Properties and specificity of a second metal chelator-sensitive proteinase in the keratinolytic larvae of the webbing clothes moth.

The properties of a second metal chelator-sensitive proteinase (metalloproteinase 2) from the larvae of the webbing clothes moth, Tineola bisselliella, have been studied. The pH optimum for casein digestion was 9-4 and the enzyme showed high stability between pH 8 and 11, but very poor stability at acid pH. The proteinase was inhibited by EDTA, but not by an EDTA-calcium complex. EDTA inhibition could be reversed by addition of a slight excess of calcium or zinc ions. The cleavage specificity of metalloproteinase 2 against the A and B chains of S-carboxymethyl insulin was almost identical to that found previously for metalloproteinase 1.     

The mosquito Aedes aegypti (L.): evidence for three new proteinases

The effect of inhibitors was studied on the proteolytic activity of crude extracts of the mosquito Aedes aegypti (L.), prepared 25 h after blood intake. This activity is only partially inhibited by the lima bean trypsin inhibitor (LBTI) or by EDTA. Experiments with mixed inhibitors are used to indicate that, apart from the well characterized trypsin-like enzymes, at least three other proteinases are present. These are an LBTI-resistant serine proteinase, a metal chelator-sensitive proteinase, and a proteinase which can only be inhibited by the ovomucoid inhibitor. These newly described proteinases are inactive against several model substrates developed for mammalian enzymes. They are partially separable by ion-exchange chromatography.     

Isolation of individual proteases from protofradin]

An electrophoretically homogeneous trypsin-like proteinase, two homogeneous proteases (presumably metal-containing) and two elastases, possessing the ATEE-esterase activity, were isolated from protofradin, a protease preparation from Actinomyces fradiae 119, using fractionation on KM-cellulose K-32. The trypsin like proteinase of protofradin possesses the esterase activity, equal to the activity of pancreatic trypsin. Protofradin elastases differ in their pH optima, response to EDTA, stability upon storage and the degree of elastin hydrolysis. The specificity of elastase is probably the same, since in elastin both enzymes hydrolyze the peptide bonds, formed by the NH2-group of glycine and alanine residues, found in elastin in large amounts. The end products of elastin hydrolysis are tripeptides.     

Properties and substrate specificity of proteinase from buckwheat seeds]

A thiol proteinase was isolated from buckwheat seeds and purified 300-fold, using ammonium sulfate, acetone fractionation ion-exchange chromatography on Sephadex CM-50 and electrofocussing. The proteinase preparation obtained was found homogenous after polyacrylamide gel electrophoresis at pH 4.5. The molecular weight of the enzyme (75.000) was determined by gel-filtration through Sephadex G-100. The activation of proteinase by cysteine, 2-mercaptoethanol and dithiothreitol, its inhibition by p-chloromercurybenzoate and the absence of inhibition by diisopropyl fluorophosphate and EDTA suggest that the enzyme isolated is a thiol proteinase. The enzyme hydrolyzed many peptide bonds in the B-chain of insulin, showing high substrate specificity. The glutelin and globulin fractions of buckwheat seed proteins were hydrolyzed by the enzyme. It is assumed that the hydrolysis of reserve proteins of buckwheat seeds is the main function of the proteinase isolated.     

Neutral metal chelator-sensitive protease in insect moulting fluid

Proteolytic activity in moulting fluid from the sphingid Manduca sexta has at least two pH optima; these occur at pH 7 and at pH 7·7. The latter activity is shown to be trypsin-like in that it is susceptible to inhibition by diisopropylfluorophosphate. By contrast, the peak at neutral pH consists of proteolytic activity not hitherto described in invertebrates. This activity shows little or no inhibition with diisopropylfluorophosphate or p-hydroxymercuribenzoate but is strongly inhibited by chelators such as 1,10-phenanthroline, 8-hydroxyquinoline, and EDTA. The neutral metal chelator-sensitive activity requires calcium but the inhibitor data permit the conclusion that the metal ion inhibited by the chelators belongs to the first transition series and thus cannot be calcium. The neutral protease appears to be similar to proteases previously characterized from bacteria and snake venom. In moulting fluid from Manduca, proteolytic activity in vitro is very low in the presence of 1,10-phenanthroline at every pH studied except pH 7·7; in vivo, ecdysis is inhibited in Manduca larvae fed on diet containing a sufficient level (0·02 per cent or higher) of 1,10-phenanthroline. The metal chelator-sensitive proteolytic activity appears to be an essential moulting protease in Manduca.     

