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JBC, Vol. 250, Issue 19, 7780-7787, Oct, 1975

Bovine renal glomerular basement membrane. Isolation and characterization of a glycoprotein component

M. Oho, P. Riquetti and B. G. Hudson

Bovine glomerular basement membrane was extracted with 6 M guanidinium chloride and the soluble material fractionated on a Bio-Gel A-1.5m column in 1% Na dodecyl-SO4. A single component was obtained by reduction of a selected column fraction with 2-mercaptoethanol followed by chromatography on an analytical Bio-Gel A-1.5m column and shown to be homogenous by electrophoresis and ultracentrifugation. It consists of 90% protein and 8.6% carbohydrate by weight. The amino acid composition is characterized by the presence of low amounts of hydroxyproline and hydroxylysine, and substantial amounts of aspartic acid, glutamic acid, half-cystine, and glycine. It contains all the monosaccharide constituents present in the whole basement membrane indicating the presence of both heteropolysaccharide and disaccharide units; the presence of the latter unit was demonstrated unequivocally by ion exchange chromatography. The component contains 1 heteropolysaccharide unit and 4 dissaccharide units/molecule of Mr equals 70,000. The molecular weight of component VII was determined by several methods. Molecular weight values of 68,000 +/- 3,000 and 72,000 +/- 2,000 were determined in 6 M guanidinium chloride by the methods of sedimentation equilibrium and gel filtration chromatography, respectively, and values of 136,000 +/- 3,100 and 140,000 +/- 2,000 were determined in 1% Na dodecyl-SO4 by the methods of polyacrylamide gel electrophoresis and gel filtration chromatography, respectively. Circular dichroism spectra indicate that component VII assumes a random coil conformation in 6 M guanidinium chloride and a more disordered conformation in 1% Na dodecyl-SO4 than standard proteins used in calibration of polyacrylamide gels and gel filtration column. These results indicate that the minimal molecular weight of component VII is about 70,000 and that the anomalous behavior in Na dodecyl-SO4 is due in part to its conformation.
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