[Studies of progestin specific binding protein in the human prostate (II): The possibility of non-specific binding protein disturbance for the accurate quantitative assay of 4S specific binding protein].
The R5020 specific binding protein in the human prostate was studied to elucidate the cause for the poorer reproducibility of 4S high affinity complex sediment than that of 7-8S high affinity complex sediment. The sucrose density gradient, with a low ionic strength buffer including sodium molybdate, was used. Charcoal assay is one of the most common methods used in steroid receptor studies. However, there is a possibility that the high amount of non-specific binding protein common in the human prostate disturbs the charcoal function which removes the free and also the loosely bound steroids from low affinity protein. In the sucrose density gradient process, charcoal treatment is necessary to obtain the apparent 7-8S peak from the 4S one because 7-8S is covered with huge 4S when the charcoal treatment is not performed. 4S complex is proved to be more sensitive than 7-8S to this form of treatment in this study. In addition, the R5020 specific binding protein is found in not only 7-8S complex but also 4S. The dissociation constant of 4S protein is identified with that in 7-8S in the low range of [3H]-R5020(0.4-5.2 nM) using Scatchard plots, but not in the high range (1.3-10.4 nM). Thus, it seems possible that the high amount of non-specific binding protein disturbs the accurate quantification of specific binding protein in 4S. In conclusion, the poor reproducibility of 4S complex through charcoal treatment is caused by the presence of the high amount of non-specific binding protein in the human prostate.
K
K
Imai
E
E
Takahashi
Y
Y
Takahashi
M
M
Kobayashi
F
F
Kumasaka
H
H
Yamanaka
D000818:Animals; D002352:Carrier Proteins / Q000032:analysis* / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002606:Charcoal; D006224:Cricetinae; D005260:Female; D006801:Humans; D008297:Male; D008722:Methods; D009649:Norpregnadienes / Q000378:metabolism*; D011372:Progestins / Q000378:metabolism; D011397:Promegestone / Q000032:analysis / Q000378:metabolism*; D011467:Prostate / Q000032:analysis*; D011470:Prostatic Hyperplasia / Q000378:metabolism
D002352:Carrier Proteins; D009649:Norpregnadienes; D011372:Progestins; D002606:Charcoal; D011397:Promegestone