Adenyl Cyclase in Bordetella pertussis Vaccines E. L. Hewlett, C. R. Manclark,* and

J. \Volff

From the National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland

Adenyl cyclase- activity was detected in a merthiolate-preserved, whole-cell pertussis vaccine obtained from Eli Lilly and Company (Indianapolis, Ind.) [1]. This observation prompted the studies that are presented herein. The subsequent discovery that supernatant culture medium of exponentially growing Bordetella pertussis contained adenyl cyclase [2] suggested that it might be an exoenzyme, thus differing from previously studied bacterial adenyl cyclases, which are intracellular and either membrane-associated (Escherichia coli) or soluble in cell lysates (Brevibacterium liquefaciens) [3-7]. We purified this adenyl cyclase from B. pertussis culture medium to a single band on sodium dodecyl sulfate (SDS) disk gel electrophoresis and found it to have the following properties [2]: (1) a molecular weight of 70,000 as determined by sucrose density gradient centrifugation, G-200 Sephadex chromatography, and SDS disk gel electrophoresis; (2) a pH optimum of 7-8; (3) a temperature optimum of 30 C; (4) lability to heat (56 C) and trypsin; (5) requirement for a divalent cation (Mg++ > Mn "): (6) insensitivity to a-keto acids, nonsubPlease address requests for reprints to Dr. Erik L. Hewlett, Building 10, Room 8N308, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014. ,. Present address: Division of Bacterial Products, Bureau of Biologics, Food and Drug Administration, Bethesda, Maryland 20014. lThe Committee on Biochemical Nomenclature of the International Union of Pure and Applied Chemistry has recommended the name "adenylate cyclase" rather than "adenyl cyclase" for this enzyme. However, because "adenyl cyclase" has been used consistently by the Journal in the past, the editors decided, with the permission of Dr. Hewlett, to retain the name "adenyl cyclase" in this article as well as throughout this issue of the Journal.

strate nucleotides, and mercury compounds; and (7) mild inhibition by monovalent salts (NaCl, NaBr, NaI, KCl, KI, and LiCl) and marked inhibition by fluoride (50% at 4 mxr). Thus, as shown in table 1, this enzyme differs significantly from other bacterial adenyl cyclases in its biochemical characteristics as well as by its presence in the culture medium. While investigating the source of the culture medium enzyme, we discovered that intact B. pertussis organisms were able to catalyze the formation of [32P]adenosine 3':5'-cyclic phosphate (cyclic AMP) when incubated with exogenous [a-32PJadenosine triphosphate (200-1,200 nmol of cyclic AMP formed per min per g of wet weight) [8]. To study the localization of this enzyme activity, we used malate dehydrogenase as an intracellular marker enzyme, and its activity was compared with that of adenyl cyclase in whole cells and cell lysates. As expected, only 2% of the total malate dehydrogenase activity was measurable with the organisms intact. In contrast, 40% of the adenyl cyclase activity was measurable before disruption of the organisms, a fact suggesting that a substantial proportion of the enzyme might be located external to the cytoplasmic membrane. If the B. pertussis adenyl cyclase is indeed extracytoplasmic, it should be sensitive to destruction by a proteolytic enzyme that would be unable to affect intracellular components during a mild, brief treatment. We exposed intact B. pertussis to trypsin (20 p.,g/ml for 5 min), stopped the reaction with trypsin inhibitor, and measured malate dehydrogenase and adenyl cyclase activities before sonication and after each 20-sec pulse with the Sonifier cell disruptor (Heat Systems-Ultrasonic, Plainview, N.Y.). The results 5216

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Bordetella- pertussis organisms have a large amount of extracytoplasmic adenyl cyclase, part of which is found in the supernatant culture medium during exponential growth. The enzyme differs from previously studied bacterial adenyl cyclases in biochemical characteristics as well as in location. Several commercial pertussis vaccines were found to contain adenyl cyclase activity; this activity is probably due to the extracytoplasmic enzyme associated with and released from the whole cells in the vaccine.


Adenyl Cyclase in B. pertussis Vaccines

Table 1. Location and biochemical characteristics of bacterial adenyl cyclases. (Data for enzymes from Escherichia coli and Brevibacterium liquefaciens are from [3-8, 12, 13] ; data for enzyme from Bordetella pertussis are from [2,8] .) B. liquefaciens

B. pertussis

No Intracellular (105,000 g supernatant)

Yes Extracytoplasmic and extracellular

E. coli


No Intracellular (30,000 g pellet)

Present in culture medium Predominant location Molecular weight




pH optimum


7-9 (without pyruvate); 9-10 (with pyruvate)


Enzyme induction, flagella formation


=inhibition of enzyme activity; and


2 ~


600 m I

o: UJ


o ~ o


-< o :JJ o G)



u r

m 20~

u 200






uUJU « 40






40 ~






-l ----ua..



