Purification and properties of a protease inhibitor from crayfish hemolymph

A protease inhibitor from the hemolymph of crayfish, Astacus astacus, has been purified by differential centrifugation, acid precipitation and preparative isoelectric focusing. The inhibitor was apparent homogenous in SDS-electrophoresis and had a molecular weight of 23,000. pI was determined to be 4.7 by isoelectric focusing. No inhibitory activity was lost when the inhibitor was incubated in a pH range of 1–11.5. The purified inhibitor was heat stable. Urea (6 m) had no effect upon the inhibitor. The inhibitor was active against subtilisin and a partly purified protease from the fungus Aphanomyces astaci. Pronase was slightly inhibited whereas trypsin, chymotrypsin, papain, Arthrobacter protease, and extracellular proteases from the fungi Aphanomyces stellatus and A. laevis were unaffected. The importance of protease inhibitors in pathogenesis between the parasitic fungus, A. astaci, and its crayfish host, A. astacus is discussed.     

Isolation and partial characterization of two alkaline proteases of the greater wax moth Galleria mellonella (L.)

Two alkaline proteases were isolated from whole-body extracts of Galleria mellonella larvae. The two proteases were separated by cation-exchange chromatography on CM-Sepharose CL6B and further purified by gel filtration on Ultrogel ACA 54. The optimal pH of activity using Azocoll as substrate was 10.5 for protease P-1 and 11.2 for protease P-2. The molecular weights of the two enzymes determined by gel filtration were respectively 12,500 and 10,500. Protease P-1 was inhibited by soybean trypsin inhibitor, TPCK, TLCK and activated by non-ionic detergents. Protease P-2 was inhibited by soybean trypsin inhibitor, 4-aminobenzamidine, ovomucoid and activated by dithiothreitol. Both enzymes were partially inhibited by PMSF.Distribution studies suggested that the two proteases were digestive enzymes.     

Proteases from the fungus Metarhizium anisopliae toxic for Galleria mellonella larvae

The high molecular fraction of the extract from Metarhizium anisopliae grown on wheat bran contains proteolytic enzymes which are toxic for Galleria mellonella larvae. The complex of proteases was fractionated using precipitation with ammonium sulfate, gel filtration, and electrofocusing. Two components have been found: one with the optimum of activity on hemoglobin at pH 6.5, and the second with the optimum around pH 9. The prevailing protease acting at pH 6.5 was inhibited by phenylmethylsulfonyl fluoride and the inhibition was followed by decrease of toxicity. The molecular weights of the enzymes are 35 × 10³ and 71 × 10³.     

The proteases and the protease inhibitor in the midgut of Leucophaea maderae

The caseinolytic enzymes of the midgut lumina and epithelia of Leucophaea were purified through precipitation by 60% saturated (NH₄)₂SO₄, followed by gel permeation on Sephadex G-200 and subsequent DEAE anionexchange chromatography. At least four peaks with enzyme activity were eluted from anionexchange chromatography columns. Gregarines of the midgut lumen apparently do not contribute to the caseinolytic activity within the midgut. Elution profiles of lumen and epithelial enzymes were nearly identical. The same enzymes were identified in the lumina of epithelial microsomal vesicles. This allows the conclusion that these enzymes are produced by the midgut epithelia.Practically all protease activity of the midgut was found in the posterior half, both in the lumen and epithelium. Feeding stimulated protease production primarily in the posterior midgut. The pH optimum of the proteases lay between 9.0 and 9.5 which was closely matched by the observed pH of the posterior midgut where most of the activity is seen. The anterior midgut pH was determined to be around 8.0.The anterior midgut of Leucophaea contained a heatstable protease inhibitor with characteristics of a competitive inhibitor. This inhibitor was precipitable by 60% saturated (NH₄)₂SO₄ and eluted from a Sephadex G-200 column more or less together with the proteases. From a DEAE anionexchange column it was eluted by 0.8 M NaCl, i.e. after the main portion of the proteases. The biological significance of the protease inhibitor in the anterior portion of the midgut is obscure.     

Isolation and biological activity of the proteases released by sea urchin eggs following fertilization.

