Biological and biophysical characterization of recombinant soluble human E-selectin purified at large scale by reversed-phase high-performance liquid chromatography.

A first step in the development of a high-throughput screening assay for antagonists of human E-selectin is the purification and characterization of the selectin. In the present paper we describe a single-step, rapid, reversed-phase HPLC purification protocol for the recombinant, soluble form of human E-selectin (rshE-selectin) produced in Chinese hamster ovary cells. The procedure resulted in high protein yields with recoveries of greater than 98%. Characterization of the reversed-phase purified rshE-selectin showed this product to be analogous to rshE-selectin purified using conventional chromatographic techniques with respect to biological activity and molecular shape. However, the carbohydrate composition of reversed-phase purified rshE-selectin, which had been variable with conventionally purified material, was found to be constant across several isolations. The protocol described herein eliminated the high mannose component associated with previously purified rshE-selectin and provided a uniform carbohydrate composition for additional experimental studies, such as NMR. This fact, coupled with the high yield and simplicity of the present purification scheme are distinct advantages over those previously published. It is expected that other mammalian selectins, such as P-selectin and L-selectin, would also be amenable to reversed-phase HPLC purification.

Determination of the monomer-dimer equilibrium of interleukin-8 reveals it is a monomer at physiological concentrations.

Interleukin-8 has been shown by X-ray crystallography and NMR to be a homodimer, suggesting that this is the form which binds to its receptor. Here we measure, for the first time, the monomer-dimer equilibrium of interleukin-8 using analytical ultracentrifugation and titration microcalorimetry and find that it dissociates readily to monomers with an equilibrium dissociation constant of 18 +/- 6 microM at 37 degrees C. The present findings suggest that the monomer is the form which binds to the receptor. Comparison of experimental and structure-based calculated thermodynamics of interleukin-8 dimerization argues for limited subunit conformational changes upon dissociation to monomer.

Hepatitis B surface antigen and polymerized albumin binding activity in sheep serum.

Sera from sheep and other domestic animals contain a substance that gives a strongly positive test for antibody to hepatitis B virus surface antigen by the accepted radioimmunoassay procedure. We have purified this substance from sheep serum to near homogeneity by ion-exchange, affinity, and molecular exclusion chromatography and have identified it to be an IgM. We present evidence that this sheep IgM is an antibody to polymerized sheep albumin. This antibody may arise due to infection by hepatitis B virus, hepatitis B virus-like viruses, or other pathological agents and may react with hepatitis B virus surface antigen by combining with polymerized albumin bound to the hepatitis B virus receptor for this polymer.

Alloalbuminemia in North India.

Electrophoretic screening of sera from 550 individuals from Punjab, North India, revealed four cases of alloalbuminemia. Two albumin variants migrated slower and two migrated faster than the common albumin A. These variants were further analyzed by electrophoresis of their cyanogen bromide fragments to localize their molecular differences. One of the slow variants appears similar to, if not identical with, albumin B, with an altered cyanogen bromide fragment CNBr VII. The other slow variant appears to be a new variant (proposed name albumin Punjab) differing from albumin A in an altered fragment CNBr VI (which also occurs in albumins Kashmir and Adana) and in an altered fragment CNBr I. Among the fast variants, one has the same altered fragment CNBr V as albumin Naskapi, while the other appears to be a new variant (proposed name albumin Patiala) having an altered fragment CNBr VI. The presence of albumin Naskapi in Punjabis, North American Indians, and Eti Turks (previously reported) is consistent with the existence of a common ancestral population in which the mutation to Naskapi occurred before the migrations eastward and westward.

Strategic planning: determining the impact of environmental characteristics and uncertainty.

This paper continues a line of research into the effect of environmental characteristics and uncertainty on strategic planning. Results suggest that measures of uncertainty and the environment need to be analyzed independently, and that uncertainty is a strong moderator of environmental characteristics and industry groupings but does not consistently affect strategic planning activities.

Albumin Naskapi variant in North American Indians and Eti Turks.

