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Biomedical subjects

A Spector

Publications and source records attributed to A Spector.

At least 73 records · Page 4Linked to original sources
PubMed1989

Raman spectroscopic evidence for a disulfide bridge in calf gamma II crystallin.

Laser Raman spectroscopy has been applied to native and dithiothreitol-treated bovine cortical gamma II crystallin to examine the state of the thiol groups and the presence of a putative disulfide bridge. The data reveal significant differences in two key spectral regions. In the thiol stretching region (2500-2600 cm-1), the dithiothreitol-reduced form shows a 25% increase in the integrated Raman signal as compared to the native form. The magnitude of this increase corresponds to the presence of 1 mol of disulfide/mol of gamma II as determined both by the Raman data and the previous biochemical analysis from this laboratory. In the disulfide stretching region (500-540 cm-1), the native form shows a line near 511 cm-1 which is absent in the reduced form. Both native and reduced forms show a triple-banded thiol signal with one or more distinct shoulders, suggesting at least three and perhaps five different environments for the cysteine residues. The difference spectrum, obtained by a 1:1 computer subtraction of the native from the reduced form, indicates that the increase in thiol signal is centered around 2572 cm-1. In every other spectral region, both native and reduced gamma II forms are closely similar. These results strongly support the biochemical data reported earlier and indicate that the reduction of the single disulfide bridge is accompanied by minimal changes in secondary structure in solution.

Animals↗
PubMed1989

UV laser photodamage to whole lenses.

Lenses from rat or calf were exposed in vitro to UV radiation from a nitrogen laser operated at 337.1 nm or from an excimer laser operated at 3.8 nm. Visible light transmission was monitored during calf lens irradiations at 308 nm and found to decrease. Proteins were extracted from the irradiated rat or calf lenses, separated into water soluble and insoluble fractions, and analysed using SDS-PAGE. Comparison of these gels with dark controls showed that, following photolysis, there was loss of polypeptide material in the 20-30 kDa region and concomitant formation of polymers at 40 and 60 kDa, and at greater than 100 kDa in calf lens (308 nm irradiation) and rat lenses (337.1 nm irradiation) in vitro. In addition, there was evidence for formation of lower molecular weight polypeptides at 10 kDa in the protein from irradiated rat lenses. The rat SDS-PAGE gels were challenged against anti-calf gamma crystallin serum. There was clear evidence that the polymeric material, in the water insoluble protein fraction from the 337.1 nm photolyzed rat lenses was derived in part from gamma crystallin. The macromolecular changes detected in these photolyzed rat and calf lens proteins were similar to those previously reported to accompany aging in the human lens. Biochemical changes of the type observed in UV irradiated rat and calf lenses may be responsible for the loss of visible light transmission seen in calf lenses.

Animals↗
PubMed1989

Repair of H2O2-induced DNA damage in bovine lens epithelial cell cultures.

H2O2 concentrations only slightly higher than normal physiological levels found in the lens and aqueous fluid produce a significant number of DNA single-strand breaks in lens epithelial cell cultures. In this investigation, the repair of DNA damaged by short-term, H2O2-induced oxidation was examined in bovine lens epithelial cell cultures. Repair was rapidly initiated and was almost completed in 30 min. A drop in NAD concentration was associated with the DNA damage. 3-Aminobenzamide inhibition of poly(ADP-ribose) polymerase, an enzyme believed to be stimulated by DNA oxidation and involved in DNA repair, prevented the loss of NAD. In contrast, a similar drop in ATP concentration was only slightly lessened by the presence of this inhibitor. Inhibition of the polymerase by 3-aminobenzamide primarily affected only the early recovery period. Overall, recovery occurred almost as effectively in the presence of the inhibitor as in its absence. Preincubation of lens cultures with o-phenanthroline, an iron chelator, prevented the drop in NAD levels associated with DNA damage. Since a hydroxyl radical is produced from H2O2 by a Fenton type reaction, this result supports the concept that the H2O2-induced oxidation of DNA is caused by hydroxyl radical. In contrast, peroxide-induced loss of activity of a cytosolic enzyme, glyceraldehyde-3-phosphate dehydrogenase, was unaffected by the presence of o-phenanthroline, suggesting direct H2O2 oxidation of this enzyme. The results of these experiments suggest that lens epithelium contains enzymes that rapidly repair single-strand DNA breaks induced by H2O2 insult.

Animals↗
PubMed1989

Differential synthesis and phosphorylation of the alpha-crystallin A and B chains during bovine lens fiber cell differentiation.

[14C]-amino acids and [32P]-orthophosphate incorporation experiments were carried out in bovine lenses in culture to study the synthesis and phosphorylation of alpha-crystallin A and B polypeptides during differentiation of the lens fiber cells. Following culture, the [14C] or [32P]-labelled alpha-crystallin was isolated by gel filtration chromatography from four regions of the lens corresponding to: A) quiescent epithelial cells, B) dividing epithelial cells and early stages of elongation, C) young elongating fibers, and D) mature fibers from the superficial cortex. The incorporation of label into the alpha-crystallin primary gene products alpha A2 and alpha B2 and their respective phosphorylated forms alpha A1 and alpha B1 was determined by isoelectric focusing and radioautography. Different synthesis and phosphorylation patterns were observed in alpha A and alpha B polypeptides. Synthesis and phosphorylation of the alpha B chain occurs most actively in the epithelial cells, both processes decrease during differentiation and there is no net accumulation of the phosphorylated form alpha B1 in the mature fiber cell. In contrast to the B chain, the A chain synthesis, minimal in the epithelial cell, increases with differentiation. Most striking, the A chain phosphorylation, not detectable in the epithelial cells, increases with differentiation. In the mature fiber cell, the phosphorylated form alpha A1 accounts for one third of the A chain. These observations indicate that the two chains may have different functions. the synthesis and phosphorylation patterns of alpha A suggest a lens-specific function of this polypeptide in the fiber cell and in the terminal differentiation process.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1989

Purification of a 43,000 dalton aggregate generated from alpha-crystallin.

A procedure is presented for the purification of an aggregate of covalently linked polypeptides of alpha-crystallin. Using either iron catalyzed oxidation or UV irradiation, 6-12% of calf lens alpha-crystallin can be converted to a 43,000 Da aggregate containing non-reducible cross-linked polypeptides as indicated by SDS-PAGE under reducing conditions. The 43,000 Da aggregate generated by Fe2+ oxidation can be isolated to approximately 85% homogeneity with respect to its relative molecular weight on SDS-PAGE. The procedure can be performed in two steps; 1) gel filtration of the oxidized alpha-crystallin under deaggregating and reducing conditions, and 2) preparative SDS-PAGE of the 43,000 Da aggregate-enriched peak obtained by filtration. The 43,000 Da aggregate band can be recovered from the polyacrylamide gels using a new preparative electroelution technique. 1.0 +/- 0.3 mg of purified 43,000 Da aggregate can be obtained from 100 mg of Fe2+ oxidized alpha-crystallin. The described methodology will facilitate further characterization of this 43,000 Da alpha-crystallin aggregate.

Animals↗
PubMed1989

The synthesis of glutathione peroxidase analogs.

While it is recognized that oxidation has a major role in the development of cataract, few compounds have been developed which effectively reduce the potential for oxidative insult. A major oxidant confronting the lens is H2O2. Few compounds capable of metabolizing H2O2 have been synthesized. This communication reports that a number of compounds with glutathione peroxidase activity have been developed. Some of these compounds are 10 fold more active than Ebselen, previously recognized as the synthetic compound with the greatest GSH peroxidase activity.

Azoles↗
PubMed1988

Fe2+ oxidation of alpha-crystallin produces a 43,000 Da aggregate composed of A and B chains cross-linked by non-reducible covalent bonds.

The structure of a 43,000 Da aggregate generated from bovine lens alpha-crystallin polypeptides of 20,000 Da using Fe2+ catalyzed oxidation, was studied by sequence analysis of a 30,000 Da proteolytic fragment. Three polypeptide components were simultaneously sequenced in the electroblotted 30,000 Da fragment, corresponding to Phe114-Ser130... of the alpha A chain, and His 111-Ser135... and Ser 35-Leu49... of the alpha B chain. The relative proportions of the components suggests that the three polypeptides are present in equimolar amounts. It is concluded that the 30,000 Da fragment and, therefore, the 43,000 Da aggregate is constructed of both the A and B polypeptide chains covalently cross-linked with non-reducible bonds. At least one of these cross-links is present towards the carboxy-terminus of the A and B chains after Phe114 and His111, respectively.

