The multicatalytic proteinase. Multiple proteolytic activities

The multicatalytic proteinase is a high molecular weight nonlysosomal proteinase which has been isolated from a variety of mammalian tissues and has been suggested to contain several distinct catalytic sites. The enzyme degrades protein and peptide substrates and can cleave bonds on the carboxyl side of basic, hydrophobic, and acidic amino acid residues. The three types of activity have been referred to as trypsin-like, chymotrypsin-like, and peptidyl-glutamyl peptide bond hydrolyzing activities, respectively. All of these proteolytic activities are associated with a single band on native polyacrylamide gels. The pH optimum of the proteinase (pH 7.5-9.5) depends on the substrate. Using synthetic peptide substrates it was possible to demonstrate two distinct activities. Trypsin-like activity is inhibited at concentrations of the peptide aldehyde inhibitors leupeptin and antipain or of N-ethylmaleimide which have little or no effect on chymotrypsin-like activity. Results of mixed-substrate experiments also suggest that there are at least two distinct types of catalytic sites. All proteolytic activity is lost following dissociation by urea or by acid treatment. Polyclonal antibodies raised against the intact multicatalytic proteinase precipitate the complex but have little effect on its proteolytic activities.     

Purification and characterization of proteolytic enzymes of Leishmania mexicana mexicana amastigotes and promastigotes

Leishmania mexicana mexicana amastigote and promastigote soluble proteinases were purified using gel filtration and ion exchange chromatography. For the amastigotes, two main proteinase activity peaks were separated with both methods. These accounted for approximately 10% and 90% of the total activity. Characterization of the two activities for substrate specificity and sensitivity to inhibitors indicated that the major peak from both column methods contained enzymes with the characteristics of cysteine proteinases. SDS-polyacrylamide gel electrophoresis of the enzyme from the major peak purified by gel filtration revealed one polypeptide with a molecular weight in the region of 31 000. In contrast, the activity of the minor peak eluted from the columns was of higher molecular weight (67 000) and was similar to metalloproteinases. Purification of the soluble proteinases in the promastigote of L. m. mexicana produced only one activity peak from both column techniques. This activity (mol. wt 67 000) corresponded to the high molecular weight proteinase of the amastigote. The purified proteinases were active on 4-nitroanilide and 7-amino-4-methylcoumarin derivatives of various small peptides. The high molecular weight proteinases of both amastigotes and promastigotes were similarly active against most of the peptides, suggesting a low specificity of the enzymes. In contrast, the low molecular weight amastigote proteinases were particularly active against two of the substrates, namely BZ-Pro-Phe-Arg-Nan and Z-Phe-Arg-MCA. These results indicate that a highly active, substrate-specific, soluble proteinase, with characteristics of a cysteine proteinase, is produced upon transformation of the L. m. mexicana promastigote to amastigote. The discovery and characterization of this enzyme offers opportunities for the development of new antileishmanial agents.     

Characterization and partial purification of the digestive proteases of the black field cricket,Teleogryllus commodus (Walker): Elastase is a major component

The major proteases of the black field cricket, Telleogryllus commodus, digestive system have been identified, partially purified and characterized. Classification of proteases into different classes of endo- and exopeptidases was made on the ability to hydrolyse specific synthetic substrates, pH optima and their interaction with a range of specific chemical and proteinaceous inhibitors. The major activities detected were trypsin, elastase, an uncharacterized proteinase (proteinase Tc), leucine aminopeptidase and carboxypeptidases A and B. Chymotrypsin activity was very low and neither cysteine endopeptidase nor metalloendopepitidase activities were found. Elastase is a newly discovered protease activity for insects.Trypsin, elastase and proteinase Tc have molecular weights of 24,300, 19,500 and 23,600, respectively; show alkaline pH optima and chemical inhibition indicative of serine endopeptidases; and interact most strongly with their characteristic class of proteinaceous inhibitors. Elastase and proteinase Tc are inhibited by a very similar spectrum of specific inhibitors, but the latter lacks activity against all specific synthetic substrates tested. Leucine aminopeptidase and carboxypeptidase A have molecular weights of 94,000 and 39,700, respectively, and show optimum activity at pH 8 and pH 9, respectively.The equilibrium dissociation constants for trypsin, elastase and proteinase Tc with 25 serine proteinase inhibitors were measured. Values spanning a 1000-fold range were obtained in each case.     

