Nonionic amphiphilic polymers derived from Tris(hydroxymethyl)-acrylamidomethane keep membrane proteins soluble and native in the absence of detergent

The UV-visible, circular dichroism (CD), and resonance Raman (RR) spectra of the wild type yeast iso-1-cytochrome c (WT) and its mutant F82H in which phenylalanine-82 (Phe-82) is substituted with His are measured and compared for oxidized and reduced forms. The CD spectra in the intrinsic and Soret spectral region, as well as RR spectra in high, middle, and low frequency regions, are discussed. From the analysis of the spectra, it is determined that in the oxidized F82H the two axial ligands to the heme iron are His-18 and His-82 whereas in the reduced form the sixth ligand switches from His-82 to Met-80 providing the coordination geometry similar to that of WT. Based on the spectroscopic data, the conclusion is that the porphyrin macrocycle is less distorted in the oxidized F82H compared to the oxidized WT. Similar distortions are present in the reduced form of the proteins. Frequency shifts of Raman bands, as well as the decrease of the alpha-helix content in the CD spectra, indicate more open conformation of the protein around the heme.     

ATP10, a yeast nuclear gene required for the assembly of the mitochondrial F1-F0 complex

A yeast nuclear gene (ATP10) is reported whose product is essential for the assembly of a functional mitochondrial ATPase complex. Mutations in ATP10 induce a loss of rutamycin sensitivity in the mitochondrial ATPase but do not affect respiratory enzymes. This phenotype has been correlated with a defect in the F0 sector of the ATPase. The wild type ATP10 gene has been cloned by transformation of an atp 10 mutant with a yeast genomic library. The gene codes for a protein of Mr = 30,293. The primary structure of the ATP10 product is not related to any known subunit of the yeast or mammalian mitochondrial ATPase complexes. To further clarify the role of this new protein in the assembly of the ATPase, an antibody was prepared against a hybrid protein expressed from a trpE/ATP 10 fusion gene. The antibody recognizes a 30-kDa protein present in wild type mitochondria. The protein is associated with the mitochondrial membrane but does not co-fractionate either with F1 or with the rutamycin-sensitive F1-F0 complex. These data suggest that the ATP10 product is not a subunit of the ATPase complex but rather is required for the assembly of the F0 sector of the complex.     

The function of mitochondrial F(O)F(1) ATP-synthase from the whiteleg shrimp Litopenaeus vannamei muscle during hypoxia

The effect of hypoxia and re-oxygenation on the mitochondrial complex F(O)F(1)-ATP synthase was investigated in the whiteleg shrimp Litopenaeus vannamei. A 660 kDa protein complex isolated from mitochondria of the shrimp muscle was identified as the ATP synthase complex. After 10h at hypoxia (1.5-2.0 mg oxygen/L), the concentration of L-lactate in plasma increased significantly, but the ATP amount and the concentration of ATPβ protein remained unaffected. Nevertheless, an increase of 70% in the ATPase activity was detected, suggesting that the enzyme may be regulated at a post-translational level. Thus, during hypoxia shrimp are able to maintain ATP amounts probably by using some other energy sources as phosphoarginine when an acute lack of energy occurs. During re-oxygenation, the ATPase activity decreased significantly and the ATP production continued via the electron transport chain and oxidative phosphorylation. The results obtained showed that shrimp faces hypoxia partially by hydrolyzing the ATP through the reaction catalyzed by the mitochondrial ATPase which increases its activity.     

A kinetic study on the hydrolytic activity of mitochondrial ATPase (F0-F1 complex) from pig heart

A kinetic study of mitochondrial ATPase (F0-F1 complex) from pig heart reported in this paper shows that when it was incubated with free Mg2+ (0-2mM), the hydrolytic activity of the ATPase was competitively activated by the Mg2+ and revealed no cooperativity. In the case of incubation with free ATP the hydrolytic activity was competitively inhibited and revealed positive cooperativity. These results are quite different from those of free F1 as obtained by Gautheron and coworkers (1). This indicates that either Mg2+ or ATP produces different effects on F1 when it is in different states, i.e., free state and membrane bound state. This may be considered to mean that the conformation of F1 in membrane bound state, which is influenced by F0 and membrane lipids is different from that of F1 in free state, thus exhibiting different catalytic site cooperativity between subunits, which is the fundamental feature of the mechanism of the enzyme action.     

Mitochondrial F(0)F(1) ATP synthase. Subunit regions on the F1 motor shielded by F(0), Functional significance, and evidence for an involvement of the unique F(0) subunit F(6)

Studies reported here were undertaken to gain greater molecular insight into the complex structure of mitochondrial ATP synthase (F(0)F(1)) and its relationship to the enzyme's function and motor-related properties. Significantly, these studies, which employed N-terminal sequence, mass spectral, proteolytic, immunological, and functional analyses, led to the following novel findings. First, at the top of F(1) within F(0)F(1), all six N-terminal regions derived from alpha + beta subunits are shielded, indicating that one or more F(0) subunits forms a "cap." Second, at the bottom of F(1) within F(0)F(1), the N-terminal region of the single delta subunit and the C-terminal regions of all three alpha subunits are shielded also by F(0). Third, and in contrast, part of the gamma subunit located at the bottom of F(1) is already shielded in F(1), indicating that there is a preferential propensity for interaction with other F(1) subunits, most likely delta and epsilon. Fourth, and consistent with the first two conclusions above that specific regions at the top and bottom of F(1) are shielded by F(0), further proteolytic shaving of alpha and beta subunits at these locations eliminates the capacity of F(1) to couple a proton gradient to ATP synthesis. Finally, evidence was obtained that the F(0) subunit called "F(6)," unique to animal ATP synthases, is involved in shielding F(1). The significance of the studies reported here, in relation to current views about ATP synthase structure and function in animal mitochondria, is discussed.     

All three subunits are required for the reconstitution of an active proton channel (F0) of Escherichia coli ATP synthase (F1F0).

The membrane-integrated, proton-translocating F0 portion of the ATP synthase (F1F0) from Escherichia coli is built up from three kinds of subunits a, b and c with the proposed stoichiometry of 1:2:10 +/- 1. We have dissociated the F0 complex by treatment with trichloroacetate (3 M) at pH 8.0, in the presence of deoxycholate (1%) and N-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate (Zwittergent 3-14, 5%). The subunits were separated by gel filtration with trichloroacetate (1 M) included in the elution buffer. The homogeneity of the fractions was checked by rechromatography and SDS-gel electrophoresis. After integration into phospholipid vesicles each subunit alone as well as all possible combinations were tested for H+ translocating activity and binding of F1. A functional H+ channel could only be reconstituted by the combination a1b2c10 which corresponds to that of native F0.     

Design and synthesis of highly active Alzheimer's beta-secretase (BACE1) inhibitors, KMI-420 and KMI-429, with enhanced chemical stability

Recently, we reported potent and small-sized BACE1 inhibitors KMI-358 and KMI-370 in which the Glu residue is replaced by a beta-N-oxalyl-DAP (l-alpha,beta-diaminopropionyl) residue at the P(4) position. The beta-N-oxalyl-DAP group is important for enhancing BACE1 inhibitory activity, but these inhibitors isomerized to alpha-N-oxalyl-DAP derivatives in solvents. Hence, we used a tetrazole moiety as a bioisostere of the free carboxylic acid of the oxalyl group. KMI-420 and KMI-429, containing a tetrazole ring, showed improved stability and potent enzyme inhibitory activity.     

One-step purification of Escherichia coli H(+)-ATPase (F0F1) and its reconstitution into liposomes with neurotransmitter transporters.

About 30% of the protein in the inner membrane of Escherichia coli strain DK8/pBWU13 is H(+)-ATPase (F0F1), and practically homogeneous F0F1 could be obtained by gradient centrifugation after solubilization of these membranes. The recombinant plasmid pBWU13 carries the unc operon for F0F1. When reconstituted into liposomes, F0F1 formed an ATP-dependent proton gradient and membrane potential. Proteoliposomes reconstituted with F0F1 and solubilized transporters from chromaffin granules or synaptic vesicle membranes could transport serotonin, dopamine, and norepinephrine dependent on ATP hydrolysis. F0F1 can be obtained rapidly from DK8/pBWU13, and its reconstitution into liposomes with transporters may be useful for monitoring these transporters during their purification.     

Chemical modification of the F0 part of the ATP synthase (F1F0) from Escherichia coli. Effects on proton conduction and F1 binding

The purified F0 part of the ATP synthase complex from Escherichia coli was incorporated into liposomes and chemically modified by various reagents. The modified F0-liposomes were assayed for H+ uptake and, after reconstitution with F1, for total and dicyclohexylcarbodiimide-sensitive ATPase activity. The water-soluble carbodiimide, 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide methiodide, (1.2 mM), inhibited H+ uptake to a great extent. Binding of F1 was almost unaffected, but the hydrolysis of ATP was uncoupled from H+ transport. This is reflected by the inhibition of dicyclohexylcarbodiimide-sensitive ATPase activity. Woodward's reagent K, N-ethyl-5-phenylisoxazolium-3'-sulfonate, inhibited both H+ uptake and total ATPase activity. Modification of arginine residues by phenylglyoxal (20 mM) was followed by inhibition of the F1 binding activity by 80% of the control. H+ translocation was reduced to 70%. Diethylpyrocarbonate (3 mM) exhibited a strong inhibiting effect on H+ uptake but not on F1 binding. Modification of tyrosine (by tetranitromethane) as well as lysine residues (by succinic anhydride) did not affect F0 functions. From the data presented we conclude that carboxyl-groups, different from the dicyclohexylcarbodiimide-binding site, are involved in H+ translocation through F0 and, in part, in the functional binding of F1. Furthermore, for the latter function, also arginine residues seem to be important. The role of histidine residues remains unclear at present.     

Distances between the b-subunits in the tether domain of F(0)F(1)-ATP synthase from E. coli

The arrangement of the b-subunits in the holo-enzyme F(0)F(1)-ATP synthase from E. coli is investigated by site-directed mutagenesis spin-label EPR. F(0)F(1)-ATP synthases couple proton translocation with the synthesis of ATP from ADP and phosphate. The hydrophilic F(1)-part and the hydrophobic membrane-integrated F(0)-part are connected by a central and a peripheral stalk. The peripheral stalk consists of two b-subunits. Cysteine mutations are introduced in the tether domain of the b-subunit at b-40, b-51, b-53, b-62 or b-64 and labeled with a nitroxide spin label. Conventional (9 GHz), high-field (95 GHz) and pulsed EPR spectroscopy reveal: All residues are in a relatively polar environment, with mobilities consistent with helix sites. The distance between the spin labels at each b-subunit is 2.9 nm in each mutant, revealing a parallel arrangement of the two helices. They can be in-register but separated by a large distance (1.9 nm), or at close contact and displaced along the helix axes by maximally 2.7 nm, which excludes an in-register coiled-coil model suggested previously for the b-subunit. Binding of the non-hydrolysable nucleotide AMPPNP to the spin-labeled enzyme had no significant influence on the distances compared to that in the absence of nucleotides.     

The yeast F(1)F(0)-ATP synthase: analysis of the molecular organization of subunit g and the importance of a conserved GXXXG motif

The F(1)F(0)-ATP synthase enzyme is located in the inner mitochondrial membrane, where it forms dimeric complexes. Dimerization of the ATP synthase involves the physical association of the neighboring membrane-embedded F(0)-sectors. In yeast, the F(0)-sector subunits g and e (Su g and Su e, respectively) play a key role in supporting the formation of ATP synthase dimers. In this study we have focused on Su g to gain a better understanding of the function and the molecular organization of this subunit within the ATP synthase complex. Su g proteins contain a GXXXG motif (G is glycine, and X is any amino acid) in their single transmembrane segment. GXXXG can be a dimerization motif that supports helix-helix interactions between neighboring transmembrane segments. We demonstrate here that the GXXXG motif is important for the function and in particular for the stability of Su g within the ATP synthase. Using site-directed mutagenesis and cross-linking approaches, we demonstrate that Su g and Su e interact, and our findings emphasize the importance of the membrane anchor regions of these proteins for their interaction. Su e also contains a conserved GXXXG motif in its membrane anchor. However, data presented here would suggest that an intact GXXXG motif in Su g is not essential for the Su g-Su e interaction. We suggest that the GXXXG motif may not be the sole basis for a Su g-Su e interaction, and possibly these dimerization motifs may enable both Su g and Su e to interact with another mitochondrial protein.     

Identification of a nuclear gene (FMC1) required for the assembly/stability of yeast mitochondrial F(1)-ATPase in heat stress conditions

We have identified a yeast nuclear gene (FMC1) that is required at elevated temperatures (37 degrees C) for the formation/stability of the F(1) sector of the mitochondrial ATP synthase. Western blot analysis showed that Fmc1p is a soluble protein located in the mitochondrial matrix. At elevated temperatures in yeast cells lacking Fmc1p, the alpha-F(1) and beta-F(1) proteins are synthesized, transported, and processed to their mature size. However, instead of being incorporated into a functional F(1) oligomer, they form large aggregates in the mitochondrial matrix. Identical perturbations were reported previously for yeast cells lacking either Atp12p or Atp11p, two specific assembly factors of the F(1) sector (Ackerman, S. H., and Tzagoloff, A. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4986--4990), and we show that the absence of Fmc1p can be efficiently compensated for by increasing the expression of Atp12p. However, unlike Atp12p and Atp11p, Fmc1p is not required in normal growth conditions (28--30 degrees C). We propose that Fmc1p is required for the proper folding/stability or functioning of Atp12p in heat stress conditions.     

Role of the carboxyl terminal region of H(+)-ATPase (F0F1) a subunit from Escherichia coli.

The effects of amino acid substitutions in the carboxyl terminal region of the H(+)-ATPase a subunit (271 amino acid residues) of Escherichia coli were studied using a defined expression system for uncB genes coded by recombinant plasmids. The a subunits with the mutations, Tyr-263----end, Trp-231----end, Glu-219----Gln, and Arg-210----Lys (or Gln) were fully defective in ATP-dependent proton translocation, and those with Gln-252----Glu (or Leu), His-245----Glu, Pro-230----Leu, and Glu-219----His were partially defective. On the other hand, the phenotypes of the Glu-269----end, Ser-265----Ala (or end), and Tyr-263----Phe mutants were essentially similar to that of the wild-type. These results suggested that seven amino acid residues between Ser-265 and the carboxyl terminus were not required for the functional proton pathway but that all the other residues except Arg-210, Glu-219, and His-245 were required for maintaining the correct conformation of the proton pathway. The results were consistent with a report that Arg-210 is directly involved in proton translocation.     

Solubilization of active complement receptor type 1 (CR1) from human erythrocyte stroma with trypsin and its purification

Mild trypsinization of human erythrocyte stroma solubilized CR1 (complement receptor type 1, C3b/C4b receptor) without significant loss of decay-accelerating activity to C5 convertases on hemolytic intermediate cells (EAC 1-3b, P). The solubilized CR1 was purified using DEAE-Sephacel, C3-Sepharose, and anti-CR1-Sepharose column chromatographies. The purified material showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing conditions, and its molecular weight was determined to be 175K, about 20K smaller than native CR1. Because the purified sample was separated into the several segments by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, the molecule is considered to be nicked and those segments are associated by disulfide bonds. These results mean that a large portion of the CR1 molecule is present outside of the plasma membrane of erythrocytes, and the intramembranous and cytoplasmic domains are not necessary for decay-accelerating activity.     

