豌豆根胞质V1-ATPase的鉴定

利用免疫印迹、免疫电镜和ATP水解活性的测定对豌豆(Pisum sativum L.)根细胞胞质中V1-ATPase复合物的存在进行鉴定.用兔抗绿豆V-type H+-ATPase 的A、B亚基的抗体进行的immuno-blotting和胶体金电镜结果都表明,胞质中存在有A、B亚基.活性测定结果进一步表明胞质具有ATP水解活性.这些结果说明豌豆根胞质具有有活性的V1-ATPase复合物.这是首次直接证明植物中有胞质V1-ATPase的存在.     

盐胁迫对豌豆根液泡膜H^＋—ATPase活性及含量的影响

为了阐明液泡膜H^ -ATPase在盐胁迫下的作用和适应性调节机制,对豌豆（Pisum sativum L.）植株进行不同盐浓度和不同盐胁迫时间（1－3d）的处理后,分别测定液泡膜H^ -ATPase的H^ 转运活性、水解性和蛋白含量（A亚基）的变化。结果表明,100mmol/L和200mmol/L NaCl 处理1dH^ -ATPase的水解活性没有变化,而250mmol/L NaCl处理1d引起水解活性降低约25％。100mmol/L NaCl处理2d内水解活性没有变化,而第3天活性下降约20％。但是上述盐胁迫均能提高液泡膜H^ -ATPase的质子转运活性,说明盐胁迫后H^ -ATPase的水解活性和质子转运活性的变化不成比例,盐胁迫可能导致偶联比率的改变。Western blot研究发现,上述盐胁迫对液泡膜H^ -ATPase（A亚基）的含量基本无影响,仅100mmol/L NaCl处理3d后A亚基的量略有下降,这些结果证明,盐胁迫能刺激提高豌豆根液泡膜H^ -ATPase的H^ 泵效率,且泵效率的提高是源于偶联比率的改变,而不是由于ATP水解活性的提高和蛋白含量的增加。     

苦皮藤素V对东方粘虫中肠V-ATP酶AB亚基复合物水解ATP活性的抑制作用

苦皮藤素V是一种对昆虫具有毒杀活性的化合物,从植物苦皮藤（Celastrus angulatus Max）中分离出来。目前,已发现苦皮藤素V可与粘虫中肠液泡型ATP酶（V-ATPase）的H、B和a亚基结合,但是其具体作用机理还尚不清楚。本研究将大肠杆菌(Escherichia coli)中表达得到的东方粘虫中肠V-ATPase A亚基突变体TSCA和V-ATPase B亚基包涵体洗涤、溶解后进行复性,获得可溶性AB亚基复合物后采用亲和层析纯化。将纯化好的AB亚基复合物测定H+K+-ATPase活性,证明其有ATP水解活性。随后,测定苦皮藤素V对复合物ATPase的抑制活性,发现加入苦皮藤素后,复合物ATPase活性降低。因此,其可能是通过抑制了AB亚基复合物的ATPase活性,从而产生了杀虫效果,证明AB亚基复合物为苦皮藤素V的潜在靶点之一。这为了解苦皮藤素与V ATPase相互作用机制打下了基础,也为进一步开发新型杀虫药物奠定了基础。     

苦皮藤素V对东方粘虫中肠V-ATP酶AB亚基复合物水解ATP活性的抑制作用北大核心CSCD

苦皮藤素V是一种对昆虫具有毒杀活性的化合物,从植物苦皮藤(Celastrus angulatus Max)中分离出来。目前,已发现苦皮藤素V可与粘虫中肠液泡型ATP酶(V-ATPase)的H、B和a亚基结合,但是其具体作用机理还尚不清楚。本研究将大肠杆菌(Escherichia coli)中表达得到的东方粘虫中肠V-ATPase A亚基突变体TSCA和V-ATPase B亚基包涵体洗涤、溶解后进行复性,获得可溶性AB亚基复合物后采用亲和层析纯化。将纯化好的AB亚基复合物测定H^+K^+-ATPase活性,证明其有ATP水解活性。随后,测定苦皮藤素V对复合物ATPase的抑制活性,发现加入苦皮藤素后,复合物ATPase活性降低。因此,其可能是通过抑制了AB亚基复合物的ATPase活性,从而产生了杀虫效果,证明AB亚基复合物为苦皮藤素V的潜在靶点之一。这为了解苦皮藤素与VATPase相互作用机制打下了基础,也为进一步开发新型杀虫药物奠定了基础。     

