Chymotrypsin-like Proteolytic Activity Research Articles

Variants in KLK11, affecting signal peptide cleavage of kallikrein-related peptidase 11, cause an autosomal-dominant cornification disorder.

Mendelian disorders of cornification (MeDOC) are a group of heterogeneous genodermatoses with different genetic bases. The pathogenesis of a substantial group of MeDOC remains to be elucidated. To identify a new causative gene and the pathogenesis of a previously undescribed autosomal-dominant cornification disorder. Whole-exome sequencing was performed in three families with the novel cornification disorder to identify the disease-causing variants. As the variants were located around the signal peptide (SP) cleavage site of a kallikrein-related peptidase, SP cleavage, subcellular localization and extracellular secretion of the variants were evaluated in eukaryotic overexpression systems by Western blotting or immunocytochemistry. Then the trypsin-like and chymotrypsin-like proteolytic activity of the peptidase and degradation of its catalytic substrate were assayed using the patients' stratum corneum (SC) samples. The morphology of the lamellar bodies and corneodesmosomes (CDs) in the patients' SC was ultrastructurally examined. A mouse model harbouring the equivalent variant was constructed and evaluated histologically. We identified two heterozygous variants affecting Gly50 in kallikrein-related peptidase (KLK)11 in a familial case and two sporadic cases with the new disorder, which is characterized by early-onset ichthyosiform erythroderma or erythrokeratoderma. KLK11 belongs to the family of kallikrein-related peptidases participating in skin desquamation by decomposing CDs, a process essential for shedding of the SC. In vitro experiments demonstrated that the variants perturbed the SP cleavage of KLK11, leading to subcellular mislocalization and impaired extracellular secretion of the KLK11 Gly50Glu variant. Both trypsin-like and chymotrypsin-like proteolytic activities were significantly decreased in the patients' SC samples. Reduced proteolysis of desmoglein 1 and delayed degeneration of CDs were detected in patients' SC, indicating delayed skin desquamation. Consistently, the patients showed a thickened, dense SC, indicating abnormal skin desquamation. Mice harbouring the homozygous c.131G>A (p.Gly44Glu) Klk11 variant, which is equivalent to KLK11 c.149G>A (p.Gly50Glu) in humans, exhibited hyperkeratosis and abnormal desquamation, partially recapitulating the phenotype. We provide evidence that variants at Gly50 affecting the SP cleavage of KLK11 cause a new autosomal-dominant cornification disorder with abnormal desquamation. Our findings highlight the essential role of KLKs in maintaining homeostasis of skin keratinization and desquamation.

Cytotoxicity and Proteasome Inhibition by Alkaloid Extract from Murraya koenigii Leaves in Breast Cancer Cells-Molecular Docking Studies.

Murraya koenigii (curry tree) leaves are rich in bioactive compounds such as flavonoids, alkaloids, and coumarins. Alkaloids from M. koenigii leaves have antianalgesic, antiulcerogenic, antiobesity, and antitumor activities. In this study, we tested the cytotoxic and proteasome-inhibitory potential of a total alkaloid extract (TAE) from M. koenigii leaves in the breast cancer cell line MDA-MB-231. The TAE decreased cell viability with an IC50 of 14.4 μg/mL and altered growth kinetics of breast cancer cells. TAE (32 μg/mL) arrested cells (35%) in the "S" phase of the cell cycle and induced apoptosis. The 26S proteasome, a multicatalytic protease complex, promotes tumor cell proliferation and protects tumor cells from apoptosis. The TAE and mahanine, a carbazole alkaloid present in M. koenigii leaves, preferentially inhibited the trypsin-like, but not the chymotrypsin-like proteolytic activity of the proteasome with an IC50 of 162 μg/mL and 287 μM, respectively. In silico analysis of 26 compounds from M. koenigii leaves revealed significant docking scores for mahanine and two other carbazole alkaloids with the β2 and β5 subunits of the catalytic 20S proteasome. Taken together, this study demonstrates that inhibition of the proteasome is an important biological activity of M. koenigii alkaloids, which may lead to cancer cell death.

Proteolytic activity of prostate-specific antigen (PSA) towards protein substrates and effect of peptides stimulating PSA activity.

