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Effects of the protease inhibitor leupeptin on proteolytic activities and regeneration of mouse skeletal muscles after exercise injuries
Abstract
Leupeptin, a nontoxic thiol protease inhibitor, has been proposed to have therapeutic use in hereditary muscular dystrophies. The purpose of this study was to characterize the in vivo changes in proteolytic activity of skeletal muscles induced by the repeated administration of leupeptin. Further, whether the modulation of proteolytic capacity by leupeptin affects the repair process of muscle injuries caused by heavy exercise was studied. Leupeptin was administered in mice intraperitoneally at a dose level of 15.5 mg/kg twice a day for 9 days. Leupeptin, known to be an inhibitor of cathepsin B both in vitro and after a single injection in vivo, paradoxically induced an increase of cathepsin B activity in mouse skeletal muscles after repeated administration. In addition, leupeptin administration for 9 days increased the activities of cathepsins C and D, as well as the rate of acid autolysis. The activity of beta-glucuronidase also increased, while those of arylsulfatase, ribonuclease, and alkaline protease were unaffected. No histopathologic changes were observed. At the low dosage used, leupeptin had no effect on the repair process of skeletal muscle after exercise injuries, although several proteolytic processes occur during the regeneration. It is suggested that the increase of acid protease activities in skeletal muscles is an adaptive response to the administration of the proteolytic inhibitor leupeptin and that leupeptin can be administered without prevention or delay of regenerative processes after the onset of myopathic changes.
... Braca et al. [10] , was used for determining the anti-oxidant property. Test solutions in different concentrations (20,40,60,80 and 100) μg/l and 6.34 μM solution of DPPH were prepared in methanol. 100 μl test solution, 100 μl DPPH and 800 μl methanol were taken in a test tube and mixed well. ...
... Dysregulation of proteases has been implicated in cancer development and progression. Leupeptin inhibits several proteases, including serine, cysteine and thiol proteases, thereby affecting multiple pathways involved in cancer [20] . Potential mechanisms through which leupeptin may exert its anti-cancer effects are apoptosis induction, cell cycle arrest, angiogenesis inhibition and metastasis suppression [21] . ...
... These observations and other results from the research community have supported the concept of autophagy inhibition as a potential therapy for muscle wasting conditions (Selsby et al., 2010;Childers et al., 2011;De Palma et al., 2012). Therefore, we treated mdx male mice via intraperitoneal injections of saline (mdx + saline) or the autophagy inhibitor, leupeptin (mdx + LPT) for 5 weeks, as described previously (Salminen, 1984;Haspel et al., 2011;Esteban-Martínez and Boya, 2015), starting at 9 weeks of age in an attempt to preserve mitochondrial degradation. Within quadriceps muscles, mitochondrial DNA copy number was not changed in mdx + LPT vs. salinetreated mdx mice ( Figure 4A). ...
Duchenne muscular dystrophy (DMD) is characterized by rapid wasting of skeletal muscle. Mitochondrial dysfunction is a well-known pathological feature of DMD. However, whether mitochondrial dysfunction occurs before muscle fiber damage in DMD pathology is not well known. Furthermore, the impact upon heterozygous female mdx carriers (mdx/+), who display dystrophin mosaicism, has received little attention. We hypothesized that dystrophin deletion leads to mitochondrial dysfunction, and that this may occur before myofiber necrosis. As a secondary complication to mitochondrial dysfunction, we also hypothesized metabolic abnormalities prior to the onset of muscle damage. In this study, we detected aberrant mitochondrial morphology, reduced cristae number, and large mitochondrial vacuoles from both male and female mdx mice prior to the onset of muscle damage. Furthermore, we systematically characterized mitochondria during disease progression starting before the onset of muscle damage, noting additional changes in mitochondrial DNA copy number and regulators of mitochondrial size. We further detected mild metabolic and mitochondrial impairments in female mdx carrier mice that were exacerbated with high-fat diet feeding. Lastly, inhibition of the strong autophagic program observed in adolescent mdx male mice via administration of the autophagy inhibitor leupeptin did not improve skeletal muscle pathology. These results are in line with previous data and suggest that before the onset of myofiber necrosis, mitochondrial and metabolic abnormalities are present within the mdx mouse.
... Since consistent muscle contractions have been theorized as needed to express CatB, it is possible that greater total training volume or more repetitions per set are needed than were implemented in the present study to facilitate this response. However, Salminen failed to observe acute changes in CatB expression following 8 hours of treadmill running in mice (Salminen 1984). Despite no study to date observing any (Hebisz et al. 2019); however, with RE the acute response has previously been shown to be augmented after 5 weeks (Yarrow et al. 2010) and 6 weeks of training (Church et al. 2016;Quiles et al. 2019) compared to week 1 of training. ...
This study examined if acute multi-joint resistance exercises (RE; back squat, bench press, and deadlift) to volitional failure elicited a postexercise increase in the circulating response of biomarkers associated with neuroprotection. Thirteen males (age: 24.5 ± 3.8 years, body mass: 84.01 ± 15.44 kg, height: 173.43 ± 8.57 cm, training age: 7.1 ± 4.2 years) performed 4 sets to failure at 80% of a 1-repetition maximum on the squat, bench press, and deadlift in successive weeks. The measured biomarkers were brain-derived neurotrophic factor (BDNF), insulin-like growth factor 1 (IGF-1), cathepsin B (CatB), and interleukin 6 (IL-6). Biomarkers were assessed immediately before and 10-min after exercise. There was a main time effect (pre-exercise: 24.00 ± 0.61 to postexercise: 27.38 ± 0.48 ng/mL; p < 0.01) for BDNF with increases in the deadlift (p = 0.01) and bench press (p = 0.01) conditions, but not in the squat condition (p = 0.21). There was a main time effect (pre-exercise: 0.87 ± 0.16 to postexercise: 2.03 ± 0.32 pg/mL; p < 0.01) for IL-6 with a significant increase in the squat (p < 0.01), but not the bench press (p = 0.88) and deadlift conditions (p = 0.24). No main time effect was observed for either CatB (p = 0.62) or IGF-1 (p = 0.56). In summary, acute multi-joint RE increases circulating BDNF. Further, this investigation is the first to report the lack of a transient change of CatB to an acute RE protocol.
NoveltyLow-volume RE to failure can increase BDNF.
Resistance training does not confer an acute Cat B response.
... These observations and other results from the research community have supported the concept of autophagy inhibition as a potential therapy for muscle wasting conditions (39,56,57). Therefore, we treated mdx male mice via intraperitoneal injections of saline (mdx + saline) or the autophagy inhibitor, leupeptin (mdx + LPT) for five weeks, as described previously (58)(59)(60), starting at 9 weeks of age in an attempt to preserve mitochondrial degradation. ...
