Purification and hydrophobic properties of the δ-endotoxin in the parasporal crystal produced by Bacillus thuringiensis var. entomocidus

Parasporal crystals of Bacillus thuringiensis var. entomocidus were separated from spores and other cell debris by the water-chloroform biphase procedure. The solubilization and fractionation were carried out under mild conditions at 4°C. Crystals were solubilized in 0.01 M dithiothreitol and 0.2 M glycine NaOH buffer at pH 10.0. The solution was treated overnight with 0.01 M Tris-HCl buffer, pH 5.5, containing 0.1% Triton N-101 and 0.1% sodium cholate, and then placed on a Sepharose 6B column, equilibrated, and later developed with the same buffer. Under these conditions, four fractions were obtained, one of which had a molecular weight ranging from 60,000 to 70,000, and demonstrated a high insecticidal activity on second instar larvae of Spodoptera litioralis. The LC₅₀ value of this fraction was a half of that of the solubilized crystals. The other three fractions had a lower activity. The active fraction was further fractionated on an octyl-Sepharose 4B resin. Elution of this column with the same buffer separated the proteins into two fractions. The first eluted fraction was highly active, while the second demonstrated a very low activity. The active fraction was further purified by loading on a short column of octyl-Sepharose 4B and eluted with a linear gradient of the same detergents. Under these conditions, the highly active fraction gave a sharp and symmetrical peak that revealed five close bands at the pH range of 6.1–6.5 on isoelectric focusing gel electrophoresis.     

Equilibrium kinetics of substrate-enzyme interactions in single crystals of cytoplasmic aspartate aminotransferase

Orthorhombic single crystals of cytoplasmic aspartate aminotransferase were examined alone or in the presence of substrates or inhibitors to quantitatively compare the interaction of ligands with the active-site chromophore between soluble and crystalline enzyme. As in enzyme solutions, equilibrium kinetic measurements can be made between substrates and single crystals of cytoplasmic aspartate aminotransferase. The absorption spectra of ligand-free enzyme forms and of enzyme-substrate or-inhibitor complexes are as distinctive as when the enzyme is in solution. The dissociation constants for glutamate with the pyridoxal form of the enzyme are identical to those in solution. The substrate analog erythro--hydroxyaspartate also binds with equal affinity to the active site in enzyme crystals as in solution; and the affinity of -ketoglutarate to bind in nonproductive complexes with the pyridoxal form of the enzyme is also unimpaired in the crystal (K _d =2 mM). In contrast to the affinity constants, the stoichiometry of the interactions does not appear to correlate to those in solution. In the presence of an amino acid plus keto acid substrates pair, the absorbance values of the enzyme-substrate complex(es) could be interpreted as for occupany of only half the available sites in the crystals. Yet an amino acid, cysteine sulfinate, and -keto acids such as , -difluorooxalacetate convert all active sites in the crystal to the pyridoxamine or pyridoxal form when added to the pyridoxal or pyridoxamine forms, respectively. This ability to completely undergo substrate-induced half-transamination and the apparently conflicting results in trapping half the sites in enzyme-substrate complexes are incorporated into a proposed reciprocating mechanism applicable only to the crystalline state of the enzyme and dictated by crystal packing forces rather than an intrinsic property of the enzyme. Active-site bound pyridoxal phosphate continues to behave as a pH indicator; nevertheless, the pK value of the single crystals is a pH unit (pK=7.15) higher than that in solution. This variation is interpreted as indication of a difference in the environment of the chromophore between the crystal and solution states. While the environmental difference does not significantly alter the affinity for substrates, it could account for the reduced rates in transformation of the enzyme-substrate complexes in half-transamination reactions in the crystalline state.     

Crystallization and preliminary X-ray investigation of lipoxygenase-3 from soybeans.

Soybean lipoxygenase-3 has been crystallized by the vapor diffusion method in 16-20% polyethylene glycol (average M(r), 3,400), 0.2 M sodium acetate buffer, pH 5.7, at 21 degrees C, at a protein concentration of 8-15 mg/mL. The crystals, which diffract to 3-A spacings, belong to the monoclinic space group C2. Cell constants are a = 111.9, b = 136.4, and c = 61.6 A and beta = 95.7 degrees. The calculated value of Matthews's constant, Vm = 2.48 A3/kDa, is consistent with the presence of one molecule of lipoxygenase per crystallographic asymmetric unit (Z = 4).     