A cold-adapted extracellular serine proteinase of the yeast<Emphasis Type="Italic"> Leucosporidium antarcticum</Emphasis>

An extracellular serine proteinase, lap2, from the psychrophilic antarctic yeast Leucosporidium antarcticum 171 was purified to homogeneity and characterized. The enzyme is a glycoprotein with a molecular mass of 34.4 kDa and an isoelectric point of pH 5.62. The proteinase is halotolerant, and its activity and stability are dependent neither on Ca²⁺ nor on other metal ions. Lap2 is a true psychrophilic enzyme because of low optimal temperature (25°C), poor thermal stability, relatively small values of free energy, enthalpy and entropy of activation, and high catalytic efficiency at 0–25°C. The 35 N-terminal amino acid residues of lap2 have homology with subtilases of the proteinase K subfamily (clan SB, family S8, subfamily C). The proteinase lap2 is the first psychrophilic subtilase in this family.Communicated by K. Horikoshi     

Specific cleavages of arginyl peptide bonds at basic amino acid pairs by a serine proteinase from the microsomal membranes of rat liver

The specificity of action of a serine proteinase from the microsomal membranes of rat liver was investigated at pH 7.5 and 37 degrees C using various peptides as substrates. HPLC analyses of the peptides produced followed by their amino acid analyses have revealed that the enzyme is a unique endopeptidase specifically cleaving arginyl peptide bonds at paired basic amino acid residues. Thus, the enzyme is suggested to be a kind of processing proteinase involved in the conversion of proproteins to their mature forms. Indeed, the enzyme cleaved specifically the NH2-terminal 20-residue peptide of proalbumin at the Arg-Arg sequence.     

A new subtilisin-like proteinase from roots of the dandelion Taraxacum officinale Webb S. L.

A serine proteinase from roots of Taraxacum officinale Webb S. L. was isolated by affinity chromatography and gel-filtration on Superose 6R using FPLC. The enzyme is a 67-kD glycoprotein containing 54% carbohydrate which we have named taraxalisin. The substrate specificity of taraxalisin toward synthetic peptides and oxidized insulin B-chain is comparable with that of cucumisin from Cucumis melo and the subtilisin-like serine proteinase macluralisin from Maclura pomifera. The proteinase is inactivated by DFP and PMSF. Taraxalisin exhibits maximal activity at pH 8.0. The pH range for stability of the enzyme is narrow--6.0-9.0. The temperature optimum for the subtilisin-like activity is 40 degrees C. The N-terminal sequence of taraxalisin has 40% of its residues identical to those of subtilisin Carlsberg. Thus, the serine proteinase from dandelion roots is a member of the subtilisin family, which is evidently widespread in the plant kingdom.     

Isolation and thermal stability studies of two novel serine proteinases from the fungus Tritirachium album Limber.

A number of serine proteinases are secreted into the culture medium when Tritirachium album Limber is supplied with protein as the only nitrogen source. From this population of proteinases, we have isolated two novel proteolytic enzymes, designated as proteinase R and T. We have compared the thermal stability of these two proteinases with that of subtilisin BPN' and proteinase K. Both of these proteinases were thermally stable in the absence of detergents in buffers of low (4.0) and high (10.0) pH. The thermal stability of proteinase T was not affected by the presence of 1.0% SDS either at pH 8.0 or 10.0 in contrast to proteinase R which became heat labile. At low pH, the presence of SDS was detrimental to the stability of all the proteinases.     

