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1.
Vacuolar-type H+-ATPases (V-ATPases) is a large multi-protein complex containing at least 14 different subunits, in which subunits A, B, C, D, E, F, G, and H compose the peripheral 500-kDa V1 responsible for ATP hydrolysis, and subunits a, c, c′, c″, and d assembly the 250-kDa membrane-integral V0 harboring the rotary mechanism to transport protons across the membrane. The assembly of V-ATPases requires the presence of all V1 and V0 subunits, in which the V1 must be completely assembled prior to association with the V0, accordingly the V0 failing to assemble cannot provide a membrane anchor for the V1, thereby prohibiting membrane association of the V-ATPase subunits. The V-ATPase mediates acidification of intracellular compartments and regulates diverse critical physiological processes of cell for functions of its numerous functional subunits. The core catalytic mechanism of the V-ATPase is a rotational catalytic mechanism. The V-ATPase holoenzyme activity is regulated by the reversible assembly/disassembly of the V1 and V0, the targeting and recycling of V-ATPase-containing vesicles to and from the plasma membrane, the coupling ratio between ATP hydrolysis and proton pumping, ATP, Ca2+, and its inhibitors and activators. 相似文献
2.
Effect of NH+4 and K+ on the activity of the ribosomal subunits in the EF-G- and EF-T-dependent GTR hydrolysis 总被引:2,自引:0,他引:2
J Voigt G Sander K Nagel A Parmeggiani 《Biochemical and biophysical research communications》1974,57(4):1279-1286
50S ribosomal subunits show in the absence of 30S subunits and at low NH4+ or K+ high turnover activity in EF-G-dependent GTP hydrolysis which is inhibited by increasing concentrations of monovalent cations. At 80 mM NH4+ or K+ this activity is already 70–80% inhibited. This effect is reversed by 30S which are stimulatory with an optimum at about 80 mM for NH4+ and 20–40 mM for K+. At low NH4+ or K+ (<5 mM) stimulation by 30S of maximal 50S activity depends on the [EF-G]/[50S]. Unlike EF-G, EF-T does not show any Phe-tRNA-dependent GTPase activity with 50S alone even at low concentrations of NH4+ or K+. 相似文献
3.
David G. Beer Martin S. Butley Alvin M. Malkinson 《Archives of biochemistry and biophysics》1984,228(1):207-219
Regulatory subunits (R subunits) of mouse lung cAMP-dependent protein kinases undergo age-dependent changes in endogenous proteolysis, with the greatest amount of the major Mr = 37,000 proteolytic fragment detectable during fetal and neonatal development. Homogenization of lung in the presence of various protease inhibitors does not affect this age-related difference, suggesting that the observed quantitative change in R subunit proteolysis occurs in vivo. Mechanisms were sought to account for this age-dependent change. The production of a Mr = 37,000 proteolytic fragment can be stimulated in lung extracts by the addition of exogenous calcium and is due to the action of an endogenous Ca2+-stimulated protease. Neonatal lung extracts show more Ca2+-stimulated proteolysis of R subunits than adult extracts, although only slight agerelated differences in either the Ca2+-stimulated protease or its specific endogenous inhibitor were observed. Age-dependent differences in R subunits which may affect sensitivity to proteases were also examined. Analysis of the two-dimensional patterns of adult and neonatal 8-N3-[32P]cAMP-labeled R subunits before or after limited proteolysis with trypsin suggests that the R subunits are structurally similar. Differences are found, however, in the relative proportions of adult and neonatal Type I R subunits (RI) in the holoenzyme or dissociated forms. An increased proportion of neonatal R subunits exist in the dissociated state, whereas adult R subunits exist primarily in the holoenzyme form. Dissociated R subunits from mouse lung are more susceptible than the holoenzyme to limited proteolysis by the partially purified lung Ca2+-stimulated protease. Dissociation of the holoenzyme in vivo may be a major factor in the age-dependent proteolytic changes observed in mouse lung protein kinases. 相似文献
4.
