Measuring enzyme hydration in nonpolar organic solvents using NMR

A very sensitive NMR method has been developed for measuring deuterated water bound to proteins suspended in nonpolar solvents. This has been used to determine the amount of bound water as a function of water activity for subtilisin Carlsberg suspended in hexane, benzene, and toluene and for alpha-chymotrypsin in hexane. The adsorption isotherms for subtilisin in the three solvents are very similar showing that water activity can be usefully employed to predict the amount of water bound to proteins in nonpolar organic media. Comparison of the degree of enzyme hydration reached in nonpolar solvents with that obtained in air shows that adsorption of strongly bound water is hardly affected by the low dielectric medium, but adsorption of loosely bound water is significantly reduced. This suggests that the hydrophobic regions of the protein surface are preferentially solvated by solvent molecules, and that in a nonpolar environment formation of a complete monolayer of water over the protein surface is thermodynamically unfavorable. (c) 1995 John Wiley & Sons, Inc.     

Pressure affects enzyme function in organic media

Pressure affects enzyme function in nonaqueous media. Activation volumes have been determined and provide evidence that the primary effect of pressure is to enhance the stripping of water off an enzyme in polar organic solvents and leads to decreased enzymatic activity. Activation volumes of subtilisin Carlsberg in organic solvents, particularly with the enzyme hydrated, have a larger magnitude than activation volumes determined in aqueous solutions. This study provides further evidence that enzymatic activity in polar organic solvents is dominated by the interaction of enzyme-bound water with the solvent. From a practical standpoint, however, the results of this study suggest that enzymatic catalysis in organic solvents may be controlled by the combined effects of pressure and enzyme hydration. (c) 1993 John Wiley & Sons, Inc.     

Solid-state enzyme deactivation in air and in organic solvents

Thermal deactivation of solid-state acid phosphates (E.C. 3.1.3.2, from potato) is analyzed, both in the presence and in the absence of organic solvents. The thermal deactivation profile departs from first order kinetics and shows an unusual activity. The process is described by a phenomenological equation, whose theoretical implications are also discussed. The total amount of buffer salts in the enzyme powder dramatically affects enzyme stability in the range 70xC to 105xC. The higher salt/protein ratio increases the rate of thermal deactivation. The deactivation rate is virtually unaffected by the presence of organic solvents, independent of their hydrophilicity. (c) 1994 John Wiley & Sons, Inc.     

Biocatalytic synthesis of acrylates in organic solvents and supercritical fluids: I. optimization of enzyme environment

Biocatalytic transesterification of methylmethacrylate is possible in many different solvents. The reaction rate is readily controlled by variation in solvent physical properties. The reaction proceeds better in hydrophobic solvents, and activity can be restored in hydrophilic solvents by the addition of water. We have now demonstrated that supercritical carbon dioxide is not a good solvent for the reaction between 2-ethlhexanol and methylmethacrylate. It apperars that the supercritical carbon dioxide may either alter the pH of the microaqueous environment associated with the protein or reversibly form covalent complexes with free amine groups on the surface of the enzyme. Although supercritical carbon dioxide is a poor solvent for acrylate transesterification, many other supercritical fluids (ethane, ethylene, sulfur hexafluoride, and fluoroform) are better than most conventional solvents. In supercritical ethane it is possible to control the activity of the enzyme by changing pressure, and the enzyme appears to follow Michaelis-Menten Kinetics. We find that sulfur hexafluoride, the first anhydrous inorganic solvent in which biocatalytic activity has been reported, is a better solvent than any conventional or supercritical organic fluid tested.     

