Main Proteinases Research Articles

Transcriptional expression profiles of the main proteinases and their regulators in coronary artery ectasia patients' mononuclear cells.

Proteolytic enzymes might contribute to coronary artery ectasia (CAE) through the destruction of extracellular matrix (ECM) vessel components. This study aimed to find out if peripheral blood mononuclear cells (PBMCs) served as a source of those proteinases and their regulators. In this study, transcriptional expression profiles of the main proteinases and their regulators were detected in the PBMCs of CAE patients as follows: (1) matrix metalloproteinases (MMP) 1, 2, 3, 8, 9, 10, 12 and 13; (2) the serine proteinases elastase: cathepsin G and proteinases 3; (3) the cysteine proteinases: cathepsin L and cathepsin S; (4) the main endogenous inhibitors for the above proteinases: tissue inhibitors of metalloproteinase (TIMP) 1 and 2, α1-antitrypsin (α1-PI) and α2-macroglobulin (A2M); (5) twelve cytokines that could regulate the above proteinases. The characteristic changes in CAE were: (1) MMP1 and MMP9 increased while the serine and cysteine families did not change; (2) the four proteinase inhibitors did not change in the CAE group; (3) among the 12 cytokines, interleukin-1 alpha (IL1A), platelet-derived growth factor (PDGF), interferon gamma (IFNγ) and growth differentiation factor 15 (GDF15) were elevated. Partial correlation analysis showed that MMP1 significantly correlated with IL1A and with IFNγ, and MMP9 correlated with IFNγ and with GDF 15. PBMCs might participate in the pathological process of CAE by the increased expression of MMP1, MMP9, IL1A, IFNγ and GDF15.

Efficacy of horse chestnut leaf extract ALH-L1005 as a matrix metalloproteinase inhibitor in ligature-induced periodontitis in canine model.

Matrix metalloproteinases (MMPs) are the main proteinases associated with periodontal tissue destruction and remodeling. Therefore, inhibition of host-derived MMPs has a key role in the prevention and reduction of periodontitis progression. Horse chestnut (Aesculus hippocastanum L.) extracts have been used as treatments for inflammatory disease, traditionally. This study assessed the clinical effect as a MMP inhibitor of horse chestnut leaf extract ALH-L1005 on periodontitis. ALH-L1005 was obtained from horse chestnut leaf and its MMP inhibitory activities estimated. Periodontitis was induced in beagles assigned to 4 groups and medicated for 6 weeks: low dose test (LT; ALH-L1005, 100 mg/kg/day), high dose test (HT; ALH-L1005, 200 mg/kg/day), positive control (PC; doxycycline, 10 mg/kg/day), or negative control (NC; placebo). Before and after administration, clinical indices of the teeth and MMP quantity in gingival tissues using zymography were measured. Clinical conditions of the LT, HT, and PC groups were significantly improved after 6 weeks. In zymographic evaluations, gelatinolytic and caseinolytic activities were suppressed in LT, HT, and PC groups but not in the NC group. The results suggest that ALH-L1005 could be an effective agent for clinical prevention and treatment of periodontitis by inhibiting the gelatinase and collagenase activities, which can detach periodontal ligaments from alveolar bone.

Transcriptional expression profiles of the main proteinases and their regulators in coronary artery ectasia patients’ mononuclear cells

