Biomedical subjects

R C Augusteyn

Publications and source records attributed to R C Augusteyn.

At least 55 records · Page 3Linked to original sources

PubMed1988

On the structure of alpha-crystallin: the minimum molecular weight.

alpha-crystallin can be isolated in two forms depending on the temperature at which the lens is extracted. At 4 degrees C, alpha c-crystallin is obtained while at 37 degrees C, a smaller molecule, alpha m-crystallin can be isolated. The apparent molecular weight of bovine foetal alpha m- and alpha c-crystallins were determined in 5 different ways using sedimentation velocity, sedimentation equilibrium and intensity fluctuation spectroscopy analyses and the experimentally determined diffusion coefficients, intrinsic viscosity and partial specific volume. Values ranged from 291,000 to 369,000 for alpha m and from 604,000 to 760,000 for alpha c due to the differential effects of the protein's polydispersity on the different methods. Subfractionation of the protein by gel filtration yielded much less polydisperse minimum species populations with molecular weights of 280,000 and 529,000 for alpha m and alpha c respectively. It was concluded that alpha-crystallin is probably synthesized as a symmetrical dodecamer and that the polydispersity of most preparations can be attributed to age-related modification in vivo as well as in vitro supra-aggregation due to variations in experimental conditions.

Animals↗

PubMed1988

Protein distribution patterns in concentric layers from single bovine lenses: changes with development and ageing.

Protein distribution patterns were determined in concentric layers removed from 24 bovine lenses ranging in age from about 6 months before birth to 180 months post-natal. It was possible to distinguish alterations in protein synthesis patterns during development and changes due to ageing, i.e., prolonged existence of the proteins. It was found that alpha-crystallin represents a constant 50% of the proteins synthesized by the fibre cells throughout life. However, the protein becomes progressively less soluble with increasing age. Beta-crystallin synthesis increases from 30% of the total proteins during prenatal development to around 40% in post-natal fibre cells. This increase is due to increased production of the beta-crystallin. In old tissues, beta H-crystallin is converted to a high molecular weight from (HMW beta) gamma-crystallins account for 22% of the proteins synthesized in the earliest prenatal fibre cells. This level decreases rapidly through prenatal development until they represent about 4% of the total at birth. Beta S-crystallin synthesis commences around this time and in the post-natal fibre cells is essentially the only low molecular weight protein. The possible significance of some of these changes is discussed with regard to the functional requirements of the lens.

Aging↗

PubMed1988

Electron microscopy of native and reconstituted alpha crystallin aggregates.

The size and shape of native alpha crystallin aggregates extracted at either 4 degrees C (alpha c-crystallin) or 37 degrees C (alpha m-crystallin), were compared with each other, as well as with aggregates reconstituted from either pure alpha A or alpha B subunits using electron microscopy. The alpha B aggregates were the most uniform in size (about 9 nm in diameter) and the best stained. alpha A particles were about the same size as the alpha B, but the population distribution was broader and some indication of interparticle association was observed. alpha c particles exhibited a bimodal distribution, with one peak greater than the reconstituted particles and the other about the same size; alpha m was smaller than the reconstituted structures.

Animals↗

PubMed1988

Quenching of tryptophan fluorescence in bovine lens proteins by acrylamide and iodide.

The microenvironments of tryptophan residues in bovine alpha-, beta H-, beta L and gamma-crystallins have been examined using acrylamide and KI quenching of fluorescence. From a consideration of the differential effects of the two quenchers, the quenching efficiencies and spectral changes, it was possible to distinguish tryptophans in different environments and to assign these to specific residues in the sequence. Two classes of tryptophan were identified in gamma-crystallin, one buried and one moderately accessible. The buried class contained tryptophans 42A and 125 which lie in the angles of the wedge-shaped domains of the protein. These residues, which had emission maxima at 326 nm, were not accessible to quenching by iodide. The more accessible residues, emitting at 334 nm, corresponded to tryptophans 64 and 148 which are in the widest part of the wedge-shaped subunit and close to the surface of the protein. The two beta-crystallins were virtually indistinguishable. They contained two buried tryptophans, probably residues 58 and 150, and three close to the surface, residues 81, 84 and 166. The quenching efficiencies for these two classes were lower than those observed with gamma-crystallin. Since the three-dimensional structures of the beta- and gamma-crystallins are probably very similar, this suggests that the polymeric nature of the beta-crystallins is responsible for the decreased accessibility of the tryptophans to the quenchers. alpha-crystallin demonstrated unusually high static quenching which made it difficult to distinguish different classes of tryptophan.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrylamide↗

