Strongyloides ransomi: proteolytic enzymes from larvae

The filariform larvae of Strongyloides ransomi can infect their hosts by penetration through skin. In this report, homogenates of these organisms were prepared and their proteolytic enzymes examined. Homogenates prepared in 0.2 M citrate, pH 4.0, contain two thiol-dependent proteinases with molecular weights of approximately 32,000 and 28,000. These proteinases have an acidic pH optimum and show substrate preferences and inhibitor susceptibilities similar to the vertebrate acidic cysteinyl proteinases. Homogenates prepared in 0.1 M Tris, pH 7.5, contain multiple proteolytic enzymes, active against both Azocoll and synthetic substrates. These enzymes do not require thiols for activity and they have an alkaline pH optimum. The enzymes are inhibited by both chelating agents and heavy metals, but not by serine-proteinase inhibitors. Extracts prepared in 0.1 M Tris-HCl, pH 7.5, contain endogenous proteinase inhibitors.     

Purification and characterization of an elastinolytic proteinase secreted by cercariae of Schistosoma mansoni

An elastinolytic proteinase secreted by tissue-invasive larvae of Schistosoma mansoni has been purified to homogeneity. Size-exclusion chromatography and chromatofocusing were used to purify the enzyme 18-fold from crude larval secretions. The native enzyme has a molecular weight of 30,000, a pI of 8, a pH optimum of 9, and a calcium dependence of 2 mM. A second Mr 17,000 form of the enzyme was present in crude secretions and appears to be an autoproteolysis product. The enzyme is a serine proteinase that preferentially binds tetrapeptide inhibitors or substrates with an aromatic or hydrophobic residue at the P-1 site. In addition to being active against elastin, the enzyme degrades Azocoll, gelatin, laminin, fibronectin, keratin, and type IV collagen.     

Purification of a neutral proteinase, associated with the actomyosin complex, from uterine myometrium.

We have purified and characterized a neutral proteinase activity from pig uterine myometrium. The proteinase co-purified with the actomyosin complex and could only be separated from it by a high concentration of a chaotropic ion, 3M-NaBr. The proteinase was further purified by gel filtration and affinity chromatography. The purified protein showed a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis corresponding to an Mr of 28 000. Gel filtration on Sephadex G-100 in a buffer containing 3M-NaBr gave an Mr of 27 500. Without the addition of the chaotropic Br- ion, the proteinase aggregates to high-Mr forms of more than 10(6)Da. The proteinase has optimum hydrolytic activity with casein as substrate at pH 7.5-8.0. The thiol-group-blocking reagents p-chloromercuribenzoate, p-chloromercuribenzenesulphonate and Hg2+, as well as soya-bean trypsin inhibitor and 4-aminobenzamidine, inhibited the proteinase. Other bivalent cations, chelating agents and the serine-specific reagents 7-amino-1-chloro-3-tosylamido-L-heptan-2-one and phenylmethanesulphonyl fluoride were without any effect on proteinase activity. The proteinase degraded myosin very rapidly at a molar ratio of proteinase to myosin of 1:50, concomitant with the rate of loss of the ATPase activity. Compared with myosin, actin was only a poor substrate and was degraded at a much lower rate, even at a high molar ratio of the proteinase to actin.     

The application of fluorescein isothiocyanate and high-performance liquid chromatography for the microsequencing of proteins and peptides

Azocoll, an insoluble, ground collagen to which a bright-red azodye is attached has been widely used for the assay of proteolytic enzymes. Earlier studies showed that hydrolysis of azocoll progressed linearly as a function of proteinase concentration but in an exponentially increasing manner as a function of time. No explanation for the latter behavior has been offered. We have found that assays of both crude extracts of Bacillus subtilis and commercial preparations of subtilisin BPN' gave linear rates of hydrolysis of azocoll as a function of protease concentration; however, both gave increasing rates of hydrolysis of azocoll as a function of time. In attempting to improve and standardize proteolytic assays using azocoll we have found: (a) the absorption maximum of solubilized azocoll at pH 7.8 is 516 nm and is not significantly altered at acid pH; (b) assays which are perfectly linear as a function of time can be obtained by using azocoll that has been vigorously prewashed with buffer; (c) the soluble filtrate removed by prewashing can regenerate the nonlinear time courses previously observed; and (d) the rate of hydrolysis of azocoll can be varied by a factor of 3 by varying the rates of agitation of the assay tubes. In summary, to obtain reproducible, linear assays it was essential to prewash commercial azocoll and agitate reaction tubes vigorously.     