Amino Acid Sequence↗
PubMed1988

Phosphorylation of beta-crystallin B2 (beta Bp) in the bovine lens.

Three major 32P-labeled polypeptides were found in the soluble fraction of bovine lenses cultured in a medium containing [32P]orthophosphate. Two of the polypeptides corresponded to the phosphorylated A and B chains of alpha-crystallin. In this communication, the third polypeptide is now identified. This polypeptide is characterized by a molecular weight of 27,000 and a pI of 6.6, eluted exclusively in the beta Low fraction of a CL-6B gel filtration separation of lens soluble material, and could be further purified by DE52 anion exchange chromatography. The only 32P-labeled amino acid detected was phosphoserine. A single 32P-labeled peptide was observed after tryptic digestion and two-dimensional mapping. The amino acid sequence of the purified peptide is Gly-Ala-Phe-His-Pro-Ser-Ser. This sequence exactly matches the expected C-terminal tryptic fragment, residues 198-204, of the bovine beta-crystallin B2. The results of carboxypeptidase A digestion of the 32P-labeled peptide suggest that only Ser203 is phosphorylated. By using the catalytic subunit of the cAMP-dependent protein kinase, purified beta B2 was phosphorylated in vitro, generating a single 32P-labeled polypeptide with the identical pI as the phosphorylated polypeptide obtained from lens culture. On the basis of these data, the Mr 27,000 32P-labeled polypeptide is identified as the phosphorylated form of the beta-crystallin B2.

Animals↗
PubMed1988

The disulfide content of calf gamma-crystallin.

The disulfide content of calf gamma-crystallin polypeptides has been investigated. The gamma-crystallin fraction of the soluble lens proteins was separated into five distinct polypeptides and characterized by isoelectric focusing, amino acid composition, and N-terminal sequence analysis to 25 residues. It has been demonstrated that 7 cysteines are present in gamma II, 4 to 5 cysteines in gamma IIIa, gamma IIIb, and gamma IV, and 6 cysteines in gamma I (beta s). Reduction of the total gamma-crystallin fraction with DTT resulted in an increase of approximately 1 to 1.5 mol of free SH per mole of protein. This increase in sulfhydryls was demonstrated to be contributed primarily by gamma II, the major polypeptide representing 50% of the total gamma-crystallin, which showed an increase of approximately 2.5 mol of sulfhydryl per mole of protein upon reduction. Insignificant disulfide content was present in gamma III and gamma IV and only a slight amount of disulfide was found in gamma I (beta s). The observed increase in sulfhydryl content upon reduction was not due to the presence of mixed disulfides of 2-mercaptoethanol, glutathione, or cysteine. The data are consistent with approximately 1 mol of intramolecular disulfide per mole of protein being present in gamma II. X-ray crystallography of gamma II has shown that the spatial location of Cys18 and Cys22 in the tertiary structure permits disulfide bond formation. Sequence analysis of the four major polypeptides of gamma-crystallin, gamma II, gamma IIIa, gamma IIIb, and gamma IV indicates that only gamma II has both Cys18 and Cys22. Cys18 is present in gamma IIIa, gamma IIIb, and gamma IV but Cys22 is replaced by His22. It is probable that the lack of disulfide in gamma IIIa, gamma IIIb, and gamma IV is due to the absence of Cys22.

Amino Acid Sequence↗
PubMed1988

The effect of H2O2 upon thioredoxin-enriched lens epithelial cells.

Thioredoxin, a dithiol polypeptide, has been examined as a potential contributor to the recovery of lens epithelial cells from oxidative insult. It is reported that Escherichia coli thioredoxin can (a) effectively reduce lens-soluble protein disulfide bonds generated by H2O2, (b) restore to its initial activity H2O2-inactivated glyceraldehyde-3-phosphate dehydrogenase, (c) act as an effective source of reducing potential for lens methionine sulfoxide peptide reductase, and (d) act as a free radical quencher based on studies with a stable free radical system generated by ascorbic acid and 2,6-dimethoxy-p-benzoquinone. Thioredoxin is much more effective than dithiothreitol in restoring glyceraldehyde-3-phosphate dehydrogenase activity and as a cofactor for methionine sulfoxide peptide reductase. Upon incubation with epithelial cells, thioredoxin can be observed in the cell using rocket immunoelectrophoresis. These cells recover from H2O2 insult more rapidly than control cell preparations based upon 1) analyses of plasma membrane-related activities: leucine and 86Rb uptake and 2) analyses of parameters primarily related to the internal cell metabolism: ATP concentration and glyceraldehyde-3-phosphate dehydrogenase activity. Analysis of thioredoxin in cell preparations indicates that only about 9% is in the reduced state implying a low effective concentration of the polypeptide. The experiments suggest that low levels of thioredoxin may significantly increase the ability of lens epithelial cells to recover from exposure to H2O2.

