Identification and purification of a 10-kilodalton protein associated with mitochondrial benzodiazepine receptors

J Biol Chem. 1995 Sep 1;270(35):20285-91. doi: 10.1074/jbc.270.35.20285.

Abstract

The isoquinoline carboxamide photoaffinity probe PK14105, a ligand with selectivity for mitochondrial benzodiazepine receptors, has been established to photolabel an 18-kDa protein. When this radioactive probe is used to photolabel rat mitochondrial preparations, a protein of 10 kDa, in addition to the 18-kDa protein, is identified following electrophoretic separation and extended autoradiography. These proteins are referred to herein as pk10 and pk18, respectively. Both proteins exhibited the same specificity to a series of ligands used in competition photolabeling studies and are mutually present at apparently similar ratios across multiple tissues. Subcellular fractionation of rat adrenals indicated that pk10 and pk18 comigrated with the mitochondrial marker enzyme cytochrome c oxidase. In numerous paradigms examining specificity, photolabeling of pk18 invariably coincided with photolabeling of pk10. In detergent-solubilized extracts of rat adrenal mitochondria, pk18 and pk10 coimmunoprecipitated when using antisera raised against pk18. Furthermore, purification of the photolabeled proteins using nondenaturing conditions demonstrated that pk18 and pk10 copurify substantiating their intimate association. A set of three antisera, specific to different regions of pk18, did not recognize pk10 on Western blots. Likewise, partial amino acid sequence of peptide fragments indicate that pk10 is not derived from proteolytic cleavage of pk18. These data suggest that pk10 represents another component of mitochondrial benzodiazepine receptors whose identity is not apparent with any known protein.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adrenal Cortex / metabolism*
  • Affinity Labels
  • Amino Acid Sequence
  • Animals
  • Antibodies
  • Benzodiazepinones / metabolism
  • Binding, Competitive
  • Blotting, Western
  • Cattle
  • Convulsants / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Isoquinolines / metabolism*
  • Kinetics
  • Macromolecular Substances
  • Mitochondria / metabolism*
  • Molecular Sequence Data
  • Molecular Weight
  • Organ Specificity
  • Peptides / chemical synthesis
  • Peptides / chemistry
  • Peptides / immunology
  • Rats
  • Rats, Sprague-Dawley
  • Receptors, GABA-A / chemistry*
  • Receptors, GABA-A / isolation & purification
  • Receptors, GABA-A / metabolism
  • Substrate Specificity
  • Temperature

Substances

  • Affinity Labels
  • Antibodies
  • Benzodiazepinones
  • Convulsants
  • Isoquinolines
  • Macromolecular Substances
  • Peptides
  • Receptors, GABA-A
  • 4'-chlorodiazepam
  • PK 14105