Biomedical subjects

J C Guillemot

Publications and source records attributed to J C Guillemot.

At least 19 recordsLinked to original sources

PubMed2001

Exploring the acceptor substrate recognition of the human beta-galactoside alpha 2,6-sialyltransferase.

Human beta1,4-galactoside alpha2,6-sialyltransferase I (ST6GalI) recognition of glycoprotein acceptors has been investigated using various soluble forms of the enzyme deleted to a variable extent in the N-terminal half of the polypeptide. Full-length and truncated forms of the enzyme have been investigated with respect to their specificity for a variety of desialylated glycoproteins of known complex glycans as well as related proteins with different carbohydrate chains. Differences in transfer efficiency have been observed between membrane and soluble enzymatic forms, indicating that deletion of the transmembrane fragment induces loss of acceptor preference. No difference in substrate recognition could be observed when soluble enzymes of similar peptide sequence were produced in yeast or mammalian cells, confirming that removal of the membrane anchor and heterologous expression do not alter enzyme folding and activity. When tested on free oligosaccharides, soluble ST6GalI displayed full ability to sialylate free N-glycans as well as various N-acetyllactosaminyl substrates. Progressive truncation of the N terminus demonstrated that the catalytic domain can proceed with sialic acid transfer with increased efficiency until 80 amino acids are deleted. Fusion of the ST6GalI catalytic domain to the N-terminal half of an unrelated transferase (core 2 beta1,6-N-acetylglucosaminyltransferase) further showed that a chimeric form of broad acceptor specificity and high activity could also be engineered in vivo. These findings therefore delineate a peptide region of approximately 50 amino acids within the ST6GalI stem region that governs both the preference for glycoprotein acceptors and catalytic activity, thereby suggesting that it may exert a steric control on the catalytic domain.

Animals↗

PubMed2001

Ep-CAM transfection in thymic epithelial cell lines triggers the formation of dynamic actin-rich protrusions involved in the organization of epithelial cell layers.

Thymic epithelium is organized in a highly connected three-dimensional network through which thymocytes differentiate. The molecular mechanisms underlying this organization are still unknown. In thymic medulla, a major site of tolerance induction, the development of the epithelial cell net is tightly regulated by the needs of thymocyte selection. These reticulated epithelial cells express high levels of the Ep-CAM molecule. Using different thymic epithelial cell lines as a model system, we found that transfection of Ep-CAM enhances cell growth and leads to a rapid reorganization of the actin cytoskeleton by inducing the formation of numerous stress fibers and long cell protrusions. Finally, the crosslinking of the extracellular domain of a chimeric CD25ec/Ep-CAMic molecule is sufficient to trigger the formation of protrusions. These results suggest that expression of Ep-CAM might balance the organizing capacity of cadherin molecules and may be participating in the formation of a dynamic stromal cell network in the thymus.

Actins↗

PubMed2000

Innate recognition of bacteria in human milk is mediated by a milk-derived highly expressed pattern recognition receptor, soluble CD14.

Little is known about innate immunity to bacteria after birth in the hitherto sterile fetal intestine. Breast-feeding has long been associated with a lower incidence of gastrointestinal infections and inflammatory and allergic diseases. We found in human breast milk a 48-kD polypeptide, which we confirmed by mass spectrometry and sequencing to be a soluble form of the bacterial pattern recognition receptor CD14 (sCD14). Milk sCD14 (m-sCD14) concentrations were up to 20-fold higher than serum sCD14 from nonpregnant, pregnant, or lactating women. In contrast, lipopolysaccharide (LPS)-binding protein was at very low levels. Mammary epithelial cells produced 48-kD sCD14. m-sCD14 mediated activation by LPS and whole bacteria of CD14 negative cells, including intestinal epithelial cells, resulting in release of innate immune response molecules. m-sCD14 was undetectable in the infant formulas and commercial (cows') milk tested, although it was present in bovine colostrum. These findings indicate a sentinel role for sCD14 in human milk during bacterial colonization of the gut, and suggest that m-sCD14 may be involved in modulating local innate and adaptive immune responses, thus controlling homeostasis in the neonatal intestine.

Amino Acid Sequence↗

PubMed2000

Factor Xa activates endothelial cells by a receptor cascade between EPR-1 and PAR-2.

