[Preparation of human placenta microvillous membrane and the study of its transferrin receptor].
Human placenta microvillous membrane (PMM) was prepared by differential centrifugation and by sucrose gradient centrifugation, and the transferrin receptor (TfR) was studied using the receptor radioassay with 125I-transferrin as a radioligand. The factors affecting ligand-receptor binding reaction and the characteristics of TfR were also studied. The result showed that there was no significant differences (P > 0.1) in specific binding rates of 125I-transferrin to its receptor between the two membrane preparations, indicating that both of them could be used for TfR analysis, but the method of differential centrifugation was more simple and less time consuming. The study of TfR in sixty cases showed that the TfR binding sites on PMM and Bmax were 3.53 +/- 1.98 x 10(12) sites/mg membrane protein and 6.33 +/- 4.21 x 10(-12) mol/mg membrane protein, respectively. The Kd was 4.95 +/- 3.39 x 10(-9) mol/L, and the highest specific binding was 26% with nonspecific binding less than 3%. The conditions for ligand-receptor binding reaction were optimized when the concentrations of membrane protein and the 125I-transferrin in each test tube were 50 micrograms and 50,000 cpm (specific radioactivity being 2109 kBq/micrograms transferrin) respectively the incubation time was 30 min, and the concentration of polyethylene glycol added for separating B/F was 12% (W/V). transferrin binding to its receptor was characterized by high affinity, high specificity and being saturated.
Li
Q
Q
Research Laboratory of Pediatric Hematologic and Oncologic Disease, Second Affiliated Hospital.
Li
F
F
Liao
Q
Q
Luo
C
C
Yang
C
C
D002462:Cell Membrane / Q000737:chemistry; D002499:Centrifugation, Density Gradient; D002824:Chorionic Villi / Q000737:chemistry*; D005260:Female; D006801:Humans; D011869:Radioligand Assay; D011990:Receptors, Transferrin / Q000378:metabolism*
D011990:Receptors, Transferrin