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• W2071366123 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. 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. Leaf peroxisomes belong to the microbodies, a group of small multipurpose cell organelles which are found in all, except some very primitive, eukaryotic cells(1Cavalier-Smith T. Nature. 1987; 326: 332-333Crossref PubMed Scopus (263) Google Scholar). They are surrounded by a single membrane. Peroxisomes usually, although not always, contain H2O2 producing enzymes and catalase to eliminate the H2O2(2Borst P. Biochim. Biophys. Acta. 1989; 1008: 1-13Crossref PubMed Scopus (141) Google Scholar). They all contain the enzymes for β-oxidation of fatty acids. Present evidence suggests that peroxisomes are not formed de novo but grow and divide like plastids and mitochondria, although they have no genome of their own. There are indications that all peroxisomes, e.g. from fungi, higher plants, and animals, have a common origin, possibly an endosymbiotic event(2Borst P. Biochim. Biophys. Acta. 1989; 1008: 1-13Crossref PubMed Scopus (141) Google Scholar). Leaf peroxisomes play a vital role in photosynthesis. They are involved in the recycling of glycolate formed as an unavoidable by-product of CO2 fixation due to oxygen reacting instead of CO2 with ribulose bisphosphate. In a leaf the ratio of oxigenation/carboxylation during photosynthesis is between 0.2 and 0.5, resulting in very high metabolic fluxes through the leaf peroxisomes. The peroxisomal reaction chains are strictly compartmentalized. When leaf peroxisomes were subjected to an “osmotic shock” the peroxisomal membrane was damaged extensively, but surprisingly the peroxisomal matrix did not disintegrate and its metabolic function, including the high compartmentation of metabolism, was unaltered(3Heupel R. Markgraf T. Robinson D.G. Heldt H.W. Plant. Physiol. 1991; 96: 971-979Crossref PubMed Scopus (44) Google Scholar, 4Reumann S. Heupel R. Heldt H.W. Planta. 1994; 193: 167-173Crossref Scopus (35) Google Scholar). The intermediates of the reaction chains did not leak out(5Heupel R. Heldt H.W. Eur. J. Biochem. 1994; 220: 165-172Crossref PubMed Scopus (53) Google Scholar). From these findings we concluded that the compartmentation of metabolism in leaf peroxisomes is not due to a boundary function of the peroxisomal membrane but is the result of the properties of the peroxisomal matrix which allow metabolite channelling(3Heupel R. Markgraf T. Robinson D.G. Heldt H.W. Plant. Physiol. 1991; 96: 971-979Crossref PubMed Scopus (44) Google Scholar). Our results suggest that specific translocators are not essential for the functional compartmentation of metabolism. Nonspecific pores seem sufficient for the metabolite transfer into and out of the leaf peroxisomes. General diffusion channels, called porins, exist in the outer membrane of Gram-negative eubacteria (for review, see (6Benz R. Ghuysen J.-R. Hakenbeck R. Bacterial Cell Wall. Elsevier Science B.V., Amsterdam1994: 397-423Google Scholar)), mitochondria(7Benz R. Biochim. Biophys. Acta. 1994; 1197: 167-196Crossref PubMed Scopus (388) Google Scholar), and plastids(8Flügge U.I. Benz R. FEBS Lett. 1984; 169: 85-89Crossref Scopus (100) Google Scholar, 9Fischer K. Weber A. Brink S. Arbinger B. Schünemann D. Borchert S. Heldt H.W. Popp B. Benz R. Link T.A. Eckerskorn C. Flügge U.I. J. Biol. Chem. 1994; 269: 25754-25760Abstract Full Text PDF PubMed Google Scholar). Bacterial porins occur as trimers of three identical subunits. Each subunit contains one diffusion channel for small molecules. They are formed entirely of amphipathic β-sheets arranged in a barrel-like structure(6Benz R. Ghuysen J.-R. Hakenbeck R. Bacterial Cell Wall. Elsevier Science B.V., Amsterdam1994: 397-423Google Scholar, 7Benz R. Biochim. Biophys. Acta. 1994; 1197: 167-196Crossref PubMed Scopus (388) Google Scholar). The results are conflicting as to whether other types of peroxisomes contain a pore-forming protein or specific translocators. Liver peroxisomes were found to be permeable to sucrose and nucleotides(10van Veldhoven P.P. Debeer L.J. Mannaerts G.P. Biochem. J. 1983; 210: 685-693Crossref PubMed Scopus (44) Google Scholar). Upon reconstitution experiments a channel forming activity was attributed to a 22-kDa membrane popypeptide(11van Veldhoven P. Just W.W. Mannaerts G.P. J. Biol. Chem. 1987; 262: 4310-4318Abstract Full Text PDF PubMed