Dec 5, 2015 - pertussis was purified either as a free enzyme or as a complex with ..... ate cyclase antibodies precipitated 99% of the active enzyme since less ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 by The American Society of Biological Chemists,
Val. 261, No. 34, Issue of December 5,pp. 16264-16269,1986 Printed in U.S.A.
Inc.
Bordetella pertussis Adenylate Cyclase PURIFICATION,CHARACTERIZATION,
ANDRADIOIMMUNOASSAY* (Received for publication, June 30, 1986)
Daniel Ladant$, Colette Brezins, Jean-Michel Alonsos, Isabelle CrenonS, and Nicole Guiso$ From the $Unite de Biochimie desRegulations Cellulaires and the $Unite d’Ecolagie Bacterienne, Institut Pasteur, 28 rue du Docteur Roun, 75724 Paris Ceden 15,France
The extracellular adenylate cyclase of Bordetella pertussis was purified either as a free enzyme or as a complex with calmodulin. The purified enzyme has a specific activityof 1600 pmol of cAMP min“.mg“ and exists under two molecular forms of 45 and 43 kDa which are apparently structurally related. Calmodulin increased considerably the resistance of adenylate cyclase to inactivation by trypsin. Although trypsin cleaved the adenylate cyclase-calmodulin complex, the digested fragments remained associated by noncovalent interactions in an active conformation. Specific mouse anti-adenylate cyclase antibodies inhibit adenylatecyclaseactivity and were used to develop a specific radioimmunoassay that allows detection of as little as 5 ng of adenylate cyclase in culture supernatants.
(1600 kmol of cAMP min” .mg-’). Antibodies raised against the purified adenylate cyclase inhibit enzymatic activity and allow detection of less than 5 ng of protein in culture supernatants using a radioimmunoassay (RIA)’procedure. MATERIALS ANDMETHODS
Chemicals Cyclodextrin was a kind gift of Dr. Suzuki (Teijin Institute of Biochemical Research, Tokyo, Japan). ATP, CAMP, bovine brain calmodulin, TPCK-trypsin, andsoybean trypsin inhibitor were from Sigma. DEAE-Sephacel was a product of Pharmacia(Uppsala). Hydroxylapatite, Affi-Gel/calmodulin, and molecular weight standards for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were from Bio-Rad. Urea (fluorimetrically pure) was a product of Schwarz/Mann. Rabbit anti-mouse immunoglobulin antibodies were a kind gift of Dr. Grassi (Section de Pharmacologie et d’Immunologie, CEN, Saclay, France). [w3‘P]ATP (3000 Ci/mmol), [3H]cAMP (40 Ci/mmol), and NaIZ5I(1000 Ci/mmol) were obtained from the Radiochemical Centre (Amersham, United Kingdom). [3H] Dinitrofluorobenzene (20.4 Ci/mmol) wasfrom New England Nuclear. X-Omat AR films were from Kodak.
Bordetella pertussis, the bacterium responsible for whooping cough, releases several proteins into culture media which Growth of Bacteria and Concentration of Adenylate Cyclasefrom playa role in the virulence of thisorganism,one of the Culture Supernatant proteins is an adenylatecyclase (1-3). This adenylate cyclase exhibits unusual properties, such as heat stability and calB. pertussis, 18323, PhaseI(type strain ATCC9797) andan modulin-dependent activation (4). Since its first identifica- avirulent, Phase IV, spontaneous stable variant of this strain, were tion by Hewlett and Wolff (2) in culture supernatants of B. grown for 48 h a t 36 “C on modified Stainer-Scholte agar medium pertussis, several studies concerning the biological effects of supplemented with cyclodextrin (11) and transferred to modified Stainer-Scholte liquid medium without cyclodextrin. The liquid culthis adenylatecyclase on mammaliancells have been reported. tures, in a l-liter Erlenmeyer flask containing 250mlof medium, Shattuck and Storm (5), using partially purified and highly were shaken at 150 rpm for 24 h a t 36 “C andgrown until theyreached active adenylatecyclase preparations, showed that theenzyme an A,,, = 1.2 k 0.2. Bacteria were then removed by centrifugation at is internalized by eukaryotic cells resulting in an increase in 5,000 X g for 25 min. The culture supernatants were either