Purification of dog VIP from a single animal

VIP, a potent vasodilator peptide, is reported to be identical in pig, cow, human and rat but to differ in four amino acids in chicken. This report describes the purification of dog VIP from the small intestine of a single animal. The purification method is based on tissue extraction with a sequence of organic solvents. The extracted VIP is concentrated onto cation-exchange cellulose and brought to purity by three HPLC steps. A 30% final yield of pure VIP was obtained from the original extract. Dog VIP was found to have the following sequence: His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala -Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn. Thus the amino acid sequence of dog VIP is identical with all the mammalian VIP's which have been reported. This suggests that a high degree of conservation throughout the molecule may be required for VIP bioactivity.     

Human serum hemopexin: direct evidence for change of its isoelectric point upon heme binding. A new serum protein fractionation

Human serum was submitted to a one step displacement-ligand exchange chromatography. Displacement removed serum albumin and part of gamma-globulins. Ligand exchange furnished an enriched heme-hemopexin fraction. An original, non denaturing human heme-hemopexin preparation is proposed.     

Ostreopexin: A hemopexin fold protein from the oyster mushroom, Pleurotus ostreatus

Proteins with hemopexin repeats are widespread in viruses, prokaryotes and eukaryotes. We report here for the first time the existence of a protein in fungi with the four-bladed β-propeller fold that is typical for hemopexin-like proteins. This protein was isolated from the edible basidiomycetous fungus Pleurotus ostreatus and is named ostreopexin. It binds to Ni^2 +-NTA-agarose, and is structurally and functionally very similar to PA2 albumins isolated from legume seeds and the hemopexin fold protein from rice. Like these plant proteins, ostreopexin shows reversible binding to hemin with moderate affinity, but does not bind to polyamines. We suggest that ostreopexin participates in intracellular management of metal (II or III)-chelates.     

Amyloid-related serum protein SAA from three animal species: comparison with human SAA.

The amyloid-relates serum protein SAA has been isolated by gel filtration in 10% formic acid from three animal species: mink, mouse, rabbit. Sera used in the isolation procedure were obtained from animals in which high concentrations of SAA had been induced by treatment with LPS. The isolated SAA proteins had a subunit size similar to that of human SAA, with m.w. values ranging from 10,000 to 11,700 (estimated by gel filtration in 6 M guanidine-HC1) or 12,400 to 15,000 (estimated by SDS-PAGE). The m.w. studies and amino acid sequence data indicated that SAA and the amyloid fibril protein AA in the mouse, and probably also the mink, are related in the same way as in man, the two proteins having common NH2-terminal amino acid sequences and SAA being extended by 20 to 40 residues at the COOH-terminal end of the molecule.     

A comparison of the structure and properties of serum transferrin from 17 animal species

1. A comparison of the chemical and physical properties of the iron transport protein transferrin, purified from the following seventeen animal sera, is reported; human, rhesus monkey, dog, cat, rabbit, guinea pig, mouse, rat, cow, sheep, goat, horse, pig, turkey, duck, turtle and rattlesnake. 2. Similarities and differences in molecular weight, isoelectric point, antibody specificity, effect of pH on iron release, number of sialic acid residues, amino acid composition and the N-terminal amino acid residue, are discussed. 3. The results are compared with the commonly accepted evolutionary origins of the 17 species.     

Amino acid structures of multiple forms of amyloid-related serum protein SAA from a single individual

Multiple forms of the acute-phase serum protein SAA were isolated from the lipoprotein fraction of plasma from a single individual. These protein forms were purified by size-exclusion, ion-exchange, and reverse-phase high-pressure liquid chromatography, and then the tryptic peptides were subjected to amino acid sequence analysis. A total of three distinct 104-residue proteins were identified. Two of these proteins differed only by having either an arginine or a histidine at position 71 while the third protein had seven amino acid differences. Each of these proteins has a 103-residue companion protein where the amino-terminal arginine has been removed. Two of these protein sequences match the two human SAA cDNA structures reported in the literature. The presence of three unique amino acid sequences in one individual is proof that there must be a minimum of two genes for SAA in humans.     

Genetic mapping of bovine mitochondrial DNA from a single animal

Using a physical map of bovine mitochondrial DNA derived from the liver of a single Holstein cow, we have determined the location of the genes specifying the large and small riibosomal RNAs by hybridization analysis and electron microscopic observations of R-loop forms. Also, the position of the origin of DNA replication (D-loop) has been located by electron microscopy. Additionally, the direction of D-loop expansion and the polarity of the large and small ribosomal RNA genes were determined.     

