SEARCH · Search PubMed

Results for “PROTEASES”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 955 records · Page 53Linked to original sources

PubMed2003

Human eosinophils are activated by cysteine proteases and release inflammatory mediators.

BACKGROUND: Recent studies suggest that serine proteases are involved in various biological responses through activation of protease-activated receptors (PARs). However, the functions of other proteases, such as cysteine proteases, are poorly understood and need elucidation. OBJECTIVE: We examined the effects of an authentic cysteine protease, papain, and a protease derived from the mite allergen, Der f 1, on functions of human eosinophils. METHODS: Purified eosinophils were incubated with papain or Der f 1. Eosinophil activation was monitored by superoxide anion generation and by degranulation. Intracellular signaling pathways were investigated through use of pharmacologic approaches. RESULTS: We found that papain potently induces human eosinophils to degranulate and to produce superoxide anion. A cysteine protease inhibitor, E-64, abolished the stimulatory effects of papain, which suggests that the protease activity of papain is necessary to trigger eosinophil responses. The eosinophil's response to papain was enhanced by IL-5 and mediated by activation of the phosphatidylinositol 3-kinase/Akt pathway. Interestingly, whereas a serine protease, trypsin, activated eosinophils through PAR2, the effects of papain were not inhibited by an antibody to PAR2, which suggests another novel mechanism for the eosinophils' response to cysteine proteases. It is likely that these observations are clinically important, because eosinophils were activated by a natural cysteine protease allergen, Der f 1, and released granule proteins. CONCLUSION: Human eosinophils are probably equipped with machineries that recognize and respond to cysteine proteases, such as those found at allergic inflammation sites; the result is active release of proinflammatory mediators.

Antigens, Dermatophagoides↗

PubMed2002

Secreted proteases from pathogenic fungi.

Many species of human pathogenic fungi secrete proteases in vitro or during the infection process. Secreted endoproteases belong to the aspartic proteases of the pepsin family, serine proteases of the subtilisin family, and metalloproteases of two different families. To these proteases has to be added the non-pepsin-type aspartic protease from Aspergillus niger and a unique chymotrypsin-like protease from Coccidioides immitis. Pathogenic fungi also secrete aminopeptidases, carboxypeptidases and dipeptidyl-peptidases. The function of fungal secreted proteases and their importance in infections vary. It is evident that secreted proteases are important for the virulence of dermatophytes since these fungi grow exclusively in the stratum corneum, nails or hair, which constitutes their sole nitrogen and carbon sources. The aspartic proteases secreted by Candida albicans are involved in the adherence process and penetration of tissues, and in interactions with the immune system of the infected host. For Aspergillus fumigatus, the role of proteolytic activity has not yet been proved. Although the secreted proteases have been intensively investigated as potential virulence factors, knowledge on protease substrate specificities is rather poor and few studies have focused on the research of inhibitors. Knowledge of substrate specificities will increase our understanding about the action of each protease secreted by pathogenic fungi and will help to determine their contribution to virulence.

Arthrodermataceae↗

PubMed1991

Proteases and Their Involvement in the Infection and Immobilization of Nematodes by the Nematophagous Fungus Arthrobotrys oligospora.

The nematophagous fungus Arthrobotrys oligospora produced extracellular proteases when grown in a liquid culture, as revealed by measuring the hydrolysis of the chromogenic substrate Azocoll. The extracellular protease activity was inhibited by phenylmethylsulfonyl fluoride (PMSF) and other serine protease inhibitors and partly inhibited by the aspartate protease inhibitor pepstatin and by a cysteine protease inhibitor [l-trans-epoxysuccinyl-leucylamide-(4-guanidino)-butane, or E-64]. Substrate gel electrophoresis showed that the fungus produced several different proteases, including multiple serine proteases. The function of proteases in the infection of nematodes was examined by treating the fungus with various protease inhibitors. None of the inhibitors tested affected the adhesion of nematodes to the traps, but incubating trap-bearing mycelium with a serine protease inhibitor, PMSF, antipain, or chymostatin, or the metalloprotease inhibitor phenanthroline significantly decreased the immobilization of nematodes captured by the fungus. Inhibitors of cysteine or aspartic proteases did not affect the immobilization of captured nematodes. The effects of PMSF on the immobilization of nematodes were probably due to serine proteases produced by the fungus, since the effects were observed when unbound inhibitor was washed away from the fungus before the nematodes were added to the system. No effects were observed when the nematodes only were pretreated with PMSF.

