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PubMed1998

Characterization of the initial steps of precursor import into rat liver mitoplasts.

Mitochondria have two independent protein-import machineries, one in the outer membrane (the Tom system) and the other in the inner membrane (the Tim system). Here, we have characterized the initial steps of precursor import into rat liver mitoplasts. The import reaction was separated into two stages, consisting of precursor binding to the mitoplasts at 0-10 degreesC, and a subsequent chase reaction at 30 degreesC. This assay revealed four distinct precursor-import steps: DeltaPsi-dependent initial binding of the precursor, precursor transfer to the Tim23-Tim17 stage, DeltaPsi-dependent translocation of the presequence across the inner membrane, and the complete translocation of the mature portion of the precursor. Antibodies against the intermembrane space domain of Tim23 inhibited neither the precursor binding nor the subsequent translocation of the presequence across the inner membrane. In contrast, the antibodies inhibited the complete translocation of the mature domain of the precursor across the inner membrane. Immunoprecipitation with anti-Tim23 IgGs revealed that the precursor-Tim23 complex increased with time and temperature after the initial targeting of the precursor to the mitoplasts. These results suggest that the precursor is first targeted to the inner membrane component DeltaPsi-dependently, then transferred to the Tim system consisting of Tim23-Tim17, and finally imported into the matrix.

Amino Acid Sequence↗
PubMed1976

Assembly of ATPase protein in sarcoplasmic reticulum membranes.

Three specimen preparation techniques for electron microscopy were used to investigate the incorporation of the ATPase polypeptide chains in the membranes of fragmented sarcoplasmic reticulum (SR) obtained from rabbit skeletal muscle. Observations were made of both normal vesicles and vesicles exposed to trypsin, which is known to cleave the ATPase protein and to alter the ultrastructure of the vesicles in predictable ways. Freeze-fracture replicas reveal the typical 90-A particles on the concave (PF) faces with a density of 5,730 +/- 520/mum2. On the other hand both negatively stained and deeply etched preparations display outer projections, which are absent on trypsin-incubated vesicles. The etched specimens afford for the first time top views of the vesicles in the absence of any stain. These views reveal outer projections on the PS surface with a density of 21,000 +/- 3,900/mum2, a value nearly approximating the density of the ATPase polypeptide chains (106,000 mol wt) calculated on the basis of protein and membrane area determinations. On the other hand, this value is three to four times higher than that found for the density of the 90-A particles on the concave fracture faces. Since both outer projections and 90-A particles are identified with the ATPase protein, it is suggested that the ATPase polypeptide chains are amphiphilic molecules, with polar ends protruding individually as outer projections on the surface of the vesicles, and hydrophobic ends appearing as 90-A particles on the concave fracture faces. The discrepancy between the densities of the outer projections and the 90-A particles may be attributed either to variable penetration of the polypeptide chains into the membrane bilayer, or to formation of oligomers containing three or four hydrophobic ends and appearing as single 90-A particles. Each ATPase chain forms a complex with 20-30 phospholipid molecules. The remaining phospholipids (approximately 70% of the total SR phospholipids) account for less than half the membrane volume. It is proposed that the outer leaflet of the SR membrane is prevalently composed of the ATPase lipoprotein complex, and the inner leaflet is mostly a phospholipid monolayer.

Adenosine Triphosphatases↗
PubMed1981

Intrinsic dichlorophenolindophenol reductase activity associated with the superoxide-generating oxidoreductase of human granulocytes.

NADPH-dependent dichlorophenolindophenol (DCIP) reductase activity cosediments with NADPH-dependent O(2-)-generating activity in subcellular particulate fractions of zymosan-stimulated human polymorphonuclear leukocytes (PMN's). Subcellular fractions derived from unstimulated PMN's were devoid of both activities, as were fractions from zymosan-stimulated PMN's of a patient known to have chronic granulomatous disease. NADPH-dependent DCIP reduction associated with the oxidoreductase-rich subcellular fractions was unaffected by addition of excess superoxide dismutase sufficient to abolish all traces of O2- production as measured by conversion of ferricytochrome c to its ferrous form. Moreover, DCIP inhibited NADPH-dependent production of O2- in subcellular fractions derived from normal donors. In contrast to the subcellular studies, whole cell suspensions were ineffective in reducing extracellular DCIP despite their capacity to generate O2-, albeit at a lesser rate, in the presence of this electron-accepting dye. These results demonstrate that DCIP reductase activity is associated with the oxidoreductase complex and suggest that it is located on the inner side of the PMN's plasma membrane. The stability of the oxidoreductase complex is markedly improved by storage in glycerol. Both overall O(2-)-generating activity and DCIP reductase activity exhibit a similar pH optimum of 7.0. The Km of the oxidoreductase complex for DCIP is 33 micro M.

