Proteases of the nematode Caenorhabditis elegans

Crude homogenates of the soil nematode Caenorhabditis elegans exhibit strong proteolytic activity at acid pH. Several kinds of enzyme account for much of this activity: cathepsin D, a carboxyl protease which is inhibited by pepstatin and optimally active toward hemoglobin at pH 3; at least two isoelectrically distinct thiol proteases (cathepsins Ce1 and Ce2) which are inhibited by leupeptin and optimally active toward Z-Phe-Arg-7-amino-4-methylcoumarin amide at pH 5; and a thiol-independent leupeptin-insensitive protease (cathepsin Ce3) with optimal activity toward casein at pH 5.5. Cathepsin D is quantitatively most significant for digestion of macromolecular substrates in vitro, since proteolysis is inhibited greater than 95% by pepstatin. Cathepsin D and the leupeptin-sensitive proteases act synergistically, but the relative contribution of the leupeptin-sensitive proteases depends upon the protein substrate.     

Purification and characterization of a neutral serine protease from non-sulfur purple photosynthetic bacterium

A neutral serine protease was purified as a homogeneous protein from the culture broth of photosynthetic bacterium T-20 by sequential chromatographies on columns of DEAE-cellulose, Toyopearl HW 55F, hydroxyapatite, and CM-cellulose. The molecular weight was estimated to be approximately 44,000 by SDS-PAGE, while the value of approximately 80,000 was obtained when the Hedrick-Smith method was used; this suggested that the enzyme consists of two identical subunits. The isoelectric point was determined to be 6.3 by isoelectric focusing. The enzyme had a pH optimum at 7.8. Maximal enzyme activity was detected at 50°C, and the activity was stable up to 50°C for 5 min at pH 7.0–7.2. The substrate specificity of the protease was investigated with a series of synthetic peptidyl-p-nitroanilide. The best substrate examined was Suc-Ala-Ala-Pro-Phe-pNA. The protease activity was inhibited by various inhibitors of serine protease such as chymostatin, PMSF, and DFP. EDTA, which is an inhibitor of metal protease, also inhibited the protease activity, whereas inhibitors of thiol and aspartic proteases had no significant effect.     

Studies on extracellular proteases of Streptococcus sanguis. Purification and characterization of a human IgA1 specific protease.

Extracellular caseinolytic activity was found in the culture fluid of Streptococcus sanguis ATCC 10556 grown in a dialyzed culture medium. This activity was due to multiple proteases that differed in their elution from hydroxyapatite, sensitivity to enzyme inhibitors, specificity and optimum pH. IgA protease, which splits human immunoglobulin A1 into intact Fc and Fab could be effectively separated from these relatively non-specific proteases and purified to apparent homogeneity in 20% yield by a five-step procedure. Although the bulk of the dextran sucrase activity was separated from the IgA protease, a small amount of sucrase activity remained with the final IgA protease preparation. In polyacrylamide gel electrophoresis at pH 9.5 both activities were located in the single protein band detected in this preparation. A quantitative method for the assay of IgA protease was developed, based on radial immunodiffusion to quantitate the Fab produced. This was used to follow the specific activity and yield during purification, and to characterize some of the catalytic properties of the enzyme. At an enzyme/substrate ratio of 1: 400 (w/w) the protease could effect 50% proteolysis of IgA in overnight incubation at 37 degrees C. The optimum activity was at pH 8.0, and 50% inhibition was achieved at 4 . 10(-4) M o-phenanthroline or 8 . 10(-4) M ethylene diamine tetraacetate. Concentrations of diisopropyl phosphofluoridate, phenylmethyl-sulfonyl fluoride, iodoacetate and p-chloromercuribenzoate up to 10(-2) M were without effect on the IgA protease activity. Full reactivation of the chelator inhibited enzyme could be achieved by the addition of Mg2+, Mn2+ or Ca2+.     

