Biomedical subjects

J Peisach

Publications and source records attributed to J Peisach.

At least 91 records · Page 5Linked to original sources

PubMed1984

Determination of copper in biological materials by atomic absorption spectroscopy: a reevaluation of the extinction coefficients for azurin and stellacyanin.

A new method for the determination of copper in biological materials by graphite furnace atomic absorption spectroscopy is presented. This new procedure is an extension of the classic method of standard additions, where the analyte concentration is determined in a series of identical samples to which various amounts of metal standard have been added. The concentration of metal in the original sample is determined from an extrapolation of a plot of absorbance versus added analyte. In the new method, the amount of copper is determined by the method of standard additions for different concentrations of the sample under investigation as well. From an extrapolation of the data, the concentration of copper in the absence of interfering matrix is obtained. Studies with fetal bovine serum demonstrate that the new extrapolation technique is precise. Furthermore, considerably more copper is detected than by the classic method of standard additions applied to a nitric acid treated sample. The matrix effects of phosphate, nitrate, albumin, and serum were also examined. Both phosphate and serum, at physiological pH, decrease the detectability of added copper, while nitrate and albumin were without effect. The accuracy of this method has been verified by determining the extinction coefficients of stellacyanin and azurin. The values obtained, 4.33 X 10(3) and 3.75 X 10(3) M-1 cm-1, respectively, are considerably different from those determined by the method of standard additions on nitric acid digests of these proteins, but were close to values previously reported and determined colorimetrically.

Animals↗

PubMed1984

Direct spectrophotometric analysis of hemoglobin in isoelectric focusing tube gels.

Methods are described for the direct spectrophotometric analysis of human oxyhemoglobin, deoxyhemoglobin, and methemoglobin focused in polyacrylamide tube gels. Visible absorption spectra (350-650 nm) of electrofocused hemoglobin bands were recorded using a diode array rapid-scan spectrophotometer. Direct optical sampling of gels allowed the identification of focused hemoglobin valency hybrids which contain two oxidized monomers per tetramer.

Gels↗

PubMed1984

Induction of hepatic microsomal drug metabolism by azo compounds: a structure-activity relationship.

The structure-activity relationship of 40 azo compounds in their ability to induce cytochrome P-448 and associated monooxygenase activities, as well as UDP-glucuronyltransferase (UDPGT) activity, was investigated. Regardless of their structure, hydrophilic azo dyes and lipophilic azobenzene derivatives were not able to induce these enzyme activities. Only those lipophilic azo dyes with 1-azo-2-naphthol or 1-azo-2-naphthylamine moieties were able to induce cytochrome P-448 and related monooxygenase activities, as well as UDPGT activity. The extent of induction is comparable to or greater than that caused by 3-methylcholanthrene (3-MC). It is suggested that those azo dyes capable of inducing P-450 type cytochromes can form coplanar structures with three fused, 6-membered rings through intramolecular hydrogen bonding. These structures are analogous to polycyclic aromatic hydrocarbons that can also induce.

Animals↗

PubMed1984

The effect of Sudan III on drug metabolizing enzymes.

We have examined the induction of drug metabolizing enzymes in rat liver microsomes by azo dye, 1-(p-phenylazophenylazo)-2-naphthol (Sudan III). Marked increases were observed in the levels of cytochrome P-448 as well as in p-nitroanisole O-demethylase (p-NAD), amaranth (AR) and neoprontosil reductases (NPR) and 7-ethoxycoumarin O-deethylase (ECD) activities. On the other hand, aminopyrene N-demethylase activity was not significantly increased. Further, induced ECD activity was inhibited 90% by a specific antibody against cytochrome P-448 while the inhibition observed with an antibody against cytochrome P-450 was less than 25%. Simultaneous administration of Sudan III and 3-methylcholanthene (3-MC) induced cytochrome P-448 up to a level brought about by either Sudan III or 3-MC treatment alone. In contrast, Sudan III did not induce cytochrome P-448 in the 3-MC insensitive DBA/2 mouse. Solubilized microsomes from Sudan III-treated rats showed an identical sodium dodecyl sulfate polyacrylamide gel electrophoretic (SDS-PAGE) pattern with those from 3-MC-treated animals. It is concluded that the cytochrome P-448 induced in liver by Sudan III is very similar to that induced by 3-MC. Sudan III also induced UDP-glucuronyltransferase activity towards 1-naphthol and estradiol. It did not induce NADPH-cytochrome c reductase, nor any of the enzymes which constitute the microsomal electron transport chain except for cytochrome P-448.

Animals↗

PubMed1984

The accessibility of type I Cu(II) centers in laccase, azurin, and stellacyanin to exchangeable hydrogen and ambient water.

