The proteolytic susceptibility of chicken gizzard myosin light chain kinase, a calmodulin-dependent enzyme, has been utilized to define the relative location of the catalytic and regulatory domains of the enzyme. Myosin light chain kinase isolated from this source exhibits a M(r) of 130,000 and is extremely sensitive to trypsin at 24°C; however, the molecule is divided into susceptible and resistant domains such that proteolysis proceeds rapidly and at multiple sites in the sensitive regions even at 4°C while the rest of the molecule remains relatively resistant to digestion. One of these sensitive areas is the calmodulin-binding domain. On the other hand, Staphylococcus aureus V8 protease digestion generates a calmodulin-binding fragment (M(r) = 70,000) that retains Ca2+/calmodulin-dependent enzymatic activity and both of the phosphorylation sites recognized by cAMP-dependent protein kinase. In contrast, treatment with chymotrypsin produces a 95,000 M(r) calmodulin-binding fragment that contains only the calmodulin-modulated phosphorylation site. Sequential proteolytic digestion studies demonstrated that the chymotryptic cleavage site responsible for the generation of this 95,000 M(r) peptide is within 3,000 M(r) of the V8 protease site which produces the 70,000 M(r) fragment. Moreover, the non-calmodulin-modulated phosphorylation site must exist in this 3,000 M(r) region. A calmodulin-Sepharose affinity adsorption protocol was developed for the digestion and used to isolate both the 70,000 and 95,000 M(r) fragments for further study. Taken together, our results are compatible with a model for chicken gizzard myosin light chain kinase in which there is no overlap between the active site, the calmodulin-binding region, and the two sites phosphorylated by cAMP-dependent protein kinase with regard to their relative position in the primary sequence of the molecule.
|Original language||English (US)|
|Number of pages||10|
|Journal||Journal of Biological Chemistry|
|State||Published - 1985|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology