Proteolysis of the calcium-dependent protease inhibitor by myocardial calcium-dependent protease

Bovine heart peak II calcium-dependent protease was capable of hydrolyzing its specific inhibitor protein at high molar ratios of protease to inhibitor. The proteolysis was inhibited by leupeptin and required millimolar calcium. Thus, it appeared to be attributable to the calcium-dependent protease and not to possible contaminating proteases in the purified preparations of inhibitor or calcium-dependent protease. Incubation of the purified inhibitor with the calcium-dependent protease produced a discrete pattern of inhibitor fragments on Western blots developed with an inhibitor-specific monoclonal antibody. Traces of similar or identical lower molecular weight immunoreactive material could be observed in Western blots of bovine heart extracts, and the immunoreactivity present as these lower molecular weight forms could be increased by incubation of the extracts with calcium ion. These results suggest that the inhibitor can be proteolyzed to low molecular weight forms which can be detected in cardiac tissue extracts, and that calcium-dependent protease(s) may be responsible for this phenomenon.     

Purification and characterization of the protease enzymes of Streptomyces thermovulgaris grown in rapemeal-derived media

When grown in a particulate-free, protein-rich medium derived from rapemeal (termed medium B), Streptomyces thermovulgaris produced multiple protease enzymes. The main protease activity was attributed to two types of serine protease, denoted as SV1 and SV2. A metallo protease component (SV3) and an azocaseinase component (SV4) were also present. Protease SV1 had a molecular weight of 30 kDa and a pI of 5.8. Protease SV2 was characterized by a high thermostability in the presence of calcium ions and had a pI of 8.4. This enzyme had a molecular weight of 60 kDa, but we suggest that this is the dimeric form, with 30 kDa being the monomer unit. The method chosen for initial downstream processing influenced both the yield and type of protease purified. When cell-free supernatant fluid was concentrated using ultrafiltration, rather than acetone precipitation, a higher percentage and a greater range of proteases were recovered. The medium used for the growth of Strep. thermovulgaris also appeared to affect the type of protease produced. A more diverse range of proteases were produced on rapemeal-derived medium when compared to yeast extract medium.     

Regulation of smooth muscle myosin light chain kinase. Allosteric effects and co-operative activation by calmodulin

The activation of smooth muscle myosin light chain kinase (MLCKase) by calcium and calmodulin (CM) was investigated over a wide range of concentrations of the enzyme using myosin (MY) or its isolated phosphorylatable light chain (L20) as substrates. The enzyme showed allosteric behavior. The specific phosphorylation activity was dependent on the concentration of MLCKase as well as on the concentrations of both substrates. However, at the lower (nanomolar) range of kinase the corresponding substrate rate relationships were hyperbolic. A high positive level of co-operativity of kinase was also observed for activation by CM in the presence of Ca2+. There was a pronounced CM/Ca-dependent inhibition of MLCKase activity when its molar ratio to CM was four to one or more. These kinetic data suggested that MLCKase could exist in several oligomeric forms, with an inactive high molecular size form and an active low molecular size form (protomers and/or dimers). This conclusion was confirmed by gel filtration studies. CM was not directly involved in the oligomerization process but instead, the oligomeric kinase shared an increased affinity for CM.     

Cytoplasmic proteases of rat liver parenchymal cells

Soluble extracts of isolated rat liver parenchymal cells contained three proteases with alkaline pH optima. One protease was a high molecular weight (Mr = 500,000) enzyme which was stimulated by ATP. The other two proteases were totally dependent on calcium for activity and displayed different calcium concentration requirements. One was half-maximally activated by 150 μM Ca²⁺ while the other required only 10 μM Ca²⁺ for half-maximal activation.     

The crystal structures of the psychrophilic subtilisin S41 and the mesophilic subtilisin Sph reveal the same calcium-loaded state

We determine and compare the crystal structure of two proteases belonging to the subtilisin superfamily: S41, a cold-adapted serine protease produced by Antarctic bacilli, at 1.4 A resolution and Sph, a mesophilic serine protease produced by Bacillus sphaericus, at 0.8 A resolution. The purpose of this comparison was to find out whether multiple calcium ion binding is a molecular factor responsible for the adaptation of S41 to extreme low temperatures. We find that these two subtilisins have the same subtilisin fold with a root mean square between the two structures of 0.54 A. The final models for S41 and Sph include a calcium-loaded state of five ions bound to each of these two subtilisin molecules. None of these calcium-binding sites correlate with the high affinity known binding site (site A) found for other subtilisins. Structural analysis of the five calcium-binding sites found in these two crystal structures indicate that three of the binding sites have two side chains of an acidic residue coordinating the calcium ion, whereas the other two binding sites have either a main-chain carbonyl, or only one acidic residue side chain coordinating the calcium ion. Thus, we conclude that three of the sites are of high affinity toward calcium ions, whereas the other two are of low affinity. Because Sph is a mesophilic subtilisin and S41 is a psychrophilic subtilisin, but both crystal structures were found to bind five calcium ions, we suggest that multiple calcium ion binding is not responsible for the adaptation of S41 to low temperatures.     

