Characterization of bovine kappa-casein fractions and the kinetics of chymosin-induced macropeptide release from carbohydrate-free and carbohydrate-containing fractions determined by high-performance gel-permeation chromatography.
Bovine kappa-casein was fractionated at pH 8.0 on DEAE-Sepharose with an NaCl gradient, followed by DEAE-cellulose chromatography using a decreasing pH gradient from pH 6.0 to 4.5. At least ten components could be identified, each differing in N-acetylneuraminic acid (NeuAc) and/or phosphorus content. Two components appeared to be multiply-phosphorylated, but did not contain NeuAc. The possible significance of this finding in relation to the mode of phosphorylation and glycosylation in vivo is discussed. A carbohydrate-free fraction as well as two NeuAc-containing fractions were compared in their substrate behaviour towards the action of the milk-clotting enzyme chymosin at pH 6.6 and 30 degrees C. To this end the trichloroacetic acid-soluble reaction products were analysed by high-performance gel-permeation chromatography. In order of increasing carbohydrate content the kcat. values found ranged from 40 to 25 s-1 and the Km values from 9 to 3 microM; the overall substrate properties of these components as reflected by the kinetic parameter kcat./Km ranged from 5 to 8 microM-1 X S-1. Irreversible polymerization of the carbohydrate-free fraction brought about a more-than-2-fold increase in Km, the kcat. value remaining virtually constant. The kcat./Km found for the cleavage of whole kappa-casein at pH 6.6 was of the same magnitude as the kcat./Km found for the polymerized carbohydrate-free fraction (i.e. about 3 microM-1 X S-1). No indication of substrate inhibition was found for the carbohydrate-free fraction.
D000818:Animals; D002364:Caseins / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002850:Chromatography, Gel / Q000379:methods*; D012085:Chymosin / Q000494:pharmacology*; D003486:Cyanides / Q000378:metabolism; D006020:Glycopeptides / Q000302:isolation & purification / Q000378:metabolism*; D006031:Glycosylation; D007700:Kinetics; D019158:N-Acetylneuraminic Acid; D010446:Peptide Fragments / Q000032:analysis; D010455:Peptides / Q000032:analysis; D010766:Phosphorylation; D012794:Sialic Acids / Q000494:pharmacology; D012995:Solubility; D014238:Trichloroacetic Acid / Q000494:pharmacology
D002364:Caseins; D003486:Cyanides; D006020:Glycopeptides; D010446:Peptide Fragments; D010455:Peptides; D012794:Sialic Acids; C020251:kappa-casein glycomacropeptide; D014238:Trichloroacetic Acid; D012085:Chymosin; D019158:N-Acetylneuraminic Acid