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Biomedical subjects

J Chaloupka

Publications and source records attributed to J Chaloupka.

At least 55 records · Page 3Linked to original sources
PubMed1981

Synthesis of exocellular proteins during the exponential and stationary phase of growth of Bacillus megaterium.

Synthesis of exocellular metalloprotease and cellular and exocellular proteins in the sporogenic strain Bacillus megaterium J-27 and asporogenic strain KM 1 was investigated. Both organisms excrete the enzyme into the medium during growth and during the stationary phase. In the asporogenic strain the excretion decreases at the end of the exponential phase. In the sporogenic strain it continues during the transition to the stationary phase at the original rate and proteolytic activity in the medium increases two to three times during 2 h after the end of the exponential phase. Both organisms synthesize relatively more exocellular proteins during the exponential phase than during the stationary phase. The proportion of exocellular protein synthesized during the exponential phase does not exceed 3 % of total proteins, during the stationary phase this proportion usually decreases to less than 1 %.

Bacillus megaterium↗
PubMed1981

Protein turnover in growing cultures of Bacillus megaterium.

A growing population of Bacillus megaterium, prelabelled with 14C-amino acids displays protein turnover, the rate of which is influenced by the growth rate. The size of the pool of short-lived proteins, degraded with a half-life of less than 1 h, is directly proportional to the mass doubling time during subsequent growth of the population. However, their degradation constant is almost the same under all conditions. The degradation constant of residual long-lived proteins is influenced by the growth rate. The labile fraction can be replenished by a combination of shift-up and shift-down treatments. Tetracycline decreases the size of the labile (short-lived) fraction and almost stops the degradation of long-lived proteins. Correlation of protein turnover and intracellular proteolytic activity indicates that other factors in addition to the proteinase are necessary for the degradation of short-lived, as well as long-lived proteins.

Bacillus megaterium↗
PubMed1980

Turnover of proteins in asporogenic Bacillus megaterium. Evidence for a gradual decrease of the turnover rate.

The rate of protein turnover in asporogenic Bacillus megaterium decreases continuously during incubation in a sporulation medium. The capability of equilibration of external amino acids with amino acids in the metabolic pool of non-growing cells was retained for at least 5 h. Leucine, while repressing the synthesis of the exocellular protease, does not significantly influence the course of protein degradation in vivo. Transfer of non-growing cells after 4 h to a fresh sporulation medium does not influence the rate of protein degradation. The gradual decrease of the rate of protein turnover in non-growing cells of the asporogenic variant is thus not an artifact caused by a decreased uptake of amino acids by cells or by conditions under which the protein turnover is determined.

Amino Acids↗
PubMed1978

Functional half-life of the exocellular protease mRNA of Bacillus megaterium.

Functional half-life of the exocellular protease mRNA was determined in in exponentially growing and stationary cells of the asporogenic strain of Bacillus megaterium KM and in the sporogenic strain of B. megaterium 27 during sporulation. No reserve of the protease mRNA could be detected in the cells and the half-lives were determined to be 6--7 min in the exponential and stationary cells of B. megaterium KM and 7.5--8.5 min in B. megaterium 27. The mean half-life of mRNA for cell proteins was determined to be 3.5--4.5 min. Thus, as compared with the mean half-life of mRNA for cell proteins that of mRNA for the exocellular protease is slightly longer.

Bacillus megaterium↗
PubMed1977

Intracellular proteolytic activity during sporulation of Bacillus megaterium.

Intracellular proteolytic activity increased during incubation of the sporogenic strain of Bacillus megaterium KM in a sporulation medium together with excretion of an extracellular metalloprotease. The exocellular protease activity in a constant volume of the medium reached a 100-fold value with respect to the intracellular activity. Maximal values of the activity of both the extracellular and intracellular enzyme were reached after 3-5 h of incubation. After 7 h 20-50% cells formed refractile spores. The intracellular proteolytic system hydrolyzed denatured proteins in vitro at a rate up to 150 mug mg-1 h-1 and native proteins at a rate up to 70 mug mg-1 h-1. Degradation of proteins in vivo proceeded from the beginning of transfer to the sporulation medium at a constant rate of 40 mug mg-1 h-1 and the inactivation of beta-galactosidase at a rate of 70 mug mg-1 h-1. The intracellular proteolytic activity was inhibited to 65-88% by EDTA, to 23-76% by PMSF. Proteolysis of denatured proteins was inhibited both by EDTA and PMSF more pronouncedly than proteolysis of native proteins; 50-65% of the activity were localized in protoplasts. Another strain of Bacillus megaterium (J) characterized by a high (up to 90%) and synchronous sporulation activity was found to behave in a similar way, but the rate of protein turnover in this strain was almost twice as high. The asporogenic strain of Bacillus megaterium KM synthesized the exocellular protease in the sporulation medium, but its protein turnover was found to decrease substantially after 3-4 h. The intracellular proteolytic system of the sporogenic strain J and the asporogenic strain KM were also inhibited by EDTA and PMSF.

