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首页> 外文学位 >Biochemistry of the cytosolic isozymes of fructose-1,6-bisphosphatase and fructose-1,6-bisphosphate aldolase from germinated castor oil seeds.
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Biochemistry of the cytosolic isozymes of fructose-1,6-bisphosphatase and fructose-1,6-bisphosphate aldolase from germinated castor oil seeds.

机译：来自发芽的蓖麻油种子中的果糖-1,6-双磷酸酶和果糖-1,6-双磷酸醛缩酶的胞质同工酶的生物化学。

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摘要

Cytosolic fructose-1,6-bisphosphatase (FBPase{dollar}{bsol}sb{lcub}{bsol}rm c{rcub}{dollar}) is the first regulated step in the gluconeogenic conversion of phosphoenolpyruvate (PEP) to hexose-phosphates in germinating castor oil seed (COS). FBPase{dollar}{bsol}sb{lcub}{bsol}rm c{rcub}{dollar} was purified 3000-fold from COS endosperm to a specific activity of 72 units/mg protein and electrophoretic homogeneity. Polyacrylamide gel electrophoresis and gel filtration indicated that FBPase{dollar}{bsol}sb{lcub}{bsol}rm c{rcub}{dollar} exists as a heterotetramer composed of 41- and 39-kDa subunits. Experiments on the effect of polyethylene glycol (PEG) on the structure and activity of FBPase{dollar}{bsol}sb{lcub}{bsol}rm c{rcub}{dollar} in dilute solution revealed that PEG: (i) stabilized the active 140-kDa heterotetrameric structure, (ii) led to a 3-fold decrease in the enzyme's {dollar}K{bsol}sb{lcub}m{rcub}{dollar} (fructose-1,6-bisphosphate (F-1,6-P{dollar}{bsol}sb2{dollar})), (iii) increased substrate inhibition, and (iv) enhanced intrinsic fluorescence.; Cytosolic F-1,6-P{dollar}{bsol}sb2{dollar} aldolase (Ald{dollar}{bsol}sb{lcub}{bsol}rm c{rcub}{dollar}) co-purified with FBPase{dollar}{bsol}sb{lcub}{bsol}rm c{rcub}{dollar} to near homogeneity and was homogeneous following separation from FBPase{dollar}{bsol}sb{lcub}{bsol}rm c{rcub}{dollar} during hydrophobic chromatography. Co-purification of Ald{dollar}{bsol}sb{lcub}{bsol}rm c{rcub}{dollar} and FBPase{dollar}{bsol}sb{lcub}{bsol}rm c{rcub}{dollar} and an observed decrease in COS Ald{dollar}{bsol}sb{lcub}{bsol}rm c{rcub}{dollar}'s intrinsic fluorescence in the presence of FBPase suggested a specific interaction between the two sequential enzymes. However, analytical multi-angle laser light scattering (MALLS), gel filtration, protease protection and 'far western' analyses did not detect formation of a high molecular weight complex when the purified enzymes were recombined at their estimated in vivo concentrations in the COS cytosol. The 40 kDa Ald{dollar}{bsol}sb{lcub}{bsol}rm c{rcub}{dollar} subunit cross-reacted with anti-(carrot Ald{dollar}{bsol}sb{lcub}{bsol}rm c{rcub}{dollar}) IgG. Gel filtration and MALLS indicated the enzyme is 160-175 kDa homotetramer. Specific activities of 2.50 and 3.23 units/mg protein for cleavage and synthesis of F-1,6-P{dollar}{bsol}sb2{dollar} (respectively), give this enzyme unusually high relative glycolytic activity. Time-course studies indicated a close correlation