鸡蛋果叶片细胞质丙酮酸激酶的部分纯化及其调节性质

鸡蛋果叶片细胞质丙酮酸激酶(PK_c)纯化92.6倍.其最适pH为7.2,对热较稳定。PEP的K_m为0.037 mmol/L,ADP的K_m为0.05 mmol/L。ASP、Asn、Cys、α—酮戊二酸和苹果酸均对PK?有轻微的激活作用,但草酸、ATP、CaCl_2则具强烈的抑制作用。     

照光对鸡蛋果绿色组织细胞质丙酮酸激酶活性的影响

鸡蛋果叶片照光１５ｍｉｎ后,其成长叶ＰＫ和ＰＫｃ活性降低,幼叶ＰＫ和ＰＫｃ活性提高。鸡蛋果成长叶ＰＫｃ受［ＡＴＰ］／［ＡＤＰ］比值调节,受ＤＴＴ轻微抑制。ＤＴＴ和生理浓度ＡＴＰ对鸡蛋果幼叶ＰＫｃ活性无明显影响。这表明鸡蛋果成长叶ＰＫｃ光下受抑制可能与光下［ＡＴＰ］／［ＡＤＰ］比值提高有关。     

鸡蛋果幼叶细胞质丙酮酸激酶的调节特性

鸡蛋果幼叶ＰＫｃ的Ｋｍ（ＰＥＰ）为０．０３７ｍｍｏｌ／Ｌ,Ｋｍ（ＡＤＰ）为０．０５ｍｍｏｌ／Ｌ,与成长叶相同。幼叶ＰＫｃ的效应物与成长叶的有较大差异,草酸和Ｃａ２＋对幼叶ＰＫＣｃ抑制机制与成长叶相同,但亲和力不同,这可能反映了酶的非活性部位具有不同的构象。     

Glycosidic Inhibitors of Melanogenesis from Leaves of Passiflora edulis

A new flavonoid glycoside, chrysin 6‐C‐β‐rutinoside (chrysin α‐L ‐rhamnopyranosyl‐(1→6)‐C‐β‐glucopyranoside; 2 ), and two new triterpene glycosides, (31R)‐31‐O‐methylpassiflorine ( 7 ) and (31S)‐31‐O‐methylpassiflorine ( 8 ), along with 14 known glycosides, including three flavonoid glycosides, 1, 3 , and 4 , six triterpene glycosides, 5, 6 , and 9 – 12 , three cyano glycosides, 13 – 15 , and two other glycosides, 16 and 17 , were isolated from a MeOH extract of the leaves of Passiflora edulis (passion flower; Passifloraceae). The structures of new compounds were elucidated on the basis of extensive spectroscopic analysis and comparison with literature data. Upon evaluation of compounds 1 – 17 against the melanogenesis in the B16 melanoma cells induced with α‐melanocyte‐stimulating hormone (α‐MSH), three compounds, isoorientin ( 1 ), 2 , and (6S,9R)‐roseoside ( 17 ), exhibited inhibitory effects with 37.3–47.2% reduction of melanin content with no, or almost no, toxicity to the cells (90.8–100.2% cell viability) at 100 μM . Western blot analysis showed that compound 2 reduced the protein levels of MITF, TRP‐1, and tyrosinase, in a concentration‐dependent manner while exerted almost no influence on the level of TRP‐2, suggesting that this compound inhibits melanogenesis on the α‐MSH‐stimulated B16 melanoma cells by, at least in part, inhibiting the expression of MITF, followed by decreasing the expression of TRP‐1 and tyrosinase. In addition, compounds 1 – 17 were evaluated for their inhibitory effects against the Epstein? Barr virus early antigen (EBV‐EA) activation induced by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) in Raji cells.     

Zn^2＋对鸡蛋果细胞质丙酮酸激酶的抑制作用

Zn2 抑制鸡蛋果 ( Passiflora edulis Smis)长成叶与幼叶细胞质丙酮酸激酶 ( PKC)活性 ,抑制程度决定于 Mg2 浓度大小。Zn2 的抑制作用在长成叶 PKC与幼叶 PKC之间表现出一定差异 :当改变 PEP或 ADP浓度时 ,以双倒数作图 ,Zn2 对长成叶和幼叶 PKC均表现为反竞争性抑制 ,但抑制常数不同。Zn2 对长成叶 PKC的抑制常数为 Ki ( PEP) =0 .0 5 9mmol/L,Ki ( ADP) =0 .0 74 mmol/L,对幼叶 PKC的抑制常数 Ki ( PEP) =0 .0 38mmol/L,Ki ( ADP) =0 .0 2 mmol/L。Zn2 对长成叶 PKC与幼叶 PKC的 Ki( Mg2 )亦不同 ,分别为 0 .0 1 6mmol/L和0 .0 0 8mmol/L。这些结果进一步证明长成叶 PKC与幼叶 PKC的酶学性质不同     

