Interaction of creatine kinase with phosphorylating rabbit heart mitochondria and mitoplasts

This paper demonstrates that the mitochondrial isoenzyme of creatine kinase (CKm) can be solubilized from rabbit heart mitochondria, the outer membrane of which has been removed or at least broken by a digitonin treatment or a short hypotonic exposure, but which has retained an important part of the capacity to phosphorylate ADP. Phosphate, ADP, or ATP, at concentrations which are used to study oxidative phosphorylation and creatine phosphate synthesis, solubilize CKm; the same is true with MgCl2 and KCl. The effect of adenine nucleotides does not seem to be due to their interaction with the adenine nucleotide translocase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that CKm is the main protein released in the described conditions; however, it does not amount to more than 1% of the total protein content of the mitoplasts. When the apparent Km for ATP of CKm was estimated by measuring creatine phosphate synthesis, the values obtained using water-treated mitochondria (0.21 mM) were slightly higher than those of intact mitochondria (0.12 mM) but the difference was not significant. In the former preparation 77% of CKm was in a soluble state. If we can extrapolate these results to intact mitochondria and suppose that in this case a fraction of CKm is also soluble in the intermembrane space, this does not support the theory of functional association between CKm and the adenine nucleotide translocase.     

Cholinergic synaptic vesicles from Torpedo marmorata contain an atractyloside-binding protein related to the mitochondrial ADP/ATP carrier

Atractyloside is known to bind to the ADP/ATP translocase of the inner mitochondrial membrane, a complex formed by two basic protein subunits of relative molecular mass around 30 000. We found that synaptic vesicles from the electric organ of Torpedo marmorata, which store acetylcholine and ATP, bind atractyloside as well. Similarly to mitochondria, a protein-atractyloside complex could be solubilized from vesicle membranes with Triton X-100. Characterization of the complex by gel filtration, isoelectric focusing and gel electrophoresis revealed that atractyloside was bound to protein V11, earlier described as a major vesicle membrane component with a relative molecular mass around 34 000 and a basic isoelectric point. Since earlier experiments have already shown that uptake of ATP into isolated vesicles in vitro is inhibited by atractyloside, we can conclude now that V11 constitutes the nucleotide carrier of this secretory organelle. The structural and functional relationship of the mitochondrial and vesicular nucleotide translocases suggest a common evolutionary origin.     

Inhibition of ADP/ATP exchange in receptor-interacting protein-mediated necrosis

Receptor-interacting protein (RIP) has been implicated in the induction of death receptor-mediated, nonapoptotic cell death. However, the mechanisms remain to be elucidated. Here we show that tumor necrosis factor alpha induced RIP-dependent inhibition of adenine nucleotide translocase (ANT)-conducted transport of ADP into mitochondria, which resulted in reduced ATP and necrotic cell death. The inhibition of ADP/ATP exchange coincided with the loss of interaction between ANT and cyclophilin D and the inability of ANT to adopt the cytosolic conformational state, which prevented cytochrome c release. Neither overexpression of Bcl-xL nor inhibition of reactive oxygen species prevented necrosis. In contrast, the ectopic expression of ANT or cyclophilin D was effective at preventing cell death. These observations demonstrate a novel mechanism initiated through death receptor ligation and mediated by RIP that results in the suppression of ANT activity and necrosis.     

In vitro alteration of the size of the liver mitochondrial adenine nucleotide pool: Correlation with respiratory functions

Mitochondrial respiration was studied as a function of the total adenine nucleotide content of rat liver mitochondria. The adenine nucleotide content was varied by treating isolated mitochondria with pyrophosphate or by incubating pyrophosphate-treated mitochondria with ATP. Mitochondria with at least 4 nmol adenine nucleotides/mg protein maintained at least 80% of the State 3 activity of control mitochondria, which had approximately 10 nmol/mg protein. However, State 3 decreased rapidly once the adenine nucleotide content fell below 4 nmol/mg protein. Between 2 and 4 nmol adenine nucleotides/mg, State 3 was not limited by the maximal capacity of electron flow as measured by the uncoupled respiration. However, at very low adenine nucleotide levels (<2 nmol/mg), the uncoupled rates of respiration were markedly depressed. State 4 was not affected by changes in the mitochondrial adenine nucleotide content. Adenine translocase activity varied in almost direct correlation with changes in the adenine nucleotide content. Therefore, adenine translocase activity was more sensitive than State 3 to changes in total adenine nucleotides over the range of 4 to 10 nmol/mg protein. The results suggest that (i) State 3 is dependent on the level of intramitochondrial adenine nucleotides, particularly in the range below 4 nmol/mg protein, (ii) adenine translocase activity is not rate-limiting for oxidative phosphorylation in mitochondria with the normal complement of adenine nucleotides, however, at low adenine nucleotide levels, depressed State 3 rates may be explained in part by the low rate of ADP translocation, and (iii) a mechanism of net ATP uptake exists in mitochondria with low internal adenine nucleotides.     