Kinetics of oxidative phosphorylation in Paracoccus denitrificans. 1. Mechanism of ATP synthesis at the active site(s) of F0F1-ATPase

(1) The rate of ATP synthesis during NADH-driven aerobic respiration has been measured in plasma membrane vesicles from Paracoccus denitrificans as a function of the concentration of the substrates, ADP and inorganic phosphate (Pi). In both cases, the response of the reaction to changes in the degree of saturation of the F0F1-ATPase generated a perfect Micaelian dependence which allowed the determination of the corresponding Michaelis constants, KmADP and KmPi. (2) These kinetic parameters possess a real mechanistic significance, as concluded from the partial reduction of the rate of phosphorylation by the energy-transfer inhibitor venturicidin and the consequent analysis of the results within the framework of the theory of metabolic control. (3) The same membrane vesicles, which catalyze very high rates of ATP synthesis, have been shown to support much lower rates of the exchange ATP in equilibrium Pi and negligible rates of ATP hydrolysis. Under similar conditions, the preparations are also capable of generating phosphorylation potentials, delta Gp, of 60-61 kJ.mol-1. (4) These properties have allowed analysis of the synthetic reaction in the presence of significant concentrations of the product, ATP, using integrated forms of the Michaelis-Menten rate equations. (5) It has been shown that ATP produces pure competitive product inhibition of the forward reaction with a value of KiATP = 16 +/- 1 microM, thus indicating that the affinity of the nucleotide for the active site(s) of the F0F1-ATPase, during net ATP synthesis, is significantly higher than previously thought. (6) The order of binding of the substrates, ADP and Pi, to the active site(s) has been determined as random. (7) At very low concentrations of ADP, a second and much smaller Michaelis constant for this substrate has been identified, with an estimated value of KmADP approximately equal to 50 nM, associated with a maximal rate of only 2% of that measured at a higher range of concentrations. (8) The results obtained are discussed in relation to the presence of two or three equivalent catalytic sites operating in the cooperative manner explicitly described by the binding change mechanism.     

Stoichiometry of the oligomycin-sensitivity-conferring protein (OSCP) in the mitochondrial F0F1-ATPase determined by an immunoelectrotransfer blot technique

The ratio between the amount of oligomycin-sensitivity-conferring protein (OSCP) and the amount of the alpha and beta subunits of F1-ATPase in the mitochondria has been determined by a method combining electrophoresis, electrotransfer and immunotitration with monoclonal antibodies. The peptides separated in SDS-polyacrylamide gel electrophoresis were blotted to nitrocellulose sheets by electrotransfer. The nitrocellulose sheets were incubated with 125I-labelled purified monoclonal antibodies specific to various peptides. The 125I-labelled immune complexes were located by immunodecoration using peroxidase-conjugated second antibodies and the blotted peptides were revealed with H2O2 and alpha-naphthol. The amount of immune complex present on the nitrocellulose was determined by counting the radioactivity present on the spots. The amount of peptide blotted is directly proportional to the amount of protein loaded on the electrophoresis. By comparing standard curves made with the isolated proteins to the values obtained in the presence of various amounts of the membrane-protein complex, one can calculate the content of this peptide in the membrane. It was found that the mitochondrial membrane contains 2 mol of OSCP per mol of F1.     

Deletions in hydrophilic domains of subunit a from the Escherichia coli F1F0-ATP synthase interfere with membrane insertion or F0 assembly.

The a subunit is a membrane component of the F1F0-ATP synthase from Escherichia coli. Regions of a which appear important for membrane insertion or F0 assembly have been identified by analysis of both deletion mutants and fusion proteins which link the mutant a subunits to alkaline phosphatase. This analysis suggests the hydrophilic, amino-terminal domain of a is required for proper membrane targeting and/or insertion of the nascent polypeptide. In addition, the subcellular fractionation of four different a subunit-beta-galactosidase fusion proteins suggests this domain is localized to the periplasm, in agreement with a proposed topological model of the protein (Lewis, M.J., Chang, J.A., and Simoni, R.D. (1990) J. Biol. Chem. 265, 10541-10550). Deletions within the next three putative loops of a appear to have no significant effect on membrane targeting or insertion. Rather, they seem to interfere with the subsequent assembly of a functional enzyme.     