The purification and characterisation of novel dipeptidyl peptidase IV-like activity from bovine serum

The discovery of a potentially novel proline-specific peptidase from bovine serum is presented which is capable of cleaving the dipeptidyl peptidase IV (DPIV) substrate Gly-Pro-MCA. The enzyme was isolated and purified with the use of Phenyl Sepharose Hydrophobic Interaction, Sephacryl S-300 Gel Filtration, and Q-Sephacryl Anion Exchange, producing an overall purification factor of 257. SDS PAGE resulted in a monomeric molecular mass of 158kDa while size exclusion chromatography generated a native molecular mass of 328kDa. The enzyme remained active over a broad pH range with a distinct preference for a neutral pH range of 7-8.5. Chromatofocusing and isoelectric focusing (IEF) revealed the enzyme's isoelectric point to be 4.74. DPIV-like activity was not inhibited by serine protease inhibitors but was by the metallo-protease inhibitors, the phenanthrolines. The enzyme was also partially inhibited by bestatin. Substrate specificity studies proved that the enzyme is capable of sequential cleavage of bovine beta-Casomorphin and Substance P. The peptidase cleaved the standard DPIV substrate, Gly-Pro-MCA with a K(M) of 38.4 microM, while Lys-Pro-MCA was hydrolysed with a K(M) of 103 microM. The DPIV-like activity was specifically inhibited by both Diprotin A and B, non-competitively, generating a K(i) of 1.4 x 10(-4) M for both inhibitors. Ile-Thiazolidide and Ile-Pyrrolidide both inhibited competitively with an inhibition constant of 3.7 x 10(-7) and 7.5 x 10(-7) M, respectively. It is concluded that bovine serum DPIV-like activity share many biochemical properties with DPIV and DPIV-like enzymes but not exclusively, suggesting that the purified peptidase may play an important novel role in bioactive oligopeptide degradation.     

Enzymatic properties of a bee venom serine protease from the bumblebee Bombus ignitus

We previously reported that bumblebee (Bombus ignitus) venom serine protease (Bi-VSP) acts as a prophenoloxidase-activating factor in arthropods and a fibrin(ogen)olytic enzyme in mammals. In the present study, we characterized the enzymatic properties of Bi-VSP purified from B. ignitus venom. The 34-kDa active form of Bi-VSP was purified from the venom of B. ignitus worker bees. Glycoprotein staining showed that approximately 20% of the total molecular mass of Bi-VSP is due to carbohydrate moieties. Bi-VSP had an optimal pH and temperature of pH 9.0 and 40 °C, respectively, and was stable at 50 °C for at least 10 min. Bi-VSP activity decreased abruptly below pH 6.0, indicating that Bi-VSP activity is almost completely inhibited at pH 5.4 of B. ignitus venom. The protease activity of Bi-VSP was strongly inhibited by typical serine protease inhibitors such as phenylmethanesulfonyl fluoride, leupeptin, and soybean trypsin inhibitor.     

Production,optimization and purification of a novel extracellular protease from the moderately halophilic bacterium <Emphasis Type="Italic">Halobacillus karajensis</Emphasis>

The production of a protease was investigated under conditions of high salinity by the moderately halophilic bacterium Halobacillus karajensis strain MA-2 in a basal medium containing peptone, beef extract, maltose and NaCl when the culture reached the stationary growth phase. Effect of various temperatures, initial pH, salt and different nutrient sources on protease production revealed that the maximum secretion occurred at 34°C, pH 8.0–8.5, and in the presence of gelatin. Replacement of NaCl by various concentrations of sodium nitrate in the basal medium also increased the protease production. The secreted protease was purified 24-fold with 68% recovery by a simple approach including a combination of acetone precipitation and Q-Sepharose ion exchange chromatography. The enzyme revealed a monomeric structure with a relative molecular mass of 36 kDa by running on SDS-PAGE. Maximum caseinolytic activity of the enzyme was observed at 50°C, pH 9.0 and 0.5 M NaCl, although at higher salinities (up to 3 M) activity still remained. The maximum enzyme activity was obtained at a broad pH range of 8.0–10.0, with 55 and 50% activity remaining at pH 6 and 11, respectively. Moreover, the enzyme activity was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), Pefabloc SC and EDTA; indicating that it probably belongs to the subclass of serine metalloproteases. These findings suggest that the protease secreted by Halobacillus karajensis has a potential for biotechnological applications from its haloalkaline properties point of view.     