Effect of Pb on leaf antioxidant enzyme activities and ultrastructure of the two ecotypes of <Emphasis Type="Italic">Sedum alfredii</Emphasis> Hance

Hydroponic experiments were conducted to study the effect of Pb on growth, leaf antioxidant enzyme activities, and ultrastructure of the accumulating ecotype (AE) and non-accumulating ecotype (NAE) of Sedum alfredii Hance. AE was found to be more tolerant to excessive Pb levels in growth medium. Concentrations of Pb in the shoots of the AE were 1.98 times higher than those in the NAE when 0.2 mM Pb was supplied. Both chlorophyll a and b did not decrease significantly in AE plants after Pb treatment, while a significant decrease was noted in chlorophyll a and b of NAE plants treated with Pb concentrations greater than 0.05 mM. The results showed that activities of superoxide dismutase (SOD) and catalase (CAT) were elevated in the leaves of AE under Pb stress. However in NAE, Pb-caused enhancement was noticed only in the activity of SOD while activity of CAT was declined as compared to the control plants. With increased Pb level, malondialdehyde (MDA) content increased significantly in both ecotypes of S. alfredii, indicating that Pb toxicity led to lipid peroxidation and membrane damage, but MDA content in the leaves of NAE was always higher than in AE plants. The ultrastructural analysis of the spongy mesophyll cells revealed that excessive Pb concentrations obviously damaged the cell membrane, chloroplasts, and mitochondria of both the ecotypes but damage was more severe in NAE. Although growth, leaf physiology, and ultrastructure of both the ecotypes were affected by Pb treatment, deleterious effects were more pronounced in NAE. This text was submitted by the authors in English.     

Effect of bilayer morphology on the subgel phase formation

We examined how morphology of bilayer assemblies affects the kinetics of the subgel phase formation in dimyristoylphosphatidylglycerol (DMPG) bilayers, which change their morphology depending on NaCl concentration. Quantitative analysis of the kinetics revealed that in flat sheet-like structures (bilayer sheets) the subgel phase forms in a simple two-state manner with the relaxation time of about 3 min at -10 degrees C while in vesicles it forms much slower under a multi-step process. Freeze-etch electron microscopic observations suggested that the kinetics of the subgel phase formation is directly correlated with the morphology of bilayer assemblies. It is likely that the bilayer sheet structure is more favorable to the subgel phase formation in DMPG bilayers than the vesicular structure.     

Solid-state nuclear magnetic resonance investigation of solvent dependence of tyrosyl ring motion in an enzyme

Tyrosyl ring motions in alpha-lytic protease were investigated by solid-state deuterium nuclear magnetic resonance (NMR) spectroscopy in lyophilized enzyme powder, in powder suspended in organic solvents, and in aqueous crystals. Ring flipping rates were determined by examining deuterium quadrupole echo line shapes. Of the four Tyr residues in the enzyme, one was flipping at the slow (< or =10(3) s(-1)) and one at the fast (> or =10(7) s(-1)) exchange limit of the line shape experiment in all the environments tested. Flipping rates of the remaining two Tyr residues depended markedly on the solvent, with the lowest flipping rates (< or =10(3) s(-1) for both residues) observed in the enzyme powder, whether dry or suspended in hydrophobic tert-butyl methyl ether. In hydrophilic dioxane and acetonitrile, the mobility of these residues increased to 10(4) and 10(5) s(-1). The latter rate rose further to 10(6) s(-1) in the hydrated hydrophilic solvents and to > or =10(7) s(-1) in aqueous crystals. The deuterium spectrum of native alpha-lytic protease was compared with that of the enzyme whose active center was covalently modified with an inhibitor, which binds next to Tyr-123, constraining its ring. This experiment revealed that water addition to acetonitrile specifically increased the flipping rate of this active center residue. Librational motions ("wobbling"), estimated by their effect on spin-lattice relaxation times, were slowest in the anhydrous solvents, intermediate in the hydrated solvents, and fastest in the aqueous crystals. Thus, alpha-lytic protease is more rigid in organic solvents than in water, as judged by mobility of its tyrosyl residues. Water stripping by hydrophilic solvents did not increase enzyme rigidity, nor were there clear correlations between mobility and either enzymatic activity or solvent dielectric constant.     

Effect of support material and enzyme pretreatment on enantioselectivity of immobilized subtilisin in organic solvents

Subtilisin Carlsberg was covalently attached to five macroporous acrylic supports of varying aquaphilicity (a measure of hydrophilicity). Kinetic parameters of the transesterification of S and R enantiomers of secphenethyl alcohol with vinyl butyrate, catalyzed by various immobilized subtilisins, were determined in anhydrous dioxane and acetonitrile. Enzyme enantioselectivity in acetonitrile, but not in dioxane, correlated with the aquaphilicity of the support; a mechanistic rationale for this phenomenon was proposed. Although the catalytic activity of immobilized subtilisin in anhydrous solvents strongly depended on enzyme pretreatment, the enantioselectivity was essential conserved. (c) 1994 John Wiley & Sons, Inc.     

Aggregation-induced alterations in fibroblast morphology

Summary The different stages during aggregation of diploid human skin fibroblasts have been examined by transmission and scanning electron microscopy. As a result of aggregation, fibroblasts form a complex tissue configuration. Numerous intercellular junctions can be observed, while the cells remain polygonal and do not develop an organised intracellular cytoskeleton. Cell division occurs only rarely. After aggregation, signs of progressive auto-digestion develop.Adhesion to a substrate results in outgrowth of the cells and monolayer formation, even when extensive cell damage had occurred. The morphology of fibroblasts in aggregates and in the monolayers, from which they were derived, is compared and the contribution of the aggregate system to the study of fibroblast behavior is discussed.J.J. Cassiman is Aangesteld Navorser van The National Foundation for Scientific Research, Belgium     

A comparison of lipase-catalyzed ester hydrolysis in reverse micelles, organic solvents, and biphasic systems

The performance of lipases from Candida rugosa and wheat germ have been investigated in three reaction media using three acetate hydrolyses as model reactions (ethyl acetate, allyl acetate, and prenyl acetate). The effect of substrate properties and water content were studied for each system (organic solvent, biphasic system, and reverse micelles). Not unexpectedly, the effect of water content is distinct for each system, and the optimal water content for enzyme activity is not always the same as that for productivity. A theoretical model has been used to simulate and predict enzyme performance in reverse micelles, and a proposed partitioning model for biphasic systems agrees well with experimental results. While the highest activities observed were in the micellar system, productivity in microemulsions is limited by low enzyme concentrations. Biphasic systems, however, support relatively good activity and productivity. The addition of water to dry organic solvents, combined with the dispersion of lyophilized enzyme powders in the solvent, resulted in significant enzyme aggregation, which not surprisingly limits the applicability of the "anhydrous" enzyme suspension approach. (c) 1995 John Wiley & Sons, Inc.     

Enzyme stabilization: state of the art

Enzyme stabilization is one of the most important fields in basic and applied enzymology. In basic enzymology, it is of particular relevance to understand enzyme stabilization principles first elucidating how and why the enzymes lose their biological activity and then deriving structure-stability relationships existing in enzymatic molecules. In applied enzymology, the most significant goal is to achieve useful compounds by biocatalysis. Enzymes are good catalysts in terms of high catalytic and specific activity with ability to function under mild conditions. However, they are not always ideal catalysts for practical applications because they are generally unstable and they inactivate rapidly through several mechanisms. In order to enhance enzyme stability, many strategies have been pursued in recent years. The present article is an attempt to provide detailed information about these strategies.     

The pollen morphology of Vicia (Leguminosae)

The pollen morphology of 32 species of the genus Vicia was studied using scanning and transmission electron microscope (SEM and TEM). The interstitia of the pollen of the examined species were found to be either regular columellate, irregular columellate, or granular. The granular pattern was recognized to be apomorphy against the columellate patterns in the tribe Vicieae by comparison with the pollen in the tribes Vicieae, Cicereae, Galegeae, Hedysareae, and Trifolieae. The group of Vicia species having the apomorphy was congruent with that having a known apomorphy of the pistil character. This group with such synapomorphies may be monophyletic, though it is treated as polyphyletic in the present infrageneric system of Vicia.     

Cells of Pseudomonas putida and Enterobacter sp. adapt to toxic organic compounds by increasing their size

The phenol-degrading solvent-tolerant bacterium Pseudomonas putida P8 changed its cell shape when grown in the presence of aromatic compounds such as phenol and 4-chlorophenol. The sizes of cells that had been growing after addition of different concentrations of the toxic compounds were measured using a coulter counter that calculates the sizes of the rod-shaped bacteria to diameters of virtual spheres. The cells showed an increase in the diameter depending on the toxic effects of the applied concentrations of both solvents. The same effect was measured for an alkanol degrading bacterium, Enterobacter sp. VKGH12, in the presence of n-butanol. The reaction of the cells to different concentrations of n-butanol was examined by scanning electron microscopy. With this technique it could be shown that the size of the bacteria increased with increasing concentrations of n-butanol. These changes in cell size were dependent on the cellular activity and occurred only after addition of non-lethal concentrations. In the presence of lethal concentrations that completely inhibited cell growth, the cell sizes were similar to those of cells without intoxication. Taking into account the mathematical formula for spherical and cylindrical diameter and surface, respectively, the cells reacted to the presence of organic solvents by decreasing the ratio between surface and volume of the cells and therefore reducing their relative surfaces. As the cell surface and especially the cytoplasmic membrane are the major targets for the toxic effects of membrane-active compounds, this reduction of the relative surface represents an adaptive response to the presence of such compounds.     

Multiple solvent crystal structures of ribonuclease A: An assessment of the method

The multiple solvent crystal structures (MSCS) method uses organic solvents to map the surfaces of proteins. It identifies binding sites and allows for a more thorough examination of protein plasticity and hydration than could be achieved by a single structure. The crystal structures of bovine pancreatic ribonuclease A (RNAse A) soaked in the following organic solvents are presented: 50% dioxane, 50% dimethylformamide, 70% dimethylsulfoxide, 70% 1,6‐hexanediol, 70% isopropanol, 50% R,S,R‐bisfuran alcohol, 70% t‐butanol, 50% trifluoroethanol, or 1.0M trimethylamine‐N‐oxide. This set of structures is compared with four sets of crystal structures of RNAse A from the protein data bank (PDB) and with the solution NMR structure to assess the validity of previously untested assumptions associated with MSCS analysis. Plasticity from MSCS is the same as from PDB structures obtained in the same crystal form and deviates only at crystal contacts when compared to structures from a diverse set of crystal environments. Furthermore, there is a good correlation between plasticity as observed by MSCS and the dynamic regions seen by NMR. Conserved water binding sites are identified by MSCS to be those that are conserved in the sets of structures taken from the PDB. Comparison of the MSCS structures with inhibitor‐bound crystal structures of RNAse A reveals that the organic solvent molecules identify key interactions made by inhibitor molecules, highlighting ligand binding hot‐spots in the active site. The present work firmly establishes the relevance of information obtained by MSCS. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Contributions to the pollen morphology and taxonomy of the Liliaceae

Pollen of 34 species from 7 genera of the Liliaceae were examined by LM and SEM with respect to the taxonomy of the family. Detailed pollen­morphological characteristics are given for all genera in the family on the basis of the results presented here together with data from the literature. The genera Tulipa and Lilium are heterogeneous in both aperture type and exine ornamentation. Pollen of Tulipa is monosulcate, 3-aperturate or inaperturate, with a microreticulate-striate, reticulate-implecto-striate, scabrate, perforate-rugulate, perforate-striate exine surface. Pollen of Lilium is monosulcate and 3-porate with a macroreticulate exine. The other genera are homogeneous in possessing of single longitudinal aperture (type monosulcate). The pattern of exine ornamentation and the structure of the aperture and its membrane are peculiar features for species and genera. Pollen of Erythronium and Tulipa are occasionally operculate, while in other representatives of the Liliaceae an operculum is lacking. Pollen morphological data support the division of the family into 3 tribes, namely Lloydieae, Lilieae, and Tulipeae.     

