Thermal sensitivity of mares' milk proteins.
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Animals↗
Biomedical subjects
F Bonomi
Publications and source records attributed to F Bonomi.
Myohemerythrin from the sipunculid, Phascolopsis gouldii: purification, properties and amino acid sequence.
Two previously unknown isoforms, labelled iso I and iso II, of the oxygen-carrying protein, myohemerythrin, have been isolated from carcasses of the sipunculid worm, Phascolopsis gouldii. The two isoforms have non-identical N-terminal amino acid sequences and slightly different absorption spectra in the met form. Far-ultraviolet circular dichroism shows that iso I contains approximately 69% alpha-helix. The complete amino acid sequence for iso I was obtained. The molecular weight calculated from this amino acid sequence and including the active site Fe-O-Fe unit, is 13,829. All of the physical and chemical properties of iso I noted above, including the amino acid sequence, are very similar to those of T. zostericola myohemerythrin. Except for the amino acid sequence, these properties are also very similar to that of a subunit in hemerythrin, the octameric analog found in hemerythrocytes. Only 58 of the 113 residues in P. gouldii hemerythrin are conserved in iso I. Sequence comparisons were used to help identify residues responsible for maintaining the common tertiary and diiron site structures in hemerythrin and myohemerythrin. The seven iron ligand residues previously identified in crystal structures of hemerythrin and myohemerythrin are conserved in iso I. However, none of the ten residue pairs previously identified as engaging in direct salt-bridge or hydrogen bond interactions between subunits in the hemerythrin octamer are conserved in iso I.
Amino Acid Sequence↗
1H NMR studies on the oxidized ferredoxin from Clostridium pasteurianum.
The 8Fe-8S ferredoxin from Clostridium pasteurianum was investigated by 1D and 2D 1H NMR. Spectra of a well-structured, full native preparation of the oxidized protein in 1 M NaCl at pH 8.0 are presented. Assignments of non-isotropically shifted resonances in the diamagnetic region of the spectrum, namely those of the unique aromatic residues F30 and Y2, are presented for the first time.
Clostridium↗
Uptake of iron by apoferritin from a ferric dihydrolipoate complex.
A study was made on the uptake of iron by horse spleen apoferritin, by using as an iron source the same ferric dihydrolipoate complex which represents the major product in the anaerobic removal of ferritin-bound iron by dihydrolipoate at neutral pH. The ferric dihydrolipoate complex was chemically synthesized and used as an iron donor to apoferritin. Iron uptake was studied, at slightly alkaline pH and in anaerobic conditions, as a function of the concentration of both the iron donor and apoferritin. Isolation of ferritin from mixtures of ferric dihydrolipoate and apoferritin, and subsequent identification of the oxidation state of ferritin-bound iron, showed that the first metal atoms were taken up in the ferrous form and that this early step was accompanied by accumulation of ferric iron. Total iron uptake increased with the molar ratio of complex to apoprotein and ranged over 25-40% of the iron being supplied. The amount of ferrous iron found inside the protein did not exceed 50-60 mol iron/mol ferritin after a 48-h incubation. At this time, ferric iron represented a significant fraction of the iron found in the isolated ferritin. Analytical and spectroscopic data indicated that fractional rates and equilibria for disassembly of the ferric complex in the presence of apoferritin were independent of the concentration of the protein and of the complex itself.
Animals↗
Interaction of Zn2+ with the bovine-heart mitochondrial bc1 complex.
A study is presented of the effect of Zn2+ on the enzymatic properties of the bovine-heart cytochrome-bc1 complex. Micromolar concentrations of Zn2+ reversibly inhibit the cytochrome-c reductase activity of either the cholate-solubilized or liposome-reconstituted complex. Kinetic analysis of the redox reactions of the cytochromes indicate that Zn2+ affects the activity of the complex at the quinol oxidation site. The following have been determined: (a) Zn2+ inhibits the pre-steady-state reduction of cytochrome c1 by duroquinol either in the absence or in the presence of antimycin, (b) it does not inhibit the reduction of b cytochromes in the absence of antimycin or in the presence of myxothiazol, (c) it inhibits cytochrome-b reduction in the presence of antimycin. Furthermore Zn2+ inhibits the antimycin-promoted oxidant-induced extrareduction of b cytochromes. Addition of Zn2+ to reduced bc1 complex causes a red shift in the absorption spectrum of cytochrome b566 and a substantial decrease in the signal intensity of the EPR spectrum of the Fe-S protein. This is interpreted as an interaction of Zn2+ with the 2Fe-2S-cluster region of the Fe-S protein, thus giving rise to inhibition of the reductase activity and of the antimycin-insensitive reduction route of b cytochromes. A Scatchard-plot of 65Zn2+ binding to the native isolated complex gave a straight line from which a value of three binding sites and a single dissociation constant of 3 x 10(-6) M can be calculated, which is practically equal to the concentration causing 50% inhibition of electron flow.
