Tartrate-resistant acid phosphatase: three-dimensional structure and structure-based functional studies : studies on the enzyme using recombinant protein produced by baculovirus expression vector system in insect cells
1University of Oulu, Faculty of Medicine, Research Center for Molecular Endocrinology
2University of Oulu, Biocenter Oulu
|Online Access:||PDF Full Text (PDF, 1.1 MB)|
|Persistent link:|| http://urn.fi/urn:isbn:9514267761
|Publish Date:|| 2002-09-13
|Thesis type:||Doctoral Dissertation
|Defence Note:||Academic Dissertation to be presented with the assent of the Faculty of Medicine, University of Oulu, for public discussion in the Auditorium 9 of the University Hospital of Oulu, on September 13th, 2002, at 12 noon.
Professor Raili Myllylä
Professor Juha Rouvinen
Osteoporosis is a disease characterized by abnormalities in the amount and architectural arrangement of bone tissue, which leads to impaired skeletal strength and increased susceptibility to fractures. Type 5 tartrate-resistant acid phosphatase (TRACP, AcP5) has been suggested to participate directly in bone resorption.
In this study, baculovirus expression vector system in insect cells was used to gain large amounts of recombinant type 5 acid phosphatase for structure determination, structure-based functional studies and production of monoclonal antibodies. Active and inactive forms of the enzyme were separated from each other by cation-exchange chromatography, and characterized. The enzyme was crystallized and the three-dimensional structure was determined. Based on the three-dimensional structure of the active site five different enzyme variants were constructed, produced in insect cells, and purified. The wild type enzyme and the mutated forms were characterized, and their kinetic parameters were determined. The importance of amino acids that were expected to be essential for the acid phosphatase activity was confirmed. The acid phosphatase activity and reactive oxygen species generating activity of this dual enzyme proved to exploit different amino acids in their reaction mechanisms.
Further studies are needed to clarify the physiological substrates of TRACP in vivo. The findings of this study could form a base for construction of inhibitors for TRACP that could be useful therapeutic agents for osteoporosis and related bone disorders.
Acta Universitatis Ouluensis. D, Medica
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