tosyl-L-arginine Methyl Ester Research Articles

BML-260 promotes the growth of cord blood and mobilized peripheral blood hematopoietic stem and progenitor cells with improved reconstitution ability.

Hematopoietic stem cells (HSCs), which are multipotent and have the ability to self-renew, are frequently used in the treatment of hematological diseases and cancer. Small molecules that target HSC quiescence regulators could be used for ex vivo expansion of both mobilized peripheral blood (mPB) and umbilical cord blood (UCB) hematopoietic stem and progenitor cells (HSPC). We identified and investigated 35 small molecules that target HSC quiescence factors. We looked at how they affected HSC activity, such as expansion, quiescence, multilineage capacity, cycling ability, metabolism, cytotoxicity, and genotoxicity. A transplantation study was carried out on immunocompromised mice to assess the expanded cells' repopulation and engraftment abilities. 4-[(5Z)-5-benzylidene-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]benzoic acid (BML)-260 and tosyl-l-arginine methyl ester (TAME) significantly increased both mPB and UCB-HSPC content and activated HSC re-entry into the cell cycle. The improved multilineage capacity was confirmed by the colony forming unit (CFU) assay. Furthermore, gene expression analysis revealed that BML-260 and TAME molecules aided HSC expansion by modulating cell cycle kinetics, such as p27, SKP2, and CDH1. In addition to these in vitro findings, we discovered that BML-260-expanded HSCs had a high hematopoietic reconstitution capacity with increased immune cell content after xenotransplantation into immunocompromised mice. In addition to the BML-260 molecule, a comparison study of serum-containing and serum-free chemically defined media, including various supplements, was performed. These in vitro and xenotransplantation results show that BML-260 molecules can be used for human HSC expansion and regulation of function. Furthermore, the medium composition discovered may be a novel platform for human HSPC expansion that could be used in clinical trials.

Effects of tosyl-l-arginine methyl ester (TAME) on the APC/c subunits: An in silico investigation for inhibiting cell cycle

The anaphase-promoting complex/cyclosome (APC/c) is requisite for controlling mitosis, which is activated by Cdh1 and Cdc20 activators. Dysregulation of APC/c is observed in many cancers and is known as a targeted drug particularly in cancer drug resistance. It was shown that tosyl-l-arginine methyl ester (TAME), via mimicking isoleucine-arginine (IR) tail of co-activators, inhibits APC/c functions. However, structure details and interaction of TAME with APC/c are poorly defined. In the current study, a well-established set of computational methods was used to identify the best binding pocket in order to inhibit APC activity. Therefore, the interaction of IR tail and Cbox of co-activators, as well as TAME as an inhibitor, as an inhibitor, with APC3 and APC8 subunits of APC/c were analyzed, regarding structure, molecular docking, molecular dynamics, and free binding energy. The results indicated that TAME bound to APC3 with a higher binding affinity (∼−7.3 kcal/mol) than APC8 (∼−5.7 kcal/mol). Also, the binding free energy value obtained for the APC3-TAME was −22.25 ± 1.12 kcal/mol. According to binding free energies, van der Waals energy was the major favorable contributor to the ligand binding. These results offer that TAME had more affinity to interact with the APC3 subunit, at the IR binding pocket than the APC8 subunit at the Cbox binding pocket. In conclusion, IR binding pocket can serve as an appropriate potential target for TAME as an inhibitor of APC/c.