stored at -30 “C until use, or concentrated directly. For this, 3 litersof culture their adenosine 3‘-5‘-monophosphate (CAMP) content, thus containing about 10 pg ofprotein and 0.1 unit of enzyme/ confirmingearlierresults of Confer and Eaton (6, 7) and supernatant ml were filtered through Millipore HAWP 0.45-pm filters (500 ml/ Hanski and Farfel (8) obtained with crude preparations of filter). More than 90% of the enzyme activity was retained on the bacterial adenylate cyclase. Moreover, Weiss e t al. (9) showed filters. About 80% of this activity can be recovered by incubating the that B. pertussis mutants lacking the extracellular adenylate filters in a total of 40 ml of 50 mM Tris’HCl (pH8) containing 6mM cyclase are avirulent,suggesting that this enzyme might have MgC1, and 0.1% Triton X-100 (buffer A). Insoluble material was asignificantrole inpathogenesis. However,very little is removed by 30-min centrifugation a t 15,000 X g and 4 “C. The specific of this “concentratedculture supernatant was 115 units/mg of this enzyme. Several activity known about the structural properties of protein as shown in Table I. studiespointedtowardthe molecular heterogeneity of B. pertussis adenylate cyclase (8, lo), the highest “toxicity” being Purification of ExtracytoplasmicAdenylate Cyclase attributed to thehigh molecular weight species (8). Procedure 1”Forty ml of concentrated culture supernatant were In this paperwe describe the calmodulin-binding properties added to 0.8-1 ml of Affi-Gel/calmodulin and themixture was gently of the extracellular adenylatecyclase and the productionof a shaken at 4 “C for 18 h. More than two-thirds of the enzyme activity homogeneous enzymepreparation witha high specificactivity was retained on the gel. The Affi-Gel/calmodulin was sedimented by centrifugation at 300 X g for 1 min then washed several times with *This workwas supported by Grant UA 1129 from the Centre 0.5 M NaCl in buffer A. Adenylate cyclase was recovered from the gpl National de la Recherche Scientifique, Grant 831025 from the Institut with 2.5 ml of 8.8 M urea in buffer A. Urea was removed by filtration National de la SantQ etde la Recherche MQdicale,Grant 84-V-0812 on a Sephadex G-25 column equilibrated with buffer A. This enzyme from the Ministkre de la Recherche et de la Technologie, and the preparation, having a specific activity of 1100 units/mg of protein, Fondation pour la Recherche MBdicale. The costs of publication of The abbreviations used are: RIA, radioimmunoassay; SDSthis article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TPCK, L-1-tosylamido-2-phenylethylchloromethyl ketone. accordance with 18 U.S.C. Section 1734 solelyto indicate this fact.
~
.~
16264
Adenylate B. pertussis TABLE I Purification of B pertussis adenylate cyclase from 3 liters of culture Step mg
30 300 Culture supernatant 230 2 Concentrated supernatant 0.121100 134 Affi-Gel/calmodulin purification DEAE-Seuhacel uurification 371600 1100.07
onitslrng
10 115
76
100 75 40
was stored at -80 "C with no loss of activity for several weeks. Procedure 2-The concentrated culture supernatant was adjusted to pH 7.0 with 1.5 M imidazole (pH 6.5), 40 ml of this material were loaded onto a 5-ml DEAE-Sephacel column equilibrated with 25 mM imidazole (pH 7.0) (buffer B) a t a rate of 10 ml/h. Adenylate cyclase activity was recovered in the flow-through, to which calmodulin (0.2 p~ final concentration) was added. The mixture was loaded onto a 3-ml DEAE-Sephacel column equilibrated in buffer B. Under these conditions 95% of the adenylate cyclase activity were retained on the column. After washing with 0.1 M NaCl in 25 mM Tris (pH 7.3) (buffer C), the adenylate cyclase-calmodulin complex was eluted with 0.3 M NaCl in buffer C a t a rate of 3 ml/h. The specific activity of the adenylate cyclase was 1600units/mg of protein. The enzyme was stored at -80 "C with no loss of activity for several weeks. The yield of the different steps of the purification procedure are listed in Table I.
