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PubMed1975

Proteolytic activities in yeast.

Studies on the mechanism and time course of the activation of proteinases A (EC 3.4.23.8), B (EC 3.4.22.9) and C (EC 3.4.12.--) in crude yeast extracts at pH 5.1 and 25 degrees C showed that the increase in proteinase B activity is paralleled with the disappearance of proteinase B inhibitor. Addition of purified proteinase A to fresh crude extracts accelerates the inactivation of the proteinase B inhibitor and the appearance of maximal activities of proteinases B and C. The decrease of proteinase B inhibitor activity and the increase of proteinase B activity are markedly retarded by the addition of pepstatin. Because 10-minus 7 M pepstatin completely inhibits proteinase A without affecting proteinase B activity, this is another indication for the role of proteinase A during the activation of proteinase B. Whereas extracts of yeast grown on minimal medium reached maximal activation of proteinases B and C after 20 h of incubation at pH 5.1 and 25 degrees C, extracts of yeast grown on complete medium had to be incubated for about 100 h. In the latter case, the addition of proteinas A results in maximal activation of proteinases B and C and disappearance of proteinase B inhibitor activity only after 10--20 h of incubation. With the optimal conditions, the maximal activities of proteinases A, B and C, as well as of the proteinase B inhibitor, were determined in crude extracts of yeast that had been grown batchwise for different lengths of time either on minimal or on complete medium. Upon incubation, all three proteinases were activated by several times their initial activity. This reflects the existence of proteolytically degradable inhibitors of the three proteinases and together with the above mentioned observations it demonstrates that the "activation" of yeast proteinases A, B and C upon incubation results from the proteolytic digestion of inhibitors rather than from activation of inactive zymogens by limited proteolysis.

Culture Media↗
PubMed1990

Conversion of porcine big endothelin to endothelin by an extract from the porcine aortic endothelial cells.

Conversion of porcine big endothelin (big ET) to endothelin (ET) by an extract from cultured porcine aortic endothelial cells was investigated using a radioimmunoassay (RIA) specific for ET and reverse-phase high performance liquid chromatography (RP-HPLC). When big ET was incubated with the extract at an acid pH in the presence of E-64, a cysteine protease inhibitor, the amount of immunoreactive-ET (IR-ET) in the incubation mixture was greatly increased and the optimum pH for this increased reaction was 4.0. The extract-induced increase in IR-ET was completely inhibited by pepstatin-A. These immunoreactive alterations correlated with those in the vasoconstrictor activity. When the incubation mixture of big ET with the cell extract was applied to the RP-HPLC, the IR-ET eluted at the same retention time as seen with synthetic porcine ET. We suggest that a pepstatin-sensitive aspartic protease is involved in the conversion of big ET to ET in vascular endothelial cells.

Animals↗
PubMed1992

Effect of cell density on endothelin release from endothelial cells and phosphoramidon dependent inhibition.

The modulation of endothelin (ET) release from endothelial cells was investigated as a function of cell density. The present study examined the release of ET from bovine pulmonary artery endothelial cells (BPAEC) and bovine aortic endothelial cells (BAEC) at various cell densities, as well as the effects of phosphoramidon, thiorphan and pepstatin on ET release at different densities. ET release from BPAEC and BAEC decreased as cell density increased. This cell density effect was not observed with prostacyclin release from either BPAEC or BAEC. Phosphoramidon (1 mM) inhibited ET release at every density examined for both BPAEC and BAEC. Thiorphan (1 mM) inhibited ET release from BPAEC weakly at low density and had no effect on ET release from BAEC. Pepstatin (1 mM) slightly inhibited ET release in BPAEC at the lowest density and had no effect at any other cell density for either cell type. These protease inhibitors had no effect on cell viability as determined by trypan blue exclusion and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide conversion assay. This study supports the concept that ET release is modulated by endothelial cell density. In addition, these data demonstrate that phosphoramidon, which presumably inhibits the endothelin converting enzyme, can inhibit ET release over a range of cell densities without affecting cell viability.

