Netropsin inhibits the increase of intracellular Ca2+-dependent serine proteinase activity in sporulatingBacillus megaterium

Netropsin suppressed the increase of intracellular proteolytic activity when added toB. megaterium incubated in a sporulation medium. The inhibited enzyme was a Ca²⁺-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.     

Netropsin suppresses development of intracellular serine proteinase activity during postexponential growth ofBacillus megaterium

The antibiotic netropsin decreased only slightly the exponential growth rate in a medium with mineral salts and glucose but its effect was increased during postexponential growth. Protein turnover and the basal intracellular serine proteinase (ISP) activity in the crude cytoplasmic fraction were not significantly affected by netropsin during exponential growth. However, ISP activity increased during postexponential growth and its rise was inhibited by the antibiotic. The population in the postexponential phase was not committed to sporulation.     

Intracellular serine proteinase behaves as a heat-stress protein in nongrowing but as a cold-stress protein in growing populations of Bacillus megaterium

A temperature increase from 35° to 40–42°C enhances the rise of cytoplasmic serine proteinase (ISP1) activity in Bacillus megaterium incubated in a sporulation medium. A temperature shift from 27°C in the growth medium to 35°C in the sporulation medium has the same effect. Elevated temperature stimulates the increase of ISP1 level when applied immediately after the transfer of cells from the growth to the sporulation medium (at T₀) or at T₃, when sporulation becomes irreversible. The cytoplasmic PMSF-resistant activity or the proteolytic activity associated with the membrane fraction is stimulated only slightly or not at all. A temperature increase to 45–47°C suppresses the rise of proteolytic activities in all cell fractions. In addition to the elevation of the ISP1 activity by an upward temperature shift, the rise of this enzyme in nongrowing cells is also stimulated by osmotic stress. In growing populations, in contrast to the rise of the ISP1 activity caused by elevated temperature in nongrowing cells, this proteinase is induced by low temperatures (24–27°C). The ISP1 activity roughly correlates with the enzyme protein concentration determined by immunoblotting.     

Dual effect of amino acids on the development of intracellular proteolytic activity in the irreversible sporulation phase ofBacillus megaterium

Amino acids added to a population ofBacillus megaterium immediately after its transfer to a sporulation medium stimulated growth, delayed sporulation by 1 h, and delayed the development of intracellular cytoplasmic serine proteinase (ISP) activity. However, the ISP activity in late sporulation stages exceeded twice that of the control population. Amino acids supplemented at T₃, i.e., at the time when engulfed forespores were developing, caused a decrease of specific ISP activity. The course of the phenylmethane sulfonyl fluoride (PMSF)-resistant activity in the cytoplasm was not affected by amino acids. Intracellular degradation of proteins prelabeled at the end of the growth phase was decreased by amino acids during the reversible sporulation phase but was only slightly affected later.     

Dependence of intracellular proteolytic enzymes in growing and sporulating cells ofBacillus megaterium on Ca2+ concentration

Intracellular proteolytic activities of growing and sporulating cells ofBacillus megaterium were compared after their separation by size-exclusion high-performance liquid chromatography. In both types of cells four soluble proteolytic activities, PI, PII, PIII and E, were found withM of more than 300, about 62, 36, and 20 kDa, respectively. PI was probably a complex of enzyme(s) or an enzyme associated with some structure. It was activated by Ca²⁺ but not by Mg²⁺ and stabilized by both divalent cations. PII, a major proteinase in both types of cells, was a serine enzyme with the same Ca²⁺ and Mg²⁺ dependence as PI. PIII was a divalent-cation-independent enzyme with a very low activity. E corresponded to the extracellular metalloenzyme produced by this organism. Possible regulation of proteolytic activity and protein degradationin vivo by Ca²⁺ during sporulation is discussed.     

Interference with sporulation can stimulate the rate of a proteinase synthesis inBacillus 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.     

