Summary
Human urinary Tamm-Horsfall glycoprotein, which contains 28% carbohydrate, has a monomeric molecular weight of about 80,000 but is isolated from urine in the form of intertwining helical suprastructures with molecular weights greater than 107. The native glycoprotein was dissociated and denatured with 6 M guanidinium chloride and was subsequently renatured by dialysis against a Tris-HCl buffer. Using sedimentation equilibrium, the renatured glycoprotein was characterized by a\(M_{w_{cell} } \) of 256,800 and a\(M_{z_{cell} } \) of 356,000. The ratio,M z/M w, of 1.39 indicates some polydispersity with regard to molecular size. There was no evidence of helical suprastructures in the renatured glycoprotein as judged by electron microscopy. Ca2+ concentrations of up to 50 mM failed to precipitate the renatured glycoprotein; in contrast, the native glycoprotein is precipitated by Ca2+ concentrations between 5–10 mM. The circular dichroic spectrum of renatured Tamm-Horsfall glycoprotein was obtained, resolved, and tentative band assignments made. The spectrum, which is quite similar to that of native Tamm-Horsfall glycoprotein, exhibited negative extrema at 269 nm (due in large part to disulfides and tyrosines) and at 215 nm (due to proteinβ-structure and the N-acetylated hexosamines). Theα-helical content of the glycoprotein was estimated to be no more than 10% and the amount ofβ-structure to be about 33%; these values were not affected by the presence of Ca2+ (1 mM). A glycopeptide fraction (ca. 90% carbohydrate), prepared by extensive pronase digestion of the reduced, S-carboxymethylated glycoprotein, exhibited an ellipticity extremum at 212 nm of +4,750 deg · cm2/dmole, referred to the concentration of (N-acetylated) hexosamines and neuraminic acid.
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Research Career Development Awardee (AM-00055).
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Puett, D., Holladay, L.A. & Robinson, J.P. Circular dichroism of human urinary Tamm-Horsfall glycoprotein. Mol Cell Biochem 15, 109–116 (1977). https://doi.org/10.1007/BF01793332
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DOI: https://doi.org/10.1007/BF01793332