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Results for “alpha-Crystallin A Chain”

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At least 19 recordsLinked to original sources
PubMed1976

The amino-acid sequence of the alpha-crystallin A chains of red kangaroo and Virginia opossum.

The amino acid sequence of the A chain of the eye lens protein alpha-crystallin from the red kangaroo (Macropus rufus) was completely determined by manual Edman degradation of tryptic, thermolytic and cyanogen bromide peptides. The sequence of the alpha-crystallin A chain from the Virginia opossum (Didelphis marsupialis) was deduced from amino acid analyses and partial Edman degradation of peptides. The 173-residue A chains of kangaroo and opossum differ in six positions, whereas comparison with the bovine alpha-crystallin A chain reveals 17 and 22 substitutions, respectively. Most substitutions occur in the COOH-terminal part of the chain.

Amino Acid Sequence↗
PubMed1992

Structural organization and stability of a thermoresistant domain generated by in vivo hydrolysis of the alpha-crystallin B chain from calf lens.

A protein fragment (M(r) approximately 9000) isolated from the cortex of nonpathological calf lenses has been structurally characterized. The polypeptide structure was well organized (39% alpha-helix, 33% beta-structure, and 28% remainder) according to the far-ultraviolet circular dichroism. The fluorescence was heterogeneous for the presence of two tryptophan classes. Structure perturbation by pH and denaturant revealed cooperative structural transitions which are characteristics of a globular organization. A single-step unfolding curve induced by Gdn-HCl (midpoint = 1.38 M Gdn-HCl) was monitored by emission maximum shift as well as by far-ultraviolet circular dichroism. This transition was analyzed as a two-state process. The standard free energy of unfolding in the absence of the denaturant, delta Go (H2O), was found to be 10.80 +/- 0.25 kJ/mol at 20 degrees C and pH 7.4. The fragment also shows an unusual thermal resistance. Its structure was unperturbed up to 90 degrees C according to the fluorescence and dichroism. This last property, its peculiar amino acid composition, and the sequence of a small segment are shared, among crystallins, only with the N-terminal region of the alpha-crystallin B chain. A search for proteolysis sites along the alpha-crystallin B chain sequence revealed that it possesses specific points for proteinase attack. These sites are particularly exposed and clustered in a very flexible region in the middle of the protein sequence. They are also well represented in the C-terminal extension of the molecule while a few are buried in the N-terminal region.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1976

Isolation and chracterization of rat-lens messenger RNAs. Comparison of lens proteins, synthesized in lens culture and in homologous and heterologous cell-free systems.

Messenger RNA has been isolated from rat lens tissue. The mRNA species which codes for the A2 chain of alpha-crystallin, revealed the same extremely high sedimentation value (14S) as the corresponding messenger from calf lens. However, it has been shown that in rat lens the 14-S messenger preparation directs the synthesis of an additional alpha-crystallin chain, designated as alpha-X with an approximate molecular weight of 24000. For comparative purpose crystallin synthesis has also been studied as well in cultured rat lens cells as in the rat lens cell-free system.

Animals↗
PubMed1978

Rat alpha-crystallin A chain with an insertion of 22 residues.

Rat lens alpha-crystallin contains, besides the usual alphaA and alphaB subunits, an additional minor chain. This subunit was purified by ion-exchange chromatography and its primary structure studied. It appeared to be an elongated alphaA-like chain, having an insertion of 22 residues between position 63 and 64 of an otherwise normal alphaA2 chain. Therefore this subunit was called alphaAIns, i.e. an alphaA chain with an inserted sequence. This inserted region, which contains three methionyl, five basic and no acidic residues, apparently results in an adequately functioning alphaAIns chain. The alphaAIns chain may be the product of a gene which has originated, after duplication of the alphaA gene, by insertion in one of the copies of a stretch of 66 nucleotides of unknown origin, or alternatively be the result of unusual transcription or processing of precursor mRNA (pre-mRNA), leaving an extra 66 nucleotides internally in the mRNA to be translated.

Amino Acid Sequence↗
PubMed1977

Primary structures of alpha-crystallin A chains of elephant, whale, hyrax and rhinoceros.

