Localization of enzyme for heme attachment to apocytochrome c in yeast mitochondria

Fractionation of yeast mitochondria by controlled hypotonic treatment revealed that the enzyme for heme attachment to apocytochrome c was localized in mitochondrial inner membrane. Trypsin digestion of mitoplasts resulted in a considerable loss of enzymatic activity, whereas the enzyme in intact mitochondria resisted the digestion. Triton X-100 solubilized the enzyme from the membrane but high concentration of salt did not. These results reveal that the enzyme for heme attachment is localized in mitochondrial inner membrane facing the cytoplasmic surface.     

Direct evidence for internalization of mitochondrial aspartate aminotransferase into mitoplasts.

Mitochondrial aspartate aminotransferase, an enzyme localized on the inner face of the inner mitochondrial membrane, is released into the intermembrane space upon addition of a "movement effector" (succinate, fumarate, pyruvate, or glutamate) [Waksman, A., & Rendon, A. (1974) Biochimie 56, 907-924]. After removal of the movement effector, 90% of the released enzyme rebound to mitoplasts. Lubrol fractionation showed that this bound activity was associated with the inner membrane. Internalization was demonstrated by using both enzymatic and molecular approaches. It was found that 70% of the reassociated enzyme became inaccessible from the outside of the mitoplast either to a nonpermeating substrate (NADH), to mild protease hydrolysis, or to recognition by a specific antibody. In contrast, in inside-out vesicles, the enzyme remained accessible to NADH, protease, and antibodies. Latency measurements performed at different temperatures on whole intact mitochondria confirmed the existence of reversible intermembrane movement of the enzyme in situ.     

Intramitochondrial localization of alanine aminotransferase in rat-liver mitochondria: comparison with glutaminase and aspartate aminotransferase

Summary The removal of the outer mitochondrial membrane and hence of constituents of the intermembrane space in rat-liver mitochondria using digitonin showed that phosphate-dependent glutaminase, alanine and aspartate aminotransferase were localized in the mitoplasts. Further fractionation of mitoplasts following their sonication resulted in 90% of glutaminase, 98% of alanine aminotransferase and 48% of aspartate aminotransferase being recovered in the soluble fraction while the remainder of each enzyme was recovered in the sonicated vesicles fraction. These results indicated that glutaminase and alanine aminotransferase were soluble matrix enzymes, the little of each enzyme recovered in the sonicated vesicles fraction being probably due to entrapment in the vesicles. Aspartate aminotransferase had dual localization, in the inner membrane and matrix with the high specific activity in sonicated vesicles confirming its association with the membrane. Activation experiments suggested that the membrane-bound enzyme was localized on the inner side of the inner mitochondrial membrane.     

Protein kinase activity and endogenous phosphorylation in subfractions of rat liver mitochondria

Rat liver mitochondria were subfractionated into outer membrane, intermembrane and mitoplast (inner membrane and matrix) fractions. Of the recovered protein kinase activity, 80-90% was found in the intermembrane fraction, while the rest was associated with mitoplasts. The intermembrane protein kinase was stimulated by cyclic AMP, while the mitoplast enzyme was stimulated by the nucleotide only after treatment with Triton X-100. Extracted protein kinase resolved into three peaks on DEAE-cellulose chromatography. All three peaks were present both in the intermembrane fraction and in mitoplasts. One peak corresponded to the catalytic subunit of cyclic AMP-dependent protein kinases, one was a cyclic AMP-independent enzyme, and the third was the cyclic AMP-dependent type II enzyme. The endogenous incorporation of phosphate was particularly high in the outer mitochondrial membrane, and occurred also in the mitoplast fraction. The incorporation in mitoplasts was to a double band of Mr 47 500, and in outer membranes to apparently heterogeneous material of comparatively low molecular weight.     

The orientation of phosphate-dependent glutaminase on the inner membrane of rat renal mitochondria

