Lu-Ku Li

Age-dependent changes in the distribution and concentration of human lens cholesterol and phospholipids

Analyses of total lipid in individual lenses 1.8-63 years of age indicate that both the cholesterol and the phospholipid concentrations have reached a high level of 10 and 14 micrograms/mg lens dry weight, respectively, after the first ten years of growth. Thereafter, the rate of phospholipid accumulation was greatly reduced to a value of 0.05 microgram/mg per year while that of cholesterol reduced to 0.19. Analyses of the distribution of lipid in successive lens fiber layers indicate that both the cholesterol and phospholipid levels increase in the entire lens between the age of 1.8 and 9 years. Older lenses showed a continuous increase in the accumulation of cholesterol in the deep cortical fibers, while little or no increase in phospholipid concentration was observed. These results indicate that the accumulation of lipids is greater than that of lens dry mass (protein) during the first decade of lens growth. Since more than 90% of lenticular lipids are associated with fiber cell membranes, these data suggest a gradual change in the differentiation of the newly formed secondary fibers from the epithelium during this period. Analyses of the phospholipid composition of the successive fiber fractions indicate that the major phospholipids of phosphatidyl ethanolamine (PE), phosphatidylserine (PS) and sphingomyelin maintained a uniform distribution in the 1.8- and 5-year-old lenses. While no change was observed with the cortical fibers, older lenses showed a gradual loss of PE and PS in the nuclear fiber up to 63 years of age. By the late teen years, nuclear PS can no longer be detected, while high levels of PE are maintained in lens nucleus. The disappearance of nuclear PE begins in the teen years and is completed by the age of 40. The decrease in PE and PS resulted in a continuous increase in the cholesterol/phospholipid ratio, a measure of membrane rigidity in the nuclear fiber in lenses 20 years of age and older. This decrease is also responsible for the exceedingly high rigidity of the nuclear fibers of lenses 60 years of age and older. Possible lamellar cholesterol organization in the lens fiber membrane is discussed.

Circular dichroism and optical rotatory dispersion of the aggregates of purified polypeptides of alpha-crystallin

The circular dichroism (CD) and optical rotary dispersion (ORD) of the aggregates of the individual polypeptides of low molecular weight alpha-crystallin (LMW alpha), A1, A2, B1 and B2 as well as deaggregated and reaggregated alpha-crystallin were compared to LMW alpha. The results indicate that reaggregated alpha-crystallin, A1 and A2 aggregates, all have spectra very similar to native alpha-crystallin. The B1 and B2 aggregates have a somewhat different spectra with the CD minima being between 206–209 nm in comparison to 216 nm for the other aggregates. This data suggests that the B chains aggregates may have a slightly smaller abundance of beta structure. When B2 and A2 polypeptides in a ratio of 1:2 were deaggregated and then reaggregated, spectra similar to native alphacrystallin were obtained. Such results suggest that A and B polypeptides interact to form macromolecules with a structure similar to the native protein. Analyses of the data were undertaken using poly(L-lys) as well as a group of proteins with known structures as references. Irrespective of the reference used, it was found that the amount of helical contribution was less than 0·01% for all of the aggregates studied. The beta structure was estimated to contribute approximately 55–63% of the total conformation.

The reaggregation of purified subunits of alpha-crystallin

Abstract Ultracentrifuge studies have been carried out with reaggregated macromolecules obtained from purified subunits of low MW calf lens alpha-crystallin. The subunits were isolated by affinity chromotography with para-amino-phenyl mercuric acetate-Agarose columns followed by chromatography with DEAE-cellulose in 7 m urea. The results suggest the following conclusions: each of the polypeptide chains of alpha-crystallin is capable of reaggregating independently. Similar to the non-interacting size heterogeneity of alphacrystallin, the aggregates of polypeptide chains A 2 and B 2 display non-equilibrium heterogeneous populations of macromolecules. Aggregates of A 1 appear to be homogeneous with respect to the minimum MW species. The reaggregated polypeptide chains, as well as deaggregated and reaggregated alpha-crystallin, all give significantly lower molecular weight populations of macromolecules than the native alpha-crystallin preparation. These results suggest that other components removed by the deaggregation-reaggregation procedure may be involved in determining the size and heterogeneity of alpha-crystallin.

Changes in lens protein in concentric fractions from individual normal human lenses

The water soluble (WS), urea soluble (US) and urea insoluble (UI) fractions from individual human lenses 1.8 to 65 years of age were isolated from concentric fiber layers. In lenses younger than 19 years, a uniform distribution in the amount of WS, US and UI fractions was found throughout the entire lens. These fractions represent 83, 11.5 and 5.5%, respectively, of the lens dry weight. This composition was observed with the cortical fibers of all lenses examined up to the 65-year old. In the nuclear fiber layers, the proportion of US protein gradually increases in the third to fourth decade of lens growth and appears to have reached a maximum representing 22-24% of the nuclear fiber mass in 50-year and older lenses. A large increase in the amount of the UI fraction to 30% of the fiber mass was observed in lenses between the 5th and 6th decade of lens growth. The change from the cortical to nuclear composition occurs in a narrow region of the lens which becomes more peripheral with aging. The cortical WS fractions were characterized by well defined polyacrylamide gel bands in sodium dodecyl sulfate (SDS). Those of the nuclear fibers were broadened, especially in the 27/29 and 16/18 kilodalton (KD) region. The disappearance of the 20/22 KD bands in the inner cortical and nuclear fibers cannot be accounted for by the small increase in protein insolubilization in these regions of lenses 40 years or younger.