Google Scholar), but the subsequent analysis of the amino acid sequence did not show any similarity to known porin structures(12Kaldi K. Diestelkötter P. Stenbeck G. Auerbach S. Jäkle U. Mägert H.J. Wieland F.T. Just W.W. FEBS Lett. 1993; 315: 217-222Crossref PubMed Scopus (36) Google Scholar). Other experiments incorporating membrane preparations of liver peroxisomes into liposomes, using the patch clamp technique(13Lemmens M. Verheyden K. van Veldhoven P. Vereecke J. Mannaerts G.P. Carmeliet E. Biochim. Biophys. Acta. 1989; 984: 351-359Crossref PubMed Scopus (44) Google Scholar), and into planar lipid bilayers (14Labarca P. Wolff D. Soto U. Necochea C. Leighton F. J. Membr. Biol. 1986; 94: 285-291Crossref PubMed Scopus (46) Google Scholar) indicated that large cation-selective voltage-gated pores with an estimated diameter of 1.5-3.0 nm may be responsible for the high permeability of liver peroxisomes. In the yeast Hansenula polymorpha, a 31-kDa peroxisomal integral membrane protein, with a structure similar to the 31-kDa mitochondrial porin of Saccharomyces cerevisiae, was claimed to be responsible for the in vitro permeability of the peroxisomes(15Sulter G.J. Verheyden K. Mannaerts G. Harder W. Veenhuis M. Yeast. 1993; 9: 733-742Crossref PubMed Scopus (21) Google Scholar). Some results are not consistent with the presence of a free diffusion channel in peroxisomes. Yeast peroxisomal membranes contain a proton-translocating ATPase(16Douma A.C. Veenhuis M. Sulter G.J. Harder W. Arch. Microbiol. 1987; 147: 42-47Crossref PubMed Scopus (55) Google Scholar), and a proton gradient across the peroxisomal membrane has been observed(17Nicolay K. Veenhuis M. Douma A.C. Harder W. Arch. Microbiol. 1987; 147: 37-41Crossref PubMed Scopus (94) Google Scholar). An ATPase activity was also found in the membranes of liver peroxisomes(18Kamijo K. Taketani S. Yokota S. Osumi T. Hashimoto T. J. Biol. Chem. 1990; 265: 4534-4540Abstract Full Text PDF PubMed Google Scholar). An integral membrane protein showing a high structural homology with mitochondrial anion translocators (19Jank B. Habermann B. Schweyen R.J. Trends Biochem. Sci. 1993; 18: 427-428Abstract Full Text PDF PubMed Scopus (3) Google Scholar) has been identified in the peroxisomal membrane of the yeast Candida boidinii. This suggests that these peroxisomes contain a specific metabolite translocator. The boundary function of the peroxisomal membrane is thus still a matter of debate. We set out to investigate whether leaf peroxisomal membranes contain channel forming activity. This report shows that leaf peroxisomal membranes contain channels allowing the passage of the metabolites of the photorespiratory metabolism and which are distinctly different from the porin channels of leaf mitochondria and chloroplasts. Peroxisomes, mitochondria, and chloroplasts were isolated from leaves of spinach (Spinacia oleracea L., U.S Hybrid 424; Ferry-Morse Seed Company, Mountain View, CA). The plants were grown and harvested as described(4Reumann S. Heupel R. Heldt H.W. Planta. 1994; 193: 167-173Crossref Scopus (35) Google Scholar). Peroxisomes were isolated by modifying to the method of Yu and Huang(20Yu C. Huang A.H.C. Arch. Biochem. Biophys. 1986; 245: 125-133Crossref PubMed Scopus (23) Google Scholar). The scale was increased by a factor of five up to 350 g of leaves, and the homogenization procedure was intensified. The final peroxisomal pellet was resuspended and stored at −20°C. The yield of peroxisomes was about 6 mg of protein with a specific activity of hydroxypyruvate reductase of about 19 εmol•(min•mg)−1 (see Table 1). The intactness measured as latency of hydroxypyruvate reductase (3Heupel R. Markgraf T. Robinson D.G. Heldt H.W. Plant. Physiol. 1991; 96: 971-979Crossref PubMed Scopus (44) Google Scholar) was about 95%.Tabled 1 Open table in a new tab The highly aggregated structure of the matrix enzymes was destroyed by subjecting the organelles (12 mg of protein) to five freeze-thaw cycles (freezing in liquid nitrogen, thawing at room temperature) and intensive homogenization in a Potter homogenizer. The suspension was adjusted to 30% (w/w) sucrose, loaded on a linear sucrose gradient (35-60% (w/w) sucrose in 10 mM HEPES, pH 7.5, 0.8 mM MgCl2), and centrifuged in a swing-out rotor (240,000 × g, 15 h, Sorvall AH 650). The gradient was fractionated from the top (12 fractions of 0.4 ml). The fractions were diluted to a sucrose