Granulocytes↗
PubMed2004

Rapid purification and mass spectrometric characterization of mitochondrial NADH dehydrogenase (Complex I) from rodent brain and a dopaminergic neuronal cell line.

Oxidative stress and mitochondrial dysfunction signify important biochemical events associated with the loss of dopaminergic neurons in Parkinson's disease (PD). Studies using in vitro and in vivo PD models or tissues from diseased patients have demonstrated a selective inhibition of mitochondrial NADH dehydrogenase (Complex I of the OXPHOS electron transport chain) that affects normal mitochondrial physiology leading to neuronal death. In an earlier study, we demonstrated that oxidative stress due to glutathione depletion in dopaminergic cells, a hallmark of PD, leads to Complex I inhibition via cysteine thiol oxidation (Jha et al. (2000) J. Biol. Chem. 275, 26096-26101). Complex I is a approximately 980-kDa multimeric enzyme spanning the inner mitochondrial membrane comprising at least 45 protein subunits. As a prerequisite to investigating modifications to Complex I using a rodent disease model for PD, we developed two independent rapid and mild isolation procedures based on sucrose gradient fractionation and immunoprecipitation to isolate Complex I from mouse brain and a cultured rat mesencephalic dopaminergic neuronal cell line. Both protocols are capable of purifying Complex I from small amounts of rodent tissue and cell cultures. Blue Native gel electrophoresis, one-dimensional and two-dimensional SDS-PAGE were employed to assess the purity and composition of isolated Complex I followed by extensive mass spectrometric characterization. Altogether, 41 of 45 rodent Complex I subunits achieved MS/MS sequence coverage. To our knowledge, this study provides the first detailed mass spectrometric analysis of neuronal Complex I proteins and provides a means to investigate the role of cysteine oxidation and other posttranslational modifications in pathologies associated with mitochondrial dysfunction.

Animals↗
PubMed2005

Structural and functional studies of the enteropathogenic Escherichia coli type III needle complex protein EscJ.

The type III secretion system (TTSS) is a macromolecular structure that spans the cell wall of Gram-negative bacterial pathogens, enabling delivery of virulence effector proteins directly to the membranes and cytosol of host eukaryotic cells. TTSS consists of a conserved needle complex (NC) that is composed of sets of inner and outer membranes rings connected by a periplasmic rod. Enteropathogenic Escherichia coli (EPEC) is an extracellular diarrhoeagenic pathogen that uses TTSS to induce actin polymerization and colonizes the intestinal epithelium. In EPEC, EscJ is predicted to be targeted to the periplasm, in a sec-dependent manner, and to bridge the TTSS membrane-associated rings. In this study we determined the global fold of EscJ using Nuclear Magnetic Resonance spectroscopy. We show that EscJ comprises two subdomains (D1 - amino acid residues 1-55 in the mature protein, and D2 - amino acid residues 90-170), each comprising a three-stranded beta-sheet flanked by two alpha-helices. A flexible region (residues 60-85) couples the structured regions D1 and D2. Periplasmic overexpression of EscJ(D1) and EscJ(D2) in a single escJ mutant bacterium failed to restore protein secretion activity, suggesting that the flexible linker is essential for the rod function. In contrast, periplasmic overexpression of EscJ(D1) and EscJ(D2) in the same wild-type bacterium had a dominant-negative phenotype suggesting defective assembly of the TTSS and protein translocation.

Amino Acid Sequence↗
PubMed2005

Gene delivery of Tim44 reduces mitochondrial superoxide production and ameliorates neointimal proliferation of injured carotid artery in diabetic rats.