Prostate-specific antigen (PSA or kallikrein-related peptidase-3, KLK3) exerts chymotrypsin-like proteolytic activity. The main biological function of PSA is the liquefaction of the clot formed after ejaculation by cleavage of semenogelins I and II in seminal fluid. PSA also cleaves several other substrates, which may explain its putative functions in prostate cancer and its antiangiogenic activity. We compared the proteolytic efficiency of PSA towards several protein and peptide substrates and studied the effect of peptides stimulating the activity of PSA with these substrates. An endothelial cell tube formation model was used to analyze the effect of PSA-degraded protein fragments on angiogenesis. We showed that PSA degrades semenogelins I and II much more efficiently than other previously identified protein substrates, e.g., fibronectin, galectin-3 and IGFBP-3. We identified nidogen-1 as a new substrate for PSA. Peptides B2 and C4 that stimulate the activity of PSA towards small peptide substrates also enhanced the proteolytic activity of PSA towards protein substrates. Nidogen-1, galectin-3 or their fragments produced by PSA did not have any effect on endothelial cell tube formation. Although PSA cleaves several other protein substrates, in addition to semenogelins, the physiological importance of this activity remains speculative. The PSA levels in prostate are very high, but several other highly active proteases, such as hK2 and trypsin, are also expressed in the prostate and may cleave protein substrates that are weakly cleaved by PSA.

Reactive oxygen species play a role in muscle wasting during thyrotoxicosis.

The role of reactive oxygen species (ROS) in muscle protein hydrolysis and protein oxidation in thyrotoxicosis has not been explored. This study indicates that ROS play a role in skeletal muscle wasting pathways in thyrotoxicosis. Two experimental groups (rats) were treated for 5days with either 3,3',5-triiodothyronine (HT) or HT with α-tocopherol (HT + αT). Two controls were used, vehicle (Control) and control treated with αT (Control + αT). Serum T3, peritoneal fat, serum glycerol, muscle and body weight, temperature, mitochondrial metabolism (cytochrome c oxidase activity), oxidative stress parameters and proteolytic activities were examined. High body temperature induced by HT returned to normal when animals were treated with αT, although total body and muscle weight did not. An increase in lipolysis was observed in the HT + αT group, as peritoneal fat decreased significantly together with an increase in serum glycerol. GSH, GSSG and total radical-trapping antioxidant parameter (TRAP) decreased and catalase activity increased in the HT group. The glutathione redox ratio was higher in HT + αT than in both HT and Control + αT groups. Carbonyl proteins, AOPP, mitochondrial and chymotrypsin-like proteolytic activities were higher in the HT group than in the Control. HT treatment with αT restored mitochondrial metabolism, TRAP, carbonyl protein, chymotrypsin-like activity and AOPP to the level as that of the Control + αT. Calpain activity was lower in the HT + αT group than in HT and Control + αT and superoxide dismutase (SOD) activity was higher in the HT + αT group than in the Control + αT. Although αT did not reverse muscle loss, ROS was involved in proteolysis to some degree.

(19)F NMR monitoring of the eukaryotic 20S proteasome chymotrypsin-like activity: an investigative tool for studying allosteric regulation.

The proteasome displays three distinct proteolytic activities. Currently, proteasome inhibitors are evaluated using specific fluorescent substrates for each of the individual active sites. However, the photophysical properties of the commonly used fluorophores are similar and thus, the simultaneous monitoring of the three proteolytic activities is not possible. We have developed a bimodal fluorescent fluorinated substrate as a novel tool to study the chymotrypsin-like (ChT-L) proteolytic activity and its regulation by inhibitors and by substrates of trypsin-like (T-L) and caspase-like sites (PA). We demonstrate that this substrate is reliable to evaluate the ChT-L inhibitory activity of new molecules either by fluorescence or (19)F NMR spectroscopy. We have found that the ChT-L activity is dramatically reduced in the presence of T-L and PA substrates. This work provides a proof of concept that the fluorinated substrate enables investigation of the allosteric regulation of the ChT-L activity.

Oxidative status and chymotrypsin-like activity in right and left ventricle hypertrophy in an experimental model of emphysema

