-100 ~1), and stored in liquid nitrogen. The cytosolic and plasma membrane fractions from rabbit heart, brain and liver were isolated in the same manner.

Purification of lung membrane A C activator(s) from lung cytosolic frarlio~ Lung 235000 x g cytosolic fraction (600-700 ml) with a protein concentration 12-14 mg/ml was applied to a 500 ml DEAE trisacryl M (LKB) column equilibrated with buffer C (20 mM Tris-HCl (pH 8.0 at 4°C), 1 mM EDTA and 2 mM fl-mercaptoethanol). The column was washed with buffer C and protein was eluted using a linear 8 h ionic strength gradient (0-400 mM NaCI) ('Ultrograd' LKB). The elution was performed at 400 m l / h and 50 ml fractions were collected. All fractions eluted from the column were assayed for AC activator activity. Active fractions were pooled and applied to a 500 ml CM-Sepharose CL-6B (Pharmacia Fine Chemicals) column equilibrated with buffer C. The column was washed with buffer C and protein was eluted with a linear 16 h gradient of NaCI (0-200 raM) at 200 ml/h. Fractions (30 nil) were collected and assayed for AC activator activity. Fractions containing the maximum AC activator activity were pooled, concentrated by ultra-filtration through a PM 10 filter (AMICON) to a final volume of 20 ml and applied to a 500 ml Ultro6el AcA 44 (LKB) column equilibrated from buffer C containing 200 mM NaCI. Elution was

perfo~'med at 30 ml/h. Fractions (15 ml) were collected and assayed for AC activator activity. To calibrate the ,lumn for molecular mass determination, purified proteins from Dalton standards MS-11 kit (Serva): rabbit muscle aldolase (160 kDa), bovine serum albumin (67 kDa), egg albumin (45 kDa), chymotrypsinogen (25 kDa), horse myoglobin (17.8 kDa) and DNP-L-alanine (255.5 Da) were chromatographed and their elution volumes were determined. Active fractions were combined, adjusted with 4 M NaCI to a final salt concentration of 400 mM and applied to a 50 ml phenyl-Sepharose CL-4B (Pharmacia) column equilibrated with buffer C containing 400 mM NaCI. The colu.mn was washed with the same buffer and protein was eluted by an 8 h linear reversal NaCI gradient (400-0 mM) at 50 ml/h. The peaks of AC activator activity were concentrated by ultra-filtration through AMICON PM 10 filter to a final protein concentration of 0.5-1.0 mg/ml, dialyzed against buffer C, divided into small aliquots (100-200 #1), and stored in liquid nitrogen.

Determination of A C activity Adenylate cyclase activity was determined in an incubation mixture (final volume 50-60/~1) which cont~.ined 50 mM Tris-HCl (pH "7.5, 37 ° C), 1 mM cAMP, 20 mM creatine phosphate, 0.5 mg/ml creatine kinase, 5 mM MgCI 2, 1 mM EGTA, 1 mM DTT, 0.1-0.2 mM ATP, 1.0-1.5 pCi [a-32p]ATP, 7-20 pg of membrane protein. All assays were performed in triplicate. When isoproterenol, GTP, GTP~,S, GDPflS or forskolin were used these effectors were added directly to assay tubes to 100 pM final concentration. Tubes were equilibrated at 37 °C for 1 min prior to starting the reaction by the addition of ATP plus [a-32P]ATP. The incubation time was 15 min. The reaction was terminated by addition of 200/~l 0.5 M HCI. The samples were boiled in water bath for 7 rain and neutralized with 1.5 M imidazole (200/~l/tube). cAMP was purified by chromatography on alumina columns as described by White [26]. The recovery of the cAMP was not less than 807o as determined using [3H]cAMP. When kinetic experiments were performed the final volume of incubation mixture was 0.8 to 1.0 ml. The formation of cAMP was initiated by the addition of ATP plus [a-32p]ATP mixture. At distinct times the reaction was stopped by transferring 50-60/~1 aliquots in duplicate into 200/~1 0.5 M HCI solution.

