Abstract
Spinach leaf peroxisomes were purified by Percoll density gradient centrifugation. After several freeze-thaw cycles, the peroxisomal membranes were separated from the matrix enzymes by sucrose density gradient centrifugation. The purity of the peroxisomal membranes was checked by measuring the activities of marker enzymes and by using antibodies. Lipid bilayer membrane experiments with the purified peroxisomal membranes, solubilized with a detergent, demonstrated that the membranes contain a channel-forming component, which may represent the major permeability pathway of these membranes. Control experiments with membranes of other cell organelles showed that the peroxisomal channel was not caused by the contamination of the peroxisomes with mitochondria or chloroplasts. The peroxisomal channel had a comparatively small single channel conductance of 350 pS in 1 M KCl as compared with channels from other cell organelles. The channel is slightly anion selective, which is in accordance with its physiological function. The single channel conductance was found to be only moderately dependent on the salt concentration in the aqueous phase. This may be explained by the presence of positive point net charges in or near the channel or by the presence of a saturable binding site inside the channel. The possible role of the channel in peroxisomal metabolism is discussed.
Highlights
The peroxisomal channel had a comparatively small single channel conductance of 350 pS in 1 M KCI as compared with channels from other cell organelles
Because of the very low contamination of the peroxisomal suspension by other Acyl-CoA synthetase (ACS)-containing membranes (Table I) the ACS could be used as marker for the peroxisomal membrane
The channel shows a single channel conductance of 350 pS
Summary
Vol 270, No 29, Issue of July 21, pp. 17559-17565, 1995 Printed in U.S.A. (Received for publication, February 14, 1995, and in revised form, April 27, 1995). The intermediates of the reaction chains did not leak out [5] From these findings we concluded that the compartmentation of metabolism in leaf peroxisomes is not due to a boundary function ofthe peroxisomal membrane but is the result ofthe properties of the peroxisomal matrix which allow metabolite channelling [3]. Each subunit contains one diffusion channel for small molecules They are formed entirely of amphipathic l3-sheets arranged in a barrel-like structure [6, 7]. An integral membrane protein showing a high structural homology with mitochondrial anion translocators [19] has been identified in the peroxisomal membrane of the yeast Candida boidinii This suggests that these peroxisomes contain a specific metabolite translocator. We set out to investigate whether leaf peroxisomal membranes contain channel forming activity.
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