Parkin Regulates the Activity of Pyruvate Kinase M2

Parkin, a ubiquitin E3 ligase, is mutated in most cases of autosomal recessive early onset Parkinson disease. It was discovered that Parkin is also mutated in glioblastoma and other human malignancies and that it inhibits tumor cell growth. Here, we identified pyruvate kinase M2 (PKM2) as a unique substrate for parkin through biochemical purification. We found that parkin interacts with PKM2 both in vitro and in vivo, and this interaction dramatically increases during glucose starvation. Ubiquitylation of PKM2 by parkin does not affect its stability but decreases its enzymatic activity. Parkin regulates the glycolysis pathway and affects the cell metabolism. Our studies revealed the novel important roles of parkin in tumor cell metabolism and provided new insight for therapy of Parkinson disease.     

Antihypertensive Effect of an Extract of Passiflora edulis Rind in Spontaneously Hypertensive Rats

Orally administered methanol extract of Passiflora edulis rind (10 mg/kg or 50 mg/kg) or luteolin (50 mg/kg), which is one of consistent polyphenols of the extract, significantly lowered systolic blood pressure in spontaneously hypertensive rats (SHRs). Quantitative analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) showed that the extract contained 20 μg/g dry weight of luteolin and 41 μg/g dry weight of luteolin-6-C-glucoside. It also contained γ-aminobutyric acid (GABA, 2.4 mg/g dry weight by LC-MS/MS or 4.4 mg/g dry weight by amino acid analysis) which has been reported to be an antihypertensive material. Since the extract contained a relatively high concentration of GABA, the antihypertensive effect of the extract in SHRs might be due mostly to the GABA-induced antihypertensive effect and partially to the vasodilatory effect of polyphenols including luteolin.     

小球藻对水溶液中Zn2+、Cd2+的吸附

对小球藻生物吸附Zn^2 ,Cd^2 的影响因素进行了研究,发现小球藻对Zn^2 ,Cd^2 的生物吸附主要经历了快速的物理吸附和缓慢的化学吸附两个步骤;pH值是影响Zn^2 ,Cd^2 生物吸附的一个重要因素,pH值为6-7时,小球藻对Zn^2 ,Cd^2 的去除率较高,在实验条件下去除率可达87%以上;研究还表明,小球藻干粉比新鲜藻能富集更多的Zn^2 ,Cd^2 。用Freundlich方程模拟吸附等温线,拟合良好。     

Cd2+ 、Zn2+单一及复合胁迫对凤眼莲根质膜3种氧化还原酶活性的影响

采用水培法研究了不同浓度Cd2+ 、Zn2+单一及复合胁迫(0.05和0.50 mmol·L-1 Cd2+,0.5和5.0 mmol·L-1 Zn2+)对凤眼莲[Eichhornia crassipes (Mart. ) Solms]幼苗根质膜3种氧化还原酶[NADH氧化酶、Fe(CN)63-还原酶和EDTA-Fe3+还原酶]活性的影响. 结果表明, Cd2+ 、 Zn2+单一及复合胁迫对凤眼莲根质膜NADH氧化酶、Fe(CN)63-还原酶及EDTA-Fe3+还原酶活性的影响效应有明显的差异.0.05或0.50 mmol·L-1 Cd2+单一胁迫处理20 h可使凤眼莲根质膜3种氧化还原酶活性均较对照显著降低(P≤0.05),0.5或5.0 mmol·L-1 Zn2+单一胁迫20 h可导致凤眼莲根质膜NADH氧化酶和EDTA-Fe3+还原酶活性也均较对照显著降低(P≤0.05).随胁迫时间的延长,Cd2+ 、 Zn2+单一及复合胁迫处理均可使凤眼莲根质膜氧化还原酶活性增强,胁迫处理20 d时凤眼莲根质膜3种氧化还原酶活性均高于胁迫20 h的活性.Cd2+-Zn2+复合胁迫对凤眼莲根质膜3种氧化还原酶活性的影响效应因作用时间和胁迫浓度的不同而有一定的差异;在胁迫20 h时,Cd2+与Zn2+之间对凤眼莲根质膜3种氧化还原酶活性的作用关系较复杂,因胁迫浓度的不同表现出协同或拮抗的关系;胁迫20 d时,Cd2+与Zn2+对凤眼莲根质膜3种氧化还原酶活性的复合影响表现出明显的拮抗关系.研究结果显示,Cd2+ 、 Zn2+对凤眼莲根质膜氧化还原酶的复合作用效应与Cd2+和Zn2+的浓度比例及胁迫时间均相关.     

