Jacques G. Bindels

The interrelationship between monomeric, oligomeric and polymeric α-crystallin in the calf lens nucleus

Calf lens nuclear α-crystallin was separated into the three known molecular weight populations on Ultrogel AcA22. Electron microscopy illustrates that the two higher molecular weight classes, which are formed upon aging of the lens, are chain-like oligomers and polymers of the spherical monomeric α-crystallin molecules of 15 nm average diameter and 1·1 × 106 average molecular weight which form the lowest molecular weight class. Our combined sedimentation and electron microscopy data make the alleged non-equilibrium state within these populations questionable. From the polypeptide chain composition, determined by isoelectric focusing, it can be inferred that the total amount of C-terminal shortened chains increases progressively from 33 to 53% from monomeric to polymeric α-crystallin. Reassociation of different types of isolated shortened A-chains in various mixtures indicates a tendency for formation of higher molecular weight reaggregates with increasing relative amount of degraded chains. The involvement of degraded chains in polymerization is postulated although it is not likely to be the only factor causing aggregation.

A Model for the Architecture of α-Crystallin

Knowledge of the quaternary structure of α-crystallin is required to understand the age-dependent superaggregation processes, eventually leading to lens opacification. Different approaches, e.g. sulfhydryl modification, chemical cross-linking, limited proteolysis and dissociation studies revealed new information, providing a basis for further studies of aging and higher-order structures. A model for the architecture of native α-crystallin from calf lens cortex, featuring subunit arrangement and surface exposure, will be presented.

Structural aspects of bovine β-crystallins: Physical characterization including dissociation-association behavior*

Bovine cortical lens extracts were fractionated on Bio-Gel A-5m or Sephacryl S-200 and revealed three and four size classes for the β -crystallins, respectively. The greatest aggregates, β H , turned out to be subject to reversible, concentration-dependent dissociation into smaller aggregates. It was inferred that the association-dissociation equilibrium is mainly affected by hydrophilic interactions. On Sephacryl S-200 a β -crystallin size class with mol. wt 71 000, determined at sedimentation equilibrium, was isolated. Moreover, a β L -crystallin fraction (mol. wt 48000) and β S -crystallin (mol. wt 28 500) could be isolated. The polypeptide compositions of all β -crystallin aggregates have been determined by sodium dodecylsulfate (SDS) gel electrophoresis and isoelectric focusing in urea. On SDS gels β S -crystallin revealed a molecular weight of 22 000 contrasting sharply with the value determined at sedimentation equilibrium.

Stepwise dissociation/denaturation and reassociation/renaturation of bovine α-crystallin in urea and guanidine hydrochloride: Sedimentation, fluorescence, near-ultraviolet and far-ultraviolet circular dichroism studies

Abstract Quaternary, tertiary and secondary structure changes of calf α-crystallin were studied as a function of urea and guanidine hydrochloride concentration at pH 7·3 and 20°C. Sedimentation velocity analysis shows that native α-crystallin (19 s, 850 000 M r ) dissociates in a step-wise fashion to single subunits (1·4 s, 20 000 M r ). Complete dissociation is reached above 5 m -urea or 2·5 m -guanidine hydrochloride. The most stable dissociation intermediate (12 s) is observed around 2–3 m -urea or 1–1·5 m -guanidine hydrochloride. Fluorescence and near-ultraviolet circular dichroism analyses indicate that the exposure of aromatic residues is correlated with step-wise dissociation. Far-ultraviolet circular dichroism analysis shows that unfolding of subunits occurs over a very wide range of denaturant concentrations. Unfolding begins as subunits are released from the 12 s dissociation intermediate and may not be complete even in 8 m -urea or 6 m -guanidine hydrochloride. Dissociation of native α-crystallin to 12 s intermediate is irreversible, but subsequent dissociation steps are reversible. Dissociated and denatured subunits can be reassembled in a 12 s ‘reassociated α-crystallin’ molecule which is identical to the 12 s dissociation intermediate in nearly every aspect of secondary, tertiary and quaternary structure. The results are discussed in relation to our earlier proposed three-layer subunit model of calf α-crystallin and are found to support this model.

