Benzoylarginine Ethyl Ester Research Articles

Cryoenzymology in aqueous media: Micellar solubilized water clusters

Amphiphilic compounds dissolved in nonpolar organic solvents form inverted micelles whose aqueous centers can solubilize relatively large amounts of enzymes. The solutions are homogeneous and optically transparent and have low viscosity and freezing points; they provide unique and favorable systems to perform the main cryoenzymologic studies previously carried out in mixed solvents. The preparation and properties of such micelles are described. The absorbance spectra of cytochrome c at various temperatures from -38 degrees C to 20 degrees C are given. The pH dependence of trypsin-catalyzed hydrolysis of benzoylarginine ethyl ester in inverted micelles, as well as kinetic properties and activation energy, is described. Some problems are analyzed.

High molecular weight kininogen: Its inability to correct the clotting of kininogen-deficient plasma after cleavage of bradykinin by plasma kallikrein, plasmin or trypsin

Human high molecular weight (HMW)-kininogen was cleaved with trypsin, plasmin or plasma kallikrein to release bradykinin. ‡ ‡ ACD, acid citrate dextrose; BAEe, benzoyl arginine ethyl ester; HMW, high molecular weight; LBTI, lima beam trypsin inhibitor; LMW, low molecular weight; PKA, prekallikrein activator; PTT, partial thromboplastin time; QAE, quarternary aminoethyl; SBTI, soy bean trypsin inhibitor; SDS, sodium dodecyl sulfate. When an excess of enzyme was used and all releasable peptide was released, the residual HMW-kininogen could not correct the clotting deficiency of kininogen-deficient plasma. There was an inverse relationship between the amount of bradykinin released and the capacity of the kininogen to correct the abnormal partial thromboplastin time of kininogen-deficient plasma. These findings and earlier ones indicate that the intact HMW-kininogen molecule is required for contact mediated clotting and activation of factor XII.

The activation of proacrosin in spermatozoa from ram, bull and boar

Acrosin activity was estimated in fractions from washed ram, bull and boar spermatozoa that had been disrupted using a Stansted Cell Disruptor. When p-aminobenzamidine was included in the medium during disruption, all the acrosin (acrosomal proteinase, EC 3.4.21.10) was recovered as its inactive zymogen form, proacrosin. But if spermatozoa were damaged before disruption, or were disrupted in the absence of p-aminobenzamidine, considerable amounts of active acrosin were detectable. It was concluded that conversion of proacrosin to acrosin takes place in spermatozoa only after the acrosome has been ruptured. In a sucrose medium, all the proacrosin was bound to the sperm heads. Conversion to acrosin took place readily with all components in a bound state. Using ram sperm heads, the conversion was found to be relatively insensitive to pH, proceeding rapidly above pH 6.5; the rate of conversion was not affected by physiological levels of Ca 2+, Mg 2+ or Zn 2+, although elevated ionic strength caused a solubilization of the acrosin activity and some slowing of the rate. Electrophoretic analysis revealed that several active forms of acrosin were involved, but the final product was a single stable form. Final levels of the active acrosin (expressed as μmol N-α- benzoyl- l-arginine ethyl ester utilised/min per 10 9 heads) were: ram 26.2; bull, 15.9; boar, 133.8. But active site titration revealed that these different levels were not reflected in the numbers of active enzyme molecules on the sperm head; boar acrosin appears to be about three times more active towards benzoyl-arginine ethyl ester than do the acrosins from the other species.

Effect of bile acids and ionic strength on trypsinogen activation by human enteropeptidase

1. (1) Increasing ionic strength diminishes the rate of enteropeptidase-catalyzed activation of trypsinogen by increasing dissociation of the Michaelis complex and, thus, K m of the reaction. 2. (2) The effect of ionic strength depends not only on the magnitude of this parameter, but also on the nature of the anion. Enzyme activity is diminished progressively by isoionic concentrations of ClO 4 −, Cl − and SO 4 2− in this order. The influence of cations of the alkali series is comparatively insignificant. 3. (3) Bile acids increase the rate of trypsinogen activation by human enteropeptidase (EC 3.4.21.9). Derivatives of cholic acid are less active than those of deoxy- and chenodeoxycholic acid. Triton X-100 is inactive. 4. (4) The effect of bile acids on enteropeptidase-catalyzed trypsinogen activation is independent of the ionic strength except at very low I values where anomalous kinetics are observed. Bile acids probably decrease dissociation of the Michaelis complex since 2.5 mM glycodeoxycholic acid has no significant effect on K m , but increases V about 2-fold over a wide range of ionic strengths (0.016–0.073). 5. (5) Bile acids have no effect on enteropeptidase-catalyzed hydrolysis of the synthetic substrate N- benzoylarginine ethyl ester, suggesting that their mode of action may involve an enzyme subsite with specific affinity for the trypsinogen molecule.