Animals↗
Purification and properties of a membrane-bound NADH-cytochrome-b5 reductase from erythrocytes of the sipunculid worm, Phascolopsis gouldii.
The purification to homogeneity of the membrane-bound NADH-cytochrome-b5 reductase from erythrocytes of the sipunculid, Phascolopsis gouldii is reported. This highly purified reductase has allowed more detailed characterizations of its molecular and kinetic properties than was possible in a previous study (Utecht, R.E. and Kurtz, D.M., Jr. (1988) Biochim. Biophys. Acta 953, 164-178). The reductase has a molecular weight of 34,000 and contains FAD as the prosthetic group. In aqueous solution containing 0.5 vol% Triton X-100, the reductase forms an aggregate of Mr approximately 220,000. A higher purity preparation of P. gouldii erythrocyte b5 was also obtained. The combination of purified, solubilized reductase and cytochrome b5 was shown to catalyze the quantitative two-electron reduction of [Fe(III),Fe(III)]methemerythrin to [Fe(II),Fe(II)]deoxyhemerythrin by NADH. The P. gouldii NADH-cytochrome b5 reductase is the first from hemerythrin-containing erythrocytes to be purified and characterized. This methemerythrin reduction system appears to be analogous to methemoglobin reductases from vertebrate erythrocytes.
Animals↗
Molecular aspects of the removal of ferritin-bound iron by DL-dihydrolipoate.
The removal of ferritin-bound iron by the physiologic dithiol DL-dihydrolipoate was studied over the pH range 5.5-9.0. A novel method was devised for the determination of iron removal, making it possible to study the actual release of iron from ferritin, regardless of the oxidation state or complexation form. The overall iron-removal process appears to depend upon a balance between the deprotonation of the dithiol and the protolytic dissolution of the iron core inside the ferritin molecule. The amount of iron removed at equilibrium increases with the pH, at any of the dihydrolipoate/ferritin iron ratios tested. The formation of the binuclear iron-dithiol complex [Fe2(dihydrolipoate)3]-3 is not strictly required for iron mobilization, but it seems to affect the efficiency of the dithiol in iron mobilization by providing a stable complexation form for the released iron outside the ferritin protein shell. Comparison of the release of ferritin-bound iron by free and immobilized dihydrolipoate indicates that mobility of the dithiol is mandatory for the removal process to take place.
Chemical Phenomena↗
Removal of ferritin-bound iron by DL-dihydrolipoate and DL-dihydrolipoamide.
The naturally occurring dithiols DL-dihydrolipoate and DL-dihydrolipoamide were tested for their ability in the removal of ferritin-bound iron. Both compounds remove the iron stored inside the protein by complexing it in the ferric form. The iron can be reduced to the ferrous form by excess dithiol, but this is not necessary for complete removal. Reaction is complete in few hours and, at molar ratios of chelator to metal higher than 10, more than 60% of the ferritin-bound iron was removed. The amount of iron stored in the ferritin molecule does not affect the rate and the yield of the removal reaction. The iron-removing ability of DL-dihydrolipoate was found to be identical to that of an equimolar solution of sodium dithionite, and to be pH-dependent. Results are discussed in terms of the molecular architecture of ferritin and of the chelators, and their possible physiological relevance is pointed out.
Chromatography, Gel↗
Enzymic synthesis of the 4Fe-4S clusters of Clostridium pasteurianum ferredoxin.