Cyclophilin D Modulates Mitochondrial F0F1-ATP Synthase by Interacting with the Lateral Stalk of the Complex

Blue native gel electrophoresis purification and immunoprecipitation of F₀F₁-ATP synthase from bovine heart mitochondria revealed that cyclophilin (CyP) D associates to the complex. Treatment of intact mitochondria with the membrane-permeable bifunctional reagent dimethyl 3,3-dithiobis-propionimidate (DTBP) cross-linked CyPD with the lateral stalk of ATP synthase, whereas no interactions with F₁ sector subunits, the ATP synthase natural inhibitor protein IF1, and the ATP/ADP carrier were observed. The ATP synthase-CyPD interactions have functional consequences on enzyme catalysis and are modulated by phosphate (increased CyPD binding and decreased enzyme activity) and cyclosporin (Cs) A (decreased CyPD binding and increased enzyme activity). Treatment of MgATP submitochondrial particles or intact mitochondria with CsA displaced CyPD from membranes and activated both hydrolysis and synthesis of ATP sustained by the enzyme. No effect of CsA was detected in CyPD-null mitochondria, which displayed a higher specific activity of the ATP synthase than wild-type mitochondria. Modulation by CyPD binding appears to be independent of IF1, whose association to ATP synthase was not affected by CsA treatment. These findings demonstrate that CyPD association to the lateral stalk of ATP synthase modulates the activity of the complex.     

Protein phosphorylation in intact bovine epididymal spermatozoa: identification of the type II regulatory subunit of cyclic adenosine 3',5'-monophosphate-dependent protein kinase as an endogenous phosphoprotein

An obstacle to the study of protein phosphorylation in mammalian spermatozoa has been the inability to incorporate sufficient amounts of 32Pi into cellular adenosine triphosphate (ATP) (Babcock et al., 1975). We report conditions under which 32Pi is effectively incorporated into the ATP of intact bovine spermatozoa. In the presence of a bicarbonate-buffered medium containing glucose, spermatozoa incorporated 32P into intracellular ATP in a time-dependent manner; after 2 h of incubation, the specific activity of [gamma-32P]ATP (2.3 X 10(4) cpm/nmol ATP) was estimated to be 50-65% of the specific activity of the intracellular phosphate pool. In the absence of glucose or other added substrates, the specific activity of [gamma-32P]ATP was 10-25% that of the specific activity observed in the presence of glucose. Washed spermatozoa incubated in carrier-free 32Pi for 2 h at 37 degrees C, and solubilized in a solution containing final concentrations of 6.8 M urea, 6% NP4O, and 5% beta-mercaptoethanol contained in excess of 40 32Pi-labeled proteins as assessed by two-dimensional polyacrylamide gel electrophoresis. Major phosphoproteins had approximate molecular weights of 93,000, 40,000, and 22,000. A different two-dimensional gel pattern was observed when cells were extracted with a solution containing 38.5 mM 2[N-cyclohexylamino] ethanesulfonic acid (CHES), pH 9.5/1.5% sodium dodecyl sulphate (SDS) at 100 degrees C. In contrast to the urea/Nonidet P-40 (NP40)/beta-mercaptoethanol extract, a 56,000 Mr phosphoprotein represented a major component while the 40,000 Mr and several of the 22,000 Mr polypeptides were markedly reduced in radioactive intensity. The 56,000 Mr species present in the CHES/SDS extract comigrated with the purified, phosphorylated regulatory subunit (RII) of cyclic adenosine 3',5'-monophosphate-dependent protein kinase from bovine heart. Antibodies to RII immunoprecipitated a 56,000 Mr, 32P-labeled polypeptide from the CHES/SDS extract that comigrated with purified, [32P] RII after two-dimensional electrophoresis. RII, then, appears to represent one of the endogenous phosphoproteins of intact bovine epididymal spermatozoa.     

