Detection of a proteinase inhibitor in Aspergillus nidulans

The activity of proteinases in mycelial extracts of Aspergillus nidulans increased during storage. The rate of activation increased with temperature. Three separate proteinase activities, differing in their electrophoretic mobilities on polyacrylamide gels, were readily detected at pH 6.5. Inhibitory activity, effective against all three proteinase activities, was also detected in fractions prepared from fresh mycelial extracts. The inhibitory factor(s) were heat-stable and non-dialysable. The inhibitory activity was lost during storage of mycelial extracts. It is proposed that the inhibitory factor(s) are digested by the proteinases during storage.     

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.     

Proteinase Enzyme System of Lactic Streptococci: I. Isolation and Partial Characterization1

Proteinase activity of Streptococcus lactis cells was maximal at pH 6.0, but after storage at 3 C the minimal activity was observed at this pH. Activity lost as a result of storage could be restored by adding glutathione. Whole cells were fractionated into soluble (intracellular) and particulate fractions by sonic disruption; both fractions contained enzymatic activity. Activity of the soluble (intracellular) fraction was found to be stable to storage at 3 C, but was inhibited progressively with increasing concentrations of p-hydroxymercuribenzoate (PHMB). The enzymatic activity of this fraction was not activated by ferrous or magnesium ions or by cysteine. In contrast, activity of the particulate fraction was labile to storage at 3 C, and the reduction was comparable to that of stored cells. Furthermore, proteinase activity in the cells and the particulate fraction was not affected by addition of PHMB. The particulate fraction was activated by ferrous and magnesium ions and by cysteine. After storage, only ferrous ion and cysteine promoted reactivation; magnesium ion was totally ineffective. The enzyme(s) contained in the particulate fraction may be involved in decreased proteinase activity observed in whole cells and in the effect on growth of cells after storage.     

Inhibitory mechanism of a cross-class serpin,the squamous cell carcinoma antigen 1

The squamous cell carcinoma antigen (SCCA) 1 and its homologous molecule, SCCA2, belong to the ovalbumin-serpin family. Although SCCA2 inhibits serine proteinases such as cathepsin G and mast cell chymase, SCCA1 targets cysteine proteinases such as cathepsin S, K, L, and papain. SCCA1 is therefore called a cross-class serpin. The inhibitory mechanism of the standard serpins is well characterized; those use a suicide substrate-like inhibitory mechanism during which an acyl-enzyme intermediate by a covalent bond is formed, and this complex is stable against hydrolysis. However, the inhibitory mechanism of cross-class serpins remains unresolved. In this article, we analyzed the inhibitory mechanism of SCCA1 on a cysteine proteinase, papain. SCCA1 interacted with papain at its reactive site loop, which was then cleaved, as the standard serpins. However, gel-filtration analyses showed that SCCA1 did not form a covalent complex with papain, in contrast to other serpins. Interaction with SCCA1 severely impaired the proteinase activity of papain, probably by inducing conformational change. The decreased, but still existing, proteinase activity of papain was completely inhibited by SCCA1 according to the suicide substrate-like inhibitory mechanism; however, papain recovered its proteinase activity with the compromised level, when all of intact SCCA1 was cleaved. These results suggest that the inhibitory mechanism of SCCA1 is unique among the serpin superfamily in that SCCA1 performs its inhibitory activity in two ways, contributing the suicide substrate-like mechanism without formation of a covalent complex and causing irreversible impairment of the catalytic activity of a proteinase.     

Evaluation of chemical constituents from Glehnia littoralis for antiproliferative activity against HT-29 human colon cancer cells

In order to investigate the potential of Glehnia littoralis as a cancer chemopreventive food, antiproliferative effects of both its crude extracts and solvent-partitioned fractions (n-hexane, 85% aq. MeOH, n-BuOH, and water) were evaluated in HT-29 human colon cancer cells. Its crude extracts and solvent-partitioned fractions exhibited dose-dependent inhibitory effects on the cell proliferation. Especially, n-hexane and 85% aq. MeOH fractions exhibited a high antiproliferative effect, induced apoptosis as determined by 4,6-diamidino-2-phenylindole (DAPI) staining, and reduced mRNA expression of Bcl-2, cyclooxygenase (COX-2) and inducible nitric oxide synthase (iNOS). Systematic separation of n-hexane and 85% aq. MeOH fractions by diverse chromatographic methods led to the isolation of furanocoumarins (1–4) and polyacetylene alcohols (5–7). All compounds exhibited dose-dependent inhibitory effects on the cell proliferation. These results indicated that potent inhibitory activity of G. littoralis on proliferation of cancer cells can be significantly traceable to furanocoumarines and polyacetylenic alcohols contained in G. littoralis.     

