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Biomedical subjects

A Spector

Publications and source records attributed to A Spector.

At least 37 records · Page 2Linked to original sources
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

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↗
PubMed1987

Pro-oxidant activation of ocular reductants. 2. Lens epithelial cell cytotoxicity of a dietary quinone is associated with a stable free radical formed with glutathione in vitro.

The ability of 2,6-dimethoxyquinone (DMQ) to impair 86Rb uptake by bovine lens epithelial cells was found to be independent of exogenous ascorbate in contrast to the impairment induced by Fe/Cu or riboflavin plus light. The cytotoxicity was associated with an electron spin resonance (ESR) detectable singlet radical (g = 2.0062) which also formed on incubation of DMQ with glutathione (GSH) or gamma-crystallin in vitro. Formation of the stable free radical appeared to require conjugation of DMQ by peptidyl thiol and required transition metal catalysis. A structure for the DMQ-glutathione free radical conjugate is proposed. Redox activity of quinone conjugates is suggested to be of relevance to an oxidative damage hypothesis of cataract.

Animals↗
PubMed1987

Does elevated glutathione protect the cell from H2O2 insult?

Utilizing glutathione ethyl ester (GSH-EE), the glutathione (GSH) level of lens epithelial cells can be increased as much as 1.9-fold. The epithelial cells maintain the additional GSH in the reduced form. This system was utilized to examine the relative effectiveness of cells with elevated GSH to withstand H2O2 insult. Three parameters were investigated, 86Rb accumulation, a measure of membrane function, ATP levels, an indication of overall metabolism and glyceraldehyde-3-phosphate dehydrogenase (GPD) activity, indicating intracellular enzyme susceptibility to oxidative insult. Under oxidative stress, much of the GSH is in the oxidized form but upon removal of the stress, rapidly returns to the reduced state. However, a loss of approximately 20% in GSH equilibrium levels has been consistently observed. Elevated GSH does not significantly increase the cells' ability to withstand or recover from oxidative stress. Indeed, elevated GSH was found to be somewhat deleterious, causing a decreased ability to recover from oxidative insult. However, in the case of GPD, a significant protection of activity was observed. The overall conclusion is that elevating intracellular GSH concentration does not increase the cells' overall ability to withstand oxidative damage.

Adenosine Triphosphate↗
PubMed1987

Stimulation of glucosylated lens epithelial Na,K-ATPase by an aldose reductase inhibitor.

In diabetes, glucosylation of the Na,K-ATPase of the lens epithelium makes the pump inefficient. K+ transport and ATP hydrolysis (at near saturating ATP concentrations) are inhibited and the kinetics of ATP hydrolysis become substrate inhibition type. The AR inhibitor (AL1576, Alcon Laboratories) stimulates K+ transport and ATP hydrolysis by glucosylated bovine lens Na,K-ATPase. This inhibitor has a slight stimulatory effect upon the unmodified enzyme function also. The AR inhibitor is not able to prevent glucosylation of the pump in high-glucose-containing medium.

Adenosine Triphosphate↗
PubMed1987

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.

Aldehyde Reductase↗
PubMed1987

Identification of the specific phosphorylated serine in the bovine alpha crystallin A1 chain.

Previous work (1,2,3) has indicated that the in vivo post-translational modification of the alpha crystallin primary gene product A2 is due to a specific phosphorylation process involving a serine residue located in a chymotryptic fragment with the sequence ARG-LEU-PRO-SER-ASN-VAL-ASP-GLN-SER-ALA-LEU which corresponds to the residues 119 to 129 of the polypeptide chain. To define which of the two serines is phosphorylated, the present experiments were carried out. The 32P-labeled chymotryptic fragment was obtained from alpha crystallin isolated from the outer cortex of calf lenses incubated in the presence of [32P]-orthophosphate. By analyses of the products obtained after Edman degradation, utilizing electrophoresis in cellulose TLC plates and radioautography, it was possible to locate the phosphate in the serine residue at position 122 in the polypeptide chain. No phosphate could be detected in the serine residue at position 127.

Amino Acid Sequence↗
PubMed1986

ATP hydrolysis kinetics of 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.

Adenosine Triphosphate↗
PubMed1986

Sodium-23 magnetic resonance imaging of the eye and lens.

In order to develop a better understanding of cataract and to evaluate the effectiveness of potential drugs, noninvasive techniques must be devised to detect early metabolic changes. As a prelude to these goals, sodium-23 imaging experiments operating at 29.8 MHz (2.7 teslas) were performed on the bovine eye and lens. A spatially localized transverse relaxation time (T2)-weighted spin-density map of the sodium-23 within the lens is presented, with a resolution better than 250 micron. Due to the presence of short-T2 (3 msec) components within the lens, only the use of the planar-integral projection reconstruction (PPR) imaging scheme allowed sufficiently short echo-times (1 msec) to permit sodium-23 signal detection. These noninvasive imaging results show differences in the apparent sodium concentration within the lens that are consistent with separate, invasive measurements of sodium concentration. Separate analysis (with no spatial localization) at 79.4 MHz (7.2 teslas), using a shift reagent (dysprosium) to distinguish extracellular from intracellular sodium, indicates that approximately 62% of the detected sodium-23 signal is intracellular. These results are consistent with observations based on invasive measurements and further support the existence of the pump-leak system and a sodium gradient within the lens.

Animals↗