Isolation and characterization of pepsin-solubilized human basement membrane (type IV) collagen peptides.
Native type IV collagen was isolated from human placental tissue by pepsin digestion, fractional salt precipitation, reduction and alkylation, a second pepsin digestion, and chromatography on diethylaminoethyl- and carboxymethyl-cellulose. After denaturation, 10 distinct peptides were isolated from this material by molecular sieve, ion-exchange, and high-performance liquid chromatography. All of the peptides were found to have amino acid compositions characteristic of type IV collagen. Analysis of the eight major peptides by amino-terminal amino acid sequencing and by cyanogen bromide and tryptic peptide mapping has revealed the manner in which they are derived from type IV collagen. Pepsin liberates two large peptides by attacking non-triple-helical regions, one derived from the alpha 1 (IV) chain (F2, Mr 90 000) and one derived from the alpha 2 (IV) chain (F3, Mr 75 000). The alpha 1 (IV)-derived F2 peptide is also represented in the pepsin digest by amino-terminal and carboxy-terminal subfragments [F4c (Mr 41 000) and F4a (Mr 60 000)], as is the alpha 2 (IV)-derived F3 peptide [F5 (Mr 28 000) and F4b (Mr 50 000), respectively]. These findings indicate that the molecular regions from which the larger peptides are derived in themselves contain pepsin-sensitive (non-triple-helical) domains. In addition, several of the peptides examined were found to be present in two slightly different forms, suggesting that closely adjacent pepsin-sensitive sites often exist within the type IV collagen molecules. The methods outlined here provide a reliable means by which identifiable type IV collagen peptides can be isolated.(ABSTRACT TRUNCATED AT 250 WORDS)
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001485:Basement Membrane / Q000032:analysis*; D003094:Collagen / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D046911:Macromolecular Substances; D010434:Pepsin A; D010446:Peptide Fragments / Q000032:analysis; D010455:Peptides / Q000302:isolation & purification*; D010920:Placenta / Q000032:analysis*; D011247:Pregnancy; D012995:Solubility; D014357:Trypsin
D000596:Amino Acids; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010455:Peptides; D003094:Collagen; D014357:Trypsin; D010434:Pepsin A