Adenosine Triphosphate↗
PubMed1988

Thioredoxin fragment 31-36 is reduced by dihydrolipoamide and reduces oxidized protein.

The thioredoxin peptide Trp-Cys-Gly-Pro-Cys-Lys, which contains the redox active dithiol, was found to be reduced by lipoamide in a coupled reaction with lipoamide dehydrogenase and NADH. The reduced peptide in turn was shown to reduce insulin, oxidized lens protein and glyceraldehyde-3-phosphate dehydrogenase. While the peptide is not as effective a catalyst for utilizing pyridine nucleotides to reduce protein disulfides as thioredoxin, it offers a system which may be developed to provide more efficient disulfide reduction. This is particularly relevant since no thioredoxin peptides have been found to be active with thioredoxin reductase.

Bacterial Proteins↗
PubMed1988

Definition and comparison of the phosphorylation sites of the A and B chains of bovine alpha-crystallin.

The major phosphorylation sites of bovine alpha-crystallin Ser122 in the A chain, Ser59 and Ser43 and/or Ser45 in the B chain have been previously characterized. Further analysis of total alpha-crystallin, isolated from the cortex of calf lenses incubated in the presence of [32P]orthophosphate, demonstrated the presence of additional phosphorylation sites in both chains. At least three additional phosphorylation sites were found in the A chain and at least one in the B chain. These additional sites accounted for approximately 25% of the radioactivity incorporated in the protein. Two general sequences were found in most phosphorylation sites of both chains of alpha-crystallin: (Arg/Lys)-(X)-Pro-Ser and Ser-(X)-Ser-Leu-Ser. In spite of the 57% homology in the sequences of the A and B chains, the phosphorylation sites are located, in the A polypeptide, at the C-terminal third and in the B polypeptide, at the N-terminal third. The alignment of the regions containing the phosphorylation sites of both chains (C-terminal third of the A and N-terminal third of the B chain) revealed an unexpected similarity in the relative positions of the sites in each chain.

Amino Acid Sequence↗
PubMed1987

The phosphorylation sites of the B2 chain of bovine alpha-crystallin.

The B2 chain of bovine lens alpha-crystallin is phosphorylated in a cAMP-dependent reaction. By analysis of 32P-labelled chymotryptic peptides isolated from alpha-crystallin obtained from lenses labelled in organ culture, two phosphorylated B2 chain fragments were found. Sequence analysis of the fragments gave the following results: Arg-Ala-Pro-Ser-Trp-Ile-Asp-Thr-Gly-Leu and Ser-Leu-Ser-Pro-Phe corresponding to residues 56 to 65 and 43 to 47, respectively. It is established by this work that B1 is a phosphorylated post-translational product of B2. Both the A2 and B2 chains of alpha-crystallin are phosphorylated at a similar site with the sequence Arg-(X)-Pro-Ser. This is an unusual site for cAMP-phosphorylation since the phosphorylated serine is preceded by a proline residue. It may also be of significance that the other B2 chain phosphorylation site even more radically differs from previously reported cAMP-dependent phosphorylation sites.

Amino Acid Sequence↗
PubMed1987

The phosphorylation of the primary gene products of alpha-crystallin.

