Biomedical subjects

E Kominami

Publications and source records attributed to E Kominami.

At least 19 recordsLinked to original sources

PubMed2001

Preoviposition activation of cathepsin-like proteinases in degenerating ovarian follicles of the mosquito Culex pipiens pallens.

Within developing ovaries of many insects, some developing follicles or oocytes usually degenerate (follicular atresia or oosorption), while the others may continue to grow to maturity, thus maintaining the balance between the number of eggs and reproductive circumstances such as available nutrients. To help clarify the phenomenon of follicular atresia during ovarian development, we examined cysteine proteinases stored in mosquito Culex pipiens pallens ovaries. First, analysis using synthesized substrates showed that cathepsin B- and L-like proteinases gradually accumulated in the developing ovaries after a blood meal, which required more than 10 min of preincubation under acidic conditions to reach their maximum activities. However, homogenates of degenerating follicles 3 days after feeding showed proteolytic activities without acid treatment, suggesting that the proteinases had already been activated, while the extract of normally developing follicles collected from the same ovaries required more than 10 min of acid preincubation to reach the optimum activities, suggesting that the enzymes remained as inactive forms. Chemical and immunohistochemical analyses showed that more proteinases are located in the cytoplasm, rather than being associated with yolk granules. Ovarian proteinases, which are believed to become activated at the onset of embryogenesis, should also be activated during oogenesis, presumably to enhance oosorption.

Animals↗

PubMed2001

Molecular characterization of mammalian homologues of class C Vps proteins that interact with syntaxin-7.

Vesicle-mediated protein sorting plays an important role in segregation of intracellular molecules into distinct organelles. Extensive genetic studies using yeast have identified more than 40 vacuolar protein sorting (VPS) genes involved in vesicle transport to vacuoles. However, their mammalian counterparts are not fully elucidated. In this study, we identified two human homologues of yeast Class C VPS genes, human VPS11 (hVPS11) and human VPS18 (hVPS18). We also characterized the subcellular localization and interactions of the protein products not only from these genes but also from the other mammalian Class C VPS homologue genes, hVPS16 and rVPS33a. The protein products of hVPS11 (hVps11) and hVPS18 (hVps18) were ubiquitously expressed in peripheral tissues, suggesting that they have a fundamental role in cellular function. Indirect immunofluorescence microscopy revealed that the mammalian Class C Vps proteins are predominantly associated with late endosomes/lysosomes. Immunoprecipitation and gel filtration studies showed that the mammalian Class C Vps proteins constitute a large hetero-oligomeric complex that interacts with syntaxin-7. These results indicate that like their yeast counterparts, mammalian Class C Vps proteins mediate vesicle trafficking steps in the endosome/lysosome pathway.

Amino Acid Sequence↗

PubMed2001

Splenic cathepsin L is maturated from the proform by interferon-gamma after immunization with exogenous antigens.

The processing of foreign protein antigens into peptides requires the participation of various endo/lysosomal proteases in antigen-presenting cells (APCs). In this study, a proenzyme of cathepsin L, procathepsin L, was found to be present in the spleens of naive mice, as demonstrated by immunoblotting. Interestingly, the maturation of cathepsin L from procathepsin L was strongly induced when the host BALB/c mice were immunized with ovalbumin or soluble leishmanial antigen, despite the fact that mouse albumin, a kind of self-antigen, did not have such a potential. Furthermore, foreign antigens, but not self-antigens, could increase the activity of cathepsin L, probably being mediated by interferon-gamma, as demonstrated by in vivo and in vitro experiments. As cathepsin L matured, the efficiency of antigen processing was increased in APCs. These results suggest that endo/lysosomal cathepsin L plays an important role in the immune regulation via antigen processing even in peripheral lymphoid tissues as well as in the thymus.

Animals↗

PubMed2001

The C-terminal region of an Apg7p/Cvt2p is required for homodimerization and is essential for its E1 activity and E1-E2 complex formation.

