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W Bode

Publications and source records attributed to W Bode.

At least 199 records · Page 11Linked to original sources
PubMed1987

Improved radioimmunoscintigraphy of human mammary carcinoma xenografts after injection of an anti-antibody.

The low tumor-to-background ratio obtained after administration of radiolabeled whole monoclonal antibodies (MAbs) is one of the major problems in immunoscintigraphy and -therapy. To reduce the blood pool label caused by the circulation of radiolabeled MAb we have investigated the advantage of injecting an anti-antibody after administration of a tumor-specific MAb in nude mice bearing human mammary carcinoma xenografts. The MAb MA 10-11 of rat origin, used in these studies, had shown a high affinity to human mammary carcinoma tissue on frozen sections and low cross-reactivity with various normal human tissues. 24 h after injection of 1.5 MBq 131I-labeled MAb containing 10 micrograms IgG2a one group of mice received an additional injection of 100 micrograms anti-rat antibody. Blood taken 2 min after the second antibody injection showed nearly the whole activity bound to antibody aggregates, that cleared very rapidly from the circulation and accumulated in liver and spleen. The transitory high liver activity decreased within several hours because of rapid deiodination of the antibody-complex in this organ. The release of radioactivity from the spleen, however, was found to be much slower. The rapid excretion of the radioactivity from the blood pool combined with a nearly constant tumor activity allowed early tumor detection with tumor-to-blood ratios of 250:1 at 48 h after anti-antibody injection compared to 1.1:1 obtained for the control animals. In addition the results may explain the reported reduction of imaging quality and high uptake of radioactivity in the spleen of patients having repeated injections of mouse MAbs due to complex formation after development of human anti-mouse antibodies.

Animals↗
PubMed1986

Crystal structure analysis and refinement at 2.5 A of hexameric C-phycocyanin from the cyanobacterium Agmenellum quadruplicatum. The molecular model and its implications for light-harvesting.

The crystal structure of the light-harvesting protein-pigment complex C-phycocyanin from the cyanobacterium Agmenellum quadruplicatum has been determined by Patterson search techniques on the basis of the molecular model of C-phycocyanin from Mastigocladus laminosus. The crystal unit cell (space group P321) contains three (alpha beta)6 hexamers centred on the crystallographic triads. The hexamer at the origin of the unit cell exhibits crystallographic 32 point symmetry. The other two hexamers (independent of the former) show crystallographic 3-fold and local 2-fold symmetry. The 3-fold redundancy of the asymmetric unit of the crystal cell was used in the refinement process, which proceeded by cyclic averaging, model building and energy-restrained crystallographic refinement. Refinement was terminated with a conventional crystallographic R-value of 0.20 with data to 2.5 A resolution. The two independent hexamers of the unit cell are identical within the limits of error at all levels of aggregation. Two trimers, which closely resemble the M. laminosus C-phycocyanin, are aggregated head-to-head to form the hexamer. Both trimers fit complementarily and are held together by polar and ionic interactions. Conservation of the amino acid residues involved in protein-chromophore and intermonomer interactions suggests common structural features for all biliproteins. Most probably, the hexameric aggregation form present in the crystals is closely related to the discs of native phycobilisome rods. All tetrapyrrole chromophores are extended but with different geometries enforced by different protein surroundings. In particular, interactions of the propionic side-chains with arginine residues and of the pyrrole nitrogen atoms with aspartate residues define configuration and conformation of the chromophores. Relative chromophore distances and orientations have been determined and a preferential pathway for the energy transfer suggested. Accordingly, within a hexamer the absorbed energy is funneled to chromophore B84 and then transduced via B84 chromophores along the phycobilisome rods.

Amino Acid Sequence↗
PubMed1986

X-ray crystallographic and biochemical characterization of single crystals formed by proteolytically modified human fibrinogen.

Large single crystals (0.7 mm X 0.4 mm X 0.3 mm) of human fibrinogen, modified with a crude exoprotease from Pseudomonas aeruginosa, have been obtained. The crystals are orthorhombic, space group P212121, with a = 9.5 +/- 0.1 nm, b = 11.1 +/- 0.1 nm, c = 44.0 +/- 0.4 nm. Their X-ray diffraction patterns extend to beyond 1.0 nm resolution. The asymmetric unit contains one fragment of 245 kDa molecular mass made up of an intact gamma chain, a slightly shortened beta chain and an N-terminal part (about one-third) of the alpha chain. In electron micrographs of rotary-shadowed samples the crystallized particles are very similar in size and shape to the well-known trinodular form of native fibrinogen. From the unit-cell dimensions and the intensity pattern a model is proposed in which the molecules consist of two halves related by a local twofold rotation axis, and are aligned with a displacement of multiples of 1/4 of their length giving a pseudohexagonal packing scheme.

Chromatography, High Pressure Liquid↗
PubMed1986

Refined 1.2 A crystal structure of the complex formed between subtilisin Carlsberg and the inhibitor eglin c. Molecular structure of eglin and its detailed interaction with subtilisin.

The crystal structure of the complex formed between eglin c, an elastase inhibitor from the medical leech, and subtilisin Carlsberg has been determined at 1.2 A resolution by a combination of Patterson search methods and isomorphous replacement techniques. The structure has been refined to a crystallographic R-value of 0.18 (8-1.2 A). Eglin consists of a four-stranded beta-sheet with an alpha-helical segment and the protease-binding loop fixed on opposite sides. This loop, which contains the reactive site Leu45I--Asp46I, is mainly held in its conformation by unique electrostatic/hydrogen bond interactions of Thr44I and Asp46I with the side chains of Arg53I and Arg51I which protrude from the hydrophobic core of the molecule. The conformation around the reactive site is similar to that found in other proteinase inhibitors. The nine residues of the binding loop Gly40I--Arg48I are involved in direct contacts with subtilisin. In this interaction, eglin segment Pro42I--Thr44I forms a three-stranded anti-parallel beta-sheet with subtilisin segments Gly100--Gly102 and Ser125--Gly127. The reactive site peptide bond of eglin is intact, and Ser221 OG of the enzyme is 2.81 A apart from the carbonyl carbon.

Amino Acid Sequence↗
PubMed1986

X-ray crystal structure of the complex of human leukocyte elastase (PMN elastase) and the third domain of the turkey ovomucoid inhibitor.

Orthorhombic crystals diffracting beyond 1.7 A resolution, have been grown from the stoichiometric complex formed between human leukocyte elastase (HLE) and the third domain of turkey ovomucoid inhibitor (OMTKY3). The crystal and molecular structure has been determined with the multiple isomorphous replacement technique. The complex has been modeled using the known structure of OMTKY3 and partial sequence information for HLE, and has been refined. The current crystallographic R-value is 0.21 for reflections from 25 to 1.8 A resolution. HLE shows the characteristic polypeptide fold of trypsin-like serine proteinases and consists of 218 amino acid residues. However, several loop segments, mainly arranged around the substrate binding site, have unique conformations. The largest deviations from the other vertebrate proteinases of known spatial structure are around Cys168. The specificity pocket is constricted by Val190, Val216 and Asp226 to preferentially accommodate medium sized hydrophobic amino acids at P1. Seven residues of the OMTKY3-binding segment are in specific contact with HLE. This interaction and geometry around the reactive site are similar as observed in other complexes. It is the first serine proteinase glycoprotein analysed, having two sugar chains attached to Asn159 and to residue 109.

Animals↗
PubMed1986

The primary structure of bdellin B-3 from the leech Hirudo medicinalis. Bdellin B-3 is a compact proteinase inhibitor of a "non-classical" Kazal type. It is present in the leech in a high molecular mass form.

A proteinase inhibitor was isolated from extracts of the leech Hirudo medicinalis by gel filtration and anion exchange chromatography. This inhibitor is similar to the bdellins in that it blocks the activity of trypsin, plasmin and sperm acrosin but has a molecular mass, as estimated by SDS polyacrylamide electrophoresis, of about 20 kDa, whereas the bdellins have molecular masses in the range 5-6 kDa. It is therefore designated as high-molecular mass bdellin B-3 (HMB). The amino-acid sequence of the inhibitor was elucidated as far as position 56. This revealed that the molecule consists of a bdellin B-3 moiety, corresponding to the N-terminal 46 residues, which is then extended at the C-terminus by a polypeptide chain of the composition Asx15, Glx25, Gly6, Val, His26-27 and Lys4. It has been formerly concluded from a partial amino-acid sequence that bdellin B-3 is a Kazal-type inhibitor. However, the complete sequence of bdellin B-3, represented by the N-terminal 46 residues of HMB, discloses that bdellin B-3 is a non-classical Kazal-type inhibitor when the number of amino-acid residues between half-cystines are considered. Presuming that formation of disulfide bridges principally follows the same pattern as in classical Kazal-type inhibitors the bdellin B-3 molecule was modeled based on the known three-dimensional structure of the third ovomucoid domains. This showed that a compact arrangement of the peptide chain of bdellin B-3 is conceivable.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1985

Crystallization of phycoerythrocyanin from the cyanobacterium Mastigocladus laminosus and preliminary characterization of two crystal forms.

The light-harvesting protein phycoerythrocyanin from the cyanobacterium Mastigocladus laminosus Cohn has been crystallized in two different crystal forms by vapour diffusion. In 5% (w/v) polyethylene glycol at pH 8.5, hexagonal crystals of space group P63 with cell constants a = b = 158 A, c = 40.6 A were obtained, which turned out to be almost isomorphous with the hexagonal crystals of C-phycocyanin from the same organism. Consequently, the conformation of both phycobiliproteins must be very similar. From 1.5 M-ammonium sulfate (pH 8.5), orthorhombic crystals of space group P2221 with cell constants a = 60.5 A, b = 105 A, c = 188 A could be grown. Density measurements of these crystals indicate that the unit cell contains 18 (alpha beta)-units. A detailed packing scheme is proposed that is consistent with the observed pseudo-hexagonal X-ray intensity pattern and with the known size and shape of (alpha beta)3-trimers of C-phycocyanin. Accordingly, disc-like (alpha beta)3-trimers are associated face-to-face and stacked one upon another in rods with a period of 60.5 A, corresponding to the cell dimension a.

Crystallography↗
PubMed1985

Bovine chymotrypsinogen A X-ray crystal structure analysis and refinement of a new crystal form at 1.8 A resolution.

The X-ray structure of a new crystal form of chymotrypsinogen A grown from ethanol/water has been determined at 1.8 A resolution using Patterson search techniques. The crystals are of orthorhombic space group P212121 and contain two molecules in the asymmetric unit. Both independent molecules (referred to as A and B) have been crystallographically refined to a final R value of 0.173 with reflection data to 1.8 A resolution. Owing to different crystal contacts, both independent molecules show at various sites conformational differences, especially in segments 33-38, 142-153 and 215-222. If these three loops are omitted in a comparison, the root-mean-square (r.m.s.) deviation of the main-chain atoms of molecules A and B is 0.32 A. If segments 70-79, 143-152 and 215-221 are omitted, a comparison of either molecule A or molecule B with the chymotrypsinogen model of Freer et al. (1970) reveals an r.m.s. deviation of the alpha-carbon atoms of about 0.7 A. Compared with the active enzyme, four spatially adjacent peptide segments, in particular, are differently organized in the zymogen: the amino-terminal segment 11-19 runs in a rigid but strained conformation along the molecular surface due to the covalent linkage through Cys1; also segment 184-194 is in a rigid unique conformation due to several mutually stabilizing interactions with the amino-terminal segment; segment 216-222, which also lines the specificity pocket, adapts to different crystal contacts and exists in both chymotrypsinogen molecules in different, but defined conformations; in particular, disulfide bridge 191-220, which covalently links both latter segments, has opposite handedness in molecules A and B; finally, the autolysis loop 142 to 153 is organized in a variety of ways and in its terminal part is completely disordered. Thus, the allosteric activation domain (Huber & Bode, 1978) is organized in defined although different conformations in chymotrypsinogen molecules A and B, in contrast to trypsinogen, where all four homologous segments of the activation domain are disordered. This reflects the structural variability and deformability of the activation domain in serine proteinase proenzymes. If the aforementioned peptide segments are omitted, a comparison of our chymotrypsinogen models with gamma-chymotrypsin (Cohen et al., 1981) yields an r.m.s. deviation for alpha-carbon atoms of about 0.5 A. The residues of the "active site triad" are arranged similarly, but the oxyanion hole is lacking in chymotrypsinogen.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1985

X-ray crystallographic structure of the light-harvesting biliprotein C-phycocyanin from the thermophilic cyanobacterium Mastigocladus laminosus and its resemblance to globin structures.

The structure of the biliprotein C-phycocyanin from the thermophilic cyanobacterium Mastigocladus laminosus has been determined at 3 A resolution by X-ray diffraction methods. Phases have been obtained by the multiple isomorphous replacement method. The electron density map could be improved by solvent flattening and has been interpreted in terms of the amino acid sequence. The protein consists of three identical (alpha-beta)-units which are arranged around a threefold symmetry axis to form a disc of approximate dimensions 110 A X 30 A with a central channel of 35 A in diameter. This aggregation form is supposed to be the same as that found in the rods of native phycobilisomes. Both subunits, alpha and beta, exhibit a similar structure and are related by a local twofold rotational axis. Each subunit is folded into eight helices and irregular loops. Six helices are arranged to form a globular part, whereas two helices stick out and mediate extensive contact between the subunits. The arrangement of the helices of the globular part resembles the globin fold: 59 equivalent C alpha-atoms have a root-mean-square deviation of 2 X 9 A. The chromophores attached to cystein 84 of the alpha- and beta-subunits are topologically equivalent to the haem. All three chromophores of C-phycocyanin, open-chain tetrapyrroles, are in an extended conformation. alpha 84 and beta 84 are attached to helix E (globin nomenclature), beta 155 is linked to the G--H loop. The shortest centre-to-centre distance between chromophores in trimer is 22 A.

Amino Acid Sequence↗
PubMed1985

Thrombin-induced fibrinopeptide release from a fibrinogen variant (fibrinogen Sydney I) with an Aalpha Arg-16----His substitution.

Fibrinogen, purified from a recently identified case of dysfibrinogenaemia, fibrinogen Sydney I, was shown by thrombin digestion, high-performance liquid chromatography (HPLC) and amino acid analysis to be a heterozygous case of an A alpha Arg-16----His substitution. Kinetic studies have been carried out on the thrombin-induced release of fibrinopeptide A (FPA), fibrinopeptide B (FPB) and the variant peptide [His16]FPA. When thrombin was added to fibrinogen Sydney I at a concentration of 0.2 U/ml release of FPA was rapid and there was a 79-fold reduced rate of release of [His16]FPA, but the rate of release of FPB was not appreciably reduced. In contrast, at lower thrombin concentrations the rate of FPB release was reduced in proportion to the rate of total FPA release, supporting the view that release of fibrinopeptides is a sequential process. The second-order kinetic constant kcat/Km for hydrolysis of the abnormal A alpha chain by thrombin was calculated from Lineweaver-Burk plots to be 16-30-fold less than that for the normal A alpha chain. Molecular modelling studies, using a refined model of the trypsin-pancreatic-trypsin-inhibitor complex have been used to suggest how the histidine at the P1 site can be accommodated within the enzyme hydrophobic active-site pocket.

Amino Acids↗
PubMed1985

The crystal and molecular structure of the third domain of silver pheasant ovomucoid (OMSVP3).

OMSVP3 and OMTKY3 (third domains of silver pheasant and turkey ovomucoid inhibitor) are Kazal-type serine proteinase inhibitors. They have been isomorphously crystallized in the monoclinic space group C2 with cell dimensions of a = 4.429 nm, b = 2.115 nm, c = 4.405 nm, beta = 107 degrees. The asymmetric unit contains one molecule corresponding to an extremely low volume per unit molecular mass of 0.0017 nm3/Da. Data collection was only possible for the OMSVP3 crystals. Orientation and position of the OMSVP3 molecules in the monoclinic unit cells were determined using Patterson search methods and the known structure of the third domain of Japanese quail ovomucoid (OMJPQ3) [Papamokos, E., Weber, E., Bode, W., Huber, R., Empie, M. W., Kato, I. and Laskowski, M., Jr (1982) J. Mol. Biol. 158, 515-537]. The OMSVP3 structure has been refined by restrained crystallographic refinement yielding a final R value of 0.199 for data to 0.15 nm resolution. Conformation and hydrogen-bonding pattern of OMSVP3 and OMJPQ3 are very similar. Large deviations occur at the NH2 terminus owing to different crystal packing, and at the C terminus of the central helix, representing an intrinsic property and resulting from amino acid substitutions far away from this site. The deviation of OMSVP3 from OMTKY3 complexed with the Streptomyces griseus protease B is very small [Fujinaga, M., Read, R. J., Sielecki, A., Ardelt, W., Laskowski, M., Jr and James, M. N. G. (1982) Proc. Natl Acad. Sci. USA, 79, 4868-4872].

Animals↗
PubMed1985

Crystallization of chicken egg white cystatin, a low molecular weight protein inhibitor of cysteine proteinases, and preliminary X-ray diffraction data.

The shorter-chain form of chicken egg white cystatin has been crystallized in 1.6 M-phosphate buffer at pH 4.0 by vapour diffusion. The crystals are of trigonal space group P3121 (or P3221), have cell constants a = b = 47.9 A, c = 87.5 A, alpha = beta = 90 degrees, gamma = 120 degrees, and contain one molecule of 12,191 molecular weight per asymmetric unit. They diffract well to about 2.0 A resolution and are suitable for X-ray crystal structure analysis.

Animals↗
PubMed1985

Structure and biology of the globular domain of basement membrane type IV collagen.