Targeting the Anaphase Promoting Complex/Cyclosome (APC/C) in Multiple Myeloma

The discovery of novel agents such as the proteasome inhibitor bortezomib has significantly increased the survival of multiple myeloma (MM) patients. However MM remains an incurable disease mainly due to relapse, associated with significant resistance to therapy including bortezomib. Therefore further investigation to elucidate the disease and the mechanisms leading to drug resistance is necessary.The success of bortezomib highlights the importance of the ubiquitin-proteasomal system (UPS) in MM. The UPS regulates protein turnover and plays a key role is several cellular processes such as apoptosis, cell cycle progression, cell proliferation and DNA replication. The Anaphase Promoting Complex/Cyclosome (APC/C) is an E3 ubiquitin ligase protein complex involved in controlling cell cycle progression. The regulation of APC/C is dependent on 2 co-activators: Cdc20 and Cdh1. The APCCdc20 complex is controlling the metaphase to anaphase transition in mitosis, while APCCdh1 controls mitotic exit and early G1 phase. During metaphase, the activity of APCCdc20 is inhibited by the spindle assembly checkpoint. When all kinetochores are properly attached, the spindle assembly checkpoint is silenced and APCCdc20 becomes activated. When APCCdc20is active, cell cycle proteins are targeted for degradation by the proteasome such as securin and cyclin A and B leading to mitotic exit. Recent studies described that spindle assembly checkpoint is defective in MM cells and that patient samples after chemotherapy and at relapse displayed an increased chromosomal instability signature including Cdc20.The aim of our study is to elucidate the importance and therapeutic potential of APC/C and its co-activators Cdc20 and Cdh1 in MM. Analysis of gene expression in the data of Zhan et al. (Blood 108, 2020-8, 2006) revealed that the co-activator Cdc20 was higher expressed in certain MM sub-groups (PR, MS, CD1, MF) compared to healthy bone marrow plasma cells. Moreover, high Cdc20 expression is correlated with poor prognosis. Cdh1 on the other hand was significantly lower expressed in all MM sub-groups compared to healthy bone marrow plasma cells. Interestingly, lower Cdh1 expression is correlated with poor prognosis. Next, we analyzed whether blocking APC/C would affect MM cells. For this study the pro-drug of TAME (tosyl-L-arginine methyl ester) that has been described as an inhibitor of the APC/C, was used. When the human myeloma cell lines LP-1 and RPMI-8226 were treated with proTAME, an accumulation of the APCCdc20 substrate cyclin B1 was seen already after 6 hours. However the levels of Skp2, an APCCdh1 substrate, were not affected by proTAME treatment. This suggests that proTAME inhibits the APCCdc20 complex but not the APCCdh1complex. We morphologically assessed the effect on number of metaphases on May-Grünwald Giemsa stained cytospins. ProTAME clearly induced an accumulation of LP-1 and RPMI-8226 cells in metaphase. Since a metaphase arrest can lead to cell death, we investigated the effect of proTAME on the viability and apoptosis. A significant dose-dependent decrease in viability and increase in apoptosis was observed after treatment with proTAME of human myeloma cell lines and primary MM cells purified from human and 5T33MM diseased mice. In contrast, other cells from the bone marrow microenvironment were not affected upon proTAME treatment. The induction of apoptosis was accompanied with caspase 3, 8, 9 and PARP cleavage. Western Blot analysis also showed phosphorylation of H2AX suggesting DNA damage upon proTAME treatment. Previous studies showed that MM is a heterogeneous disease consisting of a bulk CD138+ population and a minor CD138- population which is less sensitivity to drugs such as bortezomib. Interestingly, treatment of CD138+/- 5T33MM cells with proTAME demonstrated an equal targeting of both populations.From these results we can conclude that overexpression of Cdc20 by MM cells is correlated with a bad prognosis. Inhibition of APCCdc20 results in a metaphase arrest in MM cells which is associated with reduced viability and induction of apoptosis. Moreover, APC/C inhibition equally targets CD138+ and the more resistant CD138- 5T33MM cells. This study suggests that APC/C and its co-activator Cdc20 could be a new and promising target in MM. DisclosuresNo relevant conflicts of interest to declare.

