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W Harder

Publications and source records attributed to W Harder.

At least 55 records · Page 3Linked to original sources
PubMed1992

Unique and overlapping pollutant stress proteins of Escherichia coli.

Exposure of growing batch cultures of Escherichia coli to nine different "model micropollutants" (benzene, cadmium chloride, chlorpyrivos, 2,4-dichloroaniline, dioctylphtalate, hexachlorobenzene, pentachlorophenol, trichloroethylene, and tetrapropylbenzosulfonate) led to the induction of 13 to 39 proteins, as analyzed by two-dimensional gel electrophoresis. Some of these proteins overlapped with heat shock and carbon starvation proteins, but at least 50% were unique to a given chemical. The stress protein induction showed a temporal pattern, indicating sequential gene expression. Chemical stress protein synthesis occurred even at concentrations that had no effect on growth. Thus, the synthesis of these proteins can be a sensitive index of stress and the nature of environmental pollution.

Bacterial Proteins↗
PubMed1992

Development of multipurpose peroxisomes in Candida boidinii grown in oleic acid-methanol limited continuous cultures.

We have studied the development and metabolic significance of peroxisomes in the yeast Candida boidinii following adaptation of the organism to cultivation conditions which require the simultaneous presence and activity of two independent peroxisome-mediated pathways for growth. After the addition of methanol to oleic acid-grown cells at late exponentional growth, a number of new small peroxisomes developed which, apart from the presence of beta-oxidation enzymes, were characterized by the presence of enzymes involved in methanol metabolism (alcohol oxidase and dihydroxyacetone synthase). The latter proteins, however, were absent in the larger organelles which were originally present in the oleic acid-grown cells prior to the addition of methanol and which contained only enzymes of the beta-oxidation pathway. Subsequent experiments on cells from continuous cultures grown on a mixture of oleic acid and methanol at steady-state conditions revealed that both the enzymes of the beta-oxidation pathway and those involved in methanol metabolism were found in one and the same compartment. Thus, under these conditions the cells contained peroxisomes which were concurrently involved in the metabolism of two different carbon sources simultaneously used for growth. Our results indicated that the heterogeneity in the peroxisomal population of a single cell, observed in the transient state following the addition of methanol, is only temporary and due to heterogeneity among these organelles with respect to their capacity to incorporate newly synthesized matrix proteins.

Acetyl-CoA C-Acetyltransferase↗
PubMed1991

Physiological studies on the utilization of oleic acid by Saccharomyces cerevisiae in relation to microbody development.

We studied the influence of specific growth conditions on the induction of beta-oxidation enzymes and rate of microbody proliferation in S. cerevisiae by oleic acid. Of all conditions tested, highest enzyme levels and microbody numbers were achieved in glucose-limited continuous cultures, supplemented with oleic acids second carbon source. Comparable enzyme levels were observed in identical cultures of peroxisome-deficient (pas) mutants of S. cerevisiae. These experiments showed chemostat cultures on glucose/oleic acid mixtures to be a method of choice for future studies on microbody biogenesis/assembly in constructed, conditional pas mutants.

Cell Division↗
PubMed1991

Purification and characterization of an activator protein for methanol dehydrogenase from thermotolerant Bacillus spp.

All thermotolerant methanol-utilizing Bacillus spp. investigated by us possess a NAD-dependent methanol dehydrogenase (MDH) activity which is stimulated by a protein present in the soluble fraction of Bacillus sp. C1 cells. This activator protein was purified to homogeneity from Bacillus sp. C1 cells grown at a low dilution rate in a methanol-limited chemostat culture. The native activator protein (Mr = 50,000) is a dimer of Mr = 27,000 subunits. The N-terminal amino acid sequence revealed no significant similarity with any published sequences. Stimulation of MDH activity by the activator protein required the presence of Mg2+ ions. Plots of specific MDH activity versus activator protein concentration revealed Michaelis-Menten type kinetics. In the presence of activator protein, MDH displayed biphasic kinetics (v versus substrate concentration) toward C1-C4 primary alcohols and NAD. The data suggest that in the presence of activator protein plus Mg2+ ions, MDH possesses a high affinity active site for alcohols and NAD, in addition to an activator- and Mg2(+)-independent low affinity active site. The activation mechanism remains to be elucidated.

