Calf Lens α-Crystallin, a Molecular Chaperone, Builds Stable Complexes with β_s- and γ-Crystallins

Abstract

Calf water-soluble (WS) crystallins consist of high-molecular weight (HM), α-, β_H-, β_L-, β_s- and γ-crystallins. α-Crystallin as a molecular chaperone forms a structure with a central hole, known as Carver’s model. The only crystallins fitting into this central cavity are β_s- and γ-crystallins, with native molecular weights of 28 and 18.5-21 kD, respectively. The β_s-crystallin is loosely bound to this structure, whereas with the application of 7M urea in the sample, β_s-, may be some β_L-components and all γ-crystallins emerge from the central hole. Although WS and water-insoluble (WI) crystallins display the same immunologic determinants, there is an appreciable difference in electrophoretic mobility already in the young calf lens, α-Crystallins from the WI part demonstrate a clear cathodic shift. WI β- and γ-crystallins show smaller but well perceptible cathodic shifts. The chaperone function of α-crystallin preserves the original structures of β_s- and γ-crystallins for aggregation and other influences.

Higher Glycation of β_L- and β_S-Crystallins in the Anterior Lens Cortex and Maximum Glycation of γ-Crystallins in the Bovine Lens Nucleus, Demonstrated by Frozen Sectioning, Isoelectric Focusing and Lectin Staining

“…1, 3) nor in the human fetal lens [28]. In the bovine lens, however, we have found by immunoelectrophoresis at pH 8.9 [16] that there is a cathodic shift of water-insoluble ·-crystallins, and an anodic shift for water-soluble ß-and Á-crystallins in the aged, compared to the fetal human lens [29]. It is indeed remarkable that in diabetic rat lenses, the isofocused bands of ß L -, ß S -and Á-crystallins remain, compared to CBB staining, at the same isoelectric points, notwithstanding the increased glycation of these crystallins in diabetes, compared to the normal case [14].…”

“…One gel contained 1 ml Ampholine carrier ampholytes covering a pH range of (3.5-5):(5-7):(7-9) = 0.15:0.45:0.40 ml. After the run, the gel plates were stained in two ways: (i) for proteins by Coomassie brilliant blue (CBB) [16], with aliquot crystallin quantities of 0.4 mg; (ii) for glycated proteins by lectin staining with Con-A, with aliquot crystallin quantities of 1.2 mg. Direct lectin staining with Con-A was carried out according to Allen et al [11]: IEF was performed with subsequent fixation in 10% trichloroacetic acid for 10 min.…”

Isoelectric Focusing of Crystallins in Microsections of Calf and Adult Bovine Lens

“…Comparisons in the position and num ber of the focused bands of the samples stud ied revealed apparent differences in isoelec tric points of all the focused components. The identification of the crystallins was undertak en on the basis of isoelectric point and coeva luated in separate studies with results from immunoelectrophoresis of WS and WI crys tallins of the ageing bovine lens [9,19], The classification of the components of each class of crystallins is a useful procedure that has previously been described [6][7][8][9][10][11], The rela tive concentrations of the a-, (3-and y-classes were measured by densitometry. a-Crystallin was found to be composed of several minor components differing in isoelectric points.…”

“…The dissolution with urea of the WI crystallin fractions is useful for resolution of the protein constituents in different lens re gions. Adjustment to extremes of pH during IEF provokes better solubilization and sepa ration of crystallins [7,10,11], with preserva tion of the immunologic properties of the var ious crystallins [9,19], Statistical inference on age-related differ ences in crystallin concentrations has been based on rank tests. This was mandatory since the assumptions underlying a classical analy sis of variance based on t tests and F tests [30] are clearly not satisfied in the present case, although all crystallin differences (except HM, Pl5-6 and yi) were significant with these tests.…”

Analysis of tear proteins by one- and two-dimensional thin-layer iosoelectric focusing, sodium dodecyl sulfate electrophoresis and lectin blotting. Detection of a new component: cystatin C

“…The run was continued at 2000 V, 7.5 mA and 15 W for 1 h. During this time the current decreased to 4±5 mA. The gel was stained by 0.1% Coomassie brilliant blue for 1 h at 60C, according to Bours [7]. Eleven reference proteins (1±11) were applied as offered with the IEF calibration kit of Pharmacia (No.…”