In vitro cleavage specificity of the adenovirus type 2 proteinase

Two in vitro proteinase assay systems were developed and used to study the peptide bond specificity and substrate specificity of the adenovirus endoproteinase. Five adenovirus precursor proteins (PVI, PVII, PVIII, 87K, 11K), all found in the virion of the ts1 mutant grown at the nonpermissive temperature, were digested by the proteinase. All, except 11K, were cleaved to their mature counterparts. Some of the proteins, particularly the 87K terminal protein, were processed via cleavage intermediates similar to those found in vivo. The data suggest that the proteinase specifically hydrolyses Gly-Ala bonds. The high specificity for the natural substrates and the failure to cleave foreign proteins suggest that cleavage activity is determined not only by primary sequence but also by other physical features of the substrate. Enzyme activity was inhibited by diisopropylfluorophosphate, showing that it is a serine proteinase.     

Purification and Properties of a Highly Thermostable, Sodium Dodecyl Sulfate-Resistant and Stereospecific Proteinase from the Extremely Thermophilic Archaeon Thermococcus stetteri

The cultivation of the extremely thermophilic archaeon Thermococcus stetteri in a dialysis membrane reactor was paralleled by the production of an extremely heat-stable proteinase(s). By applying preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, an SDS-resistant proteinase was purified 67-fold in one step with a yield of 34%. The purified enzyme, which was composed of a single polypeptide chain with a molecular mass of 68 kDa, showed a broad temperature and pH profile (50 to 100(deg)C; pH 5 to 11). The optimal activity with substantial thermal stability was measured with casein at 85(deg)C and pH 8.5 to 9. Inhibition by phenylmethylsulfonyl fluoride and diisopropylfluorophosphate demonstrated that the enzyme was a serine proteinase. The enzyme displayed a relatively narrow substrate specificity, catalyzing the hydrolysis only of N-protected p-nitroanilides or p-nitrophenyl esters of basic (Arg or Lys) or hydrophobic (Phe or Tyr) l-amino acids. l-Phenylglycine amide was also attacked by the proteinase, but with a lower specificity constant. Within the detection limit, no hydrolysis of d-amino acid derivatives was observed. The catalytic efficiency of the enzyme at 80(deg)C (k(infcat)/K(infm) for benzoyl-Arg-p-nitroanilide, 10(sup4)) is the same order of magnitude when compared with that of functionally similar mesophilic enzymes. The proteinase also acts as a transferase, catalyzing the acyl transfer from protected amino acid ester or amide to amino acid amide. The observed thermostability, SDS resistance, relatively narrow substrate specificity, high stereospecificity, and limited catalytic efficiency probably reflect the tighter packing of the thermostable protein molecule and its limited flexibility. This was supported by fluorescence spectra of the enzyme, mainly due to tryptophan residues, in the temperature range of 30 to 90(deg)C. Structural reorganization was observed at temperatures over 100(deg)C. The results obtained could be of relevance for the better understanding of the structure-function relationship of enzymes from extreme thermophiles and suggest possible biotechnological application of the proteinase for resolution of racemic mixtures.     

Peptidylglutamyl-peptide hydrolase activity of the multicatalytic proteinase complex: evidence for a new high-affinity site, analysis of cooperative kinetics, and the effect of manganese ions.