Intracellular proteases during sporulation and enterotoxin formation byClostridium perfringens type A

Intracellular proteolytic activity was detected in cell-free extracts ofClostridium perfringens NCTC 10239 and NCTC 8798. The kinetics of protease, enterotoxin, and spore formation as well as growth of the wild type at elevated temperature and the use of sporulation mutants indicated that most protease activity was related to sporulation. Intracellular protease activity was inhibited by a mixture of tetrasodium ethylenediaminetetraacetic acid and phenylmethylsulfonyl fluoride; this indicated the presence of an alkaline serine protease and a neutral metallo-protease. Stage 0 sporulation mutants produced only metallo-sensitive proteases; this indicated that only the serine protease was sporulation-specific.     

Characterization of Proteinases Excreted by Bacillus subtilis Marburg Strain during Sporulation

S ummary . This study has characterized 3 proteolytic enzymes during sporulation by Bacillus subtilis Marburg strain when grown in nutrient broth. A method of purification is described which permits the separation of 2 different proteinases: one belonging to the metal enzyme group and the other to the serine enzyme group. The third enzyme, probably an esterase, showed a high esterolytic activity, but only low proteolytic activity. Determination of the 3 enzymes in a mixture was accomplished by using specific substrates and inhibitors. They were excreted simultaneously between the end of the growth phase until the appearance of the prespores. During this entire period, 20% of the total proteolytic activity was due to the metal proteinase; 80% of the proteolytic activity and 15% of the esterolytic activity was due to the serine proteinase; 85% of the esterolytic activity was the result of the esterase. These findings will contribute to a more complete phenotypic characterization of those mutants of sporulation that appear to be involved in the production of extracellular hydrolytic enzymes.     

Protein degradation during sporulation ofBacillus megaterium: Effect of actinomycin D

The first acceleration of protein degradation in cells ofBacillus megaterium was found at the stage 0–I of sporulation, the second one at the stage II–III, where the sporulation process became irreversible. These accelerations were reduced by actinomycin D inhibiting RNA and protein syntheses by more than 95%. In the presence of the antibiotic, only 8% of prelabeled proteins were degraded. Actinomycin D did not lower either the concentration of ATP or the proteolytic activity in the homogenate prepared from sporulating cells. This indicates that the inhibition of protein catabolism by actinomycin D was not owing to the absence of ATP or proteolytic enzymes. Actinomycin probably inhibited an unknown step preceding the proteolytic attack of the protein molecules during sporulation, because it had no significant effect on proteolysis during vegetative growth.     

Cell viability and protein turnover in nongrowingBacillus megaterium at sporulation suppressing temperature

InBacillus megaterium, a temperature that suppresses sporulation (43°C) only slightly exceeds both the optimum growth temperature and the temperature still permitting sporulation (40–41°C). Here we show that, when cells grown at 35°C and transferred to a sporulation medium, were subjected to shifts between 35°C and the sporulation suppressing temperature (SST, 43°C), their development and proteolytic activities were deeply affected. During the reversible sporulation phase that took place at 35°C for 2–3 h (T₂–T₃), the cells developed forespores and their protein turnover was characterized by degradation of short-lived proteins and proteins made accessible to the proteolytic attack because of starvation. During the following irreversible sporulation phase refractile heat-resistant spores appeared at T₄–T₅. Protein turnover rate increased again after T₂ and up to T₈ 60–70% prelabelled proteins were degraded. The SST suppressed sporulation at its beginning; at T₃ no asymmetric septa were observed and the amount of heat-resistant spores at T₈ was by 4–5 orders lower than at 35°C. However, the cells remained viable and were able to sporulate when transferred to a lower temperature. Protein degradation was increased up to T₃ but then its velocity sharply dropped and the amount of degraded protein at T₈ corresponded to slightly more than one-half of that found at 35°C. The cytoplasmic proteolytic activity was enhanced but the activity in the membrane fraction was decreased. When a temperature shift to SST was applied at the beginning of the irreversible sporulation phase (T_2.5), the sporulation process was impaired. A portion of forespores lyzed, the others were able to complete their development but most spores were not heat-resistant and their coats showed defects. Protein degradation increased again because an effective proteolytic system was developed during the reversible sporulation phase but the amount of degraded protein was slightly lower than at 35°C. A later (T₄) shift to SST had no effect on the sporulation process.     

