Biomedical subjects

J H Freedman

Publications and source records attributed to J H Freedman.

27 records · Page 2Linked to original sources

PubMed1987

Electron paramagnetic resonance and magnetic susceptibility studies of dimanganese concanavalin A. Evidence for antiferromagnetic exchange coupling.

The double Mn2+ complex of concanavalin A with bound saccharide (SMMPL) was examined by electron paramagnetic resonance (EPR) spectroscopy and magnetic susceptibility measurements. A room temperature X-band (9 GHz) EPR spectrum of SMMPL revealed a relatively weak, broad resonance in contrast to the spectrum with a six-line hyperfine-split pattern observed for the mononuclear, high-spin Mn2+ complex found in Ca2+-Mn2+-concanavalin A with saccharide present (SCMPL). The EPR spectrum of SMMPL at 77 K, however, consisted of a series of overlapping patterns of 11 hyperfine-split lines near g = 2.0 with members of each pattern separated by 47 G, half the value of the hyperfine splitting of SCMPL. These 11-line patterns are preserved at Q-band (35 GHz), indicating that the manganese ions in SMMPL form a spin-coupled, binuclear center. As expected for an exchange-coupled system, the EPR signal of SMMPL at 77 K saturates at a higher microwave power than those for SCMPL or Mn2+ aquoion. There is also a marked loss of EPR signal intensity for SMMPL between 4.2 and 1.4 K, which supports the view that the pair of manganese ions is exchanged-coupled. The temperature dependence of both the magnetic susceptibility and the low-temperature EPR spectral intensity can be explained by a model in which the two high-spin Mn2+ ions of SMMPL are antierromagnetically exchanged-coupled with an isotropic coupling constant J = 1.8 cm-1 (for the spin Hamiltonian Hex = JS1.S2). Zero-field splitting D' was estimated to be 375 G from the EPR spectrum.(ABSTRACT TRUNCATED AT 250 WORDS)

Concanavalin A↗

PubMed1986

Resistance to copper toxicity of cultured hepatoma cells. Characterization of resistant cell lines.

A series of four cell lines resistant to the toxic effect of copper were developed from Morris rat hepatoma cells by gradually increasing the concentration of copper in the growth medium. The EC50, that concentration of copper that kills and/or inhibits the growth of 50% of the cells after 72 h, increased 4-fold over that for wild type cells in the most resistant cell line. These cells were also resistant to zinc, cadmium, and mercury toxicity, but not to nickel or cobalt. The amount of copper in the soluble protein pool of the resistant cells increased proportionally with the concentration of copper in the medium in which they were maintained. Associated with copper accumulation was the production of an 18-kDa cysteine-rich protein which complexes a significant amount of the metal. It is suggested that resistance to copper toxicity is due to sequestration of the metal by this protein. When resistant cells were removed from the copper-enriched environment, cellular copper levels rapidly fell to that observed for wild type cells, but no reduction in either the EC50 or the level of the cysteine-rich protein was noted. This suggests that a permanent change responsible for copper resistance had occurred which is maintained in the absence of the metal.

Animals↗

PubMed1986

Structure of the copper cluster in canine hepatic metallothionein using X-ray absorption spectroscopy.

The metal binding site in the lysosomal copper metallothionein from canine liver (LyCuLP) was examined with X-ray edge and extended X-ray absorption fine structure (EXAFS) spectroscopies. The k-absorption edge spectrum of LyCuLP was consistent with the coordination of univalent copper. The Fourier transform of the EXAFS data showed four resolved shells of backscattering atoms. Comparisons between the phase and amplitude functions derived from the isolated shells to those of Cu-Cu, Cu-S, and Cu-N model compounds showed that each copper was coordinated by four sulfur atoms at a distance of 2.27 +/- 0.02 A. Analysis of the outer shell data indicated backscattering copper atoms at 2.74 +/- 0.05, 3.32 +/- 0.05, and 3.88 +/- 0.05 A. Interatomic distances determined from the EXAFS data were compared to the distances observed by X-ray crystallographic analysis of adamantane-like clusters containing four and five copper atoms and a cubic cluster containing four copper atoms, structurally similar to the 4Fe-4S clusters in some ferredoxins. The results of these comparisons suggest that the copper complexed in LyCuLP is arranged in an adamantane-like cluster. The structure derived for this protein may be conserved in other copper metallothioneins.

Animals↗

PubMed1985

Comparison of the spectroscopic and saccharide binding properties of lentil and pea isolectins.

The lentil isolectins, CMLcH A and CMLcH B, and pea isolectins, CMPSA A and CMPSA B, are compared in terms of their spectroscopic and saccharide binding properties. The paramagnetic contribution to the solvent proton magnetic relaxation dispersion profiles of solutions of the isolectins of each protein are found to be essentially identical. Electron paramagnetic resonance spectra suggest a high degree of octahedral symmetry at the Mn2+ site for both pairs of isolectins. The near-ultraviolet absorption spectra of CMLcH A and CMLcH B are identical, as are the spectra of CMPSA A and CMPSA B. Carbohydrate binding activities of the isolectins of each protein are compared using hemagglutination, precipitation, and precipitation-inhibition assays, and are found to be identical, although the activities of CMLcH and CMPSA differ somewhat. These results demonstrate that the spectroscopic and saccharide binding properties of the isolectins of CMLcH are essentially identical, as are those of the isolectins of CMPSA, and suggest that native mixtures of the isolectins may be treated as single proteins in further studies.

Disaccharides↗

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↗

PubMed1983

Purification and characterization of a novel abundant protein in rat bile that binds azo dye metabolites and copper.

Following intravenous administration to rats of the azo dye hepatocarcinogen 3'-methyl-N,N-dimethyl-4-aminoazo-[14C]benzene, 60-70% of the injected dose was recovered in bile in 2 h. Approximately 10% of bile radioactivity was trichloroacetic acid-precipitable, not extracted by n-butanol and non-dialyzable. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of bile followed by fluorography revealed two major and several minor proteins to which radiolabelled azo dye metabolites were bound; one of these major proteins (50 kDa) was purified from bile and shown to be homogeneous by SDS-polyacrylamide gel electrophoresis, isoelectric focusing (pI 7) under denaturing conditions and N-terminal analysis. The protein (MBP) comprises 15% of the total bile protein. Amino acid analysis revealed a preponderance of acidic and hydrophobic amino acids. The absorption spectrum of the native protein had a major peak at 280 nm and minor peaks at 345, 400, 600 and 650 nm. The fluorescence spectrum showed a major excitation maxima at 285 and 350 nm and corresponding emission maxima at 345 and 440 nm. Atomic absorption spectroscopy revealed 5 atoms of Cu per mol protein. Approximately 90% of the Cu was EPR silent. MBP did not react with antisera directed against rat serum IgA, albumin or ceruloplasmin; nor did these proteins react against antisera to MBP. Seven distinct peptide bands ranging from 5 to 18 kDa were obtained when MBP was subjected to CNBr cleavage and the digests were analyzed by SDS-polyacrylamide gel electrophoresis. The [14C]-Me-DAB derived radioactivity was present in only two of the peptides, indicating specific binding sites for azodye metabolites.

Animals↗

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

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↗

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↗