Alcohol Oxidoreductases↗
PubMed1991

Biosynthesis and assembly of alcohol oxidase, a peroxisomal matrix protein in methylotrophic yeasts: a review.

Alcohol oxidase (AO) catalyses the first step of methanol metabolism in yeasts. In vivo the enzyme is compartmentalized in special cell compartments, called peroxisomes. The enzyme along with the organelles are induced during growth of methylotrophic yeasts on methanol as the sole carbon source. Like all other peroxisomal matrix proteins, AO is encoded by a nuclear gene. Expression of the protein is regulated by a repression/derepression mechanism, but also by induction. Inactive monomeric precursor protein is synthesized in the cytosol and subsequently imported post-translationally into peroxisomes without further processing. Assembly into the active homo-octameric enzyme and binding of the prosthetic group flavin adenine dinucleotide occurs inside the organelle. When enhanced concentration of octameric alcohol oxidase are present in the organelles, the enzyme may form a crystalloid. Oligomerization is not dependent on translocation of AO precursors into their target organelle since octameric, active AO is detected in the cytosol and nucleus of peroxisome-deficient mutants of Hansenula polymorpha: at high expression rates large cytosolic AO crystalloids are formed, which occasionally are also encountered inside the nucleus of such mutants. This paper summarizes recent findings and views on the mechanisms involved in synthesis, import, assembly and crystallization of this important peroxisomal enzyme.

Alcohol Oxidoreductases↗
PubMed1991

Selective inactivation of alcohol oxidase in two peroxisome-deficient mutants of the yeast Hansenula polymorpha.

We have studied selective inactivation of alcohol oxidase (AO) in two peroxisome-deficient (PER) mutants of the yeast Hansenula polymorpha. In these mutants high activities of cytosolic AO are induced by different growth conditions. At enhanced expression rates AO is arranged in large crystalloids in the cytosol, whereas smaller crystalloids are often observed inside the nucleus. Transfer of cells of the PER mutant 125-2E, which completely lacks peroxisomes, to glucose-excess conditions did not lead to degradative inactivation of AO and catalase as observed in wild-type (WT) cells used as a control. The gradual decrease in enzyme activities in the PER mutant could be accounted for by dilution of existing enzyme into newly formed cells as a result of growth. Morphologically, degradation of the cytosolic crystalloids was also not observed. Similar results were obtained with a second PER mutant (strain 124-2D), impaired in the import of peroxisomal matrix proteins. This mutant is characterized by the presence of small peroxisomes and large cytosolic AO crystalloids. Upon a shift of cells to glucose-excess conditions only part of the small peroxisomes present in these cells were degraded by mechanisms similar to those observed in WT cells placed under identical conditions. These results indicate that degradative inactivation of AO in H. polymorpha is strictly dependent on the localization of the enzyme inside peroxisomes and furthermore suggests that the mechanisms triggering this process are not directed against AO protein, but instead, to the membrane surrounding the organelle. Transfer of cells to methanol- or ethanol-containing media both resulted in modification inactivation of AO.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol Oxidoreductases↗
PubMed1991

Methanol metabolism in a peroxisome-deficient mutant of Hansenula polymorpha: a physiological study.

We have studied methanol-utilization in a peroxisome-deficient (PER) mutant of Hansenula polymorpha. In spite of the fact that in carbon-limited chemostat cultures under induced conditions the enzymes involved in methanol metabolism were present at wild-type (WT) levels, this mutant is unable to grow on methanol as a sole carbon and energy source. Addition of methanol to glucose-limited (SR = 12.5 mM) chemostat cultures of the PER mutant only resulted in an increase in yield when small amounts were used (up to 22.5 mM). At increasing amounts however, a gradual decrease in cell density was observed which, at 80 mM methanol in the feed, had dropped below the original value of the glucose-limited culture. This reduction in yield was not observed when increasing amounts of formate instead of methanol were used as supplements for the glucose-limited mutant culture and also not in WT cells, used as control in these experiments. The effect of addition of methanol to a glucose-limited PER culture was also studied in the transient state during adaptation of the cells to methanol. The enzyme patterns obtained suggested that the ultimate decrease in yield observed at enhanced methanol concentrations was due to an inefficient methanol-metabolism as a consequence of the absence of peroxisomes. The absence of intact peroxisomes results in two major problems namely i) in H2O2-metabolism, which most probably is no longer mediated by catalase and ii) the inability of the cell to control the fluxes of formaldehyde, generated from methanol. The energetic consequences of this metabolism, compared to the WT situation with intact peroxisomes, are discussed.