Hyperglycemia induces the production of reactive oxygen species (ROS) from mitochondria, which is closely related to diabetic vascular complications. Mammalian translocase of inner mitochondrial membrane (Tim)44 was identified by upregulation in streptozotocin (STZ)-induced diabetic mouse kidneys; Tim44 functions as a membrane anchor of mtHsp70 to TIM23 complex and is involved in the import of preproteins with mitochondria-targeted presequence into mitochondrial matrix. The process is dependent on inner membrane potential (Delta psi) and ATP hydrolysis on ATPase domain of mtHsp70. Here, we show that the gene delivery of Tim44 using pcDNA3.1 vector (pcDNA3.1/TIM44) into the balloon injury model of STZ-induced diabetic rats ameliorated neointimal proliferation. ROS production, inflammatory responses, and cell proliferation in injured carotid artery were diminished by delivery of pcDNA3.1/TIM44. In vitro experiments using human aortic smooth muscle cells (HASMCs) revealed that the gene delivery of Tim44 normalized high-glucose-induced enhanced ROS production and increased ATP production, alterations in inner membrane potential, and cell proliferation. Transfection of siRNA and pcDNA3.1/TIM44 using HASMC culture clarified that import of antioxidative enzymes such as superoxide dismutase and glutathione peroxidase was facilitated by Tim44. Tim44 and its related molecules in mitochondrial import machinery complex are novel targets in the therapeutic interventions for diabetes and its vascular complications.

Adenosine Triphosphate↗
PubMed1999

Interactions of Al(acac)3 with cell membranes and model phospholipid bilayers.

Aluminum is a neurotoxic agent; however, little information has been obtained regarding its molecular cytotoxicity and the effects on the stability of biological membranes. This is mainly due to the ill-defined chemical speciation of the metal compounds. For this reason, the present study used aluminum acetylacetonate, (Al(acac)3), a neutral, chemically well-defined, hydrolytically stable and lipophilic compound. To understand the molecular mechanism of its interaction with cell membranes, Al(acac)3 was incubated with human erythrocytes, isolated toad skin and molecular models of biomembranes. The latter consisted of multilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoyphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. The results showed that Al(acac)3 interacted with the erythrocyte membrane modifying its normal discoid morphology to both echinocytic and stomatocytic shapes. This finding indicates that the Al complex was inserted in both the outer and inner layers of the red cell membrane, a conclusion supported by X-ray diffraction analyses of DMPC and DMPE bilayers. Electrophysiological measurements performed on toad skin revealed a significant decrease in the potential difference and short-circuit current responses after application of Al(acac)3, effects interpreted to reflect inhibition of the active transport of ions. Al(acac)3 was active on both surfaces of the skin suggesting that the membrane was permeated by the metal complex. It is concluded that Al(acac)3 both alters the molecular structure of the lipid bilayer, thereby modifying the biophysical properties of the cell membrane, and changes its physiological properties.

Animals↗
PubMed1999

The mitochondrial permeability transition pore and its role in cell death.

This article reviews the involvement of the mitochondrial permeability transition pore in necrotic and apoptotic cell death. The pore is formed from a complex of the voltage-dependent anion channel (VDAC), the adenine nucleotide translocase and cyclophilin-D (CyP-D) at contact sites between the mitochondrial outer and inner membranes. In vitro, under pseudopathological conditions of oxidative stress, relatively high Ca2+ and low ATP, the complex flickers into an open-pore state allowing free diffusion of low-Mr solutes across the inner membrane. These conditions correspond to those that unfold during tissue ischaemia and reperfusion, suggesting that pore opening may be an important factor in the pathogenesis of necrotic cell death following ischaemia/reperfusion. Evidence that the pore does open during ischaemia/reperfusion is discussed. There are also strong indications that the VDAC-adenine nucleotide translocase-CyP-D complex can recruit a number of other proteins, including Bax, and that the complex is utilized in some capacity during apoptosis. The apoptotic pathway is amplified by the release of apoptogenic proteins from the mitochondrial intermembrane space, including cytochrome c, apoptosis-inducing factor and some procaspases. Current evidence that the pore complex is involved in outer-membrane rupture and release of these proteins during programmed cell death is reviewed, along with indications that transient pore opening may provoke 'accidental' apoptosis.

Animals↗
PubMed1999

Caspase-dependent proteolysis of integral and peripheral proteins of nuclear membranes and nuclear pore complex proteins during apoptosis.