Human Leukocyte Inhibitory Factor (LIF): Use of Benzoyl L-Arginine Ethyl Ester to Detect LIF Activity

Abstract Human leukocyte inhibitory factor (LIF), a mediator elaborated by activated lymphocytes, has recently been shown to have the properties of an esterase. Further more, the LIF preparation contains an esteratic principle that hydrolyzes the amino acid ester, benzoyl arginine ethyl ester (3H-BAEE). In the present experiments, we sought to determine whether the esterase and biologic activities in the LIF preparation were due to the same or different molecules. This was accomplished by means of two series of experiments. With one approach, LIF was initially subjected to gel filtration and subsequently to polyacrylamide gel electrophoresis, sucrose density gradient electrophoresis, and affinity chromatography. The fractions obtained from each procedure were analyzed for biologic and esterase activities. The results indicate that both activities co-chromatographed with each technique. In addition, there are contaminating esterases present in the LIF preparation that can hydrolyze 3H-BAEE, some activity(ies) of which are eliminated by purification. The other approach directly demonstrated whether BAEE could serve as a substrate for LIF by substrate-competition experiments. BAEE and other structurally related amino acid esters were co-incubated with LIF and the ability of the former to protect the latter against inactivation by esterase inhibitors, or block biologic LIF activity on polymorphonuclear leukocytes was ascertained. It was shown that BAEE specifically prevented the destruction of LIF by diisopropylphosphofluoridate (DFP) and phenyl methyl sulfonyl fluoride (PMSF). Moreover, BAEE in a dose-dependent fashion, blocked LIF activity on PMN leukocytes in vitro when added to the culture supernatants. Taken together, these results strongly suggest that BAEE may serve as a substrate for LIF and point to the eventual development of a biochemical assay for this lymphocyte mediator.

Evidence of an acrosin-like enzyme in sea urchin sperm

An enzyme which hydrolyzes benzoyl arginine ethylester has been demonstrated in sperm of the sea urchin Strongylocentrotus purpuratus using a sensitive assay employing tritiated substrate. Eighty percent of the enzyme is exposed but not solubilized by treatment with either 30 m M CaCl 2 or solubilized egg jelly coat. The enzymatic activity is masked in control sperm which have not been treated with these agents. The exposed enzyme is inhibited by diisopropyl phosphofluoridate (DFP), soybean trypsin inhibitor, or phenylmethane sulfonyl fluoride, suggesting that it is a serine protease and the invertebrate counterpart of vertebrate acrosin. Inhibition of the exposed enzyme with DFP prevents subsequent fertilization.

Studies on papain catalysis with substrates containing the trans-p-phenylazobenzoyl group as a probe for hydrophobic interaction.

The susceptible bonds of substrates of the type PABz-GLYn-Arg-R (R=OCH3, n=0,1,2; R=OH, n=1,2) to papain [EC 3.4.22.2] have been identified. PABz-Arg-OMe was found to be approximately 30 times more reactive than benzoylarginine ethyl ester, due to a Km(app) value 25 times smaller. The finding that PABz-Gly2-Arg-OMe was specifically hydrolyzed at the ester bond with a second-order rate constant comparable with that of PABz-Arg-OMe indicated that the S4 subsite of the enzyme is capable of accommodating a large hydrophobic group. Other substrates were cleaved at the peptide bond one residue removed from the PABz group, in accord with the well-known fact that the S2 subsite interacts preferentially with a hydrophobic P2 residue. A negative charge on the substrate at the P2' or P3' position greatly decrease the acylation rate and also the noncovalent binding affinity. The hydrolysis of PABz-Arg-OMe and PABz-Gly2-Arg-OMe was little affected by the acridine dye proflavine.

