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.
Kawada
A
A
Department of Dermatology, Kinki University School of Medicine, Ohno-Higashi 377-2, Osaka-Sayama, Osaka, Japan. kawada@med.kindai.ac.jp
Hara
K
K
Kominami
E
E
Tezuka
T
T
Takahashi
M
M
Takahara
H
H
D000595:Amino Acid Sequence; D000818:Animals; D056668:Cathepsin L; D002403:Cathepsins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004792:Enzyme Precursors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004817:Epidermis / Q000033:anatomy & histology / Q000201:enzymology*; D007150:Immunohistochemistry; D008099:Liver / Q000033:anatomy & histology / Q000201:enzymology; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational; D051381:Rats; D017207:Rats, Sprague-Dawley
D004792:Enzyme Precursors; D002403:Cathepsins; C063905:procathepsin L; D056668:Cathepsin L