Ca2+/calmodulin-binding proteins in Dictyostelium discoideum.
We have initiated a systematic study of Ca2+/calmodulin-regulated enzymes in the cellular slime mold Dictyostelium discoideum. Using 125I-labelled D. discoideum calmodulin (CaM) as a functional probe, several Ca2+/CaM-binding proteins were detected in crude cell lysates. Proteins with apparent molecular weights of 22 kDa and 78-80 kDa, respectively, were found in the soluble fraction. In addition, membrane-bound high molecular weight CaM-binding proteins were identified. Binding of CaM to all of the proteins required the presence of Ca2+ ions and competed efficiently with nonradioactive CaM from both Dictyostelium and bovine brain. The CaM antagonists melittin, W-7 and R24571 inhibited CaM binding. With a functional cloning approach, we previously obtained cDNA clones by screening a lambda gt11 lysogen expression library; in this paper, we report the analysis of CaM-binding activity by one of the recombinant cDNA clones in Escherichia coli. When rabbit antiserum was raised against it, the antiserum recognized a 78-80-kDa protein in Dictyostelium extracts which comigrated on SDS-polyacrylamide gels with 78-80-kDa CaM-binding activity.
Winckler
T
T
Fakultät für Biologie, Universität Konstanz, Germany.
Dammann
H
H
Mutzel
R
R
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D004023:Dictyostelium / Q000201:enzymology / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D008970:Molecular Weight; D011485:Protein Binding / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis; D013960:Thyroid Function Tests
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D011994:Recombinant Proteins; D002118:Calcium