Characterization of the human calmodulin-like protein expressed in Escherichia coli.
The protein-coding region of an intronless human calmodulin-like gene [Koller, M., & Strehler, E. E. (1988) FEBS Lett. 239, 121-128] has been inserted into a pKK233-2 expression vector, and the 148-residue, M(r) = 16,800 human protein was purified to apparent homogeneity by phenyl-Sepharose affinity chromatography from cultures of Escherichia coli JM105 transformed with the recombinant vector. Several milligrams of the purified protein were obtained from 1 L of bacterial culture. A number of properties of human CLP were compared to those of bacterially expressed human calmodulin (CaM) and of bovine brain CaM. CLP showed a characteristic Ca(2+)-dependent electrophoretic mobility shift on SDS-polyacrylamide gels, although the magnitude of this shift was smaller than that observed with CaM. CLP was able to activate the 3',5'-cyclic nucleotide phosphodiesterase to the same Vmax as normal CaM, albeit with a 7-fold higher Kact. In contrast, the erythrocyte plasma membrane Ca(2+)-ATPase could only be stimulated to 62% of its maximal CaM-dependent activity by CLP. CLP was found to contain four Ca(2+)-binding sites with a mean affinity constant of 10(5) M-1, a value about 10-fold lower than that for CaM under comparable conditions. The highly tissue-specifically-expressed CLP represents a novel human Ca(2+)-binding protein showing characteristics of a CaM isoform.
Rhyner
J A
JA
Laboratory for Biochemistry, Swiss Federal Institute of Technology, Zurich.
Koller
M
M
Durussel-Gerber
I
I
Cox
J A
JA
Strehler
E E
EE
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000097:blood; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004912:Erythrocytes / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology; D009994:Osmolar Concentration; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D014169:Transformation, Bacterial
D002147:Calmodulin; D011994:Recombinant Proteins; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D000252:Calcium-Transporting ATPases; D002118:Calcium