William H. Garner

Glucosylation of human lens protein and cataractogenesis

Abstract Examination of glucosylation of lens protein was conducted utilizing tritiated BH4−. The overall results indicate that approximately 0.20 moles of tritium were incorporated per mole of protein. Similar results were obtained with normal and senile cataractous lenses with varying degrees of opacity. Furthermore no difference in the 3H incorporation was observed between soluble and insoluble protein fractions derived from these lenses. Investigation of selected polypeptides isolated from the senile cataracts gave comparable results. Protein isolated from diabetic lenses had only slightly higher levels of tritium incorporation, giving an average value of 0.27 moles per mole of protein. Analyses of the tritiated products indicate that approximately 50% of the incorporation is probably due to reduction of other types of compounds. These results suggest that glucosylation does not appear to be a primary factor in cataract formation.

Gamma-crystallin, a major cytoplasmic polypeptide disulfide linked to membrane proteins in human cataract

Abstract Examination of human cataract has revealed the presence of a number of unique complexes containing cytosol and membrane components: the high molecular weight disulfide linked aggregates and membrane preparations containing disulfide linked cytosol polypeptides. It is now shown that a major cytosol species associated with these complexes is gamma-crystallin. This conclusion is based upon investigation of polypeptides released by reduction and comparisons based on amino acid, immunochemical and sequence analyses. It is suggested that two types of complexes may be closely related.

Comparison of the 10 000 and 43 000 dalton polypeptide populations isolated from the water soluble and insoluble fractions of human cataractous lenses

Abstract The 10 000 and 43 000 dalton polypeptides from pooled human cataractous lenses were isolated from the water soluble fraction and were compared with the respective polypeptides isolated from the water insoluble fraction. The water soluble proteins were first separated with Sephadex G-200 under non-deaggregating conditions. The 10 000 dalton polypeptide was found principally in the last major protein component eluting from the column. The 43 000 dalton polypeptide was found apparently complexed non-covalently primarily within three aggregate populations ranging in size from 4 to 15 × 10 4 daltons. The 43 000 as well as the 10 000 dalton polypeptides from the G-200 column were purified in acetic acid-urea on Sephadex G-100 and G-150, respectively. The 10 000 dalton polypeptide can also be purified after the initial column fractionation without dissociation. The polypeptides from the water insoluble fraction were isolated by methodologies previously reported. Characterization of the 10 000 and 43 000 dalton polypeptides isolated from the water soluble and insoluble fractions indicated marked similarities. However, with both polypeptide populations the water insoluble component contained a greater negative charge, lower abundance of certain amino acids (e.g. tryptophan, tyrosine, methionine), and a greater atypical fluorescence. Immunochemical data indicates that the 10 000 dalton population from both soluble and insoluble fractions contains components arising from a number of different polypeptides including soluble crystallins and the extrinsic 43 000 dalton polypeptide. It was found that there is complete immunochemical identity between the soluble 43 000 dalton polypeptide and its counterpart in the disulfide high molecular weight species and the species isolated from the urea extracted nonreduced water insoluble fraction. However, the urea extracted reduced water insoluble fraction reacted only to the extent of 30% with the anti-water soluble 43 000 dalton polypeptide suggesting at least one other 43 000 dalton component which is present only in the water insoluble fraction.

Evolution of the amino acid substitution in the mammalian myoglobin gene

SummaryMultivariate statistical analyses were applied to 16 physical and chemical properties of amino acids. Four of these properties; volume, polarity, isoelectric point (charge), and hydrophobicity were found to explain adequately 96% of the total variance of amino acid attributes. Using these four quantitative measures of amino acid properties, a structural discriminate function in the form of a weighted difference sum of squares equation was developed. The discriminate function is weighted by the location of each particular residue within a given tertiary structure and yields a numerical discriminate or difference value for the replacement of these residues by different amino acids. This resulting discriminate value represents an expression of the perturbation in the local positional environment of a protein when an amino acid substitution occurs. With the use of this structural discriminate function, a residue by residue comparison of the known mammalian myoglobin sequences was carried out in an attempt to elucidate the positions of possible deviations from the known tertiary structure of sperm whale myoglobin. Only 11 of the 153 residue positions in myoglobin demonstrated possible structural deviations. From this analysis, indices of difference were calculated for all amino acid exchanges between the various myoglobins. All comparisons yielded indices of difference that were considerably lower than would be expected if mutations had been fixed at random, even if the organization of the genetic code is taken into consideration. On the basis of these results, it is inferred that some form of selection has acted in the evolution of mammalian myoglobins to favor amino acid substitutions that are compatible with the retention of the original conformation of the protein.

A Preliminary Study of the Dynamic Aspects of Age Dependent Changes in the Abundances of Human Lens Polypeptides

Growth of normal human lenses is maintained at a relatively constant rate of 0.4 mg per year after the first years of life. Water insoluble material increases at a similar constant rate, while the amount of water soluble components remains constant. Early growth stages’ are reflected by significant changes in relative abundance of all the major subunits in these fractions. However, from approximately age 40, the relative concentrations of the different subunits of lens proteins are fairly constant, except for a continued marked decrease in the 20,000 and increase in the 10,000 dalton water insoluble components. The relative stability in the concentrations of the polypeptides suggests that the rates of synthesis are comparable to the rates of degradation and/or insolubilization. The 10,000 dalton polypeptide is the result of degradation, probably of both soluble and insoluble 20,000 dalton components, which appears to be more rapid in the water insoluble fraction.