Alcohol Dehydrogenase↗
PubMed1991

Modification of flavin adenine dinucleotide in alcohol oxidase of the yeast Hansenula polymorpha.

Alcohol oxidase, a major peroxisomal protein of methanol-utilizing yeasts, may possess two different forms of flavin adenine dinucleotide, classical FAD and so-called modified FAD (mFAD). Conversion of FAD into mFAD was observed both in purified preparations of the enzyme and in cells grown in batch and continuous culture. The relative amount of mFAD in the enzyme varied from 5 to 95%, depending on the growth or storage conditions. The presence of mFAD led to a slight decrease in Vmax and a significant (about one order) decrease in the Km of alcohol oxidase with respect to methanol. The kinetics of modification measured in purified preparations of the enzyme obeyed first-order kinetics (k = 0.78 h-1). The modification process was strongly inhibited by methanol, formaldehyde or hydroxylamine. Modification observed in continuous culture under steady state conditions depended on the dilution rate and could also be described as a spontaneous first-order reaction (kapp = 0.27 h-1). FAD modification could only be detected in alcohol oxidase and not in other yeast peroxisomal flavoenzymes, such as D-amino acid oxidase from Candida boidinii.

Alcohol Oxidoreductases↗
PubMed1990

Immunocytochemical evidence for the acidic nature of peroxisomes in methylotrophic yeasts.

The possible acidic nature of the peroxisomal matrix present in intact yeast cells was studied immunocytochemically, using the weak base DAMP as a probe. Spheroplasts of methanol-grown Candida boidinii and Hansenula polymorpha were regenerated and incubated with DAMP. After immunogold labelling, using antibodies against DAMP, a specific accumulation of gold particles was observed on the peroxisomal profiles. This labelling was absent in controls, performed in the presence of ionophores or chloroquine. These results support earlier observations, that in intact cells a pH-gradient exists across the peroxisomal membrane. Experiments, carried out on osmotically swollen spheroplasts indicated that maintenance of this pH-gradient is strongly related to the cell's integrity.

Candida↗
PubMed1990

Permeability properties of peroxisomal membranes from yeasts.

We have studied the permeability properties of intact peroxisomes and purified peroxisomal membranes from two methylotrophic yeasts. After incorporation of sucrose and dextran in proteoliposomes composed of asolectin and peroxisomal membranes isolated from the yeasts Hansenula polymorpha and Candida boidinii a selective leakage of sucrose occurred indicating that the peroxisomal membranes were permeable to small molecules. Since the permeability of yeast peroxisomal membranes in vitro may be due to the isolation procedure employed, the osmotic stability of peroxisomes was tested during incubations of intact protoplasts in hypotonic media. Mild osmotic swelling of the protoplasts also resulted in swelling of the peroxisomes present in these cells but not in a release of their matrix proteins. The latter was only observed when the integrity of the cells was disturbed due to disruption of the cell membrane during further lowering of the concentration of the osmotic stabilizer. Stability tests with purified peroxisomes indicated that this leak of matrix proteins was not associated with the permeability to sucrose. Various attempts to mimic the in vivo situation and generate a proton motive force across the peroxisomal membranes in order to influence the permeability properties failed. Two different proton pumps were used for this purpose namely bacteriorhodopsin (BR) and reaction center-light-harvesting complex I (RCLH1 complex). After introduction of BR into the membrane of intact peroxisomes generation of a pH-gradient was not or barely detectable. Since this pump readily generated a pH-gradient in pure liposomes, these results strengthened the initial observations on the leakiness of the peroxisomal membrane fragments.(ABSTRACT TRUNCATED AT 250 WORDS)

Candida↗
PubMed1990

Structure/function relationships in methylotrophic yeasts.

This symposium marks the 15th anniversary of the discovery of microbodies in methylotrophic yeasts. In the intervening years much has been learned about the structure, function and biogenesis of these organelles and these advances are described. As our endeavours continued, unexpected results have confused commonly held views. This was for instance the case when microbody-minus mutants of yeasts became available which showed that some microbody matrix enzymes may be functional when present in the cytosol while others are not. At the molecular level, our understanding of structure/function relationships is also expanding. Examples are structural elements which relate to protein topogenesis and function of enzymes in different cell compartments. Other, perhaps more unusual, adaptations have also been encountered; some involve protein-protein interactions or even modified cofactors which possibly have helped methylotrophic yeasts to establish and/or maintain themselves in natural ecosystems.