Journal Article↗

PubMed1986

Disruption of respiratory cilia by proteases including those of Pseudomonas aeruginosa.

Pseudomonad proteases disrupted the function and structure of demembranated cilia (axonemes) extracted from porcine tracheae. Proteolytic degradation by the two pseudomonad proteases elastase and alkaline protease and by trypsin and subtilisin impaired motility of ATP-activated axonemes. In addition, electron microscopic observation of negatively stained axonemes indicated that exposure to proteases caused dissociation into individual doublet or singlet microtubules. Inhibition of motility and axonemal fraying occurred when axonemes were treated with less than 5 U of proteolytic activity of any of the four proteases tested. When the effects of 2 U of each protease were compared, trypsin and subtilisin were able to produce immotility in less time than pseudomonad elastase and alkaline protease, while alkaline protease and subtilisin caused the most axonemal fraying in 10 min. Proteolytic digestion of axonemal proteins was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All four proteases cleaved dynein proteins (proteins necessary for motility), though treatment with trypsin resulted in the most extensive solubilization of axonemal proteins. Trypsin and subtilisin both produced more changes in the protein profiles of treated axonemes, using fewer units of proteolytic activity, than the pseudomonad proteases. However, the limited alteration of only a few axonemal proteins by pseudomonad proteases indicates that cleavage need not be extensive to produce dysfunction. Thus, ciliary axonemes are susceptible to proteolytic attack. Degradation of axonemal proteins by pseudomonad proteases, which are released during active infection, may contribute to the impaired ciliary function associated with pseudomonad colonization of the respiratory tract.

Animals↗

PubMed1994

Recombinant expression and antigenic properties of a 32-kilodalton extracellular alkaline protease, representing a possible virulence factor from Aspergillus fumigatus.

A 32-kDa nonglycosylated alkaline protease (EC 3.4.1.14) with elastolytic activity, secreted by the opportunistic pathogen Aspergillus fumigatus ATCC 42202, is suggested to be a virulence factor of this fungus. The enzyme is a serine protease of the subtilisin family, and its cDNA nucleotide sequence has recently been reported. We have cloned the cDNA encoding the mature protease into a high-level Escherichia coli expression plasmid and produced the recombinant protease as a fusion protein with a six-adjacent-histidine affinity tag at the carboxy terminus. Subsequently, the recombinant protease was purified to homogeneity, with affinity chromatography yielding 30 to 40 mg of recombinant protease per liter of E. coli culture. Refolded recombinant protease, in comparison with native protease, demonstrated weak enzymatic activity but similar immunochemical characteristics as analyzed by antigen-specific enzyme-linked immunosorbent assay (ELISA), competition ELISA, and immunoblotting assays. To assess the allergenic potential of the protease, sera from patients with allergic bronchopulmonary aspergillosis and sera from healthy control individuals were analyzed by ELISA and immunoblotting techniques. Sera from patients with allergic bronchopulmonary aspergillosis did not have protease-specific immunoglobulin E (IgE) antibodies and, remarkably, did not show significantly elevated protease-specific IgG antibody levels compared with those in sera from healthy control individuals. This suggests that the alkaline protease from A. fumigatus does not elicit IgE antibodies and has weak immunogenicity, a property which may explain fungus persistence in allergic individuals.

Amino Acid Sequence↗

PubMed1996

Separate functional domains of the herpes simplex virus type 1 protease: evidence for cleavage inside capsids.

