Peter D. Jeffrey

The direct analysis of sedimentation equilibrium results obtained with polymerizing systems

Theory is presented in relation to sedimentation equilibrium results obtained with polymerizing systems, which permits evaluation of the activity of the monomer as a function of total weight concentration. In contrast to established methods, the suggested procedure does not involve the solution of simultaneous equations which are sums of exponentials or the determination of weight-average molecular weights. A major advantage of the method is that it avoids errors inherent in differentiation and integration steps. An extrapolation to infinite filution is involved, but this is to a defined limit and is uncomplicated by the existence of critical points in the relevant plot. The method is capable of detecting possible volume changes inherent on polymer formation, of treating systems where activity coefficients of solute species are functions of total concentration and of describing the system in terms of relevant equilibrium constants. These points and comparisons with existing methods of analysis are illustrated with numerical examples and with results obtained with lysozyme at pH 6.7. The lysozyme results are interpretable in terms of either a non-ideal monomer-dimer system or a monomer-dimer-trimer system.

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.

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.

The self-association of zinc-free bovine insulin. A single model based on interactions in the crystal that describes the association pattern in solution at pH 2, 7 and 10

Sedimentation equilibrium studies are used to establish that a new pattern for the self-association of zinc-free insulin in solution is applicable over a wide range of conditions of pH, ionic strength and temperature. In this pattern, which is based on information from the existing literature on the X-ray crystal structure of insulin, the insulin monomer is viewed as having two distinct faces both capable of self-interaction. Sedimentation equilibrium experiments were analysed using expressions formulated for this association pattern that describe the dependence of weight average molecular weight and monomer concentration on total protein concentration. It has thereby been possible to obtain values for the two association constants which govern the system for each set of conditions studied, due allowance having been made for composition dependent non-ideality effects. Furthermore, by relating the pH, temperature and ionic strength dependence of the association constants with properties of various amino acid residues on the surface of the insulin monomer, it has also been possible to assign tentatively each constant to a particular reaction domain.

Improved detection of anti-Jo-1 antibody, a marker for myositis, using purified histidyl-tRNA synthetase

The increased detection of anti-Jo-1 antibody afforded by the use of the purified antigen, histidyl-tRNA synthetase, in counterimmunoelectrophoresis is demonstrated. Using purified antigen, anti-Jo-1 antibody was detected in the sera of 16/33 (48.5%) patients with confirmed myositis and in 20/45 (44.5%) patients with confirmed or possible myositis. This rate is approximately double that obtained with commercial thymus extracts both in this study and seven others reported in the literature. The presence of antibody shows marked correlation with the activity of myositis at the time of serum sampling and with the presence of interstitial lung disease. Detection rates are similar in patients with polymyositis and dermatomyositis both with and without additional connective tissue diseases.

Escherichia coli PII protein: purification, crystallization and oligomeric structure

The Escherichia coli signal transduction protein PII, product of the glnB gene, was overproduced and purified. The predicted molecular weight of the protein based on the correct nucleotide sequence is 12,427 and is very close to the value 12,435 obtained by matrix‐assisted laser desorption mass spectrometry. Hexagonal crystals of the unuridylylated form of PII with dimensions 0.2 × 0.2 × 0.3 mm were grown and analysed by X‐ray diffraction. The crystals belong to space group P63 with a=b=61.6Å,c= 56.3 Å and V m of 2.5 for one subunit in the asymmetric unit. A low‐resolution electron density map showed electron density concentrated around a three‐fold axis, suggesting the molecule to be a trimer. A sedimentation equilibrium experiment of the meniscus depletion type was used to estimate a molecular weight of 35,000 ± 1,000 for PII in solution. This result is consistent with the native protein being a homotrimer.