Isolation of the hemopexin receptor from human placenta

A hemopexin receptor detected in detergent-solubilized placental membranes was purified from the human placenta, using hemopexin-Sepharose affinity chromatography. The solubilized membranes exhibited binding sites of 2.77 pmol of hemopexin/mg of protein with a dissociation constant (Kd) of 6.6 X 10(-8) M. The purified receptor has a molecular weight of 80,000, determined on sodium dodecyl sulfate-gel electrophoresis. Immunoinhibition experiments using the antibody against the placental receptor revealed inhibition of binding of 125I-hemopexin to human leukemia K562 and HL 60 cells, thereby strongly supporting that the polypeptide isolated from the human placenta was the hemopexin receptor.     

Bovine serum brevin. Purification by hydrophobic chromatography and properties

Brevin, an actin-severing protein present in serum from numerous mammals, has been purified to homogeneity from bovine serum, using hydrophobic chromatography as the last purification step. The physicochemical parameters of brevin have been established and some of them studied in the absence and presence of Ca2+. Brevin exhibits an apparent Stokes radius, Rs, of 3.4 nm, an intrinsic sedimentation coefficient S degrees 20, W, of 4.8 S and 4.4 S in the absence and presence of Ca2+ respectively, indicative of calcium-induced conformational change. The native molecular mass of brevin was found to be 68 kDa and the hydrodynamic data suggest that the protein is an asymmetric molecule. Sedimentation equilibrium studies demonstrated that Ca2+ affects the shape (asymmetry) of brevin without altering its molecular mass. Limited tryptic and chymotryptic digestion of brevin distinguishes the Ca2+-induced conformation from the EGTA one. No change in the electrophoretic migration of brevin was seen upon Ca2+ addition. Several isoforms were detected by two-dimensional gel electrophoresis. Brevin increases the rate of nucleation of actin but decreases the rate of elongation of the filaments and the steady-state viscosity of F-actin in substoichiometric amounts, as measured by viscometric assays under high shear conditions. Electron microscopic examination documents these effects. Brevin produces shorter actin filaments and binds to the 'barbed' end of filaments to which monomers add preferentially during elongation, as demonstrated by indirect immunogold staining of antibodies against brevin. Filament elongation occurs only at the slowly growing end. An enzyme-linked immunosorbent assay was developed and used to detect and quantify brevin and related proteins in extracts of different bovine cells and tissues. Liver and smooth muscles were found to contain the highest amounts of the severing protein.     

Bovine serum albumin--haloperidol as a tool for the study of dopaminergic transmission: behavioural and neurochemical effects following a single injection in the nucleus accumbens

Bovine serum albumin haloperidol (BSA-Hal) is a macromolecular complex with 14 molecules of haloperidol immobilized on the BSA protein backbone. This compound produces a selective, long lasting and reversible blockade of dopamine receptor activity. Its action was demonstrated by the ability to trigger a high rate of ipsilateral amphetamine-induced rotation up to 6 days after a single unilateral injection of the conjugate into the striatum. In the present study, the effect of the blockade of dopamine transmission in the nucleus accumbens (n.Acc) on spontaneous and learned behaviors was tested. The results indicate that the specific and long lasting blockade of dopamine receptors by bilateral injection of BSA-Hal in the n.Acc (1.6 micrograms/2 microliters) induced deficits in spontaneous alternation in a Y-maze on the 2nd and 5th days after the injection but not on the 11th day of the experiment, impaired acquisition but not retention in a radial 8-arm maze, increased latency to escape during learning in the place navigation task. These findings confirm the involvement of the n.Acc system in processes that have been generally attributed to the limbic system.     

Isolation and physical chemical properties of rat hemopexin]

Rat hemopexin was purified by a procedure involving three different steps : ammonium sulfate precipitation, rivanol precipitation and DEAE-cellulose chromatography with concave gradient of molarity. Purity of the preparation was checked by three different methods : analytical ultracentrifugation, immunoelectrophoresis and acrylamide gel electrophoresis. The principal physical properties were studied. The amino acid and carbohydrate composition was determined and compared with that of human and rabbit hemopexin.     

Anaphylactogenic, antigeni and avid properties of the antibotulin serum from the blood of various animal species]

It was shown that purified and concentrated by the "Diaferm" method antibotulin sera from horse and cattle blood failed to differ by anaphylactogenic properties; at the same time in sensitization of the organism to protein of one animal species the use of the sera of another species provided a lesser reactogenicity of the preparation. The antigenic activity of the purified and concentrated sera from the blood or horses and cows in testing on rabbits was identical, but in response to cow alpha-globulin the animals responsed by a more intensive production of precipitins. The activity of cow and horse antibotulin serum (determined by the rate and stability of their association with the corresponding toxin) proved to be identical.     