o o ~



UJ Cf)


s: m

~u 600

= no effect.

clase, on the other hand, was markedly different in that (1) trypsin treatment of intact organisms reduced the measurable adenyl cyclase activity by >95% (from 351 to 14 nmol of cyclic AMP per min per g of wet weight); (2) sonication of tryp-





:t> r :t>




are demonstrated in figure I. The magnitude and time course of release of malate dehydrogenase activity was the same in control and trypsin-treated cells, a finding indicating that the trypsin had no effect on this intracellular enzyme. The adenyl cy-




"'(+) = stimulation of enzyme activity; (-)

~ 400





4:UJ -l-l

















80 100 120 140



20 40 60 80 100 120 140 TOTAL TIME OF SONICATION (SEC)

Figure 1. Effects of sonication and trypsin treatment on activities of adenyl cyclase and malate dehydrogenase in Bordetella pertussis organisms. Adenyl cyclase and malate dehydrogenase were assayed before sonication and after each 20-sec pulse with the Sonifier cell disruptor. Control (untreated) B. pertussis organisms (left) were compared with B. pertussis organisms exposed to trypsin (20 pgJml for 5 min) and then to trypsin inhibitor (right). Adenyl cyclase activity (~) is expressed as nmol of adenosine 3' :5'-cyclic phosphate (cAMP) formed per mm per g of wet weight. Note the IO-fold reduction in scale for the trypsin-treated cells. Malate dehydrogenase activity (0----0) is expressed as ,umol of reduced nicotinamide adenine dinucleotide oxidized per min per g of wet weight. For further details of experimental procedure see [8].

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Effects of" Nucleotides a-Keto acids Fluoride Mercury compounds Function

Hewlett, Mane/ark, and Wolff


sin-treated cells for 140 sec revealed a residual adenyl cyclase activity equal to 9% of that of the control, sonicated cell value (63 vs. 696 nmol of cyclic AMP per min per g of wet weight; this residual 9% activity appeared to represent true in-

tracellular enzyme in that it was easily destroyed by trypsin treatment after sonication); (3) the release of adenyl cyclase activity from control cells by sonication was different in magnitude and kinetics from that of trypsin-treated cells.

Table 2. Adenyl cyclase activity and concentrations of adjuvant and bacteria in different Bordetella pertussis vaccines.

Lot 1 2 3 4 5 Lot 1 2 3 4 5 6 Lot 1 2 3 4 5 6 Lot 1 2 3 4 5 Lot

Adenyl cyclase

Concentration of Adjuvant

pmol/min per 10 J.LI


B. pertussis

AIP04 AIP04 AIP04 AIP0 4 AlP04

0.24 0.27 0.26 0.23 0.25

6.2 12.5 8.6 10 12.5

Alum Alum Alum Alum Alum Alum

0.14 0.14 0.19 0.21 0.20 0.12

15 15 15 15 15 15

0.069 0.028 0.031 0.058 0.067 0.003

AIP04 AIP04 AIP04 AIP04 AIP04 AlP04

0.07 0.07 0.07 0.82 0.08

16 16 16 16 16

0.010 0..002 0.002 0.022 0.010 0


pmol/min per SHD

A 1.01 18.0 9.72 1.78 9.88

±0.18 ± 8.3 ± 2.0 ±0.09 ± 1.7

50.7 899 486 89.0 494



D None Alum Alum Alum

0.19 0.23 0.19


0.066 0.066 0.003 0.007 0


0.15 0.15 0.08 0.15

10 10 15 10

0.041 0 0 0


0.55 0.59

10 10

0.028 0.036





1 2 3 4 Lot F 1 2 Lot G 1

Control (nonpertussis vaccines) Eli Lilly and Company pertussis vaccine [1]

0 0.022-0.032 7.38 (1971) 0.708 (1976)

NOTE. Commercial lots of pertussis vaccine from eight manufacturers were sampled and assayed for adenyl cyclase activity. Ten microliters of vaccine were used in the assay, and results are expressed as pmol of adenosine 3' :5'·cyclic phosphate (cyclic AMP) formed per min/ l O J.L1. For those vaccines containing enzyme activity, the results presented are means ± SE from at least two assays done in triplicate and are expressed as activity per single human dose (SHD; 0.5 ml), A range of values is shown for the nonpertussis vaccines used as controls, and the 1971 and 1976 activities are listed for the Eli Lilly and Company (Indian. apolis, Ind.) pertussis vaccine [1]. Adjuvant content is expressed as mg of elemental aluminum per SHD, and bacterial organism content is expressed as opacity units per SHD.