Phosphate-dependent glutaminase is associated with the inner membrane of rat renal mitochondria. The orientation of this enzyme was characterized by comparing its sensitivity in isolated mitochondria and in mitoplasts to two membrane impermeable inhibitors. Mitoplasts were prepared by repeated swelling of mitochondria in a hypotonic phosphate solution. This procedure released greater than 70% of the adenylate kinase from the intermembrane space, but less than 10 and 25% of the marker activities characteristic of the inner membrane and matrix compartments, respectively. The addition of 20 microM p-chloromercuriphenylsulfonate (pCMPS) caused a rapid inactivation of the purified glutaminase. In contrast, the glutaminase contained in isolated mitochondria and mitoplasts was only slightly affected by the addition of up to 2 mM pCMPS. Similarly, the activity in mitochondria and mitoplasts was not inhibited by the addition of an excess of inactivating Fab antibodies. However, a similar extent of inactivation occurred when either membrane fraction was incubated with concentrations of octylglucoside greater than 0.35%. Mitochondria were also treated with increasing concentrations of digitonin. At 0.4 mg digitonin/mg protein, all of the adenylate kinase was released but the glutaminase activity was either slightly inhibited or unaffected by the addition of pCMPS or the Fab antibodies, respectively. These studies establish that the glutaminase is localized on the inner surface of the inner membrane. Therefore, mitochondrial catabolism of glutamine must occur only within the matrix compartment.     

The mitochondrial location of protoporphyrinogen oxidase

Using the digitonin method and subsequent fractionation of rat liver mitochondria, protoporphyrinogen oxidase (penultimate enzyme in the heme biosynthesis pathway) was found to be closely associated with the mitochondrial inner membrane fraction. Chemical treatment with non-specific probes (trypsin and diazobenzene sulfonate) of either intact or inverted mitoplasts, indicated that protoporphyrinogen oxidase was anchored within the lipid bilayer of the inner membrane. Protoporphyrinogen had an equal access to the active site of the enzyme from both sides of the inner membrane and its transformation to protoporphyrin did not appear to be energy-dependent. Studies of protoporphyrinogen synthesis from exogenously added coproporphyrinogen in either intact or hypoosmotically treated mitochondria underlined the importance of the peculiar submitochondrial location of coproporphyrinogen oxidase and protoporphyrinogen oxidase for the transfer of substrates to the inner membrane.     

Orientation of ferrochelatase in bovine liver mitochondria

The orientation of ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1), the terminal enzyme of the heme biosynthetic pathway, was examined in bovine liver mitochondria. The ability of a membrane-impermeable sulfhydryl reagent, 4,4'-dimaleimidylstilbene-2,2'-disulfonic acid, to inactivate ferrochelatase in intact or disrupted mitochondria and mitoplasts was examined. Using succinate dehydrogenase as an internal marker, it was found that ferrochelatase was inactivated only in disrupted mitochondria and mitoplasts, suggesting an internal location for the active site of the enzyme. In addition, antibodies raised against purified ferrochelatase were found to inhibit activity only in disrupted but not in intact mitoplasts. These data demonstrate that in bovine liver mitochondria ferrochelatase is located on the matrix side of the inner mitochondrial membrane. Data obtained with the membrane-impermeable amino reagent isethionyl acetimidate indicate that ferrochelatase physically spans the inner mitochondrial membrane with portions of the protein exposed on both sides of the membrane.     

Influence of mitochondrial creatine kinase on the mitochondrial/extramitochondrial distribution of high energy phosphates in muscle tissue: evidence for a leak in the creatine shuttle

The influence of mitochondrial creatine kinase on subcellular high energy systems has been investigated using isolated rat heart mitochondria, mitoplasts and intact heart and skeletal muscle tissue.In isolated mitochondria, the creatine kinase is functionally coupled to oxidative phosphorylation at active respiratory chain, so that it catalyses the formation of creatine phosphate against its thermodynamic equilibrium. Therefore the mass action ratio is shifted from the equilibrium ratio to lower values. At inhibited respiration, it is close to the equilibrium value, irrespective of the mechanism of the inhibition. The same results were obtained for mitoplasts under conditions where the mitochondrial creatine kinase is still associated with the inner membrane.In intact tissue increasing amounts of creatine phosphate are found in the mitochondrial compartment when respiration and/or muscle work are increased. It is suggested that at high rates of oxidative phosphorylation creatine phosphate is accumulated in the intermembrane space due to the high activity of mitochondrial creatine kinase and the restricted permeability of reactants into the extramitochondrial space. A certain amount of this creatine phosphate leaks into the mitochondrial matrix.This leak is confirmed in isolated rat heart mitochondria where creatine phosphate is taken up when it is generated by the mitochondrial creatine kinase reaction. At inhibited creatine kinase, external creatine phosphate is not taken up. Likewise, mitoplasts only take up creatine phosphate when creatine kinase is still associated with the inner membrane. Both findings indicate that uptake is dependent on the functional active creatine kinase coupled to oxidative phosphorylation.Creatine phosphate uptake into mitochondria is inhibited with carboxyatractyloside. This suggests a possible role of the mitochondrial adenine nucleotide translocase in creatine phosphate uptake.Taken together, our findings are in agreement with the proposal that creatine kinase operates in the intermembrane space as a functional unit with the adenine nucleotide translocase in the inner membrane for optimal transfer of energy from the electron transport chain to extramitochondrial ATP-consuming reactions.     

