兔α-晶状体蛋白的分子伴娘功能的研究

目的 了解兔α-品状体蛋白是否具有分子伴娘功能。方法凝胶过滤法分离水溶性晶状体蛋白。在550C高温条件下,观察α-晶状体蛋白是否能抑制β-low晶状体蛋白的凝集和变性;观察α-晶状体蛋白是否能抑制糖基化诱导的葡萄糖-6-磷酸脱氢酶(G6PD)的失活,均以牛血清白蛋白(BSA)作为对照。结果 α-晶状体蛋白能抑制热诱导的p—low晶状体蛋白的凝集和变性;α-晶状体蛋白亦能抑制糖基化诱导的G6PD的失活;而牛血清白蛋白却无此作用。结论 α-晶状体蛋白具有分子伴娘功能。     

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。     

紫外线辐射对α-晶状体蛋白分子伴侣活性的作用

目的 研究在老化和白内障形成过程中紫外线（UV）辐射对α-晶状体蛋白分子伴侣活性的作用。方法 新生Sprague-Dawley大鼠皮下注射亚硒酸钠诱导硒性白内障（SC），采用SephacrylS-300HR分离幼年和老年兔晶状体皮质和核以及SC大鼠α-晶状体蛋白。以α-晶状体蛋白对过氧化氢酶热凝聚的抑制作为分子伴侣活性指标，观察UV-B（300nm)辐射α-晶状体蛋白蛋白后，其伴侣活性的变化。结果 UV辐射新生和老年兔晶状体核αL-晶状体蛋白后30h，与辐射前比较伴侣活性降低约18%和14%，而皮质的变化不显著；至100h时，老年兔皮质，核和新生兔核αL-晶状体蛋白比αL-晶状体蛋白敏感，UV辐射可进一步降低硒性白内障α-晶状体蛋白的分子伴侣活性。     

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]     

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。     

翻译后修饰对α—晶体蛋白分子伴侣活性的影响及白内障形成机制

翻译后修饰（post translational modification,PTM）是蛋白质翻译后通过酶的催化或酶控制下的反应而发生修饰，包括糖基化、氨甲酰化、氧化、磷酸化、乙酰化、脱酰胺和切除作用等。白内障和老化被认为是一类结构性疾病（conformational disease）。PTM可造成蛋白质结构改变。α－晶体蛋白作为晶状体主要的结构蛋白质，具有分子伴侣（molecular chaperone）活性，可抑制蛋白质的凝聚和酶的失活。PTM可诱导α－晶体蛋白分子内部或分子之间的交联，导致其分子伴侣活性降低，加速白内障形成。赖氨酸基因PTM最敏感，封闭赖氨酸的ε－氨基基团，可阻止和延迟蛋白质交联。寻找抑制或阻断PTM的因子，有助于药物治疗此类疾病方法的突破。     

阿司匹林对萘性白内障α-晶状体蛋白分子伴侣活性的保护作用

目的探讨低剂量阿司匹林对萘性白内障α-晶状体蛋白分子伴侣活性的保护作用。方法将45只150～160g雌性sD大鼠随机分为空白对照组（无特殊处理）、萘性白内障组（每日0．5mg／kg萘灌胃3d后改为每日1mg／kg）和阿司匹林组（萘灌胃4h后每日100mg／kg阿司匹林灌胃）。应用定期图像分析和高效液相色谱（HPLC）法分离纯化α-晶状体蛋白,紫外分光光度法测定其抑制热诱导的β—L晶状体蛋白变性的能力。结果3周时萘性白内障组晶状体开始出现混浊,程度逐步加重,阿司匹林组混浊早期进展较萘性白内障慢,6周后进展程度接近于萘性白内障组。实验2周时3组晶状体混浊程度的比较差异无统计学意义（F：0．032,P=0．969）。实验第4、6、8、10周时3组晶状体混浊程度比较差异均有统计学意义（F=5031．130,P=0．000：F=115964．000,P=0．000;F=169846．500,P=0．000;F=195431．200,P=0．000）,且空白对照组与萘性白内障组、空白对照组与阿司匹林组、阿司匹林组与萘性白内障组之间的差异均有统计学意义（P=0．000）。阿司匹林组的α-晶状体蛋白分子伴侣活性高于萘性白内障组。结论低剂量阿司匹林通过保护n一晶状体蛋白分子伴侣活性延缓萘性白内障大鼠晶状体混浊的进展,此作用在白内障早期尤为明显。     

