A modified fibrin plate for rapid detection of proteinases and proteinase inhibitors in fractions after liquid chromatography

A rapid procedure for detecting proteolytic enzymes and proteinase inhibitors in fractions after liquid chromatography has been developed. The detection is based on the proteolysis of a very thin layer of fibrin. Proteinase (or proteinase inhibitor) positive and negative fractions can be distinguished in a short period of time.     

Enzymatic degradation of formaldehyde-crosslinked gelatin

A general method is proposed for characterizing the enzymatic degradability of formaldehyde-crosslinked gelatin which is simple and uses subtilisin as a readily available, commercial alkaline proteinase. Solubilization of gelatin involved dissolution of species not chemically bonded to the crosslinked network, as well as the soluble fractions resulting to the enzymatic degradation. There was a linear relationship between the complete solubilization time of the gelatin and its exposure time to the formaldehyde crosslinking procedure. © Rapid Science Ltd. 1998     

Rapid detection of proteolytic enzymes and their inhibitors

Summary A rapid procedure for detecting proteolytic enzymes and proteinase inhibitors in various solutions has been developed. The detection is based on the proteolysis of a thin laver of dved gelatin laying on a disk prepared from a photographic film. Proteinase (or proteinase inhibitor) fositive and negative samples can be distinguished in a short period of time.     

Proteolytic enzymes; further studies on protein,polypeptide, and other inhibitors of serum proteinase,leucoproteinase, trypsin,and papain

1. Serum proteinase precursor was found in plasma protein fractions I and III of Cohn. Inhibitors of serum proteinase, leucoproteinase, trypsin, and papain were found in fractions IV-1 and IV-4, and to a lesser extent in fractions V and I. 2. Pancreatic, soy bean, lima bean, and egg white inhibitors inhibited trypsin stoichiometrically. Pancreatic inhibitor had comparable inhibitory activity against serum proteinase; soy bean inhibitor had somewhat less, lima bean inhibitor even less, and egg white inhibitor very little. None of these inhibitors appreciably inhibited leucoproteinase or papain. 3. Serum and fractions IV - 1 and IV - 4 had marked inhibitory activity against trypsin and leucoproteinase, and somewhat less against serum proteinase and papain. The inhibitory activity of the plasma proteins against trypsin and leucoproteinase was due almost entirely to fractions IV - 1 and IV - 4; against serum proteinase and papain fraction V was slightly more important. The "reconstituted plasma proteins" accounted for 8 to 25 per cent of the proteinase-inhibitory activity of whole serum or plasma. 4. The proteinase-inhibitory activity of serum, plasma protein fractions, and soy bean inhibitor was heat labile, while that of pancreatic, lima bean, and egg white inhibitors was relatively heat stable. 5. Reducing and oxidizing agents, in very high concentration, inhibited serum proteinase, as well as trypsin and leucoproteinase. These proteinases were not influenced by mercurial sulfhydryl inhibitors, indicating that free sulfhydryl groups do not play an important part in their activity.     

Purification and characterization of an intracellular aspartyl acid proteinase (pumAi) from Ustilago maydis

The intracellular proteinase pumAi in Ustilago maydis has been associated with yeast-mycelium dimorphic transition. The proteinase was purified from a cell-free extract by ammonium sulfate fractionation and chromatographic steps including hydrophobic interactions on a Phenyl Superose column, ion exchange on a Mono Q column, and gel filtration on Superose 12 columns. The enzyme has a mass of 35.3-36.6 kDa, a pH and temperature optimum of 4.0 and 40 degrees C, respectively, and a pI of 5.5. The enzyme degraded hemoglobin, gelatin, albumin, and casein, but not collagen, and the enzymatic activity was strongly inhibited by pepstatin A, an aspartyl proteinase-specific inhibitor. The biochemical characteristics of pumAi are similar to other fungal intracellular aspartyl proteinases, however, this is the first biochemical characterization of a basidiomycete proteinase probably associated with dimorphic yeast-mycelium transition.     

