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At least 19 recordsLinked to original sources
PubMed1992

Biochemical characterization of crystallins from pigeon lenses: structural and sequence analysis of pigeon delta-crystallin.

Crystallins from pigeon eye lenses were isolated and purified by gel-permeation chromatography and characterized by gel electrophoresis, amino-acid composition and sequence analysis. Alpha- and beta-crystallins could be obtained in relatively pure forms by single-step size-exclusion chromatography whereas an extra step of ion-exchange chromatography was needed for the separation of delta-crystallin from the beta-crystallin fraction. In contrast to most characterized vertebrate species, a large amount of glycogen is eluted as a high molecular form in the first peak of the gel filtration column. Pigeon delta-crystallin, similar to duck and reptilian delta-crystallins, exists as a tetrameric structure of about 200 kDa in the native form and is composed of one major subunit of 50 kDa with heterogeneous isoelectric points spreading in a range of 4.7 to 6.8. In contrast to those obtained from duck, goose and caiman, delta-crystallin isolated from the pigeon lens possessed very little argininosuccinate lyase activity. However, pigeon delta-crystallin can still cross-react with the antibody against enzymically active duck delta-crystallin as revealed by the sensitive immunoblotting technique. It was also shown that the delta-crystallin content of the total pigeon soluble proteins decreased with the age of the animal. Structural analysis of purified delta-crystallin fraction was made with respect to its amino-acid composition and protein primary sequence. N-terminal sequence analysis indicated the presence of blocked amino-termini in all crystallin fractions of pigeon lenses. Therefore, a sequence analysis of PCR (polymerase chain reaction) amplified delta-crystallin cDNA was employed to deduce the protein sequence of this crystallin. Structural comparison of delta-crystallin sequences from pigeon, chicken and duck lenses casts some doubts on the recent claim that His-89-->Gln mutation in the chicken delta-crystallin may account for the loss of argininosuccinate lyase activity in this avian species, as compared to high enzymic activity in the duck crystallin (Barbosa et al. (1991) J. Biol. Chem. 266, 5286-5290).

Amino Acid Sequence↗
PubMedDate not supplied

Immunohistochemical study of crystallin synthesis during morphogenesis of the crystalline lens in mice.

Using indirect immunofluorescence, the sequence of the synthesis of various classes of crystallins during normal morphogenesis of the crystalline lens in mice was shown: The alpha- begin to be synthesized first, then the gamma-, and finally the beta-crystallins. Using mice with hereditary anophthalmia (genotype ey-1/ey-1 ey-2/ey-2) permitted it to be established that the synthesis of alpha-crystallins occurs even when there is no morphogenesis of the crystalline lens. In mice of this genotype, the lens placode, which is reduced as compared to the norm, is resorbed as a rule, and does not develop into the crystalline lens vesicle. Separate cells containing alpha-crystallins were found in cranial epithelium on serial cross sections of the eye area of 13-day-old mutant embryos. Consequently, in ey-1/ey-1 ey-2/ey-2 embryos, alpha-crystallin synthesis takes place even in cells of the resorbed lens placode after brief inducing influence of the optic vesicle. As opposed to alpha-crystallins, synthesis of gamma- and beta-crystallins is detected only when lens fibers have formed. This is characteristic for embryos of ey-1/ey-1 ey-2/ey-2 genotype, as well as for mouse embryos homozygous for the fi gene. Data of the present work indicate that the inducing influence of the optic vesicle is necessary for activation of the genes controlling alpha-crystallin synthesis, while the influence of the retinal rudiment is necessary for derepression of the genes for gamma- and beta-crystallin synthesis.

Animals↗
PubMed1992

Sequence analysis of pigeon delta-crystallin gene and its deduced primary structure. Comparison of avian delta-crystallins with and without endogenous argininosuccinate lyase activity.

delta-Crystallin is a major lens protein present in the avian and reptilian lenses. To facilitate the cloning of the delta-crystallin gene, cDNA was constructed from the poly(A)+ RNA of pigeon lenses, amplified by the polymerase chain reaction (PCR). The PCR product was then subcloned into pUC19 vector and transformed into E. coli strain JM109. Plasmids purified from the positive clones were prepared for nucleotide sequencing by the dideoxynucleotide chain-termination method. Sequencing two clones, containing 1.4 kb DNA inserts coding for delta-crystallin allowed the construction of a complete, full-length reading frame of 1,417 bp covering a deduced protein sequence of 466 amino acids, including the universal translation-initiating methionine. The pigeon delta-crystallin shows 88, 83 and 69% sequence identity to duck delta 2, chicken delta 1 crystallins and human argininosuccinate lyase respectively. It is also shown that, in contrast to duck delta 2 crystallin which has a high argininosuccinate lyase activity, pigeon delta-crystallin appears to contain very low activity of this enzyme, despite the fact that they share a highly homologous structure. A structural comparison of delta-crystallins with or without enzymatic activity suggested several amino acid replacements which may account for the loss of argininosuccinate lyase activity in the lenses of certain avian species.

Amino Acid Sequence↗
PubMed1976

Reiteration frequency of delta-crystallin DNA in lens and non-lens tissues of chick embryos. delta-Crystallin gene is not amplified during lens cell differentiation.

