Purification and Characterization of Serine Proteinase 2 from Bacillus intermedius 3-19

A proteinase secreted in the late stationary phase was isolated from the culture fluid of Bacillus intermedius 3-19 by ion-exchange chromatography on CM-cellulose followed by FPLC on a Mono S column. The enzyme was completely inhibited by the serine proteinase inhibitors diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride. The maximum proteolytic activity against the synthetic chromogenic substrate Z-Ala-Ala-Leu-pNA was observed at pH 9.0. The molecular weight of the enzyme is 28 kD and its isoelectric point is 9.2. We have also determined pH- and thermostability and Km and kcat of this proteinase. The enzyme has been classified as a thiol-dependent serine proteinase. N-Terminal amino acid sequence (10 residues) and amino acid composition of the protein were also determined. By the mode of hydrolysis of peptide bonds in the oxidized B-chain of insulin, this enzyme is similar to the thiol-dependent serine proteinase 1 from B. intermedius 3-19 secreted during vegetative growth.     

Thiol-dependent serine proteinase from <Emphasis Type="Italic">Paecilomyces lilacinus</Emphasis>: Purification and catalytic properties

An extracellular thiol-dependent serine proteinase was isolated from culture medium filtrate of the microscopic fungus Paecilomyces lilacinus with a yield of 33%. The enzyme is inactivated by specific inhibitors of serine proteinases, phenylmethylsulfonyl fluoride, as well as by chloromercuribenzoate and mercury acetate, but is resistant to chelating agents. The proteinase has broad specificity, hydrolyzes proteins and p-nitroanilides of N-acylated tripeptides, exhibiting maximal activity in hydrolysis of substrates containing long hydrophobic and aromatic residues (norleucine, leucine, phenylalanine) as well as arginine at the P1 position. The enzyme has a molecular weight of 33 kD. The enzyme is most active at pH 10.0-11.5; it is thermostable and is characterized by broad optimum temperature range (30-60 degrees C), displaying about 25% of maximal activity at 0 degrees C. The N-terminal sequence of the enzyme (Gly-Ala-Thr-Thr-Gln-Gly-Ala-Thr-Gly/Ile-Xxx-Gly) has no distinct homology with known primary structures of serine proteinases from fungi and bacilli. Based on its physicochemical and enzymatic properties, the serine proteinase from P. lilacinus can be classified as a thiol-dependent subtilisin-like enzyme.     

Effect of Staphylococcus aureus serine proteinase on human lymphocyte impairment

Assessment of lymphocyte surface membrane integrity by means of trypan exclusion test and longer term survival studies based on 3HTdR uptake and Con A stimulation test were used to estimate effect of Staphylococcus aureus serine proteinase on human lymphocytes in vitro. At concentration of proteinase as high as 10 micrograms/ml, the percentage of trypan-stained lymphocytes increase with enzyme level. In contrast, decrease of 3HTdR level and Con A stimulation is already apparent at concentrations of proteinase ten times lower. Autologous serum exerts strong protective effect. Observed impairment is discussed as significant for local response in inflammation.     

Thiol-dependent serine proteinase from Streptomyces thermovulgaris

The cultural filtrates of S. thermovulgaris contain a proteinase which is active towards the chromogenic subtilisin substrate, Z-Ala-Ala-Leu-pNa, and azocasein. Pure enzyme preparations were obtained by affinity chromatography on bacitracin-Sepharose with subsequent rechromatography on the same adsorbent. The proteinase was completely inactivated by PMSF and DFP, the specific inhibitors for serine proteinase, by thiol reagents (HgCl2, PCMB) and by the protein inhibitor from S. jantinus. The pH activity optimum for the enzyme is 7.8-8.2, temperature optimum is 55 degrees C. The enzyme is stable at pH 6-9, has a pI of 5.0 and a molecular mass of 32 kDa. When tested against the peptide substrate, the enzyme shows a specificity characteristic for subtilisins. The N-terminal sequence of the enzyme, Tyr-Thr-Pro-Asn-Asp-Pro-Tyr-Phe-Ser-Ser-Arg-Gln-Tyr-Gly, shows a 100% homology with that of terminase, a thiol-dependent serine proteinase. On the basis of the above considerations the enzyme may be related to the subfamily of thiol-dependent serine proteinases.     