Preferential degradation of the oxidatively modified form of glutamine synthetase by intracellular mammalian proteases

Four intracellular proteases partially purified from liver preferentially degraded the oxidatively modified (catalytically inactive) form of glutamine synthetase. One of the proteases was cathepsin D which is of lysosomal origin; the other three proteases were present in the cytosol. Two of these were calcium-dependent proteases with different calcium requirements. The low-calcium-requiring type (calpain I) accounted for most of the calcium-dependent activity of both mouse and rat liver. The calcium-independent cytosolic protease, referred to as the alkaline protease, has a molecular weight of 300,000 determined by gel filtration. Native glutamine synthetase was not significantly degraded by the cytosolic proteases at physiological pH, but oxidative modification of the enzyme caused a dramatic increase in its susceptibility to attack by these proteases. In contrast, trypsin and papain did degrade the native enzyme and the degradation of modified glutamine synthetase was only 2- to 4-fold more rapid. Adenylylation of glutamine synthetase had little effect on its susceptibility to proteolysis. Although major structural modifications such as dissociation, relaxation, and denaturation also increased the rate of degradation, the oxidative modification is a specific type of covalent modification which could occur in vivo. Oxidative modification can be catalyzed by a variety of mixed function oxidase systems present within cells and causes inactivation of a number of enzymes. Moreover, the presence of cytosolic proteases which recognize the oxidized form of glutamine synthetase suggests that oxidative modification may be involved in intracellular protein turnover.     

Extracellular and membrane-bound proteases from Bacillus subtilis.

Bacillus subtilis YY88 synthesizes increased amounts of extracellular and membrane-bound proteases. More than 99% of the extracellular protease activity is accounted for by an alkaline serine protease and a neutral metalloprotease. An esterase having low protease activity accounts for less than 1% of the secreted protease. These enzymes were purified to homogeneity. Molecular weights of approximately 28,500 and 39,500 were determined for the alkaline and neutral proteases, respectively. The esterase had a molecular weight of approximately 35,000. Amino-terminal amino acid sequences were determined, and the actions of a number of inhibitors were examined. Membrane vesicles contained bound forms of alkaline and neutral proteases and a group of previously undetected proteases (M proteases). Membrane-bound proteases were extracted with Triton X-100. Membrane-bound alkaline and neutral proteases were indistinguishable from the extracellular enzymes by the criteria of molecular weight, immunoprecipitation, and sensitivity to inhibitors. The M protease fraction accounted for approximately 7% of the total activity in Triton X-100 extracts of membrane vesicles. The M protease fraction was partially fractionated into four species (M1 through M4) by ion-exchange chromatography. Immunoprecipitation and sensitivity to inhibitors distinguished membrane-bound alkaline and neutral proteases from M proteases. In contrast to alkaline and neutral proteases, proteases M2 and M3 exhibited exopeptidase activity.     

Purification from Synaptosomal Plasma Membranes of Calpain I, a Thiol Protease Activated by Micromolar Calcium Concentrations

Synaptosomal plasma membranes (SPMs) were prepared from whole rat brain and assayed for calcium-stimulated proteolytic activity. Addition of calcium to SPMs caused a dose-dependent increase in trichloroacetic acid-soluble protein. Two peaks of protease activity directed against a casein substrate were detectable when SPMs were incubated with low-ionic-strength buffer and the extract was fractionated on DEAE-cellulose. The enzyme in peak 1 required less than 1/10 the calcium concentration for activation as the peak 2 protease (Kact1 = 35 microM; Kact2 = 500 microM). The specific thiol-protease inhibitors leupeptin and antipain and the alkylator iodoacetate blocked enzyme activity. The low-sensitivity protease was converted to a high-sensitivity enzyme (Kact = 20 microM) by substrate affinity chromatography in the presence of calcium. This protease was purified 550-fold from SPMs. The high- and low-sensitivity membrane-associated calcium-dependent proteases are part of a family of enzymes, the calpains, previously reported in cytosolic fractions of several tissues.     