A procedure was developed for purifying the globular domain NC1 of basement membrane collagen from collagenase digests of a variety of tissues. The globule (Mr = 170,000) is a hexameric structure originating from two collagen IV molecules that are cross-linked at their COOH-terminal ends. Dissociation into subunits derived from alpha 1(IV) and alpha 2(IV) chains occurs at a pH below 4 and after denaturation (8 M urea). The subunits obtained include monomers (Mr = 28,000) and two different dimers (Da,Db) which are connected by disulfide bonds (Db) and/or nonreducible bonds (Da). Almost perfect reconstitution to hexamers is obtained in neutral buffer with mixtures of the subunits or purified dimers but not with purified monomers. Stabilization by dimer formation and other physical data suggest conformationally distinct segments within the subunits, which is also supported by a repeating subdomain structure deduced from cDNA sequences. Monocline crystals of NC1 give a sufficiently detailed X-ray diffraction pattern that should permit elucidation of the three-dimensional structure of the hexamer. Antibodies raised against the globular domain react with all subunits and mainly recognize epitopes stabilized by internal disulfide bridges and/or the hexameric assembly. Immunoprecipitation tests with these antibodies demonstrated a slightly larger subunit size of NC1 in PYS-2 cell culture and the rapid release of precursor-specific segments prior to secretion from the cells. Autoantibodies against mouse tumor NC1 were produced in mice and were detected both in the blood and as tissue-bound forms (kidney, lung). The autoantibody response is accompanied by certain pathological alterations mimicking Goodpasture's syndrome. The possible relationship between the two diseases is substantiated by reaction of Goodpasture antisera with the globular domain obtained from various tissue sources.

Animals↗
PubMed1985

Determination of the protein content of crystals formed by Mastigocladus laminosus C-phycocyanin, Chroomonas spec. phycocyanin-645 and modified human fibrinogen using an improved Ficoll density gradient method.

We demonstrated for several protein crystals of known protein content that the simple Ficoll density gradient method for crystal density determination as described by Westbrook (1976) often leads to quite erroneous results. In particular, the apparent density of loosely packed crystals can show a tremendous change within the first minutes of measurement. In order to derive the correct protein content the apparent crystal density must be followed as a function of time and has to be extrapolated back to the time of insertion of the crystal into the gradient. The packing densities of four novel protein crystals, formed by Mastigocladus laminosus C-phycocyanin, Chroomonas spec. phycocyanin-645 (two forms), and modified human fibrinogen have been determined and that of proteinase II of Crotalus adamanteus has been corrected. The C-phycocyanin crystals were found to contain (in contrast to earlier results reported by others) only one (alpha beta)-monomer, the phycocyanin-645 crystals two and three (alpha alpha' beta 2)-monomers, respectively, and the fibrinogen crystals one fibrinogen molecule per asymmetric unit.

Centrifugation, Density Gradient↗
PubMed1984

The refined 2.2-A (0.22-nm) X-ray crystal structure of the ternary complex formed by bovine trypsinogen, valine-valine and the Arg15 analogue of bovine pancreatic trypsin inhibitor.

Large orthorhombic crystals of the complex formed by bovine trypsinogen and a semisynthetic homologous bovine pancreatic trypsin inhibitor with the reactive-site lysine residue replaced by an arginine residue [( Arg15]PTI) have been obtained which are isomorphous with the crystals of PTI-trypsinogen [Bode, W., Schwager, P. and Huber, R. (1978) J. Mol. Biol. 118, 99-112]. The X-ray crystal structure of the ternary complex of trypsinogen-[Arg15]PTI with the dipeptide Val-Val has been determined by X-ray data to 2.2-A (0.22-nm) resolution by means of difference Fourier methods and has been crystallographically refined to a final R-value of 0.17. Replacement of the reactive-site Lys15 by an arginine residue is accompanied in the complex by small movements of polar side groups of trypsin and enclosed solvent molecules within the specificity pocket. Only solvent molecule 414 OH which mediates the hydrogen bond interactions between Lys15 NZ and Asp189 carboxylate is expelled, thus allowing the bulkier guanidyl group to approach this carboxylate. The dipeptide Val-Val binds in the pocket accepting the Ile-Val N-terminus in trypsin. The cavity left by the CD-methyl group of Ile16 upon replacement by a valine residue is only partially filled by slight rearrangements of neighbouring peptide side chains. Part of the positive free energy change observed upon replacement of Ile-Val may allow for the maintenance of this cavity.

Animals↗