Evidence that Human Leukocyte Inhibitory Factor (LIF) Is an Esterase

Abstract There are known to be two distinct lymphocyte mediators which inhibit cell migration in vitro, migration inhibitory factor (MIF), which inhibits macrophage migration, and leukocyte inhibitory factor (LIF), which influences the migration of polymorphonuclear leukocytes (PMNL). The present studies were carried out to clarify conflicting reports concerning the sensitivity of MIF activity to a serine esterase inhibitor, diisopropyl phosphofluoridate (DFP). Human MIF and LIF activities were generated by stimulating blood lymphocytes with concanavalin A, filtering the supernatants on Sephadex G-100 gels, and collecting the active fractions of each. MIF and LIF preparations were incubated for 30 min at 37°C with varying concentrations of DFP, dialyzed extensively to remove the inhibitor, and tested on their respective indicator cells. The results showed that LIF activity, but not MIF activity, was irreversibly abolished by DFP (10-3 to 10-5 M). Another irreversible terase inhibitor m-[o-(2-chloro-5-fluorosulfonyl-phenylureido)-phenoxybutoxy] benzamidine, in a dose-dependent fashion, abolished LIF activity. The inactivation of LIF by DFP was concentration, time, pH, and temperature dependent, indicating that DFP served as a substrate for LIF. In addition, LIF preparations (but not MIF) were found to contain hydrolytic activity for certain artificial substrates such as benzoyl arginine ethyl ester and tosyl arginine methyl ester but not glysyl lysyl methyl ester. The response of human monocytes to MIF was not altered by pretreatment with DFP or soybean trypsin inhibitor (STI). In contrast, pretreatment of PMNL with DFP and STI prevented their response to LIF. These studies suggest that LIF, but not MIF, is an esterase.

FUNCTIONALLY ABNORMAL α2-MACROGLOBULIN (α2M) IN CYSTIC FIBROSIS (CF)

Previous studies in our laboratory have demonstrated the absence of an α2M-protease complex in activatad plasma of patients with CF (Padlat. Res. 10:812, 1976). Recently we have documented decreasad complex formation of CF α2M with various andoproteases as compared to normal α2M; CF heterozygotes gave intermediate values (Biochem. Biophys. Res. Commun. 71:864, 1976). This study was undertaken in ordar to furthar characterize the differences in the α2M from CF patients as compared to normal controls. The kinetic properties of purified α2M from 3 healthy donors were compared to those of α2M from 3 patients with CF. The binding affinity of α2M to bovine trypsin was determined from its inhibition of benzoyl-arginine ethyl ester hydrolysis by trypsin. A typical competitive inhibition was obtained with a Ki value of 6 × 10−7 M for normal α2M and of 3 × 10−6 M for CF α2M. The Km value for α2M-trypsin complex for this substrate was 5 × 10−4 M for normal α2M-trypsin complex and 4 × 10−5 M for the CF α2M complex. Upon incubation at 38° C, the normal α2M-trypsin complex gradually regained partial activity towards high molecular weight substrate and susceptibility to soybean trypsin inhibitor inhibition. In contrast, this phenomenon could not be demonstrated with the CF α2M-trypsin complexes. These studies provide additional evidence that the α2M in CF is functionally abnormal and provide an explanation for tha presence of the various CF factors of a polypeptide nature.

Arginine esterase in cerebrospinal fluid and pro-arginine esterase in plasma from patients with migraine.

Estimates of prekallikrein levels in plasma specimens from patients with migraine and from healthy individuals were obtained by determining the benzoyl-arginine ethyl ester (BAEe) esterase activities developed on activation with kaolin, as suggested by Costerase level- 'n the patients and in the control material, and kinetic data provided no evidence of a difference in inhibitor levels. Only very low BAEe esterase activity was registered in samples of cerebrospinal fluid obtained from the patients and no significant difference between attacks and free intervals was detected. When citrated EDTA-treated plasma was activated with acetone-incubated normal plasma containing prekallikrein activator (factor XIIf), no significant difference in BAEe esterase activity was noticed between plasma from the patients and that from the control persons. When, however, citrated plasma without EDTA was used, a significantly higher peak level of esterase activity was registered in the patient plasma. This observation might suggest the presence of a factor positioned between active factor XII and prekallikrein, and present in higher amounts in plasma from patients with migraine than in healthy individuals.

Factor X activating enzyme from Russell's viper venom: isolation and characterization.