Cu2+和Zn2+对西藏拟溞存活、生长和生殖的影响

在温度(150.5)C,盐度15.50.5的条件下,研究了Cu2+和Zn2+对西藏拟溞(DaphniopsistibetanaSars)存活、生长和生殖的影响。结果表明,西藏拟溞在各Cu2+活度组中的存活率差异不显著,而在10-8.60mol/L组中的体长增长率显著高于其他各组。当水环境中Cu2+活度为10-8.30和10-8.13mol/L时,西藏拟溞的内禀增长率(rm)为0.2211和0.2171/d,显著高于对照组,而西藏拟溞在不同Cu2+活度组中的产卵率均高于对照组,为0.9705-1.1742。西藏拟溞在各Cu2+活度组中的存活率和生长率差异均不显著。当Zn2+活度为10-7.04-10-6.82mol/L时,西藏拟溞的rm为0.2249-0.2296/d,显著高于对照组。西藏拟溞在Zn2+活度为10-7.04mol/L组中的产卵率最大,为1.0178,10-6.82mol/L组次之,为0.867。当Zn2+活度为10-7.04-10-6.82mol/L时,西藏拟溞一生生殖次数显著高于对照组(1.58),为1.92-2.17次。因此,综合来看,当水环境中的Cu2+活度为10-8.60-10-8.13mol/L、Zn2+活度为10-7.04-10-6.82mol/L时能明显的促进西藏拟溞的种群增长、发育和生殖。论文讨论了Cu2+和Zn2+对西藏拟溞的促长机理。       

Covalent Inhibition of Pyruvate Kinase M2 Reprograms Metabolic and Inflammatory Pathways in Hepatic Macrophages against Non-alcoholic Fatty Liver Disease

Warburg effect of aerobic glycolysis in hepatic M1 macrophages is a major cause for metabolic dysfunction and inflammatory stress in non-alcoholic fatty liver disease (NAFLD). Plant-derived triterpene celastrol markedly inhibited macrophage M1 polarization and adipocyte hypertrophy in obesity. The present study was designed to identify the celastrol-bound proteins which reprogrammed metabolic and inflammatory pathways in M1 macrophages. Pyruvate kinase M2 (PKM2) was determined to be a major celastrol-bound protein. Peptide mapping revealed that celastrol bound to the residue Cys³¹ while covalent conjugation altered the spatial conformation and inhibited the enzyme activity of PKM2. Mechanistic studies showed that celastrol reduced the expression of glycolytic enzymes (e.g., GLUT1, HK2, LDHA, PKM2) and related signaling proteins (e.g., Akt, HIF-1α, mTOR), shifted aerobic glycolysis to mitochondrial oxidative phosphorylation and skewed macrophage polarization from inflammatory M1 type to anti-inflammatory M2 type. Animal experiments indicated that celastrol promoted weight loss, reduced serum cholesterol level, lipid accumulation and hepatic fibrosis in the mouse model of NAFLD. Collectively, the present study demonstrated that celastrol might alleviate lipid accumulation, inflammation and fibrosis in the liver via covalent modification of PKM2.     

New perspectives on S100 proteins: a multi-functional Ca 2+ -, Zn 2+ - and Cu 2+ -binding protein family