The protease activity released from sea urchin egg cortical granules into the surrounding seawater at fertilization is involved in vitelline layer elevation and the block to polyspermy. The cortical granule protease components were isolated by isoelectric precipitation and affinity chromatography on p-aminobenzamidine-Sepharose columns. Elution profiles from affinity columns suggested heterogeneity of the proteases, and polyacrylamide-gel electrofocusing of affinity-purified preparations established the presence of two proteins. Dramatically different biological activities were resolved by affinity chromatography. Early-eluting fractions of low specific activity delaminated the vitelline layer from the egg plasma membrane; this activity is termed vitelline delaminase. Late-eluting fractions of high specific activity modified the egg vitelline layer surface such that sperm could not bind or fertilize them; this activity is referred to as sperm receptor hydrolase. The biological activities of the sea urchin proteases are apparently the result of limited action on the vitelline layer, unlike bovine trypsin which simply digests the vitelline layer. The cortical granule proteases lost biological specificity when stored at 0°C at pH 8.0. Esterase activity increased, and the preparation acquired the ability to digest the vitelline layer. Increase of the esterase activity in protease preparations was prevented by storage at low pH.The molecular weight of both enzymes was estimated by sucrose gradient centrifugation to be 47,000, whereas multiple components with molecular weights between 10⁵ and 10⁶ were demonstrated by gel filtration.     

Immunolocalization and characterization of two novel proteases in Leishmania donovani: Putative roles in host invasion and parasite development

Two novel intracellular proteases having identical molecular mass (58 kDa) were purified from virulent Indian strain of Leishmania donovani by a combination of aprotinin-agarose affinity chromatography, ion exchange chromatography and finally continuous elution electrophoresis. Both of these proteases migrate in SDS-PAGE as a single homogeneous bands suggesting monomeric nature of these proteases. The enzyme activity of one of the proteases was inhibited by serine protease inhibitor aprotinin and another one was inhibited by metalloprotease inhibitor 1, 10 phenanthroline. The purified enzymes were thus of serine protease (SP-Ld) and metalloprotease (MP-Ld) type. The optimal pH for protease activity is 8.0 and 7.5 for SP-Ld and MP-Ld respectively. The temperature optimum for SP-Ld is 28 °C and for MP-Ld is 37 °C showing their thermostability upto 60 °C. Broad substrate (both natural and synthetic) specificity and the effect of Ca²⁺ upon these enzymes suggested novelty of these proteases. Kinetic data indicate that SP-Ld is of trypsin like as BAPNA appears to be the best substrate and MP-Ld seems to be collagenase type as it degrades azocoll with maximum efficiency. Both immunofluorescence and immune-gold electron microscopy studies revealed that the SP-Ld is localized in the flagellar pocket as well as at the surface of the parasite, whereas MP-Ld is located extensively near the flagellar pocket region. This work also suggests that the uses of anti SP-Ld and anti MP-Ld antibodies are quite significant in interfering with the process of parasite invasion and multiplication respectively. Thus the major role of SP-Ld could be predicted in invasion process as it down regulates the phagocytic activity of macrophages, and MP-Ld appears to play important roles in parasitic development.     

Purification and characterization of an acidic trypsin/subtilisin inhibitor from tortoise egg white

Egg whites of three species of tortoise and turtle have been compared by gel chromatography for inhibitory activity against proteases. The egg white of Geomda trijuga trijuga Schariggar contains trypsin/subtilisin inhibitor while the egg white of Caretta caretta Linn. contains both trypsin and chymotrypsin inhibitors. No protease inhibitory activity has been detected in the egg white of Trionyx gangeticus Cuvier. An acidic trypsin/subtilisin inhibitor has been purified to homogeneity from the egg white of tortoise (G. trijuga trijuga). It is a single polypeptide chain of 100 amino acid residues, having a molecular weight of 11 700. It contains six disulphide bonds and is devoid of methionine and carbohydrate moiety. Its isoelectric point is at pH 5.95 and is stable at 100°C for 4 h at neutral pH. The inhibitor inhibits both trypsin and subtilisin by forming enzyme-inhibitor complexes at a molar ratio close to unity. Their dissociation contants are 7.2·10^?9 M for bovine trypsin adn 5.5·10^?7 M for subtilisin. Chemical modification of amino groups with trinitrobenzene sulfonate has reduced its inhibitory activities against both trypsin and subtilisin, but the loss of its trypsin inhibitory activity is faster than of its subtilisin inhibitory activity. It has independent binding sites for inhibition of trypsin and subtilisin.     

