Isoelectric focusing and immunochemistry of lens crystallins

Abstract

This is a review of 11 published articles with some conclusive results on the isoelectric focusing and immunochemistry of lens crystallins. The techniques used throughout the work are isoelectric focusing and immunological methods.The isoelectric focusing technique is performed on an analytical scale to realize the separation of proteins by isoelectric focusing in thin-layer polyacrylamide gels. The importance of carrying out all isofocusing techniques at 4 ~ C, and also of measuring the pH gradients established at the same temperature is noticed. Free isoelectric focusing is used as a preparative step to isolate uncontaminated crystallin fractions. Both analytical and preparative isoelectric focusing experiments of crystallins performed throughout the work are in absence of urea or any other dissociating medium.The purity of proteins isolated by free iseelectric focusing is examined by criteria of homogeneity which are founded on three different techniques, e.g. thin-layer isoelectric focusing, immunoelectrophoresis and antigen/antibody crossed electrophoresis.The existence of various crystallins in the chick lens, like ~z-, ~-crystallin and FISC, related to own data, is reviewed and nomenclatures of chick lens crystallins are compared with present data from literature. The antigenic properties of proteins from chick intra-ocular tissues are also discussed. Anti-total iris serum contains antibodies against crystallins and serum proteins. The presence of proteins immunologically identical to lens crystallins in chick iris is shown by indirect immunofluorescence experiments: brilliant fluorescence of the lens is observed due to the presence of crystallin antibodies in anti-total iris serum. It is concluded that proteins of all intra-ocular tissues which are immunologically identical to lens crystallins are essential structural elements. Contrary to this statement several authors support the theory that the presence of proteins immunologically identical to lens crystallins in the intra-ocular tissues is due to a post-mortem diffusion of these antigens from the lens. The presence of several erum proteins in all intra-ocular tissues excepting lens is shown. Evidence is presented that the number of serum proteins in intra-ocular tissues is restricted by the various blood/ocular barriers, and that the blood/retinal barrier is of the same nature as the blood/brain barrier.The presence ~-, ~-, ,,,-and pre-~-crystallins is shown in various stages of the bovine embryonic lens by thin-layer isoelectric focusing and various immunological methods. It is defined that the bovine -~,-crystallins have isoelectric points higher than pI --7.0. It is shown by immuno-osmophoresis that the embryonic ~-crystallins consist of two molecular forms with different electrophoretic mobilities. The ~-crystallins are forming half the total amount of the crystallins present in the embryonic lens. It is concluded that the "c-crystallins are more prominent in embryonic cow lens and in adult lens nucleus and less prominent in adult lens cortex. The...

Water-Soluble and Insoluble Crystallins of the Developing Human Fetal Lens, Analyzed by Agarose/Polyacrylamide Thin-Layer Isoelectric Focusing

“…The human fetal ycrystallins in figure la-h showed a slightly diffuse appearance, like polyclonal human IgG after isofocusing [Bours, 1976] In the bovine lens the pre-a-crystallin is focused at pi = 5.25 ( fig. lj) [Bours, 1975], In human fetal lens one band focused at pi = 5.00 ( fig. Id), which was fairly constant at 1.5% ( fig.…”

“…Id), which was fairly constant at 1.5% ( fig. 2g), represents pre-a-crystallin [Bours et al, 1987], This crystallin was also detected by isotachophoresis of the same samples [Bours and Fodisch, 1986c], Pre-acrystallin cannot be considered as the most basic isofocused band of a-crystallin, be cause a-crystallin precipitates during isofo cusing [Bours, 1975] and is identifiable ac cording to its appearance.…”

Changes in the Electrophoretic Mobility of α-Crystallin during Lens Cell Differentiation

“…Investigations with fetal a-crystallin demonstrate that differences in the electrophoretic mobility may be attributed to differences in polypeptide chain composition [Bours and Brahma, 1973;Bours, 1974;Hoenders, 1976], On the other hand, a-crystallin aggregates from lens cortex show the same electro phoretic mobility ( fig. 4) despite of large differences in size [Kramps el al., 1975].…”

Lactate Dehydrogenase Composition of the Rabbit Retina and Choroid after Ischemia Caused by Ligature of the Optic Nerve

“…The homogenate was centrifuged at 38,000# for 1 h at 4°C. The supernatants were used for the detection of lactate dehydrogenase isoenzymes by tétra zolium staining according to D ietz and Lubrano [1967] after thin-layer isoelectric focus ing at low voltage [Bours, 1975[Bours, , 1977, The quantities of the LDH-isoenzymes are given by aid of densitométrie measurement of the photographed bands of staining with a Chromo scan recording reflectance densitometer (Joyce, Loebl & Co., Ltd., London, England) [Brahma and Bours, 1972]. The patterns were run with wedge A and B, with a reproduci bility of 5%.…”