年龄和离体翻译后修饰对兔α-晶状体蛋白分子伴娘活性的影响

目的：探讨年龄和离体翻译后翻译对兔α－晶状体蛋白分子伴娘活性的影响。方法：（1）用凝胶过滤法分离幼年、成年和老年3个组兔水溶性晶状体蛋白,观察不同年龄兔α－晶状体蛋白蛋白抑制β-low晶状体蛋白凝集和变性作用的程度差异。（2）孵育α－晶状体蛋白使之氧化和糖基化,观察修饰和未修饰的α－晶状体蛋白抑制β-low晶状体蛋白凝集和变性作用的程度差异。结果：（1）3个年龄组α－晶状体蛋白抑制热诱导β-low晶状体蛋白抑制凝集和变性作用的程度差异有显著意义（F＝29．100,P＜0．01）,成年组兔α－晶状体蛋白的抑制作用强于老年组,弱于幼年组,差异有显著意义（q＝4．1924,4．339,P＜0．05）。（2）离体氧化和糖基化修饰的α－晶状体蛋白抑制β-low晶状体蛋白凝集和变性的作用均弱于未修饰的α－晶状体蛋白,差异有显著意义（q＝26．9171,11．6404;P＜0．01）。结论：（1）随着年龄的增加,兔α－晶状体蛋白的分子伴娘活性呈下降趋势。（2）离体翻译后修饰可使兔α－晶状体蛋白的分子伴娘活性下降。     

糖基化对牛α-晶状体蛋白分子伴娘功能的作用

α-晶状体蛋白作为晶状体的主要结构蛋白，是维持晶状体结构和屈光特性的重要物质，其分子伴娘功能对保护晶状体透明具有决定性作用。糖基化可修饰α-晶状体蛋白，影响其分子伴娘功能，参与年龄相关性白内障和糖尿病性白内障的形成。本研究通过观察α-磷酸果糖(fructose-6-phosphate，F6P)和核糖对α-晶状体蛋白的修饰和影响，证实糖基化在白内障形成中的作用机制。     

α-晶状体蛋白的结构和功能及基因学研究进展

α-晶状体蛋白包括αA-,αB-晶状体蛋白,是哺乳动物晶状体的主要蛋白,属于小热休克蛋白家族成员,具有分子伴侣功能,可以抑制变性蛋白质的凝聚和酶的失活。α-晶状体蛋白基因突变可导致白内障和肌病。本文对α-晶状体蛋白的分布、结构、功能以及基因学等方面综述。     

ATP对α-晶状体蛋白分子伴侣功能及构象的影响

目的 探讨α-晶状体蛋白分子伴侣功能的作用机制.方法将柠檬酸合酶(CS)、盐酸胍和不同浓度的α-晶状体蛋白及3 mmol/L的ATP温育,分光光度计测定CS的活性.α-晶状体蛋白与ATP温育后用荧光分光光度计检测其荧光发射光谱. 结果α-晶状体蛋白不能使变性的CS复性,但能抑制盐酸胍诱导的CS凝聚,保护CS的失活且呈浓度依赖性.ATP可使这些作用增强.ATP浓度增高可导致α-晶状体蛋白色氨酸荧光强度降低. 结论 ATP可增强α-晶状体蛋白的分子伴侣功能且能改变α-晶状体蛋白的分子构象.     

糖基化对α-晶状体蛋白的修饰和分子伴侣活性的降低

目的　研究糖基化对 α-晶状体蛋白分子伴侣活性的作用。方法　分离牛晶状体α-晶状体蛋白 ,分别与 0 .5 mol· L- 1 果糖和 0 .5 m ol· L- 1 葡萄糖在 37℃温育 ,在第 0、2 4、32天测定 380 nm的光吸收值和 40 0 nm非色氨酸荧光值 ,高压液相色谱 ( high performanceliquid chrom atography,HPL C)和 SDS- PAGE评价蛋白质交联程度 ,采用过氧化氢酶( catalase,CAT)和βL-晶状体蛋白的热凝聚光散射值 ,作为α-晶状体蛋白的分子伴侣活性指标。结果　果糖和葡萄糖可导致时间依赖性 α-晶状体蛋白在 380 nm吸收值的增加 ,非色氨酸荧光值升高 ;HPL C和 SDS- PAGE提示糖与α-晶状体蛋白形成交联复合物。果糖较葡萄糖作用明显。糖基化导致 α-晶状体蛋白抑制 CAT和 βL-晶状体蛋白热凝聚作用降低 ,孵育第 2 4天 ,葡萄糖组α-晶状体蛋白分子伴侣活性分别降低约 12 %和 19% ,果糖组约7%和 9% .结论　糖基化导致 α-晶状体蛋白形成高分子聚合物、凝聚、交联 ,分子伴侣活性丧失 ,这在老化和糖尿病性白内障形成过程中起重要作用。     

