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Biomedical subjects

J Peisach

Publications and source records attributed to J Peisach.

At least 109 records · Page 6Linked to original sources
PubMed1980

Growth-modulating plasma tripeptide may function by facilitating copper uptake into cells.

The plasma tripeptide glycyl-L-lysine (GHL), when added at nanomolar concentrations to a wide group of cultured systems, produces a disparate set of responses ranging from the stimulation of growth and differentiation to outright toxicity. Such diverse actions imply that this tripeptide mediates some basic biochemical function common to many types of cells and organisms. During the isolation of GHL we found the compound to co-isolate through a number of steps with approximately equimolar copper and about 1/5 molar iron. Maximal effects on hepatoma cells (HTC4) were seen when the peptide was added with copper and iron to the growth medium. Structure-function studies revealed that several tripeptides with a histidyl-lysyl linkage were nearly as active as GHL. The association of GHL with copper and a homology similarity between the tripeptide and the copper transport sites on albumin and alpha-fetoprotein, where the cupric atom is bound to a histidyl residue adjacent to a basic residue, suggested that GHL may act as a copper transport factor. We report here that the tripeptide readily forms complexes with copper(II) and enhances the uptake of the metal into cultured hepatoma cells.

Animals↗
PubMed1980

Origin of malondialdehyde from DNA degraded by Fe(II) x bleomycin.

Ferrous bleomycin is known to break DNA efficiently in vitro in the presence of O2, giving rise to ologonucleotides, bases, and compounds resembling malondialdehyde in their chromogenic reaction with 2-thiobarbituric acid. Chromatography of radiolabeled DNA reaction mixtures resolves three kinds of malondialdehyde-like products, related by sequential conversions. The first chromogenic product is linked to DNA, and its formation does not entail the release of a base. It decomposes readily to the second product, a compound containing the base and deoxyribose carons 1'-3'. Hydrolysis of either product yields the third, which is indistinguishable from authentic malondialdehyde. These findings suggest that the oxygen-dependent cleavage of DNA by Fe(II) x bleomycin can begin with the rupture of the deoxyribose 3'-4'-carbon bond. The initiation of these events is concurrent with the initiation of another mode of DNA degradation, involving the release of free base alone, in a yield similar to that of chromogen.

Bacteriophage lambda↗
PubMed1980

Macrophage variants in oxygen metabolism.

Whereas phagocytic cells from normal individuals have the capacity to kill ingested bacteria and parasites, those from patients with several uncommon genetic deficiency diseases are known to be defective in bactericidal activity. Studies on neutrophils of these patients have revealed fundamental defects in their ability to reduce molecular oxygen and metabolize it to superoxide anion, hydrogen peroxide, and oxygen radicals. In the present experiments, we describe a clone of a continuous murine macrophage-like cell line, J774.16, that, upon appropriate stimulation, activates the hexose monophosphate shunt, and produces superoxide anion and hydrogen peroxide. With nitroblue tetrazolium to select against cells capable of being stimulated by phorbol myristate acetate to reduce the dye to polymer--formazan--which is toxic fot cells, we have selected for variants that are defective in oxygen metabolism. Four of these subclones have been characterized and found to be lacking in the ability (a) to generate superoxide anion, as measured by cytochrome c reduction; (b) to produce hydrogen peroxide, as measured by the ability to form complex I with cytochrome c peroxidase; and (c) to be stimulated to oxidize glucose via the hexose monophosphate shunt. These variants appear to represent a useful model for studying the molecular basis for macrophage cytocidal activity.

Animals↗
PubMed1980

Electron spin echo studies of cytochrome c oxidase.

We have studied the linear electric field effect in pulsed EPR of the "EPR-detectable copper" signal of beef heart cytochrome c oxidase and have compared our results with those for a variety of square planar and tetrahedral Cu(II) model compounds and with Cu(II) proteins containing either type 1 or type 2 copper. The electric field induced g shifts (linear electric field effect) for cytochrome oxidase are comparable in magnitude to those for simple Cu(II) complexes and for some copper proteins containing type 2 sites. The shifts are smaller than those for tetrahedral copper complexes and for type 1 copper sites. However, the magnetic field dependence of the linear electric field effect does not resemble that observed for any Cu(II) complex studied nor for type 1 copper. These findings cannot be reconciled with the tetrahedral Cu(II) model proposed by Greenaway, Chan, and Vincow ((1977) Biochim. Biophys. Acta 490, 62-78, 1977) to explain the unusual EPR spectrum of cytochrome oxidase.

Animals↗
PubMed1980

Purification and characterization of hydroxyindole oxidase from the gills of Mytilus edulis.

An hydroxyindole oxidase has been purified 100-fold from the gill plates of Mytilus edulis. This preparation yields a single band on acrylamide gel electrophoresis and isoelectric focusing. Metal analysis and spectral data show that both heme iron and copper are present in the purified enzyme in a 1:1 molar ratio. Potassium cyanide, sodium azide, various copper chelators, and carbon monoxide inhibit the enzyme-catalyzed reaction. These inhibition studies, as well as the photoreversibility of the CO inhibition, lend support to the conclusion that metal ions function as necessary cofactors for substrate oxidation. The Mytilus gill enzyme oxidizes a variety of substrates, among them biogenic amines. For all substrates tested, 1 dioxygen molecule is consumed/mol of substrate oxidized. When p-coumaric acid is hydroxylated, caffeic acid is formed in quantitative yield. H2O2 has been identified as a second product of the oxidation reaction. The addition of catalase prior to the onset of reaction is inhibitory while H2O2 or ascorbate activate the enzyme. These findings suggest that the hydroxyindole oxidase may be a monooxygenase which does not require reduced pyridine nucleotide for reductive activation.

