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Biomedical subjects

A P Minton

Publications and source records attributed to A P Minton.

At least 19 recordsLinked to original sources
PubMed1992

Calcium-linked self-association of human complement C1s.

The weight-average molecular weight of C1s, an activated serine protease subcomponent of human complement C1, has been measured by means of sedimentation equilibrium over a wide range of both protein and calcium ion concentrations. The combined data may be accounted for quantitatively by a simple model for Ca(2+)-dependent self-association of C1s to a dimer. According to this model, the monomer contains a single Ca2+ binding site with K approximately equal to 3 x 10(5) M-1, and the dimer contains three independent Ca binding sites, two having a Ca2+ affinity lower than that of the monomer (K approximately equal to 3 x 10(4) M-1). The third binding site in the dimer, which presumably lies at the interface between the two amino-terminal alpha domains, has a higher Ca2+ affinity (K approximately equal to 1 x 10(8) M-1) and provides the driving force for C1s dimerization in the presence of calcium.

Binding Sites↗
PubMed1992

Model for the role of macromolecular crowding in regulation of cellular volume.

A simple model is proposed to account for large increases in transporter-mediated ion flux across cell membranes that are elicited by small fractional changes of cell volume. The model is based upon the concept that, as a result of large excluded volume effects in cytoplasm (macromolecular crowding), the tendency of soluble macromolecules to associate with membrane proteins is much more sensitive to changes in cell water content than expected on the basis of simple considerations of mass action. The model postulates that an ion transporter may exist in either an active dephosphorylated state or an inactive phosphorylated state and that the steady-state activity of the transporter reflects a balance between the rates of phosphatase-catalyzed activation and kinase-catalyzed inactivation. Cell swelling results in the inhibition of kinase relative to phosphatase activity, thereby increasing the steady-state concentration of the active form of the transporter. Calculated volume-dependent stimulation of ion flux is comparable to that observed experimentally.

Animals↗
PubMed1992

Confinement as a determinant of macromolecular structure and reactivity.

The confinement of macromolecules within enclosures or "pores" of comparable dimensions results in significant size- and shape-dependent alterations of macromolecular chemical potential and reactivity. Calculations of the magnitude of this effect for model particles of different shapes in model enclosures of different shapes were carried out using hard particle partition theory developed by Giddings et al. (J. Phys. Chem. 1968. 72:4397-4408). Results obtained indicate that the equilibrium constants of reactions, such as isomerization, self-association, and site binding, that result in significant change in macromolecular size, shape, and/or mobility may be altered within pores by as much as several orders of magnitude relative to the value in the unbounded or bulk phase. Confinement also produces a substantial size-dependent outward force on the walls of an enclosure. These results are likely to be important within the fluid phase of biological media, such as the cytoplasm of eukaryotic cells, containing significant volume fractions of large fibrous structures (e.g., the cytomatrix).

Biopolymers↗
PubMed1991

Interactions between globular proteins and F-actin in isotonic saline solution.

Solutions of each of three different globular proteins (cytochrome c, chromophorically labeled serum albumin, and chromophorically labeled aldolase), mixed with another unlabeled globular protein or with fibrous actin, were prepared in pH 8.0 Tris-HCl buffer containing 0.15 M NaCl. Each solution was centrifuged at low speed, at 5 degrees C, until unassociated globular protein in solution achieved sedimentation equilibrium. Individual absorbance gradients of both macrosolutes in the mixtures subsequent to centrifugation were obtained via optical scans of the centrifuge tubes at two wavelengths. The gradients of each macrosolute in mixtures of two globular proteins revealed no association of globular proteins under the conditions of these experiments, but perturbation of the gradients of serum albumin, aldolase, and cytochrome c in the presence of F-actin indicated association of all three globular proteins with F-actin. Perturbation of actin gradients in the presence of serum albumin and aldolase suggested partial depolymerization of the F-actin by the globular protein. Analysis of the data with a simple phenomenological model relating free globular protein, bound globular protein, and total actin concentration provided estimates of the respective equilibrium constants for association of serum albumin and aldolase with F-actin, under the conditions of these experiments, of the order of 0.1 microM-1.

Actins↗
PubMed1990

A model for the interaction of muscarinic receptors, agonists, and two distinct effector substances.

