Inhibitory Effect Of Leupeptin Research Articles

Inhibitory effect of leupeptin on the intracellular maturation of lysosomal cathepsin L in primary cultures of rat hepatocytes.

To investigate the intracellular processing event for lysosomal cathespin L, we examined the effect of leupeptin, a non-covalent cysteine proteinase inhibitor, on the intracellular processing kinetics of cathepsin L as analyzed by pulse-chase experiments in vivo with [35S]methionine in primary cultures of rat hepatocytes. This revealed that cathepsin L was initially synthesized as proenzyme of molecular weight 39 kDa and the proenzyme was subsequently processed to the mature form of the enzyme, 30 and 25 kDa. In the leupeptin-treated cells, the proteolytic conversion of cellular procathepsin L, of molecular weight 39 kDa, to the mature enzyme was significantly inhibited and considerable amounts of proenzyme were found in the cell after 8 h chase periods. Furthermore, the subcellular fractionation experiment demonstrated that the intracellular processing of procathepsin L in the high density lysosomal fraction was significantly inhibited and that considerable amounts of the procathepsin L form were still observed in the dense lysosomal fraction after a 2 h chase period. These results suggest that leupeptin treatment caused significant inhibition of the intracellular maturation of cathepsin L. These findings show that cysteine proteinase plays an important role in the intracellular proteolytic processing and activation of lysosomal cathepsin L in vivo and that this processing event occurs within the lysosomes.

Leupeptin inhibits phospholipases D and C activation in rat hepatocytes

The relationship between phospholipase D and C activation was studied in intact rat hepatocytes and rat liver plasma membranes. In intact hepatocytes, in the presence of ethanol, vasopressin, phorbol ester, and calcium independently stimulated phosphatidylethanol (PETH) formation, a specific marker of phospholipase D activity. Leupeptin (10–1500 μM) inhibited PETH formation induced by vasopressin, but was ineffective in response to phorbol ester or calcium. Leupeptin also inhibited the formation of inositol phosphates in intact cells in response to vasopressin. In liver plasma membranes, GTP[S] induced the production of phosphatidic acid and, in the presence of ethanol, PETH. Plasma membrane-associated phospholipase D did not require calcium and was insensitive to protein kinase C inhibitors. Leupeptin inhibited PETH formation in response to GTP[S]. The inhibition by leupeptin could be overcome by increasing the concentration of GTP[S]. In plasma membranes, the inhibitory effects of leupeptin on phospholipase D occurred at doses that far exceed those required to maximally inhibit proteolysis. These data highlight a central role for phospholipase C in the activation of phospholipase D, and a minor role for a direct G-protein activation. The findings also demonstrate a novel use of leupeptin as an inhibitor of phospholipases D and C perhaps at the level of a G protein.

Effect of leupeptin on the lateral mobility of proteins in the plasma membrane of hepatocytes of C57BL/6 mice: FRAP studies on liver smears

Four groups of male C57BL/6 mice (ages 2, 3.8, 11.8–12.5 and 24 months) were administered leupeptin at 5 mg/100 g body weight/day via intraperitoneally implanted osmotic minipumps. Both untreated and saline-treated mice served as controls. The time and dose-dependence of the effects of leupeptin on the lateral mobility of proteins in hepatocyte surface membranes were analyzed first in mice treated for 2, 5, 8, 11 and 14 days, respectively, by using the fluorescence recovery after photobleaching (FRAP) technique. The age dependence of the response to this treatment was also studied after 14 days of treatment in mice of various ages. The average lateral diffusion constant (D) and the fractional recovery (FR) were measured, and D × FR calculated. Leupeptin treatment at a daily dose of 5 mg/100g for 2 weeks increased plasma glutamic-pyruvlc transaminase levels 2-fold in all age groups. Leupeptin treatment caused a linear increase of D and a decrease of FR with respect to the duration of the treatment in adult mice. In all age groups about 30% of the membrane proteins became immobile after 14 days of treatment, whereas the still mobile fraction displayed a large increase of D. The values of D × FR in the leupeptin-treated groups were slightly higher than those in the untreated mice until the age of 15 months but decreased after this age. The results are interpreted in terms of the known inhibitory effect of leupeptin on cytoplasmic and lysosomal thiol proteases, causing a general slowing down of the protein turnover and, specifically, of proteins in the hepatocyte membrane.

Biosynthesis of Lipoprotein Lipase in Cultured Mouse Adipocytes: II. Processing, subunit assembly, and intracellular transport