The characteristic deuterium modulation pattern was observed in the electron spin-echo envelopes for laccase, decupro laccase (from which Type 2 copper had been removed), stellacyanin, and azurin that had been exchanged against D2O. From the decay rate of the modulation pattern and from a quantitative analysis of the modulation depth, we conclude that the Cu(II) sites in these proteins are directly accessible to solvent. Similar results were obtained for laccase and decupro laccase.

Azurin↗

PubMed1984

Oxygen metabolism in cloned macrophage cell lines: glucose dependence of superoxide production, metabolic and spectral analysis.

The requirements of a cloned macrophage-like cell line, J774.16, for oxygen metabolism, and the nature of the defect in oxidative metabolism in a variant clone derived from it, J774.C3C, were studied. Upon stimulation with phorbol myristate acetate (PMA), the parental clone produced approximately 1 nmol O2-/min/10(6) cells, whereas the variant clone produced no detectable O2- under the same conditions. Sustained O2- production by J774.16 was totally dependent on extracellular glucose; in glucose-free medium, the cells initiated O2- production but could not sustain it. When cells were stimulated with PMA, glucose-C-1 oxidation of J774.16 cells increased 20-fold while that of J774.C3C remained at resting levels. O2- production in J774.16 cells was inhibited by some agents known to block mitochondrial electron transport before coenzyme Q, such as rotenone and tetrathiafulvalene, whereas antimycin A enhanced O2- production. A dissociation between O2- production and glucose-C-1 oxidation was observed when J774.16 was treated with certain metabolic inhibitors. Quinacrine, 2,4-dinitrophenol, chlorpromazine, and trifluoperazine inhibited O2- production completely under conditions in which glucose-C-1 oxidation was reduced only by 30%. Rotenone inhibited O2- production with no effect on glucose-C-1 oxidation whereas antimycin A augmented O2- production 50% but inhibited glucose oxidation by 20%. Glucose transport studies, with 2-deoxy-D-glucose, showed that the Km for glucose transport of both clones was about 1 mM, indicating that cells could effectively transport glucose even at low concentrations. The Vmax for glucose transport in both J774.16 and variant J774.C3C cells doubled after PMA stimulation, indicating that the variant was effectively stimulated by PMA, even though O2- was not produced. Similarly, PMA induced protein phosphorylation in both clones. No differences between clones J774.16 and J774.C3C in hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glutathione reductase, or glutathione peroxidase activities could be found. When dithionite-reduced and -oxidized difference spectra of plasma membranes of these clones were compared, comparable levels of b-type cytochrome were found in both clones. However, CO difference spectra indicated that CO was bound to a b-type cytochrome (presumed to be b-245) in clone J774.16 but not in J774.C3C.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1983

Characterization of 3-iron ferredoxins by means of the linear electric field effect in EPR.

We describe a method for the differentiation of 3-iron from 2-iron and 4-iron Fe/S proteins based on consideration of both the magnetic field dependence of shifts in g induced by an externally applied electric field (LEFE) and the continuous wave EPR spectra properties. The magnetic field dependence and the magnitude of the LEFE for 3-iron ferredoxins are similar to those for 4-iron ferredoxins but differ considerably from those for 2-iron ferredoxins or for high potential iron proteins. Furthermore, as 3-iron ferredoxins and high potential iron proteins are EPR-active when oxidized while 2-iron and 4-iron ferredoxins are only EPR-active when reduced, the differentiation among all of them can be made on the basis of both continuous wave EPR and LEFE properties, but not by each individually.

Aconitate Hydratase↗

PubMed1983

Oxidation state dependence of proton exchange near the iron-sulfur centers in ferredoxins and high-potential iron-sulfur proteins.

For both the [2Fe-2S] and the [4Fe-4S] ferredoxins, dialysis against 2H2O prior to single electron reduction leads to the appearance of a deuterium modulation pattern in the electron spin echo decay envelope indicative of deuteron-proton exchange very near the paramagnetic center. In contrast, if the ferredoxin is exposed to 2H2O after its reduction in H2O, far less deuterium exchange near the metal center takes place. Thus, proton exchange with solvent is in part dependent on the redox state of the protein. For high potential iron-sulfur proteins, this type of proton-deuteron exchange near the metal center does not occur unless the protein is partially unfolded in dimethylsulfoxide in 2H2O.

Adrenodoxin↗

PubMed1983

Mössbauer study of iron bleomycin and its activation intermediates.