The herpes simplex virus type 1 (HSV-1) protease (Pra) and related proteins are involved in the assembly of viral capsids and virion maturation. Pra is a serine protease, and the active-site residue has been mapped to amino acid (aa) 129 (Ser). This 635-aa protease, encoded by the UL26 gene, is autoproteolytically processed at two sites, the release (R) site between amino acid residues 247 and 248 and the maturation (M) site between residues 610 and 611. When the protease cleaves itself at both sites, it releases Nb, the catalytic domain (N0), and the C-terminal 25 aa. ICP35, a substrate of the HSV-1 protease, is the product of the UL26.5 gene. As it is translated from a Met codon within the UL26 gene, ICP35 cd are identical to the C-terminal 329-aa sequence of the protease and are trans cleaved at an identical C-terminal site to generate ICP35 e,f and a 25-aa peptide. Only fully processed Pra (N0 and Nb) and ICP35 (ICP35 e,f) are present in B capsids, which are believed to be precursors of mature virions. Using an R-site mutant A247S virus, we have recently shown that this mutant protease retains enzymatic activity but fails to support viral growth, suggesting that the release of N0 is required for viral replication. Here we report that another mutant protease, with an amino acid substitution (Ser to Cys) at the active site, can complement the A247S mutant but not a protease deletion mutant. Cell lines expressing the active-site mutant protease were isolated and shown to complement the A247S mutant at the levels of capsid assembly, DNA packaging, and viral growth. Therefore, the complementation between the R-site mutant and the active-site mutant reconstituted wild-type Pra function. One feature of this intragenic complementation is that following sedimentation of infected-cell lysates on sucrose gradients, both N-terminally unprocessed and processed proteases were isolated from the fractions where normal B capsids sediment, suggesting that proteolytic processing occurs inside capsids. Our results demonstrate that the HSV-1 protease has distinct functional domains and some of these functions can complement in trans.

Animals↗

PubMed1985

Purification and characterization of heat-stable proteases from Bacillus stearothermophilus RM-67.

The extracellular proteases of Bacillus stearothermophilus RM-67 were purified by ammonium sulfate fractionation (40 to 70% saturation), gel filtration through Sephadex G-100, and diethylaminoethyl-Sephadex A-50 ion-exchange chromatography. Gel filtration resulted in separation of the enzyme preparation into one minor (protease I) and one major (protease II) peak. The three-step purification scheme resulted in 39.5-fold purification and an overall recovery of 8.1% of protease I and 87.8-fold purification and 59.7% recovery of protease II. Purified proteases had pH and temperature optima of 8.0 and 70 degrees C. Protease I and II, when together, retained 100% activity at 60 degrees C for 30 min. Manganese imported 100% stability to the pooled proteases at 65 degrees C for 30 min. Amino acid analysis of the major peak (protease II) revealed the absence of half cystine and methionine. Protease I and II had molecular weights of 67,610 and 19,950 and Michaelis-Menten constants (casein) of 1.33 and 2.0 mg/ml. Energy of activation was 14,300 cal/mol for protease I and 11,150 cal/mol for protease II. Corresponding heat of activation was l3,620 and 10,470 cal/mol.

Animals↗

PubMed1991

The scrapie-associated form of PrP is made from a cell surface precursor that is both protease- and phospholipase-sensitive.

A common feature of scrapie and related transmissible spongiform encephalopathies is the accumulation of an abnormal protease-resistant form of PrP which may be the major component of the infectious agent. While it is known that both the normal (protease-sensitive) PrP and protease-resistant PrP are encoded by the same endogenous gene, the nature of the disease-associated modification of PrP is not understood. To study the cellular events leading to the formation of protease-resistant PrP, we have compared its biosynthesis to that of its normal isoform in scrapie-infected mouse neuroblastoma cells. In pulse-chase labeling experiments, the protease-resistant PrP was synthesized and degraded much more slowly than the normal PrP, suggesting that protease-resistant PrP is made from a protease-sensitive precursor. More significantly, we found that the precursor of protease-resistant PrP was eliminated from intact cells by treatments with phosphatidylinositol-specific phospholipase C and trypsin. This demonstrated that, unlike the protease-resistant PrP itself, the precursor is phospholipase- and protease-sensitive and at least transiently found on the cell surface. By these criteria, the precursor of protease-resistant PrP is indistinguishable from the normal PrP isoform. These results indicate that the conversion of PrP to the protease- and phospholipase-resistant state is a post-translational event that occurs after the precursor reaches the cell surface.