Animals↗
PubMed1978

Mechanism of the dipsogenic action of tetradecapeptide renin substrate.

The mechanism of the dipsogenic action of synthetic tetradecapeptide renin substrate (TDP) was studied in rats with chronically implanted lateral ventricular cannulae. All hormones and drugs were injected via the ventricular cannulae. The dipsogenic action of TDP was unaffected by the renin inhibitor pepstatin but was markedly reduced by the angiotensin converting enzyme inhibitor SQ 20881. Homogenates of rat brain readily formed angiotensin II from TDP in vitro and this was likewise unaffected by pepstatin but was reduced or abolished by SQ 20881 or by chelating agents. Natural renin substrate did not cause drinking and did not generate angiotensin II when incubated with brain homogenates. These results demonstrate that rat brain converting enzyme can generate angiotensin II from TDP and that this effect is responsible for the dipsogenic action of TDP.

Angiotensin II↗
PubMed1980

Altered protein synthesis and creatine kinase in breast muscle cell cultures from dystrophic chick embryos.

The total protein synthesis (TPS), myosin synthesis (MS) and creatine kinase (CK) levels in muscle cell cultures obtained from 400 normal (strain 454) and 400 dystrophic chick embryos (strain 455) were investigated. The cultures were obtained from breast muscles of 12 day chick embryos by dissociation in 0.25% trypsin, preplating and plating of 5 x 10(5) floating cells on gelatin coated dishes in Minimal Essential Medium, 10% horse serum and 2% chick embryo extract. After 6 days, when electron-microscopic studies demonstrated good muscle differentiation, cell cultures were labeled with [3H]leucine. TPS and MS, respectively, showed 85% and 65% increases in breast muscle cell cultures from dystrophic chick embryos. The half-life times for total protein and myosin from dystrophics were 19 and 32 hr, respectively as compared with 36 and 48 hr from controls. Noncollagen protein content (NCP) showed 27% decrease in postfusion stage (12 days) of cell cultures from dystrophics. The CK level showed 30% lower values in the cells from dystrophics but 50% higher values in their culture medium. The addition of leupeptin plus pepstatin (50 microgram/ml) to these cultures resotred NCP content, total protein and myosin turnover to normal values and significantly increased TPS and MS. The addition of diphenylhydantoin (DPH) (20 microgram/ml) to cell cultures from dystrophics did not change the NCP content nor the turnover for total protein and myosin but significantly increased TPS, MS and CK while medium CK significantly decreased. The addition of leupeptin plus pepstatin or DPH to muscle cell cultures from normal chick embryos also significantly stimulated TPS and MS.

Animals↗
PubMed1992

Role of hepatic lysosomes in the degradation of metallothionein.

The degradation of metallothionein (MT) by rat liver was examined. Degradation of MT by liver homogenate was greater than by cytosol. In addition, MT degradation by the homogenate at pH 5.5 was more than that at pH 7.2. Because lysosomal proteases function at acidic pH, these findings suggest the importance of lysosomes in MT degradation. The degradation by the lysosomal fraction was about 400-fold greater than that by the cytosol. Because cathepsins are the principal lysosomal proteases, we used cathepsin-specific inhibitors, such as leupeptin, E-64 and pepstatin, to determine the relative importance of different cathepsins in degrading MT. The study reveals that cathepsin B and/or L is (are) probably the most important enzyme(s) in degrading hepatic MT, because leupeptin, which blocks cathepsin B and L activity, inhibited the degradation of apo-MT by about 80%. Cathepsin D appears to be of least importance in MT degradation, because inhibition of this enzyme by pepstatin reduced degradation by only 20%. Studies on the degradation of apo-MT, ZnMT, and CdMT indicated that apo-MT is about 1500-fold more sensitive to degradation than ZnMT and CdMT. These data suggest that metals protect MT from degradation. This is further supported by a reconstitution experiment, which shows that with a progressive decrease of MT: metal ratio following titration of apo-MT by metals, there is a concomitant reduction in degradation. At a lysosomal pH of around 4.7, about 60% of Zn and 20% of Cd are displaced from MT, thereby making it susceptible to degradation. We propose, therefore, that lysosomes are probably important for MT degradation in vivo and that metal release is a prerequisite for degradation. With the release of metals, MT becomes susceptible to degradation, which is probably accomplished by the lysosomal cathepsins, in particular cathepsins B and L.