Assessment of proteasome activity in cell lysates and tissue homogenates using peptide substrates

The ubiquitin-proteasome pathway is a major route of degradation of cell proteins. It also plays an essential role in maintaining cell homeostasis by degrading many rate-limiting enzymes and critical regulatory proteins. Alterations in proteasome activity have been implicated in a number of pathologies including Parkinson's disease, Alzheimer's disease and diabetes. The eukaryotic proteasome is a multicatalytic protease characterized by three activities with distinct specificities against peptide substrates. Although substrates were identified which could selectively measure the individual activities in the purified proteasome little data is available on how specific those substrates are for proteasomal activity when used with biological samples which may contain many other active peptidases. Here we examine the three major peptidase activities in lysates of two cell types and in a liver cytosol fraction in the presence of specific proteasome inhibitors and after fractionation by gel permeation chromatography. We demonstrate that other proteinases present in these preparations can degrade the commonly used proteasome substrates under the standard assay conditions. We develop a simple method for separating the proteasome from the lower molecular weight proteases using a 500kDa molecular weight cut-off membrane. This allows proteasome activity to be accurately measured in crude biological samples and may have quite broad applicability. We also identify low molecular weight tryptic activity in both the cell and tissue preparations which could not be inhibited by the proteasome inhibitor epoxomycin but was inhibitable by two cysteine proteinase inhibitors and by lactacystin suggesting that lactacystin may not be completely proteasome specific.     

Characterization of different proteolytic activities in Trypanosoma brucei brucei

The variant surface glycoprotein of African trypanosomes is released after overnight incubation of parasites at 4°C in pH 5.5 phosphate glucose buffer and may be purified by Concanavalin A Sepharose affinity chromatography [1]. The addition of proteinase inhibitors during the parasite incubation is necessary to prevent the proteolysis of the variant surface glycoproteins by the trypanosomal released proteinases. Using this procedure without the addition of proteinase inhibitors, the proteolytic activities, released from the bloodstream forms Trypanosoma brucei brucei variant AnTat 1.1, were separated by Concanavalin-A Sepharose affinity chromatography. The unretained material (F1) shows hydrolytic activity against the two synthetic substrates Z-Phe-Arg-AMC and Z-Arg-Arg-AMC, which is stimulated by dithiothreitol, but not inhibited by E-64, and characterized by and alkaline pH optimum and an estimated molecular mass of 80–100 kDa. The Michaelis constant for the substrates Z-Arg-Arg-AMC and Z-Phe-Arg-AMC was, respectively, 2.8 and 6.7 μM. The retained material eluted by addition of 1% methyl-α-D-mannopyranoside (F2) shows hydrolytic activity against the synthetic substrate Z-Phe-Arg-AMC, which is stimulated by dithiothreitol, inhibited by E-64, active between pH 6.0 and 8.0, and could be separated into two peaks of activity by HPLC, one peak of high molecular mass (> 70 kDa) and the other peak of lower molecular mass (30–70 kDa). By electrophoresis in gels containing gelatin as substrate, this fraction contains several proteins with gelatinolytic activity, whereas the unretained fraction F1 did not have any gelatinolytic activity.     

Effects of yeast proteinase A, proteinase B and carboxypeptidase Y on yeast phosphofructokinase.

Incubation of a crude yeast extract containing phosphofructokinase with proteinase A, proteinase B or carboxypeptidase Y gave the following results: Proteinase B and carboxypeptidase Y did not change the activity of phosphofructokinase during incubation. On the other hand, incubation with proteinase A resulted in a 40-100% activation; continued incubation, however, led to an inactivation of the enzyme. Addition of allosteric effectors did not change the activation or inactivation process. The activated phosphofructokinase was not changed with respect to pH optimum and ATP inhibition. Molecular weight determination of phosphofructokinase in crude extracts in the presence of inhibitors of proteinase A indicated a molecular weight of 700000. Without inhibitors of proteinase A, the molecular weight was determined to be 600 000, while after 40-100% activation by proteinase A, a molecular weight of 500 000 was obtained. The activity profile of proteinase A in density gradients indicated that this enzyme is bound to variety of cellular proteins.     