Objective Proteolytic enzymes might contribute to coronary artery ectasia (CAE) through the destruction of extracellular matrix (ECM) vessel components. This study aimed to find out if peripheral blood mononuclear cells (PBMCs) served as a source of those proteinases and their regulators.Method In this study, transcriptional expression profiles of the main proteinases and their regulators were detected in the PBMCs of CAE patients as follows: (1) matrix metalloproteinases (MMP) 1, 2, 3, 8, 9, 10, 12 and 13; (2) the serine proteinases elastase: cathepsin G and proteinases 3; (3) the cysteine proteinases: cathepsin L and cathepsin S; (4) the main endogenous inhibitors for the above proteinases: tissue inhibitors of metalloproteinase (TIMP) 1 and 2, α 1-antitrypsin (α 1-PI) and α 2-macroglobulin (A2M); (5) twelve cytokines that could regulate the above proteinases.Result The characteristic changes in CAE were: (1) MMP1 and MMP9 increased while the serine and cysteine families did not change; (2) the four proteinase inhibitors did not change in the CAE group; (3) among the 12 cytokines, interleukin-1 alpha (IL1A), platelet-derived growth factor (PDGF), interferon gamma (IFNγ) and growth differentiation factor 15 (GDF15) were elevated. Partial correlation analysis showed that MMP1 significantly correlated with IL1A and with IFNγ, and MMP9 correlated with IFN γ and with GDF 15.Conclusion PBMCs might participate in the pathological process of CAE by the increased expression of MMP1, MMP9, IL1A, IFN γ and GDF15.

Growth Differentiation Factor 15 Inhibits Elevated Expression of Matrix Metalloproteinase 1 Induced by Pooled Serum in Patients with Coroanry Artery Ectasia

To observe the effects of coroanry artery ectasia (CAE) patients' pooled serum on the main proteinases and extracellular matrix (ECM) synthesis and explore whether the growth differentiation factor 15(GDF 15) can regulate the characteristic changes induced by CAE patients' pooled serum. Serum samples were collected from 32 CAE patients, 30 patients with coronary heart disease (CHD), and 31 subjects with normal coronary arteries (CON) and then mixed in the same volumes by groups. Then human umbilical vein smooth muscle cells were cultured with the media containing 25% pooled serum. After having been disposed, proteinase system and ECM synthesis system were detected in the cell and culture media samples. GDF15 or GDF15 antibodies was added into the 25% pooled serum in each group to observe if GDF 15 could impact the characteristic changes induced by CAE patients' pooled serum. The expression of matrix metalloproteinases (MMP) 1 mRNA in CAE group was significantly higher than CON group (P=0.002) and CHD group (P=0.000), the secretory MMP1 protein and total MMPs activity in culture media were also upregulated in CAE group (both P<0.01). After adding GDF 15 into the culture media (GDF15+CAE group), the MMP1 mRNA ,secretory MMP1 protein, and total MMPs activity were significantly lower than CAE group (all P<0.01), while in the GDF15 antibody+CAE group, the MMP1 mRNA and total MMPs activities were significantly higher than in GDF15+CAE group (both P<0.01), but the secretory MMP1 protein was not different from GDF 15+CAE group (P>0.05). The vascular smooth muscle cells may participate in the CAE process mainly by regulating MMPs system but not the elastase 2 or ECM synthesis system, and GDF15 may be an compensatory factor to prohibit the over-destruction of coronary ECM induced by MMPs.

Partial characterization of alkaline proteases from viscera of vermiculated sailfin catfish Pterygoplichthys disjunctivus Weber, 1991

Vermiculated sailfin catfish (Pterygoplichthys disjunctivus, Weber, 1991), a member of the Loricariidae family and an invasive species of several inland waters around the world, possess an enormous digestive tract representing about 10% of fish weight. Thus, the aim of this study was to partially characterize proteases from their digestive tracts. Azocasein digestion of the crude extract of intestine at different pH values and temperatures revealed the presence of alkaline proteases with optimum activities at pH 9.0 and 50°C. Incubation assays of the crude extract with inhibitors such as phenyl methyl sulfonyl fluoride, N-α-p-tosyl-l-lysine chloromethyl ketone, N-tosyl-phenyalanine chloromethyl ketone, benzamidine, pepstatin A and ethylenediamine tetra-acetic acid showed that trypsin and chymotrypsin are the main alkaline proteinases present. Zymography showed that the crude extract of Pterygoplichthysdisjunctivus viscera contained proteases with molecular masses ranging from 21.5 to 116 kDa. Trypsin and chymotrypsin were inhibited by the following ions in decreasing order: Hg2+, Fe2+, Cu2+, Li+, Mg2+, K+, while Mn2+, and Ca2+ had no effect. Activities decreased continuously as the NaCl concentration increased from 0 to 30%. These results constitute important background information for future studies and for the potential biotechnological use of the crude digestive extract from this invasive species.