Although cardiac muscle hypertrophy has been studied in association with several diseases, its mechanism in patients with emphysema, in particular in relation to oxidative stress and proteolysis, remains unknown. The role of oxidative stress and proteolysis in right and left ventricle hypertrophy was investigated in hamsters with emphysema induced by 2 different doses of papain (20mg/mL, E20 and 40mg/mL, E40). The thickness of the ventricles, total and cardiac weight, lipid peroxidation, carbonyl proteins, total antioxidant capacity (TAC), and proteasomal proteolytic activity were evaluated in the right ventricle (RV) and the left ventricle (LV) of control and emphysema hamsters. RV thickness was increased by 12% in the E20 group and by 29% in the E40 group. Lipid peroxidation measured by chemiluminescence was increased in the E40 group (from 3350.68±392.44URL/g tissue to 4696.63±1076.70URL/g tissue, p<0.05). TAC also increased only in the E40 group. In the LV, chemiluminescence values increased from 4044.77±503.39 to 5517.10±388.27 in the E20 group and to 8169.14±1748.77URL/g tissue in the E40 group (p<0.05, both). TAC significantly increased in the E20 and E40 groups. No differences were detected in substances reactive to thiobarbituric acid or carbonyl proteins when comparing ventricles or doses. Chymotrypsin-like proteolytic activity significantly decreased in both groups and ventricles. Emphysema can induce right and left ventricle lipid peroxidation and result in antioxidant mobilization. These data together support the idea that cardiac hypertrophy in response to emphysema is mediated in part by proteolytic pathways with involvement of reactive species.

Lung injury-dependent oxidative status and chymotrypsin-like activity of skeletal muscles in hamsters with experimental emphysema

BackgroundPeripheral skeletal muscle is altered in patients suffering from emphysema and chronic obstructive pulmonary disease (COPD). Oxidative stress have been demonstrated to participate on skeletal muscle loss of several states, including disuse atrophy, mechanical ventilation, and chronic diseases. No evidences have demonstrated the occurance in a severity manner.MethodsWe evaluated body weight, muscle loss, oxidative stress, and chymotrypsin-like proteolytic activity in the gastrocnemius muscle of emphysemic hamsters. The experimental animals had 2 different severities of lung damage from experimental emphysema induced by 20 mg/mL (E20) and 40 mg/mL (E40) papain.ResultsThe severity of emphysema increased significantly in E20 (60.52 ± 2.8, p < 0.05) and E40 (52.27 ± 4.7; crossed the alveolar intercepts) groups. As compared to the control group, there was a reduction on body (171.6 ± 15.9 g) and muscle weight (251.87 ± 24.87 mg) in the E20 group (157.5 ± 10.3 mg and 230.12 ± 23.52 mg, for body and muscle weight, respectively), which was accentuated in the E40 group (137.4 ± 7.2 g and 197.87 ± 10.49 mg, for body and muscle weight, respectively). Additionally, the thiobarbituric acid reactive substances (TBARS), tert-butyl hydroperoxide-initiated chemiluminescence (CL), carbonylated proteins, and chymotrypsin-like proteolytic activity were elevated in the E40 group as compared to the E20 group (p < 0.05 for all comparisons). The severity of emphysema significantly correlated with the progressive increase in CL (r = −0.95), TBARS (r = −0.98), carbonyl proteins (r = −0.99), and chymotrypsin-like proteolytic activity (r = −0.90). Furthermore, augmentation of proteolytic activity correlated significantly with CL (r = 0.97), TBARS (r = 0.96), and carbonyl proteins (r = 0.91).ConclusionsTaken together, the results of the present study suggest that muscle atrophy observed in this model of emphysema is mediated by increased muscle chymotrypsin-like activity, with possible involvement of oxidative stress in a severity-dependent manner.

Dysfunction of the ubiquitin–proteasome system in the cerebellum of aging Ts65Dn mice

In the cerebellum of adult-aging Ts65Dn mice, a murine model of Down syndrome, Purkinje cells undergo degeneration. Searching for the cause of Purkinje cell degeneration, we have studied the ubiquitin–proteasome system (UPS) in the cerebellum of aging Ts65Dn mice. Inhibition of UPS is sufficient to induce neuron degeneration and death. Proteasome chymotrypsin-like proteolytic activity was reduced by 35% in the cerebellum of Ts65Dn mice in comparison with euploid animals. Accordingly, Western blot analysis of ubiquitin showed an increase in ubiquitinated proteins. Immunocytochemistry for ubiquitin revealed strongly positive intranuclear inclusions in Purkinje cells and large neurons of cerebellar nuclei. The Western blot analysis of ubiquitin in nuclear protein extracts confirmed the increase of ubiquitinated proteins in the cell nuclei. After FUS immunocytochemistry, large intranuclear inclusions were visible in Purkinje cells and large neurons of cerebellar nuclei in Ts65Dn mice. Together, data indicate a possible role for proteasome inhibition in the cerebellar neurodegeneration in Ts65Dn mice.

