Pepsin-solubilized collagen of human nucleus pulposus and annulus fibrosus.
Human nucleus pulposus and annulus fibrosus, obtained at autopsy from patients 7-30 years of age, were extracted with 2 M guanidine-HCl (pH 5.82) to remove proteoglycans, then stirred with pepsin in 0.5 M acetic acid, followed by three 24-h extractions with 1 M NaCl (pH 7.5) and one 24-h extraction with 2 M KSCN (potassium thiocyanate) (pH 7.2). Pepsin and NaCl solubilized an average of about 30% of nucleus pulposus collagen and 18% of annulus fibrosus collagen. KSCN extracted a further 34% of nucleus pulposus collagen and only 4% of annulus fibrosus collagen. CM-cellulose chromatography of nucleus and annulus collagen purified from the pepsin, NaCl and KSCN supernatants consistently revealed only one peak, always appearing slightly ahead of the alpha1 position for rat tail tendon type I collagen. Polyacrylamide and SDS-gel electrophoresis consistently revealed only one band with the mobility of alpha1 chains. Amino acid composition of collagen from nucleus and annulus is comparable to those of mammalian and avian cartilage type II collagen, and distinctly different from those of rat tail tendonand guinea pig skin type I collagens. Periodate oxidation of nucleus and annulus collagens showed that 81% and 67%, respectively, of the hydroxylysine residues survive treatment, compared to 71% for bovine articular cartilage collagen and 17% for guinea pig skin collagen. Total hexose analysis revealed 1.8 muM and 2.0 muM hexose per muM periodate-stable hydroxylysine in nucleus and annulus collagens, respectively. Ion exchange chromatography showed the presence of glucose and galactose in a ratio of 0.92:1 in nucleas collagen and 1.07:1 in annulus collagen. Pepsin-solubilized, NaCl-extracted collagen from nucleus and annulus formed native-type fibrils in vitro. The banding patterns of ATP-induced segment-long-spacing precipitates of nucleus and annulus collagens were identical to each other and indistinguishable from those of cartilage (type II) collagen, but distinctly different from those of rat tail tendon (type I) collagen. These data suggest that the collagen which can be extracted after limited pepsin attack of human nucleus and annulus is of the form [alpha1 (II)]3.
D000293:Adolescent; D000328:Adult; D000367:Age Factors; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001842:Bone and Bones; D002356:Cartilage / Q000032:analysis; D002417:Cattle; D002648:Child; D003094:Collagen / Q000302:isolation & purification; D006601:Hexoses / Q000032:analysis; D006801:Humans; D006909:Hydroxyproline / Q000032:analysis; D007403:Intervertebral Disc / Q000032:analysis*; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D009928:Organ Specificity; D010434:Pepsin A; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D013710:Tendons / Q000032:analysis
D000596:Amino Acids; D006601:Hexoses; D046911:Macromolecular Substances; D003094:Collagen; D010434:Pepsin A; D006909:Hydroxyproline