JouRNAL OF BACTURIOLOGY, Aug. 1976, p. 890-898 Copyright © 1976 American Society for Microbiology

Vol. 127, No. 2 Printed in U.S.A.

Soluble Adenylate Cyclase from the Culture Medium of Bordetella pertussis: Purification and Characterization E. HEWLETT*

AND

J. WOLFF

National Institute ofArthritis, Metabolism and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014 Received for publication 17 February 1976

Culture medium of exponentially growing Bordetella pertussis (strain 114) contains significant quantities of soluble (100,000 x g for 1 h) adenylate cyclase. The enzyme was purified by chromatography on diethylaminoethyl-cellulose and Sephadex G-200. The purest material yielded a single band on sodium dodecyl sulfate-disc gel electrophoresis. It is heat labile, has a temperature optimum of 30°C, a pH optimum of pH 7 to 8, and a K. for adenosine 5'-triphosphate of 0.4 mM, and requires Mg2+ for maximum activity. The molecular weight, by sodium dodecyl sulfate-disc gel electrophoresis and sucrose density gradient, is approximately 70,000. The enzyme is markedly inhibited by fluoride and weakly inhibited by monovalent salts, but its activity is not altered by a-keto acids or nonsubstrate nucleoside triphosphates. Thus, by its presence in the culture supernatant, its smaller molecular weight, and its insensitivity to a-keto acids and nucleotides, this enzyme differs from the bacterial adenylate cyclases previously described.

Bacterial products have been shown to stimulate (3, 10, 19, 21, 29), inhibit (9), and alter the hormone responsiveness (2, 10) of the adenylate cyclase of eucaryotic cells. Similarly, toxic components of Bordetella pertussis alter the responsiveness of adenylate cyclase systems in animals and man (1, 4, 18). In the evaluation of the influence of products of B. pertussis on mammalian cells, the presence of adenylate cyclase activity was detected in the 100,000 x g supernatant of a Merthiolate-preserved wholecell Pertussis Vaccine obtained from Eli Lilly (28). In addition to the presence of this enzymatic activity in the whole-cell vaccine, a significant amount of adenylate cyclase can be demonstrated in the supernatant culture medium of exponentially growing B. pertussis organisms. Although adenylate cyclase has been purified and characterized from lysates of three other bacterial species (11, 12, 15, 23-25), no evidence for the presence of this enzyme in their culture supernatants has thus far been reported. Moreover, because of the adenylate cyclase-stimulating effects of cholera toxin, Vibrio cholerae culture media have been examined for the presence of soluble adenylate cyclase, but none has been found (W. B. Greenough III, personal communication). In this paper we describe the purification and characterization of a soluble adenylate cyclase not previously characterized in B. pertussis nor reported in the culture supernatant of any bacteria.

MATERIALS AND METHODS Chemicals. Radioisotopes used were la32P]adenosine 5'-triphosphate (ATP) (10 Ci/mmol) and La-32P]adenylyl 5'-imidodiphosphate tAMPP(NH)P] (5-6 Ci/mmol), from ICN; and [3H]cylic adenosine 3',5'-monophate (cAMP) (0.044 Ci/mmol), from New England Nuclear. Nucleotides were obtained from Sigma. Purification procedures and adenylate cyclase assays were carried out in tricine [N-tris(hydroxymethyl)methyl-glycine] buffer, purchased from Calbiochem. Phosphocreatine and creatine phosphokinase (crystalline) were obtained from Sigma. Purified beef heart cAMP phosphodiesterase (0.15 U/mg) was obtained from Gallard-Schlesinger. Thimerosal (Merthiolate sodium) was obtained from National Biochemicals Corp. Whatman DEAE (microgranular diethylaminoethyl-cellulose, DE52) was obtained from Reeve Angel. Growth of the organism. B. pertussis (strain 114) was grown by Marie Urban and Charles Manclark (Laboratory of Bacterial Products, Bureau of Biologics, Food and Drug Administration), and culture supernatant was kindly provided for enzyme studies. The organism was grown in 500-ml flasks containing 250 ml of modified Stainer-Scholte medium (22; C. R. Manclark, M. A. Urban, and E. P. Summers, manuscript in preparation) consisting of: sodium glutamate, 10.72 g; NaCl, 2.5 g; KH2PO4, 0.5 g; KCl, 0.2 g; MgCl2-6H20, 0.1 g; CaCl2, 0.02 g, L-proline, 0.24 g; tris(hydroxymethyl)aminomethane base, 1.525 g; plus membrane filter (Millipore)sterilized supplement of L-cystine, 40 mg; FeSO4' 7H20, 10 mg; ascorbic acid, 20 mg; nicotinic acid, 4 mg; and glutathione, 100 mg; in a total volume of 1 liter. The pH of the medium was 7.6. Cultures 890