The Localization of a 43K Polypeptide in Normal and Cataractous Lenses by Immunofluorescence

Abstract The distribution of the 43K polypeptide within the human lens has been examined by immunofluorescence. The results indioate that in the mature lens fiber this polypeptide is located in the region of the inner surface of the fiber cell membrane. It is not present in the epithelial cell but is found throughout the developing fiber cell. In fetal lenses it is located throughout the fiber cytoplasm in the first trimester but by six months the distribution of the polypeptide is similar to that found in the adult. In cataractous lenses there are amorphous areas devoid of 43K and globules containing 43K throughout their structure. The study indicates that immunofluorescence provides a valuable tool for examining cataract and the distribution of lens polypeptides. The location of the 43K in the mature fiber cell at the intracellular membrane surface suggests that it may be involved in some aspect of membrane biology.

Validity and Reproducibility of the Cooperative Cataract Research Group (CCRG) Cataract Classification System

The validity and reproducibility with which six classifiers [one experienced (L.T.C.), and five novices (W.G., F.G., W.W., J.W. and O.W.)] used the CCRG cataract classification system was assessed. The validity of index classifications was assessed by computing sensitivities and pairwise interclass correlations between experienced and novice classifiers using the formers classification as the standard. The number of unordered combinations of terms in the CCRGs classification was reduced by combining cortical terms according to the CCRGs accepted system of staged simplification. The number of combinations of terms at each stage is as follows: Stage I (greater than 1000); II (127); III (63); IV (15); V (7); VI and VII (3) and VIII (2). Excellent agreement was obtained between the experienced and novice classifiers for Stages VII and VIII of the classification, good agreement for Stages V and VI and poor agreement for Stages IV, III and II (sensitivities of 97, 96, 72, 59, 40, 24 and 20% respectively). Good agreement was also achieved for the classifications of single lenticular regions, except for subcapsular regions. The intra- and interobserver reproducibility was assessed by computing the Kappa statistic to (1) compare classifications between novice observers and (2) compare repeat classifications made by the same observer by viewing the same cataract once on each of three different days. The novice classifiers had excellent intraobserver reproducibility for Stages VII and VIII (Kappas of 0.87 and 0.97 respectively), good reproducibility for Stages IV, V and VI (Kappas of 0.53, 0.62 and 0.62, respectively) and marginal reproducibility for stages II and III (Kappas of 0.39 and 0.40, respectively). The intraobserver reproducibility of the experienced classifier was superior to the others for virtually all characteristics with excellent reproducibility for Stages IV, V, VI, VII and VIII with Kappas of 0.79, 0.90, 1.0, 1.0 and 1.0, respectively and good reproducibility for Stages II and III (Kappas of 0.55 and 0.64, respectively). These results indicate that the simplified CCRG cataract classification system (Stages IV-VIII) passes the minimum standards for reproducibility. The performance of the experienced classifier far exceeds the minimum standards and indicates the feasibility of improving classifier performance with training and practice.

Protein Disulfide Levels and Lens Elasticity Modulation: Applications for Presbyopia

Purpose The purpose of the experiments described here was to determine the effects of lipoic acid (LA)-dependent disulfide reduction on mouse lens elasticity, to synthesize the choline ester of LA (LACE), and to characterize the effects of topical ocular doses of LACE on mouse lens elasticity. Methods Eight-month-old mouse lenses (C57BL/6J) were incubated for 12 hours in medium supplemented with selected levels (0–500 μM) of LA. Lens elasticity was measured using the coverslip method. After the elasticity measurements, P-SH and PSSP levels were determined in homogenates by differential alkylation before and after alkylation. Choline ester of LA was synthesized and characterized by mass spectrometry and HPLC. Eight-month-old C57BL/6J mice were treated with 2.5 μL of a formulation of 5% LACE three times per day at 8-hour intervals in the right eye (OD) for 5 weeks. After the final treatment, lenses were removed and placed in a cuvette containing buffer. Elasticity was determined with a computer-controlled instrument that provided Z-stage upward movements in 1-μm increments with concomitant force measurements with a Harvard Apparatus F10 isometric force transducer. The elasticity of lenses from 8-week-old C57BL/6J mice was determined for comparison. Results Lipoic acid treatment led to a concentration-dependent decrease in lens protein disulfides concurrent with an increase in lens elasticity. The structure and purity of newly synthesized LACE was confirmed. Aqueous humor concentrations of LA were higher in eyes of mice following topical ocular treatment with LACE than in mice following topical ocular treatment with LA. The lenses of the treated eyes of the old mice were more elastic than the lenses of untreated eyes (i.e., the relative force required for similar Z displacements was higher in the lenses of untreated eyes). In most instances, the lenses of the treated eyes were even more elastic than the lenses of the 8-week-old mice. Conclusions As the elasticity of the human lens decreases with age, humans lose the ability to accommodate. The results, briefly described in this abstract, suggest a topical ocular treatment to increase lens elasticity through reduction of disulfides to restore accommodative amplitude.