In addition to its pivotal role in hemostasis, factor Xa binds to human umbilical vein endothelial cells through the recognition of a protein called effector cell protease receptor (EPR-1). This interaction is associated with signal transduction, generation of intracellular second messengers, and modulation of cytokine gene expression. Inhibitors of factor Xa catalytic activity block these responses, thus indicating that the factor Xa-dependent event of local proteolysis is absolutely required for cell activation. Because EPR-1 does not contain proteolysis-sensitive sites, we investigated the possibility that signal transduction by factor Xa requires proteolytic activation of a member of the protease-activated receptor (PAR) gene family. Catalytic inactivation of factor Xa by DX9065 suppressed factor Xa-induced increase in cytosolic free Ca(2+) in endothelial cells (IC(50)=0.23 micromol/L) but failed to reduce ligand binding to EPR-1. In desensitization experiments, trypsin or the PAR-2-specific activator peptide, SLIGKV, ablated the Ca(2+) signaling response induced by factor Xa. Conversely, pretreatment of endothelial cells with factor Xa blocked the PAR-2-dependent increase in cytosolic Ca(2+) signaling, whereas PAR-1-dependent responses were unaffected. Direct cleavage of PAR-2 by factor Xa on endothelial cells was demonstrated by cleavage of a synthetic peptide duplicating the PAR-2 cleavage site and by immunofluorescence with an antibody to a peptide containing the 40-amino acid PAR-2 extracellular extension. These data suggest that factor Xa induces endothelial cell activation via a novel cascade of receptor activation involving docking to EPR-1 and local proteolytic cleavage of PAR-2.

Amino Acid Sequence↗

PubMed1999

Human 76p: A new member of the gamma-tubulin-associated protein family.

The role of the centrosomes in microtubule nucleation remains largely unknown at the molecular level. gamma-Tubulin and the two associated proteins h103p (hGCP2) and h104p (hGCP3) are essential. These proteins are also present in soluble complexes containing additional polypeptides. Partial sequencing of a 76- kD polypeptide band from these complexes allowed the isolation of a cDNA encoding for a new protein (h76p = hGCP4) expressed ubiquitously in mammalian tissues. Orthologues of h76p have been characterized in Drosophila and in the higher plant Medicago. Several pieces of evidence indicate that h76p is involved in microtubule nucleation. (1) h76p is localized at the centrosome as demonstrated by immunofluorescence. (2) h76p and gamma-tubulin are associated in the gamma-tubulin complexes. (3) gamma-tubulin complexes containing h76p bind to microtubules. (4) h76p is recruited to the spindle poles and to Xenopus sperm basal bodies. (5) h76p is necessary for aster nucleation by sperm basal bodies and recombinant h76p partially replaces endogenous 76p in oocyte extracts. Surprisingly, h76p shares partial sequence identity with human centrosomal proteins h103p and h104p, suggesting a common protein core. Hence, human gamma-tubulin appears associated with at least three evolutionary related centrosomal proteins, raising new questions about their functions at the molecular level.

Amino Acid Sequence↗

PubMed1999

Inducible recruitment of Cdc42 or WASP to a cell-surface receptor triggers actin polymerization and filopodium formation.

BACKGROUND: Cdc42, a GTP-binding protein of the Rho family, controls actin cytoskeletal organization and helps to generate actin-based protruding structures, such as filopodia. In vitro, Cdc42 regulates actin polymerization by facilitating the creation of free barbed ends - the more rapidly growing ends of actin filaments - and subsequent elongation at these ends. The Wiskott- Aldrich syndrome protein, WASP, which has a pleckstrin-homology domain and a Cdc42/Rac-binding motif, has been implicated in cell signaling and cytoskeleton reorganization. We have investigated the consequences of local recruitment of activated Cdc42 or WASP to the plasma membrane. RESULTS: We used an activated Cdc42 protein that could be recruited to an engineered membrane receptor by adding rapamycin as a bridge, and added antibody-coupled beads to aggregate these receptors. Inducible recruitment of Cdc42 to clusters of receptors stimulated actin polymerization, resulting in the formation of membrane protrusions. Cdc42-induced protrusions were enriched in the vasodilator-stimulated phosphoprotein VASP and the focal-adhesion-associated proteins zyxin and ezrin. The Cdc42 effector WASP could also induce the formation of protrusions, albeit of different morphology. CONCLUSIONS: This is the first demonstration that the local recruitment of activated Cdc42 or its downstream effector, WASP, to a membrane receptor in whole cells is sufficient to trigger actin polymerization that results in the formation of membrane protrusions. Our data suggest that Cdc42-induced actin-based protrusions result from the local and serial recruitment of cytoskeletal proteins including zyxin, VASP, and ezrin.