A rapid and sensitive assay has been developed for the factor-dependent dissociation of eukaryotic ribosomes. This assay takes advantage of the observation that initiation factor eIF-2 will bind Met-tRNAfmet to 40 S subunits but not to 80 S ribosomes. Incubation of wheat germ ribosomes at 1 mm Mg2+ results in their dissociation into 40 S subunits. These subunits spontaneously reassociate when the Mg2+ concentration is raised to 4 mm. However, if the incubation at 1 mm Mg2+ is carried out in the presence of an extract containing a ribosome dissociation factor, a certain portion of the subunits will fail to reassociate when the Mg2+ concentration is raised to 4 mm. The 40 S subunits remaining due to the presence of the dissociation factor can bind [35S]Met-tRNAfmet in the presence of wheat germ eIF-2. The [35S]Met-tRNAfmet bound to the 40 S subunits is readily detected by its retention on a Millipore filter. 相似文献
5.
6.
Action of methanol on the assocation of ribosomal subunits and its effect on the GTPase activity of elongation factor G 总被引:1,自引:0,他引:1
Methanol causes association of 30S and 50S ribosomal subunits from at MgCl2 concentrations in which they are normally completely dissociated. The 70S ribosome formed under these conditions shows a lower sedimentation velocity and is functionally active in the EF-G GTPase. Association of ribosomal subunits in the presence as well as absence of methanol is affected by washing the ribosomes with 0.5 M NH4Cl. Methanol reduces the Mg2+ concentration required for subunit association as well as for EF-G GTPase activity. The basic requirement for EF-G GTPase activity both with and without alcohol is shown to be the association of 30S and 50S subunits. 相似文献
7.
Michael Forgac 《Journal of bioenergetics and biomembranes》1992,24(4):341-350
Clathrin-coated vesicles play an important role in both receptor-mediated endocytosis and intracellular membrane traffic in eukaryotic cells. The coated vesicle (H+)-ATPase functions to provide the acidic environment within endosomes and other intracellular compartments necessary for receptor recycling and intracellular membrane traffic. The coated vesicle (H+)-ATPase is composed of nine different subunits which are divided into two distinct domains. The peripheral V1 domain, which has the structure 733:583:401:341:331, possesses the nucleotide binding sites of the (H+)-ATPase. The integral V0 domain, which has the composition 1001:381:191:176, contains the pathway for proton conduction across the membrane. Topographical analysis indicates a structure for the coated vesicle (H+)-ATPase very similar to that of the F-type ATPases. Reassembly studies have allowed us to probe the function of particular subunits in this complex and the activity properties of the separate domains. These studies have led to insights into possible mechanisms of regulating vacuolar acidification. 相似文献
8.
H+-transporting, F1Fo-type ATP synthases utilize a transmembrane H+ potential to drive ATP formation by a rotary catalytic mechanism. ATP is formed in alternating β subunits of the extramembranous F1 sector of the enzyme, synthesis being driven by rotation of the γ subunit in the center of the F1 molecule between the alternating catalytic sites . The H+ electrochemical potential is thought to drive γ subunit rotation by first coupling H+ transport to rotation of an oligomeric rotor of c subunits within the transmembrane Fo sector. The γ subunit is forced to turn with the c-oligomeric rotor due to connections between subunit c and the γ and ε subunits of F1. In this essay we will review recent studies on the Escherichia coli Fo sector. The monomeric structure of subunit c, determined by NMR, shows that subunit c folds in a helical hairpin with the proton carrying Asp61 centered in the second transmembrane helix (TMH). A model for the structural organization of the c10 oligomer in Fo was deduced from extensive cross-linking studies and by molecular modeling. The model indicates that the H+-carrying carboxyl of subunit c is occluded between neighboring subunits of the c10 oligomer and that two c subunits pack in a “front-to-back” manner to form the H+ (cation) binding site. In order for protons to gain access to Asp61 during the protonation/deprotonation cycle, we propose that the outer, Asp61-bearing TMH-2s of the c-ring and TMHs from subunits composing the inlet and outlet channels must turn relative to each other, and that the swiveling motion associated with Asp61 protonation/deprotonation drives the rotation of the c-ring. The NMR structures of wild-type subunit c differs according to the protonation state of Asp61. The idea that the conformational state of subunit c changes during the catalytic cycle is supported by the cross-linking evidence in situ, and two recent NMR structures of functional mutant proteins in which critical residues have been switched between TMH-1 and TMH-2. The structural information is considered in the context of the possible mechanism of rotary movement of the c10 oligomer during coupled synthesis of ATP. 相似文献
9.