A TRYPSIN‐LIKE PROTEINASE IN THE MIDGUT OF Ectomyelois ceratoniae ZELLER (LEPIDOPTERA: PYRALIDAE): PURIFICATION,CHARACTERIZATION, AND HOST PLANT INHIBITORS

A trypsin‐like proteinase was purified and characterized in the midgut of Ectomyelois ceratoniae. A purification process that used Sepharyl G‐100 and DEAE‐cellulose fast flow chromatographies revealed a proteinase with specific activity of 66.7 μmol/min/mg protein, recovery of 27.04 and purification fold of 23.35. Molecular weight of the purified protein was found to be 35.8 kDa. Optimal pH and temperature were obtained 9 and 20°C for the purified trypsin proteinase, respectively. The purified enzyme was significantly inhibited by PMSF, TLCK, and SBTI as specific inhibitors of trypsins in which TLCK showed the highest inhibitory effect. Trypsin proteinase inhibitors were extracted from four varieties of pomegranate including Brait, Torsh‐Sabz, May‐Khosh, and Shirin by ion exchange chromatography. It was found that fractions 17–20 of Brait; fractions 18 and 21–26 of Torsh‐Sabz; fractions 1–7, 11–17, and 19–21 of May‐Khosh and fraction 8 for Shirin showed presence of trypsin inhibitor in these host. Comparison of their inhibitory effects on the purified trypsin proteinase of E. ceratoniae demonstrated that fractions from May‐khosh variety had the highest effect on the enzyme among other extracted fractions. Characterization of serine proteinases of insects mainly trypsins is one of the promising methods to decrease population and damages via extracting their inhibitors and providing resistant varieties.     

Extracellular proteolytic eyzymes of microscopic fungi from thermal springs of the Barguzin Valley (Northern Baikal region)

Production of extracellular proteolytic enzymes was studied in thermophilic fungi Paecelomyces variotii and Aspergillus carneus, isolated from thermal springs of the Barguzin Valley. Protease synthesis in these fungi requires protein in the ambient medium. The composition of the enzymes secreted by A. carneus depends on the kind of carbohydrate present in the medium. The proteinase of this fungus digests synthetic substrates and gelatin (optimum pH 7.7). It belongs to neutral serine proteases. Extracellular P. variotii proteases digests gelatin (optimum pH 9.7-10.4). According to inhibitor analysis data, they are assigned to alkaline metalloproteinases and serine proteinases.     

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.     

Inhibition of human granulocyte elastase and kininogenase

Using an inhibitory analysis, the different enzymatic nature of kininogenase and elastase activities in serine proteinase fractions (pI 8.3-10.75) isolated from human granulocyte lysates by isoelectrofocusing was demonstrated. The thermo- and acid-stable serine proteinase inhibitor from rabbit serum was shown to completely inhibit the kininogenase activity in these fractions but to have no inhibiting action on the elastase activity. On the contrast, the specific granulocyte elastase inhibitor, N-3-carbomethoxypropanoyl-L-alanyl-L-alanyl-L-prolyl-L-valyl-chloromethylketone , inhibits granulocyte elastase and does not inhibit the kininogenase activity in lysate fractions. The efficiency of granulocyte elastase inhibition by this chloromethylketone is evaluated by the kinetic parameters k3, Ki. The values of k3/Ki for granulocyte elastase forms with pI of 10.75, 8.9 and 8.0 are 1430, 670 and 360 M-1 S-1, respectively and show effective inhibition of the three forms by this inhibitor. Based on the different degree of inhibition of the three elastase forms by chloromethylketone inhibitor the existence of the family of elastaselike enzymes in human granulocytes is postulated.     

Proteolytically active complexes of cathepsin L and a cysteine proteinase inhibitor; purification and demonstration of their formation in vitro.

Proteolytically active complexes of the proteinase cathepsin L, with an endogenous inhibitor of cysteine proteinases, were purified from sheep liver. The complexes were active against the synthetic substrate Z-Phe-Arg-NHMec and also the proteins azocasein and gelatin. The composition of the complexes was demonstrated by Western blotting, after reducing and nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis with monospecific antibodies raised against purified sheep liver cathepsin L and purified sheep liver cysteine proteinase inhibitor (probably stefin B). Similar complexes could be formed in vitro, by coincubation of purified sheep liver cathepsin L with the purified sheep liver cystatin at a pH of 5.5 or higher.     

Isolation and properties of a metal-dependent endopeptidase from human uterus hydrolysing synthetic collagenase substrates.