content of 30% (w/w) and stored at −80°C. All the data of enzyme activities (Fig. 1), SDS-PAGE1 1The abbreviation used is: PAGEpolyacrylamide gel electrophoresis. and Western blot analysis (Fig. 2), and porin activity (Fig. 6) were obtained with the same gradient and confirmed by additional experiments.Figure 2:Western blot analysis as control for membrane contamination of the peroxisomal membrane fractions and SDS-PAGE. Fractions of the sucrose gradient were subjected to SDS-PAGE (A) and immunoblotted with a polyclonal antibody against the 24-kDa protein of the outer envelope membrane of spinach chloroplasts (B) or with a polyclonal antibody against the 30-kDa porin of pea root plastids, showing strong cross-reactivity with the 30-kDa porin of the outer membrane of spinach mitochondria (C) as described under “Experimental Procedures.” For Western blot analysis in each lane, 20 εg of protein and for silver stain 3 εg of protein were separated by SDS-PAGE. Fractions 8+9 and 10+11 were pooled in a protein ratio of 1:1 because of their minor protein content. As a control 5 and 20 εg of protein of spinach chloroplast envelope membranes and mitochondrial membranes were blotted. P, peroxisomal suspension.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6:Comparison of porin activity and content of peroxisomal membrane in the fractions of the sucrose gradient. The concentration of peroxisomal membrane was measured on the basis of the specific ACS activity. The channel-forming activity was determined as explained in the text by using membranes from diphytanoyl phosphatidylcholine/n-decane. The voltage applied was 10 mV; T = 25°C.View Large Image Figure ViewerDownload Hi-res image Download (PPT) polyacrylamide gel electrophoresis. Chloroplasts were isolated according to Heldt and Sauer (21Heldt H.W. Sauer F. Biochim. Biophys. Acta. 1971; 234: 83-91Crossref PubMed Scopus (227) Google Scholar) and chloroplast envelope membranes according to Douce et al.(22Douce R. Holtz R.B. Benson A.A. J. Biol. Chem. 1973; 248: 7215-7222Abstract Full Text PDF PubMed Google Scholar). Mitochondria were purified according to the method of Neuburger et al.(23Neuburger M. Journet E.-P. Bligny R. Carde J.P. Douce R. Arch. Biochem. Biophys. 1982; 217: 312-323Crossref PubMed Scopus (249) Google Scholar). The mitochondria were disrupted osmotically by incubation for 30 min in distilled water on ice and sedimented afterward (160,000 × g, 45 min, Kontron TFT 65.13). If not stated otherwise, measurements of marker enzyme activities were carried out at 25°C in a final volume of 1 ml. Hydroxypyruvate reductase and catalase were measured as described previously(3Heupel R. Markgraf T. Robinson D.G. Heldt H.W. Plant. Physiol. 1991; 96: 971-979Crossref PubMed Scopus (44) Google Scholar). Acyl-CoA synthetase (ACS) was measured as described by Fischer et al.(9Fischer K. Weber A. Brink S. Arbinger B. Schünemann D. Borchert S. Heldt H.W. Popp B. Benz R. Link T.A. Eckerskorn C. Flügge U.I. J. Biol. Chem. 1994; 269: 25754-25760Abstract Full Text PDF PubMed Google Scholar). The reaction was stopped after incubation for 10 min at 30°C. The measurement of NADH- ferricyanide reductase (700 εl)(24Douce R. Mannella C.A. Bonner Jr., W.D. Biochim. Biophys. Acta. 1973; 292: 105-116Crossref PubMed Scopus (148) Google Scholar), NADH-cytochrome c reductase(25Sauer A. Robinson D.G. Planta. 1985; 166: 227-233Crossref PubMed Scopus (20) Google Scholar), cytochrome c oxidase (700 εl) (24Douce R. Mannella C.A. Bonner Jr., W.D. Biochim. Biophys. Acta. 1973; 292: 105-116Crossref PubMed Scopus (148) Google Scholar), and NADP-glycerinaldehyde-3-phosphate dehydrogenase (26Gerhardt R. Heldt H.W. Plant Physiol. 1984; 75: 542-547Crossref PubMed Google Scholar) was performed as described. Protein was determined according to Peterson (27Peterson G.L. Anal. Biochem. 1977; 83: 346-356Crossref PubMed Scopus (7132) Google Scholar). Precipitation of protein fractions was carried out using chloroform-methanol(28Wessel D. Flügge U.-I. Anal. Biochem. 1984; 138: 141-143Crossref PubMed Scopus (3169) Google Scholar). SDS-PAGE was performed on 12.5% gels according to Laemmli(29Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207159) Google Scholar). Polypeptide bands were made visible by silver staining(30Blum H. Beier H. Gross H.J. Electrophoresis. 