The protease from Russell's viper venom that activates factor X (Stuart factor), factor IX (Christmas factor), and protein C was purified by gel filtration on Sephadex G-150 and QAE-Sephadex A-50 column chromatography. The purified enzyme migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 79 000. A minimal molecular weight of 78 500 +/- 800 was determined by sedimentation equilibrium in the presence of 6 M guanidine hydrochloride. Upon reduction with 2-mercaptoethanol, a heavy chain (mol wt 59 000) and a light chain were observed. The light chain migrated as a single band (mol wt 19 000) in 7.5% polyacrylamide-sodium dodecyl sulfate gels but appeared as a doublet (mol wt 18 000 and 20 000) in 10% polyacrylamide-sodium dodecyl sulfate gels. The amino-terminal end of the heavy chain was heterogeneous and contained isoleucine, valine and serine. The amino-terminal sequence of the light chain was Val-Leu-Asp. The factor X activator contained 13% carbohydrate including 6.0% hexose, 1.7% N-acetyleneuraminic acid, and 5.3% galactosamine. Most of the carbohydrate was found to be present in the heavy chain, although some was also observed in both forms of the light chain. The factor X activator had no esterase activity toward benzoyl-Phe-Val-Arg-p-nitroanilide or benzoylarginine ethyl ester and was not inhibited by 0.05 M diisopropyl phosphorofluoridate. These data indicate that factor X activator from Russell's viper venom is a highly specific protease composed of one heavy chain and one light chain, and these chains are held together by a disulfide bond(s).

The effect of calcium ions on the hydrolysis of benzoylarginine ethyl ester by porcine enteropepridase

Calcium ions are shown to have a marked pH-dependent effect on the kinetics of benzoyllarginine ethyl ester hydrolysis by porcine enteropeptidase (EC 3.4.21.9). Below pH 6.0, calcium ions stimulate benzoylarginine ethyl ester hydrolysis but inhibit this activity above pH 6.0. This effect is mainly on the K m for benzoylarginine ethyl ester. At pH 5.3, 2 mM calcium ions reduce the K m for benzoylarginine ethyl ester from 0.31 mM to 0.26 mM while at pH 6.5 the K m increases four-fold from 0.035 mM to 0.12 mM in the presence of calcium ions. Enteropeptidase activity is not inhibited by ethylenediaminetetraacetate indicating that calcium ions are a non-essential cofactor for benzoylarginine ethyl ester hydrolysis.

Immunochemical studies on pinguinain, a sulfhydryl plant protease

Pinguinain, a sulfhydryl plant protease was purified by Sephadex G-100 gel filtration, tested for homogeneity by polyacrylamide gel electrophoresis, and tested immunochemically for structural similarities with papain and fruit bromelain. This study demonstrated appreciable structural similarities with fruit bromelain but no gross similarity to papain. Chemical modification of the sulfhydryl groups by 5,5′ dithio-bis-2-nitrobenzoic acid and N-ethyl maleimide markedly diminish its enzymatic activity but do not alter appreciably its immunochemical behavior. Combination of antibodies with the native enzyme does not abolish its enzymic activity on the low molecular weight substrates benzoyl arginine ethyl ester and benzoyl arginine amide but lowers its enzymatic activity on human serum albumin. From this data we have concluded that the active site is structurally distinct from the antigenic determinant sites.

The activating system of chitin synthetase from Saccharomyces cerevisiae. Purification and properties of the activating factor.

The yeast proteinase that causes activation of the chitin synthetase zymogen has been purified by a procedure that includes affinity chromatography on an agarose column to which the proteinaceous inhibitor of the enzyme had been covalently attached. The purified enzyme yielded a single band upon disc gel electrophoresis at pH 4.5 in the presence of urea. At the same pH, but without urea, a faint band was detected in coincidence with enzymatic activity, whereas at pH 9.5, either in the absence or in the presence of sodium dodecyl sulfate, no protein zone could be seen. From sedimentation and gel filtration data, a molecular weight of 44,000 was estimated. The proteinase was active within a wide range of pH values, with an optimum between pH 6.5 AND 7. Titraton of the activity with the protein inhibitor from yeast required 1 mol of inhibitor/mol of enzyme. A similar result was obtained with phenylmethylsulfonyl fluoride, an indication that 1 serine residue is required for enzymatic activity. The enzyme exhibited hydrolytic activity with several proteins and esterolytic activity with many synthetic substrates, including benzoylarginine ethyl ester and acetyltyrosine ethyl ester.A comparison of the properties of the enzyme with those of known yeast proteinases led to the conclusion that the chitin synthestase activating factor is identical with the enzyme previously designated as proteinase B (EC 3.4.22.9). This is the first time that a homogeneous preparation of proteinase B has been obtained and characterized.

