Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1986 Nov 15;240(1):87–97. doi: 10.1042/bj2400087

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.

H J Vreeman, S Visser, C J Slangen, J A Van Riel
PMCID: PMC1147380  PMID: 3103611

Abstract

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.

Full text

PDF
87

Images in this article

88Fig. 1.
on p.88
90Fig. 2.
on p.90
92Fig. 4.
on p.92

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Brignon G., Chtourou A., Ribadeau-Dumas B. Preparation and amino acid sequence of human kappa-casein. FEBS Lett. 1985 Aug 19;188(1):48–54. doi: 10.1016/0014-5793(85)80872-3. [DOI] [PubMed] [Google Scholar]
  2. Carles C., Ribadeau Dumas B. Kinetics of the action of chymosin (rennin) on a peptide bond of bovine alpha s1-casein. Comparison of the behaviour of this substrate with that of beta- and kappa o-caseins. FEBS Lett. 1985 Jun 17;185(2):282–286. doi: 10.1016/0014-5793(85)80923-6. [DOI] [PubMed] [Google Scholar]
  3. DOWD J. E., RIGGS D. S. A COMPARISON OF ESTIMATES OF MICHAELIS-MENTEN KINETIC CONSTANTS FROM VARIOUS LINEAR TRANSFORMATIONS. J Biol Chem. 1965 Feb;240:863–869. [PubMed] [Google Scholar]
  4. Doi H., Kawaguchi N., Ibuki F., Kanamori M. Susceptibility of kappa-casein components to various proteases. J Nutr Sci Vitaminol (Tokyo) 1979;25(1):33–41. doi: 10.3177/jnsv.25.33. [DOI] [PubMed] [Google Scholar]
  5. Foltmann B., Pedersen V. B., Jacobsen H., Kauffman D., Wybrandt G. The complete amino acid sequence of prochymosin. Proc Natl Acad Sci U S A. 1977 Jun;74(6):2321–2324. doi: 10.1073/pnas.74.6.2321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. GIBBONS R. A., CHESSEMAN G. C. Action of rennin on casein: the function of the neuraminic acid residues. Biochim Biophys Acta. 1962 Jan 29;56:354–356. doi: 10.1016/0006-3002(62)90575-9. [DOI] [PubMed] [Google Scholar]
  7. Garnier J., Mocquot G., Ribadeau-Dumas B., Maubois J. L. Coagulation du lait par la présure: aspects scientifiques et technologiques. Ann Nutr Aliment. 1968;22(2):B495–B552. [PubMed] [Google Scholar]
  8. Hanes C. S. Studies on plant amylases: The effect of starch concentration upon the velocity of hydrolysis by the amylase of germinated barley. Biochem J. 1932;26(5):1406–1421. doi: 10.1042/bj0261406. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Mercier J. C., Maubois J. L., Poznanski S., Ribadeau-Dumas B. Fractionnement préparatif des caséines de vache et de brebis par chromatographie sur D.E.A.E. cellulose, en milieu urée et 2-mercaptoéthanol. Bull Soc Chim Biol (Paris) 1968;50(3):521–530. [PubMed] [Google Scholar]
  10. Mercier J. C. Phosphorylation of caseins, present evidence for an amino acid triplet code posttranslationally recognized by specific kinases. Biochimie. 1981 Jan;63(1):1–17. doi: 10.1016/s0300-9084(81)80141-1. [DOI] [PubMed] [Google Scholar]
  11. SCHMIDT D. G. STARCH-GEL ELECTROPHORESIS OF K-CASEIN. Biochim Biophys Acta. 1964 Aug 19;90:411–414. doi: 10.1016/0304-4165(64)90210-7. [DOI] [PubMed] [Google Scholar]
  12. Sanner T., Kovács-Proszt G. Kinetic studies on the aggregation of kappa-casein by Mucor pusillus protease. Biochim Biophys Acta. 1973 Mar 23;303(1):68–76. doi: 10.1016/0005-2795(73)90149-9. [DOI] [PubMed] [Google Scholar]
  13. Sinkinson G., Wheelock J. V. Carbohydrates of the glycopeptides released by the action of rennin on whole milk. Biochim Biophys Acta. 1970 Sep 22;215(3):517–521. doi: 10.1016/0304-4165(70)90102-9. [DOI] [PubMed] [Google Scholar]
  14. Slattery C. W. Variation in the glycosylation pattern of bovine kappa-casein with micelle size and its relationship to a micelle model. Biochemistry. 1978 Mar 21;17(6):1100–1104. doi: 10.1021/bi00599a025. [DOI] [PubMed] [Google Scholar]
  15. Visser S., Rollema H. S. Quantification of chymosin action on nonlabeled kappa-casein-related peptide substrates by ultraviolet spectrophotometry: description of kinetics by the analysis of progress curves. Anal Biochem. 1986 Mar;153(2):235–241. doi: 10.1016/0003-2697(86)90087-4. [DOI] [PubMed] [Google Scholar]
  16. Visser S., Van Rooijen P. J., Schattenkerk C., Kerling K. E. Peptide substrates for chymosin (rennin). Kinetic studies with bovine kappa-casein-(103-108)-hexapeptide analogues. Biochim Biophys Acta. 1977 Mar 15;481(1):171–176. doi: 10.1016/0005-2744(77)90148-6. [DOI] [PubMed] [Google Scholar]
  17. Visser S., Van Rooijen P. J., Schattenkerk C., Kerling K. E. Peptide substrates for chymosin (rennin). Kinetic studies with peptides of different chain length including parts of the sequence 101-112 of bovine k-casein. Biochim Biophys Acta. 1976 Jun 7;438(1):265–272. doi: 10.1016/0005-2744(76)90242-4. [DOI] [PubMed] [Google Scholar]
  18. Vreeman H. J., Both P., Brinkhuis J. A., van der Spek C. Purification and some physicochemical properties of bovine kappa-casein. Biochim Biophys Acta. 1977 Mar 28;491(1):93–103. doi: 10.1016/0005-2795(77)90044-7. [DOI] [PubMed] [Google Scholar]
  19. Vreeman H. J., Brinkhuis J. A., van der Spek C. A. Some association properties of bovine SH-kappa-casein. Biophys Chem. 1981 Oct;14(2):185–193. doi: 10.1016/0301-4622(81)85018-1. [DOI] [PubMed] [Google Scholar]
  20. WARREN L. The thiobarbituric acid assay of sialic acids. J Biol Chem. 1959 Aug;234(8):1971–1975. [PubMed] [Google Scholar]
  21. Yamauchi K., Azuma N., Kobayashi H., Kaminogawa S. Isolation and properties of human kappa-casein. J Biochem. 1981 Oct;90(4):1005–1012. doi: 10.1093/oxfordjournals.jbchem.a133552. [DOI] [PubMed] [Google Scholar]
  22. van Halbeek H., Dorland L., Vliegenthart J. F., Fiat A. M., Jolles P. A 360-MHz 1H-NMR study of three oligosaccharides isolated from cow kappa-casein. Biochim Biophys Acta. 1980 Jun 26;623(2):295–300. doi: 10.1016/0005-2795(80)90257-3. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES