The leaf peroxisomal form (MFP IV) of multifunctional protein functioning in fatty-acid β-oxidation

We have purified for the first time from green leaves a multifunctional protein (MFP) involved in fatty acid -oxidation. The protein, designated MFP IV, was extracted from green leaves of three-week-old cucumber (Cucumis sativus L.) plants. Chromatography on cation exchanger, separation on hydroxylapatite, and fast-protein liquid chromatography on Phenylsuperose led to a more than 7000-fold purification and to the isolation of an apparently homogeneous 80-kDa monomeric protein. This protein is immunologically related to the glyoxysomal MFP II, as evidenced by immunodecoration with antiserum raised against MFP II. Comparison of molecular masses of all MFPs presently known revealed that the MFP prepared from green leaves (MFP IV) is distinct from MFP II (76.5 kDa) and MFP I (74 kDa) from dark-grown cotyledons. By including other properties in this comparison, we demonstrated that MFP IV can also be distinguished from the glyoxysomal MFP III (81 kDa) and the bacterially expressed MFP-a (80 kDa). Moreover, MFP IV is a constituent of leaf peroxisomes and contains the activities of 2-enoyl-CoA hydratase (EC 4.2.1.17),l-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) and 3-hydroxyacyl-CoA epimerase.Abbreviation MFP multifunctional protein This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.     

Resolution and determination of enantiomeric purity of the enantiomers of felodipine using chiral-AGP® as stationary phase

A direct chiral chromatographic reversed phase method for the determination of the enantiomers of felodipine is described. The influence of charged and uncharged modifiers as well as the effect of the mobile phase pH on the enantiomeric resolution is discussed. A high mobile phase pH and the addition of 2-propanol as organic modifier gave the highest separation factor (α = 1.3). The high mobile phase pH (pH = 7.6) is outside the recommended pH limit of silica based columns but was necessary to achieve baseline resolution of (R)- and (S)-felodipine. Improvement of column efficiency by increasing column temperature was utilized for optimization of the enantiomeric resolution (Rs = 1.7). The enantiomers of felodipine and three related compounds were separated within 15 min. The enantiomeric purity of (R)- and (S)-felodipine in injections and (R)-felodipine in bulk substance was higher than 99.5% and no racemization was observed after storage at accelerated conditions. A poor Chiral-AGP® column used for a long period was restored using a simple wash step together with repacking the top of the chromatographic column. © 1995 Wiley-Liss, Inc.     

Growth,nitrogen fixation and relative efficiency of nitrogenase inAlnus incana grown in different cultivation systems

Summary Three cultivation systems were compared. In one system the alders were grown hydroponically. In the two other systems the alders were planted in gravel and either given water and nutrients at intervals or the nutrient solution was continuously supplied. Alders continuously supplied with nutrients and water showed a significantly more rapid growth, higher biomass production and higher nitrogen content than did alders given nutrients and water at intervals or alders hydroponically grown. Alders continuously supplied with water and nutrients had a constant RE (relative efficiency of nitrogenase) of about 0.80 throughout the experimental period while alders supplied with water and nutrients at intervals showed a slight decrease in RE at the end of the experimental period. No strict relationship was found between RE and nitrogen content or between RE and plant productivity.     

Neural crest cell migration in relation to extracellular matrix organization in the embryonic axolotl trunk

Temporal and regional aspects of early neural crest cell migration in relation to extracellular matrix (ECM) organization and distribution in the embryonic axolotl trunk were studied by light microscopy, TEM, and SEM. The dominating structure of the interstitial ECM is a complex network of fibrils, which are indicated by ruthenium red staining to consist of collagen in association with ruthenium red-positive components, probably including glycosaminoglycans. The ECM fibrils, which are largely used as substratum for locomotion by the crest cells, have a temporally and regionally specific organization and distribution. Increase in ECM fibrils on the neural tube, ahead of the crest cell front, is correlated with initiation of crest cell emigration, and it is suggested that the fibrils may stimulate this process by providing a suitable substratum for cell locomotion. An increase in ECM fibrils in extracellular spaces surrounding the crest cell population is correlated with an expansion of these spaces and with progressing crest cell migration into them. It is proposed that the spatial organization of the ECM fibrils influences crest cell shape and orientation during early migration.     

Epidermal growth factor modulates extracellular Ca requirement for multiplication of normal human skin fibroblasts

Epidermal growth factor (EGF) at 10 ng/ml reduces by over 50-fold the extracellular Ca²⁺ required for multiplication of normal human skin fibroblasts. Therefore, a Ca²⁺-related process may play a central role in the mechanism by which EGF exerts its effect on cell multiplication.     

