Human platelet 20S proteasome: inhibition of its chymotrypsin-like activity and identification of the proteasome activator PA28. A preliminary report.

Earlier studies have demonstrated that human platelets contain the 20S proteasome, and its protein activator. However, understanding the potential role of the proteasome in human platelets requires a detailed knowledge about its chymotryptic-like activity, a crucial one for protein degradation in all eukaryotic cells. In this communication we have shown that human platelet 20S proteasome exhibited chymotryptic-like activity towards succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin as substrate at a broad pH range, with optimum between pH 7.

Lactacystin inhibits cathepsin A activity in melanoma cell lines.

We describe the inhibitory effect of the proteasome inhibitor, lactacystin, on cathepsin A activity in murine melanoma cell lines. In vitro lactacystin metabolite, beta-lactone, at a concentration of 1 microM, significantly suppressed cathepsin A activity in B78 melanoma cell lysates by about 50%. Exposure of three murine melanoma cell lines with different metastatic potential to lactacystin at a concentration of 5 microM for 6 h caused a significant reduction in the carboxypeptidase activity of this enzyme, while the inhibitory activity remained unchanged for at least 12 h.

Separation of cathepsin A-like enzyme and the proteasome: evidence that lactacystin/beta-lactone is not a specific inhibitor of the proteasome.

Previous studies have described a human platelet cathepsin A-like enzyme with a number of similarities to the "acidic" and "neutral" chymotrypsin-like activities of the proteasome. This includes its strong inhibition by the highly specific proteasome inhibitor Lactacystin/beta-lactone, suggesting that either the Cbz-Phe-Ala-hydrolyzing activity attributed to cathepsin A was due to the chymotrypsin-like activity of the proteasome or that lactacystin was not a specific inhibitor of the proteasome. In the present study we discard the first possibility on the basis of the following findings: (a) human platelet cathepsin A, unlike proteasome, binds to concanavalin A, and does not bind to Heparin-Sepharose at pH 7.

Effects of epsilon-aminocaproylaminoacids on prothrombin activation and thrombin activity.

Influence of four epsilon-aminocaproylaminoacids on prothrombin activation and thrombin activity was examined. Only epsilon-aminocaproylnorleucine markedly inhibited the prothrombin activation in an extrinsic system.

Inhibitors of pepsin, trypsin and chymotrypsin in seeds of plants consumed by humans and animals. I. Evaluation of pepsin, trypsin, and chymotrypsin inhibitors activity in seeds of 26 plant species.

Antipepsin, antitrypsin and antichymotrypsin activity was determined in seed extracts of 26 plants consumed by humans and animals (small bean, broad bean, pumpkin, kidney bean, charlock, pea, buckwheat, barley, maize, flax, lupine, poppy, almond, peanut, hazel, walnut, oat, millet, wheat, rice, rape, sunflower, lentils soya bean, vetch, rye). Antipepsin activity is found in the seeds of small bean, pumpkin, flax, peanut, walnut, oat, wheat, sunflower, lentils and soya bean. Antitrypsin and antichymotrypsin activities are of different intensity in seed extracts of all examined plants.

The influence of epsilon-aminocaproic acid derivatives on the anticoagulant activity of heparin.

Derivatives of epsilon-aminocaproic acid with antifibrinolytic activity, at low concentration, do not influence the anticoagulant activity of heparin under the heparin-thrombin test conditions. At concentrations higher than 0.002 M tested compounds slightly enhance the anticoagulant action of heparin.

Lactacystin, a specific inhibitor of the proteasome, inhibits human platelet lysosomal cathepsin A-like enzyme.

Lactacystin, the most specific inhibitor of the proteasome, strongly inhibited at pH 5.5 the activity of human platelet lysosomal cathepsin A-like enzyme. At a concentration as low as 1-5 microM it almost completely decreased the hydrolysis rate of cathepsin A specific substrates: Cbz-Phe-Ala and FA-Phe-Phe.

Activity of liver proteases in experimental methanol intoxication.

Intoxication of rats with methanol (1.5 and 3.0 g/kg body weight) led to a significant, time- and dose-dependent decrease in the activities of cathepsins A, B and C, while the activity of cathepsin D was unaffected.

Prolidase activity and beta 1 integrin expression in moderately and poorly differentiated lung adenocarcinomas.

Primary human lung adenocarcinomas were divided into two groups according to the degree of histologic differentiation: G2-moderately and G3-poorly differentiated tumors. Each group was compared with normal lung tissue in respect to prolidase activity, its ability to interact with specific antibody, free proline and beta 1 integrin subunit content as well as ability of beta 1 integrin subunit to interact with specific antibody. It was found that prolidase activity in lung adenocarcinomas G3, was significantly elevated in comparison to normal lung tissue.

Plasminogen activators and plasmin in lung cancer.

Urokinase plasminogen activator and plasmin contribute to detach neoplastic cells from solid tumor and facilitate the movement of these cells through interstitium and capillary walls as well as infiltration of the surrounding structures. Plasminogen activators inhibitors fulfill a regulatory function in these processes. Determining activity and concentration, finding subcellular, cellular and zonal localization of every component of plasminogen activation system has diagnostic and prognostic importance in different lung cancer types.