首先被果糖糖基化攻击的晶状体蛋白质的分离和鉴定

目的鉴定早期糖基化晶状体蛋白质。方法新鲜牛晶状体匀浆与[^14C]-果糖孵育24h，凝胶过滤层析（Sephacryl S-300HR）分离水溶性蛋白质，并进一步分离脱氢酶；采用亲合层析（Affi-gel601）ff离糖基化蛋白质；SDS-聚丙烯酰胺凝胶电泳分离和鉴定富含放射活性组份的性质。结果α、β-晶状体蛋白和某些脱氢酶为早期糖基化蛋白质。再次分离表明αA2和βA3-晶状体蛋白为首攻糖基化蛋白质；亲合层析结果显示分子质量约为20kin的可能为α和γ-晶状体蛋白，36kin的多肽可能为苹果酸盐脱氢酶或乳酸脱氢酶，这些均为早期糖基化蛋白质。结论αA2、βA3、γ-晶状体蛋白和某些脱氢酶是最容易被糖基化的晶状体蛋白质。糖基化导致晶状体蛋白质的结构发生改变和蛋白质凝聚。早期糖基化诱导酶（如脱氢酶）的失活，可能参与白内障的形成。[眼科新进展21105；25（5）：404—407]     

α-晶状体蛋白分子伴娘功能的初步研究

α-晶状体蛋白作为晶状体的主要结构蛋白，具有分子伴娘特性，对长期维持晶状体的透明具有重要意义。为探讨其作用机制，我们对比观察了透明晶状体皮质和核、年龄相关性白内障不同年龄患者晶状体和不同混浊程度晶状体中α--晶状体蛋白对过氧化氢酶热凝聚的抑制作用。     

α晶状体蛋白的研究进展

α晶状体蛋白包括αA和αB晶状体蛋白，属于小分子热休克蛋白家族成员。α晶状体蛋白在晶状体中为主要的屈光介质，同时具有分子伴娘的功能。αA晶状体蛋白主要存在于晶状体，在脾和胸腺有微量表达。αB晶状体蛋白在许多器官、组织、细胞均有结构性表达，在抵御内、外环境应激发挥重要作用。本文对α晶状体蛋白的分子量及结构，在晶状体内、外中的作用，与神经系统疾病的关系及α晶状体蛋白基因敲除研究等方面作一综述。     

α-晶体蛋白的分子伴娘样作用与白内障

分子伴娘的功能是介导其它蛋白质的正确折叠和装配,但它本身却不是有功能的最终装配产物的组成成分。α晶体蛋白作为晶体中重要的成分蛋白,具有分子伴娘样活性,对各种变性剂包括加热、紫外线照射和化学处理造成的蛋白质非特异性的凝聚具有抑制作用,对糖基化诱导的重要的代谢和抗氧化酶的失活具有保护作用。α晶体蛋白分子伴娘作用的丧失与白内障发病密切相关。本文就其研究现状及前景作扼要综述。     

肌肽对激素诱导晶状体α-晶状体蛋白修饰的作用

目的：研究肌肽对糖皮质激素诱导的α-晶状体蛋白修饰和分子伴侣功能降低的保护作用，以及肌肽与糖皮质激素的直接反应。 方法：采用SephacryIS-300HR凝胶柱分离牛αL-晶状体蛋白。αL-晶状体蛋白分别与不同浓度的泼尼松龙-21-半琥珀酸（P-21-H）及肌肽孵育不同时间。以α1-晶状体蛋白对热凝聚的抑制作为分子伴侣活性指标。采用SDS-聚丙烯酰胺凝胶电泳和荧光光度仪来监测被修饰的αL-晶状体蛋白，并观察肌肽和泼尼松龙-21-半琥珀酸作用后的光谱变化。 结果：泼尼松龙-21-半琥珀酸可导致浓度和时间依赖性αL-晶状体蛋白分子伴侣活性的降低，但是肌肽加剧了这种效应。被泼尼松龙-21-半琥珀酸修饰后的较未被修饰的αL-晶状体蛋白色氨酸荧光强度显著降低，但是其非色氨酸荧光强度向较长波长移位，并呈现时间一剂量依赖的增强，提示有新的荧光色素物的形成。SDS-聚丙烯酰胺凝胶电泳结果显示肌肽能与泼尼松龙-21-半琥珀酸迅速反应，从而抑制激素诱导的蛋白质修饰。时间依赖性的光吸收强度增加提示肌肽与泼尼松龙-21-半琥珀酸反应可能形成了新的加合物。 结论：肌肽能够与泼尼松龙-21-半琥珀酸直接反应，肌肽具有潜在的抗糖皮质激素作用。     