1987; 8: 93-99Crossref Scopus (3741) Google Scholar). In Western blot analysis the bound antibodies were made visible with a peroxidase-coupled second antibody using an ECL-kit (Amersham-Buchler, Braunschweig, Germany). The methods used for the bilayer experiments have been described in detail(31Benz R. Janko K. Boos W. Läuger P. Biochim. Biophys. Acta. 1978; 511: 305-319Crossref PubMed Scopus (413) Google Scholar, 32Schmid A. Krömer S. Heldt H.W. Benz R. Biochim. Biophys. Acta. 1992; 1112: 174-180Crossref PubMed Scopus (22) Google Scholar). Peroxisomal membranes were solubilized in 0.5% Genapol X-80 (Fluka, Neu-Ulm) and added to the aqueous phases at one or both sides of the black membranes. From measurement of the activities of hydroxypyruvate reductase and catalase as marker enzymes for the peroxisomal matrix, the yield of the peroxisomes obtained from spinach leaves was evaluated as about 5 and 11% (Table 1). This low yield reflects the difficulties of peroxisomal isolation and is inherent to all published preparation procedures(20Yu C. Huang A.H.C. Arch. Biochem. Biophys. 1986; 245: 125-133Crossref PubMed Scopus (23) Google Scholar, 33Schmitt M.R. Edwards G.E. Plant Physiol. 1983; 72: 728-734Crossref PubMed Google Scholar). The contamination of the peroxisomal preparation with chloroplasts, mitochondria, and ER is quite low, as the recoveries of the corresponding marker enzyme activities were 0.05, 0.2, and 0.01% as compared to the starting homogenate (Table 1). From this the contamination of the peroxisomal fraction by mitochondria, chloroplasts, and ER can be evaluated as less than 1, 4, and 0.2%, respectively. The purity of the peroxisomal suspension has been checked earlier by electron microscopy(3Heupel R. Markgraf T. Robinson D.G. Heldt H.W. Plant. Physiol. 1991; 96: 971-979Crossref PubMed Scopus (44) Google Scholar). The most used method for the isolation of peroxisomal membranes of rat liver (11van Veldhoven P. Just W.W. Mannaerts G.P. J. Biol. Chem. 1987; 262: 4310-4318Abstract Full Text PDF PubMed Google Scholar, 34Fujiki Y. Fowler S. Shio H. Hubbard A.L. Lazarow P.B. J. Cell Biol. 1982; 93: 103-110Crossref PubMed Scopus (288) Google Scholar) and glyoxysomes (35Chapman K.D. Trelease R.N. Plant Physiol. Biochem. 1992; 30: 1-10Google Scholar, 36Wolins N.E. Donaldson R.P. J. Biol. Chem. 1994; 269: 1149-1153Abstract Full Text PDF PubMed Google Scholar) is treating the peroxisomes with 100 mM sodium carbonate at pH 11.5(37Fujiki Y. Hubbard A.L. Fowler S. Lazarow P.B. J. Cell Biol. 1982; 93: 97-102Crossref PubMed Scopus (1382) Google Scholar). This method was unsuitable because our measurements indicated a destruction of the porin activity. After various attempts to solubilize the highly aggregated matrix enzymes, we found it best to disrupt the peroxisomes mechanically (see “Experimental Procedures”) and to separate the membranes by centrifugation in a sucrose density gradient. At present, unfortunately, there is no specific marker enzyme for the peroxisomal membrane of leaves. ACS, which has been shown to be closely associated with the membranes of leaf peroxisomes (38Gerhardt B. Physiol. Vég. 1986; 24: 397-410Google Scholar) and glyoxysomes (a differentiation form of plant peroxisomes), is present in the outer membrane of the chloroplast envelope (39Andrews J. Keegstra K. Plant Physiol. 1983; 72: 735-740Crossref PubMed Google Scholar, 40Block M.A. Dorne A.J. Joyard J. Douce R. FEBS Lett. 1983; 153: 377-381Crossref Scopus (55) Google Scholar, 41Joyard J. Stumpf P.K. Plant Physiol. 1981; 67: 250-256Crossref PubMed Google Scholar) and also in the microsomal membranes(42Lessire R. Cassagne C. Plant Sci. Lett. 1979; 16: 31-39Crossref Scopus (13) Google Scholar, 43Sukumar V. Sastry P.S. Biochem. Int. 1987; 14: 719-726Google Scholar, 44Ichihara K. Nakagawa M. Tanaka K. Plant Cell Physiol. 1993; 34: 557-566Google Scholar). It is possible that the outer membranes of plant mitochondria also contain this enzyme(45Frentzen M. Neuburger M. Joyard J. Douce R. Eur. J. Biochem. 