Extracellular alkaline proteinase of Colletotrichum gloeosporioides

The main proteinase of the filamentous fungus Colletotrichum gloeosporioides causing anthracnoses and serious problems for production and storage of agricultural products has molecular mass of 57 kD and was purified more than 200-fold to homogeneity with the yield of 5%. Maximal activity of the proteinase is at pH 9.0-10.0, and the enzyme is stable at pH 6.0-11.5 (residual activity not less than 70%). The studied enzyme completely kept its activity to 55 degrees C, with a temperature optimum of 45 degrees C. The purified C. gloeosporioides proteinase is stable at alkaline pH values, but rapidly loses its activity at pH values lower than 5.0. Addition of bovine serum albumin stabilizes the enzyme under acidic conditions. Data on inhibitor analysis and substrate specificity of the enzyme allow its classification as a serine proteinase of subtilisin family. It is demonstrated that the extracellular proteinase of C. gloeosporioides specifically effects plant cell wall proteins. It is proposed that the studied proteinase--via hydrolysis of cell wall--provides for penetration of the fungus into the tissues of the host plant.     

Grass group I pollen allergens (beta-expansins) lack proteinase activity and do not cause wall loosening via proteolysis.

Group I grass pollen allergens make up a subgroup of the beta-expansin family of cell wall loosening proteins in plants. A recent study reported that recombinant Phl p 1, the group I allergen from timothy grass pollen, was associated with papain-like proteinase activity and suggested that expansins loosen the plant cell wall via proteolysis. We tested this idea with three experimental approaches. First, we evaluated three purified native group I allergens from timothy grass, ryegrass and maize (Phl p 1, Lol p 1, Zea m 1) using five proteinase assays with a variety of substrates. The proteins had substantial wall loosening activity, but no detectable proteolytic activity. Thus we cannot confirm proteolytic activity in the pollen allergen class of beta-expansins. Second, we tested the ability of proteinases to induce cell wall extension in vitro. Tests included cysteine proteinases, serine proteinases, aspartic proteinases, metallo proteinases, and aggressive proteinase mixtures, none of which induced wall extension in vitro. Thus, wall proteins are unlikely to be important load-bearing components of the plant cell wall. Third, we tested the sensitivity of beta-expansin activity and native wall extension activity to proteinase inhibitors. The results show that a wide range of proteinase inhibitors (phenylmethanesulfonyl fluoride, N-ethylmaleimide, iodoacetic acid, Pefabloc SC, and others) inhibited neither activity. From these three sets of results we conclude proteolysis is not a likely mechanism of plant cell wall loosening and that the pollen allergen class of beta-expansins do not loosen cell walls via a proteolytic mechanism.     

Streptococcus thermophilus cell wall-anchored proteinase: release, purification, and biochemical and genetic characterization

Streptococcus thermophilus CNRZ 385 expresses a cell envelope proteinase (PrtS), which is characterized in the present work, both at the biochemical and genetic levels. Since PrtS is resistant to most classical methods of extraction from the cell envelopes, we developed a three-step process based on loosening of the cell wall by cultivation of the cells in the presence of glycine (20 mM), mechanical disruption (with alumina powder), and enzymatic treatment (lysozyme). The pure enzyme is a serine proteinase highly activated by Ca(2+) ions. Its activity was optimal at 37 degrees C and pH 7.5 with acetyl-Ala-Ala-Pro-Phe-paranitroanilide as substrate. The study of the hydrolysis of the chromogenic and casein substrates indicated that PrtS presented an intermediate specificity between the most divergent types of cell envelope proteinases from lactococci, known as the PI and PIII types. This result was confirmed by the sequence determination of the regions involved in substrate specificity, which were a mix between those of PI and PIII types, and also had unique residues. Sequence analysis of the PrtS encoding gene revealed that PrtS is a member of the subtilase family. It is a multidomain protein which is maturated and tightly anchored to the cell wall via a mechanism involving an LPXTG motif. PrtS bears similarities to cell envelope proteinases from pyogenic streptococci (C5a peptidase and cell surface proteinase) and lactic acid bacteria (PrtP, PrtH, and PrtB). The highest homologies were found with streptococcal proteinases which lack, as PrtS, one domain (the B domain) present in cell envelope proteinases from all other lactic acid bacteria.     

