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Biomedical subjects

H Decker

Publications and source records attributed to H Decker.

At least 55 records · Page 3Linked to original sources
PubMed1994

GAS TRANSPORT IN THE HAEMOLYMPH OF ARACHNIDS - OXYGEN TRANSPORT AND THE PHYSIOLOGICAL ROLE OF HAEMOCYANIN

Oxygen equilibrium curves and the relationships between the partial pressure of CO2 and pH were determined for the haemolymph of the arachnids Eurypelma californicum, Pandinus imperator and Cupiennius salei. A new type of experimental apparatus was constructed, tested and used to make these measurements on small undiluted cell-free haemolymph samples. Most of its components were made in our workshop and were inexpensive. The apparatus proved to be very reliable as demonstrated by control (oxygen concentration) experiments. In previous reports on O2 equilibrium curves of E. californicum haemocyanin, Tris­HCl buffer was used to set the pH, whereas in our experiments, pH was adjusted by altering the partial pressure of CO2. The O2 concentration measurements demonstrate an increase in oxygen affinity of E. californicum haemocyanin when using Tris­HCl buffer. The position and shape of the O2 equilibrium curves of E. californicum and P. imperator haemocyanin show a distinct dependency on pH. Oxygen affinity is lower in E. californicum and cooperativity changes with pH in E. californicum, but not in P. imperator. Oxygen transport in the haemolymph of E. californicum during rest, activity and recovery was calculated on the basis of the O2 equilibrium curves of undiluted haemolymph. Apart from oxygen transport, there are indications that haemocyanin is also involved in other physiological processes. For example, it may function as a storage protein.

Journal Article↗
PubMed1994

Overproduction of the acyl carrier protein component of a type II polyketide synthase stimulates production of tetracenomycin biosynthetic intermediates in Streptomyces glaucescens.

The development of microorganisms with improved antibiotic production is an important goal in the commercialization of new pharmaceuticals or in lowering the cost of established drugs. We report a way to achieve this for biosynthetic intermediates of an antibiotic made by the polyketide pathway whose earliest steps involve a Type II multienzyme complex. Introduction of the tcmKLM beta-ketoacyl: ACP synthase and acyl carrier protein (ACP) genes or just the tcmM ACP gene into the tetracenomycin (Tcm) C-producing Streptomyces glaucescens wild-type strain, or its tcmN or tcmO blocked mutants, on high copy vectors under the control of strong promoters caused a 2 to 30-fold overproduction of Tcm D3 and some other biosynthetic intermediates (or shunt products) and a 25 to 30% increase in Tcm C production relative to the control strains carrying the plasmid vector only. However, Tcm C production was not greater than that obtained with the vector-free wild-type strain. The unexpected effect of increased ACP on Tcm D3 production suggests that the level of this protein can influence either the activity or level of the three other components of the Tcm polyketide synthase.

Acyl Carrier Protein↗
PubMed1993

Nucleotide sequences and heterologous expression of tcmG and tcmP, biosynthetic genes for tetracenomycin C in Streptomyces glaucescens.

The nucleotide sequence of the tcmIII, tcmIc, and tcmVII region of the tetracenomycin (TCM) C gene cluster of Streptomyces glaucescens ETH 22794 (GLA.0) revealed the presence of two genes, tcmP and tcmG. The deduced product of tcmG resembles flavoprotein hydroxylases found in several other bacteria, whereas the predicted amino acid sequence of tcmP is not significantly similar to those of any known proteins in the available data bases. Southern blot hybridization revealed an approximately 180-bp deletion in a tcmIII (tcmG) mutant and a 1,800-bp insertion in a tcmVII (tcmP) mutant. Heterologous expression of tcmG and tcmP in Streptomyces lividans and tcmP in Escherichia coli established that tcmP encodes an O-methyltransferase, catalyzing the methylation of the C-9 carboxy group of TCM E to yield TCM A2, and that tcmG is responsible for the hydroxylation of TCM A2 at positions C-4, C-4a, and C-12a to give TCM C. These are the final two steps of TCM C biosynthesis.

Alleles↗
PubMed1993

Transcriptional analysis of the Streptomyces glaucescens tetracenomycin C biosynthesis gene cluster.

A 12.6-kb DNA fragment from Streptomyces glaucescens GLA.0 containing the 12 genes for tetracenomycin (TCM) C biosynthesis and resistance enabled Streptomyces lividans to produce TCM C. Transcriptional analysis of the tcmPG intergenic region in this cluster established the presence of two divergent promoters. The tcmIc mutation, a T-to-G transversion in the -10 region of the tcmG promoter, decreased promoter activity drastically at the stationary growth stage and time of maximum TCM C accumulation. This promoter may direct the transcription of a tcmGHIJKLMNO operon, while the other promoter is for tcmP.

