Biomedical subjects

J van Oostrum

Publications and source records attributed to J van Oostrum.

29 records · Page 2Linked to original sources

PubMed1992

Identification of a reactive lysyl residue (Lys103) of recombinant human interleukin-1 beta. Mechanism of its reactivity and implication of its functional role in receptor binding.

Recombinant human interleukin-1 beta (h-IL-1 beta) was chemically modified with 4-(N,N-dimethylamino)-4'-isothiocyanatoazobenzene-2'-sulfonic acid (S-DABITC), a water-soluble color reagent specific for lysine labeling. Modified h-IL-1 beta was digested by lysyl endopeptidase. Peptides containing labeled lysines were detected at the visible wavelength (436 nm) and isolated by HPLC. The modification sites were eventually determined by sequence analysis. The results revealed that Lys103, Lys92, Lys93, and Lys94 of h-IL-1 beta reacted selectively with S-DABITC. A 1-h incubation with 1 mM S-DABITC at room temperature resulted in a quantitative modification of Lys103, 22% of Lys92, 27% of Lys93, and 18% of Lys94, respectively. This modification was accompanied by a 20-fold decrease of the protein's ability to bind to the receptor. Furthermore, a mutant of h-IL-1 beta (M9, Glu105 substituted by Lys) exhibits markedly impaired receptor binding, and the S-DABITC reactivity of its Lys103 was found to be reduced by 90%. These findings suggest that Lys103 of h-IL-1 beta might play an important role in the h-IL-1 beta/receptor interaction.

Amino Acid Sequence↗

PubMed1992

Purification, analysis, and enzymatic activity of recombinant human synovial fluid phospholipase A2 and N-terminal variants.

Recombinant human synovial fluid phospholipase A2 (rPLA2) and several variants with N-terminal sequences modified by addition or deletion of one or two amino acid residues (ala or Met; Des-Asn1, Leu2) have been expressed in mammalian cells and in Escherichia coli, respectively, purified to homogeneity, and characterized. The observed values for the molecular mass of rPLA2 and variants are in complete agreement with the predicted values for a correctly folded structure containing seven disulfide bridges. Moreover, the relative proportions of the various types of secondary structures of the variants of rPLA2, as measured by CD spectroscopy, are similar to that found for native porcine pancreatic PLA2, indicating that the recombinant proteins are correctly folded. Enzymatic activities of rPLA2 with modified N-termini decreased to 1.3-0.005% of the activity of the mature rPLA2, emphasizing a key role of the N-terminus for catalytic activity.

Amino Acid Sequence↗

PubMed1991

Mode of action of the lanthionine-containing peptide antibiotics duramycin, duramycin B and C, and cinnamycin as indirect inhibitors of phospholipase A2.

Effects of the lanthionine-containing peptide antibiotics duramycin, duramycin B, duramycin C and cinnamycin on the activity of phospholipase A2 from six different sources were studied, and their mode of action was investigated. The four antibiotics inhibited potently all tested phospholipases A2, with IC50 values of around 1 microM, using phosphatidylethanolamine or [1-14C]oleate-labelled Escherichia coli, whose phospholipids are rich in phosphatidylethanolamine, as substrates. No inhibition was observed when the substrate was phosphatidylcholine. Binding of the antibiotics to the lipid fraction of E. coli could be demonstrated by co-sedimentation with whole, but not with lipid-depleted E. coli. In addition, preincubation of duramycin B with vesicles of phosphatidylethanolamine, but not those of phosphatidylcholine, prevented the inhibition of phospholipase A2 activity. The interaction of duramycin B and C, but not that of the biologically inactive compounds actagardine and the duramycin B trisulphoxide, with phosphatidylethanolamine was demonstrated using circular dichroism studies. On the other hand, no interaction of duramycin B with phosphatidylcholine could be demonstrated. A strict correlation between the physico-chemical interaction of the studied lantibiotics, demonstrated by circular dichroism spectroscopy, and their inhibition of phospholipase A2 was observed. These results suggest that lanthionine-containing peptide antibiotics inhibit phospholipase A2 indirectly by specifically sequestering the substrate phosphatidylethanolamine. This mode of action is analogous to the one described for the protein lipocortin.

Alanine↗

PubMed1991

The structure of murine interleukin-1 beta at 2.8 A resolution.

The three-dimensional structure of recombinant murine interleukin-1 beta has been solved by X-ray crystallographic techniques to 2.8 A resolution and refined to a crystallographic R factor of 0.192. Although murine interleukin-1 beta crystallizes in the same space group as human interleukin-1 beta with almost identical unit cell dimensions, the packing of the molecules is quite different. The murine interleukin-1 beta structure was solved by molecular replacement using the refined structure of human interleukin-1 beta as trial structure, and found to be related to the human structure by a nearly perfect twofold rotation about the crystallographic y-axis and a 14 degrees rotation about the z-axis, with no translation. The folding of murine interleukin-1 beta is similar to that found for the human variant, consisting of 12 beta strands wrapped around a core of hydrophobic side chains in a tetrahedron-like fashion. Significant differences with respect to the human structure are seen at the N terminus and in 4 of the 11 loops connecting the 12 beta strands.