A metal-dependent peptidase was isolated from the homogenate of human uterus by standard chromatographic techniques and purified to apparent homogeneity. The peptidase hydrolysed the synthetic vertebrate collagenase substrate 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg (Dnp-peptide), the synthetic bacterial collagenase substrate 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (PZ-peptide) and gelatinolytic peptides of gelatin, but was inactive against collagen type I, gelatin and casein. The cleavage site for the Dnp-peptide was the Gly-Ile bond. The enzyme was not only inhibited by metal chelators, such as EDTA, 1,10-phenantroline and dithiothreitol but also by thiol reagents, such as mersalylic acid and N-ethylmaleimid. However, E-64, an inhibitor for thiolproteinases, and leupeptin, an inhibitor for thiol- and serine proteases, did not exhibit any inhibitory activity. Pepstatin, an inhibitor for aspartate proteinases, and inhibitors for serine proteinases like phenylmethanesulfonyl fluoride and Trasylol were ineffective as well. The purified peptidase displayed a single band in the SDS-PAGE with an apparent molecular mass of 65 kDa. Employing isoelectric focusing an IP of 5.0 could be determined. The enzyme's properties are discussed in relation to the proteinase EC 3.4.24.11 and to proteinases of the collagenase family as well as the possibility to discriminate these three metalloproteinase classes by employing the Dnp-peptide.     

Low molecular weight serine proteinase inhibitors of the human intervertebral disc

Human lumbar disc tissue when extracted with 4M GuHCl and subjected to dissociative CsCl density gradient ultracentrifugation yielded trypsin inhibitor activity in the low bouyant density fractions (rho less than or equal to 1.38 g/ml). Disc proteoglycans sedimented in the high bouyant density fractions (rho greater than or equal to 1.5 g/ml). Sephadex G75F gel filtration of the low bouyant density protein fractions afforded a major low molecular weight (Kav = 0.5) trypsin inhibitor pool which was further purified by trypsin affinity chromatography. This latter step facilitated separation of the trypsin inhibitors from neutral proteinase activity also present. The trypsin inhibitor fraction so isolated was shown to possess potent inhibitory activity against a range of human serine proteinases including leukocyte elastase and cathepsin G, urokinase, kallikrein, plasmin and thrombin. Significantly this serine proteinase inhibitor preparation effectively prevented degradation of proteoglycans by a neutral proteinase also isolated from the human intervertebral disc.     

FRACTIONATION OF GELATIN

1. It is possible to fractionate gelatin by means of reprecipitation at 23°C. of a salt-free solution of pH 4.7 into two fractions, one of which is soluble in water at any temperature, and a second one which does not dissolve in water even when heated to 80°C. 2. The proportion of the soluble fraction in gelatin is much greater than of the insoluble one. 3. The insoluble fraction of gelatin does not swell when mixed with water, but it does swell in the presence of acid and alkali which finally dissolve it. 4. Blocks of concentrated gel made by dissolving various mixtures of the soluble and insoluble fractions of gelatin in dilute NaOH swell differently when placed in large volumes of dilute buffer solution pH 4.7 at 5°C. The gel consisting of the insoluble material shows only a trace of swelling, while those containing a mixture of soluble and insoluble swell considerably. The swelling increases rapidly as the proportion of the soluble fraction increases. 5. A 5 per cent gel made up by dissolving the insoluble fraction of gelatin in dilute NaOH loses about 70 per cent of its weight when placed in dilute buffer pH 4.7 at 5°C. A similar gel made up of ordinary gelatin loses only about 20 per cent of its weight under the same conditions. 6. It was not found possible to resynthesize isoelectric gelatin from its components. 7. An insoluble substance similar in many respects to the one obtained by reprecipitation of gelatin is produce on partial hydrolysis of gelatin in dilute hydrochloric acid at 90°C.     

Gelatinases and serine proteinase inhibitors of seminal plasma and the reproductive tract of turkey (Meleagris gallopavo)

This study examined proteolytic enzymes and serine proteinase inhibitors in turkey seminal plasma with relation to their distribution within the reproductive tract and to yellow semen syndrome (YSS). Proteases of blood plasma, extracts from the reproductive tract, and seminal plasma were analyzed by gelatin zymography. We found a clear regional distribution of proteolytic enzymes in the turkey reproductive tract. Each part was characterized by a unique profile of serine proteolytic enzymes of molecular weights ranging from 29 to 88 kDa. The ductus deferens was found to be a site of very intense proteolytic activity. Two metalloproteases of 58 and 66 kDa were detected in all parts of the reproductive tract and seminal plasma. Using electrophoretic methods for detection of anti-trypsin activity, we found three serine proteinase inhibitors in turkey seminal plasma. Two inhibitors were found in the testis and epididymis and a third in the ductus deferens and seminal plasma. Blood plasma was characterized by the presence of two metalloproteinases and one serine proteinase inhibitor (of low migration rate) that were also detected in the reproductive tract. Amidase and anti-trypsin activities (expressed per gram of protein) differed for yellow and white seminal plasma. We concluded that turkey seminal plasma contains metalloproteases, serine proteinases, and serine proteinase inhibitors. The metalloproteases and one proteinase inhibitor are related to blood proteinases but the other two inhibitors and serine proteinases seem to be unique for the reproductive tract.     

