Peaks Of Proteolytic Activity Research Articles

Production, partial characterization, and immobilization in alginate beads of an alkaline protease from a new thermophilic fungus Myceliophthora sp.

Thermophilic fungi produce thermostable enzymes which have a number of applications, mainly in biotechnological processes. In this work, we describe the characterization of a protease produced in solidstate (SSF) and submerged (SmF) fermentations by a newly isolated thermophilic fungus identified as a putative new species in the genus Myceliophthora. Enzyme-production rate was evaluated for both fermentation processes, and in SSF, using a medium composed of a mixture of wheat bran and casein, the proteolytic output was 4.5-fold larger than that obtained in SmF. Additionally, the peak of proteolytic activity was obtained after 3 days for SSF whereas for SmF it was after 4 days. The crude enzyme obtained by both SSF and SmF displayed similar optimum temperature at 50 degrees C, but the optimum pH shifted from 7 (SmF) to 9(SSF). The alkaline protease produced through solid-state fermentation (SSF), was immobilized on beads of calcium alginate, allowing comparative analyses of free and immobilized proteases to be carried out. It was observed that both optimum temperature and thermal stability of the immobilized enzyme were higher than for the free enzyme. Moreover, the immobilized enzyme showed considerable stability for up to 7 reuses.

HtrA2 is up‐regulated in the rat testis after experimental cryptorchidism

The aim of the present study was to elucidate the role of high temperature requirement A2 (HtrA2) in germ cell loss in the heat-stressed testis. We examined the expression of HtrA2, caspase-9 activity and proteolytic activity of HtrA2 in the rat testis, and their in vivo responses to experimental cryptorchid treatment. Northern analysis revealed the expression of HtrA2 mRNA peaked at days 1 and 7 after cryptorchid treatment. While expression of HtrA2 mRNA was seen in the spermatogonium, spermatocytes and some spermatids in normal adult rat testis, experimental cryptorchidism treatment resulted in a marked increase in its signal intensity in spermatocytes and some spermatids, and the layers of spermatogonium and early primary spermatocytes became negative at days 1 and 7 after the treatment. However, the spermatogonium, Sertoli cells and interstitial cells appeared to have strong intensities at days 14, 28 and 56 after the treatment. Western analysis revealed the expression of HtrA2 protein peaked at day 2 coinciding with the increase of positive spermatogonium, the appearance of protein-positive interstitial cells, and day 28 coinciding with the reappearance of protein-positive interstitial cells. Caspase-9 activity peaked at day 2 and HtrA2 proteolytic activity peaked at day 28. Consequently, the first peak of HtrA2 mRNA expression was followed by the peak of caspase-9 activity and the second peak was followed by the peak of proteolytic activity; however, the second peak of mRNA expression had considerable chronological difference from that of the protein. These findings suggest the probabilities that the heat stress results in germ cell death by a caspase-independent manner with the elevation of HtrA2 proteolytic activity, as well as a caspase-dependent manner with the elevation of caspase-9 activity.

Effect of ethylene producer ethrel and antioxidant ionol (BHT) on the proteolytic apparatus in coleoptiles of wheat seedlings during apoptosis.

It was established that total proteolytic activity in etiolated wheat seedlings changes in ontogenesis in cycles: peaks of proteolytic activity correspond to the 3rd, 5th, and 8th days of seedling growth, respectively. The maximum of proteolytic activity preceded the maximum of nuclease activity, which may be due to activation of nucleases by proteolytic enzymes. According to inhibitory analysis the cysteine and serine proteases play the main role in apoptosis in wheat coleoptiles. Growing of seedlings in the presence of ethrel stimulated apoptosis in the coleoptile, and it increased (almost 6-fold) the proteolytic activity in its cells. On the other hand, the antioxidant ionol (BHT) suppressed the induction of proteases, particularly at the second stage of coleoptile development, and it slowed down the increase in the nuclease activity after 6th day of the seedling life. It is suggested that phytohormones and antioxidants participate in regulation of apoptosis in the ageing coleoptile, directly or indirectly effecting the proteolytic apparatus in the coleoptile cells.

