Localization, Purification, and Characterization of the Rabbit Sarcoplasmic Reticulum Associated Calmodulin-Dependent Protein Kinase

M. Pelosi^* and A. Donella-Deana

Department of Biological Chemistry, University of Padova, viale G. Colombo 3, 35121 Padova, Italy; fax: +39-049-807-3310; E-mail: mpelosi@civ.bio.unipd.it

^* To whom correspondence should be addressed.

Received June 29, 1999

---

The Ca²⁺/calmodulin dependent protein kinase associated with the sarcoplasmic reticulum membranes (SR CaM kinase) plays a specific and important role in the modulation of both Ca²⁺ uptake and release functions of the sarcoplasmic reticulum itself. In this work we have localized a 60 kD SR CaM kinase in slow and fast twitch rabbit skeletal muscle fractions; the kinase was present in both the longitudinal and the junctional sarcoplasmic reticulum. We then developed a procedure for the purification of the active kinase from the longitudinal sarcoplasmic reticulum and performed biochemical and functional characterization of the enzyme. Differently from what was previously suggested, our analysis shows that the biochemical properties of the purified SR CaM kinase (Ca²⁺ sensitivity, K_0.5 for calmodulin, K_m for ATP, IC₅₀ for the specific inhibitory peptide (290-309), autophosphorylation properties) are not significantly different from those of the soluble multifunctional CaM kinase II. Moreover, we show that the purified SR CaM kinase retains the ability to autophosphorylate in a Ca²⁺/calmodulin-dependent manner, becoming a Ca²⁺-independent enzyme. In the light of the knowledge of the rabbit SR CaM kinase biochemical properties, we propose and discuss the possibility that, under physiological conditions, the activity of the autophosphorylated kinase persists when the Ca²⁺ transient is over.

---

KEY WORDS: calmodulin-dependent protein kinase, sarcoplasmic reticulum, muscle proteins, calcium, phosphorylation

---