ABSTRACT
The superoxide-dismutase (SOD) enzyme, isolated from the halophilic halotolerant bacterium Ba1, was found to be a dimer with a molecular weight of 40 kD and a subunit weight of 23.5 kD. The partial N-terminal sequence showed significant homology to SODs isolated from various sources. Metal analysis showed that SOD from Ba1 contains manganese and iron with the following stoichiometries: 0.9 +/- 0.4 Mn/dimer and 0.6 +/- 0.2 Fe/dimer. Two bands were obtained by isoelectric-focusing, at pI of 4.45 and at 4.40. Native SOD from Ba1 at room temperature was ESR silent. An ESR spectrum of hydrated Mn(II) was obtained from denaturated enzyme. Native enzyme cooled to 97 K showed an ESR spectrum identified as being due to Fe(III). The spectrum was pH-independent. SOD from Ba1 was not inactivated by H2O2. On the basis of these observations, SOD from Ba1 was characterized as MnSOD. The excitation fluorescence spectrum of SOD from Ba1 showed four main peaks in the visible region. The effects on the spectra of KSCN, NaN3, NaF, and ascorbate were examined. Measurements of H2(17)O-nmr relaxation times T1 and T2, for solutions containing E. coli MnSOD and FeSOD, showed no paramagnetic contribution. These results support the assumption that the water molecule at the active site is strongly bound.
Subject(s)
Escherichia coli/enzymology , Gram-Negative Aerobic Bacteria/enzymology , Sodium Chloride/metabolism , Superoxide Dismutase/isolation & purification , Amino Acid Sequence , Binding Sites , Gram-Negative Aerobic Bacteria/metabolism , Linear Models , Molecular Sequence Data , Sensitivity and Specificity , Sequence Homology, Amino Acid , Species Specificity , Spectrometry, Fluorescence , Spectrophotometry , Superoxide Dismutase/chemistryABSTRACT
The intracellular sodium concentration and intracellular volume of human red blood cells were determined from 23Na and 1H NMR spectra. It is shown that sodium dissolved in the intracellular water has a concentration higher than that previously published. The intracellular sodium concentration measured was 11.4 +/- 3.1 mM. A comparison of different NMR methods used to determine sodium concentration is given.
Subject(s)
Erythrocytes/chemistry , Magnetic Resonance Spectroscopy/methods , Sodium/analysis , Erythrocyte Volume , Humans , Sodium/metabolismABSTRACT
A novel spectroscopic method is described for following the kinetics of resealing of hemolysed erythrocyte ghosts. The procedure is based on the broadening of the EPR spectrum of nitroxyl radicals by paramagnetic ions. The method is used to study the effect of Ca2+, Mg2+ and dimethonium ion on the kinetics of resealing.
Subject(s)
Electron Spin Resonance Spectroscopy/methods , Erythrocyte Membrane/physiology , Bis-Trimethylammonium Compounds/pharmacology , Calcium/pharmacology , Humans , Kinetics , Magnesium/pharmacologyABSTRACT
The hydrophobic interaction between spin-labelled stearic acid and spectrin was studied by electron paramagnetic resonance (EPR) and fluorescence quenching. The results are quantitatively interpreted in terms of two types of binding site on spectrin. A comparison between the results of the EPR and fluorescence experiments show the drawback of the fluorescence method in binding studies.
Subject(s)
Spectrin/metabolism , Stearic Acids/metabolism , Binding Sites , Electron Spin Resonance Spectroscopy , Fluorescence , Water/metabolismABSTRACT
Evidence is presented that electrostatic forces play a major role in the interaction between the cell membrane and cytoskeleton of human erythrocytes. Experiments were carried out on the effects of ionic strength variation, Ca2+ and Mg2+ ions, dimethonium ion and lipophilic ions on the release of spectrin from the erythrocyte ghost. In addition it was shown that the release of spectrin for fixed Ca2+ or Mg2+ concentration shows a maximum as a function of Na+ concentration. All results are consistent with the existence of a repulsive electrostatic force between membrane and cytoskeleton.
Subject(s)
Cytoskeleton/metabolism , Erythrocyte Membrane/metabolism , Erythrocytes/metabolism , Aspirin/pharmacology , Bis-Trimethylammonium Compounds/metabolism , Calcium/metabolism , Chlorides/metabolism , Electrochemistry , Humans , Iodine/metabolism , Magnesium/metabolism , Sodium/metabolism , Spectrin/metabolismABSTRACT
Sonicated vesicles of 20-50 nm in diameter consisting of neutral phospholipids and a variety of acidic phospholipids were interacted with polylysine, cytochrome c, Ca2+ and Mg2+. The addition of polycations caused massive aggregation accompanied by an increase of membrane permeability as determined by leakage of fluorescent dye. Aggregation was followed by fusion of the vesicles into structures that in some cases exceeded 1 micron in diameter. Polylysine induced aggregation and appreciable fusion at charge ratios (polylysine/phospholipid) of 0.5-2, while divalent cations did so only at charge ratios (cation/phospholipid) greater than 10. Aggregation and fusion induced by polylysine were dependent also on the size of the polycation, i.e., the longer the molecule the less needed to induce similar aggregation. It appears that, due to the concentration of charges on a single molecule, polylysine is at least an order of magnitude more effective than divalent cations at inducing fusion of membranes. Cytochrome c induced fusion of similar vesicles at moderately acidic pH (pH 4.2).