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L W Nichol

Publications and source records attributed to L W Nichol.

At least 37 records · Page 2Linked to original sources
PubMed1981

Binding of aldolase to actin-containing filaments. Quantitative reappraisal of the interactions.

Previously reported results of equilibrium-partition experiments on the interaction of aldolase with actin-containing filaments [Walsh, Winzor, Clarke, Masters & Morton (1980) Biochem. J. 186, 89-98] have been subjected to a more rigorous theoretical analysis involving consideration of the consequences of cross-linking interactions between enzyme and filament. The experimental results obtained with F-actin-tropomyosin are best described by a model with one binding site per heptameric repeat unit of filament and a value of 39000 M-1 for the site binding constant, k. Similar analyses of the influence of Ca2+ on aldolase binding to F-actin--tropomyosin--troponin substantiate the existence of two equivalent binding sites (k = 14900 M-1) for the enzyme on each repeat unit of the thin filament. The Ca2+-sensitivity of this interaction reflects either a decrease in the strength of aldolase binding to these two sites (k = 8200 M-1) or the elimination of one site.

Actins↗
PubMed1981

Exclusion chromatography of concentrated hemoglobin solutions. Comparison of the self-association behavior of the oxy and deoxy forms of the alpha 2 beta 2 species.

Theory pertaining to the interpretation of partition chromatography results obtained with self-associating protein systems studied at high total concentrations is extended to permit consideration of situations in which both monomeric and dimeric states partition. This development, which includes considerations of thermodynamic nonideality effects, permits a quantitative correlation of human oxyhemoglobin results reported previously and obtained in this work employing a different stationary matrix of controlled-pore glass beads. The two sets of results, obtained at pH 7.3 and 20 degree C, indicate that the alpha 2 beta 2 species of oxyhemoglobin self-associates. Two types of association pattern, discrete dimerization and an indefinite self-association, are examined. This is done for a realistic range of values for the radius, r. of the effective hard sphere appropriate to the calculation of the covolume of the alpha 2 beta 2 species in the assessment of the thermodynamic nonideality contribution. Assessed values of the isodesmic association constant range from 66 +/- 23 M-1 (r - 2.84 nm) to 154 +/- 26 M-1 (r = 3.13 nm). This mode of indefinite association is marginally favored over dimerization when the larger value of r is considered, the two patterns becoming virtually indistinguishable for the lower value of r. Partition chromatography results are also presented for human deoxyhemoglobin up to a total concentration of 225 g/l, and are analyzed in a similar fashion to show that the indefinite self-association pattern is favored, governed by an isodesmic constant in the range 91 +/- 9 M-1 (r = 2.84 nm) to 223 +/- 84 M-1 (r = 3.13 nm). Comparison of the constants assessed for the oxy and deoxy systems permits discussion of the concept that oxygen binds preferentially to the alpha 2 beta 2 species of deoxyhemoglobin in comparison with its polymers.

Chromatography, Gel↗
PubMed1980

The indefinite self-association of lysozyme: consideration of composition-dependent activity coefficients.

An improved iterative method for computing association constants from sedimentation equilibrium results obtained with self-interacting protein systems is presented which accounts for the composition-dependence of the activity coefficients of all oligomeric species. The method is based on the calculation of viral coefficients from covolume and charge considerations, the statistical mechanical basis of which is discussed in relation to the DLVO theory. The method is applied to results obtained with lysozyme in diethylbarbiturate buffer of pH 8.0 and ionic strength 0.15 at 15 degrees C. It is shown that these results, encompassing a range of total solute concentration up to 19.7 g/liter are consistent with self-association patterns comprising either a monomer--dimer--trimer system or an isodesmic indefinite self-association of the monomer, the latter being favored. A firmer distinction between these possibilities is sought on the basis of the dependence of the weight-average partition coefficient, determined by frontal gel chromatography, on total solute concentration (up to 56.6/liter). This analysis accounts for the composition-dependence of the ratio of the activity coefficients of partitioning monomer in mobile and stationary phases. It is concluded that all results are consistent with an indefinite self-association of lysozyme governed by a single association constant of 4.61 x 10(2) liter/mole.

