6 Biochimica et Biophysica Acta, 1035 (1990)6-11 Elsevier BBAGEN 23338 Properties of the ATPase activity associated with peroxisome-enriched fractions from rat liver: comparison with mitochondrial FIF0-ATPase Ernst J. Wolvetang 1, Ronald J.A. Wanders 1, Ruud B.H. Schutgens 1, Jan A. Berden 2 and Joseph M. Tager 2 Department of Pediatrics, Academic Medical Centre and 2 E.C. Slater Institute for Biochemical Research, University of Amsterdam, Academic Medical Centre, Amsterdam (The Netherlands) (Received 16 October 1989) (Revised manuscript received13 March 1990) Key words: Peroxisome; ATPase; Permeability;ATPase, FIF0 - Highly purified peroxisomal fractions from rat liver contain ATIPase activity (18.8 + 0.1 mnol/min per mg, n -- 6). This activity is about 2% of that found in purified mitochondrial fractions. Measurement of marker enzyme activities and immunobiotting of the peroxisomai fraction with an antiserum raised against the fl-subunit of mitochondrial ATIPase indicates that the ATPase activity in the peroxisomal fractions can not be ascribed to contamination with mitochondria or other subeeilular organelles. From the sensitivity of the ATPase present in the peroxisomal fraction towards a variety of ATPase inhibitors, we conclude that it displays both V-type and F-type features and is distinguishable from both the mitochondrial FiF0-ATPase and the lysosomal V-type ATPase. Introduction The osmotic behavior of isolated peroxisomes in sucrose solutions and the equilibrium density of these organdies in sucrose density gradients led de Duve and collaborators to conclude that isolated peroxisomes are permeable to sucrose [1]. Furthermore, peroxisomal en- zymes such as D-amino-acid oxidase and L-a-hydroxy- acid oxidase A do not exhibit latency, indicating that their substrates can readily permeate through the mem- brane of peroxisomes [2,3] at least when the organdies are isolated. The absence of cofactor pools in isolated peroxisomes is in line with the concept of a freely permeable peroxisomal membrane [4,5]. Mannaerts and co-workers suggested that a 22 kDa peroxisomal in- tegral membrane protein forms a porin which is re- sponsible for the permeation of hydrophilic molecules with a molecular mass of up to 800 Da [6]. The concept Abbreviations: DCCD, N,N'-dicyclohexylcarbodiimide;NEM, N- ethylmaleimide; SDS-PAGE, sodium dodecyl sulphate polyacryl- amide gel electrophoresis Correspondence: R.J.A. Wanders, Department of Pediatrics(FO-224), Academic Medical Centre, Meibergdreef9, 1105 AZ Amsterdam, The Netherlands. of a peroxisome freely permeable to low molecular weight compounds is difficult to reconcile with recent findings by Nicolay et al. [7] and Douma et al. [8] in yeast. Nicolay et al. [7] recently presented presumptive evidence based upon 31p-NMR experiments indicating that peroxisomes in the yeast Hansenula polymorpha have an acidic interior. From the chemical shift of the phosphate pool, presumed to be present in peroxisomes of intact methanol-induced cells, Nicolay et al. [7] calculated that yeast peroxisomes in vivo have an inter- nal pH of 5.8-6.0. In a parallel study, Douma et al. [8] presented evidence for the association of an ATPase with the peroxisomal membrane in H. polymorpha. The authors suggested that this ATPase is responsible for the acidification of the peroxisomes. The properties of the ATPase closely resembled those of the mitochon- drial F1F0-ATPase of H. polymorpha. Recently, del Valle et al. [9] reported the presence of ATPase activity in peroxisomes from the fiver of rats treated with the peroxisome proliferator cipofibrate. In contrast to the results of Douma et al. [8], del Valle et al. [9] concluded that this ATPase activity exhibits properties of a neutral vacuolar type of ATPase. In the present paper, we report that highly purified peroxisomal fractions from rat fiver contain ATPase activity. The properties of this ATPase activity are described. 0304-4165/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)