Purification of calmodulin-stimulated cyclic nucleotide phosphodiesterase by monoclonal antibody affinity chromatography

Methods Enzymol. 1988;159:543-57. doi: 10.1016/0076-6879(88)59053-5.

Abstract

Immobilized ACC-1 and ACAP-1 antibodies are effective tools for the purification of active calmodulin-dependent phosphodiesterases. ACC-1 antibody binds all bovine and rat brain isozymes in a Ca2+-dependent manner and has been used for their purification. Since ACC-1 binds both bovine brain isozymes (61- and 63-kDa forms) and ACAP-1 recognizes only the 61-kDa isozyme, ACAP-1 can be used to separate and purify the two brain isozymes. The procedures described here for phosphodiesterase isolation from brain are rapid and require few enzymatic assays, resulting in preparations of good purity, specific activity, and yield (Tables II, III). The procedures for brain tissue can be easily adapted for use with larger amounts of tissue. The cross-reactivity of ACP-1 for rat brain phosphodiesterase suggests that this antibody may recognize isozymes from other mammalian tissues.

Publication types

  • Comparative Study

MeSH terms

  • 3',5'-Cyclic-AMP Phosphodiesterases / isolation & purification*
  • 3',5'-Cyclic-AMP Phosphodiesterases / metabolism
  • Animals
  • Antibodies, Monoclonal / isolation & purification
  • Brain / enzymology*
  • Calmodulin / physiology
  • Cattle
  • Chromatography, Affinity / methods
  • Chromatography, DEAE-Cellulose / methods
  • Chromatography, High Pressure Liquid / methods
  • Cyclic Nucleotide Phosphodiesterases, Type 1
  • Indicators and Reagents
  • Isoenzymes / isolation & purification
  • Isoenzymes / metabolism
  • Kinetics
  • Molecular Weight
  • Myocardium / enzymology*
  • Rats

Substances

  • Antibodies, Monoclonal
  • Calmodulin
  • Indicators and Reagents
  • Isoenzymes
  • 3',5'-Cyclic-AMP Phosphodiesterases
  • Cyclic Nucleotide Phosphodiesterases, Type 1