Altered cysteine proteinase activity in insecticide-resistant strains of the maize weevil: Purification and characterization

Insecticide resistance is usually associated with fitness costs, but such costs may be mitigated by increased energy and amino acid accumulation and mobilization as has been suggested in the maize weevil Sitophilus zeamais (Coleoptera: Curculionidae). To address this adaptation, cysteine proteinases (E.C. 3.4.22), one of the main proteinases in weevils, was purified from an insecticide-susceptible and two insecticide-resistant strains of the maize weevil (one with fitness costs, referred as resistant-cost, and the other without it, referred to as resistant no-cost) using thiol-sepharose affinity chromatography. Purification of the cysteine proteinases revealed a single 74,000 Da molecular mass band in the susceptible strain, two bands of 72,000 and 83,000 Da in the resistant cost strain, and two bands of 68,000 and 74,000 Da in the resistant no-cost strain. Purified cysteine proteinases of the three strains behaved differently regarding casein degradation and inhibition; the proteinases least sensitive to inhibition by the specific cysteine proteinase inhibitor E-64 were those from the resistant no-cost strain as indicated by their highest I(50) value. The pH and temperature profile of cysteine proteinase activity differed among strains and although substrate affinity (i.e. K(M)) of the cysteine proteinases was similar, the V(max) value for cysteine-proteinases from the resistant cost strain was 3-fold and 5-fold higher than V(max) values for the resistant no-cost and susceptible strains respectively. Cysteine proteinase activity was highest for the resistant cost strain rather than the resistant no-cost. Therefore enhanced cysteine proteinase activity is unlikely to be playing significant role in mitigating the costs usually associated with insecticide resistance.

Digestive enzymes and in-vitro digestibility of different species of phytoplankton for culture of the freshwater pearl mussel, Hyriopsis (Hyriopsis) bialatus

Hyriopsis (Hyriopsis) bialatus has been cultured during the mussel life cycle from glochidia to the adult stage with a low total survival of 6% up to 130-day-old juveniles. The main digestive enzymes (amylase and proteinases) were not detectable in one-day-old juveniles, and increased during development. The stomach, including diges- tive glands, was the major digestive organ for both carbohydrate and protein. The optimum conditions for amylase activity were 40C and pH 7; for acidic proteinases they were 60C and pH 5. Two main alkaline proteinases were found in the intestine, with optimum conditions of 30C and pH 8 and 60C and pH 8. To improve mussel survival by finding suitable phytoplankton species and age as food for juveniles and adults, an in-vitro digestibility test was performed on ten algal species three and seven days old using amylase and proteinases in crude enzyme extracts from different mussel life stages. Among the phytoplankton selected, the three most efficiently digested by juveniles were seven- day-old Chlorella sp.2, seven-day-old Chlorococcum sp. and seven-day-old Kirchneriella incurvata, in the ratio 1:1:3 for 30-day-old juveniles and 3:1:1 for 130-day-old juveniles. For the adult mussel, three-day-old Chlorella sp.2, seven-day-old Coccomyxa sp., and seven-day-old Monoraphidium sp., in the ratio 3:1:1, were the most digestible phyto- plankton. Levels of in-vitro digestibility were related to the quality (not the concentrations) of carbohydrate and protein in the phytoplankton mixtures, and protein digestibility seemed to be the key factor determining food quality for the mussel.