Purification, assay and kinetic features of HIV-1 proteinase

1) The aspartic proteinase of the human immunodeficiency virus type 1 (HIV-1) was purified from cultures of recombinant E. coli. The enzyme preparation is homogeneous as judged by SDS-polyacrylamide gel electrophoresis and isoelectric focusing. 2) A rapid assay procedure for the proteinase was established which makes use of the cleavage of a radiolabeled decapeptide and the separation of substrate and labeled product by ion-exchange resin. 3) Activity of the enzyme is optimal at an ionic strength of 2.5-3.5M; also, the inhibitor pepstatin is a more potent inhibitor at higher ionic strength. This can be attributed to a tighter binding of both substrate and inhibitor in high-salt buffer. 4) The Km value of the decapeptide substrate is independent of the pH in the range of 3.5-7.5, while kcat shows a bell-shaped curve with a maximum at pH 5.2. The shape of the curve can be attributed to pKa values of 4.2 and 6.2 of groups on the enzyme. Pepstatin inhibition is optimal below pH 5.5, but becomes weak above pH 6.     

Processing of an NH2-terminally extended thermostable alpha-amylase by Bacillus subtilis alkaline protease

To analyze the processing of extracellular enzymes of Bacillus subtilis, an NH2-terminally extended hybrid alpha-amylase [pTUBE638-alpha-amylase (E24)] was purified from the periplasm of E. coli(pTUBE638) as the substrate for the in vitro processing reaction, in which a 21-amino-acid extra-peptide was added at the NH2-terminus of the mature thermostable alpha-amylase. The extended peptide in pTUBE638-alpha-amylase (E24) was completely processed by the extracellular alkaline protease of B. subtilis alone at pH 7.5 to 10.0. The processing was inhibited by 2 mM PMSF. In contrast, the neutral protease did not process the extended peptide. The processing activity of the purified alkaline protease was fully active in 100 mM phosphate and glycine-NaCl-NaOH buffer while it was partially active in 100 mM Tris-HCl or MOPS buffer. The optimum pH of the activity ranged from 8.0 to 9.0, although the optimum pH of the alkaline protease activity toward casein and Azocoll was 10.5. The NH2-terminal amino acid sequences of the enzymes processed in vitro coincided with those of the mature extracellular thermostable alpha-amylases in the culture medium of B. subtilis (pTUBE638). The appearance of the processing activity of alkaline protease was correlated with the changes of the pH in the culture medium.     

The simultaneous release by bone explants in culture and the parallel activation of procollagenase and of a latent neutral proteinase that degrades cartilage proteoglycans and denatured collagen.

1. A latent neutral proteinase was found in culture media of mouse bone explants. Its accumulation during the cultures is closely parallel to that of procollagenase; both require the presence of heparin in the media. 2. Latent neutral proteinase was activated by several treatments of the media known to activate procollagenase, such as limited proteolysis by trypsin, chymotrypsin, plasmin or kallikrein, dialysis against 3 M-NaSCN at 4 degrees C and prolonged preincubation at 25 degrees C. Its activation often followed that of the procollagenase present in the same media. 3. Activation of neutral proteinase (as does that of procollagenase) by trypsin or plasmin involved two successive steps: the activation of a latent endogenous activator present in the media followed by the activation of neutral proteinase itself by that activator. 4. The proteinase degrades cartilage proteoglycans, denatured collagen (Azocoll) and casein at neutral pH; it is inhibited by EDTA, cysteine or serum. Collagenase is not inhibited by casein or Azocoll and is less resistant to heat or to trypsin than is the proteinase. Partial separation of the two enzymes was achieved by gel filtration of the media but not by fractional (NH4)2SO4 precipitation, by ion exchange or by affinity chromatography on Sepharose-collagen. These fractionations did not activate latent enzymes. 5. Trypsin activation decreases the molecular weight of both latent enzymes (60 000-70 000) by 20 000-30 000, as determined by gel filtration of media after removal of heparin. 6. The latency of both enzymes could be due either to a zymogen or to an enzyme-inhibitor complex. A thermostable inhibitor of both enzymes was found in some media. However, combinations of either enzyme with that inhibitor were not reactivated by trypsin, indicating that this inhibitor is unlikely to be the cause of the latency.     