Actins↗

PubMed1999

Antibody neutralization of HIV-1 and the potential for vaccine design.

Neutralisation by antibody is, for a number of viruses, an in vitro correlate for protection in vivo. For HIV-1 this is controversial. However, the induction of a potent anti-HIV neutralising antibody response remains one of the principal goals in vaccine development. A greater knowledge of the fundamental mechanisms underlying the neutralisation process would help direct research towards suitable vaccine immunogens. The primary determinant of HIV neutralisation appears to be antibody affinity for the trimeric envelope glycoprotein spike on the virion, suggesting that epitope-specific effects are secondary and implying a single, dominant mechanism of neutralisation. Antibody interference with virion attachment to the target cell appears to be a major mechanism of neutralisation by gp120-specific antibodies. This is probably achieved both by antibody-induced dissociation of gp120 from gp41 and by direct inhibition of virus binding to receptor-coreceptor complexes. A gp41-specific antibody neutralises by interfering with post-attachment steps leading to virus membrane fusion. Recent advances in structural analyses of the HIV envelope glycoproteins coupled with data obtained from antibody mapping and neutralisation studies allow a greater understanding of Env function and its inhibition. This in turn should lead to a more rational basis for vaccine design aimed at stimulating highly effective neutralising antibodies.

AIDS Vaccines↗

PubMed1998

Fc receptor-mediated phagocytosis requires CDC42 and Rac1.

At the surface of phagocytes, antibody-opsonized particles are recognized by surface receptors for the Fc portion of immunoglobulins (FcRs) that mediate their capture by an actin-driven process called phagocytosis which is poorly defined. We have analyzed the function of the Rho proteins Rac1 and CDC42 in the high affinity receptor for IgE (FcepsilonRI)-mediated phagocytosis using transfected rat basophil leukemia (RBL-2H3) mast cells expressing dominant inhibitory forms of CDC42 and Rac1. Binding of opsonized particles to untransfected RBL-2H3 cells led to the accumulation of F-actin at the site of contact with the particles and further, to particle internalization. This process was inhibited by Clostridium difficile toxin B, a general inhibitor of Rho GTP-binding proteins. Dominant inhibition of Rac1 or CDC42 function severely inhibited particle internalization but not F-actin accumulation. Inhibition of CDC42 function resulted in the appearance of pedestal-like structures with particles at their tips, while particles bound at the surface of the Rac1 mutant cell line were enclosed within thin membrane protrusions that did not fuse. These phenotypic differences indicate that Rac1 and CDC42 have distinct functions and may act cooperatively in the assembly of the phagocytic cup. Inhibition of phagocytosis in the mutant cell lines was accompanied by the persistence of tyrosine-phosphorylated proteins around bound particles. Phagocytic cup closure and particle internalization were also blocked when phosphotyrosine dephosphorylation was inhibited by treatment of RBL-2H3 cells with phenylarsine oxide, an inhibitor of protein phosphotyrosine phosphatases. Altogether, our data show that Rac1 and CDC42 are required to coordinate actin filament organization and membrane extension to form phagocytic cups and to allow particle internalization during FcR-mediated phagocytosis. Our data also suggest that Rac1 and CDC42 are involved in phosphotyrosine dephosphorylation required for particle internalization.

Animals↗

PubMed1998

Characterization of brain PCTAIRE-1 kinase immunoreactivity and its interactions with p11 and 14-3-3 proteins.