Serine proteinase from the higher basidiomycetes of Coprinus genus

A thiol-dependent serine proteinase has been isolated for the first time from a higher basidiomycete Coprinus 7N culture filtrate by affinity chromatography on bacitracin-Sepharose combined with ion-exchange chromatography on DEAE-Sepharose. This procedure resulted in a homogeneous enzyme with 32-fold purification and 55% yield. The enzyme has a molecular mass of 33,000 Da and pI of 8.5; its amino acid composition appears as follows: Lys7, His7, Arg10, Asx29, Thr24, Ser30, Glx19, Pro13, Gly39, Ala40, Cys2-3, Val23, Met1-2, Ile14, Leu13, Tyr6, Phe7. The enzyme shows the optimal activity towards Z-Ala-Ala-Leu-pNA at 8.5 and is stable at pH 6-9. The temperature optimum of the enzyme activity lies at 37 degrees C. The proteinase is completely inactivated by the specific inhibitors of serine proteinases, diisopropylfluorophosphate and phenylmethylsulfonylfluoride, as well as by the SH-group reagent, p-chloromercuribenzoate. The Coprinus 7N proteinase hydrolyzes, azocasein, azoalbumin, hemoglobin, fibrin and synthetic chromogenic peptide substrates, e. g., Z-Ala-Ala-Leu-pNA, Z-Gly-gly-Leu-pNA. Some properties of the Coprinus 7N proteinase are very similar to those of thiol-dependent serine proteinases from bacilli, actinomycetes, fungi and plants which form a subfamily of thiol-dependent serine proteinases within the family of subtilisins.     

New functions for an old enzyme: nonhemostatic roles for tissue-type plasminogen activator in the central nervous system

Tissue-type plasminogen activator (tPA) is a highly specific serine proteinase that activates the zymogen plasminogen to the broad-specificity proteinase plasmin. Tissue-type plasminogen activator is found not only in the blood, where its primary function is as a thrombolytic enzyme, but also in the central nervous system (CNS), where it promotes events associated with synaptic plasticity and acts as a regulator of the permeability of the neurovascular unit. Tissue-type plasminogen activator has also been associated with pathological events in the CNS such as cerebral ischemia and seizures. Neuroserpin is an inhibitory serpin that reacts preferentially with tPA and is located in regions of the brain where either tPA message or tPA protein are also found, indicating that neuroserpin is the selective inhibitor of tPA in the CNS. There is a growing body of evidence demonstrating the participation of tPA in a number of physiological and pathological events in the CNS, as well as the role of neuroserpin as the natural regulator of tPA's activity in these processes. This review will focus on nonhemostatic roles of tPA in the CNS with emphasis on its newly described function as a regulator of permeability of the neurovascular unit and on the regulatory role of neuroserpin in these events.     

Activation of purified cytoplasmic 17 beta-hydroxysteroid dehydrogenase from human placenta by human albumin and globulin

The cytoplasmic 17 beta-hydroxysteroid dehydrogenase of human placenta, purified more than 2500-fold, was activated by small amounts of human albumin and globulin. This activation was dependent on substrate concentration. At 20 microM estradiol (10 X KM) and two different concentrations of enzyme (0.01 and 2 micrograms/ml), the activation was greatest at albumin or globulin concentrations between 0 and 30 micrograms/ml. At "low" concentrations of estradiol (20 nM = 10(-2) X KM) and enzyme (0.01 microgram/ml), maximal activity occurred at approximately 10 micrograms/ml. Higher concentrations of albumin and globulin led to a decline in activity.     

Purification to homogeneity of protein kinase C from bovine brain--identity with the phorbol ester receptor

The calcium- and phospholipid-dependent kinase activity (protein kinase C) was isolated from bovine brains by a combination of DEAE-cellulose chromatography, gel filtration and hydrophobic chromatography on octyl-Sepharose and phenyl-Sepharose. The phorbol ester receptor co-purifies with the protein kinase C throughout the procedure yielding a homogeneous protein of 79 500 daltons on SDS-polyacrylamide gels. The purified kinase incorporated approximately 5000 nmol phosphate into substrate/min/mg protein at saturating concentrations of Ca2+ and phosphatidyl serine. Reciprocal plots of protein kinase activity at varying phosphatidyl serine concentrations were biphasic and yielded two apparent Ka values for phosphatidyl serine of 0.6-2 and 35-80 micrograms/ml). These apparent Ka values were reduced 2- to 3-fold by either diolein (20 micrograms/ml) or phorbol-12,13-dibutyrate (10 micrograms/ml). The protein binds [3H]phorbol-12,13-dibutyrate ( [3H]PDB) with high affinity (Ka = 15 nM) in a phosphatidyl serine-dependent manner. At saturating phosphatidyl serine concentrations 0.89 mol [3H]PDB are bound per mol protein. The identification of protein kinase C as the phorbol ester receptor is discussed with respect to the function and regulation of this protein.     