Characteristics of intracellular proteolytic activities ofBacillus megaterium

Intracellular proteolytic activities ofB. 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 C_I was stable, had a relative molecular mass ofM _r=105000 (M=105 kg/mol) and was inhibited by EDTA and PMSF, whereas the second, designated C_II, was labile and had a relative molecular mass ofM _r=46000 (M=46 kg/mol). Because of its lability it could not be characterized in detail. In the “periplasm” only a single proteolytic enzyme P (M _r=28000;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 C_I, could be demonstrated.     

PURIFICATION AND CHARACTERIZATION OF A PROTEINASE FROM THE PROBIOTIC Lactobacillus rhamnosus OXY

A proteinase produced by the human gastrointestinal isolate Lactobacillus rhamnosus strain OXY was identified and characterized. The prtR2 gene coding for proteinase activity was detected in the examined strain. The PCR primers used were constructed on the basis of the sequence of the prtR2 proteinase gene from Lactobacillus rhamnosus GG. The enzyme was purified by fast protein liquid chromatography (FPLC) using CM-Sepharose Fast Flow and Sephacryl S-300 columns. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the enzyme had a relatively low molecular mass of 60 kD. Protease activity was observed at a pH range from 6.5 to 7.5 with optimum k _cat/K _m values at pH 7.0 and 40°C. Maximum proteolytic activity (59 U mL^?1) was achieved after 48 hr of cultivation. The activity of the enzyme was inhibited only by irreversible inhibitors specific for serine proteinases (PMSF and 3,4-dichloro-isocumarine), suggesting that the enzyme was a serine proteinase. Proteinase activity was increased by Ca²⁺ and Mg²⁺, and inhibited by Cu²⁺, Zn²⁺, Cd²⁺, and Fe^2+.     

Identification of a soluble enzyme from C3H/10T1/2 cells which is inhibited by the Bowman-Birk proteinase inhibitor

The anticarcinogenic Bowman-Birk proteinase inhibitor (BBI) inhibits a 70-kDa serine proteinase in C3H/10T1/2 transformed fibroblasts. Two serine proteinases, the proline endopeptidase and a novel neutral proteolytic activity, both having a mass of approximately 70-kDa, were isolated from the cytoplasm of C3H/10T1/2 cells. BBI did not inhibit diisopropylfluorophosphate binding to the proline endopeptidase or its ability to hydrolyze peptides. However, BBI blocked the binding of diisopropylfluorophosphate and inhibited the cleavage of peptides by the novel cytoplasmic enzyme. Thus BBI does not inhibit the proline endopeptidase but another soluble 70-kDa serine proteinase from C3H/10T1/2 cells.     

Opposite regulation by temperature of the intracellular Ca2+-dependent serine proteinase in growing and nongrowingBacillus megaterium

Specific activity of the cytoplasmic Ca²⁺-dependent serine proteinase (ISP1) in exponentially growing cultures decreased with increasing growth temperature. On the other hand, a temperature shift-up applied to a non-growing population incubated in a sporulation medium induced a rise of its activity. The ISP1 activity was assayed in the presence of 30 mmol/L CaCl₂ to release the enzyme inhibition and/or stimulate its processing. Immunoblotting applied to the 1-D SDS-PAGE electrophoretogram detected the ISP1 in growing cells mainly in bands withM of 41 and 38 kDa. The intensity of the latter decreased with increasing growth temperature. In nongrowing cells another intensively reacting band ofM 40 kDa appeared. In contrast to the commonly accepted opinion that starvation brings about a rise of the ISP1 synthesis or activation, its increase during incubation in sporulation medium was found only in cells pregrown at 35 and 42°C, where the enzyme activity in growing culture was low. No increase of the ISP1 specific activity in sporulation medium was detected in cells pregrown at 24 or 31°C, where the activity in growing cells was high.     