The alpha-crystallin primary gene product A2 and its post-translational modified counterpart A1 were isolated from calf lens cortex. The amino acid compositions determined from both chains were almost identical and in excellent agreement with that calculated from the reported sequence of A2. Chemical analysis of phosphate revealed 1 mol/mol of A1 and was negative in A2. Phosphoamino acid analysis demonstrated the presence of phosphoserine only in A1. Chymotryptic peptide maps of A2 and A1 resolved approximately 50 peptides and were strikingly similar. An apparent change in the relative mobility of one peptide was the only difference observed between A1 and A2. Phosphate analysis of this peptide obtained from A1 and A2 was positive only in the peptide from A1. Identical amino acid composition and the sequence Arg-Leu-Pro-Ser-Asn-Val-Asp-Gln-Ser-Ala-Leu was found for the peptide isolated from both chains, corresponding to residues 119 to 129 in the reported sequence of A2. These results indicate that the post-translational modification of A2 to A1 is the result of a phosphorylation reaction rather than a spontaneous nonenzymatic deamidation as previously suggested.

Amino Acid Sequence↗
PubMed1987

Age-dependent changes in the distribution and concentration of human lens cholesterol and phospholipids.

Analyses of total lipid in individual lenses 1.8-63 years of age indicate that both the cholesterol and the phospholipid concentrations have reached a high level of 10 and 14 micrograms/mg lens dry weight, respectively, after the first ten years of growth. Thereafter, the rate of phospholipid accumulation was greatly reduced to a value of 0.05 microgram/mg per year while that of cholesterol reduced to 0.19. Analyses of the distribution of lipid in successive lens fiber layers indicate that both the cholesterol and phospholipid levels increase in the entire lens between the age of 1.8 and 9 years. Older lenses showed a continuous increase in the accumulation of cholesterol in the deep cortical fibers, while little or no increase in phospholipid concentration was observed. These results indicate that the accumulation of lipids is greater than that of lens dry mass (protein) during the first decade of lens growth. Since more than 90% of lenticular lipids are associated with fiber cell membranes, these data suggest a gradual change in the differentiation of the newly formed secondary fibers from the epithelium during this period. Analyses of the phospholipid composition of the successive fiber fractions indicate that the major phospholipids of phosphatidyl ethanolamine (PE), phosphatidylserine (PS) and sphingomyelin maintained a uniform distribution in the 1.8- and 5-year-old lenses. While no change was observed with the cortical fibers, older lenses showed a gradual loss of PE and PS in the nuclear fiber up to 63 years of age. By the late teen years, nuclear PS can no longer be detected, while high levels of PE are maintained in lens nucleus. The disappearance of nuclear PE begins in the teen years and is completed by the age of 40. The decrease in PE and PS resulted in a continuous increase in the cholesterol/phospholipid ratio, a measure of membrane rigidity in the nuclear fiber in lenses 20 years of age and older. This decrease is also responsible for the exceedingly high rigidity of the nuclear fibers of lenses 60 years of age and older. Possible lamellar cholesterol organization in the lens fiber membrane is discussed.

Adolescent↗
PubMed1987

Pro-oxidant activation of ocular reductants. 1. Copper and riboflavin stimulate ascorbate oxidation causing lens epithelial cytotoxicity in vitro.

Ascorbic acid may not only be involved as an antioxidant in the aqueous humour and lens but under certain conditions may also undergo 'pro-oxidant conversion' leading to the generation of oxidants. It is demonstrated that the bovine aqueous humour contains electron spin resonance (ESR) detectable levels of ascorbyl semiquinone free radical associated with the transition metal-catalysed oxidation of ascorbate. Cu2+ was found to be a more efficient catalyst of ascorbate oxidation than Fe2+ and was similarly more potent in inhibiting 86Rb uptake of lens epithelial cells in the presence of ascorbate. The metal-chelating agent diethylenetriaminepentaacetic acid (DETAPAC) inhibited metal-catalysed ascorbate oxidation and lens epithelial cytotoxicity. Riboflavin in the presence of light stimulates the formation of ESR-detectable ascorbyl semiquinone radicals and stimulates O2 consumption (oxidation) by ascorbate in a manner dependent upon the concentration of riboflavin. It is proposed that photoexcited riboflavin can perform a one-electron oxidation of ascorbate generating a riboflavin radical. This radical may then autoxidize generating superoxide anion. The riboflavin-mediated photoimpairment of 86Rb uptake of bovine lens epithelial cells was found to be dependent upon the presence of ascorbate. This 'pro-oxidant' activation of ascorbate by riboflavin is discussed in the context of previous studies on riboflavin phototoxicology.

Animals↗