Isolation and characterization of a metal ion-dependent alkaline protease from a halotolerant Bacillus aquimaris VITP4

A halotolerant bacterium Bacillus acquimaris VITP4 was used for the production of extracellular protease. Fractional precipitation using ammonium chloride was used to obtain the enzyme. The protease exhibited optimum activity at pH 8.0 and 40 degrees C and retained 50% of its optimal proteolytic activity even in the presence of 4 M NaCl, suggesting that it is halotolerant. The molecular mass of protease, as revealed by SDS-PAGE was found to be 34 kDa and the homogeneity of the enzyme was confirmed by gelatin zymography and reverse-phase HPLC. Upon purification, the specific activity of th enzyme increased from 533 U/mg to 1719 U/mg. Protease inhibitors like phenyl methane sulphonyl fluoride and 2-mercaptoethanol did not affect the activity of the enzyme, but EDTA inhibited the activity, indicating the requirement of metal ions for activity. Cu2, Ni2+ and Mn2+ enhanced the enzyme activity, but Zn2+, Hg2+ and Fe2+ decreased the activity, while Mg2+, Ca2+ and K+ had no effect on the enzyme activity. The protease was quite stable in the presence of cationic (CTAB), anionic (SDS) and neutral detergents (Triton X-100 and Tween-20) and exhibited antimicrobial activity against selected bacterial and fungal strains. The stability characteristics and broad spectrum antimicrobial activity indicated the potential use of this protease in industrial applications.     

Leishmania (Leishmania) amazonensis: purification and characterization of a promastigote serine protease

Pathogenic protozoan proteases play crucial roles in the host-parasite interaction, and its characterization contributes to the understanding of protozoan disease mechanisms. A Leishmania amazonensis promastigote protease was purified 36-fold, using aprotinin-agarose affinity chromatography and gel filtration high performance liquid chromatography, yielding a total recovery of 49%. The molecular mass of active enzyme obtained from native gel filtration HPLC and SDS-PAGE under conditions of reduction and non-reduction was 68 kDa, suggesting that the enzyme may exist as a monomer. The protease isoelectric point (pI) was around 4.45 and, as demonstrated by deglycosylation assay, it did not have any carbohydrate content. The optimal pH and temperature of the enzyme were 8.0 and 28 degrees C, respectively, determined using alpha-N-rho-tosyl-L-arginyl-methyl ester (L-TAME) as substrate. Assays of thermal stability indicated that 50% of the enzymatic activity was preserved after 4 min of pre-treatment at 42 degrees C and after 24 h of pre-treatment at 37 degrees C, both in the absence of substrate. Hemoglobin, bovine serum albumin (BSA), ovalbumin, and both gelatin and peptide substrates containing arginine in ester bound were hydrolyzed by 68 kDa protease. The insulin beta-chain was also hydrolyzed by the protease, and four peptidic bonds (L11-V12, E13-A14, L15-Y16, and Y16-L17) were susceptible to the 68-kDa protease action. Inhibition studies suggested that the enzyme belonged to a serine protease class inhibited by calcium ions and activated by manganese ions. These findings demonstrate that the L. amazonensis 68-kDa serine protease differs from those of other protozoan parasites.     

Physiological and nutritional factors affecting synthesis of extracellular metalloproteases by Clostridium bifermentans NCTC 2914.