Animals↗
PubMed1994

Both cathepsin B and cathepsin D are necessary for processing of ovalbumin as well as for degradation of class II MHC invariant chain.

The effect of highly selective inhibitors of cathepsins on the processing of ovalbumin (OVA) and the presentation of an OVA-derived antigenic peptide (OVA323-339) by antigen presenting cells (APC) was investigated. Both CA-074 (a specific inhibitor of cathepsin B) and pepstatin A (a specific inhibitor of cathepsin D) showed an inhibitory effect on the IL-2 production from an OVA-specific, I-Ad-restricted helper T (Th) cell clone upon stimulation with OVA presented by the I-Ad-positive APC. In contrast, the presentation of the antigenic epitope, OVA323-339, to the same Th clone was not inhibited by either CA-074 or pepstatin A alone, nor even by the mixture of both inhibitors. When APC were treated with cathepsin inhibitor for 24 h, and then antigen and Th were added to the culture, the presentation of not only OVA but also an OVA-derived antigenic peptide was inhibited by either cathepsin inhibitor alone. In addition, the expression of invariant chain on APC was significantly augmented by the pretreatment of APC with either cathepsin inhibitor. Two main conclusions are drawn from these results. First, not only aspartyl protease, such as cathepsin D, but also thiol protease, such as cathepsin B, is involved in antigen processing by APC. Second, both cathepsin B and cathepsin D are necessary for degradation of the invariant chain (Ii) from the MHC class II alpha beta heterodimer in endosomes in order to express functional MHC class II molecules for binding antigenic peptides.

Animals↗
PubMed1982

The effect of short-term and chronic immunosuppression on Theiler's virus demyelination.

Theiler's virus (TV)-infected mice were treated with antithymocyte serum (ATS), cyclophosphamide or pepstatin (a protease inhibitor) to determine the effect on demyelination. When ATS and cyclophosphamide were begun at the time of infection there was significantly less demyelination at 2.5-3.5 weeks than in pepstatin or non-treated infected controls. When immunosuppression was continued for 5 weeks, or when it was not started until 5 weeks post-infection, no significant decrease in demyelination was seen compared to controls. The findings indicate that timing of immunosuppression is critical in determining the extent of TV demyelination. Such demyelination may occur by different mechanisms that are active at different times. The "bystander effect' may be important in early demyelination, but late demyelination may be due to other causes, such as oligodendrocyte lytic infection.

Animals↗
PubMed1983

Purification of Plasmodium lophurae cathepsin D and its effects on erythrocyte membrane proteins.

The cathepsin D of Plasmodium lophurae was purified using a combination of CM-Sephadex, pepstatin-agarose and Sephadex G-100 chromatography. The plasmodial enzyme was distinct from that of the host red cell and bovine spleen in its low isoelectric point (pI 4.3). The cathepsin D of P. lophurae, as well as plasmodial extracts demonstrating such proteinase activity, were able to digest the membrane proteins of duckling and human red cells at pH 7.4; proteolysis was not inhibited in phosphate-buffered saline by 100 microM pepstatin. Membrane proteins most susceptible to proteolysis were those of the cytoskeleton, notably bands 1 and 2 (spectrin), bands 2.1-2.6 (spectrin-binding proteins) and band 3. Membrane protein degradation by crude plasmodial extracts was partially inhibited by a combination of 10 mM FeCl3, and 10 mM phenylmethylsulfonyl fluoride in phosphate-buffered saline. The changes induced in erythrocyte membrane proteins by exposure to plasmodial cathepsin D parallel the alterations observed in red cell membranes obtained from malaria infected cells. Since the action of the plasmodial protease was confined to the inner surface of the red cell membrane, it is possible that protease-induced modifications in the red cell cytoskeleton could lead to merozoite release.

