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 language | English (US) |
|---|---|
| Pages (from-to) | 129-134 |
| Number of pages | 6 |
| Journal | Infection and Immunity |
| Volume | 55 |
| Issue number | 1 |
| State | Published - 1987 |
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ASJC Scopus subject areas
- Immunology
Cite this
Bordetella pertussis adenylate cyclase : Isolation and purification by calmodulin-sepharose 4B chromatography. / Friedman, Richard L.
In: Infection and Immunity, Vol. 55, No. 1, 1987, p. 129-134.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Bordetella pertussis adenylate cyclase
T2 - Isolation and purification by calmodulin-sepharose 4B chromatography
AU - Friedman, Richard L
PY - 1987
Y1 - 1987
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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UR - http://www.scopus.com/inward/citedby.url?scp=0023074159&partnerID=8YFLogxK
M3 - Article
C2 - 2878883
AN - SCOPUS:0023074159
VL - 55
SP - 129
EP - 134
JO - Infection and Immunity
JF - Infection and Immunity
SN - 0019-9567
IS - 1
ER -