Digestive proteinases of larvae of the corn earworm, Heliothis zea: characterization, distribution, and dietary relationships.

Proteinases and peptidases from the intestinal tract of fifth-instar larvae of Heliothis (= Helicoverpa) zea (Boddie) (Lepidoptera:Noctuidae) were characterized based on their substrate specificity, tissue of origin, and pH optimum. Activity corresponding to trypsin, chymotrypsin, carboxypeptidases A and B, and leucine aminopeptidase was detected in regurgitated fluids, midgut contents, and midgut wall. High levels of proteinase activity were detected in whole midgut homogenates, with much lower levels being observed in foregut and salivary gland homogenates. In addition, enzyme levels were determined from midgut lumen contents, midgut wall homogenates, and regurgitated fluids. Proteinase activities were highest in the regurgitated fluids and midgut lumen contents, with the exception of leucine aminopeptidase activity, which was found primarily in the midgut wall. Larvae fed their natural diet of soybean leaves had digestive proteinase levels that were similar to those of larvae fed artificial diet. No major differences in midgut proteinase activity were detected between larvae reared under axenic or xenic conditions, indicating that the larvae are capable of digesting proteins in the absence of gut microorganisms. The effect of pH on the activity of each proteinase was studied. The pH optima for the major proteinases were determined to be pH 8.0-8.5 for trypsin, when tosyl-L-arginine methyl ester was used as the substrate; and pH 7.5-8.0 for chymotrypsin, when benzoyl-L-tyrosine ethyl ester was used as the substrate.     

Purification and characterization of the free form of the lactococcal extracellular proteinase and its autoproteolytic cleavage products

In addition to the cell wall proteinase, Lactococcus lactis subsp. cremoris produced significant amounts of a free extracellular proteinase. The free proteinase activity was highest in the late exponential and early stationary phase of growth, whereas the cell wall activity was highest in the last half of the exponential phase. Both proteinase forms had a pH optimum between 4-6 and 5-8, and they behaved similarly upon anion exchange and hydrophobic interaction chromatography, chromatofocusing and gel filtration, indicating that they were related. Purification to homogeneity, as judged by SDS-PAGE, resulted in a 50,000-80,000-fold increase in the specific activity of the free proteinase. It contained two major protein species (termed pro150 and pro115) with proteinase activity. As judged by SDS-PAGE, the Mr values of pro150 and pro115 were 150,000 and 115,000, respectively, and by chromatofocusing the isoelectric points were 4.3 and 4.1, respectively. Upon gel filtration, pro150 and pro115 had Mr values of 300,000 and 125,000, respectively, indicating that pro150 was a dimer and pro115 a monomer. Pro115 was an autodegradation product of pro150. Other distinct autodegradation products had Mr values of 90,000 (p90), 53,000 (p53), 37,000 (p37) and 30,000 (p30). These had little if any proteinase activity. Pro115, p90 and p53 had a common N-terminal sequence with that reported for the cell wall proteinase. Judging from its N-terminal sequence and Mr, p30 was derived from the C-terminal half of p53. Cleavage of pro150 to pro115 generated p37.     

Vascular smooth muscle cells efficiently activate a new proteinase cascade involving plasminogen and fibronectin

The plasminogen/plasmin system is involved in vascular wall remodeling after injury, through extracellular matrix (ECM) degradation and proteinase activation. Vascular smooth muscle cells (VSMCs) synthesize various components of the plasminogen/plasmin system. We investigated the conversion of plasminogen into plasmin in primary cultured rat VSMCs. VSMCs efficiently converted exogenous plasminogen into plasmin in a time- and dose-dependent manner. We measured plasmin activity by monitoring the hydrolysis of Tosyl-G-P-R-Mca, a fluorogenic substrate of plasmin. Cell-mediated plasmin activation was associated with the degradation of ECM, as revealed by fibronectin proteolysis. Plasmin also activated a proteinase able to hydrolyze Mca-P-L-G-L-Dpa-A-R-NH(2), a fluorogenic substrate of matrix metalloproteinases (MMPs). However, this proteinase was not inhibited by an MMP inhibitor. Furthermore, this proteinase displayed similar biochemical and pharmacological properties to fibronectin-proteinase, a recently identified zinc-dependent metalloproteinase located in the gelatin-binding domain of fibronectin. These results show that VSMCs convert exogenous plasminogen into plasmin in their pericellular environment. By hydrolyzing matrix protein plasmin activates a latent metalloproteinase that differs from MMP, fibronectin-proteinase. This metalloproteinase may participate to vascular wall remodeling, in concert with other proteinases.     

