Acidic phospholipids, unsaturated fatty acids, and limited proteolysis mimic the effect of calmodulin on the purified erythrocyte Ca2+ - ATPase

The purified Ca2+-pumping ATPase of human erythrocyte membranes (Niggli, V., Adunyah, E. S., Penniston, J. T., and Carafoli, E. (1981) J. Biol. Chem. 256, 395-401) can be stimulated, in the absence of calmodulin, by other treatments. 1. A variety of acidic phospholipids (phosphatidylserine, cardiolipin, phosphatidylinositol, and phosphatidic acid) stimulate the Vmax and decrease the Km (Ca2+) of the isolated enzyme to the same extent as calmodulin. Unsaturated fatty acids (oleic and linoleic acid) have the same effect as phospholipids but at lower concentrations. Neutral phospholipids (phosphatidylcholine, sphingomyelin, and phosphatidylethanolamine) have no effect on the enzyme. The minimal proportion of acidic phospholipids in the environment of the enzyme necessary for full stimulation is about 40%. 2. The isolated enzyme, after reconstitution in phosphatidylcholine liposomes in the absence of calmodulin, can be activated by limited proteolysis. The trypsinized enzyme has the same high Vmax and high affinity for Ca2+ of the enzyme in the presence of calmodulin.     

The role of phospholipid acyl chains in the activation of mitochondrial ATPase complex.

1. The role of length and unsaturation of phospholipid acyl chains in the activation of ATPase complex was studied with synthetic phosphatidylcholines and a phospholipid-dependent preparation obtained after cholate-extraction of submitochondrial particles (Kagawa, Y. and Racker, E. (1966) J. Biol. Chem. 241, 2467--2474). 2. Micelle-forming, short-chain phosphatidylcholines produced activation only at critical micellar concentration. The reactivated complex was cold-stable but the oligomycin sensitivity was low. 3. Bilayer-forming saturated phosphatidylcholines produced activation which was maximal at 9 carbon atoms in each chain but decreased sharply as the chain-length was increased and essentially disappeared at 14 carbon atoms. By contrast the oligomycin-sensitivity increased with the increase in chain length. 4. Activation of ATPase complex reappeared when bilayers were formed with long-chain unsaturated phosphatidylcholines. The activity was oligomycin sensitive. Significant inhibition of activity was observed also after incorporation of cholesterol into the bilayers. 5. By contrast the activation induced by negatively charged liposomes of diacylphosphatidylglycerol was independent on acyl-chain composition and occurred at very low amounts of phospholipid. 6. The discontinuity in the Arrhenius plot of activity of the ATPase complex reactivated with saturated phospholipids was found at temperatures close to the gel-to-liquid crystalline transition of the lipid showing that the activity of ATPase complex was sensitive to the physical state of membrane phospholipids. 7. It is concluded that (a) reactivation of ATPase complex by isoelectric phospholipids is an interfacial activation, the minimum requirement for the lipid effect being micelle formation. (b) In order to gain the properties of the native complex a stable lamellar phase is needed. Both activity and oligomycin sensitivity are regulated by the chain length and degree of unsaturation of phospholipid acyl chains.     

Assembly of the F0 proton channel of the Escherichia coli F1F0 ATPase: low proton conductance of reconstituted Fo sectors synthesized and assembled in the absence of F1.

We have previously proposed that during assembly of the Escherichia coli F1F0 ATPase, the proton permeability of the Fo sector of the E. coli F1F0 ATPase is increased significantly by interactions with F1 subunits [Pati, S., & Brusilow, W.S.A. (1989) J. Biol. Chem 264, 2640-2644]. To test this model for Fo assembly, we purified F0 sectors synthesized in the presence and absence of F1 subunits and measured the abilities of these different preparations to bind purified F1 ATPase and to conduct protons when reconstituted into liposomes. The results of these studies demonstrated significant differences in proton-conducting abilities of the different Fo preparations. Fo sectors synthesized in the presence of F1 subunits were more permeable to protons than those synthesized in the absence of F1 subunits.     

Reconstitution of the proton translocating ATPase from bovine heart mitochondria into planar phospholipid bilayer membranes

Proton translocating ATPase (F0F1) from bovine heart mitochondria was reconstituted into planar phospholipid bilayers, and its electrogenicity was directly demonstrated. The F0F1 ATPase was solubilized using 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid (CHAPS) as a detergent followed by sucrose density gradient centrifugation according to the method originally described by McEnery et al. for rat liver mitochondria (McEnery et al. (1986) J. Biol. Chem. 259, 4642-4651), with minor modifications. The purified ATPase was reconstituted into proteoliposomes and then reconstituted into planar phospholipid bilayers by the modified fusion method (Hirata et al. (1986) J. Biol. Chem. 261, 9839-9843). A short-circuit current of up to 0.4 pA was induced by adding ATP, and this current was suppressed by the F1 ATPase inhibitor NaN3 or by a specific mitochondrial F0 inhibitor, oligomycin. The direction of the current corresponded to the flow of positive charges from the F1 side to the F0 side. All these facts clearly demonstrate that the mitochondrial F0F1 ATPase was successfully reconstituted into planar phospholipid bilayers, and the current was generated by the ATPase.     

Biosynthesis of novel acidic phospholipid analogs in Escherichia coli.

When cultured in the presence of 600 mM D-mannitol, Escherichia coli K-12 cells synthesized two novel phospholipids. The identities of these compounds are postulated to be phosphatidylmannitol and diphosphatidylmannitol, the sugar alcohol analogs of phosphatidylglycerol and cardiolipin, respectively. The nonacylated glycerol moieties of the normal acidic phospholipids were substituted by D-mannitol. The formation of the analogs was significantly enhanced when strains harboring the pss-1 allele, a temperature-sensitive mutation in phosphatidylserine synthase (Ohta and Shibuya, J. Bacteriol. 132:434-443, 1977), were grown at 42 degrees C, and the accumulation of the analogs was maximum in late stationary phase; more than 90% of the total cellular lipids were these novel phospholipids. Strains with a defective cardiolipin synthase (Pluschke et al., J. Biol. Chem. 253:5048-5055, 1978) failed to form the analog lipids, whereas cells with increased cardiolipin synthase activity due to the presence of a pBR322-derived recombinant plasmid containing the structural gene for cardiolipin synthase produced more mannitol lipids than wild-type strains. These observations and the structures of the analog lipids indicated that cardiolipin synthase participates in the formation of these novel phospholipids. We suggest that reversible alcoholysis and condensation, in addition to low substrate specificity of the enzyme, are the mechanisms involved in this process. Addition to the medium of other straight-chain alditols, D-arabitol, ribitol, xylitol, erythritol, and L-threitol also yielded pairs of novel phospholipids, whereas sorbitol or galactitol produced only one analog in small quantities. These acidic phospholipid analogs have not been reported in any living system. They should be useful in the study of structure-function relationships of phospholipids and in manipulating the structures of various membrane systems.     

Identification and primary structure of the cardiolipin-binding domain of mitochondrial creatine kinase

It was recently shown that the mitochondrial isozyme of heart creatine kinase binds to cardiolipin on the outer half of the inner membrane [Müller, M., et al. (1985) J. Biol. Chem. 260, 3839-3843]. The enzyme has now been extracted and purified to homogeneity from rat heart mitochondria, and cleaved with CNBr. The fragments have been separated on an FPLC system using a Mono Q HR 5/5 column. Only one of these binds to cardiolipin-containing liposomes and has thus been identified as the cardiolipin-binding domain of the enzyme. Its amino acid sequence has been determined. The fragment contains 25 amino acids and corresponds to the N-terminal region of the protein. The binding of the fragment of cardiolipin-containing liposomes was inhibited by adriamycin. Another and larger CNBr fragment could be specifically labelled with periodate-oxidized (di-aldehyde) ATP and has thus been identified as the ATP-binding domain. Chemical modification of the basic amino acids Lys and Arg of the enzyme abolished its binding to cardiolipin.     