Animals↗

PubMed1995

In vivo bacterial protease production during Pseudomonas aeruginosa corneal infection.

PURPOSE: To establish if active pseudomonal proteases are present in vivo during corneal infection with Pseudomonas aeruginosa and to determine if the mouse strains used in these and previous studies have the ability to mount a nonocular antibody response to the purified proteases because antibodies to the bacterial proteases were not detected previously during in vivo ocular infection. METHODS: At certain times after corneal infection with P. aeruginosa, corneas were harvested and supernatants from the corneal homogenates were analyzed for proteolytic activity by zymography and immunoreactivity by immunoblotting. The efficiency of the extraction procedures used in these studies was determined by incubating uninfected corneal homogenates with the purified proteases. The resultant supernatants were analyzed for alkaline protease and elastase activity. Additionally, mice were immunized intraperitoneally with the purified proteases with and without adjuvant to determine if the animals could mount a nonocular antibody response. RESULTS: Corneas infected with P. aeruginosa demonstrated the presence of alkaline protease, but not elastase, by the two methods examined. The kinetics of the in vivo alkaline protease response closely parallels previously reported bacterial clearance studies in that peak alkaline protease activity was detected in corneal tissue when peak bacterial numbers also were observed in the eye, and it was absent when the eyes were sterile or nearly sterile. In addition, C57BL/6J mice were capable of mounting a nonocular antibody response to microgram quantities of both proteases only in the presence of adjuvant. CONCLUSIONS: In the model described, enzymatically active alkaline protease, but not elastase, was demonstrated in corneal tissues during in vivo infection. Concentrations of these proteases were much lower than those required to stimulate an antibody response.

Animals↗

PubMed2000

Inhibition of HIV-1 protease by a boron-modified polypeptide.

Six boronated tetrapeptides with the carboxy moiety of phenylalanine replaced by dihydroxyboron were synthesized, and their activities against human immunodeficiency virus 1 (HIV-1) protease subsequently investigated. The sequences of these peptides were derived from HIV-1 protease substrates, which included the C-terminal part of the scissile bond (Phe-Pro) within the gag-pol polyprotein. Enzymatic studies showed that these compounds were competitive inhibitors of HIV-1 protease with K(i) values ranging from 5 to 18 microM when experiments were performed at high enzyme concentrations (above 5 x 10(-8) M); however, at low protease concentrations inhibition was due in part to an increase of the association constants of the protease subunits. Ac-Thr-Leu-Asn-PheB inhibited HIV-1 protease with a K(i) of 5 microM, whereas the non-boronated parental compound was inactive at concentrations up to 400 microM, which indicates the significance of boronation in enzyme inhibition. The boronated tetrapeptides were inhibitory to an HIV-1 protease variant that is resistant to several HIV-1 protease inhibitors. Finally, fluorescence analysis showed that the interactions between the boronated peptide Ac-Thr-Leu-Asn-PheB and HIV-1 protease resulted in a rapid decrease of fluorescence emission at 360 nm, which suggests the formation of a compound/enzyme complex. Boronated peptides may provide useful reagents for studying protease biochemistry and yield valuable information toward the development of protease dimerization inhibitors.

Boron Compounds↗

PubMed2003

A major role for a set of non-active site mutations in the development of HIV-1 protease drug resistance.

