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Biomedical subjects

J Chaloupka

Publications and source records attributed to J Chaloupka.

At least 37 records · Page 2Linked to original sources
PubMed1993

Turnover of canavanine-containing proteins in Saccharomyces cerevisiae.

Saccharomyces cerevisiae grown for 2 h in the presence of 0.5 mmol/L canavanine in a synthetic medium with ethanol as the sole carbon source (OEC) exhibited a slowing down of protein synthesis for 3-4 h after a shift to fresh ethanol-based medium containing 1.0 mmol/L arginine (OEA) in comparison with untreated cells grown on OEA. The change of carbon source from ethanol to glucose (OGA) after growth in the OEC medium resulted in an even deeper decline of protein synthesis. The degradation of canavanine-containing proteins in cells pregrown and labelled in an OEC medium after transfer to OEA was more rapid than in the OGA medium. The initial rate of protein degradation during the first hour in the OGA medium was less than 1%/h whereas in the OEA medium it reached almost 10%/h. The fraction of proteins with high turnover (half-life 0.46 h) constituted 8.3% on OEA, while during subsequent growth on OGA it was only 0.75% with a half-life of 0.12 h.

Canavanine↗
PubMed1993

Netropsin inhibits the increase of intracellular Ca(2+)-dependent serine proteinase activity in sporulating Bacillus megaterium.

Netropsin suppressed the increase of intracellular proteolytic activity when added to B. megaterium incubated in a sporulation medium. The inhibited enzyme was a Ca(2+)-dependent serine proteinase. Sporulation and protein turnover in later sporulation phases were inhibited as well. Different concentrations of netropsin affected various aspects of protein catabolism differently.

Bacillus megaterium↗
PubMed1993

Sporulation and synthesis of extracellular proteinases in Bacillus subtilis are more temperature-sensitive than growth.

Bacillus subtilis 115 grew in a medium with amino acids and glucose with the maximum specific growth rates mu of 1.20-1.10/h in the temperature range of 45-48 degrees C. Activity of the extracellular neutral proteinase excreted by 1.3 mg/mL dry mass during 8 h of the postexponential and stationary growth phases decreased from its maximum value of 0.23 TU/mL at 40 degrees C to 0.13 and 0.06 TU/mL at 45 and 48 degrees C, respectively. Formation of the extracellular serine proteinase decreased even more - from 0.18 TU/mL at 40 degrees C to 0.06 and 0.03 TU/mL at 45 and 48 degrees C, respectively. Sporulation, expressed as the portion of sporangia with refractile spores at the 6th h of the stationary phase decreased from 46% at 40 degrees C to 17 and 3% at 45 and 48 degrees C, respectively.

Bacillus subtilis↗
PubMed1993

Heat shock applied early in sporulation affects heat resistance of Bacillus megaterium spores.

Cells of Bacillus megaterium 27 were challenged by a 30-min heat shock at 45 degrees C during various sporulation stages and then shifted back to a temperature permissive for sporulation (27 degrees C), at which they developed spores. Heat shock applied at 120 min after the end of the exponential phase induced synthesis of heat shock proteins (HSPs) in the sporangia and delayed the inactivation of spores at 85 degrees C. Several HSPs, mainly HSP 70, could be detected in the cytoplasm of these spores. An analogous HSP, the main HSP induced by increased temperature during growth, belongs to the GroEL group according to its N-terminal sequence. The identity of this protein was confirmed by Western blot (immunoblot) analysis with polyclonal antibodies against B. subtilis GroEL. Sporangia treated by heat shock immediately or 240 min after exponential phase also synthesized HSPs, but none of them could be detected in the spores in an appreciable amount. These spores showed only a slightly increased heat resistance.

Amino Acid Sequence↗
PubMed1990

Effect of actinomycin D on viability, sporulation and nucleotide pool of Bacillus megaterium.

A transient 7-fold rise of ppGpp concentration, 2-3-fold increase of pppGpp concentration and 50% drop of the concentration of GTP in Bacillus megaterium cells immediately after their transfer to the sporulation medium were observed. Actinomycin D, in concentrations inhibiting RNA synthesis by 95%, blocked the rise of the (p)ppGpp pool and caused an instant several-fold increase of the GTP level. When the cells were exposed to actinomycin D in the sporulation medium for a 1-h period (time 0-1 h, 1-2 h or 2.20-3.20-h), they were able to form colonies on nutrient agar after being kept, in addition for 1-2 h in the sporulation medium free of the antibiotic. The ability of sporulation was, however, markedly limited. The share of cells that could sporulate increased when the irreversible sporulation phase was reached.

