Bordetella pertussis adenylate cyclase: Isolation and purification by calmodulin-sepharose 4B chromatography

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Abstract

Purified preparations of adenylate cyclase were obtained from crude urea extracts of Bordetella pertussis by a one-step calmodulin affinity chromatography technique. Diluted extract was loaded onto the column and washed, and adenylate cyclase was eluted with 10mM EGTA [ethylene glycol-bis(β-aminoethyl ether)-N,N,N', N'-tetraacetic acid]. A 104-fold purification was accomplished in one step. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the affinity-purified adenylate cyclase was dissociated into one major protein band with an apparent molecular weight of 60,000 and a minor band at 200,000. The affinity-purified adenylate cyclase was observed (i) to have adenylate cyclase enzymatic activity which was activated by calmodulin, (ii) to bind 125I-calmodulin, and (iii) to be free of pertussis toxin as determined by in vivo and in vitro assays.

Original languageEnglish (US)
Pages (from-to)129-134
Number of pages6
JournalInfection and Immunity
Volume55
Issue number1
StatePublished - 1987

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Bordetella pertussis
Agarose Chromatography
Calmodulin
Adenylyl Cyclases
Sepharose
Ethylene Glycol
Egtazic Acid
Pertussis Toxin
Complex Mixtures
Affinity Chromatography
Sodium Dodecyl Sulfate
Ether
Urea
Polyacrylamide Gel Electrophoresis
Molecular Weight
Acids
Proteins

ASJC Scopus subject areas

  • Immunology

Cite this

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abstract = "Purified preparations of adenylate cyclase were obtained from crude urea extracts of Bordetella pertussis by a one-step calmodulin affinity chromatography technique. Diluted extract was loaded onto the column and washed, and adenylate cyclase was eluted with 10mM EGTA [ethylene glycol-bis(β-aminoethyl ether)-N,N,N', N'-tetraacetic acid]. A 104-fold purification was accomplished in one step. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the affinity-purified adenylate cyclase was dissociated into one major protein band with an apparent molecular weight of 60,000 and a minor band at 200,000. The affinity-purified adenylate cyclase was observed (i) to have adenylate cyclase enzymatic activity which was activated by calmodulin, (ii) to bind 125I-calmodulin, and (iii) to be free of pertussis toxin as determined by in vivo and in vitro assays.",
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T1 - Bordetella pertussis adenylate cyclase

T2 - Isolation and purification by calmodulin-sepharose 4B chromatography

AU - Friedman, Richard L

PY - 1987

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N2 - Purified preparations of adenylate cyclase were obtained from crude urea extracts of Bordetella pertussis by a one-step calmodulin affinity chromatography technique. Diluted extract was loaded onto the column and washed, and adenylate cyclase was eluted with 10mM EGTA [ethylene glycol-bis(β-aminoethyl ether)-N,N,N', N'-tetraacetic acid]. A 104-fold purification was accomplished in one step. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the affinity-purified adenylate cyclase was dissociated into one major protein band with an apparent molecular weight of 60,000 and a minor band at 200,000. The affinity-purified adenylate cyclase was observed (i) to have adenylate cyclase enzymatic activity which was activated by calmodulin, (ii) to bind 125I-calmodulin, and (iii) to be free of pertussis toxin as determined by in vivo and in vitro assays.

AB - Purified preparations of adenylate cyclase were obtained from crude urea extracts of Bordetella pertussis by a one-step calmodulin affinity chromatography technique. Diluted extract was loaded onto the column and washed, and adenylate cyclase was eluted with 10mM EGTA [ethylene glycol-bis(β-aminoethyl ether)-N,N,N', N'-tetraacetic acid]. A 104-fold purification was accomplished in one step. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the affinity-purified adenylate cyclase was dissociated into one major protein band with an apparent molecular weight of 60,000 and a minor band at 200,000. The affinity-purified adenylate cyclase was observed (i) to have adenylate cyclase enzymatic activity which was activated by calmodulin, (ii) to bind 125I-calmodulin, and (iii) to be free of pertussis toxin as determined by in vivo and in vitro assays.

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