Isolation and biological activity of the proteases released by sea urchin eggs following fertilization.

The protease activity released from sea urchin egg cortical granules into the surrounding seawater at fertilization is involved in vitelline layer elevation and the block to polyspermy. The cortical granule protease components were isolated by isoelectric precipitation and affinity chromatography on p-aminobenzamidine-Sepharose columns. Elution profiles from affinity columns suggested heterogeneity of the proteases, and polyacrylamide-gel electrofocusing of affinity-purified preparations established the presence of two proteins. Dramatically different biological activities were resolved by affinity chromatography. Early-eluting fractions of low specific activity delaminated the vitelline layer from the egg plasma membrane; this activity is termed vitelline delaminase. Late-eluting fractions of high specific activity modified the egg vitelline layer surface such that sperm could not bind or fertilize them; this activity is referred to as sperm receptor hydrolase. The biological activities of the sea urchin proteases are apparently the result of limited action on the vitelline layer, unlike bovine trypsin which simply digests the vitelline layer. The cortical granule proteases lost biological specificity when stored at 0°C at pH 8.0. Esterase activity increased, and the preparation acquired the ability to digest the vitelline layer. Increase of the esterase activity in protease preparations was prevented by storage at low pH.The molecular weight of both enzymes was estimated by sucrose gradient centrifugation to be 47,000, whereas multiple components with molecular weights between 10⁵ and 10⁶ were demonstrated by gel filtration.     

Role of bound calcium ions in thermostable, proteolytic enzymes. Separation of intrinsic and calcium ion contributions to the kinetic thermal stability.

The total kinetic thermal stability of a protein molecule, expressed as the total free energy of activation in thermal denaturation reactions, can be separated into an intrinsic contribution of the polypeptide chain and a contribution due to the binding of calcium ions. The theory for this procedure is applied to thermal denaturation data, obtained at the pH of optimum stability, for the serine proteases, thermomycolase and subtilisin types Carlsberg and BPN', and for the zinc metalloendopeptidases, thermolysin and neutral protease A. The results, obtained from Arrhenius plots at high and low free calcium ion concentrations, reveal a considerable variation in the calcium ion contribution to the total kinetic thermal stability of the various enzymes. In the serine protease group, at 70 degrees C, the stability is largest for thermomycolase, mainly due to a relatively high intrinsic contribution. For the metalloendopeptidases the total kinetic thermal stability is largest for thermolysin, the difference between thermolysin and neutral protease A being dominated by bound calcium ion contributions. The intrinsic kinetic thermal stability of the polypeptide chain of thermolysin is considerably smaller than that of any of the serine proteases and is probably of the same order of magnitude as that of neutral protease A. Thus, the well known total kinetic thermal stability of thermolysin is due mainly to a single calcium ion (Voordouw, G., and Roche, R. S. (1975), Biochemistry 14, 4667) that binds with high affinity even at very high temperatures (K congruent to 6 X 10(7) M-1 at 80 degrees C).     

Developmentally regulated proteases from the basidiomycete Schizophyllum commune

The basidiomycete Schizophyllum commune produces three chromatographically distinguishable proteases which are capable of attack on a variety of other enzymes from S. commune and other sources. These proteases, which are produced during a specific phase of the development cycle, exhibit typical enzyme kinetic patterns, are active in the neutral to weakly alkaline pH range and are inhibited by phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, and ovomucoid. No pattern of specificity toward the test enzymes could be discerned. The proteases co-purify with the activity which causes the increase in cold lability of S. commune phosphoglucomutase reported previously. In addition, one of the protease enzymes could be purified to the point where it had no significant ability to release trichloroacetic acid products from denatured substrates at pH 3 or pH 7. When undenatured hemoglobin was used as a substrate, the purified protease releases a relatively large molecular weight nonheme peptide. Relatively large peptides are also formed after proteolysis of rabbit muscle phosphoglucomutase. These results suggest that the protease carries out only limited proteolysis.     

The structure and function of acid proteases. VII. Distribution and some properties of acid proteases in monkey tissues.