Natural selection of protein structural and functional properties: a single nucleotide polymorphism perspective

Background

The rates of molecular evolution for protein-coding genes depend on the stringency of functional or structural constraints. The Ka/Ks ratio has been commonly used as an indicator of selective constraints and is typically calculated from interspecies alignments. Recent accumulation of single nucleotide polymorphism (SNP) data has enabled the derivation of Ka/Ks ratios for polymorphism (SNP A/S ratios).

Results

Using data from the dbSNP database, we conducted the first large-scale survey of SNP A/S ratios for different structural and functional properties. We confirmed that the SNP A/S ratio is largely correlated with Ka/Ks for divergence. We observed stronger selective constraints for proteins that have high mRNA expression levels or broad expression patterns, have no paralogs, arose earlier in evolution, have natively disordered regions, are located in cytoplasm and nucleus, or are related to human diseases. On the residue level, we found higher degrees of variation for residues that are exposed to solvent, are in a loop conformation, natively disordered regions or low complexity regions, or are in the signal peptides of secreted proteins. Our analysis also revealed that histones and protein kinases are among the protein families that are under the strongest selective constraints, whereas olfactory and taste receptors are among the most variable groups.

Conclusion

Our study suggests that the SNP A/S ratio is a robust measure for selective constraints. The correlations between SNP A/S ratios and other variables provide valuable insights into the natural selection of various structural or functional properties, particularly for human-specific genes and constraints within the human lineage.     

Bovine conglutinin is a collagen-like protein

Conglutinin is a bovine plasma protein which is relatively large and asymmetric with elevated contents of glycine and, to some extent, proline. Although its physiologic function is unknown, conglutinin is known to bind, in the presence of calcium, to yeast cell walls and to the solid-phase-inactivated third component of complement. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, isolated conglutinin appeared to consist of a single polypeptide chain (Mr 48 000). Unreduced conglutinin consisted of a single stained band with an apparent molecular weight of approximately 300 000. Cross-linking experiments with glutaraldehyde and dimethyl suberimidate suggested that this Mr 300 000 molecule consists of six of the disulfide-linked polypeptide chains. Amino acid composition revealed hydroxylysine and hydroxyproline together with elevated glycine and proline contents. Digestion of reduced, alkylated conglutinin with bacterial collagenase resulted in formation of a precipitate which consisted of an Mr 24 000 peptide which was digested to Mr 21 000 with large quantities of collagenase. These peptides contained less glycine, proline, hydroxylysine, and hydroxyproline than did the intact protein. The supernatant from this digestion mixture was, however, enriched in these four amino acids, with glycine making up nearly one-third of the total. Prolonged digestion with pepsin at 37 degrees C resulted in an Mr 20 000 peptide which was enriched in glycine, proline, hydroxyproline, and hydroxylysine. Amino-terminal sequence analysis showed that the glycine-X-Y repeating sequence begins at residue 26.(ABSTRACT TRUNCATED AT 250 WORDS)     

SANS studies of concentrated protein solutions. I. Bovine serum albumin

Small-angle neutron scattering (SANS) was used to examine concentrated bovine serum albumin solutions of up to 20% protein w/v. At higher protein concentrations, scattering data show distinct features that can be ascribed to strong intermolecular interactions. Differential scattering cross-sections are fitted to a theoretical model of interparticle potential consisting of a hard core plus an exponentially decaying “tail.” For moderate ionic strength (0.03M K Acetate, pH 5.9), the intermolecular interaction agrees with the double-layer repulsive part of the well-known DLVO (Derjaguin, Landau, Verwey, Overbeek) theory for interacting colloidal particles. We thus demonstrate that it is possible to determine size parameters and the surface charge of protein molecules in dense solutions. At high salt concentrations (≥0.2M NaCl) data can be fitted by the same potential model, although interpretation in terms of DLVO theory is not possible. Even in this case, however, “effective” molecular size and potential parameters can be determined.     

Latrotoxin-like properties of a protein from brain

In bovine brain cortex cytoplasm we have identified a soluble protein (L-protein) of M_r 90 kDa interacting with polyclonal antibodies to -latrotoxin. The L-protein forms potential-dependent and cation-selective ion channels in BLM, which are blocked by Cd²⁺. The fusogenic activity of the L-protein was demonstrated on liposomes. We have arrived at the conclusion that the action mechanisms of the L-protein and -latrotoxin are similar.