Mechanisms of a conversion from membrane associated lysosomal acid phosphatase to content forms.
We reported that membrane-associated APase (M-APase) is anchored in the lipid bilayer through its hydrophobic sequence close to the COOH-terminus [Biochem. Biophys. Res. Commun. (1989) 162, 1044-1053] and is released from lysosomal membranes into the lysosomal contents by limited proteolysis with cathepsin D [J. Biochem. (1990) 108, 287-291]. We here report the conversion process of M-APase to three forms of the content enzyme (C-APase I, II, and III) by assigning the COOH-terminus of each APase in lysosomes. The purified M-APase (67 kDa) was subjected to COOH-terminal determination after digestion with cathepsin D. The COOH-terminus of cathepsin D-digested M-APase (65 kDa) ended at the position of the 382nd leucine residue. The COOH-termini of C-APase I (48 kDa) and III (64 kDa) were also determined. Since the two enzymes ended at the same position of the 373rd alanine residue, this COOH-terminal is 9 amino acid residues shorter than that of cathepsin D-digested M-APase. Then, we compared NH2-terminal sequences of the three enzymes, and found that those of three enzymes are exactly the same. Therefore, protein portions of C-APase I and III proved to be identical. The above results indicate that in lysosomes M-APase is first hydrolyzed between amino acid residues 382 and 383 by cathepsin D, and after solubilization, the enzyme is converted to C-APase III by losing 9 amino acid residues by lysosomal carboxypeptidase(s). Molecular weight differences among three C-APases (III, 64 kDa; II, 55 kDa; I, 48 kDa) probably are due to different degrees of carbohydrate chain degradations as reported previously [J. Biochem. (1989) 105, 449-456].
Himeno
M
M
Division of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
Nakamura
K
K
Tanaka
Y
Y
Yamada
H
H
Imoto
T
T
Kato
K
K
D000135:Acid Phosphatase / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002402:Cathepsin D / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D007425:Intracellular Membranes / Q000201:enzymology*; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D008051:Lipid Bilayers; D008099:Liver / Q000201:enzymology*; D008247:Lysosomes / Q000201:enzymology*; D008961:Models, Structural; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D011487:Protein Conformation; D051381:Rats
D007527:Isoenzymes; D008051:Lipid Bilayers; D010446:Peptide Fragments; D000135:Acid Phosphatase; D002402:Cathepsin D