Background:
Muscular dystrophies are a diverse family of genetic and hereditary disorders manifested primarily by the progressive wasting of skeletal muscle. Duchenne muscular dystrophy (DMD), the most common muscular dystrophy, has no cure, with most treatments seeking to mitigate symptoms. Emerging gene or stem cell therapies hold promise, although widespread clinical adoption may not occur for quite some time. There remains a need for alternative strategies, including drug and lifestyle combination-based therapies, and to continue furthering understanding the physiological effects of dystrophin gene mutations. Mitochondrial dysfunction is well known as a pathological feature of DMD. However, whether mitochondrial dysfunction is a cause or the consequence of DMD is not well known. We hypothesized that dystrophin deletion would lead to mitochondrial and metabolic abnormalities prior to the onset of observable muscle damage.
Methods:
Utilizing the commonly employed muscular dystrophy mouse model, C57BL/10ScSn-Dmdmdx/J (mdx), we sought to determine how the loss of dystrophin effects mitochondria and metabolism in both male and female mdx mice. We also treated male mdx mice with an autophagy inhibitor, leupeptin, to investigate its potentially impact on mdx pathology.
Results:
We detected, via electron microscopy, aberrant mitochondrial morphology, reduced cristae numbers per area of mitochondria, and large mitochondrial vacuoles from both two-week-old male and 24-week-old female mdx carrier mice, prior to the onset of visible muscle fiber damage. We systematically characterized mitochondria during disease progression starting before the onset of gross muscle fiber damage noting changes in mitochondrial DNA copy number and regulators of mitochondrial size. We further detected mild metabolic and mitochondrial impairments in female mdx carrier mice (heterozygous mdx/+) that was exacerbated with high-fat diet feeding. Lastly, we found autophagy inhibition did not improve pathology in mdx male mice.
Conclusions:
Our results suggest that prior to the onset of visible muscle damage, mitochondrial and metabolic abnormalities are present within the mdx mouse.
... Though our study did not replicate Moon et al. (2016) who observed an increase in plasma CTHB following four months of aerobic exercise, our intervention was much shorter in duration. It is worth noting that the animal literature has reported reduced CTHB gene expression in the cardiac muscle of mice following five days of treadmill running (Smuder et al., 2013) and no change in CTHB activity in skeletal muscle following 8 h of exhaustive exercise (Salminen, 1984). However, little is known about the role that CTHB plays in cognitive function in humans. ...
Objective
The benefits of exercise on brain health is well known in aging and psychiatric populations. However, the relationship between habitual exercise in young and healthy adults remains unclear. This study explored the effects an eight-week exercise prescription on cognitive function, brain-derived neurotrophic factor (BDNF) and cathepsin B (CTHB) in young and healthy adults.
Methods
A total of 22 low-active, young and healthy adults were recruited from a local university. A total of 12 participants performed an eight-week exercise prescription and 12 participants served as controls. Cognitive assessments, cardiorespiratory fitness and plasma BDNF and CTHB concentrations were measured at baseline and eight weeks.
Results
Results showed exercise improved cardiorespiratory fitness ( p = 0.044, d = 1.48) with no improvements in cognitive function or no changes in plasma BDNF and CTHB concentrations.
Conclusion
We provide evidence that a short-term course of moderate exercise does not improve cognitive function or change plasma biochemical markers concentrations in young and healthy adults, despite mild improvements in cardiorespiratory fitness. These results suggest that cognitive health may peak during early adulthood leaving little room for improvement throughout this period of the lifespan.
... PADK causes increased proenzyme expression in the transgenic mouse brains, likely related to the paradoxical findings in which the protease inhibitor leupeptin increased cathepsin activities in different tissues. The broad-acting inhibitor, as high as 200 mg/kg, caused significant inhibition of cathepsins B and L, but after clearance of the administered leupeptin not only did cathepsin activities recover to control levels they continued to increase more than two-fold [48][49][50][51]. Note that high levels of PADK administered to mice (60 mg/ kg i.p. in 100% DMSO) or to 3T3 fibroblasts (100 mM applied daily) found no such increases in cathepsin B and L activities [48,51]. ...
Alzheimer's disease (AD) is an age-related neurodegenerative pathology in which defects in proteolytic clearance of amyloid β peptide (Aβ) likely contribute to the progressive nature of the disorder. Lysosomal proteases of the cathepsin family exhibit up-regulation in response to accumulating proteins including Aβ(1-42). Here, the lysosomal modulator Z-Phe-Ala-diazomethylketone (PADK) was used to test whether proteolytic activity can be enhanced to reduce the accumulation events in AD mouse models expressing different levels of Aβ pathology. Systemic PADK injections in APP(SwInd) and APPswe/PS1ΔE9 mice caused 3- to 8-fold increases in cathepsin B protein levels and 3- to 10-fold increases in the enzyme's activity in lysosomal fractions, while neprilysin and insulin-degrading enzyme remained unchanged. Biochemical analyses indicated the modulation predominantly targeted the active mature forms of cathepsin B and markedly changed Rab proteins but not LAMP1, suggesting the involvement of enhanced trafficking. The modulated lysosomal system led to reductions in both Aβ immunostaining as well as Aβ(x-42) sandwich ELISA measures in APP(SwInd) mice of 10-11 months. More extensive Aβ deposition in 20-22-month APPswe/PS1ΔE9 mice was also reduced by PADK. Selective ELISAs found that a corresponding production of the less pathogenic Aβ(1-38) occurs as Aβ(1-42) levels decrease in the mouse models, indicating that PADK treatment leads to Aβ truncation. Associated with Aβ clearance was the elimination of behavioral and synaptic protein deficits evident in the two transgenic models. These findings indicate that pharmacologically-controlled lysosomal modulation reduces Aβ(1-42) accumulation, possibly through intracellular truncation that also influences extracellular deposition, and in turn offsets the defects in synaptic composition and cognitive functions. The selective modulation promotes clearance at different levels of Aβ pathology and provides proof-of-principle for small molecule therapeutic development for AD and possibly other protein accumulation disorders.
... Salminei~ and Vihko (198 1) found that prolonged submaximal exercise caused an increase in cathepsin D activity and exhaustive exercise increased the activity of several acid hydrolases in skeletal muscle (Vihko et al., 1978). Salminen (1984) showed that physical activity to exhaustion induced an increase in both cathepsin C and D activity, but no change for cathepsin B. Exercise has also been found to cause necrotic lesions in mouse skeletal muscle and stimulate the lysoso-ma1 system of the muscle fibers adjacent to the necrotic foci (Vihko et al., 1978;Salminen and Vihko, 1984). ...