PSMB8 Encoding the β5i Proteasome Subunit Is Mutated in Joint Contractures, Muscle Atrophy, Microcytic Anemia, and Panniculitis-Induced Lipodystrophy Syndrome

We performed homozygosity mapping in two recently reported pedigrees from Portugal and Mexico with an autosomal-recessive autoinflammatory syndrome characterized by joint contractures, muscle atrophy, microcytic anemia, and panniculitis-induced lipodystrophy (JMP). This revealed only one homozygous region spanning 2.4 Mb (5818 SNPs) on chromosome 6p21 shared by all three affected individuals from both families. We directly sequenced genes involved in immune response located in this critical region, excluding the HLA complex genes. We found a homozygous missense mutation c.224C>T (p.Thr75Met) in the proteasome subunit, beta-type, 8 (PSMB8) gene in affected patients from both pedigrees. The mutation segregated in an autosomal-recessive fashion and was not detected in 275 unrelated ethnically matched healthy subjects. PSMB8 encodes a catalytic subunit of the 20S immunoproteasomes called β5i. Immunoproteasome-mediated proteolysis generates immunogenic epitopes presented by major histocompatibility complex (MHC) class I molecules. Threonine at position 75 is highly conserved and its substitution with methionine disrupts the tertiary structure of PSMB8. As compared to normal lymphoblasts, those from an affected patient showed significantly reduced chymotrypsin-like proteolytic activity mediated by immunoproteasomes. We conclude that mutations in PSMB8 cause JMP syndrome, most probably by affecting MHC class I antigen processing.

Enzymic, Phylogenetic, and Structural Characterization of the Unusual Papain-like Protease Domain of Plasmodium falciparum SERA5

Serine repeat antigen 5 (SERA5) is an abundant antigen of the human malaria parasite Plasmodium falciparum and is the most strongly expressed member of the nine-gene SERA family. It appears to be essential for the maintenance of the erythrocytic cycle, unlike a number of other members of this family, and has been implicated in parasite egress and/or erythrocyte invasion. All SERA proteins possess a central domain that has homology to papain except in the case of SERA5 (and some other SERAs), where the active site cysteine has been replaced with a serine. To investigate if this domain retains catalytic activity, we expressed, purified, and refolded a recombinant form of the SERA5 enzyme domain. This protein possessed chymotrypsin-like proteolytic activity as it processed substrates downstream of aromatic residues, and its activity was reversed by the serine protease inhibitor 3,4-diisocoumarin. Although all Plasmodium SERA enzyme domain sequences share considerable homology, phylogenetic studies revealed two distinct clusters across the genus, separated according to whether they possess an active site serine or cysteine. All Plasmodia appear to have at least one member of each group. Consistent with separate biological roles for members of these two clusters, molecular modeling studies revealed that SERA5 and SERA6 enzyme domains have dramatically different surface properties, although both have a characteristic papain-like fold, catalytic cleft, and an appropriately positioned catalytic triad. This study provides impetus for the examination of SERA5 as a target for antimalarial drug design.

Biochemical Analysis of the 20 S Proteasome of Trypanosoma brucei

We describe here biochemical characterization of the 20 S proteasome from the parasitic protozoan Trypanosoma brucei. Similar to the mammalian proteasome, the T. brucei proteasome is made up of seven alpha- and seven beta-subunits. Of the seven beta-type subunits, five contain pro-sequences that are proteolytically removed during assembly, and three of them are predicted to be catalytic based on primary sequence. Affinity labeling studies revealed that, unlike the mammalian proteasome where three beta-subunits were labeled by the affinity reagents, only two beta-subunits of the T. brucei proteasome were labeled in the complex. These two subunits corresponded to beta2 and beta5 subunits responsible for the trypsin-like and chymotrypsin-like proteolytic activities, respectively. Screening of a library of 137,180 tetrapeptide fluorogenic substrates against the T. brucei 20 S proteasome confirmed the nominal beta1-subunit (caspase-like or PGPH) activity and identified an overall substrate preference for hydrophobic residues at the P1 to P4 positions in a substrate. This overall stringency is relaxed in the 11 S regulator (PA26)-20 S proteasome complex, which shows both appreciable activities for cleavage after acidic amino acids and a broadened activity for cleavage after basic amino acids. The 20 S proteasome from T. brucei also shows appreciable activity for cleavage after P1-Gln that is minimally observed in the human counterpart. These results demonstrate the importance of substrate sequence specificity of the T. brucei proteasome and highlight its biochemical divergence from the human enzyme.

Proteolytic activity of 26s proteasomes isolated from muscles of the tobacco hornworm, Manduca sexta: differences between surviving muscles and those undergoing developmentally programmed cell death.