Substratum acidification and proteinase activation by murine B16F10 melanoma cultures

Murine B16F10 melanoma cells, cultured within 0.7% agarose gels containing the fluorescent proteinase substrate acetamidofluorescein-BSA, catalyze the hydrolysis of the substrate in the region immediately surrounding the cell. Fluorescence ratio measurements on hydrolyzed substrate correlate with an average pH of 5.5 ± 0.2 in the adjacent substratum region. Enzymatic activity within the gel is partially inhibited by leupeptin, pepstatin, phenylmethylsulfonyl fluoride, EDTA and by anti-human cathepsin B, suggesting potential roles for thiol-, aspartic- and metalloproteinases. The time-course of fluorescence intensity, correlated with substratum pH measurements, suggest that substrate hydrolysis is catalyzed by enzymes with pH optima of < 5.5. Invasion by these cells through thin barriers of reconstituted basement membrane gel (Matrigel) is totally blocked by the thiol proteinase inhibitor, leupeptin. It is suggested that secreted or cell-surface acid proteinase enzymes, activated by the cell-mediated local hyperacidity, are involved in substrate hydrolysis and that these enzymes may be important in invasiveness by this cell-line.     

Proteinase activity and agglutination of leukemia cells.

The proteinase activity was assayed in the leukemia cells L 1210 and in the ascites fluid with [3H] acetylated haemoglobin as a substrate. The proteinase activity at pH 4.1 increased in cells and in the ascites fluid with age of the tumor. The proteinase activity at pH 7.8 was low, but the enzyme activity in the cell homogenate increased between 5th and 7th day of the tumor growth and it was also present in the ascites fluid. It was observed that the leukemia cells aggregate in vivo and in vitro at pH values of the ascites fluid above pH 7.0. It was suggested, that the aggregation of leukemia cells is due to the tumor cell proteinase activity released to the ascites fluid.     

Enzymes secreted by filamentous fungi: regulation of secretion and purification of an extracellular protease of Trichoderma harzianum

Extracellular proteases secreted by the filamentous fungus Trichoderma harzianum have been identified. A proteinase active towards Z-Ala-Ala-Leu-pNa--the substrate of subtilisin-like proteases--dominated in the culture medium. This proteinase is synthesized de novo in response to addition of a protein substrate to the medium. Changing the carbohydrate in the culture medium changed the quantitative and qualitative spectrum of secreted enzymes. The most active extracellular proteinase of Trichoderma harzianum was purified 322-foldfrom the culture medium and obtained with a yield of 7.2%. The molecular mass of this proteinase is 73 kD and its pI is 5.35. The isolated enzyme has two distinct activity maxima, at pH 7.5 and 10.0, and is stable in the pH range 6.0-11.0. The temperature optimum for enzyme activity is 40 degrees C at pH 8. 0. The proteinase is stable up to 45-50 degrees C (depending on the substrate used). Calcium ions stabilized the enzyme at 55-60 degrees C. According to data on the study of functional groups of the active center and substrate specificity, the enzyme isolated from the culture medium of Trichoderma harzianum is a subtilisin-like serine proteinase.     

Isolation and characterization of the proteinase inhibitor-inducing factor from tomato leaves. Identity and activity of poly- and oligogalacturonide fragments

Mild acid hydrolysis of a small (Mr = 6 kDa) pectic polysaccharide isolated from tomato leaves, an inducer of the synthesis and accumulation of two proteinase inhibitors in excised tomato plants, yielded a alpha-D-polygalacturonic acid polymer with degree of polymerization = 20 that retained proteinase inhibitor-inducing activity. Enzymic and acid hydrolysis of this polygalacturonan yielded a series of alpha-1,4-D-galacturonic acid oligomers with degrees of polymerization from 2 to 6 which were purified to homogeneity and assayed for proteinase inhibitor-inducing activity in young excised tomato plants. All of the oligomers exhibited activity. The hexagalacturonide possessed the highest activity and the trimer the lowest. The evidence supports a possible role for plant cell wall fragments as systemic messengers that regulate the expression of proteinase inhibitor genes in plant leaves in response to pest attacks.     

