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L L Ding

Publications and source records attributed to L L Ding.

10 recordsLinked to original sources
PubMed1999

Lens alpha-crystallin: function and structure.

alpha-Crystallin is a major lens protein, comprising up to 40% of total lens proteins, where its structural function is to assist in maintaining the proper refractive index in the lens. In addition to its structural role, it has been shown to function in a chaperone-like manner. The chaperone-like function of alpha-crystallin will help prevent the formation of large light-scattering aggregates and possibly cataract. In the lens, alpha-crystallin is a polydisperse molecule consisting of a 3:1 ratio of alpha A to alpha B subunits. In this study, we expressed recombinant alpha A- and alpha B-crystallin in E. coli and compared the polydispersity, structure and aggregation state between each other and native bovine lens alpha-crystallin. Using gel permeation chromatography to assay for polydispersity, we found native alpha-crystallin to be significantly more polydisperse than either recombinant alpha A- or alpha B-crystallin, with alpha B-crystallin having the most homogeneous structure of the three. Reconstructed images of alpha B-crystallin obtained with cryo-electron microscopy support the concept that alpha B-crystallin is an extremely dynamic molecule and demonstrated that it has a hollow interior. Interestingly, we present evidence that native alpha-crystallin is significantly more thermally stable than either alpha A- or alpha B-crystallin alone. In fact, our experiments suggest that a 3:1 ratio of alpha A to alpha B subunit composition in an alpha-crystallin molecule is optimal in terms of thermal stability. This fascinating result explains the stoichiometric ratios of alpha A- and alpha B-crystallin subunits in the mammalian lens.

Aging↗
PubMed1997

Subunit exchange of alphaA-crystallin.

alpha-Crystallin, the major protein in the mammalian lens, is a molecular chaperone that can bind denaturing proteins and prevent their aggregation. Like other structurally related small heat shock proteins, each alpha-crystallin molecule is composed of an average of 40 subunits that can undergo extensive reorganization. In this study we used fluorescence resonance energy transfer to monitor the rapid exchange of recombinant alpha-crystallin subunits. We labeled alphaA-crystallin with stilbene iodoacetamide (4-acetamido-4'-((iodoacetyl)amino)stilbene-2,2'-disulfonic acid), which serves as an energy donor and with lucifer yellow iodoacetamide, which serves as an energy acceptor. Upon mixing the two populations of labeled alphaA-crystallin, we observed a reversible, time-dependent decrease in stilbene iodoacetamide emission intensity and a concomitant increase in lucifer yellow iodoacetamide fluorescence. This result is indicative of an exchange reaction that brings the fluorescent alphaA-crystallin subunits close to each other. We further showed that the exchange reaction is strongly dependent on temperature, with a rate constant of 0.075 min-1 at 37 degrees C and an activation energy of 60 kcal/mol. The subunit exchange is independent of pH and calcium concentration but decreases at low and high ionic strength, suggesting the involvement of both ionic and hydrophobic interactions. It is also markedly reduced by the binding of large denatured proteins. The degree of inhibition is directly proportional to the molecular mass and the amount of bound polypeptide, suggesting an interaction of several alphaA-crystallin subunits with multiple binding sites of the denaturing protein. Our findings reveal a dynamic organization of alphaA-crystallin subunits, which may be a key factor in preventing protein aggregation during denaturation.

Animals↗
PubMed1995

Interaction of alpha-crystallin with spin-labeled peptides.

alpha-Crystallin is a major protein of the vertebrate lens once thought to be highly specialized for conferring transparency. However, recent work has revealed a wide tissue distribution and a sequence homology to small heat shock proteins, suggesting a more general role for the protein. Like other molecular chaperons, alpha-crystallin is known to bind to unfolded proteins and suppress nonspecific aggregation in vitro. In the present work, spin-labeled derivatives of the insulin B chain and melittin were used to investigate the state of these proteins bound to alpha-crystallin. Insulin was selected since unfolding can be triggered by reduction of the interchain disulfide bonds, a treatment that does not affect alpha-crystallin. Upon reduction of insulin, the separated B chains aggregate. In the presence of alpha-crystallin, the B chains bind to alpha-crystallin and aggregation is suppressed. Melittin, a 26 amino acid peptide from bee venom, was selected for study since it is a random coil under physiological conditions, and its interaction with alpha-crystallin can be directly studied. EPR analysis of the spin-labeled peptides shows that the nitroxide side chains are immobilized in a polar environment on alpha-crystallin and that they are separated by 25 A or more in the complex, indicating that the bound proteins are not clustered. The bound B chains of insulin are not in a fully extended conformation, and melittin does not appear to bind to a hydrophobic surface in alpha-crystallin as an amphipathic helix, as it does to membranes and some other proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Crystallins↗
PubMed1993

Enzyme activities and crystallin profiles of clear and cataractous lenses of the RCS rat.