Heart mitochondrial creatine kinase revisited: the outer mitochondrial membrane is not important for coupling of phosphocreatine production to oxidative phosphorylation

The state of mitochondrial creatine kinase (CKmi-mi) in intact dog heart mitochondria and mitoplasts and the mechanism of its functional coupling with the oxidative phosphorylation system have been reinvestigated under different osmotic conditions and ionic compositions of the medium. It has been established that in a medium which mimics the cardiac cell cytoplasma, dissociation of CKmi-mi from the membrane of mitoplasts increases when the mitoplasts are swollen due to hypoosmotic treatment. It was shown by EPR that hypoosmotic treatment results in the enhancement of the mobility of phospholipids in the membrane bilayer. It has been also shown that when CKmi-mi is detached from the inner membrane in intact mitochondria in isotonic KCl solution, the effects of the coupling between CKmi-mi and oxidative phosphorylation via ATP/ADP translocase disappear in spite of the presence of CKmi-mi in the intermembrane space and intactness of the outer mitochondrial membrane. Therefore, this coupling cannot be explained by the "compartmented coupling" mechanism or "dynamic adenine nucleotide compartmentation" in the intermembrane space due to diffusion limitation for adenine nucleotides through the outer mitochondrial membrane, as has been supposed by several authors (F.N. Gellerich et al. (1987) Biochim. Biophys. Acta 890, 117-126; S.P.J. Brooks and C.H. Suelter (1987) Arch. Biochem. Biophys. 253, 122-132). The data obtained show that the displacement of the enzyme from the membrane results in significantly increased sensitivity of the coupled processes of aerobic phosphocreatine synthesis to inhibition by the product, phosphocreatine. Thus, all results show that under physiological osmotic and ionic conditions CKmi-mi remains firmly attached to the inner mitochondrial membrane and effectively coupled with ATP/ADP translocase due to intimate dynamic interaction between those proteins.     

Long-chain acyl CoA synthetase,carnitine and β-oxidation in the pea-seed mitochondrion

Mitochondria from pea (Pisum sativum L.) seeds were separated into two fractions, mitoplasts (intact inner membrane) and the outer-membrane fraction. The mitoplasts only oxidised palmitate in the presence of carnitine and added outermembrane fraction. Mitoplasts were able to oxidise palmitoylCoA in the presence of carnitine and added outer-membrane fraction had no effect on this oxidation. It was concluded that a long-chain acylCoA synthetase (EC 6.2.1.3) was located on the outer membrane and that the activity of this enzyme in assays was more than sufficient to account for any observed rate of O₂ uptake during palmitate oxidation by pea mitochondria. The location of carnitine long-chain acyltransferase (carnitine palmitoyl transferase EC 2.3.1.21) would appear to be the mitoplast i.e. the inner mitochondrial membrane, and confirms the previous work at Newcastle.Abbreviation Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Dicarboxylate transport in the inner membrane matrix fraction obtained from rat liver mitochondria

Dicarboxylate transport was studied in the inner membrane matrix fraction (mitoplasts) and compared to that in intact rat-liver mitochondria from which the former was obtained.It is concluded that, kinetics of dicarboxylate exchange measured in mitoplasts, are very similar to those observed with mitochondria. These results would indicate that the preparation technique preserves the integrity of the inner membrane and that neither the outer membrane nor the components of the peripheral space affect these results.     

Intramitochondrial fatty acylation of a cytoplasmic imported protein in animal cells

Mitochondrial digitonin particles from mouse liver (and also from other tissues) incorporate [3H]myristic acid into a 52-kilodalton (kDa) protein in an energy-dependent manner. The 52-kDa N-myristylated protein is located inside the mitochondrial inner membrane since it is protected against proteolytic degradation in intact mitoplasts. Disruption of mitochondrial inner membrane by sonication results in severalfold higher labeling of the 52-kDa protein, further confirming that the enzyme system for protein fatty acylation as well as the 52-kDa target protein are compartmentalized inside the mitochondrial inner membrane matrix. The results of in vitro labeling of submitochondrial fractions suggest that both the 52-kDa target protein and the enzyme system for fatty acylation are in the matrix fraction, although the N-myristylated protein is found loosely associated with the inner membrane. Finally, immunoprecipitation of cytoplasmic free polysome translation products and in vitro transport of proteins into isolated mitochondria show that the 52-kDa protein is of cytoplasmic translation origin. These results demonstrate that the intramitochondrial N-myristylation of the 52-kDa protein is not translationally linked.     