Can size distributions of European lake fish communities be predicted by trophic positions of their fish species?

An organism''s body size plays an important role in ecological interactions such as predator–prey relationships. As predators are typically larger than their prey, this often leads to a strong positive relationship between body size and trophic position in aquatic ecosystems. The distribution of body sizes in a community can thus be an indicator of the strengths of predator–prey interactions. The aim of this study was to gain more insight into the relationship between fish body size distribution and trophic position in a wide range of European lakes. We used quantile regression to examine the relationship between fish species'' trophic position and their log‐transformed maximum body mass for 48 fish species found in 235 European lakes. Subsequently, we examined whether the slopes of the continuous community size distributions, estimated by maximum likelihood, were predicted by trophic position, predator–prey mass ratio (PPMR), or abundance (number per unit effort) of fish communities in these lakes. We found a positive linear relationship between species'' maximum body mass and average trophic position in fishes only for the 75% quantile, contrasting our expectation that species'' trophic position systematically increases with maximum body mass for fish species in European lakes. Consequently, the size spectrum slope was not related to the average community trophic position, but there were negative effects of community PPMR and total fish abundance on the size spectrum slope. We conclude that predator–prey interactions likely do not contribute strongly to shaping community size distributions in these lakes.     

Structural and Mechanistic Studies Reveal the Functional Role of Bicovalent Flavinylation in Berberine Bridge Enzyme

Berberine bridge enzyme (BBE) is a member of the recently discovered family of bicovalently flavinylated proteins. In this group of enzymes, the FAD cofactor is linked via its 8α-methyl group and the C-6 atom to conserved histidine and cysteine residues, His-104 and Cys-166 for BBE, respectively. 6-S-Cysteinylation has recently been shown to have a significant influence on the redox potential of the flavin cofactor; however, 8α-histidylation evaded a closer characterization due to extremely low expression levels upon substitution. Co-overexpression of protein disulfide isomerase improved expression levels and allowed isolation and purification of the H104A protein variant. To gain more insight into the functional role of the unusual dual mode of cofactor attachment, we solved the x-ray crystal structures of two mutant proteins, H104A and C166A BBE, each lacking one of the covalent linkages. Information from a structure of wild type enzyme in complex with the product of the catalyzed reaction is combined with the kinetic and structural characterization of the protein variants to demonstrate the importance of the bicovalent linkage for substrate binding and efficient oxidation. In addition, the redox potential of the flavin cofactor is enhanced additively by the dual mode of cofactor attachment. The reduced level of expression for the H104A mutant protein and the difficulty of isolating even small amounts of the protein variant with both linkages removed (H104A-C166A) also points toward a possible role of covalent flavinylation during protein folding.Since the discovery of the first known example of a covalent bond between a flavin cofactor and an amino acid side chain occurring in enzymes in the 1950s (1), a number of different types of linkages have been identified: 8α-histidylation (either to N1 or to N3), 8α-O-tyrosylation, 8α-S-cysteinylation, and 6-S-cysteinylation. For current reviews relating to these modes of flavin attachment, see Refs. 2 and 3. Recently, another way of covalent tethering of FAD to proteins was discovered in x-ray crystallographic studies on glucooligosaccharide oxidase (GOOX)⁴ from Acremonium strictum (4). The mode of flavin linkage observed in this case employs both 8α-histidylation and 6-S-cysteinylation to form a bicovalently attached cofactor. Representative members of all these groups have been studied in detail, and several explanations for the role of the covalent flavinylation have been put forward. Some of the suggestions tend to be rather specific for the system being studied, e.g. prevention of cofactor inactivation at the C-6 position for trimethylamine dehydrogenase (5) or facilitation of electron transfer from the flavin to the cytochrome subunit for p-cresol methylhydroxylase (6). Other explanations including the increase of the flavin redox potential due to the covalent linkage (7–9) and the prevention of cofactor dissociation (10, 11) were found for several enzymes also harboring different types of cofactor attachments. Taking into account that protein stability (12) and optimal binding of substrate molecules (11, 13) are also positively influenced by covalent tethering of the flavin, one might speculate that no generally applicable explanation for the covalent attachment of flavins to proteins exists. Therefore, it seems likely that the large variety of systems operating with one of the above mentioned modes of cofactor tethering might have evolved to also adapt to a diversity of enzymatic challenges.Berberine bridge enzyme (BBE) from Eschscholzia californica is a plant enzyme involved in alkaloid biosynthesis, catalyzing the challenging oxidative cyclization of (S)-reticuline to (S)-scoulerine (Scheme 1). This enzyme was recently shown to belong to the group of flavoenzymes with a bicovalently attached FAD (14). After the discovery of this unusual mode of linkage in the crystal structure of GOOX (4), several members of this group, all belonging to the vanillyl-alcohol oxidase family (15), were identified by biochemical methods (16–18) and also structural studies (19). Because some of the suggested benefits of a covalent cofactor attachment can easily be brought about by a single linkage, e.g. prevention of cofactor dissociation or stabilization of the tertiary structure, the two amino acids attached to FAD might have different and individual functions as well as an additive effect on physicochemical properties such as redox potentials or substrate binding and oxidation. To elucidate the relative importance for the overall enzymatic functioning of members of this group, more detailed studies have been performed on GOOX (11), chito-oligosaccharide oxidase (ChitO) from Fusarium graminearum (17), and BBE (20). Common results of these analyses show that the bicovalent FAD has a redox potential of about +130 mV, which is among the highest potentials reported for flavoenzymes. Replacement of one of the amino acids involved in anchoring of the cofactor generally reduces the rate of cofactor reduction and the steady-state turnover rate, but whether this can be directly linked to reduced redox potentials of these mutant proteins has been under debate (11).Open in a separate windowSCHEME 1.Overall reaction catalyzed by BBE.To address these issues further, we report the expression of the H104A mutant protein of BBE. A biochemical characterization of this protein variant with respect to the redox potential, transient kinetics, and steady-state analysis is combined with the structural analysis of both the H104A and the C166A mutant proteins. In addition, a structure of wild type (WT) BBE in complex with the product of the enzyme-catalyzed reaction is presented, which provides further insights toward the involvement of active site amino acids during the course of the reaction. Together with the recently reported x-ray crystal structure of WT BBE with and without substrate bound (21) and the biochemical characterization of the C166A mutant protein (20), these results provide interesting insights into the role of bicovalent FAD attachment in enzymes.     

Detecting Invasions of Marine Organisms: Kamptozoan Case Histories

Detecting marine invasions can be challenging, especially for lesser-known taxa, and requires (a) thorough field surveys of the region of interest for members of the taxon, (b) systematic analyses to identify all species found, (c) literature searches for the worldwide distribution of these species and for previous records of the taxon in this region, and (d) application of rigorous criteria to assess whether each species found is native or introduced. We carried out these steps in order to detect and document kamptozoan (entoproct) invasions on the American mid-Atlantic coast. We report on the occurrence of two colonial kamptozoans (Barentsia benedeni, Loxosomatoides laevis) in Chesapeake Bay (Maryland and Virginia, USA). On the American Atlantic coast, B. benedeni had previously only been reported from Massachusetts, although this species has a worldwide distribution in bays and harbors. The genus Loxosomatoides had not previously been reported from North America and L. laevis was known only from India. Since the genus Loxosomatoides was very poorly characterized, we briefly review all four of its species, which differ only slightly from each other. We have also synonymized L. japonicum with L. laevis. We did not find any of the kamptozoan species previously recorded in surveys of Chesapeake Bay and the American Atlantic coast. This is the first detailed consideration of anthropogenic influences on kamptozoan distributions, and we emphasize that most kamptozoan species are cryptogenic pending further investigation.     

Identifying protein construct variants with increased crystallization propensity--a case study

This study describes an efficient multiparallel automated workflow of cloning, expression, purification, and crystallization of a large set of construct variants for isolated protein domains aimed at structure determination by X-ray crystallography. This methodology is applied to MAPKAP kinase 2, a key enzyme in the inflammation pathway and thus an attractive drug target. The study reveals a distinct subset of truncation variants with improved crystallization properties. These constructs distinguish themselves by increased solubility and stability during a parallel automated multistep purification process including removal of the recombinant tag. High-throughput protein melting point analysis characterizes this subset of constructs as particularly thermostable. Both parallel purification screening and melting point determination clearly identify residue 364 as the optimal C terminus for the kinase domain. Moreover, all three constructs that ultimately crystallized feature this C terminus. At the N terminus, only three amino acids differentiate a noncrystallizing from a crystallizing construct. This study addresses the very common issues associated with difficult to crystallize proteins, those of solubility and stability, and the crucial importance of particular residues in the formation of crystal contacts. A methodology is suggested that includes biophysical measurements to efficiently identify and produce construct variants of isolated protein domains which exhibit higher crystallization propensity.