Degradation of insect cuticle by Paecilomyces farinosus proteases

The entomopathogenic fungus Paecilomyces farinosus showed proteolytic activity in both solid and semi-liquid culture with gelatin as sole N and C source. Semi-liquid cultures were used to characterise proteases. Zymography of crude culture filtrates showed several bands of gelatin degradation in electrophoresis gels. Gel filtration chromatography of these filtrates revealed two peaks of proteolytic activity. Ion-exchange absorption eliminated gelatin from culture filtrates while retaining activity and was used to semipurify P. farinosus proteases. Semipurified culture filtrates had basic pH (8.5 approx.) optimum for proteolytic activity. Treatment of these filtrates with effectors revealed that P. farinosus proteases are serine proteases containing sulphydryl groups. Isoelectrofocusing combined with zymography revealed the presence of several active basic isoforms. Larvae of the lepidopteran Galleria mellonella showed cuticle damage and protein release 1h after incubation with semipurified extracts of P. farinosus. These results indicate that proteolytic enzymes could be involved in insect host penetration by P. farinosus.

Recombinant cysteine proteinase can activate splenic T cells to produce IL-5 in Spirometra-infected mice

The somatic extract of L. intestinalis plerocercoids reveals hydrolytic activity against N-Benzoyl-l-tyrosine ethyl ester (BTEE) and Azocoll, and inactivates the esterolysis by mammalian trypsin and chymotripsin. The proteolytic enzyme activity and the inhibitory effect were completely separated by Sephadex G-100 column chromatography. Gel chromatography of the somatic extract revealed two peaks of proteolytic activity : one is bound to macromolecular substances, the other appears to be in free form and has a molecular weight of approx 60,000–65,000. The proteolytic activity showed the following characteristics : Tris-HCl buffer provided the highest activity against BTEE, the pH optimum was 7·4–7·8; the enzyme was activated by 10−5m-Ca2+, Mg2+ or Mn2+, it was inhibited by 10−5m-Cu2+, but not by 10−5m-Zn2+. 0.001% soybean trypsin inhibitor, 2 × 10−3m-EDTA, 1 mm-tosyl-l-phenylalanyl chloromethane, 1000 KIU/ml Trasylol did not inhibit the proteolytic activity, but it was inhibited by 1 mm-phenylmethyl-sulphonyl fluoride. The enzyme activity completely ceased upon 5 % TCA treatment or incubation at 56°C for 30 min. The trypsin and chyrnotrypsin inhibitor activities were eluted from the Sephadex G-100 column in a single peak with an estimated molecular weight of 6700–7200. The inhibitory effect was not sensitive to pH changes, and treatment by 5% TCA or incubation at 80°C for 15 min was ineffective. The proteolytic activity of plerocercoid extract was not effected ‘in vitro’ by the inhibitors isolated from this parasite.

Proteolytic activity in the fat body during the pupal-adult metamorphosis of the silkworm,Bombyx mori

Proteolytic activity with an optimum pH of 4.0 was found in fat bodies of 5 to 6 day-old female pupae of the silkworm, Bombyx mori, using azocoll as a synthetic substrate. The activity was strongly inhibited by p-chloromercuric benzoate at 0.5 mM, although inhibitory effect of several kinds of compounds on proteolytic activity was examined. Developmental changes in the activity were associated with two peaks of proteolytic activity related to the larval-pupal and pupal-adult transformation, the activity in the fat body of females was clearly higher than that of males. The profile of the proteolytic activity after chromatography on a column of Sephacryl S-300 showed that, in the female fat body, the highest activity was in the fraction with a molecular weight of about 232,000 daltons while, in male fat body, the highest activity was in the fraction with a molecular weight of about 105,000 daltons. The storage proteins were digested when extracts from fat bodies, which contain storage proteins and proteases, were incubated and only when the pH of the extract was adjusted to 4.0. In the case of female fat body, storage protein 1 was degraded earlier than storage protein 2. Furthermore, the administration of 20-hydroxyecdysone (5 μg/animal) to the isolated abdomens of female pupae (dauer-pupae) induced the proteolytic activity in the fat body in parallel with ovarian development. These results suggest that the proteases in female and male fat bodies are responsible for the utilization of storage proteins during pupal-adult development.