Animals↗
PubMed1979

Studies of the electron-nuclear coupling between Fe(III) and 14N in cytochrome P-450 and in a series of low spin heme compounds.

We have observed the nuclear modulation pattern in the envelope of electron spin echoes for various low spin paramagnetic heme proteins including cytochrome c, myoglobin hydroxide, myoglobin mercaptoethanol, and cytochrome P-450, using the three-pulse-stimulated echo method. We have also carried out similar experiments with model compounds containing either [14N]- or [15N]imidazole. In many of the compounds studied, we have been able to identify the nuclear modulation effects arising from 14N of the porphyrin ring and have been able to characterize and interpret the modulation effects due to 14N of various nitrogenous axial ligands. We have found that the heme of low spin ferric cytochrome P-450 is coordinated to a nitrogenous ligand, probably imidazole. We have also demonstrated that the remote 14N of the imidazole ligand in a [14N]imidazole-heme-NO-model compound is coupled differently than in myoglobin nitroxide, demonstrating the direct effect of the protein of metal ligand bonding.

Binding Sites↗
PubMed1979

Oxygenated iron bleomycin. A short-lived intermediate in the reaction of ferrous bleomycin with O2.

The reaction of Fe(II) . bleomycin with O2 to yield Fe(III) . bleomycin has been resolved into two kinetic events by stopped-flow spectrophotometry. The first event is first order with respect to both bleomycin and O2 and may be regarded as a second order reaction (k = 6.1 x 10(3) M-1s-1 at 2 degrees C). The first product has no EPR spectrum. The optical spectrum resembles those of Fe(II) . bleomycin complexes with CO, NO, and ethyl isocyanide. We propose that the first product is an Fe(II) . bleomycin . O2 complex. The second kinetic event is first order with respect to the first accumulated product (k = 0.11 s-1 at 2 degrees C) and independent of oxygen concentration. The product of this reaction is indistinguishable from Fe(III) . bleomycin by optical and EPR spectroscopy.

Bleomycin↗
PubMed1979

Iron-bleomycin interactions with oxygen and oxygen analogues. Effects on spectra and drug activity.

Despite extensive structural dissimilarities, iron . bleomycin complexes and heme-containing oxygenases display remarkable similarities in binding oxygen antagonists and in spectral properties deriving from bound iron. Fe(II)-bleomycin reversibly forms a complex with either CO or isocyanide (lambda max = 384 and 497 nm, respectively), either of which interfere with its oxygen-dependent cleavage of DNA. A similar but paramagnetic complex forms with NO (lambda max = 470 nm; AN = 24 G). In contrast, cyanide enhances bleomycin activity against DNA. Complexes of bleomycin and FE(III), formed either by direct association or by autoxidation of the Fe(II) . bleomycin complex, exhibit indistinguishable EPR and visible spectra, which change characteristically with pH. At neutral pH, Fe(III) . bleomycin is a low spin complex (g = 2.45, 2.18, 1.89; lambda max = 365, 384 nm) and, at low pH, it is a high spin rhombic complex (geff = 9.4, 4.3; lambda max = 430 nm). These complexes are interconvertible (pK 4.3). Fe(II) . bleomycin oxidation, although reversible by spectral criteria, is accompanied by drug inactivation unless DNA is present.

Animals↗
PubMed1979

Measurement of 14N superhyperfine frequencies in stellacyanin by an electron spin echo method.

We have measured the 14N superhyperfine frequencies for weakly coupled nitrogen in stellacyanin and in a model compound Cu(II)-diethylenetriamine-imidazole using a 3-pulse spin echo technique. By making computer simulations of the superhyperfine spectrum, we have been able to show that these frequencies result from the interaction of the remote protonated nitrogen of metal-bound imidazole with Cu(II).

Electron Spin Resonance Spectroscopy↗
PubMed1979

The nature of the copper atoms of cytochrome c oxidase as studied by optical and x-ray absorption edge spectroscopy.

X-ray absorption edge spectroscopy has been used to study the copper of 1--2 mM cytochrome c oxidase in the resting oxidized, mixed-valence, and fully reduced states. A comparison was made of this protein with copper complexes and with natural and artificial copper proteins. Spectra were obtained with synchrotron radiation from the SPEAR storage ring using highly sensitive fluorescence detectors. Temperatures of -80 to -120 degrees C were employed further to improve the stability of the samples and to avoid the possibility of either auto- or photon-induced reduction of the materials, which might have occurred in previous studies. In order to characterize the valence states of the Cu and Fe components, the samples were monitored by infrared and visible spectroscopy before and after irradiation by the X-ray beam. The combination of the optical and X-ray absorption techniques has afforded a deconvolution of the four species of copper in the various states of cytochrome c oxidase and the tentative assignment of Cu alpha, the copper redox coupled to the heme alpha of cytochrome alpha, as a highly covalent type of copper and Cu alpha 3, the copper of cytochrome alpha 3, as a more ionic 'blue' type I copper. The implications of these findings upon the mechanism of action of cytochrome oxidase are briefly outlined.

Absorptiometry, Photon↗