Animals↗
PubMed1993

Purification and partial characterization of an acid proteinase from Dirofilaria immitis.

An acid proteinase of Dirofilaria immitis worms was purified 437-fold by gel filtration on Sephadex G-75 followed by pepstatin-agarose gel affinity chromatography. The enzyme with a molecular weight of 42,000 and specific activity of 384 units (mg protein)-1 was homogeneous as judged by both affinity chromatography and SDS-polyacrylamide gel electrophoresis. However, polyacrylamide disc electrophoresis at pH 8.9 revealed that the enzyme is composed of 5 multiforms, all carrying proteinase activity. Optimum pH of the enzyme was in the range of pH 2.8-3.4, and its isoelectric point ranged between 5.8 and 6.4. The purified proteinase showed a potent activity against hemoglobin and myoglobin releasing acid soluble peptides, but not free amino acids. Complete inhibition of the proteolytic activity by pepstatin in the order of 10(-7) M strongly suggests that the enzyme belongs to the carboxyl-proteinase family.

Amino Acids↗
PubMed2004

A new pepsin assay to detect pulmonary aspiration of gastric contents among newly intubated patients.

Aspiration of gastric contents by endotracheally intubated patients is associated with significant morbidity and mortality. Previous studies suggest that pepsin in tracheal aspirates may be a valuable marker of occult aspiration. We sought to show the sensitivity and specificity of a new, pepsin-specific assay in humans. A prospective, case-controlled study was conducted with subjects serving as their own controls. After planned endotracheal and nasogastric intubation for elective surgery, 20 participants had tracheal and gastric aspirates withdrawn. A blinded investigator tested samples for the presence of pepsin using the assay. Positive samples were then tested with pepstatin, a specific pepsin inhibitor, to ensure that positive results were due to pepsin. All tracheal aspirates tested negative and all gastric aspirates tested positive for pepsin. Pepstatin halted pepsin activity in all positive samples, ensuring that positive results were due to pepsin. A pepsin-specific assay is extremely reliable for detecting gastric contents in humans.

Adult↗
PubMed2005

Characterization of plasmepsin V, a membrane-bound aspartic protease homolog in the endoplasmic reticulum of Plasmodium falciparum.

Aspartic proteases participate in a wide variety of cellular processes in eukaryotic organisms. The genome of the human malaria parasite Plasmodium falciparum encodes 10 aspartic protease homologs. Functions have been assigned to four of these: plasmepsins I, II, IV and histo-aspartic protease are key players in the catabolism of hemoglobin in the food vacuole. The functions of the other six remain obscure. To better understand the roles of aspartic proteases in blood stage growth and asexual reproduction of P. falciparum, we have characterized the biosynthesis, cellular location and pepstatin-binding properties of plasmepsin V (PM V). PM V is expressed over the course of asexual intraerythrocytic development. The amount of PM V in the parasite is lowest in the ring stage and increases steadily through schizogony. The proregion of this aspartic protease homolog exhibits remarkable interspecies diversity and appears not to be removed following biosynthesis. In intraerythrocytic parasites, PM V is located in the endoplasmic reticulum but not in ERD2-associated Golgi structures. Fractionation and solubilization experiments demonstrate that PM V is an integral membrane protein, a result that is consistent with the presence of a C-terminal putative transmembrane domain in the PM V sequence. In contrast to the food vacuole plasmepsins, detergent-solubilized PM V does not bind the aspartic protease inhibitor pepstatin. Together, these results strongly suggest that the role of PM V in P. falciparum is distinct from those of previously characterized plasmepsins.

Amino Acid Sequence↗
PubMed1997

Cathepsin D displays in vitro beta-secretase-like specificity.

