Lens membranes. IV. Preparative isolation and characterization of membranes and various membrane proteins from calf lens.

A scheme is proposed for the preparative isolation of matrix-bound and matrix-free calf lens membranes, and the conditions and recoveries of the procedures have been studied. Gas chromatographic analyses have been carried out to characterize the sugars of the intrinsic glycoproteins. Galactose and N-acetyl-glucosamine are dominant sugar components and fucose, glucose and N-acetyl-galactosamine are minor constituents.Urea-treated membranes, prepared from cortex plus nucleus, contain only very small amounts of polypeptides which immunologically cross-react with α- and γ-crystallin and which can probably be classified as remnants. The weight ratio protein : cholesterol : phospholipid of these membranes amounted to 4·4 : 1 : 1·9, and the molar ratio cholesterol : phospholipid was 1·0, which is equal to those of the original lens tissue.A water-extractable protein fraction can be recovered from urea-treated membranes, which gives six bands in SDS-gel electrophoresis (mol. wt. 54 000, 47 000, 34 000, 27 000, 19 000 and 16 000). This fraction contains some α- and γ-crystallin according to immunological and electrophoretical criteria. Its major component has a molecular weight of 47 000.     

Lens membranes vi. Some characteristics of the EDTA-extractable protein (EEP) from bovine lens fiber membranes.

After urea-treatment of calf lens fiber membranes, a well defined extrinsic membrane protein fraction (EEP) can be extracted by EDTA-solution. It contains polypeptides of mol. wt. 32K and 35K (SDS-gel electrophoresis). Thin-layer gel filtration shows that without detergent no oligomers are formed. It is easily lost by its adsorption characteristics, however, it can be purified from contaminating α crystallin by gel filtration. The 35K fraction comprises components with isoelectric points at 4·6–4·7 (double band) and between 6·2 and 7·2. The 32K fraction shows components at 4·6–4·7 and 6·2 as was found by two-dimensional gel electrophoresis. The method of isolation, the isoelectric focusing pattern, and the amino acid and mol. wt. composition distinguish EEP clearly from the soluble crystallins. EEP comprises two distinct antigenic components with isoelectric points between 6·2 and 7·2. This was revealed by immunoelectrofocusing of EEP vs. anti-EEP antiserum from rabbit. Combining the results of two-dimensional electrophoresis and immunoelectrofocusing it was found that the 32K and 35K polypeptide have different antigenic properties. Traces of crystallins are difficult to remove. However, by means of specific anti-crystallin antisera it has been shown that EEP has a determinant in common with γ crystallin.     

Change of Water—Soluble—Protein,Urea—Soluble—Protein and Membrane Intrinsic Protein in Human Senile Cataract

Purpose:To analyze the change of water-soluble-protein(WSP),urea-soluble-protein(USP)and membrane intrinsic protein(MIP)in human senile catarct.Methods:The water-soluble-fractions(WSF)were prepared basically according to the method of Kibbelear,et al.But in this study,5mmol/LB-mercaptoethanol was added to the buffer solution.The urea-soluble-fractions(USF)were pre-pared basically according to the method of Kibbelear,et al.Lens fiber cell mem-branes were purified basically according to the method of Russell,et al.SDS-PAGE were performed according to the procedure of Laemmili,et al.using re-solving gel13%and3%stacking gel.Results:The WSPwas fractionated intoHM^ α^-,β1-3^-andγ-crystallin compo-nents.In nuclear cataractous lenses HM^ α^-and B-crystallin increase,while r-crystallin decrease.The USP from clear lenses contains mainlyαβchains of22KD,whereas in cataractous lenses,especially in nuclear cataractous lenses,the relative amount of the 28-and23KDpolypeptide(the components of β-crys-tallin)increased markedly.Lens fiber cell MIP,clear lens and cataract lens con-tained the main polypeptide of 27KD(MIP)and23KD(MP23).Conclusion:The water-insolube protein,whether in quantity or in quality,plays an important role in cataract formation.Eye Science 1995,11:124-127.     