Adenosine Triphosphate↗
PubMed1990

Characteristics of intracellular proteolytic activities of Bacillus megaterium.

Intracellular proteolytic activities of B. megaterium KM occur soluble in the cytoplasm and periplasm and insoluble in the membrane. Two proteolytic enzymes were found in the cytoplasmic fraction by gel filtration on Sephadex G 150 and by polyacrylamide gel electrophoresis. The first enzyme called CI was stable, had a relative molecular mass of Mr = 105,000 (M = 105 kg/mol) and was inhibited by EDTA and PMSF, whereas the second, designated CII, was labile and had a relative molecular mass of Mr = 46,000 (M = 46 kg/mol). Because of its lability it could not be characterized in detail. In the "periplasm" only a single proteolytic enzyme P (Mr = 28,000; M = 28 kg/mol) inhibited by EDTA could be demonstrated. The extracellular enzyme exhibited similar properties. The membrane proteolytic activity was sensitive to PMSF and EDTA. The membrane enzymes have not yet been solubilized. In cells of the mutant KM 12 that does not produce the extracellular proteinase, only one type of proteinase, in all its properties identical with the cytoplasmic proteinase CI, could be demonstrated.

Bacillus megaterium↗
PubMed1989

Regulation of extracellular proteins and alpha-amylase secretion by temperature in Bacillus subtilis.

alpha-Amylase was found to be the main protein secreted by Bacillus subtilis, corresponding to 90, 87 and 60% of total extracellular proteins at 30, 40 and 45 degrees C, respectively. A change in temperature can affect the pattern of proteins secreted as detected by gel electrophoresis. 14C-Leucine incorporation into extracellular proteins and their proportion at the end of the growth phase was higher at 30 degrees C than that at 40 or 45 degrees C. The effect of temperature on alpha-amylase synthesis as determined by its enzymic activity and on the extracellular protein synthesis followed a similar pattern.

Bacillus subtilis↗
PubMed1988

Netropsin increases formation of mRNA coding for a neutral metalloproteinase in Bacillus megaterium.

The anticancer drug netropsin increases the synthesis of an exocellular metalloproteinase during exponential growth as well as in the stationary phase of a sporulating strain of Bacillus megaterium. Its effect is due to a stimulation of the synthesis of the mRNA coding for the proteinase, determined as a residual synthesis of the enzyme in the presence of actinomycin D. The half-life of the proteinase mRNA (5-6 min at 35 degrees C) is not affected by netropsin. Netropsin relieves partially the repression of the proteinase mRNA caused by amino acids, whereas the repression brought about by an increased temperature is almost unaffected by the drug.

Amino Acids↗
PubMed1988

Effect of temperature on alpha-amylase formation and DNA replication in Bacillus subtilis.

The effect of temperature on extracellular alpha-amylase synthesis and chromosomal and plasmid DNA replication in Bacillus subtilis A18 carrying plasmid pMI10 was studied. The specific growth rate mu increased with elevated temperature up to 42.5 degrees C, while the activities of alpha-amylase per population dry mass decreased. No obvious quantitative changes of 14C-thymidine incorporation per dry mass increase and no basic differences in plasmid copy number in the range of temperatures from 25 to 40 degrees C were found.

Bacillus subtilis↗
PubMed1986

Turnover of abnormal proteins in Bacillus megaterium and Saccharomyces cerevisiae: differences between in vivo and in vitro degradation.

Degradation of abnormal proteins in Bacillus megaterium and Saccharomyces cerevisiae in vivo was compared with that in cell-free extracts. Protein degradation in vivo, when the cells were labelled with 14C-leucine during growth in the presence of ethionine, was affected by the concentration of the analogue used. Proteins synthesized in the presence of 0.2-1 mM ethionine were degraded most rapidly in both organisms. The proteolytic enzyme system of yeast degraded the analogue-containing proteins in vitro faster than the normal proteins. This holds also for proteins synthesized in the presence of 5 mM ethionine, whose degradation in vivo was impaired. The proteolytic system of B. megaterium, on the other hand, was unable in vitro to differentiate between normal and abnormal proteins. Denatured proteins underwent preferential degradation over normal and ethionine-containing proteins.