Purification and characterization of proteinase In, a trypsin-like proteinase, in Escherichia coli.

We previously found a trypsin-like proteinase which momentarily appears immediately before DNA synthesis in the cell cycle of Escherichia coli synchronized by phosphate starvation and which is closely related to the initiation of DNA replication (Kato, M., Irisawa, T., Morimoto, Y. and Muramatu, M., unpublished results). The proteinase was named proteinase In. It was purified approximately 2880-fold with a recovery of 15%. The isolated enzyme appeared homogeneous by gel filtration and electrophoresis. Its molecular mass was estimated by analytical gel filtration and SDS/PAGE as approximately 66 kDa. The isoelectric point of proteinase In is 4.9 and its optimal pH is approximately 9. Although protein In hydrolyzes fluorogenic substrate for trypsin, its hydrolytic activity seems markedly affected by amino-acid sequence lying towards the N-terminal from the P1 (lysine, arginine) residue. The proteinase does not hydrolyze N2-benzoyl-D,L-arginine-4-nitronanilide and fluorogenic substrates for chymotrypsin and elastase. The proteinase activity is inhibited by leupeptin, antipain and 4-nitrophenyl 4-guanidinobenzoate, but the effects of tosyl-L-lysine chloromethane, diisopropylfluorophosphate, benzamidine and pentamidine isethionate on the proteinase activity are weak or not inhibitory. Its activity is strongly affected in the presence of NaCl and KCl, and at a concentration of 1.5 M, these increase the activity 14-fold and 13-fold, respectively, above that without salt. Proteinase In was strongly inhibited by various esters of trans-4-guanidinomethylcyclohexanecarboxylic acid, and their inhibitory effects were roughly correlated with those on growth of E. coli. Proteinase activity was found in the cytoplasmic fraction.     

Studies on a proteinase B mutant of yeast.

Yeast mutant lacking proteinase B activity have been isolated [Wolf, D. H. and Ehmann, C. (1978) FEBS Lett. 92, 121--124]. One of these mutants (HP232) is characterized in detail. Absence of the vacuolar localized enzyme is confirmed by checking for proteinase B activity in isolated mutant vacuoles. Defective proteinase B activity segregates 2:2 in meiotic tetrads. The mutation is shown to be recessive. Mutant proteinase B activity is not only absent against the synthetic substrate. Azocoll, but also against the physiological substrate pre-chitin synthetase, cytoplasmic malate dehydrogenase and fructose-1,6-bisphosphatase. The mutant shows normal vegetative growth, a phenomenon not consistent with the idea that proteinase B might be the activating principle of chitin synthetase zymogen in vivo. Fluorescence microscopy shows normal chitin insertion. Enzymes underlying carbon-catabolite inactivation in wild-type cells (a mechanism proposed to be possibly triggered by proteinase B) such as cytoplasmic malate dehydrogenase, fructose-1,6-bisphosphatase, phosphoenolpyruvate carboxykinase and isocitrate lyase, are inactivated also in the mutant. NADP-dependent glutamate dehydrogenase, which is found to be inactivated in glucose-starved wild-type cells, proceeds normally in the mutant. Mutant cells show more than 40% reduced protein degradation under starvation conditions. Sporulating diploids, homozygous for proteinase B absence, also exhibit an approximately 40% reduced protein degradation as compared to homozygous wild-type diploids or diploids heterozygous for the mutant gene. The time of the appearance of the first ascospores of diploid cells, homozygous for proteinase B deficiency, is delayed about 50% and sporulation frequency is reduced to about the same extent as compared to homozygous wild-type diploids or diploids heterozygous for the mutant gene.     