α-晶体蛋白分子伴侣活性在白内障发病中作用的研究进展

α－晶体蛋白是晶状体中重要的成份蛋白质，是由αＡ和αＢ亚基组成的四聚体蛋白。长期被认为其功能主要是维持晶体的结构和屈光性，自１９９２年Ｈｏｒｗｉｔｚ证实α－晶体蛋白具有分子伴侣（ｍｏｌｅｃｕｌａｒ ｃｈａｐｅｒｏｎｅ）作用之后，大量的研究表明α－晶体蛋白能够抑制其它晶状体蛋白和酶的化学性修饰和热凝聚而保护其活性，对长期维持晶体状体的透明性具有重要作用。αＡ亚基Ｃ－端序列和αＢ亚基Ｃ－端的伸展的变化     

晶状体蛋白的翻译后修饰

晶状体蛋白（α-、β-、γ-晶状体蛋白）可发生翻译后修饰,且在不同的位点发生不同类型的翻译后修饰,从而影响晶状体蛋白的功能.部分翻译后修饰不仅增加晶状体蛋白的不溶性,也改变晶状体蛋白的构象,最终促进白内障的形成;有些翻译后修饰则与白内障的形成无关,可能与晶状体蛋白的发育或保护作用有关.研究晶状体蛋白的翻译后修饰及调控,对深入了解晶状体发育、衰老、白内障形成等过程有重要意义.就其在蛋白质水平上阐明有关晶状体的生理及病理过程进行综述.     

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。     

Reverse-phase HPLC analysis of human alpha crystallin

A rapid and highly sensitive reverse-phase HPLC (RP-HPLC) method was used to separate crystallin subunits from human alpha crystallin. Three distinct peaks were separated; by electrophoretic and immunological analyses the first and second peaks were identified as alpha B and alpha A respectively. On the other hand, peak 3 appeared to be a modified form of alpha crystallin. The ratio of alpha A and alpha B proteins was 3:1 in 1 day old lenses which gradually changed to 2:1 in 17 year old lenses and to 1:1 in the 50 and 82 year old whole lenses and 82 year old lens cortex, with a concomitant increase in the modified alpha, suggesting that alpha A subunits are relatively more involved in aggregation. Analysis of the 82 year old lens nucleus also supported this conclusion. The RP-HPLC analysis of the HMW aggregate fraction showed substantial enrichment of the modified alpha. The alpha A and alpha B subunits independently reassociated to form polymeric alpha crystallin whereas the modified alpha reassociated to form HMW aggregates as shown by molecular sieve HPLC. Hence it appears that the HMW aggregate peak was constituted by modified alpha crystallin. Only in the peak 3 material the 280 nm absorbance was about 2-fold higher than what was expected from the actual protein content. The data suggest that the changes induced by post-translational modifications may have some role in the formation of modified alpha. The present RP-HPLC method is useful in separating these modified alpha from the unmodified alpha A and alpha B subunits.     

Influence of the activity of glutathione reductase on the response of cultured lens epithelial cells from young and old rabbits to hydrogen peroxide

Our previous studies on cultured rabbit lens epithelial cells from 4-day-old rabbits showed that the glutathione redox cycle plays an important role in detoxifying H2O2, a potentially damaging oxidant present in the aqueous humor. Here we report the effect of donor age and cell density on the ability of cultured rabbit lens epithelial cells to detoxify H2O2. Lens epithelial cells (8 x 10(5] from a 4-day-old and an 8-year-old rabbit were cultured for 3 hr in minimal essential medium (MEM) or in MEM containing 0.01-0.1 mM H2O2 maintained with glucose oxidase. We determined the effect of H2O2 on the level of reduced glutathione (GSH), hexose monophosphate shunt activity, cell growth, and morphology. For growth studies, cells were exposed to the desired concentration of H2O2 for 3 hr and then cultured in MEM plus 10% rabbit serum for 7 days and counted. Young and old untreated cells contained high levels (30-40 nmol/8 x 10(5) cells) of GSH. Cells from 4-day-old rabbits tolerated 0.03 mM H2O2 with no effect on GSH and a minimal decrease in subsequent cell growth. However, in the older cells, GSH and growth were substantially diminished following treatment with 0.03 mM H2O2. Cells plated out at high density (8 x 10(5] were more tolerant of 0.03 mM H2O2 than cells plated out at low density (5 x 10(4]. Maximum shunt activity in the younger cells exposed to H2O2 was twice that of the older cells and occurred at a higher level of H2O2 (0.04 compared with 0.03 mM). Enzyme activities in untreated young and old cells were comparable for hexokinase, glucose-6-phosphate dehydrogenase, and glutathione peroxidase. However, glutathione reductase activity was 50% lower in the cells from the 8-year-old rabbit. The toxicity of H2O2 to cultured lens epithelial cells was directly related to donor age and inversely related to cell density. The damage in the older lens epithelial cells at 0.03 mM H2O2 was apparently due, in part, to a diminished response of the glutathione redox cycle to oxidative challenge.     