Anti-Bacterial Agents↗
PubMed1992

Quaternary and subunit structure of Calliphora arylphorin as deduced from electron microscopy, electrophoresis, and sequence similarities with arthropod hemocyanin.

Arylphorin was purified from larvae of the blowfly Calliphora vicina and studied in its oligomeric form and after dissociation at pH 9.6 into native subunits. In accordance with earlier literature, it was electrophoretically shown to be a 500 kDa hexamer (1 x 6) consisting of 78 kDa polypeptides (= subunits). Electron micrographs of negatively stained hexamers show a characteristic curvilinear, equilateral triangle of 12 nm in diameter (top view) and a rectangle measuring 10 x 12 nm (side view). Alternatively, particles in the top view orientation exhibit a roughly circular shape 12 nm in diameter. Crossed immunoelectrophoresis revealed the presence of a major subunit type; the nature of a very minor and a third immunologically separated component remains unclear. A novel 2 x 6 arylphorin particle was detected and isolated. It comprises less than 10% of the total arylphorin material and shows a long, narrow interhexamer bridge in the electron microscope. An arylphorin dissociation intermediate identified as a trimer (1/2 x 6) was isolated; its possible quaternary structure is discussed on the basis of electron micrographs. The epitope of monoclonal antibody Ec-7 directed against tarantula (Eurypelma californicum) hemocyanin subunit d and also reactive to Calliphora arylphorin was traced to a highly conserved peptide of 27 amino acids localized in the center of the protein. The primary structure of Calliphora arylphorin as published in our preceding paper (Naumann and Scheller 1991) is compared in detail to the sequences of spider and spiny lobster hemocyanin. This revealed a basic framework of 103 strictly conserved amino acids. Isofunctional exchanges are proposed for another 76 positions. On the basis of these similarities, and the published three-dimensional model of spiny lobster hemocyanin, a detailed model of the quaternary structure of Calliphora arylphorin is presented. A second larval storage protein previously termed protein II was purified from Calliphora hemolymph. It was demonstrated to be a 500 kDa hexamer of 83 kDa subunits. In the electron microscope it shows a cubic view 9 nm in length with a large central hole and a rectangular view (9 x 10 nm) with a large central cavity. A morphologically very similar hemolymph protein was detected in Drosophila melanogaster larvae. From its structural appearance it is uncertain whether protein II belongs to the hemocyanin superfamily or not.

Amino Acid Sequence↗
PubMed1991

Structure-activity relationships of the nikkomycins.

The structure-activity relationships of different nikkomycins were studied to evaluate the structural requirements for a potent chitin synthase inhibitor. We investigated the transport of the nikkomycins via the peptide transport system of the yeast Yarrowia lipolytica and determined the kinetic parameters for nikkomycin Z uptake [Km = 24 microM, Vmax = 2.2 nmol min-1 (mg dry wt)-1]. We demonstrated that the beta-methyl group of the N-terminal amino acid of dipeptide nikkomycins protects the molecule against peptidase activity in crude cell-extracts of different fungi. Furthermore, the relationship between inhibition constants for chitin synthase, transport of the nikkomycins via the peptide transport system, susceptibility to degradation by cellular proteases and whole-cell activity of the nikkomycins are discussed.

Aminoglycosides↗
PubMed1991

Metabolic products of microorganisms. 258. Enzymatic bromination of nikkomycin Z.

Two brominated nikkomycins were produced by enzymatic halogenation of nikkomycin Z in the presence of a nonheme bromoperoxidase isolated from Streptomyces aureofaciens Tü 24. The monobrominated and dibrominated nikkomycin Z derivatives were substituted at the hydroxypyridyl moiety of the N-terminal amino acid of nikkomycin Z at position C-6"' (ZBr) or C-4"' and C-6"' (ZBr2). The brominated nikkomycin Z derivatives had a decreased affinity to chitin synthase of Coprinus cinereus as compared to nikkomycin Z and exhibited a low inhibitory activity towards various fungi and yeasts.

Aminoglycosides↗
PubMed1990

Nested allostery in scorpion hemocyanin (Pandinus imperator).

The oxygen-binding behavior of the 24-meric hemocyanin of the scorpion Pandinus imperator and its dependence on allosteric effectors such as protons can be successfully described by the nesting model; the MWC model is not acceptable. The affinities of the four assumed conformations of the allosteric unit, the 12-meric half-molecule, are not dependent on pH whereas the three allosteric equilibrium constants decrease with decreasing proton concentration. Comparison with the oxygen-binding behavior of the 24-meric tarantula hemocyanin (Eurypelma californicum) reveals that the affinity values for the various conformations seem to be conserved for chelicerata hemocyanin.