1990; 187: 395-402Crossref PubMed Scopus (24) Google Scholar). The results of Table 1 show that only a minor portion of the cellular ACS activity is associated with the peroxisomes. Despite this, because of the very low contamination of the peroxisomal suspension by other ACS-containing membranes (Table 1) the ACS could be used as marker for the peroxisomal membrane. If 50% of the ACS activity were associated with the chloroplasts, the ACS activity from contaminating chloroplasts in the peroxisomal preparation would be less than 25% of the measured ACS activity. The same calculation with ER results in 1%. In the experiment shown in Fig. 1, the activities of marker enzymes for the various subcellular components have been measured in the different fractions of the sucrose density gradient. Most of the protein (about 80%) and the marker enzyme for the peroxisomal matrix catalase and hydroxypyruvate reductase are found in the upper part of the gradient. These fractions represent the peroxisomal matrix proteins. The ACS activity forms a distinct peak at a density of 1.21-1.23 g/ml. Control experiments by measuring specific marker enzymes and Western blot analysis of all fractions of the sucrose gradient excluded that this ACS peak resulted from nonperoxisomal sources. 1) One of the major constituents of the outer envelope membrane of spinach chloroplasts is a 24-kDa protein which function is unknown until now(46Fischer K. Weber A. Arbinger B. Brink S. Eckerskorn C. Flügge U.I. Plant Mol. Biol. 1994; 25: 167-177Crossref PubMed Scopus (35) Google Scholar). Using an antibody against this protein, we could localize the contaminating outer envelope membrane in the first fraction of the sucrose gradient (Fig. 2B) at its low equilibrium density concurring with earlier results(39Andrews J. Keegstra K. Plant Physiol. 1983; 72: 735-740Crossref PubMed Google Scholar, 47Block M.A. Dorne A.J. Joyard J Douce R. J. Biol. Chem. 1983; 258: 13273-13280Abstract Full Text PDF PubMed Google Scholar). The 24-kDa protein was not detectable in the fractions containing ACS activity indicating that the outer envelope membrane is absent in these fractions. 2) NADH-cytochrome c reductase, a marker enzyme for ER membranes(25Sauer A. Robinson D.G. Planta. 1985; 166: 227-233Crossref PubMed Scopus (20) Google Scholar), was found at low activities on the top of the gradient at a density of 1.15 g/ml as shown earlier(25Sauer A. Robinson D.G. Planta. 1985; 166: 227-233Crossref PubMed Scopus (20) Google Scholar). 3) The outer membrane of mitochondria was detected with the marker enzyme NADH-ferricyanide reductase (22Douce R. Holtz R.B. Benson A.A. J. Biol. Chem. 1973; 248: 7215-7222Abstract Full Text PDF PubMed Google Scholar) and in addition by using polyclonal antibodies against the 30-kDa porin of non-green pea root plastids(9Fischer K. Weber A. Brink S. Arbinger B. Schünemann D. Borchert S. Heldt H.W. Popp B. Benz R. Link T.A. Eckerskorn C. Flügge U.I. J. Biol. Chem. 1994; 269: 25754-25760Abstract Full Text PDF PubMed Google Scholar). As the porins of plant non-green plastids and mitochondria are relatively homologous proteins(9Fischer K. Weber A. Brink S. Arbinger B. Schünemann D. Borchert S. Heldt H.W. Popp B. Benz R. Link T.A. Eckerskorn C. Flügge U.I. J. Biol. Chem. 1994; 269: 25754-25760Abstract Full Text PDF PubMed Google Scholar), these antibodies show cross-reaction with the mitochondrial 30-kDa porin of spinach (Fig. 2C) and are thus a useful marker for the detection of the outer membrane of spinach mitochondria. Using both methods we were able to show that the content of the outer mitochondrial membrane in that part of the gradient where the maximal ACS activity is localized is rather low. The cross-reaction with a polypeptide of 66 kDa in fractions 10-12 could be an nonspecific artifact. The activity of NADH-ferricyanide reductase in the upper part of the gradient (fractions 1-4) may be due to peroxisomal activity as potato tuber peroxisomes were shown to possess this enzyme(48Struglics A. Fredlund K.M. Rasmusson A.G.M.I.M. Physiologia Plantarum. 1993; 88: 19-28Crossref Scopus (77) Google Scholar). Thylakoid membranes were enriched at 1.17-1.18 g/ml (data not shown) as reported earlier(49Ludwig B. Kindl H. Hoppe-Seyler's Z. Physiol. Chem. 1976; 357: 177-186Crossref PubMed Scopus (12) Google Scholar). As the ACS peak contained neither outer chloroplast envelope membranes, outer mitochondrial membranes, nor ER membranes to any detectable extent, it can be concluded that the ACS peak represents the peroxisomal membrane and that this membrane is not contaminated with outer membranes of chloroplasts or mitochondria to any appreciable amount. As the outer mitochondrial and outer chloroplast envelope membranes both contain porins, the absence of these membranes in the peroxisomal membrane fraction is essential. In gradients with incompletely disrupted peroxisomes, the peroxisomal membrane could be identified from the adhering activities of the peroxisomal enzymes catalase, hydroxypyruvate reductase, and malate dehydrogenase, forming a second smaller peak at 1.215 g/ml. Glyoxysomal membranes have been reported to equilibrate at this density(50Preisig-Müller R. Muster G. Kindl H. Eur. J. Biochem. 