Studies on the reaggregation of isolated subunits of calf lens alpha-crystallin

Abstract The subunits of low molecular weight alpha-crystallin have been studied with Sephadex G-100 columns in the presence of 5 m guanidine · HCl. Such experiments indicate that more than 50% of the material is present in the dimer form and a small fraction (5–9%) is of higher molecular weight, the remainder being represented by the monomer fraction. The relative amounts of these fractions vary somewhat depending on the method used for the isolation of alpha-crystallin. Analysis of the major fractions suggest that the dimers are composed of A chains while the monomers are predominantly B chains. No disulphides are present in the dimer, but sulfhydryl groups appear to be involved in the dimerization. A chelatable component has been shown to participate in the dimerization process. The A chains can be purified by DEAE-cellulose chromatography in the presence of 7 m urea and the purified polypeptides appear similar to A chains isolated by other methods. Reaggregation studies in the ultracentrifuge after the removal of guanidine · HCl from these Sephadex fractions suggest the following conclusion: combination of all the fractions, i.e. the minor high molecular weight fraction, the dimer and the monomer fractions, resulted in reaggregation to an s value expected for alpha-crystallin. Reaggregation of the starting alpha-crystallin after treatment with 5 m guanidine · HCl indicated no change in the s value of the material (S20w = 17·4 s). The dimer material alone reaggregated to give a large S20w value of 29·2 s while the monomer material reaggregated to a value of 18·1 s, which approximates the size of the precursor alpha-crystallin. Recombination of mixtures of the dimer and monomer give S20w values reflecting the relative proportion of the components; e.g. a mixture of 55% dimer and 45% monomer, a composition similar to alpha-crystallin, gives an S20w value of 24·4 s. Since the control experiments with all the fractions give an S20w value of 17·0 s, the minor subunit component must be of fundamental importance in controlling the size of the alpha-crystallin aggregates.

Effects of sucrose on interactions of calf lens soluble proteins

Abstract Sucrose was used to simulate the dense in vivo environment during the extraction and isolation of soluble proteins (crystallins) from the cortical and nuclear regions of the calf lens. Addition of sucrose to the eluting buffer markedly affects the βH-crystallin region during gel filtration with Sepharose 6B-CL in a Tris buffer alone: the region is progressively diminished by levels of 0·25 m and 0·50 m -sucrose and disappears completely in buffered 1·0 m -sucrose. Concomitantly, decreases in the βH-crystallin region are accompanied by increases in the βL-crystallin region of the chromatogram developed with the Tris buffer alone. Compared to 10% of the total weight of proteins that elute as γ-crystallin in Tris buffer, only 3% are recovered in the presence of 0·25 m -sucrose. A poorly resolved peak immediately preceding the γ-crystallin region and a small increase in α-crystallin are observed with 1·0 m -sucrose. The changes in the crystallins were further characterized and demonstrated by equilibrium ultracentrifugation as well as by polyacrylamide gel electrophoreses in the presence and absence of urea. Weight average molecular weights ( M w) determined in Tris buffer containing 1·0 m -sucrose used for isolation of the three major cortical crystallin fractions gave values of 840 000, 55 000 and 23 500 daltons. They correspond, respectively, to those for α-, βL- and γ-crystallins isolated with Tris buffer alone. Results from gel electrophoresis in 7·0 m -urea indicate that the β-crystallin fraction isolated in 1·0 m -sucrose contains the sum of polypeptides present in βH- and βL-crystallins. In the absence of urea, however, polyacrylamide gel pattern of the native, undissociated sucrose β-crystallin fraction is distinetly different from and cannot be accounted for by a combination of those of βH- and βL-. With or without urea present the gel patterns for α- and γ-crystallin fractions isolated in buffered 1·0 m -sucrose are indistinguishable from those for the respective crystallins obtained in the Tris buffer alone. The observed changes in βH- and γ-crystallins upon the introduction of sucrose are shown to be reversible upon the removal of the reagents. These results suggest that the interaction or aggregation of lens proteins in a dense medium of reduced activity of water is different from that in a dilute buffer.