Pharmacologic Inhibition of the Anaphase-Promoting Complex Induces A Spindle Checkpoint-Dependent Mitotic Arrest in the Absence of Spindle Damage

Microtubule inhibitors are important cancer drugs that induce mitotic arrest by activating the spindle assembly checkpoint (SAC), which, in turn, inhibits the ubiquitin ligase activity of the anaphase-promoting complex (APC). Here, we report a small molecule, tosyl-L-arginine methyl ester (TAME), which binds to the APC and prevents its activation by Cdc20 and Cdh1. A prodrug of TAME arrests cells in metaphase without perturbing the spindle, but nonetheless the arrest is dependent on the SAC. Metaphase arrest induced byaproteasome inhibitor is also SAC dependent, suggesting that APC-dependent proteolysis is required to inactivate the SAC. We propose that mutual antagonism between the APC and the SAC yields a positive feedback loop that amplifies the ability of TAME to induce mitotic arrest.

Kinetic Properties of Three Isoforms of Trypsin Isolated from the Pyloric Caeca of Chum Salmon (Oncorhynchus keta)

Three isoforms of anionic chum salmon trypsin (ST-1, ST-2, and ST-3) were purified from the pyloric caeca of chum salmon (Oncorhynchus keta). The molecular weights of the three isoforms were about 24 kDa as determined by SDS-PAGE. The isoelectric points of ST-1, ST-2, and ST-3 were 5.8, 5.4, and 5.6, respectively. The apparent K(m) values of two isoforms (ST-1 and ST-2) for BAPA (benzoyl-L-arginine-p-nitroanilide) hydrolysis at 5, 15, 25 and 35 degrees C were slightly higher than that of the main isoform ST-3, depending on temperature. The turnover numbers, k(cat), of ST-1 and ST-2 were about twice as high as that of ST-3. Consequently, the catalytic efficiencies (k(cat)/K(m)) of ST-1 and ST-2 were more efficient than ST-3. There were marked differences in both apparent K(m) and k(cat) values of three anionic chum salmon trypsins as compared to bovine cationic trypsin. K(m) values of all chum salmon trypsins were approximately 10 times lower than those of bovine trypsin, depending on the temperature. The k(cat) values of all chum salmon trypsins were about 2- to 5-fold higher than those of bovine trypsin; therefore, the catalytic efficiencies (k(cat)/K(m)) of chum salmon trypsin were 20- to 40-fold more efficient than those of bovine trypsin. On the other hand, k(cat)/K(m) values of ST-1 for TAME (tosyl-L-arginine methyl ester) hydrolysis were lower than those of bovine trypsin, whereas k(cat)/K(m) values of ST-2 and ST-3 were comparable to those of bovine trypsin, depending on the temperature.

A Bradykinin Antagonist Modifies Allergen-induced Mediator Release and Late Bronchial Responses in Sheep

We assessed the role of bradykinin (BK) in allergen-induced early and late bronchial responses, airway inflammation, mediator release, and antigen-induced airway hyperresponsiveness in allergic sheep by studying the effects of the BK B2 receptor antagonist, NPC-567 (D-Arg-[Hyp3, D-Phe7]-BK), on these parameters. Antigen challenge was performed on two occasions greater than 3 wk apart, once with placebo (control) and once after high-dose (10 mg/ml) and low-dose (5 mg/ml) treatments with aerosol NPC-567. In the control trials (n = 14) antigen challenge resulted in an early and late increase in specific lung resistance (SRL). The early response was associated with increases (p less than 0.05) in prostaglandin (PG) D2, immunoreactive kinin, tosyl-L-arginine methyl ester (TAME)-esterase, and PGE2 in bronchoalveolar lavage (BAL) fluid. The late response was associated with increases (p less than 0.05) in leukotrienes (LT) B4 and C4, thromboxane (TX) B2, 6-keto-PGF10, and PGE2. There was a significant influx of neutrophils in the BAL fluid during the late response, and airway hyperresponsiveness to carbachol aerosol was apparent 4 h after challenge. In six sheep the high-dose NPC-567 treatment (given before, during, and 4 h after antigen challenge) did not attenuate the early bronchoconstrictor response or the early release of mediators but caused a significant reduction in the late response (p less than 0.05). This protective effect was accompanied by reductions (p less than 0.05) in both the concentrations of all the mediators associated with the late response and the severity of the BAL neutrophilia. High-dose NPC-567 did not attenuate the airway hyperresponsiveness or the cellular inflammatory response seen 24 h after challenge. In eight sheep treated with the low dose of NPC-567 (given before, during, and 4, 8, and 24 h after challenge) the early response was not blocked but the late response was again inhibited, as were the mediators associated with the late response. At the low dose the drug did not prevent the airway inflammation at 8 or 24 h. The additional treatments did, however, prevent the 24 h hyperresponsiveness. These data suggest that kinin generation during antigen-induced airway anaphylaxis may be important for controlling the release of arachidonic acid metabolites from airway inflammatory cells that contribute to the development of the late response in the allergic sheep model.