Bacillus megaterium↗
PubMed1977

Relationships between intracellular proteolytic activity and protein turnover in Bacillus megaterium.

When incubated in a sporulation medium, the sporogenous strains of Bacillus megaterium degrade proteins at a rate of 4-10% X h-1. The maximal rate of protein turnover is reached after 3-4 hrs at the time of development of forespores and then decreases again. The rate of protein turnover in the asporogenous strain decreases steadily under similar conditions from 3-8% X h-1 at the beginning of incubation to 1% X h-1 after 5-6 hrs in the sporulation medium. The rate of degradation of proteins in vitro in protoplast lysates is similar or higher than the rate of protein turnover. The exocellular, as well as periplasmic proteolytic activity, is suppressed by amino acids more severely than the activity in protoplasts. Mutants devoid of the exocellular proteolytic enzyme contain also less proteolytic activity in the periplasm than in the protoplasts, in contrast to the wild strain. However, their rate of protein turnover, as well as the degradation of abnormal proteins is similar to that in the wild strain. This supports a view that the proteolytic system in protoplasts is involved in intracellular protein catabolism. The periplasmic enzyme can be considered as a kind of the exocellular proteinase.

Bacillus megaterium↗
PubMed1976

Exocellular proteases of Serratia marcescens and their toxicity to larvae of Galleria mellonella.

Out of 18 strains of Serratia marcescens producing exocellular proteases the strain Serratia marcescens CCEB 415 was selected according to preliminary experiments. It could be shown that the train exhibits proteolytic activity reaching up to 10 TU per 1 ml of the culture filtrate in a medium with gelatine and peptone. Two proteolytic enzyme could be demonstrated by means of specific inhibitors EDTA and diisopropyfluorophosphate: metalprotease with optimum activity at pH 7.5 and serine protease with pH optimum of 10.9. The enzymes were purified on Sephadex and DEAE cellulose columns and by means of gel electrophoresis. However, it was not possible to separate them. The optimum temperature for activity of the mixture of the two enzymes was 50degrees C, molecular weight varied around 37000 (according to gel filtration); certain kinetic characteristics of their activity were determined. Excess subtrate (casein) inhibited activity of the enzyme mixture. Toxicity of proteases expressed as LD50 units equals 78 - 10(3) TU per larva of Galleria mellonella.

Animals↗
PubMed1976

The effect of substitution of diaminopimelic acid by 4-hydroxy-diaminopimelic acid on the synthesis and degradation of murein in Escherichia coli 173-25.

Defects in the formation of the septum and gradually autolysis of cells occur when the dap-dependent mutant of Escherichia coli is grown in a medium with 4-hydroxy-diaminopimelic acid. When the culture grown in the presence of the labelled analogue is supplemented with the non-radioactive diaminopimelic acid a portion of the TCA-soluble radioactivity is released from the cells during 20 min after the addition of diaminopimelic acid. During this time interval the elongated forms formed in the presence of the analogue divide, however, only on the condition that the above forms are not irreversibly damaged. The increased concentration of the analogue in the medium substantially suppresses the irregularities in the development of the septum as well as the degradation of analogue containing cell wall. However, the growth rate in the presence of the analogue is always slightly lower than that in the presence of diaminopimelic acid. The cell wall pulse-labelled with diaminopimelic acid or its analogue for a time interval shorter than 1/4 of the generation time exhibits the same or only slightly higher rate of diaminopimelic acid is probably utilized less effectively for the synthesis of murein than diaminopimelic acid. However, its incorporation into the wall does not result in pronounced damage of the cell.

Cell Division↗
PubMed1975

Protease activity in cells of Bacillus megaterium during derepression.

A proteolytic activity hydrolyzing denatured proteins of Bacillus megaterium labelled with 35S or 14C amino acids was detected in cells of the asporogenic strain of Bacillus megaterium. The substrate is hydrolyzed by the enzyme or enzymes at optimum pH around 7, their activity being almost completely inhibited by EDTA and o-phenanthroline. PMSF, the inhibitor of serine proteases, is slightly inhibitory. Gel filtration on a Sephadex column separated the protease activity to two or three fractions. The protease activity in cells with the repressed synthesis of protease corresponds to 5-20 mug of substrate degraded per hour by 1 mg of protein at 37 degrees C. It increases five to ten-fold during the derepression. When the intracellular protease activity increases the extracellular enzyme begins to be excreted into the medium. The intracellular protease activity rapidly decreases after the addition of chloramphenicol or of a mixture of amino acids to the derepressed culture. Half or even more of the protease activity is released from the cells during their conversion to protoplasts by means of lysozyme. This "periplasmic" activity remains mostly in the supernatant also after mesosomes have been centrifuged down from the periplasm. A portion of the activity bound in protoplasts sediments together with membrane fraction after their lysis.

Amino Acids↗