DNA recombination activity in soybean mitochondria

Mitochondrial genomes in higher plants are much larger and more complex as compared to animal mitochondrial genomes. There is growing evidence that plant mitochondrial genomes exist predominantly as a collection of linear and highly branched DNA molecules and replicate by a recombination-dependent mechanism. However, biochemical evidence of mitochondrial DNA (mtDNA) recombination activity in plants has previously been lacking. We provide the first report of strand-invasion activity in plant mitochondria. Similar to bacterial RecA, this activity from soybean is dependent on the presence of ATP and Mg(2+). Western blot analysis using an antibody against the Arabidopsis mitochondrial RecA protein shows cross-reaction with a soybean protein of about 44 kDa, indicating conservation of this protein in at least these two plant species. mtDNA structure was analyzed by electron microscopy of total soybean mtDNA and molecules recovered after field-inversion gel electrophoresis (FIGE). While most molecules were found to be linear, some molecules contained highly branched DNA structures and a small but reproducible proportion consisted of circular molecules (many with tails) similar to recombination intermediates. The presence of recombination intermediates in plant mitochondria preparations is further supported by analysis of mtDNA molecules by 2-D agarose gel electrophoresis, which indicated the presence of complex recombination structures along with a considerable amount of single-stranded DNA. These data collectively provide convincing evidence for the occurrence of homologous DNA recombination in plant mitochondria.     

Dependence of mitochondrial oxidative phosphorylation on activity of the adenine nucleotide translocase

The coupled reactions of electron transport and ATP synthesis for the first two sites of mitochondrial oxidative phosphorylation have been previously reported to be near equilibrium in isolated respiring pigeon heart (Erecińska, M., Veech, R. L., and Wilson, D. F. (1974) Arch. Biochem. Biophys. 160, 412-421) and rat liver mitochondria (Forman, N. G., and Wilson, D. F. (1982) J. Biol. Chem. 257, 12908-12915). Measurements are presented in this paper which demonstrate that the same relationship exists for both forward and reverse electron transport in rat heart mitochondria. This conclusion implies that adenine nucleotide translocation, a partial reaction of the system, is also near equilibrium, contrasting with proposals that the translocase is rate-limiting for oxidative phosphorylation. To resolve this controversy, the respiratory rates of suspensions of isolated rat liver and rat heart mitochondria were controlled by varying either the added [ATP]/[ADP][Pi] ratios ratios or [ADP] (by varying hexokinase in a regenerating system). Titrations with carboxyatractyloside, a high affinity inhibitor of the translocase which is noncompetitive with ADP, were carried out to assess the dependence of the respiratory rate on translocase activity. Plots of respiratory rate versus [carboxyatractyloside] were all strongly sigmoidal. In liver mitochondria, 40%-70% and in heart mitochondria 66% of the sites could be blocked with carboxyatractyloside before a 10% decrease in the respiratory rate was observed. Further analysis showed that liver and heart mitochondria have translocase/cytochrome a ratios of 1.52 and 3.20, respectively, and that at 23 degrees C the maximal turnover numbers for the translocases were 65 s-1 and 23 s-1. In all states of controlled respiration (no added inhibitor), a substantial excess of translocase activity was present, suggesting that the translocase was not normally rate-limiting in oxidative phosphorylation.     

棉铃虫腺苷酸转移酶基因的克隆与分析

腺苷酸转移酶(ANT)是线粒体内膜上负责能量分子传导的转运蛋白, 在细胞凋亡调控网络中有重要作用。本研究以棉铃虫幼虫组织的mRNA为模板, 根据鳞翅目昆虫ant基因编码区保守序列设计引物, 进行RT-PCR分析, 同时结合5′、3′ RACE方法扩增出棉铃虫ant基因的全长cDNA序列, cDNA全长为1 190 bp (GenBank登录号AY253868), 具有完整的开放阅读框架(ORF, 133~1 033 bp), 编码蛋白为300个氨基酸, 其中N端22个氨基酸为信号肽, 引导ANT蛋白定位于线粒体内膜。该蛋白具有3个保守的线粒体穿膜功能结构域, 形成能量分子传导的转运通道, 催化细胞质中ADP和线粒体内ATP间进行跨膜交换。通过与其他昆虫的腺苷酸转移酶蛋白序列比较, 发现该基因具有高度的保守性, 氨基酸序列同源性都在90%左右。     