Isolation and characterization of hemorrhagic toxin from the venom of Trimeresurus elegans

1. 1. Hemorrhagic toxin from the venom of Trimeresurus elegans was purified in a homogeneous form using gel filtration and ion exchange chromatographies. 2. 2. Hemorrhagic toxin possessed hemorrhagic and TAME (tosyl- l-arginine methyl ester) hydrolytic activities. These activities were inhibited when hemorrhagic toxin was incubated with benzamidine or N-bromosuccinimide. 3. 3. Its mol. wt was 28,500 and the isoelectric point was 7.4. 4. 4. This toxin contains ca 1.5 mol Ca per mol of protein.

β-Fibrinogenase from the venom of Vipera lebetina

An arginine esterase was purified from the venom of Vipera lebetina by gel filtration on Sephadex G-100 and by affinity and DEAE-cellulose chromatography. The enzyme has a mol. wt of 52,500 and p I ∼ 3. It is a glycoprotein containing 23% of neutral sugars, and has extremely high thermostability. The esterase activity is inhibited by diisopropylfluorophosphate (DFP) and phenylmethylsulfonyl fluoride (PMSF). The K m and k cat values are for α-N- benzoyl- l-arginine ethyl ester (BAEE) 7.7 × 10 −5M and 43.8 sec −1, for p- tosyl- l-arginine methyl ester (TAME) 3.6×10 −4M and 39.8 sec −1 (pH 8.5, 25°C, and for α-N- benzoyl- dl-arginine -4- nitroanilide (BAPNA) 1.8×10 −4M and 0.94 sec −1 (pH 8.3, 25°C), respectively. Lysine esters are not hydrolyzed. The enzyme has weak caseinolytic activity and hydrolyzes glucagon at the sites Lys 12-Tyr 13, Arg 17-Arg 18 and Arg 18-Ala 19. In fibrinogen it cleaves Bβ-chain first and later also the Aα-chain.

Production and characterization of monoclonal antibodies specific for human mast cell tryptase

Human mast cell tryptase was purified from lung tissue by high salt extraction, ammonium sulphate precipitation, octyl Sepharose and heparin-agarose chromatography. The tryptase isolated was a tetramer with a molecular weight of 132 kD on gel filtration, and on SDS-polyacrylamide gel electrophoresis was reduced to a single diffuse band with a mean molecular weight of 32.5 kD. Purified tryptase catalysed the cleavage of the tryptic substrates tosyl L-arginine methyl ester and benzoyl DL-arginine p-nitroanilide; enzymatic activity was enhanced in the presence of heparin but markedly decreased in the presence of 2 M sodium chloride. Rabbit antisera and three new monoclonal antibodies (AA1, AA3 and AA5) were produced which were specific for tryptase in indirect ELISAs, immunoenzymatic overlay in crossed immunoelectrophoresis and by Western blotting. Additive and competitive ELISA experiments suggested that the three monoclonal antibodies all recognized epitopes within a single highly immunogenic area of the tryptase molecule, and enzyme assays indicated that this site was distant from the active site. Binding of monoclonal antibodies to tryptase was not affected by the presence of heparin, or by periodate treatment of the antigen suggesting that carbohydrate epitopes were not recognized. Western blotting indicated that some heterogeneity in molecular weight for monomeric tryptase was not reflected in antigenic differences. An immunofluorescence procedure with cytocentrifuge preparations of enzymatically dispersed lung, colon and skin revealed highly specific localization of tryptase to the granules of all mast cells, but there was no binding to other cells in these preparations, to cultured keratinocytes, to basophils or to any other blood leucocyte.