The main proteinases in Dermatobia hominis second and third instars larvae are serine-proteinases

We performed a combination of proteinase assay, either in solution or immobilized in sodium dodecyl sulfate-polyacrylamide gel copolymerized with gelatin, to detect and quantify proteinases of Dermatobia hominis second (L2) and third (L3) instar larvae. In the quantitative assay, we examined proteinase activity by hydrolysis of a panel of peptide bonds specific for the main proteinase classes. We verified that the pGlu-Phe-Leu p-nitroanilide substrate was hydrolyzed by crude extracts of L2 (3.0 ± 0.2 nmol h −1 mg of protein −1) and L3 (7.7 ± 0.1 nmol h −1 mg of protein −1) and that both activities were partially inhibited by trans-epoxysuccinyl- l-leucylamido-(4-guanidino)butane, 15% and 3%, respectively. Also, we demonstrated that the Nα- p-Tosyl- l-Arg methyl ester substrate was hydrolyzed by crude extracts of L2 (117 ± 24 nmol h −1 mg of protein −1) and L3 (111 ± 10 nmol h −1 mg of protein −1), suggesting a predominance of esterase activity in the crude larval preparation. Interestingly, the specific activity of serine-proteinases was totally inhibited by phenylmethylsulphonyl fluoride in the L3 crude extract, while only 10% of this enzyme class activity was inhibited in the L2 crude extract. The results of the qualitative assays with substrate gels suggested that L2 and L3 larvae express serine-proteinases with similar (13 and 22 kDa) and distinct (50 kDa in L2 and 30 kDa in L3) relative molecular masses. These findings contribute to the biochemical characterization of D. hominis L2 and L3 larvae.

Expression of extracellular matrix metalloproteinase inducer and matrix metalloproteinases during mouse embryonic development

Mouse embryo implantation is a highly invasive and controlled process that involves remodeling and degradation of the extracellular matrix of the uterus. Matrix metalloproteinases (MMPs) are the main proteinases facilitating this process. Extracellular matrix metalloproteinase inducer (EMMPRIN) can stimulate the production of MMPs and is required for successful implantation in the mouse. The aims of the present study were to examine the expression profiles of mRNA and proteins for EMMPRIN and MMPs in the developing mouse embryo in vitro, and to study whether EMMPRIN protein induces the production of MMPs by mouse blastocysts. EMMPRIN mRNA, detected by RT-PCR, was present at all stages of embryo development from the one-cell to the blastocyst outgrowth. EMMPRIN protein, observed by confocal microscopy, was present on the cell surface at the same stages of development as was the mRNA. Of seven MMPs studied, murine collagenase-like A (Mcol-A), murine collagenase-like B (Mcol-B) and gelatinase A (MMP-2) mRNAs were detected only in blastocyst outgrowths by RT-PCR. Gelatinase B (MMP-9) mRNA was detected both in expanded blastocysts and blastocyst outgrowths. MMP-2 and -9 proteins were detected in the cytoplasm of outgrowing trophoblast cells. Collagenase-2 (MMP-8), collagenase-3 (MMP-13), or stromelysin-1 (MMP-3) mRNAs were not present at any stage of pre- or peri-implantation mouse embryo development. Quantitative RT-PCR analyses showed that recombinant EMMPRIN protein did not stimulate MMP-2 or -9 expression by mouse blastocyst outgrowths. These data suggest that EMMPRIN may regulate physiological functions other than MMP production by mouse embryos during implantation.

Characterization of a torovirus main proteinase.

Viruses of the order Nidovirales encode huge replicase polyproteins. These are processed primarily by the chymotrypsin-like main proteinases (M(pro)s). So far, M(pro)s have been studied only for corona-, arteri-, and roniviruses. Here, we report the characterization of the M(pro) of toroviruses, the fourth main Nidovirus branch. Comparative sequence analysis of polyprotein 1a of equine torovirus (EToV) strain Berne, identified a serine proteinase domain, flanked by hydrophobic regions. Heterologous expression of this domain resulted in autoprocessing at flanking cleavage sites. N-terminal sequence analysis of cleavage products tentatively identified FxxQ downward arrow(S, A) as the substrate consensus sequence. EToV M(pro) combines several traits of its closest relatives. It has a predicted three-domain structure, with two catalytic beta-barrel domains and an additional C-terminal domain of unknown function. With respect to substrate specificity, the EToV M(pro) resembles its coronavirus homologue in its preference for P1-Gln, but its substrate-binding subsite, S1, more closely resembles that of arteri- and ronivirus M(pro)s, which prefer P1-Glu. Surprisingly, in contrast to the M(pro)s of corona- and roniviruses, but like that of arterivirus, the torovirus M(pro) uses serine instead of cysteine as its principal nucleophile. Under the premise that the M(pro)s of corona- and toroviruses are more closely related to each other than to those of arteri- and roniviruses, the transition from serine- to cysteine-based proteolytic catalysis (or vice versa) must have happened more than once in the course of nidovirus evolution. In this respect, it is of interest that a mutant EToV M(pro) with a Ser165-->Cys substitution retained partial enzymatic activity.