Although the proteolytic events accompanying acute and chronic perturbations in striated muscle protein turnover remain to be fully elucidated, the purpose of this paper is to (a) review the chemistry of the nonlysosomal calpain-calpastatin system, and (b) provide evidence for the involvement of a nonlysosomal, calcium-activated neutral protease (calpain) in the response of skeletal muscle protein breakdown to altered nutritional status (diet composition; energy restriction) and increased periods of contractile activity (exercise). In reviewing the literature, it is apparent that calpain is involved in the protein catabolism which accompanies alterations in diet composition and/or energy restriction. The precise mechanism of calpain action remains to be elucidated; however, the role of altered metabolic status contributing to calcium imbalances is discussed relative to increasing protein degradation. Hypotheses for further investigation are provided in regard to identifying the targeting of selected proteins (and organelles) for degradation by calpain.
... Salminei~ and Vihko (198 1) found that prolonged submaximal exercise caused an increase in cathepsin D activity and exhaustive exercise increased the activity of several acid hydrolases in skeletal muscle (Vihko et al., 1978). Salminen (1984) showed that physical activity to exhaustion induced an increase in both cathepsin C and D activity, but no change for cathepsin B. Exercise has also been found to cause necrotic lesions in mouse skeletal muscle and stimulate the lysoso-ma1 system of the muscle fibers adjacent to the necrotic foci (Vihko et al., 1978;Salminen and Vihko, 1984). ...
The purpose of this study was to investigate whether changes in substrate oxidation that are caused by energy restriction influenced muscle function and skeletal muscle calcium stimulated protease activity in female athletes. Endurance athletes were randomly assigned to maintenance energy (100% kcal) or energy restricted (75% kcal) diet treatment groups for 14 days while maintaining regular activity. Body weight significantly decreased in the 75% diet group (-1.7 +/- 0.3 kg; p < .05), while fat oxidation increased (p < .05). Minimal changes in quadriceps function (assessed using the Kin/Com isokinetic dynamometer) were observed following diet treatment, except selected loss of muscle function in the 75% diet group at a movement velocity of 120 deg/s. These results suggest that increased fat oxidation that is induced by an acute energy restriction does not promote loss of general muscle function and activation of calcium-sensitive muscle proteases.
... Dox was added to the animal's drinking water starting on day 2. Animals were sacrificed on day 12, at which time BAL was performed, lung volumes were assessed, histologies were evaluated, and morphometric evaluations were undertaken. Studies evaluating the role(s) of cysteine proteases were performed in an identical fashion except that comparisons were made of the cysteine protease inhibitors E-64 (7.5 mg/kg) and leupeptin (15 mg/kg) and saline vehicle controls (34,35). ...
Cigarette smoke exposure is the major cause of chronic obstructive pulmonary disease (COPD). However, only a minority of smokers develop significant COPD, and patients with asthma or asthma-like airway hyperresponsiveness or eosinophilia experience accelerated loss of lung function after cigarette smoke exposure. Pulmonary inflammation is a characteristic feature of lungs from patients with COPD. Surprisingly, the mediators of this inflammation and their contributions to the pathogenesis and varied natural history of COPD are not well defined. Here we show that IL-13, a critical cytokine in asthma, causes emphysema with enhanced lung volumes and compliance, mucus metaplasia, and inflammation, when inducibly overexpressed in the adult murine lung. MMP-2, -9, -12, -13, and -14 and cathepsins B, S, L, H, and K were induced by IL-13 in this setting. In addition, treatment with MMP or cysteine proteinase antagonists significantly decreased the emphysema and inflammation, but not the mucus in these animals. These studies demonstrate that IL-13 is a potent stimulator of MMP and cathepsin-based proteolytic pathways in the lung. They also demonstrate that IL-13 causes emphysema via a MMP- and cathepsin-dependent mechanism(s) and highlight common mechanisms that may underlie COPD and asthma.
... The regeneration process includes muscle fibre degeneration followed by fibre reconstruction, which occurs through the activation of satellite cells (Schmalbruch, 1976; Schultz et al., 1985). Tissue remodelling, which takes place during muscle regeneration is a complex process involving proteolytic enzymes (Maltz and Oron, 1990; Salminen, 1984). Among proteinases that are believed to be of major importance in the regeneration context, is the calciumactivated neutral cysteine proteinase, referred to as calpain (Murachi, 1984; Melloni and Pontremoli, 1989; Johnson, 1990 ). ...
Skeletal muscle regenerates after injury. Tissue remodelling, which takes place during muscle regeneration, is a complex process involving proteolytic enzymes. It is inferred that micro and milli calpains are involved in the protein turnover and structural adaptation associated with muscle myolysis and reconstruction. Using a whole-crush injured skeletal muscle, we previously have shown that in vivo muscle treatment with synthetic heparan sulfate mimetics, called RGTAs (for ReGeneraTing Agents), greatly accelerates and improves muscle regeneration after crushing. This effect was particularly striking in the case of the slow muscle Soleus that otherwise would be atrophied. Therefore, we used this regeneration model to study milli and micro calpain expressions in the regenerating Soleus muscle and to address the question of a possible effect of RGTAs treatment on calpain levels. Micro and milli calpain contents increased by about five times to culminate at days 7 and 14 after crushing respectively, thus during the phases of fibre reconstruction and reinnervation. After 64 days of regeneration, muscles still displayed higher levels of both calpains than an intact uninjured muscle. Milli calpain detected by immunocytochemistry was shown in the cytoplasm whereas micro calpain was in both nuclei and cytoplasm in small myofibres but appeared almost exclusively in nuclei of more mature fibres. Interestingly, the treatment of muscles with RGTA highly reduced the increase of both milli and micro calpain contents in Soleus regenerating muscles. These results suggest that the improvement of muscle regeneration induced by RGTA may be partly mediated by minimising the consequences of calpain activity.