Allosteric Site↗
PubMed1990

Conformational changes of tarantula (Eurypelma californicum) haemocyanin detected with a fluorescent probe, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole.

Different fluorescent labels were tested in order to monitor conformational transitions of the four-hexamer haemocyanin from the tarantula Eurypelma californicum during the oxygenation process. When the four-hexamer was labelled with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, the maximum wavelength lambda max of the fluorescence emission spectrum was significantly shifted up to 5 nm, depending on pH and the degree of oxygenation. The values for lambda max of the fully oxygenated haemocyanin were 531.5 nm (pH less than 7.8) and 530.0 nm (pH greater than 7.8). For deoxygenated haemocyanin the values were 533.5 nm (pH less than 7.2) and 535.2 nm (pH greater than 7.2). The occurrence of four distinct emission maxima supports the hypothesis of four conformational species for the tarantula haemocyanin, which have been predicted by the nesting model [Robert, C. H., Decker, H., Richey, B., Gill, S. J. & Wyman, J. (1987) Proc. Natl Acad. Sci. USA 84, 1891-1895]. Only four amino acids of the four-hexamer were labelled with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. They were identified as lysine 484 on the purified peptide Leu-Arg-Lys-Phe-His-Arg. This amino acid is located on the surface of the four copies of subunit d. The sharp shift of the maxima of the emission wavelengths during oxygenation indicates that the four copies of subunits d synchronously take part in the conformational switch. This points to a concerted mechanism for the conformational transitions of the tarantula haemocyanin.

4-Chloro-7-nitrobenzofurazan↗
PubMed1990

Nested allostery of arthropodan hemocyanin (Eurypelma californicum and Homarus americanus). The role of protons.

Continuous oxygen binding curves for two arthropodan hemocyanins were performed at different pH values ranging from 7.0 to 8.7 and in the presence of physiological concentrations of the bivalent ions Ca2+ and Mg2+. The arthropods Eurypelma californicum and Homarus americanus are classified as chelicerata and crustaceans, respectively. Their structurally well-characterized hemocyanins are composed of, in the case of E. californicum 24 subunits, and in the case of H. americanus 12 subunits. The role of protons as allosteric effectors of the oxygen binding was analysed in terms of the nesting model, which assumes hierarchies of allosteric equilibria that are based on obvious structural hierarchies. For each hemocyanin, the smallest structural repeating unit, the 12-mer or the 6-mer, respectively, was regarded as the "allosteric unit". Two allosteric units are allosterically coupled within the native molecules. The analysis revealed that in accordance with the postulations of the classical Monod-Wyman-Changeux model protons as allosteric effectors do not change the oxygen affinities of the four postulated conformations, but influence the allosteric equilibria between them at two different hierarchical levels. Model-independent determination of the affinity constants for the binding of the first and the last oxygen molecule to the native hemocyanins and to the isolated half-molecules confirmed the affinities calculated according to the nesting model. The stepwise establishment of new conformations during the assembly process from monomers to the structurally identical repeating unit and further on to the native molecule is shown. Possible physiological advantages of allosterically coupled allosteric units in contrast to allosterically uncoupled ones are thought to be (1) the option to regulate oxygen binding on different levels of structural hierarchy and (2) the increase of the oxygen-carrying capacity.

Allosteric Site↗
PubMed1990

[Hierarchies in the structure and function of oxygen-binding proteins].

The structures of respiratory proteins such as hemoglobins and hemocyanins show obvious hierarchies. They belong to the class of allosteric macromolecules with cooperative functional properties, as do key metabolic enzymes. When examining the molecular mechanisms on which allostery and cooperativity are based, it could be shown for arthropod hemocyanins that the structural hierarchy of these macromolecules is reflected in a functional hierarchy. This relationship is described quantitatively in the "nesting" model. This model also offers explanations for the physiological significance of the prominent hierarchy of these molecules.

Animals↗
PubMed1990

Metabolic products of microorganisms. 255. Nikkomycins Wz and Wx, new chitin synthetase inhibitors from Streptomyces tendae.

Two new dipeptidyl nikkomycins of the Z and X type were isolated from the culture broth of Streptomyces tendae TU 901/395-11/32 and characterized. They show a variation in the amino acid moiety of the molecule. Nikkomycin Wz is composed of L-tyrosine and 5-amino-5-deoxy-D-allo-furanuronic acid N-glycosidally bound to uracil, whereas nikkomycin Wx is composed of L-tyrosine and 5-amino-5-deoxy-D-allo-furanuronic acid N-glycosidally bound to 4-formyl-4-imidazolin-2-one. The new nikkomycins are good inhibitors of chitin synthetase from Coprinus cinereus but they did not inhibit growth of fungi and yeasts.