Cyclase
16265 RESULTS
Interaction of Extracellular AdenylateCyclase with Calmoddin-Adenylate cyclase obtained from culture supernatants of B. pertussis previously grown on solid Stainer-Scholte medium containing cyclodextrin, could be activated 20- to 50fold by calmodulin. The enzyme in crude culture supernatants was concentrated approximately 100-fold by adsorption/desorption on filter membranes. This procedure resultedin a 10-fold purification of the adenylatecyclase and the production of a homogeneous form that sedimented on sucrose density gradient with an apparent S value of 3.6 (see below). To investigate the interaction of adenylate cyclase with calmodulin, we incubated different amounts of enzyme with a constant amount of lZ5I-labeled calmodulinand the mixture was separated bysucrose density gradient centrifugation.The molar ratio of adenylate cyclase to calmodulin was empirically chosen to be 5:l (Fig. M), 1:l (Fig. lB), or 15 (Fig. lC), assuming for adenylate cyclase a specific activity of 1,600 units/mg of protein and a molecular weight of 45,000 (see below), and for bovine brain calmodulin a specific radioactivity of 20 Ci/mmol and a molecular weight of 18,000. When
I
Iodinations All iodinations were performed a t room temperature using the chloramine-T method (12). The iodinated calmodulin had a specific radioactivity of 20 Ci/mmol and was stable for 2 monthswhen stored a t -20 "C. Prior to iodination, the adenylate cyclase-calmodulin complex obtained after purification on DEAE-Sephacel (see above, "Procedure 2'7, was chromatographed on hydroxylapatite in order to remove the detergent. The '251-labeledadenylate cyclase preparation was stable for 2 months at -20 "C. Immunization Procedures Four-week-old female BALB/c mice (purchased from the Ferme Experimentale de Rennemoulin, Institut Pasteur) were immunized subcutaneously three times at weekly intervals with 10 pl of the AffiGel/calmodulin/adenylate cyclase preparation. Mice were boosted 1 month later, using an adenylate cyclase preparation obtained from DEAE-Sephacel, and sera were collected 3 days after the booster shot. Anti-adenylate cyclase antibodies were detected by using a RIA procedure. Radioimmunoassay All immunological reactions were performed in buffer A. The reaction mixture contained 100 pl of lZ5I-labeledadenylate cyclase (2 X lo4 cpm and 4 X units of enzyme activity), 100 p1 of diluted anti-adenylate cyclase mouse immune serum, and 100 p1 of unlabeled adenylate cyclase or concentrated culture supernatant. After 18 h of incubation at 4 "C, 100 pl of rabbitanti-mouse immunoglobulin antibodies and 100 p1 of 8% polyethylene glycol, diluted in 1% preimmune mouse serum, were added to each tube. After another incubation of 30 min at 4 "C, the tubes were centrifuged at 2000 X g for 30 min, the supernatants were discarded, and the radioactivity of the pellet measured in a y counter. Analytical Procedures Adenylate cyclase activity was measured using the procedure of White (13) as modified by Hanoune et al. (14). The reaction was performed in a medium containing 50 mM Tris. HCl (pH 8), 1 mM [n-"P]ATP (5 X lo5cpm/assay), 6 mMMgC12, bovine serum albumin (100 pg), 0.12 mM CaC12,and 0.1 pM calmodulin (when added). One unit of adenylate cyclase corresponds to 1 pmol of CAMP formed in 1 min a t 30 "C at pH 8. Proteins were measured either according to Bradford (15) or Schultz et al. (16). SDS-PAGE was performed as described by Laemmli (17). The gelswere either silver stained as described by Morrissey (18) or autoradiographed after exposure on X-Omat AR films at -70 " C .
20 10
-
Sample no top FIG. 1. Calmodulin-bindingproperties of adenylate cyclase analyzed on sucrose gradient. 0.5 ml of buffer A, containing 20 nM 'Z51-labeledcalmodulin (20 Ci/mmol) and three dilutions of concentrated culture supernatant (A, 5 units/ml; B , 1 unit/ml; C, 0.2 unit/ml), was incubated 1 h at 4 "C followedby5-20% sucrose gradient centrifugation during 16 h at 35,000 rpm in a SW41 rotor. Fractions of 0.5 ml were collected and analyzed for radioactivity and adenylate cyclase activity (measured in the presence of calmodulin and expressed inunits/ml). Standards in each experiment were: a, pyruvate kinase (s20,w= 10 S); b, lactate dehydrogenase (szo,Lu= 7 S); c , creatine kinase (sz0. = 5 S).