A major problem in the chemotherapy of HIV-1 infection is the appearance of drug resistance. In the case of HIV-1 protease inhibitors, resistance originates from mutations in the protease molecule that lower the affinity of inhibitors while still maintaining a viable enzymatic profile. Drug resistance mutations can be classified as active site or non-active site mutations depending on their location within the protease molecule. Active site mutations directly affect drug/target interactions, and their action can be readily understood in structural terms. Non-active site mutations influence binding from distal locations, and their mechanism of action is not immediately apparent. In this paper, we have characterized a mutant form of the HIV-1 protease, ANAM-11, identified in clinical isolates from HIV-1 infected patients treated with protease inhibitors. This mutant protease contains 11 mutations, 10 of which are located outside the active site (L10I/M36I/S37D/M46I/R57K/L63P/A71V/G73S/L90M/I93L) and 1 within the active site (I84V). ANAM-11 lowers the binding affinity of indinavir, nelfinavir, saquinavir, and ritonavir by factors of 4000, 3300, 5800, and 80000, respectively. Surprisingly, most of the loss in inhibitor affinity is due to the non-active site mutations as demonstrated by additional experiments performed with a protease containing only the 10 non-active site mutations (NAM-10) and another containing only the active site mutation (A-1). Kinetic analysis with two different substrates yielded comparable catalytic efficiencies for A-1, ANAM-11, NAM-10, and the wild-type protease. These studies demonstrate that non-active site mutations can be the primary source of resistance and that their role is not necessarily limited to compensate deleterious effects of active site mutations. Analysis of the structural stability of the proteases by differential scanning calorimetry reveals that ANAM-11 and NAM-10 are structurally more stable than the wild-type protease while A-1 is less stable. Together, the binding and structural thermodynamic results suggest that the non-active site mutants affect inhibitor binding by altering the geometry of the binding site cavity through the accumulation of mutations within the core of the protease molecule.

Binding Sites↗

PubMed1998

Structural basis for specificity of retroviral proteases.

The Rous sarcoma virus (RSV) protease S9 variant has been engineered to exhibit high affinity for HIV-1 protease substrates and inhibitors in order to verify the residues deduced to be critical for the specificity differences. The variant has 9 substitutions (S38T, I42D, I44V, M73V, A100L, V104T, R105P, G106V, and S107N) of structurally equivalent residues from HIV-1 protease. Unlike the wild-type enzyme, RSV S9 protease hydrolyzes peptides representing the HIV-1 protease polyprotein cleavage sites. The crystal structure of RSV S9 protease with the inhibitor, Arg-Val-Leu-r-Phe-Glu-Ala-Nle-NH2, a reduced peptide analogue of the HIV-1 CA-p2 cleavage site, has been refined to an R factor of 0.175 at 2.4-A resolution. The structure shows flap residues that were not visible in the previous crystal structure of unliganded wild-type enzyme. Flap residues 64-76 are structurally similar to residues 47-59 of HIV-1 protease. However, residues 61-63 form unique loops at the base of the flaps. Mutational analysis indicates that these loop residues are essential for catalytic activity. Side chains of flap residues His 65 and Gln 63' make hydrogen bond interactions with the inhibitor P3 amide and P4' carbonyl oxygen, respectively. Other interactions of RSV S9 protease with the CA-p2 analogue are very similar to those observed in the crystal structure of HIV-1 protease with the same inhibitor. This is the first crystal structure of an avian retroviral protease in complex with an inhibitor, and it verifies our knowledge of the molecular basis for specificity differences between RSV and HIV-1 proteases.

Aspartic Acid Endopeptidases↗

PubMed2001

Naturally occurring sequence polymorphisms within HIV type 1 group O protease.

Mutations within the protease gene associated with reduced susceptibility to protease inhibitors have been well documented for HIV-1 group M subtype B strains. In contrast, limited genotypic and phenotypic information is available for the genetically diverse HIV-1 group O strains. Preexisting resistance-associated polymorphisms have the potential to contribute to a poor virological response to antiviral drug treatment in group O-infected patients. In the present study, the protease genes of 28 protease inhibitor-naive HIV-1 group O-infected patients were analyzed to identify any naturally occurring amino acid polymorphisms associated with drug resistance. Comparison of the consensus group O protease sequence with subtype B of group M indicated that both groups have almost identical sequences in the protease active site, the flap and the substrate-binding site. Analysis of the 28 individual protease sequences revealed polymorphisms at 34% of the positions within the protease gene, but no primary mutations associated with protease inhibitor resistance. In contrast, each of the strains harbored multiple secondary or accessory mutations associated with resistance to protease inhibitors in group M viruses. Residues 10I, 15V, 36I, 41K, 62V, 63T/A/K/I, 64V, 71V, and 93L were identified in most strains. The presence of multiple natural sequence polymorphisms associated with drug resistance in the protease gene of group O viruses may contribute to a more rapid emergence of drug resistance phenotype and treatment failure in group O-infected patients.