Inhibition of digestive proteinases of stored grain Coleoptera by oryzacystatin, a cysteine proteinase inhibitor from rice seed

Electrophoresis of midgut extracts from the rice weevil, Sitophilus oryzae, and the red flour beetle, Tribolium castaneum, in polyacrylamide gels containing sodium dodecyl sulfate and gelatin revealed there was one major proteinase (apparent molecular mass = 40,000) in the rice weevil and two major proteinases (apparent molecular masses = 20,000 and 17,000) in the red flour beetle. The pH optima using [3H]casein as substrate were about pH 6.8 for the rice weevil and pH 5.2 for the red flour beetle. Use of specific inhibitors, including L-trans-epoxysuccinyl-leucylamino-(4- guanidino)-butane (E-64), p-chloromercuriphenylsulfonic acid (PCMS), and oryzacystatin, indicated that nearly all of the proteinase activity against casein was contributed by cysteine proteinases. The estimated IC50 values for oryzacystatin were 2 x 10(-6) M and 4 x 10(-7) M when tested against midgut extracts from T. castaneum and S. oryzae, respectively.     

Digestive proteinases of the larger black flour beetle, Cynaeus angustus (Coleoptera: Tenebrionidae)

Digestion in the larger black flour beetle, Cynaeus angustus (LeConte), was studied to identify new control methods for this pest of stored grains and grain products. The physiological pH of the larval gut, as measured with extracts in water, was approximately 6.1, and the pH for optimal hydrolysis of casein by gut extracts was 6.2 when buffers were reducing. However, under non-reducing conditions, hydrolysis of casein and synthetic serine proteinase substrates was optimal in alkaline buffer. Three major proteinase activities were observed in zymograms using casein or gelatin. Caseinolytic activity of C. angustus gut extracts was inhibited by inhibitors that target aspartic and serine proteinase classes, with minor inhibition by a cysteine proteinase inhibitor. In particular, soybean trypsin and trypsin/chymotrypsin inhibitors were most effective in reducing the in vitro caseinolytic activity of gut extracts. Based on these data, further studies are suggested on the effects of dietary soybean inhibitors of serine proteinases, singly and in combination with aspartic and cysteine proteinase inhibitors, on C. angustus larvae. Results from these studies can be used to develop new control strategies to prevent damage to grains and stored products by C. angustus and similar coleopteran pests.     

Proteolytic enzymes in sea urchin eggs: characterization, localization and activity before and after fertilization

The pH versus proteinase activity curve (casein or hemoglobin plus urea substrate) for homogenates of unfertilized Lytechinus eggs reveals two regions of maximum activity: one between pH 3.5 and 4.3, and another of far greater magnitude from pH 8.0 to 11.0. The two classes of proteinases can be separated on a sucrose density gradient. Both the acid and alkaline proteinases in homogenates prepared in isotonic monovalent salt solutions are remarkably stable at pH 7.4 and 0°C. Using synthetic peptide substrates, an enzyme with the specific esterase activity of chymotrypsin was demonstrated; this enzyme accounts for the major part of the proteinase activity at alkaline pH. In addition, an enzyme with specific esterase activity of trypsin was shown to be present, but of low activity. The proteinase activity at acid pH is largely due to an enzyme resembling cathepsin D. The data also suggest the presence of cathepsin B and cathepsin IV (or catheptic carboxypeptidase). When eggs are homogenized in isotonic NaCl plus KCl at pH 7.4, 0.02 M tris buffer at 0°C, all of the alkaline proteinase, and 85–90% of the acid proteinase activity is sedimented at 10,000 g. The presence of any proteinase activity in the supernatant phase represents an artifact of the preparative procedures used. The granules which possess the proteinase activity are contained entirely in the yolk fractions; and the acid proteinase is contained in a population of granules which sediment more readily than those which contain the alkaline proteinase. The acid proteinase resembles the lysosomal acid hydrolases in that it is readily released from the particulates; in contrast, the alkaline proteinase is bound relatively firmly. In contradistinction to reports in the literature, no changes in proteinase activity nor intracellular distribution could be detected following fertilization.     