Amino Acid Sequence↗

PubMed1990

Sialylated carbohydrate chains of recombinant human glycoproteins expressed in Chinese hamster ovary cells contain traces of N-glycolylneuraminic acid.

HPLC analysis of sialic acids released from recombinant variants of human tissue plasminogen activator, human chimeric plasminogen activator, human erythropoietin, and human follitropin, expressed in Chinese hamster ovary cells, demonstrates for each glycoprotein the presence of N-acetylneuraminic acid and N-glycolylneuraminic acid in a ratio of 97:3. Structural analysis by 500 MHz1H-NMR spectroscopy, of the enzymatically released N-linked carbohydrate chains of chimeric plasminogen activator and of erythropoietin, showed that alpha 2-3 linked N-glycolylneuraminic acid can occur in different N-acetyllactosamine type antennary structures.

Animals↗

PubMed1990

10,20-Methanorhodopsins: (7E,9E,13E)-10,20-methanorhodopsin and (7E,9Z,13Z)-10,20-methanorhodopsin. 11-cis-locked rhodopsin analog pigments with unusual thermal and photo-stability.

Synthesis of the retinal analog, 10,20-methanoretinal (R6), where the 11Z conformation is locked in a six-membered ring, yielded four stereoisomers (7E,9E,13E, 7E,9E,13Z, 7E,9Z,13E and 7E,9Z,13Z). These four isomers were separated by straight-phase isocratic HPLC and identified by 1H-NMR and NOE analysis. All isomers smoothly recombined with bovine opsin at a relatively high rate (5-10% of that of the natural chromophore 11Z-retinal). The corresponding 13E and 13Z isomers yielded identical analog pigments, probably due to rapid thermal isomerization around the C13 = C14 double bond. The (7E,9E)-isomers produced a pigment with maximal absorbance at 510 nm, while the pigment produced from the (7E,9Z)-isomers had maximal absorbance at 494 nm. Based upon kinetic considerations, the chromophore structure in the 510-nm-absorbing pigment should be (7E,9E,13E), i.e. equivalent to 11Z-retinal and rhodopsin, while the chromophore structure in the 494-nm-absorbing pigment should be (7E,9Z,13Z), i.e. equivalent to (9Z,11Z,13Z)-rhodopsin, an isorhodopsin analog. In analogy to the 11-cis-locked rhodopsin analogs Rh5 and Rh7, the 510-nm-absorbing pigment, (7E,9E,13E)-10,20-methanorhodopsin, was dubbed Rh6 and the 494-nm-absorbing pigment. (7E,9Z,13Z)-10,20-methanorhodopsin, was dubbed Iso6. The opsin shift of Rh6 (2660 cm-1) is practically identical to that of rhodopsin itself (2650 cm-1). Rh6 and Iso6 are nearly as stable as rhodopsin towards hydroxylamine and solubilization in detergent solution and could be easily purified and reconstituted into proteoliposomes by established procedures. Due to the 11-cis-lock, Rh6 is much less photolabile (bleaching rate less than 1%) than rhodopsin, but it is not completely photostable, probably since photoisomerization around the C7 = C8, C9 = C10 and C13 = C14 bonds is allowed. Illumination of either Rh6 or Iso6 does not generate the common photointermediates but instead produces a complex pattern of photochemical transitions, which during continuous illumination leads to the same final steady state, absorbing at 498 nm. This process is accompanied by a slow but steady loss of pigment, probably due to hydrolytic release of chromophore, which is markedly accelerated in the presence of hydroxylamine. In a physiological assay (light-dependent activation of rod cGMP phosphodiesterase) Rh6 is only marginally active and this probably reflects conformational changes accompanying the above-mentioned photochemical transitions. This supports the concept that normal rhodopsin-based phototransduction requires 11Z to all-E isomerization.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1989

Adenovirus polypeptide IX revealed as capsid cement by difference images from electron microscopy and crystallography.

Particles of adenovirus type 2 (ad2), when disassembled, consistently yield groups-of-nine (GON) hexons, which are the major virion shell component. The location of a minor component (6%) of the GON has been determined using a novel combination of electron microscopy and X-ray crystallography. The Brookhaven Scanning Transmission Electron Microscope (STEM) was used to estimate the distribution of protein in the GON to a resolution of 15-18 A. The relative hexon positions then were determined to within 1 A using a model of the hexon derived from the X-ray crystal structure to search the STEM image. The difference image between the STEM image and a model hexon group reveals individual monomers of polypeptide IX extending along the hexon--hexon interfaces. The distribution confirms our earlier proposal that four trimers of polypeptide IX are embedded in the large cavities in the upper surface of the GON to cement hexons into a highly-stable assembly.