Extracellular protease production by Clostridium bifermentans NCTC 2914 occurred throughout the growth phase in batch culture. In both glucose-excess and -limited chemostats, protease formation was inversely related to the dilution rate, over the range D = 0.03 to 0.70 h-1. At high dilution rates (D greater than 0.25 h-1), protease activities were greatest under excess glucose conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of chemostat culture effluents showed the presence of up to 18 bands of protease activity at low dilution rates, with apparent molecular masses ranging from about 36 to 125 kDa. High-performance liquid chromatography gel filtration of culture supernatants gave four peaks of activity at 34, 42, 60, and 102 kDa. Glucose, peptone, and phosphate stimulated protease formation, but ammonia concentrations up to 10 g liter-1 had little effect on the process. Culture pH in glucose-excess chemostats strongly influenced protease synthesis, which was maximal during growth at pH 6.4. The optimal pH of protease activity was 7.0. Although a wide variety of proteins were hydrolyzed by C. bifermentans proteases, none of the enzymes were collagenolytic. Of 21 different p-nitroanilide, beta-naphthylamide, and N-carbobenzoyl substrates tested, none were hydrolyzed. With the exception of Ca2+, divalent metal ions inhibited proteolysis. Experiments with protease inhibitors demonstrated that 1 mM EDTA inhibited protease activities in culture supernatants by over 90%, indicating that the enzymes were principally of the metalloprotease type.     

Physiological and nutritional factors affecting synthesis of extracellular metalloproteases by Clostridium bifermentans NCTC 2914.

Extracellular protease production by Clostridium bifermentans NCTC 2914 occurred throughout the growth phase in batch culture. In both glucose-excess and -limited chemostats, protease formation was inversely related to the dilution rate, over the range D = 0.03 to 0.70 h-1. At high dilution rates (D greater than 0.25 h-1), protease activities were greatest under excess glucose conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of chemostat culture effluents showed the presence of up to 18 bands of protease activity at low dilution rates, with apparent molecular masses ranging from about 36 to 125 kDa. High-performance liquid chromatography gel filtration of culture supernatants gave four peaks of activity at 34, 42, 60, and 102 kDa. Glucose, peptone, and phosphate stimulated protease formation, but ammonia concentrations up to 10 g liter-1 had little effect on the process. Culture pH in glucose-excess chemostats strongly influenced protease synthesis, which was maximal during growth at pH 6.4. The optimal pH of protease activity was 7.0. Although a wide variety of proteins were hydrolyzed by C. bifermentans proteases, none of the enzymes were collagenolytic. Of 21 different p-nitroanilide, beta-naphthylamide, and N-carbobenzoyl substrates tested, none were hydrolyzed. With the exception of Ca2+, divalent metal ions inhibited proteolysis. Experiments with protease inhibitors demonstrated that 1 mM EDTA inhibited protease activities in culture supernatants by over 90%, indicating that the enzymes were principally of the metalloprotease type.     

Monoclonal antibodies against Pseudomonas aeruginosa elastase: a neutralizing antibody which recognizes a conformational epitope related to an active site of elastase.

We have established seven murine hybridoma cell lines which produce monoclonal antibodies (mAbs) against Pseudomonas aeruginosa elastase. The seven mAbs recognized at least six different epitopes on the elastase molecule. All mAbs inhibited both enzymatic activities of elastase and protease, in which elastin fluorescein and hide powder azure were used as substrates, respectively. One of them, mAb E-4D3, strongly neutralized enzymatic activities of peptidase in which furylacryloyl-glycyl-leucinamide was used as a substrate, as well as of elastase and protease. In contrast, the other six mAbs did not neutralize peptidase activity at all. The Ki value for furylacryloyl-glycl-leucinamide of E-4D3, as well as its Fab fragment, was comparable to those for metalloprotease inhibitors such as phosphoramidon and Zincov inhibitor. The binding of mAb E-4D3 was inhibited by phosphoramidon and Zincov inhibitor, but not by metal chelators such as EDTA and o-phenanthroline. A line of evidence suggests that mAb E-4D3 directly interacts with active site and highly neutralizes enzymatic activity of P. aeruginosa elastase. Data of Western blotting and ELISA suggest that mAb E-4D3 is likely to recognize an elastase molecule in a conformation-dependent manner as an epitope. In contrast, the neutralizing activity of the other mAbs against elastase and protease seems to be caused by a low accessibility of an enzyme to insoluble and high-molecular-mass substrates through the binding and steric hindrance of the mAbs to an enzyme.     

