Abstract
The beta 1-adrenergic receptor of turkey erythrocytes has been purified by a combination of affinity and high performance steric exclusion chromatography. These procedures provide preparations with specific activities of greater than 15,000 pmol/mg of protein with an overall recovery of approximately 30% of the receptor activity solubilized from membrane preparations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radioiodinated purified receptor reveals two bands of labeled protein with apparent Mr = 40,000 +/- 2,000 and 45,000 +/- 3,000 in a 3-4:1 ratio. These same two peptides can also be labeled specifically and in approximately the same ration in both membranes and purified preparations using the photoaffinity probe 125I-labeled p-azidobenzylcarazolol. When the two purified polypeptides are completely separated by high performance liquid chromatography and subjected to detailed ligand binding studies, identical beta 1-adrenergic specificities are found for the two receptor forms. Preliminary characterization of these two proteins by partial protease digestion suggests a large degree of similarity between them, albeit with some significant differences. These results demonstrate that both purification and photoaffinity labeling identify two polypeptides in turkey erythrocyte membranes as containing a beta 1-adrenergic receptor binding site. The functional and structural relationships of these two forms of the receptor remain to be elucidated.
Highlights
From the Howard Hughes Medical Institute Laboratory, Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina27710
We have and the samples were electrophoresed on 12% acrylamide gels as demonstrated that thetechniques of affinity chromatography described under "Methods." Untreated receptor preparations (Mr= 40,000 and 45,000) are shown in the two furthermost lefthand lanes
Untreated samples are shown in the and high performance liquid chromatography which have been successfullyapplied previously to thepurification of the &adrenergic receptor of the frog erythrocyte can be successfully used as well to purify the P1-adrenergic receptor from two lefthand lanes
Summary
Identical P1-adrenergic specificities are found for the two receptor forms. Sepharose CL-4B-alprenolol was preparedas described labeling identify two polypeptides in turkey erythro- earlier [8].Premixed SDS'-polyacrylamide gel electrophoresis standcyte membranesas containing a &adrenergic receptorards Using combinations of affinity chromatography and high performance liquid chromatography on steric exclusion columns, we have been successful in purifying the Pa-adrenergic receptor from frog erythrocytes [3, 4]. It consists entirely of M , = 58,000 subunits. $ To whom requests for reprints should be addressed at BOX3287, Duke University Medical Center, Durham, NC 27710
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