Adenoviruses, Human↗

PubMed1987

The structure of the adenovirus capsid. III. Hexon packing determined from electron micrographs of capsid fragments.

The orientation and relative positions of all 240 hexons in the icosahedral outer capsid of adenovirus have been determined. Two types of capsid fragments, obtained after selective disruption of the virion, were analyzed using electron microscopy and image-processing techniques. Planar inverted groups-of-nine, arising from the central region of the capsid facet, were minimally stained to reveal the morphology of restricted regions of their component hexons. Images shown to be related by correspondence analysis were averaged and features of the individual hexon molecule, known from an X-ray crystallographic investigation, were used in their interpretation. The study confirms earlier observations that the hexons in the group-of-nine are distributed on a p3 net, shows that the hexons form a close-packed array using the pseudo-hexagonal shape of the hexon base, and provides their relative positions. Twenty interlocking groups-of-nine account for 180 of the 240 hexons present in the viral capsid. The orientation of the remaining 60 peripentonal hexons was obtained from a rotationally averaged image of a quarter-capsid, a novel viral fragment comprising five complete facets. Each peripentonal hexon forms planar asymmetric interactions with two neighbors in an adjacent group-of-nine so that it lies on an extension of the p3 net. The complete facet thus consists of 12 hexons arranged on a planar p3 net, with a shape that permits interlocking of hexons at the capsid edge. The relative positions of the hexons have been determined to within 5 A using the molecular model, and indicate that the pseudo-hexagonal basal regions are close-packed in a manner that maximizes the hexon-hexon contacts. The results confirm the model proposed earlier for the arrangement of hexons within the adenovirus capsid (Burnett, 1985), and show the power of the inter-disciplinary approach.

Adenoviridae↗

PubMed1985

Isolation and crystallization of lambda exonuclease.

lambda Exonuclease is a deoxyribonuclease induced by bacteriophage lambda. Mutations in the structural gene for the protein affect general recombination and indicate a possible function for the enzyme. A large scale isolation procedure was employed to purify enough enzyme from a heat-induced lambda lysogen for X-ray crystallographic analysis. Analytical ultracentrifugation and SDS-polyacrylamide electrophoresis revealed that lambda exonuclease is a tetramer with molecular mass 107,000 Da. Crystallization trials produced morphologically perfect crystals of a size suitable for X-ray diffraction studies. Cubic crystallographic symmetry was indicated by the lack of birefringence when the crystals were inspected with polarized light. X-ray precession photographs indicated that lambda exonuclease crystallizes in a space group of P4(1)32, or its enantiomorph P4(3)32, with 24 tetramers in the unit cell of edge 210 A.

Bacteriophage lambda↗

PubMed1985

Molecular composition of the adenovirus type 2 virion.

The representation of the different structural polypeptides within the adenovirus virion has been accurately determined, and the particle molecular weight has been derived. A stoichiometric analysis was performed with [35S]methionine as a radiolabel, and analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to separate the polypeptides. The recently available sequence of the adenovirus type 2 genome was used to determine the number of methionines in each polypeptide. The resulting relative representation was placed on an absolute scale by using the known number of hexon polypeptides per virion. The analysis provides new information on the composition of the vertex region, which has been the subject of some controversy. Penton base was found to be present in 60 copies, distributed as pentamers at each of the 12 vertices. Three fiber monomers were associated with one penton base to form the penton complex. Polypeptide IX was present in 240 copies per virion and 12 copies per group-of-nine hexons, supporting a model proposed earlier for the distribution of this protein. The location of polypeptide IX explains the dissociation of the virus outer capsid into groups-of-nine hexons. The penton base was microheterogeneous, and the relative amounts suggest that the symmetry mismatch, which occurs within the penton complex between base and fiber, is resolved by the synthesis of penton base polypeptides from two closely spaced start codons.

Adenoviruses, Human↗

PubMedDate not supplied

Interpretation of electron micrographs of adenovirus hexon arrays using a crystallographic molecular model.

Two types of two-dimensional arrays of purified adenovirus type 2 hexon have been obtained and analyzed by Fourier filtration of their electron micrographs. One array contained continuously close-packed hexons, distributed on a hexagonal p3 lattice, with a unit cell dimension of 94 +/- 2 A. The other array contained close-packed hexons with a regular absence, so that rings of six hexons related by sixfold symmetry formed a p6 unit cell. The cell dimension of the hexagonal array was 153 +/- 3 A, with neighboring hexons separated by 88 +/- 2 A. Smaller p6 arrays were also formed by hexons freed from complete virions on the microscope grid by treatment with distilled water. A molecular model of hexon, known from the X-ray crystallographic structure, was used to interpret Fourier-filtered images of the arrays, and to determine the relative orientations of the hexon molecules. The hexon-hexon interaction in the p3 array is that found in the virion facet, whereas that in the p6 array is a planar form of the interaction between peripentonal hexons around the vertex.

Adenoviridae↗