The lysosomal cysteine proteinase cathepsin L is synthesized in cultured mouse NIH 3T3 cells as a 39 kDa precursor and processed intracellularly into active 29 kDa and 20 kDa + 5 kDa lysosomal forms. Addition to culture media of the peptidyl aldehyde leupeptin, a non-covalent inhibitor of cathepsin L, results in the accumulation of the 20 kDa mature form of the enzyme, resulting in increased activity of cathepsin L as measured in an in vitro assay system in the absence of leupeptin. The more potent irreversible cathepsin L inhibitors benzyloxycarbonyl-Phe-Ala-diazomethane and L-transepoxysuccinyl-L-leucylamino-(4-guanidino)butane, when added to living cells at low concentrations, result in accumulation of all partially processed forms of cathepsin L, especially the 29 kDa form, suggesting that cathepsin L is responsible for its own processing. Exogenous procathepsin L introduced into CHO cells by endocytosis via the mannose 6-phosphate receptor is processed in a manner similar to endogenous procathepsin L. We conclude that the major intracellular pathway for processing of procathepsin L, either endogenous or exogenous, probably requires active cathepsin L.
A large body of evidence has been assembled to indicate the substantial importance of proteolytic processes in various physiological functions. It has recently become clear too that endo-acting peptide bond hydrolases provisionally characterized and classified at present as serine, cysteine, aspartic and metallo together with unknown catalytic mechanism proteinases sometimes act in cascades. They are controlled by natural proteinase inhibitors present in cells and body fluids. In the first part of the present monograph the author was concerned to present an overview on the morphological and physiological approach to localization, surveying reaction principles and methods suitable for visualization of proteolytic enzymes and their natural and synthetic inhibitors. In the second part the roles played by proteinases have been summarized from the point of view of cell biology. The selection of earlier and recent data reviewed on the involvement of proteolysis in the behavior of individual cells reveals that enzymes, whether they be exogeneous or intrinsic, can be effective and sensitive modulators of cellular growth and morphology. There exists a close correlation between malignant growth and degradation of cells. It appears likely that as yet unknown or at least so far inadequately characterized factors that influence the survival or the death of cells may turn out to be proteinases. The causal role of extracellular proteolysis in cancer cell metastases, in stopping cancer cell growth and in cytolysis remains for further investigated. Ovulation, fertilization and implantation are basic biological functions in which proteolytic enzymes play a key role. The emergence of new approaches in reproductive biology and a growing factual basis will inevitably necessitate a reevaluation of present knowledge of proteolytic processes involved. The molecular aspects of intracellular protein catabolism have been discussed in terms of the inhibition of lysosomal and/or non-lysosomal protein breakdown. Peptide and protein hormone biosynthesis and inactivation are still at the centre of interest in cell biology, and a number of proteinases have been implicated in both processes. A number of conjectures partly based on the author's own work have been discussed which suggest the possibility of the involvement of proteolysis in exocytosis and endocytosis. The author's optimistic conclusion is that through the common action of biochemists, cell biologists, cytochemists, and pharmacologists the mystery of cellular proteolysis is beginning to be solved.
Suramin-induced lysosomal storage disease reproduced in the rat was extended to the mouse with the attempt to characterize enzymatically and morphologically heterogeneous responses of various organs to the drug. Suramin administration strikingly decreased (3-6 days afterward) the activity of beta-glucuronidase in all tissues studied (kidney, liver, heart, and skeletal muscle). The enzymatic responses were small in the activities of beta-N-acetyl-glucosaminidase. The activity of arylsulfatase A decreased to a varying degree in mouse tissues, but in rats the activity increased in liver and skeletal muscle. The activity of cathepsin D increased in rat tissues. Suramin induced morphological changes characteristic to lysosomal storage diseases in kidney and liver but not in heart and skeletal muscle of both mice and rats. Kidney was appreciably more susceptible to suramin than liver. The occurrence of lysosomal accumulations, membranous lamellar inclusions, and granular material were most prominent in tubular cells of kidney and in Kupffer cells of liver. These cells also presented intensive Alcian blue staining. Interestingly, the enzymatic and morphological responses did not correlate with each other, which may reflect differences in the regulation of lysosomal functions in various cell types.
We injected 12 New Zealand white rabbits intraperitoneally with 15 mg/kg Leupeptin on alternative days for about 4 months. After 1 week of Leupeptin treatment, they were challenged with purified acetylcholine receptor (AChR) from Torpedo californica in Freund's complete adjuvant. All control animals died within 60 days. Six animals treated with Leupeptin did not develop EAMG in spite of repeated AChR injections. Three animals developed clinical signs of EAMG after 65 days. The clinical course was short in the one that survived and prolonged in the 2 that finally died. All animals (Leupeptin-treated and controls) had circulating anti-AChR antibodies. Among the survivors, titers were slightly lower and EMG repetitive stimulation tests were normal. Leupeptin (0.02-200 mM) did not prevent curaremimetic [3H]toxin binding to AChR in membranes or in solution, nor dissociate AChR-toxin-antibody complexes. Immune response to antigens other than receptor remained intact in Leupeptin-treated animals. Leupeptin was not toxic at the doses given. The mechanism of this protection is not well understood. Leupeptin seems to decelerate the turnover rate of AChR induced by anti-AChR antibodies and/or to decrease the complement-mediated immune attack against the muscle end-plate.
The perception of muscle soreness is probably due to the activation of free nerve endings around muscle fibers. These nerve endings serve as receptors of noxious stimuli associated with muscle damage. Modulation of soreness may take place at the peripheral receptor sites or at a central or spinal level. This multilevel modulation may explain the large intersubject variation in the perception of muscle soreness. The type of exercise that produces the greatest degree of soreness is eccentric exercise, although isometric exercise may also result in soreness. Eccentric exercise has been shown to produce muscle cellular damage and decrements in motor performance as well. Although training is considered to prevent muscle soreness, even trained individuals will become sore following a novel or unaccustomed exercise bout. Thus, training is specific to the type of exercise performed. Our laboratories have shown that the performance of a single exercise bout will have an effect on a subsequent similar bout given up to 6 weeks later. Thus, when a second bout of downhill running was given to subjects 6 weeks after the first bout, with no intervening exercise, less soreness developed, and muscle damage was estimated to be reduced. The explanation for this long-lasting prophylactic or "training effect" is currently under investigation in our laboratories.