P. Wilhelm P. M. Abuja O. Meisenberger I. Pilz B. Bowien G. P. Pal U. Hahn W. Saenger 《European biophysics journal : EBJ》1986,14(2):93-96
The quaternary structure of ribulose-1,5-bisphosphate carboxylase-oxygenase (rubisco) from Rhodospirillum rubrum, an enzyme consisting of two large subunits, L2, was investigated by small-angle X-ray scattering. In the presence of HCO
3
-
and Mg2+, rubisco is in the active state and displays a radius of gyration of 2.96 nm, a maximum diameter of 9.5 nm and a volume of 170 nm3. A model is presented where the subunits are arranged back-to-back, rotated relative to each other by 90°, and shifted by 1.3 nm. Upon inactivation by removal of HCO
3
-
and Mg2+, the model swells slightly without any distinct changes in configuration. This contrasts with our previous observations with rubisco from Alcaligenes eutrophus, an enzyme composed of small (S) and large (L) subunits, L8S8, where inactivation gives rise to substantial changes in configuration.Abbreviations RuBP
Ribulose-1,5-bisphosphate
- 3-PGA
3-phosphoglyceric acid 相似文献
10.
Michael Forgac 《FEBS letters》1998,440(3)
The vacuolar (H+)-ATPases (or V-ATPases) function to acidify intracellular compartments in eukaryotic cells, playing an important role in such processes as receptor-mediated endocytosis, intracellular membrane traffic, protein degradation and coupled transport. V-ATPases in the plasma membrane of specialized cells also function in renal acidification, bone resorption and cytosolic pH maintenance. The V-ATPases are composed of two domains. The V1 domain is a 570-kDa peripheral complex composed of 8 subunits (subunits A–H) of molecular weight 70–13 kDa which is responsible for ATP hydrolysis. The V0 domain is a 260-kDa integral complex composed of 5 subunits (subunits a–d) which is responsible for proton translocation. The V-ATPases are structurally related to the F-ATPases which function in ATP synthesis. Biochemical and mutational studies have begun to reveal the function of individual subunits and residues in V-ATPase activity. A central question in this field is the mechanism of regulation of vacuolar acidification in vivo. Evidence has been obtained suggesting a number of possible mechanisms of regulating V-ATPase activity, including reversible dissociation of V1 and V0 domains, disulfide bond formation at the catalytic site and differential targeting of V-ATPases. Control of anion conductance may also function to regulate vacuolar pH. Because of the diversity of functions of V-ATPases, cells most likely employ multiple mechanisms for controlling their activity. 相似文献
11.
Anne M. Smardon Negin Dehdar Nasab Maureen Tarsio Theodore T. Diakov Patricia M. Kane 《The Journal of biological chemistry》2015,290(46):27511-27523
The RAVE complex (regulator of the H+-ATPase of vacuolar and endosomal membranes) is required for biosynthetic assembly and glucose-stimulated reassembly of the yeast vacuolar H+-ATPase (V-ATPase). Yeast RAVE contains three subunits: Rav1, Rav2, and Skp1. Rav1 is the largest subunit, and it binds Rav2 and Skp1 of RAVE; the E, G, and C subunits of the V-ATPase peripheral V1 sector; and Vph1 of the membrane Vo sector. We identified Rav1 regions required for interaction with its binding partners through deletion analysis, co-immunoprecipitation, two-hybrid assay, and pulldown assays with expressed proteins. We find that Skp1 binding requires sequences near the C terminus of Rav1, V1 subunits E and C bind to a conserved region in the C-terminal half of Rav1, and the cytosolic domain of Vph1 binds near the junction of the Rav1 N- and C-terminal halves. In contrast, Rav2 binds to the N-terminal domain of Rav1, which can be modeled as a double β-propeller. Only the V1 C subunit binds to both Rav1 and Rav2. Using GFP-tagged RAVE subunits in vivo, we demonstrate glucose-dependent association of RAVE with the vacuolar membrane, consistent with its role in glucose-dependent V-ATPase assembly. It is known that V1 subunit C localizes to the V1-Vo interface in assembled V-ATPase complexes and is important in regulated disassembly of V-ATPases. We propose that RAVE cycles between cytosol and vacuolar membrane in a glucose-dependent manner, positioning V1 and V0 subcomplexes and orienting the V1 C subunit to promote assembly. 相似文献
12.