K^＋营养对NaCl胁迫下盐地碱蓬生长及叶片液泡膜V—H^＋—ATP和V—H^＋—PPase活性的影响

对溶液培养的盐地碱蓬（Suaeda salsa L.)幼苗进行不同浓度NaCl胁迫并改变培养液中K^ 浓度,以了解K^ 营养对NaCl胁迫下盐地碱蓬幼苗生长及叶片液泡膜V－H^ -ATPase、V－H^ -PPase活性的影响。提高培养液K^ 浓度可明显增加盐胁迫下碱蓬植株的鲜重、干重,促进盐地碱蓬叶片及根部组织K^ 积累。盐地碱蓬叶片液泡膜V－H^ -ATPase至少由A、B、C、D、E及c亚基组成,其表达量在缺K^ 处理（12μmol/L K^ )下随盐胁迫浓度的增加而减小,而在正常K^ （6mmol/L)培养下则随盐胁迫浓度的增加而增加;盐地碱蓬叶片液泡膜V－H^ -PPase分子量为72kD,在缺K^ 和正常K^ 供应情况下,V－H^ -PPase均有较高表达。V－H^ -ATPase及V－H^ -PPase活性变化与其亚基表达量变化基本成正相关。结果表明：K^ 对盐生植物碱蓬的耐盐性有重要作用,盐胁迫下,K^ 可能参与了V－H^ -ATPase和V－H^ -PPase活性调控。     

玉米根V-ATPase的A亚基磷酸化位点的鉴定

以玉米 (Zea mays L.) 根的高纯度液泡膜为材料进行的磷酸化反应表明,液泡膜蛋白的磷酸化可明显提高V型H -ATPase (V-ATPase) 的ATP水解活性和H 转运活性。进一步研究表明,纯化的液泡膜蛋白能被硫代磷酸化,用V-ATPase的A亚基抗体将一条约69 kD的条带鉴定为A亚基。为了测定V-ATPase的A亚基的磷酸化位点,从硫代磷酸化的凝胶中切下A亚基条带并用胰蛋白酶彻底消化。用RP-HPLC分离纯化酶解片断,收集纯化的硫代磷酸化肽段进行质谱分析所测定的分子量为573.83 Da。A亚基胰蛋白酶彻底消化后能产生61个肽段,只有F56肽段的分子量573.66 Da与573.83 Da最接近,而且F56肽段上只有第525位的丝氨酸可以被磷酸化。因此可以确定,玉米根V-AT-Pase A亚基的潜在磷酸化位点为Ser525。就我们所知,这是首次确定植物V-ATPase A亚基的磷酸化位点。     

水分胁迫对抗旱性不同的荔枝叶片细胞壁H+-ATPase活性的影响

以抗旱性较强的荔枝东刘1号和抗旱性较弱的陈紫1－2年分盆栽实生幼苗为试验材料。研究了水分胁迫对荔枝（Litchi chinensis Sonn）叶片细胞胞质以及以离子键和共价键与细胞壁结合的H^ -ATPase活性的影响。结果表明：（1）叶片相对含水量随水分胁迫程度的增加而减少,抗旱性强的东刘1号下降的幅度小于抗旱性弱的陈紫。（2）H^ -ATPase在细胞中的分布是：细胞胞质H^ -ATPase占绝大多数,其次是共价键结合H^ -ATPase,离子键结合H^ -ATPase最少,品种间差异不明显。（3）在水分胁迫下,荔枝叶片H^ -ATPase活性（比活性）均上升,抗旱性强的品种上升的幅度均大于抗旱性弱的品种。     