VOL. 127, 1976

BORDETELLA PERTUSSIS ADENYLATE CYCLASE

were begun with a 5% inoculum of exponentially growing cells, grown at 35.5°C on an oscillating shaker (150 rpm) with a room-air gas phase, and harvested at 24 ± 4 h by centrifugation (6,000 x g for 30 min). Bacterial cells were used for other studies, and the culture supernatant was used for enzyme purification. Enzyme assay. Adenylate cyclase activity was measured using [a-32P]ATP conversion to [32p]_ cAMP. The reaction mix consisted of three parts: (i) tricine buffer plus any additives (20 ul); (ii) substrate, Mg2+, and regenerating system (30 ,l); and (iii) enzyme protein (10 ,ul). The concentrations of components in 60 ,ul (final volume) were tricine (60 mM, pH 8.0), MgCl2 (10 mM), ATP (1 mM with 106 to 1.5 x 106 cpm of [a-32P]ATP), creatine phosphate (6.7 mM), crystalline creatine phosphokinase (20 ,ug), and approximately 300 ng of enzyme protein. The reaction was initiated by addition of the enzyme, and incubation was at 30°C for 10 min. It was terminated by the addition of 100 ,ul of "stopping solution" containing 1% sodium dodecyl sulfate (SDS), 20 mM ATP, and 6.25 mM cAMP (including 15,000 to 20,000 cpm of [3HIcAMP per tube for calculation of recovery). The cAMP was separated from contaminating nucleotides by the double-column method of Salomon et al. (20). Final recovery of cAMP was 65 to 80%. Radioactivity was detected in a Packard Tri-Carb scintillation counter using Aquasol (12 ml) as scintillant. Counting error was approximately 1%. Assay blanks were obtained by incubation of reaction mix plus buffer for 10 min in the absence of enzyme. All experimental points (except those from column effluents) were assayed in triplicate. The production of cAMP was linear for at least 20 min, using 360 ng of enzyme protein, the standard amount used per reaction tube. cAMP accumulation was proportional to enzyme concentration over a range of 64 to 960 ng per assay tube, the maximum concentration being three times that used in the standard assay. All proteins were determined by the method of Lowry et al. (16) using a serum albumin standard, except those from column eluates, on which relative protein concentrations were determined fluorometrically by excitation at 280 nm and emission at 340 nm. To confirm that the reaction product was cAMP, purified beef heart cAMP phosphodiesterase was added to a reaction mix after completion of the incubation. A 10-min incubation at 300C with 0.4 MAg of phosphodiesterase per ml (80 ug total) in the presence of 10 mM Mg2+ yielded a loss of 95% of the counts from the cAMP fraction, as compared with a loss of 92% of counts from a standard of [3H]cAMP. In addition, the product was assayed for cAMP content by the cAMP binding protein determination by the method of Gilman (8). The values for cAMP production obtained by the binding assay were equivalent to those calculated from the [32P]ATP assay. Although an ATP-regenerating system of creatine phosphate and creatine phosphokinase was used, there was no difference in cAMP formation at 10 min with or without its use. There was a small (~30-

Soluble adenylate cyclase from the culture medium of Bordetella pertussis: purification and characterization.

JouRNAL OF BACTURIOLOGY, Aug. 1976, p. 890-898 Copyright © 1976 American Society for Microbiology Vol. 127, No. 2 Printed in U.S.A. Soluble Adenylat...
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