Animals↗
PubMed1979

Chromatographic evidence of the self-association of oxyhemoglobin in concentrated solutions: its biological implications.

Expressions that take into account the effects of thermodynamic non-ideality, described in terms of a high-order virial expansion, are derived for the concentration-dependence of the weight-average partition coefficient in exclusion chromatography of a single solute and of a solute undergoing reversible self-association. Comparison of the concentration-dependences predicted by those expressions with results obtained for bovine and human oxyhemoglobins on CPG-10-120 porous glass beads in 0.156 I phosphate-chloride buffer, pH 7.3, shows that neither oxyhemoglobin conforms with the concept of it being a single alpha 2 beta 2 entity with Stokes radius of 3.13 nm, the experimental value. Previously published osmotic pressure and sedimentation equilibrium results are also shown to be inconsistent with this concept. On the other hand, both sets of exclusion chromatography results are consistent with the joint operation of thermodynamic non-ideality and reversible association of the alpha 2 beta 2 species. From the magnitude of the equilibrium constant, derived for either of two possible modes of association, it is calculated that only half of the oxyhemoglobin would be in the alpha 2 beta 2 states under conditions of oxygen saturation and a concentration of 320 g/liter, that pertaining in the red blood cell. The consequences of this association phenomenon are discussed in relation to the oxygen binding curves obtained by others in the presence and absence of 2,3-diphosphoglycerate (DPG). An explanation is provided of the observed dependence on hemoglobin concentration of oxygen-binding in the presence of DPG, and of the absence of such an effect in DPG-free solutions. It is concluded that the control of oxygen binding to hemoglobin in the physiological situation involves the joint operation of self-association and allosteric effects.

Animals↗
PubMed1979

Studies of macromolecular heterogeneous associations involving cross-linking: a re-examination of the ovalbumin-lysozyme system.

A system is considered in which a multivalent acceptor interacts with a bivalent ligand in solution to form an array of complexes via multiple binding and cross-linking reactions. With the use of reacted site probability functions expressions are derived in terms of a site binding constant which are of potential use in the interpretation of sedimentation equilibrium and binding results obtained with such systems. Their potential use is explored in relation to results obtained on the interacting ovalbumin-lysozyme system at pH 6.80, ionic strength 0.02. A comparison is made of this interpretation with that based on an interaction pattern involving only multiple binding of ligand in the absence of cross-linking effects. While both interpretations quantitatively describe certain results, it is shown, by invoking further experimental observations on apparent weight-average molecular weight and precipitation behavior, that the more favored interpretation is that involving the operation of a spectrum of forces leading to a large array of ovalbumin-lysozyme complexes, including those of the cross-linked type. It is stressed that the particular ovalbumin-lysozyme system is but one example of interaction between oppositely charged macromolecules and therefore that the derived equations may find wider application to such systems and those known to involve more specific cross-linking interactions.

Chemical Phenomena↗
PubMed1978

The role of zinc in the stabilization of the dimeric form of bacterial alpha-amylase.

Bacterial alpha-amylase was shown by equilibrium and velocity-sedimentation studies to be a monomer-dimer equilibrium system in 0.10M-NaCl/0.015M-calcium acetate/0.010M-EDTA, pH7.0; an association constant of 2.4 X 10(3)M-1 is obtained. Studies of the binding of Zn2+ to alpha-amylase in 0.10M-NaCl/0.005M-calcium acetate, pH7.0, yielded binding curves that exhibit dependence on the concentration of alpha-amylase (Zn2+-free) used in the equilibrium-dialysis experiments. Results are described very satisfactorily by a reaction scheme in which Zn2+ binds exclusively to the dimer of the above monomer--dimer system with an association constant of 1.0 X 10(6)M-1. The present results refute the earlier scheme for dimer stabilization by Zn2+ in which the metal ion formed a cross-link between two non polymerizing monomer units.

