Mass-spectrometric evidence for quinonoid-lysine coupling products in cigar protein

On subjecting hydrolysates of hydrogenated cigar protein to derivatization and preparative GLC, two fractions were obtained which had volatilities and mass spectra consistent with an origin from oxidative-condensation products of chlorogenic acid with lysine residues. The mass-spectral evidence was chiefly from high-resolution mass spectrometry of the heaviest fragment ions; there was also high-resolution comparison of the lighter fragment ions with those from relevant model compounds. New compounds prepared were the hydantoin from 6-(1-trans-octahydroquinol-2-onyl)-DL-norleucine and the heptafluorobutyrylated n-propyl esters of four other less-common amino acids. Details of the mass spectra are provided in a Supplementary Publication 4 or have been sent to the Mass Spectrometry Data Centre.     

Polyamine changes during the development of zygotic embryos in Prunus avium

A study of the polyamine profile was carried out during zygotic embryo development in Prunus avium. Zygotic embryos were collected from 2 donor trees and sorted into 3 size classes: C1 [2.5 to 3.5 mm]; C2 [3.6 to 4.5 mm] and C3 [5.5 to 7 mm]. Evolution of the various polyamines was similar for the two donor trees. Changes in the relative amount of the various free polyamines were observed during zygotic embryo development. Agmatine and spermine levels increased from C1 to C3. Spermidine, the predominant polyamine, showed a two-fold decrease in C3 compared with C1 and C2; the evolution of putrescine was opposed, showing an increase in the last developmental stage. The putrescine/spermidine ratio could be a marker for these 3 developmental stages with a higher ratio in C3 compared with C1 and C2. Polyamine changes in cotyledons from class C1 were investigated during in vitro culture. A 10-day induction on a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin caused a strong decline in free spermidine levels and a dramatic increase in free putrescine. The formation of conjugated putrescine occurred simultaneously, and twenty days after removal of growth regulators, the various polyamine contents were still at the same level.Abbreviations Agm agmatine - Dap diaminopropane - 2,4-D 2,4-dichlorophenoxyacetic acid - Put putrescine - Spd spermidine - Spm spermine     

Echinococcus granulosus: antigen characterization by chemical treatment and enzymatic deglycosylation.

Parasite antigenic fractions obtained by biochemical purification of sheep hydatid fluid were subjected to enzymatic digestion. The relative mobilities of the 5 and B antigens, before and after treatment, were analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Antigenic fractions transferred to nitrocellulose were also treated with sodium metaperiodate and concanavalin A. The results indicate that antigen 5 contains a substantial amount of carbohydrates covalently linked to a polypeptide backbone, which strongly bind to concanavalin A and is removed by N-glycosidase F (PNGase F). Antigen 5 possesses complex N-linked oligosaccharides (PNGase F sensitive), without terminal N-acetyl-D-glucosamine residues (N-acetyl-D-glucosaminidase nonsensitive) and has no high-mannose oligosaccharides (endo-beta-N-acetylglucosaminidase H nonsensitive). In contrast, the antigen B of low molecular weight is not susceptible to either enzymatic digestions (PNGase F, Endo H, and N-acetyl-D-glucosaminidase) or sodium metaperiodate oxidation and it does not bind to concanavalin A. Polyclonal antibodies prepared against the two antigens reacted with the deglycosylated antigen 5 in Western blot. The dominant epitopes are, therefore, polypeptides, although the presence of carbohydrate epitopes in the native glycoproteins cannot be excluded.     