The binding of the agonist carbamylcholine to muscarinic receptors in rat heart myocytes from young and aged cultures and in rat atrial membranes has been measured in the absence and presence of GppNHp, pertussis toxin, and/or batrachotoxin. The effect of each of the added substances upon agonist binding was accounted for by a model according to which the receptor may form an equilibrium complex with agonist and either of two distinct effector substances, one of which is postulated to increase the affinity of receptor for agonist and the other of which is postulated to decrease the affinity of receptor for agonist.

Aging↗
PubMed1990

Quantitative characterization of reversible molecular associations via analytical centrifugation.

The ultracentrifuge provides several techniques for the quantitative characterization of reversible small molecule-macromolecule and macromolecule-macromolecule interactions in solution. The nature of the association to be studied determines the preferred technique. High speed centrifugation is the method of choice for characterizing reversible heteroassociations between species of greatly different mass (i.e., sedimentation coefficient). This technique provides a relatively rapid, artifact-free, and thermodynamically rigorous means of quantifying the amount of nonsedimenting or slowly sedimenting free ligand in equilibrium with rapidly sedimenting acceptor-bound ligand at one particular solution composition. Results obtained over a broad range of ligand and/or acceptor concentrations lead to model-independent binding isotherms that may subsequently be analyzed in the context of models for ligand-acceptor association. Lower speed centrifugation to sedimentation equilibrium is the method of choice for characterizing reversible selfassociations and for characterizing heteroassociations between components that cannot be well separated on the basis of sedimentation velocity. In the dilute limit, this technique can provide model-free information about the dependence of weight-average molecular weight of each component upon solution composition, which can subsequently be analyzed in the context of equilibrium models for self- or heteroassociation. At higher concentrations, the models must be generalized to allow for the effect of nonspecific (nonideal) interactions upon sedimentation and association. The use of tracers provides a means for greatly extending the range of solute concentrations and solution compositions over which both types of measurements may be applied, providing enhanced ability to discriminate between alternative proposed mechanisms for self- or heteroassociations.

Chemistry Techniques, Analytical↗
PubMed1989

Hidden self-association of proteins.

Sedimentation equilibrium measurements were carried out on solutions of bovine serum albumin, aldolase, and ovalbumin in phosphate-buffered saline, pH 7.2, at 10 degrees C. The data obtained for each protein were analyzed to yield the dependence of apparent weight-average molecular weight upon protein concentration, over a concentration range of ca 1-200 g/L. Using the approximate theory of Chatelier and Minton [1987) Biopolymers 26, 507-524), models are formulated for the dependence of apparent weight-average molecular weight upon concentration in non-ideal solutions containing proteins which may self-associate according to a monomer/n-mer or a monomer/dimer/tetramer scheme. The concentration dependence data for serum albumin may be accounted for, assuming either no self-association or weak monomer/dimer association. The data for aldolase may be accounted for assuming either weak monomer/dimer or weak monomer/trimer association. The data for ovalbumin may be accounted for assuming either weak monomer/trimer or weak monomer/dimer/tetramer association. The associations do not approach saturation at the highest concentrations studied, and the standard-state free energy changes accompanying self-association amount to less than 4 kcal/mol of intermolecular contacts, suggesting that non-specific clustering of protein molecules at high concentration rather than the formation of specific complexes is being observed.

Animals↗
PubMed1989

Lateral diffusion of membrane proteins in protein-rich membranes. A simple hard particle model for concentration dependence of the two-dimensional diffusion coefficient.

A model for the effect of protein concentration on the rate of lateral diffusion of integral membrane proteins is presented, in which the proteins are represented by equivalent hard circular particles on a surface. As the density of particles increases, the probability of finding a vacancy immediately adjacent to a tracer particle into which it may diffuse decreases, resulting in a concomitant reduction of the tracer diffusion coefficient. Using scaled particle theory to calculate the concentration-dependent probabilities, a simple approximate result is obtained in closed form, that is compared with the results of previously published Monte Carlo lattice simulations and experimental observations.

Diffusion↗
PubMed1988

An automated method for rapid determination of diffusion coefficients via measurements of boundary spreading.

