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M V Gonchar

Publications and source records attributed to M V Gonchar.

At least 19 recordsLinked to original sources
PubMed2006

Purification and characterization of alcohol oxidase from a genetically constructed over-producing strain of the methylotrophic yeast Hansenula polymorpha.

Alcohol oxidase (AOX) has been purified 8-fold from a genetically constructed over-producing strain of the methylotrophic yeast Hansenula polymorpha C-105 (gcr1 catX) with impaired glucose-induced catabolite repression and completely devoid of catalase. The final enzyme preparation was homogeneous as judged by polyacrylamide gel electrophoresis and HPLC. Some physicochemical and biochemical properties of AOX were studied in detail: molecular weight (approximately 620 kD), isoelectric point (pI 6.1), and UV-VIS, circular dichroism (CD), and fluorescence spectra. The content of different secondary structure motifs of the enzyme has been calculated from the CD spectra using a computer program. It was found that the native protein contains about 50% alpha-helix, 25% beta-sheet, and about 20% random structures. The kinetic parameters for different substrates, such as methanol, ethanol, and formaldehyde, were measured using a Clark oxygen electrode. The rate of enzymatic oxidation of formaldehyde by alcohol oxidase from H. polymorpha is only twice lower compared to the best substrate of the enzyme, methanol.

Alcohol Oxidoreductases↗
PubMed2000

Isolation and physico-chemical characterization of a cytochrome c from the methylotrophic yeast Hansenula polymorpha.

Cytochrome c from the methylotrophic yeast Hansenula polymorpha was isolated and purified to homogeneity for the first time. The final yield of the highly purified protein from 1.4 kg (wet weight) cells was about 20 mg. The hemoprotein has an apparent molecular mass of 12 kDa and isoelectric point (pI) of 9.3. The purified protein was characterized by electronic, EPR and NMR spectroscopies. The redox potential of the cytochrome, E degrees, measured by cyclic voltammetry measurements at neutral pH, is 0.302 V. Both NMR spectroscopy and electrochemical measurements confirm the presence in the solution of several acid-base equilibria, the most pronounced being characterized by a pK(a) of 8.3. The latter pK(a) was attributed to the detachment of the iron(III) ion-coordinated methionine and its replacement by a lysine residue. The electrochemically derived thermodynamic parameters for neutral and alkaline protein species (DeltaS degrees (rc) and DeltaH degrees (rc)) were obtained from the temperature dependence of the redox potential.

Cytochrome c Group↗
PubMed2000

Efficient bioconversion of ethanol to acetaldehyde using a novel mutant strain of the methylotrophic yeast Hansenula polymorpha.

We report the isolation of mutant strains of the methylotrophic yeast Hansenula polymorpha that are able to efficiently oxidize ethanol to acetaldehyde in an intact cell system. The oxidation reaction is catalyzed by alcohol oxidase (AOX), a key enzyme in the methanol metabolic pathway that is typically present only in H. polymorpha cells growing on methanol. At least three mutations were introduced in the strains. Two of the mutations resulted in high levels of AOX in glucose-grown cells of the yeast. The third mutation introduced a defect in the cell's normal ability to degrade AOX in response to ethanol, and thus stabilizing the enzyme in the presence of this substrate. Using these strains, conditions for bioconversion of ethanol to acetaldehyde were examined. In addition to pH and buffer concentration, we found that the yield of acetaldehyde was improved by the addition of the proteinase inhibitor phenylmethylsulfonyl fluoride (PMSF) and by permeabilization of the cells with digitonin. Under optimal shake-flask conditions using one of the H. polymorpha mutant strains, conversion of ethanol to acetaldehyde was nearly quantitative.

Acetaldehyde↗
PubMed2000

Development of highly selective and stable potentiometric sensors for formaldehyde determination.

