Identification and primary structure of calmodulin binding domains in the phosphorylase kinase holoenzyme

TY - JOUR

T1 - Identification and primary structure of calmodulin binding domains in the phosphorylase kinase holoenzyme

AU - James, Peter

AU - Cohen, Philip

AU - Carafoli, Ernesto

PY - 1991/12/1

Y1 - 1991/12/1

N2 - Fast twitch skeletal muscle phosphorylase kinase was isolated and incubated with a radioactive, bifunctional, photoactivable, and cleavable cross-linker conjugated to calmodulin. Incubation of the holoenzyme only resulted in the labeling of the α-subunit in the presence of Ca2+. After cleavage with CNBr (and subdigestion with Asp-N protease), a sequence was identified (residues 1069-1087) in the α-subunit which had the predominant basic character and the propensity to form an amphiphilic helix like other calmodulin binding domains. If cross-linked calmodulin was incubated with the isolated subunits of phosphorylase kinase, radioactivity was recovered in seven CNBr peptides: three came from the α-subunits, one of them corresponding to the sequence labeled in the holoenzyme. Three came from the β-subunit, and one came from the γ-subunit. The latter contained the two adjacent calmodulin binding domains recently identified in the γ-subunit (Dasgupta, M., Honeycutt, T., and Blumenthal, D. K. (1988) J. Biol. Chem. 264, 17156-17163).

AB - Fast twitch skeletal muscle phosphorylase kinase was isolated and incubated with a radioactive, bifunctional, photoactivable, and cleavable cross-linker conjugated to calmodulin. Incubation of the holoenzyme only resulted in the labeling of the α-subunit in the presence of Ca2+. After cleavage with CNBr (and subdigestion with Asp-N protease), a sequence was identified (residues 1069-1087) in the α-subunit which had the predominant basic character and the propensity to form an amphiphilic helix like other calmodulin binding domains. If cross-linked calmodulin was incubated with the isolated subunits of phosphorylase kinase, radioactivity was recovered in seven CNBr peptides: three came from the α-subunits, one of them corresponding to the sequence labeled in the holoenzyme. Three came from the β-subunit, and one came from the γ-subunit. The latter contained the two adjacent calmodulin binding domains recently identified in the γ-subunit (Dasgupta, M., Honeycutt, T., and Blumenthal, D. K. (1988) J. Biol. Chem. 264, 17156-17163).

UR - https://www.scopus.com/pages/publications/0025873914

M3 - Article

C2 - 2016317

AN - SCOPUS:0025873914

SN - 0021-9258

VL - 266

SP - 7087

EP - 7091

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 11

ER -