Barbarín Arreguín

Hevein: NMR assignment and assessment of solution-state folding for the agglutinin-toxin motif.

The first high-resolution solution-state structure of a member of the toxin-agglutinin folding motif with the WGA disulfide linkage is presented. The 1H NMR spectrum of hevein has been 100% assigned from residue 2 through residue 43, the C-terminus, using two-dimensional correlation and NOE spectroscopy. During the course of the NOESY analysis, the three-dimensional structural features of hevein were derived, using nonstereospecific distance constraints (with tight bounds) for XPLOR simulated annealing followed by unconstrained relaxation in the CHARMm force field, at two levels of long-range constraint density. In addition, a large number of low-bound-only constraints, corresponding to unobserved NOEs, were used in both refinements. The first structure elucidation employed a total of 180 distance constraints (60 of which were medium or long range, i/i+n with n < or = 2). The second refinement employed 244 (101 medium or long range) constraints: some conformation-insensitive intraresidue constraints were deleted, two misassigned long-range constraints were corrected, and 41 new i/i+n (n > or = 2) constraints were added. The average bounds precisions of the two refinements were comparable (+/- 0.44 A) and significantly tighter than those that result when a universal low bound corresponding to the sum of the van der Waals radii was used. (The more conservative treatment of NOEs gave the same final structure but required a higher constraint density before assignment errors would stand out during the refinement.) Constraint density also has a significant influence on convergence and accuracy using tight constraints. The study demonstrates that convergence within an ensemble of solution structures is not a dependable criterion for either the accuracy or precision of the derived structure. The best fitting conformers from the refinement at the higher constraint density bear a greater similarity to the solid-state structure of the domains of wheat germ agglutinin (0.95 A rmsd over residues 2-32) than to the recently reported 2.8-A X-ray structure of hevein (1.25 A rmsd over residues 2-32, 2.83 A rmsd over residues 2-42). The consensus conformer from the solution data is defined to a backbone rmsd of < 0.6 A over the full sequence for which NMR data could be collected.(ABSTRACT TRUNCATED AT 400 WORDS)

Purification and properties of a lipase from Cephaloleia presignis (Coleoptera, Chrysomelidae)

A novel lipase from the insect Cephaloleia presignis was purified by a procedure involving ammonium sulphate precipitation, and Phenyl Toyopearl 650M, DEAE‐5PW and hydrophobic‐interaction column chromatographies. The purified lipase was homogeneous with a molecular mass of 31000 Da by SDS/PAGE and of 29000 Da by gel filtration on a Superose 12 column. The enzyme was indentified as a glycoprotein with a pI of 6.9. The enzyme unspecifically liberated short‐chain to long‐chain fatty acids from p‐nitrophenyl esters, methyl esters and triglycerides. The N‐terminal 28 amino acid residues were determined as AGTLGYATRHVLPIFTLDDYTGSNEMWG, which showed no similarity with known proteins, suggesting that the purified lipase may belong to a novel class of hydrolases.

13C-N.M.R. characterizations of the sarsasapogenin disaccharides, the filiferins a and b: 2-O-(β-d-xylopyranosyl)- and 2-O(β-d-glucopyranosyl)-β-d-galactopyranosides

Abstract Filiferin B is identical to timosaponin A-III, which had previously been shown to be 3- O -[2- O -(β- d -glucopyranosyl)-β- d -galactopyranosyl]sarsasapogenin. A larger-scale isolation of filiferin B from the seeds of Yucca filifera led to the isolation of filiferin A, now shown to be 3- O -[2- O -β- d -xylopyranosyl)-β- d -galactopyranosyl]-sarsasapogenin. The presence of the xylose residue was established by way of hydrolysis. 8-Methoxycarbonyloctyl 2- O -(β- d -glucopyranosyl)-β- d -galactopyranoside was synthesized to serve as a model for interpretation of the 13 C-n.m.r. spectrum of filiferin B. The information thus gained, together with the 13 C-n.m.r. spectra of other, simple model-compounds, permitted assignment of the structure for filiferin A. 8-Methoxycarbonyloctyl 2- O -(α- d -glucopyranosyl)-β- d -galactopyranoside was also synthesized.