Intracellular proteolytic activity during sporulation ofBacillus megaterium

Intracellular proteolytic activity increased during incubation of the sporogenic strain ofBacillus 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 respeot 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 proteinsin vitro at a rate up to 150 μg mg_-1 h_-1 and native proteins at a rate up to 70 μg mg^-1 h^-1. Degradation of proteinsin vivo proceeded from the beginning of transfer to the sporulation medium at a constant rate of 40 μg mg^-1 h^-1 and the inactivation of beta-galactosidase at a rate of 70 μg 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 ofBacillus 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 ofBacillus megaterium KM synthesized the exocellular protease in the sporulation medium, but its protein turnover was found to decrease substantially after 3 – 4 h. The intraeellular proteolytic system of the sporogenic strain J and the asporogenic strain KM were also inhibited by EDTA and PMSF.     

Comparison of the multicatalytic proteinases isolated from the nucleus and cytoplasm of chicken red blood cells.

1. Two chromatographically distinct multicatalytic proteinases (MCP's) were isolated from the cytoplasm of chicken red blood cells and one MCP was purified from the nuclei. 2. The nuclear and the majority (97-99%) of the cytoplasmic multicatalytic proteolytic activity were chromatographically similar and differed from the minor cytoplasmic activity in their elution from hydroxylapatite, number of subunits on 2D-SDS-PAGE, and in their sensitivity to proteinase inhibitors. 3. Dichloroisocoumarin, a serine proteinase inhibitor, inhibited the hydrolysis of fluorogenic peptides but stimulated the degradation of casein by the multicatalytic proteinases suggesting that this enzyme has distinct active sites for protein and peptide hydrolysis.     

PURIFICATION AND CHARACTERIZATION OF A PROTEINASE FROM THE PROBIOTIC Lactobacillus rhamnosus OXY

A proteinase produced by the human gastrointestinal isolate Lactobacillus rhamnosus strain OXY was identified and characterized. The prtR2 gene coding for proteinase activity was detected in the examined strain. The PCR primers used were constructed on the basis of the sequence of the prtR2 proteinase gene from Lactobacillus rhamnosus GG. The enzyme was purified by fast protein liquid chromatography (FPLC) using CM-Sepharose Fast Flow and Sephacryl S-300 columns. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the enzyme had a relatively low molecular mass of 60 kD. Protease activity was observed at a pH range from 6.5 to 7.5 with optimum k (cat)/K (m) values at pH 7.0 and 40°C. Maximum proteolytic activity (59?U mL(-1)) was achieved after 48?hr of cultivation. The activity of the enzyme was inhibited only by irreversible inhibitors specific for serine proteinases (PMSF and 3,4-dichloro-isocumarine), suggesting that the enzyme was a serine proteinase. Proteinase activity was increased by Ca(2+) and Mg(2+), and inhibited by Cu(2+), Zn(2+), Cd(2+), and Fe(2+.).     

In Vivo and In Vitro Function of the Intracellular Proteolytic Apparatus in Nongrowing Bacillus megaterium Under Heat Stress

In Bacillus megaterium sporulating at 35°C, up to 90% of 10-min pulse-labeled proteins were degraded. Degradation proceeded in two waves. Short-lived proteins, i.e., intrinsically labile proteins and proteins made short-lived because of starvation, were mostly degraded during the reversible sporulation phase. Their amount corresponded to 20% or slightly more during 2 h. The second wave of protein degradation, which followed during the irreversible sporulation phase at 35°C, increased the amount of total degradable pulse-labeled proteins to about 90%. This wave was absent in the isogenic asporogenic mutant 27-36 or in the wild strain, whose sporulation was inhibited by increased temperature. The proportion of degradable proteins was thus reduced to less than 40% in the asporogenic mutant incubated at 35°C and to 46% in the wild strain whose sporulation was suppressed by the temperature of 47°C. Unlike sporulating cells, these cells were thus capable of degrading short-lived and denatured proteins, but were not able to degrade most of other proteins. The in vitro protein degradation was substantially enhanced by increasing the Ca²⁺ concentration, suggesting a role of Ca²⁺-dependent proteinase(s) in the process. Received: 23 July 1998 / Accepted: 19 August 1998     

Protease activities of rumen protozoa.