1994; 219: 57-63Crossref PubMed Scopus (33) Google Scholar). SDS-PAGE was performed with the fractions 1-12 of the same gradient (Fig. 2A). The dominant proteins of the peroxisomal membrane had a subunit molecular mass of about 50, 48, 45, 43, 39, 32, and 13 kDa. In further experiments we investigated whether peroxisomes contain any pore forming activity. The sedimented peroxisomes (protein concentration about 1 mg/ml) were treated with the detergent Genapol X-80 (final concentration 0.5%) to solubilize the peroxisomal membrane. The detergent extract was added to the aqueous phase, bathing a lipid bilayer, and the membrane current was measured. Fig. 3 demonstrates that there is indeed a pore forming activity in these detergent extracts. Each conductance step of Fig. 3 corresponds to the incorporation of one channel-forming unit into the membrane. The average single channel conductance of these channels is only 350 pS in 1 M KCl (see the histogram in Fig. 4). The occurrence of a single channel conductance of 600 pS probably indicates the incorporation of a dimer. The conductance of the peroxisomal channel is rather small as compared with those channels formed by mitochondrial or chloroplast porins under otherwise identical conditions (see also below).Figure 4:Histogram of the probability of the occurrence of certain conductivity units observed with membranes formed of diphytanoyl phosphatidylcholine/n-decane in the presence of 1 εg/ml detergent-solubilized spinach leaf peroxisomes. The aqueous phase contained 1 M KCl. The applied membrane potential was 10 mV; T = 25°C. The average single channel conductance was 350 pS for 344 single channel events. The data were collected from 10 different membranes. P(G), probability of the single channel conductance G.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The channel formed by the detergent extracts from whole peroxisomes had a completely different single channel conductance as compared with the porins from other plant cell membranes. To give further direct evidence that the origin of the novel channel-forming protein is the peroxisomal membrane, we investigated the channel forming activity of the various fractions of the sucrose gradient as follows: small quantities of the fractions were dissolved in Genapol X-80 and added to the aqueous phase on both sides of an artificial bilayer (final protein concentration in the aqueous phase 0.6 εg/ml). After a lag time of a few minutes, probably caused by slow aqueous diffusion of the protein, the conductance of the membrane, caused by the insertion of channels into the membrane, started to increase. The time course of the increase was similar to that described previously for porins of mitochondrial or bacterial origin(51Roos N. Benz R. Brdiczka D. Biochim. Biophys. Acta. 1982; 686: 204-214Crossref PubMed Scopus (145) Google Scholar, 52Benz R. Ishii J. Nakae T. J. Membr. Biol. 1980; 56: 19-29Crossref PubMed Scopus (60) Google Scholar). The number of inserted pores with a single channel conductance of less than 1 nS in a given time (20 min) was counted. The distribution of these conductance steps in a histogram (data not shown) was similar to that shown for whole peroxisomes (Fig. 4). The mean values of three measurements were taken as a semiquantitative measure for the peroxisomal channel forming activity of the sample. After the insertion of more than 30-50 pores in one black membrane, the single channel conductance could not be clearly identified (see Figure 3:, Figure 4:, Figure 5:). As the channel incorporation was so frequent for the fractions containing the peroxisomal membranes, several bilayers had to be painted during the measuring period. Control experiments showed that the addition of the detergent Genapol X-80 alone at a similar concentration to that used with the protein did not lead to any appreciable increase in the membrane conductance. We were able to identify three different types of channels in the protein samples taken from the sucrose density gradient. The light fractions contained (besides the 350 pS channel; see Fig. 5A) a channel with a giant single