The assembly of high voltage-activated Ca2+ channels with different β subunits influences channel properties and possibly subcellular targeting. We studied β subunit expression in the somata and axon terminals of the magnocellular neurosecretory cells, which are located in the supraoptic nucleus (SON) and neurohypophysis, respectively. Antibodies directed against the 4 CaVβ subunits (CaVβ1-CaVβ4) were used for immunoblots and for immunostaining of slices of these two tissues. We found that all 4 β subunits are expressed in both locations, but that CaVβ2 had the highest relative expression in the neurohypophysis. These data suggest that the CaVβ2 subunit is selectively targeted to axon terminals and may play a role in targeting and/or regulating the properties of Ca2+ channels. 相似文献
13.
A. N. Noskov 《Russian Journal of Bioorganic Chemistry》2013,39(6):600-606
AB5 toxins are pore-forming protein complexes, which destroy eukaryotic target cells through ADP-ribosylation or N-glycosylation of intracellular enzyme complexes by A1 subunits. In this paradigm, B subunit pentamer interacts with the target-cell receptors and forms a pore in the cell membrane. Then receptor-mediated endocytosis is induced, and A subunit is translocated into the cytosol. In the present article, we propose a new model of A1 subunit translocation as a globular structure. It is based on those endosome properties that present it as a phospholipid bilayer “ball” with 3D structure as opposed to planar “unfolding-folding” 2D model. Furthermore, the proposed model accounts for membrane phospholipid physical and chemical properties and the activity of membrane-bound K+/Na+- and H+-ATPases. A subunit translocation (together with the B subunit) from the endosome to the cytosol is driven by the proton potential difference generated by H+-ATPases. This is followed by the reduction of A1-A2 disulphide bond by intracellular enzymes, and subunits B and A2 return back into the endosome, where they are destroyed by endosomal/lysosomal proteases; the membrane pore is closed. Endosome integrates into the cellular membrane (endosome recycling), and membrane-bound enzymatic complexes (ATPases and others) return back to their initial position. The proposed model of receptor-mediated endocytosis is a universal mechanism of membrane reparation and translocation of effector toxin subunits or any other pore-forming proteins into the target cell. 相似文献
14.
《Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology》1996,1292(1):140-144
The small ribosomal subunit of the halophilic archaeon Haloferax mediterranei has been reconstituted from its dissociated rRNA and protein components. Efficient reconstitution of particles, fully active in poly(U)-dependent polyphenylalanine synthesis, occurs after 2 h of incubation at 36°C in the presence of l.5 M of (NH4)2SO4 100 mM of MgAc2, 20 mM Tris-HCI (pH 8.2) and 6 mM 2-mercaptoethanol. Important differences in the optimal ionic conditions for the reconstitution of the 30S and the 50S ribosomal subunits from Haloferax mediterranei have been found. K+ and NH4+ ions have differing abilities to promote the reconstitution of the particles. The assembly of 30S ribosomal subunits of H. mediterranei has a higher tolerance to ionic strength than the assembly of the 50S subunits and it is independent of the Mg2+concentration present in the system. 相似文献
15.