Leupeptin is an established, reversible inhibitor of cathepsin B, a lysosomal cysteine proteinase. Yet, in rat fibroblasts as well as in foetal mouse calvaria, we observed an increase of the activity of cathepsin B in homogenates of cells and tissue harvested after culture in the presence of leupeptin. This effect was also seen for other lysosomal hydrolases, namely sphingomyelinase, N-acetyl-beta-glucosaminidase, arylsulphatase A and phospholipase A1 in fibroblasts, and beta-glucuronidase in mouse calvaria. In calvaria, antipain, another reversible cysteine proteinase inhibitor, caused a similar effect, whereas E-64, an irreversible inhibitor, was consistently inhibitory of the cathepsin B activity; yet it also caused an increase of beta-glucuronidase activity. The effect of leupeptin in fibroblasts was dose and time-dependent, required the continuous presence of the inhibitor, and was not dependent from protein synthesis. Actually, addition of cycloheximide caused a severe loss of activity of cathepsin B and of sphingomyelinase. In the presence of both cycloheximide and leupeptin, however, these two activities were retained to a value corresponding to that found in excess in cells cultivated with leupeptin alone. The data therefore suggests that leupeptin exerts the effects described in this paper by preventing the degradation of cathepsin B, sphingomyelinase and probably several other lysosomal hydrolases by cysteine proteinases. We therefore propose that cysteine proteinases play a key role in the control of the steady-state levels of these enzymes in normal conditions.
To investigate the effects of chronic oxidative stress on lysosomal function in trabecular meshwork (TM) cells.
Confluent cultures of porcine TM cells were grown for 2 weeks in physiological (5% O(2)) or hyperoxic conditions (40% O(2)) in the presence or absence of the protease inhibitor leupeptin (10 microM). The following parameters were quantified using the fluorogenic probes indicated within parentheses: autofluorescence, intracellular reactive oxygen species (ROS; H(2)DCFDA), mitochondrial membrane potential (JC-1), mitochondrial content (Mitotracker Red; Invitrogen-Molecular Probes, Eugene, OR), lysosomal content (acridine orange and Lysotracker Red [Invitrogen-Molecular Probes]), autophagic vacuole content (MDC), SA-beta-galactosidase (FDG), and cathepsin activities (z-FR-AMC). Cathepsin levels were quantified by qPCR and Western blot analysis. Ultrastructural analysis was performed by transmission electron microscopy.
Prolonged exposure of porcine TM cells to a hyperoxic environment led to an increase in ROS production and oxidized material. Electron micrographs revealed the cytoplasmic accumulation of lipofuscin-loaded lysosomes. Augmented lysosomal and autophagic vacuole content was confirmed with specific fluorophores. The mRNA and protein levels of several cathepsins were upregulated with oxidative stress. This upregulated expression did not correlate with increased lysosomal activity.
The results indicate that chronic exposure of TM cells to oxidative stress causes the accumulation of nondegradable material within the lysosomal compartment, leading to diminished lysosomal activity. Since the lysosomal system is responsible for the continuous turnover of cellular organelles, impaired lysosomal activity may lead to progressive failure of cellular TM function with age.
Potential usefulness of leupeptin and bestatin for treatment of muscle dystrophy has been suggested by their effects on dystrophy mice. Therefore, we examined the in vivo effects of these inhibitors on catabolic enzymes in muscles and other organs of dystrophy mice. The actions of these inhibitors were more extensive than were expected from their in vitro actions. Leupeptin tended to decrease most of aminopeptidase activities in muscles. Bestatin and forphenicinol tended to decrease not only aminopeptidase activities but also the activities of endopeptidases and glycosidases in muscles. There were significant correlations among the response patterns of each organ to inhibitors. These correlations were higher than those among responses of different organs to an inhibitor. These observations seem to be important in explaining the effects of enzyme inhibitors in whole body.
1.1. Activity of endogenous proteases in the muscles of various vertebrates was measured over a wide pH range. Three distinct regions of autolysis was found at acid, neutral and alkaline pH range. The activity in the latter pH was observed only in rat. mouse, and to a smaller extent in dog and human skeletal muscles.2.2. In the rat muscle the amount and size of peptides formed during autolysis as well as distribution of autolytic activity in the subcellular fractions was determined.3.3. Proteases working at acid pH range and localized in lyzosomes seem to degrade chiefly soluble sarcoplasmic and not myofibrillar proteins.4.4. Autolytic activity at alkaline pH is present in 0–600 g fractions. This protease degrades all myofibrillar proteins. Its activity is inhibited almost completely by diisopropyl-fluorophosphate and soy-bean trypsin inhibitor, partially by l-I-tosylamidophenylethylchloromethylketone and by injection of rats with the preparation “48/80”. All the results indicate that the alkaline protease is a chymotrypsin-like protease of mast-cell origin.5.5. The results suggest that at neutral pH range another proteolytic system is active in muscle. although its activity is rather low.
Adult, untrained NMRI mice were exhausted on a motor-driven treadmill by an intermittent-type running programme. Serial cryostate sections for the staining of NADH-tetrazolium reductase, beta-glucuronidase, beta-N-acetylglucosaminidase, and beta-glycerophosphatase activities and for making hematoxylin-eosin staining were cut from m. quadriceps femoris 1, 2, 3, 5, 7, and 15 days after physical exhaustion. A strong increase in the activities of beta-glucuronidase and beta-N-acetylglucosaminidase was observed 7 days after exhaustion and the activity changes, which were similar for the both glycosidases, were more prominent in the highly oxidative red compared to less oxidative white fibres. Activity granules were more numerous in the perinuclear than the interfibrillar area of red fibres. Spots were arranged like longitudinal chains between myofibrils. Activity in connective tissue was usually observed only in animals exhausted 3--7 days earlier. Simultaneous activity in fibres exceeded that in connective tissue. beta-Glycerophosphatase activity was not, by the method used, seen in histologically "healthy" or normal-looking fibres. In samples taken 2--5 days after exhaustion some degenerating and necrotic fibres were observed. Inflammatory reaction was also observed being at its strongest five days after loading when mononuclear cells were seen inside necrotic fibres. The number of regenerating muscle cells was most abundant 7 days after exhaustion. It is suggested that temporary hypoxia, which accompanies exhaustive physical exercise in skeletal muscle, upsets the energy metabolism and homeostasis of fibres and causes the observed histological and histochemical alterations, which possess features typical of both lethal and sublethal acute cell injury.
The activities of β-glucuronidase, β-N-acetylglucosaminidase, arylsulphatase, ribonuclease,p-nitrophenylphosphatase, and malate dehydrogenase together with protein content were assayed from representative mixed (m. rectus femoris), predominantly red (proximal heads ofm. vastus lateralis, m.v. medius andm. v. intermedius), and predominantly white (distal head ofm. vastus lateralis) muscle homogenates of mice during a two-week period following one single exposure to exhausting intermittent running on a treadmill. The activities of cathepsin D and β-glycerophosphatase were assayed from mixed muscle only. In all three muscle types, particularly in red muscle, the activities of β-glucuronidase, β-N-acetylglucosaminidase, arylsulphatase, and ribonuclease progressively increased between one to five days after the exercise; thereafter the activities began to decrease, being near the control values 15 days after the exercise. In mixed muscle, cathepsin D activity increased. No corresponding changes were observed in the activities of acid phosphatases.