We have studied the fate of the nuclear envelope (NE) in different human cells committed to apoptosis by different chemical agents. Using a battery of antibodies against marker proteins of the three domains of the nuclear envelope, namely lamin B (LB) for the lamina, transmembrane proteins LBR and LAP2 for the inner nuclear membrane, and nucleoporins p62, Nup153 and gp210 for the nuclear pore complexes (NPCs), we observed a selective and conserved cleavage of LB, LAP2 and Nup153. In lymphoid cells, the rate of cleavage of these markers was independent of the apoptosis inducing agent, actinomycin D or etoposide, and more rapid than in attached epithelial cells. While lamin B is cleaved by caspase 6, the protease responsible for the cleavage of LAP2 and Nup153 was probably caspase 3, since (1) cleavage of both proteins was specifically prevented by in vivo addition of caspase 3 inhibitor Ac-DEVD-CHO and (2) consensus sites for these caspases are present in both proteins. As LB, LAP2 and Nup153 are exposed at the inner face of the nuclear envelope and all interact with chromatin, we suggest that their cleavage allows both the detachment of NE from chromatin and the clustering of NPCs in the plane of the membrane, two conserved morphological features of apoptosis observed in this study.

Apoptosis↗
PubMed1993

Autoreactive liver-infiltrating T cells in primary biliary cirrhosis recognize inner mitochondrial epitopes and the pyruvate dehydrogenase complex.

Primary biliary cirrhosis (PBC) is characterized by lymphoid infiltrates in the portal tracts of the liver and the occurrence of antimitochondrial autoantibodies in serum directed against components of the pyruvate dehydrogenase complex and the other alpha-keto acid dehydrogenase complexes. These enzymes are located on the inner mitochondrial membrane. The destruction of the biliary tract in PBC is thought to be mediated by autoreactive liver-infiltrating T cells exerting cytotoxic activity or releasing certain lymphokines. In this study the reactivity of liver infiltrating T cells was shown to a bovine pyruvate dehydrogenase complex (PDH), a purified E2 subunit (PDH-E2) and a crude preparation of human liver mitoplasts (HLM), i.e. mitochondria depleted of their outer membranes. Peripheral blood lymphocytes (PBL) from 11 of 15 patients (73.3%) with PBC showed a HLA class II-restricted proliferative response to the PDH complex whereas PBL from patients with chronic viral hepatitis, autoimmune hepatitis or extrahepatic cholestatic icterus (n = 20) and healthy controls (n = 5) did not. In addition 13 of 15 PBL from patients with PBC (86.6%) and three of nine PBL from patients with autoimmune hepatitis (33.3%) reacted with the crude HLM preparation whereas no reactivity was found with PBL from eight patients with chronic viral hepatitis, three patients with extrahepatic cholestasis or five healthy controls. Clonal analysis of 115 liver-infiltrating T cells derived from two diagnostic liver biopsies of patients with PBC revealed a predominance of activated CD4+CD8- T helper cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1999

The preprotein translocase of the mitochondrial inner membrane: function and evolution.

Growing mitochondria acquire most of their proteins by the uptake of mitochondrial preproteins from the cytosol. To mediate this protein import, both mitochondrial membranes contain independent protein transport systems: the Tom machinery in the outer membrane and the Tim machinery in the inner membrane. Transport of proteins across the inner membrane and sorting to the different inner mitochondrial compartments is mediated by several protein complexes which have been identified in the past years. A complex containing the integral membrane proteins Tim17 and Tim23 constitutes the import channel for preproteins containing amino-terminal hydrophilic presequences. This complex is associated with Tim44 which serves as an adaptor protein for the binding of mtHsp70 to the membrane. mtHsp70, a 70 kDa heat shock protein of the mitochondrial matrix, drives the ATP-dependent import reaction of the processed preprotein after cleavage of the presequence. Preproteins containing internal targeting information are imported by a separate import machinery, which consists of the intermembrane-space proteins Tim9, Tim10, and Tim12, and the inner membrane proteins Tim22 and Tim54. The proteins Tim17, Tim22, and Tim23 have in common a similar topology in the membrane and a homologous amino acid sequence. Moreover, they show a sequence similarity to OEP16, a channel-forming amino acid transporter in the outer envelope of chloroplasts, and to LivH, a component of a prokaryotic amino acid permease, defining a new PRAT-family of preprotein and amino acid transporters.

Adenosine Triphosphatases↗
PubMed2000

Combinatorial synthesis of novel and potent inhibitors of NADH:ubiquinone oxidoreductase.