A Rat Skin Acid Esteroprotease Hydrolyzing Benzolarginine Ethylester: Purification And Properties

A benzoylarginine ethylester hydrolyzing enzyme from rat skin has been purified 54-fold by chromatography on arginine methylester-CH-Sepharose and Sephadex G-200 prior to isoelectrofocusing. The molecular weight of the enzyme is approximately 125,000. The enzyme hydrolyzes bensoylarginine ethylester, acetyltyrosine ethylester, benzoylarginine methylester, benzoyllysine methylester, and benzoylalanine methylester. Casein is slightly hydrolyzed at an optimum pH of 6.5. The enzyme is inhibited by diisopropylfluorophosphate, and p-chloromercuribenzoic acid. KC1 enhances the extraction of the enzyme, increased its activity, and is essential for its stability.

Adrenergic and cholinergic control of the activation of the kalli-krein-kinin system in the rat blood.

Sympathomimetic amines activate the kallikrein system of rat blood, causing up to 50% lowering of kininogen, transient kinin release and transient benzoyl arginine ethyl ester (BAEE) hydrolase activity. The action of catecholamines mimics and could be a model for the study of kallikrein activation and kinin release observed following nerve stimulation (Hilton & Lewis [1], Gautvik, Kriz, Lund-Learsen and Waaler [2], Inoki, Toyoda & Yamamoto [3], Turker & Turker [4], Inoki, Hayashi, Kudo, Matsumoto & Yamamoto [5]). Catecholamine activation of the kininogen-consuming process has the following characteristics: a) reproducible in vitro in rat whole oxaleted blood (6); b) not obtained in cell-free plasma nor in plasma containing either platelets, eosinophils, lymphocytes or erythrocytes (7); c) effectiveness: epinephrine > nor-epinephrine; isoprenaline ineffective, does not block the action of epinephrine (6); d) inhibited by either alpha or beta-adrenergic antagonists, the former being more potent (6); e) inhibited by acetylsalicylic acid (Aspirin), bovine kallikrein inhibitor (Trasylol) or soy bean trypsin inhibitor (SBTI) (6,7). f) reproducible in rat plasma treated with rat peritoneal fluid cell (PFC) suspensions exposed to epinephrine for 2 min.; not obtained with untreated PFC; after activation by epinephrine, PFC can be centrifuged and washed without loss of their acquired activity (8); g) not obtained when PFC free of mast cells are added to plasma in the presence of epinephrine (8); h) inhibited by dibutyril cyclic-AMP, 5 × 10−5M (9); i) possibly associated with mast cell vacuolization and intracellular granule retraction, a phenomenon observed in epinephrine or nor-epinephrine-treated rat mesentery mast cells (10) which is inhibited by Aspirin (8), at the same concentrations which block kininogen depletion; j) epinephrine activation of kallikrein in the presence of PFC is not accompanied by the release of histamine from such cells. Increased amounts of histamine are however observed in the tissues of epinephrine-treated rats (11, 12); they are to be attributed to enhanced, short-term synthesis of histamine induced by epinephrine, (Rosengren & Svensson) (13); k) kininogen depletion and mast cell vacuolization apparently identical to those produced by catecholamines, are shown in rats treated with carbamylcholine, a cholinergic agonist. The effect on kininogen can also be shown in vitro, in oxalated blood incubated for 5 min. with carbamylcholine. This excludes reflexly released catecholamines as mediators of kallikrein activation in carbamylcholine treated rats.