Interaction of the mitochondrial ATPase complex with phospholipids

The interaction of bovine heart mitochondrial oligomycin-sensitive ATPase (Serrano, R., Kranner, B. L., and Racker, E. (1976) J. Biol. Chem. 251, 2453-2461) with phospholipids has been examined by labeling the subunits exposed to lipids with photoreactive radioactive phospholipids. A subunit of Mr = 29,000 and some polypeptides in the range of 6,000 to 13,000 daltons were labeled. F1-ATPase subunits did not interact with the photoactive probes. This result is compared with the different pattern of labeling obtained with another mitochondrial ATPase preparation (Galante, Y.M., Wong, S. Y., and Hatefi, Y. (1979) J. Biol. Chem. 254, 12372-12378), which is devoid of the 29,000 component.     

Studies on the activation of rat liver pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase by adrenaline and glucagon. Role of increases in intramitochondrial Ca2+ concentration.

The association of different phospholipids with a lipid-depleted oligomycin-sensitive ATPase from bovine cardiac mitochondria [Serrano, Kanner & Racker (1976) J. Biol. Chem. 251, 2453-2461] has been examined using three approaches. First, reconstitution of the ATPase with different synthetic diacyl phospholipids resulted in a 2-10-fold stimulation of ATPase specific activity depending upon the particular phospholipid employed. The phospholipid headgroup region displayed the following order of ATPase reactivation potential: dioleoylphosphatidylglycerol greater than dioleoylphosphatidic acid greater than dioleoylphosphatidylcholine. Furthermore, the ATPase showed higher levels of specific activity when reconstituted with dioleoyl phospholipid derivatives compared with dimyristoyl derivatives. Second, examination of the phospholipid remaining associated with the lipid-depleted ATPase upon purification showed that phosphatidylcholine, phosphatidylethanolamine, and diphosphatidylglycerol were present. No relative enrichment of any of these phospholipids (compared with their distribution in submitochondrial particles) was noted. Therefore, no preferential association between the ATPase and any one phospholipid could be found in the mitochondrial ATPase. Third, the sodium cholate-mediated phospholipid exchange procedure was employed for studying the phospholipid requirements of the ATPase. Replacement of about 50% of the mitochondrial phospholipid remaining with the lipid-depleted ATPase could be achieved utilizing either synthetic phosphatidic acid or phosphatidylcholine. Examination of the displaced mitochondrial phospholipid showed that phosphatidylcholine, phosphatidylethanolamine, and diphosphatidylglycerol were replaced with equal facility.     

Removing the brakes to cardiomyocyte cell cycle

Comment on: Di Stefano V, et al. J Biol Chem 2011; 286:8644-54.     

Role of cardiolipin in the functioning of mitochondrial L-glycerol-3-phosphate dehydrogenase

Adriamycin was used in situ, in isolated liver mitochondria of hyperthyroid rats to study the role of cardiolipin in the functioning of FAD-linked L-glycerol-3-phosphate dehydrogenase. The apparent kinetic parameters of the reaction catalyzed by the enzyme were affected by adriamycin. The effect of adriamycin was dependent on the electron acceptor, suggesting the existence of distinct binding sites for hydrophobic and hydrophilic acceptors. Assuming a correlation between the two plateaus observed upon binding of adriamycin to the mitochondria and the penetration of the drug into the two leaflets of the inner membrane [Cheneval et al. (1985) J. Biol. Chem. 260, 13003-13007], we can deduce that cardiolipin in both leaflets influences predominantly the electron acceptor binding site(s).     

Fusion of alphaviruses with liposomes is a non-leaky process

It has been reported that low-pH-induced fusion of influenza virus with liposomes results in rapid and extensive release of both low- and high-molecular-weight substances from the liposomes [Günther-Ausborn et al., J. Biol. Chem. 270 (1995) 29279-29285; Shangguan et al., Biochemistry 35 (1996) 4956-4965]. Here, we demonstrate retention of encapsulated water-soluble compounds during fusion of Semliki Forest virus (SFV) or Sindbis virus with liposomes at low pH. Under conditions allowing complete fusion of the liposomes, a limited fluorescence dequenching of liposome-encapsulated calcein was observed, particularly for SFV. Also, radioactively labeled inulin or sucrose were largely retained. Freezing and thawing of the viruses in the absence of sucrose resulted in an enhanced leakiness of fusion. These results support the notion that the alphavirus fusion event per se is non-leaky and may well involve a discrete hemifusion intermediate.     