The differentiation of embryonic chick lens fibers is characterized by an increased rate of synthesis of the lens protein, delta-crystallin, and accumulation of delta-crystallin mRNA. In the present study, the number of delta-crystallin genes in lens and non-lens tissues of embryonic chicks has been determined to test whether the accumulation of delta-crystallin mRNA during lens fiber differentiation is associated with an amplification of delta-crystallin genes. DNA from embryonic chick lens fibers, embryonic chick lens epithelia, or whole chick embryos with the eyes removed, was annealed with [3H]DNA complementary to delta-crystallin mRNA. Analysis of the annealing reactions indicated that the sequences for delta-crystallin are in the unique fraction of the chick genome, and are not amplified in the lens during embryonic development.

Animals↗
PubMed1976

Human alpha-crystallin. I. The isolation and characterization of newly synthesized alpha-crystallin.

Studies of the incorporation of 14C amino acids into human lens proteins demonstrate that an alpha-crystallin fraction takes up more than six times as much radioactivity as any other lens protein. Based on analyses with a calibrated Bio-Gel A-1.5 m column, a molecular weight of 4.9 x 10(5) +/- 5 per cent was obtained for this protein while sedimentation equilibrium analyses indicated a weight average molecular weight, Mw, of 7.5 x 10(5) +/- 4 per cent at 10,000 r.p.m. Gel electrophoresis in sodium dodecyl sulfate revealed two components with molecular weights of 22,000 and 20,000, values similar to those found with calf alpha-crystallin. Alkaline urea gel electrophoresis indicated one major polypeptide with a mobility similar to the B2 chain of calf alpha-crystallin and two major bands with mobilities between those of the calf alpha-crystallin A2 and A1 chains. Amino acid analyses of this newly synthesized alpha-crystallin gave a composition which with a few exceptions is very similar to that of calf alpha-crystallin. All three major polypeptides contained 14C amino acids. However, from the present data, it cannot be determined whether the three polypeptides were independently synthesized or a rapid transformation produced one of the labeled polypeptides in the A region. There appears to be between three and four times as many presumptive A as B polypeptides.

Amino Acids↗
PubMed1992

Alpha-crystallin exists in a non-spherical form. A study on the rotational properties of native and reconstituted alpha-crystallin.

Native alpha-crystallin, obtained from the cortex of calf lenses with FPLC (Pharmacia) was characterized by means of transient-electric-birefringence measurements and ultraviolet linear-dichroism spectroscopy. These techniques were also performed on 6-M-urea-dissociated and reconstituted alpha-crystallin. Transient-electric-birefringence measurements offer the possibility to characterize the often observed, but usually neglected, non-spherical occurrences of alpha-crystallin in more detail. Although not distinguishable with size-exclusion chromatography, we could identify at least two different classes of both native and reconstituted alpha-crystallin, from which at least one consists of non-spherical molecules. The results are compared with those obtained with electron microscopy using different staining methods. From the three independent techniques used we find evidence that a fraction of the alpha-crystallin exists in a more extended quaternary structure. The results are difficult to explain with a concentric three-layer model for alpha-crystallin as proposed by Tardieu et al. [Tardieu, A., Laporte, D., Licinio, P., Krop, B. & Delaye, M. (1986) J. Mol. Biol. 192, 711-724].

Animals↗
PubMed1992

Protein interactions in the calf eye lens: interactions between beta-crystallins are repulsive whereas in gamma-crystallins they are attractive.

Non-specific interactions in beta- and gamma-crystallins have been studied by solution X-ray scattering and osmotic pressure experiments. Measurements were carried out as a function of protein concentration at two ionic strengths. The effect of temperature was tested between 7 degrees C and 31 degrees C. Two types of interactions were observed. With beta-crystallin solutions, a repulsive coulombic interaction could be inferred from the decrease of the normalized X-ray scattering intensity near the origin with increasing protein concentration and from the fact that the osmotic pressure increases much more rapidly than in the ideal case. As was previously observed with alpha-crystallins, such behaviour is dependent upon ionic strength but is hardly affected by temperature. In contrast, with gamma-crystallin solutions, the normalized X-ray scattering intensity near the origin increases with increasing protein concentration and the osmotic pressure increases less rapidly than in the ideal case. Such behaviour indicates that attractive forces are predominant, although we do not yet know their molecular origin. Under our experimental conditions, the effect of temperature was striking whereas no obvious contribution of the ionic strength could be seen, perhaps owing to masking by the large temperature effect. The relevance of the different types of non-specific interactions for lens function is discussed.

Animals↗
PubMedDate not supplied

[Planar charged clusters--structural invariants of the gamma-crystallin family of the crystalline lens: functional role and evolutionary conservatism].

Plane charge clusters from the calf eye lens protein gamma-crystallin are considered. The clusters consist of four to six side chain charged groups with interatomic distances in ionic pairs from 4 to 7 A. The charge clusters appear to decrease the hydrophilic potential of the molecular surface which maintains the transparent refracting lens medium of vertebrates with a very high protein concentration. It is shown that the charge pattern for different gene products of one species is conservative as well as for whole set of 25 sequences of vertebrates, including carp, frog, mouse, rat, calf and human. Taking into account "neutral mutations", Asp-Glu and Arg-Lys the homology of those charge positions is equal to 95-100%. Functionally important charge clusters are absent in the ancient structural motifs of gamma-crystallin.

Amino Acid Sequence↗