Royal College of Surgeons (RCS) rats have hereditary retinal degeneration in association with posterior subcapsular opacities. Cataract formation is thought to be correlated with an increase in lipid peroxidation products in the vitreous (Zigler and Hess, 1985). In order to examine the possibility that parallel changes in enzyme activity are occurring within the lens, we analysed the activity of four key enzymes and the crystallin protein profile. We compared RCS rat lenses at three different stages of cataract formation to clear lenses of the nonpigmented RCS rat, lenses from pigmented RCS rat and to normal (Fisher) rat. Our data shows that concomitant with the appearance of the RCS cataract, the ratio of the crystallins beta 1, beta H and gamma to the total lens protein was reduced. The crystallin profile of a clear RCS lens was similar to that of a normal (Fisher) lens. No significant difference in the activity of the enzymes hexokinase and glucose-6-phosphate dehydrogenase (G6PD) was found among the lenses, however the activity of glutathione reductase and aldolase was reduced in the cataractous lenses.

Animals↗
PubMed1986

Age-dependent changes in the heat-stable crystallin, beta Bp, of the human lens.

The present study examined the effects of aging and cataractogenesis on the biochemical properties of the uniquely heat-stable lens crystallin, beta basic principle polypeptide (beta Bp). Using the techniques of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western-blot immunoassay, we analyzed cortical and nuclear lens sections from normal lenses of individuals aged 0 to 91 years for beta Bp content in the soluble fraction, and retention of heat stability with aging. In addition, we compared the characteristics of beta Bp in cataractous lenses with those of normal lenses of approximately the same age. While beta Bp is synthesized in new cortical cells throughout life, the beta Bp of the nucleus, which had been laid down early in life, decreased significantly in both absolute concentration and in its proportion of the total soluble protein fraction during the normal aging process. In addition, posttranslational changes in the nuclear soluble beta Bp result in a gradual loss of approximately 3000 d in the apparent mass of the beta Bp molecule; i.e., as a result of the aging process, the single heat-stable band of an apparent mass of 26 kd on SDS-PAGE of the young lens nucleus becomes two heat-stable bands, one at 26 kd and one at 23 kd. Normal lenses up to 91 years of age always retain some of the 26 kd subunit, whereas lenses with severe nuclear cataracts had only the 23 kd subunit in the soluble fraction.

Adolescent↗
PubMed1985

Spatial and temporal mapping of the age-related changes in human lens crystallins.

Using the techniques of high performance liquid chromatography (HPLC), gel electrophoresis in the presence of sodium dodecyl sulfate (SDS), and immunoblotting, we have analyzed the age-related changes in soluble crystallins of the human lens. A 3 mm core along the optical axis of each lens was frozen-sectioned and the sections were biochemically analyzed for distribution and quantity of the various soluble protein species. Both cortical and nuclear samples show a monotonic decrease in the concentration of the 19 000 and 21 000 MW proteins with age. We find that these proteins behave anomalously on SDS-polyacrylamide gels, running near the top of the gel when the samples are not boiled before loading; this permitted us to observe the gradual, age-related loss of these bands from the gels of both nuclear and cortical samples. The high molecular weight, or TSK-3000 void volume, fraction (greater than 350,000) of the cortex contained alpha crystallin at all ages. However, in the nucleus, while this fraction is primarily composed of alpha crystallin early in life (i.e. before 15 years of age), there is a gradual incorporation of other crystallins into the void volume. This change in the composition of the high-molecular-weight, soluble protein fraction is reflected in: a change in the subunit mobility on SDS-polyacrylamide gels; reactivity of the fraction to crystallin antibodies, i.e. in the young nucleus there is reactivity to anti-alpha crystallin only, with a gradual increase in reactivity to anti-beta and anti-gamma crystallins. The void volume fraction of the nucleus persists as a major component of the soluble protein pool until 42-44 years of age, at which time the proportion of the total soluble protein represented by this void volume fraction decreases precipitously. These changes in the soluble protein profile are discussed in terms of their potential influence on the functioning of the lens.

Adolescent↗
PubMed1984

Clinicobiochemical correlations in aging-related human cataract. The Pan American Association and American Journal of Ophthalmology lecture.

In a series of 50 consecutive adult patients (24 men and 26 women) we studied aging-related cataract by means of comprehensive presurgical examination of the patient, the eyes, and the cataractous lens; cataract sample collection from predetermined sites during surgery; biochemical analyses with high-performance liquid chromatography of each individual lens sample; and computer-assisted biostatistical data analysis. Compared with normal values established on clear human lenses obtained from a donor eye program, the ratios of 23,000 (23K) crystallin to total beta, gamma, and low molecular weight soluble proteins were statistically significantly lower in the anterior axial lens cortex (P less than .001) and in the lens nucleus (P less than .001). Increased grades of cortex opacification and nucleus opacification were generally associated with progressively decreased 23K crystallin ratios. In this series of patients with diverse cataract-associated systemic diseases, family histories, ophthalmic disorders, and drug-use patterns, decreased 23K crystallin ratios were a sensitive marker for aging-related cataract. Similar biochemical abnormalities in patients with multiple cataract-associated factors may have important clinical implications.

Adult↗
PubMed1983

Presence of low molecular weight polypeptides in human brunescent cataracts.

Microdissected sections from opaque & brunescent lens nuclei contain low molecular weight (4,000-8,000 dalton) polypeptides not found in microdissected sections of transparent lens nuclei. Tryptic digestion of these polypeptides from different cataracts reveal similar peptide maps. Together, these results support the involvement of proteolysis in human cataractogenesis, and suggest the possibility of similar molecular mechanisms occurring in cleavage of lens polypeptides during formation of the opaque & brunescent human cataract.

Aged↗