Subcellular distribution of histone-degrading enzyme activities from rat liver.

Chromatin prepared from liver tissue contains a histone-degrading enzyme activity with a pH optimum of 7.5-8.0, whereas chromatin isolated from purified nuclei is devoid of it. The histone-degrading enzyme activity was assayed with radioactively labelled total histones from Ehrlich ascites tumor cells. Among the different subcellular fractions assayed, only lysosomes and mitochondria exhibited histone-degrading enzymes. A pH optimum around 4.0-5.0 was found for the lysosomal fraction, whereas 7.5-8.0 has been found for mitochondria. Binding studies of frozen and thawed lysosomes or mitochondria to proteinase-free chromatin demonstrate that the proteinase associated with chromatin isolated from frozen tissue originates from damaged mitochondria. The protein degradation patterns obtained after acrylamide gel electrophoresis are similar for the chromatin-associated and the mitochondrial proteinase and different from that obtained after incubation with lysosomes. The chromatin-associated proteinase as well as the mitochondrial proteinase are strongly inhibited by 1.0 mM phenylmethanesulfonyl fluoride. Weak inhibition is found for lysosomal proteinases at pH 5. Kallikrein-trypsin inhibitor, however, inhibits lysosomal proteinase activity and has no effect on either chromatin-associated or mitochondrial proteinases. The higher template activity of chromatin isolated from a total homogenate compared to chromatin prepared from nuclei may be due to the presence of this histone-degrading enzyme activity.     

Tissue distribution, inhibition and activation of gelatinolytic activities in Atlantic cod (Gadus morhua)

Gelatinolytic activities in fish tissues with properties like matrix metalloproteinases (MMPs) have been paid little attention. However, they have been proposed to participate in post mortem degradation during storage and the disintegration of pericellular connective tissue during spawning. In this paper the distribution of gelatinolytic activities in liver, heart, muscle, gill, and male and female gonad of Atlantic cod (Gadus morhua) was studied by using gelatin SDS-PAGE, proteinase inhibitors, gelatin and lentil lectin Sepharose affinity chromatography. The amount of gelatin degrading enzymes varied from tissue to tissue. Most of the gelatin binding enzymes were found to be matrix metalloproteinases by adding galardin, a broad range MMP inhibitor, to the incubation buffer. A 72 kDa form of cod gelatin degrading enzyme had properties similar to human proMMP-2, as it could be activated by p-aminophenylmercuric acetate and trypsin. Like the human MMP-2 it did not bind to lentil lectin. An 83 kDa cod gelatin degrading enzyme had properties similar to the 92 kDa progelatinase B (proMMP-9). These properties were also similar to that of the 72 kDa form, except that the 83 kDa cod gelatinase was bound to lentil lectin, showing that it is a glycoprotein like MMP-9.     

THE SWELLING PRESSURE OF GELATIN AND THE MECHANISM OF SWELLING IN WATER AND NEUTRAL SALT SOLUTIONS

1. A method is described for measuring the swelling pressure of solid gelatin. 2. It was found that this pressure increases rapidly between 15° and 37°C., and that the percentage change is nearly independent of the concentration of gelatin. 3. It is suggested that this pressure is due to the osmotic pressure of a soluble constituent of the gelatin held in the network of insoluble fibers, and that gelatin probably consists of a mixture of at least two substances or groups of substances, one of which is soluble in cold water, does not form a gel, and has a low viscosity and a high osmotic pressure. The second is insoluble in cold water, forms a gel in very low concentration, and swells much less than ordinary gelatin. 4. Two fractions, having approximately the above properties, were isolated from gelatin by alcohol precipitation at different temperatures. 5. Increasing the temperature and adding neutral salts greatly increase the pressure of the insoluble fraction and have little effect on that of the soluble fraction. 6. Adding increasing amounts of the soluble fraction to the insoluble one results in greater and greater swelling. 7. These results are considered as evidence for the idea that the swelling of gelatin in water or salt solutions is an osmotic phenomenon, and that gelatin consists of a network of an insoluble substance enclosing a solution of a soluble constituent.     