PURIFICATION AND CHARACTERIZATION OF MULTIPLE FORMS OF ASCLEPAIN G FROM ASCLEPIAS GLAUCESCENS H.B.K.

Abstract Ten asclepain forms were found to be present in the latex of milkweed (Asclepias glaucescens H.B.K.) plants. Four of them were purified by high performance liquid chromatography on a cation exchange column, and characterized. Asclepains Ag3, Ag6, Ag7 and Ag8 were isolated as homogeneous proteins of very similar molecular weights (approx. 23 000) with isoelectric points greater than 9.0. These forms possess nearly identical secondary structures, as judged from their circular dichroism spectra. Interestingly, one of these forms (Ag3) markedly differed from the other three forms with respect to its specificity toward ester and amide substrates.

Unusual far-ultraviolet circular dichroism of wheat germ agglutinin and hevein originated from cystine residues

Abstract The conformation of wheat germ agglutinin and hevein was studied by means of circular dichroism. The spectra of these proteins were remarkably similar below 250 nm, indicating the presence of common structural characteristics in their polypeptide chains. Besides, both spectra displayed a positive extremum around 221–223 nm which is rather unusual among globular proteins, this dichroic signal was only slightly modified in the presence of 6 M guanidine hydrochloride, but it was completely cancelled in 0.05 M dithiothretol. Thus, it seems that in both proteins disulfide bonds are responsible for this singular circular dichroism band. Kinetic studies of disulfide-bridge reduction showed different reactivities of these groups for hevein and wheat germ agglutinin.

Sterol composition and biosynthesis in the sponge Spheciospongia vesparia

The sterol composition of Spheciospongia vesparia from the Caribbean Sea is reported. Three groups of sterols, Δ 5 , Δ 0 , Δ 5,7 , were found. The isolated 5α,8α-epidioxy sterols appeared to be predominantly, if not exclusively, artifacts. The auto-oxidation of native Δ 5,7 -sterols to 5α,8α-epidioxy sterols has been investigated. A transformation of dietary Δ 5 -sterols to Δ 5,7 -sterols has been demonstrated in Spheciospongia vesparia.

Isolation and characterization of a protease from the marine sponge Spheciospongia vesparia.

A protein that showed activity against proteic (casein and hide powder azure) and synthetic (BAEE and HLPA) substrates was isolated from the marine sponge Spheciospongia vesparia. The protease was purified from an aqueous extract by ammonium sulfate precipitation, gel filtration, hydrophobic and HPLC‐anion exchange chromatographies. The purified protease showed a single band in SDS‐PAGE minigels and had a molecular weight of 29,600, but when submitted to isoelectric focusing it showed 2 bands with isoelectric points of 4.56 and 4.43. Its catalytic action was inhibited by EDTA and 1,10‐phenanthroline, so it seemed to be a metalloprotease.

Oxidative reactions of the pentose pathway and the origin of reducing equivalents for the biosynthesis of rubber in Hevea brasiliensis latex.

The presence of the oxidative reactions of the pentose phosphate pathway in hevea latex and their interaction with the biosynthesis of rubber are demonstrated using radioisotopes. During the second oxidation–reduction reaction of the pentose phosphate pathway the tritium in C3 of glucose-6-phosphate is transported as tritiated nicotinamide adenine dinucleotide phosphate and serves to reduce [3-14C] 3-hydroxy-3-methylglutaryl coenzyme A to mevalonate. Reduced nicotinamide adenine dinucleotide phosphate is the inter-connection between these two metabolic routes present in hevea latex, thus the hydrogens of glucose end up in rubber. © 2000 Society of Chemical Industry