H. Garrett Wada Philip E. Hass Howard H. Sussman 《Journal of cellular biochemistry》1979,10(3):287-305
The sialoglycoprotein subunits of human placental brush border membranes were labeled by sequential treatment with periodate and (3H)-sodium borohydride, which trititates sialic acid, and by lactoperoxidase-catalyzed (125I) iodination of tyrosine residues. The labeled subunits were characterized with respect to their affinity for antisera raised against Triton X-100 extracts of placental brush border membranes. The immunochemically reactive components were analyzed by two-dimensional electrophoresis according to a modification of the O'Farrell technique [20] enabling the assignment of estimated Mr? and pI. Of the 33 3H-labeled brush border subunits present in Triton X-100-solubilized membrane preparations, 18 subunits reacted with antiplacental brush border antisera insolubilized on CNBr-activated Sepharose or in immunoprecipitates. Fourteen of these tritiated subunits were also labeled with 125I, confirming that these are glycoproteins. The plasma membranes of normal human liver and microsomes from kidney were examined for the placental brush border glycoprotein subunits by reaction with insolubilized antiplacental brush border antisera and two-dimensional electrophoresis of the reacting tritium-labeled subunits. Comparison of the two-dimensional electrophoretic maps of the immunochemically reacting glycoproteins from liver, kidney, and placenta resulted in the identification of seven placental subunits in common with liver and kidney on the basis of antigenic cross-reactivity, Mr?, and pI. Four placental glycoproteins were not found in the other tissues and are potentially specific to the placenta. Three of the placental subunits were only seen in placenta and kidney. Three of the subunits ran at the dye front and could not be assigned molecular weights. One of the subunits was poorly labeled by tritiation of sialic acid and was not considered. 相似文献
16.
Specific mgi mutations in the α, β or γ subunits of the mitochondrial F1-ATPase have previously been found to suppress ρ0 lethality in the petite-negative yeast Kluyveromyces lactis. To determine whether the suppressive activity of the altered F1 is dependent on the F0 sector of ATP synthase, we isolated and disrupted the genes KlATP4, 5 and 7, the three nuclear genes encoding subunits b, OSCP and d. Strains disrupted for any one, or all three of these genes are
respiration deficient and have reduced viability. However a strain devoid of the three nuclear genes is still unable to lose
mitochondrial DNA, whereas a mgi mutant with the three genes inactivated remains petite-positive. In the latter case, ρ0 mutants can be isolated, upon treatment with ethidium bromide, that lack six major F0 subunits, namely the nucleus-encoded subunits b, OSCP and d, and the mitochondrially encoded Atp6, 8 and 9p. Production of
ρ0 mutants indicates that an F1-complex carrying a mgi mutation can assemble in the absence of F0 subunits and that suppression of ρ0 lethality is an intrinsic property of the altered F1 particle.
Received: 7 April 1998 / Accepted: 10 June 1998 相似文献
17.
Jeffrey B. Smith Paul C. Sternweis Leon A. Heppel 《Journal of cellular biochemistry》1975,3(3):248-255
We have partially purified active delta and epsilon subunits of the E. coli membranebound Mg2+ -ATPase (ECF1). Treating purified ECF1 with 50% pyridine precipitates the major subunits (α, β, and γ) of the enzyme, but the two minor subunits (δ and ϵ), which are present in relatively small amounts, remain in solution. The delta and epsilon subunits were then resolved from one another by anion exchange chromatography. The partially purified epsilon strongly inhibits the hydrolytic activity of ECF1. The epsilon fraction inhibits both the highly purified five-subunit ATPase and the enzyme deficient in the δ subunit. The latter result indicates that the delta subunit is not required for inhibition by epsilon. By contrast, two-subunit enzyme, consisting chiefly of the α and β subunits, was insensitive to the ATPase inhibitor, suggesting that the γ subunit may be required for inhibition by epsilon. The partially purified delta subunit restored the capacity of ATPase deficient in delta to recombine with ATPase-depleted membranes and to reconstitute ATP-dependent transhydrogenase. Previously we reported (Biochem. Biophys. Res. Commun. 62:764 [1975]) that a fraction containing both the delta and epsilon subunits of ECF1 restored the capacity of ATPase missing delta to recombine with depleted membranes and to function as a coupling factor in oxidative phosphorylation and for the energized transhydrogenase. These reconstitution experiments using isolated subunits provide rather substantial evidence that the delta subunit is essential for attaching the ATPase to the membrane and that the epsilon subunit has a regulatory function as an inhibitor of the ATPase activity of ECF1. 相似文献
18.