The intersegmental muscles (ISMs) of tobacco hornworm,Manduca sexta are a well-characterised model system for examining the biochemical changes that accompany programmed cell death during development. When the ISMs become committed to die, there are dramatic increases in both the ubiquitin-expression, and ubiquitin-dependent proteolysis. Since the 26S proteasome is responsible for ATP/ubiquitin-dependent proteolysis in cells, we examined its enzymatic properties. Specific chymotrypsin-like proteolytic activity of 26S proteasomes isolated from ISM is four times higher than that of surviving flight muscle (FM). However, specific activity does not change between developmental stages within ISM or FM. The difference between proteolytic capacity of the two kinds of muscles is even higher when the ISM become committed to die because 26S proteasome content of ISM increases just before cell death. These observations underline the role of 26S proteasome in programmed cell death.

The Ubiquitin-Proteasome Proteolytic Pathway in Heart vs Skeletal Muscle: Effects of Acute Diabetes

The ubiquitin-proteasome system is thought to play a major role in normal muscle protein turnover and to contribute to diabetes-induced protein wasting in skeletal muscle. However, its importance in cardiac muscle is not clear. We measured heart muscle mRNA for ubiquitin and for the C2 and C8 proteasomal subunits, the amount of free ubiquitin and the proteasome chymotrypsin-like proteolytic activity in control and diabetic rats. Results were compared to those in skeletal muscle (rectus). Heart ubiquitin, C2 and C8 subunit mRNA and proteolytic activity were significantly greater than in skeletal muscle (P ≤ 0.05). This suggests that the ubiquitin proteasomal pathway may also be important for normal heart muscle turnover. Diabetes increased ubiquitin mRNA by ∼50% in heart (P < 0.03) and by ∼100% in skeletal muscle (P < 0.005). It remained high after 3 days of insulin treatment in both tissues. C2 and C8 subunit mRNA did not change with diabetes or insulin treatment. Diabetes did not change the amount of free ubiquitin or the proteasomal (lactacystin-inhibitable) chymotrypsin-like peptidase activity in heart or skeletal muscle. In conclusions, gene expression for several components of the ubiquitin-proteasome proteolytic pathway is significantly higher in cardiac than in skeletal muscle, as is the proteasome chymotrypsin-like peptidase activity. Diabetes increases the expression of ubiquitin but not C2 or C8 subunit mRNA, nor does it significantly alter the amount of free ubiquitin or the proteasome chymotrypsin-like peptidase activity. The rate-limiting step of enhanced protein degradation in diabetic rat heart and skeletal muscle may be located at ubiquitin conjugation and/or its binding to proteasome, not at the ubiquitin availability or the proteasome itself.

A New Structural Class of Proteasome Inhibitors that Prevent NF-κB Activation

The multicatalytic proteinase or proteasome is a highly conserved cellular structure that is responsible for the ATP-dependent proteolysis of many proteins involved in important regulatory cellular processes. We have identified a novel class of inhibitors of the chymotrypsin-like proteolytic activity of the 20S proteasome that exhibit ic 50 values ranging from 0.1 to 0.5 μg/mL (0.1 to 1 μM). In cell proliferation assays, these compounds inhibit growth with an ic 50 ranging from 5 to 10 μg/mL (10–20 μM). A representative member of this class of inhibitors was tested in other biological assays. CVT-634 (5-methoxy-1-indanone-3-acetyl-leu- d-leu-1-indanylamide) prevented lipopolysaccharide (LPS), tumor necrosis factor (TNF)-, and phorbol ester-induced activation of nuclear factor κB (NF-κB) in vitro by preventing signal-induced degradation of IκB-α. In these studies, the IκB-α that accumulated was hyperphosphorylated, indicating that CVT-634 did not inhibit IκB-α kinase, the enzyme responsible for signal-induced phosphorylation of IκB-α. In vivo studies indicated that CVT-634 prevented LPS-induced TNF synthesis in a murine macrophage cell line. In addition, in mice pretreated with CVT-634 at 25 and 50 mg/kg and subsequently treated with LPS, serum TNF levels were significantly lower (225 ± 59 and 83 ± 41 pg/mL, respectively) than in those mice that were treated only with LPS (865 ± 282 pg/mL). These studies suggest that specific inhibition of the chymotrypsin-like activity of the proteasome is sufficient to prevent signal-induced NF-κB activation and that the proteasome is a novel target for the identification of agents that may be useful in the treatment of diseases whose etiology is dependent upon the activation of NF-κB.