X-Prolyl-Dipeptidyl Aminopeptidase of Lactobacillus delbrueckii subsp. bulgaricus: Characterization of the Enzyme and Isolation of Deficient Mutants

Lactobacillus delbrueckii subsp. bulgaricus CNRZ 397 is able to hydrolyze X-proline-para-nitroanilides and X-proline-β-naphthylamides (X for alanyl- or glycyl-). A single metal-independent cytoplasmic enzyme with a molecular weight estimated to be 82,000 is responsible for these activities and was named X-prolyl-dipeptidyl aminopeptidase (X-Pro-DPAP). Isolation and analysis of mutants totally deficient for X-Pro-DPAP activity showed that a total lack of this enzyme induces (i) a decrease in the growth rate; (ii) an increase in cell wall proteinase activity; (iii) the loss of three cell wall proteins with respective molecular masses of 16, 40, and 52 kilodaltons; and (iv) enhancement of a cell wall protein with a molecular mass of 150 kilodaltons. The involvement of X-Pro-DPAP in casein catabolism is discussed.     

Various physico-chemical properties of lytic proteinase L2 of the enzyme preparation lysoamidase isolated from bacteria of Pseudomonadaceae family

Some physico-chemical properties of lytic proteinase L2 isolated from the enzymatic microbial preparation of lysoamidase were studied. The molecular mass of the enzyme is 15 000 Da, pI is 5.3. The enzyme hydrolyzes casein as well as the cells and cell walls of Staphylococcus aureus 209-P. The pH optimum of casein hydrolysis lies at 9.5; that for cell wall hydrolysis at 8.0. The temperature optimum for casein hydrolysis and cell lysis lies at 55 degrees C and 65 degrees C, respectively. The enzyme proteolytic activity is inhibited by serine proteinase inhibitors in a greater degree than the lytic activity. 50% of the proteolytic and lytic activities is lost upon enzyme heating for 15 min at 65 degrees C.     

Genetic Characterization of a Cell Envelope-Associated Proteinase from Lactobacillus helveticus CNRZ32

A cell envelope-associated proteinase gene (prtH) was identified in Lactobacillus helveticus CNRZ32. The prtH gene encodes a protein of 1,849 amino acids and with a predicted molecular mass of 204 kDa. The deduced amino acid sequence of the prtH product has significant identity (45%) to that of the lactococcal PrtP proteinases. Southern blot analysis indicates that prtH is not broadly distributed within L. helveticus. A prtH deletion mutant of CNRZ32 was constructed to evaluate the physiological role of PrtH. PrtH is not required for rapid growth or fast acid production in milk by CNRZ32. Cell surface proteinase activity and specificity were determined by hydrolysis of alpha(s1)-casein fragment 1-23 by whole cells. A comparison of CNRZ32 and its prtH deletion mutant indicates that CNRZ32 has at least two cell surface proteinases that differ in substrate specificity.     

Fusion expression of Escherichia coli prlC gene and preparation of PrlC proteinase affinity column

Escherichia coli PrlC is a trypsin-like proteinase regulating the cell cycle. The Escherichia coli prlC gene has been cloned into the pET28a prokaryotic expression vector. The recombinant fusion protein was produced mostly in the soluble, active form and the expression level amounted to approximately 70% of total protein. The recombinant proteinase was efficiently adsorbed to a resin containing immobilized Ni²⁺ via its amino terminal fusion hexahistidine tail to give a PrlC proteinase affinity column. The adsorbed fusion proteinase hydrolyzed 4-methylcoumaryl-7-amide of tert-butoxycarbonyl-l-valyl-l-prolyl-l-arginine (Boc-Val-Pro-Arg-NH-Mec), the specific substrate for the trypsin-like proteinase activity of E. coli PrlC.     

Carbohydrate utilization affects Lactobacillus delbrueckii subsp. lactis 313 cell-enveloped-associated proteinase production

The effect of different sugars (glucose, glycerol, maltose, galactose and lactose) on cell-membrane-associated proteinase production by Lactobacillus delbrueckii subsp. lactis 313 (LDL 313) was investigated. The experimental results showed that aside glycerol and galactose, all the other sugars supported high growth levels of LDL 313, with glucose displaying the maximum biomass concentration of 0.85 mg/mL dry cell weight for cells harvested at the mid-exponential phase of ??12 h after inoculation. The specific proteinase yield, a measure of the rate of proteinase production relative to cell wall biosynthesis, was used to evaluate the preferential degree of proteinase metabolism as induced by the consumption of different sugar substrates by LDL 313. It was found that maltose displayed the highest specific proteinase yield of 12.59 U/mg sugar consumed. Further, molecular differences were observed in the SDS electrophoretic profile of cell surface proteins generated for the different carbon substrates. This is a preliminary study which supports the inference that different sugars stimulate the production of different cell-surface proteins with a significant effect on cell proteinase activity.     