Interaction of rabbit liver cathepsin M and fructose 1,6-bisphosphatase converting enzyme with their endogenous inhibitors

The stoichiometry of complex formation between two lysosomal proteinases from rabbit liver, cathepsin M and fructose 1,6-bisphosphatase converting enzyme (CE), and their respective endogenous inhibitors was studied by the equilibrium gel penetration method. In each case the molecular weight of the complex was found to be the sum of the molecular weights of the proteinase and its inhibitor, indicating the formation of 1:1 complexes. From the reappearance of proteinase activity on dilution, it is concluded that complex formation is reversible. Localization of the proteinase activities on the outer surface of the lysosomes was confirmed in these experiments by the inhibition of this proteinase activity on addition of inhibitors to intact lysosomes. The digestion by subtilisin of rabbit liver aldolase and rabbit liver fructose 1,6-bisphosphatase, the endogenous substrates for the lysosomal proteinases, was unaffected by the inhibitors.     

Purification and Characterization of Serine Proteinase 2 from Bacillus intermedius 3-19

A proteinase secreted in the late stationary phase was isolated from the culture fluid of Bacillus intermedius 3-19 by ion-exchange chromatography on CM-cellulose followed by FPLC on a Mono S column. The enzyme was completely inhibited by the serine proteinase inhibitors diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride. The maximum proteolytic activity against the synthetic chromogenic substrate Z-Ala-Ala-Leu-pNA was observed at pH 9.0. The molecular weight of the enzyme is 28 kD and its isoelectric point is 9.2. We have also determined pH- and thermostability and Km and kcat of this proteinase. The enzyme has been classified as a thiol-dependent serine proteinase. N-Terminal amino acid sequence (10 residues) and amino acid composition of the protein were also determined. By the mode of hydrolysis of peptide bonds in the oxidized B-chain of insulin, this enzyme is similar to the thiol-dependent serine proteinase 1 from B. intermedius 3-19 secreted during vegetative growth.     

Characterization of different proteolytic activities in Trypanosoma brucei brucei.

The variant surface glycoprotein of African trypanosomes is released after overnight incubation of parasites at 4 degrees C in pH 5.5 phosphate glucose buffer and may be purified by Concanavalin A Sepharose affinity chromatography. The addition of proteinase inhibitors during the parasite incubation is necessary to prevent the proteolysis of the variant surface glycoprotein by the trypanosomal released proteinases. Using this procedure without the addition of proteinase inhibitors, the proteolytic activities, released from the bloodstream forms Trypanosoma brucei brucei variant AnTat 1.1, were separated by Concanavalin-A Sepharose affinity chromatography. The unretained material (F1) shows hydrolytic activity against the two synthetic substrates Z-Phe-Arg-AMC and Z-Arg-Arg-AMC, which is stimulated by dithiothreitol, but not inhibited by E-64, and characterized by an alkaline pH optimum and an estimated molecular mass of 80-100 kDa. The Michaelis constant for the substrates Z-Arg-Arg-AMC and Z-Phe-Arg-AMC was, respectively, 2.8 and 6.7 microM. The retained material eluted by addition of 1% methyl-alpha-D-mannopyranoside (F2) shows hydrolytic activity against the synthetic substrate Z-Phe-Arg-AMC, which is stimulated by dithiothreitol, inhibited by E-64, active between pH 6.0 and 8.0, and could be separated into two peaks of activity by HPLC, one peak of high molecular mass (greater than 70 kDa) and the other peak of lower molecular mass (30-70 kDa). By electrophoresis in gels containing gelatin as substrate, this fraction contains several proteins with gelatinolytic activity, whereas the unretained fraction F1 did not have any gelatinolytic activity.     