High-performance gel permeation chromatography of bovine eye lens proteins in combination with low-angle laser light scattering : Superior resolution of the oligomeric β-crystallins☆

Calf lens extracts were subjected to high-performance gel permeation chromatography on TSK GEL G4000 SW and G3000 SW columns (fractionation range: 5 x 10(6)-10(4) daltons) and resolved into thirteen crystallin fractions: HM-, alpha-, six beta H-, two beta L-, beta S- and two gamma-crystallins. Molecular weights were determined using a low-angle laser light scattering detection system. The weight average and number average molecular weights for cortical alpha-crystallin, 860,000 and 740,000, respectively, reveal a polydispersity factor of 1.16 for this heterogeneous protein. The eight different beta-crystallin fractions could be found with practically all possible oligomeric structures from dimers to aggregates larger than dodecamers. Different structures are found for the predominant beta H-crystallin fractions, viz., hexamers and pentamers, in the extracts from cortex and nucleus. Additional identification of the fractions by sodium dodecyl sulphate gel electrophoresis and isoelectric focusing in the presence of urea also indicated that semi-preparative application of this high-performance technique is possible. The co-elution of putative cytoskeletal proteins with some beta-crystallins was remarkable; moreover, co-elution of FM-crystallin with beta S-crystallin is discussed. A 23,000-dalton fraction, mainly found in the cortical region, most likely corresponds to the 24,000-dalton gamma-crystallin preparation obtained from cattle lens cortices. It is questioned whether the similarities between this fraction and beta S-crystallin are merely coincidental.

Comparative and age-dependent aspects of crystallin size and distribution in human, rabbit, bovine, rat, chicken, duck, frog and dogfish lenses

Lens extracts obtained from eight vertebrate species were analyzed using high-performance gel permeation chromatography with differential refractive index and low-angle laser light scattering detection. The elution patterns enabled the direct determination of the proportions of the crystallin classes as well as the molecular weights of the numerous size fractions in a more rapid and accurate way than previously. Concerning the mammalian species, also age-dependent aspects could be assessed by comparing the data obtained from the extracts of differently aged lenses and/or lens parts.

Age-dependent variations in the distribution of rat lens water-soluble crystallins, size fractionation and molecular weight determination

In order to obtain deeper insights into the mechanisms that are responsible for the age-related changes in the eye lens, the water-soluble proteins of 6-day-old to over 3-year-old rat lenses were analyzed by high-performance gel-permeation chromatography. Using this technique eleven crystallin fractions could be discerned: HM-, alpha-, three beta H-, two beta L-, beta S- and three gamma-crystallins. The concentrations of the higher molecular weight crystallins (HM-, alpha- and beta H-crystallin) seem to increase with age while those of the lower molecular weight (beta L- and gamma-crystallin) decrease. Taking into account the gradual increase of water-insoluble protein with aging, the relative amount of alpha-crystallin decreases from an age of 0.5 year after an initial increase. Additionally, an age-dependent increase in its molecular weight was found: from 7 . 10(5) to over one million. It appears that the gamma-crystallins are directly involved in the insolubilization process, while alpha- and beta L-crystallin first take part in aggregation processes leading to HM- and beta H-crystallin aggregates. These aggregation and insolubilization processes proceed gradually with increasing age. A steep decrease in gamma-crystallin concentration in the early phase of life, which also causes the relative increase in alpha-crystallin content in this period, may originate from a decrease in biosynthesis of certain gamma-crystallins.

Artificial peaks in gel permeation chromatography due to combining column supports with different pore sizes

Abstract Contrary to the elution pattern of bovine eye lens proteins chromatographed on a TSK GEL G5000 PW type column, an additional peak, representing 4.10 6 dalton aggregates, was observed on a combined G5000 PW–G4000 SW type system. This extra peak, found between the G5000 PW total exclusion fraction and α-crystallin (M r  1.1 · 10 6 ), appeared to correspond to the G4000 SW void volume. Simulation calculations substantiated that this peak is the result of coupling the two columns and does not originate from a distinct sub-population of molecules. Such artificial peaks may be expected whenever a broad molecular weight distribution extends beyond the fractionation limits of at least one of the constituents of a combined gel permeation system. It is inferred that the hypothetical exclusion limit for compact, globular, symmetrical proteins on TSK GEL G5000 PW is near 10 8 dalton.

Protein patterns obtained from support-bound gels by combining the images at different stages of destaining.

A more informative method for the visualization of proteins on thin-layer gels, based on combining the images of the gel at different stages of destaining, is presented. It is useful whenever important information seems to be lost after prolonged destaining. The method, which makes it unnecessary to run different loads in different channels, has been developed utilizing isoelectric focusing on polyester film-bound agarose gels. The strongly destained gel is superimposed on a negative image of the same gel made at an earlier phase of destaining, thus showing white spots on a gray background for minor components and dark bands in a white field surrounded by the grey background for the abundant ones. In general, the method may be applied to gel images obtained by different staining procedures.