An antibody directed against the C-terminal part of PCTAIRE-1 recognized three proteins in rodent brain. The high-molecular-mass band is most abundant in the cerebellum, hippocampus and cortex. It migrated at the same apparent molecular mass as recombinant PCTAIRE-1 and interacted, like recombinant PCTAIRE-1, with p11 and 14-3-3 proteins. Combination of p11 or 14-3-3 affinity resins with immunoprecipitation and peptide elution allowed us to obtain a purified full-length PCTAIRE-1 preparation having significant kinase activity. These results suggest that PCTAIRE-1 is an active kinase in brain. The catalytic core region of PCTAIRE-1 which is common for all cyclin-dependent kinases, does not interact with p11 and 14-3-3 proteins in the two-hybrid assay. Full interaction with p11 and 14-3-3 proteins requires both, the N-terminal and C-terminal ends of PCTAIRE-1, suggesting that complex three-dimensional arrangements are responsible for these interactions. A low-molecular-mass protein (migrating at about 30 kDa) that was also recognized by the antibody directed against the carboxy-terminal part of PCTAIRE-1, is abundant and almost homogeneously distributed in all brain areas investigated. Database searches starting with the amino acid sequences of two peptides obtained by tryptic digestion of this protein yielded cDNA and genomic (a gene of about 10 kb on human chromosome 1q24-1q25 and clone 262D12) sequences, allowing us to compose a DNA sequence coding for a putative 26 kDa protein containing both peptides. This protein has no important sequence similarity with any other known protein. But many DNA sequences are found in databases with an almost 100% identity with parts of the 26 kDa protein coding sequence. Our results allow us to attribute these widely distributed cDNA sequences to an existing 26-kDa protein and to localize a gene within two recently published genomic sequences.

14-3-3 Proteins↗

PubMed1998

Tyrosine phosphorylation of the Wiskott-Aldrich syndrome protein by Lyn and Btk is regulated by CDC42.

The Wiskott-Aldrich syndrome (WAS) is a rare immunodeficiency disease affecting mainly platelets and lymphocytes. Here, we show that the WAS gene product, WASp, is tyrosine phosphorylated upon aggregation of the high affinity IgE receptor (Fc epsilonRI) at the surface of RBL-2H3 rat tumor mast cells. Lyn and the Bruton's tyrosine kinase (Btk), two protein tyrosine kinases involved in Fc epsilonRI-signaling phosphorylate WASp and interact with WASp in vivo. Interestingly, expression of a GTPase defective mutant form of CDC42, that interacts with WASp, is accompanied by a substantial increase in WASp tyrosine phosphorylation. This study suggests that activated CDC42 recruits WASp to the plasma membrane where it becomes phosphorylated by Lyn and Btk. We conclude that WASp represents a connection between protein tyrosine kinase signaling pathways and CDC42 function in cytoskeleton and cell growth regulation in hematopoietic cells.

Agammaglobulinaemia Tyrosine Kinase↗

PubMed1998

Microsomal epoxide hydrolase of rat liver is a subunit of theanti-oestrogen-binding site.

A tritiated photoaffinity labelling analogue of tamoxifen, [(2-azido-4-benzyl)-phenoxy]-N-ethylmorpholine (azido-MBPE), was used to identify the anti-oestrogen-binding site (AEBS) in rat liver tissue [Poirot, Chailleux, Fargin, Bayard and Faye (1990) J. Biol. Chem. 265, 17039-17043]. UV irradiation of rat liver microsomal proteins incubated with tritiated azido-MBPE led to the characterization of two photolabelled proteins of molecular masses 40 and 50 kDa. The amino acid sequences of proteolytic products from the 50 kDa protein were identical with those from rat microsomal epoxide hydrolase (mEH). Treatment of hepatocytes with anti-sense mRNA directed against mEH abolished AEBS in these cells. In addition we found that tamoxifen and N-morpholino-2-[4-(phenylmethyl)phenoxy]ethanamine, a selective ligand of AEBS, were potent inhibitors of the catalytic hydration of styrene oxide by mEH. However, functional overexpression of the human mEH did not significantly modify the binding capacity of [3H]tamoxifen. Taken together, these results suggest that the 50 kDa protein, mEH, is necessary but not sufficient to reconstitute AEBS.

Affinity Labels↗

PubMed1998

Intramolecular masking of nuclear import signal on NF-AT4 by casein kinase I and MEKK1.

T cell activation requires the import of NF-AT transcription factors to the nucleus, a process promoted by calcineurin-dependent dephosphorylation and inhibited by poorly understood protein kinases. Here, we report the identification of two protein kinases that oppose NF-AT4 nuclear import. Casein kinase Ialpha directly binds and phosphorylates NF-AT4, resulting in the inhibiton of NF-AT4 nuclear translocation. MEKK1 indirectly suppresses NF-AT4 nuclear import by stabilizing the interaction between NF-AT4 and CKIalpha. CKIalpha thus acts to establish an intramolecular masking of the nuclear location signal on NF-AT4, while MEKK1 augments this mechanism, and may further provide a link to signal transduction pathways regulating NF-AT4.

