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Biomedical subjects

A Spector

Publications and source records attributed to A Spector.

At least 91 records · Page 5Linked to original sources
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↗
PubMed1986

A new HPLC method to determine glutathione-protein mixed disulfide.

A sensitive method for measuring glutathione-protein mixed disulfides is described. The method is based on cleavage of the protein disulfides with performic acid followed by reaction with dinitrofluorobenzene and HPLC analysis with a Bondapak-amine column. Samples containing 0.1 nmoles or more of glutathione-protein mixed disulfide can be detected. The method has been used to demonstrate (a) the presence of low levels of glutathione mixed disulfide in gamma crystallin isolated from bovine lenses, (b) a dramatic increase in such mixed disulfides after exposure of denatured gamma crystallin to O2 in the presence of glutathione, and (c) the formation of glutathione-protein mixed disulfide in lens epithelial cells exposed to 0.6 mM H2O2 for one hour.

Animals↗
PubMed1986

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.

Adult↗
PubMed1986

H2O2-modification of Na,K-ATPase. Alterations in external Na+ and K+ stimulation of K+ influx.

Studies, at steady state, of the Na,K-ATPase dependent influx of K+ into bovine lenses in organ culture are used to characterize further the H2O2-modification of the Na+ pump. Control lenses display constants for interaction with external Na+ and K+ similar to those obtained for the erythrocyte. H2O2 treatment of the bovine lens leads to total loss of external Na+ stimulation and alteration of external K+ stimulation.

Adenosine Triphosphate↗
PubMed1985

Glucose-6-phosphate modification of bovine renal Na,K-ATPase: a model for changes occurring in the human renal medulla in diabetes.

The kinetics of hydrolysis of ATP were determined for the renal Na,K-ATPase, in the K+ conformation, modified with glucose-6-phosphate. There was a shift in the ATP hydrolysis kinetics from negative kinetic co-operativity for the control enzyme preparations to substrate inhibition kinetics for the modified enzyme preparations. The effect was reversible and stabilized after NaBH4 reduction. Approximately 4 moles of glucose-6-phosphate were incorporated per mole of Na,K-ATPase (based on MW of 150,000 daltons). Similar substrate inhibition kinetics were observed for the renal Na,K-ATPase isolated from several human subjects with mature onset diabetes.

Adenosine Triphosphate↗
PubMed1985

The photolysis of lens fiber membranes.

Calf lens fiber membranes were photolyzed in the presence and absence of sensitizers and scavengers. Photolytic damage was assessed by SDS-polyacrylamide gel electrophoresis, UV and fluorescence spectra and amino acid analyses. With irradiation, there is an apparent polymerization of the major membrane polypeptide (MP26) and the formation of material which does not enter SDS-polyacrylamide gels. Some degradation was also observed. These changes are accompanied by losses of histidine and tryptophan and changes in the UV spectra. The rate of photolysis is enhanced in the presence of the glucoside of 3-hydroxykynurenine (3-OH-KYN), a compound endogenous to the lens. The reaction is retarded in the presence of sulfhydryl-containing compounds such as glutathione.

Amino Acids↗
PubMed1985

cAMP-dependent phosphorylation of bovine lens alpha-crystallin.

This communication reports that the A1 and B1 chains of bovine lens alpha-crystallin are phosphorylated. The conclusion is based on the following evidence: (i) When soluble preparations from lens cortex are incubated with [gamma-32P]ATP, a cAMP-dependent labeling of a high molecular weight protein is obtained. (ii) After NaDodSO4/PAGE, the label is found in two bands with Mr 22,000 and 20,000, corresponding to the B and A chains of alpha-crystallin, respectively. (iii) Isoelectric focusing indicates that the radioactivity is almost exclusively in bands with pI values of 5.58 and 6.70, corresponding to the A1 and B1 chains, respectively. (iv) Similar results are obtained in experiments of [32P]orthophosphate incorporation in lens organ culture. (v) Analyses of the digested protein indicate the label is exclusively in phosphoserine. (vi) 31P NMR analyses of native, proteolytically digested, and urea-treated alpha-crystallin gives a chemical shift of 4.6 ppm relative to 85% H3PO4 at pH 7.4, suggesting that the phosphate is covalently bound to a serine in the protein. An abundance of approximately one phosphate per four or five monomer units was found. (vii) Similar results were obtained by chemical analyses of independently prepared alpha-crystallin samples. The results are consistent with the view that the A1 and B1 chains arise as result of the phosphorylation of directly synthesized A2 and B2 polypeptides. It is suggested that this metabolically controlled phosphorylation may be associated with the terminal differentiation of the lens epithelial cell and the intracellular organization of the lens fiber cell.

Animals↗
PubMed1985

The effect of H2O2 on lens epithelial cell glutathione.