Aggregation of deamidated human βB2-crystallin and incomplete rescue by α-crystallin chaperone

Aging of the lens is accompanied by extensive deamidation of the lens specific proteins, the crystallins. Deamidated crystallins are increased in the insoluble proteins and may contribute to cataracts. Deamidation has been shown in vitro to alter the structure and decrease the stability of human lens βB1, βB2 and βA3-crystallin. Of particular interest, βB2 mutants were constructed to mimic the effect of in vivo deamidations at the interacting interface between domains, at Q70 in the N terminal domain and at Q162, its C-terminal homologue. The double mutant was also constructed. We previously reported that deamidation at the critical interface sites decreased stability, while preserving the dimeric 3D structure. In the present study, dynamic light scattering, differential scanning calorimetry and small angle X-ray scattering were used to investigate the effect of deamidation on stability, thermal unfolding and aggregation. The bovine βLb fraction was used for comparative analysis. The chaperone requirements of the various samples were determined using bovine α-crystallins as the chaperone. Deamidation at both interface Gln residues or at Q70, but not Q162, significantly lowered the temperature for unfolding and aggregation, which was rapidly followed by precipitation. This deamidation-induced aggregation and precipitation was not completely prevented by α-crystallin chaperone. A potential mechanism for cataract formation in vivo involving accumulation of deamidated β-crystallin aggregates is discussed.     

离体氨甲酰化诱导α-晶体蛋白分子伴侣活性的丧失

目的:研究氨甲酰化作为晶状体蛋白质重要的翻译后修饰,对α-晶体蛋白分子伴侣活性的作用。方法:分离牛晶状体αL和βL-晶状体蛋白。αL-晶状体蛋白分别与不同浓度的[14C]氰酸钾温育1至7 d,采用三氯醋酸沉淀法测定αL-晶状体蛋白与[14C]氰酸基团的结合率。αL-晶状体蛋白分别与50和100 mmol.L-1氰酸钾37℃温育3至7 d,测定αL-晶体蛋白抑制βL-晶状体蛋白热凝聚的分子伴侣活性,高效液相色谱法(HPLC)分析αL-晶状体蛋白的氨甲酰化作用。结果:αL-晶状体蛋白与[14C]氰酸基团的结合率和离体氨甲酰化诱导的α-晶状体蛋白伴侣活性的降低呈剂量和时间依赖性。伴侣活性的降低与较高氰酸基团的结合相一致。与对照相比,100mmol.L-1氰酸钾温育7 d导致αL-晶体蛋白伴侣活性几乎殆尽。.HPLC结果提示与氰酸钾温育3 d后可发生剂量依赖性αL-晶体蛋白的凝聚。结论:离体氨甲酰化通过高分子量凝聚物的形成修饰α-晶体蛋白,氨甲酰化诱导α-晶体蛋白的凝聚可能导致其伴侣活性的丧失。     

The origin of some enzymes in tear fluid, determined by comparative investigation with two collection methods.

Comparative investigations of human tear fluid, collected in capillaries as well on filter paper strips, revealed that the enzymes lactate dehydrogenase (LDH), malate dehydrogenase (MDH), pyruvate kinase (PK), isocitrate dehydrogenase (ICDH), aldolase (ALD), glucose-6-phosphate dehydrogenase (G-6-PDH), sorbitol dehydrogenase (SDH), glutamate dehydrogenase (GLDH) and glutamate-oxalacetate transaminase (GOT), are not secreted by the lacrimal gland, but can be liberated into tear fluid after slight damage of the conjunctival and corneal epithelium. Hexokinase (HK) and glutamate-pyruvate transaminase (GPT) and the lysosomal enzymes α-galactosidase (α-gal) and β-hexosaminidase (β-hex), which are produced by the lacrimal gland, may be present in increased amounts after slight epithelial damage. A practical application of these findings is discussed.     