Purification and Characterization of a Keratinolytic Serine Proteinase from Streptomyces albidoflavus

Streptomyces strain K_1-02, which was identified as a strain of Streptomyces albidoflavus, secreted at least six extracellular proteases when it was cultured on feather meal-based medium. The major keratinolytic serine proteinase was purified to homogeneity by a two-step procedure. This enzyme had a molecular weight of 18,000 and was optimally active at pH values ranging from 6 to 9.5 and at temperatures ranging from 40 to 70°C. Its sensitivity to protease inhibitors, its specificity on synthetic substrates, and its remarkably high level of NH₂-terminal sequence homology with Streptomyces griseus protease B (SGPB) showed that the new enzyme, designated SAKase, was homologous to SGPB. We tested the activity of SAKase with soluble and fibrous substrates (elastin, keratin, and type I collagen) and found that it was very specific for keratinous substrates compared to SGPB and proteinase K.     

Protease activities of rumen protozoa.

Intact, metabolically active rumen protozoa prepared by gravity sedimentation and washing in a mineral solution at 10 to 15 degrees C had comparatively low proteolytic activity on azocasein and low endogenous proteolytic activity. Protozoa washed in 0.1 M potassium phosphate buffer (pH 6.8) at 4 degrees C and stored on ice autolysed when they were warmed to 39 degrees C. They also exhibited low proteolytic activity on azocasein, but they had a high endogenous proteolytic activity with a pH optimum of 5.8. The endogenous proteolytic activity was inhibited by cysteine proteinase inhibitors, for example, iodoacetate (63.1%) and the aspartic proteinase inhibitor, pepstatin (43.9%). Inhibitors specific for serine proteinases and metalloproteinases were without effect. The serine and cysteine proteinase inhibitors of microbial origin, including antipain, chymostatin, and leupeptin, caused up to 67% inhibition of endogenous proteolysis. Hydrolysis of casein by protozoa autolysates was also inhibited by cysteine proteinase inhibitors. Some of the inhibitors decreased endogenous deamination, in particular, phosphoramidon, which had little inhibitory effect on proteolysis. Protozoal and bacterial preparations exhibited low hydrolytic activities on synthetic proteinase and carboxypeptidase substrates, although the protozoa had 10 to 78 times greater hydrolytic activity (per milligram of protein) than bacteria on the synthetic aminopeptidase substrates L-leucine-p-nitroanilide, L-leucine-beta-naphthylamide, and L-leucinamide. The aminopeptidase activity was partially inhibited by bestatin. It was concluded that cysteine proteinases and, to a lesser extent, aspartic proteinases are primarily responsible for proteolysis in autolysates of rumen protozoa. The protozoal autolysates had high aminopeptidase activity; low deaminase activity was observed on endogenous amino acids.     

Protease activities of rumen protozoa

Intact, metabolically active rumen protozoa prepared by gravity sedimentation and washing in a mineral solution at 10 to 15 degrees C had comparatively low proteolytic activity on azocasein and low endogenous proteolytic activity. Protozoa washed in 0.1 M potassium phosphate buffer (pH 6.8) at 4 degrees C and stored on ice autolysed when they were warmed to 39 degrees C. They also exhibited low proteolytic activity on azocasein, but they had a high endogenous proteolytic activity with a pH optimum of 5.8. The endogenous proteolytic activity was inhibited by cysteine proteinase inhibitors, for example, iodoacetate (63.1%) and the aspartic proteinase inhibitor, pepstatin (43.9%). Inhibitors specific for serine proteinases and metalloproteinases were without effect. The serine and cysteine proteinase inhibitors of microbial origin, including antipain, chymostatin, and leupeptin, caused up to 67% inhibition of endogenous proteolysis. Hydrolysis of casein by protozoa autolysates was also inhibited by cysteine proteinase inhibitors. Some of the inhibitors decreased endogenous deamination, in particular, phosphoramidon, which had little inhibitory effect on proteolysis. Protozoal and bacterial preparations exhibited low hydrolytic activities on synthetic proteinase and carboxypeptidase substrates, although the protozoa had 10 to 78 times greater hydrolytic activity (per milligram of protein) than bacteria on the synthetic aminopeptidase substrates L-leucine-p-nitroanilide, L-leucine-beta-naphthylamide, and L-leucinamide. The aminopeptidase activity was partially inhibited by bestatin. It was concluded that cysteine proteinases and, to a lesser extent, aspartic proteinases are primarily responsible for proteolysis in autolysates of rumen protozoa. The protozoal autolysates had high aminopeptidase activity; low deaminase activity was observed on endogenous amino acids.     

