The structural bovine eye lens protein alpha-crystallin was dissociated in 7 M urea and its four subunits, A1, A2, B1, and B2, were separated by means of ion-exchange chromatography. Homopolymeric reaggregates of these subunits were prepared by removal of the denaturant via dialysis. It was found that subunits were exchanged upon incubation of mixtures of two homopolymers under native conditions. New hybrid species were formed within 24 h as demonstrated by isoelectric focusing. Moreover, native alpha-crystallin molecules also exchanged subunits when incubated with homopolymeric aggregates of B2 subunits. Subunit exchange between native alpha-crystallin molecules is postulated, and a "dynamic quaternary structure" is presented that allows the polydisperse protein to adapt to changes in cytoplasmic conditions upon aging of the lens tissue.

Aging of the eye lens is reflected by pronounced changes on the molecular level. So far, these alterations have been investigated in particular with respect to the structural lens proteins or crystallins, which comprise approximately 95% of the dry weight. It seems that virtually postsynthetic modifications are involved, although errors on the translational level cannot be excluded. A variety of chemically or physically defined processes have been observed with aging. These include formation of high-molecular-weight, eventually insoluble aggregates; formation of disulfide bridges and other covalent crosslinks; deamidation of asparaginyl and glutaminyl residues; partial degradation of polypeptides at characteristic sites; racemization of aspartyl residues; nonenzymatic glycosylation; and photooxidation of tryptophanyl residues.

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The stability of the native quaternary structure of bovine cc-crystallin was studied. by sedimentation analysis and electron microscopy, as a function of pH (7 -1 I), ionic strength (0.01 -0.5), temperature (6 -60 'C) and calcium ion concentration (0 and 10 mM).Three successive transitions are distinguished at 20 ' C. Firstly, a slow transconformation step, which is independent of pH, ionic strength or calcium ions. Secondly, an irreversible primary dissociation step, favoured by increasing pH above 8 and/or a lower ionic strength, with formation of 'alkali-modified x-crystallin', which is spherically shaped like the native protein but has a smaller average diameter, sedimentation coefficient and molecular weight. Thirdly, with further increase of pH above 9, a rapidly reversible dissociation of alkali-modified a-crystallin characterized by a single reaction boundary in sedimentation velocity analysis. In the presence of calcium ions the quaternary structure is stabilized to the extent that no dissociation is observed up to at least pH 10.3.Upon increase of temperature, at pH 7.3, a slow irreversible dissociation and swelling run parallel until a limit is reached around 37 '-C with formation of 'temperature-modified r-crystallin', which is indistinguishable from the native protein by electron microscopy, but has a higher relative viscosity and lower sedimentation coefficient and molecular weight. Calcium ions have little or no effect on this transition. Above 3 7 ' C a reversal of this transition or aggregation is indicated.These findings, together with previous structural data on microheterogeneity, reassociation from urea, and aging of a-crystallin in viva, are incorporated into a hypothetical scheme of transitions, based on a three-layer model for the quaternary structure.The crystallins are a group of structural proteins which occur in extremely high concentrations in the eye lens cells, and, as such, play a role in the refraction and focussing of light rays (reviewed in [l -31). The largest of these, a-crystallin, has a weight-average molecular weight ( M , ) of about 850000 in the bovine lens cortex, and it is a spherical assembly of A and B-type subunits, both of about 20 000 molecular weight, in 3 : 1 ratio [4,5]. Primarily in the lens nucleus agerelated structural changes affect the state of aggregation, the solubility and the interaction of x-crystallin with other crystallins and lens membranes, which in turn may affect light refraction and lens transparency, and in extreme cases may lead to visual impairment, Previous papers in this scries appeared earlier in this journal [4.5,7,8]. A preliminary report of part of this work was presented at the Workshop on Aging of the Lens, Bonn-Meckenheim, July 1977 [20]._ _ _ at least in humans [3,6]. The elucidation of the quaternary structure of a-crystallin will undoubtedly contribute to (and it may prove to be essential for) an understanding of these processes on the molecular level. In earlier papers of this series information on the qtructure has been acquir...

Cortical a-crystallin was size-fractionated by gel filtration on Ultrogel AcA22 and chargefractionated by anion-exchange chromatography on DE-52 DEAE-cellulose using gradient elution.Electron microscopy demonstrates that both native and reassociated a-crystallin are heterogeneous populations of spherical or slightly ellipsoidal molecules with diameters of 13.5 -16.0 nm (maximum at 14.0-15.0 nm) for native a-crystallin and 8.5-12.5 nm (maximum at 10.0-10.5 nm) for reassociated a-crystallin.An enormous charge heterogeneity of native a-crystallin was detected, which is shown to arise from variations in the stoichiometry of the 5 main types of subunits. The molar ratio of acidic chains (Az, A1 and to basic chains (B2 and BI) varies from 70/30-80/20 (averaging about 3/1) and the amount of deamidated chains (Al and B1) varies from 7-37%. Recombination of the subunits, after dissociation in 6 M urea, leads to a charge heterogeneity of reassociated a-crystallin very similar to that of native a-crystallin. Therefore, specific formation of pure A or B chain aggregates is not preferred. Instead, random combination of subunits is theoretically shown to be sufficient to describe the observed charge microheterogeneity of both reassociated and native a-crystallin.No obvious relationship exists between size and charge heterogeneity. Within these ranges of molecular weight and subunit composition there are more than 1000 different combinations of A2. A1, A:-151, B2 and B1 conceivable.Towards a better understanding of the eye lens architecture and its function on a molecular level, a thorough structural characterization of the main protein constituents, the crystallins, is required, in particular their quaternary structure, packing arrangement and interactions in vivo. Additional interest in one of the major multisubunit lens proteins, acrystallin, arises from its usefulness as a marker for studies of such fundamental processes as growth, differentiation and aging [l -31. We investigate various aspects of the quaternary structure of bovine low-M, a-crystallin in this and following papers in this series, with the purpose of providing a sound basis for further studies of aging and higher-order structures.The subunit composition and physical characteristics of bovine a-crystallin change with development and aging of the lens, which results, even at a very early stage, in a distinct size and charge heterogeneity.