Characterizations of recombinant human tartrate-resistant acid phosphatase from osteosarcoma: comparison study between recombinant and placental proteins.
We cloned the human tartrate-resistant acid phosphatase (TRAP) gene from human osteosarcoma cells (Saos-2), and produced recombinant human TRAP (rhTRAP) using a baculovirus vector expression system. RhTRAP from Sf9 culture medium was purified by cation exchange chromatography, gel filtration and affinity chromatography. The molecular mass and amino acid composition of the rhTRAP were consistent with the deduced amino acid composition from the TRAP gene. The N-terminal amino acid sequence of rhTRAP was identical to that of TRAP purified from osteoclastoma and hairy cell leukemia spleen. The monoclonal antibodies generated against rhTRAP also reacted to human placental TRAP (pTRAP). The optimum pH of rhTRAP and pTRAP were pH 5.0-5.5 and pH 6.0-6.5, respectively. The enzymatic activities of rhTRAP and pTRAP were activated by reducing agents such as 2-mercaptoethanol, dithiothreitol and ascorbic acid. The activities of rhTRAP and pTRAP were enhanced by Fe2+ ions, but were inhibited by Fe3+ ions. The present results indicate that rhTRAP has similar properties to the native human TRAP, and suggest that the enhancement of TRAP activity by reducing agents might be expressed via the reduction of Fe ions at the metal center.
Miyazaki
S
S
Immunology Laboratory, Diagnostic Department, Yamasa Corporation, Choshi, Japan. miyazaki@yamasa.com
Igarashi
M
M
Nagata
A
A
Komoda
T
T
D000135:Acid Phosphatase / Q000737:chemistry* / Q000302:isolation & purification; D000328:Adult; D000596:Amino Acids / Q000032:analysis / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001483:Base Sequence; D001859:Bone Neoplasms / Q000201:enzymology*; D002460:Cell Line; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007313:Insecta / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry* / Q000302:isolation & purification; D051379:Mice; D008807:Mice, Inbred BALB C; D012516:Osteosarcoma / Q000201:enzymology*; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry; D019163:Reducing Agents / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000071681:Tartrate-Resistant Acid Phosphatase
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D007527:Isoenzymes; D012333:RNA, Messenger; D011994:Recombinant Proteins; D019163:Reducing Agents; D000135:Acid Phosphatase; C000605511:Acp5 protein, mouse; D000071681:Tartrate-Resistant Acid Phosphatase