Purification and characterization of a multicatalytic proteinase from Atlantic salmon (Salmo salar) muscle

A high molecular mass alkaline proteinase was purified by DEAE-Sepharose and Mono Q chromatography. The mol. wt was estimated to be about 600,000. Under denaturing conditions, the enzyme dissociated into a cluster of subunits with mol. wt ranging from 25,000 to 30,000. The isoelectric point of the enzyme was about pH 7.3. The proteinase was able to hydrolyse N-terminal-blocked 4-methyl-7-coumarylamide substrates for either trypsin- or chymotrypsin-like activity. It was also able to hydrolyse haemoglobin and myosin at temperatures of about 60°C. The activities responded to pH and some chemicals in different ways. The trypsin-like activity was clearly inhibited by several serine protease inhibitors. These results suggest that the enzyme is multicatalytic, having at least two different active sites.     

Degradation of glyceraldehyde-3-phosphate dehydrogenase triggered by 4-hydroxy-2-nonenal and 4-hydroxy-2-hexenal

Lipid peroxidation products such as 4-hydroxy-2-nonenal (HNE) may be responsible for various pathophysiological events under oxidative stress, since they injure cellular components such as proteins and DNA. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is a key enzyme of glycolysis and has been reported to be a multifunctional enzyme, is one of the enzymes inhibited by HNE. Previous studies showed that GAPDH is degraded when incubated with acetylleucine chloromethyl ketone (ALCK), resulting in the liberation of a 23-kDa fragment. In this study, we examined whether GAPDH incubated with HNE or other aldehydes of lipid peroxidation products are degraded similarly to that with ALCK. The U937 cell extract was incubated with these aldehydes at 37 degrees C and analyzed by Western blotting using anti-GAPDH antibodies. Incubation with HNE or 4-hydroxy-2-hexenal (HHE) decreased GAPDH activity and GAPDH protein level, and increased the 23-kDa fragment, in time- and dose-dependent manners, but that with other aldehydes did not. Gel filtration using the Superose 6 showed that the GAPDH-degrading activity was eluted in higher molecular fractions than proteasome activity. The enzyme activity was detected at the basic range of pH and inhibited by serine protease inhibitors, diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, but not by other protease inhibitors including a proteasome inhibitor, MG-132, and a tripeptidyl peptidase II (TPP II) inhibitor, AAF-CMK. These results suggest that GAPDH modified by HNE and HHE is degraded by a giant serine protease, releasing the 23-kDa fragment, not by proteasome or TPP II.     

Human beta-tryptase: detection and characterization of the active monomer and prevention of tetramer reconstitution by protease inhibitors