Conformational study of N(epsilon)-(carboxymethyl)lysine adducts of recombinant alpha-crystallins.

PURPOSE: Lens proteins underwent nonenzymatic glycation, and the advanced glycation end products (AGEs) were detected by immunological assays. One of the major AGE structures is N(epsilon)-(carboxymethyl)lysine (CML). Since the involvement of AGEs in the pathogenesis of diabetic complications is speculated, the effects of CML formation on proteins were studied. METHODS: CML adducts were generated in recombinant alphaA- and alphaB-crystallins by incubation with glyoxylic acid and NaBH3CN. SDS-PAGE and size exclusion chromatography were used to detect subunit degradation and high-molecular-weight (HMW) aggregation. Conformational change was determined by fluorescence and circular dichroism (CD) measurements. The chaperone function was studied by DTT-induced aggregation of insulin. RESULTS: Lysine modification was estimated to be 60-90% depending on the conditions of incubation. No subunit degradation or HMW aggregation was observed. Fluorescence and CD measurements detected a conformational change in CML adducts. Measurements of chaperone-like activity, however, indicated that the formation of CML increased the protein's ability to protect insulin against DTT-induced aggregation. CONCLUSIONS: Although CML adducts of alphaA- and alphaB-crystallins, the major AGE structures formed in vitro, changed protein conformation, no subunit degradation and HMW aggregation were observed. Moreover, the CML adducts increased chaperone-like activity of both alphaA- and alphaB-crystallins. The results suggest that CML formation alone may not play a major role in protein aggregation and lens opacity.     

Purification and some properties of camel lens crystallins

Five major crystallin fractions were found in camel lens fractionated by Sepharose CL-6B column chromatography. These crystallin fractions were named alpha high (alpha H), alpha low (alpha L), beta high (beta H), beta low (beta L) and gamma (gamma) by comparison with the elution profiles and molecular weights of rabbit crystallins. The amino acid composition and isoelectric focusing bands for crystallins of camel and rabbit were remarkably similar, but individual differences were found. By means of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), two alpha H crystallin subunits, of 23K and 20K molecular weight, were found in camel and rabbit. Likewise, a single 21K molecular weight band was found in the gamma crystallin of camel and rabbit. In camel, the beta high crystallin consisted of five major subunits, while rabbit beta high crystallin consisted of only three subunits. On SDS-PAGE, camel and rabbit beta low crystallins both showed two major subunits of 27K and 23K molecular weight but camel beta low crystallin showed an additional 35K molecular weight subunit. Characterization of camel lens crystallins may contribute to understanding the effect of aging on aggregation of camel crystallins.     

Modulation of hydrogen peroxide induced injury to corneal endothelium by virus mediated catalase gene transfer

AIM: To examine the effect of catalase gene transfer on survival of corneal endothelial cells (EC) following challenge with hydrogen peroxide (H(2)O(2)) in an ex vivo model of oxidative stress. METHODS: A recombinant adenovirus vector (AdCL) was used to transfer human catalase cDNA into EC of whole thickness rabbit corneas ex vivo. The resulting catalase protein concentration was measured in corneal lysates by ELISA; catalase functional activity in lysates was determined using a H(2)O(2) activity assay. To examine the morphological effects of catalase gene transfer in modulation of H(2)O(2) induced injury, transduced corneas were maintained in ex vivo culture and challenged with H(2)O(2). Laser scanning confocal microscopy was used to image EC injury. Cell density, cell morphology, and ratios of viable to necrotic cells were determined. RESULTS: Following incubation with AdCL, catalase expression reached maximum at 5-7 days. Corneas transduced with AdCL showed increased EC cell survival following challenge with H(2)O(2) on day 3 when compared to null vector control or mock infected corneas. CONCLUSIONS: Ex vivo catalase gene transfer can protect EC from death mediated by H(2)O(2). This gene based approach to the protection of corneal endothelium from oxidative stress may have application in prevention of EC loss in pathological conditions in which H(2)O(2) is involved and in ex vivo donor corneal storage before transplantation.     