Investigation of lens epithelial cells indicates that under normal conditions, essentially all of the detectable cellular glutathione is in a reduced state. However, exposure to levels of H2O2 in the range found in the aqueous fluid of cataract patients causes rapid, very large changes in the glutathione redox ratios. Immediately following short-term exposure to 0.15-0.2 mM H2O2, reduced glutathione drops to 19% of its normal level and the remainder of the total glutathione is found in the oxidized form. Within the next few minutes, the redox ratio returns to normal. However, total glutathione levels remain approximately 20% below normal even one hour after exposure to H2O2. With exposure to a higher concentration of H2O2, a greater loss of glutathione is observed. The results suggest that the glutathione redox ratios change dramatically as a result of oxidative insult but quickly return to normal when the oxidative insult is removed. The formation of mixed glutathione-protein disulfide was also observed but only after long-term (1 hour) exposure to a high level (0.6 mM) of H2O2.

Animals↗
PubMed1985

Phosphorylated intermediates of two Ca++-ATPases in membrane preparations from lens epithelial cells.

By incubating preparations enriched in membranes from lens epithelial cells with [gamma 32P]-ATP and Ca++ at 0 degrees C for 15 seconds followed by SDS-PAGE analysis, it was possible to demonstrate a Ca++-dependent [32P]-phosphate incorporation in two polypeptides with Mr 105,000 and 140,000. Treatment of phosphorylated preparations with 0.06 N hydroxylamine at pH 5.4 and 25 degrees C removed the label from both polypeptides indicating that the phosphate was attached to the proteins by an anhydride linkage characteristic of the phosphorylated intermediates of the ATPases. Membrane preparations from sarcoplasmic reticulum and red blood cell studied under the same conditions showed a Ca++-dependent [32P]-phosphate incorporation into polypeptides with Mr 105,000 and 138,000, respectively, corresponding to the phosphorylated intermediates of the Ca++-ATPases present in these preparations. The results suggest the presence of two Ca++-ATPases in lens epithelial cells which, in terms of Mr, appear to be similar to those present in the sarcoplasmic reticulum and the red blood cell plasma membrane, respectively.

Animals↗
PubMed1985

Membrane cholesterol and phospholipid in consecutive concentric sections of human lenses.

Lens membrane preparations have been shown to have a remarkable rigidity which increases in the inner nuclear region of the lens and has been correlated with the cholesterol (C)/phospholipid (PL) ratio. However, the distribution of these lipids in single lenses had not been determined. Utilizing a new technique for isolating consecutive layers of a human lens, lipid composition and contents of seven pairs of normal lenses from subjects ranging from 54 to 77 years old have been analyzed. It was found that the PL content remains relatively constant at 22-24 micrograms/mg through all but the nuclear 10-15% of the lens dry weight where it drops precipitously to about 7 micrograms/mg. The C distribution is more complex; the C content is at a low level of 14 micrograms/mg in the outer cortical 15-20%, rises to 25 micrograms/mg in the inner cortical 40-60% of the total lens weight, and drops to 12 micrograms/mg upon reaching the nucleus. Thus, the continuous increase in the lens C/PL ratio is due to the increase in C in the cortex and the large decrease in PL in the nucleus. Analyses of phospholipid and fatty acid composition in the different regions of the lens indicate significant differences. However, the abundance of mono-unsaturated fatty acids contributing to the rigidity of the membrane has only minor variation. The lens has a remarkably low overall lipid content of 4% and only 2% in the nuclear region. Calculation of the surface area of the nuclear fiber cell suggests that less than one-third of the membrane is made of PL bilayer. Thus, a mosaic of PL and C patches or some other type of structure involving membrane fusion must be present. Conversion of the % dry weight occupied by the concentric fiber fractions to their location on the lens axis in mm indicates that the nuclear 15% dry weight of the tissue occupies more than 50% of the axial length. This region contains the embryonic lens and the primary lens fibers. Similarly, the metabolically active outer 20% of the dry weight accounts for less than 10% of the visual axial length and contains cells undergoing terminal differentiation. Cataractous lenses have lipid distributions similar to those of the normal lenses suggesting that membrane lipid is either not involved in cataract formation or that the primary insult is localized in an undetectable small number of fiber cells.

Aged↗
PubMed1984

Kinetic cooperativity change after H2O2 modification of (Na,K)-ATPase.

The kinetics of hydrolysis of ATP and p-nitrophenylphosphate and the action of the allosteric effectors, Na+ and K+, upon the hydrolysis of these substrates were used to study the H2O2-modified, uncoupled (Na,K)-ATPase isolated from cultured bovine lenses ( Garner , W. H., Garner , M. H., and Spector , A. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2044-2048). Pure bovine renal (Na,K)-ATPase was modified by H2O2 in 150 mM KCl and 20 mM MgCl2 to yield an enzyme with kinetic properties similar to the enzyme isolated from the H2O2-treated, cultured bovine lens. H2O2 modification changes the interaction of the ATP hydrolysis site from negative to positive kinetic cooperativity. H2O2 modification dramatically alters Na+ stimulation of ATP hydrolysis and Na+ inhibition of p-nitrophenylphosphate hydrolysis while having little effect upon K+ control of the hydrolysis of these two substrates.

Adenosine Triphosphate↗