Bacterial expression and characterization of proteins derived from the chicken calmodulin cDNA and a calmodulin processed gene.
Both normal chicken calmodulin (CaM) and a CaM-like mutant protein have been expressed in bacteria, isolated and evaluated with respect to several physical and biological properties. The mutant CaM is derived from a CaM-like gene that lacks intervening sequences and probably evolved from a CaM-processed gene (Stein, J. P., Munjaal, R. P., Lagacé, L., Lai, E. C., O'Malley, B. W., and Means, A. R. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 6485-6489). The mutant CaM protein contains 16 of the 19 amino acids encoded by the CaM-like gene. Normal chicken CaM produced in bacteria is identical to rat CaM by all criteria tested except that it is not trimethylated. The protein product of the CaM-like gene has been termed CaML and exhibits properties which are very similar to CaM despite the presence of 16 amino acid substitutions. CaML binds Ca2+ as evidenced by Ca2+-dependent binding to phenothiazine- and phenyl-Sepharose affinity resins and a Ca2+-dependent electrophoretic mobility shift which is similar to but distinct from CaM. CaML cross-reacts with a monospecific CaM antibody and has an immunodilution curve which is identical to bacterially synthesized CaM. Finally, CaML can maximally activate rat brain phosphodiesterase but with altered kinetic parameters as compared to CaM. These data suggest that the nucleotide substitutions in the putative CaM processed gene are not random but are selected to retain CaM-like functions in the encoded protein. Such a mechanism may exist for other processed genes.
Putkey
J A
JA
Slaughter
G R
GR
Means
A R
AR
D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000235:genetics* / Q000494:pharmacology; D002645:Chickens; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005786:Gene Expression Regulation; D006358:Hot Temperature; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D010957:Plasmids
D002147:Calmodulin; D004247:DNA; D010727:Phosphoric Diester Hydrolases; D002118:Calcium