Nucleotide analysis To monitor ATP and its metabolites, aliquots (1 #1) were withdrawn from the AC reaction mixture before the reaction was stopped and spotted onto PEI-cellulose thin-layer chromatography plates (Sigma). The mobile phase was 1.0 M LiCI. Spots containing radioactive ATP metabolites were visualized by autoradiography.

215

Determination of adenylate kinase activity The adenylate kinase activity was measured at 340 nm by continually measuring a conversion of NADH to NAD at 25 °C in the reaction mixture (final volume 2 ml) containing (final concentrations): 50 mM Tris-HCl (pH 7.5), 50 mM KCI, 8 mM MgC12, 100 #M EDTA, 3 mM ATP, 1 mM AMP, 5 mM potassium phosphoenolpyruvate, 0.16 mM NADH, pyruvate kinase (3.3 units per ml) and lactate dehydrogenase (3.3 units per ml). Before the reaction was started by the addition of commercially available adenylate kinase or purified cytosolic AC 'activators', the baseline for contaminating adenylate kinase activity which is present in commercially obtained pyruvate kinase and lactate dehydrogenase was recorded. Protein assays were performed by the method of Peterson [32] using bovine serum albumin as a standard. SDS-PAGE of proteins was performed in 12% gel [331. Materials [ ~. 32PIATP (10-50 Ci/mmol), [2,8- 3HlcAMP (30-50 Ci/mmol) were purchased from Amersham (U.K.); Tris, EDTA, EGTA, sucrose, fl-mercaptoethanol, (+_)-dithiotrheitol (DTT), neutral alumina, for column chromatography, Lubrol PX, imidazole, bovine serum albumin, ATP (prepared by phosphorylation of adenosine), diadenosine pentaphospha'te, cAMP, AMP, phosphoeno/pyruvate, NADH, phosphocreatine, di-Tris salt, isoproterenol, adenylate kinase (myokinase), ( - )-lactate dehydrogenase, pyruvate kinase, phenylmethylsulfonyl fluoride (PMSF), purchased from Sigma; forskolin was from Calbiochem (U.S.A.); creatine kinase, GTP, GDPflS, GTP),S from Boehringer-Mannheim (F.R.G.). All chemicals used for SDS-PAGE were electrophoresis grade. All other reagents were of highest purity available. Results and Discussion

The specific activity of AC in lung membranes is very low but the addition of lung cytosolic fraction to the incubation mixture markedly increases the enzyme activity (Table I). The 10-fold activating effect of cytosolic fraction on AC activity occurred both in the absence and in the presence of AC activators - isoproterenol, GTPTS, forskolin, GTPTS+isoproterenol or GTPTS + forskolin. GDPflS had no effect on activation of AC by cytosolic fraction (Table I). The activating effect of cytosolic fraction on lung membrane AC activity was abolished from 53.40 + 3.10 to 5.41 5:0.25 pmol cAMP/rain per mg protein after cytosolic fraction heat treatment and to 5.92 + 0.34 pmol cAMP/rain per mg protein after trypsin digestion. These data suggest that a protein(s) is involved in the activating effect of cvtosolic fraction on AC activity and this seems to be consistent

TABLE I

Effect of lung cytosolic fraction on lung membrane adenylate cyclase activity Concentration of ATP in the incubation medium (final volume 50 pl) was 0.1 mM. Assays contained 14 #g of lung membrane protein and 112 #g of lung cytosolic protein, as indicated. AC activity of cytosolic fraction was not significant. Data in all tables are mean+_S.E, of triplicate determinations. Addition

pmol cAMP/min per mg protein without cytosolic fraction

None Isoproterenoi (100 pM) GDPflS (100/~M) GDPflS (100 #M) + isoproterenol (100 #M) GTPTS (lO0/aM) GTP-yS (100 pM) + isoproterenol (100pM) Forskolin (100 #M) GDPBS (100 #M) + forskolin (100 pM) GTPTS (100/~M) +forsko!in(lOOpM)