Identification and partial purification of a heart mitochondrial membrane proteinase

Membrane-bound proteinase activity was demonstrated by a solid-phase assay system in both beef heart and rat liver mitochondria. The activity was sensitive to SH reagents and assorted proteinase inhibitors. Although stimulated by nonionic detergents, it became labile when solubilized by detergents. The proteinase activity from heart mitochondria copurified with the ADP:ATP translocator protein. Gel electrophoresis of this preparation revealed the translocator polypeptide as well as a number of minor components. In solubilized mitochondria the ADP:ATP translocator polypeptide slowly disappeared upon standing at 0°C as revealed by polyacrylamide gel electrophoresis under denaturing conditions. The loss of this polypeptide was prevented by addition of proteinase inhibitors as well as the translocator affinity ligand, carboxyatractylate. These observations confirm the presence of an integral membrane proteinase in mitochondria and suggest a structural and enzymatic interaction between the proteinase and the ADP:ATP translocator.Abbreviations PMSF phenylmethanesulfonyl fluoride - TPCK l-1-tosylamido-2-phenylethylchloromethyl ketone - TLCK 1-chloro-3-tosylamido-7-amino-l-2-heptanone - NEM N-ethylmaleimide - PCMBS p-chloromercuriphenylsulfonic acid - SDS sodium dodecyl sulfate - MOPS morpholinopropane sulfonate - [I₅₀] concentration of inhibitor required to give 50% inhibition     

Conserved structural domains among species and tissues-specific differences in the mitochondrial phosphate-transport protein and the ADP/ATP carrier

Peptide maps were generated of the CNBr-digested mitochondrial phosphate-transport protein and ADP/ATP carrier from bovine and rat heart, rat liver and blowfly flight muscle. Total mitochondrial proteins from the same sources plus pig heart were separated by SDS-polyacrylamide gel electrophoresis. The peptide maps and the total mitochondrial proteins were electroblotted onto nitrocellulose membranes and reacted with rabbit antisera raised against the purified bovine heart phosphate-transport protein and the ADP/ATP carrier. On the basis of antibody specificity, mobility in SDS-polyacrylamide gel electrophoresis, and peptide maps the following was concluded. Phosphate-transport protein alpha and phosphate-transport protein beta (pig and bovine heart) react equally with the first and also with the second of two independent phosphate-transport protein-antisera. Tissue-specific structural domains exist for both the phosphate-transport protein and the ADP/ATP carrier, i.e., one phosphate-transport protein-antiserum reacts with the phosphate-transport protein from all assayed sources, the other only with the cardiac phosphate-transport protein. These differences may reflect tissue-specific regulation of phosphate and adenine nucleotide transport. Homologies among the different species are found for the phosphate transport protein and the ADP/ATP carrier, except for the flight muscle ADP/ATP carrier. These conserved structural domains of the phosphate-transport protein may relate directly to catalytic activity. Alkylation of the purified phosphate-transport proteins and the ADP/ATP carriers by the transport inhibitor N-ethylmaleimide affects electrophoretic mobilities but not the antibody binding. Neither of the two phosphate-transport protein-antisera nor the ADP/ATP-carrier antiserum react with both phosphate transport protein and ADP/ATP carrier, even though these two proteins possess similarities in primary structure and function. Possible mechanisms for generating tissue-specific structural differences in the proteins are discussed.     

Conserved structural domains among species and tissues-specific differences in the mitochondrial phosphate-transport protein and the ADP/ATP carrier