Charge and molecular weight heterogeneity of EDTA-extractable proteins from calf lens membranes

The EDTA-extractable proteins (EEP) of calf lens fiber cell membranes have been further characterized. Fiber EEP has been purified by gel filtration and resolved into eight bands with molecular weights of 30-38 K dalton by SDS-polyacrylamide gel electrophoresis. For epithelial EEP the same range has been obtained. In agreement with these findings a value of 33 K has been determined for fiber EEP by Sephadex G200 thin-layer gel filtration, while 34 K dalton was found by high-performance gel permeation chromatography in combination with low-angle laser light scattering (HPGPC-LALLS). The isoelectric focusing patterns of fiber and epithelial EEP show considerable charge heterogeneity. By two-dimensional electrophoresis the relation molecular weight-isoelectric point has been established for most EEP components. Peptide maps of the individual protein bands of fiber EEP differ from each other and from those of the beta Bp-, beta B1a- and beta B1b-crystallin bands, which have about the same molecular weight. From our results we conclude that EEP is not an oligomeric nor a multisubunit protein, but a collection of different extrinsic membrane proteins, biochemically unrelated to lens crystallins. The fact that removal of the cytoskeleton by urea-treatment of the membranes is a prerequisite for its isolation by EDTA or EGTA suggests that EEP is bound to the inner surface of the plasma membranes, probably via calcium.     

Human lens fiber cell plasma membranes. I. Isolation,polypeptide composition and changes associated with ageing

A detailed methodology is offered for the bulk isolation of human lens fiber cell plasma membranes. The human lens fiber plasma membrane fraction is isolable as the water-insoluble and urea-insoluble but detergent-soluble material, under reducing conditions. Ultrastructural characterization confirmed the homogeneity of this fraction. Biochemical, immunological and electrophoretic analyses were conducted upon the isolated fiber plasma membranes.Analyses were conducted of whole-lens, cortical and nuclear fiber plasma membranes from fetal, newborn, 30–50, 50–60, and 60–80-year-old normal human lenses. The fiber plasma membrane fraction remained a relatively constant 0·9% of the fresh (wet) weight of the human lens throughout lifespan; the protein-lipid ratio was determined as 1:1·2 with a slight increase in the lipid factorial of older lenses (50 years and older). Thirteen (13) polypeptides, ranging in molecular weight from 12–235 kilodaltons were resolved electrophoretically for the membranes. The phospholipid-containing 25 and 27 kilodalton polypeptides were found to constitute the main intrinsic protein of the human lens fiber membranes. α-Crystallin polypeptides (20 and 22·5 kilodaltons) were consistently recovered from the membranes, and their presence confirmed immunologically only following total detergent solubilization of the membranes. Reactions of an antiserum to the main intrinsic protein of chick lens fiber membranes and the human lens membranes were negative. The major age-dependent change in the protein composition of whole-lens human fiber membranes consisted of a gradual reversal in the preponderance of the 27 kilodalton polypeptide prenatally (and at birth) by the 25 kilodalton polypeptide as the main intrinsic polypeptide of the membranes postnatally. Additional changes comprised a gradual increase in the weight fraction of the 12 kilodalton polypeptide in the whole-lens fiber membranes throughout lifespan, and a gradual decrease of the α-crystallin content of cortical fiber membranes of lenses 50 years and older.     

Lens membranes VII. MIP is an immunologically specific component of lens fiber membranes and is identical with 26K band protein.

Calf lens MIP¹ (solvent-extracted from the fiber membranes) and 26K band protein (isolated by SDS-PAGE) have been shown to have very similar amino acid compositions and molecular weights. MIP comprises two components which are immunologically identical with the two antigens detected in 26K band protein. In addition, MIP extracts contain two antigens in very small amounts which are common to urea-treated fiber and epithelial lens membranes. 26K band protein contains one of these antigens. Immunodiffusion, immunofluorescence and SDS-PAGE show MIP to be a specific component of the fiber membranes, which cannot be detected in epithelial membranes. Therefore, MIP may be considered as a marker of differentiation. MIP determinants can neither be found in other ocular tissues than the lens nor in non-ocular tissues, demonstrating the organ specificity of the protein.     