Modulation of mitochondrial F0F1 catalysis by boundary and bulk phase phospholipids

The importance of boundary and bulk phase phospholipids was studied on a mitochondrial ATPase complex isolated by AH-Sepharose chromatography as described by Dreyfus et al (1984, Anal. Biochem. 142,215-220), this preparation was devoid of the adenine nucleotide carrier. The presence of isoelectric or acidic phospholipids during the purification in the column allows the exchange of tightly bound phospholipids up to 95%. ATP hydrolysis and oligomycin sensitivity were slightly affected by the nature of boundary and bulk phase phospholipids, while Pi-ATP exchange was highly inhibited.     

Noncooperative vs. cooperative reactivation of D-beta-hydroxybutyrate dehydrogenase: multiple equilibria for lecithin binding are determined by the physical state (soluble vs. bilayer) and composition of the phospholipids

D-beta-Hydroxybutyrate dehydrogenase (BDH) is a lecithin-requiring mitochondrial enzyme that catalyzes the interconversion of beta-hydroxybutyrate and acetoacetate. The purified soluble enzyme devoid of lipid (i.e., the apodehydrogenase) can be reactivated with soluble lecithin or by insertion into phospholipid vesicles containing lecithin. Lipid activation curves have a sigmoidal shape, and two models have been proposed to explain them. We have previously reported that the kinetics of reactivation with short-chain lecithins in the soluble state is consistent with a model in which the enzyme enzyme contains two identical, noninteracting lecithin binding sites, both of which must be occupied to activate the enzyme [noncooperative mechanism; Cortese, J.D., Vidal, J.C., Churchill, P., McIntyre, J.O., & Fleischer, S. (1982) Biochemistry 21, 3899-3908]. More recently a kinetic model involving cooperative interactions between lecithin binding sites was proposed for the reactivation of the membrane-bound enzyme [Sandermann, H., Jr., McIntyre, J.O., & Fleischer, S. (1986) J. Biol. Chem. 261, 6201-6208]. This study reinvestigates the basis for the different conclusions in these two studies. The previous study with soluble lecithins was limited to about 34% of maximal activation compared with mitochondrial phospholipid, due to inactivation of the enzyme at the critical micellar concentration. We could now extend this study to 91% activation by increasing the ethanol concentration. This experimental evidence confirms that the soluble system follows a noncooperative equation. We provide a new kinetic approach to test the cooperative model. A velocity equation is derived for a Hill-type cooperative ligand binding system interacting with a mixture of ligands. This equation predicts a proportionality between an overall weighted cooperative dissociation constant [Kcoop(w)] and a dissociation constant for a single lecithin (PC) species from interacting sites (KPC), regulated by the PC molar fraction (XPC): 1/Kcoop(w) = XPC/KPC. The equation was applied to the data of Sandermann et al. [Sandermann, H., Jr., McIntyre, J.O., & Fleischer, S. (1986) J. Biol. Chem. 261, 6201-6208] as well as to newly obtained data. The results obtained over a wide range of PC molar fractions and different mixtures of bilayer phospholipids fit this equation, confirming the cooperative behavior. We conclude that BDH has a different mode of reactivation depending on the nature of the lipid environment. With soluble lecithin, the activation is noncooperative, whereas in the bilayer, mixtures of phospholipids give cooperative behavior that fits a Hill equation.(ABSTRACT TRUNCATED AT 400 WORDS)     

The interaction of Phe472 with a fluorescent inhibitor bound to the complex of myosin subfragment-1 with nucleotide

