title: The cAMP-binding proteins of Leishmania are not the regulatory subunits of cAMP-dependent protein kinase.
abstract: The most commonly used method to determine the cAMP binding activity in cytosolic extracts of promastigotes of Leishmania spp. underestimated by approximately 11.5-fold the total amount of [(3)H]cAMP bound, when compared with results obtained by the modified Millipore filter technique. Three cAMP-binding proteins (BPI, BPII and BPIII) were partially purified and characterized. The native molecular masses of BPI, BPII and BPIII were estimated to be 105, 155 and 145 kDa, respectively. The binding of [(3)H]cAMP to these proteins was affected to different extents by several cAMP analogues. Antibodies directed against the types I and II regulatory subunits of PKA did not cross-react with the leishmanial extract. Photoaffinity labeling of the cytosolic extracts with 8-N(3)-[(32)P]cAMP specifically labeled a band of M(r) 116000 and a band of M(r) 80000 partially saturable by cAMP. From these results, it is concluded that the leishmanial cAMP-binding proteins appear to belong to a different class distinct from the regulatory subunits of cAMP-dependent protein kinases.
authors.affiliation: Leishmania Division and Department of Cell Biology, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Calcutta 700 032, India.
authors.forename: C
authors.initials: C
authors.lastname: Banerjee
authors.forename: D
authors.initials: D
authors.lastname: Sarkar
mesh_terms: D000818:Animals; D000906:Antibodies / Q000378:metabolism; D001665:Binding Sites; D015153:Blotting, Western; D002352:Carrier Proteins; D002417:Cattle; D002848:Chromatography, DEAE-Cellulose; D003429:Cross Reactions; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism; D002373:Cyclic AMP Receptor Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D003600:Cytosol / Q000000981:diagnostic imaging / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007893:Leishmania donovani / Q000378:metabolism*; D008851:Micropore Filters; D018482:Muscle, Skeletal / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D019920:Photoaffinity Labels; D011485:Protein Binding; D011877:Radionuclide Imaging
chemical_list: D000906:Antibodies; D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein; D019920:Photoaffinity Labels; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases