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Glycosylated κ-caseins and the sizes of bovine casein micelles. Analysis of the different forms of κ-casein
Biochimica et Biophysica Acta
830 (1985) 213-215
213
Elsevier BBA30102
BBA Report
G l y e o s y l a t e d K-caseins and the sizes of b o v i n e casein micelles. A n a l y s i s of the different f o r m s of K-casein D.G. Dalgleish The Hannah Research Institute. dyr. KA6 5 H L ( U.K.)
(Received March 25th. 1985)
Key words: x-Casein;Casein micelle; Glycosylation;Protein separation; (Bovinemilk)
Fast protein liquid chromatography (FPLC) of the K-casein from bovine casein micelles of different sizes is used to demonstrate that the proportions of glycosylated and non-glycoslated forms of K-casein do not vary with micellar size. The results suggest that glycosylated K-casein is distributed similarly to unglycosylated r-casein within the mieellar structure.
The proportion of the stabilizing protein, Kcasein, in bovine casein micelles decreases as the diameter of the micelle increases [1-3], which provides circumstantial evidence for the surface location of K-casein. However, the position of glycosylated forms of K-casein (comprising more than half of the total x-casein) appears to be in doubt. Schmidt and Both [4] find that the K-casein is almost entirely on the surfaces of synthetic casein micelles, using gold-labelling and electron microscopy. It has been claimed that the largest native casein micelles contain a greater proportion of glycosylated K-casein than the smaller ones, that the x-casein is distributed throughout the miceilar structure [5,6], but that large rnicelles may have surfaces enriched with glyco-x-casein [6]. In one case, glycosylated x-casein was hardly detectable in small micelles fractionated on controlled-pore glass (CPG). Conversely, Slattery [8] suggested that the proportion of glycosylated x-casein was inversely related to size apart from the micelles of largest diameter, although Creamer [9] found a higher proportion of glycosylated material in the larger micelles, and Donnelly et al. [2] found no apparent difference in CPG-fractionated micelles. To attempt to resolve these differences, we have
used fast protein liquid chromatography (FPLC) to analyse the x-caseins contained in eight fractions of micelles of different sizes derived from milk by differential centrifugation. Milk was taken from the bulk tank of the Institute farm immediately after morning milking and was skimmed by centrifugation at 3000 rpm at 20°C. The fat layer was removed, and the micellar fraction was further fractionated into eight fractions, with average radii between 50 and 140 nm, by differential centrifugation at 20°C. Each micellar fraction was dispersed in ultrafiltrate from the same milk. Samples (0.5 ml) were added to 0.5 g solid urea and 10/tl of fl-mercaptoethanol, 100/~l of 1 M NaOH and 100 ~al of 0.4 M EDTA to dissociate the micelles and break down K-casein polymers [10], and were then diluted with 2.5 ml of 8 M urea/10 mM imidazole (pH 7.0). These solutions were filtered through a cellulose acetate filter (Millipore) of pore size 0.22 ~tm. These were analyzed for the x-casein using a Pharmacia FPLC system with a Mono-Q (anion exchange) HR-5 column. 200 #l of sample was applied to the column and a preliminary wash was given, using 3 ml 3.3 M urea/10 mM imidazole (pH 7.0) (buffer A). A gradient of 0 to 0.3 M NaCl in buffer A was
0167-4838/85/$03.30 ~ 1985 ElsevierScience Publishers B.V. (Biomedical Division)
214
run over 12 ml, after which the column was washed with 3 ml 1 M NaCI in buffer A followed by 3 ml of buffer A. The z-average radii of the micellar fractions which were used were determined by photon correlation spectroscopy [11], using a scattering angle of 90 ° only, with an He-Ne laser as the light source. Radii of the particles were calculated from the correlation functions by the method of cumulants [12] and the Stokes-Einstein formula. Chromatograms for samples from the micellar fractions 1 and 8 are shown in Fig. 1. The x-casein eluted as several peaks, containing both the A and B genetic variants and their glycosylated forms. The peaks A0 and B0 were identified by comparison with samples contained only x-caseins A or B. These two peaks containing unglycosylated rcasein, being the species of lowest charge. The average areas of the A0 and B0 peaks were in the ratio 1:0.28, in agreement with the expected gene frequency in the Friesian breed of 0.25 [13]. The other peaks identified as r-casein fractions (labelled 1-4) represent the glycosylated material and r-casein containing two phosphoserine residues [14]. All of the peaks A0, B0 and 1-4 were destroyed by rennet, confirming that they were all indeed r-caseins. After renneting, a small residual peak was left in the area occupied by r-casein, which is possibly the y:-casein fraction [15], and the complex of peaks which elute before the B0 peak was also unaffected by rennet: these were taken to be the 71-caseins. The chromatograms of the two fractions from the extremes of the centrifugation process were very similar (Fig. 1), apart from small but not significant differences in the r-casein peaks 3 and 4 (the most highly-charged fractions). The individual peaks in the chromatogram were integrated and the sums of A0 and B0, and peaks 1-4 were calculated and expressed as fractions of the total r-casein. Within a close tolerance, no differences were found between the different micellar fractions (Table I). Thus, over a 2.5-fold change in micellar radius, there did not appear to be a marked difference in the distribution of the glycosylated and unglycosylated forms of r-caseins. These results agree with the estimate of Donnelly et al. [2] on the invariance of the r-casein fractions, but not with the results of Slattery [8],
b
E c 0
oO 04
03 J~ u)
k,_
w
10
15
10
15
E l u t i o n V o l u m e (ml)
Fig. 1. FPLC chromatograms of x-caseins from (a) fraction 1 (largest micelles) and (b) fraction 8 (smallest micelles) on Mono-Q column (pH 7.0). The different fractions are identified as in the text. The shaded region in each chromatogram is the elution profile of exhaustively-renneted micelles in each size class.
Horisberger and Rouvet-Vauthy [5], Creamer et al. [9] or Yoshikawa et al. [7]. Of these studies, one is indirect, in so far as the conclusions derive from specifically stained electron micrographs: this is also true for the results of Kudo et al. [6]. These two studies differed in their conclusions, one [6] favouring a surface location for the glycosylated x-casein in larger micelles, but interior locations in smaller micelles, and the other [5] favouring the interior. The studies of Slattery [8] and Yoshikawa et al. [7] also differed, since Slattery estimated that, apart from the largest micelles, the proportion of glycosylated r-casein was inversely related to the miceile size, whereas Yoshikawa et al. [7] claimed that large micelles were highly glycosylated compared with small ones. Both of these studies used the scanning of unstained [8] or stained [7] polyacrylamide gels after electrophoresis of the caseins to quantitate the individual forms of x-casein. The results shown above cannot describe the location of the x-casein in the micelles: they do
215
References
TABLE I FRACTIONS OF UNGLYCOSYLATED AND GLYCOSYLATED K-CASEIN IN DIFFERENT SIZE-CLASSES OF MICELLES Micellar fraction
Radius (nm)
Unglycosylated
Glycosylated
1 2 3 4 5 6 7 8
n.d. 120 110 95 85 70 60 55
0.481 0.489 0.482 0.483 0.488 0.487 0.487 0.465
0.519 0.511 0.518 0.517 0.512 0.513 0.513 0.535
confirm that since there is no change in the glycox-casein/x-casein ratio with micellar size, the glyco-r-casein cannot be located preferentially either in the interior or on the exterior of the micelle. There may be x-casein in the interior of micelles, but any distribution of r-casein between the interior and exterior of the micelles must, according to our analyses, affect all r-casein fractions to the same extent. The author would like to thank Dr. D.T. Davies and Mr. A.J.R. Law for helpful discussions, and Miss A. Nelson for skilled technical assistance.
1 McGann, T.C.A., Donnelly, W.J., Kearney, R.D. and Buchheim, W. (1980) Biochim. Biophys. Acta 630, 261-270 2 Donnelly, W.J., McNeill, G.P., Buchheim, W. and McGann, T.C.A. (1984) Biochim. Biophys. Acta 789, 136-143 3 Davies, D.T. and Law, A.J.R. (1983) J. Dairy Res. 50, 67-75 4 Schmidt, D.G. and Both. P. (1982) Milchwissenschaft 37, 336-337 5 Horisberger, M. and Rouvet-Vauthy, M. (1984) Histochemistry 80, 523-526 6 Kudo, S., lwata, S. and Mada, M. (1979) J. Dairy Sci. 62, 916-920 7 Yoshikawa, M., Takeuchi, M., Sasaki, R. and Chibar, H. (1982) Agric. Biol. Chem. 46, 1043-1048 8 Slattery, C.W. (1978) Biochemistry 17, 1100-1104 9 Creamer, L.K., Wheelock, J.V. and Samuel, D. (1973) Biochim. Biophys. Acta 317, 202-206 10 Woychik, J.H., Kalan, A.B. and Noelken, M.E. (1966) Biochemistry 7, 2276-2282 11 Chu, B. (1974) Laser Light Scattering, Academic Press,, new York 12 Koppel, D.E. (1972) J. Chem. Phys. 57, 4814-4820 13 Aschaffenburg, R. (1968) J. Dairy Res. 35, 447-460 14 Vreeman, H.J., Both, P., Brinkhuis, J.A. and Van der Spek. C. (1977) Biochim. Biophys. Acta 491, 93-103 15 Swaisgood, H.E. (1982) in Developments in Dairy Chemistry, Vol. 1 (Fox, P.F., ed.), Ch. 1, Applied Science Publishers, Barking
830 (1985) 213-215
213
Elsevier BBA30102
BBA Report
G l y e o s y l a t e d K-caseins and the sizes of b o v i n e casein micelles. A n a l y s i s of the different f o r m s of K-casein D.G. Dalgleish The Hannah Research Institute. dyr. KA6 5 H L ( U.K.)
(Received March 25th. 1985)
Key words: x-Casein;Casein micelle; Glycosylation;Protein separation; (Bovinemilk)
Fast protein liquid chromatography (FPLC) of the K-casein from bovine casein micelles of different sizes is used to demonstrate that the proportions of glycosylated and non-glycoslated forms of K-casein do not vary with micellar size. The results suggest that glycosylated K-casein is distributed similarly to unglycosylated r-casein within the mieellar structure.
The proportion of the stabilizing protein, Kcasein, in bovine casein micelles decreases as the diameter of the micelle increases [1-3], which provides circumstantial evidence for the surface location of K-casein. However, the position of glycosylated forms of K-casein (comprising more than half of the total x-casein) appears to be in doubt. Schmidt and Both [4] find that the K-casein is almost entirely on the surfaces of synthetic casein micelles, using gold-labelling and electron microscopy. It has been claimed that the largest native casein micelles contain a greater proportion of glycosylated K-casein than the smaller ones, that the x-casein is distributed throughout the miceilar structure [5,6], but that large rnicelles may have surfaces enriched with glyco-x-casein [6]. In one case, glycosylated x-casein was hardly detectable in small micelles fractionated on controlled-pore glass (CPG). Conversely, Slattery [8] suggested that the proportion of glycosylated x-casein was inversely related to size apart from the micelles of largest diameter, although Creamer [9] found a higher proportion of glycosylated material in the larger micelles, and Donnelly et al. [2] found no apparent difference in CPG-fractionated micelles. To attempt to resolve these differences, we have
used fast protein liquid chromatography (FPLC) to analyse the x-caseins contained in eight fractions of micelles of different sizes derived from milk by differential centrifugation. Milk was taken from the bulk tank of the Institute farm immediately after morning milking and was skimmed by centrifugation at 3000 rpm at 20°C. The fat layer was removed, and the micellar fraction was further fractionated into eight fractions, with average radii between 50 and 140 nm, by differential centrifugation at 20°C. Each micellar fraction was dispersed in ultrafiltrate from the same milk. Samples (0.5 ml) were added to 0.5 g solid urea and 10/tl of fl-mercaptoethanol, 100/~l of 1 M NaOH and 100 ~al of 0.4 M EDTA to dissociate the micelles and break down K-casein polymers [10], and were then diluted with 2.5 ml of 8 M urea/10 mM imidazole (pH 7.0). These solutions were filtered through a cellulose acetate filter (Millipore) of pore size 0.22 ~tm. These were analyzed for the x-casein using a Pharmacia FPLC system with a Mono-Q (anion exchange) HR-5 column. 200 #l of sample was applied to the column and a preliminary wash was given, using 3 ml 3.3 M urea/10 mM imidazole (pH 7.0) (buffer A). A gradient of 0 to 0.3 M NaCl in buffer A was
0167-4838/85/$03.30 ~ 1985 ElsevierScience Publishers B.V. (Biomedical Division)
214
run over 12 ml, after which the column was washed with 3 ml 1 M NaCI in buffer A followed by 3 ml of buffer A. The z-average radii of the micellar fractions which were used were determined by photon correlation spectroscopy [11], using a scattering angle of 90 ° only, with an He-Ne laser as the light source. Radii of the particles were calculated from the correlation functions by the method of cumulants [12] and the Stokes-Einstein formula. Chromatograms for samples from the micellar fractions 1 and 8 are shown in Fig. 1. The x-casein eluted as several peaks, containing both the A and B genetic variants and their glycosylated forms. The peaks A0 and B0 were identified by comparison with samples contained only x-caseins A or B. These two peaks containing unglycosylated rcasein, being the species of lowest charge. The average areas of the A0 and B0 peaks were in the ratio 1:0.28, in agreement with the expected gene frequency in the Friesian breed of 0.25 [13]. The other peaks identified as r-casein fractions (labelled 1-4) represent the glycosylated material and r-casein containing two phosphoserine residues [14]. All of the peaks A0, B0 and 1-4 were destroyed by rennet, confirming that they were all indeed r-caseins. After renneting, a small residual peak was left in the area occupied by r-casein, which is possibly the y:-casein fraction [15], and the complex of peaks which elute before the B0 peak was also unaffected by rennet: these were taken to be the 71-caseins. The chromatograms of the two fractions from the extremes of the centrifugation process were very similar (Fig. 1), apart from small but not significant differences in the r-casein peaks 3 and 4 (the most highly-charged fractions). The individual peaks in the chromatogram were integrated and the sums of A0 and B0, and peaks 1-4 were calculated and expressed as fractions of the total r-casein. Within a close tolerance, no differences were found between the different micellar fractions (Table I). Thus, over a 2.5-fold change in micellar radius, there did not appear to be a marked difference in the distribution of the glycosylated and unglycosylated forms of r-caseins. These results agree with the estimate of Donnelly et al. [2] on the invariance of the r-casein fractions, but not with the results of Slattery [8],
b
E c 0
oO 04
03 J~ u)
k,_
w
10
15
10
15
E l u t i o n V o l u m e (ml)
Fig. 1. FPLC chromatograms of x-caseins from (a) fraction 1 (largest micelles) and (b) fraction 8 (smallest micelles) on Mono-Q column (pH 7.0). The different fractions are identified as in the text. The shaded region in each chromatogram is the elution profile of exhaustively-renneted micelles in each size class.
Horisberger and Rouvet-Vauthy [5], Creamer et al. [9] or Yoshikawa et al. [7]. Of these studies, one is indirect, in so far as the conclusions derive from specifically stained electron micrographs: this is also true for the results of Kudo et al. [6]. These two studies differed in their conclusions, one [6] favouring a surface location for the glycosylated x-casein in larger micelles, but interior locations in smaller micelles, and the other [5] favouring the interior. The studies of Slattery [8] and Yoshikawa et al. [7] also differed, since Slattery estimated that, apart from the largest micelles, the proportion of glycosylated r-casein was inversely related to the miceile size, whereas Yoshikawa et al. [7] claimed that large micelles were highly glycosylated compared with small ones. Both of these studies used the scanning of unstained [8] or stained [7] polyacrylamide gels after electrophoresis of the caseins to quantitate the individual forms of x-casein. The results shown above cannot describe the location of the x-casein in the micelles: they do
215
References
TABLE I FRACTIONS OF UNGLYCOSYLATED AND GLYCOSYLATED K-CASEIN IN DIFFERENT SIZE-CLASSES OF MICELLES Micellar fraction
Radius (nm)
Unglycosylated
Glycosylated
1 2 3 4 5 6 7 8
n.d. 120 110 95 85 70 60 55
0.481 0.489 0.482 0.483 0.488 0.487 0.487 0.465
0.519 0.511 0.518 0.517 0.512 0.513 0.513 0.535
confirm that since there is no change in the glycox-casein/x-casein ratio with micellar size, the glyco-r-casein cannot be located preferentially either in the interior or on the exterior of the micelle. There may be x-casein in the interior of micelles, but any distribution of r-casein between the interior and exterior of the micelles must, according to our analyses, affect all r-casein fractions to the same extent. The author would like to thank Dr. D.T. Davies and Mr. A.J.R. Law for helpful discussions, and Miss A. Nelson for skilled technical assistance.
1 McGann, T.C.A., Donnelly, W.J., Kearney, R.D. and Buchheim, W. (1980) Biochim. Biophys. Acta 630, 261-270 2 Donnelly, W.J., McNeill, G.P., Buchheim, W. and McGann, T.C.A. (1984) Biochim. Biophys. Acta 789, 136-143 3 Davies, D.T. and Law, A.J.R. (1983) J. Dairy Res. 50, 67-75 4 Schmidt, D.G. and Both. P. (1982) Milchwissenschaft 37, 336-337 5 Horisberger, M. and Rouvet-Vauthy, M. (1984) Histochemistry 80, 523-526 6 Kudo, S., lwata, S. and Mada, M. (1979) J. Dairy Sci. 62, 916-920 7 Yoshikawa, M., Takeuchi, M., Sasaki, R. and Chibar, H. (1982) Agric. Biol. Chem. 46, 1043-1048 8 Slattery, C.W. (1978) Biochemistry 17, 1100-1104 9 Creamer, L.K., Wheelock, J.V. and Samuel, D. (1973) Biochim. Biophys. Acta 317, 202-206 10 Woychik, J.H., Kalan, A.B. and Noelken, M.E. (1966) Biochemistry 7, 2276-2282 11 Chu, B. (1974) Laser Light Scattering, Academic Press,, new York 12 Koppel, D.E. (1972) J. Chem. Phys. 57, 4814-4820 13 Aschaffenburg, R. (1968) J. Dairy Res. 35, 447-460 14 Vreeman, H.J., Both, P., Brinkhuis, J.A. and Van der Spek. C. (1977) Biochim. Biophys. Acta 491, 93-103 15 Swaisgood, H.E. (1982) in Developments in Dairy Chemistry, Vol. 1 (Fox, P.F., ed.), Ch. 1, Applied Science Publishers, Barking