Hepatocyte growth factor plasma levels after myocardial infarction are not affected by recombinant tissue-type plasminogen-activator therapy

Hepatocyte growth factor (HGF), a cytokine involved in tissue regeneration, angiogenesis and lateral vessel growth, is secreted as a biological-inactive, single-chain precursor named pro-HGF. In case, of tissue injury pro-HGF is proteolytically cleaved at the extracellular locus by serine proteases. Results obtained from in vitro experiments showed that urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) can cleave single-chain HGF. In this study we measured serum HGF levels in patients with acute myocardial infarction (MCI). Two groups of patients were compared. One group (n = 7) was treated with a conventional therapy and the other group (n = 7) was subjected to a thrombolytic therapy with recombinant tissue-type plasminogen activator (rtPA). Serum samples were collected at time of admission and subsequently 12-16 hours, 20-30 hours and 50-60 hours after onset of chest pain. At admission and before administration of rtPA, serum HGF levels peaked at 16.8 +/- 2.2 ng/ml in the lysed group and at 20.7 +/- 6.5 ng/ml in the non-lysed group. Levels then continuously declined, reaching lowest values 50-60 hours after onset of chest pain (3.2 +/- 1.3 ng/ml in the group treated with rtPA versus 4.4 +/- 0.9 ng/ml in the non-lysed group). No statistical significant difference could be detected between the two groups at any time. We suggest that serine proteases other than tPA are involved in HGF activation in vivo.     

Characterization of two extracellular proteinases from Aspergillus terreus and their role in the formation of low molecular weight endoglucanases

Two extracellular proteolytic activities from the wood degrading fungus Aspergillus terreus have been characterized. Proteinase I (serine thiol-dependent enzyme) was active over a broad pH range (7·0–10·0) and at 55°C. The second proteinase (metalloproteinase) showed optimal activity at pH 6·0–7·0 and at 65–70°C. Both proteins had isoelectric points at acid pH and contained carbohydrate moieties. The metalloproteinase possessed a uniquely high content of serine and threonine and an extremely low percentage of glutamate and aspartate. The metalloproteinase was involved in the formation of the low molecular mass endoglucanases of A. terreus.     

Plasmin degradation of cartilage proteoglycan

Employing agarose gel electrophoresis, physiological concentrations of plasmin have been shown to degrade purified proteoglycan monomers and aggregates isolated from bovine articular cartilage. Proteoglycan degradation was (1) proportional to plasmin concentration, (2) dependent on the conversion of plasminogen to plasmin by plasminogen activator, (3) not displayed by plasminogen or plasminogen activator alone, and (4) inhibited by a serine proteinase inhibitor. These results, coupled with other findings, provide further support for a possible role of plasmin/plasminogen activator in cartilage destruction associated with rheumatoid arthritis.     

Mechanism of Na+/K(+)-ATPase activation by trypsin and kallikrein

The mechanism of the Na+/K(+)-ATPase activation by trypsin (from bovine pancreas) and kallikrein (from human plasma) was investigated on enzyme preparations from different sources (beef heart and dog kidney) and at different degrees of purification (beef heart). Kallikrein was effective on both beef and dog enzymes, whereas trypsin stimulated only the beef-heart Na+/K(+)-ATPase. The extent of activation by the proteinases was inversely related to the degree of purification (maximal enzyme activation about 60 and 20% on the partially purified and the more purified enzymes, respectively). Enzyme activation was observed up to 0.5-0.6 microgram/ml of proteinase. At higher concentrations the activation decreased and was converted into inhibition at proteinase concentrations above 1.0 micrograms/ml. Na+/K(+)-ATPase stimulation was due to an increase in the Vmax of the enzyme reaction. Km for ATP remained unaffected. The activating effect was favoured by sodium and counteracted by potassium. Accordingly, Na(+)-ATPase activity was stimulated to a greater extent (up to 350%), whereas K(+)-dependent p-nitrophenylphosphatase activity proved to be insensitive to the actions of the proteinases. The Na+/K(+)-ATPase stimulation by both proteinases was antagonized by either ouabain or canrenone, two drugs that bind on the extracellular side of the Na+/K(+)-ATPase molecule. On the contrary, the enzyme inactivation observed at high proteinase concentrations was not counteracted by these two drugs. The stimulation of either Na+/K(+)- or Na(+)-ATPase activity was shown to be an irreversible effect without any significant protein degradation detectable by SDS gel electrophoresis. The results obtained suggest that proteinases exert their stimulatory effects by interacting preferentially with the E2 conformation of Na+/K(+)-ATPase at site(s) located on the extracellular moiety of the enzyme.     