with cytosolic fraction

0.425 + 0.01

3.92 + 0.12

0.638 +00.02 0.301 _+0.08

5.93 ±0.24 3 13 ±0.25

0.311 +0.06 2.72 _+0.18

3.10±0.33 28.7 ±0.10

5.44 ±0.14 3.67 +00.71

58.1 +0.21 37.5 ±0.32

2.60 +0.14

26.9 ± 1.70

6.51 +0.24

66.2 ±2.43

with the earlier reported phenomenon of the cytosolic protein-dependent activation of lung membrane AC from mammalian lungs [27-31,34]. Romano and Molinoff [34] showed that the effect of the cytosolic fraction on the lung AC can be 'mimicked by commercially available adenylate kinase'. These authors demonstrated that in the standard AC assay mixture (containing the creatine kinase plus creatine phosphate as ADP ~ ATP regenerating system, but devoid of adenylate kinase) the rapid A T P - , AMP degradation occurs in the presence of lung membranes. The addition of the adenylate kinase to the standard incubation mixture induced the rapid AMP---, ATP resynthesis and supported a constant rate of cAMP synthesis by lung membrane AC for long periods of incubation. In the presence of exogenous adenylate kinase the stimulating effect of cytosolic fraction on lung membrane AC was not revealed [34]. On the other hand, as reported by Ofulue and Nijjar [29] and Whitsettet al. [30], the constant rate of cAMP synthesis by lung membrane AC was achieved in the AC assay mixture devoid of adenylate kinase. Moreover, the addition of adenylate ldnase in the incubation mixture did not increase the AC activity, whereas the addition of lung cytosolic fraction induced the pronounced activation of AC [30]. Conflicting experimental data and discrepancies [29,30,34] concerning the possible mechanism of the stimulatory effect of the heat-labile, trypsin-sensitive

216

AMP

ATP

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TABLE III

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Effect of diadenosine pentaphosphate (DAPP) and Lt,,brol PX on the lung cytosol-dependent activation of lung membrane adenylate cyclase Concentrations of ATE GTPTS and forskolin in the incubation mixture (final volume 50 ~l) were 0.1 raM, Assays contained 14 ~g of lung membrane protein and 112 /xg of lung cytosolic protein as indicated.

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1 2 3 4 5 6 7 8 9 Fig, I. Thin-layer chromatographic analysis of AI"P and its metabolites present in reaction mixtures containing lung membrane AC preparation, Lane l: original reaction mixture: lanes 2-9: incubation mixture after 15 min of incubation at 37°C with 2, 14 #g of lung membrane protein: 3, 14 ~tg of lung membrane protei, ~,:s 112 ~g of lung cytosoli¢ protein; 4, 14 ttg of lung membrane protein plus 2.5 ~tg of exogenous adenylate kinase: 5, 14 ~g of lung membrane protein plus 112 ~g of heart cytosolic protein; 6, 14 ~tg of lung membrnae protein plus t12 ~tg of brain cytosolic protein; 7, 14 ~g of lung membrane protein plus 112/~g of liver cytosolic protein: 8, 14 Itg of lung membrane protein plus 0.1% Lubrol PX; 9, 14 ~g of lung membrane protein plus 112 /tg of lung cytosolic protein plus 0.1% Lubroi PX, Original incubation mixture (final volume 50 ~l) contained 0,1 mM ATP, 100/tM GTPTS and 100 ~tM forskolin. 1 /~l aliquots were removed at the indicated time and analysed by thin-layer chromatography as described in Materials and Methods.