Purification and characterisation of a protease inhibitor from Streptomyces chromofuscus 34-1 with an antiviral activity

The aim of this study was to purify and characterise a novel protease inhibitor (PISC-2002) isolated from culture supernatants of Streptomyces chromofuscus. PISC-2002 was purified by anion-exchange chromatography, and RP-HPLC analysis. PISC-2002 had a molecular mass of 11.2 kDa and a high content of hydrophobic amino acids and proline. N-terminal sequence gave two sequences differing by one residue. The main sequence is ASLPAVSALVLTV and the shorter sequence is SLPAVSALVLTV. This shows its homology to Streptomyces subtilisin inhibitor family. Besides its large spectrum of powerful inhibitory activities against various serine proteases, PISC-2002 displayed significant antiviral effect against influenza virus A/Rostock/34 (H7N7).     

Purification and characterization of a protein inhibitor of calcium-dependent proteases from rat liver

Soluble extracts of rat liver contain a protein inhibitor of calcium-dependent proteases. The inhibitor has an apparent M_r = 250,000 and is separated from the calcium-dependent proteases by gel-filtration chromatography in the presence of EGTA. The inhibitor has been purified by affinity chromatography using a calcium-dependent protease covalently linked to Affi-Gel 15. The inhibitor specifically binds to this affinity resin in a calcium-dependent manner and elutes in the presence of EDTA or EGTA. The purified inhibitor appears as a single protein with M_r = 125,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Presumably it is a dimer under nondenaturing conditions. The inhibitor inhibits each of two calcium-dependent proteases from rat liver and from other tissues and species. However, it has no effect on any other protease tested.     

Potent platelet aggregation inhibitor from Trimeresurus gramineus snake venom

Using DEAE-Sephadex A-50 column chromatography and gel filtration, a potent platelet aggregation inhibitor from Trimeresurus gramineus venom was purified. It was an acidic phospholipase a, rich in aspartic acid, glutamic acid and half-cystine, with an isoelectric point of 3.6. At a concentration of 10 μg/ml, the purified inhibitor showed a marked inhibitory effect on platelet aggregations induced by adenosine diphosphate, collagen, sodium arachidonate and ionophore A-23187 in rabbit platelet-rich plasma, washed platelet suspension, as well as in thrombin-degranulated platelet suspension. The ID₅₀ of this venom inhibitor was about 2.5–5 μg/ml in platelet aggregations induced by all these aggregation inducers. The action of this inhibitor could be partially antagonized by phosphatidylethanolamine. High concentration of Ca²⁺ (5 mM) did not reverse the inhibitory action even in the presence of ionophore A-238187. The [¹⁴C]serotonin release induced by sodium arachidonate and thrombin was unaffected. Malonic dialdehyde formation induced by these aggregation inducers remained unchanged. Basal and prostaglandin E₁-stimulated cAMP levels were not altered by this inhibitor. No lactate dehydrogenase was released even at a concentration of 62.5 μg/ml. Polylysine-induced platelet agglutination was not affected. β-Mercaptoethanol inactivated both its phospholiase A enzymatic and platelet inhibitory activities, while p-bromophenacyl bromide only inactivated the former activity. The possibility of acting on a common final step of platelet aggregation, i.e. the intercellular adhesion between the activated platelets, was proposed.     

Detection and partial characterization of lymphoid cell surface proteases

Cultures of viable thymocytes and lymph node cells (LNC) were found to exhibit neutral protease activity toward radiolabeled protein substrates. Proteases were not actively secreted in serum-free culture. Thymocyte surface proteases were not affected by incubation of the cells in 1 mM ethylenediaminetetraacetic acid (EDTA) or 1 mM ethylene glycol bis(aminoethyl ether) N, N'-tetraacetic acid (EGTA); however, approximately 25% of lymph node cell surface protease activity was released from the cells by EDTA. It was concluded that the majority of protease activity displayed by both cell types was tightly associated with the cell surface. The inhibitor sensitivity of the cell surface proteases detected on hamster thymocytes and LNC and rat thymocytes was very similar. Cell surface protease activity was inhibited (85%) by the serine protease inhibitors diisopropylfluorophosphate (DFP) and phenylmethylsulfonylfluoride (PMSF) and was partially inhibited by l-1-tosylamide-2-phenylethylchloromethyl ketone(TPCK) and soybean trypsin inhibitor (SBTI), but not by N-α-p-tosyl-l-lysine-chloromethyl ketone (TLCK) or ?-aminocaproic acid (EACA). The bacterial protease inhibitor antipain was strongly inhibitory whereas leupeptin was less effective and elastinal did not inhibit cell surface protease activity. Thymocyte surface proteases were also inhibited (65%) by ZnCl₂, but not be several other divalent cations. In LNC, both ZnCl₂ and NiCl₂ were inhibitory to a lesser extent (32% inhibition). At least one surface protease in both thymocytes and LNC could function as a plasminogen activator.     