Amino Acid Sequence↗

PubMed1997

Purification and characterization of the human SR 31747A-binding protein. A nuclear membrane protein related to yeast sterol isomerase.

SR 31747A, defined as a sigma ligand, is a novel immunosuppressive agent that blocks proliferation of human and mouse lymphocytes. Using a radiolabeled chemical probe, we here purified a target of SR 31747A and called it SR 31747A-binding protein (SR-BP). Purified SR-BP retained its binding properties and migrated on SDS-polyacrylamide gel as a Mr 28,000 protein. Cloning of the cDNA encoding human SR-BP shows an open reading frame for a 223-amino acid protein, which is homologous to the recently cloned sigma 1 receptor. Interestingly, the deduced amino acid sequence was found to be related to fungal C8-C7 sterol isomerase, encoded by the ERG2 gene. The ERG2 gene product has been identified recently as the molecular target of SR 31747A that mediates antiproliferative effects of the drug in yeast. Northern blot analysis of SR-BP gene expression revealed a single transcript of 2 kilobases which was widely expressed among organs, with the highest abundance in liver and the lowest abundance in brain. Subcellular localization analysis in various cells, using a specific monoclonal antibody raised against SR-BP, demonstrated that this protein was associated with the nuclear envelope. When studying the binding of SR 31747A on membranes from yeast expressing SR-BP, we found a pharmacological profile of sigma 1 receptors; binding was displaced by (+)-pentazocine, haloperidol, and (+)-SKF 10,047, with (+)-SKF 10, 047 being a more potent competitor than (-)-SKF 10,047. Scatchard plot analysis revealed Kd values of 7.1 nM and 0.15 nM for (+)-pentazocine and SR 31747A, respectively, indicating an affinity of SR-BP 50-fold higher for SR 31747A than for pentazocine. Additionally, we showed that pentazocine, a competitive inhibitor of SR 31747A binding, also prevents the immunosuppressive effect of SR 31747A. Taken together, these findings strongly suggest that SR-BP represents the molecular target for SR 31747A in mammalian tissues, which could be critical for T cell proliferation.

Amino Acid Sequence↗

PubMed1997

Protein depletion and refeeding change the proportion of mouse liver glutathione S-transferase subunits.

The effect of protein depletion followed by refeeding with a normal diet on the content of mouse liver cytosolic proteins was studied. By peptide-mass fingerprinting and N-terminal sequencing, three polypeptides whose contents changed with dietary protein level were identified as glutathione S-transferases (GST) Yb1, Yc and Yf subunits. Five days of depletion caused the increase of Yb1 and Yf (21.6% and 78.5%, respectively) and the decrease of Yc (31.2%). After two days of refeeding, Yb1 and Yc were practically restored, while the neoplastic marker Yf remained higher (63.4%). None of the nutritional conditions tested induced new GSTs. While protein depletion-refeeding altered the ratios between the constitutive GST subunits, total liver GST content and activity were unaffected by depletion and slightly increased by refeeding. The increased amounts of Yb1 and Yf, and the maintenance of total GST content, indicate that during protein depletion, the GST subunits levels are controlled by mechanisms different from the majority of cytosolic proteins.

Amino Acid Sequence↗

PubMed1997

Determination of the three-dimensional structure of CC chemokine monocyte chemoattractant protein 3 by 1H two-dimensional NMR spectroscopy.

MCP-3 is a beta chemokine consisting of 76 amino acid residues. It has been described to be involved in the activation of all leukocytic cells, activation mediated by the presence of multiple binding sites on the target cells. Its three-dimensional structure has been studied by making use of two-dimensional 1H NMR spectroscopy. MCP-3 exhibits the same monomeric structure as the other chemokines, i.e., a three-stranded antiparallel beta sheet covered on one face by an alpha helix. Although it belongs to the same subfamily as RANTES (Chung et al., 1995; Faitbrother et al., 1994) and hMIP-1beta (Lodi et al., 1994), the MCP-3 dimer is folded like IL-8 with the so-called alphabeta sandwich structural motif. Structural and sequence analysis gives clear indications suggesting that the other MCP chemokines may have the same quaternary structure, contrary to the other beta chemokines.

Amino Acid Sequence↗

PubMed1997

Selective control of membrane ruffling and actin plaque assembly by the Rho GTPases Rac1 and CDC42 in FcepsilonRI-activated rat basophilic leukemia (RBL-2H3) cells.