The formation of A beta and A beta-containing fragments is likely a key event in the process of neural degeneration in Alzheimer's disease. The N-terminal residue (Asp-1) of A beta and its C-terminally extended sequences is liberated from the beta-amyloid precursor protein (beta APP) by beta-secretase(s). This activity appears highly increased by the presence (N-terminally to Asp-1) of a double-mutation (KM-->NL) found in several Swedish families affected by early onset Alzheimer's disease. By means of synthetic peptides encompassing the "normal' (N peptide) and mutated (delta NL peptide) sequences targeted by beta-secretase(s), we have detected a human brain protease displaying preferred efficiency for the delta NL peptide than for the non-mutated analog. This activity is sensitive to pepstatin, maximally active at acidic pH and hydrolyses the two peptides at the expected M/D or L/D cleavage sites. Such acidic activity is also detected in rat brain, PC12 cells and primary cultured astrocytes. The pepstatin sensitivity and pH maximum of the brain activity that appeared reminiscent of those displayed by the acidic protease cathepsin D led us to examine this enzyme as a putative beta-secretase-like candidate. Purified cathepsin D displays higher catalytic parameters for the delta NL peptide than for the non-mutated peptide, cleaves these two substrates at the expected M/D or L/D sites, and is maximally active at acidic pH. However, cathepsin D does not cleave peptides bearing mutations that were previously shown to drastically lower or fully block A beta secretion by transfected cells. Furthermore, cathepsin D hydrolyses recombinant baculoviral delta NL beta APP751 at a 6-fold higher rate than beta APP751 and gives rise to a 12-kDa C-terminal product that is recognized by antibodies fully specific of the N-terminus of A beta. Altogether, our study indicates that cathepsin D displays several in vitro beta-secretase-like properties that suggests that this protease could fulfill such a role, at least in the Swedish genetic form of Alzheimer's disease.

Alzheimer Disease↗
PubMed2001

Biochemical alteration of membrane-associated IL-6 RI (80-kDa) in adherent macrophages and vascular endothelium.

The potential biochemical mechanisms that mediate the 'shedding' of soluble IL-6 RI (80-kDa) receptor fragments in populations of adherent macrophages were explored. Stimulated macrophages displayed proportional increases in both the expression of membrane-associated IL-6 RI (80-kDa) and the release of soluble receptor fragments. The use of protease inhibitors implicated thiol/cysteine and carboxyl/aspartate proteases in this process. Cathepsin-D depleted membrane-associated IL-6 RI (80-kDa) complexes and generated soluble receptor fragments. A carboxyl/aspartate protease from activated macrophages isolated utilizing pepstatin-A affinity chromatography, was also found to affect membrane-associated IL-6 RI (80-kDa) complexes and generate soluble receptor fragments. Most likely, this fraction corresponded to cathepsin-D based upon its origin, pepstatin-A binding avidity, Hb-PAGE zymography, and hydrolysis of an enzyme-specific substrate. We conclude that cathepsin-D can generate soluble fragments of IL-6 RI (80-kDa) expressed by both macrophages and vascular endothelium.

Animals↗
PubMed2004

A heterogeneous enzymatic assay for quantification of Plasmepsin II activity and the evaluation of its inhibitors.

The emergence and worldwide spreading of Plasmodium falciparum strains that shown to be resistant to traditional drugs is considered a very serious health problem, given the high mortality and morbidity rate of Malaria. In the search for new drugs against this parasite, Hb hydrolyzing enzymes, such as Plasmepsin II (Plm II), have been classified as very promising targets for therapeutic attacks. In this work, it is developed a cheap and high-throughput heterogeneous enzymatic assay for measuring Plasmepsin II activity in order to use it as a tool in the discovery of new inhibitors of this enzyme. In this assay, Plasmepsin II acts upon a solid-phase bound synthetic peptide (DU2) whose sequence comprises the cleavage site F(33)-L(34) present in Hb alpha-chain. The peptide surface density is quantified by means of a classical ELISA-based procedure. In order to estimate the kinetic constants of the system and to quantify both, enzymatic and inhibitory activity, it was used a model for the kinetics of enzyme quasi-saturable systems previously developed by our group, that fitted very well to the experimental data. It was used Pepstatin as a model inhibitor of Plasmepsin II and the resulting dose-response relation agreed with the expected behavior for the Pepstatin-Plasmepsin II pair under the employed experimental conditions.