The time course of the activity changes closely resembled that earlier found to be caused by ischaemia in rabbit muscles. It is tentatively concluded that the two treatments, exhaustive exercise and temporary ischaemia, cause similar cell injuries, and that the lysosomal system involved seems to function similarly in the post-stress recovery of the fibres from these injuries.
This study examined the effects of certain protease inhibitors on protein turnover, atrophy, and viability of cultured fetal mouse hearts. Leupeptin (30 μM) diminished net proteolysis by about 50% (P < 0.001) and as a consequence retarded cardiac atrophy. Hearts cultured with leupeptin for 2 days contained 19% more protein than control hearts (P < 0.02). Leupeptin did not alter microscopic appearance, pattern of contraction, rates of protein synthesis and protein leakage, or levels of adenosine triphosphate, lactate dehydrogenase, and creatine kinase. Other protease inhibitors (antipain, pepstatin, chymostatin, and those from soybean and bovine lung) had little or no effect on proteolysis and did not decrease atrophy. Leupeptin inhibits intracellular cathepsin B activity. However, in hearts exposed to leupeptin for 48 h and then washed to remove the inhibitor, cathepsin B level was twice control, whereas three other lysosomal hydrolase activities changed little or not at all. Thus, prolonged exposure to its inhibitor selectively increased tissue content of this lysosomal protease. These findings illustrate the importance of protein breakdown in determining tissue mass. Leupeptin appears useful in studies of protein turnover, in maintaining tissues or organs in culture, and possibly in the therapy of certain diseases.
The protease inhibitor leupeptin decreases protein degradation in rat skeletal and cardiac muscle incubated in vitro, while protein synthesis remains unaltered. Leupeptin also lowers protein breakdown in denervated rat muscles and affected muscles from mice with hereditary muscular dystrophy. Leupeptin may thus be useful in retarding tissue atrophy. Since homogenates of leupeptin-treated muscles had decreased cathepsin B activity, this lysosomal protease may play a role in protein turnover in normal and diseased muscles.
The protease inhibitors leupeptin and pepstatin were used in vivo in genetically dystrophic chickens to determine their effects
on the histological and biochemical changes observed in this disease. These compounds appear to delay the degeneration of
muscle tissue which is characteristic of this disorder and thus may have potential therapeutic value in the treatment of muscular
dystrophy.
The method described in this communication is sensitive and allows the direct determination in crude tissue extracts of the enzyme that hydrolyzes α-N-benzoyl-dl-arginine p-nitroanilde (Bz-Arg-NPhNO2). It may be useful in determining cathepsin B activity in crude enzyme preparations when the reaction mixture is incubated with an inhibitor of trypsinlike enzymes which are also capable of hydrolyzing Bz-Arg-NPhNO2.
The effect of the proteinase inhibitors, pepstatin, leupeptin, and antipain, on dystrophic and normal embryonic chicken muscle cells growing in tissue culture was determined. The three inhibitors are effective against lysosomal cathepsins as well as other proteinases. The inhibitors appeared to delay atrophy and degeneration of dystrophic muscle fibers markedly; the effect on the normal muscle fibers was less striking. Catheptic activity and acidic autolysis are known to increase in the dystrophic chicken. These results support the suggestion that lysosomal proteases are involved, by an unknown mechanism, in the degradative process in dystrophic tissue. Delay in the process of degradation of muscle tissue suggests that these low molecular weight, nontoxic inhibitors offer some prospects as therapeutic agents for treatment of muscular dystrophy and other degenerative muscle diseases.
Leupeptins, leupeptin Pr and leupeptin Ac, strongly inhibit proteolysis by plasmin, trypsin and papain, but do not inhibit proteolysis by α-chymotrypsin. The inhibition is competitive with substrates. The inhibitory effect on esterolysis by plasmin and trypsin is weaker than on proteolysis. The results with derivatives of leupeptins which contain carboxyl or alcohol instead of aldehyde and of di-n-butyl acetals of leupeptins indicate that the free aldehyde group plays a role in the activities. Leupeptins are absorbed orally and at least about 25 % is excreted in urine. Oral administration of leupeptins exhibited an anti-inflammatory effect on edema. Leupeptins inhibited thrombokinase reaction and coagulation of blood of human and rabbit. Type of inhibition was different from heparin. Coagulation of blood of rats and dogs are not inhibited. The effects of leupeptins on thrombokinase, thrombin, plasmin, trypsin, papain, kallikrein and α-chymotrypsin were compared with those of e-aminocaproic acid, trans-4-aminomethylcyclohexanecarboxylic acid, soybean trypsin inhibitor and trasylol.
We have investigated the effect of some inhibitors on the breakdown of proteins at 38 °C and pH 5 by highly purified Triton WR-1339-filled lysosomes obtained from rat liver.When cathepsin D (EC 3.4.23.5) was completely inhibited by pepstatin, the rate of degradation of native serum albumin was not and that of carboxymethylated serum albumin only slightly influenced. Leupeptin or antipain, used at a concentration which resulted in more than 95% inhibition of cathepsin B1 (EC 3.4.22.1) activity, strongly decreased the rate of degradation of native albumin, but affected the degradation of carboxymethylated albumin or hemoglobin only to a slight degree. Leupeptin or antipain in combination with pepstatin inhibited proteolysis of the three proteins strongly, but not completely. Omission of Cl−, essential for cathepsin C (EC 3.4.14.1) activity, from incubation mixtures containing pepstatin, leupeptin and antipain had only a slight additional inhibitory effect. Addition of both pepstatin and monoiodoacetate fully suppressed the degradation of these protein substrates.Some of the experiments have also been done using Triton WR-1339-filled lysosomes obtained from purified hepatocytes.Our results indicate that (1) cathepsin D is not essential for degradation of proteins by liver lysosomal enzymes: (2) cathepsin B1 (or possibly another leupeptin-inhibited thiol protease) is the most important enzyme in the degradation of native albumin; (3) thiol enzymes, other than cathepsin B1, C or D, are involved in lysosomal protein degradation; (4) hepatocyte lysosomes contain a set of proteases sufficient for rapid and extensive degradation of albumin.