Antigen-independent leukocyte random locomotion inhibitory activity in human ultrafiltrated leukocyte extracts

Human ultrafiltrated leukocyte extracts (MW < 5000) were fractionated by Sephadex G-10 column chromatography and the effects of these fractions on leukocyte random locomotion were investigated in vitro. Fr-4, one of these fractions, had significant leukocyte random locomotion inhibitory activity, independent of the presence of mononuclear leukocytes. This inhibitory activity was not due to cytotoxic effects on leukocytes. As seen by scanning electron microscopy, the number of cell surface pseudopods on leukocytes incubated with Fr-4 was reduced. Fr-4A, one of three fractions separated from Fr-4 by Sephadex G-25 column chromatography, significantly inhibited leukocyte random locomotion. Fr-4A contained numerous components, one of which was identified as 2-deoxyribose, on the basis of thin-layer chromatography. Biologically 2-deoxyribose showed an inhibitory effect on leukocyte locomotion and a reduction of the extrusion of pseudopods on the surface of leukocytes, at the range of assayed concentrations. This inhibitory activity is probably derived from 2-deoxyribose.     

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.     

Antibacterial activity in four marine crustacean decapods

A search for antibacterial activity in different body-parts of Pandalus borealis (northern shrimp), Pagurus bernhardus (hermit crab), Hyas araneus (spider crab) and Paralithodes camtschatica (king crab) was conducted. Dried samples were extracted with 60% (v/v) acetonitrile, containing 0.1% (v/v) trifluoroacetic acid, and further extracted and concentrated on C18 cartridges. Eluates from the solid phase extraction were tested for antibacterial, lysozyme and haemolytic activity. Antibacterial activity against Escherichia coli, Vibrio anguillarum, Corynebacterium glutamicum and Staphylococcus aureus was detected in extracts from several tissues in all species tested, but mainly in the haemolymph and haemocyte extracts. V. anguillarum and C. glutamicum were generally the most sensitive micro-organisms. In P. borealis and P. bernhardus most of the active fractions were not affected by proteinase K treatment, while in H. araneus and P. camtschatica most fractions were sensitive to proteinase K treatment, indicating antibacterial factors of proteinaceous nature. In P. bernhardus the active fractions were generally heat labile, whereas in H. araneus the activities were resistant to heat. Differences between active extracts regarding hydrophobicity and sensitivity for heat and proteinase K treatment indicate that several compounds are responsible for the antibacterial activities detected. Lysozyme-like activity could be detected in some fractions and haemolytic activity against human red blood cells could be detected in haemolymph/haemocyte and exoskeleton extracts from all species tested.     

Identification and Partial Characterization of Trypsin Inhibitory Activity in Seed of Some Fruit Plants

Plant proteinase inhibitors are natural plant defense agents against pest and predators. Many plant serine proteinase inhibitors have been purified and characterized particularly from the seeds of Leguminosae family. In this study, some common fruit plant seeds were evaluated for proteinase inhibitory activity. The seed extract of six fruit plants (Prunus domestics, Prunus persica, Prunus amygdalus, Prunus armeniaca, Citrus aurentium and Aegle marmilos) showed significant inhibitory activity against trypsin. The seed extract of P. domestica showed highest trypsin inhibitory activity (133.81 TIU mg^?1 protein).The highest protein content was found in P. persica and P. armeniaca (106.90 and 105.52 mg g^?1 flour respectively). Zymogram analysis showed variable number of trypsin inhibitor isoforms ranging from single band for A. marmilos to four isoforms for P. domestica and P. armeniaca. The seed extract of all plants, except C. aurentium, exhibited trypsin inhibitory activity over a broad range of pH and temperature.The inhibitory activity in seed extract of A. marmilos was found to be the most stable at higher temperature retaining almost 60% of inhibitory activity at 90 °C.     

Antimicrobial activity in the tissues of Atlantic cod (Gadus morhua L.)