Apg7p/Cvt2p, a protein-activating enzyme, is essential for both the Apg12p-Apg5p conjugation system and the Apg8p membrane targeting in autophagy and cytoplasm-to-vacuole targeting in the yeast Saccharomyces cerevisiae. Similar to the ubiquitin-conjugating system, both Apg12p and Apg8p are activated by Apg7p, an E1-like enzyme. Apg12p is then transferred to Apg10p, an E2-like enzyme, and conjugated with Apg5p, whereas Apg8p is transferred to Apg3p, another E2-like enzyme, followed by conjugation with phosphatidylethanolamine. Evidence is presented here that Apg7p forms a homodimer with two active-site cysteine residues via the C-terminal region. The dimerization of Apg7p is independent of the other Apg proteins and facilitated by overexpressed Apg12p. The C-terminal 123 amino acids of Apg7p (residues 508 to 630 out of 630 amino acids) are sufficient for its dimerization, where there is neither an ATP binding domain nor an active-site cysteine essential for its E1 activity. The deletion of its carboxyl 40 amino acids (residues 591-630 out of 630 amino acids) results in several defects of not only Apg7p dimerization but also interactions with two substrates, Apg12p and Apg8p and Apg12p-Apg5p conjugation, whereas the mutant Apg7p contains both an ATP binding domain and an active-site cysteine. Furthermore, the carboxyl 40 amino acids of Apg7p are also essential for the interaction of Apg7p with Apg3p to form the E1-E2 complex for Apg8p. These results suggest that Apg7p forms a homodimer via the C-terminal region and that the C-terminal region is essential for both the activity of the E1 enzyme for Apg12p and Apg8p as well as the formation of an E1-E2 complex for Apg8p.

Amino Acid Sequence↗

PubMed2001

Leupeptin-induced appearance of partial fragment of betaine homocysteine methyltransferase during autophagic maturation in rat hepatocytes.

A cytosolic enzyme, betaine homocysteine methyltransferase (BHMT), and its partial fragments were discovered as autolysosomal membrane proteins from rat liver in the presence of leupeptin [Ueno et al. (1999) J. Biol. Chem. 274, 15222-15229]. The present study was undertaken to further characterize the transport and processing of BHMT from cytosol to autolysosome and to test if the fragment can be used as an in vitro probe for the maturation step of macroautophagy. Upon subcellular fractionation, BHMT (p44) was found in all fractions, while its 32-kDa fragment (p32) was found only in the mitochondrial-lysosomal (ML) fraction. Incubation of isolated hepatocytes with leupeptin induced time-dependent accumulation of p32 in the ML fraction from 30 to 90 min after the start of incubation. However, chloroquine completely inhibited the appearance of p32, indicating that the processing from p44 to p32 is lysosomal. Incubation with Bafilomycin A(1), a vacuolar H(+)-ATPase inhibitor, together with leupeptin, led to linear accumulation of p44, but not of p32. The p44 accumulation rate was calculated to be 4.9%/h, which was comparable to autophagic sequestration rate. The distribution of p44 within the ML fraction turned out to be dual, i.e., the membrane-surface attached and luminal/sedimentable forms. Amino acids and 3-methyladenine, both of which specifically suppress macroautophagy, inhibited the accumulation of p32 as well as of p44. Finally, energy-dependent appearance of p32 was demonstrated during incubation of postnucler supernatant fractions, making it possible to establish an in vitro assay system. All the results strongly support the idea that BHMT is taken up and degraded to p32 through the macroautophagic pathway, and that p32 could be a novel probe for the maturation of macroautophagy.

Animals↗

PubMed2001

Expression of proteinases and inflammatory cytokines in subchondral bone regions in the destructive joint of rheumatoid arthritis.

OBJECTIVE: We previously described abnormalities in the bone marrow of patients with rheumatoid arthritis (RA), but were able to shed little light on the pathogenic roles of inflammatory cytokines and proteinases in joint destruction in the subchondral region in RA. This is the first report to describe the co-localization of cytokines and proteinases in this area. METHODS: Decalcified paraffin-embedded sections from 10 patients with RA and five patients with osteoarthritis (OA) were examined for the immunolocalization of cathepsins B, K and L and the localization of messenger RNAs for interleukin 1beta (IL-1beta), tumour necrosis factor alpha (TNF-alpha) and matrix metalloproteinase 9 (MMP-9). The cells were double-stained with anti-CD68 or anti-prolyl 4-hydroxylase (PH) antibody. RESULTS: An immunohistochemical study confirmed the expression of cathepsins B and L by CD68-positive mononuclear cells at the sites of significant cartilage and bone erosion from the subchondral region in all RA specimens. Osteoclast-like cells showed intense staining for cathepsin K and MMP-9. Osteoblast-like cells strongly expressed MMP-9. Analysis of serial sections revealed that expression of the IL-1beta and TNF-alpha genes occurred near that of the cathepsins and MMP-9 in the subchondral region. CONCLUSION: We conclude that inflammatory cytokines and tissue-damaging proteinases play important roles in joint destruction in the subchondral region in RA.