16266
Adenylate B. pertussis
adenylate cyclase was in excess over radiolabeled calmodulin (Fig. lA),80% of the radioactivity was shifted from a sedimentation coefficient of 2 S, corresponding to thatof calmodulin, to 3.9 S. Enzyme as measured by its activity, sedimented as a nonsymmetrical peak withan S value of 3.6, corresponding to that of free adenylate cyclase and a shoulder at 3.9 S. This suggests that the peak of radioactivity with an S value of 3.9 corresponds to the adenylate cyclase-calmodulin complex. When the adenylate cyclase to calmodulin ratio was 1:l (Fig. 1B) or 1:5 (Fig. IC), 60 and lo%, respectively, of the radioactivity was recovered in the3.9 S peak. The proportion of free to bound lZ5I-labeledcalmodulin in the threeconditions allowed estimation of an association constant with adenylate cyclase of 10 nM. This value is in close agreement with that calculated ( 5 nM) from the activation curve of adenylate cyclase activity by calmodulin (data not shown; Refs. 19 and 20). Purification of Free and Calmodulin-bound Adenylate Cyclase-We have used the high affinity of adenylate cyclase for calmodulin to purify this enzyme by affinity chromatography on Affi-Gel/calmodulin as described under "Materials and Methods." In theresulting enzyme preparation, four proteins (50, 45, 43, and 40 kDa) could be detected by SDS-PAGE as shown in Fig. 2 A , lane 3. The additional bands observed were due to artifacts of silver staining. The same four proteins could also be identified when radiolabeled calmodulin was used to probe protein blotsof concentrated supernatants after SDS-PAGE (data notshown). On the other hand, a simple and efficient purification of the enzyme-calmodulin complex was based on the fact that the binding of calmodulin to adenylate cyclase altersits interaction with ion exchangers. Free adenylate cyclase does not bind to DEAE-Sephacel whereas the complex does. As shown in Fig. 2B, such a purified preparation contained two polypeptides ( M , = 45,000 and 43,000, respectively) and calmodulin in excess. After iodination the two polypeptides and calmodulin were labeled (Fig. 2B, lane 6). It must be noted that iodination of the adenylate cyclase-calmodulin complex did not affect its enzymaticactivity while free adenylate cyclase was inactivated by this iodination procedure (data not shown). Enzymatic digestion of the two iodinated polypeptides, by Staphylococcus aureus V8 protease, was performed after their separation on a preparative SDS gel. As shown in Fig. 3, the two polypeptides have similar proteolytic digestion patterns which indicate that they are structurally related. Sensitivity to Trypsin Digestion of Free and Calmodulinbound Adenylate Cyclase-The conformational differences between free and calmodulin-bound adenylate cyclases were studied by investigating their sensitivity to trypsindigestion. Incubation of free adenylate cyclase with trypsin a t 0 "C (at 1:l (w/w) ratio) resultedin complete inactivation of the enzyme within 2 min (Fig. 4A). Under the same conditions, the adenylate cyclase-calmodulin complex was much more resistant to proteolytic inactivation; 70% of enzyme activity remained after 30 min of incubation.SDS-PAGE of the digestion products of '251-labeledadenylate cyclase-calmodulin complex showed a rapid conversion of the 45-kDa polypeptide to the 43-kDa form, which was progressively cleaved to a 25-kDa peptide. This latter appeared to be much more resistant to further proteolysis. Free '251-labeledcalmodulin in excess over the '251-labeled enzyme-calmodulin complex was rapidly degraded by trypsin (Fig. 4B). These results raised the question as to the catalytic function of the 25-kDa peptide, since 70% of the original adenylate
Cyclase
A
-30K
c 2 0 K
I
1
2
3
4
B 94K" 67K-
ADENYLATE
TYCLAS~
43 K",
I 30K-
20K-9
1
2
3
4
5
6
FIG. 2. Purification of adenylate
cyclase. Purification was performed as described under "Materials and Methods" using either Affi-Gel/calmodulin ( A ) or DEAE-Sephacel ( B ) The samples were run on SDS-PAGE (10% acrylamide) and the gel was silver stained. A: lane 1, concentrated culture supernatant; lane 2 proteins unbound to Affi-Gel/calmodulin; lane 3, proteins eluted from Affi-Gel/calmodulin. B lane I, concentrated culture supernatant; lane 2, proteins unbound to thefirst DEAE-Sephacel; lane 3, proteins unbound to the second DEAE-Sephacel; lane 4, proteins eluted during washing of the second DEAE-Sephacel; lane 5, proteins eluted from the second DEAE-Sephacel column; lane 6, 9-labeled adenylate cyclase preparation purified on DEAE-Sephacel.
