Absence of NADH channeling in coupled reaction of mitochondrial malate dehydrogenase and complex I in alamethicin-permeabilized rat liver mitochondria q Alexander B. Kotlyar, * ,1 Elena Maklashina, and Gary Cecchini * Molecular Biology Division (151-S), VA Medical Center, San Francisco, CA 94121, USA Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143, USA Received 31 March 2004 Available online 7 May 2004 Abstract A simple in situ model of alamethicin-permeabilized isolated rat liver mitochondria was used to investigate the channeling of NADH between mitochondrial malate dehydrogenase (MDH) and NADH:ubiquinone oxidoreductase (complex I). Alamethicin- induced pores in the mitochondrial inner membrane allow effective transport of low molecular mass components such as NAD þ / NADH but not soluble proteins. Permeabilized mitochondria demonstrate high rates of respiration in the presence of malate/ glutamate and NAD þ due to coupled reaction between MDH and complex I. In the presence of pyruvate and lactate dehydro- genase, an extramitochondrial competitive NADH utilizing system, respiration of permeabilized mitochondria with malate/gluta- mate and NAD þ was completely abolished. These data are in agreement with the free diffusion of NADH and do not support the suggestion of direct channeling of NADH from MDH to complex I. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Substrate channeling; Mitochondrial respiration; Alamethicin; Mitochondria permeabilization; Malate dehydrogenase; NADH:ubiqui- none oxidoreductase; Complex I Mitochondria play a central role in cellular homeo- stasis as they contain major energy producing pathways. Reducing equivalents, mainly in the form of NADH, produced in the reactions of the Krebs cycle and b-ox- idation of fatty acids enzymes are utilized by the respi- ratory chain to form a transmembrane proton gradient that is used to produce ATP. Initial assumptions that soluble enzymes were randomly dispersed in the mito- chondrial matrix have been superseded by the hypoth- esis that functional separation of metabolic pathways is achieved by organized multienzyme systems (metabo- lons) [1–3]. This hypothesis supposes that the soluble multienzyme systems may be anchored to components in the inner mitochondrial membrane. It has been demonstrated that several mitochondrial soluble NAD þ -dependent dehydrogenases such as malate de- hydrogenase (MDH), b-hydroxyacyl-CoA dehydroge- nase, a-ketoglutarate dehydrogenase complex, and the pyruvate dehydrogenase complex specifically associate with NADH:ubiquinone oxidoreductase (complex I) [4–6]. Mammalian complex I is a multisubunit enzyme comprising at least 46 individual subunits (with a total mass >900 kDa) that form a membrane intrinsic domain and a peripheral hydrophilic arm extended into the mitochondrial matrix [7]. Protein–protein interactions between soluble dehydrogenases and complex I were confirmed by analytical centrifugation and fluorescence polarization electrophoretic techniques [4,6,8,9] em- ployed with gently sonicated mitochondria as well as for isolated soluble enzymes bound to submitochondrial particles. The large mass of the soluble domain of complex I and the fact that it harbors the NAD þ / q Abbreviations: SMP, submitochondrial particles; BSA, bovine serum albumin; MDH, malate dehydrogenase; LDH, lactate dehydro- genase. * Corresponding authors. Fax: 1-415-750-6959 (G. Cecchini). E-mail addresses: s2shak@post.tau.ac.il (A.B. Kotlyar), ceccini @itsa.ucsf.edu (G. Cecchini). 1 Present address: Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Israel. 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.04.131 Biochemical and Biophysical Research Communications 318 (2004) 987–991 BBRC www.elsevier.com/locate/ybbrc