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Title: Purification and assay of cell-invasive form of calmodulin-sensitive adenylyl cyclase from Bordetella pertussis

Abstract

An invasive form of the CaM-sensitive adenylyl cyclase from Bordetella pertussis can be isolated from bacterial culture supernatants. This isolation is achieved through the use of QAE-Sephadex anion-exchange chromatography. It has been demonstrated that the addition of exogenous Ca{sup 2}{sup +} to the anion-exchange gradient buffers will affect elution from the column and will thereby affect the isolation of invasive adenylyl cyclase. This is probably due to a Ca2(+)-dependent interaction of the catalytic subunit with another component in the culture supernatant. Two peaks of adenylyl cyclase activity are obtained. The Pk1 adenylyl cyclase preparation is able to cause significant increases in intracellular cAMP levels in animal cells. This increase occurs rapidly and in a dose-dependent manner in both N1E-115 mouse neuroblastoma cells and human erythrocytes. The Pk2 adenylyl cyclase has catalytic activity but is not cell invasive. This material can serve, therefore, as a control to ensure that the cAMP which is measured is, indeed, intracellular. A second control is to add exogenous CaM to the Pk1 adenylyl cyclase preparation. The 45-kDa catalytic subunit-CaM complex is not cell invasive. Although the mechanism for membrane translocation of the adenylyl cyclase is unknown, there is evidence that the adenylyl cyclase enters animalmore » cells by a mechanism distinct from receptor-mediated endocytosis. Calmodulin-sensitive adenylyl cyclase activity can be removed from preparations of the adenylyl cyclase that have been subjected to SDS-polyacrylamide gel electrophoresis. This property of the enzyme has enabled purification of the catalytic subunit to apparent homogeneity. The purified catalytic subunit from culture supernatants has a predicted molecular weight of 45,000. This polypeptide interacts directly with Ca{sup 2}{sup +} and this interaction may be important for its invasion into animal cells.« less

Authors:
; ;
Publication Date:
OSTI Identifier:
5544897
Resource Type:
Journal Article
Journal Name:
Methods in Enzymology; (USA)
Additional Journal Information:
Journal Volume: 195; Journal ID: ISSN 0076-6879
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; CYCLASES; FRACTIONATION; ATP; BACTERIA; CALCIUM COMPOUNDS; CHROMATOGRAPHY; ENZYME ACTIVITY; ION EXCHANGE CHROMATOGRAPHY; ISOTOPE DILUTION; MOLECULAR WEIGHT; PHOSPHORUS ISOTOPES; TRITIUM COMPOUNDS; VIRULENCE; ALKALINE EARTH METAL COMPOUNDS; ENZYMES; HYDROGEN COMPOUNDS; ISOTOPE APPLICATIONS; ISOTOPES; LYASES; MICROORGANISMS; NUCLEOTIDES; ORGANIC COMPOUNDS; SEPARATION PROCESSES; TRACER TECHNIQUES; 550201* - Biochemistry- Tracer Techniques

Citation Formats

Masure, H.R., Donovan, M.G., and Storm, D.R. Purification and assay of cell-invasive form of calmodulin-sensitive adenylyl cyclase from Bordetella pertussis. United States: N. p., 1991. Web. doi:10.1016/0076-6879(91)95161-C.
Masure, H.R., Donovan, M.G., & Storm, D.R. Purification and assay of cell-invasive form of calmodulin-sensitive adenylyl cyclase from Bordetella pertussis. United States. doi:10.1016/0076-6879(91)95161-C.
Masure, H.R., Donovan, M.G., and Storm, D.R. Tue . "Purification and assay of cell-invasive form of calmodulin-sensitive adenylyl cyclase from Bordetella pertussis". United States. doi:10.1016/0076-6879(91)95161-C.
@article{osti_5544897,
title = {Purification and assay of cell-invasive form of calmodulin-sensitive adenylyl cyclase from Bordetella pertussis},
author = {Masure, H.R. and Donovan, M.G. and Storm, D.R.},
abstractNote = {An invasive form of the CaM-sensitive adenylyl cyclase from Bordetella pertussis can be isolated from bacterial culture supernatants. This isolation is achieved through the use of QAE-Sephadex anion-exchange chromatography. It has been demonstrated that the addition of exogenous Ca{sup 2}{sup +} to the anion-exchange gradient buffers will affect elution from the column and will thereby affect the isolation of invasive adenylyl cyclase. This is probably due to a Ca2(+)-dependent interaction of the catalytic subunit with another component in the culture supernatant. Two peaks of adenylyl cyclase activity are obtained. The Pk1 adenylyl cyclase preparation is able to cause significant increases in intracellular cAMP levels in animal cells. This increase occurs rapidly and in a dose-dependent manner in both N1E-115 mouse neuroblastoma cells and human erythrocytes. The Pk2 adenylyl cyclase has catalytic activity but is not cell invasive. This material can serve, therefore, as a control to ensure that the cAMP which is measured is, indeed, intracellular. A second control is to add exogenous CaM to the Pk1 adenylyl cyclase preparation. The 45-kDa catalytic subunit-CaM complex is not cell invasive. Although the mechanism for membrane translocation of the adenylyl cyclase is unknown, there is evidence that the adenylyl cyclase enters animal cells by a mechanism distinct from receptor-mediated endocytosis. Calmodulin-sensitive adenylyl cyclase activity can be removed from preparations of the adenylyl cyclase that have been subjected to SDS-polyacrylamide gel electrophoresis. This property of the enzyme has enabled purification of the catalytic subunit to apparent homogeneity. The purified catalytic subunit from culture supernatants has a predicted molecular weight of 45,000. This polypeptide interacts directly with Ca{sup 2}{sup +} and this interaction may be important for its invasion into animal cells.},
doi = {10.1016/0076-6879(91)95161-C},
journal = {Methods in Enzymology; (USA)},
issn = {0076-6879},
number = ,
volume = 195,
place = {United States},
year = {1991},
month = {1}
}