Adult↗

PubMed2001

Prevention of apoptosis of mammalian cells by the CED-3-cleaved form of CED-9.

CED-9 prevents apoptosis in embryonic cells of Caenorhabditis elegans but not in mammalian cells. We show here that the prevention of apoptosis in mammalian cells requires a CED-3-cleaved form (68-280) of CED-9 which is localized in the inner mitochondrial membrane. The viability of PC12 and HeLa cells was significantly increased after death stimuli when truncated CED-9 was expressed in these cells but full-length CED-9 did not. The truncated CED-9 expressed in these cells was largely localized to the inner mitochondrial and the endoplasmic reticulum membranes, while full-length CED-9 was detected mainly in endoplasmic reticulum fractions. Moreover, truncated CED-9 in purified mitochondria was resistant to trypsin digestion, but full-length CED-9 was not. These results suggest that the CED-3-cleaved form of CED-9 prevents apoptosis in mammalian cells by localizing to the inner mitochondrial membrane.

Animals↗

PubMed2001

Specific localization of lysosomal aminopeptidases in type II alveolar epithelial cells of the rat lung.

We previously demonstrated that lysosomal cysteine proteinases, cathepsins B, H, and L were localized in lysosomes of alveolar macrophages and bronchial epithelial cells in the rat lung, while cathepsin H, a typical aminopeptidase, was additionally distributed in lamellar bodies containing surfactant in type II alveolar epithelial cells (ISHII et al., 1991). The present immunohistochemical study further examined the localization of lysosomal aminopeptidases, cathepsin C, and tripeptidyl peptidase I (TPP-I) in the rat lung. Western blotting confirmed the presence of cathepsin C and TPP-I as active forms in the pulmonary tissue, showing 25 kD and 47 kD, respectively. Immunohisto/cytochemical observations demonstrated that positive staining for cathepsin C and TPP-I was more intensely localized in alveolar epithelial regions than in bronchial or bronchiolar epithelial cells. By double immunostaining using confocal laser microscopy, immunoreactivity for cathepsin H was found to be co-localized with that for cathepsin C or TPP-I in both type II cells and macrophages. Moreover, when doubly stained with anti-cathepsin C and ED2, single-positive type II cells could be clearly distinguished from double-positive macrophages in the alveolar region. Immunoelectron microscopy revealed the gold labeling of cathepsin C or TPP-I in multivesicular and composite bodies, and lamellar bodies of Type II cells. These results showing that lysosomal aminopeptidases such as cathepsin H, cathepsin C and TPP-I are localized in lamellar bodies of type II alveolar epithelial cells strongly argue for the participation of lysosomal aminopeptidases in the formation process of surfactant containing specific proteins.

Aminopeptidases↗

PubMed2000

Interaction of the Unc-51-like kinase and microtubule-associated protein light chain 3 related proteins in the brain: possible role of vesicular transport in axonal elongation.

We identified two mammalian ULK1 (Unc-51-like kinase involved in neurite extension) binding proteins by yeast two-hybrid screening. Both proteins showed high structural similarity to microtubule-associated protein (MAP) light chain 3 (LC3). One is identical to the Golgi-associated ATPase Enhancer of 16 kDa (GATE-16), an essential factor for intra-Golgi transport [39]. The other is identical to the gamma 2-subunit of GABA-A receptor associated protein (GABARAP) which has a possible role in receptor transport [46]. Using the yeast two-hybrid system and the in vitro GST pull-down assay, we found that the N-terminal proline/serine rich (PS) domain of ULK1 (amino acid 287-416) is required for ULK1-GATE-16 and ULK1-GABARAP protein interactions. However, the kinase activity of ULK1 affected neither ULK1-GATE-16 nor ULK1-GABARAP interaction. Immunohistochemical analysis using ULK1 and GABARAP antibodies showed that the ULK1 and the GABARAP proteins co-localized to many kind of neurons such as pyramidal cells of the hippocampus, mitral cells of the olfactory bulb, and Purkinje cells of the cerebellum. In HeLa cells, endogenous ULK1 and tagged GABARAP showed punctate structures in the cytosol, and were colocalized. These results suggest that the interaction of ULK1 and GABARAP is important to vesicle transport and axonal elongation in mammalian neurons.