1. The distribution of acid protease activity in various tissues of Japanese monkey (Macaca fuscata fuscata) was investigated with hemoglobin as a substrate at pH 3.0. The activity per protein weight in crude extracts was highest in spleen and lung, and decreased in the order: spleen, lung greater than kidney, testis greater than brain greater than liver, placenta greater than thyroid gland, muscle. The activity in crude muscle extract was about one-tenth those of spleen and lung. The activity per wet tissue weight was in roughly the same order except for a lower activity per wet weight of brain. 2. Upon chromatography of each crude extract on a Sephadex G-100 column, one major activity peak was eluted at a position corresponding to a molecular weight of about 41,000. This enzyme activity is attributed to cathepsin D [EC 3.4.23.5]. In addition, a minor activity peak was eluted in the case of spleen, lung and kidney at the break-through position, corresponding to a molecular weight of more than 100,000. This activity peak is presumably due to cathepsin E. These acid protease activities were, in most cases, strongly inhibited by pepstatin, an acid protease-specific peptide inhibitor. 3. The distribution of acid protease activity was investigated in the brain of crab-eating monkey (Macaca fascicularis). The activity was fairly evenly distributed among several regions of the brain, and its distribution was similar to those of other acid hydrolases, especially N-acetyl-beta-D-glucosaminidase [EC 3.2.1.30] and acid phosphatase [EC 3.1.3.2], which are marker enzymes of lysosomes.     

Purification and characterization of two extracellular alkaline proteases from a newly isolated obligate alkalophilic Bacillus sphaericus

Two novel extracellular serine proteases were purified to homogeneity from the cell-free culture filtrate of an obligate alkalophilic Bacillus sphaericus by a combination of ultrafiltration, ammonium sulfate precipitation and chromatographic methods. The enzymes showed similar substrate specificities, but differed in hydrophobicity and molecular mass. Protease A was a monomeric protease with a relative molecular mass (M _r) of 28.7 kDa, whereas protease B, with a M _r of 68.0 kDa, apparently consisted of smaller subunits. The purified protease A had a specific activity on hemoglobin of 5.1 U/mg protein compared to 40.9 U/mg protein in the case of protease B. Both proteases were most active on SAAPF-pNa, a substrate for chymotrypsin-like serine proteases. However, the K _m values of these two proteases on SAAPF-pNa were higher than that for α-chymotrypsin, indicating a lower affinity of proteases A and B for this substrate compared to chymotrypsin. Unlike other Bacillus serine proteases, neither protease A nor B stained with Coomasie blue R-250, even with loading of a large amount of protein, and they stained poorly with the silver staining method. However, NH₂-terminal amino acid sequencing of protease B revealed a high similarity with subtilisin Carlsberg (67% homology). Almost total inhibition of both proteases by PMSF, but very little/no inhibition by trypsin and chymotrypsin inhibitors (TPCK and TLCK) or thiol reagents (PCMB and iodoacetic acid), further supported the view that the enzyme belonged to the serine protease family. Journal of Industrial Microbiology & Biotechnology (2001) 26, 387–393. Received 05 November 2000/ Accepted in revised form 23 April 2001     

Proteolysis of the protein inhibitor of calcium-dependent proteases produces lower molecular weight fragments that retain inhibitory activity

The specific inhibitor of calcium-dependent proteases was purified from soluble extracts of bovine heart. The protein had a molecular weight of 125,000 on sodium dodecyl sulfate polyacrylamide gels and migrated on gel filtration chromatography with an apparent molecular weight of 250,000. The inhibitor specifically blocked the action of the two calcium-dependent proteases, CDP-I and CDP-II, but did not influence a variety of other proteases including trypsin, chymotrypsin, or Staphylococcus aureus V8 protease. These latter enzymes extensively degraded the inhibitor to discrete lower molecular weight peptides as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by gel filtration chromatography. Under the conditions studied, proteolysis of the inhibitor had little or no effect on its inhibitory activity; isolated peptides with molecular weights as low as 17,000 retained inhibitory function. A number of various-sized inhibitor fragments were isolated by gel filtration chromatography and by SDS-PAGE. These fragments were compared with the intact inhibitor for their ability to inhibit CDPs. As suggested previously by us and others, one molecule of intact inhibitor appears to inhibit up to five molecules of calcium-dependent protease. The inhibitor fragments of decreasing size inhibited correspondingly fewer molecules of protease. These results suggest that the inhibitor protein contains multiple functional domains and may explain some of the discrepancies in reported molecular weights for this protein.     