Adenylate cyclase activity (pmol cAMP/rain per mg protein)

Without cytosolic protein With cytosolic protein

without additions

with DAPP with Lubrol (50 ~m) PX (0.1%)

5.37 + 0.23 52.9 + 1.52

3.30 + 0.08 3.84 ± 0.21

48.9 + 1.21 50.1"±3.60

--* AMP-degradation was reduced to less than 5% of original (Fig. 1). The ability of cytosolic fractions to support the constant ATP concentration in AC assay mixture correlated with their ability to 'activate' lung membrane AC (Table It). The addition of adenylate kinase inhibitor [35] diadenosine pentaphosphate to the AC assay mixture induced the complete abolition of the activating effect of cytosolic fraction on the lung membrane AC (Table Ill). Diadenosine pentaphosphate was able only to in450

factor from lung cytosolic fraction on the lung membrane AC led us to reevaluate the assay conditions employed for the measurement of AC activity in lung membranes. The thin-layer chromatographic analysis of the radioactive products formed from [o-nP]ATP in the course of AC reaction showed the complete degradation of ATP into AMP (Fig. 1). When the lung cytosolic fraction or cytosolic fractions from heart, brain and liver were included in the assay, the membrane-induced ATP

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TABLE ii

Effect of cytosolic protein from different sources on A C activity of lung membranes Concentrations of ATP, GTP¥S and forskolin in the incubation mixture (final volume 50 td) were 0.1 mM. Assays contained 14 ~tg of lung membrane protein and 112 ~g of cytosolic protein from different sources where indicated. AC activity in cytosol fractions was not significant.

Q.

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Assay condition

Adenylase cyclase activity (pmol cAMP/min per mg protein)

Membranes Membranes + lung cytosofic fraction Membranes + brain cytosolic fraction Membranes + liver cytosolic fraction Membranes + heart cytosofic fraction

6.21 + 0.2 58.2 ±2.4 77.8 ±4.1 99.9 ±8.6 123 ± 8.0

I 15

I 20

of incubation

I 25

I 30

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(rain)

Fig. 2. Time-course of cAMP formation by lung membrane AC in the absence (o - - o ) and in the presence of lung cytosolic fraction (A~A), exogenous adenylate kinase (Q O), lung cytosolic fraction plus exogenous adenylate kinase (E II). Incubation medium contained 0.2 mM ATP, 100 /~M GTPTS and 100 /~M forskolin. In each case incubation mixture (final volume 1 ml) contained 280 ltg of lung membrane protein and, where indicated, 2.24 mg of lung cytosolic protein and/or 50/Lg of exogenous adenylate kinase. Before starting of reaction by the addition of ATP+[a32p]ATP each incubation mixture was pretreated at 37 °C for 5 min.

217 hibit the ATP-regenerating activity of cytosolic fraction, but had no effect on the ATP-~ AMP-degradating activity of lung membranes (data not shown). The nonionic detergent Lubrol PX in concentration 0.1% increased 10-fold the cAMP synthesis by lung membrane AC and no cytosolic fraction-dependent activation of AC was observed in the presence of Lubrol PX (Table III). The activating effect of Lubrol PX on lung membrane AC correlated with its ability to prevent the lung membrane-induced degradation of ATP to AMP in the AC mixture (Fig. 1). 0.1% Chaps also partially prevented ATP ~ AMP degradating activity of lung membranes; however, the

effect of Chaps was less expressive than effect of Lubrol (data not shown). The kinetics of the cAMP synthesis by lung membrane AC in the standard assay mixture are shown in Fig. 2. The rate of cAMP accumulation is not linear and reaction stops after 10 rain of incubation. In the presence of lung cytosolic fraction or exogenous adenylate kinase, the reaction proceeds linearly for at least 30 min. Addition of cytosolic fraction to adenylate kinase did not increase the rate above that seen with adenylate kinase alone. Thus, the cytosolic fraction-dependent enhancement of cAMP synthesis by lung membrane AC may be adequately explained by the reversal or the

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Fig. 3. Chromatography of AC 'activator' from rabbit lung cytosolic fraction on DEAE-Trisacryl M. (A) -, eluate absorbance at 279 nm; o o, AC 'activator' activity; . . . . . . , NaCI concentration. The AC activator activity was assayed by adding 2 0 / d each fraction to the incubation medium (final volume 60/zl), containing 0.2 mM ATE 14 ~ag of lung membrane protein, 100 ~M GTPyS and 100 I~M forskolin. (B) Thin-layer chromatographic analysis of ATP and its metabolites present in lung membrane AC reaction mixtures in the absence and in the presence of DEAE-Trisacryl fractions. 1/zl from each AC reaction mixture (Fig. 3A) was spotted onto PEl-cellulose thin-layer chromatography plates and analysed as described in Materials and Methods.

