Debdutta Roy

Isolation and characterization of an age-dependent polypeptide from human lens with non-tryptophan fluorescence

With aging of the human lens there is an increase in insoluble protein which occurs primarily in the nuclear region. The character of this material changes with age from a white precipitate isolated from young lenses to a predominantly heavy yellow material obtained from older tissue. An increase in protein-bound non-tryptophan fluorescence also occurs with aging. This fluorescence has a corrected absorption maximum of 325 nm and a corrected emission maximum of 415 nm. The fluorescence is primarily associated with the insoluble yellow protein of the nuclear region, particularly in older lenses. It is also found in the soluble proteins where it is predominately in the high molecular weight components. In both the soluble and insoluble fluorescence containing protein fractions this fluorescence could be detected only in a 43 000 dalton polypeptide. This polypeptide probably contains two C-terminal residues but no N-terminal groups. Ba(OH)2 or proteolytic hydrolysis of some of the fluorescent proteins produces a low molecular weight fluorescent component containing equimolar amounts of glycine and proline but no N-terminal groups or other amino acids. It is therefore concluded that the 43 000 dalton polypeptide is composed of two smaller polypeptides linked by a fluorescent component via their N-terminal groups, glycine and proline.

Human insoluble lens protein II. Isolation and characterization of a 9600 dalton polypeptide

Abstract A polypeptide fraction of 9600 daltons has been isolated from human insoluble protein. Although this fraction is homogeneous in size, it is heterogeneous in charge. The isolated fraction self aggregates to soluble macromolecules of approximately 4 × 105 daltons indicating that it requires interaction with other polypeptides to form the insoluble protein. By DE52 chromatography this polypeptide fraction can be separated into 12 fractions. They have very similar amino acid compositions, blocked amino termini and the same carboxy terminal residues. The unfractionated material and most of the isolated fractions have a high non-tryptophan fluorescence ( 320 405 nm , uncorrected). Sequence analyses of tryptic peptides isolated from this material indicate that the major portion of the polypeptides including residues 1–70 is identical to the amino terminal sequence of the alpha crystallin A chain. The remaining sequence representing approximately 1200 daltons remains to be determined. However, no other A chain peptide has been isolated.

Sodium/potassium ATPase in normal and cataractous human lenses

Na/K ATPase activity has been determined in normal and cataractous human lenses using labeled ATP as a substrate. The enzyme is distributed throughout the lens with approximately 1/3 of the total activity in the capsule-epithelium and 1/2 of the activity in the cortex. Furthermore, the activity of Na/K ATPase decreases with increasing age and this decrease occurs primarily in the inner nuclear region. In severe cataractous lenses, a marked decrease in the Na/K ATPase activity has been demonstrated in all parts of the lens, in contrast to immature cataracts, where the decrease in the enzyme activity occurs primarily in the cortical and nuclear regions.

High molecular weight protein from human lenses

Abstract A new rapid method based upon differential centrifugation is described for the isolation of high molecular weight (HMW) protein from human lenses. Similar material is obtained with this method or by gel filtration. It was found that the HMW protein is almost identical in composition to the insoluble protein and is probably an intermediate in the formation of the insoluble fraction. There appears to be a spectrum of increasingly large components ranging from the relatively small HMW species to the largest particles in the insoluble fraction. Analyses of the polypeptides composing these proteins indicate the presence of 11 000, 20 000, 22 000, 27 000 and 45 000 dalton components. It is concluded that the human HMW and insoluble proteins are composed of a mixture of lens proteins as well as a major fraction consisting of smaller degraded polypeptides.

Changes in lens protein in concentric fractions from individual normal human lenses