Interleukin-3 Binding to the Murine βIL-3 and Human βc Receptors Involves Functional Epitopes Formed by Domains 1 and 4 of Different Protein Chains

Interleukin-3 (IL-3) is a cytokine produced by activated T-cells and mast cells that is active on a broad range of hematopoietic cells and in the nervous system and appears to be important in several chronic inflammatory diseases. In this study, alanine substitutions were used to investigate the role of residues of the human β-common (hβc) receptor and the murine IL-3-specific (βIL-3) receptor in IL-3 binding. We show that the domain 1 residues, Tyr15 and Phe79, of the hβc receptor are important for high affinity IL-3 binding and receptor activation as shown previously for the related cytokines, interleukin-5 and granulocyte-macrophage colony-stimulating factor, which also signal through this receptor subunit. From the x-ray structure of hβc, it is clear that the domain 1 residues cooperate with domain 4 residues to form a novel ligand-binding interface involving the two protein chains of the intertwined homodimer receptor. We demonstrate by ultracentrifugation that the βIL-3 receptor is also a homodimer. Its high sequence homology with hβc suggests that their structures are homologous, and we identified an analogous binding interface in βIL-3 for direct IL-3 binding to the high affinity binding site in hβc. Tyr21 (A–B loop), Phe85, and Asn87 (E–F loop) of domain 1; Ile320 of the interdomain loop; and Tyr348 (B′–C′ loop) and Tyr401 (F′–G′ loop) of domain 4 were shown to have critical individual roles and Arg84 and Tyr317 major secondary roles in direct murine IL-3 binding to the βIL-3receptor. Most surprising, none of the key residues for direct IL-3 binding were critical for high affinity binding in the presence of the murine IL-3 α receptor, indicating a fundamentally different mechanism of high affinity binding to that used by hβc.

Steady State Kinetics of Consecutive Enzyme Catalysed Reactions Involving Single Substrates: Procedures for the Interpretation of Coupled Assays

Abstract Sets of differential rate equations are written describing a linear sequence of reactions occurring in solution each catalysed by a control enzyme or one of the Michaelis-Menten type. It is shown that the solutions of these equations may be formulated as a set of Maclaurin polynomials, expressing the concentration of each reactant and of final product as a function of time. From arrays of such polynomials, general expressions are induced for the first non-zero term of the series. These are used to formulate a procedure (illustrated with an example simulated by numerical integration) by which results of coupled enzymic assays may be analysed in terms of maximal velocities and apparent Michaelis constants: correlation is made with other established methods for conducting coupled assays. The present procedure assumes a steady state of enzyme-substrate complexes but not of intermediate reactants.

The use of sedimentation coefficients to distinguish between models for protein oligomers

The sedimentation coefficients of proteins are dependent on their sizes, shapes and densities and on the density and viscosity of the solvent. However, when the sedimentation coefficients of an oligomeric protein and its protomer are measured under the same experimental conditions, the ratio of the two coefficients depends only on the protomer shape and the mode of aggregation. This property, which we shall call the sedimentation ratio, therefore provides a way of distinguishing between models for oligomeric proteins. To allow examination of the behaviour of the sedimentation ratio, sedimentation coefficients are calculated for a comprehensive range of protomer shapes and modes of aggregation in hexameric systems using equations derived by Kirkwood. As illustrations of the method the resulting sedimentation ratios are compared with experimental values for insulin and arthroped hemocyanin, which eliminates many of the possible structures for these proteins. When experimental estimates of degree of hydration and molecular dimensions are also considered, all but a group of virtually identical structures are eliminated for the insulin hexamer and a single most likely structure remains for arthropod hemocyanin. The insulin structure is in good agreement with that determined by X-ray crystallography while the hemocyanin hexameric structure is a hexagonal prism formed by the cyclic aggregation of prolate ellipsoids of axial ratio about 2.5 : 1.

The interaction of insulin with its receptor: Cross-linking via insulin association as the source of receptor clustering

SummaryThe extensive association of mammalian insulins in solution and the aggregation of insulin receptors in cell membranes are well documented. The hypothesis advanced here is that a direct connection exists between these observations. It is postulated that, after binding to its receptor, an insulin monomer can interact with another similarly bonded hormone-receptor complex through those groups on the insulin monomer faces utilized for dimer-dimer contacts in the crystal and in solution. Regarded thus, the insulin molecules are effectively bivalent as required for the formation of cross-links between receptors, with the accompanying enhancement of biological activity. A number of properties of native insulins from different animals, and of modified insulins, are considered in the light of this suggestion. It is shown to have considerable power in reconciling a diversity of such observations and to provide a plausible model for the experimentally observed receptor clustering phenomenon.