Using Mössbauer spectroscopy, we have examined iron-bleomycin in various oxidation states and in complexes with dioxygen or carbon monoxide. Ferrous bleomycin is a high spin ferrous complex. Addition of O2 converts it into an EPR-silent oxygenated complex. Mössbauer studies in strong applied magnetic fields show that oxygenated bleomycin is diamagnetic. At 4.2 K, the quadrupole splitting delta EQ = -2.96 mm/s and the isomer shift delta = 0.16 mm/s suggest that its electronic structure is best described as low spin ferric iron bound to superoxide anion. A single electron reduction yields activated bleomycin, an EPR-active form that still retains oxygen and which is kinetically competent to initiate DNA cleavage. We have produced this complex by exposing ferrous bleomycin to O2 or by reacting ferric bleomycin with H2O2. The Mössbauer spectra give convincing evidence that the iron of activated bleomycin is low spin ferric. The decay of activated bleomycin yields low spin ferric bleomycin, a complex with Mössbauer parameters nearly identical with those reported for ferric cytochrome P-450. Although iron bleomycin does not have a polyaromatic structure like heme, many features of its electronic structure at the iron are very similar to those produced by the sulfur-coordinated heme iron of ferric cytochrome P-450, a protein that catalyzes a similar oxygen-dependent reaction.

Bleomycin↗

PubMed1982

The stimulation of microsomal azoreduction by flavins.

The reduction of the azo dye, amaranth, by rat liver microsomes is inhibited about 90% by carbon monoxide, suggesting that the reaction largely depends on cytochrome P-450. Reducing equivalents for this reaction are supplied by NADPH. This reaction is stimulated by riboflavin, FMN and FAD, as well as by methylviologen. A large fraction of the stimulated reaction is not blocked by CO, indicating that there is a pathway of electron transfer which is independent of cytochrome P-450. Rat liver microsomes can reduce FAD, with reducing equivalents supplied by NADPH. The FADH2 thus produced is quickly oxidized by amaranth so that two FADH2 are oxidized for every amaranth reduced. The same stoichiometry is observed with photochemically prepared FADH2, formed in the absence of microsomes.

Amaranth Dye↗

PubMed1982

Structure of the Glycyl-L-histidyl-L-lysine--copper(II) complex in solution.

Optical, electron paramagnetic resonance, and electron spin-echo envelope spectroscopies were used to examine the structure of the Cu(II) complex of glycyl-L-histidyl-L-lysine (GHL) in solution. At neutral pH, GHL forms a mononuclear 1:1 Cu(II) compound having an EPR spectrum resembling that of Cu(II) equatorially coordinated by two or three nitrogen atoms. Electron spin-echo studies demonstrate that one of these is located in the histidyl imidazole ring. A pH titration of Cu(II)-GHL shows three optical transitions with apparent pKs of 3.6, 9.2 and 11.4 and molecularities, with respect to protons, of 2, 2, and 1, respectively. At the lowest pK, GHL binds Cu(II), forming the species present at physiological pH. At elevated pH, spectroscopic experiments suggest that an alteration of the Cu(II) structure occurs, yet the bound imidazole is retained. These solution studies are consistent with nitrogen coordination of Cu(II) in Cu(II)-GHL, but the solid-state polymeric structure, with oxygen-bridged Cu(II) pairs as previously determined by X-ray crystallographic analysis [Pickart, L., Freedman, J. H., Loker, W. J., Peisach, J., Perkins, C. M., Steinkamp, R. E., & Weinstein, B. (1980) Nature (London) 288, 715-717; C. M. Perkins, N. J. Rose, R. E. Steinkamp, L. H. Jensen, B. Weinstein, and L. Pickart, unpublished results], does not exist in solution.

Copper↗

PubMed1982

Electron spin echo studies of the copper complexes of conalbumin.

Electron spin echo envelope spectroscopy was used to probe the two metal binding sites of Cu(II)-conalbumin. The echo envelope spectrum of Cu(II)-conalbumin-oxalate, with metal ion at either one or both of the binding sites, contains lines arising from the interaction of the electron spin of Cu(II) with bound imidazole, demonstrating histidine ligation to the metal ion. The 13C superhyperfine interaction of bound [13C]oxalate, obtained from the ratio of the electron spin echo envelopes of Cu(II)-conalbumin-[13C]oxalate to that of Cu(II)-conalbumin-[12C]oxalate, is about twice the free precession frequency and indicates a contact interaction between 13C and Cu(II). This study indicates that oxalate is directly coordinated to the metal ion. Over the pH range 7.0 to 10.0, where Cu(II)-conalbumin binds carbonate as an associated anion, the echo envelope spectrum indicates that at least one imidazole ligand is coordinated to Cu(II). Below pH 6.0 and above pH 11.0, imidazole coordination is not observed.

Binding Sites↗

PubMed1982

Stoichiometry of DNA strand scission and aldehyde formation by bleomycin.