Two types of biosensors selective to formaldehyde have been developed on the basis of pH-sensitive field effect transistor as a transducer. Highly or partially purified alcohol oxidase (AOX) and the permeabilised cells of methylotrophic yeast Hansenula polymorpha (as a source of AOX) have been used as sensitive elements. The response time in steady-state measurement mode is in the range of 10-60 s for the enzyme-based sensors and 60-120 s for the cell-based sensor. When measured in kinetic mode the response time of all biosensors developed was less than 5 s. The linear dynamic range of the sensor output signals corresponds to 5-200 mM formaldehyde for highly and partially purified alcohol oxidase, and 5-50 mM formaldehyde for the cells. The operational stability of the biosensors is not less than 7 h, and the relative standard deviation of intra-sensor response is approximately 2 and 5% for the enzyme- and cell-based sensors, respectively. When stored at 4 degrees C, the enzyme and cell sensor responses have been found stable for more than 60 and 30 days, respectively. Both types of biosensors demonstrate a high selectivity to formaldehyde with no potentiometric response to primary alcohols, including methanol, or glycerol and glucose. The possible reasons of such unexpected high selectivity of AOX-based FET-sensors to formaldehyde are discussed. The influence of the biomembrane composition and the effect of different buffers on the sensor response to formaldehyde are also discussed.

Alcohol Oxidoreductases↗
PubMed1999

Arachidonic acid regulation of prostanoid synthesis in macrophages.

The dynamics of prostaglandin (PG) E2 synthesis by mouse peritoneal macrophages during the delivery of the basic substrate, arachidonic acid (AA), from different sources to the enzyme system of the cells was investigated. The dynamics of PGE2 synthesis in these cells was studied both after addition of exogenous AA and after stimulating the liberation of AA from intracellular pools with the calcium ionophore A23187. The kinetics of PGE2 synthesis when AA was supplied from intracellular and extracellular sources were absolutely different. PGE2 metabolism and the inactivation of the key enzyme of PG synthesis (PGH-synthase) during the reaction may be the regulating factors in the kinetics of PGE2 synthesis in the cells. For the different sources of AA in the cells, the rate constants of PGE2 consumption (k2) and PGH-synthase inactivation in the course of the reaction (kin) were calculated. The experimentally determined value of the apparent rate constant kin was identical to the theoretically calculated kin value for the case when AA was provided from an intracellular source. An observed deceleration in the PGE2 synthesis kinetics from exogenous AA is characterized by a 10-fold drop in the apparent kin and k2 values. The possibility of prostanoid synthesis regulation at the level of the traditional, constitutive isoenzyme PGH-synthase-1 is discussed.

Animals↗
PubMed1998

Microbial O2- and H2O2-electrode sensors for alcohol assays based on the use of permeabilized mutant yeast cells as the sensitive bioelements.

Two types of alcohol-specific microbial/electrochemical biosensors have been developed using specially constructed mutant cells of the methylotrophic yeast Hansenula polymorpha. The cells were immobilized in a calcium alginate gel, and placed between two membranes on the surface of oxygen or hydrogen peroxide-electrodes. The O2 electrode based biosensor contained mutant cells with strongly elevated alcohol oxidase activity. The peroxide electrode based biosensor consisted of catalase-defective mutant cells which produce hydrogen peroxide in the presence of alcohol. Both types of mutant cells were used in permeabilized form in order to release some components of the cellular respiration system, thus increasing the selectivity of the cellular respiration response to alcohol (cell/O2-biosensor) Permeabilization also increased sensitivity of the signal and shortened the response time (cell/H2O2-biosensor). Cell/O2 biosensors were linear up to 1.2 mM for ethanol and 0.35 mM for methanol, cell/H2O2 biosensors were linear up to 4.0 mM for ethanol, and 1.2 mM for methanol. Results were reproducible, sample pretreatment was not required, and the sensors exhibited good operational and storage stability. The use of sucrose, dulcitol or inositol during the preparation of the sensors resulted in increased stability of cells during their liophilization and storage in the dried state. Both biosensors had similar selectivity towards alcohols in the order of methanol (100%), ethanol (21%), and formaldehyde (12%). No signal was observed with glucose or glycerol as substrates.

Alcohols↗
PubMed1998

Lack of direct connection between arachidonic acid release and prostanoid synthesis upon differentiation of U937 cell.