cyclase activity persisted even after both the43- and 45-kDa polypeptides disappeared. To answer this question, trypsin digestion products of the radiolabeled adenylate cyclase-calmodulin complex were separated by sedimentation on sucrose density gradient prior to analysis by SDS-PAGE (Fig. 5 ) . As expected, in the control experiment, without trypsin treatment, the radioactivity was found in two distinct peaks, corresponding to enzymatically active complex and to free calmodulin (Fig. 5A). After 30 min of incubationin the presence of trypsin, 55% of the original activity was recovered. Analysis of this trypsin-digested enzyme showed that it had the same apparent S value as the untreatedenzyme (Fig. 5B). However, analysis of fractions corresponding to the peak of activity on a SDS gel revealed only a 25-kDa polypeptide. It seems therefore, that although trypsincleaves peptide bond(s) of the adenylate cyclase molecule, the fragments still hold together by noncovalent interactions in the same active conformation asthe undigested adenylate cyclase-calmodulin
Adenylate B. pertussis
16267
Cyclase
A
of
min
digestion
B A
B
C
D
E
F
-43K-
111
FIG. 3. Peptide mapping by limited proteolysis of the labeled adenylate cyclase. The two labeled polypeptides of 45 and 43 kDa were cut from an SDS gel (7.5% acrylamide) which had been loaded with the lZ5I-labeledadenylate cyclase-calmodulin complex. The gel was autoradiographed to visualize the positions of the two polypeptides and thegel slices were applied to a second SDS gel (15% acrylamide) in the presence of the S. aurew protease as described by Cleveland et al. (29). A , C, and E, digestion pattern of 45 kDa and B , D, and F, digestion pattern of 43 kDa with 0.075, 0.75, and 7.5pg respectively, of protease.
complex. It may also be noted that analysis by SDS-PAGE of the peak corresponding to the adenylate cyclase-calmodulin complex after separation from free ’251-labeledcalmodulin in excess revealed that only adenylate cyclase was labeled and not calmodulin (Fig. 5A). Possibly, the tyrosine residues of calmodulin were inaccessible to iodination in the complex. Radioimmunoassay of Adenylate Cyclase-The adenylate cyclase-calmodulin complex was used to generate anti-enzyme antibodies in mice. As shown in Fig. 6 (lanes A, B , and C), the mouse antiserum precipitated specifically 1251-labeledenzyme and not ‘2sI-labeledcalmodulin. These results led us to develop a RIA for determining the concentration of adenylate cyclase in crude supernatants or bacterial extracts of B. pertussis. A typical dose-response curve shown in Fig. 6 was obtained with an anti-adenylate cyclase serum dilution of 1:2000. As little as 5 ng of adenylate cyclase could be detected using this RIA. The inhibition curve obtained with the concentrated supernatant paralleled the standard curve determined with purified enzyme. No adenylate cyclase could be detected inthe culture supernatant devoid of enzyme activity obtained from the avirulent Phase IV derivative of the strain. This result was interpreted by assuming that the Phase IV variant produces undetectable levels of adenylate cyclase and not an inactive protein. Inhibition of Adenylate Cyclase Activity by Anti-adenylate Cyclase Antiserum-Incubation of adenylate cyclase with 30-40% serum albumin or preimmune serum resulted in a increase in activity whereas incubation of the enzyme with the specific antiserum resulted in 90% inhibition of the activity (Table 11). The antiserum concentration was chosen in order to be in excess of antibodies. Furthermore, anti-adenylate cyclase antibodies precipitated 99% of the active enzyme since less than 1% of the initial activity remained in the supernatant.
