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Identification and partial purification of a heart mitochondrial membrane proteinase

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Abstract

Membrane-bound proteinase activity was demonstrated by a solid-phase assay system in both beef heart and rat liver mitochondria. The activity was sensitive to SH reagents and assorted proteinase inhibitors. Although stimulated by nonionic detergents, it became labile when solubilized by detergents. The proteinase activity from heart mitochondria copurified with the ADP:ATP translocator protein. Gel electrophoresis of this preparation revealed the translocator polypeptide as well as a number of minor components. In solubilized mitochondria the ADP:ATP translocator polypeptide slowly disappeared upon standing at 0°C as revealed by polyacrylamide gel electrophoresis under denaturing conditions. The loss of this polypeptide was prevented by addition of proteinase inhibitors as well as the translocator affinity ligand, carboxyatractylate. These observations confirm the presence of an integral membrane proteinase in mitochondria and suggest a structural and enzymatic interaction between the proteinase and the ADP:ATP translocator.

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Abbreviations

PMSF:

phenylmethanesulfonyl fluoride

TPCK:

l-1-tosylamido-2-phenylethylchloromethyl ketone

TLCK:

1-chloro-3-tosylamido-7-amino-l-2-heptanone

NEM:

N-ethylmaleimide

PCMBS:

p-chloromercuriphenylsulfonic acid

SDS:

sodium dodecyl sulfate

MOPS:

morpholinopropane sulfonate

[I50]:

concentration of inhibitor required to give 50% inhibition

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Authors and Affiliations

  1. Department of Biochemistry, School of Medicine, Oregon Health Sciences University, 97201, Portland, Oregon

    James F. Hare

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  1. James F. Hare
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Hare, J.F. Identification and partial purification of a heart mitochondrial membrane proteinase. J Bioenerg Biomembr 15, 195–206 (1983). https://doi.org/10.1007/BF00743940

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  • DOI: https://doi.org/10.1007/BF00743940

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