[An improved purification of human placental transferrin receptor and biochemical properties of the receptor].
An improved method for the purification of human placental transferrin receptor (Tf-R) was developed. Fresh human placenta was homogenized in cold acetone and the acetone powder was prepared. After the acetone powder had been washed with HEPES buffer, the insoluble proteins containing Tf-R were separated by centrifugation and dissolved in Emulgen 109P-containing buffer. Tf-R was collected by affinity binding to Tf-Sepharose and extracted by consecutive treatment with 4 different kinds of buffers. Tf-R was eluted by buffer C (2 M KCl) and buffer D (0.5 M NaSCN). Tf-R was characterized as a 90-kDa monomer on gradient SDS-PAGE (4-20%) in the presence of 2-mercaptoethanol. Though there were several minor bands of 180- and above 205-kDa, all these bands were confirmed as Tf-R by Western blotting using an anti-Tf-R monoclonal antibody (OKT 9). The apparent molecular weights, measured by HPLC using a TSK-G 3,000 SW column, demonstrated that Tf-Rs eluted with buffer C were approximately 370-, 500- and above 500-kDa, but only a peak of above 500-kDa was found in Tf-R eluted with buffer D. Although the polymers of Tf-R with molecular weight of above 500-kDa were resistant to trypsin digestion, the Tf-R of 370-kDa was resistant to the enzyme only when it conjugated to the diferric Tf. The stability of the polymers of above 500-kDa to trypsin digestion suggested an advantage for the repeated use of Tf-R in the endocytosis of diferric Tf, which was performed by the translocation of Tf-R between cell surface and intracellular vesicles.
Taguchi
M
M
Second Department of Biochemistry, Nippon Medical School, Tokyo, Japan.
D002851:Chromatography, High Pressure Liquid; D005260:Female; D006801:Humans; D008970:Molecular Weight; D010920:Placenta / Q000378:metabolism*; D011108:Polymers; D011247:Pregnancy; D011990:Receptors, Transferrin / Q000302:isolation & purification* / Q000378:metabolism; D014168:Transferrin / Q000378:metabolism; D014357:Trypsin
D011108:Polymers; D011990:Receptors, Transferrin; D014168:Transferrin; D014357:Trypsin