The changes in AA incorporation and release as well as prostanoid synthesis upon differentiation of human premonocytic cell line, U937, induced by three functionally diverse agents--phorbol ester (TPA), dimethyl sulfoxide (DMSO), and retinoic acid (RA) have been investigated. The rate of AA incorporation into the cells remained unchanged whereas a 3- to 6-fold increase in AA release upon stimulation with Ca(2+)-ionophore A23187 as compared to undifferentiated cells was observed. While undifferentiated cells were incapable to metabolise AA via the cyclooxygenase pathway all three types of differentiated U937 cells produced TxB2 and PGE2. Only TPA-differentiated cells responded with a 6-fold increase of prostanoid synthesis after A23187 stimulation, whereas in DMSO-differentiated cells prostanoid synthesis was slightly stimulated by A23187 and in RA-differentiated cells it was not stimulated at all. Thus, agonist-induced prostanoid synthesis in differentiated cells is dependent on the nature of differentiating agent and does not correlate with AA liberation.

Arachidonic Acid↗
PubMed1997

Prostaglandin E2 biphasic control of lymphocyte proliferation: inhibition by picomolar concentrations.

Prostaglandins (PGs) have an important physiological role in the modulation of various cell immune functions. The main sources of PGs during immune responses are monocyte cells. We report here the ability of non-stimulated macrophages to synthesize prostanoids and show that peritoneal mouse macrophages synthesize PGE2, PGF2a and thromboxane B2, spleen macrophages produce PGE2 and PGF2a, and in a fresh medium this synthesis reaches a constant basal level in a few hours. We studied the kinetics of Con A-induced proliferation of murine splenocytes under the influence of a wide range of PGE2 concentrations (10(-14)-10(-7) M). The suppressive effect of PGE2 decreased when its concentration was lowered and disappeared at 10(-9) M PGE2 (this concentration corresponded to the basal level of non-stimulated macrophage synthesis of PGE2). Further lowering of the concentration became essential for the proliferation process once again, and at picomolar concentrations PGE2 caused a suppressive effect comparable with that for 10(-8) M PGE2. We also found that PGE2 significantly inhibited cell proliferation when it was added 1 h before the addition of mitogen, as compared with simultaneous mitogen addition. The effect was obtained for both low (10(-12) M) and high (10(-8) M) PGE2 concentrations. This phenomenon of PGE2 biphasic control of lymphocyte proliferation may play an important role in cellular homeostasis, in particular in immune cell function regulation.

Animals↗
PubMed1997

Cytochrome c peroxidase from a methylotrophic yeast: physiological role and isolation.

Mutant strains of the methylotrophic yeast Hansenula polymorpha defective in catalase (cat) and in glucose repression of alcohol oxidase synthesis (gcr1) have been isolated following multiple UV mutagenesis steps. One representative gcr1 cat mutant C-105 grows during batch cultivation in a glucose/methanol medium. However, growth is preceded by a prolonged lag period. C-105 and other gcr1 cat mutants do not grow on methanol medium without an alternative carbon source. A large collection of second-site suppressor catalase-defective (scd) revertants were isolated with restored ability for methylotrophic growth (Mth+) in the absence of catalase activity. These Mth+ gcr1 cat scd strains utilize methanol as a sole source of carbon and energy, although biomass yields are reduced relative to the wild-type strain. In contrast to the parental C-105 strain, H2O2 does not accumulate in the methanol medium of the revertants. We show that restoration of methylotrophic growth in the suppressor strains is strongly correlated with increased levels of the alternative H2O2-destroying enzyme, cytochrome c peroxidase. Cytochrome c peroxidase from cell-free extracts of one of the scd revertants has been purified to homogeneity and crystallized.

Acatalasia↗
PubMed1997

Prostaglandin H synthase of mouse macrophages: inhibiting and activating action of ibuprofen.