Molecular evolution and domain structure of plasminogen-related growth factors (HGF/SF and HGF1/MSP).

Plasminogen-related growth factors, a new family of polypeptide growth factors with the basic domain organization and mechanism of activation of the blood proteinase plasminogen, include hepatocyte growth factor/scatter factor (HGF/SF), a potent effector of the growth, movement, and differentiation of epithelia and endothelia, and hepatocyte growth factor-like/macrophage stimulating protein (HGF1/MSP), an effector of macrophage chemotaxis and phagocytosis. Phylogeny of the serine proteinase domains and analysis of intron-exon boundaries and kringle sequences indicate that HGF/SF, HGF1/MSP, plasminogen, and apolipoprotein (a) have evolved from a common ancestral gene that consisted of an N-terminal domain corresponding to plasminogen activation peptide (PAP), 3 copies of the kringle domain, and a serine proteinase domain. Models of the N domains of HGF/SF, HGF1/MSP, and plasminogen, characterized by the presence of 4 conserved Cys residues forming a loop in a loop, have been modeled based on disulfide-bond constraints. There is a distinct pattern of charged and hydrophobic residues in the helix-strand-helix motif proposed for the PAP domain of HGF/SF; these may be important for receptor interaction. Three-dimensional structures of the 4 kringle and the serine proteinase domains of HGF/SF were constructed by comparative modeling using the suite of programs COMPOSER and were energy minimized. Docking of a lysine analogue indicates a putative lysine-binding pocket within kringle 2 (and possibly another in kringle 4). The models suggest a mechanism for the formation of a noncovalent HGF/SF homodimer that may be responsible for the activation of the Met receptor. These data provide evidence for the divergent evolution and structural similarity of plasminogen, HGF/SF, and HGF1/MSP, and highlight a new strategy for growth factor evolution, namely the adaptation of a proteolytic enzyme to a role in receptor activation.     

Plasmin degradation of cartilage proteoglycan

Employing agarose gel electrophoresis, physiological concentrations of plasmin have been shown to degrade purified proteoglycan monomers and aggregates isolated from bovine articular cartilage. Proteoglycan degradation was (1) proportional to plasmin concentration, (2) dependent on the conversion of plasminogen to plasmin by plasminogen activator, (3) not displayed by plasminogen activator alone, and (4) inhibited by a serine proteinase inhibitor. These results, coupled with other findings, provide further support for a possible role of plasmin/plasminogen activator in cartilage destruction associated with rheumatoid arthritis.     

Esterase A is a proteinase from rat urine that can activate plasminogen

A proteinase which can activate human, dog and rat plasminogen to plasmin has been isolated from the urine of female rats, using affinity chromatography on benzamidine-coupled Sepharose. Inhibition by diisopropylfluorophosphate, tosyl-L-lysine chloromethylketone and benzamidine classified the enzyme as trypsin-like. The proteinase has weak activity on alpha-casein and hemoglobin, but will not lyse fibrin clots. It readily cleaves arginyl amides, including synthetic substrates specific for human glandular kallikrein and other serine proteinases. A chromogenic substrate for human urokinase (pyro Glu-Gly-Arg-pNA) is a poor substrate for the rat proteinase. Characteristics of the enzyme, such as its molecular weight (25 900), kinetic parameters and inhibition by aprotinin, indicate that this proteinase is esterase A, described by several investigators. Esterase A is shown not to be a true urinary plasminogen activator but rather is a unique arginine-specific proteinase. Urokinase-like and kallikrein-like activity are part of a broader proteolytic activity displayed by this enzyme.     