Engagement of the high affinity IgE receptor (FcepsilonRI) in mast cells elicits a series of intracellular signalling events including cytoskeletal reorganization and granule exocytosis. To analyze the coupling of receptor activation to specific cytoskeletal responses, we expressed dominant negative mutant forms of the Rho GTPases CDC42 and Rac1 in rat RBL-2H3 tumor mast cells. We show here that dominant inhibition of CDC42 function decreases cell adhesion, interferes with Fc(epsilon)RI-induced actin plaque assembly and reduced the recruitment of vinculin at the cell-substratum interface, while the inhibitory Rac1 mutant abolishes Fc(epsilon)RI-mediated membrane ruffling. The expression of trans-dominant inhibitory forms of either CDC42 or Rac1 significantly inhibited antigen-induced degranulation. Altogether, our results demonstrate that CDC42 and Rac1 control distinct pathways downstream of FcepsilonRI engagement leading either to the induction of actin plaques, or to the production of membrane ruffles. These two pathways are critically involved during the degranulation response induced by Fc(epsilon)RI aggregation.

Actins↗

PubMed1996

Targeted disruption of guanosine diphosphate-dissociation inhibitor for Rho-related proteins, GDID4: normal hematopoietic differentiation but subtle defect in superoxide production by macrophages derived from in vitro embryonal stem cell differentiation.

The Rho subfamily of small guanosine triphosphate (GTP)-binding proteins, through their role in cytoskeletal organization, is involved in diverse cellular functions, including cell motility and morphologic changes during differentiation. Rac also has a special role in the production of superoxide, a key component in phagocytic antimicrobial function. Guanosine diphosphate (GDP)-dissociation inhibitors (GDIs) belong to one of three classes of proteins that regulate the critical cycling of GTP-binding proteins between the inactive and active states. Two homologous GDIs for the Rho subfamily have been identified. GDID4 is preferentially expressed in hematopoietic cells, while RhoGDI is ubiquitously expressed. Whether different physiologic functions are subserved by the two GDIs is unknown. We have derived embryonal stem (ES) cells with targeted disruption of both alleles of the GDID4 gene and examined hematopoiesis and phagocytic functions of macrophages derived from in vitro ES-cell differentiation. GDID4-/- ES cells develop like wild-type cells into colonies that contain heterogeneous populations of progenitor cells and differentiated erythromyeloid cells. GDID4-/- cells express no GDID4 protein, but have normal levels of RhoGDI. GDID4-/- macrophages phagocytose yeasts and antibody-opsonized erythrocytes as effectively as wild-type macrophages. However, a slight but consistent reduction in their capacity to generate superoxide was observed, which suggests new insight into the cellular role of GDID4. The minimal phenotypic effect of a loss of function of GDID4 also indicates a significant redundancy of function between GDID4 and RhoGDI. Their functional repertoire may be better revealed by a disruption of both genes. The use of hematopoietic cells derived in vitro from genotypically altered ES cells avoids the difficulties inherent in generating knockout animals and is a useful complementary approach for evaluating the gene function.

Animals↗

PubMed1996

Purification and characterization of a novel protease from culture filtrates of a Streptomyces sp.

A fibrinolytic protease has been isolated from Streptomyces sp. culture filtrate by successive chromatography on Mono S and Sephadex G50. The purified protease had a molecular mass of 33 kDa and had an isoelectric point of 6.7. It showed a sharp pH optimum at 7.8 with maximal protease activity between 35 degrees C and 50 degrees C. Its amino acid composition and amino-terminal sequence (17 residues) were determined. The protein exhibited marked hydrolytic activity toward the substrates N-Succ-(Ala)2-Pro-Phe-pNA (K(m) = 0.77 mM, Vmax = 24.2 mumol mg-1 min-1) and N-Succ-(Ala)2-Pro-Leu-pNA (K(m) = 0.92 mM, Vmax = 7.7 mumol mg-1 min-1). It was totally inhibited by alpha 1-antitrypsin, D-Phe-Pro-Arg-chloromethylketone and sodium dodecyl sulfate but was insensitive to EDTA, dithiothreitol, phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, pepstatin or elastatinal. In this respect, this protease differed in its physico-chemical and biochemical properties from other extracellular proteases previously found in bacteria and fungi. The results suggest that it has properties of chymotrypsin-like serine-type proteases.

Amino Acid Sequence↗