Molecular Dynamics Simulations of Various Coronavirus Main Proteinases

In this study, two homology models (denoted as MproST and MproSH) of main proteinase (Mpro) from the novel coronavirus associated with severe acute respiratory syndrome (SARS-CoV) were constructed based on the crystal structures of Mpro from transmissible gastroenteritis coronavirus (TGEV) (MproT) and human coronavirus HcoV-229E (MproH), respectively. Both MproST and MproSH exhibit similar folds as their respective template proteins. These homology models reveal three distinct functional domains as well as an intervening loop connecting domains II and III as found in both template proteins. A catalytic cleft containing the substrate binding sites S1 and S2 between domains I and II are also observed. S2 undergoes more significant structural fluctuation than S1 during the 400 ps molecular dynamics simulations because it is located at the open mouth of the catalytic cleft, while S1 is situated in the very bottom of this cleft. The thermal unfolding of these proteins begins at domain III, where the structure is least conserved among these proteins. Mpro may still maintain its proteolytic activity while it is partially unfolded. The electrostatic interaction between Arg40 and Asp186 plays an important role in maintaining the structural integrity of both S1 and S2.

Effects of some antituberculous and anti-leprotic drugs on cathepsins B, H and L

The cysteine proteinases like cathepsins B, L and H are main hydrolytic enzymes present in lysosomes and play an important role in intracellular protein degradation. Tuberculosis and leprosy, both are tissue- destructive diseases. Main drugs used in chemotherapy of these diseases may inhibit the main lysosomal cysteine proteinases i.e. cathepsins B, L and H released during tissue destruction and thus prevent the further destruction of tissue by these enzymes. So the aim of this study is to see the effect of antituberculous and antileprotic drugs on these proteolytic enzymes. The effect of commonly used antituberculous and antileprotic drugs was screened on the activities of purified brain lysosomal cysteine proteinases namely cathepsins B [EC 3.4.22.1], L [EC 3.4.22.15] and H [EC 3.4.22.16]. Among the antileprotic drugs, only clofazimine inhibited the enzymic activities whereas dapsone had no effect whatsoever. In antituberculous drugs, rifampicin was the most inhibitory while isoniazid had little inhibitory potency. Streptomycin and pyrazinamide did not effect the activities at all. As regards the mechanism of inhibition, clofazimine and isoniazid inhibited the enzymes in a non-competitive manner with K values of 0.25 mM and 5.0 mM for cathepsin B, 0.071 mM and 0.833 mM for cathepsin L and 1.513 mM and 0.885 mM for cathepsin H, While rifampicin could effect in a competitive manner with K(i) values of 0.03 mM, 0.125 mM and 0.027 mM for cathepsin B, L and H respectively.

IN SEARCH OF SARS THERAPEUTICS

USING CRYSTAL STRUCTURES of a crucial enzyme from two related coronaviruses as a guide, German scientists claim to have identified a promising starting point for the design of a drug to combat severe acute respiratory syndrome. Many viruses, including the coronavirus that causes SARS, rely on a protein-cleaving enzyme called main proteinase to activate replication. Such viruses must replicate themselves in order to cause infection, making this enzyme a prime target for drug development. Rolf Hilgenfeld, director of the Institute for Biochemistry at the University of Lubeck, in Germany, and his coworkers determined X-ray crystal structures of the main proteinases produced by a human coronavirus and a pig coronavirus. Hilgenfeld's team used their structures to create a structural model of the SARS coronavirus main proteinase [Science, published online May 13, http://wwwsciencemag.org/cgi/rapidpdf/1085658vl]. Eidogen, based in Pasadena, Calif., made public its own model of the same enzyme on May 14. On the ...