BACKGROUND: NADH:ubiquinone oxidoreductase (complex I) is the first of three large enzyme complexes located in the cell's inner mitochondrial membrane which form the electron transport chain that carries electrons from NADH to molecular oxygen during oxidative phosphorylation. There is significant interest in developing small molecule inhibitors of this enzyme for use as biological probes, insecticides and potential chemopreventive/chemotherapeutic agents. Herein we describe the application of novel natural product-like libraries to the discovery of a family of potent benzopyran-based inhibitors. RESULTS: Initially a combinatorial library of benzopyrans, modeled after natural products, was synthesized using a solid phase cycloloading strategy. Screening of this diversity oriented library for inhibitory potency against NADH:ubiquinone oxidoreductase activity in vitro using bovine heart electron transport particles provided several lead compounds which were further refined through a series of focused libraries. CONCLUSIONS: Using this combinatorial library approach, a family of potent 2,2-dimethylbenzopyran-based inhibitors was developed with IC(50) values in the range of 18-55 nM. Cell-based assays revealed that these inhibitors were rather non-cytotoxic in the MCF-7 cell line; however, they were quite cytostatic in a panel of cancer cell lines suggesting their potential as chemotherapeutic/chemopreventive candidates.

Animals↗
PubMed1996

The nuclear-encoded 18 kDa (IP) AQDQ subunit of bovine heart complex I is phosphorylated by the mitochondrial cAMP-dependent protein kinase.

In bovine heart mitochondria a protein of M(r) 18 kDa, phosphorylated by mtPKA, is associated to the NADH-ubiquinone oxidoreductase in the inner membrane and is present in purified preparation of this complex. The 18 kDa phosphoprotein has now been isolated and sequenced. It is identified as the 18 kDa (IP) AQDQ subunit of complex I, a protein of 133 amino acids with a phosphorylation consensus site RVS at position 129-131.

Amino Acid Sequence↗
PubMed1997

Mitochondrial inner membrane bound Pet1402 protein is rapidly imported into mitochondria and affects the integrity of the cytochrome oxidase and ubiquinol-cytochrome c oxidoreductase complexes.

In vitro synthesized Pet1402 precursor protein is very rapidly and efficiently imported into isolated mitochondria. The import depends on a membrane potential and functional mtHsp70. The mitochondrial targeting sequence of the Pet1402 precursor protein is removed by the matrix processing peptidase MPP and the mature protein is firmly embedded in the inner mitochondrial membrane. The Pet1402 protein is required for the integrity of the cytochrome oxidase and ubiquinol-cytochrome c oxidoreductase complexes.

Biological Transport↗
PubMed2006

Oxygen sensing by mitochondria at complex III: the paradox of increased reactive oxygen species during hypoxia.

All eukaryotic cells utilize oxidative phosphorylation to maintain their high-energy phosphate stores. Mitochondrial oxygen consumption is required for ATP generation, and cell survival is threatened when cells are deprived of O(2). Consequently, all cells have the ability to sense O(2), and to activate adaptive processes that will enhance the likelihood of survival in anticipation that oxygen availability might become limiting. Mitochondria have long been considered a likely site of oxygen sensing, and we propose that the electron transport chain acts as an O(2) sensor by releasing reactive oxygen species (ROS) in response to hypoxia. The ROS released during hypoxia act as signalling agents that trigger diverse functional responses, including activation of gene expression through the stabilization of the transcription factor hypoxia-inducible factor (HIF)-alpha. The primary site of ROS production during hypoxia appears to be complex III. The paradoxical increase in ROS production during hypoxia may be explained by an effect of O(2) within the mitochondrial inner membrane on: (a) the lifetime of the ubisemiquinone radical in complex III; (b) the relative release of mitochondrial ROS towards the matrix compartment versus the intermembrane space; or (c) the ability of O(2) to access the ubisemiquinone radical in complex III. In summary, the process of oxygen sensing is of fundamental importance in biology. An ability to control the oxygen sensing mechanism in cells, potentially using small molecules that do not disrupt oxygen consumption, would open valuable therapeutic avenues that could have a profound impact on a diverse range of diseases.

Animals↗
PubMed2004

Tic32, an essential component in chloroplast biogenesis.