As part of a study of the evolutionary development of the eye lens protein alpha-crystallin the 173-residue A chain of this protein has been studied in elephant, whale, hyrax and rhinoceros. The primary structures were inferred mainly from amino acid compositions of peptides obtained by enzymic digestions and CNBr cleavage. The positions of substitutions, as compared to the known bovine A chain, were confirmed by Edman degradation. In accordance with the previously observed slow rate of evolution of the A chain only a small number of substitutions was found among these species. Elephant and hyrax share a number of unique substitutions, strongly indicating a common ancestry of these two species within the mammalian class.

Amino Acid Sequence↗
PubMed1978

Post-translational assembly of lens alpha-crystallin in the reticulocyte lysate and in Xenopus laevis oocytes.

Lens mRNA was translated in reticulocyte lysate predominantly into monomeric alpha-crystallin chains. Lens polyribosomes added to the cell-free system produced the same polypeptides, but these were detected predominantly in alpha-crystallin aggregates. Lens mRNA, after microinjection into Xenopus laevis oocytes, produced alpha-crystallin subunits that were exclusively found in the form of high-molecular-weight complexes. Also after injection of the purified 14-S mRNA, coding for the alphaA subuint, the synthesized alpha-A polypeptides were incorporated into high-molecular-weight aggregates. In contrast, the synthesis of alphaB subunits, directed by a 10-S mRNA, did not result in aggregate formation. The experiments thus suggest that aggregate formation of alpha-crystallin is triggered by its alphaA subunits, which are then joined by the alphaB subunits. This process occurs partly in the cell-free system and completely in Xenopus oocytes.

Animals↗
PubMed1977

The bicistronic nature of lens alpha-crystallin 14S mRNA.

The A2 and B2 polypeptide chains of calf lens alpha-crystallin are synthesized on a 14S, 1500-nucleotide mRNA and a 10S, 735-nucleotide mRNA, respectively. The 10S mRNA is theoretically compatible with the size of the B2 chain, but the 14S mRNA contains approximately twice the required number of nucleotides necessary for A2 chain synthesis. This fact raises the question of the function of the additional nucleotide sequence in the 14S mRNA. The following observations on 14S mRNA suggest that it may contain an additional cistron. (i) Under a number of denaturing conditions, 14S mRNA continues to retain its initial size characteristics. (ii) In addition to synthesis of the A2 chain, 14S mRNA directs the synthesis of another polypeptide with the same electrophoretic mobility as that of the B2 chain. (iii) Molecular hybridization of the 14S mRNA with the cDNA produced from the 10S mRNA suggests that 2 mol of the cDNA bind to 1 mol of the 14S mRNA. (iv) Examination of the nucleotide sequences of the 10S and 14S mRNAs by two-dimensional maps of RNase A and T1 digests indicates marked similarity. The overall data suggest that the additional cistronic component may carry coding information for an alpha-crystallin polypeptide or a closely related polypeptide species.

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↗
PubMed1976

Human alpha-crystallin-III isolation and characterization of protein from normal infant lenses and old lens peripheries.

Alpha-crystallin isolated from the peripheries of old normal or cataractous lenses appears to be identical, consisting of eleven polypeptides, five B, and six A chains. In contrast, alpha-crystallin isolated from normal six-week-old human lenses has only three major polypeptides, corresponding to B1, A1, and A2 of the old human lens protein as well as small amounts of some of the other components. Comparisons with bovine alpha-crystallin are also reported. Based on gel filtration experiments with Bio-Gel A-1.5m, two distinct populations of alpha-crystallin were found in old lens periphery, one containing species greater than 1.5 X 10(6) daltons and another of approximately 9 X 10(5) daltons. In the cataract preparations, the higher molecular weight fraction is predominant. This fraction is not present in young lenses.

Aged↗
PubMed1977

Evolutionary changes of alpha-crystallin and the phylogeny of mammalian orders.

The sequences of the A chains of the eye lens protein alpha-crystallin from seventeen mammalian species were compared. They showed a generally slow rate of evolution, but with marked variations in different lineages. Most substitutions have occurred in the C-terminal part of the chain, which probably forms part of the surface of the alpha-crystallin aggregate. The ancestral sequence method of Dayhoff revealed interesting indications about the phylogenetic relationships between the eleven mammalian orders that were represented by the investigated species. Most evident was the divergence of marsupial and placental orders. A notable resemblance between the hyrax and elephant sequences was observed, setting them apart from the ungulates, including whale. Primates, rodents, lagomorphs, insectivores and tupaiids seem to derive from a common stem group. These phylogenetic inferences are discussed in relation to current palaeontological and taxonomical opinions, and compared to evidence from other protein sequence data.