A pre-aspartate-specific protease from human leukocytes that cleaves pro-interleukin-1 beta

Interleukin-1 beta is a 17.4-kilodalton hormone derived from a 33-kilodalton inactive precursor produced by monocytes. We used the precursor as a substrate to detect proteolytic activities in peripheral blood mono-nuclear cell-conditioned medium that might be involved in interleukin-1 beta processing. We found that the conditioned medium, following passage through DEAE-Sephacel, generates a biologically active fragment from the precursor that runs slightly higher than the mature hormone in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The responsible activity behaved as a single protein in ion exchange chromatography. It was completely inhibited by metal ion chelators and not by inhibitors of serine, cysteine, or aspartate proteases, and it was dependent on both calcium (or magnesium) and zinc. The enzyme was not inhibited by three substrate-based metalloprotease inhibitors, phosphoramidon, benzyloxycarbonyl-Gly-Leu-NH2, and N-(2-carboxy-3-phenylpropionyl)-Leu. NH2-terminal sequence analysis showed that cleavage of the precursor occurred between a histidine and an aspartate residue, and digestion of synthetic peptides indicated that the protease is specific for pre-aspartate cleavages.     

Low temperature, gibberellin and acid lipase activity in removal of apple seed dormancy

The activity of acid lipase and the level of gibberellin A₄ (GA₄) were determined in apple embryos excised from seeds after different time periods of stratification and subsequently cultured in darkness at 4°C or at 25°C. Enzyme activity and GA₄ content were higher at 4°C. Exogenous gibberellin stimulated lipase activity, while AMO-1618, an inhibitor of gibberellin biosynthesis, inhibited, to the same degree, both the enzyme activity and the GA₄ accumulation. The involvement of GA₄ and lipolytic enzymes in cold-mediated removal of embryonal dormancy has been discussed and compared with the role of these two factors in light-stimulated germination of dormant apple embryos, described earlier (Smoleńska and Lewak 1974).     

Expression of an Anaplerotic Enzyme, Pyruvate Carboxylase, Improves Recombinant Protein Production in Escherichia coli

Anaplerotic enzyme reactions are those which replenish tricarboxylic acid intermediates that are withdrawn for the synthesis of biomass. In this study, we examined recombinant protein production in Escherichia coli containing activity in an additional anaplerotic enzyme, pyruvate carboxylase. In batch fermentations, the presence of pyruvate carboxylase resulted in 68% greater production of the model protein, β-galactosidase, 41% greater cell yield, and 57% lower acetate concentration. We discuss why these results indicate that acetate concentration does not limit cell growth and protein synthesis, as predicted by other researchers, and suggest instead that the rate of acetate formation represents an inefficient consumption of glucose carbon, which is reduced by the presence of pyruvate carboxylase.     

Experimental and theoretical studies of the three-dimensional structure of human interleukin-4

The structure of human interleukin 4 (IL-4) was predicted utilizing a series of experimental and theoretical techniques. Circular Dichroism (CD) spectroscopy indicated that IL-4 belonged to the all alpha-helix class of protein structures. Secondary structure prediction, site-directed mutagenesis, and CD spectroscopy suggested a predominantly alpha-helical structure, consistent with a four-helix bundle structural motif. A human/mouse IL-4 chimera was constructed to qualitatively evaluate alternative secondary structure predictions. The four predicted helices were assembled into tertiary structures using established algorithms. The mapping of three disulfide bridges in IL-4 provided additional constraints on possible tertiary structures. Using accessible surface contact area as a criterion, the most suitable structures were right handed all antiparallel four-helix bundles with two overhand loop connections. Successful loop closure and incorporation of the three disulfide constraints were possible while maintaining the expected shape, solvent accessibility, and steric interactions between loops and helices. Lastly, energy minimization was used to regularize the chain.     

Alcohol-induced potentiation of the neurally evoked contractions of the retractor unguis muscle of the locust, Schistocerca gregaria

Each of a series of seven monohydroxyl alcohols caused an increase in the force of the neurally evoked contractions of the innervated retractor unguis muscle isolated from the metathoracic femurs of the locust, Schistocerca gregaria. The negative logarithm of equipotent concentrations of the alcohols was proportional to the logarithm of the partition coefficient (1-octanol/water) of the alcohols: $\text{log}\text{1}\text{C}\text{= 1·282}\text{log}\text{P+1·684}$, where C is concentration, and P is the partition coefficient.Ethylene glycol and glycerol did not potentiate the contractions, possibly due to their very low lipophilic character. Acetone (10^?2 M) and benzene (10^?5 M) also potentiated the neurally evoked contractions of the insect muscle.