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Rabbit tubular basement membrane. Isolation and analysis of polypeptides
Ist Teil von
The Journal of biological chemistry, 1979-10, Vol.254 (20), p.10503-10513
Ort / Verlag
United States: American Society for Biochemistry and Molecular Biology
Erscheinungsjahr
1979
Link zum Volltext
Quelle
Free E-Journal (出版社公開部分のみ)
Beschreibungen/Notizen
Renal tubules from rabbit kidneys were isolated from thin shavings of the kidney surface. Basement membrane was then prepared
following sonication of the isolated tubules. To insure preservation of the integrity of the basement membrane polypeptides,
the protease inhibitors, diisopropyl fluorophosphate, ethylenediaminetetraacetic acid, N-ethylmaleimide, and epsilon-amino-caproic
acid were used at all stages of the preparations. The optimal conditions of sonication and centrifugation were established
and the chemical composition of basement membrane prepared under these conditions was examined in detail. Glycine, hydroxyproline,
and hydroxylysine were found in concentrations of 206, 65, and 18 residues per thousand, respectively, in basement membrane
from young kidneys. About 38% of the basement membrane was found to be soluble in sodium dodecyl sulfate upon incubation at
90 degrees C, and to possess relatively low amounts of the amino acids characteristic of collagen. Electrophoretic analysis
of this fraction revealed that the major subunits ranged in approximate molecular weight from 18,500 to greater than 10(6).
When analyzed with disulfide bonds reduced, a molecular weight range from 31,000 to 275,000 was observed for this fraction.
The sodium dodecyl sulfate-insoluble fraction could be dissolved upon reduction and alkylation and its composition was enriched
in the amino acids characteristic of collagen. Polypeptides from this fraction were analyzed by electrophoresis in agarose
and in agarose-acrylamide gels. The approximate molecular weight of the smallest component was 164,000. Additional polypeptides
were observed whose molecular weights occurred in multimers of this component, up to 1.1 x 10(6), possibly indicating covalent
cross-linked multimers of a basic collagen-like polypeptide(s).