Adaptor Proteins, Signal Transducing↗

PubMed2000

The human homolog of Saccharomyces cerevisiae Apg7p is a Protein-activating enzyme for multiple substrates including human Apg12p, GATE-16, GABARAP, and MAP-LC3.

Autophagy is a process that involves the bulk degradation of cytoplasmic components by the lysosomal/vacuolar system. In the yeast, Saccharomyces cerevisiae, an autophagosome is formed in the cytosol. The outer membrane of the autophagosome is fused with the vacuole, releasing the inner membrane structure, an autophagic body, into the vacuole. The autophagic body is subsequently degraded by vacuolar hydrolases. Taking advantage of yeast genetics, apg (autophagy-defective) mutants were isolated that are defective in terms of formation of autophagic bodies under nutrient starvation conditions. One of the APG gene products, Apg12p, is covalently attached to Apg5p via the C-terminal Gly of Apg12p as in the case of ubiquitylation, and this conjugation is essential for autophagy. Apg7p is a novel E1 enzyme essential for the Apg12p-conjugation system. In mammalian cells, the human Apg12p homolog (hApg12p) also conjugates with the human Apg5p homolog. In this study, the unique characteristics of hApg7p are shown. A two-hybrid experiment indicated that hApg12p interacts with hApg7p. Site-directed mutagenesis revealed that Cys(572) of hApg7p is an authentic active site cysteine residue essential for the formation of the hApg7p.hApg12p intermediate. Overexpression of hApg7p enhances the formation of the hApg5p.hApg12p conjugate, indicating that hApg7p is an E1-like enzyme essential for the hApg12p conjugation system. Cross-linking experiments and glycerol-gradient centrifugation analysis showed that the mammalian Apg7p homolog forms a homodimer as in yeast Apg7p. Each of three human Apg8p counterparts, i.e. the Golgi-associated ATPase enhancer of 16 kDa, GABA(A) receptor-associated protein, and microtubule-associated protein light chain 3, coimmunoprecipitates with hApg7p and conjugates with mutant hApg7p(C572S) to form a stable intermediate via an ester bond. These results indicate that hApg7p is an authentic protein-activating enzyme for hApg12p and the three Apg8p homologs.

Adaptor Proteins, Signal Transducing↗

PubMed2000

A ubiquitin-like system mediates protein lipidation.

Autophagy is a dynamic membrane phenomenon for bulk protein degradation in the lysosome/vacuole. Apg8/Aut7 is an essential factor for autophagy in yeast. We previously found that the carboxy-terminal arginine of nascent Apg8 is removed by Apg4/Aut2 protease, leaving a glycine residue at the C terminus. Apg8 is then converted to a form (Apg8-X) that is tightly bound to the membrane. Here we report a new mode of protein lipidation. Apg8 is covalently conjugated to phosphatidylethanolamine through an amide bond between the C-terminal glycine and the amino group of phosphatidylethanolamine. This lipidation is mediated by a ubiquitination-like system. Apg8 is a ubiquitin-like protein that is activated by an E1 protein, Apg7 (refs 7, 8), and is transferred subsequently to the E2 enzymes Apg3/Aut1 (ref. 9). Apg7 activates two different ubiquitin-like proteins, Apg12 (ref. 10) and Apg8, and assigns them to specific E2 enzymes, Apg10 (ref. 11) and Apg3, respectively. These reactions are necessary for the formation of Apg8-phosphatidylethanolamine. This lipidation has an essential role in membrane dynamics during autophagy.

Amino Acid Sequence↗

PubMed2000

LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing.

Little is known about the protein constituents of autophagosome membranes in mammalian cells. Here we demonstrate that the rat microtubule-associated protein 1 light chain 3 (LC3), a homologue of Apg8p essential for autophagy in yeast, is associated to the autophagosome membranes after processing. Two forms of LC3, called LC3-I and -II, were produced post-translationally in various cells. LC3-I is cytosolic, whereas LC3-II is membrane bound. The autophagic vacuole fraction prepared from starved rat liver was enriched with LC3-II. Immunoelectron microscopy on LC3 revealed specific labelling of autophagosome membranes in addition to the cytoplasmic labelling. LC3-II was present both inside and outside of autophagosomes. Mutational analyses suggest that LC3-I is formed by the removal of the C-terminal 22 amino acids from newly synthesized LC3, followed by the conversion of a fraction of LC3-I into LC3-II. The amount of LC3-II is correlated with the extent of autophagosome formation. LC3-II is the first mammalian protein identified that specifically associates with autophagosome membranes.