The fluorescent probe 3-[4-(3-phenyl-2-pyrazolin-1-yl)benzene-1-sulfonyl amido]phenylboronic acid (PPBA) acts as a fluorescent inhibitor for the ATPases of skeletal [Hiratsuka (1994) J. Biol. Chem. 269, 27251-27257] and Dictyostelium discoideum [Bobkov et al. (1997) J. Muscle Res. Cell Motil. 18, 563-571] myosins. The former paper suggested that, upon addition of excess nucleotides to the binary complex of subfragment-1 from skeletal myosin (S1) with PPBA, a stable ternary complex of S1 with PPBA and nucleotide is formed. Useful fluorescence properties of PPBA enable us to distinguish the conformation of the myosin ATPase at the ATP state from that at the ADP state. In the present paper, to determine the PPBA-binding site in the complexes, enzymatic and fluorescence properties of the S1.PPBA.nucleotide complexes were investigated. Upon formation of the ternary complex with ATP, a new peak appeared at 398 nm in the PPBA fluorescence spectrum. Experiments using model compounds of aromatic amino acid suggested that this fluorescence peak at 398 nm is originated from PPBA interacting with Phe residue(s). Taking into account differences in fluorescence spectra between complexes of S1 and those of subfragment-1 from D. discoideum myosin (S1dC), in the ternary complex of S1 formed with ATP, PPBA was suggested to interact with Phe residue(s) that is absent in S1dC. Docking simulation of PPBA on the S1.nucleotide complex revealed that Phe472 interacts with PPBA. Binding sites of PPBA and blebbistatin, an inhibitor showing high affinity and selectivity toward myosin II [Kovács et al. (2004) J. Biol. Chem. 279, 35557-35563], seem to overlap at least partly.     

Analysis of cDNA clones for Acanthamoeba profilin-I and profilin-II shows end to end homology with vertebrate profilins and a small family of profilin genes.

We have cloned and sequenced full length cDNAs for Acanthamoeba profilin-I and profilin-II. The genes and the encoded proteins are nearly identical except for the region between bp 121 and 210 where 35% of the nucleotides and 47% of amino acids differ. Most of these substitutions are conservative, although three of them are responsible for the differences in the isoelectric points of the isoforms [Kaiser et al., Cell Biol., 102:221-226, 1986]. The DNA sequence revealed six corrections in the previously published protein sequence of profilin-I [Ampe et al., J. Biol. Chem. 260:834-840, 1985] and for the first time resolved the ambiguities at the five positions where profilin-IA and -IB differ. The DNA sequence of profilin-II also allowed us to make two corrections in the protein sequence [Ampe et al., FEBS Lett. 228:17-21, 1988a]. Probes prepared from the cDNAs revealed 1 profilin-IA gene, one strongly cross-hybridizing profilin-I gene and one strongly reacting profilin-II gene on Southern blots of Acanthamoeba DNA. Weaker reactions with other genomic DNA fragments leave open the possibility of one additional gene each for profilin-I and profilin-II. Four different profilin RNAs were resolved on Northern blots. It possible to align the sequences of the three Acanthamoeba profilins with the sequences of nine other profilins from five different phyla. There are only two invariant residues in these profilin sequences, but many pairwise identities and conservative substitutions that indicate considerable divergence of this family of proteins from its ancestral precursor.     

Biochemical and immunological characterization of the flagellar-associated regulatory subunit of a type II cyclic adenosine 5'-monophosphate-dependent protein kinase