A highly efficient hydrophobic interaction chromatographic absorbent improved the purification of hepatitis B surface antigen (HBsAg) derived from Hansenula polymorpha cell

To improve the purification efficiency of recombinant hepatitis B surface antigen derived from Hansenula polymorpha (Hans-HBsAg), a serial of absorbents for hydrophobic interaction chromatography with the controllable ligand density and spacer arm were synthesized, then developed and further applied to purify Hans-HBsAg. The absorbent, Butyl-S QZT with the ligand density of 25 μmol/(g wet gel) and spacer arm of 3C, was screened out and its physical and chemical properties were evaluated. High rigidity and low backpressure (<0.06 MPa) were obtained at the flow rate up to 20 ml/min. Moreover, it has the stable chemical characteristics of subjecting to high concentrations of acid, alkali and detergents. This HIC absorbent was further applied to purify Hans-HBsAg with the recovery 94% and purification-fold 9 under the optimized operation condition at pH 6.5 and concentration of ammonium sulfate 7.5%. Finally, the HIC adsorbent of Butyl-S QZT was applied in the integrated three-step chromatographic purification process to purify Hans-HBsAg. About 140 mg of purified Hans-HBsAg was obtained from 1 l cell disruption supernatant at the total recovery of 27% and the purification-fold of 151.8. Based on the assay of SDS-PAGE and SEC-HPLC, the purity of the purified HBsAg was over 99% to meet the requirement for the further inoculation use.     

Isolation and purification of an active gamma-subunit of the F0.F1-ATP synthase from chromatophore membranes of Rhodospirillum rubrum. The role of gamma in ATP synthesis and hydrolysis as compared to proton translocation

We have earlier shown that extraction of Rhodospirillum rubrum chromatophores with LiCl removed completely the beta-subunit of their coupling factor ATPase complex leaving the other four subunits attached to the membrane (Philosoph, S., Binder, A., and Gromet-Elhanan, Z. (1977) J. Biol. Chem. 252, 8747-8752). Further treatment of these beta-less chromatophores with LiBr, under the described optimal conditions, resulted in specific removal of one additional subunit, the gamma-subunit, and both subunits were purified to homogeneity. The beta, gamma-less chromatophores as well as the beta-less ones lost their ATP-linked activities, but retained their light-induced proton uptake, resulting in the formation of an electrochemical gradient of protons composed of both a pH gradient and a membrane potential. These results indicate that the removed beta and gamma subunits cannot be an integral part of an H+ gate in the R. rubrum chromatophore membrane. Each of the removed subunits could bind to the beta, gamma-less chromatophores, but such separate reconstitution of either beta or gamma alone did not lead to restoration of any ATP-linked activity. ATP synthesis and hydrolysis could be restored to the same extent to these chromatophores by their reconstitution with both beta and gamma. It is thus concluded that the presence of both subunits is required for ATP synthesis as well as hydrolysis by the R. rubrum F0.F1 complex. The identical degree of elimination and restoration of ATP synthesis and hydrolysis upon removal and reconstitution of beta and gamma indicates that in R. rubrum at least, there seems to be no reason for suggesting the operation of different catalytic sites for the two activities.     

Binding of an intrinsic ATPase inhibitor to the F(1)FoATPase in phosphorylating conditions of yeast mitochondria

Yeast mitochondrial ATP synthase has three regulatory proteins; ATPase inhibitor, 9K protein, and 15K protein. A mutant yeast lacking these three regulatory factors was constructed by gene disruption. Rates of ATP synthesis of both wild-type and the mutant yeast mitochondria decreased with decrease of respiration, while their membrane potential was maintained at 170-160 mV under various respiration rates. When mitochondrial respiration was blocked by antimycin A, the membrane potential of both types of mitochondria was maintained at about 160 mV by ATP hydrolysis. ATP hydrolyzing activity of F(1)FoATPase solubilized from normal mitochondria decreased in proportion to the rate of ATP synthesis, while the activity of the mutant F(1)FoATPase was constant regardless of changes in the rate of phosphorylation. These observations strongly suggest that F(1)FoATPase in the phosphorylating mitochondria is a mixture of two types of enzyme, phosphorylating and non-phosphorylating enzymes, whose ratio is determined by the rate of respiration and that the ATPase inhibitor binds preferentially to the non-phosphorylating enzyme.