Question 4: Basic Questions About the Origin of Life: On Chirobiogenesis

Some experimental aspects concerning either the enantio-enrichment of α-amino acids starting from non-racemic mixtures or the formation of homochiral oligopeptides and oligonucleotides starting from racemic precursors are described. In principle, it is possible that more than one of these processes had played a role in chirobiogenesis. Presented at the International School of Complexity – 4th Course: Basic Questions on the Origins of Life; “Ettore Majorana” Foundation and Centre for Scientific Culture, Erice, Italy, 1–6 October 2006.     

Crystallization of ADP-ribosyl cyclase from Aplysia californica

ADP-ribosyl cyclase synthesizes the secondary messenger cyclic ADP-ribose from NAD⁺. Diffraction quality crystals of the enzyme from ovotestes of Aplysia californica have been obtained. Crystallographic analysis of this enzyme will yield insight into the mode of binding of the novel cyclic nucleotide and the mechanism by which NAD⁺ is cyclized.     

Structural organization of xanthine crystals in the median ocellus of a member of the ancestral insect group Archaeognatha

Biogenic purine crystals function in vision as mirrors, multilayer reflectors and light scatterers. We investigated a light sensory organ in a primarily wingless insect, the jumping bristletail Lepismachilis rozsypali (Archaeognatha), an ancestral group. The visual system of this animal comprises two compound eyes, two lateral ocelli, and a median ocellus, which is located on the front of the head, pointing downwards to the ground surface.We determined that the median ocellus contains crystals of xanthine, and we obtained insights into their function. To date, xanthine biocrystals have only been found in the Archaeognatha. We performed a structural analysis, using reflection light microscopy, cryo-FIB-SEM, microCT and cryo-SEM. The xanthine crystals cover the bottom of a bowl-shaped volume in the median ocellus, in analogy to a tapetum, and reflect photons to light-sensitive receptors that are spread in the volume without apparent order or preferential orientation. We infer that the median ocellus operates as an irregular multifocal reflector, which is not capable of forming images. A possible function of this organ is to improve photon capture, and by so doing assess distances from the ground surface when jumping by determining changes in the intensity and contrast of the incident light.     

Developmentally regulated antigen associated with calcium crystals in tobacco anthers

We have found and characterized an antigen associated with crystal-containing cells in the stomium and connective tissue of the anthers of Nicotiana tabacum L. (tobacco). The antigen, defined by the monoclonal antibody NtF-8B1, localizes to subcellular regions surrounding the crystals. At the light-microscope level, the antigen is detectable just after the first appearance of crystals in the connective tissue of the anther, and at approximately the same time as the appearance of crystals in the stomium. The antigen is not detectable on a Western blot, and gave inconclusive results on a test of periodate sensitivity. It is not the crystals themselves, nor is the presence of the crystals required for antibody recognition. The antigen is sensitive to heat and protease treatment, indicating that it is a protein. The antigen is not tightly membrane-bound, in spite of its localization closely surrounding the crystals. Chemical tests indicate that the druse crystals in the stomium are calcium oxalate.Abbreviations ELISA enzyme-linked immunosorbent assay - FITC fluorescein isothiocyanate This research was supported by a National Science Foundation postdoctoral fellowship to B.L.H., by National Science Foundation grants DMB-87-15799 and to W.E.F. BSR-88-18035, and by U.S. Department of Agriculture grant GAM-89-01056. The authors thank Phillip T. Evans (Louisiana State University, Baton Rouge, USA), Wilma L. Lingle, Harry T. Horner, Jr. (Iowa State University), and A. Jack Fowler, Jr., for advice and helpful discussions.     

Two-dimensional structure of phospholipase induced crystals of Ca2+-ATPase from sarcoplasmic reticulum

A two-dimensional projection map was computed of the Ca²⁺-ATPase molecules in sarcoplasmic reticulum, isolated from rabbit skeletal muscle. Crystalline arrays of Ca²⁺-ATPase molecules were formed by incubating the membrane vesicles with phospholipase A₂ and dialysing against Tris/HCl buffer. Ca²⁺-ATPase molecules appear as quasi-triangular blobs in the projection map and seem to form dimers. The projection map seems to indicate an enzyme conformation somewhat similar to vanadate-induced crystals but different from lanthanide-induced crystals of Ca^2*-ATPase.     