Amylases↗
PubMed1978

Uptake of n-(9-anthroyloxy) fatty acid fluorescent probes into lipid bilayers.

A description in thermodynamic terms is given of ligand-membrane interaction which may occur by either or both a binding and a partition process. Results obtained by fluorescence enhancement and polarization techniques on the uptake of n-(9-anthroyloxy) fatty acids by phospholipid bilayers are analysed to show that binding rather than partition effects primarily determine the extent of probe uptake. Liposome concentration-dependence effects are also reported which required that binding results obtained with different probes be compared at fixed lipid concentrations. On this basis it is concluded that as the separation of the anthracene and carboxyl groups within the fatty acid molecule is increased, and hence as the anthracene group moves deeper into the bilayer, the fluorescent probe is bound to the bilayer with greater affinity but is accepted by fewer binding sites within the membrane. Studies on probe uptake at high ionic strength and into negatively charged bilayers indicate that hydrophobic rather than electrostatic interactions make the dominant contribution to the free energy of binding.

Fatty Acids↗
PubMed1977

The polymerization pattern of zinc(II)-insulin at pH 7.0.

Sedimentation equilibrium experiments were conducted at pH 7.0 using solutions of bovine insulin containing 2 mol of zinc(II) ions per six base-mol of insulin. A detailed analysis of these results revealed the existence of a stable zinc-insulin hexamer together with linked polymerization reactions. Specifically these are a background polymerization of zinc-free insulin as previously described by Jeffrey et al. ((1976) Biochemistry 15, 4660--4665) and a slight tendency for the zinc-insulin hexamer to undergo indefinite self-association. Equilibrium constants governing these reactions are reported together with equations which permit calculation of the composition of the solution at any given total concentration. Comment is made on the possible biological significance of this linked polymerization pattern, and on the likely identity of the structure of the stable zinc-insulin hexamer with that previously reported from X-ray crystallographic studies.

Animals↗
PubMed1977

The simulation of the urea cycle: correlation of effects due to inborn errors in the catalytic properties of the enzymes with clinical-biochemical observations.

Steady-state rate equations are written on the basis of information obtained from the literature describing the kinetics of the four enzyme-catalysed reactions comprising the urea cycle. These equations are formulated into a set which also accounts for fluxes of input and output compounds external to the cycle. Numerical integration of this set of equations is performed employing parameters selected to approximate those pertaining to the operation of the urea cycle in normal liver. The result is a pattern of intermediate metabolite concentrations, which forms a basis for the comparison of patterns reflecting the effects of inborn errors of metabolism. The latter are calculated by varying specified kinetic parameters in the numerical integration. Each of the observed clinical syndromes. Hyperammonemia Types I and II, Hyperarginemia, Citrullinemia and Argininosuccinicaciduria, is discussed.

Amino Acid Metabolism, Inborn Errors↗
PubMed1976

Polymerization pattern of insulin at pH 7.0.

Sedimentation equilibrium results, obtained with bovine zinc-free insulin (with and without a component of proinsulin) at pH 7.0, I o.2, 25 degrees C, and up to a total concentration of 0.8 g/l., are shown to be consistent with three different polymerization patterns, all involving an isodesmic indefinite self-association of specified oligomeric species. The analysis procedure, based on closed solutions formed by summing infinite series, yields for each pattern a set of equilibrium constants, It is shown that a distinction between the possible patterns can be made by analyzing sedimentation equilibrium results obtained in a higher total concentration range (up to 4 g/1.) with insulin freed of zinc and proinsulin, account being taken of the composition dependence of activity coefficients. The favored pattern, which differs from that previously reported in the literature, involves the dimerization of monomeric insulin (mol wt 5734), governed by a dimerization constant of 11 X 10(4) M-1 and the isodesmic indefinite self-association of the dimer, described by an association constant of 1.7 X 10(4) M-1. This polymerization pattern is also shown to be consistent with the reaction boundary observed in sedimentation velocity experiments.

Hydrogen-Ion Concentration↗