Starvation for Reduced Nitrogen Stimulates the Secretion of Proteases by Maize Endosperm Cultures

Summary Maize (Zea mays L.) endosperm cultures are normally grown in a medium containing nitrate plus asparagine. The endosperm cells do not grow unless supplied with a source of reduced nitrogen. It is possible to rescue nitrogen-starved cultures by adding bovine serum albumin (BSA) to the medium. Intact BSA rapidly disappears after addition to cultures previously incubated without reduced nitrogen for four days. Extracellular BSA-degrading activity is low in cultures grown with a complete medium but increased greatly in the medium of cells cultured without reduced nitrogen. Culture growth is not rescued by the addition of poly-L-asparagine or glycoproteins to the culture medium. Results obtained with activity gels suggest the secretion of two endoproteases from nitrogen-starved cultures. When culture filtrates were assayed in vitro at varying pH values, there were two peaks of proteolytic activity, one at 4.0 and the other at 5.5. By using functional group-directed reagents, it was shown that the pH 5.5 protease has a sulfhydryl-type mechanism while the pH 4.0 protease is a zinc-metalloenzyme.

Purification and properties of extracellular proteases produced by the nematophagous fungus Verticillium suchlasporium

The fungal parasite of eggs of cyst nematodes, Verticillium suchlasporium, produced extracellular proteases when grown in semiliquid culture with gelatin as the only source of nitrogen and carbon. The proteolytic activity of culture filtrates was maximum 12–14 days after inoculation. Gel filtration chromatography in Sephadex G-100 resolved two peaks of proteolytic activity. The peak accounting for most of the activity was further purified by ion-exchange chromatography in SP-Sephadex C-25 as a single peak. This protease had a molecular mass of 32 kDa calculated by sodium dodecyl sulfate – polyacrylamide gel electrophoresis. The enzyme was an endopeptidase that degraded fibrinogen in zymograms and had an optimum pH of 8.5 using fluorescein isothiocyanate – casein as the substrate. It was inhibited by phenylmethylsulfonyl fluoride, indicating that it was a serine protease. The purified protease was able to degrade certain cyst nematode proteins suggesting the involvement and specificity of the 32-kDa protease during nematode egg penetration by Verticillium suchlasporium. Key words: nematophagous fungi, Verticillium suchlasporium, extracellular enzymes, serine proteases.

Purification and characterization of a 27,000-Mr extracellular proteinase from Trichophyton rubrum.

A proteinase of Mr 27,000 with a possible role in the metabolism and invasion of host tissues was purified from the conditioned medium of Trichophyton rubrum by concanavalin A and anion-exchange chromatography. Peaks of proteolytic activity were analyzed by substrate gel electrophoresis. The 27,000-Mr proteinase had a pH optimum of 8.0, a calcium dependence of 2 mM, and was inhibited by serine proteinase inhibitors, especially phenylmethylsulfonyl fluoride and Phe-Gly-Ala-Leu-chloromethyl ketone. By polyacrylamide gel electrophoresis, the 27,000-Mr proteinase had a reduced molecular weight of 44,000 and reacted with [3H]diisopropyl fluorophosphate. The proteinase degraded azocoll, type III collagen, type IV procollagen, laminin, fibronectin, and the peptide substrates succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (1,573 M-1 s-1) and t-butyloxy carbonyl-Ala-Ala-Leu-p-nitroanilide (1,614 M-1 s-1).

A study on the identities of the three species of chromatin-associated proteinases in a mutant of Saccharomyces cerevisiae which lacks four major vacuolar proteinases.

Using mutant strain ABYS1 of Saccharomyces cerevisiae lacking four main vacuolar proteinases, proteinase A, proteinase B, carboxypeptidase Y, and carboxypeptidase S, we examined the identities of chromatin-associated proteinases, ruling out possible contamination of the chromatin fraction by them. The chromatin of strain ABYS1 showed three peaks of proteolytic activity at pH 4, 7, and 11, and these activities were found to be derived from three species of proteinases, the aspartic, serine neutral, and serine alkaline ones. As these chromatin-associated proteinases of strain ABYS1 were identical in both quality and quantity to those of wild-type strain of yeast, we suggest that the yeast chromatin contains three species of specific proteinases as essential components.

