Biomedical subjects

H L Dekker

Publications and source records attributed to H L Dekker.

At least 19 recordsLinked to original sources

PubMed2001

A new crosslinker for mass spectrometric analysis of the quaternary structure of protein complexes.

Mass spectrometric structural analysis of crosslinked peptides is a powerful method to elucidate the spatial arrangement of polypeptides in protein complexes. Our aim is to develop bifunctional crosslinkers that, after crosslinking protein complexes followed by proteolytic digestion, give rise to crosslinked peptides that can be readily tracked down by mass spectrometry. To this end we synthesized the crosslinker N-benzyliminodiacetoyloxysuccinimid (BID), which yields stable benzyl cation marker ions upon low-energy collision-induced dissociation (CID) tandem mass spectrometry. Sensitive detection of the marker ion upon low-energy CID is demonstrated with different BID-crosslinked peptide preparations. With BID it becomes possible to retrieve crosslinked and crosslinker-adducted peptides, without the necessity of purifying crosslinked peptides prior to identification. The basic design of this crosslinker can be varied upon, in order to meet specific crosslinking needs.

Bradykinin↗

PubMed2000

Oxidation reactions catalyzed by vanadium chloroperoxidase from Curvularia inaequalis.

Vanadium haloperoxidases have been reported to mediate the oxidation of halides to hypohalous acid and the sulfoxidation of organic sulfides to the corresponding sulfoxides in the presence of hydrogen peroxide. However, traditional heme peroxidase substrates were reported not to be oxidized by vanadium haloperoxidases. Surprisingly, we have now found that the recombinant vanadium chloroperoxidase from the fungus Curvularia inaequalis catalyzes the oxidation of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), a classical chromogenic heme peroxidase substrate. The enzyme mediates the oxidation of ABTS in the presence of hydrogen peroxide with a turnover frequency of 11 s(-1) at its pH optimum of 4.0. The Km of the recombinant enzyme for ABTS was observed to be approximately 35 microM at this pH value. In addition, the bleaching of an industrial sulfonated azo dye, Chicago Sky Blue 6B, catalyzed by the recombinant vanadium chloroperoxidase in the presence of hydrogen peroxide is reported.

Chloride Peroxidase↗

PubMed1999

The sulfonium ion linkage in myeloperoxidase. Direct spectroscopic detection by isotopic labeling and effect of mutation.

The heme group of myeloperoxidase is covalently linked via two ester bonds to the protein and a unique sulfonium ion linkage involving Met(243). Mutation of Met(243) into Thr, Gln, and Val, which are the corresponding residues of eosinophil peroxidase, lactoperoxidase, and thyroid peroxidase, respectively, and into Cys was performed. The Soret band in the optical absorbance spectrum in the oxidized mutants is now found at approximately 411 nm. Both the pyridine hemochrome spectra and resonance Raman spectra of the mutants are affected by the mutation. In the Met(243) mutants the affinity for chloride has decreased 100-fold. All mutants have lost their chlorination activity, except for the M243T mutant, which still has 15% activity left. By Fourier transform infared difference spectroscopy it was possible to specifically detect the (13)CD(3)-labeled methionyl sulfonium ion linkage. We conclude that the sulfonium ion linkage serves two roles. First, it serves as an electron-withdrawing substituent via its positive charge, and, second, together with its neighboring residue Glu(242), it appears to be responsible for the lower symmetry of the heme group and distortion from the planar conformation normally seen in heme-containing proteins.

Amino Acid Substitution↗

PubMed1999

Characterization of Urtica dioica agglutinin isolectins and the encoding gene family.

Urtica dioica agglutinin (UDA) has previously been found in roots and rhizomes of stinging nettles as a mixture of UDA-isolectins. Protein and cDNA sequencing have shown that mature UDA is composed of two hevein domains and is processed from a precursor protein. The precursor contains a signal peptide, two in-tandem hevein domains, a hinge region and a carboxyl-terminal chitinase domain. Genomic fragments encoding precursors for UDA-isolectins have been amplified by five independent polymerase chain reactions on genomic DNA from stinging nettle ecotype Weerselo. One amplified gene was completely sequenced. As compared to the published cDNA sequence, the genomic sequence contains, besides two basepair substitutions, two introns located at the same positions as in other plant chitinases. By partial sequence analysis of 40 amplified genes, 16 different genes were identified which encode seven putative UDA-isolectins. The deduced amino acid sequences share 78.9-98.9% identity. In extracts of roots and rhizomes of stinging nettle ecotype Weerselo six out of these seven isolectins were detected by mass spectrometry. One of them is an acidic form, which has not been identified before. Our results demonstrate that UDA is encoded by a large gene family.