Inhibition of gut proteases and development of dengue vector, Aedes aegypti by Allium sativum protease inhibitor

The paper describes the bio efficacy of a protease inhibitor; isolated from Allium sativum ‘garlic’ (ASPI); against Aedes aegypti mosquito, a well-known transmitter of dengue and Chikungunya. The purification of protease inhibitor from Allium sativum ‘garlic’ (ASPI) was carried out by ammonium sulfate precipitation followed by Fast Protein Liquid Chromatography using akta DEAE-Cellulose column. The protein fraction demonstrating trypsin inhibitory activity was further evaluated for its insecticidal activity using gut protease inhibition assay and larvicidal assay. ASPI is an inhibitor of porcine trypsin (IC₅₀ of 650.726?μg/mL) and has molecular weight of ~15?kDa determined by SDS PAGE similar to other inhibitors of the Kunitz-type family (14–26?kDa). ASPI demonstrated 50% reduced activity of Ae. aegypti midgut proteases and showed a dose-dependent acute toxicity on Ae. aegypti 3rd instars exhibiting LC₅₀ value of ~50.827?μg/mL. After ten days of larval exposure ASPI resulted in a 24-h delay of larval development and ~72% mortality at 61.5?μg/mL. These results suggest that ASPI may serve as potent insecticidal agent and hence opens a new gateway in the field of phyto-remediation.     

Kunitz-type inhibitors in human serum. Identification and characterization

Human serum contains small amounts (approximately 0.1 mg/liter) of two protein protease inhibitors of low molecular weight (approximately 6500) and basic isoelectric point (Kunitz-type). They were purified by affinity chromatography on immobilized trypsin and ion-exchange chromatography in the fast protein liquid chromatography system. Their chemical, immunochemical, and functional properties indicate that the purified inhibitors are highly homologous with the basic pancreatic trypsin inhibitor which is widely distributed in bovids and caprids. Their inhibitory activity toward serine proteases such as plasmin and kallikrein suggests a possible regulatory role in blood clotting and fibrinolysis.     

Reversible inhibition of cell multiplication in vitro by inhibitors of arginine esteroproteases

Two classes of active-site specific inhibitors of trypsin-like proteases have been shown to inhibit reversibly the multiplication of eukaryotic cells in vitro. The competitive inhibitors p-aminobenzamidine and benzamidine were found to arrest the growth of normal and transformed mouse fibroblasts and human KB cells. The inhibition of cell multiplication occurred within 24 h and was accompanied by substantial decreases in the rates of DNA and protein synthesis. The rate of RNA synthesis was relatively unaffected by the protease inhibitors. In agreement with the known inhibition constants (K_i) for their action against trypsin, p-aminobenzamidine was a much more effective inhibitor of cell multiplication than benzamidine. In addition, tosyllysine chloromethyl ketone (Tos-LysCH₂Cl), an active-site titrant and irreversible inhibitor of trypsin, was found to cause a reversible inhibition of growth. These results suggest that an essential protease activity is necessary for cell multiplication. However, in the case of mouse L-cells, all of the inhibitors and particulary p-aminobenzamidine caused excessive accumulation of lactate in the extracellular medium. This observation, which suggests the possibility of additional sites of action of these compounds in cells, was found to depend upon the cell type and appears to be unrelated to the inhibition of growth.     

Alanine aminotransferase in the three last larval instars of Barathra brassicae infected by Nosema plodiae

During three last larval instars of Barathra brassicae the alanine aminotransferase activity pattern exhibits two maxima which do not correspond with the maxima of activity of phosphatases and proteases. In the larvae infected by Nosema plodiae a different course of activity can be seen from the 5th day of infection. The activity pattern in the diseased larvae rises above that of a normal larva by 90% shortly before the last molt and reaches a maximum at the end of normal larval development. Alanine aminotransferase in the gut and in the fat body exhibits two main pH optima of activity, 8.0 and 9.5, respectively, and a low activity peak at pH 4.5. This last activity is not present in diseased animals. The temperature and pH inactivation are also described.     

Isolation and characterization of a protease inhibitor from spinach leaves

An acid-stable and heat-labile proteinous protease inhibitor which was found in spinach leaves but not in seeds was isolated by sequential chromatography and preparative isoelectric focusing. The isoelectric point of this inhibitor was 4.5. The inhibitor had a M_r of ca 18 000 and was rich in aspartic acid and glycine; it had 4 half-cystine, 2 tryptophan and no methionine residues. Its extinction coefficient (E|_cm^%) was 13.7 at 280 nm. The inhibition was competitive and the dissociation constant was 3.32 × 10^?13 M. The inhibitor was specific to serine proteases and strongly inhibited trypsin and weakly inhibited α-chymotrypsin and kallikrein.     