Dicarboxylate transport in the inner membrane matrix fraction obtained from rat liver mitochondria.

Dicarboxylate transport was studied in the inner membrane matrix fraction (mitoplasts) and compared to that in intact rat-liver mitochondria from which the former was obtained. It is concluded that, kinetics of dicarboxylate exchange measured in mitoplasts, are very similar to those observed with mitochondria. These results would indicate that the preparation technique preserves the integrity of the inner membrane and that neither the outer membrane nor the components of the peripheral space affect these results.     

Novel FMN-containing rotenone-insensitive NADH dehydrogenase from Trypanosoma brucei mitochondria: isolation and characterization

A rotenone-insensitive NADH dehydrogenase has been isolated from the mitochondria of the procyclic form of African parasite, Trypanosoma brucei. The active form of the purified enzyme appears to be a dimer consisting of two 33-kDa subunits with noncovalently bound FMN as a cofactor. Hypotonic treatment of intact mitochondria revealed that the NADH dehydrogenase is located in the inner membrane/matrix fraction facing the matrix. The treatment of mitochondria with increasing concentrations of digitonin suggested that the NADH dehydrogenase is loosely bound to the inner mitochondrial membrane. The NADH:ubiquinone reductase activity is insensitive to rotenone, flavone, or dicumarol; however, it was inhibited by diphenyl iodonium in a time- and concentration-dependent manner. Maximum inhibition by diphenyl iodonium required preincubation with NADH to reduce the flavin. More complete inhibition was obtained with the more hydrophobic electron acceptors, such as Q(1) or Q(2), as compared to the more hydrophilic ones, such as Q(0) or dichloroindophenol. Kinetic analysis of the enzyme indicated that the enzyme followed a ping-pong mechanism. The enzyme conducts a one-electron transfer and can reduce molecular oxygen forming superoxide radical.     

Separation of outer and inner membranes of mitochondria from the brown adipose tissue of infant rats.

Digitonin treatment and the swelling-shrinkage-sonication procedure as used to separate mitochondria membranes were applied to mitochondria from the brown adipose tissue (BAT) of infant rats. Digitonin at a concentration of 0.15 mg/mg mitochondrial protein produced disruption of the outer membrane of BAT mitochondria and a complete release of adenylate kinase. However, fragments of the outer membrane remained firmly attached to the inner membrane-matrix particles (mitoplasts) and sedimented at 10 000 g, as indicated by the activity of monoamine oxidase in the pellet. Only at 0.5 mg digitonin/mg protein did outer membrane become almost entirely separated. Oxidation of external cytochrome c by mitoplasts was only 50% of the total cytochrome oxidase at 0.5 mg digitonin/mg protein, indicating an incomplete exposure of the inner membrane to the external medium. Ultrastructural studies revealed that a large proportion of mitoplasts retained the orthodox configuration under these conditions. Outer membrane fragments obtained by the swelling-shrinkage-sonication procedure were of buoyant density corresponding to 20–30% (weight/vol) sucrose. After a 10 sec sonication of mitochondria, a relatively pure outer membrane fraction could be obtained with a yield not exceeding 20%. Longer sonication increased the yield, but also increased the degree of contamination by inner membrane fragments. Optimum conditions for the separation of outer and inner membranes from brown adipose tissue mitochondria are described.     

Carnitine acetyltransferase in pea cotyledon mitochondria

Purified pea cotyledon mitochondria did not oxidise acetyl-CoA in the presence of carnitine. However, acetylcarnitine was oxidised. It was concluded that acetylcarnitine passed through the mitochondrial membrane barrier but acetyl-CoA did not. Only a sensitive radioactive assay detected carnitine acetyltransferase in intact mitochondrion or intact mitoplast preparations. When the mitochondria or mitoplasts were burst, acetyl-CoA substrate was available to the matrix carnitine acetyltransferase and a high activity of the enzyme was measured. The inner mitochondrial membrane is there-fore the membrane barrier to acetyl-CoA but acetylcarnitine is suggested to be transported through this membrane via an integral carnitine: acylcarnitine translocator. Evidence is presented to indicate that when the cotyledons from 48-h-grown peas are oxidising pyruvate, acetylcarnitine formed in the mitochondrial matrix by the action of matrix carnitine acetyltransferase may be transported to extra-mitochondrial sites via the membrane translocator.     