Digestive proteinases of larvae of the corn earworm, Heliothis zea: characterization, distribution, and dietary relationships.

Proteinases and peptidases from the intestinal tract of fifth-instar larvae of Heliothis (= Helicoverpa) zea (Boddie) (Lepidoptera:Noctuidae) were characterized based on their substrate specificity, tissue of origin, and pH optimum. Activity corresponding to trypsin, chymotrypsin, carboxypeptidases A and B, and leucine aminopeptidase was detected in regurgitated fluids, midgut contents, and midgut wall. High levels of proteinase activity were detected in whole midgut homogenates, with much lower levels being observed in foregut and salivary gland homogenates. In addition, enzyme levels were determined from midgut lumen contents, midgut wall homogenates, and regurgitated fluids. Proteinase activities were highest in the regurgitated fluids and midgut lumen contents, with the exception of leucine aminopeptidase activity, which was found primarily in the midgut wall. Larvae fed their natural diet of soybean leaves had digestive proteinase levels that were similar to those of larvae fed artificial diet. No major differences in midgut proteinase activity were detected between larvae reared under axenic or xenic conditions, indicating that the larvae are capable of digesting proteins in the absence of gut microorganisms. The effect of pH on the activity of each proteinase was studied. The pH optima for the major proteinases were determined to be pH 8.0-8.5 for trypsin, when tosyl-L-arginine methyl ester was used as the substrate; and pH 7.5-8.0 for chymotrypsin, when benzoyl-L-tyrosine ethyl ester was used as the substrate.     

Cysteine proteinase activity in various developmental stages of Clonorchis sinensis: a comparative analysis

1. Cysteine proteinase activity in acidic extracts of various developmental stages of Clonorchis sinensis (metacercariae, 1-, 2-, and 3-month old worms) was examined. All the activities were maximum at acidic pH and showed inhibitor susceptibilities similar to the vertebrate cysteine proteinases. 2. Specific activity of cysteine proteinase(s) was highest in metacercariae with either CBZ-phe-arg-AFC or Azocoll as the substrate. The immature and mature worms had similar (but less than metacercariae) levels of activity. 3. A soluble cysteine proteinase with a native molecular weight of approximately 20,000 +/- 1414 was partially purified from 1-, 2-, and 3-month worms. The molecular weight of similar activity in metacercariae was approximately 32,000. 4. Results suggest developmental regulation of cysteine proteinase activity in the life cycle of C. sinensis.     

Stromelysin, a connective tissue-degrading metalloendopeptidase secreted by stimulated rabbit synovial fibroblasts in parallel with collagenase. Biosynthesis, isolation, characterization, and substrates

Rabbit synovial fibroblasts induced to undergo a specific switch in gene expression by agents that alter cell morphology secreted the neutral proteinase precursor procollagenase (apparent Mr of 53,000 and 57,000). A major Mr = 51,000 polypeptide that was always induced coordinately with procollagenase has now been identified as the proenzyme form of a metal-dependent proteinase active at neutral pH. We have named this proteinase stromelysin. Prostromelysin and procollagenase were the most prominent [35S]methionine-labeled secreted proteins of the induced fibroblasts. By the use of casein degradation as an assay for enzyme activity, stromelysin was isolated with high yield from the conditioned culture medium of 12-O-tetradecanoylphorbol 13-acetate-treated fibroblasts and migrated as an active form of Mr = 21,000 that was immunologically identical to the proteoglycan-degrading proteinase purified from rabbit bone. Immunoglobulin G from antiserum raised to purified rabbit bone proteoglycanase immunoprecipitated the Mr = 51,000 proenzyme form from conditioned medium of induced rabbit cells and also immunoprecipitated an Mr = 55,000 polypeptide from induced human fibroblasts. When rabbit prostromelysin was activated by trypsin or 4-aminophenylmercuric acetate, the proenzyme was converted to an active form of Mr = 41,000. During the course of the purification, prostromelysin was converted to an additional activatable form of Mr = 35,000 and additional active forms of Mr = 21,000-25,000, which had related peptide maps distinct from collagenase. All of these forms were immunologically cross-reactive. Purified stromelysin degraded casein, cartilage proteoglycans, fibronectin, alpha 1-proteinase inhibitor, and immunoglobulin G2a and had limited activity on laminin, elastin, type IV collagen, and gelatin, but did not degrade type I collagen. Stromelysin was inhibited by EDTA, 1,10-phenanthroline, and the specific glycoprotein tissue inhibitor of metalloproteinases isolated from human amniotic fluid and was therefore classified as a metalloproteinase.     