Intact, metabolically active rumen protozoa prepared by gravity sedimentation and washing in a mineral solution at 10 to 15 degrees C had comparatively low proteolytic activity on azocasein and low endogenous proteolytic activity. Protozoa washed in 0.1 M potassium phosphate buffer (pH 6.8) at 4 degrees C and stored on ice autolysed when they were warmed to 39 degrees C. They also exhibited low proteolytic activity on azocasein, but they had a high endogenous proteolytic activity with a pH optimum of 5.8. The endogenous proteolytic activity was inhibited by cysteine proteinase inhibitors, for example, iodoacetate (63.1%) and the aspartic proteinase inhibitor, pepstatin (43.9%). Inhibitors specific for serine proteinases and metalloproteinases were without effect. The serine and cysteine proteinase inhibitors of microbial origin, including antipain, chymostatin, and leupeptin, caused up to 67% inhibition of endogenous proteolysis. Hydrolysis of casein by protozoa autolysates was also inhibited by cysteine proteinase inhibitors. Some of the inhibitors decreased endogenous deamination, in particular, phosphoramidon, which had little inhibitory effect on proteolysis. Protozoal and bacterial preparations exhibited low hydrolytic activities on synthetic proteinase and carboxypeptidase substrates, although the protozoa had 10 to 78 times greater hydrolytic activity (per milligram of protein) than bacteria on the synthetic aminopeptidase substrates L-leucine-p-nitroanilide, L-leucine-beta-naphthylamide, and L-leucinamide. The aminopeptidase activity was partially inhibited by bestatin. It was concluded that cysteine proteinases and, to a lesser extent, aspartic proteinases are primarily responsible for proteolysis in autolysates of rumen protozoa. The protozoal autolysates had high aminopeptidase activity; low deaminase activity was observed on endogenous amino acids.     

Protease activities of rumen protozoa

Intact, metabolically active rumen protozoa prepared by gravity sedimentation and washing in a mineral solution at 10 to 15 degrees C had comparatively low proteolytic activity on azocasein and low endogenous proteolytic activity. Protozoa washed in 0.1 M potassium phosphate buffer (pH 6.8) at 4 degrees C and stored on ice autolysed when they were warmed to 39 degrees C. They also exhibited low proteolytic activity on azocasein, but they had a high endogenous proteolytic activity with a pH optimum of 5.8. The endogenous proteolytic activity was inhibited by cysteine proteinase inhibitors, for example, iodoacetate (63.1%) and the aspartic proteinase inhibitor, pepstatin (43.9%). Inhibitors specific for serine proteinases and metalloproteinases were without effect. The serine and cysteine proteinase inhibitors of microbial origin, including antipain, chymostatin, and leupeptin, caused up to 67% inhibition of endogenous proteolysis. Hydrolysis of casein by protozoa autolysates was also inhibited by cysteine proteinase inhibitors. Some of the inhibitors decreased endogenous deamination, in particular, phosphoramidon, which had little inhibitory effect on proteolysis. Protozoal and bacterial preparations exhibited low hydrolytic activities on synthetic proteinase and carboxypeptidase substrates, although the protozoa had 10 to 78 times greater hydrolytic activity (per milligram of protein) than bacteria on the synthetic aminopeptidase substrates L-leucine-p-nitroanilide, L-leucine-beta-naphthylamide, and L-leucinamide. The aminopeptidase activity was partially inhibited by bestatin. It was concluded that cysteine proteinases and, to a lesser extent, aspartic proteinases are primarily responsible for proteolysis in autolysates of rumen protozoa. The protozoal autolysates had high aminopeptidase activity; low deaminase activity was observed on endogenous amino acids.