Detection and partial characterization of the chromatin-associated proteases of yeast Saccharomyces cerevisiae.

The chromatin fraction was prepared from yeast Saccharomyces cerevisiae free from cytoplasmic contamination except for a trace of mitochondria. When the yeast chromatin was incubated with histones as a substrate it showed three peaks of proteolytic activity as approximately pH 4, pH 7 and pH 11. These activities were separated from each other by differential extractions from chromatin and successive gel filtration through Sephadex G-100. Proteases were partially characterized by affinity labeling with [3H]diisopropylfluorophosphate (iPr2P-F) and by various protease inhibitors. The neutral and the alkaline proteases were serine proteases with a molecular mass of 35 kDa and 25 kDa respectively. The acidic protease showed a molecular size larger than 100 kDa on the gel filtration, and was probably an aspartyl protease because it was most strongly inhibited by pepstatin. A iPr2P-F-binding protein with a molecular mass of 66 kDa, found in chromatin, was likely to be converted to the alkaline protease of 25 kDa when chromatin was incubated at pH 10 or in 6 M urea/0.1 M phosphoric acid at the extraction. The distribution of proteolytic activities and iPr2P-F-binding proteins were compared among chromatins from different strains and from cells in different growth phases and it was found that these three proteases were present in all of them but with different proportions. Considering that rat liver chromatin contains equivalents to these proteases [Tsurugi, K. and Ogata, K. (1982) J. Biochem. (Tokyo) 92, 1369-1381], the results suggested that they play some important roles in the function of eukaryotic chromatin.

Nitrogen and phosphorus mobilisation in maturing/senescing wheat flag leaves

Abstract Investigations are reported on the metabolism and mobilisation of nitrogen and phosphorus by wheat flag leaves during a total lifespan of 8–9 weeks. Two separate trials are included, one with eight cultivars and a second with six cultivars, with and without late nitrogen. Parameters examined include activity of the enzyme proteinase, alkaline pyrophosphatase and nitrate reductase, as well as associated component changes such as α-amino nitrogen, protein-amino ratios, and soluble phosphate level. Because of the prime importance of post-anthesis leaf area duration in the production of grain yield, these parameters have been studied as potential and routinely assayable indicators of the onset and progress of senescence. Over the range of parameters there were several significant weekly (sampling), cultivar and treatment (late nitrogen) differences, and the significance of these in monitoring senescence is discussed. Particular attention is given to the relatively early release of simple metabolites (amino nitrogen and free phosphate) in incipient senescence, and to terminal mobilisation as manifest by a substantial late peak of proteolytic activity.

50] Proteases in Escherichia coli

Publisher Summary This chapter presents the procedure for purification and assaying of proteases in Escherichia coli. The purification procedure involves preparation of cell-free extract and fractionation of cell-free extract on DEAE-cellulose. For preparation of cell-free extract, freshly grown, or frozen E. coli K12 cells are suspended in buffer. The cell suspension is disrupted in a French press at 14,000 psi, the resulting homogenate is centrifuged at 31,000 g for 30 min, and the supernatant at 130,000 g for 3 hr. Following extensive dialysis against buffer B, the insoluble material is removed by centrifugation. In fractionation dialyzed cell-free extract is applied to a DEAEcellulose column equilibrated with buffer. The column is washed with the same buffer until the Azso of the eluate is less than 0.05. The adsorbed proteins are then eluted with 1500 ml of a linear salt gradient. Alternate fractions are assayed for proteolytic activity against [3H]casein and [125I]insulin in the presence and absence of 3 mM ATP. Different peaks of proteolytic activity against these substrates are found. For convenience, they are designated peaks I-V, according to their order of elution from the column. To obtain sufficient material for further purification, peaks from several DEAE-cellulose columns can be pooled. The following steps are described for enzyme preparations that would correspond to starting with 250-300 g of frozen cells. For various proteases in different peak individual chromatograpgy is carried out for respective proteases.