Peptide maps were generated of the CNBr-digested mitochondrial phosphate-transport protein and ADP/ATP carrier from bovine and rat heart, rat liver and blowfly flight muscle. Total mitochondrial proteins from the same sources plus pig heart were separated by SDS-polyacrylamide gel electrophoresis. The peptide maps and the total mitochondrial proteins were electroblotted onto nitrocellulose membranes and reacted with rabbit antisera raised against the purified bovine heart phosphate-transport protein and the ADP/ATP carrier. On the basis of antibody specificity, mobility in SDS-polyacrylamide gel electrophoresis, and peptide maps the following was concluded. (1) Phosphate-transport protein and phosphate-transport protein β (pig and bovine heart) react equally with the first and also with the second of two independent phosphate-transport protein-antisera. (2) Tissue-specific structural domains exist for both the phosphate-transport protein and the ADP/ATP carrier, i.e., one phosphate-transport protein-antiserum reacts with the phosphate-transport protein from all assayed sources, the other only with the cardiac phosphate-transport protein. These differences may reflect tissue-specific regulation of phosphate and adenine nucleotide transport. (3) Homologies among the different species are found for the phosphate transport protein and the ADP/ATP carrier, except for the flight muscle ADP/ATP carrier. These conserved structural domains of the phosphate-transport protein may relate directly to catalytic activity. (4) Alkylation of the purified phosphate-transport proteins and the ADP/ATP carriers by the transport inhibitor N-ethylmaleimide affects electrophoretic mobilities but not the antibody binding. (5) Neither of the two phosphate-transport protein-antisera nor the ADP/ATP-carrier antiserum react with both phosphate transport protein and ADP/ATP carrier, even though these two proteins possess similarities in primary structure and function. Possible mechanisms for generating tissue-specific structural differences in the proteins are discussed.     

Dimeric H-ATP synthase in the chloroplast of Chlamydomonas reinhardtii

H⁺-ATP synthase is the dominant ATP production site in mitochondria and chloroplasts. So far, dimerization of ATP synthase has been observed only in mitochondria by biochemical and electron microscopic investigations. Although the physiological relevance remains still enigmatic, dimerization was proposed to be a unique feature of the mitochondrion [Biochim. Biophys. Acta 1555 (2002) 154]. It is hard to imagine, however, that closely related protein complexes of mitochondria and chloroplast should show such severe differences in structural organization. We present the first evidences for dimerization of chloroplast ATP synthases within the thylakoid membrane.By investigation of the thylakoid membrane of Chlamydomonas reinhardtii by blue-native polyacrylamide gel electrophoresis, dimerization of the chloroplast ATP synthase was detected. Chloroplast ATP synthase dimer dissociates into monomers upon incubation with vanadate or phosphate but not by incubation with molybdate, while the mitochondrial dimer is not affected by the incubation. This suggests a distinct dimerization mechanism for mitochondrial and chloroplast ATP synthase. Since vanadate and phosphate bind to the active sites, contact sites located on the hydrophilic CF₁ part are suggested for the chloroplast ATP synthase dimer. As the degree of dimerization varies with phosphate concentration, dimerization might be a response to low phosphate concentrations.     

Photoaffinity labeling of a synaptic vesicle specific nucleotide transport system from Torpedo marmorata

We have employed azido derivatives of ATP and AMP to identify the ATP translocase of synaptic vesicles. Azido-AMP inhibits transport of both ATP and AMP in vitro. The affinity of the translocase for the azido derivatives is similar to that of the native ligands. Upon UV irradiation of vesicles incubated with radiolabeled azido-AMP or -ATP, a molecular weight (Mr) 34000 polypeptide is selectively modified. On two-dimensional gel electrophoresis, the single radiolabeled polypeptide has a pI of approximately 7.7. Analysis of the fractions obtained when vesicles were purified on linear sucrose density gradients reveals that the Mr 34000 polypeptide is highly enriched in the vesicle-containing fractions. The findings support the notion that this polypeptide is identical with a previously described vesicle-specific component of the same molecular size [Stadler, H., & Tashiro, T. (1979) Eur. J. Biochem. 101, 171-178], and we conclude on the basis of uptake inhibition and photoaffinity labeling results that this protein is directly involved in ATP translocation of synaptic vesicles.     

HP30‐2, a mitochondrial PRAT protein for import of signal sequence‐less precursor proteins in Arabidopsis thaliana

Chloroplasts and mitochondria contain a family of putative preprotein and amino acid transporters designated PRAT. Here,we analyzed the role of two previously characterized PRAT protein family members,encoded by At3g49560(HP30) and At5g24650(HP30-2),in planta using a combination of genetic,cell biological and biochemical approaches. Expression studies and green fluorescent protein tagging identified HP30-2 both in chloroplasts and mitochondria,whereas HP30 was located exclusively in chloroplasts. Biochemical evidence was obtained for an association of mitochondrial HP30-2 with two distinct protein complexes,one containing the inner membrane translocase TIM22 and the other containing an alternative NAD(P)H dehydrogenase subunit(NDC_1)implicated in a respiratory complex 1-like electron transport chain. Through its association with TIM22,HP30-2 is involved in the uptake of carrier proteins and other,hydrophobic membrane proteins lacking cleavable NH2-terminal presequences,whereas HP30-2's interaction with NDC1 may permit controlling mitochondrial biogenesis and activity.     