Overexpression of cysteine proteinase 2 in Entamoeba histolytica or Entamoeba dispar increases amoeba-induced monolayer destruction in vitro but does not augment amoebic liver abscess formation in gerbils.

To study the role of cysteine proteinases in the pathogenicity of Entamoeba histolytica, we have attempted to overexpress the three main cysteine proteinases (EhCP1, EhCP2, EhCP5) of this parasite in trophozoites of E. histolytica as well as in non-pathogenic Entamoeba dispar by episomal transfection. Although each of the corresponding coding sequences were cloned in identical expression plasmids, we were unable to overexpress EhCP1 and EhCP5, respectively, but could substantially induce expression of EhCP2 in both amoeba species by sevenfold, leading to a threefold increase in total cysteine proteinase activity. Overexpression of EhCP2 did not influence expression of other cysteine proteinases and could be attributed to an increase of a single 35 kDa activity band in substrate gel electrophoresis. In contrast to previous findings, which indicated that amoeba cysteine proteinases are involved in erythrophagocytosis and liver abscess formation, cells overexpressing EhCP2 showed no difference in erythrophagocytosis or liver abscess formation compared with respective controls. However, overexpression of EhCP2 in both amoeba species resulted in a marked increase of in vitro monolayer destruction.

Soybean Kunitz, C-II and PI-IV inhibitor genes confer different levels of insect resistance to tobacco and potato transgenic plants

In modern, highly intensive agriculture, the control of insect pests is basically achieved with the appli- cation of chemical pesticides. Heavy reliance on this sole strategy is associated with several drawbacks, and the de- velopment of alternative or complementary methods to chemical control is desirable. In this work, three soybean genes (KTi3, C-II and PI-IV) coding for serine proteinase inhibitors were isolated by PCR and transferred to Agro- bacterium tumefaciens EHA 105, which in turn was used for transforming tobacco leaf and potato tuber discs. Bio- chemical assays confirmed that transgenic plants synthe- sized serine proteinase inhibitors; rates of expression varied among plants. The level of insect resistance (tested with Spodoptera littoralis Boisduval) was particularly high in tobacco, where many plants caused the death of all larvae. In potatoes, larval mortality was much less fre- quently achieved, but the results were still encouraging in that larval weight gain was reduced by 50% in the pres- ence of adequate amounts of inhibitor. When 8-day-old larvae were fed different KTi 3 -expressing tobacco plants, a highly significant (P<0.01) correlation was observed be- tween inhibitor content and larval live weight. Larval weight gain was found to be dependent on midgut proteo- lytic activity. On the basis of the evidence collected, it is suggested that further work is required to identify more specific inhibitors for the main proteinases of the target in- sect.

口底へん平上皮癌細胞におけるアスパルティックプロテアーゼの発現

Cathepsin D and E are the main aspartic proteinases, suggested to be involved in the invasion and progression of cancer cells. We immunohistochemically studied the localization of cathepsins in oral carcinoma. Specimens of SCC of the oral floor, obtained from 18 patients, were prepared. Part of the serial paraffin sections were stained with hematoxylin-eosin and classified according to Anneroth's classification. Immunohistochemical staining was performed by the labeled streptavidin-biotin method, using affinity-purified polyclonal antibodies against cathepsin D and E. The expression of cathepsin D was observed in the cancer cells and was significantly increased in the T 4 category and in clinical stage III and IV. No significant difference was observed in the expression of cathepsins among the malignancy groups classified by Anneroth's method; medium and high malignancy groups were associated with shorter survival. The expression of cathepsin E in cancer cells was very low, and there was no correlation with any clinical or histopathological factor.