We have shown previously that the regulatory subunit (RII) of a type II cyclic AMP (cAMP)-dependent protein kinase is tightly associated with mammalian sperm flagella (J. A. Horowitz et al. (1984) J. Biol. Chem. 259, 832-838; J. A. Horowitz et al. (1988) J. Biol. Chem. 263, 2098-2104). In the present study the flagellar RII was compared to other well-characterized RIIs using biochemical and immunological methods. Flagellar polypeptides were screened by immunoblot analysis with monoclonal antibodies directed against the RII alpha and RII beta isoforms. An RII beta monoclonal antibody failed to cross-react with any flagellar polypeptide. In contrast, mAB 622, an RII alpha/RII beta monoclonal antibody, cross-reacted with a 57,000 Da polypeptide. However, another RII alpha/RII beta monoclonal antibody interacted weakly with the flagellar RII, suggesting that the epitope for this antibody is modified in flagellar RII. Partial peptide mapping of 8-azido-[32P]cAMP-labeled RIIs revealed that although heart and testis generated similar fragmentation patterns, there were differences in the maps from flagellar RII. Two-dimensional sodium dodecyl sulfate-gel electrophoresis of 8-azido-[32P]cAMP-labeled RII from rat flagella and bovine heart showed that the former possessed a considerably more acidic isoelectric point. Partial proteolysis of the flagellar RII by either endogenous or exogenous proteases resulted in the cleavage of RII to a 40,000 Mr fragment. Complete release of this fragment from the flagellum was achieved if proteolysis was performed in the presence of thiol reducing agents. In their absence, approximately 50% of the fragment remained bound to the flagellum. The soluble proteolytic fragment was shown to be monomeric by native high-resolution gel-permeation chromatography and contained a functional cAMP-binding site(s).     

Mitochondrial adenosinetriphosphatase inhibitor protein: reversible interaction with complex V (ATP synthetase complex)

Mitochondrial ATPase inhibitor protein (IF1) reacts reversibly with complex V and inhibits up to 90% of its ATPase activity. Both the rate and extent of inhibition are pH and temperature dependent and increase as the pH is lowered from pH 8 tp 6.7 (the lowest pH examined) or as the temperature is increased from 4 to 36 degrees C. Nucleotide triphosphates plus Mg2+ ions are required for inhibition of complex V ATPase activity by IF1. In the presence of Mg2+ ions, the effectiveness order of nucleotides is ATP greater than ITP greater than GTP greater than UTP. Highly purified complex V, which requires added phospholipids for expressing ATPase and ATP-Pi exchange activities, cannot be inhibited by IF1 plust ATP-Mg2+ unless phospholipids are also added. This indicates that the active state of the enzyme is necessary for the IF1 effect to be manifested, because F1-ATPase, which does not contain nor require phospholipids for catalyzing ATP hydrolysis, can be inhibited by IF1 plus ATP-Mg2+ in the absence of added phospholipids. The IF1-inhibited complex V, but not IF1-inhibited F1-ATPase, can be reactivated by incubation at pH greater than 7.0 in the absence of ATP-Mg2+. The reactivation rate is pH dependent and is influenced by temperature and enzyme concentration. Complex V preparations contain small and variable amounts of IF1. This endogenous IF1 behaves the same as added IF1 with respect to conditions described above for inhibition and reactivation and can result in 25-50% inhibition in different complex V preparations. However, complex V lacking endogenous IF1 can be reconstituted from F0, F1, oligomycin sensitivity conferring protein, and phospholipids. Inhibition of this reconstituted preparation in the presence of ATP-Mg2+ depends entirely on addition of IF1. In general, the ATP-Pi exchange activity of complex V is more sensitive to the chemical inhibitors of F1-AtPase tha its ATPase activity. This is not so, however, for IF1. Under conditions that IF1 caused approximately 75% inhibition of ATPase activity of complex V, no more than 10% of the ATP-Pi exchange activity was inhibited.     

Binding of a tightly folded artificial mitochondrial precursor protein to the mitochondrial outer membrane involves a lipid-mediated conformational change

An artificial mitochondrial precursor protein (the presequence of cytochrome oxidase subunit IV fused to mouse dihydrofolate reductase) binds to isolated yeast mitochondrial outer membranes and to liposomes whose phospholipid composition resembles that of outer membranes. In both cases, binding is strongly inhibited by low temperature or methotrexate (which stabilizes the dihydrofolate reductase moiety) and partly inhibited by adriamycin (which binds to acidic phospholipids). Binding is accompanied by partial unfolding of the protein. Binding of the urea-denatured fusion protein to outer membranes or liposomes is insensitive to low temperature, methotrexate, or adriamycin. These results, and those reported in the accompanying paper (Eilers, M., Endo, T., and Schatz, G. (1989) J. Biol. Chem. 264, 2945-2950) suggest that import of this fusion protein into isolated mitochondria involves at least partial unfolding by acidic phospholipids on the mitochondrial surface.