Separation of macromolecules in isotachophoresis systems involving single or multiple counterions

The isotachcophoresis principle provides unique opportunities for rational designs of fractionation procedures involving charged molecules. Theoretically any two charged molecules that are soluble under the experimental conditions involved can be physically separated if their electrophoretic net mobilities differ only slightly in the electrophoresis medium used. A theoretical and practical outline is presented that enables the reader to set up this fractionation system and on a rational basis develop fractionation procedures for a given set of charged macromolecules by isotachophoresis with simple and well characterized ampholytes as spacer substances. The planning of preparative experiments in this approach is based on results obtained from rapid analytical screens on a microgram scale. The report includes an appendix containing the theoretical basis for computation of buffer compositions in the isotachophoretic steady state with mono/polyvalent constituents in systems involving one or more counterions and controlled amounts of interferiong ions.     

Cell free synthesis of some storage protein subunits by polyribosomes and RNA isolated from developing seeds of pea (Pisum sativum L.)

Polyribosomes which have template activity in the wheat germ system have been isolated from developing pea seeds. Some of the translation products have identical mobilities to the vicilin and legumin subunits by SDS-PAGE. Certain products were specifically immunoprecipitated with antisera prepared against purified vicilin and legumin fractions. Various RNA fractions including poly A-rich RNA have also been isolated from polyribosomes and shown to direct the synthesis of polyripeptides whose properties are similar to the storage protein subunits. The results are discussed in relationship to other investigations with seed storage protein biosynthesis in vitro.Abbreviations DTT dithiothreitol - SDS-PAGE SDS-polyacrylamide gel electrophoresis - TCA tricarboxylic acid     

Isolation of a high molecular weight glycoconjugate derived from the surface of S purpuratus eggs that is implicated in sperm adhesion

Sea urchin sperm–egg adhesion is mediated by bindin, a sperm surface protein that has lectin-like activity. Bindin agglutinates eggs, and this interaction has been shown to be inhibited by glycopeptides released from the egg surface by protease treatment. In this study, we report the purification and properties of such an egg surface glycoconjugate that may be involved in sperm adhesion. The glycoconjugate was partially purified by gel filtration and affinity chromatography on bindin particles. Upon gel filtration on Sepharose CL 4-B, the glycoconjugate elutes near the void volume, suggesting that it has a molecular weight in excess of one million. In addition, we have found that the egg surface glycoconjugate agglutinates bindin particles, indicating that it is multivalent. Carbohydrate analysis indicates that the glycoconjugate is composed primarily of fucosc, xylose, galactose, and glucose. This purified egg surface component is the most potent inhibitor of bindin-mediated egg agglutination yet described.     

Application of the ‘profiles of relationship’ method to distantly related proteins

Summary An analysis is made of the applicability of the recently published profiles of relationship method for establishing evolutionary relatedness among proteins by using the distantly related proinsulin and neurotoxin protein se-quences as a test object. The method is based on a simultaneous group analysis of both the frequency of acceptance of mutations and their genetic code inter-changeability. Regularities in the patterns of the profiles, which reflect decreased similarity with the passage of time, are established for typical cases of closely related, distantly related and unrelated proteins. This makes it possible to distinguish distantly related from unrelated proteins without extensive statistical randomization procedures. New evidence is stated in favour of a previously suggested definition of interchangeability which does not consider the third base in the codon. The applicability of the profiles of relationship method is examined on the distant relationship between proinsulin and the snake and scorpion neurotoxins which has been established previously by means of conventional approaches.     

Properties of a major α-globulin of rice endosperm

Globulin was isolated from milled rice (Oryza sativa, line IR480-5-9) by 5% NACl extraction and was precipitated by (NH₄)₂SO₂ or by dialysis against water. The extract was purified by repeated isoelectric precipitation at pH 4.5. The major globulin fraction (40%) exhibited one band by electrophoresis at pH 4.5 but two bands at pH 8.3. Similarly, one sharp peak was shown by sedimentation corresponding to 1.41S (α-globulin) in acetic acid (pH 2) and NaOH (pH 11.7) but a broad asymmetric peak was obtained at pH 6.7, 8.3 and 8.9. Gel filtration of the α-globulin at pH 6.5 exhibited 2 proteins with MW 20 000 and 98 000. The results suggest a pH dependent aggregation phenomenon. The two proteins could not be separated by DEAE-cellulose chromatography. SDS-polyacrylamide electrophoresis of α-globulin revealed one subunit with MW 18 000. This α-globulin is poorer in lysine and histidine but richer in cystine, methionine, arginine, tyrosine and glutamic acid than whole milled rice proteinfa]Taken part from the M.S. thesis of AAP from the University of the Philippine at Los Baños (1977).     

Abscisic acid binding to subcellular fractions from leaves of Vicia faba

A centrifugation binding assay has been used to demonstrate the binding of [³H] (±) abscisic acid to membrane-rich fractions prepared from leaves of Vicia faba L. Kinetic analysis of this binding shows evidence of saturation of binding sites with increasing concentration of ligand. Scatchard analysis of these data yields a biphasic plot possibly indicating the presence of two types of binding sites. The dissocation constant for the high affinity site has been calculated to be 3.5×10^-8 mol 1^-1.     

Mannosephilic adhesins produced by some strains of motile Aeromonas reveal structural details of Salmonella lipopolysaccharide through the phenomenon of co-agglutination

Abstract Some strains of motile Aeromonas produce lectin-like adhesins, whose activity can be inhibited by d -mannose. Such strains can co-agglutinate with some strains of Salmonella . Whether or not co-agglutination occurs is dependent upon both the properties of the Aeromonas adhesin and the structure of the Salmonella lipopolysaccharide (LPS). These studies have enabled new structural information for Salmonella LPS to be deduced and have confirmed previous studies regarding the nature of the Aeromonas adhesin binding site. It is possible that the observed in vitro co-agglutination between Aeromonas and Salmonella is a reflection of an in vivo situation which could modify the virulence of either or both bacteria.