Isolation and properties of an inhibitor of phospholipase A from Bothrops neuwiedii venom

An inhibitor of phoapholipase A has been isolated from Bothrops neuwiedii venom after gel filtrations through Sephadex G-50 (pH 4.5), Sephadex G-25 (pH 7.6), Sephadex G-15 (pH 4.0), and chromatography on SE-Sephadex C-25 (pH 4.2–4.5). When subjected to paper electrophoresis, the inhibitor migrates as a simple compound with isoelectric point near pH 6.8. Aminoacid composition, sensitivity toward proteases, and the absorption spectrum fit in well with a polypeptide structure lacking tyrosine and tryptophan. In the absence of EDTA, an inactive, anionic derivative appears in inhibitor preparations; the reaction can be reversed by 2-mercaptoethanol. Direct interaction of enzyme and inhibitor is proved by the inhibition of enzyme activity and the chromatography of enzyme-inhibitor mixtures. Titration of inhibitor with venom phospholipases A (isoenzymes P-1 and P-2) yields sigmoid-shaped concentration-inhibition curves, with P-1 far more sensitive than P-2. The enzyme-inhibitor interaction depends on pH since it is tight at pH 4.5 but does not occur at pH 7.5. Presence of thiol groups in inhibitor is consistent with (a) characteristic spectral changes after reaction of inhibitor with PMB ⁴ and NEM; (b) the inhibitor inhibition by PMB, NEM, iodoacetate, and Hg²⁺, and (c) the reversal of PMB inhibition with reduced glutathione. Since phospholipase A is insensitive towards Hg²⁺, addition of Hg²⁺ to enzyme-inhibitor mixtures (or crude venom samples) causes an apparent enzyme activation (deinhibition). When substrate (egg-yolk lipoprotein) is added to enzyme-inhibitor mixtures, the reaction kinetics show an initial “lag-period” which is proportional to the inhibitor concentration. The “lag-period” does not occur in the absence of inhibitor or in the presence of Hg²⁺, that inactivates the inhibitor.     

Isolation and properties of carboxypeptidase from leaves of wounded tomato plants

A carboxypeptidase was purified to homogeneity from upper, unwounded leaves of tomato plants in which carboxypeptidase activity had been induced to increase over three-fold by severely wounding the lower leaves. The carboxypeptidase was purified by ammonium sulfate precipitation, affinity chromatography, and finally by gel permeation chromatography. Electrophoresis at pH 4.3 and isoelectric focusing showed only a single band. The isoelectric point was 5.2 and the MW 105 000. Tomato carboxypeptidase possessed both peptidase and esterase activities and it sequentially hydrolysed amino acids from the carboxyl-terminal end of insulin chain B. It was optimally active at pH 6–7 on peptidase substrates, and at pH 8 on esterase substrates. The enzyme was inhibited by diisopropylfluorophosphate and incorporated 1 mol of DFP-[³H]. per mol of enzyme. Both peptidase and esterase activities were strongly inhibited by HgCl₂ but not by p-hydroxymercuribenzoate or iodoacetamide. Carboxypeptidase inhibitor from potatoes did not inhibit the enzyme.     

Purification, characterization and substrate specificity of a trypsin from the Amazonian fish tambaqui (Colossoma macropomum)

An enzyme was purified from the pyloric caecum of tambaqui (Colossoma macropomum) through heat treatment, ammonium sulfate fractionation, Sephadex® G-75 and p-aminobenzamidine-agarose affinity chromatography. The enzyme had a molecular mass of 23.9 kDa, NH₂-terminal amino acid sequence of IVGGYECKAHSQPHVSLNI and substrate specificity for arginine at P1, efficiently hydrolizing substrates with leucine and lysine at P2 and serine and arginine at P1′. Using the substrate z-FR-MCA, the enzyme exhibited greatest activity at pH 9.0 and 50 °C, whereas, with BAPNA activity was higher in a pH range of 7.5-11.5 and at 70 °C. Moreover, the enzyme maintained ca. 60% of its activity after incubated for 3 h at 60 °C. The enzymatic activity significantly decreased in the presence of TLCK, benzamidine (trypsin inhibitors) and PMSF (serine protease inhibitor). This source of trypsin may be an attractive alternative for the detergent and food industry.