Proteases in egg, miracidium and adult of Fasciola gigantica. Characterization of serine and cysteine proteases from adult

Proteolytic activity of 0-12 day old eggs, miracidium and adult worm of Fasciola gigantica was assessed and proteases were partially purified by DEAE-Sepharose and CM-cellulose columns. Four forms of protease were separated, PIa, PIb, PIc and PII. Purifications were completed for PIc and PII using Sephacryl S-200 chromatography. A number of natural and synthetic proteins were tested as substrates for F. gigantica PIc and PII. The two proteases had moderate activity levels toward azoalbumin and casein compared to azocasein, while gelatin, hemoglobin, albumin and fibrin had very low affinity toward the two enzymes. Amidolytic substrates are more specific to protease activity. PIc had higher affinity toward BAPNA-HCl (N-benzoyl-arginine-p-nitroanilide-HCl) and BTPNA-HCl (N-benzoyl-tyrosine-p-nitroanilide-HCl) at pH 8.0 indicating that the enzyme was a serine protease. However, PII had higher affinity toward BAPNA at pH 6.5 in the presence of sulfhydryl groups (beta-mercaptoethanol) indicating that the enzyme was a cysteine protease. The effect of specific protease inhibitors on these enzymes was studied. The results confirmed that proteases PIc and PII could be serine and cysteine proteases, respectively. The molecular weights of F. gigantica PIc and PII were 60,000 and 25,000, respectively. F. gigantica PIc and PII had pH optima at 7.5 and 5.5 and K(M) of 2 and 5 mg azocasein/mL, respectively. For amidolytic substrates, PIc had K(M) of 0.3 mM BAPNA/mL and 0.5 mM BTPNA/mL at pH 8.0 and PII had K(M) of 0.6 mM BAPNA/mL at pH 6.5 with reducing agent. F. gigantica PIc and PII had the same optimum temperature at 50 degrees C and were stable up to 40 degrees C. All examined metal cations tested had inhibitory effects toward the two enzymes. From substrate specificity and protease inhibitor studies, PIc and PII could be designated as serine PIc and cysteine PII, respectively.     

Degradation of proteins with blocked amino groups by cytoplasmic proteases

The effect of blocking amino groups on the susceptibility of BSA and calmodulin to high molecular weight protease (HMP) and calpain, the two major cytosolic proteases, was studied. Both proteases hydrolyzed methylated vs. unmodified BSA more slowly. Methylation of BSA resulted in the accumulation of proteolytic intermediates, especially of larger sizes. However, similar fragments were generated from unmodified BSA indicating that rates of hydrolysis rather that sites of proteolytic cleavage were altered. Calmodulin from Dictyostelium discoideum was hydrolyzed rapidly by HMP whereas brain and muscle calmodulins which have a epsilon-N-trimethyl residue on the single surface lysine were relatively stable.     

The role of subunit autolysis in activation of smooth muscle Ca2+-dependent proteases

Ca2+-dependent proteases isolated from chicken gizzard and bovine aortic smooth muscle were compared with respect to subunit autolysis and the role of autolysis in modulating enzyme activity. The protease isolated from chicken gizzard was a heterodimer consisting of 80,000- and 30,000-dalton subunits. The protease isolated under identical conditions from bovine aorta consisted of 75,000- and 30,000-dalton subunits. In the presence of Ca2+, both enzymes underwent autolysis of their 30,000-dalton subunits with conversion to an 18,000-dalton species. In addition, the 80,000-dalton subunit of the gizzard protease was degraded to a 76,000-dalton form. The Ca2+ concentrations required for autolysis of the 30,000-dalton subunits were different for the two enzymes (i.e. gizzard: K0.5 Ca2+ = 335 microM; aortic: K0.5 Ca2+ = 1,250 microM) although in both cases, stimulation of autolysis by Ca2+ exhibited positive cooperativity. When compared with respect to kinetics of substrate degradation, the native forms of the smooth muscle Ca2+-dependent proteases (gizzard, GIIa = 80,000/30,000-dalton heterodimer; bovine aortic, IIa = 75,000/30,000-dalton heterodimer) exhibited a lag phase in product appearance. On the other hand, the autolyzed forms (gizzard, GIIb = 76,000/18,000-dalton heterodimer; bovine aortic, IIb = 75,000/18,000-dalton heterodimer) exhibited linear rates of substrate degradation. These results were analyzed in terms of autolysis of the 30,000-dalton subunits as determined by the conversion of this subunit to its 18,000 dalton form. For both enzymes, the time course for the autolytic transition, 30,000----18,000 daltons, and Ca2+-dependence of the apparent rate constants for this transition were found to correlate well with the lag phase in enzymatic activity. No such correlation could be established for the 80,000----76,000 dalton autolytic transition of the high molecular mass subunit of the gizzard protease. Our results suggest that catalytic activity of the Ca2+-dependent proteases isolated from gizzard and bovine aortic smooth muscle requires autolysis of the 30,000-dalton subunit. The native or unautolyzed forms of these enzymes appear to be proenzymes that can be activated by autolysis.     

