Crystallization and preliminary X-ray diffraction analysis of protein L-isoaspartyl O-methyltransferase from wheat germ.

Wheat-germ protein L-isoaspartyl O-methyltransferase (WPIMT) can initiate the conversion of L-isoaspartyl residues in a protein or peptide, which accumulate during the aging process in wheat-germ seeds, to normal L-aspartyl groups. The recombinant protein of WPIMT was overexpressed in Escherichia coli and purified to homogeneity. The protein was crystallized in the presence of S-adenosine-L-homocysteine using 2-methyl-2,4-pentanediol. Preliminary X-ray analysis indicated a tetragonal space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 77.3, c = 152.9 A for cryofrozen crystals at 90 K. The crystals diffracted to 3.3 A and contain two molecules per asymmetric unit.

Crystallization of agGST1-6, a recombinant glutathione S-transferase from a DDT-resistant strain of Anopheles gambiae.

Glutathione S-transferases (GSTs) belong to a family of detoxification enzymes that conjugate glutathione to various xenobiotics, thus facilitating their expulsion from the cell. GST activity is elevated in many insecticide-resistant insects, including the DDT-resistant malaria vector Anopheles gambiae. Crystals of the recombinant form of a GST from A. gambiae, agGST1-6, have been grown in at least five different crystal forms, with a broad range of diffraction resolution limits. A complete 2.0 A data set has been collected on a C-centered orthorhombic crystal form with unit-cell parameters a = 99.0, b = 199.4, c = 89.6 A. A search for heavy-atom derivatives has been initiated, along with phase-determination efforts by molecular replacement.

Antibacterial hydroxycinnamic esters from Piper caninum from Paluma, north Queensland, Australia. The crystal and molecular structure of (+)-bornyl coumarate.

The crude chloroform bark extract of Piper caninum (Piperaceae) exhibits antibacterial activity against the Gram-positive bacteria, Bacillus cereus, Staphylococcus aureus, and Streptococcus pneumoniae. The antibacterial agents in this extract have been isolated using bioactivity-directed chromatographic techniques and identified by NMR spectroscopy as (+)-bornyl p-coumarate and bornyl caffeate. A single-crystal X-ray structure has been carried out on (+)-bornyl p-coumarate. The compound crystallizes in the orthorhombic space group P2(1)2(1)2(1) (#19) with a = 12.659(4), b = 13.281(4), and c = 10.177(3) A. Fullmatrix least-squares refinement converged at R = 0.047, and Rw = 0.058.

The crystal structure of pea lectin at 6-A resolution.

The three-dimensional crystal structure of the mitogenic lectin from the green pea (Pisum sativum) has been determined at 6-A resolution by x-ray diffraction methods. Pea lectin was isolated by use of affinity chromatography and was crystallized from polyethylene glycol solutions. Crystals of pea lectin are orthorhombic, space group P212121, and diffract to at least 1.2-A resolution. The unit cell dimensions are a = 50.85(5), b = 61.23(5), and c = 137.3(2) A. The calculated mass of protein per asymmetric unit is 49,000 daltons, and the crystals are 44% solvent by volume. There are two pea lectin monomers per crystallographic asymmetric unit. Diffractometer data were collected from a native crystal and from a single site uranyl heavy atom derivative crystal. The position of the uranium atom, determined from three-dimensional Patterson maps, was refined by least squares techniques (R index - 0.46 for centric data). A three-dimensional electron density map was calculated by use of phases determined by isomorphous-replacement and anomalous-dispersion contributions. The boundaries of the pea lectin molecule are clearly visible in the map. The molecule appears to be a dimer, roughly peanut-shaped, formed by the close association of the two monomer units. In shape and size, it bears a striking resemblance to the concanavalin A dimer, in which monomers combine to form a dimer-wide contiguous antiparallel pleated sheet.

Hydrogen peroxide formation upon oxidation of oxalic acid in presence and absence of oxygen and of manganese(II)--III. Iron(III) and copper(II) as retarders.

The formation of considerable amounts of hydrogen peroxide upon the slow addition of various oxidizing agents to oxalic acid in dilute sulphuric acid in the presence of oxygen and Mn(II) is greatly retarded in the presence of Fe(III) or Cu(II). With hydrogen peroxide as oxidizing agent and a trace of Fe(II) there is considerable decomposition of peroxide, under a nitrogen atmosphere, after a few hours at 25 degrees in light (from a tungsten lamp), and less decomposition in the dark. This decomposition is decreased by Mn(II) and also when the original mixture contains Fe(III). With oxygen as the oxidizing agent Fe(II) is about 100 times as effective an inhibitor of peroxide formation as Fe(III). With all oxidizing agents used, Cu(II) is some 6-10 times more effective as a retarder than Fe(III). The inhibition is accounted for by the reaction Fe(III) [or Cu(II)] + CO(-)(2) --> Fe(II) [or Cu(I)] + CO(2).

Particle-size determination by low-angle light scattering: effect of refractive index.

The intensity of scattering I(theta) by transparent and absorbing spheres has been calculated from exact Mie theory for values of radius from 0.1 microm to 100 ,microm, wavelength 5461 A, and the following values of refractive index m - im': (1) m = 1.01, 1.05, 1.1 (0.1) 2.0, m' = 0; (2) m = 2, m' = 0.002, 0.02, 0.2, and 2. The calculations have been made for a range of scattering angle theta large enough to indicate the first maximum in I(theta)theta(2). According to the Sloan method for determining particle radius, the location of this maximum is inversely proportional to radius, independent of refractive index. The exact calculations show that this is accurate for radii above 10 microm, but for smaller particles the value of the refractive index affects the location to some extent.

Formation of hydrogen peroxide upon oxidation of oxalic acid in presence and absence of oxygen-II Oxygen as oxidant.

Hydrogen peroxide, m a mixture that is 0.1M in manganese(II) sulphate and sulphuric add and exposed to light from a tungsten lamp, decomposes slowly at room temperature in a nitrogen atmosphere. At 75 degrees the reaction is fairly rapid in the dark (about 20-25% in 4 hr) and about 60% faster in light. Presence of oxalic add has little effect on the rate of disappearance of peroxide, the overall reaction corresponding to H(2)C(2)O(4) + H(2)O(2) --> 2CO(2) + 2H(2)O. In the presence of oxygen [and manganese(II)] this reaction does not take place; instead, hydrogen peroxide is formed and oxalic add disappears in equimolar amounts. The extent of all reactions greatly increases with increasing concentration of manganese. Acrylonitrilc acts as a retarder. Conditions m the presence of mangancse(II) are described, under which oxalic add can be oxidized quantitatively by oxygen to carbon dioxide with formation of an amount of hydrogen peroxide equimolar to the oxalic add oxidized. Reaction mechanisms are proposed to account for the fact that in the absence of oxygen 1 mole of peroxide disappears and in the presence of oxygen 1 mote of peroxide is formed for each mole of oxalic add reacted.