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Biomedical subjects

A Colosimo

Publications and source records attributed to A Colosimo.

At least 19 recordsLinked to original sources
PubMed1991

A Hypercard program for the identification of biological specimens.

A Hypercard-based software tool developed to provide help in the identification of biological specimens is presented. The package implements a matching algorithm that compares alphanumeric strings and runs on Macintosh computers, though its simple architecture can be transferred to other computers and/or other programming environments. The overall performance of the program and its easy customization to specific problems are demonstrated by discussing at length one application in the field of earthworm identification.

Algorithms↗
PubMed1990

Interaction of hemoglobin with chloride and 2,3-bisphosphoglycerate. A comparative approach.

The equilibrium oxygen-binding properties of hemoglobins from reindeer (Rangifer tarandus tarandus), musk ox (Ovibos muschatos) and a bat (Rousettus aegyptiacus) have been investigated with special reference to the effect of heterotrophic ligands such as chloride and 2,3-bisphosphoglycerate [Gri(2,3)P2]. The results obtained with hemoglobins from reindeer and musk ox indicate that their low oxygen affinity and their insensitivity to Gri(2,3)P2 are not only an intrinsic property of the molecule, as proposed in the case of ruminant hemoglobins, but also the results of the interplay between chloride and Gri(2,3)P2 interactions. In other words, insensitivity of reindeer and musk ox hemoglobins to Gri(2,3)P2 is mainly due to a decreased affinity constant for this cofactor and to an increased affinity constant for chloride anions; this renders more effective the competition of chloride for th anion-binding site. On the other hand bat hemoglobin behaves in a completely different way and could be regarded as a type case of low-affinity hemoglobin since its functional properties are modulated neither by chloride nor by Gri(2,3)P2. The results are discussed in the light of the amino acid residues which are known to be involved in the binding of organic phosphates.

Allosteric Regulation↗
PubMed1990

What the intermediate compounds in ligand binding to hemoglobin tell about the mechanism of cooperativity.

The populations of the intermediates in concentrated solutions of hemoglobin A0 equilibrated at various PCO values, pH 7.0, 0.1 M KCl, and 20 degrees C, have been determined using cryogenic methods. Data on CO saturations and distributions of intermediates were analysed in terms of the free energies of dimer-tetramer assembly of the intermediates (G.K. Ackers and F.R. Smith, Annu. Rev. Biophys. Chem. 16 (1987) 583). The cooperative free energy value of the singly ligated species was approximately one-half the total cooperative energy. The cooperative free energy value of the doubly ligated species was not significantly different from that of carboxyhemoglobin. Because of experimental error, the observed difference in concentrations among the populations of the doubly ligated species cannot be taken as indicative of their functional heterogeneity. Additional studies on some NO intermediates have emphasized that (alpha 1 beta 1)(alpha 2 beta 2)X, a key intermediate in the formulation of the 'third-state' hypothesis in the deoxy/cyanomethemoglobin system, has a free energy value for dimer-tetramer assembly which is critically dependent on the nature of the ligand X as suggested by Ackers and Smith (reference as cited above).

Carboxyhemoglobin↗
PubMed1990

SIMFIT: a microcomputer software-toolkit for modelistic studies in biochemistry.

A software package suitable for personal computers and designed to handle simulation and fitting problems related to the study of biomolecules under pre-steady and steady state conditions is presented, and its overall architecture as well as the implemented algorithms illustrated. The peculiar features of the package are: (i) integrated capability of simulating dynamic models and fitting to them experimental data; (ii) handling of stiff problems; (iii) free use of algebraic as well as differential equations; (iv) objective comparison of models of different complexity. The above features are discussed through a number of examples taken from the direct experience of the authors in enzyme kinetics and ligand binding.

Biochemical Phenomena↗
PubMed1990

Microcomputer-assisted periodic pattern recognition in the primary structure of proteins.

Some relevant problems concerning the computational methods able to predict the higher structures of biopolymers from the sequence of their monomers will be introduced and a software package, LAPS (Look At Primary Structures) able to tackle many of those problems even on microcomputers, will be illustrated in its architecture and performances. In discussing some exemplary applications, special emphasis will be given to the recognition of repetitive patterns.

Amino Acid Sequence↗
PubMed1989

Allosteric formulation of thermal transitions in macromolecules, including effects of ligand binding and oligomerization.

We examine the effects of concentration (aggregation), buffers, and ligation, under conditions of either constant ligand activity or limited total amount of ligand, upon thermal denaturation of macromolecules as measured by scanning calorimetry. In doing so we utilize and extend an earlier generalized allosteric treatment [S. J. Gill, B. Richey, G. Bishop, and J. Wyman (1985) Biophys. Chem. 21, 1-14], applicable to ligand binding, enthalpy changes, and volume changes in a macromolecular system. The approach is contrasted with formulations based on the idea of structural domains. We show how information from the full scanning calorimetric curves can be utilized in arriving at and testing appropriate models for observed behavior in selected examples.

