Margaret Garner

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.

Sulfur oxidation in selected human cortical cataracts and nuclear cataracts

The water soluble protein, the water insoluble non-membrane protein, and the membrane fraction were isolated from both the cortex and nucleus of nuclear and cortical cataracts. Measurements of cysteine and methionine oxidation in these fractions indicated that sulfur oxidation occurs in the transparent as well as the opaque region of the lenses. Cysteic acid is found only in the opaque region. In the nucleus of nuclear cataracts, extrinsic and cytoplasmic polypeptides are found covalently linked by disulfide bonds to the intrinsic membrane. In the cortex of cortical cataracts, there is no intrinsic membrane involvement. However, a high molecular weight disulfide linked aggregate can be isolated from the cortex of cortical cataracts. This aggregate appears to contain the same extrinsic and cytoplasmic proteins as are found covalently attached to the membrane of the nuclear cataracts.

ATP hydrolysis kinetics by Na,K-ATPase in cataract

The steady-state kinetics of hydrolysis of Mg2+ ATP by the epithelial Na,K-ATPase of individual human lenses were determined. Among the cataract lens population, four distinct kinetic types were observed: negative kinetic co-operativity. Michaelis-Menten kinetics, positive kinetic co-operativity, and substrate inhibition kinetics. Negative kinetic co-operativity and Michaelis-Menten kinetics were also observed in a group of presumably clear lenses from non-diabetic individuals ages 16-42 years. Substrate inhibition kinetics were found to be prevalent in individuals with mature onset diabetes. Substrate inhibition kinetics were also observed for Na,K-ATPase isolated from lenses which had been incubated in high glucose. It would appear that this modification leads to an inhibition of Na,K-ATPase-dependent K+ influx into these cultured lenses.

Investigation of Nakano lens proteins.

Abstract Changes in the protein chemistry of the Nakano lens with age and developing cataract and comparison with normal mouse lens protein are reported. It was found that significant differences exist between the protein of the normal and the cataractous lens. In Nakano lenses high molecular weight disulfide-linked aggregates, disulfide-linked cytosol polypeptides to the fiber membrane, an apparent increase in the concentration of degraded polypeptides, disulfide crosslinking of low molecular weight species and marked differences in membrane polypeptide profiles were observed. A striking similarity was found between these observations with the Nakano cataract and previous reports of the changes in protein chemistry in the development of senile human cataract. It can be concluded that although the initiating event for induction of the cataract may differ, the sequence of events following such insult may be similar.

Direct Stimulation of Na+-K+-ATPase and Its Glucosylated Derivative By Aldose Reductase Inhibitor

In the presence of 10−8 M concentrations of the aldose reductase inhibitor AL 1576, there is a 20-30% increase in the rate of hydrolysis of near-saturating concentrations of ATP by bovine renal Na+-K+-ATPase. When bovine renal Na+-K+-ATPase is reacted with glucose 6-phosphate in the presence of 10−8 M concentrations of AL 1576 or 10−6 M concentrations of a second aldose reductase inhibitor, sorbinil, glucosylation occurs. Whereas sorbinil has no effect on ATP hydrolysis by the glucosylated Na+-K+-ATPase, 10−8 M AL 1576 causes a shift in the kinetics of hydrolysis of ATP from substrate inhibition to normal substrate activation. The aldose reductase inhibitors interact with the enzyme at the low-affinity ATP-binding site.

The photolysis of lens protein: molecular changes.

The photolysis of lens protein leads to the destruction of a number of amino acids. In addition to tryptophan; methionine, histidine and cysteine are destroyed upon irradiation. The latter three amino acids are not photolyzed directly but are degraded after an initial absorption of light by tryptophan. At least part of this mediated damage is probably due to singlet oxygen. The photo-destruction of tryptophan was found to be concentration dependent. As the concentration of lens proteins increases, the photolability decreases.

Na+-K+-ATPase and Changes in ATP Hydrolysis, Monovalent Cation Affinity, and K+ Occlusion in Diabetic and Galactosemic Rats

This study showed that steady-state kinetics of ATP hydrolysis by Na+-K+-ATPase are altered in the BB Wistar diabetic rat and experimental galactosemia. Four days after onset, this change was not evident if NaCNBH3 was omitted during enzyme preparations (indicating reversibility). Ninety days after onset, NaCNBH3 reduction was not necessary to see the change in ATP hydrolysis kinetics (indicating nonreversibility). The change in steady-state ATP hydrolysis was similar to that reported earlier for Na+-K+-ATPase of the lens epithelium and kidney medulla of diabetic individuals and for two in vitro glycosylation models. Our study also showed that the affinities of Na+-K+-ATPase for K+ are altered, and Na+-K+-ATPase-dependent K+ occlusion is inhibited in diabetic and galactosemic animals. Because K+ occlusion is required for efficient K+ transport, this finding supports previous in vitro studies that indicated that glycosylation inhibits pump-dependent K+ transport. Furthermore, our study suggested an irreversible impairment of Na+-K+-ATPase function in the diabetic BB Wistar rat as early as 15 days after onset, even when blood glucose was maintained at 6.7 mM by daily insulin injection.

Immunochemical characterization of the main intrinsic proteins of the human lens membrane

Using antibodies to both the 26000 dalton and 22000 dalton fractions of the human lens membrane, it is shown that in the normal aging process there is cleavage of the 26000 dalton chain to products with molecular weights of 22000 and 16000 daltons. Furthermore, the human lens membrane contains at least one other 22000 dalton chain which is immunochemically unrelated to the 26000 dalton component.

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.