Amino Acid Sequence↗

PubMed1999

Characterization of the Asp94 and Glu242 mutants in myeloperoxidase, the residues linking the heme group via ester bonds.

The heme group of all mammalian peroxidases is covalently linked to the protein matrix via two esterbonds, as we have recently shown by Fourier transform infrared (FTIR) difference spectroscopy [Kooter, I. M., Pierik, A.J., Merkx, M., Averill, B.A., Moguilevsky, N., Bollen, A. & Wever, R. (1997) J. Am. Chem. Soc. 119, 11542-11543]. We have examined the effects of mutation of Asp94 and Glu242, responsible for those ester bonds in myeloperoxidase, on the spectroscopic properties and catalytic activity of this enzyme. Mutation of Asp94 in myeloperoxidase results in two species. The first species has spectroscopic characteristics similar to that of wild-type myeloperoxidase. The second species has spectroscopic characteristics similar to that of Met243-->Gln mutant, and it is therefore concluded that, besides loss of the ester bond involving Asp94, this species also has lost the sulfonium ion linkage that is also characteristic of myeloperoxidase. The Asp94-->Asn mutant still has about 30% residual peroxidase activity while for the Asp94-->Val mutant only a few percentage activity is left. When Glu242 is mutated the sulfonium ion linkage is not affected, but this residue together with its neighbouring residue Met243, according to resonance Raman spectra, is responsible for the low symmetry of the heme group. Mutation of either of these residues results in loss of the bowed distortion from the planar conformation, and in a heme group with higher symmetry. For the Glu242-->Gln mutant 8% residual peroxidase activity is found.

Aspartic Acid↗

PubMed1999

Processing, targeting, and antifungal activity of stinging nettle agglutinin in transgenic tobacco.

The gene encoding the precursor to stinging nettle (Urtica dioica L. ) isolectin I was introduced into tobacco (Nicotiana tabacum). In transgenic plants this precursor was processed to mature-sized lectin. The mature isolectin is deposited intracellularly, most likely in the vacuoles. A gene construct lacking the C-terminal 25 amino acids was also introduced in tobacco to study the role of the C terminus in subcellular trafficking. In tobacco plants that expressed this construct, the mutant precursor was correctly processed and the mature isolectin was targeted to the intercellular space. These results indicate the presence of a C-terminal signal for intracellular retention of stinging nettle lectin and most likely for sorting of the lectin to the vacuoles. In addition, correct processing of this lectin did not depend on vacuolar deposition. Isolectin I purified from tobacco displayed identical biological activities as isolectin I isolated from stinging nettle. In vitro antifungal assays on germinated spores of the fungi Botrytis cinerea, Trichoderma viride, and Colletotrichum lindemuthianum revealed that growth inhibition by stinging nettle isolectin I occurs at a specific phase of fungal growth and is temporal, suggesting that the fungi had an adaptation mechanism.

Amino Acid Sequence↗

PubMed1998

The sulphoxidation of thioanisole catalysed by lactoperoxidase and Coprinus cinereus peroxidase: evidence for an oxygen-rebound mechanism.

Using both stopped-flow and conventional spectroscopy, the oxygenation of methyl phenyl sulphide by both lactoperoxidase (LPO) and Coprinus cinereus peroxidase (CiP) was monitored. Controlled continuous addition of H2O2 during turnover and monitoring the presence of native enzymes, compounds I, II and III, led to formation of the sulphoxide in high yield and enantioselectivity. Under those conditions, LPO catalysed the formation of (R) methyl phenyl sulphoxide with a yield of 85% and an enantiomeric excess (e.e.) of 80%. CiP catalysed the formation of (S) methyl phenyl sulphoxide with a yield of 84% and an e.e. of 73%. The enantioselective performance was markedly influenced by the purity of the enzymes used. Presence of compound III during turnover led to rapid inactivation of the peroxidases and, therefore, to both a lower yield of the sulphoxides and a lower enantioselectivity. Stopped-flow kinetic data show that, for both LPO and CiP, the transition of compound I to compound II depends on the concentration of the methyl phenyl sulphide, suggesting an oxygen-rebound mechanism. In line with this mechanism, a methyl phenyl sulphide radical cation was detected by EPR during turnover for LPO.

