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KMID : 1161519990030020221
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Animal Cells and Systems
1999 Volume.3 No. 2 p.221 ~ p.228
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Pepstatin-insensitive carboxyl proteinase: A biochemical marker for late lysosomes in Amoeba proteus
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Kwon Hae-Kyung
Kim Hyeon-Jung
Ahn Tae-In
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Abstract
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In order to find a biochemical marker for late lysosomes, we characterized two cDNAs which were cloned by using a monoclonal antibody (mAb) against lysosomes in Amoeba proteus as a probe. The two cDNAs, a 1.3?kb cDNA in pBSK?lys45 and a 1.6?kb cDNA in pBSK?lys60, were found to encode proteins homologous to pepstatin?insensitive carboxyl proteinases (PICPs). E. coli transformed with pBSK?lys45 produced two immunopositive polypeptides (45 and 43 kDa) and the cDNA in 1274 bases encoded a 44,733?Da protein (Lys45) of 420 amino acids containing one site for a core oligosaccharide. On the other hand, E. coli transformed with pBSK?lys60 produced several polypeptides (64, 54, 45, 41, and 37 kDa) reacting with the mAb. The cDNA contained 1629 bases and encoded a 59,231?Da protein (Lys60) of 530 amino acids containing two sites for asparagine?linked core oligosaccharides. These two cDNAs showed identities of 60.3% in nucleotide sequences and 23.6% in amino acid sequences. Lys45 and Lys60 appeared to share XXEFQK as a common antigenic domain. The amino acid sequence of the Lys45 protein showed 17.4% identity and 40.9% similarity to that of PiCP from Pseudomonas sp. 101. On the other hand, Lys60 showed a 24.3% identity and 51.9% similarity with human lysosomal PICP in the amino acid sequence. A putative active center for sertne protease, GTS*xxxxxFxG, was found to be conserved among PICP homologues. The two PICPs are the first reported enzymatic markers for late lysosomes
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KEYWORD
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Amoeba cDNA, Acid proteinase, Phagolysosome, Phagocytosis, Marker enzyme, Lysosome
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