The use of a simple device by which a layer of solvent may be deposited onto a solution of an optically absorbing solute in a cylindrical quartz tube, without substantial mixing of solution and solvent, is described. The spreading of the boundary thus formed may be monitored as a function of time using an automated absorbance scanning device previously described [A. K. Attri and A. P. Minton (1983) Anal. Biochem. 133, 142-152]. A semiautomatic procedure for determining the diffusion coefficient from the time dependence of the shape of the boundary is described and is particularly well-suited for real-time data analysis with a laboratory microcomputer. The diffusion coefficients of several proteins have been measured using the technique reported, and the results are generally in good agreement with values reported in the literature. The feasibility of using this technique in combination with a previously described method for measuring the sedimentation coefficient [A. K. Attri and A. P. Minton (1984) Anal. Biochem. 136, 407-415] to rapidly determine the molecular weight of a protein is established.

Diffusion↗
PubMed1988

Tracer diffusion of globular proteins in concentrated protein solutions.

The diffusion of tracer proteins at low concentration was measured in solutions containing "background" proteins at concentrations of up to 200 g/liter. The fractional reduction of the diffusion coefficient of tracer in the presence of a given weight/volume concentration of background species generally increases with increasing size of tracer species and with decreasing size of background species. The dependence of the diffusion constants of three out of four tracer species upon the concentrations of four background species is accounted for semiquantitatively by a simple hard particle model. Extrapolation of model calculations to higher background concentrations suggests that in solutions containing proteins at concentrations comparable to those found in biological fluid media, the diffusive transport of larger proteins and aggregates may be slower than in dilute solution by several orders of magnitude.

Algorithms↗
PubMed1987

Simultaneous determination of the individual concentration gradients of two solute species in a centrifuged mixture: application to analytical ultracentrifugation.

A procedure for determining the absolute activity of 14C-labeled and 3H-labeled solutes in a mixture from the measured counts per minute in two scintillation energy windows is described. It is shown that the method described here provides a substantially more accurate determination of 3H activity in the presence of a larger 14C activity, and a more accurate determination of 14C activity in the presence of a larger 3H activity, than does the standard dual label analysis implemented in a Beckman LS 3801 scintillation counter. The new dual label procedure is combined with the automated fractionation procedure of Attri and Minton [(1986) Anal. Biochem. 152, 319-328] to permit the gradients of each of two differently radiolabeled solute species in a mixture to be individually determined following centrifugation. It is shown that the sedimentation coefficients of each of two differently labeled noninteracting proteins in a mixture may be readily determined in a sedimentation velocity experiment, and that the molecular weights of each of two such proteins in a mixture may be readily determined in a sedimentation equilibrium experiment.

Carbon Radioisotopes↗
PubMed1986

Soluble fibrin-fibrinogen complexes as intermediates in fibrin gel formation.

Oligomer formation in fibrinogen solutions following addition of thrombin was studied by addition of thrombin inhibitor at various times subsequent to thrombin, followed by size-exclusion chromatography (SEC) on a high-performance SEC column capable of resolving species of molecular weights less than or equal to 10(6). Peaks corresponding to species with 1, 2, 3, and 4 or more times the molecular weight of fibrinogen were detected and quantified via nonlinear least-squares curve-fitting procedures. The evolution of each of these peaks with time is well accounted for by a kinetic model in which the predominant component of each oligomeric molecular weight species is a linear complex of fibrinogen and fibrin. The observed predominance of trimeric over dimeric oligomers even at short times suggests that the thrombin-catalyzed release of the two A fibrinopeptides from a single molecule of fibrinogen is highly cooperative.

Animals↗
PubMed1986

Technique and apparatus for automated fractionation of the contents of small centrifuge tubes: application to analytical ultracentrifugation.

An automated method is described for dividing the contents of small cylindrical centrifuge tubes into fractions deriving from laminae of solution as thin as 0.1 mm in the direction of the cylindrical axis. Experimental data are presented to demonstrate that fractions as small as 1 microliter may be prepared with a standard deviation of less than 3% in volume delivery and that negligible mixing occurs between the contents of adjacent fractions during the fractionation procedure. The method has been used to quantitate the gradients of a variety of radiolabeled proteins formed in sedimentation velocity and sedimentation equilibrium experiments. Sedimentation coefficients and molecular weights calculated from the gradients obtained agree well with literature values and with values obtained by optically scanning the centrifuge tubes (A.K. Attri and A.P. Minton, 1983, Anal. Biochem. 133, 142-152; 1984, Anal. Biochem. 136, 407-415). The present technique combines a spatial resolution equal to that of optical methods of gradient measurement with a sensitivity which may be several orders of magnitude greater, depending upon the specific activity of labeled solute.

Animals↗