Coprinus↗

PubMed1998

Isolation, characterization, and primary structure of the vanadium chloroperoxidase from the fungus Embellisia didymospora.

Here we describe the isolation, purification, and basic kinetic parameters of a vanadium type chloroperoxidase from the hyphomycete fungus Embellisia didymospora. The enzyme proved to possess similar high substrate affinities, a Km of 5 microM for a bromide, 1.2 mM for a chloride, and 60 microM for a hydrogen peroxide, as those of the vanadium chloroperoxidase from Curvularia inaequalis, although with lower turnover rates for both Cl- and Br-. Substrate bromide was also found to inhibit the enzyme, a feature subsequently also noted for the chloroperoxidase from C. inaequalis. The gene encoding this enzyme was identified using DNA Southern blotting techniques and subsequently isolated and sequenced. A comparison is made between this vanadium chloroperoxidase and that of the fungus C. inaequalis both kinetically and at the sequence level. At the primary structural level the two chloroperoxidases share 68% identity, with conservation of all active site residues.

Amino Acid Sequence↗

PubMed1997

The regulation of the vanadium chloroperoxidase from Curvularia inaequalis.

The effects of carbon and nitrogen source on the regulation of the vanadium chloroperoxidase secreted by the fungus Curnularia inaequalis were investigated. The addition of glucose showed a repressing effect on both the observed messenger RNA level and the measured enzyme activities, whereas the addition of glutamate as nitrogen source and the addition of both glutamate and glycerol had no effect. Addition of vanadate had no effect on the level of mRNA. Eight hundred base pairs of the upstream promoter region of vCPO were sequenced and various features of interest are highlighted. Closer inspection of the mycelium revealed that once secreted, vCPO probably remains tightly associated with the hyphae in two forms, one of which may be a proform of the enzyme. A possible cleavage event at the C-terminus may lower its potential for hyphal association and permit its disassociation into the growth medium. A putative role for the vanadium chloroperoxidase is put forward.

Base Sequence↗

PubMed1997

From phosphatases to vanadium peroxidases: a similar architecture of the active site.

We show here that the amino acid residues contributing to the active sites of the vanadate containing haloperoxidases are conserved within three families of acid phosphatases; this suggests that the active sites of these enzymes are very similar. This is confirmed by activity measurements showing that apochloroperoxidase exhibits phosphatase activity. These observations not only reveal interesting evolutionary relationships between these groups of enzymes but may also have important implications for the research on acid phosphatases, especially glucose-6-phosphatase-the enzyme affected in von Gierke disease-of which the predicted membrane topology may have to be reconsidered.

Acid Phosphatase↗

PubMed1995

Inhibition of pig kidney diamine oxidase by nazlinin and nazlinin derivatives.

Nazlinin (1-(4-butylamino)-1,2,3,4-tetrahydro-beta-carboline) (1), an alkaloid recently isolated from Nitraria schoberi, and its two derivatives, 1-(4-butylamino)-3,4-dihydro-beta-carboline (2) and 1-(4-butylamino)-beta-carboline (3), were synthesized and their interaction with pig kidney diamine oxidase (PKDO) was studied. Nazlinin appeared to be a very poor substrate while 3 was a good substrate with an apparent Km of 9.3-10(-5) M. The enzyme was inhibited by 1 and 2. With both compounds the mode of inhibition found was non-competitive and inhibition constants calculated from the slopes and intercepts of double-reciprocal plots show that 2 is a much more potent inhibitor than the natural product. The relationship between the structure of these compounds and the results found is discussed.

Amine Oxidase (Copper-Containing)↗

PubMed1995

Primary structure and characterization of the vanadium chloroperoxidase from the fungus Curvularia inaequalis.