PubMed1987

A possible structure for alpha-crystallin.

alpha-Crystallin, the major protein of the mammalian eye lens, is found in vivo as a multimeric aggregate composed of two closely related subunits whose molar ratio is widely variable from species to species. Attempts to determine the arrangement of the subunits within the aggregate, or even to determine the size of the aggregate and the number of subunits composing it, have not resulted in general agreement. Because of the variability in alpha-crystallin particle size, the apparent dependence of this parameter on certain environmental factors (e.g. temperature), the absence of a specific requirement for either alpha-crystallin isoform in aggregation, and the sharp division in the amino acid sequence between a strong hydrophobic region and a sharply hydrophilic one, it is suggested that the alpha-crystallin aggregate has the properties of a protein micelle. This hypothesis is consistent with what is known of the alpha-crystallin molecule and aggregate, and can be tested experimentally. If this hypothesis is shown to be true, then alpha-crystallin will be the first example of a naturally occurring protein micelle.

Animals↗

PubMed1987

The location of sulphydryl groups in alpha-crystallin.

The microenvironments of the sulphydryl groups in the multimeric protein, alpha-crystallin, were studied by examining: the rate of the reaction of the groups with DTNB; the effect of increasing urea concentrations on their accessibilities; and the quenching of a fluorescent probe. In foetal bovine alpha-crystallin (1 SH/alpha A subunit) both kinetic and quenching studies indicated that over 90% of the sulphydryl groups fell into a single buried class; the remainder was exposed. In the human protein (2 SH/alpha A subunit), half of the groups were buried and the other half exposed. Accessible sulphydryl groups increased gradually as the urea concentration was increased, with complete exposure at about 4.0 M. Sedimentation velocity analyses revealed that no significant dissociation of the aggregates into subunits occurred below 3.5 M urea, at which point over 80% of the sulphydryl groups were exposed. An age-dependent increase (3-35%) was found in the proportion of exposed sulphydryl groups in bovine alpha-crystallin and a decrease in the urea concentration required to expose the remainder. It was concluded that the single cysteine is buried in the newly synthesized protein, but becomes solvent-exposed as a result of age-related conformational changes. Our observations are consistent with a quaternary structure in which all alpha A subunits occupy equivalent sites.

Aging↗

PubMed1987

The refractive increments of bovine alpha-, beta-, and gamma-crystallins.

The refractive increments have been determined for purified bovine alpha-, beta- and gamma-crystallins, using three different light sources. The values obtained were used to predict the refractive index gradient in the rat lens. Excellent agreement was obtained between the predicted and observed gradients.

Animals↗

PubMed1987

Serum tryptophan and cataract.

Total serum tryptophan concentrations were measured in 74 cataractous patients and 124 controls. No significant differences were observed in the mean values for the cataractous and normal subjects, 11.4 +/- 2.6 and 11.3 +/- 2.2 micrograms/ml respectively, or in the range of values, 7.4-17.1 and 7.3-17.4 micrograms/ml respectively. These results do not support previous reports that serum tryptophan is elevated in cataractous subjects.

Adult↗

PubMed1987

Isolation of alpha-crystallin subunits by gel filtration.

In the presence of 0.1 M glycine, pH 2.5, alpha-crystallin dissociates into aggregates containing only alpha A chains plus monomeric alpha B chains. Advantage has been taken of this to develop a method for the purification of the alpha A and alpha B chains by gel filtration on Sephadex G-75. This method gives high yields of purified subunits and avoids the risk of carbamylation associated with other methods which use high concentrations of urea.

Animals↗

PubMed1987

The state of sulphydryl groups in proteins isolated from normal and cataractous human lenses.