The water soluble (WS), urea soluble (US) and urea insoluble (UI) fractions from individual human lenses 1.8 to 65 years of age were isolated from concentric fiber layers. In lenses younger than 19 years, a uniform distribution in the amount of WS, US and UI fractions was found throughout the entire lens. These fractions represent 83, 11.5 and 5.5%, respectively, of the lens dry weight. This composition was observed with the cortical fibers of all lenses examined up to the 65-year old. In the nuclear fiber layers, the proportion of US protein gradually increases in the third to fourth decade of lens growth and appears to have reached a maximum representing 22-24% of the nuclear fiber mass in 50-year and older lenses. A large increase in the amount of the UI fraction to 30% of the fiber mass was observed in lenses between the 5th and 6th decade of lens growth. The change from the cortical to nuclear composition occurs in a narrow region of the lens which becomes more peripheral with aging. The cortical WS fractions were characterized by well defined polyacrylamide gel bands in sodium dodecyl sulfate (SDS). Those of the nuclear fibers were broadened, especially in the 27/29 and 16/18 kilodalton (KD) region. The disappearance of the 20/22 KD bands in the inner cortical and nuclear fibers cannot be accounted for by the small increase in protein insolubilization in these regions of lenses 40 years or younger.

Human insoluble lens protein I. Separation and partial characterization of polypeptides

Abstract A number of new methods are described for fractionating the polypeptides of the water insoluble protein from human lenses. In most cases, three major fractions can be obtained: a high molecular weight (HMW) polypeptide fraction containing components greater than 100 000 daltons plus a 43 000 dalton fraction, an intermediate sized fraction containing components from 27 000 to 20 000 daltons and a low molecular weight fraction containing an 11 000 dalton component. Under certain conditions, the 11 000, 43 000 and > 100 000 dalton fraction can be obtained pure on the basis of SDS gel electrophoresis. Marked charge heterogeneity was observed in all samples investigated. The HMW polypeptide fraction is the major component containing 50% of the H 2 O insoluble protein with 30% represented by the 27 000–20 000 dalton fraction and 20% by the 11 000 dalton component. Investigation of the urea soluble and urea insoluble components of the water insoluble fraction indicates a close similarity in chemical and physical characteristics. On the basis of similarity in chemical and physical properties and increasing insolubility, it is proposed that a precursor relationship may exist where by HMW protein → urea soluble → urea insoluble protein. The HMW polypeptide fraction may not be stabilized by intrachain disulfide bonds. Furthermore, the marked susceptibility of this fraction to tryptic digestion where essentially only low molecular weight components rapidly appear, suggests that there may be few if any non-reducible crosslinks stabilizing the overall HMW polypeptide structure. The absence of any free NH 2 -terminal residue in the H 2 O insoluble fraction suggests that unmodified γ-crystallin may not be present.

Lens plasma membrane: Isolation and biochemical characterization

Lens plasma membrane from different animal lenses has been prepared by the acylation procedure. Using three different criteria: heat aggregation of intrinsic membrane polypeptides, immunochemistry and solubilization of intrinsic proteins at low (40 mM) LIS concentration, it has been shown that these preparations are essentially free of cytoplasmic contaminants. Using the results obtained with acylated membrane as the reference of purity, it has been shown that both sucrose gradient centrifugation of bovine WI protein and urea washing of old human lenses give impure membrane preparations. The main intrinsic polypeptides (mol. wt 26 000 and 22 000) of human lens membrane have been purified and characterized. It has been shown by enzymatic digestion and amino terminal analysis of the residual membrane-bound fragments that the amino terminal halves of the polypeptides are embedded in the lipid bilayer and are probably blocked at their amino terminal sites. Lipid analyses of human and bovine lens membranes suggest that the protein to total lipid ratio is 1:1. Carbohydrate analyses of chromatographically separated intrinsic membrane polypeptides indicate the presence of 1 mol glucose/1 mol protein.

Age dependent changes in the abundance of the major polypeptides of human lens membrane

Abstract Changes in the abundance of the major polypeptides of human lens membrane with aging and cataractogenesis have been studied. It has been shown that with aging the relative amounts of the two major membrane polypeptides, 26,000 and 22,000 dalton components, change up to the age of approximately 40. Thereafter the relative abundance of these two polypeptides remains constant. Explanations for this alteration are discussed in relation to similar age dependent changes of the proteins of the human lens.

Lens calcium activated proteinase: Degradation of vimentin

The lens has been shown to contain a Ca+2 activated proteinase specific for vimentin. The proteinase is present in the soluble fraction of the cortex but not in the epithelium. It is suggested that this proteinase is expressed during terminal differentiation of the epithelial cells and may be responsible for degradation of the intermediate filaments in the fiber cells. The proteinase is inhibited by EGTA but not by several proteinase inhibitors.

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.