大麦幼苗根系液泡膜质子泵对苗的发育和盐胁迫的响应

大麦种子露白后52h的根和芽鞘中V－PPase的水解和质子转运活性较高,并随着幼苗的生长进程而下降,V－ATPase活性逐渐增强,用NaCl处理20d龄的大麦幼苗后,耐盐品种根系的V－PPase活性升高,而不耐盐品种则下降,NaCl对离体膜徵囊V－PPase活性有抑制作用。     

玉米根V-ATPase的A亚基磷酸化位点的鉴定

以玉米(Zea mays L)根的高纯度液泡膜为材料进行的磷酸化反应表明,液泡膜蛋白的磷酸化可明显提高v型H -ATPase(V-ATPase)的ATP水解活性和H 转运活性.进一步研究表明,纯化的液泡膜蛋白能被硫代磷酸化,用V-ATPase的A亚基抗体将一条约69 kD的条带鉴定为A亚基.为了测定V-ATPase的A亚基的磷酸化位点,从硫代磷酸化的凝胶中切下A亚基条带并用胰蛋白酶彻底消化.用RP-HPLC分离纯化酶解片断,收集纯化的硫代磷酸化肽段进行质谱分析所测定的分子量为573.83 Da.A亚基胰蛋白酶彻底消化后能产生61个肽段,只有F56肽段的分子量573.66 Da与573.83 Da最接近,而且F56肽段上只有第525位的丝氨酸可以被磷酸化.因此可以确定,玉米根V-AT-Pase A亚基的潜在磷酸化位点为Ser525.就我们所知,这是首次确定植物V-ATPase A亚基的磷酸化位点.     

一种新的植物低分子量ATPase的纯化及性质研究

采用丙酮粉技术,DEAE-Sephadex A50及FPLC离子交换层析技术从玉米花粉胞质中分离纯化了一种具有ATPase活性和GTPase活性的低分子量可溶性蛋白,纯化倍数为105倍.用SDS-PAGE、二维电泳及薄层扫描技术分析了分离样品的纯度.亚基分子量约为38ku,非变性PAGE测定的全酶分子量为76ku,表明该酶分子是由两个相同亚基组成的二聚体蛋白.等电聚焦电泳测定其等电点为5.6,是酸性蛋白质.用抗牛脑dynamin或kinesin的抗体进行Western-blotting,结果表明该酶蛋白与它们无免疫交叉反应.药理学研究表明:38ku蛋白对Na₃VO₄及NEM均非常敏感.     

ATPase与植物抗盐性

本文综述了高等植物细胞ATPase在盐胁迫下的活性变化及其调控机制。V型H+_ATPase与细胞离子区隔化和植物抗盐性密切相关。盐胁迫提高抗盐植物液泡膜H+_ATPase活性,主要是通过增加V型H+_ATPase主要功能亚基的基因表达以及蛋白质合成。盐胁迫通常降低质膜H+-ATPase活性,很可能是由于酶蛋白质合成受阻,质膜H+-ATPase活性的变化与盐胁迫的强度和时间长短有关。此外,本文还对ABA和Ca2+-CaM等胁迫信号物质对ATPase活性的调控及其与植物抗盐性的关系进行了总结。研究ATPase对盐胁迫的响应和调控机制,有助于阐明植物的盐生境适应机制,也有利于植物的抗盐育种工作。     

Reconstitution in vitro of V1 complex of Thermus thermophilus V-ATPase revealed that ATP binding to the A subunit is crucial for V1 formation