Ligula intestinalis (Cestoda: Pseudophyllidae): Studies on the properties of proteolytic and protease inhibitor activities of plerocercoid larvae

The somatic extract of L. intestinalis plerocercoids reveals hydrolytic activity against N-Benzoyl- l-tyrosine ethyl ester (BTEE) and Azocoll, and inactivates the esterolysis by mammalian trypsin and chymotripsin. The proteolytic enzyme activity and the inhibitory effect were completely separated by Sephadex G-100 column chromatography. Gel chromatography of the somatic extract revealed two peaks of proteolytic activity : one is bound to macromolecular substances, the other appears to be in free form and has a molecular weight of approx 60,000–65,000. The proteolytic activity showed the following characteristics : Tris-HCl buffer provided the highest activity against BTEE, the pH optimum was 7·4–7·8; the enzyme was activated by 10 −5 m-Ca 2+, Mg 2+ or Mn 2+, it was inhibited by 10 −5 m-Cu 2+, but not by 10 −5 m-Zn 2+. 0.001% soybean trypsin inhibitor, 2 × 10 −3 m-EDTA, 1 m m-tosyl- l-phenylalanyl chloromethane, 1000 KIU/ml Trasylol did not inhibit the proteolytic activity, but it was inhibited by 1 m m-phenylmethyl-sulphonyl fluoride. The enzyme activity completely ceased upon 5 % TCA treatment or incubation at 56°C for 30 min. The trypsin and chyrnotrypsin inhibitor activities were eluted from the Sephadex G-100 column in a single peak with an estimated molecular weight of 6700–7200. The inhibitory effect was not sensitive to pH changes, and treatment by 5% TCA or incubation at 80°C for 15 min was ineffective. The proteolytic activity of plerocercoid extract was not effected ‘ in vitro’ by the inhibitors isolated from this parasite.

Protein digestion in the sea gooseberryPleurobranchia bachei A. Agassiz (Ctenophora: Tentaculata)

Peaks of proteolytic activity of pharyngeal juice occur at pH 5.75 and pH 7.5. The proteases responsible for digestion include a tryptic alkaline protease and a thiol-activated acid protease which is probably cathepsin B. Levels of proteolytic activity parallel those of other carnivorous invertebrates which feed on zooplankton.

The pepsinogens of cat gastric mucosa and the pepsins derived from them.

Fundic mucosal extracts of the cat were examined by chromatography using DEAE-cellulose at pH 5.3. The peaks of proteolytic activity located by this method were shown to be heterogeneous on electrophoresis. By electrophoresis of individual homogenous chromatographic fractions 9 separate zones of proteolytic activity were detected. One zone did not migrate from the origin and is possibly a non-pepsinogen gastric protease. Simultaneous electrophoresis of pepsinogens and pepsins indicated that each pepsinogen gave rise to one pepsin. One zymogen gives rise to a pepsin active with the synthetic substrate acetyl-L-phenylalanyl-L-diiodotyrosine (APDT).

Esterases in human neutrophil granulocytes: evidence for their protease nature.

Summary. The nature of the esterases localized in granules of human neutrophil granulocytes has been investigated. The esterases of the granule fraction were fractionated first by selective solubilization of the granule proteins with 0.15 M‐NaCl and subsequently by gel filtration of the dissolved proteins on Sephadex G‐75. The chymotrypsin‐like activity of the granule fractions was insoluble in 0.15 M‐NaCI. Part of the granule esterase activity is known to be due to this chymotrypsin‐like enzyme. The soluble esterase activity of the granule fraction could be attributed to proteases as well. Two peaks of proteolytic activity on casein at pH 8.0 were obtained by gel filtration. The peak eluted first from the Sephadex G‐75 column contained two esterase proteins with a molecular weight of about 70000. The second protease peak contained the elastase‐like activity in addition to other proteases. A molecular weight of about 33000 was estimated for these proteins. Acrylamidegel electrophoresis of this fraction revealed a pattern of esterase bands. No esterase bands were demonstrable on the gel electrophoretograms given by fractions without proteolytic activity.