Purification and characterization of a proteinase from Euphausia superba Dana (Antarctic krill)

The thiol-dependent serine proteinase (inhibited by DFP, PMSF, pCMB and iodoacetate) was isolated from the whole krill specimens and from the content of the krill digestive tract. The enzyme was purified to homogeneity using a seven-step procedure. Its specific activity with denatured haemoglobin as a substrate was about 6.0 unit/mg. The molecular weight of the enzyme, as determined by gel exclusion chromatography was 33 000 and by polyacrylamide gel electrophoresis with SDS 31 600 (12.5% gel) and 27 000 (7.5% gel). The enzyme is an acidic glycoprotein (pI below 2.9) containing about 5% of carbohydrate. The pH optimum of the enzyme with haemoglobin was 6.0 at the optimal temperature of 40 degrees C in 15-min reaction. The enzyme showed the esterase activity (hydrolysis of BAEE) and was inactive with carbobenzoxy- and benzoyl-dipeptides with the following C-terminal amino acids: Phe, Tyr, Lys, Gly and Leu.     

Purification of plasminogen activator from Rous sarcoma virus-infected chick embryo fibroblast culture medium

A new procedure for the purification of plasminogen activator secreted by cultured Rous sarcoma virus-infected chick embryo fibroblasts was described. The enzyme was isolated from culture medium containing 0.75% calf serum depleted of plasminogen by lysine-agarose affinity column chromatography and of high-molecular-weight protease inhibitors by ultracentrifugation. The culture conditions allowed convenient preparation of large amounts of culture fluid with relatively high concentrations of plasminogen activator. The purification of the enzyme was accomplished by affinity chromatography on fibrin-celite and p-aminobenzamidine-agarose columns, and by gel-filtration chromatography in the presence of urea. The activity was recovered in greater than 90% yield, and the enzyme was essentially homogeneous when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Yields from 500 ml culture fluid exceeded 500 micrograms.     

A method for determining plasmin by the rate of fibrin gel lysis

A simple and sensitive method has been developed to assess the fibrinolytic activity of plasmin from the change in the column height of fibrin gel. Two conditions were used: 1) 37 degrees C and 16 h incubation at plasmin concentrations of 0.5-50 micrograms/ml and 2) 25 degrees C and 1-2.5 h incubation at plasmin concentrations of 50-1000 micrograms/ml. The method permits to observe the kinetics of fibrinolysis at plasmin concentrations higher that 10 micrograms/ml. The results have shown that the method is applicable for quantitation of plasminogen in human plasma. The method is precise and well reproducible.     

Subtilisin-like proteinase secreted by the <Emphasis Type="Italic">Bacillus pumilus</Emphasis> KMM 62 strain at different growth stages

Proteinase secreted in the environment by bacilli on different growth stages was isolated by ion chromatography from the culture medium of Bacillus pumilus KMM 62. According to the hydrolysis character of specific chromogenic substrates and inhibition type, the enzyme belongs to subtilisin-like serine proteinases. The isolated proteinase with the molecular mass of 30 kDa displays maximum activity on hydrolysis of the peptide substrate Z-Ala-Ala-Leu-pNA at pH 8.0–8.5 and temperature 30°C. The protein is stable in the range of pH 7.5–10.0. It was shown that subtilisin-like serine proteinase from B. pumilus KMM 62 possessed thrombolytic activity.     

Characterization and partial purification of an insulinase from Neurospora crassa.

An insulin-binding metal- and thiol-dependent proteinase has been purified 1491-fold from high speed cytosolic fractions of the fungus Neurospora crassa. This enzyme resembles insulin-degrading enzymes (insulinases) present in mammalian cells and in Drosophila melanogaster in the following ways: (i) it degrades radiolabeled insulin with a specificity similar to that of rat muscle insulinase, as demonstrated by HPLC analysis of the degradation products; (ii) it is inhibited by bacitracin, EDTA, 1,10-phenanthroline, and the sulfhydryl-reactive compounds N-ethylmaleimide and p-chloromercuribenzoate, but not by inhibitors of serine proteases or by lysosomal protease inhibitors. Cross-linking with 125I-insulin labels a band of ca. 120 kDa, and several smaller bands which may represent degradation products. The N. crassa insulinase is stimulated by Mn2+ and strongly inhibited by Zn2+; Mn2+ can also reactivate the enzyme after inhibition by EDTA, but Zn2+ is ineffective. The N. crassa protein differs in this regard from mammalian and insect insulinases which are generally activated by both Mn2+ and Zn2+. This finding extends the apparent evolutionary conservation of these metal- and thiol-dependent proteases into the microbial realm.