Individual crystallins, urea-soluble and urea-insoluble proteins were isolated from the nucleus and cortex of types I-IV cataractous lenses and normal lenses. The levels of protein sulphydryls (P-SH), disulphides (S-S), as well as surface (F-SH) and buried (S-SH) in these proteins were determined by reaction with 5, 5'-dithiotris- (2-nitrobenzoic acid) or performic acid oxidation followed by amino acid analysis. During nuclear colour development there is a progressive decrease in the sulphydryl content of the crystallins. In the nuclei of advanced cataractous lenses, the P-SH decreases to 10% of the levels found in the normal nucleus. Similar but smaller changes take place in the cortex. No specific changes were found between the crystallins, with the exception of beta S crystallin. The cysteine remains constant in all lens types suggesting no higher oxidation products are formed. There is a significant shift in the distribution of cysteine in the nucleus of type III and IV lenses. Urea-insoluble proteins are the predominant species, accounting for about 70% of the total cysteine pool. This is consistent with the accumulation of modified insoluble polypeptides during senile nuclear cataract formation.

Aged↗

PubMed1987

The nature of disulphide bonds in rat lens proteins.

The nature of disulphide bonds in 3, 7 and 24 month old rat lens proteins has been determined in order to examine the possibility that there is a direct correlation between intermolecular disulphide bond formation and hardening of the lens during ageing. Significant levels of S-S were found in all of the crystallins, as well as in the insoluble proteins. However, no substantial changes in the molecular weight distribution of the proteins, or their constituent polypeptides, were observed after the reduction of these bonds. This indicates that almost all of the S-S bonds are intramolecular. It would appear that the hardening of the nucleus is not a result of intermolecular disulphide crosslinking during ageing. Instead, our observations suggest that intramolecular disulphide bonds may make the gamma-crystallin molecule more compact, thereby promoting closer packing in the nucleus where the concentration of this protein is highest.

Animals↗

PubMed1986

Immunological comparison of heat-shock proteins and alpha-crystallin.

It has been shown using ELISA and affinity chromatography, that extracts from heat-shocked Drosophila melanogaster larvae and cultured cells react with monoclonal antibodies raised against bovine alpha-crystallin. It is suggested that this cross-reactivity is associated with the region of the sequence corresponding to residues 98-108 of the bovine alpha A-crystallin chain. This region may be important for the functions of both alpha-crystallin and the heat-shock proteins.

Amino Acid Sequence↗

PubMed1986

On the antigenic relationship between the alpha A and alpha B subunits of alpha-crystallin in bovine lens.

The immunochemical reactivities of the alpha A and alpha B subunits from bovine alpha-crystallin have been compared using 5 monoclonal antibodies and 2 polyvalent antisera. Each subunit bound the same maximum amount of antibody, regardless of its source, and each subunit was able to completely displace alpha-crystallin from its antibodies. One monoclonal antibody (463-12.2) and mouse anti-alpha B polyclonal antiserum bound equally well to the two subunits; with the other monoclonal antibodies and an anti alpha-crystallin antiserum, the affinities for the alpha A chains were about 10(3) fold higher than those for the alpha beta chains. These observations indicate that the alpha A and alpha B subunits of bovine alpha-crystallin share several similar, but not necessarily identical, cross-reacting antigenic determinants. The reasons for the differences between these observations and those of other investigators are discussed.

Animals↗

PubMed1985

Ontogeny of human lens crystallins.

The soluble proteins from prenatal and neonatal human lenses were fractionated by gel filtration into four distinct size classes viz. high molecular weight alpha-crystallin (HM-alpha), alpha-crystallin, intermediate molecular weight (IMW) proteins and low molecular weight (LMW) proteins. Extinction coefficients of the isolated proteins were determined and used to calculate the proportions of each fraction on a weight basis. The distributions of polypeptides within each of these fractions were analyzed by SDS gel electrophoresis and isoelectric focussing, followed by densitometric scanning of the gels. HM-alpha is detectable as early as the 14th week of gestation and its proportions increase rapidly, to about 9% of the total protein in the 1 year postnatal lens. The alpha-crystallin, IMW and LMW fractions show concomitant decreases and by 1 year they represent about 34, 35 and 18%, respectively. However, the proportions of IMW and LMW proteins do not accurately reflect those of the beta- and gamma-crystallins, as is often assumed. Substantial levels of non-crystallin polypeptides were found in the IMW protein fractions, including a group of very basic polypeptides (VBP) which comprised up to one-third of this material in the youngest lenses. Moreover, in postnatal lenses beta s-crystallin accounted for almost half of the LMW proteins. These points considered, alpha-crystallin is the major protein in the neonatal lens (approximately 42%, including HM-alpha), followed by the beta-crystallin (approximately 36% at most and probably less), the gamma-crystallins (approximately 11%) and beta s-crystallin (approximately 9%). Substantial changes in the proportions of specific polypeptides were observed throughout early development. These appear to result from changes at the level of protein synthesis and from postsynthetic modification. The A:B subunit ratio of alpha-crystallin drops from about 12 to below 3 during early development. This coincides with increasing levels of various deamidated and degraded subunits. The major beta-crystallin polypeptide also undergoes rapid deamidation and evidence is presented suggesting that the gamma-crystallins are subject to similar modification. The most dramatic changes were observed in the constituents of the LMW proteins. The synthesis of gamma-crystallins virtually ceases at some time around birth. At the same time, the levels of beta s-crystallin undergo an explosive increase. These and other changes are discussed in terms of their possible functional significance. They are also related to the complex protein status found in old lenses.