Purification and characterization of a trypsin-like enzyme from the midgut gland of the Atlantic blue crab, Callinectes sapidus

1. 1. A trypsin-like enzyme from the midgut gland of the Atlantic blue crab, Callinectes sapidus, was purified and studied. 2. 2. Purification was achieved by a combination of molecular sieving, ion exchange, and hydrophobic chromatography. Degree of purity was assessed by SDS-polyacrylamide electrophoresis. 3. 3. Using N-p- tosyl- l-arginine methyl ester (TAME) as a substrate, the enzyme displays optimal activity in the absence of calcium at pH 8.2. 4. 4. The tryptic activity is extremely stable between pH 7 and 8.5, but is rapidly inactivated below pH 6. Thirty-minute incubations above 50°C also inactivate the enzyme. 5. 5. The tryptic activity has a molecular weight of 33,500 da and an isoelectric point of approximately 4. 6. 6. The trypsin-like protease from Callinectes sapidus is similar to crayfish and bovine trypsins in that it is inhibited by phenylmethane sulfonylfluoride, tosyl- l-lysine chloromethyl ketone, and soybean trypsin inhibitor. Its pH optimum and its specificity for TAME and N-p- tosyl- l-lysine methyl ester (TLME) are also similar. It is concluded that the enzyme characterized in this study is trypsin.

Isolation and characterization of a fibrinogen-clotting enzyme from venom of the snake, Lachesis muta muta(Peruvian bushmaster)

A. Yarleque, S. Campos, E. Escobar, F. Lazo, N. Sanchez, S. Hyslop, N. A. Marsh, P. J. Butterworth and R. G. Price. Isolation and characterization of a fibrinogen-clotting enzyme from venom of the snake Lachesis muta muta(Peruvian bushmaster). Toxicon 27, 1189–1197, 1989.—A fibrinogen-clotting enzyme from the venom of the Peruvian bushmaster snake was purified to homogeneity by gel filtration on Sephadex G-100 followed by DEAE-cellulose ion-exchange chromatography using a linear ionic strength gradient with NaCl. The specific activity of the enzyme was 866 NIH U/mg, representing a 55-fold purification, with a recovery of 45%. The amino acid composition was Asx 30, Thr 14, Ser 15, Glx 33, Pro 23, Gly 22, Ala 15, Val 22, Cys 18, Met 3, Ile 18, Leu 23, Tyr 2, Phe 13, His 8, Lys 11, Arg 11. The total carbohydrate content was 13.4%, comprised of 3.4% hexose, 8.7% hexosamine and 1.3% sialic acid. The enzyme was active against the synthetic amide substrate α- N-benzoyl- dl-arginine- p-nitroanilide (BAPNA) and against the ester substrates α- N-benzoyl- l-arginine ethyl ester (BAEE) and tosyl- l-arginine methyl ester (TAME). Kinetic parameters for TAME esterolysis were: V max , 135 μmoles/min/mg and K m , 2.5 × 10 −4M. The pH optimum was 8.0. V max for BAPNA amidolysis was 0.363 μmoles/min/mg and K m , 7.5 × 10 −5M. Enzyme activity was reduced by diethylpyrocarbonate and by photo-oxidation, suggesting that the enzyme is a serine protease with a histidine residue involved in the active site. The enzyme released fibrinopeptide A rapidly from purified human fibrinogen and fibrinopeptide B more slowly. Factor XIII was not activated and the clotting activity was not inhibited by heparin. A dose of 50 μg/kg brought about defibrinogenation in anaesthetized rats but rabbits were unaffected. A dose of 80 μg/kg defibrinogenated conscious rats after 5 hr. There were no hypotensive or haemorrhagic effects.