beta-Tryptase is a trypsin-like serine protease stored in mast cell secretory granules primarily as an enzymatically active tetramer. The current study aims to determine whether monomeric beta-tryptase also can exhibit enzyme activity, as suggested previously. At neutral pH beta-tryptase tetramers in the absence of heparin or dextran sulfate spontaneously convert to inactive monomers. Addition of a polyanion to these monomers at neutral pH fails to convert them back to a tetramer or to an enzymatically active state. In contrast, at acidic pH addition of a polyanion resurrects enzyme activity. Whether this activity is associated with tetramers or monomers depends on the concentration of beta-tryptase. Under the experimental conditions employed at pH 6 in the presence of heparin, the monomer concentration at which 50% conversion to tetramers occurs is 193 ng/mL. Activity against tripeptide substrates by monomers is detected at pH 6 but not at pH 7.4, whereas tetramer activity is greater at pH 7.4 than pH 6.0. Active monomers are inhibited by soybean trypsin inhibitor, bovine pancreatic trypsin inhibitor, antithrombin III, and alpha2-macroglobulin, whereas active tetramers are resistant to these inhibitors. Active monomers form complexes with these inhibitors and cleave both antithrombin III and alpha2-macroglobulin. These inhibitors also prevent reconstitution of monomers to tetramers, indicating that inactive monomers become active monomers before becoming active tetramers. The ability of tryptase monomers to become active at acidic pH raises the possibilities of expanded substrate specificities as well as inhibitor susceptibilities where the low-pH environments associated with inflammation or poor vascularity are encountered in vivo.     

Purification and characterization of a unique alkaline elastase from Micrococcus luteus

Micrococcus luteus isolated from human skin secretes an alkaline protease which degrades elastin. M. luteus protease (MLP) was produced in the late logarithmic and stationary phases of growth. MLP, purified to homogeneity by a three-step process, had a molecular mass of 32,812 Da and an isoelectric point of 9.3. MLP was active and highly stable in solution for 24 h from pH 6.0 to 10.5; it had maximal activity at temperatures between 57 and 59 degrees C. The presence of calcium in the solution was essential for enzyme activity and to prevent autolysis. Optimal activity occurred between pH 9.0 and 9.5, with 60% maximal activity from pH 6.5 to 11.0. The enzyme was inhibited by the serine enzyme inhibitors phenylmethylsulfonyl fluoride and chymostatin but not by the metalloenzyme inhibitor 1,10-phenanthroline or sulfhydryl enzyme inhibitors. Casein, bovine serum albumin, ovalbumin, beta-lactoglobulin, and elastin were digested by the protease while collagen and keratin were resistant to digestion. MLP demonstrated both esterase and amidase activity on synthetic peptide substrates. MLP preferentially cleaved the Leu(15)-Tyr(16) and Phe(24)-Phe(25) bonds of the oxidized beta-chain of insulin. Longer digests of insulin and the pattern of activity against synthetic substrates suggest that MLP has a cleavage specificity for bulky, hydrophobic, or aromatic amino acids in the P(1) or P(1)' positions. Amino acid sequences from the N-terminus and internal peptides of MLP were unique.     

Protease activity of 80 kDa protein secreted from the apicomplexan parasite Toxoplasma gondii

This study describes the characterization of 80 kDa protease showing gelationlytic property among three proteases in the excretory/secretory proteins (ESP) from Toxoplasma gondii. The protease activity was detected in the ESP but not in the somatic extract of RH tachyzoites. This protease was active only in the presence of calcium ion but not other divalent cationic ions such as Cu(2+), Zn(2+), Mg(2+), and Mn(2+), implying that Ca(2+) is critical factor for the activation of the protease. The 80 kDa protease was optimally active at pH 7.5. Its gelatinolytic activity was maximal at 37 degrees C, and significant level of enzyme activity of the protease remained after heat treatment at 56 degrees C for 30 min or 100 degrees C for 10 min. This thermostable enzyme was strongly inhibited by metal chelators, i.e., EDTA, EGTA, and 1,10- phenanthroline. Thus, the 80 kDa protease in the ESP secreted by T. gondii was classified as a calcium dependent neutral metalloprotease.     

Purification and characteristics of alkaline proteinase from alkalophylic Bacillus sp.]