Ex novo enzymic synthesis of the two 4Fe-4S clusters of Clostridium pasteurianum ferredoxin has been achieved by incubation of the apoprotein with catalytic amounts of the sulfurtransferase rhodanese in the presence of thiosulfate, DL-dihydrolipoate and ferric ammonium citrate. This enzymic reconstitution procedure was compared to a chemical one, in which the enzyme was replaced by sodium sulfide. A further comparison was made with the results previously obtained in the enzymic synthesis of the 2Fe-2S cluster of spinach ferredoxin, allowing the following conclusions to be drawn. The nature of the cluster to be inserted into the reconstituted iron-sulfur protein is determined by the apoprotein itself. The refolding of the structure of the iron-sulfur proteins around the newly inserted cluster is the rate-limiting step in both chemical and enzymic reconstitution. Rhodanese appears to play a role in the recovery of the native architecture of the reconstituted iron-sulfur protein(s). The extension to the 4Fe-4S centers of the rhodanese-based biosynthetic system allows this enzymic route to be proposed as a general way to the in vivo synthesis of iron-sulfur structures.
Apoproteins↗
Enzymic synthesis of the iron-sulfur cluster of spinach ferredoxin.
A biologically active spinach ferredoxin was reconstituted from the apoprotein by incubation with catalytic amounts of the sulfurtransferase rhodanese in the presence of thiosulfate, reduced lipoate and ferric ammonium citrate. Analytical and spectroscopical features of the reconstituted ferredoxin were identical to those of the native one; yield of the reconstitution reaction was 80%. Yields and kinetic parameters of the enzymic and chemical reconstitution were also compared. The higher efficiency of the enzymic system is ascribed to a productive interaction between rhodanese and apoferredoxin favouring the process of cluster build-up and insertion. The physiological relevance of this synthetic activity is discussed.
Animals↗
Chromatographic separation of extruded iron-sulfur cores from the apoproteins of Clostridium pasteurianum and spinach ferredoxins in aqueous Triton X-100/urea.
Iron-sulfur core extrusions from spinach [( 2Fe-2S]) and Clostridium pasteurianum (2[4Fe-4S]) ferredoxins in aqueous Triton X-100/urea containing excess benzenethiol yield quantitatively [FenSn(SPh)4]2- with n = 2 and n = 4, respectively. The iron-sulfur cluster can be separated from the corresponding apoprotein by rapid passage of the extrusion mixture over a small anaerobic column of Whatman DE-52 anion-exchange cellulose. Essentially quantitative recovery of [FenSn (SPh)4]2- is achieved in the eluate. The apoprotein remaining on the column can be eluted with 0.5 M NaCl. Most of the residual Triton X-100 and benzenethiol can be removed by passage of the apoprotein eluate over a small column of Bio-Beads SM-2, a hydrophobic polystyrene adsorbent. Apoprotein recovery is comparable to that obtained by other chromatographic methods. At least with spinach ferredoxin, the apoprotein prepared in this fashion can be reconstituted. The procedures developed in this work are potentially most applicable to selective removal of [2Fe-2S] and [4Fe-4S] centers from a multicenter enzyme without irreversible denaturation.
Apoproteins↗
[Analysis of diastolic heart function. Principles and considerations on the use of 2 harmonics].
In this study the authors have briefly exposed the mathematical bases about the application of the Fourier transform in cardiac radioisotopic examinations. It has pointed out the usefulness of two harmonics for clinical applications.
Diastole↗
Modification of the thermodynamic properties of the electron-transferring groups in mitochondrial succinate dehydrogenase upon binding of succinate.
The redox properties of the covalently-bound flavin and of the tetrahedral iron-sulfur center S1 of succinate dehydrogenase were studied as a function of the binding of different ligands to the enzyme. The midpoint potential of both flavin and S1 increases by some 200 mV when protein binds succinate to a site having Kdsucc = 0.8-1.0 mM, thus different from the substrate binding site. Succinate binding increases the potential of the oxidized flavin/semiquinone half-cell more than that of the semiquinone/reduced flavin one: this results in higher semiquinone formation with increasing succinate. Malonate and fumarate appear to mimic, in this regard, the effect of succinate. The increase in midpoint potential of S1 upon binding of dicarboxylic acid is related to an increase in hydrophobicity of the cluster environment. The possible molecular basis for the modulation of the flavin potential is discussed together with the significance of this shift on the catalytic behaviour of the protein.
Binding Sites↗
The inhibition of rhodanese by lipoate and iron-sulfur proteins.