The allosteric effect of salt on human mast cell tryptase

The inhibitory effect of potassium chloride and ammonium sulphate on purified human skin tryptase and bovine trypsin was studied enzyme-kinetically, using Z Gly Pro Arg pNA, Z Gly Arg AMC, benzoyl- l-arginine ethyl ester (BAEE) and tosyl- l-arginine methyl ester (TAME) as substrates. With increasing salt concentrations, the curve of reaction velocity vs. substrate concentration changed from hyperbolic to sigmoidal when anilide substrates (Z Gly Pro pNA or AMC) were used. Only the K m value increased, while the V max value remained unchanged. The trend was similar with BAEE or TAME as the substrates. However, the effect of salt on the hydrolysis of these ester substrates was not as strong as on the hydrolysis of anilide substrates, and sigmoidal kinetics were not observed even at the highest KCl concentration (0.7 M) used. Heparin, used as a stabilizer, had no influence on this phenomenon, but it did slightly decrease the apparent K m and V max values in low-salt conditions. By comparison, trypsin was not as strongly affected by salt as tryptase, and the inhibition type was mixed competitive and non-competitive. The present results indicate that the salt acts on tryptase as an allosteric effector, and this should be carefully considered when enzyme kinetic parameters and enzyme activity of skin tryptase are measured.

Purification and characterization of two trypsin-like enzymes from the digestive tract of anchovy Engraulis encrasicholus

1. 1. Two trypsin-like enzymes, designated Trypsin A and B, were purified from the pyloric caeca and intestine of anchovy by (NH 4) 2SO 4 fractionation, affinity chromatography (Benzamidine-Sepharose-6B) and ion exchange chromatography (DEAE-Sepharose). 2. 2. Both trypsins catalyzed the hydrolysis of N- benzoyl- dl-arginine p-nitroanilide (BAPNA), p- tosyl- l-arginine methyl ester (TAME), casein and myofibrillar protein and they were inhibited by several well established trypsin-inhibitors. 3. 3. The enzymes had mol. wts of 27,000 (Trypsin A) and 28,000 (Trypsin B). Their isoelectric points were about 4.9 (Trypsin A) and 4.6 (Trypsin B) and they had similar amino acid composition. 4. 4. The enzymes had a pH optimum of 8–9 for the hydrolysis of BAPNA and 9.5 for the digestion of casein and myofibrillar protein. Their activity and stability were affected by calcium ions. 5. 5. Trypsin A and B resemble other fish trypsins in their mol. wt, pI, kinetic properties and the instability at low pH and they are similar to bovine trypsin in their dependence of Ca 2+ for activity and stability.

Kallikrein-like enzyme from Crotalus ruber ruber (red rattlesnake) venom

1. 1. A kallikrein-like enzyme was isolated and characterized from the venom of Crotalus ruber ruber (red rattlesnake). 2. 2. The kallikrein-like enzyme was shown to be homogeneous as demonstrated by a single band on acrylamide gel electrophoresis, isoelectric focusing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunodiffusion and reverse-phase (RP) HPLC. 3. 3. The enzyme has a molecular weight of 31,000 and isoelectric point of 4.6. It consists of 271 total amino acid residues, 24% of which are acidic amino acids. 4. 4. Specific esterolytic activities of the kallikrein-like enzyme on N- tosyl- l- arginine methylester (TAME) and N- benzoyl- l- arginine ethylester (BAEE) are 109.5 and 23.6 μmol/min/mg, respectively. 5. 5. The enzyme differs from trypsin as the soybean trypsin inhibitor does not inhibit the enzyme's action. Diisopropylfluorophosphate (DFP) inhibits the enzyme, suggesting that the serine hydroxyl group is important for enzyme activity. 6. 6. The enzyme is not lethal at 15 μg/g in mice and has no hemorrhagic activity, yet the injection of the purified enzyme intradermally, produced capillary permeability-increasing activity as shown by the use of Evans blue dye, and immediate drop in blood pressure. It also contracted the rat uterus.