Transformation of rat liver cell line by Rous sarcoma virus causes loss of cell surface fibronectin, accompanied with secretion of metallo-proteinase that preferentially digests the fibronectin

An epithelial cell line derived from the liver of a normal Buffalo rat (BRL) was transformed by Rous sarcoma virus (RSV). The RSV-transformed cells were separated into five clones (RSV-BRL1 through 5), which were morphologically different. RSV-BRL cells exhibited the following characteristics distinct from those of BRL cells: tumorigenicity, irregular cell arrangement, loose intercellular junction, growth in soft agar (anchorage-independent growth) except for RSV-BRL3 and 5, and loss of cell surface fibronectin. When BRL cells were cultured in the standard medium supplemented with the serum-free conditioned medium of RSV-BRL cells, the amount of the cell surface fibronectin decreased significantly. It was found that RSV-BRL cells secreted a proteinase capable of hydrolyzing the fibronectin, whereas BRL cells secreted hardly any of this proteinase. The fibronectin-hydrolyzing proteinase (FNase) could also hydrolyze plasma fibronectin added as an exogenous substrate. The hydrolysis of plasma fibronectin was inhibited by ethylenediamine tetraacetate, but stimulated by rho-chloromercuribenzoate and calcium ion. This indicates that FNase is a metallo-enzyme, but not a serine or thiol enzyme. In addition to the proteinase, RSV-BRL cells secreted plasminogen activator and a proteinase inhibitor which inhibited the activity of plasmin but not FNase.     

Characterization of a Cell Envelope-Associated Proteinase Activity from Streptococcus thermophilus H-Strains

The production and biochemical properties of cell envelope-associated proteinases from two strains of Streptococcus thermophilus (strains CNRZ 385 and CNRZ 703) were compared. No significant difference in proteinase activity was found for strain CNRZ 385 when cells were grown in skim milk medium and M17 broth. Strain CNRZ 703 exhibited a threefold-higher proteinase activity when cells were grown in low-heat skim milk medium than when grown in M17 broth. Forty-one percent of the total activity of CNRZ 385 was localized on the cell wall. The optimum pH for enzymatic activity at 37°C was around 7.0. Serine proteinase inhibitors, such as phenylmethylsulfonyl fluoride and diisopropylfluorophosphate, inhibited the enzyme activity in both strains. The divalents cations Ca²⁺, Mg²⁺, and Mn²⁺ were activators, while Zn²⁺ and Cu²⁺ were inhibitors. β-Casein was hydrolyzed more rapidly than α_s1-casein. The results of DNA hybridization and immunoblot studies suggested that the S. thermophilus cell wall proteinase and the lactococcal proteinase are not closely related.     

Purification and characterization of serine proteinase from a halophilic bacterium, Filobacillus sp. RF2-5

In order to find a unique proteinase, proteinase-producing bacteria were screened from fish sauce in Thailand. An isolated moderately halophilic bacterium was classified and named Filobacillus sp. RF2-5. The molecular weight of the purified enzyme was estimated to be 49 kDa. The enzyme showed the highest activity at 60 degrees C and pH 10-11 under 10% NaCl, and was highly stable in the presence of about 25% NaCl. The activity was strongly inhibited by phenylmethane sulfonyl fluoride (PMSF), chymostatin, and alpha-microbial alkaline proteinase inhibitor (MAPI). Proteinase activity was activated about 2-fold and 2.5-fold by the addition of 5% and 15-25% NaCl respectively using Suc-Ala-Ala-Phe-pNA as a substrate. The N-terminal 15 amino acid sequence of the purified enzyme showed about 67% identity to that of serine proteinase from Bacillus subtilis 168 and Bacillus subtilis (natto). The proteinase was found to prefer Phe, Met, and Thr at the P1 position, and Ile at the P2 position of peptide substrates, respectively. This is the first serine proteinase with a moderately thermophilic, NaCl-stable, and NaCl-activatable, and that has a unique substrate specificity at the P2 position of substrates from moderately halophilic bacteria, Filobacillus sp.