Activation of fibroblast procollagenase by mast cell proteases.

Proteases capable of activating procollagenase from gingiva and from fibroblast and macrophage monolayer cultures were harvested from homogenates of canine tumor mast cells. The mast cell proteases lysed casein and Azocoll but not native collagen. In low salt concentrations the enzymes existed at high molecular weight complexes, which were dissociated by increasing the salt concentration above 1.0 M (NaCl, KCl). Gel filtration in 1.4 M KCl separated the protease activity into three peaks, all of which activated procollagenase. Two of the enzymes showed substrate specificities (hydrolysis of p-tosyl-L-arginine methyl ester and benzoyl-tyrosine ethyl ester) and reactive center reactivities similar to pancreatic trypsin and chymotrypsin. Based on gel filtration, apparent molecular weights of 160 000 (p-tosyl-L-arginine methyl ester esterase), 90 000 (main procollagenase activator) and 36 000 benzoyl-tyrosine ethyl ester esterase) were determined. Activation of procollagenase resulted in a 18-20 000 decrease of the molecular weight. The activation was directly related to the amount of activator added within certain limits. Further addition of activator resulted in proteolytic inactivation of collagenase.     

Purification and characterization of a rabbit bone metalloproteinase that degrades proteoglycan and other connective-tissue components

A metalloproteinase, 'proteoglycanase', that degrades proteoglycan and insoluble type IV collagen as well as casein was purified to homogeneity from rabbit bone culture medium. The major form of this proteinase had a final specific activity of 2400 micrograms of casein degraded/min per mg of enzyme protein, and Mr 24 500 by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis or 12 500 by gel-filtration chromatography. It was active over the pH range 5.0-9.0 against a number of substrates, and the rates of degradation were almost constant over the whole of this range. The products generated from proteoglycan-aggregate degradation by this enzyme indicated cleavage at multiple chondroitin sulphate-binding sites along the protein core. In a new assay to detect degradation of insoluble type IV collagen, the proteoglycanase generated large fragments, probably by cleavage in the non-helical regions. The enzyme degraded laminin, fibronectin and procollagen, removing the extension peptides of the last-mentioned. It also cleaved the 'weak region' of the type III collagen helix in a manner analogous to trypsin. The synthetic substrate 2,4-dinitrophenyl-Pro-Leu-Gly-Ile-Ala-Gly-Arg-NH2 was cleaved exclusively at the Gly-Ile bond. The proteoglycanase was inhibited by tissue inhibitors of metalloproteinases from rabbit bone culture medium, human amniotic fluid and bovine nasal-cartilage extracts, forming essentially irreversible inactive complexes. The importance of this tissue-derived enzyme, with such a wide-ranging degradative capacity, in normal and pathological connective-tissue matrix degradation is discussed.     

An evaluation of fluorometric proteinase assays which employ fluorescamine

The sensitivity and utility of proteinase assays employing fluorescamine, a compound which reacts with primary amines to form a fluorescent adduct, was assessed. As little as 1 ng of purified trypsin or clostridiopeptidase A could be detected within 3 h of incubation at 37 degrees C, using casein or gelatin as substrates. Increasing the incubation period to 18 h permitted the detection of 250 pg of each enzyme. When gelled collagen was utilized as substrate, the sensitivity to clostridiopeptidase A was reduced to 2.5 ng at 3 h and 500 pg at 18 h. The techniques could be used to measure the gelatinase, caseinase, and collagenase activities of culture media conditioned by synovial tissue. The main disadvantage of this assay is its susceptibility to interference by compounds which fluoresce or quench. This, in turn, necessitates additional blanks, which may render the assay tedious.     