Enantiomeric Enrichment on the Prebiotic Earth

Evaporation of an aqueous solution of a chiral, nonracemic (partially resolved) amino acid or hydroxy acid from a clay or silica surface is shown to produce crystals in six cases that possess increased concentrations of the more abundant enantiomer.The increases range from a factor of 1.2 as the low to a high greater than 6-fold above the near 8% enantiomeric excess starting levels. This newly discovered enantiomeric enrichment process is presented in the context of it having been a possible instrument in the attainment of levels of configurational homogeneity sufficient for the emergence of life on Earth. Portions of this work were presented to The 14th International Conference on the Origin of Life, Beijing, China, June 21, 2005     

Mechanical deformation enhances catalytic activity of crystalline carboxypeptidase A.

A new approach for investigating mechano-chemical interactions in enzymes is described. The catalytic activity of crystalline crosslinked enzymes subjected to uniaxial deformation has been measured. Extension of monoclinic P2(1) crystals of carboxypeptidase A along the [010] direction leads to a many-fold increase in catalytic esterase activity with no changes in the effective Michaelis constant. This increase is interpreted as due to liberation of conformational mobility associated with catalytic activity of the enzyme in the deformed crystal.     

Thermotropic and lyotropic properties of long chain alkyl glycopyranosides: part III: pH-sensitive headgroups

As part of a series of papers, the influence of carbohydrate headgroups and aliphatic chains on the mesogenic properties of glycolipids was investigated. Alkyl glycosides with different types of aliphatic chains were synthesised. Neutral glycolipids were oxidized to their uronic acid derivatives, using the well established TEMPO-oxidation. For comparison a 6-deoxy-6-amino alkylglucopyranoside was synthesised. In addition, the thermotropic and lyotropic phase behaviour of the synthesised compounds were investigated. The thermotropism was characterised by polarising microscopy, the lyotropism by the contact preparation method.     

多管藻中R－藻蓝蛋自的分离、结晶和初步晶体学研究

从青岛采集的多管藻（Ｐｏｌｙｓｉｐｈｏｎｉａｕｒｃｅｏｌａｔａ）中分离得到的Ｒ－藻蓝蛋白,在ｐＨ＝７．０的０．０５ｍｏｌ／Ｌ磷酸盐－硫酸铵缓冲液中,使用悬滴气相扩散法获得适合Ｘ光衍射分析用单晶。经Ｂｕｅｒｇｅｒ徘循照相和ＸＲＤ—１００面探测仪分析,Ｒ－藻蓝蛋白晶体属于四方晶系,空间群为Ｐ４１（３）２１２,晶胞参数：ａ＝ｂ＝１３７．５ｃ＝２１８．５α＝β＝γ＝９０°。用等比重梯度柱法测定了晶体和母液的比重分别为１．１９和１．０９。根据分子量与晶胞体积估算,一个不对称单位含有一个分子,推测它的分子聚集态形式为（αβ）３。     

Preliminary crystallographic analysis of class 3 rat liver aldehyde dehydrogenase

NAD-linked aldehyde dehydrogenases (A1DH) (EC 1.2.1.3) catalyze the irreversible oxidation of a wide variety of aldehydes to their respective carboxylic acids. Crystals of a class 3 AIDH (from an Escherichia coli expression system) suitable for X-ray analysis have been obtained. These crystals, which can be grown to a size of 0.8 x 0.3 x 0.2 mm, diffract to 2.5 A resolution. Analysis of the diffraction pattern indicates that the crystals belong to the monoclinic space group P21, with cell parameters a = 65.11 A, b = 170.67 A, c = 47.15 A, and beta = 110.5 degrees. Assuming one dimer per asymmetric unit, the value Vm is calculated to be 2.45 and the solvent content of the crystal is estimated to be 50%. A self-rotation function study produced significant rotation peaks (58% of the origin) on the kappa = 180 section at psi = 90 degrees and phi = 71 degrees and 341 degrees, indicating that the pseudo-dimer axis is (or is very nearly) perpendicular to the b-axis.     

Crystallization and preliminary X-ray crystallographic analysis of arylesterase from Pseudomonas fluorescens

Large crystals of arylesterase from Pseudomonas fluorescens have been grown at room temperature using ammonium sulfate as a precipitant. They grow to dimensions of 0.7 × 0.7 × 0.6 mm³ within a month. The crystals belong to the trigonal space group P3₁ (or P3₂), with unit cell dimensions of a= 147.12 Å and c= 131.08 Å. The asymmetric unit seems to contain six molecules of dimeric aryles-terase, with corresponding crystal volume per protein mass (VM ) of 2.53 ų/Da and solvent fraction of 51.5% by volume. The crystals diffract to at least 2.2 Å Bragg spacing when exposed to X-rays from a rotating-anode source. X-ray data have been collected to 2.9 Å Bragg spacing from native crystals. © 1993 Wiley-Liss, Inc.     