Influence de l'α1-macroglobuline du serum de rat sur l'activite enzymatique de la trypsine et de la chymotrypsine

1. 1. The rat α1-macroglobulin (α1M) can bind trypsin and chymotrypsin with the formation of an enzymatically active complex. The links are strong enough to resist gel-filtration. The effect of α1M on the esterolytic activities of trypsin and chymotrypsin was investigated using benzoylarginine-ethyl ester, p-tosyl- l-arginine methylester and acetyl-tyrosine ethyl ester as substrates. 2. 2. α1M inhibits esterolytic activity of chymotrypsin but has no action on trypsin. 3. 3. The Michaelis constant of the complex was determined by the method of Eadie (1942). The proteolytic activity was investigated using casein as a substrate. 4. 4. The ability of rat α1M to protect trypsin and chymotrypsin from soybean trypsin inhibitor was determined. One mole of α1M binds 2 moles of chymotrypsin. 5. 5. Rat α1M has numerous similarities with human α2M and rabbit α1 and α2M.

Stimulation of Proteolytic Digestion by Intestinal Goblet Cell Mucus

Intestinal goblet cell mucus (GCM) was added to incubations of casein and trypsin (or chymotrypsin) to discover whether mucus could inhibit proteolysis. Contrary to expectation, GCM stimulated casein hydrolysis, reaching a maximum effect at a GCM to casein ratio (w/w) of 0.083. GCM did not contain proteolytic enzymes or proenzymes as contaminants, nor did GCM serve as a substrate for trypsin. Stimulation was not reduced by removing 85% of the sialic acid from GCM. Harsh physical treatment (boiling and freezing) of casein decreased (50%) the GCM effect, as did partial predigestion of casein by trypsin, and elevation of trypsin concentration beyond 3 mug per ml. Thus the undegraded structure of casein appeared to be important for the stimulation of proteolysis by GCM. GCM also enhanced the hydrolysis by trypsin of intestinal brush border membrane protein, but had no effect on the hydrolysis of hemoglobin, albumin, or benzoyl arginine ethyl ester. These results suggest that GCM reacts with specific substrates, in a fashion which promotes their digestion by trypsin or chymotrypsin.

Determination of prekininogenase in rat plasma.

Abstract: Prekininogenases in rat citrated plasma were activated with acetone (20% v/v). EDTA was added after the evaporation of acetone to increase the activity of the enzyme preparation. The preparation was tested for kininogenolytic activity (release of kinin in 60$dG‐heated human citrated EDTA‐plasma), esterolytic activity (hydrolysis of benzoyl arginine ethyl ester or tosyl arginine ethyl ester) and caseinolytic activity. The decrease in urokinase activable caseinolytic activity together with the development of spontaneous caseinolytic activity which could be inhibited by lima bean trypsin inhibitor, and also experiments on the hydrolysis of lysine ethyl ester, suggested the presence of plasmin in the enzyme preparation. When enzyme preparations were prepared from plasma of rats pretreated with carrageenin, ellagic acid or serotonin, significant reductions in the above mentioned activities were observed. A good correlation was obtained between the two acyl‐arginine esterase assays, while no such correlation was obtained between the kininogenase and the esterase assays. This fact probably reflects differences in the relative amounts of kallikrein and plasmin present.

Propriétés générales et spécificité d'action d'une endopeptidase neutre intracellulaire de Streptococcus thermophilus