Interaction of insulin-like growth factor II (IGF-II) with multiple plasma proteins: high affinity binding of plasminogen to IGF-II and IGF-binding protein-3

In the circulation, most of the insulin-like growth factors (IGFs), IGF-binding proteins (IGFBPs), and IGFBP proteases are bound in high molecular mass complexes of > or =150 kDa. To investigate molecular interactions between proteins involved in IGF.IGFBP complexes, Cohn fraction IV of human plasma was subjected to IGF-II affinity chromatography followed by reversed-phase high pressure liquid chromatography and analysis of bound proteins. Mass spectrometry and Western blotting revealed the presence of IGFBP-3, IGFBP-5, transferrin, plasminogen, prekallikrein, antithrombin III, and the soluble IGF-II/mannose 6-phosphate receptor in the eluate. Furthermore, an IGFBP-3 protease cleaving also IGFBP-2 but not IGFBP-4 was co-purified from the IGF-II column. Inhibitor studies and IGFBP-3 zymography have demonstrated that the 92-kDa IGFBP-3 protease belongs to the class of serine-dependent proteases. IGF-II ligand blotting and surface plasmon resonance spectrometry have been used to identify plasminogen as a novel high affinity IGF-II-binding protein capable of binding to IGFBP-3 with 50-fold higher affinity than transferrin. In combination with transferrin, the overall binding constant of plasminogen/transferrin for IGF-II was reduced 7-fold. Size exclusion chromatography of the IGF-II matrix eluate revealed that transferrin, plasminogen, and the IGFBP-3 protease are present in different high molecular mass complexes of > or =440 kDa. The present data indicate that IGFs, low and high affinity IGFBPs, several IGFBP-associated proteins, and IGFBP proteases can interact, which may result in the formation of binary, ternary, and higher molecular weight complexes capable of modulating IGF binding properties and the stability of IGFBPs.     

Purification of new calcium activated protease (low calcium requiring form) and comparison to high calcium requiring form

A new calcium activated protease which requries low concentration of calcium was purified to almost homogeneity from porcine heart muscle. The protease was composed of two polypeptide chains of approximately 90 K and 30 K. The 90 K subunit was larger than the large subunit of the high calcium requring form of calcium activated protease, therefore we concluded that the low calcium requiring form is different from the high calcium requring form and its auto-digested protease. The low calcium reqiring form of calcium activated protease was also activated by manganese and balium, and was very stable even at pH 9.0     

Studies on new intracellular proteases in various organs of rat. 1. Purification and comparison of their properties.

1. Specific proteases which inactivate the apo-proteins of many pyridoxal enzymes were found in skeletal muscle, liver and small intestine of rats. The protease from these three organs were purified and their properties were compared. 2. The purified proteases from liver and skeletal muscle appeared homogeneous on acrylamide gel electrophoresis. Two different proteases were separated from small intestine. A homogeneous, crystalline enzyme was obtained from the muscle layer while enzyme from the mucosa was partially purified. 3. They showed substrate specificity for pyridoxal enzymes. Their pH optima were in an alkaline region. They showed activity with the substrate of chymotrypsin, N-acetyl-L-tyrosine ethyl ester, but not with that of trypsin, p-toluenesulfonyl-L-arginine ethyl ester. They were inhibited by pyridoxal phosphate or pyridoxamine phosphate and seryl residues were involved in their active center. 4. The four enzymes differed in the following characters: (a) molecular weights; (b) patterns of elution from a CM-Sephadex column; (c) rates of inactivation of substrate enzymes; (d) rates of cleavage of N-acetyl-L-tyrosine ethyl ester; (e) reactivities with antiserum against the enzyme from the muscle layer of small intestine; (f) specific activities. 5. The amino acid composition and effect of chemical modifications of the crystalline enzyme from the muscle layer of small intestine were examined to elucidate its active sites and mode of action. Serine and histidine residues were found to be essential for protease activity. A tyrosine residue was also necessary for activity. Modifications of its sulfhydryl group, amino residues and carboxyl group had no effect on its activity.