Dependence of alpha-synuclein aggregate morphology on solution conditions

Alpha-synuclein is the major component of Lewy bodies and Lewy neurites, which are granular and filamentous protein inclusions that are the defining pathological features of several neurodegenerative conditions such as Parkinson's disease. Fibrillar aggregates formed from alpha-synuclein in vitro resemble brain-derived material, but the role of such aggregates in the etiology of Parkinson's disease and their relation to the toxic molecular species remain unclear. In this study, we investigated the effects of pH and salt concentration on the in vitro assembly of human wild-type alpha-synuclein, particularly with regard to aggregation rate and aggregate morphology. Aggregates formed at pH 7.0 and pH 6.0 in the absence of NaCl and MgCl(2) were fibrillar; the pH 6.0 fibrils displayed a helical twist, as clearly evident by scanning force and electron microscopy. Incubations at pH 7.0 remained transparent during the process of aggregation and exhibited strong thioflavin-T and weak 8-anilino-1-naphthalenesulfonate (ANS) binding; furthermore, they were efficient in seeding fibrillization of fresh solutions. In contrast, incubating alpha-synuclein at low pH (pH 4.0 or pH 5.0) resulted in the rapid formation of turbid suspensions characterized by strong ANS binding, reduced thioflavin-T binding and reduced seeding efficiency. At pH 4.0, fibril formation was abrogated; instead, very large aggregates (dimensions approximately 100 microm) of amorphous appearance were visible by light microscopy. As with acidic conditions, addition of 0.2M NaCl or 10mM MgCl(2) to pH 7.0 incubations led to a shorter aggregation lag time and formation of large, amorphous aggregates. These results demonstrate that the morphology of alpha-synuclein aggregates is highly sensitive to solution conditions, implying that the fibrillar state does not necessarily represent the predominant or most functionally significant aggregated state under physiological conditions.     

Efficient multiple-solvent suppression for the study of the interactions of organic solvents with biomolecules

A modification of the excitation sculpting sequence [Hwang, T.-L. and Shaka, A.J. (1995) J. Magn. Reson., A112, 275–279] for achieving efficient multiple-solvents suppression in 1D and 2D ¹H NMR experiments is presented. Implementation of this scheme in the ePHOGSY experiment allows rapid and sensitive detection of weak interactions between organic solvents or small molecules and biomolecules. Application of the techniques to the peptide Sandostatin® dissolved in H₂O and DMSO is demonstrated.     

The expansion of maize root-cap mucilage during hydration. 2. Observations on soil-grown roots by cryo-scanning electron microscopy

Expansion of root-cap mucilage during hydration was followed by cryo-scanning analytical microscopy of soil-grown roots of diploperennis and Zea mays. Roots examined directly from the soil have no expanded mucilage. Their condensed, unexpanded mucilage is in three domains, periplasmic, intercellular and peripheral to the cap tissue. Carbon concentration is the same in the three domains. During hydration there is no change in carbon concentration as the condensed mucilage moves through these three domains; however there is a sharp drop at the periphery where a gel phase transition occurs. The rate of expansion of the mucilage blob around the root tip is limited by the rate of this gel phase transition.     

Observations on morphology and hydrolytic enzyme histochemistry of excysted metacercariae of Himasthla rhigedana (Trematoda: Echinostomatidae)

The morphology of in vitro excysted metacercariae of Himasthia rhigedana was studied by light microscopy, enzyme histochemistry, and scanning electron microscopy (SEM). The body surface is covered with longitudinal rows of pointed spines which extend from the anterior end to just below the acetabulum. Histochemical methods for acid and alkaline phosphatases, β-glucoronidasc, leucine aminopeplidase, nonspecific esterase, acetylcholinesterase, butyrylcholincsterase, chymotrypsin-like protease, α-galactosidase and β-galactosidase were applied to the excysted metacercariae. Certain systems of the metacercaria containing these enzymes showed positive reactions to the appropriate methods and were selectively visualized. Reactions for alkaline phosphatase occur throughout most of the excretory system while acid phosphatase occurs in the gut, oral, and ventral suckers. Reactions for nonspecific esterases and cholinesterascs are found throughout the nervous system, in the gut, and in the oral and ventral suckers. The results of including specific inhibitors or activators in the incubation medium after preineubation m 10^?5m diethyl p-nitrophenyl phosphate (E-600) suggested that A-and C-typc esterases are present in the gut. Inclusion of 10^?4m eserine in the incubation medium abolished cholinesterase activity in the nervous system.