Vacuolar-type H(+)-ATPase (V-ATPase or V-type ATPase) is a multisubunit complex comprised of a water-soluble V(1) complex, responsible for ATP hydrolysis, and a membrane-embedded V(o) complex, responsible for proton translocation. The V(1) complex of Thermus thermophilus V-ATPase has the subunit composition of A(3)B(3)DF, in which the A and B subunits form a hexameric ring structure. A central stalk composed of the D and F subunits penetrates the ring. In this study, we investigated the pathway for assembly of the V(1) complex by reconstituting the V(1) complex from the monomeric A and B subunits and DF subcomplex in vitro. Assembly of these components into the V(1) complex required binding of ATP to the A subunit, although hydrolysis of ATP is not necessary. In the absence of the DF subcomplex, the A and B monomers assembled into A(1)B(1) and A(3)B(3) subcomplexes in an ATP binding-dependent manner, suggesting that ATP binding-dependent interaction between the A and B subunits is a crucial step of assembly into V(1) complex. Kinetic analysis of assembly of the A and B monomers into the A(1)B(1) heterodimer using fluorescence resonance energy transfer indicated that the A subunit binds ATP prior to binding the B subunit. Kinetics of binding of a fluorescent ADP analog, N-methylanthraniloyl ADP (mant-ADP), to the monomeric A subunit also supported the rapid nucleotide binding to the A subunit.     

Distinct expression patterns of different subunit isoforms of the V-ATPase in the rat epididymis

In the epididymis and vas deferens, the vacuolar H(+)ATPase (V-ATPase), located in the apical pole of narrow and clear cells, is required to establish an acidic luminal pH. Low pH is important for the maturation of sperm and their storage in a quiescent state. The V-ATPase also participates in the acidification of intracellular organelles. The V-ATPase contains many subunits, and several of these subunits have multiple isoforms. So far, only subunits ATP6V1B1, ATP6V1B2, and ATP6V1E2, previously identified as B1, B2, and E subunits, have been described in the rat epididymis. Here, we report the localization of V-ATPase subunit isoforms ATP6V1A, ATP6V1C1, ATP6V1C2, ATP6V1G1, ATP6V1G3, ATP6V0A1, ATP6V0A2, ATP6V0A4, ATP6V0D1, and ATP6V0D2, previously labeled A, C1, C2, G1, G3, a1, a2, a4, d1, and d2, in epithelial cells of the rat epididymis and vas deferens. Narrow and clear cells showed a strong apical staining for all subunits, except the ATP6V0A2 isoform. Subunits ATP6V0A2 and ATP6V1A were detected in intracellular structures closely associated but not identical to the TGN of principal cells and narrow/clear cells, and subunit ATP6V0D1 was strongly expressed in the apical membrane of principal cells in the apparent absence of other V-ATPase subunits. In conclusion, more than one isoform of subunits ATP6V1C, ATP6V1G, ATP6V0A, and ATP6V0D of the V-ATPase are present in the epididymal and vas deferens epithelium. Our results confirm that narrow and clear cells are well fit for active proton secretion. In addition, the diverse functions of the V-ATPase may be established through the utilization of specific subunit isoforms. In principal cells, the ATP6V0D1 isoform may have a physiological function that is distinct from its role in proton transport via the V-ATPase complex.     

Structure of the yeast vacuolar ATPase

The subunit architecture of the yeast vacuolar ATPase (V-ATPase) was analyzed by single particle transmission electron microscopy and electrospray ionization (ESI) tandem mass spectrometry. A three-dimensional model of the intact V-ATPase was calculated from two-dimensional projections of the complex at a resolution of 25 angstroms. Images of yeast V-ATPase decorated with monoclonal antibodies against subunits A, E, and G position subunit A within the pseudo-hexagonal arrangement in the V1, the N terminus of subunit G in the V1-V0 interface, and the C terminus of subunit E at the top of the V1 domain. ESI tandem mass spectrometry of yeast V1-ATPase showed that subunits E and G are most easily lost in collision-induced dissociation, consistent with a peripheral location of the subunits. An atomic model of the yeast V-ATPase was generated by fitting of the available x-ray crystal structures into the electron microscopy-derived electron density map. The resulting atomic model of the yeast vacuolar ATPase serves as a framework to help understand the role the peripheral stalk subunits are playing in the regulation of the ATP hydrolysis driven proton pumping activity of the vacuolar ATPase.     