The multicatalytic proteinase (MCP) complex or proteasome is a major nonlysosomal proteinase of eukaryotic cells. The proteinase can cleave peptide bonds on the carboxyl side of hydrophobic, basic, or acidic amino acid residues. These activities have been referred to as "chymotrypsin-like", "trypsin-like", and "peptidylglutamyl-peptide hydrolase" activities, respectively, and have been shown to be catalyzed at distinct sites. The latter activity is often assayed with the synthetic peptide substrate Z-Leu-Leu-Glu-beta-naphthylamide (LLE-NA). N-tBoc-Ala-Ala-Asp-SBzl is also a substrate for the rat liver MCP, suggesting a broader specificity for cleavage on the carboxyl side of acidic residues than the peptidylglutamyl-peptide hydrolase activity previously reported. The pH optimum is in the range of pH 7.0-7.5. Studies of the dependence of velocity on LLE-NA concentration show (a) that there is a high-affinity site (LLE1) which obeys Michaelis-Menten kinetics with a Km value of approximately 100 microM and (b) that at higher substrate concentrations (LLE2) the curve is sigmoidal, suggesting either allosteric activation of the proteinase at a second site or the involvement of multiple catalytic sites which display positive cooperativity. Activity at the high-affinity site (LLE1) can be distinguished from that of the activity of the LLE2 component by the effect of inhibitors, divalent metal ions, and KCl, as well as by its response to heat treatment. The addition of 1 mM MnCl2 stimulates both LLE1 and LLE2 activities and also permits saturation of MCP with substrate at concentrations of LLE-NA below the solubility limit of this peptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Substrate specificity of carboxyl proteinase fromPycnoporus coccineus,a wood-deteriorating fungus

A carboxyl proteinase was purified from submerged-culture filtrate of a wood-deteriorating basidiomycete,Pycnoporus coccineus. The purified enzyme was found to be essentially homogeneous in disc gel electrophoresis tests at pH 9.4 and 2.3. The specificity and mode of action ofP. coccineus carboxyl proteinase I_a were investigated with the oxidized B-chain of insulinP. coccineus carboxyl proteinase I_a hydrolyzed primarily three peptide bonds, Ala¹⁴-Leu¹⁵, Tyr¹⁶-Leu¹⁷, and Phe²⁴-Phe²⁵ bonds, in the oxidized B-chain of insulin.     

Purification and characterization of the heat-stable serine proteinase from Thermomonospora fusca YX.

The proteinase secreted from Thermomonospora fusca YX grown on cellulose was purified by (NH4)2SO4 fractionation and cation-exchange chromatography. The isolated proteinase readily hydrolysed several proteins and demonstrated activity towards casein from 35 to 95 degrees C (at pH 8.0) with maximum activity at 80 degrees C. It exhibited broad pH and ionic-strength optima centered at pH 9.0 and 0.2 M-NaCl respectively, and it retained high activity in the presence of 2% (w/v) SDS, 20 mM-dithiothreitol and 1.0 M-NaCl. The proteinase, which was fully inhibited by phenylmethanesulphonyl fluoride, had an Mr of 14,500 and an isoelectric point at 9.21. A measurement of proteinase thermal stability demonstrated a T50% (15 min) of 85 degrees C at pH 4.5.     

Serine proteinase from Cucurbita ficifolia seeds

A new serine proteinase was isolated from Cucurbita ficifolia seeds by the purification procedure, which includes: extraction, salting out with ammonium sulphate, chromatography on CM-cellulose. Sephacryl S-300 gel filtration and h.p.l.c. on DEAE-2SW TSK column. The enzyme was homogeneous both in native and SDS PAGE. Three independent methods showed its molecular mass to be approximately 77 kDa. The enzyme was inhibited by specific serine proteinase organic inhibitors, and was active in the presence of inhibitors specific for other proteinase classes. Surprisingly, squash proteinase exhibited a very high and broad pH optimum with a maximum at 10.7. It hydrolysed many different peptide bonds in B-chain of insulin and was able to cleave four bonds in endogenous serine proteinase inhibitor (CMTI).     

A latent form of cathepsin B in pleural effusions. I. Characterization of the enzyme in breast cancer patients

A latent cysteine proteinase has been found in the pleural effusion fluid of patients with breast cancer. It can be converted by pepsin to an active form, the properties of which, including the pH optimum, pH stability, substrate specificity, and sensitivity to various proteinase inhibitors, were found to be closely related to those of cathepsin B. Unlike the pepsin-generated enzyme, which was rapidly inactivated above pH 7.0, the latent enzyme showed substantially higher stability in the region around and above neutral pH. The apparent Mr values of the latent and pepsin-generated enzyme forms were approximately 45,000 and 32,000, respectively. Both enzyme forms exhibited heterogeneous binding affinity to concanavalin A-Sepharose 4B. Altogether, our results demonstrate that a latent cathepsin B form occurs in vivo in pleural effusions of breast cancer patients.     