0
2
4
&LE
8
min
12 20 30
of
digestion FIG. 4. Sensitivity to trypsin digestion of free and calmodulin-bound adenylate cyclase. A , 50 pl of adenylate cyclase purified on Affi-Gel/calmodulin (0)or adenylate cyclase-calmodulincomplex purified on DEAE-Sephacel(0) (1.5 units/ml, 1 pg/ml in buffer A) were incubated a t 0 “C with 50 pl of TPCK-trypsin (1 pg/ml in buffer A). At the indicated times, aliquots of 10 pl were diluted in 20 pl of soybean trypsin inhibitor (0.2 mg/ml in buffer A). Activity was measured in the presence of calmodulin as described under “Materials and Methods.” B, ’Z51-labeledadenylate cyclase-calmodulincomplex (1.5 units/ml) was treated under the same conditions; aliquots were either assayed for adenylate cyclase activity or run on SDS-PAGE (10% acrylamide). DISCUSSION
B. pertussis adenylate cyclase appears to be a unique bacterial enzyme since it has theability to enter eukaryotic cells and, subsequently, to induce an important increase of intracellular CAMPlevels (5,6, 8). Moreover it is highly activated by calmodulin, a protein not found in bacteria (4,20). Several reports emphasized the molecular heterogeneity of B. pertussis adenylate cyclase (8,lO). Kessin and Franke (10) suggested that hydrophobic interactions may be responsible, at least in part, for aggregating the enzyme in high molecular weight forms. Adsorption of adenylate cyclase on Millipore filters as well as the requirement of nonionic detergents for its “desorption” are inagreement with such an interpretation. Moreover, this “solubilization” convertsadenylate cyclase into a homogeneous form with an apparent S value of3.6. Since 80% of the adenylate cyclase activity was recovered by this procedure one could suppose that a single species of low molecular weight accounted for all the catalytic activity.
B. pertussis Adtmylate Cyclase
16268
I
”1 20
43 43 KK-
10
b
1
1
A
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Q
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.-
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10
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-
10
Sample
20
no
30
top
-.
1
10
lo00
100
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FIG. 6. Radioimmunoassay of adenylate cyclase. Bo, initial binding corresponding to 50% of lZ5I-labeledadenylate cyclase bound to the antiserum in the absence of unlabeled adenylate cyclase. B, percent of residual ‘2’I-labeled adenylate cyclase bound in the presence of different amounts of unlabeled adenylate cyclase (0)or concentratedculture supernatant of strain 18323 (H) or culture supernatant of the avirulent Phase IV variant (*). Inset, ’251-labeled adenylate cyclase preparation was incubated with preimmune serum or anti-adenylate cyclase mouse antiserum 18 h at 4 “C and the lZ5Ilabeled adenylate cyclase-antibody complexes precipitated as described under “Materials and Methods.” The pellets were solubilized in sample buffer and run on SDS-PAGE (10% acrylamide). Lane A , 1251-labeledadenylate cyclase preparation as control; lane B, immunoprecipitation with preimmune serum; lune C, immunoprecipitation with anti-adenylate cyclase mouse antiserum.
FIG. 5. Sucrose gradient analysis of tryptic digests of ‘“1TABLE I1 labeled adenylate cyclase-calmodulin complex. After 16 h of Inhibition of adenylate c y c h e activity by antibodies centrifugation at 52,000 rpm in a SW60 rotor at 4 “C on 5-20% Three hundred pl of adenylate cyclase (60 X units/ml), purified sucrose gradients, fractions of 120 pl were collected and adenylate cyclase activity (in thepresence of calmodulin) and radioactivity were on Affi-Gel/calmodulin, was incubated in the absence or presence of measured. Insets, autoradiography of SDS-PAGE (10% acrylamide) 250 pg of bovine serum albumin or 5 pl of serum. After 18 h a t 0 ‘C, of indicated fractions. A, untreated lZ5I-labeledadenylate cyclase- aliquots wereremoved and assayed for adenylate cyclase (in the units; 6.8 X lo5 cpm). B, lZ5I- presence of calmodulin). Antigen-antibody complexes were precipicalmodulin preparation (12 X labeled adenylate cyclase-calmodulin preparation digested by trypsin tated asdescribed under “Materialsand Methods” and theenzymatic activity assayed in the supernatant. for 30 min a t 0 “C (initial activity in the absence of trypsin: 2 x units, 1.15 X 10’ cpm; after 30 min of digestion: 1.1X loM3 units, 1.15 Adenylate cyclase activity X 10’ cpm). The results were expressed in percentage of initial Additions Before After activity. Standards were: a, creatine kinase (s20,ru= 5 S ) ; b, adenylate precipitation precipitation . ~S). kinase ( ~ ~ =0 2.4 unitsjrnl