Amino Acid Sequence↗

PubMed2000

Alteration of substrate and inhibitor specificity of feline immunodeficiency virus protease.

Feline immunodeficiency virus (FIV) protease is structurally very similar to human immunodeficiency virus (HIV) protease but exhibits distinct substrate and inhibitor specificities. We performed mutagenesis of subsite residues of FIV protease in order to define interactions that dictate this specificity. The I37V, N55M, M56I, V59I, and Q99V mutants yielded full activity. The I37V, N55M, V59I, and Q99V mutants showed a significant increase in activity against the HIV-1 reverse transcriptase/integrase and P2/nucleocapsid junction peptides compared with wild-type (wt) FIV protease. The I37V, V59I, and Q99V mutants also showed an increase in activity against two rapidly cleaved peptides selected by cleavage of a phage display library with HIV-1 protease. Mutations at Q54K, I98P, and L101I dramatically reduced activity. Mutants containing a I35D or I57G substitution showed no activity against either FIV or HIV substrates. FIV proteases all failed to cut HIV-1 matrix/capsid, P1/P6, P6/protease, and protease/reverse transcriptase junctions, indicating that none of the substitutions were sufficient to change the specificity completely. The I37V, N55M, M56I, V59I, and Q99V mutants, compared with wt FIV protease, all showed inhibitor specificity more similar to that of HIV-1 protease. The data also suggest that FIV protease prefers a hydrophobic P2/P2' residue like Val over Asn or Glu, which are utilized by HIV-1 protease, and that S2/S2' might play a critical role in distinguishing FIV and HIV-1 protease by specificity. The findings extend our observations regarding the interactions involved in substrate binding and aid in the development of broad-based inhibitors.

Amino Acid Sequence↗

PubMed1985

Protease nexin. Properties and a modified purification procedure.

The present paper describes chemical and functional properties of protease nexin, a serine protease inhibitor released from cultured human fibroblasts. It is shown that protease nexin is actually synthesized by fibroblasts and represents about 1% of their secreted protein. Analysis of the amino acid composition of purified protease nexin indicates that it is evolutionarily related to antithrombin III and heparin cofactor II. Protease nexin contains approximately 6% carbohydrate, with 2.3% amino sugar, 1.1% neutral sugar, and 3.0% sialic acid. The Mr calculated from equilibrium sedimentation analysis is 43,000. Protease nexin is a broad specificity inhibitor of trypsin-like serine proteases. It reacts rapidly with trypsin (kassoc = 4.2 +/- 0.4 X 10(6) M-1 s-1), thrombin (kassoc = 6.0 +/- 1.3 X 10(5) M-1 s-1), urokinase (kassoc = 1.5 +/- 0.1 X 10(5) M-1 s-1), and plasmin (kassoc = 1.3 +/- 0.1 X 10(5) M-1 s-1), and slowly inhibits Factor Xa and the gamma subunit of nerve growth factor but does not inhibit chymotrypsin-like proteases or leukocyte elastase. In the presence of heparin, protease nexin inhibits thrombin at a nearly diffusion-controlled rate. Two heparin affinity classes of protease nexin can be detected. The present characterization pertains to the fraction of protease nexin having the higher affinity for heparin. The low affinity material, which is the minor fraction, is lost during purification.

Amino Acids↗

PubMed1987

Studies of three major proteases associated with guinea pig sperm acrosomes.