Inhibition of neutrophil-endothelial cell adhesion by a neutrophil product, cathepsin G

In the present study we investigated the modulation of the polymorphonuclear neutrophil (PMN)-endothelial cell adhesion process by the two main proteinases released from activated PMN during their adhesion to endothelium. Our results showed that, in contrast with elastase, cathepsin G was a powerful inhibitor of PAIN adhesion to interleukin-1 (IL-1)-treated human umbilical vein endothelial cells. This inhibitory effect was linked to the enzymatic activity of the proteinase and was selectively directed against PMN. Because the viability and the reactivity of PMN were not modified by cathepsin G, we looked for a possible effect on adhesion molecules. L-selectin was not cleaved by cathepsin G, whereas it was by chymotrypsin, a closely related proteinase. Cathepsin G blocked PMN adhesion to activated endothelial cells, but also to serum- or fibrinogen-coated plates, three adhesion processes mediated by CD11b/CD18. However, by FACScan analysis or by immunoprecipitation, we failed to find evidence of modifications of CD11b/CD18 expression. Although the precise molecular target(s) of cathepsin G remain(s) to be defined, these data indicate that this proteinase, which is known as an inflammatory mediator, can also be considered as a potential down-regulator of adhesion reactions involved in the inflammatory process.

Purification of cysteine proteinases from trichomonads using bacitracin-sepharose

Bacitracin affinity chromatography has been used to purify proteinases of the parasitic protozoon Tritrichomonas foetus. It proved superior to other affinity chromatography methods we have tested for the purification of trichomonad proteinases and should prove a useful procedure for purifying cysteine proteinases from these parasites and other parasitic protozoa. The main cysteine proteinases of T. foetus were purified over 100-fold to be free from the majority of other cell proteins. About 90 micrograms of protein containing 1.56-fold more proteinase activity than was detectable in the original cell lysate was obtained from 10(9) cells (7.2 mg protein). SDS-PAGE revealed that the eluate contained two main Coomassie blue-staining bands. N-terminal amino acid sequence analysis of these proteins confirmed that one of them was a cysteine proteinase with unusual features. Cysteine proteinases were also purified from cell lysates of Trichomonas vaginalis and a N-terminal sequence determined. This is the first amino acid sequence information that has been obtained for trichomonad cysteine proteinases. The method was also used to purify proteinases from the medium of T. foetus cultures. Some selectivity in binding of the proteinases to the affinity column was found.

Hydrolysis of l-leucine methyl ester by Leishmania mexicana mexicana amastigote cysteine proteinases

The main cysteine proteinases of the amastigote form of Leishmania mexicana mexicana were partially purified by gel filtration and ion exchange chromatography. The latter procedure resulted in the separation of some individual cysteine proteinases, as demonstrated by gelatin-sodium dodecyl sulphatepolyacrylamide gel electrophoresis. Fractions containing the partially purified proteinases rapidly hydrolysed l-leucine methyl ester to leucine. The activity towards this compound co-eluted with and resembled the parasite's cysteine proteinase activity. The results suggest that amastigotes of L.m.mexicana are susceptible to l-leucine methyl ester because this compound is rapidly hydrolysed by cysteine proteinases that occur in abundance in the megasomes of this stage.

Proteinase activities in bovine atrium and the possible role of mast cell tryptase in the processing of Atrial Natriuretic Factor (ANF)

1. 1. Using low salt, Triton X-100 and high salt extracts of bovine atria, two main proteinases were identified by means of fluorogenic oligopeptide substrates. 2. 2. An acidic proteinase, extracted in low salt and Triton X-100 was identified as cathepsin B, but it caused little hydrolysis of the Z-Gly-Pro-Arg-containing substrate that resembles the cleavage site for activation of pro-ANF. 3. 3. An alkaline proteinase was extracted only with high salt and had characteristics of the serine proteinase tryptase. It cleaved Z-Gly-Pro-Arg-containing substrates more efficiently than others tested and was localized in and around mast cells histochemically. Previously, Imada et al., 1988 ( J. biol. Chem. 263, 9515–9519) found an identical enzyme would cleave ANF from pro-ANF. 4. 4. These results suggest therefore that mast cell typtase may be involved in the activation of ANF from pro-ANF.