Chloroplast protein import across the inner envelope is facilitated by the translocon of the inner envelope of chloroplasts (Tic). Here we have identified Tic32 as a novel subunit of the Tic complex. Tic32 can be purified from solubilized inner envelope membranes by chromatography on Tic110 containing affinity matrix. Co-immunoprecipitation experiments using either Tic32 or Tic110 antisera indicated a tight association between these polypeptides as well as with other Tic subunits, e.g. Tic40, Tic22, or Tic62, whereas the outer envelope protein Toc75 was not found in this complex. Chemical cross-linking suggests that Tic32 is involved late in the overall translocation process, because both the precursor form as well as the mature form of Rubisco small subunit can be detected. We were unable to isolate Arabidopsis null mutants of the attic32 gene, indicating that Tic32 is essential for viability. Deletion of the attic32 gene resulted in early seed abortion because the embryo was unable to differentiate from the heart stage to the torpedo stage. The homology of Tic32 to short-chain dehydrogenases suggests a dual role of Tic32 in import, one as a regulatory component and one as an important subunit in the assembly of the entire complex.

Amino Acid Sequence↗
PubMed2003

On the involvement of mitochondrial intermembrane junctional complexes in apoptosis.

The voltage dependent anion channel and the adenine nucleotide translocase are the principal proteins found in the mitochondrial outer and inner membranes, respectively. The two proteins can associate to form a junctional complex that establishes contact sites between the two membranes. This complex in turn recruits a range of proteins depending on the function to be executed. Among these, the junctional complexes can bind Bax and other proapoptotic proteins. Bax regulates the involvement of mitochondria in the apoptotic signalling pathway by controlling the the release of apoptogenic proteins from the mitochondrial intermembrane space to the cytosol. Another protein recruited to ANT is cyclophilin-D. Cyclophilin-D is a peptidylprolyl cis-trans-isomerase located in the intramitochondrial (matrix) compartment which stabilizes a "deformed" conformation of ANT in which its native gating properties are lost. Although the deformed state, when extensive, is lethal, cells can tolerate this conformational change when it occurs transiently. There is now a large body of data that implicates the voltage dependent anion channel, the adenine nucleotide translocase and cyclophilin-D, both separately and together, in the mitochondrial reactions of apoptosis. But there is no consensus over how, or indeed if, they are involved. This article examines the data relevant to this question and considers why a complex of these three proteins may be essential for the action of Bax and other proapoptotic proteins in permeabilizing the outer membrane to intermembrane space proteins.

Animals↗
PubMed1987

ACTH control of cholesterol side-chain cleavage at adrenal mitochondrial cytochrome P-450scc. Regulation of intramitochondrial cholesterol transfer.

Rat adrenal mitochondria exhibit a linear 2-fold accumulation of cholesterol for 20 min following either in vivo ether stress or ACTH administration, providing cholesterol metabolism is inhibited by aminoglutethimide (AMG). Additional cycloheximide (CX) pretreatment only slightly decreases this increase, but the location of accumulation shifts from the inner membrane to the outer membrane, implying a decreased cholesterol transfer from outer to inner membrane. Although the capacity of outer mitochondrial membranes was saturated after a 10-min treatment with CX, a 20-min treatment resulted in further retention of cholesterol in intact mitochondria that was not recovered in the isolated membranes. An additional pool of loosely bound cholesterol is proposed for CX mitochondria. These studies provide evidence that the CX-sensitive step of adrenal steroidogenesis attributed to loss of a labile ACTH regulatory protein (Pedersen, R.C. and Brownie, A.C. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 1882-1886) involves cholesterol transfer from the outer to the inner mitochondrial membrane. ACTH also enhances the PI and PE content of the outer membranes by a CX-sensitive mechanism that may contribute to intramitochondrial cholesterol transport. CX treatment does not affect cholesterol uptake by the inner membrane from phospholipid vesicles. The initial rate of endogenous metabolism in isolated inner membranes is insensitive to pretreatment (2 nmol/nmol P-450/min). The duration of this linear rate was increased 4-fold by AMG treatment while this increase was prevented by CX treatment. The kinetics indicate differences in inner membrane reactive cholesterol levels. Inner membranes also contained a fraction of unreactive cholesterol that is insensitive to pretreatment. Cholesterol-P-450scc complex formation for all pretreatments fits a single hyperbolic function of the reactive cholesterol content of the inner mitochondrial membrane (Kd = 0.025 mol cholesterol/mol phospholipid), and is activated over 5-fold upon mitochondrial disruption. All changes in inner membranes caused by CX can, therefore, be attributed solely to the restricted cholesterol access in vivo.

Adrenal Glands↗