Amino Acid Sequence↗

PubMed2000

Regulation of cell surface expression of CTLA-4 by secretion of CTLA-4-containing lysosomes upon activation of CD4+ T cells.

CTLA-4 is expressed on the surface of activated T cells and negatively regulates T cell activation. Because a low-level expression of CTLA-4 on the cell surface is sufficient to induce negative signals in T cells, the surface expression of CTLA-4 is strictly regulated. We previously demonstrated that the association of CTLA-4 with the clathrin-associated adaptor complex AP-2 induces internalization of CTLA-4 and keeps the surface expression low. However, the mechanism to induce high expression on the cell surface upon stimulation has not yet been clarified. To address this, we investigated the intracellular dynamics of CTLA-4 by analyzing its localization and trafficking in wild-type and mutant CTLA-4-transfected Th1 clones. CTLA-4 is accumulated in intracellular granules, which we identified as lysosomes. CTLA-4 is degraded in lysosomes in a short period, and the degradation process may serve as one of the mechanisms to regulate CTLA-4 expression. Upon TCR stimulation, CTLA-4-containing lysosomes are secreted as proven by the secretion of cathepsin D and beta-hexosaminidase in parallel with the increase of surface expression of CTLA-4 and lysosomal glycoprotein 85, a lysosomal marker. These results suggest that the cell surface expression of CTLA-4 is up-regulated upon stimulation by utilizing a mechanism of secretory lysosomes in CD4(+)T cells.

Abatacept↗

PubMed2000

Cathepsin D deficiency induces lysosomal storage with ceroid lipofuscin in mouse CNS neurons.

Cathepsin D-deficient (CD-/-) mice have been shown to manifest seizures and become blind near the terminal stage [approximately postnatal day (P) 26]. We therefore examined the morphological, immunocytochemical, and biochemical features of CNS tissues of these mice. By electron microscopy, autophagosome/autolysosome-like bodies containing part of the cytoplasm, granular osmiophilic deposits, and fingerprint profiles were demonstrated in the neuronal perikarya of CD-/- mouse brains after P20. Autophagosomes and granular osmiophilic deposits were detected in neurons at P0 but were few in number, whereas they increased in the neuronal perikarya within days after birth. Some large-sized neurons having autophagosome/autolysosome-like bodies in the perikarya appeared in the CNS tissues, especially in the thalamic region and the cerebral cortex, at P17. These lysosomal bodies occupied the perikarya of almost all neurons in CD-/- mouse brains obtained from P23 until the terminal stage. Because these neurons exhibited autofluorescence, it was considered that ceroid lipofuscin may accumulate in lysosomal structures of CD-/- neurons. Subunit c of mitochondrial ATP synthase was found to accumulate in the lysosomes of neurons, although the activity of tripeptidyl peptidase-I significantly increased in the brain. Moreover, neurons near the terminal stage were often shrunken and possessed irregular nuclei through which small dense chromatin masses were scattered. These results suggest that the CNS neurons in CD-/- mice show a new form of lysosomal accumulation disease with a phenotype resembling neuronal ceroid lipofuscinosis.

Action Potentials↗

PubMed2000

Generation of both MHC class I- and class II-restricted antigenic peptides from exogenously added ovalbumin in murine phagosomes.

The phagosome fraction derived from a murine macrophage cell line (J774.1), which had internalized ovalbumin (OVA)-coated latex beads, was isolated. The peptides recovered from the phagosome fraction were separated on reverse phase HPLC and each fraction was analyzed for the content of either major histocompatibility complex (MHC) class I- or class II-restricted OVA-derived peptide. Both peptides were detected in the phagosome fraction after less than 15 min of internalization. It was also indicated that phagosomes degrade OVA protein into both MHC class I- and class II-restricted antigenic peptides by employing the same types of cathepsins. Furthermore, the results suggest that the MHC class I-restricted peptide rapidly exits from the phagosome to the cytosol. These findings illustrate a potential role for phagosomes not only in MHC class II-restricted but also in MHC class I-restricted exogenous antigen presentation pathways. Our results also point to the vital role of phagosomes in non-cytosolic antigen presentation pathway, in which further degradation of antigens by the proteasome is dispensable.

