TY - JOUR
T1 - Purification of Calmodulin-Stimulated Phosphodiesterase by Affinity Chromatography on Calmodulin Fragment 1–77 Linked to Sepharose
AU - Draetta, Giulio
AU - Klee, Claude B.
N1 - Copyright:
Copyright 2018 Elsevier B.V., All rights reserved.
PY - 1988/1
Y1 - 1988/1
N2 - Because of the presence of many calmodulin-binding proteins in brain, affinity chromatography based on binding to calmodulin lacks specificity. Additional conventional purification steps are needed, prolonging the length of the purification procedure, causing a reduction in yield and increasing the probability of proteolysis and subsequent decrease of calmodulin stimulation. The lack of specificity of the calmodulin affinity chromatography step can be overcome by the use of calmodulin fragments. The N-terminal half-fragment of calmodulin, fragment 1-77, binds cyclic nucleotide phosphodiesterase but does not interact with most of the other calmodulin binding proteins present in brain extracts. Thus, affinity chromatogramaphy on fragment 1-77 coupled to Sepharose can be used to selectively purify the phosphodiesterase. A three-step method, based on the specific interaction of the enzyme with calmodulin 1-77, allows the rapid (2–3days) purification of cyclic nucleotide phosphodiesterase to a high specific activity and with retention of a large stimulation by calmodulin.
AB - Because of the presence of many calmodulin-binding proteins in brain, affinity chromatography based on binding to calmodulin lacks specificity. Additional conventional purification steps are needed, prolonging the length of the purification procedure, causing a reduction in yield and increasing the probability of proteolysis and subsequent decrease of calmodulin stimulation. The lack of specificity of the calmodulin affinity chromatography step can be overcome by the use of calmodulin fragments. The N-terminal half-fragment of calmodulin, fragment 1-77, binds cyclic nucleotide phosphodiesterase but does not interact with most of the other calmodulin binding proteins present in brain extracts. Thus, affinity chromatogramaphy on fragment 1-77 coupled to Sepharose can be used to selectively purify the phosphodiesterase. A three-step method, based on the specific interaction of the enzyme with calmodulin 1-77, allows the rapid (2–3days) purification of cyclic nucleotide phosphodiesterase to a high specific activity and with retention of a large stimulation by calmodulin.
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U2 - 10.1016/0076-6879(88)59055-9
DO - 10.1016/0076-6879(88)59055-9
M3 - Article
C2 - 2842620
AN - SCOPUS:0023708192
SN - 0076-6879
VL - 159
SP - 573
EP - 581
JO - Methods in enzymology
JF - Methods in enzymology
IS - C
ER -