A simple vertebrate collagenase assay using soluble radioactive collagen substrate

A highly sensitive assay for vertebrate collagenase has been developed using [14C]proline- or [3H]proline-labeled collagen as soluble substrate. The substrate was easy to prepare, gave high specific activity (1.4 X 10(6) cpm/mg collagen), and was stable at -20 degrees C for a long period. The digestion reaction for the assay was done at 21 degrees C to minimize the cleavage of collagen by proteases other than collagenase and to protect the 3/4 and 1/4 cleavage fragments of collagen from being further attacked by proteases. The cleaved products were denatured and then separated from undigested native collagen by precipitation with 1 M NaCl at pH 3.5. The conditions selected for denaturation and separation gave better discrimination between the cleaved products and uncleaved substrate than did conditions used in some other assays. The digestion products can be examined further by gel electrophoresis at the end of the assay to confirm the activity of vertebrate collagenase. This assay can also be adapted to assess telopeptidase activity independently of collagenase activity.     

Substrate specificity of enzymes from Bacillus mesentericus

The proteolytic enzymes of the sporogenous Bacillus mesentericus strains 64 and 8 were tested for their ability to hydrolyse different protein substrates. The enzymes were isolated using affinity chromatography on bacillichine-silochrome, and eluted with 25% isopropanol in 0.05 M Tris-HCl buffer, pH 8.0-8.4, containing 0.01 M CaCl2. Casein, hemoglobin, elastin, albumin and synthetic peptides, Z-L-Ala-Ala-Leu-pNa and Z-L-Ala-Gly-Leu-pNa, were used as substrates. The activity of esterase was assayed in terms of indophenyl acetate cleavage. The proteinases were compared with terrilytin, a commercial preparation. The proteinase of strain 64 was active in the hydrolysis of casein, hemoglobin and elastin; its specificity was close to that of terrilytin. The proteinase of strain 8 differed from them in a higher thrombolytic and fibrinolytic activity, and had a high esterase activity.     

Neutral proteinase activity ofCandida albicans

Neutral proteinase activity (pH optimum 6–7.5) was demonstrated in cell extracts of the yeast form ofCandida albicans H-317. Proteolytic activity which required activation by treatment with sodium dodecyl sulfate was measured by a colorimetric assay for Azocoll hydrolysis. The activity was inhibited by antipain, chymostatin, phenylmethylsulfonyl fluoride, andp-chloromercuribenzoate, but was not affected by ethylenediaminetetraacetate, leupeptin, or pepstatin A. These results give the first evidence of a neutral proteinase inC. albicans.     

Degradation of cartilage proteoglycans by a neutral proteinase secreted by rabbit bone-marrow macrophages in culture.

When cultivated together with pieces of cartilage biosynthetically labelled with 35S in their proteoglycans, rabbit macrophages, differentiated in vitro from bone-marrow cells, cause the release of soluble 35S-labelled material into the culture medium. This process is inhibited by killing the macrophages or by cycloheximide treatment, and is due to the secretion by the cells of a metal-dependent neutral proteinase capable of degrading cartilage proteoglycan subunits into fragments of high molecular weight. Enzyme activity is optimum at about pH7, and is inhibited by EDTA, o-phenanthroline, cysteine or serum, but not by di-isopropyl phosphorofluoridate nor by 4-hydroxymercuribenzoate. The effect of EDTA is partially reversed by Co2+ or Zn2+ ions. The enzyme is eluted from Sephadex G-150 columns as a single peak of material (apparent mol.wt. 17000) that contains also most of the proteolytic activity exerted by culture media on Azocoll (denatured collagen) or on casein. The possible role of this metalloproteinase in chronic inflammatory processes is discussed, particularly in connection with joint erosions in rheumatoid arthritis.