Antimicrobial peptides (AMP) are important components of the innate immune system in metazoans. They have been studied widely in several fishes, but little is known about these defence factors in Atlantic cod, which is thought to have a less sophisticated adaptive immune system compared to other teleosts. The aim of the present study was to screen for potential AMPs in various tissues of Atlantic cod and to examine their spectra of activity. Acidic crude extracts were prepared from thirteen tissues (i.e. mucus, gills, skin, intestine, rectum, head kidney, spleen, blood, gall bladder, liver, ovary, muscle and peritoneal wall). Following partial purification by solid-phase extraction, 78 fractions were obtained and these were assayed for antimicrobial activity using a two-layer radial diffusion assay. Some of the fractions prepared from several tissues examined had potent activity against the test bacteria. In general, acetonitrile rich fractions displayed higher antibacterial activity than the aqueous ones. The most potent fractions were obtained from the gall bladder and they exhibited potent antimicrobial activity against 8 of the 9 test bacteria, including the cod pathogen Vibrio anguillarum. Antibacterial activity was completely eliminated or reduced upon treatment with proteinase K in most fractions. Protein profiles obtained by SDS-PAGE and two-dimensional gel electrophoresis showed that antimicrobial activity of the partially purified tissue extracts might be due to cationic, low molecular weight peptides.     

Proteinase Enzyme System of Lactic Streptococci II. Role of Membrane Proteinase in Cellular Function

The effect of several environmental conditions on the structure and activity of a membrane-associated proteinase from Streptococcus lactis was investigated. The activity of the enzyme varied with pH. Before storage at 3 C, maximal activity occurred at pH 6.0, but was minimal at this pH after storage. At all pH values tested, the enzyme was inactivated after storage. After storage at 3 C, the enzyme showed gross structural alterations with a concomitant loss of activity. Gel filtration and sedimentation velocity data indicated that inactivation of the enzyme was the result of aggregation to higher molecular weight forms. p-Hydroxymercuribenzoate prevented inactivation of the enzyme during storage by preventing aggregation. Activity was correlated with disaggregation of polymer forms of the enzyme to an active monomer. The storage-inactivated enzyme could be reactivated by treatment of the enzyme with cysteine, glutathione, or ferrous ion. Glutathione enabled stored cells to produce acid at their original rate when subcultured in milk. This was attributed to the effect of glutathione on the membrane proteinase. The data suggested that the biological activity of stored cells may be dependent upon the activity of the membrane proteinase.     

小桐子枝叶提取物对植物病原真菌的生物活性

采用菌丝生长抑制法,测定了小桐子枝叶6种不同溶剂提取物对小麦赤霉病菌、稻瘟病菌、烟草疫霉菌和辣椒疫霉菌的抑制作用,从中筛选出抑制作用最强的粗提物进行进一步的活性组分分离和抑菌活性测定。结果表明小桐子枝叶的乙醇提取物对4种植物病原菌抑制活性最高,在浓度为0.8 g·L^-1时,小桐子枝叶的乙醇提取物对小麦赤霉病菌、稻瘟病菌、烟草疫霉菌、辣椒疫霉菌菌丝生长抑制率分别为：87.1%、90.3%、86.4%、77.9%,其抗菌活性与农药世高均无显著差异;在小桐子枝叶乙醇提取物的不同溶剂萃取物对稻瘟病菌和烟草疫霉病菌进行生物活性追踪测试中发现,石油醚和水萃取物都具有较好的活性,当浓度为0.8 g·L^-1时,石油醚和水萃取物对两种病菌抑制率都达50%以上。表明小桐子枝叶含有丰富的抗植物病原真菌活性物质,且主要存在于小桐子枝叶乙醇提取物的石油醚相和水相中。     

A flower-inducing substance of high molecular weight from higher plants

The flower-inducing activities of aqueous extracts of several plants were fractionated by gel filtration. Three major peaks, corresponding to molecular weights of about 120, 20 to 30, and 5 to 10 kilodaltons, were detected in extracts of Lemna, Pharbitis, and Brassica. The latter two peaks may be degradation products generated during the extraction procedure. In extracts of soybean seeds, only the peak of material of 120 kilodaltons was detected. This is the first published report of a high molecular mass substance with florigenic activity in Lemna plants. The florigenic substance had some properties associated with proteins (or polypeptides), but the activity was unaffected by treatment with proteinase K.     