Purification and characterization of a metallo-endoproteinase from mouse kidney.

A metallo-endoproteinase was purified from mouse kidney. The enzyme was solubilized from the 100 000 g sediment of kidney homogenates with toluene and trypsin, and further purified by fractionation with (NH4)2SO4. DEAE-cellulose chromatography and gel filtration. The molecular weight of the metalloproteinase was estimated by gel filtration on Sepharose 6B to be 270 000--320 000. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in the presence of 2-mercaptoethanol, a single major protein with a mol.wt. of 81 000 was observed. Thus the active enzyme is an oligomer, probably a tetramer. It is a glycoprotein and has an apparent isoelectric point of 4.3. Kidney homogenates and purified preparations of the metalloproteinase degraded azocasein optimally at pH 9.5 and at I 0.15--0.2. The activity was not affected by inhibitors of serine proteinases (di-isopropyl phosphorofluoridate, phenylmethanesulphonyl fluoride), cysteine proteinases (4-hydroxymercuribenzoate, iodoacetate), aspartic proteinases (pepstatin) or several other proteinase inhibitors from actinomycetes (leupeptin, antipain and phosphoramidon). Inhibition of the enzyme was observed with metal chelators (EDTA, EGTA, 1,10-phenanthroline), and thiol compounds (cysteine, glutathione, dithioerythritol, 2-mercaptoethanol). The metalloproteinase degraded azocasein, azocoll, casein, haemoglobulin and aldolase, but showed little or no activity against the synthetic substrates benzoylarginine 2-naphthylamide, benzoylglycylarginine, benzyloxycarbonylglutamyltyrosine or acetylphenylalanyl 2-naphthyl ester. This metalloproteinase from mouse kidney appears to be distinct from previously described kidney proteinases.     

Isolation and properties of cathepsins A from chicken liver]

Four highly purified enzyme preparations, which belongs to the cathepsine A group in their substrate specificity, are isolated from the extract of a hen liver acetone powder. The preparations are designated as A1, A2, A3 and A4 according to their electrophoretic mobility. The A1 component is a protein with molecular weight of about 200 000, it degrades a synthetic substrate and, to a small degree, hemoglobin. This protein is suggested to be the fragment of a liver enzyme complex. The A2 component has molecular weight of about 70 000 and the highest activity. The A3 component has molecular weight 100 000 and the lowest activity. The A2 and A3 components are similar to cathepsine A from other tissues. THe A4 component is characterized by a high electrophoretic mobility, a resistance in neutral and weakly alkaline media and a low molecular weight (of about 60 000). Cu2+, Ag+ and diisopropylphosphofluoridate completely inhibited the activity of all the enzyme preparation studied. Tosyl-alpha-amino-phenylethyl-chloremethylketone inhibited the enzymes activity only by 50-70%.     

Purification of a high-molecular-weight inhibitor of the calcium-activated proteinase

An inhibitor of the muscle calcium-activated proteinases has been purified from porcine skeletal muscle by using DEAE-cellulose column chromatography, thermal treatment, Sephacryl S-400 column chromatography in 6 M urea and Sephacryl S-300 column chromatography in 6 M urea. Sodium dodecyl sulfate polyacrylamide slab gel electrophoresis shows that the purified inhibitor is homogeneous and has a subunit molecular weight of 172 000. The inhibitor inactivates both the low- and high-calcium-requiring forms of the calcium-activated proteinase but does not inhibit other proteinases against which it has been tried. It thus appears that the inhibitor is specific for the calcium-activated proteinase. Studies using homogeneous inhibitor and high-calcium-requiring proteinase show that one molecule of the inhibitor can inactivate up to eight molecules of the calcium-activated proteinase. Inactivation of the calcium-activated proteinase by the inhibitor cannot be reversed by calcium concentrations as high as 25 mM, thus eliminating the possibility that the inhibitor functions by chelating calcium. The inhibitory peptide appears to be extremely susceptible to proteolysis during its isolation. Even in the presence of synthetic proteinase inhibitors different inhibitor preparations yield homogeneous inhibitory peptides ranging in molecular weight from 145 000 to 172 000. Preparative electrophoresis and column chromatography have been used to isolate putative proteolytic breakdown products of the 172 kDa peptide at 145, 114, 41 and 29 kDa.     

A metalloproteinase associated with gelonin, a "ribosome inactivating protein"

Gelonin, a single-chain protein which inactivates eukaryotic ribosomes, becomes split into peptides when incubated with SDS. During the chromatographic purification of gelonin on carboxymethylcellulose three overlapping peaks emerge in the gelonin elution region, containing three proteins with small differences in apparent molecular weight (31,500, 30,000 and 29,200). All three proteins are endowed with inhibitory activity on protein synthesis and with proteinase activity, although with different specific activities, and all three give rise to the same peptides upon incubation with SDS, suggesting that they are isoforms of gelonin. The gelonin-associated proteinase acts only on gelonin, while it is inactive on the most common substrates for endoproteinases. The proteolytic activity is not inhibited by inhibitors of serine- or SH-proteinases, while it is completely abolished by chelating agents. Divalent cations restore the proteolytic activity inhibited by EDTA. The stability of the proteinase activity on exposure of gelonin to extreme values of pH or to prolonged incubation has been investigated. The inhibitory activity on protein synthesis and the proteinase activity are differently affected by these treatments.     

Inhibition of plant cell proteolytic activities that degrade tubulin

The requirement for proteinase inhibitors during the chromatographic isolation of tubulin from cultured cells of rose (Rosa sp. cv. Paul's scarlet) was examined by NadodecylSO4-polyacrylamide gel electrophoresis, electron microscopy and immunoblotting. Tubulin fractions isolated in the absence of proteinase inhibitors showed substoichiometric ratios of alpha-subunit to beta-subunit, and low molecular weight polypeptides, one (approximately 32 Kd) of which coassembled with polymers. Electron microscopy revealed polymorphic structures, including C- and S-shaped ribbons and free protofilaments. Immunoblotting experiments with IgGs to the individual alpha- and beta-subunits showed that some of the low molecular weight polypeptides were fragments of proteolytically degraded subunits. The use of low micromolar concentrations of the synthetic proteinase inhibitors leupeptin hemisulfate and pepstatin A protected tubulin from endogenous proteolytic activities during the isolation procedure and resulted in increased tubulin purity.     

Extracellular proteinases from the phytopathogenic fungus Fusarium culmorum

The growth of Fusarium culmorum fungus on a medium containing thermostable proteins from potato tubers was accompanied by the production of proteinases, exhibiting activity over a broad pH range (from 6.0-10.0). When studied by SDS-PAGE in the presence of beta-mercaptoethanol, extracellular proteinases were represented by at least five species with a molecular weight of 30-60 kDa. Inhibitor analysis and studies of enzyme activities with synthetic substrates demonstrated that the culture liquid of Fusarium culmorum contained serine proteinases of various classes. The amount of subtilisin-like proteinases was the highest. A near-complete inhibition of the enzymes was caused by proteinaceous proteinase inhibitors from potato tubers. These data suggest that proteinases of the phytopathogen Fusarium culmorum serve as a metabolic target for natural inhibitors of potato proteinases.     

Trypsin-like proteinase from Actinomyces fradiae 119

Using electrophoresis and ultracentrifugation, a homogeneous proteinase was isolated from protofradine, a protease Act. fradiae 119 preparation. The purification procedure included filtration on DEAE-cellulose, gel filtration through Arcylex P-10, CM-chromatography and desalting on Sephadex G-15. The proteinase under study is an endopeptidase which hydrolyzes low molecular weight synthetic trypsin substrates as well as casein and denaturated collagen. Diisopropylfluorophosphate and soya bean trypsin inhibitor completely inhibit the proteinase activity, whereas pCMB and EDTA have no such effect. The stability maximum is observed at pH of 2.5-3.5, the action maximum at pH 8.7-9.5. The amino acid composition of the enzyme is similar to that of trypsin from Str. griseus. The molecular weights of the proteinase as determined by gel filtration and sedimentation equilibrium method are equal to 25400 and 26500, respectively. The isoelectric point lies at 5.3. The data obtained suggest that the proteinase can be attributed to the family of trypsin proteinases.