Stabilizing effect of lysosomal beta-galactosidase on the catalytic activity of protective protein/cathepsin A secreted by human platelets.
The 32/20-kDa two-chain form of protective protein/cathepsin A (CathA) secreted by human platelets was thermostable in the aggregation supernatant at acidic pH, but was denatured at neutral pH. Leupeptin partly protected the CathA against denaturation, which was not observed in the supernatant after depletion of the cosecreted lysosomal acid beta-galactosidase (beta-Gal) by affinity separation with p-aminophenylthiogalactose (PATG)-agarose beads even at pH 4.8. The purified recombinant human beta-Gal proteins, the 84-kDa precursor and 64-kDa mature-like enzyme (the tryptic product of the 84-kDa precursor), also protected the CathA against denaturation at neutral pH in part. Biospecific interaction analysis revealed that the CathA secreted by platelets dose dependently bound to the immobilized recombinant beta-Gal proteins. The association rate constant of CathA with the 64-kDa mature-like beta-Gal was 4.0 x 10(6) (M-1 s-1) at acidic pH, which was three times larger than that with the 84-kDa beta-Gal precursor. The calculated affinity constants for the enzyme molecules at acidic pH were approximately 1 x 10(9) (M-1), and those at neutral pH were two orders lower. These results first demonstrated that beta-Gal stabilizes the catalytic activity of CathA through direct binding in vitro. The affinity was shown to increase with removal of the carboxy-terminal domain of the beta-Gal precursor.
Itoh
K
K
Department of Clinical Genetics, The Tokyo Metropolitan Institute of Medical Science, Japan. itou@rinshoken.or.jp
Naganawa
Y
Y
Kamei
S
S
Shimmoto
M
M
Sakuraba
H
H
D001792:Blood Platelets / Q000201:enzymology* / Q000378:metabolism*; D002268:Carboxypeptidases / Q000097:blood* / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D043402:Cathepsin A; D002474:Cell-Free System; D004795:Enzyme Stability; D005110:Extracellular Space / Q000201:enzymology; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008247:Lysosomes / Q000201:enzymology*; D010974:Platelet Aggregation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D011994:Recombinant Proteins; D001616:beta-Galactosidase; D002268:Carboxypeptidases; C468629:CTSA protein, human; D043402:Cathepsin A