The effect of low (10(-10)-10(-14 M) ibuprofen concentrations on the release of labeled arachidonic acid metabolites by mouse peritoneal macrophages containing a constitutive isoform of prostaglandin H synthase was investigated. It was found that during the activation the cells metabolized AA through the cyclooxygenase pathway, synthesizing PGE2 (110 +/- 10 ng per 10(6) cells), PGF(2 alpha) (120 +/- 15 ng per 10(6) cells), and TxB2 (48 +/- 5 ng per 10(6) cells). Incubation of the macrophages with 10(-12) M ibuprofen leads to a sharp increase of PGf2 and PGF(2 alpha) synthesis (315 +/- 84 and 320 +/- 20 ng per 10(6) cells, respectively). A microsomal preparation of macrophage PGH-synthase was compared with PGH-synthase of ram seminal vesicles and analogy of their properties was shown (Km = 5.3.10(-5) M, IC50, ibuprofen = 15 microM). Some particularities of the PGH-synthase consecutive isoform (PGH-synthase-1) cellular regulation are discussed.

Animals↗
PubMed1997

Oxidation of exogenous formaldehyde in methylotrophic and nonmethylotrophic yeast cells.

Nonmethylotrophic (Candida maltosa and Saccharomyces cerevisiae) and methylotrophic (Hansenula polymorpha) yeast cells acidified their incubation media in the presence of formaldehyde. This was associated with the release of formate. We studied the formaldehyde-dependent production of formic acid and the enzymatic properties of these strains grown on media containing various carbon sources. The acidifying potential was considerably lower in formaldehyde dehydrogenase-deficient cells of mutant strains of H. polymorpha. The rates of acidification by C. maltosa and S. cerevisiae depended on the activity of their nonspecific aldehyde dehydrogenases. We suggest that accumulation of formate by yeast cells incubated in the presence of formaldehyde is caused by the total activity of formaldehyde dehydrogenase and nonspecific aldehyde dehydrogenase in methylotrophic yeasts or aldehyde dehydrogenase only in nonmethylotrophic yeasts. This is probably an additional mechanism for detoxification of formaldehyde.

Aldehyde Dehydrogenase↗
PubMed1995

Low concentrations of nonsteroidal anti-inflammatory drugs affect cell functions.

The effect of 10(-14)-10(-4)M ibuprofen and aspirin both on arachidonic acid metabolism in peritoneal murine macrophages and on the concanavalin A-induced proliferation of murine splenocytes were investigated. It was shown that 10(-7)-10(-4)M ibuprofen inhibits the arachidonic acid metabolism. On the other hand, 10(-12)-10(-11)M ibuprofen causes pronounced activation of arachidonic acid metabolism. The low concentration (10(-14)-10(-10)M) effects also take place when non-steroidal anti-inflammatory drugs influence other functions of the immune system: that is, they activate the splenocyte mitogen-induced proliferative response. These results are in accord with our suggestion that the low concentration effects of these drugs do not depend upon cell types and may have an important physiological significance.

Animals↗
PubMed1993

A cell biosensor specific for formaldehyde based on pH-sensitive transistors coupled to methylotrophic yeast cells with genetically adjusted metabolism.

A cell biosensor specific for formaldehyde was developed using double-mutant cells of the methylotrophic yeast Hansenula polymorpha A3-11. The activities of some of the enzymes in the metabolic pathway of the wild-strain cells were deliberately suppressed by introducing respective genetic blocks to optimize the selectivity and acidification rate. Mutant yeast cells produced in this way were immobilized in Ca-alginate gel on the gate of a pH-sensitive field effect transistor. The local acidification of the extracellular medium due to specific conversion of formaldehyde was recorded. The steady-state response time of the biosensor was 2-3 min, i.e., about 10 times shorter than the response time for the alcohol-specific cell biosensors described earlier. The linear dynamic range of the sensor's response corresponds to formaldehyde concentrations of 2 to 200 mM. The operational stability of the sensor was not less than 4 h. The biosensor demonstrated high specificity to formaldehyde with no response to several organic acids, methanol, and other alcohols, except for low sensitivity to ethanol. The influence of sample buffer capacity and pH on the sensor response, as well as thermostability, was investigated.