Allosteric Regulation↗
PubMed1987

A kinetic model for lipoxygenases based on experimental data with the lipoxygenase of reticulocytes.

A comprehensive kinetic model for lipoxygenase catalysis is proposed which includes the simultaneous occurrence of dioxygenase and hydroperoxidase activities and is based on the assumption of a single binding site for substrate fatty acid and product. The aerobic reaction of purified lipoxygenase from rabbit reticulocytes with 9,12(Z,Z)-octadecadienoic acid (linoleic acid) as substrate was studied. The rate constants and the dissociation constants of this enzyme were calculated for the model from progress curves; the model describes correctly the experimental data. The following kinetic features of the reticulocyte enzyme are assumed to apply generally to lipoxygenases. (a) The enzyme shows autoactivation by its product. (b) The rate-limiting step is the hydrogen abstraction. (c) Both substrate fatty acid and its product are competitive inhibitors of the lipoxygenase. (d) Lowering the oxygen concentration enhances the degree of substrate inhibition, whereas product inhibition is not influenced. (e) If substrate is in excess the oxygen concentration determines the share of dioxygenase and hydroperoxidase activities of the enzyme. As predicted from the model it was found that at low concentrations of oxygen the regio- and stereo-specificities of the dioxygenation are diminished. During the autoactivation phase the steady-state approximation does not hold.

Animals↗
PubMed1985

Cytochrome c-551 and azurin oxidation catalysed by Pseudomonas aeruginosa cytochrome oxidase. A steady-state kinetic study.

The kinetics of oxidation of azurin and cytochrome c-551 catalysed by Pseudomonas aeruginosa cytochrome oxidase were re-investigated, and the steady-state parameters were evaluated by parametric and non-parametric methods. At low concentrations of substrates (e.g. less than or equal to 50 microM) the values obtained for Km and catalytic-centre activity are respectively 15 +/- 3 microM and 77 +/- 6 min-1 for azurin and 2.15 +/- 0.23 microM and 66 +/- 2 min-1 for cytochrome c-551, in general accord with previous reports assigning to cytochrome c-551 the higher affinity for the enzyme and to azurin a slightly higher catalytic rate. However, when the cytochrome c-551 concentration was extended well beyond the value of Km, the initial velocity increased, and eventually almost doubled at a substrate concentration greater than or equal to 100 microM. This result suggests a 'half-hearted' behaviour, since at relatively low cytochrome c-551 concentrations only one of the two identical binding sites of the dimeric enzyme seems to be catalytically active, possibly because of unfavourable interactions influencing the stability of the Michaelis-Menten complex at the second site. When reduced azurin and cytochrome c-551 are simultaneously exposed to Ps. aeruginosa cytochrome oxidase, the observed steady-state oxidation kinetics are complex, as expected in view of the rapid electron transfer between cytochrome c-551 and azurin in the free state. In spite of this complexity, it seems likely that a mechanism involving a simple competition between the two substrates for the same active site on the enzyme is operative. Addition of a chemically modified and redox inactive form of azurin (Hg-azurin) had no effect on the initial rate of oxidation of either azurin and cytochrome c-551, but clearly altered the time course of the overall process by removing, at least partially, the product inhibition. The results lead to the following conclusions: (i) reduced azurin and cytochrome c-551 bind at the same site on the enzyme, and thus compete; (ii) Hg-azurin binds at a regulatory site, competing with the product rather than the substrate; (iii) the two binding sites on the dimeric enzyme, though intrinsically equivalent, display unfavourable interactions. Since water is the product of the reduction of oxygen, point (iii) has important implications for the reaction mechanism.

Apoproteins↗
PubMed1985

Mechanism of control of cytochrome oxidase activity by the electrochemical-potential gradient.

Cytochrome c oxidation by bovine cytochrome oxidase embedded into liposomal vesicles with high respiratory control ratio (RCR = 6-10) has been studied by rapid-mixing experiments in the presence and absence of different ionophores. Kinetic analysis of the reaction indicates a linkage between the intrinsic activity of the enzyme, the efficiency of coupling and the electrochemical potential across the membrane. A simple model, based on two allosteric states with different catalytic properties in rapid equilibrium, is presented and successfully applied in the simulation of the observed time-course.

Animals↗
PubMed1985

Kinetics of redox-linked proton pumping activity of native and subunit III-depleted cytochrome c oxidase: a stopped-flow investigation.