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Manufacturer lot, sample


Adenyl Cyclase in B. pertussis Vaccines

protective antigen [9-10], the production of an optimal vaccine necessitates manipulation of the organism in order to maximize the protective components and minimize the toxicities. We are currently attempting to determine whether this adenyl cyclase exoenzyme is associated with any of the well-known toxic effects of B. pertussis [11] and what its role may be in regulating the metabolism of the organism.

References 1. Wolff, j., Cook, G. H. Activation of thyroid membrane adenylate cyclase. J. BioI. Chern. 248:350-355, 1973. 2. Hewlett, E. L., Wolff, J. Soluble adenylate cyclase from the culture medium of Bordetella pertussis. J. Bacteriol. 127:800--898, 1976. 3. Ide, M. Adenyl cyclase of Escherichia coli. Biochern. Biophys. Res. Commun. 36:42-46, 1969. 4. Tao, M., Lipmann, F. Isolation of adenyl cyclase from Escherichia coli. Proc. Natl. Acad. Sci. (U.S.A.) 63:86-92, 1969. 5. Tao, M., Huberman, A. Some properties of Escherichia coli adenyl cyclase. Arch. Biochem. Biophys. 141: 236-240, 1970. 6. Hirata, M., Hayaishi, O. Adenyl cyclase of Breoibacterium liqueiaciens. Biochem. Biophys. Acta 149: 1-11,1967. 7. Takai, K., Kurashina, Y., Suzuki-Hod, C., Okamoto, H., Hayaishi, O. Adenylate cyclase from Breoibacterium liquetaciens. I. Purification, crystallization and some properties. J. BioI. Chern. 249:1965-1972,1974. 8. Hewlett, E. L., Urban, M., Manclark, C., Wolff, J. Extracytoplasmic adenylate cyclase of Bordetella pertussis. Proc. Natl, Acad. Sci. (U.S.A.) 73:1926-1930, 1976. 9. Munoz, J. J. Symposium on relationship of structure of microorganisms to their immunological properties. I. Immunological and other biological activities of Bordetella pertussis antigens. Bacteriol. Rev. 27: 325-340, 1963. 10. Munoz, J. J. Pertussigen: a substance from Bordetella pertussis with many biological activities [abstract no. 3354]. Fed. Proc 35:813,1976. II. Olson, L. C. Pertussis. Medicine 54:427-469, 1975. 12. deCrombrugghe, B., Perlman, R. L., Varmus, H. E.• Pastan, I. Regulation of inducible enzyme synthesis in Escherichia coli by cyclic adenosine 3',5'-monophosphate. J. BioI. Chern. 244:5828-5835, 1969. 13. Yokota, T., Gots, J. S. Requirement of adenosine 3',5'cyclic phosphate for flagella formation in Escherichia coli and Salmonella typhimurium. J. Bacteriol. 103: 513-516,1970.

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These data support our hypothesis that B. pertussis has a large pool of extracytoplasmic adenyl cyclase (all of which is sensitive to treatment of the intact cells with trypsin and part of which is measurable without disruption of cells), soluble enzyme that is present in the culture medium, and a small amount of intracellular enzyme [8]. When the original observation of adenyl cyclase activity in a whole-cell pertussis vaccine was reconsidered in light of the present information, it appeared likely that the activity consisted of extracytoplasmic enzyme associated with the whole organism and possibly some enzyme solubilized into the medium of the vaccine during storage and handling. The persistence of the enzyme activity after storage at 4 C for months to years was striking. In fact, we recently assayed the original vaccine again and found that it contained adenyl cyclase activity equivalent to 10% of the value measured five years ago (table 2, bottom). In the present study, we measured adenyl cyclase activity in a number of commercial lots of pertussis vaccine submitted to the Bureau of Biologies (Food and Drug Administration, Bethesda, Md.j for testing and release. The results of this screening process are shown in table 2. Although most of the vaccines tested were inactive in the adenyl cyclase assay (essentially the same as control, nonpertussis vaccines), all of the samples tested from manufacturer A had significant activity. The values ranged from 50 to almost 900 pmol of cyclic AMP formed per min per single dose (human) of vaccine. Since all of the vaccines studied met the required limits for potency, toxicity, adjuvant content, and organism concentration, we were unable to explain the differences in activity on the basis of anyone of these parameters. It has become clear from further study, however, that there are a number of variables that influence the adenyl cyclase activity expressed by the intact B. pertussis organisms and several other factors that determine its persistence during the handling of the organisms in the preparation of vaccine. Since it appears that some of the toxic properties of B. pertussis are closely associated with the

Adenyl cyclase in Bordetella pertussis vaccines.

Adenyl Cyclase in Bordetella pertussis Vaccines E. L. Hewlett, C. R. Manclark,* and J. \Volff From the National Institute of Arthritis, Metabolism,...
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