Collagen cross-linking in human bone and articular cartilage. Age-related changes in the content of mature hydroxypyridinium residues.

Non-immune activation of the first component of complement (C1) by the heart mitochondrial inner membrane has been investigated. Cardiolipin, the only strong activator of C1 among phospholipids, is present in large amounts in the heart mitochondrial inner membrane. We therefore studied its contribution to C1 activation by mitochondria. The proteins of the mitochondrial inner membrane were found to activate C1 only weakly, in contrast with the phospholipid fraction which induces strong C1 activation. Furthermore, the digestion of mitochondrial inner membranes with proteolytic enzymes did not affect C1 activation. Additional support in favour of cardiolipin being the responsible activator came from competition experiments with mitochondrial creatine kinase (mt-CPK) and adriamycin, known to bind to cardiolipin. Both mt-CPK and adriamycin displaced C1q from the mitochondrial inner membrane. In addition, C1q displaced mt-CPK bound to mitoplasts.     

Studies on the structure of NADH: ubiquinone oxidoreductase complex: topography of the subunits of the iron-sulfur protein component

Resolution of the mitochondrial NADH:ubiquinone oxidoreductase complex (Complex I) by chaotropic agents result in the separation of three building blocks of the enzyme, designated FP (flavoprotein), IP (iron-sulfur protein), and HP (hydrophobic protein). FP contains three subunits of Mr 51, 24, and 9 kDa; one FMN; and two iron-sulfur clusters. Immunochemical studies with monospecific antibodies to the FP subunits have indicated that all three subunits of FP protrude from the inner mitochondrial membrane on the matrix side, whereas no reactive epitopes from these subunits were found exposed on the cytosolic side [A.-L. Han, T. Yagi, and Y. Hatefi (1988) Arch. Biochem. Biophys. 267, 490-496]. IP contains six subunits of Mr 75, 49, 30, 18, 15, and 13 kDa and four iron-sulfur clusters. In the present study, immunochemical experiments (enzyme-linked immunosorbent assays and 125I-protein A labeling) were carried out with monospecific antibodies to the above IP subunits and with bovine Complex I, submitochondrial particles, mitoplasts, and intact mitochondria as sources of antigens. Results have indicated that all six IP subunits protrude from the inner mitochondrial membrane into the matrix, and that the 75-kDa subunit, and possibly the 15-kDa subunit, protrude in mitoplasts from the cytosolic side as well. No epitopes reactive toward the monospecific antibodies to the 49-, 30-, 18-, and 13-kDa subunits were detected in mitoplasts.     

Interaction of a surface-active base with the fraction of membrane-bound Williams’ protons

According to the Williams model, the work of mitochondrial respiratory H⁺ pumps gives rise to a fraction of membrane-bound protons (R-protons) that have excess free energy, which is used in the reaction of ATP synthesis. We have earlier managed to detect such a fraction in mitochondria and mitoplasts and to rigorously show (for mitoplasts) that the non-equilibrium R-proton fraction is localized on the surface of the inner membrane. Here we show that a surface-active compound 2,4,6-trichloro-3-pentadecylphenol anion (TCP-C₁₅) selectively interacts with the R-proton fraction, and describe in detail its influence on mitochondrial respiration under conditions of R-proton generation. We also report endogenous regulation of the R-proton fraction volume, which is performed by the phosphate transport system. The results are discussed in terms of the local coupling model.     

Protein kinase activity and endogenous phosphorylation in subfractions of rat liver mitochondria

Rat liver mitochondria were subfractionated into outer membrane, intermembrane and mitoplast (inner membrane and matrix) fractions. Of the recovered protein kinase activity, 80–90% was found in the intermembrane fraction, while the rest was associated with mitoplast. The intermembrane prostimulated kinase was stimulated by cyclic AMP, while the mitoplast enzyme was stimulated by the nucleotide only after treatment with Triton X-100. Extracted protein kinase resolved into three peaks on DEAE-cellulose chromatography. All three peaks were present both in the intermembrane fraction and in mitoplast. One peak corresponded to the catalytic subunit of cyclic AMP-dependent protein kinase, one was a cyclic AMP-independent enzyme, and the third was the cyclic AMP-dependent type II enzyme. The endogenous incorporation of phosphate was particularly high in the outer mitochondrial membrane, and occurred also in the mitoplast fraction. The incorporation in mitoplasts was to a double band of Mr 47 500, and in outer membranes to apparently heterogeneous material of comparatively low molecular weight.