S100 proteins (16 members) show a very divergent pattern of cell- and tissue-specific expression, of subcel-lular localizations and relocations, of post-translational modifications, and of affinities for Ca 2+ , Zn 2+ , and Cu 2+ , consistent with their pleiotropic intra- and extracellular functions. Up to 40 target proteins are reported to interact with S100 proteins and for S100A1 alone 15 target proteins are presently known. Therefore it is not surprising that many functional roles have been proposed and that several human disorders such as cancer, neurodegenerative diseases, cardiomyopathies, inflammations, diabetes, and allergies are associated with an altered expression of S100 proteins. It is not unlikely that their biological activity in some cases is regulated by Zn 2+ and Cu 2+ , rather than by Ca 2+ Despite the numerous putative functions of S100 proteins, their three-dimensional structures of, e.g., S100B, S100A6, and S100A7 are surprisingly similar. They contain a compact dimerization domain whose conformation is rather insensitive to Ca 2+ binding and two lateral a-helices III and III, which project outward of each subunit when Ca 2+ is bound. Target docking depends on the two hydrophobic patches in front of the paired EF-hand generated by the binding of Ca 2+. The selec-tivity in target binding is assured by the central linker between the two EF-hands and the C-terminal tail. It appears that the S100-binding domain in some target proteins contains a basic amphiphilic a-helix and that the mode of interaction and activation bears structural similarity to that of calmodulin.© Kluwer Academic Publishers     

拟南芥叶细胞游离钙离子的测定

低温(4℃)条件下将钙离子荧光探针Fluo-3／AM导入拟南芥叶细胞,利用激光共聚焦显微技术检测了胞内钙离子荧光强度的分布。实验证明,低温导入Fluo-3／AM法测定拟南芥叶细胞中钙离子荧光强度的变化切实可行。茉莉酸(JA)处理能够诱导胞内游离钙离子浓度的升高。     

Cu2+和Zn2+对普通小麦幼苗生长的影响

Zn^2＋在植物体中参与生长素的合成和某些酶系统的活动;Cu^2＋在催化氧化还原反应中起作用,是植物体内多种氧化酶的组成部分,与光合作用密切相关,在脂肪代谢、蛋白质分解中有一定的作用。当植物体内Cu^2＋和Zn^2＋含量超过一定浓度时对细胞有较大的毒害,危害植物的生长和发育,并可经食物链富集危害人的健康。本文研究了相同浓度的Cu^2＋和Zn^2＋对普通小麦幼苗生理生化特征的影响,以期为防止金属离子污染和培育抗性品种提供参考数据。     

Pyruvate Kinase Activity in Crude Extracts of Leaves of Cynodon dactylonand Other C4Plants

A new extraction procedure and an LDH-coupled assay method are presented for the study of pyruvate kinase (PK) in leaf crude extracts from Cynodon dactylon(L.) Pers and other C₄plants. Extraction at pH 6.8 and assay at pH 6.2 facilitated the measuring of PK activity by eliminating phosphoenolpyruvate carboxylase interference more effectively than the thermal inactivation or chemical inhibition previously used. The method suggested did not affect the kinetic properties of PK as compared to the purified enzyme from C. dactylon.     

丙酮酸激酶前mRNA可变剪接的调控在肿瘤代谢中的作用和意义

丙酮酸激酶是糖酵解的关键酶之一,丙酮酸激酶m基因前mRNA(pre-mRNA)通过可变剪接产生M1和M2型两种丙酮酸激酶异构体,2种异构体的选择性表达决定肿瘤细胞的代谢表型,改变肿瘤细胞的增殖和生长。因此,调控丙酮酸激酶可变剪接,对于控制肿瘤细胞的生长代谢十分重要。研究发现,核不均一核糖核蛋白(hnRNP)A1/A2及多聚嘧啶结合蛋白(PTB,又称hnRNPⅠ)具有调控丙酮酸激酶前mRNA可变剪接的作用,并且致癌转录因子c-Myc与hnRNP A1/A2及PTB在肿瘤细胞中的过表达密切相关。我们结合相关研究进展,简要综述丙酮酸激酶可变剪接调控机制。     