老化过程中人晶状体蛋白质的改变

用凝胶过滤和SDS-PAGE对不同年龄人透明晶状体水溶性蛋白、尿素溶蛋白及纤维细胞膜内在蛋白进行了分析。随着年龄的增长,HM+α-晶体蛋白的相对含量增加,而β-、γ-晶体蛋白降低。四岁儿童晶状体含有较多的β_1-晶体蛋白,成人晶状体则含量明显降低。成人晶状体尿素溶蛋白的主要成分为α-晶体蛋白,而四岁儿童晶状体尿素溶蛋白中α-晶体蛋白及23kDa的β-晶体蛋白成分含量均较高并含有较多的细胞骨架蛋白。细胞膜主要内在蛋白(MIP)的相对含量随年龄的增长而下降,而MP23升高。     

Status of caveolin-1 in various membrane domains of the bovine lens

Recent studies of the distribution and relative concentration of caveolin-1 in fractions of bovine lens epithelial and fiber cells have led to the novel concept that caveolin-1 may largely exist as a peripheral membrane protein in some cells. Caveolin-1 is typically viewed as a scaffolding protein for caveolae in plasma membrane. In this study, membrane from cultured bovine lens epithelial cells and bovine lens fiber cells were divided into urea soluble and insoluble fractions. Cytosolic lipid vesicles were also recovered from the lens epithelial cells. Lipid-raft domains were recovered from fiber cells following treatment with detergents and examined for caveolin and lipid content. Aliquots of all fractions were Western blotted for caveolin-1. Fluorescence microscopy and double immunofluorescence labeling were used to examine the distribution of caveolin-1 in cultured epithelial cells. Electron micrographs revealed an abundance of caveolae in plasma membrane of cultured lens epithelial cells. About 60% of the caveolin-1 in the epithelial-crude membrane was soluble in urea, a characteristic of peripheral membrane proteins. About 30% of the total was urea-insoluble membrane protein that likely supports the structure of caveolae. The remaining caveolin was part of cytosolic lipid vesicles. By contrast, most caveolin in the bovine lens fiber cell membrane was identified as intrinsic protein, being present at relatively low concentrations in caveolae-free lipid raft domains enriched in cholesterol and sphingomyelin. We estimate that these domains occupied 25-30% of the fiber cell membrane surface. Thus, the status of caveolin-1 in lens epithelial cells appears markedly different from that in fiber cells.     

Cytoplasmic filaments in the crystalline lens of various species: functional correlations.

The distribution of cytoplasmic filaments in lenses of five species was studied with the electron microscope. Two distinct patterns emerged. One pattern, in which filaments are grouped in characteristic bundles around the nucleus, in processes, and throughout the subcortical cytoplasm of epithelial cells, is typical of spherical, non-accommodating lenses of mice and rats. The second pattern is associated with anteriorly-flattened, accommodating lenses of infant human, squirrel and frog. In these, filaments are scattered in epithelial cells, but are accumulated on either side of the plasma membrane junction between epithelial cells and lens fibers. They are especially dense on the lens fiber side of the junction, and form a lattice associated with the lens fiber plasma membrane. The lattice is less extensive along the sides of lens fibers not in contact with epithelial cells. In spherical lenses the epithelial-fiber lattice is greatly reduced. Filaments in both types of lenses ranged in diameter between 5 and 11 nm. The filaments are thought to be a mixture of thin and intermediate filaments.It is hypothesized that the role of cytoplasmic filaments in lens, depending on the pattern present, is either to structurally support a spherical shape, or to provide a contractile force or elasticity to return the flattened anterior surface to the accommodated state in conjunction with the elasticity of the lens capsule.     

接触镜相关性角膜敏感性变化的机制研究

目的:针对配戴不同类型接触镜后角膜敏感性变化的机制进行研究,以期对接触镜的临床验配和并发症的早期发现与处理提供帮助。方法:采用问卷的形式收集基本信息,应用Cochet and Bonnet aesthesiometer测量49例98眼角膜敏感性,按照不同分组进行比较分析。结果:分别配戴RGP和软镜,戴镜1～12mo组,1～5a组中央部角膜敏感性均较未戴镜组明显下降(P=0.000),而二组之间差异无显著意义(RGP镜片P=0.921,软镜P=0.685);配戴Dk为36和82的RGP镜片1～12mo,二者差异无显著意义(P=0.263);分别配戴RGP镜片和软镜1～12mo,1～5a,两组中央角膜敏感性差异无显著意义(1～12mo组P=0.263,1～5a组P=0.366)。结论:配戴接触镜导致的角膜敏感性下降是多种机制共同作用的结果,不同类型镜片的机制略有不同。     