Abstract The biosynthesis and turnover of lipoprotein lipase (LPL) have been investigated in adipose 3T3-F442A cells labeled with [35S]methionine. Pulse-chase experiments, endo-beta-N-acetylglucosaminidase H treatment, and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis have indicated that LPL is synthesized in the endoplasmic reticulum as a glycoprotein of Mr = 55,500 bearing two N-oligosaccharide side chains of the high mannose-type. This precursor form of LPL is transported within 10 min to the Golgi apparatus, and this event is accompanied by the formation of a mature species of Mr = 58,000. Treatment of the Mr = 58,000 species with glycopeptidase F yielded a Mr = 51,000 protein similar to that observed after treatment of the Mr = 55,500 precursor form or after inhibition of N-glycosylation in tunicamycin-treated cells. The precursor form of LPL of Mr = 55,500 does not accumulate in the cells since, after a labeling period of 2 h, only the Mr = 58,000 species is detected. It is shown that only 20% of the newly synthesized molecules of Mr = 58,000 are constitutively secreted, whereas 80% are degraded, most likely in lysosomes, as indicated by the inhibitory effect of leupeptin upon the degradation process. Under heparin stimulation, quantitative secretion of the mature form of LPL takes place whereas the intracellular degradation is arrested. Heparin is able to mobilize intracellular LPL without changing the rate of LPL export from the endoplasmic reticulum to the cell surface. Sucrose gradient centrifugation of the material from intracellular cisternae shows that the Mr = 55,500 precursor form is present as a monomer (s = 4.1 S), whereas the Mr = 58,000 mature form is present as a homodimer (s = 6.8 S) to which LPL activity is associated. The results are interpreted as LPL being transiently stored under a dimeric form before its degradation. A sorting process of LPL in the Golgi apparatus, followed by its entry either mainly in a regulated pathway or in a constitutive pathway, is proposed.

Leupeptin does not affect the normal signal transduction mechanism in platelets

Calpains are Ca 2+-dependent serine proteases that can regulate protein kinase C-mediated cellular events by cleaving the membrane-bound native enzyme to yield an activated cytosolic fragment. Inhibition of calpain by leupeptin may cause enhancement or inhibition of cellular functions depending on the nature of the protein kinase C reaction involved. We have studied the effects of leupeptin on platelet responses (aggregation, secretion, thromboxane B 2 formation and intracellular Ca 2+ and pH changes) induced by either thrombin, collagen or phorbol 12-myristate 13-acetate (TPA), which are known to activate protein kinase C by different mechanisms. Only thrombin-induced responses were inhibited by leupeptin. This suggests that the inhibitory effect of leupeptin is not due to antagonism of calpain in this system, but to direct interference with the proteolytic effect of thrombin.

Competitive inhibition of [3H]dexamethasone binding to mammary glucocorticoid receptor by leupeptin.

The inhibitory effect of leupeptin on [3H]dexamethasone binding to the glucocorticoid receptor from lactating goat mammary cytosol has been studied. Leupeptin (10 mM) caused a significant (about 35%) inhibition of [3H]dexamethasone binding to glucocorticoid receptor. Binding inhibition is further increased following filtration of unlabeled cytosolic receptor through a Bio-Gel A 0.5-m column. Binding inhibition was partially reversed by monothioglycerol at 10 mM concentration. A double reciprocal plot revealed that leupeptin appears to be a competitive inhibitor of [3H]dexamethasone binding to the glucocorticoid receptor. Low salt sucrose density gradient centrifugation revealed that the leupeptin-treated sample formed a slightly larger (approximately 9 S) receptor complex (leupeptin-free complex sediments at 8 S).

Differential effects of proteinase inhibitors and amines on the lysosomal and non-lysosomal pathways of protein degradation in isolated rat hepatocytes

Ammonia, which like other lysosomotropic amines inhibits protein degradation in isolated rat hepatocytes by 70–80%, was utilized as a diagnostic tool to distinguish between the relative effects of various proteinase inhibitors on the lysosomal and non-lysosomal pathways of intracellular protein degradation. Leupeptin was found to inhibit lysosomal protein degradation by 80–85%, and non-lysosomal degradation by about 15%. Antipain had a similar, but somewhat weaker effect. Pepstain, bestatin and aprotinin (Traysylol) produced minor inhibitory effects (possibly on both degradation, pathways), whereas bacitracin and soybean trypsin inhibitor wre ineffective. Chymostatin inhibited lysosomal protein degradation by about 45%, whereas the non-lysosomal pathway was inhibited by more than 50%. Chymostatin was unique among the inhibitors tested in causing such a pronounced effect on non-lysosomal protein degradation, and appeared to selectively inhibit the energy-dependent portion of this pathway. The effects of the various inhibitors were additive to the extent expected on the basis of their kwown actions on lysosomal and non-lysosomal protein degradation. Thus, a combination of methylamine, leupeptine and chymostatin inhibited overall protein degradation by about 90%, resulting in a substantial improvement of the cellular nitrogen balance. The degradation inhibitors caused a partial inhibition of protein synthesis, apparently mainly by shutting down the supply of amino acids from the lysosome. The inhibitory effects of leupeptin and antipain were completely reversed by amino acid addition, whereas some inhibition remained in the case of chymostatin and the lysosomotropic amines, possibly reflecting a certain nonspecific toxicity.

Involvement of proteolytic activity in early events in lymphocyte transformation by phytohemagglutinin

The stimulation by phytohemagglutinin (PHA) of DNA synthesis in cultured blood lymphocytes of guinea pig was markedly inhibited by addition of leupeptin, a well-characterized, powerful protease inhibitor of tripeptide nature. About 30 to 40 per cent inhibition was observed at 40 μg/ml of leupeptin when leupeptin was added 30 min prior to or together with PHA. Per cent inhibition by the appropriate amount of leupeptin was proportional to the amount of PHA added in the range of 0.6 to 3.0 μg of PHA at which the per cent inhibition reached maximum. This inhibitory effect of leupeptin on PHA stimulation was abolished when the lymphocytes were preincubated with PHA for more than 10 min before addition of leupeptin or preincubated with leupeptin for more than 60 min prior to PHA addition.