between total Ald activity and Ald{dollar}{bsol}sb{lcub}{bsol}rm c{rcub}{dollar} concentration during COS development and germination. The results suggest that the synthesis of ALD{dollar}{bsol}sb{lcub}{bsol}rm c{rcub}{dollar} is highly regulated in germinated COS, and that this regulation follows a preset developmental program.; Quantitative changes in COS FBPase{dollar}{bsol}sb{lcub}{bsol}rm c{rcub}{dollar} intrinsic fluorescence due to ligand binding were used to elucidate a model for the regulation of FBPase{dollar}{bsol}sb{lcub}{bsol}rm c{rcub}{dollar} by its ligands. It is hypothesized that a pronounced allosteric transition mediated by AMP binding increases access of F-1,6-P{dollar}{bsol}sb2{dollar} and F-2,6-P{dollar}{bsol}sb2{dollar} to a common inhibitory binding site that overlaps with the enzyme's catalytic site.
机译：胞质果糖-1,6-双磷酸酶（FBPase {dollar} {bsol} sb {lcub} {bsol} rm c {rcub} {dollar}）是磷酸烯醇丙酮酸（PEP）糖异生转化为磷酸己糖的第一步。发芽蓖麻油种子（COS）。从COS胚乳中纯化FBPase {dollar} {bsol} sb {lcub} {bsol} rm c {rcub} {dollar} 3000倍至比活度为72单位/ mg蛋白和电泳均一性。聚丙烯酰胺凝胶电泳和凝胶过滤表明FBPase {dollar} {bsol} sb {lcub} {bsol} rm c {rcub} {dollar}作为由41-和39-kDa亚基组成的异四聚体存在。聚乙二醇（PEG）对FBPase {dollar} {bsol} sb {lcub} {bsol} rm c {rcub} {dollar}的结构和活性影响的实验表明，PEG：（i）稳定了活跃的140 kDa异四聚体结构，（ii）导致该酶的{美元} K {bsol} sb {lcub} m {rcub} {美元}（果糖1,6-双磷酸（F-1 ，6-P {dollar} {bsol} sb2 {dollar}）），（iii）增加底物抑制，和（iv）增强固有荧光。与FBPase {美元一起纯化的胞质F-1,6-P {美元} {bsol} sb2 {美元}醛缩酶（Ald {美元} {bsol} sb {lcub} {bsol} rm c {rcub} {美元}） } {bsol} sb {lcub} {bsol} rm c {rcub} {dollar}接近均一，并且与FBPase分离后是均匀的{dollar} {bsol} sb {lcub} {bsol} rm c {rcub} {dollar}在疏水色谱法中。共纯化Ald {dollar} {bsol} sb {lcub} {bsol} rm c {rcub} {dollar}和FBPase {dollar} {bsol} sb {lcub} {bsol} rm c {rcub} {dollar}和在存在FBPase的情况下，观察到的COS Ald {dollar} {bsol} sb {lcub} {bsol} rm c {rcub} {dollar}固有荧光的降低表明这两种连续酶之间存在特定的相互作用。但是，当纯化的酶以其估计的体内浓度在COS胞质溶胶中重组时，分析性多角度激光散射（MALLS），凝胶过滤，蛋白酶保护和“远西”分析未检测到高分子量复合物的形成。 。 40 kDa Ald {dollar} {bsol} sb {lcub} {bsol} rm c {rcub} {dollar}亚基与抗胡萝卜胡萝卜素Ald {dollar} {bsol} sb {lcub} {bsol} rm c交叉反应{rcub} {dollar}）IgG。凝胶过滤和MALLS表明该酶是160-175kDa的同四聚体。 2.50和3.23单位/ mg蛋白的比活性分别切割和合成F-1,6-P {sb2} sb2 {美元}，使该酶异常高的相对糖酵解活性。时程研究表明，在COS发育和萌发期间，总Ald活性与Ald {dollar} {bsol} sb {lcub} {bsol} rm c {rcub} {dollar}浓度之间密切相关。结果表明，在发芽的COS中ALD {dollar} {bsol} sb {lcub} {bsol} rm c {rcub} {dollar}的合成受到高度调控，并且该调控遵循预设的发育程序。由于配体结合而导致的COS FBPase {dollar} {bsol} sb {lcub} {bsol} rm c {rcub} {dollar}内在荧光的定量变化被用于阐明FBPase {dollar} {bsol} sb调节模型。 {lcub} {bsol} rm c {rcub} {dollar}由其配体组成。假设由AMP结合介导的明显的变构过渡增加了F-1,6-P {dollar} {bsol} sb2 {dollar}和F-2,6-P {dollar} {bsol} sb2 {dollar}与酶催化位点重叠的常见抑制性结合位点。

著录项

  • 作者

    Hodgson, Richard John.;

  • 作者单位

    Queen's University at Kingston (Canada).;

  • 授予单位 Queen's University at Kingston (Canada).;
  • 学科 Chemistry Biochemistry.; Biology Plant Physiology.
  • 学位 Ph.D.
  • 年度 1998
  • 页码 p.3412
  • 总页数 184
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物化学;
  • 关键词

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