Inhibition of Mediator Release in Allergic Rhinitis by Pretreatment with Topical Glucocorticosteroids

Patients with allergic rhinitis often have immediate symptoms after antigen challenge (the early-phase response), followed several hours later by a recurrence of symptoms (the late-phase response). Systemic glucocorticosteroids are known to inhibit the late-phase but not the early-phase response. We studied the effect of one week of pretreatment with topical (rather than systemic) glucocorticosteroids on the response to nasal challenge with antigen in a double-blind, randomized, placebo-controlled crossover study of 13 allergic patients who had previously had a dual response to nasal challenge. The patients were challenged with three 10-fold increments of allergen, producing an early response, and were then followed for 11 hours, encompassing the late response, before they were rechallenged with the lowest dose of allergen. We monitored their responses by means of symptom scores and measurements of the levels of histamine, tosyl-L-arginine methyl ester (TAME)-esterase activity, and kinins in nasal lavages. Topical glucocorticosteroids significantly reduced both the symptoms and the levels of histamine, TAME-esterase activity, and kinins in the early, late, and rechallenge allergic reactions. The fact that, in contrast to treatment with systemic glucocorticosteroids, prolonged pretreatment with topical glucocorticosteroids inhibited the early-phase response to antigen suggests that the route and duration of administration affect the mechanisms of action of the steroids. We conclude that inhibition of the early-phase as well as the late-phase response by topical glucocorticosteroids may provide an advantage over treatment with systemic glucocorticosteroids in patients with allergic rhinitis.

A preliminary characterization of alkaline digestive proteases in the posterior midgut of the face fly, Musca autumnalis De Geer

Alkaline proteases in the posterior midgut of the face fly, Musca autumnalis De Geer, were identified using specific synthetic substrates and nonspecific substrate azocasein. Identification was confirmed with potential protease activators and inhibitors. Optimal azocasein hydrolysis occurred at pH 8.0 and substrate hydrolysis was inhibited by EGTA (ethyleneglycol-bis-(2-aminoethyl ether)-N,N-tetraacetic acid), tosyl-L-lysine chloromethyl ketone (TLCK), and soybean trypsin inhibitor (STI). Tryptic proteolysis was identified by maximal hydrolysis, at pH 8.0, of trypsin specific substrates BAPNA (benzoyl-DL-arginine-p-nitroanilide) and TAME (tosyl-L-arginine methyl ester). TAME hydrolysis was inhibited by PMSF (phenylmethylsulfonyl fluoride), STI, pepstatin A, and dithiothreitol (DTT), but BAPNA hydrolysis was inhibited only by STI and DTT. Chymotrypsin was demonstrated by maximal hydrolysis of BTEE (benzoyl-L-tyrosine ethyl ester) at pH 8.0 and DTT, PMSF, and STI inhibited hydrolysis of this substrate. Aminopeptidase activity was demonstrated by maximal hydrolysis of leucine-p-nitro-anilide at pH 8.0 and the peptidase was inhibited by o-phenanthroline. Carboxypeptidase A-like enzyme hydrolyzed hippuryl-DL-phenyllactic acid maximally at pH 7.5 and carboxypeptidase B showed maximum hydrolysis of hippuryl-L-arginine at pH 7.0. Both carboxypeptidases were inhibited by EDTA (ethylene diamine tetraacetic acid), EGTA, and DTT and carboxypeptidase A was also inhibited by PMSF.

A preliminary characterization of digestive proteases in the posterior midgut of the stable fly Stomoxys calcitrans (L.) (Diptera:Muscidae)