Physical and chemical changes in alpha-crystallin during maturation of lens fibers

Abstract A mechanical separation of calf lens into three fractions composed of outer cortex, inner cortex and nucleus provided material representing three phases of fiber maturation. Separation of the lenticular proteins in each fraction was achieved by gel filtration on agarose 15 m . The amount of high mol. wt. alpha-crystallin aggregates was found to increase appreciably as the fibers age. The amount of low mol. wt. alpha-crystallin, however, decreases while the average size gradually increases as the fibers are displaced toward the center of the lens. An increase in sedimentation coefficient corresponding to an increase of 50% in mol. wt. was found when the low mol. wt. alpha-crystallin from the outer cortex was compared to that from the nuclear region. No detectable change in the amino acid or the subunit composition accompanied this increase in size. The circular dichroism (CD) of these crystallins also revealed no conformation changes of the polypeptide backbone. Unique to the CD spectrum for the nuclear alpha-crystallin are minima at 235, 214 and 206 nm. This observation was corroborated by far u.v. spectroscopy to 195 nm. The loss of these CD minima as well as changes in the u.v. profile that can be effected by 7 m -urea suggests the presence of chromophore(s) attached non-covalently to the nuclear protein. One or more other chromophore(s) common to all low mol. wt. alpha-crystallins were revealed by a 40% reduction in the extinction at 195 nm after urea treatment of the cortical alpha-crystallins with no associated change in the CD spectrum. The 20% reduction in extinction for the nuclear protein suggests the presence of similar chromophore(s). Deaggregation followed by reaggregation not only markedly diminished the size of the low mol. wt. alpha-crystallins but also drastically reduced the disparities in size of this protein among the three fiber fractions. These results suggest that chromophore(s) removed by urea treatment may play a decisive role in determining the size of the alpha-crystallin marcromolecule during the maturation of the lens fibers.

Age dependent lipid and protein changes in individual bovine lenses.

Successive fiber fractions isolated from individual bovine lenses were fractionated to examine changes in lens proteins and membrane lipids as a function of age. In calf lens of about 1.2 gm wet weight, cholesterol (C) is maintained at a level of 3.3 microgram/mg dry weight in the outer cortical 30% of the lens. In the inner cortex, a C content of 2.4 micrograms/mg was found that decreased somewhat to 2.1 micrograms/mg in the inner nuclear 20% of the lens. The almost linear decrease in phospholipid (P) content from 11.6 in the cortex to 1.7 micrograms/mg in the nucleus resulted in a cortex to nucleus increase in C/P ratio from 0.5 to about 2.0 (mol/mol). Compared to calf lenses, a low C level of 2.4 micrograms/mg was observed in the outer cortex of cow lenses (approximately 3.0 gm wet weight). No significant difference in C level was found between the calf and cow lenses either in the inner cortical or nuclear regions. The P level was reduced to 6 and 1.2 micrograms/mg in the outer cortex and nucleus of the cow lens, respectively. The low nuclear P content is responsible for the observed high C/P value of 3.6. The lower lipid content found in the cortex of older lens suggests an age dependent decrease in the amount of available membrane lipid to envelope the newly formed fibers. A cortex to nucleus increase in the amount of urea-soluble (US) protein fraction from about 6 to 14% of total fiber mass was observed with the calf lens. In the cow lenses, the nuclear US fraction accounts for almost 30% of the fiber.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of temperature, concentration and carboxymethylation on interactions of calf lens crystallins.

Abstract Concentration and temperature dependent interactions of calf cortical crystallins were studied by means of gel filtration and disc gel electrophoretic analyses. Low temperature (8°C) gel filtration of either low or high levels of cortical crystallin extracts (5–59 A 280 units) yields elution profiles that are indistinguishable in the distributions of α-, β H -, β L - and γ-crystallins. Gel filtration of low levels of extracts between 22 and 44°C causes a gradual but ultimately complete temperature-dependent disappearance of the region of β H -crystallins and to a lesser degree in the γ-crystallin peak. Chromatography of high levels of extracts at 44°C results in the reappearance of a high mol. wt. β-crystallin peak with electrophoretic property and polypeptide composition different from those of β H -crystallin isolated at the low temperature of 8°C. Preincubation of high levels of extracts at 44°C causes some small but irreversible changes in the elution profile obtained at 8°C. Taken together these results suggest that the presence of high mol. wt. β-crystallin is the result of largely reversible equilibria between β-crystallins and other lenticular components. The reaction of calf cortical extracts with increasing amounts of iodoacetic acid results in a progressive loss in the β H -crystallin region of the 8°C filtration profile. Results from amino acid analyses and sodium dodecylsulfate (SDS) gel electrophoreses of the respective α-, β L - and γ-crystallin fractions indicate that the SH moieties of the cysteine residues are essential to the formation of high mol. wt. β-crystallins.

Fractionation and characterization of the polypeptide chains of low molecular weight calf lens alpha-crystallin.

Abstract Purified polypeptides of low molecular weight calf alpha-crystallin have been obtained by a combination of affinity chromatography upon a p-aminophenylmercuric acetate-Bio-Gel A-5m column followed by DEAE cellulose chromatography. The affinity chromatography provides a very rapid procedure for obtaining A chains completely free of B polypeptides. Subsequent fractionation upon DEAE cellulose yields pure B2, B1, A2 and A1. Atypical A chains have also been observed and fractionated. Characterization of the isolated polypeptides indicates that on the basis of size, amino acid composition and fingerprinting of tryptic and chymotryptic peptides, B2 and B1 are very closely related as are A2 and A1. The data suggest a ratio of approximately 2 : 1 for A to B chains.