Isolation and Characterization of Ovochymase, a Chymotrypsin-like Protease Released during Xenopus laevis Egg Activation

A chymotrypsin-like protease contained in the perivitelline space of unactivated Xenopus eggs is released during egg activation and appears to participate in vitelline envelope conversion. This 30-kDa protease, which we have termed ovochymase, was isolated from the exudate of activated eggs using a soy bean trypsin inhibitor-agarose affinity column. The column eluant contained only two proteins, the 30-kDa ovochymase plus a 78-kDa chymotrypsin-like proteolytic activity, The 78-kDa protease was not usually observed in fresh egg exudate samples and thus was activated during the purification process and may represent the proposed precursor of the 30-kDa protease. The 30- and 78-kDa proteases were separated by gel filtration HPLC or by SDS-PAGE. The N-terminal amino acid sequence of SDS-PAGE-isolated ovochymase was determined to be VVGGQQAAPR. This conserved amino acid sequence, plus active site specific inhibition and substrate specificity studies, places ovochymase in the serine protease I family of enzymes. A two-dimensional protease activity gel revealed that ovochymase is present as several isozymes with a wide range of pI's.

Formation of neutrophil-activating peptide 2 from platelet-derived connective-tissue-activating peptide III by different tissue proteinases

Neutrophil-activating peptide 2 (NAP-2) is generated by cleavage of two inactive precursors, connective-tissue-activating peptide III (CTAP-III) and platelet basic protein (PBP), which are stored in the alpha-granules of blood platelets. Using highly purified CTAP-III as the substrate we studied the generation of NAP-2 by several neutral tissue proteinases. CTAP-III was rapidly cleaved by chymotrypsin, cathepsin G and trypsin, yielding products with neutrophil-stimulating activity. This activity remained unchanged for 24 h in the presence of chymotrypsin, decreased only slowly in the presence of cathepsin G, but was rapidly destroyed by trypsin. CTAP-III was also degraded by human neutrophil elastase and porcine pancreatic elastase, but no active fragments were obtained. By contrast, no degradation of CTAP-III was observed with thrombin, plasmin or 'granzymes' from cytolytic T-lymphocyte granules. Two active fragments of CTAP-III, generated by chymotrypsin or cathepsin G, were purified and partially sequenced, and were found to have the same N-terminal sequence as NAP-2. These results indicate that both proteinases cleave preferentially the bond between amino acids 15 (Tyr) and 16 (Ala) of CTAP-III. We conclude that chymotrypsin-like proteolytic activity in the vicinity of activated platelets may generate NAP-2 intravascularly. Due to its presence in the primary granules of neutrophils and monocytes cathepsin G is likely to be involved in this process.

カテプシンＧのリンパ球活性化作用

Cathepsin G is one of serine proteases with chymotrypsin-like proteolytic activity and known to exist in neutrophils and spleens. We purified the protease from human bone marrow cells by extraction with 1 M potassium phosphate buffer (pH 7.0) and ap-plying to column chromatography of DEAF-cellulose, Sephadex G-100, CM-cellulose, and Mono-S. We further separated it into three isozymes by employing hydrophobic chromatography, and named them cathepsin.Purified cathepsin G potentiated the effect of mitogens such as Con A and PHA on human lymphocytes. The protease further stimulated synthesis of both DNA and RNA of lymphocytes in the absence of mitogens. Effect of cathepsin G on the lymphocyte activity was prevented by inhibitors of cathepsin G. Therefore, the effect of it on lymphocyte activity is considered to be due to its proteolytic activity. The effect of cathepsin G on the function of lymphocytes differed among isozymes of the protease. From these results, we suppose that cathepsin G plays an important role in biophylaxis by regulating functions of lymphocytes.

Changes of a high molecular mass proteinase activity during Bufo bufo development

1. 1. A chymotrypsin-like proteolytic activity was found both in unfertilized eggs and in embryos of Bufo bufo. 2. 2. A dramatical change of activity can be observed in the course of embryonic development. The activity rapidly increases after fertilization up to the stage 9 followed by a fall to a level close to unfertilized eggs. 3. 3. Gel chromatography analysis reveals, in all stages of development, the presence of a single peak of proteinase activity characterized by a very high molecular mass. 4. 4. Proteinase activity, found change during the development of Bufo bufo, was characterized by substrate specificity, protease inhibitor and pH effect. All results obtained suggest that the chymotrypsin-like activity can be assigned to the multicatalytic proteinase.