Crystallins↗

PubMed1985

Reconstructing normal alpha-crystallin from the modified cataractous protein.

alpha-Crystallin from human cataractous lenses is so extensively modified that it is unsuitable for structural studies. We have disassembled the protein and, from the remaining traces of unmodified polypeptides, reconstructed a more homogeneous and conformationally discrete molecule. We suggest that this approach may be particularly useful to studies of enzymology and molecular gerontology.

Cataract↗

PubMed1985

Monoclonal antibodies to bovine alpha-crystallin.

Monoclonal antibodies to bovine alpha-crystallin have been produced using hybridoma technology. Five selected hybridoma clones were maintained as ascites tumours in mice and gram quantities of the antibodies were purified from the ascites fluids by affinity chromatography on alpha m-crystallin covalently bound to Sepharose CL-2B. Preliminary characterization studies suggest that the antibodies are pure and monospecific. The antibodies could be divided into two groups on the basis of their reactivities towards iodinated alpha-crystallin, their ability to bind to Protein A-Sepharose, their immunoglobulin subclass, their immunoelectrophoretic patterns and their abilities to react with chicken and opossum alpha-crystallin. The availability of these monoclonal antibodies will greatly facilitate studies on the surface topography of alpha-crystallin.

Amino Acids↗

PubMed1984

On the structure of alpha m-crystallin. The reversibility of urea dissociation.

Fetal calf alpha-crystallins were denatured using various concentrations of urea and analyzed by gel filtration and ultracentrifugation. alpha c-Crystallin (18.4 S) irreversibly dissociates to alpha m-crystallin (11.8 S) at low urea concentrations. These findings substantiate our previous proposal that alpha c-crystallin is an artefactual aggregate produced by low isolation temperatures (Thomson, J.A., and Augusteyn, R.C. (1983) Exp. Eye Res. 37, 367-377). The sedimentation coefficient of alpha m-crystallin decreases steadily with increasing urea, due to a parallel change in the diffusion coefficient and not to a decrease in molecular weight. The aggregate dissociates directly to subunits, with no evidence for stable intermediate species. Complete dissociation, in 8 M urea, is accompanied by gross disruption of the secondary and tertiary structures. Following removal of the urea, the reassociated alpha r-crystallin is indistinguishable from native alpha m-crystallin in secondary, tertiary, and quaternary structure, as judged from the sedimentation and diffusion coefficients, subunit contents, near- and far-UV CD spectra, the microenvironments of cyteine and aromatic amino acids, and from their immunochemical properties. We have concluded that the native structure of alpha-crystallin can be completely recovered after dissociation and denaturation in urea.

Animals↗

PubMed1984

Pyridine nucleotides in normal and cataractous human lenses.

The levels of NAD+, NADH, NADP+ and NADPH have been determined in each cortex and nucleus of about 80 normal and cataractous human lenses and in a small number of calf, rabbit and rat lenses. Cataractous lenses had lower levels of all nucleotides than the normal lenses but no progressive decreases were associated with the development of nuclear colour, or with cortical, diabetic, traumatic and steroid cataracts. It was concluded that a lack of reduced pyridine nucleotides was not responsible for a decrease in the ability of the lens to protect itself against oxidative damage.

Aged↗