Using reverse transcription of messenger RNA followed by amplification using the polymerase chain reaction, three overlapping cDNA fragments encompassing the encoding sequence of the vanadium chloroperoxidase from the fungus Curvularia inaequalis were isolated and sequenced. The sequence was confirmed by DNA sequence analysis of genomic DNA. The deduced amino acid sequence predicts a protein of 609 residues with a mass of 67488 Da. Competitive reverse-transcription polymerase chain reaction analysis indicates that vanadium chloroperoxidase expression takes place in the secondary-growth phase initiated by nutrient depletion. Southern-blot analysis of genomic DNA indicates that there is only a single gene encoding the vanadium chloroperoxidase and that no isoenzymes are present. The N-terminal amino acid residue was blocked and could not be determined by amino acid sequencing, although evidence is presented showing that the N-terminal region starts very close to the first encoded methionine residue. Although the vanadium chloroperoxidase is secreted, it was not possible to assign a leader peptide. The protein contains two putative N-glycosylation sites but experiments indicate that the protein is non-glycosylated. Two cysteine residues are present in the protein both as free thiols: no disulphide bridging was found. Metal analysis revealed that iron, copper, and calcium do not constitute part of the protein. Zinc was found at a ratio of 0.3 +/- 0.04 mol/mol protein. Boiling and subsequent SDS/PAGE of the protein sample showed a typical degradation pattern of the enzyme. Amino acid sequence analysis of the resulting peptides showed that the cleavage took place at Asp-Pro bonds of which six are located throughout the protein. No sequence similarity with other known peroxidases was found except for one small region, sharing limited similarity with bacterial haloperoxidases and other alpha/beta-hydrolase-fold enzymes. In the case of the bacterial bromoperoxidases from this group, a methionine located in this region was suggested to have a role in catalysis. Methionine, however, was not involved in the catalysis of the vanadium chloroperoxidase.

Amino Acid Sequence↗

PubMed1993

Regulation of the expression of mitochondrial proteins: relationship between mtDNA copy number and cytochrome-c oxidase activity in human cells and tissues.

The relationship between the relative amounts of nuclear and mitochondrial genes for cytochrome-c oxidase subunits and their transcripts and cytochrome-c oxidase activity was investigated in several human tissues and cell lines to get more insight into the regulation of the expression of this mitochondrial enzyme complex. The results show: (1) a wide range of mtDNA copy numbers; (2) constant ratios between the steady-state levels of the transcripts for the various cytochrome-c oxidase subunits, and (3) large variations in cytochrome-c oxidase activity in different tissues and cell lines that could not be related to the differences in mtDNA copy number. We conclude that the transcription of genes for both mitochondrial and nuclear cytochrome-c oxidase subunits is regulated coordinatedly, but also that the mtDNA copy number plays a minor role in determining differences in cytochrome-c oxidase activity between different cell and tissue types.

Cell Nucleus↗

PubMed1993

Mitochondria in cultured human muscle cells depleted of mitochondrial DNA.

Cultured human muscle cells were depleted of mitochondrial DNA (mtDNA) by prolonged treatment with ethidium bromide (EB). In these respiration-deficient muscle cells neither cytochrome c oxidase activity nor mtDNA were detectable. However, mitochondrial matrix enzymes remained present and were localized in mitochondria-like organelles, as shown by subcellular fractionation. Metabolic labeling showed synthesis of cytochrome c oxidase subunits coded by nuclear DNA (nDNA). These results indicate that depletion of mtDNA in cultured human myoblasts does not inhibit expression of nDNA-coded mitochondrial proteins. The characteristic thread-like pattern of mitochondria was lost in mtDNA-depleted myoblasts, as shown by immunofluorescence with antibodies against cytochrome c oxidase and the F1 part of the mitochondrial ATP synthase (F1-ATPase) and by fluorescence of the carbocyanine dye, 3,3'-dipentyloxacarbocyanine iodide (DiOC5(3)). The organelles visualized by these methods were round and swollen and had a localization different from lysosomes as shown by double-labeling with mitochondrial and lysosomal antibodies. These results indicate that not only synthesis, but also import of mitochondrial proteins into mitochondria-like organelles remains possible in respiration-deficient cells.

Cell Nucleus↗

PubMed1992

Presteady-state and steady-state kinetic properties of human cytochrome c oxidase. Identification of rate-limiting steps in mammalian cytochrome c oxidase.