氨甲酰化诱导的牛晶状体3-磷酸甘油醛脱氢酶和硫醇转移酶的失活(英文)

目的:3-磷酸甘油醛脱氢酶(GAPDH)是糖酵解过程中的一个关键酶,与老化和白内障发生密切相关,硫醇转移酶(TTase)可断裂晶状体蛋白质氧化形成的二硫键使硫醇化的蛋白质脱硫醇,对于维持晶状体透明性极为重要。氨甲酰化为重要的翻译后修饰。本研究观察氨甲酰化是否能诱导晶状体GAPDH和TTase的失活。方法:透明新鲜的晶状体取自2周岁健康牛,与100mmol/L氰酸钾于37℃水孵箱分别振动温育7d和12d。设透明晶状体为正常对照组。所有晶状体孵育在50mmol/LDMEM液中,然后每只晶状体去囊膜,匀浆,提取水溶性蛋白质。用分光光度计分别测量GAPDH和TTase的活性。结果:与氰酸钾孵育后GAPDH的活性与正常对照组相比显著降低(P<0.01),其中12d组与7d组相比,活性明显下降。TTase的活性在孵育7d后与对照组相比有统计学差异(P<0.05),12d组的活性与对照组相比有显著差异(P<0.01),7d组活性与12d组相比无统计学差异(P=0.19296)。结论:研究结果显示,分别与100mmol/L氰酸钾孵育7d和12d后,氨甲酰化组的GAPDH和TTase活性与对照组相比有统计学差异,证实氨甲酰化可以诱导GAPDH和TTase酶的失活,为TTase和GAPDH在白内障发病机制中的进一步研究提供了新思路。     

Decreased chaperone activity of alpha-crystallin in selenite cataract may result from selenite-induced aggregation

PURPOSE: To investigate the role of chaperone activity of alpha-crystallin in selenite-induced cataract formation. METHODS: Selenite cataract was induced in Sprague-Dawley rats by five subcutaneous injections of sodium selenite over a 20-day period starting at 8-10 days postpartum. alpha-Crystallin was separated from the rat lenses by size-exclusion chromatography. Bovine alpha(L)-crystallin and beta(L)-crystallin were isolated for studies in vitro, and for the chaperone assays. The protective effects of both alpha(H)- and alpha(L)-crystallin were measured spectrophotometrically in four different assay procedures including the thermally induced aggregation of catalase and beta(L)-crystallin, and the fructation- and heat-induced inactivation of catalase. The bovine alpha(L)-crystallin was incubated with different concentrations of sodium selenite for 72 h and then its chaperone activity against heat-induced beta(L)-crystallin aggregation was assayed. The aggregation of selenite-treated alpha(L)-crystallin was analysed by molecular sieve high-performance liquid chromatography (HPLC). RESULTS: The protection of alpha(H)-crystallin was less than that of alpha(L)-crystallin in both normal and cataractous lenses. The chaperone activities of both alpha(H)- and alpha(L)-crystallin in selenite cataract were decreased compared with normal lenses. The protection provided by both alpha(H)-crystallin and alpha(L)-crystallin against the thermal aggregation of catalase was much greater than their protection against thermally and chemically induced inactivation. HPLC analysis demonstrated aggregation of alpha-crystallin by sodium selenite after 24 h incubation in a dose-dependent fashion. CONCLUSION: The chaperone activity of alpha-crystallin presented parallel patterns of activity with different methods, further supporting the view that the different assays measure essentially the same property. The decreased chaperone activity of alpha-crystallin in selenite cataract may result from selenite-induced aggregation.     

The effect of copper on human and bovine lens and on human cultured lens epithelium enzymes.

The effect of physiological levels of copper on the glycolytic and shunt pathway enzymes of glucose metabolism in human and bovine lens and on cultured lens epithelium has been studied. In human and bovine lens homogenates, hexokinase, phosphofructokinase, pyruvate kinase, glyceraldehydephosphate dehydrogenase, phosphoglycerate kinase and 6-phosphogluconate dehydrogenase were almost completely inhibited in the presence of 50 μm-copper. Aldolase, glucose-6-phosphate dehydrogenase and triose phosphate isomerase were inhibited 78, 50 and 36% respectively. The inhibition of most of the enzymes was not significantly reversed by the addition of EDTA. The inhibition of enzyme activity was also not reversed by dialysis of the copper-treated enzyme against phosphate buffer. Similar results were obtained when human cultured lens epithelium was used as the enzyme source. The studies indicate that if the increased amount of copper present in senile cataractous lenses is in the free form, it can significantly impair glucose metabolism in the lens.     