The high affinity of the bacterial enzyme for calmodulin was confirmed once again in our experiments. Unlike the free enzyme the complex binds to DEAE-Sephacel, probably because of the acidic properties of calmodulin. This property of adenylate cyclase was used to purify it as a complex with calmodulin with an approximately 40% yield. The specific activity of the complex was 1600 rmol of CAMP min” .mg” of protein, the highest value ever reported for adenylate cyclase. Our purest preparation consists of two polypeptides (45 and 43 kDa) that are apparentlystructurally related. Apparent S values of free (3.6 S) and calmodulin-bound adenylate cyclase (3.9 S) suggest that the active enzyme is monomeric and that each polypeptide can bind 1 calmodulin molecule. The presence of these two polypeptides raise questions about the mechanism which governs their formation. We do not know as yet whetherthe 43-kDa polypeptide arises by proteolysis during concentration of culture supernatants or isa product of physiological processing during secretion of the enzyme. Preliminary results showed that adenylate cyclase extracted from the periplasmic space of the bacteria is active and hasa molecular mass of 45 kDa. The high sensitivity of B. pertussis adenylate cyclase to
Bovine serum albumin Preimmune serum 66 Anti-adenvlate cvclase serum
62 100 92 9
62 64.5 0.7
inactivation by trypsin was already known from experiments on crude enzyme preparations (3, 8). The “protective” effect exerted by calmodulin could be due to the maintenance of “nicked” adenylate cyclase through noncovalent interactions with calmodulin into anactive conformation which resembles the uncleaved protein. Although not an unusual mechanism of protection towards proteolytic enzymes, this effect of calmodulin on adenylate cyclase is quite different from what is known for other calmodulin-dependent enzymes such as phosphodiesterase, ATPases, or kinases (21-25). Calmodulin-sensitive enzymes are generally activated by limited proteolysis and converted into a lower molecular weight calmodulinindependent species. This mechanism certainly does not apply to B. pertussis adenylate cyclase. Antibodies elicited in mice against the 45- and 43-kDa polypeptides inhibit adenylate cyclase activity. These antibodies are probably active site directed. These specific anti-
B. pertussis AdenylateCyclase bodies were used to set up a RIA allowing the detection of low amounts of adenylate cyclase protein (5 ng). This assay be to study B'pertussis mutants lacking cyclase activity by determining whether they are deficient in the synthesis of the enzyme or produce antigenic but nonactive material. Likewise, the antibodies could be used to study potential cross-reactivity of B. pertussis adenylate cyclase with adenylate cyclases produced by other organisms or cells. Since B. pertussis adenylate cyclase has the original property, in common with the adenylate cyclase of another pathogen, Bacillus anthracis (26), and eukaryotic adenylate cyclases (21, 27, 28), to be activatable by calmodulin, it is tempting to speculate about its evolutionary origin, which might be either endogeneous and adapted to the host or eukaryotic. Acknowledgments-We thank Dr. Grassi for the gift of rabbit antimouse immunoglobulin antibodies and Dr. Y. Suzuki for providing cyclodextrin. We are grateful to Dr. A. Ullmann for her constant interest and fruitful criticisms. The helpful advice of Dr. 0. Biirzu, Dr. M. E. Goldberg, and Dr. M. VCron is deeply acknowledged. We thank R. Predeleanu and M. Rocancourt for expert technical assistance and M. Ferrand for typing the manuscript. REFERENCES 1. Hewlett, E. L., Weiss, A. L., Crane, J. K., Pearson, R. D., Anderson, H. J., Myers, G. A., Evans, W. S., Hantske, L. L., Kay, H. D., and Cronin, M. J. (1985) Deu. Biol. Standard 6 1 , 21-26 2. Hewlett, E. L., and Wolff, J. (1976) J. Bacteriol. 127, 890-898 3. Hewlett, E. L., Urban, M. A., Manclark, C. R., and Wolff, J. (1976) Proc. Natl. Acad. Sci. U. S. A. 7 3 , 1926-1930 4. Wolff, J., Cook, H., Goldhammer, A. R., and Berkowitz, S. A. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 3841-3844 5. Shattuck, R. L., and Storm, D. R. (1985) Biochemistry 24,63236328 6. Confer, D. L., and Eaton, J. W. (1982) Science 217,948-950
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