channel conductance of 7-9 nS in 1 M KCl. This channel was similar to that found in reconstitution experiments with the outer membrane of the chloroplast envelope (see Fig. 5C; 8). In other fractions (preferentially fractions 2-3), we sometimes observed an additional channel, which was indistinguishable from mitochondrial porin from plant and other sources (see Fig. 5B; 7). The most prominent channel, however, in all fractions was the 350 pS channel. Fig. 6 shows the distribution of this channel within the fraction of the sucrose density gradient. It is noteworthy that the peroxisomal porin activity measured as the number of channels (20 min after the protein addition) in a membrane with a surface area of 1 mm2 corresponded to the 350 pS channel only. When one of the other channels did happen to incorporate into the lipid bilayer membrane, the experiment was stopped and a new experiment was started. This was necessary because of the much higher single channel conductance of the mitochondrial and chloroplast porins. Fig. 6 shows also the specific activity of ACS, i.e. the content of peroxisomal membrane in the fractions of the sucrose density gradient. There is a correlation between the specific ACS activity and the pore forming activity showing that the 350 pS channel resides within the peroxisomal membrane. After the addition of whole peroxisomes or peroxisomal membranes, which had been solubilized with Genapol X-80, step increases in membrane conductance could be resolved (see Figure 3:, Figure 4:, Figure 5:). The average single channel conductance with 1 M KCl was 350 pS (see the histogram of Fig. 4). Figure 3:, Figure 4:, Figure 5: show also that most of the steps were directed upwards. Only a few downward steps were observed which means that the peroxisomal channels had a long lifetime. Even at higher transmembrane potentials of about 50 mV the closing events did not become more frequent. This demonstrates that the peroxisomal porin is not voltage-regulated at these potentials. The channel formed by peroxisomal porin was permeable for a variety of ions. Table 2 summarizes the single channel conductance for different salt solutions. The nature of the anions had a substantial influence on the single channel conductance, whereas the influence of the cations was rather small. Thus, the single channel conductance in 1 M KCl was approximately the same as in 1 M LiCl, but it was considerably smaller in 1 M potassium acetate (K+ and Cl− and Li+ and acetate have the same aqueous mobility)(53Castellan G.W. Physical Chemistry. Addison-Wesley, Reading MA1983: 769-780Google Scholar). This result suggests that the channel had a certain preference for anions. We found that a variety of organic anions, such as formiate, glycerate, and glycolate are permeating this channel. It is noteworthy, that the conductance of the channel was not a linear function of the bulk aqueous concentration, which may indicate that positive point net charges are localized in or near the channel mouth. On the other hand, it is also possible that the channel contains a binding site for organic anions, which facilitates the diffusion of these ions in a similar way as specific bacterial porins do for certain substrates such as sugars, nucleosides, and phosphate(6Benz R. Ghuysen J.-R. Hakenbeck R. Bacterial Cell Wall. Elsevier Science B.V., Amsterdam1994: 397-423Google Scholar).Tabled 1 Open table in a new tab The single channel data suggested that the peroxisomal channel is anion-selective. We performed zero current membrane potential measurements to study its ion selectivity in more detail. Membranes were formed in 100 mM KCl, and detergent-solubilized peroxisomal membrane was added to the aqueous phase when the membranes were in the black state. After incorporation of 100-1000 channels into a membrane, salt gradients were established by addition of small amounts of 3 M KCl solution to one side of the membrane. Under these conditions, the more diluted side of the membrane became negative which indicated indeed preferential movement of chloride over potassium. However, the potential and the permeability ratio between potassium and chloride (as calculated from the Goldman-Hodgkin-Katz equation; 54) differed considerably from experiment to experiment, which probably means that other channels such as mitochondrial porin or the chloroplast porin interfered with the measurements and led to irreproducible