Analysis of Calcium-Binding Sites in Calcium-Activated Neutral Protease
Calcium-activated neutral protease (CANP) is a typical intracellular protease responsible for the processing and turnover of various intracellular proteins.1–5 Uncontrolled, CANP can destroy various cellular components, leading to various disease processes like muscular dystrophy. Hence the activity must be tightly regulated. Because CANP absolutely requires calcium for activity, calcium plays a pivotal role in its regulation. Although many of the important steps of CANP activity regulation have been clarified, most studies have not provided any insight into the binding of calcium to CANP6. Structural analyses of CANP have revealed the existence of a calmodulin-like domain in the C-terminal region of each of the two constituent, subunits (80K and 30K subunits).7–9 These domains are presumed to bind calcium and determine the calcium sensitivity of CANP. Nevertheless, the following questions remain unsolved: whether the calmodulin domains indeed bind calcium; whether calcium-binding sites exist only in the calmodulin domains; and, if so, how many calcium ions bind to each domain and which EF hand structures in the calmodulin domains are functional calcium-binding sites.
KeywordsCalcium Binding Regulatory Light Chain Double Reciprocal Plot Calcium Binding Site Calcium Coordination
Unable to display preview. Download preview PDF.
- 4.T. Murachi, M. Hatanaka, and T. Hamakubo, Calpains and neuropeptide metabolism, in “Neuropeptides and their peptidases,” Turner, ed., Ellis Horwood Ltd., Chichester (1987).Google Scholar
- 5.R. Mellgren, Calcium-dependent proteases: an enzyme system active at cellular membrane? FASEB J., 2:110 (1988).Google Scholar
- 9.T. Sakihama, H. Kakidani, K. Zenita, N. Yumoto, T. Kikuchi, T. Sasaki, R. Knnagi, S. Nakanishi, M. Ohmori, K. Takio, K. Titani, and T. Murachi, A putative Ca2+-binding protein: structure of the light subunit of porcine calpain elucidated by molecular cloning and protein sequence analysis, Proc. Natl. Acad. Sci.USA, 82:6075 (1985).PubMedCrossRefGoogle Scholar
- 14.Y. Minami, Y. Emori, S. Imajoh, H. Kawasaki, and K. Suzuki, Carboxy-terminal truncation and site-directed mutagenesis of the small subunit of rabbit calcium-dependent protease, J. Biochem., 103: in press (1988).Google Scholar
- 17.O.H. Lowry, N.I. Rosenbrough, A.L. Farr, and R.J. Randall, Protein measurement with the Folin-phenol reagent, J. Biol. Chem., 193: 265 (1955).Google Scholar