Methanol↗
PubMed1989

Use of electron microscopy in the examination of lattice defects in crystals of alcohol oxidase.

Alcohol oxidase, purified from the yeast Hansenula polymorpha, was crystallized in vitro for the purpose of determining its structure at atomic resolution by X-ray diffraction methods. The crystals obtained yielded only extremely weak diffraction patterns: the maximal resolution observed was in the best case 6 A. Electron microscopy of thin sections indicated that most crystals showed lattice defects which might explain the poor diffraction patterns: most surprising was the appearance of large holes interrupting an otherwise regular lattice in one of the crystal forms examined. Our results indicate that transmission electron microscopy is a suitable tool for the inspection of crystals to be used in X-ray crystallography. The method allows rapid determination of lattice defects and enables optimization of crystallization conditions.

Alcohol Oxidoreductases↗
PubMed1989

Heterologous expression of alcohol oxidase in Saccharomyces cerevisiae: properties of the enzyme and implications for microbody development.

Alcohol oxidase (AO) expressed in transformed oleic acid-grown Saccharomyces cerevisiae, accumulated into microbodies to up to 8% of the total protein content of the organelles. This led to a small increase in volume fraction of the organelles, but not in their number. Most of the AO protein was present in large aggregates in the cytosol. The AO synthesized was inactive, irrespective of its subcellular localization and did not contain FAD. When the same AO gene was expressed in fused protoplasts of transformed S. cerevisiae and Hansenula polymorpha, the enzyme was properly assembled and activated in H. polymorpha microbodies.

Alcohol Oxidoreductases↗
PubMed1989

An improved method for light- and electron microscopial studies of nematode/fungal interactions.

A method is presented that enables studies to be made of single nematode-fungal interactions under conditions where fungal growth at the expense of external nutrients is prevented. The nematophagous fungus Arthrobotrys oligospora was used as a model organism in these studies. The method is based on removal of the traps from the vegetative mycelium, immediately after a nematode was captured and transfer of the trap with the captured nematode into a droplet of sterile distilled water placed in a moisture chamber. In the absence of external nutrients, such isolated traps of A. oligospora were fully effective in penetrating and subsequently digesting the captured nematode. Solely vegetative mycelium was formed at the expense of the digested nematode; this developed from the trap that originally had captured the nematode. One advantage of the present method is that studies on various stages of the nematode-fungal interaction can now be performed under conditions that exclude major influences of external nutrients which otherwise could be communicated to the trap cells by way of the vegetative mycelium.

Animals↗
PubMed1989

Methanol metabolism in thermotolerant methylotrophic Bacillus strains involving a novel catabolic NAD-dependent methanol dehydrogenase as a key enzyme.

The enzymology of methanol utilization in thermotolerant methylotrophic Bacillus strains was investigated. In all strains an immunologically related NAD-dependent methanol dehydrogenase was involved in the initial oxidation of methanol. In cells of Bacillus sp. C1 grown under methanol-limiting conditions this enzyme constituted a high percentage of total soluble protein. The methanol dehydrogenase from this organism was purified to homogeneity and characterized. In cell-free extracts the enzyme displayed biphasic kinetics towards methanol, with apparent Km values of 3.8 and 166 mM. Carbon assimilation was by way of the fructose-1,6-bisphosphate aldolase cleavage and transketolase/transaldolase rearrangement variant of the RuMP cycle of formaldehyde fixation. The key enzymes of the RuMP cycle, hexulose-6-phosphate synthase (HPS) and hexulose-6-phosphate isomerase (HPI), were present at very high levels of activity. Failure of whole cells to oxidize formate, and the absence of formaldehyde- and formate dehydrogenases indicated the operation of a non-linear oxidation sequence for formaldehyde via HPS. A comparison of the levels of methanol dehydrogenase and HPS in cells of Bacillus sp. C1 grown on methanol and glucose suggested that the synthesis of these enzymes is not under coordinate control.

Alcohol Oxidoreductases↗