Kaspar H. Winterhalter

Localization of organic anion transporting polypeptide 4 (Oatp4) in rat liver and comparison of its substrate specificity with Oatp1, Oatp2 and Oatp3.

Organic anion transporting polypeptides (rodents: Oatps; human: OATPs) are involved in the absorption and elimination of a wide variety of structurally unrelated amphipathic organic compounds. Several members of this protein family mediate the uptake of substrates across the basolateral membrane of hepatocytes as the first step in hepatic elimination. In contrast to the well-characterized Oatp1 and Oatp2, the localization and substrate specificity of the recently cloned Oatp4 have not been investigated in detail. Therefore, we raised an antibody against the C-terminal end of Oatp4 and localized this 85-kDa protein to the basolateral membrane of rat hepatocytes. Similar to Oatp1 and Oatp2, Oatp4 is a multispecific transporter with high affinities for bromosulfophthalein, dehydroepiandrosterone sulfate, leukotriene C4, and anionic peptides. In addition, we compared the substrate specificity of Oatp4 to that of Oatp3, which so far has mainly been shown to mediate intestinal bile acid transport. Oatp3 had a similar broad substrate specificity, but in general much lower affinities than Oatp4. Thus, while Oatp4 seems to work in concert with Oatp1 and Oatp2 in the basolateral membrane of rat hepatocytes, Oatp3 is a multispecific transport system in the small intestine.

Low pH crystal structure of glycosylated lignin peroxidase from Phanerochaete chrysosporium at 2.5 Å resolution

The heme‐containing glycoprotein lignin peroxidase (pI 4.15) has been crystallized at pH 4.0. The structure of the peroxidase from the orthorhombic crystals has been determined by multiple isomorphous replacement. The model comprises all 343 amino acids, one heme molecule, and three sugar residues. It has been refined to an R‐factor of 20.3%. The chain fold of residues 15 to 275 is in general similar to those of cytochrome c peroxidase. Despite binding of the heme to the same region and a similar arrangement of the proximal and distal histidine as in cytochrome c peroxidase a significantly larger distance of the iron ion to the proximal histidine is observed. Distinct electron density extending from Asn‐257 and at the distal side of the heme indicates ordered sugar residues in the crystal.

Microsomal lipid peroxidation causes an increase in the order of the membrane lipid domain

The microsomal fraction isolated from rat liver can undergo an enzymatically catalyzed lipid peroxidation when incubated in the presence of NADPH, concomitant with an enhanced oxygen uptake and oxidation of NADPH [ 1 ]. The mechanism of microsomal lipid peroxidation has been extensively studied [2-8]. The first step is the abstraction of a hydrogen atom from an allylic methylen group. This leads to the formation of a lipid radical. Subsequent chain reactions result in a breakdown of poly-unsaturated fatty acids and the production of stable endproducts including ethane, propane, pentane, and also to the production of malondialdehyde. Several biochemical changes concomitant with, or due to, lipid peroxidation have been investigated [9,10]. However, little is known about the biophysical consequences of lipid peroxidation in biomembranes. Spectral studies of fluorescent probes [ 11 ] indicated that lipid peroxidation decreases the fluidity of microsomal membranes, whereas a spin label study showed that 7 radiation-induced lipid peroxidation decreases the degree of order in membrane lipids [ 12]. Using steady state fluorescence anisotropy of diphenylhexatriene we now show that lipid peroxidation in rat liver microsomes increases the order of the microsomal phospholipid acyl chains. This increase in order might be due to the formation of covalent bonds between adjacent lipid radicals. This is the first quantitative study of the change in membrane structure induced by lipid peroxidation.

High-resolution native and complex structures of thermostable beta-mannanase from Thermomonospora fusca - substrate specificity in glycosyl hydrolase family 5.

BACKGROUND . beta-Mannanases hydrolyse the O-glycosidic bonds in mannan, a hemicellulose constituent of plants. These enzymes have potential use in pulp and paper production and are of significant biotechnological interest. Thermostable beta-mannanases would be particularly useful due to their high temperature optimum and broad pH tolerance. The thermophilic actinomycete Thermomonospora fusca secretes at least one beta-mannanase (molecular mass 38 kDa) with a temperature optimum of 80 degreesC. No three-dimensional structure of a mannan-degrading enzyme has been reported until now. RESULTS . The crystal structure of the thermostable beta-mannanase from T. fusca has been determined by the multiple isomorphous replacement method and refined to 1.5 A resolution. In addition to the native enzyme, the structures of the mannotriose- and mannohexaose-bound forms of the enzyme have been determined to resolutions of 1.9 A and 1.6 A, respectively. CONCLUSIONS . Analysis of the -1 subsite of T. fusca mannanase reveals neither a favourable interaction towards the axial HO-C(2) nor a discrimination against the equatorial hydroxyl group of gluco-configurated substrates. We propose that selectivity arises from two possible mechanisms: a hydrophobic interaction of the substrate with Val263, conserved in family 5 bacterial mannanases, which discriminates between the different conformations of the hydroxymethyl group in native mannan and cellulose; and/or a specific interaction between Asp259 and the axial hydroxyl group at the C(2) of the substrate in the -2 subsite. Compared with the catalytic clefts of family 5 cellulases, the groove of T. fusca mannanase has a strongly reduced number of aromatic residues providing platforms for stacking with the substrate. This deletion of every second platform is in good agreement with the orientation of the axial hydroxyl groups in mannan.

Screening of Thiol Compounds: Depolarization-Induced Release of Glutathione and Cysteine from Rat Brain Slices

Superfusates from rat brain slices were screened for thiol compounds after derivatization with monobromo‐bimane by reversed‐phase HPLC. Only glutathione and cysteine were detected. The Ca2+‐dependent release of these compounds from slices of different regions of rat brain was investigated, applying a highly sensitive and reproducible quantification method, based on reduction of superfusates with dithiothreitol, reaction of thiols with iodoacetic acid, precolumn derivatization with o‐phthalaldehyde reagent solution, and analysis with reversed‐phase HPLC. This methodology allowed determination of reduced and total thiols in aliquots of the same superfusates. Mostly reduced glutathione and cysteine were released upon K+ depolarization and the Ca2+ dependency suggests that they originate from a neuronal compartment. The GSH release was most prominent in the mesodiencephalon, cortex, hippocampus, and striatum and lowest in the pons‐medulla and cerebellum. This underscores a physiologically significant role for glutathione in CNS neurotransmission.

Type VI collagen is a major component of the human cornea

Collagen type VI is shown to be present in the human cornea. This finding is based on comparative peptide mapping relative to type VI collagen isolated from placenta and on immunoblotting using antibodies specific for human type VI collagen. Scanning of polyacrylamide gels indicates that type VI collagen comprises as much as one quarter of the dry weight of the cornea. Indirect immunofluorescence shows this collagen to be distributed throughout the corneal stroma. Thus, type VI collagen must be considered a major component of the extracellular matrix of the human cornea.

The Glypiated Neuronal Cell Adhesion Molecule Contactin/F11 Complexes with src-Family Protein Tyrosine Kinase Fyn

Glycosyl phosphatidylinositol-anchored glycoproteins of the immunoglobulin superfamily play an important role in the formation of neuronal networks during development. The mechanism whereby neuronal GPI-linked molecules transduce recognition signals remains to be established. Analysis of detergent-resistant immune-complexes reveals that the glypiated neuronal cell adhesion molecule contactin/F11 specifically complexes with the cytoplasmic, nonreceptor type src-family tyrosine kinase Fyn. Antibody-mediated cross-linking of contactin/F11 on embryonic chick neuronal cells leads to an increase of the Fyn-activity coprecipitated with contactin/F11, and elevates phosphorylation of an additional 75/80 K component within the contactin/F11-immune-complex. Additionally, binding of ligands, i.e., contactin/F11-specific antibody or tenascin-R, a natural ligand of contactin/F11, to the surface of HeLa transfectants expressing contactin/F11, causes capping of contactin/F11 and a concomitant change in the distribution of the intracellular kinase Fyn, thus confirming their physical association. This indicates that contactin/F11-mediated signaling requires Fyn.

Artificial intermediates in the reaction of haemoglobin: Functional and conformational properties of the cyanmet intermediates☆

Artificial cyanmet intermediates of haemoglobin were obtained by mixing cyanmet chains with their oxygenated partners. At pH 7 and 20 °C, the oxygen affinity of the two intermediates is similar and lies between that of haemoglobin and the isolated chains; the value of n in the Hill equation is equal to 1 for (α + β+ CN) but may be slightly higher (~1.1 to 1.2) for (α+ N + β). The alkaline Bohr effect is very similar for the two intermediates, but the acid Bohr effect is lacking in (α + β+ CN). In the reaction with carbon monoxide, the intermediates are somewhat faster than haemoglobin but very much slower than the isolated chains. Conformational changes upon ligand binding to haemoglobin are implied by changes in reactivity of the β93 sulphydryl groups and by changes in the rotational strength at 233 nm. Addition of oxygen to (α+ CN + β) is followed by a change in the time-course of p-mercuribenzoate binding by the reactive sulphydryl groups, whereas oxygenation of the other intermediate does not modify the rate of mercuribenzoate binding. A change in the value of [R′] at 233 nm as a result of oxygen binding is observed, which is about the same in both cases (Δ [R′]233 ∼- 3%).

Comparative studies of collagens in normal and keratoconus corneas

In this paper we present strong evidence that the aberrations in keratoconus corneas are not directly related to alterations in collagen composition and distribution. This conclusion is based on comparative studies of collagen types I, III, IV, V and the recently described collagen types VI and VII in keratoconus and normal corneas. The data are derived from biochemical analysis of collagen fractions sequentially extracted with pepsin and sodium-dodecylsulphate, from amino acid analysis of hydrolysates of entire corneal tissues as well as from immunoblotting of the extracted collagens with specific antibodies. These antibodies were also used to examine the distribution of the collagens in immunofluorescence experiments on corneal sections. The yields of the collagen extractions were demonstrated to be age dependent but were not altered in keratoconus samples. Apart from one case associated with osteogenesis imperfecta type I, comparative studies of keratoconus and normal corneas showed no differences in collagen composition of the extracts. This was confirmed by amino acid analysis of tissue-hydrolysates. The distributions of collagen types I, III, IV, V, VI and VII in corneal sections were in general unchanged in keratoconus corneas, the only differences being in scar tissues observed in the Bowman layer of some keratoconus samples.

Purification, crystallisation and X-ray diffraction study of fully functional laccases from two ligninolytic fungi

Laccase isozymes from the white-rot basidiomycete fungi Trametes versicolor and Pycnoporus cinnabarinus were purified to apparent iso-electric homogeneity and crystallised. T. versicolor laccase crystallises in two crystal forms, both with the orthorhombic space group P2(1)2(1)2(1), which diffract to 1.9 and 2.95 A resolution, respectively. The crystals of P. cinnabarinus laccase belong to the monoclinic space group C2 and diffract to at least 2.2 A resolution. All the laccase crystals are suitable for X-ray structure determination and contain a full complement of copper ions.