A study was made on the effects of DL-dihydrolipoate, lipoate and iron-sulfur proteins on the activity of rhodanese (EC 2.8.1.1) with dihydrolipoate or cyanide as acceptors. DL-Dihydrolipoate inactivates rhodanese, lipoate does not, and the opposite occurs with the sulfur-free form of the transferase. The observed effects vary with the sulfane sulfur acceptor from rhodanese (i.e., dihydrolipoate or cyanide) and depend on intramolecular oxidation of the catalytic sulfhydryl or on formation of a mixed disulfide with dihydrolipoate. Thiosulfate protects against inactivation by reloading the active-site cysteine with persulfide sulfur. The inhibition of sulfur transfer by iron-sulfur proteins appears related to the amount of native iron-sulfur structure interacting with rhodanese. The implications of the results for a possible biological role of rhodanese are considered.
Animals↗
Competing models for the analysis of red cell survival obtained with 51Cr labelling technique.
A computer program for the analysis of 51Cr labelled red cell survival data was used in 84 patients and 8 normal subjects to estimate the parameters of 7 competing models derived from different hypotheses of the red cell destruction process. The purpose was to establish the optimal complexity of a red cell survival model to be used. Senescence or random destruction were the most common identified patterns of red cell death (79% of the cases). Models assuming different levels of death probability according to the age of cells, or 2 populations of cells with different death probabilities, were selected in 21% of the cases. These latter patients were characterized by the lowest values of MRCL. The conclusion of the work was that an automatic procedure for selecting the best model of red cell destruction is necessary when information concerning the mechanism of red cell death is required. For the more practical aim of obtaining an accurate measurement of the MRCL, the minimal set of competing models should account of at least 2 different populations of cells in addition to the random or senescence destruction models.
Anemia, Hemolytic↗
Kinetics and equilibria of active site core extrusion from spinach ferredoxin in aqueous N,N-dimethylformamide/Triton X-100 solutions.
The nature and reactivity of each species participating in core extrusion of the [2Fe-2S] cluster of spinach ferredoxin by benzenethiol have been investigated in a novel aqueous medium containing 10-40% (v/v) N,N-dimethylformamide (DMF) plus 5-6% (v/v) of the nonionic detergent, Triton X-100. By use of visible absorption and circular dichroism spectroscopies, it is found that prior to addition of benzenethiol, modifications of the ferredoxin induced by DMF are reversible. Both the extent and rate of modification are dependent on DMF concentration and on ionic strength. At ferredoxin concentrations near 0.1 mM, complete modification by DMF is shown to be unnecessary for quantitative core extrusion provided that the benzenethiol concentration is greater than or equal to 50 mM and the DMF concentration is greater than or equal to 10% (v/v). When benzenethiol is added after DMF, core extrusion occurs in two phases. Our interpretation is that the rapid phase corresponds to core extrusion of that portion of the ferredoxin already modified by DMF and that the slower phase corresponds to modification of the remaining portion of the ferredoxin by DMF. When DMF solutions containing benzenethiol and Triton X-100 in various ratios are mixed with aqueous solutions of spinach ferredoxin, the rate of core extrusion appears to be determined both by the rate at which the ferredoxin is modified and by the Triton/benzenethiol ratio. Under all conditions examined we observe significantly faster rates of core extrusion from spinach ferredoxin in aqueous Triton/DMF than in 80/20 (v/v) hexamethylphosphoramide/water, a previously used core extrusion medium. Our results suggest a catalytic role for the micellar phase.
Binding Sites↗
Role of flavin and iron sulfur centers in the transition of succinate dehydrogenase from the activated to the non-activated form.
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Circular Dichroism↗
Catalytic and molecular modifications of succinate dehydrogenase by monovalent inorganic anions.
The enzymatic activity and the oxidation state of soluble, activated, substrate-reduced succinate dehydrogenase are modified by the presence of bromide. The anion inhibits the enzyme by two different mechanisms which depend on the ratio of bromide to succinate. At high ratios binding of two bromide ions is required and a competitive inhibition is observed: removal of succinate from the substrate binding site (Kd = 0.1 mM) leads to oxidation of the flavin. At lower ratios but with sufficient succinate to saturate a site with Kd = 1.52 mM, uncompetitive inhibition by a single bromide ion is observed. Mechanisms, as well as the possible physiological significance of the novel type of regulation of succinate dehydrogenase, are discussed.
Anions↗