Regulation of k influx in barley : effects of low temperature

The proteinases present in dark-germinated flax seeds (Linum usitatissimum) were studied as a function of germination at 25°C. A majority of activity was present in basic proteinases with an acidic pH optimum and a temperature optimum of 45°C in the digestion of hemoglobin. Electrophoresis in a sodium dodecyl sulfate-polyacrylamide mixture which had been polymerized with gelatin was used to separate proteins in extracts of seedlings. Subsequent activation of proteinases with Triton X-100 and resultant digestion of gelatin proved to be very reproducible and afforded detection and good quantification of various proteinase zones. An ethylenediaminetetraacetate-sensitive proteinase zone, P4 (about 60,000 daltons), appeared at day 3 after imbibition and attained maximum activity at day 4. This correlates with a rapid loss in vivo of the glyoxysomal enzyme, isocitrate lyase (EC 4.1.3.1). Ethylenediaminetetraacetate also slowed the loss of isocitrate lyase activity in extracts of 4-day seedlings in a dose-dependent manner. The addition of leupeptin, α-tolylsulfonyl fluoride, Pepstatin A, p-chloromercuribenzoate, or 1,10-phenanthroline prior to, during, or after exchange of Triton X-100 for sodium dodecyl sulfate had almost no inhibitory effect upon proteinases in 4-day seedlings.     

Hormone-responsive alkaline proteinase in rat skeletal muscle is not a mast cell-derived enzyme

Proteinase activity was determined in myofibrils from intact rat skeletal muscle and from skeletal muscle myocytes grown in culture. In vivo administration of the mast cell degranulator compound 48/80 abolished the alkaline proteinase activity in myofibrils obtained from normal or streptozotocin-diabetic rats. Exposure of myocytes to compound 48/80 in cell cultures had no effect on their myofibrillar proteinase activity, nor did it affect the rate of overall protein degradation in these cells. Co-incubation of cultured mast cells (line P815Y) with myocytes followed by sonication of the cell mixture resulted in a marked reduction of the proteinase activity in the pellet fraction, suggesting that the mast cells contain inhibitor(s) of myofibrillar proteinase activity. It is suggested that the myofibril-bound alkaline proteinase activity is not a mast cell-derived enzyme but a genuine component of muscle cells. The in vivo 48/80-induced reduction of muscle myofibrillar proteinase activity appears to be due to release of a soluble inhibitory activity rather than removal of mast cell proteinase from the tissue by degranulation.     

2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one, an Inhibitor from Zea mays with Differential Activity against Soft Rotting Erwinia Species

[2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one] DIMBOA was extracted with ethyl acetate from acidified water homogenates of corn (Zea mays L.) seedlings. Pure DIMBOA or ethyl acetate extracts of corn tissue were added to bacterial growth medium at five concentrations (measured as hydroxamates). DIMBOA and corn extracts were more inhibitory to soft rot bacteria (Erwinia spp.) that are nonpathogenic to corn than to soft rot bacteria that are corn pathogens. The inhibitory activity of DIMBOA was similar to that of the ethyl acetate extracts. Both corn extracts and DIMBOA prolonged the lag phase of bacterial growth without significantly changing log phase growth rates. At various concentrations of the inhibitor, 50 to 100% of the activity of corn extracts inhibitory to different bacterial isolates was attributable to DIMBOA. Extracts of DIMBOA-deficient plants (genotype bxbx) were not inhibitory to Erwinia spp. It was concluded that DIMBOA is the major active component in those corn extracts which are inhibitory to soft rot Erwinia species.     

Proteolysis in Euglena gracilis

Endoproteolytic activities (EC 3.4.22. and 23.) of cell-free extracts of Euglena gracilis, measured by autolysis and azocaseinolysis, vary considerably during the culture growth cycle. They are high in the lag phase, drop sharply up to the mid-logarithmic phase, and then rise again reaching the initial high levels in the stationary phase. This pattern has been observed for both the soluble and the particulate proteolytic activities of four cell types differing with regard to the developmental state of the chloroplast: dark-grown, light-induced, and light-grown wild-type cells, as well as light-grown apoplastic W₃BUL mutant cells, all on a glucose-based medium. Therefore, the activity of the main intracellular proteinases is neither directly nor indirectly light-regulated, but seems to be controlled by the availability of nutrients. Endogenous inhibitors of proteinases could not be detected. Cysteine proteinase activity has been found in the soluble and the particulate fractions, but aspartic proteinase activity in the latter ones only. Different cysteine proteinases may be present in the two fractions, during the different growth phases, and in the four cell types studied.Abbreviations CBB Coomassie Brilliant Blue G-250 - DFP diisopropyl fluorophosphate - EDTA disodium ethylendiaminetetraacetic acid - E-64 l-transepoxysuccinyl-leucyl-amido(4-guanidino)butane - Iog phase logarithmic growth phase - MET 2-mercaptoethanol - PMSF phenylmethylsulfonyl fluoride - Z benzyloxycarbonyl Paper I of this series is Krauspe and Scheer (1986). A preliminary publication appeared (Krauspe et al. 1982)     