α-晶状体蛋白在眼科疾病中的作用研究进展

α-晶状体蛋白是晶状体中重要的成分蛋白质,是由αA-和αB-亚基组成的四聚体蛋白质。近年来研究表明α-晶状体蛋白具有很多特性,包括分子伴侣活性、自激酶活性、磷酸化丝氨酸残基-19、45、59模式及通过抑制细胞凋亡蛋白酶前体-3的活化抗凋亡等,但其主要作用在于维持晶状体的结构和屈光性,此外,它还可与视网膜神经节细胞（RGCs）的细胞膜结合促进RGCs的存活和轴突的再生,部分改善视功能。α-晶状体蛋白分子伴侣活性的下降可导致其他晶状体蛋白的凝聚和酶的失活,与眼科疾病的发生密切相关。就α-晶状体蛋白的研究进展及其与眼科疾病的关系进行综述。     

Distribution and some properties of cathepsin D in the retinal pigment epithelium.

Cathepsin D in the retinal pigment epithelium of the bovine eye was studied using standard enzymological techniques. In the bovine eye the retinal pigment epithelium revealed the highest specific activity, the activity being highest in the lysosomal fraction. The enzyme activity in the lysosomal extract of the retinal pigment epithelium was proportional to both the enzyme concentration and incubation time. When the enzyme was heated, the enzyme activity was lost completely. The initial rate of the tyrosine release was dependent on the concentration of the bovine serum albumin, exhibiting a hyperbolic saturation curve. The optimal pH of the enzyme was about 4·0. The enzyme activity was not affected by thiol agents.     

Methylglyoxal inhibits glycation-mediated loss in chaperone function and synthesis of pentosidine in alpha-crystallin

Alpha-crystallin is a major protein in the eye lens and it functions as a molecular chaperone by preventing aggregation of mildly denatured proteins. Glycation, the reaction of sugars and ascorbate with proteins, causes covalent cross-linking and reduces the chaperone function of alpha-crystallin. We demonstrated that methylglyoxal (MGO), a metabolic alpha-dicarbonyl compound, modifies arginine residues in alpha-crystallin and enhances its chaperone function. We wanted to determine whether modification by MGO could protect alpha-crystallin from glycation-mediated cross-linking and loss of chaperone function. Our results show that MGO-modification of isolated bovine lens alpha-crystallin inhibits formation of pentosidine, a glycation-derived protein crosslink. Proteins in organ cultured rat lenses were similarly protected from pentosidine formation. Glycation by sugars and ascorbate resulted in almost complete loss of chaperone function of alpha-crystallin. Surprisingly, addition of MGO during or before glycation not only inhibited the loss of chaperone function, but it actually enhanced the chaperone function of alpha-crystallin. Together, these data suggest that in the aging lens, MGO inhibits glycation-mediated pentosidine synthesis and the loss of chaperone function of alpha-crystallin.     

Systemic human diseases as oxidative risk factors in cataractogenesis. II. Chronic renal failure

In this study we have investigated the oxidative metabolism of red cells (RBC), plasma, serum and aqueous humour of healthy subjects and of age-matched cataractous patients with and without chronic renal failure (CRF). Reduced glutathione (GSH) levels in RBC were lower in CRF patients than in the other groups. Oxidized glutathione (GSSG) plasma levels in CRF patients were higher than those of controls and cataractous subjects. The activity of the enzyme glucose-6-phosphate dehydrogenase in RBC was significantly reduced in CRF patients with respect to the other two groups. The levels of malondialdehyde (MDA) in RBC and in lens were about twice in CRF patients compared with the other two groups. The plasma levels of vitamin E were diminished in CRF patients; on the contrary, the biological liquid oxidant activity (BLOA) of serum in CRF patients was significantly higher than in controls and in cataractous patients without CRF. Cataractous patients with and without CRF showed similar levels of GSH in aqueous humour; on the contrary, the content of GSSG was significantly higher in CRF patients. Our findings seem to demonstrate that CRF patients are exposed to oxidative stresses that could probably act synergistically with uraemia and carbamylation of lens proteins. This synergism could explain why CRF represents a relatively high risk factor for cataract.