Subunit H of the vacuolar (H+) ATPase inhibits ATP hydrolysis by the free V1 domain by interaction with the rotary subunit F

The vacuolar (H+) ATPases (V-ATPases) are large, multimeric proton pumps that, like the related family of F1F0 ATP synthases, employ a rotary mechanism. ATP hydrolysis by the peripheral V1 domain drives rotation of a rotary complex (the rotor) relative to the stationary part of the enzyme (the stator), leading to proton translocation through the integral V0 domain. One mechanism of regulating V-ATPase activity in vivo involves reversible dissociation of the V1 and V0 domains. Unlike the corresponding domains in F1F0, the dissociated V1 domain does not hydrolyze ATP, and the free V0 domain does not passively conduct protons. These properties are important to avoid generation of an uncoupled ATPase activity or an unregulated proton conductance upon dissociation of the complex in vivo. Previous results (Parra, K. J., Keenan, K. L., and Kane, P. M. (2000) J. Biol. Chem. 275, 21761-21767) showed that subunit H (part of the stator) inhibits ATP hydrolysis by free V1. To test the hypothesis that subunit H accomplishes this by bridging rotor and stator in free V1, cysteine-mediated cross-linking studies were performed. Unique cysteine residues were introduced over the surface of subunit H from yeast by site-directed mutagenesis and used as the site of attachment of the photo-activated cross-linking reagent maleimido benzophenone. After UV-activated cross-linking, cross-linked products were identified by Western blot using subunit-specific antibodies. The results indicate that the subunit H mutant S381C shows cross-linking between subunit H and subunit F (a rotor subunit) in the free V1 domain but not in the intact V1V0 complex. These results indicate that subunits H and F are proximal in free V1, supporting the hypothesis that subunit H inhibits free V1 by bridging the rotary and stator domains.     

The H subunit (Vma13p) of the yeast V-ATPase inhibits the ATPase activity of cytosolic V1 complexes

V-ATPases are composed of a peripheral complex containing the ATP-binding sites, the V(1) sector, attached to a membrane complex containing the proton pore, the V(o) sector. In vivo, free, inactive V(1) and V(o) sectors exist in dynamic equilibrium with fully assembled, active V(1) V(o) complexes, and this equilibrium can be perturbed by changes in carbon source. Free V(1) complexes were isolated from the cytosol of wild-type yeast cells and mutant strains lacking V(o) subunit c (Vma3p) or V(1) subunit H (Vma13p). V(1) complexes from wild-type or vma3Delta mutant cells were very similar, and contained all previously identified yeast V(1) subunits except subunit C (Vma5p). These V(1) complexes hydrolyzed CaATP but not MgATP, and CaATP hydrolysis rapidly decelerated with time. V(1) complexes from vma13Delta cells contained all V(1) subunits except C and H, and had markedly different catalytic properties. The initial rate of CaATP hydrolysis was maintained for much longer. The complexes also hydrolyzed MgATP, but showed a rapid deceleration in hydrolysis. These results indicate that the H subunit plays an important role in silencing unproductive ATP hydrolysis by cytosolic V(1) complexes, but suggest that other mechanisms, such as product inhibition, may also play a role in silencing in vivo.     

The F subunit of Thermus thermophilus V1-ATPase promotes ATPase activity but is not necessary for rotation

V(1)-ATPase from the thermophilic bacterium Thermus thermophilus is a molecular rotary motor with a subunit composition of A(3)B(3)DF, and its central rotor is composed of the D and F subunits. To determine the role of the F subunit, we generated an A(3)B(3)D subcomplex and compared it with A(3)B(3)DF. The ATP hydrolyzing activity of A(3)B(3)D (V(max) = 20 s(-1)) was lower than that of A(3)B(3)DF (V(max) = 31 s(-1)) and was more susceptible to MgADP inhibition during ATP hydrolysis. A(3)B(3)D was able to bind the F subunit to form A(3)B(3)DF. The C-terminally truncated F((Delta85-106)) subunit was also bound to A(3)B(3)D, but the F((Delta69-106)) subunit was not, indicating the importance of residues 69-84 of the F subunit for association with A(3)B(3)D. The ATPase activity of A(3)B(3)DF((Delta85-106)) (V(max) = 24 s(-1)) was intermediate between that of A(3)B(3)D and A(3)B(3)DF. A single molecule experiment showed the rotation of the D subunit in A(3)B(3)D, implying that the F subunit is a dispensable component for rotation itself. Thus, the F subunit binds peripherally to the D subunit, but promotes V(1)-ATPase catalysis.     

