Methods in Enzymology Vol.139 Cellular Regulators, Part A: Calcium- and Calmodulin-Binding Proteins

This chapter outlines analytical and preparative details for use of affinity matrix. An affinity matrix of immobilized melittin is prepared for purification of calmodulin (CaM). Melittin, the major component of bee venom, is a 26 amino acid peptide that is amphipathic. It is noted that melittin inhibited cyclic nucleotide phosphodiesterase and that inhibition is dependent on Ca2+ and competitive with CaM. The affinity of melittin for CaM is estimated to be in the nanomolar range. Because of this high binding constant, as well as its well-defined properties and availability, it seems as an appropriate ligand for the Ca2+-dependent affinity chromatography of CaM and perhaps other Ca2+-binding proteins. Studies have shown that melittin-Sepharose is suitable for large-scale purification of CaM and S-100 protein from brain and that, in some tissues, purification of CaM to an essentially homogeneous state is accomplished in a single step. An important step in preparation of an affinity matrix is to establish the optimum relationship between substituent concentration on the gel and binding capacity/recovery. The speed, reproducibility, and scale-up potential of this procedure, prove useful for investigators involved in physical and structural studies of the two proteins where large quantities of protein are necessary.

• Series: Methods in Enzymology (Book 139)
• Hardcover: 916 pages
• Publisher: Academic Press;
  
• Language: English
• ISBN-10: 0121820394
• ISBN-13: 978-0121820398
• Product Dimensions: 6 x 9 inches

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