Proteolytic enzymes in proliferation and neoplastic metastases formation.

Metalloproteases, plasminogen urokinase activator, plasmin and cathepsins enable the expansion of neoplastic tumors, leading to metastases formation. They cause neoplastic cells to detach from tumor, facilitate cell movement, implantation and participate in tumor vascularization. The regulation of these processes is accomplished during the synthesis and activation of proenzymes.

Tripeptide methylketones: synthesis and antiplasmin activity.

A series of tripeptide methylketones with C-terminal lysine was obtained via Dakin-West method and tested for their antiplasmin activity with the use of antifibrinolytic and antiamidolytic tests. The tripeptide methylketones has been found to inhibit plasmin, however with much lower potency than respective aldehydes.

Synthesis and activity of N epsilon-aminocaproyl-L-norleucine derivatives.

Five new derivatives of epsilon-aminocaproyl-L-norleucine were synthesized. Antifibrinolytic and anticaseinolytic activities were tested. All tested compounds were inhibitors of plasmin.

Influence of dipeptide derivatives of S-substituted cysteines on the time of fibrinolysis.

Amino acids containing sulphur, dipeptide derivatives of methionine and S-substituted derivatives of cysteine are potent antifibrinolytic agents. The structural moiety of the substances responsible for the effect on the clot formation is not known. Present study was undertaken in order to evaluate the effect of some analogues of dipeptides containing S-substituted derivatives of cysteine with the formula A-Cys(S-X)-Y (where A-amino acid, X-benzyl, butyl, hexyl, nonyl and Y-OH or OMe) on clot dissolution under the antifibrinolytic test conditions.

Synthesis and activity of N alpha-(epsilon-aminocaproyl)-alanines and N epsilon-(epsilon-aminocaproyl)-caproic acid.

Four new dipeptides containing epsilon-aminocaproic acid were synthesized. Antibrinolytic, antiacaseinolytic activity and influence on activation of plasminogen by streptokinase were tested.

The effect of activated alveolar macrophages on experimental lung emphysema development. I. Protease and antiprotease activities in the culture medium of alveolar macrophages.

Aim of the present study was to evaluate cathepsin D, base protease, antiplasmin, antitrypsin and antichymotrypsin activities and protein content in the 24h culture medium of the alveolar macrophages (AM) deriving from the rats treated BCG-vaccine and from rats with papain-induced emphysema. In the culture medium of cells isolated from the rats which were given BCG or papain and BCG+papain we observed an increase of base protease activity and a decrease of cathepsin D activity comparing with control group. Increased antitrypsin activity in BCG and BCG+papain-treated rats and decreased antitrypsin activity in papain-treated rats were observed.

Cathepsin D activity and protein degradation products in organs and blood during experimental declamping shock.

Aortic cross-clamping in dogs for 60 min causes an increase in cathepsin D activity in the kidney, liver, lung, heart, skeletal muscle and the blood serum. It causes no changes in the content of protein and its degradation products of the examined organs, apart from the lungs, where the above parameters are higher. The intensity of the observed changes in the kidney and other organs does not depend on the level of aortic cross-clamping (above or below renal arteries).

Activity of membrane, cytosol and lysosome enzymes in organs and blood serum during declamping shock.

The aim of the study was to evaluate the enzyme activity of cellular membranes (GGT), cytosol (ALT, AST) and lysosome (AP, AcP) in the cytosol, whole homogenate and blood serum during declamping shock, following release of abdominal aorta cross-clamping. The aorta was clamped for 60 minutes. An increase in GGT, AP and AcP activities in the cytosol and whole homogenate of the renal cortex, renal medulla, liver, lung, heart and the skeletal muscle occurs after declamping.

Hemostatic activity of organs in declamping shock.

The aim of the study was to determine the haemostatic components activity of organs in declamping shock. The abdominal aorta was cross-clamped below or above renal arteries. No significant changes in tromboplastic and antithrombin activities were found in the kidney, liver, lung, heart and skeletal muscle.

Disturbances of hemostasis during declamping shock.

The aorta, above or below renal arteries was clamped for 60 minutes, in a canine model. The blood was taken for testing from above the aorta bifurcation before clamping, after 30 minutes of its duration, directly after declamping and every 30 minute during next 4 hours. Irrespective of clamping level, the platelet count, clot retraction and prothrombin consumption do not undergo significant changes.

Cathepsin D activity in the intestinal wall in experimental untreated hemorrhagic shock.

The total cathepsin D activities in the intestinal wall and in venous mesenteric and arterial systemic blood were investigated on the rats in untreated hemorrhagic shock lasting 60 minutes. We observed a decrease in cathepsin D activity in homogenates of respective segments of small and large bowels and an increase in the enzyme activity in blood serum of both origin. The shock resulted in lowering protein concentration in the intestinal wall and its increase in the mesenteric blood.