Immobilized Soybean Trypsin Inhibitor in the Stabilization, Resolution and Purification of Adenosine‐3′: 5′‐monophosphate‐Dependent Protein Kinases

Eytan ALHANATY, Shmuel SHALTIEL, Nava BASHAN, Shimon MOSES

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5 Scopus citations

Abstract

The protein kinase dependent on adenosine 3′: 5′‐monophosphate (cAMP) in homogenates of rat intestinal mucosa was found to be remarkably unstable during its purification due (at least in part) to proteolysis. Since soybean trypsin inhibitor was found to protect the kinase from inactivation, an attempt was made to use this inhibitor, coupled to Sepharose, in order to extract and remove the proteolytic enzyme(s) responsible for the instability at an early stage of the purification. Indeed, this column excluded the undissociated form of cAMP‐dependent protein kinase (R2C2) in a considerably more stable form, illustrating the use of immobilized proteolytic inhibitors for stabilizing proteins in crude extracts containing potent proteases. But unexpectedly the same column retarded a protein kinase which was not activated by cAMP. The latter enzyme was identified as the free catalytic subunit (C) of cAMP‐dependent protein kinase since it was found to be inhibited by the Walsh‐Krebs specific protein inhibitor. On the basis of these observations, a method was developed for the purification of C from rabbit muscle which is based on (a) passage of the crude extract on the column and (b) biospecific dissociation of R2C2 by cAMP and rechromatography of the extract on the same column. This procedure yields > 300‐fold purification of C in the last step (with a recovery of 34%) and a rapid resolution between R2C2 and C. The same column can also be used for the resolution of type I and type II cAMP‐dependent protein kinases, and for > 40 fold purification of the type II form of the enzyme, in one step. The theoretical and practical implications of these results with regard to the use and abuse of biorecognition in chromatography are discussed.

Original languageEnglish
Pages (from-to)283-290
Number of pages8
JournalEuropean Journal of Biochemistry
Volume101
Issue number1
DOIs
StatePublished - 1 Jan 1979

ASJC Scopus subject areas

  • Biochemistry

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