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R C Augusteyn

Publications and source records attributed to R C Augusteyn.

At least 73 records · Page 4Linked to original sources
PubMed1984

The purification and properties of human lens glutathione reductase.

A very simple and rapid method for the purification of human lens glutathione reductase has been developed. The method involves only two steps--affinity chromatography on 2',5'-ADP-Sepharose 4B and gel filtration on Sephacryl S-200. With whole lenses, the purification achieved is over 18000-fold and 80% of the activity in the tissue homogenate is recovered as an enzyme with a specific activity of 218 IU/mg-1. Glutathione reductase was purified from the nucleus and cortex of type I cataractous human lenses and the properties of the two enzymes were compared. No differences could be detected between these enzymes in their specific activities, molecular weights, pH-activity profiles, heat labilities, reactivity towards various substrates and kinetic parameters (Vmax and Km) for glutathione, NADPH2 and NADH2. Therefore, it was concluded that specific alterations in the properties of nuclear glutathione reductase were not responsible for the decreased ability of the lens nucleus to protect itself against oxidative insults. Several different glutathione reductase preparations were examined for their ability to cleave mixed disulphides of lens proteins and glutathione. Only crude tissue extracts (wheat germ and whole lens) were able to cleave the mixed disulphides: no activity was observed with purified lens or yeast glutathione reductases. Therefore, it was concluded that glutathione reductase does not cleave mixed disulphides.

Cataract↗
PubMed1984

The effects of hydrogen peroxide on lens proteins: a possible model for nuclear cataract.

Soluble proteins from the human lens nucleus were incubated for 12 weeks with 0, 10, 100 and 1000 microM-H2O2. Treatments were monitored for alterations in cysteine (sulphydryl), methionine, tryptophan and other amino acids; protein size, solubility and conformation; polypeptide sizes; and non-tryptophan fluorescence. Progressive changes were observed in several of these parameters. Cysteine (up to 100%) and methionine (up to 45%) were rapidly oxidized but no significant alterations were found in any other amino acids. No new chromophores were generated but the non-tryptophan fluorescence was enhanced three-fold. The gradually increasing solvent accessibility of tryptophan residues indicated that the proteins were undergoing conformational alterations. This was accompanied by the insolubilization of protein (up to 75%). The insoluble proteins consisted largely of covalently cross-linked polymers of the lens polypeptides. Disulphide bonds and dityrosine were shown not to be involved in the cross-linking. The modifications observed, as well as their order and extent, are very similar to those found in the cataractous lens. Our observations suggest that low concentrations of H2O2 may be responsible for the oxidative modification of lens proteins during the development of senile nuclear cataracts.

Amino Acids↗
PubMed1984

Conformational changes in soluble lens proteins during the development of senile nuclear cataract.

Soluble crystallins were isolated, by gel filtration, from the nucleus and cortex of types I-IV cataractous lenses and neonatal lenses. Extinction coefficients were determined and used to calculate the proportion of the crystallins. alpha Crystallins were the major proteins in the cortex of the cataractous lens whereas beta crystallins predominate in the nucleus and in the neonatal lens. Electrophoretic and immunological data, indicated that there was very little soluble gamma crystallin in the cataractous tissue. Highly specific competitive radioimmunoassays were used to estimate the crystallin contents of the various extracts. Values obtained for the neonatal extracts agreed closely with those from gel filtration but, in general, the values found for the cataractous proteins were low. The immunological reactivity of beta crystallin decreased progressively with the development of nuclear colour whereas alpha crystallin reactivity decreased sharply with the onset of nuclear cataract (type II lens) and then partially recovered. No significant alterations were found in the micro-environments of tryptophan residues in any of the proteins indicating that there are no gross conformational changes in the soluble proteins during cataract development. It would appear that the losses of immunochemical reactivity are due to chemical and/or conformational alterations which are restricted to the surface of the protein molecules.

Aged↗
PubMed1983

Immunochemical properties of vertebrate alpha-crystallins.

A competitive radioimmunoassay was used to determine the reactivities of alpha-crystallins from 13 species with antibodies directed toward calf alpha-crystallin. The results indicate that species as diverse as human and dogfish share the same number of crossreacting antigenic determinants. The various alpha-crystallins can be distinguished only on the basis of their differing affinities for the antiserum. Hydrophilicity profiles for alpha A and alpha B polypeptides of all species were found to be remarkably similar. On the basis of these, four major sequential determinants could be predicted for each polypeptide. The location and sequence of these determinants were found to be essentially conserved in all alpha-crystallins examined. These results are in agreement with the observed crossreactivities. However, there was little obvious correlation between substitutions in determinants and observed variations in respective alpha-crystallin/antibody affinities. Conservation of antigenic determinants over such a wide evolutionary range may reflect stringent constraints on the overall surface and three-dimensional structure of vertebrate alpha-crystallins.

Amino Acid Sequence↗
PubMed1983

Superoxide dismutase, catalase and glutathione peroxidase in the human cataractous lens.

The activities of the protective enzymes, superoxide dismutase, catalase and glutathione peroxidase have been measured in the cortical and nuclear sections of 76 human cataractous lenses as well as in calf, rabbit and rat lenses. No changes was observed in the activity of catalase with the progressive development of cataract. However, a precipitous decrease (70%) in both superoxide dismutase and glutathione peroxidase in the nuclear region of the lens was found at the onset of nuclear cataract. Further decreases accompanied the progression of the cataract and similar, but less marked, decreases were observed in the cortical region of the lens. It is suggested that the inactivation of these enzymes may result in an elevation of the H2O2 and O2.- levels in the lens and that this may be responsible for the oxidative modification of lens proteins observed in nuclear cataracts.

Aged↗
PubMed1983

alpha m-Crystallin: the native form of the protein?

Evidence is presented that alpha-crystallin isolated at 37 degrees C exists as a species, alpha m, which has a minimum molecular weight of about 320000 and a sedimentation coefficient of about 12 S. The amino acid composition, subunit distribution, near- and far-UV CD spectra and immunochemical properties were identical to those of the previously studied, 19 S protein, alpha c-crystallin (minimum molecular weight, 635000). It was demonstrated that only alpha m-crystallin was present in 37 degrees C lens extracts and that cooling of lenses or extracts resulted in a conversion of alpha m- to alpha c-crystallin. This conversion appears to be a general phenomenon, independent of age or species. It was concluded that alpha c-crystallin is an aggregate, produced by cooling, and that alpha m-crystallin is more likely to represent the in vivo form of the protein.

Amino Acids↗
PubMed1977

Oxidative changes in human lens proteins during senile nuclear cataract formation.

1. Proteins from the cortex and nucleus of the human lens were studied to determine if any changes could be detected in their amino acids during senile cataract formation. 2. Senile nuclear cataract formation was found to be accompanied by a progressive oxidation of cysteine and methionine. The oxidation of methionine and changes in the distribution of the nuclear proteins did not appear to start until about 60% of the cysteine had been oxidized. 3. In the advanced nuclear cataractous lens, about 90% of the cysteine has been oxidized and 45% of the methionine is present as the sulphoxide in the nuclear proteins. The levels of other amino acids appeared to remain constant. 4. Similar, but smaller, changes were found in the cortical proteins in advanced nuclear cataractous lenses, suggesting that the oxidation spreads from the nucleus to the cortex. 5. These changes were discussed with regard to current views on cataract formation and it was concluded that they are probably the result of simple oxidation of the proteins with O2 or H2O2.

Adult↗