J. Biochem, 1989, Vol. 106, No. 1 133-138
© 1989 Japanese Biochemical Society
research-article |
Topological Investigations. Study of the Trypsin Sensitivity of the iV-Acetylglucosaminyl and Mannosyl-Transferase Activities Located in the Outer Mitochondrial Membrane1
*Department of Biochemistry, University of Lyon INSERM-CNRS U. 189
**Lyon-Sud Medical School 69921 Oullins Cedex, France
2To whom correspondence should be addressed.
The trypsin sensitivity of the mitochondrial N-acetylglucosaminyl and mannosyltransfer-ase activities involved in the N-glycoprotein biosynthesis through dolichol intermediates as well as the N-acetylglucosaminyl-transferase activity involved in direct N-glycosyla-tion were examined in mitochondria and isolated outer mitochondrial membrane preparations. The trypsin action on mitochondrial membrane was checked by measuring the activities of marker enzymes (rotenone-insensitive NADH cytochrome c reductase, adenyl-ate kinase, and monoamine oxidase). Glycosyl-transferase activities of both N-glycosyla-tion pathways were insensitive to trypsin action and consequently were located in the outer mitochondrial membrane. Based on the activator effect of the trypsin on these enzyme activities, the results suggested two distinct orientations of their active sites. As regards the N-glycoprotein biosynthesis pathway through dolichol intermediates, the dolichol-phosphoryl-mannose and dolichol-pyrophosphoryl-di-N-acetylchitobiose synthases would be oriented outside while the oligomannosyl-synthase and the oligomannosyl-transferase would be rather oriented inside in the outer membrane. The N-acetylglucosami-nyl-transferase involved in the direct transfer of N-acetylglucosamine from its nucleotide donor to a proteinic acceptor would be oriented outside in the outer membrane.
1This work was supported by the Institut National de la Sante et de la Recherche Medicate (U. 189), the Centre National de la Recherche Scientifique (SDI n-15720 I), and the University of Lyon (Lyon-Sud Medical School).