Human cytochrome c oxidase was purified in a fully active form from heart and skeletal muscle. The enzyme was selectively solubilised with octylglucoside and KCl from submitochondrial particles followed by ammonium sulphate fractionation. The presteady-state and steady-state kinetic properties of the human cytochrome c oxidase preparations with either human cytochrome c or horse cytochrome c were studied spectrophotometrically and compared with those of bovine heart cytochrome c oxidase. The interaction between human cytochrome c and human cytochrome c oxidase proved to be highly specific. It is proposed that for efficient electron transfer to occur, a conformational change in the complex is required, thereby shifting the initially unfavourable redox equilibrium. The very slow presteady-state reaction between human cytochrome c oxidase and horse cytochrome c suggests that, in this case, the conformational change does not occur. The proposed model was also used to explain the steady-state kinetic parameters under various conditions. At high ionic strength (I = 200 mM, pH 7.4), the kcat was highly dependent on the type of oxidase and it is proposed that the internal electron transfer is the rate-limiting step. The kcat value of the 'high-affinity' phase, observed at low ionic strength (I = 18 mM, pH 7.4), was determined by the cytochrome c/cytochrome c oxidase combination applied, whereas the Km was highly dependent only on the type of cytochrome c used. Our results suggest that, depending on the cytochrome c/cytochrome c oxidase combination, either the dissociation of ferricytochrome c or the internal electron transfer is the rate-limiting step in the 'high-affinity' phase at low ionic strength. The 'low-affinity' kcat value was not only determined by the type of oxidase used, but also by the type of cytochrome c. It is proposed that the internal electron-transfer rate of the 'low-affinity' reaction is enhanced by the binding of a second molecule of cytochrome c.

Animals↗

PubMed1992

Isoforms of cytochrome c oxidase in tissues and cell lines of the mouse.

The subunit pattern of immunopurified cytochrome c oxidase from cultured mouse cells and mature tissues of the mouse was investigated by electrophoretic analysis. In mature tissues two forms of cytochrome c oxidase could clearly be identified on the basis of differences in morbidity or staining intensity of subunits VIa and VIII. One form was present in muscle and heart, and the other in liver, kidney and spleen. In lung both forms were found. In the thymus, subunit VIII showed the characteristics of subunit VIII found in muscle and heart, whereas subunit VIa resembled subunit VIa found in liver. This suggest the existence of a third cytochrome c oxidase isoform. The subunits of cytochrome c oxidase from cultured cell lines showed no differences between the various cell lines and resembled those of mature mouse liver tissue. The cytochrome c oxidase isoform from cultured proliferating cells might therefore be the same as the one found in liver. Alternatively, it might represent either a normally occurring fetal isoform, or a form specific for poorly differentiated cultured cells.

3T3 Cells↗

PubMed1992

Relationship between culture conditions and the dependency on mitochondrial function of mammalian cell proliferation.

In cultured mammalian cells, the relationship was investigated between mitochondrial function and proliferation under various culture conditions. Continuous inhibition of the expression of the mitochondrial genome was used to reduce the activity of enzymes involved in oxidative phosphorylation by 50% at every cell division. Under these conditions, culturing in relatively poor media resulted in arrest of the proliferation of most cell lines after 1 cell division. This was preceded by decreasing levels of ATP and increasing levels of ADP, suggesting that the ATP-generating capacity of the cells was limiting. Culturing in richer media led to arrest of the proliferation after 5 to 6 divisions, but accumulation of ADP was not observed. Addition of pyruvate to rich culture media and, at least for 1 cell line, increasing the CO2 levels, completely prevented proliferation arrest. Inability to synthesise metabolic precursors via mitochondrial intermediary metabolism probably explains growth arrest of cells cultured in rich media. Pyruvate and CO2 were, however, without effect on the proliferation arrest of cells cultured in relatively poor media. Therefore, pyruvate dependency for growth of cells without functional mitochondria holds true only under culture conditions where the ATP-generating capacity of the cells is not limiting.

3T3 Cells↗

PubMed1991

Quantification of mitochondrial proteins in cultured cells by immuno-flow cytometry.

Immuno-flow cytometry was tested as a tool to estimate the cellular concentration of mitochondrial proteins in cultured cells, using cytochrome c oxidase as a model enzyme. Cells labelled with antibodies against cytochrome c oxidase, in which the amount of the enzyme was reduced by various extents, showed a linear relationship between the size of the signal obtained by immuno-flow cytometry and the amount of the enzyme. The determination by immuno-flow cytometry resulted in data comparable to the results obtained by immunoprecipitation and activity measurements. Since immuno-flow cytometry requires only limited numbers of cells, the method could especially be of value for diagnostic purposes. This is illustrated by the results obtained by comparing activity measurements and immuno-flow cytometry in the initial screening of cell lines derived from patients with deficiencies in the activity of cytochrome c oxidase.

Cells, Cultured↗