Proteinases contained in the posterior midgut of the stable fly, Stomoxys calcitrans (L.), have been tentatively identified by posterior midgut hydrolysis of synthetic substrates. Trypsin was identified by maximal hydrolysis of benzoyl- dl-arginine -p- nitroanilide (BAPNA) and tosyl- l-arginine methyl ester (TAME) at pH 8.0 and chymotrypsin, by maximal hydrolysis of benzoyl- l-tyrosine ethyl ester (BTEE) at pH 8.0. Carboxypeptidase A and B were identified by their maximal hydrolysis of hippuryl- dl-phenyllactic acid and hippuryl- l-arginine at pH 8.0 and 7.5 respectively and aminopeptidase was identified by maximal hydrolysis of leucine- p-nitroanilide at pH 7.5. Molecular weights, determined using gel exclusion chromatography, for proteinases were 19,500 for trypsin, 27,500 and 64,000 for chymotrypsin. Hydrolytic activity for the protein substrate haemoglobin occurred as two peaks with molecular weights of 19,500 and 27,500. Molecular weights for the peptidases were; carboxypeptidase A 26,000, carboxypeptidase B, 28,500 and greater than 1,500,000 for aminopeptidase. These results indicate that the posterior midgut of the stable fly contains both proteinases and peptidases necessary for proteolytic breakdown of the ingested blood meal.

A comparative study of clothing factors from the venom of Agkistrodon acutus collected in China and Taiwan

1. 1. Two clotting factors, Cf-1(C) and Cf-2(C) were isolated from Agkistrodon acutus (collected in China) venom by gel filtration, ion-exchange chromatography and affinity chromatography. Using the same procedure, two clotting factors, Cf-1(T) and Cf-2(T), were isolated from Agkistrodon acutus (collected in Taiwan) venom and their characteristics were compared with Cf-1(C) and Cf-2(C). 2. 2. Molecular weights of Cf-1(C), Cf-2(C), Cf-1(T) and Cf-2(T) were determined to be 44,000, 70,000, 25,000 and 44,000 respectively. The factors were not immunologically related. 3. 3. The four clotting factors possessed tosyl- l-arginine methyl ester (TAME) hydrolyzing activity and coagulated fibrinogen to fibrin. Only Aα chain was cleaved when fibrinogen was incubated with each factor. 4. 4. Agkistrodon acutus is not classified by geographical location, however it is obvious that venom components vary between the Chinese and Taiwanese forms.

Studies on proteolytic activity in the midgut homogenate of the blowfly Chrysomia chloropyga (Wied.) (Diptera: Calliphoridae)

Hydrolysis of the substrate Hide Powder Azure was accelerated by increasing concentration of the midgut enzyme extract of Chrysomyia chloropyga. A linear relationship between proteolytic activity and substrate concentration was established. Optimum temperature of substrate hydrolysis at pH 7.9 was shown to be 400C and activity at different temperatures and at various incubation periods did not alter the optimum. At the pH range (3 to 11) and at temperatures of 350, 450 and 550C, the pH activity of the enzyme extract showed two peaks, a low peak at pH 5 and a high one at pH 8. The crude enzyme extract was resolved into six protein ‘bands by polyacrylamide gel electrophoresis. The partially-purified proteolytic fractions hydrolysed N -p- Tosyl-L-arginine methylester (TAME) and N-Benzoyl-L-tyrosine ethylester (BTEE) at pH 8.0. Trypsin and Chymotrypsin were demonstrated in the enzyme preparation

血中における酸安定性トリプシン‐プラスミンインヒビター（ＡＳＴＩ）の酵素による活性出現

An acid stable trypsin-plasmin inhibitor (ASTI) was found to be produced when several SH-proteases were incubated with human plasma. The maximal activity produced with bromelain was approximately 40U/ml plasma, which represented about a 10-fold increase compared with that of the normal human plasma (less than 5U/ml). On the other hand, lesser activity was observed with several serine proteases, trypsin, elastase, followed by chymotrypsin. Neither the plasma fibrinolytic enzymes including plasmin and urokinase, nor plasma kallikrein had any effect on the ASTI production in the same condition.An increase in the ASTI acativity was also demonstrated in plasma derived from patients with acute pancreatitis (21U/ml), and the attivity was almost parallel to that of tosyl-L-arginine methylester (TAMe) hydrolyzing activity in plasma. In an experimental anaphylaxis of the rabbit, the ASTI activity in plasma also increased more than 5 times after antigen (bovine serum albumin) was injected.