Response of mitochondrial ATPase activity to uncouplers in isolated organelles and whole cells of Zajdela hepatoma

ATPase activity of coupled Zajdela hepatoma mitochondria was rendered uncoupler-sensitive by decreasing free fatty acids content in mitochondria or by preincubation of mitochondria with ATP prior to the addition of an uncoupler. The latter treatment resulted in an accelerated transport of ATP into the organelles. The effect of carbonylcyanide-m-chlorophenylhydrazone and oligomycin on the decrease of the ATP content in whole Zajdela hepatoma cells indicated that the hepatoma mitochondrial ATPase is stimulated by uncouplers $\text{in}\text{vivo}$. The conclusion is that the uncoupler-insensitive ATPase activity of coupled Zajdela hepatoma mitochondria is exhibited only by isolated organelles and results from a reduced $\text{ATP}\text{ADP}$ translocase activity.     

Purification and properties of ATPase inhibitor from rat liver mitochondria.

(1) The ATPase inhibitior protein has been isolated from rat liver mitochondria in purified form. The molecular weight determined by sodium dodecyl sulfate gel electrophoresis is approximately 9500, and the isoelectric point is 8.9. (2) The protein inhibits both the soluble ATPase and the particle-bound ATPase from rat liver mitochondria. It also inhibits ATPase activities of soluble F1, and inhibitor-depleted submitochondrial particles derived from bovine heart mitochondria. (3) On particle-bound ATPase the inhibitor has its maximal effect if incubated in the presence of Mg2+. ATP at slightly acidic pH. (4) The inhibitor has a minimal effect on Pi-ATP exchange activity in sonicated submitochondrial particles. However, unexpectedly the inhibitor greatly stimules Pi-ATP exchange activity in whole mitochondria while the low ATPase activity of the mitochondria is not affected. The possible mechanism of action of the inhibitor on intact mitochondria is offered.     

Adenine nucleotide pool size,adenine nucleotide translocase activity,and respiratory activity in newborn rabbit liver mitochondria

The adenine nucleotide content (ATP+ADP+AMP) of newborn rabbit liver mitochondria was 6.0±0.5 nmol/mg mitochondrial protein at birth, increased rapidly to 14.5±1.7 nmol/mg protein by 2 h postnatal, peaked at 6 h, then decreased gradually to 7.8±0.6 nmol/mg protein by 4 days postnatal. There was a strong positive correlation (r=0.82) between the total adenine nucleotide pool size and adenine nucleotide translocase activity in these mitochondria. In contrast, glutamate + malate-supported State 3 respiratory rates remained constant from birth through the first week of life. State 4 rates also remained constant, as did the respiratory control index and uncoupled respiratory rates. The following conclusions are suggested: (1) The maximum rate of translocase activity is limited by the intramitochondrial adenine nucleotide pool size. (2) In newborn rabbit liver mitochondria, the State 3 respiratory rate is not limited by either the adenine pool size or the maximum capacity for translocase-mediated adenine exchange. (3) In contrast to rat, rabbit liver mitochondria are fully functional at birth with regard to respiratory rates and oxidative phosphorylation. (4) The rapid postnatal accumulation of adenine nucleotides by liver mitochondria, now documented in two species, may be a general characteristic of normal metabolic adjustment in neonatal mammals.     

Cardiolipin is the membrane receptor for mitochondrial creatine phosphokinase

Treatment of rat heart mitochondria with phosphate or mersalyl releases a number of proteins, including the mitochondrial creatine kinase (mt-CK). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the released proteins showed that phosphate is more selective than mersalyl in releasing mt-CK. The rebinding of mt-CK to mitochondria was selectively inhibited by adriamycin, which complexes membrane-bound cardiolipin. mt-CK activity and binding experiments have shown that intact mitochondria are able to bind approximately twice the amount of mt-CK they originally contain. Liver mitochondria bound heart mitochondria mt-CK to the same extent as creatine kinase-depleted heart mitochondria. mt-CK was bound by liposomes but only if they contained cardiolipin. The binding of mt-CK to cardiolipin-containing liposomes was inhibited by adriamycin. Phosphatidylcholine liposomes reconstituted with the purified ADP/ATP translocator failed to bind mt-CK.