Amino Acid Sequence↗
PubMed1976

Human alpha-crystallin: characterization of the protein isolated from the periphery of cataractous lenses.

alpha-Crystallin has been isolated from the peripheral region of old cataractous lenses. It was found to be closely related to bovine alpha-crystallin and to human newly synthesized alpha-crystallin in terms of its amino acid composition, the size of its polypeptide chains and the lack of free NH2-terminal groups. However, in contrast to the simple urea gel electrophoretic polypeptide patterns obtained with the reference proteins, 11 polypeptides were detected in the preparation. Ten of the polypeptides were isolated and shown to be either A or B chains on the basis of their amino acid compositions and comparison of the peptide maps of their tryptic hydrolysates. The four B chains as well as the six A chains were closely related, with most of the tryptic peptides being common to all members of their respective group. A nomenclature based upon the urea gel electrophoretic mobilites of the polypeptides has been proposed to define each chain. It was found that this alpha-crystallin preparation is composed of at least two populations of macromolecules, one of which contains macromolecules greater than 5 X 10(6) daltons on the basis of gel filtration with Bio-Gel A-5m. The compositions of the two fractions were found to be essentially identical.

Aged↗
PubMed1976

Intracellular degradation and deamidation of alpha-crystallin subunits.

The subunites AA2 and N2, present in alpha-crystallin from the nucleus of young and old bovine lenses, were isolated and characterized. It was found that AA2 is identical to a shorter A-chain and hence it was designated as A1-101. The subunit N2 turned out to be identical with a shorter B-chain and was designated as b 1/2-170. Characterization of the subunit N1, present only in alpha-crystallin from the cortex of old bovine lenses, suggested that his subunit is a modified B-chain, probably a deamidation product; it was designated as B0. Con relation to the age of the fiber cells in old bovine lenses with that in calf lenses revealed that the observed specific limited degradation of the subunits of alpha-crystallin increased with older age of the tissue. The deamidation process was found not to be related to the aging of the tissue. Eventually, a clear picture concerning the heterogeneity and fickleness of the alpha-crystallin subunit structure was obtained.

Age Factors↗
PubMed1978

The quaternary structure of bovine alpha-crystallin. Size and charge microheterogeneity: more than 1000 different hybrids?

Cortial alpha-crystallin was size-fractionated by gel filtration on Ultrogel AcA22 and charge-fractionated by anion-exchange chromatography on DE-52 DEAE-cellulose using gradient elution. Electron microscopy demonstrates that both native and reassociated alpha-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 alpha-crystallin and 8.5--12.5 nm (maximum at 10.0--10.5 nm) for reassociated alpha-crystallin. An enormous charge heterogeneity of native alpha-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 (A2, A1 and A1/2-151) to basic chains (B2 and B1) varies from 70/30--80/20 (averaging about 3/1) and the amount of deamidated chains (A1 and B1) varies from 7--37%. Recombination of the subunits, after dissociation in 6 M urea, leads to a charge heterogeneity of reassociated alpha-crystallin very similar to that of native alpha-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 alpha-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, A1/2-151, B2 and B1 conceivable.

Animals↗
PubMed1977

Isolation and characterization of initiation fragments from lens 10S and 14S alpha-crystallin messenger ribonucleic acids.

The alpha-crystallin 10S and 14S messenger ribonucleic acids (mRNAs) for the B and A chains, respectively, were isolated from calf lenses. Initiation complexes were formed with both mRNAs after which the unprotected regions were digested with ribonuclease T1. A single fragment of approximately 45 nucleotides was obtained from both the 10S and 14S mRNAs. The fragments retained the ability to reform initiation complexes under standard conditions. Two-dimensional fractionation of ribonuclease T1 digests indicated considerable similarity between the 10S and 14S fragments. However, marked differences in the (U)G region were observed. The addition of the methylating agent S-adenosyl-L-methionine to the mRNA initiation system increases complex formation form two to five times, suggesting that methylation may be required for initiation.

Animals↗