Aminoglycosides↗
PubMed1989

Allosteric oxygen-binding properties of reassembled tarantula (Eurypelma californicum) hemocyanin with incorporated apo- or met-subunits.

4x6-meric hemocyanin of the tarantula Eurypelma californicum was dissociated into subunits; one type of subunit was removed by immunoaffinity chromatography and replaced by its apo- or met-form. The mixture was reassembled and the reconstituted 4x6-mers were isolated. This was performed for subunits a, bc, d, e, f and g, respectively. It was verified by crossed immunoelectrophoresis that each type of subunit including the modified one, was incorporated in the reassembled 4x6-mers. Oxygen binding curves of the purified reconstituted 4x6-mers were recorded at different pH values (pH 7.0-9.0; 20 degrees C). Half-saturation pressures (P50) and the cooperativities were calculated using unmodified, reassembled 4x6-mers as reference. In all cases, incorporation of a met-subunit increased oxygen affinity. In contrast, incorporation of an apo-subunit either slightly decreased oxygen affinity (bc, f and d) or had no detected influence (others). The Bohr effect remained more or less unchanged in every case. Cooperativity was generally decreased. The met-modification of subunit d had the strongest effect. No significant differences could be observed between the respective met- and apomodification, except for experiments with subunit d. Generally, the value of hmax exceeded h50 by a factor of 1.3 to 1.5. pH-sensitivity of cooperativity was distinctly influenced depending on the modified subunit. The strongest effect was observed for subunit bc. Our results demonstrate, for the first time, that each subunit of tarantula hemocyanin is involved in the allosteric processes. Apparently, they uniformly contribute to oxygen affinity and Bohr effect, but distinctly to cooperativity and pH sensitivity of the latter.

Allosteric Regulation↗
PubMed1989

Metabolic products of microorganisms. 252. Isolation of new nikkomycins from Streptomyces tendae.

Two new nikkomycins were isolated from the culture broth of Streptomyces tendae Tü 901/PF 53+-3. The new compounds are the dipeptide nikkomycin pseudo-Z (psi Z) and tripeptide nikkomycin pseudo-J (psi J), which are analogues to nikkomycins Z and J. Nikkomycins pseudo-Z and pseudo-J have a C-glycosidic linkage between uracil and 5-amino-5-deoxy-D-allo-furanuronic acid, which is comparable to the C-glycosidic bond in pseudouridine. The new Cc-nucleoside nikkomycins exhibit a lower biological activity than the CN-nucleoside nikkomycins.

Aminoglycosides↗
PubMed1989

Metabolic products of microorganisms. 254. Structure of the new nikkomycins pseudo-Z and pseudo-J.

Two new nikkomycins were isolated from the fermentation broth of Streptomyces tendae Tü 901/PF 53+-3. These new metabolites, nikkomycins pseudo-Z (psi-Z,1) and pseudo-J (psi-J, 2) differ from the corresponding nikkomycins Z and J by a C-glycosidic bond between C-5 of uracil and C-1' of 5-amino-5-deoxy-D-allo-furanuronic acid instead of an N-glycosidic bond. The structure elucidation was achieved by two-dimensional NMR techniques and mass spectrometry.

Aminoglycosides↗
PubMed1988

Nested allosteric interaction in tarantula hemocyanin revealed through the binding of oxygen and carbon monoxide.

We have examined the competitive binding of oxygen and carbon monoxide to the multisubunit hemocyanin of the tarantula Eurypelma californicum. Employment of high-precision thin-layer methods has enabled detailed characterization of the pure oxygen and pure carbon monoxide binding curves, as well as binding curves performed under mixed-gas conditions. The pure oxygen binding curve and the displacement of oxygen by carbon monoxide at full ligand saturation are highly cooperative, but in the absence of oxygen, carbon monoxide binds noncooperatively. The results were analyzed globally within the framework of a nested allosteric model [Robert, C.H., Decker, H., Richey, B., Gill, S.J., & Wyman, J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1891-1895] which takes into account the hierarchy of subunit structure present in the macromolecule. The use of two ligands enables one to recognize two distinct levels of allosteric interaction functioning in the protein assembly. The binding characteristics of the allosteric states demonstrated for Eurypelma follow a similar pattern as those found earlier for Homarus americanus.

Allosteric Site↗
PubMed1987

Nesting: hierarchies of allosteric interactions.

A generalization of the allosteric model is presented that incorporates a hierarchy of conformational equilibria. Such a formulation draws upon structural organization already seen in many large macromolecular systems. The functional binding properties of the macromolecule reflect conformational equilibria at each structural level. Appropriate "nested" models are used to interpret structural features and functional aspects of two hemocyanin systems with a large number (12 and 24) of binding sites.

Allosteric Regulation↗