Characterization of casein phosphopeptides prepared using alcalase: determination of enzyme specificity.
Tryptic casein phosphopeptides containing the cluster sequence-Ser(P)-Ser(P)-Ser(P)-Glu-Glu- have been shown to stablize amorphous calcium phosphate at neutral and alkaline pH and be anticariogenic in various in vitro, animal and human experiments. Furthermore, metal ion complexes of the casein phosphopeptides (CPPs) have potential as dietetic supplements to increase the bioavailability of calcium, iron, and other essential metal ions. In this study, we have used a Ca2+/ethanol selective precipitation procedure to produce a range of phosphopeptides from an alcalase digest of whole casein. The CPPs released by alcalase were truncated relative to those which are released by trypsin. The peptides could be grouped into those containing the cluster sequence as well as the group of tri-, di-, and monophosphorylated peptides. The two groups contained a number of homologous peptides of varying lengths resulting from the broad specificity of alcalase. Alcalase was observed to cleave peptide bonds on the carboxyl side of Glu, Met, Leu, Tyr, Lys, and Gln; however, of the twenty-six different cleavage sites, seventeen contained a Glu in the P1 position and of these, fifteen contained a hydrophobic residue in either the P2' or P3' positions. Furthermore, of the twenty-six cleavage sites identified, twenty-two contained a hydrophobic residue in either the P2' or P3' positions. Of the four other sites cleaved by alcalase, two contained a hydrophobic residue in the P1' position and one a hydrophobic residue in the P1 position.
Adamson
N J
NJ
Biochemistry and Molecular Biology Unit, School of Dental Science, University of Melbourne, Australia.
Reynolds
E C
EC
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001682:Biological Availability; D002118:Calcium / Q000494:pharmacology; D002364:Caseins / Q000737:chemistry* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D019075:Electrophoresis, Capillary; D000431:Ethanol / Q000494:pharmacology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D010748:Phosphopeptides / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D013379:Substrate Specificity; D013381:Subtilisins / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
D000596:Amino Acids; D002364:Caseins; D010446:Peptide Fragments; D010748:Phosphopeptides; D000431:Ethanol; D013381:Subtilisins; D014357:Trypsin; D002118:Calcium