Purification of the Ca2+-dependent proteinase inhibitor from bovine cardiac muscle and its interaction with the millimolar Ca2+-dependent proteinase

A protein inhibitor of the Ca2+-dependent proteinase has been purified from bovine cardiac muscle by using the following steps in succession: salting out 17,600 X gmax supernatants from muscle homogenates in 50 mM Tris acetate, pH 7.5, 4 mM EDTA between 25 and 65% ammonium sulfate saturation; eluting between 25 and 120 mM KCl from a DEAE-cellulose column at pH 7.5; salting out between 30 and 60% ammonium sulfate saturation; Ultrogel-22 gel permeation chromatography at pH 7.5; heating to 80 degrees C followed by immediate cooling to 0 degree C; 6% agarose gel permeation chromatography in 4 M urea, pH 7.5; and elution from a phenyl-Sepharose hydrophobic column between 0.7 and 0.5 M ammonium sulfate. Approximately 1.16-1.69 mg of purified Ca2+-dependent proteinase inhibitor are obtained from 1 kg of bovine cardiac muscle, fresh weight. Bovine cardiac Ca2+-dependent proteinase inhibitor has an Mr of 115,000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a pI of 4.85-4.95, very little alpha-helical structure, a very low specific absorbance of 1.647 (A1% 280), and very low contents of histidine, tryptophan, phenylalanine, and tyrosine. Bovine cardiac Ca2+-dependent proteinase inhibitor probably contains a single polypeptide chain in nondenaturing solvents. One 115-kDa inhibitor polypeptide inactivates 10 110-kDa millimolar Ca2+-requiring proteinase (millimolar Ca2+-dependent proteinase) molecules in assays of purified proteins. Inhibition of millimolar proteinase by the proteinase inhibitor did not change in the pH range 6.2-8.6. The inhibitor requires Ca2+ to bind to millimolar Ca2+-dependent proteinase. The Ca2+ concentration required for one-half-maximum binding of millimolar Ca2+-dependent proteinase to the inhibitor was 0.53 mM, compared with a Ca2+ concentration of 0.92 mM required for one-half maximum activity of millimolar Ca2+-dependent proteinase in the absence of the proteinase inhibitor. Unless millimolar Ca2+-dependent proteinase is located subcellularly in a different place than the proteinase inhibitor or unless the proteinase/inhibitor interaction is regulated, millimolar proteinase could never be active in situ.     

Purification and Characterization of Extracellular Proteinases of Aspergillus oryzae

The extracellular proteinases of Aspergillus oryzae EI 212 were separated into two active fractions by (NH4)2SO4 and ethanol fractionation followed by diethylaminoethyl-Sephadex A-50 and hydroxyapatite chromatography. The molecular weight was estimated by gel filtration to be about 70,000 and 35,000 for proteinases I and II, respectively. Optimum pH for casein and hemoglobin hydrolysis was 6.5 at 60 C for proteinase I and 10.0 at 45 C for proteinase II, and for gelatin hydrolysis it was 6.5 at 45 C for both enzymes. The enzymes were stable over the pH range 6 to 8 at 30 C for 60 min. The enzyme activity for both the proteinases was accelerated by Cu2+ and inhibited by Fe2+, Fe3+, Hg2+, and Ag+. Halogenators (e.g., N-chlorosuccinimide) and diisopropyl fluorophosphate inhibited proteinase II. Sulfhydryl reagents such as p-chloromercuribenzoate and iodoacetate inhibited proteinase I. Sulfhydryl compounds accelerated the action of both enzymes.     

Cd(II), Pb(II) and Zn(II) ions regulate expression of the metal-transporting P-type ATPase ZntA in Escherichia coli

Feeding bioassay results established that the soybean cysteine proteinase inhibitor N (soyacystatin N, scN) substantially inhibits growth and development of western corn rootworm (WCR), by attenuating digestive proteolysis [Zhao, Y. et al. (1996) Plant Physiol. 111, 1299-1306]. Recombinant scN was more inhibitory than the potent and broad specificity cysteine proteinase inhibitor E-64. WCR digestive proteolytic activity was separated by mildly denaturing SDS-PAGE into two fractions and in-gel assays confirmed that the proteinase activities of each were largely scN-sensitive. Since binding affinity to the target proteinase [Koiwa, H. et al. (1998) Plant J. 14, 371-380] governs the effectiveness of scN as a proteinase inhibitor and an insecticide, five peptides (28-33 kDa) were isolated from WCR gut extracts by scN affinity chromatographic separation. Analysis of the N-terminal sequence of these peptides revealed similarity to a cathepsin L-like cysteine proteinase (DvCAL1, Diabrotica virgifera virgifera cathepsin L) encoded by a WCR cDNA. Our results indicate that cathepsin L orthologs are pivotal digestive proteinases of WCR larvae, and are targets of plant defensive cystatins (phytocystatins), like scN.