Isolation and identification of RANKL-induced osteoclast differentiation inhibitor from Pleurotus citrinopileatus

The purpose of this study was to investigate the effects of water extracts from edible mushroom, Pleurotus citrinopileatus fruiting body on RANKL-induced osteoclast differentiation. The osteoclast differentiation inhibitor from water extracts of P. citrinopileatus was purified by ultrafiltration, size exclusion column chromatography, ethanol precipitation, and treatment with various enzymes. The water extracts from P. citrinopileatus showed significant osteoclast differentiation inhibitory activity, and its 50 kDa above fraction from ultrafiltration showed the highest osteoclast differentiation inhibitory activity. Fractions 2 and 3 from size exclusion column chromatography clearly inhibited RANKL-induced osteoclast differentiation. When these fractions were treated by ethanol precipitation, the precipitates showed high osteoclast differentiation inhibitory activity. Finally, we obtained the precipitates as the purified osteoclast differentiation inhibitor, and it was identified as β-glucan. However, further studies are required to be used as candidate of anti-osteoporotic agent.     

A low-molecular-weight inhibitor of the neutral proteinase from rat intestinal smooth muscle.

1. Rat intestinal smooth muscle was shown to contain endogenous inhibitory activity towards the neutral trypsin-like muscle proteinase described previously [Beynon & Kay (1978) Biochem. J. 173, 291--298]. 2. Comtamination of the muscle tissue by mucosal, blood and pancreatic inhibitors was shown to be unlikely. 3. The inhibitory activity was resolved into high- and low-molecular-weight components. 4. The low-molecular-weight component was purified to homogeneity. It has a molecular weight of approx. 9000 and was stable over the pH range 3--11. 5. It inhibited the muscle proteinase competitively (Ki congruent to t microM), but had no effect on any of the other proteinases tested. 6. Leupeptin also inhibited the muscle proteinase competitively (Ki congruent to 0.3 microM), whereas the low-molecular weight proteins gastrin, glucagon and insulin B-chain had very little effect. 7. A role for a weakly binding inhibitor in modulating the influence of the neutral proteinase on intracellular protein degradation is considered.     

Inhibitors of endogenous proteinases in the seeds of Scots pine, Pinus sylvestris

Extracts of resting pine seeds inhibited the proteinase activities present in extracts of endosperms of germinating seeds (hydrolysis of haemoglobin at pH 3.7 and hydrolysis of casein at pH 5.4 and 7.0). Heating the extracts of resting seeds at 60°C destroyed their own proteinase activity but their proteinase inhibitor activity decreased by only 25 to 30%. Some properties of the inhibitor(s) were studied using extracts treated at 60°C. The inhibitor activities were non-dialysable. the inhibition increased linearly with increasing inhibitor concentration up to 80% of total proteinase activity, and the maximal inhibition was 80% at pH 3.7. 90% at pH 5.4. and 97% at pH 7.0. The extracts of resting seeds did not inhibit the pepsin-like acid pine proteinase that accounts for a minor part of the proteolytic activity of endosperm extracts at pH 3.7. Neither did they have any effect on the acid pine carboxypeptidase or trypsin and chymotrypsin. Fresh extracts of endosperms of germinating seeds contained relatively high proteinase activity (assayed directly) and moderate inhibitor activity (assayed after treatment at 60°C). When fresh extracts were dialysed at 50°C for 48 h their proteinase activities increased considerably while the corresponding inhibitor activities disappeared. It is concluded that the decrease of inhibitors during dialysis is due to enzymatic inactivation and that the corresponding increase of proteinase activities is at least partly due to the destruction of the inhibitors.