Animals↗
PubMed2001

Lysosomal involvement in hepatocyte cytotoxicity induced by Cu(2+) but not Cd(2+).

Previously we showed that the redox active Cu(2+) was much more effective than Cd(2+) at inducing reactive oxygen species ("ROS") formation in hepatocytes and furthermore "ROS" scavengers prevented Cu(2+)-induced hepatocyte cytotoxicity (Pourahmad and O'Brien, 2000). In the following it is shown that hepatocyte cytotoxicity induced by Cu(2+), but not Cd(2+), was preceded by lysosomal membrane damage as demonstrated by acridine orange release. Cytotoxicity, "ROS" formation, and lipid peroxidation were also readily prevented by methylamine or chloroquine (lysosomotropic agents) or 3-methyladenine (an inhibitor of autophagy). Hepatocyte lysosomal proteolysis was also activated by Cu(2+), but not Cd(2+), as tyrosine was released from the hepatocytes and was prevented by leupeptin and pepstatin (lysosomal protease inhibitors). Cu(2+)-induced cytotoxicity was also prevented by leupeptin and pepstatin. A marked increase in Cu(2+)-induced hepatocyte toxicity also occurred if the lysosomal toxins gentamicin or aurothioglucose were added at the same time as the Cu(2+). Furthermore, destabilizing lysosomal membranes beforehand by preincubating the hepatocytes with gentamicin or aurothioglucose prevented Cu(2+)-induced hepatocyte cytotoxicity. It is proposed that Cu(2+)-induced cytotoxicity involves lysosomal damage that causes the release of cytotoxic digestive enzymes as a result of lysosomal membrane damage by "ROS" generated by lysosomal Cu(2+) redox cycling.

Acridine Orange↗
PubMed1999

Lysosomal release of cathepsin D precedes relocation of cytochrome c and loss of mitochondrial transmembrane potential during apoptosis induced by oxidative stress.

Apoptosis was induced in human foreskin fibroblasts by the redox-cycling quinone naphthazarin (5,8-dihydroxy-1,4-naphthoquinone). Most of the cells displayed ultrastructure typical of apoptosis after 8 h of exposure to naphthazarin. Apoptosis was inhibited in fibroblasts pretreated with the cathepsin D inhibitor pepstatin A. Immunofluorescence analysis of the intracellular distribution of cathepsin D revealed a distinct granular pattern in control cells, whereas cells treated with naphthazarin for 30 min exhibited more diffuse staining that corresponded to release of the enzyme from lysosomes to the cytosol. After 2 h, release of cytochrome c from mitochondria to the cytosol was indicated by immunofluorescence. The membrane-potential-sensitive probe JC-1 and flow cytometry did not detect a permanent decrease in mitochondrial transmembrane potential (delta psi(m)) until after 5 h of naphthazarin treatment. Our findings show that, during naphthazarin-induced apoptosis, lysosomal destabilization (measured as release of cathepsin D) precedes release of cytochrome c, loss of delta psi(m), and morphologic alterations. Moreover, apoptosis could be inhibited by pretreatment with pepstatin A.

Apoptosis↗
PubMed1979

Synthesis of all the stereoisomers of statine (4-amino-3-hydroxy-6-methylheptanoic acid). Inhibition of pepsin activity by N-carbobenzoxy-L-valyl-L-valyl-statine derived from the four stereoisomers.

Synthesis of all four stereoisomers of the novel amino acid statine, 4-amino-3-hydroxy-6-methylheptanoic acid, found in pepstatin, a potent acid protease inhibitor, has been accomplished. Carbobenzoxy-L-valyl-L-valyl-statine tripeptides derived from all four stereoisomers have been prepared and their effect on pepsin activity is compared to that of pepstatin.

Amino Acids↗