Bacillus megaterium↗
PubMed1986

Interference with sporulation can stimulate the rate of a proteinase synthesis in Bacillus megaterium.

Bacillus megaterium, in which sporulation was blocked either by mutation or with netropsin, synthesizes during the stationary phase more exocellular proteinase than the sporulating culture. The asporogenic mutant synthesizes the enzyme at a higher rate and for a longer time than does the sporulating population. The culture, whose sporulation was inhibited by netropsin, produces the proteinase at a higher rate, although for only a limited time interval.

Bacillus megaterium↗
PubMed1985

Temperature as a factor regulating the synthesis of microbial enzymes.

Temperature affects the formation of several enzymes permanently during long-term growth (e.g. penicillinases, proteases, respiratory enzymes) or transiently immediately after a heat or cold shock (RNA polymerase, aminoacyl-t-RNA synthetases, ribonucleases and others). The synthesis of developmentally regulated enzymes may be suppressed by temperatures permissive for growth but not for differentiation (e.g. sporulation). The control of enzyme formation by temperature may be operating at the level of template multiplication (plasmid DNA), transcription, translation or formation of low-molecular-weight effectors.

Bacteria↗
PubMed1984

Mutants of Bacillus megaterium with altered synthesis of an exocellular neutral proteinase.

Germinated spores of Bacillus megaterium were mutagenized with ethyl methanesulphonate and spread on test agar with caseinate. Colonies with altered proteolytic zones or morphology were isolated and tested in liquid media. The mutants can be divided into four groups: A) those producing more proteinase in both growth and sporulation media, B) those producing the same amount of the enzyme in growth medium but higher amount in sporulation medium, C) those producing less proteinase in the growth medium and more in the sporulation one, D) those producing less or no enzyme. Clones of the first three groups were phenotypically asporogenic. All mutants producing more enzyme during growth retained their sensitivity to repression by amino acids. Isolation of mutants of types B) and C) supports the idea of differences in the control of proteinase synthesis during growth and during sporulation.

Bacillus megaterium↗
PubMed1984

Repression of the synthesis of exocellular and intracellular proteinases in Bacillus megaterium.

The synthesis of exocellular proteinase decreases with increasing concentration of amino acids in the growth medium. After removal of amino acids the enzyme synthesis is gradually restored to normal values. The presence of inhibitors of transcription (actinomycin D) or translation (chloramphenicol) blocks the restoration of enzyme synthesis. No active or inactive precursors of the exocellular enzyme could be detected in the cell. It is likely that the enzyme synthesis is regulated by amino acids at the level of specific mRNA synthesis rather than at the translation level or at the level of secretion. The activity of the enzyme that has already been secreted to the external medium is partially inhibited by amino acids. The periplasmic proteinase is repressed by amino acids to the same extent as the exocellular enzyme. The content of the enzyme(s) inside the protoplast is also decreased during growth in the presence of amino acids.

Amino Acids↗
PubMed1983

Suppression of an exocellular proteinase synthesis in Bacillus megaterium by increased temperature.

During cultivation of Bacillus megaterium at 42 degrees C the amount of the exocellular protease produced by growing cells sharply decreases as compared with temperatures of 28 and 35 degrees C. Within the above range the growth rate and incorporation of amino acids increase with increasing temperature. The culture adapted to 42 degrees C does not produce more proteinase at this temperature than the non-adapted culture. The high temperature does not induce accumulation of the enzyme in the cells. Total protein excretion was slightly lower at 42 degrees C than at 28 and 35 degrees C.

Bacillus megaterium↗
PubMed1983

Combined effect of temperature and nutrients on protein turnover in Bacillus megaterium.

Protein turnover was followed in populations of Bacillus megaterium growing in temperature range of 17-48 degrees C in different media. Higher temperature stimulated the protein turnover (expressed as the amount of protein degraded during 3.3 h) in all the media tested up to the optimal growth temperature (40-42 degrees C). Protein turnover in a medium containing amino acids continued to be stimulated by temperature even above this point; no further significant increase of turnover was found in the other media.

Amino Acids↗