《BBA》1986,849(1):121-130
The binding of 3′-O-(1-naphthoyl)adenosinetriphosphate (1-naphthoyl-ATP), ATP and ADP to TF1 and to the isolated α and β subunits was investigated by measuring changes of intrinsic protein fluorescence and of fluorescence anisotropy of 1-naphthoyl-ATP upon binding. The following results were obtained. (1) The isolated α and β subunits bind 1 mol 1-naphthoyl-ATP with a dissociation constant (KD(1-naphthoyl-ATP)) of 4.6 μM and 1.9 μM, respectively. (2) The KD(ATP) for α and β subunits is 8 μM and 11 μM, respectively. (3) The KD(ADP) for α and β subunits is 38 μM μM and 7 μM, respectively. (4) TF1 binds 2 mol 1-naphthoyl-ATP per mol enzyme with KD = 170 nM. (5) The rate constant for 1-naphthoyl-ATP binding to α and β subunit is more than 5 · 104 M−1s−1. (6) The rate constant for 1-naphthoyl-ATP binding to TF1 is 6.6 · 103 M−1 · s−1 (monophasic reaction); the rate constant for its dissociation in the presence of ATP is biphasic with a fast first phase (kA−1 = 3 · 10−3s−1) and a slower second phase (kA−2 < 0.2 · 10−3s−1). From the appearance of a second peak in the fluorescence emission spectrum of 1-naphthoyl-ATP upon binding it is concluded that the binding sites in TF1 are located in an environment more hydrophobic than the binding sites on isolated α and β subunits. The differences in kinetic and thermodynamic parameters for ligand binding to isolated versus integrated α and β subunits, respectively, are explained by interactions between these subunits in the enzyme complex. 相似文献
19.
Ribosome activity and modification of 16S RNA are influenced by deletion of ribosomal protein S20 总被引:5,自引:1,他引:4
Monica Rydén-Aulin Zhang Shaoping Per Kylsten† Leif A. Isaksson 《Molecular microbiology》1993,7(6):983-992
A spontaneous mutant of Escherichia coli K-12 was isolated that shows an increased misreading ability of all three nonsense codons together with an inability to grow at 42° C. It is demonstrated that the mutation is a deletion of the gene rpsT, coding for ribosomal protein S20. The loss of this protein not only influences the decoding properties of the ribosome; the modification pattern of 16S ribosomal RNA is also changed. This leads to a deficiency in the ability of the mutant to associate its 30S subunits with 50S subunits to form 70S ribosomes. It is suggested that two modified bases, m5C and m62A, are directly or indirectly essential for association of subunits to functional ribosomes in the rpsT mutant strain. Two other modifications were also studied; m2G which is not affected at all and m3U which is undermodified in both active and inactive subunits and, therefore, not involved in subunit association. 相似文献
20.
Rat liver 40 S ribosomal subunits, in the presence of magnesium ions, bind homologous, resolved Met-tRNAs in the absence of added exogenous proteins. The interaction of the aminoacyl-tRNAs with the particle is dependent on the concentration of magnesium ions in the incubation. At various Mg2+ concentrations examined, binding of the putative initiator Met-tRNAi to 40 S subunits is greater than that observed with Met-tRNAm. Also, binding of Met-tRNAi to 40 S subunits is greater than that obtained with 40 S plus 60 S particles. The initial rate of formation of the 40 S·Met-tRNAi complex is greater at 25 °C than at 37 or 4 °C; decay of the complex, which is observed after 15 min of incubation, is greater at 37 °C but it is slower if 60 S subunits are added after the complex has been formed. If 60 S subunits are added to the incubation with 40 S subunits at the start of the reaction, binding of Met-tRNAi is inhibited; inhibition is also obtained if elongation (binding) factor EF-1 or stripped tRNAs (particularly tRNAMet) are present in the incubation mixture containing 40 S subunits. Acetyl-Met-tRNAi binds to 40 S·ApUpG complex to the same extent as unacetylated Met-tRNAi and, after addition of 60 S subunits, reacts extensively with puromycin; the addition of elongation (translocation) factor EF-2 and GTP do not affect the extent of the puromycin reaction, suggesting that the acMet-tRNAi is bound to a site on the 40 S subunits which becomes the P site on 80 S ribosomes. 相似文献