RGP镜片对儿童角膜敏感性的影响

目的:研究儿童配戴RGP镜片后角膜敏感性的变化.方法:采用问卷的形式收集基本信息,应用Cochet and Bonnet aesthesiometer测量67例134眼角膜敏感性,按照不同分组进行比较分析.结果:儿童戴镜1～12mo组,戴镜1～5a组中央部和下方角膜敏感性明显下降(中央P=0.000,下方P=0.002),而2组之间的差异无显著意义(中央P=0.997,下方P=0.056).儿童戴镜1～5a组颞侧,鼻侧,上方角膜敏感性明显下降(颞侧P=0.000,鼻侧P=0.000,上方P=0.001),戴镜1～5a组与戴镜1～12mo组(颞侧P=0.133,鼻侧P=0.125,上方P=O.205),未戴镜组与戴镜1～12mo组(颞侧P=0.06,鼻侧P=0.200,上方P=0.207),差异均无显著意义.儿童与成年人分别配戴RGP镜片1～12mo和1～5a,中央角膜敏感性无显著差异(1～12mo.P=0.343,1～5aP=0.105).结论:RGP镜片对于儿童和成人的中央角膜敏感性的影响是相似的.但由于儿童自身的特点,为儿童验配RGP仍应慎重.     

Lens membranes III. Freeze fracture morphology and composition of bovine lens fibre membranes in relation to ageing

Ageing of the fibre membranes of calf lens causes a shift in their cholesterol-phospholipid and protein-lipid ratios to higher values because of phosphoglyceride loss. The electrophoretic polypeptide patterns of urea-treated equatorial, cortical and nuclear membranes are similar, although the relative amounts of the higher molecular weight polypeptides decrease towards deeper layers. It is shown that these biochemical changes are associated with alterations in freeze fracture morphology. Intramembrane particles become more and more aggregated, which has been interpreted as the result of a change in the lipid environment during ageing. Both fracture faces greatly differ in density of particle distribution, the A face exhibiting about 1300 particles per μm² and the B face about 400 particles per μm². The particles are uniform in size (about 70 Å in diameter) and spherical in shape. In the B face pits have been found of about 50 Å in diameter, probably complementary to the particles in the A face.Deoxycholate-insoluble, junction-like structures isolated from fibre membranes probably contain no specific junctional protein(s). Most or all of the intrinsic proteins of these membranes seem to be present and contain MIP (main intrinsic polypeptide of fibre membranes, mol. wt. 26 500) as the main component.     

不同人工晶体材料及设计方式对兔眼后囊混浊影响的实验研究

目的 对比3种不同材料及设计的人工晶体对兔眼晶状体后囊混浊的影响.方法 将实验动物48只随机分成A、B、C、D4个组,超声乳化摘除术后在囊袋内分别植入不同的人工晶体.A组植入三片式聚甲基丙稀酸甲酯(PMMA)材料、光学部边缘为圆形设计的人工晶体;B组植入三片式硅胶(Silicon)材料、光学部边缘也为圆形设计的人工晶体;c组植入丙烯酸酯(Acrylic)材料、光学部边缘为直角方型侧缘设计的人工晶体,D组作为空白对照组只行超声乳化晶体摘除术,未植入人工晶体.术后1 d、1周、1月、3月分别进行了术眼的裂隙灯检查及照像、术后3月行病理及电镜检查.结果 术后第1天,(1)前节炎症反应:A、B、C、组瞳孔区都有膜形成.角膜轻度水肿,D组瞳孔区未见膜形成,角膜轻度水肿.术后1周时A、B、C组前房闪光阳性,三组瞳孔区渗出膜均较前吸收,D组前房闪光消失,A、B、C组后囊膜清亮,D组(空白对照组)后囊膜已有明显混浊.术后1月时,A、B组瞳孔区渗出膜仍未完全吸收,前房闪光均阳性.C组瞳孔区渗出膜完全吸收.前房闪光消失.(2)后囊膜及人工晶体在囊袋内位置情况:术后1月A、B组中央视区后囊膜轻度混浊,并发现2组中各有10只眼人工晶体在囊袋内偏位和囊膜夹持.C组后囊膜清亮,有2只眼发生了囊膜夹持,其余10只眼的人工晶体在囊袋内具有良好的居中性,D组(空白对照组)后囊膜已完全混浊.术后3月时,A、B组中央视区后囊膜混浊情况相似,均较前更加明显,C组只有2例中央视区后囊膜有轻度的混浊,其它10例术眼均保持清亮.结论 C组单片式丙烯酸酯材料、光学部边缘为直角方型侧缘的人工晶体,可能对减少后囊膜混浊的发生,维持人工晶体正位有积极重要的作用.