Both conventional polyacrylamide gel electrophoresis and a new type of electrophoretic screening procedure indicate that the polymorphic albumin variants Naskapi, found chiefly in the Naskapi Indians of Quebec, and Mersin, found in the Eti Turks of southeastern Turkey, are molecularly identical or very similar and that the amino acid substitution site in these variants is located between residues 330 and 446. This discovery is consistent with a genetic relationship between the Eti Turks and American Indians. We also report a new variant found in the Eti Turks, albumin Adana, which migrates similarly to albumin B on conventional gels but which our new system shows to differ from the common albumin A and albumin B by a substitution between residues 549 and 585.

Localization of the amino acid substitution site in a new variant of human serum albumin, albumin Mexico-2.

Using an electrophoretic screening procedure, we have discovered that two species of human serum albumin Mexico occur that are indistinguishable by conventional electrophoretic methods. We suggest that these species be referred to as albumins Mexico-1 and Mexico-2. Isolation and determination of the partial sequence of the cyanogen bromide fragment of albumin Mexico-2 that differs from the corresponding fragment of the common albumin A revealed this variant to arise from at least a glycine/aspartic acid substitution at position 550. This region of the albumin molecule is involved in the binding of the fatty acid, palmitate.

Isolation and characterization of the histone variants in chicken erythrocytes.

Chicken erythrocyte histones 2A, 2B, and 3 can be resolved into nonallelic primary structure variants by polyacrylamide gel electrophoresis in the presence of Triton X-100. These variants were isolated and characterized by analysis of their tryptic and thermolytic peptides. The major variants of chicken H2A and H2B differ from the analogous component of calf thymus by a small number of conservative amino acid substitutions in the basic terminal regions, which interact with DNA. This moderate rate of allelic evolution of the slightly lysine-rich histones contrasts with the complete conservatism found in the arginine-rich histones. Chicken H4 and both chicken H3 variants are identical with their corresponding components in mammals. The amino acid substitutions distinguishing histone variants are located within the highly conserved hydrophobic regions, which are involved in histone--histone interactions.

The resolution of membrane proteins based upon size, charge, and hydrophobicity.

Currently available systems for resolving membrane proteins are based only on size and charge differences. Recently, it has been shown that Triton-urea-acetic acid gels which separate proteins on the basis of charge, size and hydrophobicity are capable of resolving proteins differing only by the substitution of a single neutral amino acid. We have applied this new method to the resolution of bacterial envelope proteins. Conditions for optimal resolution of different bacterial envelope proteins were determined by electrophoresis through transverse urea and Triton X-100 gradient gels. We have also correlated the components resolved in this system with those resolved by classical sodium dodecyl sulfate-gel electrophoresis by using two-dimensional slab gels combining the two systems. Furthermore, envelope protein fractions from different species and strains of bacteria were compared to identify specific proteins. This system appears to be a promising method for investigating envelope proteins which are due to missense mutations.

Biochemical and immunological characterization of two distinct variants of histone H2A in Friend leukemia.

Changes in the relative amount of two histone H2A subfractions have been observed in cells at different proliferative stages of Friend leukemia. Biochemical analyses of the purified H2A subfractions reveal them to be different in primary structure, and not the result of postsynthetic modifications of the same parent protein. Antibodies raised against the purified H2A.2 subfraction cross react with H2A.1 and H2A.2, but show high specificity for the immunizing subfraction at higher sera dilutions. Only H2A.2 contains a methionine which appears critical to an antigenic difference that immunologically distinguishes H2A.2 from H2A.1. The observed change in the relative amounts of two nonallelic variants of a histone coincident with changes in the physiologic states of the cell may indicate a correlation between genome structure and function.

Chromatographic evidence for the existence of multiple forms of cathepsin B1.

Preparations of ox spleen cathepsin B1 have been found to give multiple peaks of activity upon chromatography on DEAE-cellulose in NaCl gradients or by equilibrium chromatography in 0.05m-NaCl. CM-cellulose gradient chromatography also shows several cathepsin B1 peaks. This evidence indicates that ox spleen cathepsin B1 can exist in at least three to five forms. All forms have the same molecular weight, are thiol-activated and are inhibited by typical thiol inhibitors. The possible sources of this multiplicity of activity are discussed and a possible physiological role for cathepsin B2 is suggested.