Effect of UV-A light on the chaperone-like properties of young and old lens alpha-crystallin

PURPOSE: To study the damaging effect of UV-A irradiation on the chaperone-like properties of alpha-crystallin and the subsequent recovery process of young and old bovine lenses. METHODS: Young and old bovine lenses were kept in organ culture. After 24 hours of incubation they were irradiated with UV-A at 365 nm, and optical quality measurements were performed during the experiments (192 hours). alpha-Crystallin and alpha1-, alphaA2-, alphaB1-, and alphaB2-crystallin subunits were analyzed, separated by gel filtration and cation exchange chromatography, respectively, after different culture times. Protein patterns were obtained after two-dimensional (2-D) gel electrophoresis. Chaperone-like activity was determined on the basis of insulin B-chain and betaL-crystallin aggregation assays. Aggregation of alpha-crystallin was analyzed, tryptophan fluorescence measurements were performed, and alpha-crystallin mRNA levels were determined. RESULTS: The water-soluble alpha-crystallin obtained from old lenses compared with young lenses after UV irradiation had decreased chaperone activity, a higher molecular weight, and increased loss of tryptophan fluorescence. Moreover, alpha-crystallin mRNA virtually disappeared, whereas extra spots on the 2-D protein pattern appeared, possibly because of deamidation. CONCLUSIONS: alpha-Crystallin obtained from old lenses is more affected by irradiation than alpha-crystallin derived from young lenses. Moreover, it appeared that alphaB-crystallin from UV-treated old lenses compared with control lenses was less susceptible to UV-A than alphaA-crystallin. It may well be that alphaB-crystallin protects alphaA-crystallin in vivo.     

The effects of hyperbaric oxygen on the crystallins of cultured rabbit lenses: a possible catalytic role for copper

Oxidative effects on lens proteins have been linked with the formation of human age-related cataract, particularly nuclear cataract. This study investigated the effects of hyperbaric oxygen (HBO)-induced oxidative stress on nuclear and cortical alpha-, beta- and gamma-crystallins of cultured rabbit lenses, using high performance liquid chromatography (HPLC). The lenses were treated with 50 atm of either 100% N(2)(control) or 100% O(2)(experimental) for 3, 6, 16 and 48 hr. The levels of reduced glutathione (GSH) and water-soluble (WS) protein decreased more rapidly in the nucleus of the O(2)-treated lens than in the cortex. The first significant loss of WS protein in each of the two regions occurred when levels of GSH had decreased by at least 90% in either the nucleus (at 6 hr) or the cortex (at 16 hr). HPLC analysis of the nuclear WS proteins indicated that beta-crystallins were the first proteins affected by the oxidative stress. Soon after HBO-treatment was initiated (at 6 hr) and prior to insolubilization of protein, nuclear beta- and gamma-crystallins moved to the higher molecular weight alpha-crystallin fraction; 2-D gel electrophoresis and Western blotting indicated the presence of disulfide-crosslinked and non-crosslinked beta- and gamma-crystallins in this fraction. Significantly different HBO-induced effects were observed on lens cortical crystallins compared to those for the nucleus. For example, gamma-crystallins in the cortex shifted very soon after HBO-treatment (at 3 hr) to slightly higher molecular weights, possibly the result of protein/glutathione mixed disulfide formation; however, this phenomenon was not observed in the nucleus. Cortical beta- and gamma-crystallins remained in solution longer than nuclear proteins following HBO-treatment of the lenses, presumably the result of protection from the four-fold higher level of GSH (22 vs 6 m M) present in the lens periphery. Surprisingly, there was no movement of beta- and gamma-crystallins to alpha(H)- and alpha-crystallin fractions in the cortex of the O(2)-treated lens, in contrast to that observed for the nucleus. Cortical crystallins appeared to go directly from being soluble to being insoluble with no high molecular weight intermediate stage. The data suggested a possible chaperone-like function for alpha-crystallin in the nucleus of the stressed lenses, but not in the cortex. HBO-induced effects on lens nuclear supernatants, which mimicked those observed for intact lenses, could be nearly completely prevented by the copper-chelator bathocuproine, but not by the iron-chelator deferoxamine. Overall, the results provide additional evidence demonstrating an increased susceptibility of the lens nucleus to oxidative stress; the greater protective ability of the cortex may be linked to a higher capacity for beta- and gamma-crystallin/glutathione mixed disulfide formation, inhibiting disulfide-crosslinked insolubilization. The data also implicate copper as a catalyst for the autoxidation of -SH groups in the lens, and suggest that alpha-crystallin chaperone-like activity may play a greater role in the lens nucleus than in the cortex in preventing oxidative insolubilization of crystallins.     

The anticomplementary activity of the rabbit lens.

We wished to determine if inhibitors of the complement system are present in the normal rabbit lens. Soluble less extract in various dilutions was incubated with normal human serum (as source of complement). The residual hemolytic activity was measured using sheep erythrocytes sensitized with antisheep rabbit hemolysin. The lens extract was found to contain two heat stable anticomplementary factors of different molecular weights capable of cleaving C2 and C4. Gel filtration showed that one of these factors was located in the alpha-crystalline region. It is suggested that the anticomplementary factors in the lens may be related to a natural mechanism for the modulation of complement mediated lens injury.