Zn对荇菜叶片保护酶活性、渗透调节物质含量和Ca2+定位分布的影响

本文以不同浓度Zn（0、5、10、15、20mg/L）处理9d的荇菜(Nymphoides peltatum (Gmel.)O. Kuntze)为实验材料,分析了Zn对叶片超氧化物歧化酶（SOD）和过氧化物酶（POD）活性、渗透调节物质（脯氨酸和可溶性糖）含量的影响,并用焦锑酸钙沉淀的细胞化学方法观察了Zn胁迫条件下叶肉细胞内Ca2+水平和分布的动态变化,以揭示水生植物对Zn胁迫的应答机制。研究结果表明,Zn明显抑制了SOD活性和刺激POD活性上升;脯氨酸和可溶性糖积累显著。电镜观察发现,正常条件下叶细胞中的Ca2+主要定位在胞间隙和液泡中,细胞基质和细胞核中较少。添加Zn后,胞间隙和液泡中的Ca2+逐渐进入细胞质,使细胞质中Ca2+浓度明显升高,特别是在质膜内侧和细胞核中出现大量较大的呈圆环状的钙沉淀颗粒。作者认为与保护酶活性紊乱相比,脯氨酸和可溶性糖在荇菜对Zn胁迫的适应中发挥更大的作用。同时细胞内Ca2+水平的增加,可能与许多生理生化过程的改变有关,其在质膜和细胞核等局部区域的大量分布,将会引发对植物的伤害,直至最终死亡。由此可见,荇菜体内多种防御系统同时对Zn胁迫做出反应,包括诱导胁迫相关酶（POD）活性,增加渗透调节物质（脯氨酸和可溶性糖）合成或含量以及改变疏松结合钙的亚细胞分布和含量等。     

Cytosolic Ca2+ and H+ buffers in green algae: A comment

Summary Recently Plieth et al. [Protoplasma (1997) 198: 107–124; 199: 223] gave a quantitative picture of the Ca²⁺ and H⁺ buffers in green algae which we would like to comment. In that paper a mechanistic model was derived which describes the relationship between cytosolic Ca²⁺ and H⁺ assuming that Ca²⁺ and H⁺ interact with the same binding site of a Ca²⁺-H⁺-exchange buffer. But the increase of the cytosolic free Ca²⁺ concentration observed upon acidification can alternatively be described by a co-operative (n=2) protonation of a Ca²⁺/H⁺-binding buffer pointing to an allosteric mechanism of Ca²⁺ liberation. Furthermore we present evidences that the cytosolic buffer capacities for H⁺ (90 mM/pH) and Ca²⁺ (20 mM/pCa) given for Eremosphaera viridis were overestimated by a factor of three and three orders of magnitude, respectively.Abbreviations [Ca²⁺]_c free cytosolic - Ca²⁺ concentration     

发育中脑惊厥性损伤与海马Zn2+转移

Zn^2＋是一种新的调节神经系统兴奋毒性损伤的离子型介质。积聚于海马苔藓纤维（MF）通路突触前膜囊泡内的Zn^2＋,通过特定的自稳态机制向突触后神经元转运,以此实现对大脑兴奋-抑制平衡和认知功能的调节作用。发育中长程或反复惊厥造成海马MF通路Zn^2＋的自稳态破坏,Zn^2＋在细胞内和突触间发生异常转移,并有再生性发芽等病理损伤现象。Zn^2＋转运体、Ca^2＋通透性α-氨基-3-羧基-5-甲基异恶唑-4-丙酸（AMPA）／红藻氨酸通道（Ca-A／K通道）、金属结合蛋白和线粒体等共同参与发芽过程中Zn^2＋的异常转移。除此之外,Zn^2＋亦可作为神经调质,激活信号转导通路,对突触的功能或可塑性产生微妙的影响。这一独特的离子型跨突触信使作用可能具有重要的生理和病理意义。     

Hydrogen Peroxide Induces a Long-Lasting Inhibition of the Ca2+-Dependent Glutamate Release in Cerebrocortical Synaptosomes Without Interfering with Cytosolic Ca2+

Abstract: We studied the action of H₂O₂ on the exocytosis of glutamate by cerebrocortical synaptosomes. The treatment of synaptosomes with H₂O₂ (50–150 µ M ) for a few minutes results in a long-lasting depression of the Ca²⁺-dependent exocytosis of glutamate, induced by KCl or by the K⁺-channel inhibitor 4-aminopyridine. The energy state of synaptosomes, as judged by the level of phosphocreatine and the ATP/ADP ratio, was not affected by H₂O₂, although a transient decrease was observed after the treatment. H₂O₂ did not promote peroxidation, as judged by the formation of malondialdehyde. In indo-1-loaded synaptosomes, the treatment with H₂O₂ did not modify significantly the KCl-induced increase of [Ca²⁺]_i. H₂O₂ inhibited exocytosis also when the latter was induced by increasing [Ca²⁺]_i with the Ca²⁺ ionophore ionomycin. The effects of H₂O₂ were unchanged in the presence of superoxide dismutase and the presence of the Fe³⁺ chelator deferoxamine. These results appear to indicate that H₂O₂, apparently without damaging the synaptosomes, induces a long-lasting inhibition of the exocytosis of glutamate by acting directly on the exocytotic process.