Alkaline protease was purified from Bacillus sp. isolated from soil. The pH optimum was 11.5 at 37 degrees C. Calcium divalent cation was effective to stabilize the enzyme especially at higher temperatures. The proteolytic activity was inhibited by active site inhibitors of PMSF (Phenylmethylsulfonyl fluoride), and ions of Mg, Mn, Pb, Li, Zn, Ag, Hg. The enzyme was stable in the presence of some detergents, such as Triton-X-100, Tween-80, SDS (sodium dodecyl sulfate) and EDTA (ethylendiaminetetraacetic acid), pH 11.5 and 37 degrees C for 30 min. The optimum pH was 11.5 at 37 degrees C and the optimum temperature was 62 degrees C at pH 11.5.     

An extracellular--pepstatin insensitive acid protease produced by Thermoplasma volcanium

In this study, some parameters for the production and caseinolytic activity of an extracellular thermostable acid protease from a thermoacidophilic archaeon Thermoplasma volcanium were determined. The highest level of growth and enzyme production were detected at pH 3.0 over an incubation period of 192 h at 60 degrees C. The pH optimum for the acid protease activity was 3.0 and the enzyme was fairly stable over a broad pH range (pH 3.0-8.0). The temperature for maximum activity of the enzyme was 55 degrees C and activity remained stable between 50 degrees C and 70 degrees C. These features could be of relevance for various biotechnological applications of this enzyme. Serine-(PMSF), cysteine-(DTT), metallo-(EDTA) and aspartate-(pepstatin) protease inhibitors did not inhibit the caseinolytic activity of the enzyme. Therefore, Tp. volcanium acid protease could be a member of the pepstatin-insensitive carboxyl proteinases.     

Interplay between acid phosphatase and cysteine proteases in mediating vitellin degradation during early embryogenesis of Periplaneta americana

In this work, we characterized the activities of two classes of proteases and AcP during early embryogenesis of Periplaneta americana. AcP activity was first detected at day 6 and reached a maximum level at day 10 of development. Using phosphoamino acids, phosphatase activity was shown to be directed only against phosphotyrosine at day 6 while at day 10 it was also active against phosphoserine. In parallel, two classes of proteases were detected and located within yolk granules: a clan CA-cysteine protease, which was inhibited by E-64, insensitive to CA 074 and activated by acidic pH at day 3; and a neutral serine protease, which was inhibited by aprotinin at day 6. Assays of vitellin (Vt) degradation evidenced that incubations at neutral pH induced slight proteolysis, while the incubations at acidic pH did not result in Vt degradation. However, pre-incubations of Vt with AcP increased the levels of Vt acidic proteolysis and this could be inhibited by the addition of phosphatase inhibitors. On the other hand, the same pre-incubations showed no effects on the profile of degradation at neutral pH. We propose that AcP and cysteine protease cooperate to assure Vt breakdown during early embryogenesis of P. americana.     

Dirofilaria immitis: proteases produced by third- and fourth-stage larvae.

A model of cutaneous extracellular matrix was used to determine if live Dirofilaria immitis larvae secrete proteases which are active at physiological pH and capable of degrading macromolecules found in cutaneous tissue. After 72 hr, 100 third-stage larvae (L3) degraded 24% of the total matrix, while fourth-stage larvae (L4) degraded 10%. A sharp increase in the amount of matrix degraded by L3 corresponded with the onset of the molting process. L3 and L4 degraded comparable amounts of the glycoprotein and elastin components of the matrix, but molting L3 degraded nearly twice the amount of the collagen component (62% vs 35%). Characterization of proteases present in larval-soluble extracts and excretory-secretory products using synthetic substrates and protease inhibitors demonstrated cysteine-protease and metalloprotease activity. Cysteine protease activity was found in whole worm extracts of both L3 and L4. Metalloprotease was secreted at higher levels by molting L3, but was also secreted by L4. Partial separation of the metalloprotease by size-exclusion chromatography indicated that the molecular weight of the native enzyme was in the 49-54 kDa range. The cysteine protease activity was demonstrated in fractions corresponding to 34-39 kDa. The biological function of the D. immitis larval proteases remains to be conclusively determined; however, these data suggest that they are involved in degradation of components of cutaneous tissue and in the molting process.