218 prevention of membrane-induced ATP--* AMP degradation, rather than direct specific activation of AC itself. The ability of exogeneous adenylate kinase to imitate the 'stimulatory' effect of cytosolic fraction and the potency of adenylate kinase inhibitor diadenosine pentaphosphate to block this 'activation', supports the AMP-~ATP regeneration 'mechanism' of cytosolic fraction effect. Similarly, the inhibitory effect of Lubrol PX on lung membrane ATP -* AMP degradation activity and its 'activating' effect on AC activity allow to s,ggest the ATP preservation 'mechanism' of cytosolic fraction effect. To investigate the possibility that cytosol contained adenylate kinase responsible for the apparent activation of AC, we undertook purification of the AC'activator(s)' from lung cytosolic fraction, The activity of 'activator' during purification was tested by its ability to 'activate' lung membrane AC in the standard incubation mixture (devoid of exogenous adenylate kinase) and to preserve the ATP depletion in AC assay mixture. Since basal AC specific activity in lung membranes is very low under these conditions (Table I), the AC activators GTPyS + forskolin were added. As follows from data presented in Table I, neither GTPyS nor forskolin had any masking effect on cytosolic fraction-dependent 'activation' of lung membrane AC. The chromatography of lung cytosolic protein on DEAE-Trisacryl resulted in the elution of AC 'activator' activity and its effective separation from the acidic cytosolic proteins tightly bound to the matrix (Fig. 3A). 'Activator' containing fractions were able to maintain the constant level of ATP in the AC assay mixture (Fig. 3B). The DEAE fractions, containing maximum 'activator' activity were chromatographed on CM-Sepharose. The elution of 'activator' activity was performed at

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Fig. 5. Chromatography of pooled AC 'activator' fraction from CMSepharose CL-6B on UItrogel AcA44. - - , eluate absorbance at 279 nm; o .. O. AC 'activator' activity. The AC 'activator' activity in eluate fractions was assayed as described in the legend to Fig. 3A.

NaC! concentration 40-60 mM (Fig. 4). The peak of AC 'activator' from CM-Sepharose was applied on the Ultrogel AcA44 column (Fig. 5). The 'activator' activity was eluted in an asymmetrical peak in the volume corresponding to the elution volume of 25 kDa chymotrypsinogen A. The Ultrogel fractions were applied on the Phenyl-Sepharose column (Fig. 6). About 50% of the total AC 'activator' activity (peak 1) passed through the matrix, whereas another part of 'activator' activity was eluted from the column by the reversal linear NaCI gradient at salt concentration 300-250 mM (peak 2). According to SDS-PAGE analysis, AC 'activators' in the 1 and 2 peaks were presented by the homogeneous proteins with the molecular masses 23 kDa and 29 kDa, respectively (Fig. 7).

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Fig. 4. Chromatography of DEAE-Trisacryl M A C 'activator' fraction on CM-Sepharose CL-6B. ~ , eluate absorb=ace at 279 nm; o O, AC 'activator' activity; . . . . . . , NaCI concentration. The AC "activator' activity in eluate fractions was measured as described in the legend to Fig. 3A.

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Fig. 6. Chromatography of pooled AC 'activator' fraction from Ultrogel AcA44 on Phenyl-Sepharose CL-4B. ~ , eluate optical density at 2"/9 rim; o . o AC 'activator' activity; . . . . . . , NaCl concentration. The AC activator activity in eluate fractions was assayed as described in the legend to Fig. 3A.

219 start - - , ,

membranes without AK (9

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Apparent activation of rabbit lung membrane adenylate cyclase by cytosolic proteins possessing adenylate kinase activity.

Lung cytosolic fraction (23500 x g supernatant) activates cAMP synthesis by lung membrane adenylate cyclase (AC). 23 kDa and 29 kDa proteins were isol...
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