Strenuous physical exercise induces necrosis of skeletal muscle fibers and increases lysosomal enzyme activities in surviving muscle fibers. This study examines the ultrastructural basis of the stimulation of the lysosomal system in mouse vastus medialis muscle during the appearance and repair of exercise-induced (9 h of running) injuries. Necrotic fibers appeared the day after exercise and an inflammatory response with the replacement of necrotic fibers by phagocytes was highest 2-3 days after exertion. Ultrastructural study of surviving muscle fibers revealed numerous autophagic vacuoles, residual bodies, and spheromembranous structures at the periphery of myofibers, especially in fibers adjacent to necrotic fibers. The autophagic response was most prominent between 2 and 7 days after exertion. Autophagic vacuoles with double or single limiting membranes contained mitochondria at various stages of degradation. Vacuolar and multilamellar structures were also observed in regenerating muscle fibers. The structure of injured skeletal muscle fibers returned to normal within 2 weeks. It is proposed that increased autophagic activity could be related to the breakdown of cellular constituents of surviving muscle fibers to provide structural elements for regenerating muscle fibers.
The administration to rats of leupeptin produced prominent numbers of enlarged and irregularly shaped autolysosomes in hepatocytes. Percoll density equilibration of crude lysosomal fractions from rat livers showed that most lysosomal enzyme migrated form normal lysosomal density fractions toward higher density fractions within 30 min after leupeptin injection. The denser particles were ultrastructurally identified with the autolysosomes. In analysis by differential centrifugation of the liver homogenates, lysosomal enzymes became sedimentable with particles of greater sedimentation rate within 30 min after leupeptin injection. These changes in physical properties of lysosomes reverted to normal after 24 h, and concomitant disappearance of autolysosomes in hepatocytes was observed by electron microscopy. When the time course of distribution of leupeptin in subcellular fractions was analyzed, particle-bound leupeptin shifted with time to larger particle fractions in parallel with the shift of lysosomal enzyme distribution. Upon injection with leupeptin, cathepsin B activity was inhibited by more than 80% for about 3 h and was gradually restored to a normal level after 24 h. Leupeptin treatment caused marked delay of the degradation of endocytosed FITC-labeled asialofetuin in hepatic lysosomes, and its inhibitory effect lasted for over 9 h after the injection. It was suggested that autophagy is probably a normal process of protein degradation in hepatocytes, and because of a retarded digestion of sequestered materials, autolysosomes persisted for a long period and made up the majority of the lysosome population in the leupeptin-treated cells.
beta-Galactosidase was normalized by a serine-thiol protease inhibitor, leupeptin with concentration of 10 micrograms/ml in cultured skin fibroblasts from patients with beta-galactosidase-alpha-neuraminidase deficiency (beta-Gal-/Neu-). The induction of this enzyme was not observed in normal cells. Because the enzymic activity of cathepsin B1 increased significantly both in beta-Gal-/Neu- and normal cells by leupeptin loading, the restoration of beta-galactosidase in beta-Gal-/Neu- cells can not be explained by the theory that leupeptin inhibited intracellular degradation of beta-galactosidase molecules. The effects of leupeptin and sucrose on lysosomal hydrolase induction were compared.
Specific inhibitors of three lysosomal proteinases are present in the cytosolic and lysosomal compartments of rabbit liver. The cytosolic inhibitors, purified by chromatography on DEAE-Trisacryl and Sephadex G-75, show specificities toward cathepsin M, cathepsins B and L, and fructose 1,6-bisphosphatase converting enzyme (CE), respectively, and are designated IM, IB/L, and ICE. Inhibitors with similar specificities have been isolated from the intralysosomal compartment. Two of these inhibitors, IM and ICE, are also present in the lysosomal membranes. The lysosomal distribution parallels that of the respective proteinases. The inhibitors are polypeptides with molecular weights of 5,000-10,000 for the two forms of IB/L, 12,500 for IM, and 10,000-40,000 for the ICE species.
Leupeptin is a potent inhibitor of cathepsin B in vitro and is presumed to act in a similar manner in vivo. It is currently being used in several laboratories to examine the role of lysosomal proteinases such as cathepsin B in mouse models of muscular dystrophy. This report clearly demonstrates that leupeptin in adequate concentrations in vivo, is a potent stimulator of cathepsin B activity in striated muscle, heart, liver and kidney of the mouse. This paradoxical effect indicates that care is required in the interpretation of the results of the use of leupeptin as a cathepsin B inhibitor in vivo and that its use as an antiprotease for therapeutic purposes may be limited. Studies on CBZ-Phe-Ala-CHN2 demonstrated that this agent, when administered in vivo, inhibited Cathepsin B in the tissues assayed.
Mice with genetic muscular dystrophy were treated with intraperitoneal injections of the proteinase inhibitor leupeptin, beginning before the onset of weakness. A significant number of the treated animals failed to develop histological evidence of dystrophy, compared with controls. Leupeptin treatment prevented (or delayed) the onset of muscular dystrophy in this experiment.
Male NMRI-mice, aged 3, 6, 9, and 12 months, were made to run for a period of 4 4 at a speed of 13.5 m/min on a motor-driven treadmill, 5 days after exertion, selected enzymatic estimates of acid and alkaline proteolytic as well as energy metabolic capacities were analyzed from the cardiac muscle and from the red and white parts of m. quadriceps femoris (MQF). The activities of alkaline and myofibrillar proteases increased most considerably in skeletal muscles with age. Cathepsin D and beta-glucuronidase activities were less affected in both muscles. Prolonged running increased the activities of cathepsin D, dipeptidyl aminopeptidase I and beta-glucuronidase in the white and, especially in the red part of MQF. This stimulation of acid hydrolytic capacity was more prominent at the ages of 3 and 6 months than in the older animals. The estimates of alkaline proteolytic or energy metabolic capacities were not affected by prolonged running. In cardiac muscle, no significant changes were recorded in acid hydrolytic or energy metabolic capacity. Histological observation showed no necrosis or other pathological phenomena in the proximal part of m. rectus femoris after excretion. We suggest that the increased acid proteolytic capacity is involved in subcellular regenerative processes of skeletal muscle fibres. The smaller lysosomal response of older mice may indicate a reduced potential capacity for cellular repair.
The proteinase inhibitors leupeptin and chymostatin are inactivated by preparations of perfused mouse liver. The inhibitors are degraded maximally at neutral or alkaline pH values. The inactivating enzymes are inhibited by Dip-F and pms-F but not by EDTA, 1,10 phenanthroline or iodoacetic acid. The significance of these results is discussed in terms of the potential of the inhibitors as antiproteolytic drugs.