results. This is because the single channel conductance of the peroxisomal porin (350 pS) is about 6-12 times smaller than that of mitochondrial porin (2.4 or 4.0 nS) and about 23 times smaller than that of chloroplast porin (8 nS) under otherwise identical conditions (1 M KCl). The peroxisomal membrane of leaf peroxisomes contains a channel-forming protein that is different from those of other cell organelles. The channel shows a single channel conductance of 350 pS (1 M KCl) which is much lower than those formed by the porins of mitochondria and chloroplasts under otherwise identical conditions (see Table 3). Furthermore, the single channel conductance in salts containing organic anions is much smaller as expected from the mobility of these ions in the aqueous phase. Apparently the peroxisomal channel is not a wide, water-filled channel like the channels of the mitochondrial and the chloroplast porins. The single channel conductance is not a linear function of the bulk aqueous concentration, caused either by positive net charges in or near the channel mouth or by a binding site for anions. This is another indication that the peroxisomal channel has specialized channel properties. The size of the peroxisomal channel is somewhat difficult to obtain from the single channel measurements since only the size of wide, water-filled channels may be obtained from their conductance(6Benz R. Ghuysen J.-R. Hakenbeck R. Bacterial Cell Wall. Elsevier Science B.V., Amsterdam1994: 397-423Google Scholar, 7Benz R. Biochim. Biophys. Acta. 1994; 1197: 167-196Crossref PubMed Scopus (388) Google Scholar). As shown in Table 2 the K+ salts of formiate, acetate, glycerate, and glycolate are able to penetrate the channels, but the channel conductance with these ions is much smaller than with KCl. It appears from this result that the channels are just large enough to let these organic anions pass through. From the size of the permeating molecules, a channel diameter of about 1 nm may be estimated. The channel appears to be well suited to enable the transfer of metabolites in and out of the peroxisomes during photorespiratory metabolism.Tabled 1 Open table in a new tab Our data strongly suggest that plant peroxisomes contain an ion-permeable channel. The question arises whether pores are a common principle of the permeability properties of peroxisomal membranes. We mentioned in the Introduction that peroxisomes from yeast and liver may contain pores that have very similar properties to those in the outer membrane of the mitochondria(11van Veldhoven P. Just W.W. Mannaerts G.P. J. Biol. Chem. 1987; 262: 4310-4318Abstract Full Text PDF PubMed Google Scholar, 12Kaldi K. Diestelkötter P. Stenbeck G. Auerbach S. Jäkle U. Mägert H.J. Wieland F.T. Just W.W. FEBS Lett. 1993; 315: 217-222Crossref PubMed Scopus (36) Google Scholar, 15Sulter G.J. Verheyden K. Mannaerts G. Harder W. Veenhuis M. Yeast. 1993; 9: 733-742Crossref PubMed Scopus (21) Google Scholar). In particular, the channels reconstituted from liver peroxisomes show the same high voltage dependence, commencing at about 20-30 mV, as mitochondrial porins(7Benz R. Biochim. Biophys. Acta. 1994; 1197: 167-196Crossref PubMed Scopus (388) Google Scholar, 11van Veldhoven P. Just W.W. Mannaerts G.P. J. Biol. Chem. 1987; 262: 4310-4318Abstract Full Text PDF PubMed Google Scholar). We did not observe any voltage dependence up to 70 mV, indicating that the channel from leaf peroxisomes described here is not voltage regulated within the physiological range of membrane potentials. Obviously the channels attributed to liver peroxisomes have completely different properties than the channel described in our study. It remains to be studied whether the relatively small peroxisomal pores characterized here to be completely different from mitochondrial and chloroplast pores are a special feature of leaf peroxisomes, or whether they are also present in peroxisomes from other cells. We thank Dr. S. Borchert for useful discussions and M. Raabe for her excellent help in preparation of peroxisomes. The antibodies were kindly provided by Prof. Dr. Flügge (Universität Köln)." @default.
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• W2071366123 date "1995-07-01" @default.
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• W2071366123 title "The Membrane of Leaf Peroxisomes Contains a Porin-like Channel" @default.
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