Surface labeling of thyrocytes isolated by a new method combining enzymatic digestion and percoll gradient centrifugation.
Thyrocytes were isolated from porcine thyroid glands using a new method entailing two-step collagenase digestion and Percoll gradient centrifugation. Good yield and a high percentage of viable thyrocytes free from other contaminating cells was achieved. Proteins present on the surface of thyroid cell plasma membranes were then specifically labeled using Iodogen and 125I-. Membrane lysates were separated by electrophoresis on 10% polyacrylamide gel, under reducing and nonreducing conditions, followed by autoradiography. When the gels were stained with silver nitrate some 30 bands were visualized in both the presence and absence of reductant. Only 9 bands were found to be labeled under nonreducing conditions and 12 in the presence of reductant. Two bands involved in the thyrotropin receptor structure--Mr = 66,000 and Mr = 70,000, respectively--were visualized in the absence of reductant. Upon reduction the Mr 66,000 band was retained and a new band (Mr = 33,000) was seen. The mild enzymatic treatment used in isolating thyrocytes and the lack of contamination with other cells allowed the consistent labeling of exposed plasma membrane components by the Iodogen method such that the orientation of thyrotropin receptor components in the plasma membrane could be deduced.
D000818:Animals; D002462:Cell Membrane / Q000032:analysis; D002469:Cell Separation / Q000379:methods; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D007457:Iodine Radioisotopes; D007553:Isotope Labeling / Q000379:methods*; D008565:Membrane Proteins / Q000032:analysis*; D003012:Microbial Collagenase; D013552:Swine; D013961:Thyroid Gland / Q000032:analysis* / Q000166:cytology; D014508:Urea / Q000031:analogs & derivatives
D007457:Iodine Radioisotopes; D008565:Membrane Proteins; C012385:1,3,4,6-tetrachloro-3 alpha,6 alpha-diphenylglycoluril; D014508:Urea; D003012:Microbial Collagenase