Leupeptin was found to be a slow, tight-binding inhibitor of cathepsin B from human spleen and rabbit liver. During the enzyme-catalyzed reaction in the presence of inhibitor a concentration-dependent transient state, lasting several minutes, preceded the attainment of the steady state and was characterized by a concave upward or a concave downward lag phase depending on whether the enzyme had been preincubated with the inhibitor or not, respectively. From the pre-steady-state phase of the curves both k on and k off for the formation of the enzyme-inhibitor complex could be calculated. Ki, as the ratio k off/k on, was in good agreement with the inhibition constant obtained using a steady-state treatment. k on was 1.8 X 10(5) M-1 s-1 and 2.0 X 10(5) M-1 s-1 for the human and rabbit enzyme, respectively and the slowness of the binding process fitted into the general concept of enzyme hysteresis. The activation of the essential cysteine residue of cathepsin B by dithiothreitol was also a very slow process characterized by a second-order rate constant of 4.1 M-1 s-1. The kinetic features of leupeptin binding allow the prediction of the possible efficiency of this inhibitor on cathepsin B in vivo. It is shown that in order for leupeptin to be a physiologically significant inhibitor of cathepsin B, its concentration at the target site must exceed 10 microM, at least. This contrasts with the predictions drawn from the value of Ki (approximately 5 nM), which would suggest an effective inhibition of the enzyme already at a concentration of 0.05 microM.
Addition of pepstatin, an inhibitor of acid protease, to 2-day cultures of rat hepatocytes rapidly inhibited the activity to hydrolyze hemoglobin (Hb), but did not affect the activity to hydrolyze α-N-benzoyl-dl-arginine-β-naphthylamide (BANA). On the other hand, addition of leupeptin, an inhibitor of thiol protease, inhibited the activity of BANA hydrolase and caused a sixfold increase in the activity of Hb hydrolase within 1 day. Neither protease inhibitor affected the rate of protein synthesis. Release of amino acids from hepatocytes into Hanks' salt solution was measured by the ninhydrin method. Pepstatin inhibited the release only 15% within 2 days, but leupeptin inhibited it 65% within 10 h. These two inhibitors had additive inhibitory effects on the release, suggesting that they inhibit the degradations of different groups of proteins. The inhibitory effect of leupeptin gradually decreased after 10 h, which is consistent with the observed induction of a protease activity mentioned above. A preferential involvement of leupeptin-sensitive protease in the degradation of proteins with longer half-lives was suggested from studies on [14C]leucine release from hepatocytes prelabeled for 30 h. On the other hand, the two inhibitors had similar effects on the release of [14C]leucine from hepatocytes labeled for only 1 h. Their inhibitory effects were again additive, but there was no reduction in the inhibition by leupeptin on prolonged incubation, suggesting that proteins with short half-lives were not substrates for the induced protease. These results suggest that in hepatocytes, proteins with longer half-lives are degraded more by cathepsin B than by cathepsin D, while those with short half-lives are degraded equally by these two proteases.
Induction of P-galactosidase in P-galactosidase-a-neuraminidase deficiency: Effects of leupeptin and sucrose Reduction of protein degradation and atrophy in cultured fetal mouse hearts by leupeptin
- T Kato
- S Okada
- T Yutaka
- Yabuuchi
- P Libby
- Js Ingwall
- Goldberg
Kato T, Okada S, Yutaka T, Yabuuchi H: Induction of P-galactosidase in P-galactosidase-a-neuraminidase deficiency: Effects of leupeptin and sucrose. Biochem Int 1983, 6:267-273 14. Libby P, Ingwall JS, Goldberg AL: Reduction of protein degradation and atrophy in cultured fetal mouse hearts by leupeptin. Am J Physiol 1979, 237:E35-E39
Effects of leupeptin and pepstatin on protein turnover in adult rat hepato-70 SALMINEN AJP * October 1984 cytes in primary culture Salminen A, Vihko V: Effects of age and prolonged running on proteolytic capacity in mouse cardiac and skeletal muscles
- K Tanaka
- N Ikegaki
- Ichihara
Tanaka K, Ikegaki N, Ichihara A: Effects of leupeptin and pepstatin on protein turnover in adult rat hepato-70 SALMINEN AJP * October 1984 cytes in primary culture. Arch Biochem Biophys 1981, 208:296-304 16. Salminen A, Vihko V: Effects of age and prolonged running on proteolytic capacity in mouse cardiac and skeletal muscles. Acta Physiol Scand 1981, 112:89-95
Endogenous inhibitors of lysosomal proteinases Beynon RJ, Brown CP, Butler PE: The inactivation of Streptomyces-derived proteinase inhibitors by mammalian tissue preparations
- S Pontremoli
- E Melloni
- F Salamino
- B Sparatore
- M Michetti
- Horecker
Pontremoli S, Melloni E, Salamino F, Sparatore B, Michetti M, Horecker BL: Endogenous inhibitors of lysosomal proteinases. Proc Natl Acad Sci USA 1983, 80:1261-1264 22. Beynon RJ, Brown CP, Butler PE: The inactivation of Streptomyces-derived proteinase inhibitors by mammalian tissue preparations. Acta Biol Med Ger 1981, 40:1539-1546
Activities ofproteinase inhibi-tors ofmicrobialorigin,Proteinases in Mammalian Cells and Tissues
- Umezawah
UmezawaH, AoyagiT: Activities ofproteinase inhibi-tors ofmicrobialorigin,Proteinases in Mammalian Cells and Tissues. EditedbyAJ Barrett.Amsterdam, North-Holland, 1977, pp637-662
Role of individualcathepsinsinlysosomal protein digestionas testedby specificinhibitors
- Huismanw
- L Lanting
- Hj Doddema
- Jmw Bouma
- Gruber
HuismanW, Lanting L, Doddema HJ, Bouma JMW, Gruber M: Role of individualcathepsinsinlysosomal protein digestionas testedby specificinhibitors. Biochim BiophysActa 1974, 370:297-30
The slowtight-bindinginhi-bition ofcathepsinBby leupeptin.Ahystereticeffect
- Baicia
- Gyger
BaiciA, Gyger-MarazziM: The slowtight-bindinginhi-bition ofcathepsinBby leupeptin.Ahystereticeffect. Eur J Biochem 1982, 129:33-4
Biologicalactivi-ties ofleupeptins
- T Aoyagi
- S Miyata
- Nanbom
- F Kojima
- Matsuzakim
- Ishizukam
- Takeuchit
- Umezawa
Aoyagi T, Miyata S,NanboM, Kojima F,MatsuzakiM, IshizukaM, TakeuchiT,Umezawa H:Biologicalactivi-ties ofleupeptins.J Antibiot(Tokyo) 1969,22:1815-182
cytes in primary culture
cytes in primary culture. Arch Biochem Biophys 1981,
208:296-304