This communication describes the purification, main properties and substrate specificity of an intracellular endopeptidase from S. thermophilus. Preliminary steps of the purification procedure of the intracellular extract and the isolation of the endopeptidase by DEAE-cellulose and ECTEOLA-cellulose chromatographies have been described in a previous communication [3]. Endopeptidase purification was finished by two successive chromatographies on microgranular DEAE-cellulose and by gel filtration on Sephadex G 100. The molecular weight of this enzyme was found to be 39000 daltons by gel filtration on Sephadex G 100. It was maximally active around pH 6.50 and 40°C (substrate = glucagon); its apparent energy of activation was 10 000 cal/mole. Endopeptidase was most stable at temperatures less than 40°C. EDTA 5 × 10−4 M induced a very significant reduction — and O-phenanthroline a slight reduction — in enzyme activity. Metal ions such as Mn++ can reactivate it to a total degree, metal ions such as Co++, Zn++, Mg++ or Ca++ to a partial degree after the inactivation by a chelating agent such as EDTA. p-CMB induced a slight reduction in enzyme activity but iodo-acetic acid and DFP were not inhibitors. This enzyme attacked insulin-carboxymethylated B chain more rapidly than glucagon. Attention must be drawn to the fact that it did not appear to hydrolyse proteins (insulin, cytochrome C, ribonuclease, β-lactoglobuline, isoelectric casein). It splits neither leucine-p-nitroanilide, Leu-Leu nor benzoyl arginine ethyl ester, N-benzoyl tyrosine ethyl ester, tosyl arginine methyl ester; moreover there is no free amino acid in insulin B-chain or glucagon digest. Therefore this peptidase has to be considered as an endopeptidase. The specificity of the endopeptidase was determined by analysis of the peptides of the digests of glucagon and insulin B-chain. These peptides were isolated on columns of Bio-Rad AG 50 W × 2, Bio-Gel P 2, by paper chromatography and high tension electrophoresis. The localization of the peptides and the extent to which the various peptide bonds in the chains had been split indicate that the two peptide bonds in insulin B-chain Tyr18-Leu17 and Gly23-Phe24 are very sensitive to the action of this endopeptidase; the peptide honds in insulin B-chain His5-Leu6, His10-Leu11, Glu13-Ala14, Ala14-Leu15, Leu15-Tyr16 and Tyr26-Th27 or glucagon Thr5-Phe6, Asp21-Phe22, Val23-Gln24 and Leu26-Met27 are less sensitive. All of the bonds readily cleaved were those involving the α-amino group of hydrophobic residues, i.e. X-Leu or X-Phe. Therefore this intracellular enzyme is a «neutral endopeptidase. La purification, les principales propriétés et la spécificité d'action d'une endopeptidase intracellulaire de S. thermophilus sont décrites dans ce mémoire. Les étapes préliminaires de purification de l'extrait endocellulaire ont été décrites précédemment ainsi que l'isolement de l'endopeptidase après chromatographie sur DEAE-cellulose et ECTEOLA-cellulose [3]. La purification de l'endopeptidase était achevée par deux chromatographies successives sur DEAE-cellulose microgranulaire et par filtration sur gel Sephadex G 100. Son poids moléculaire était estimé à 39 000 daltons par filtration sur gel Sephadex G 100. Son activité était maximum à pH 6,50 et à 40°C (substrat glucagon); l'énergie apparente d'activation était de 10 000 cal./mole. L'endopeptidase était stable pour les température inférieures à 40°C. L'EDTA 5 × 10−4 M inhibait fortement l'enzyme et l'O-phénanthroline faiblement. Les ions Mn++ la réactivaient totalement, les ions Co++, Zn++, Mg++ ou Ca++ partiellement, après l'inactivation par un chélateur tel que l'EDTA. Le p-CMB l'inhibait faiblement mais l'acide iodoacétique et le DFP n'étaient pas inhibiteurs. Cette enzyme hydrolyse la chaîne B carboxyméthylée de l'insuline plus fortement que le glucagon, mais elle ne semble pas hydrolyser les protéines natives (insuline, cytochrome C, ribonucléase, β-lactoglobuline, caséines). Elle n'hydrolyse pas non plus ni la LNA, ni la Leu-Leu, ni la BAEE, ni la BTEE, ni la TAEE. De plus, aucun acide aminé libre n'était non plus retrouvé après hydrolyse du glucagon ou de la chaîne B de l'insuline. Donc cette peptidase doit être considérée comme une endopeptidase stricte. Sa spécificité était déterminée par analyse des peptides des hydrolysats de glucagon et de chaîne B de l'insuline. Ces peptides étaient isolés sur colonne de Bio-Rad AG 50 W × 2 et Bio-Gel P 2 et par chromatographie sur papier et électrophorèse sous haute tension. La position des peptides dans ces chaînes et l'estimation de leur rendement montrent que les deux liaisons peptidiques Tyr16-Leu17 et Gly23Phe24 de la chaîne B de l'insuline sont très sensibles à l'action de l'endopeptidase; les liaisons His5-Leu6, His10-Leu11, Glu13-Ala14, Ala14-Leu15, Leu15-Tyr16, et Tyr26-Thr27 de la chaîne B de l'insuline et Thr5-Phe6, Asp21-Phe22, Val23-Gln24, et Leu26-Met27 du glucagon sont moins rapidement hydrolysées. La plupart des liaisons peptidiques et, spécialement celles le plus rapidement clivées, impliquent le groupe α-aminé d'un résidu hydrophobe, tel que X-Leu ou X-Phe. Donc cette enzyme intracellulaire appartient au groupe des endopeptidases «neutres.