A colorimetric assay of DNA breakage by bleomycin has been standardized and indicates that strand scission is stoichiometric with the formation of a single equivalent of an aldehyde compound consisting of base plus deoxyribose carbons 1' to 3'. Both strand scission and aldehyde formation require the presence of O2. An alternate DNA lesion inflicted by bleomycin, alkali labilization, is O2-dependent, as is the accompanying release of free bases.

Aldehydes↗

PubMed1982

Reduction of copper(II)-bleomycin: a model for in vivo drug activity.

The effect of aliphatic thiols, including glutathione, cysteine, and dithiothreitol, on the anaerobic reduction of Cu(II)-bleomycin was examined. At neutral pH, cysteine is more efficient in reducing Cu(II)-bleomycin than either dithiothreitol or glutathione, while at alkaline pH the rate of reduction with all three reagents increases substantially. A kinetic treatment suggests that 2 equiv of thiol is required for each mole of Cu(II)-bleomycin reduced. Material balance studies verify this stoichiometry. If anaerobic reduction of Cu(II)-bleomycin is carried out in the presence of Fe(II), iron is chelated by the drug. This metal-drug complex is capable of degrading DNA when O2 is introduced. The extent of DNA degradation, as measured by the release of malondialdehyde-like chromogens from the DNA, is directly dependent on the amount of Cu(II)-bleomycin reduced.

Animals↗

PubMed1982

Effects of O2 on the reactions of activated bleomycin.

The antitumor drug, bleomycin, interacts with either Fe(II) and O 2 or Fe(III) and H2O2 to form an activated complex which attacks DNA. Under aerobic conditions, both reactions yield similar quantities of free bases and products consisting of base plus deoxyribose carbon atoms 1 to 3. Under anaerobic conditions, activated bleomycin releases only free base. The yield of free base is the same under aerobic or anaerobic conditions, provided DNA is furnished in excess. When the DNA concentration is limiting, more base is released under anaerobic than under aerobic conditions. Drug self-destruction proceeds as quickly and completely in the presence or absence of O2.

Aerobiosis↗

PubMed1982

Stellacyanin. Studies of the metal-binding site using x-ray absorption spectroscopy.

Stellacyanin is a mucoprotein of molecular weight approximately 20,000 containing one copper atom in a blue or type I site. The metal ion can exist in both the Cu(II) and Cu(I) redox states. The metal binding site in plastocyanin, another blue copper protein, contains one cysteinyl, one methionyl, and two imidazoyl residues (Colman et al. 1978. Nature [Lond.]. 272:319-324.), but an exactly analogous site cannot exist in stellacyanin as it lacks methionine. The copper coordination in stellacyanin has been studied by x-ray edge absorption and extended x-ray absorption fine structure (EXAFS) analysis. A new, very conservative data analysis procedure has been introduced, which suggests that the there are two nitrogen atoms in the first coordination shell of the oxidized [Cu(II)] protein and one in the reduced [Cu(I)] protein; these N atoms have normal Cu--N distances: 1.95-2.05 A. In both redox states there are either one or two sulfur atoms coordinating the copper, the exact number being indeterminable from the present data. In the oxidized state the Cu--S distance is intermediate between the short bond found in plastocyanin and those found in near tetragonal copper model compounds. Above -140 degree C, radiation damage of the protein occurs. At room temperature the oxidized proteins is modified in the x-ray beam at a rate of 0.25%/s.

Binding Sites↗

PubMed1981

Activated bleomycin. A transient complex of drug, iron, and oxygen that degrades DNA.

"Activated bleomycin" is an oxygenated iron drug complex which embodies the drug's DNA-cleaving activity. This activity is exercised on DNA, if present, but if DNA is absent, the drug itself is inactivated. Hyperfine interactions in the EPR spectra of activated bleomycin prepared with 57Fe(II) and 17O2 demonstrate the presence of iron as Fe(III) and of bound oxygen originating in dioxygen. Bleomycin can also be activated with Fe(III) and either H2O2 or ethyl hydroperoxide. These latter reactions do not produce a ferrous intermediate nor do they require O2. But O2 is required for the reaction of activated bleomycin with DNA to yield the malondialdehyde-like chromogens used to monitor DNA degradation. The attack on DNA is quantitatively concurrent with the decay of activated bleomycin, however generated.

Aerobiosis↗

PubMed1981

Pulsed EPR studies of peroxide-activated cytochrome c peroxidase and of the mercaptoethanol derivative of Neurospora tyrosinase.

We have measured the linear electric field effect for peroxide-activated yeast cytochrome c peroxidase and for the mercaptoethanol derivative of Neurospora tyrosinase. Although both of these materials have EPR spectra resembling those of free radicals, the linear electric field effect measurements demonstrate that there is a metal ion associated with the paramagnetic centers. In addition, we have also observed superhyperfine interactions with 14N nuclei.

Catechol Oxidase↗