Purification and Some Properties of Metal Proteinases from Lentinus edodes

To investigate the function of proteinases in the fruiting of Basidiomycetes, we purified the neutral proteinase in vegetative mycelium of Lentinus edodes. About 1.6 mg of purified enzyme was obtained from 1.5 kg of mycelium. The purified enzyme was confirmed to be monodispersive on disc electrophoresis.

The neutral proteinase was most active around pH 7.5 toward hemoglobin and 7.0 toward casein and was extremely labile with temperature. The enzyme was strongly inhibited by EDTA or Talopeptin (MK-I). The molecular weight and isoelectric point of the enzyme were 45,000 and pH 5.3, respectively. The enzyme contained no methionine residues. The enzyme hydrolyzed the bonds involving hydrophobic or bulky amino acid residues of oxidized insulin B-chain such as His-Leu (10–11 and 5–6), Leu (17)-Val (18) and Ala (14)-Leu (15).

These characteristics are compared with those of the metal proteinase in the fruit-body of the same fungus, which was purified and characterized at the same time as in vegetative mycelium. We also compare it with proteinases from other microbes.     

Role of calcium in activity and stability of the Lactococcus lactis cell envelope proteinase

The mature lactococcal cell envelope proteinase (CEP) consists of an N-terminal subtilisin-like proteinase domain and a large C-terminal extension of unknown function whose far end anchors the molecule in the cell envelope. Different types of CEP can be distinguished on the basis of specificity and amino acid sequence. Removal of weakly bound Ca2+ from the native cell-bound CEP of Lactococcus lactis SK11 (type III specificity) is coupled with a significant reversible decrease in specific activity and a dramatic reversible reduction in thermal stability, as a result of which no activity at 25 degrees C (pH 6.5) can be measured. The consequences of Ca2+ removal are less dramatic for the CEP of strain Wg2 (mixed type I-type III specificity). Autoproteolytic release of CEP from cells concerns this so-called "Ca-free" form only and occurs most efficiently in the case of the Wg2 CEP. The results of a study of the relationship between the Ca2+ concentration and the stability and activity of the cell-bound SK11 CEP at 25 degrees C suggested that binding of at least two Ca2+ ions occurred. Similar studies performed with hybrid CEPs constructed from SK11 and Wg2 wild-type CEPs revealed that the C-terminal extension plays a determinative role with respect to the ultimate distinct Ca2+ dependence of the cell-bound CEP. The results are discussed in terms of predicted Ca2+ binding sites in the subtilisin-like proteinase domain and Ca-triggered structural rearrangements that influence both the conformational stability of the enzyme and the effectiveness of the catalytic site. We argue that distinctive primary folding of the proteinase domain is guided and maintained by the large C-terminal extension.     

Stability studies on the cathepsin L proteinase of the helminth parasite, Fasciola hepatica

Fasciola hepatica, the liver fluke, secretes a cathepsin L cysteine proteinase. The enzyme is active over the pH range 5-9 and is remarkably stable at 37 degrees C, pH 7.0, in contrast to mammalian cathepsin Ls that are active in the acidic pH range and are inactivated within 15 min at neutral pH. The liver fluke proteinase is also very tolerant of organic solvents, particularly dimethylformamide. However, it is completely inactivated by 1 mM Hg(2+) and adversely affected by other heavy metals and divalent cations. Addition of glycerol and EDTA enhanced the liver fluke enzyme's stability at 50 degrees C, while glucose and glycerol protected the enzyme from inactivation by repeated freeze-thawing. The high stability of liver fluke cathepsin L suggests that it may have potential for use in bioindustrial applications.