Amino Acid Sequence↗

PubMed2000

Characterization of endopeptidase activity of tripeptidyl peptidase-I/CLN2 protein which is deficient in classical late infantile neuronal ceroid lipofuscinosis.

Endopeptidase activities of the CLN2 gene product (Cln2p)/tripeptidyl peptidase I (TPP-I), purified from rat spleen, were studied using the synthetic fluorogenic substrates. We designed and constructed decapeptides, based on the known sequence cleavage specificities of bacterial pepstatin-insensitive carboxyl proteases (BPICP). MOCAc-Gly-Lys-Pro-Ile-Pro-Phe-Phe-Arg-Leu-Lys(Dnp)r-NH(2) is readily hydrolyzed by Cln2p/TPP-I (K(cat)/K(m) = 7.8 s(-1) mM(-1)). The enzyme had a maximal activity at pH 3.0 for an endopeptidase substrate, but at pH 4.5 with respect to tripeptidyl peptidase activity. Both endopeptidase and tripeptidyl peptidase activities were strongly inhibited by Ala-Ala-Phe-CH(2)Cl, but not inhibited by tyrostatin, an inhibitor of bacterial pepstatin-insensitive carboxyl proteases, pepstatin, or inhibitors of serine proteases. Fibroblasts from classical late infantile neuronal ceroid lipofuscinosis patients have less than 5% of the normal tripeptidyl peptidase activity and pepstatin-insensitive endopeptidase activity. Cln2p/TPP-I is a unique enzyme with both tripeptidyl peptidase and endopeptidase activities for certain substrate specificity.

Amino Acid Sequence↗

PubMed2000

CLIP-derived self peptides bound to MHC class II molecules of medullary thymic epithelial cells differ from those of cortical thymic epithelial cells in their diversity, length, and C-terminal processing.

Medullary thymic epithelial cells (mTEC) are able to present soluble antigens to CD4+ helper T cell lines, whereas cortical thymic epithelial cells (cTEC) are not (Mizuochi, T., et al., J. Exp. Med. 1992. 175: 1601-1605). In addition, class II heterodimers from mTEC migrated with apparently less relative molecular mass in SDS-PAGE than those from cTEC (Kasai, M., et al., Eur. J. Immunol. 1998. 28:1867-1876). To investigate the cause of the distinct migration profiles of class II heterodimers in both TEC types, class II heterodimer-associated peptides were analyzed by matrix-assisted laser desorption ionization mass spectrometry. Self peptides from cTEC were shown to vary moderately in length and to be highly diverse, including low amounts of CLIP (class II-associated invariant chain peptide) variants. On the other hand, self peptides from two mTEC consisted predominantly of two CLIP variants with exceptional C-terminal extensions. C-terminally overhanging residues of CLIP in mTEC may be responsible for the distinct migration of class II heterodimers in SDS-PAGE. Differences in migration of class II heterodimers on SDS gels was also observed in H2-DM+ vesicles isolated from both TEC. The possible contribution of self peptides bound to class II heterodimers in TEC to positive or negative selection of T cells in the thymus is discussed.

Amino Acid Sequence↗

PubMed2000

Precursor of rat epidermal cathepsin L: purification and immunohistochemical localization.

Immunoblot study using anti-rat cathepsin L antibody revealed almost only a precursor present in a crude extract of homogenized lower epidermis of rat skin, while the precursor and mature form were detected in the upper epidermis. To elucidate mechanisms of synthesis of cathepsin L, we have purified a precursor of cathepsin L from rat epidermis and investigated its localization in the skin. The precursor was purified after separation from the mature form in the final purification step of active fraction for N-benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methylcoumari n. The precursor showed a single protein band with Mr 39 kDa on SDS/polyacrylamide gel electrophoresis and was immunoreacted with the anti-rat cathepsin L antibody. Two types of NH(2)-terminal sequences obtained were identical to the amino acid residues from -96 to -86 and those from -93 to -87 deduced from cDNA of the precursor of cathepsin L. The precursor was processed to mature form of the enzyme and the enzyme activity remarkably increased after 48-h incubation of the whole epidermis in 1 M acetate buffer (pH 3. 5) at 20 degrees C. In histological sections of the skin, a thin and diffuse staining pattern was present in the spinous layer and a dense and linear staining in the granular layer of the epidermis. In contrast, rat liver showed more mature form than the precursor by immunohistological findings. These results suggest that cathepsin L may have some roles in the terminal differentiation.

Amino Acid Sequence↗