草莓果实成熟过程中细胞Ca2+-ATPase活性的变化

‘春星’草莓果实成熟时,总糖和花青苷含量增加,呼吸速率也显著升高;同时,细胞溶质Ca2 -ATPase活性和微粒体膜的Ca2 ．ATPase总活性变化具有相似的特点,即先升高,至粉红期达到高峰,全红期又下降,在微粒体膜中以质膜Ca2 -ATPase占的比例最高。抑制质膜Ca2 -ATPase活性的Na3VO4能促进草莓果实花青苷积累、降低可溶性总糖含量,但对呼吸速率的影响则因草莓果实成熟度不同而异。     

Mutational analysis of the stator subunit E of the yeast V-ATPase

Subunit E is a component of the peripheral stalk(s) that couples membrane and peripheral subunits of the V-ATPase complex. In order to elucidate the function of subunit E, site-directed mutations were performed at the amino terminus and carboxyl terminus. Except for S78A and D233A/T202A, which exhibited V(1)V(o) assembly defects, the function of subunit E was resistant to mutations. Most mutations complemented the growth phenotype of vma4Delta mutants, including T6A and D233A, which only had 25% of the wild-type ATPase activity. Residues Ser-78 and Thr-202 were essential for V(1)V(o) assembly and function. The mutation S78A destabilized subunit E and prevented assembly of V(1) subunits at the membranes. Mutant T202A membranes exhibited 2-fold increased V(max) and about 2-fold less of V(1)V(o) assembly; the mutation increased the specific activity of V(1)V(o) by enhancing the k(cat) of the enzyme 4-fold. Reduced levels of V(1)V(o) and V(o) complexes at T202A membranes suggest that the balance between V(1)V(o) and V(o) was not perturbed; instead, cells adjusted the amount of assembled V-ATPase complexes in order to compensate for the enhanced activity. These results indicated communication between subunit E and the catalytic sites at the A(3)B(3) hexamer and suggest potential regulatory roles for the carboxyl end of subunit E. At the carboxyl end, alanine substitution of Asp-233 significantly reduced ATP hydrolysis, although the truncation 229-233Delta and the point mutation K230A did not affect assembly and activity. The implication of these results for the topology and functions of subunit E within the V-ATPase complex are discussed.     

Reconstitution of vacuolar-type rotary H+-ATPase/synthase from Thermus thermophilus

Vacuolar-type rotary H(+)-ATPase/synthase (V(o)V(1)) from Thermus thermophilus, composed of nine subunits, A, B, D, F, C, E, G, I, and L, has been reconstituted from individually isolated V(1) (A(3)B(3)D(1)F(1)) and V(o) (C(1)E(2)G(2)I(1)L(12)) subcomplexes in vitro. A(3)B(3)D and A(3)B(3) also reconstituted with V(o), resulting in a holoenzyme-like complexes. However, A(3)B(3)D-V(o) and A(3)B(3)-V(o) did not show ATP synthesis and dicyclohexylcarbodiimide-sensitive ATPase activity. The reconstitution process was monitored in real time by fluorescence resonance energy transfer (FRET) between an acceptor dye attached to subunit F or D in V(1) or A(3)B(3)D and a donor dye attached to subunit C in V(o). The estimated dissociation constants K(d) for V(o)V(1) and A(3)B(3)D-V(o) were ～0.3 and ～1 nm at 25 °C, respectively. These results suggest that the A(3)B(3) domain tightly associated with the two EG peripheral stalks of V(o), even in the absence of the central shaft subunits. In addition, F subunit is essential for coupling of ATP hydrolysis and proton translocation and has a key role in the stability of whole complex. However, the contribution of the F subunit to the association of A(3)B(3) with V(o) is much lower than that of the EG peripheral stalks.