The major proteases associated with guinea pig sperm were investigated by using immunological and electrophoretic techniques. Three major proteases were detected following sodium dodecyl sulfate-polyacryl-amide gel electrophoresis in gels containing 0.1% gelatin. These enzymes had molecular weights of 47,000, 34,000, and 32,000 relative to reduced protein standards and 58,500, 40,000, and 37,500 relative to unreduced standards. All three protease activities were present in acid extracts of sperm, detergent extracts of sperm, and the soluble acrosomal components of sperm released following induction of the acrosome reaction with the Ca2+-ionophore A23187. As determined by indirect immunofluorescence, an antibody to purified boar acrosin specifically cross-reacted with the acrosomes of guinea pig sperm. Decreased fluorescence was associated with sperm that had lost their acrosomes. Immunoblot analysis demonstrated that this antibody reacted with the 47,000 Mr protease but not the 32,000 and 34,000 Mr proteases. All three proteases were maximally active in the pH 7.0-8.5 region and were inhibited by classical inhibitors of acrosin activity. During a 3-hour incubation period, MgCl2 (10 mM) inhibited the activities of the 32,000 and 34,000 Mr proteases while the 47,000 Mr protease was stimulated. Although these proteases shared properties that would classify them as trypsin-like proteases, only the 47,000 Mr protease could be definitely classified as a member of the proacrosin-acrosin family based upon cross-reaction with an antibody to purified boar acrosin.

Acrosin↗

PubMed1999

Analysis of the effects of Perkinsus marinus proteases on plasma proteins of the Eastern oyster (Crassostrea virginica) and the Pacific oyster (Crassostrea gigas).

We employed two in vitro buffer systems to determine the potential pathogenic effects of Perkinsus marinus serine proteases on the plasma proteins of the eastern oyster (Crassostrea virginica) and the Pacific oyster (Crassostrea gigas). Specifically, this study characterized the oyster plasma protein targets of P. marinus proteases. Additionally, protease-specific inhibitory activity was revealed upon comparison of artificial (PBS) and endogenous (plasma-based) diluents employed during protease digestions. It was found that a C. virginica plasma protein of approximately 35 kDa was eliminated when a standard buffer (PBS) was used as a diluent; however, this protein was preserved when a low-molecular-weight, plasma-based, diluent was used. The results strongly indicate that low-molecular-weight inhibitors of P. marinus proteases are present in oyster plasma. A control (nonparasitic) serine protease, alpha-chymotrypsin, was employed to ascertain the specificity of the protease inhibitors. Although alpha-chymotrypsin possesses ample proteolytic activity for C. virginica plasma proteins, the anti-proteases could specifically inhibit only P. marinus proteases. Such specificity of anti-protease activity is not uncommon among low-molecular-weight serine proteases. The hemolymph target protein was isolated by 2D electrophoresis and isoelectrically isolated for further characterization by N-terminal amino acid sequencing.

Animals↗

PubMed1995

Elevated protease activities in human amnion and chorion correlate with preterm premature rupture of membranes.

OBJECTIVES: The mechanism(s) of preterm premature rupture of fetal membranes resulting in preterm birth remains unknown. Studies suggest that fetal membranes are susceptible to weakening by protease attack and that collagenases may be active at the site of rupture. In this study fetal membranes from women delivered after preterm premature rupture of membranes were compared with control membranes and analyzed qualitatively and quantitatively for protease activities. STUDY DESIGN: Fourteen membranes from women with preterm premature rupture of membranes and nine membranes from women delivered preterm without premature rupture of membranes or otherwise normal women delivered at term vaginally or by cesarean section were studied. Zymogram gel electrophoresis with gelatin incorporation was used to assess the number and apparent molecular weights of protease activities. Functional and quantitative studies of protease activity were measured by fluorescent substrate cleavage. RESULTS: Zymogram gel electrophoresis studies demonstrated the presence of five to seven different protease bands in preterm premature rupture of membranes samples, whereas control membranes demonstrated only one to three protease bands. Fluorescent studies of protease activity demonstrated a 10- to 40-fold increase in activity in membranes from women with preterm premature rupture of membranes compared with normal control membranes. Studies with protease inhibitors suggest that most of the activity is due to metalloproteinases. CONCLUSION: In membranes from women with preterm premature rupture of membranes there appears to be a general increase in the amount of protease activity and increased numbers of putatively different proteases. Increased activity or deregulated protease control may mediate preterm premature rupture of membranes and be a potentially remediable cause of preterm birth.

Amnion↗