Biosensing Techniques↗
PubMed1988

Genetic control of methanol utilization in yeasts.

Considered are our own data and those found in literature on the properties of yeast mutants impaired in their ability to utilize methanol as sole carbon and energy source; hypotheses about the role of alcohol oxidase and citrate synthase in biogenesis of peroxisomes are proposed. It has been proved that formaldehyde reductase participates in the control of the formaldehyde level in the cell. Properties of mutants defective in the catabolite repression and inactivation of enzymes of methanol metabolism are described. The existence of several autonomous mechanisms of the catabolite repression of alcohol oxidase has been shown. It has been found, that the induction of glyoxysomal enzymes of C2-metabolism is repressed by methanol in the ecr1 mutant of Pichia pinus with the affected repression of alcohol oxidase by ethanol. Data are presented on the regulatory properties of the recently discovered acidification system of the medium induced by methanol. Such acidification occurs due to symport extrusion of protons and formate anions from the cells.

Biotechnology↗
PubMed1984

[Multiple forms of horse pepsin].

Using ion-exchange and affinity chromatography and isoelectrofocusing, eight forms of pepsin with pI 1.6, 1.8, 2.1, 2.3, 2.6, 2.8, 3.2 and 3.6, were isolated from horse gastric juice. The molecular weights, amino acid composition, N-terminal sequence and functional activity of these multiple forms were determined. Partial primary structure of tryptic peptides of pepsin with pI 2.3 was investigated. The analyzed partial sequences of the forms with pI 1.8, 2.1, 2.3, and 2.6 have identical structures which differ from the amino acid sequence of pepsin with pI 3.2 by four substituents. In terms of their functional activity, horse pepsins differ only insignificantly. Presumably, the pepsins under study (at least the forms with pI 1.8, 2.1, 2.3, 2.6 and 3.2) arose comparatively recently as a result of duplication of the common precursor gene and exist at an early stage of structural and functional divergence. As far as their primary structure and functional properties are concerned, these pepsins are more related to pepsin A than to other isoenzymes of gastric aspartyl proteinases of mammalia, e. g., gastricsin or chymosin.

Amino Acid Sequence↗
PubMed1976

[Biospecific chromatography of chymosin].

Chromatography of commercial rennet was studied on biospecific sorbents obtained by means of coupling of activated Sepharose 4B with epsilon-aminocapronyl-D-phenylalanine methyl ester and amide, epsilon-aminocapronyl-L-phenylalanyl-D-phenylalanine methyl ester, gramicidin S and N-2,4-dinitrophenylhexamethylenediamine. A mixture of two similar on their specificity enzymes chymosin and bovine pepsin was isolated from rennet by the chromatography on these sorbents. The individual enzymes might be isolated by chromatography on immobilized ribonuclease at pH 3,0, or by means of electrofocusing in pH gradient 4-6. Coloured inhibitor of acid proteases, N-diazoacetyl-N'-2,4-dinitrophenyl-ethylenediamine (DDE) is found to inactivate chymosin at pH 5,6 in the presence of Cu2+,one residue of the inhibitor being attached to the enzyme molecule. Unlike pig pepsin, chymosin is not inhibited with DDE at pH 4,7 and at the enzyme:DDE:Cu2+ ratio being 1:40:40. a synthesis of peptide sorbents is described.

Chromatography↗
PubMed1976

[Equine pepsins].

6 forms of pepsin are found in horse gastric juice. Amino acid sequence is determined of N-terminal (most variable) part of polypeptide chain of main pepsin components. Equine pepsines, which have pI 2.1 and 2.3, are found to have identical amino acid sequence at least for 31 amino acid residues. The same sequence is observed in the component with pI 2.6 for 10 first residues. The sequence of equine pepsin with pI 3.2 has 3 substitutions for 33 amino acids, when compared with pepsines having pI 2.1 and 2.3. The forms of equine pepsin studied are more similar than the other isoenzyme pair, human pepsin and gastricsin.

Amino Acid Sequence↗