J. Biochem, 1994, Vol. 115, No. 2 367
© 1994 Japanese Biochemical Society
Amino Acid Sequence of Calmodulin from Wheat Germ
Subsequent to the publication of this paper, Ling and Zielinski cloned and sequenced cDNAs encoding calmodulin (CaM) from barley (Hordeum vulgare L.) (1). They reported that barley and spinach CaMs (2) have no insertion/deletion events. Thus, we reinvestigated amino acid sequence of wheat germ CaM, especially around the blocked amino- and carboxyl-termini.
The carboxymethylated CaM was digested with trypsin and the resulting peptides were separated by high-performance liquid chromatography, with assignment of individual peptides by amino acid analysis and/or peptide sequencing. The amino-terminal blocked peptide T1 (the composition: D 2.0, T 0.9, E 4.1, A 2.0, 11.0, L 1.0, F 1.0, K 0.9) and carboxyl-terminal peptide T12 (the composition: A 1.1, V 1.0, M 1.3, K 1.0) were analyzed.
The amino acid sequence of T1 was identified to be D-Q-L-T-D-E-Q-I-A-E-F-K, after removal of the N-terminal acetylamino acid by digestion with acylamino-acid-releasing enzyme. The N-terminal blocked amino acid of T1 was confirmed to be acetyl-Ala by fast atom bombardment (FAB) mass spectrometric analysis: [M+H]+ of T1 was found to be 1, 549.69 (the calculated value is 1, 549.70). Thus the amino acid sequence of T1 was confirmed to be Ac-A-D-Q-L-T-D-E-Q-I-A-E-F-K. T12 was sequenced directly to be V-M-M-A-K.
From the present results we report here the corrected sequence of wheat germ CaM without insertion of Asn residue between residues 8 and 9, and deletion of Thr residue at position 146 in Fig. 2.