ARAGONITE CRYSTALS IN SPIROGYRA SP. (CHLOROPHYTA)

Intracellular crystals of aragonite have been identified by selected area electron diffraction (SAED) in a species of the freshwater filamentous alga Spirogyra from the Thames River, Ontario, Canada. The crystals are 2 to 24 μm in diameter, and characterized by a unique cross-shaped morphology, in which needle-like, or prismatic outgrowths develop from a common axis. Crystals may be dispersed throughout filaments, but tend to cluster as aggregates towards the centre .     

Computer Simulation of Liquid Crystal Films

Abstract

We present the results of extensive Monte Carlo simulations of liquid crystal films of various thicknesses. A simple nearest-neighbour lattice model, the Lebwohl-Lasher model, is employed, with periodic boundaries in two directions and free, planar, surfaces in the third. Particular attention is devoted to locating the temperature of the order-disorder (nematic-isotropic) phase transition. Weak first-order behaviour apparently persists in systems as thin as 8 layers across, but below this the transition cannot be detected. The shift of the transition temperature from its bulk value approaches the expected asymptotic linear dependence on inverse thickness, but significant deviations from this are seen for films of 10 layers thickness and less. These results enable an accurate estimate to be made of the bulk phase transition temperature in the thermodynamic limit, and the result is consistent with that extrapolated from systems with full periodic boundaries.     

Cytotoxicity of two proteins isolated from Bacillus thuringiensis var. israelensis crystals to insect and mammalian cell lines

Abstract Two toxic proteins (proteins A and B) were isolated from Bacillus thuringiensis var. israelensis crystals using ion-exchange chromatography. A cell cytotoxicity assay based on the uptake of [³H]uridine by tissue culture cells following exposure to the toxic proteins, was used to measure the viability of the cells. Proteins A and B were cytotoxic to Aedes aegypti and MA104 cells. Protein B was also cytotoxic to Spodoptera frugiperda cells.     

苏云金杆菌4.071 8菌株杀虫晶体性质的研究

研究了苏云金杆菌4．071 8菌株晶体的分离及其在电镜下的形态结构,并通过SDS－PAGE对其杀虫晶体蛋白进行了分析,证明了4．071 8菌株晶体有立方体型和双金字塔型两种,分别由原毒素CryⅠ及CryⅡ组成,该菌对鳞翅目和双翅目昆虫均有毒杀作用。     

Crystallization and preliminary X-ray crystallographic analysis of phospholipid transfer protein from maize seedlings

Phospholipid transfer protein from maize seedlings has been crystallized using trisodium citrate as precipitant. The crystal belongs to the orthorhombic space group P2₁2₁2₁ with unit cell dimensions of a = 24.46 Å, b = 49.97 Å, and c = 69.99 Å. The presence of one molecule in the asymmetric unit gives a crystal volume per protein mass (V_m) of 2.36 Å ³/Da and a solvent content of 48% by volume. The X-ray diffraction pattern extends at least to 1.6 Å Bragg spacing when exposed to both CuK_α and synchrotron X-rays. A set of X-ray data to approximately 1.9 Å Bragg spacing has been collected from a native crystal. © 1994 Wiley-Liss, Inc.     

ELECTRON MICROSCOPIC OBSERVATIONS ON TUBULAR STRUCTURES INVOLVED IN CRYSTAL FORMATION IN THE RED ALGA PLEONOSPORIUM SQUARRULOSUM (HARV.) ABBOTT1

Hexagonal or angular crystalline inclusions in Pleonosporium (Naeg.) Hauck vegetative cells were examined using electron microscopy. Ultrastructural analysis reveals that the inclusions initially contain tubular elements resembling microtubules but, with continued differentiation, are transformed into rod containing crystals. The tubular structures initially measure 25 nm in diameter. Scattered tubules become arranged in a parallel and alternate pattern and undergo subsequent enlargement to approximately 29 nm. Following enlargement, each tubule apparently disaggregates into rods that form a crystal having hexagonally arranged rod-like subunits. It is suggested that these tubules may represent microtubules and the resultant crystals are composed of tubulin.