The kinetics of oxidation of reduced cytochrome c by cytochrome c oxidase reconstituted into unilamellar vesicles (COV) has been followed by stopped-flow method in the time range 3 msec-1 sec. In the presence of valinomycin, the oxidation of cytochrome c is linked to proton ejection in the external medium, with an apparent stoichiometry (H+/e-) of 0.93 +/- 0.22, under conditions in which the enzyme is in the more active "pulsed" state (i.e., having undergone oxidation-reduction cycles). The time course of reaction indicates that the conformational change(s) involved in coupling the redox reaction to proton translocation is fast. Similar experiments carried out with cytochrome c oxidase depleted of subunit III show that proton-pumping is maintained, although with a lower efficiency (H+/e- = 0.5). The number of protons ejected per electron appears to be correlated to the value of the respiratory control ratio; although this result is partly due to an increase in the rate of diffusion back into the vesicles, a relationship between the respiratory control ratio and the efficiency of the proton pump may be inferred, suggesting a control of the H+/e-ratio.

Animals↗
PubMed1979

A re-evaluation of some basic structural and functional properties of Pseudomonas cytochrome oxidase.

Determinations of iron content and dry-weight measurements on samples of Pseudomonas cytochrome oxidase were coupled with sodium dodecyl sulphate/polyacrylamide-gel-electrophoresis studies of both the native protein and covalently cross-linked oligomers in order to estimate the enzyme's molecular weight and spectral absorption coefficients. A value of epsilon(ox.) (410)=282x10(3) litre.mol(-1).cm(-1) was calculated for a dimeric protein molecule having a total molecular weight of 122000 (based on iron analysis). Steady-state kinetic observations of the enzyme-catalysed oxidation of reduced azurin by nitrite indicated a marked increase in enzyme inactivation as the pH was raised from 5.7 to 7.2. Since NO, a product of the nitrite reductase activity of Pseudomonas cytochrome oxidase, is known to bind to the enzyme, a study was undertaken to try to assess the potential of NO as a product inhibitor. Investigations showed that samples of the oxidized protein at pH values 4, 5 and 6 bound NO to both haem c and d(1) components, but oxidized enzyme samples at pH7 and above formed their reduced ligand-bound forms when placed under an atmosphere of the gas. Ascorbate-reduced enzyme samples at pH4, 5, 6 and 7 were also found to bind NO at both haem components, although at pH7 the rate of haem c binding was very slow. At pH8 and 9 only the ferrohaem d(1) bound NO. Titration experiments on the reduced protein over the pH range 5-7, with nitrite as a precursor of NO, showed that the haem d(1) had a much higher affinity than the haem c: experiments at pH5.2 and 5.9 with NO-equilibrated solutions revealed the same pattern of behaviour with the oxidized enzyme.

Chemical Phenomena↗
PubMed1978

The reaction of Pseudomonas aeruginosa cytochrome c-551 oxidase with oxygen.

The reaction of ascorbate-reduced Pseudomonas cytochrome oxidase with oxygen was studied by using stopped-flow techniques at pH 7.0 and 25 degrees C. The observed time courses were complex, the reaction consisting of three phases. Of these, only the fastest process, with a second-order rate constant of 3.3 X 10(4) M-1.S-1, was dependent on oxygen concentration. The two slower processes were first-order reactions with rates of 1.0 +/- 0.4s-1 and 0.1 +/- 0.03s-1. A kinetic titration experiment revealed that the enzyme had a relatively low affinity constant for oxygen, approx. 10(4)M-1. Kinetic difference spectra were determined for all three reaction phases, showing each to have different characteristics. The fast-phase difference spectrum showed that changes occurred at both the haem c and haem d1 components of the enzyme during this process. These changes were consistent with the haem c becoming oxidized, but with the haem d1 assuming a form that did not correspond to the normal oxidized state, a situation that was not restored even after the second kinetic phase, which reflected further changes in the haem d1 component. The results are discussed in terms of a kinetic scheme.

Chemical Phenomena↗
PubMed1978

Properties of cytochrome c modified by attachment to a carbohydrate polymer.

By enzymic digestion of the polysaccharide part of the covalent complex between cytochrome c and Sephadex G-200, a new water-soluble cytochrome c derivative is obtained (called cytochrome cr). Measurement of the free amino groups of this derivative indicates that on average the molar ratio between cytochrome c and polysaccharide is close to 1. Chemical determination of the sugar content gives a value of approx. 24000 for the molecular weight of cytochrome cr. On these bases the soluble cytochrome cr complex may be thought of as a folded protein to which a long polysaccharide tail is covalently bound. The functional behaviour of cytochrome cr is much more similar to that of the native molecule than to that of the insoluble complex (cytochrome ci). In particular the kinetics of the reaction of cytochrome cr and cytochrome cn (native) with ascorbate, ferrocyanide-ferricyanide, O2 and cytochrome c oxidase were investigated in considerable detail. The results of these experiments, together with the observation that the insoluble complex of cytochrome c is a very poor substrate of cytochrome c oxidase [Colosimo, Brunori & Antonini (1976) Biochem. J. 153, (657-661], indicate that hindrance effects constraining the approach between cytochrome cr and its oxidase are of greater importance than specific chemical modifications in determining the functional behavior of the protein.

Cytochrome c Group↗