THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 259, No. 8, Issue of April 25, pp. 5132-5138, 1984 Prmted in U S.A

Human ,&Casein AMINO ACID SEQUENCE AND IDENTIFICATION OF PHOSPHORYLATION SITES*

(Received for publication, October 12, 1983)

Rae Greenberg, Merton L. Groves, and Harold J. Dower From the Eastern Regional Research Center, United States Department of Agriculture, Philadelphia, Pennsylvania 19118

The primary structure of human &casein has been determined by automated Edman degradation of the intact protein and of peptides derived therefrom by hydrolysis with trypsin and by chemical cleavage with cyanogen bromide. For each form of this multiphos- phorylated protein (0-5 P/molecule), phosphorylated sites at specific seryl and threonyl residues have been identified. These are located near the amino terminus, within the first 10 residues of this 212-amino acid molecule. Sequence comparison of human &casein with the bovine and ovine proteins reveals 5090 iden- tity and a 10-residue shifted alignment relationship. Locations of prolyl and charged residues are generally conserved for the three homologues. The sequence data indicate the existence of genetic polymorphism involv- ing uncharged residues in human ,&casein.

Mature human milk, the food widely recommended for the full term infant by the nutritional and pediatric communities (1, a), contains 0.9% protein (3), of which the casein fraction comprises 30-35% (4). The major component of human casein has an apparent electrophoretic and compositional similarity to @-casein from the well characterized bovine milk system (5,6) which contains 3.3% protein, 80% casein, 38% of which is @-casein. Detailed analyses of the human casein fraction including structure and physicochemical properties of 0-cas- ein are lacking. At the molecular level, human and bovine @- casein differ significantly, the most obvious difference being the contrast in phosphorylation state. Human @-casein occurs in multiphosphorylated forms having 0-5 phosphate groups/ molecule (5, 6), while the bovine homologue is usually found as a fully phosphorylated molecule. Since milk caseins repre- sent the prime source of calcium and phosphorus for the neonate, differences in the concentration and availability of these elements may have implications in the feeding of banked human milk to preterm or at-risk infants (7-10).

To characterize more fully human @-casein at the molecular level, this report presents the complete amino acid sequence of the 212-residue protein and the identification of phosphor- ylation sites in the individual forms of this multiphosphory- lated protein. A comparison of the primary structures of human, bovine ( l l ) , and ovine (12) @-caseins reveals both sequence homology and an interesting shifted alignment re- lationship. The sequence data also indicate the existence of genetic polymorphism in human 0-casein. Brief accounts of portions of this work have appeared (13, 14).

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

51

EXPERIMENTAL PROCEDURES]

RESULTS

The p-casein fraction of human milk, isolated by Groves and Gordon ( 5 ) , contains six electrophoretic bands as shown in Fig. 1. This complex was fractionated and after purification by DEAE-cellulose column chromatography, each of the in- dividual components was found to be identical in amino acid composition ( 5 ) . These proteins differed only in phosphorus content, values ranging from 0 to 5 mol/mol as indicated in Fig. 1. Previous results from this laboratory (14) had shown that the nonphosphorylated form contained a sequence cluster identical to the known phosphorylated regions of bovine cys1-

and @-caseins (15). This suggested that the phosphorylated residues were located near the amino terminus of the mole- cule. Sequence examinations of the individual 2-P and 4-P 0- casein forms confirmed this (14). I t was thus determined that each of the multiphosphorylated forms contained phosphate groups on specific seryl or threonyl residues and were not mixtures of species having a certain number of phosphate groups randomly distributed. Using the 0-P amino-terminal sequence as a guide, phosphorus was located in each of the other forms as described under “Experimental Procedures.” In all five cases phosphate groups were quantitatively located at specific sites within the first 10 residues (Fig. 2). A complete explanation of the 1-P case is presented in the discussion.

The amino acid.sequence of human @-casein and the strat- egy employed in its elucidation are shown in Fig. 3. The basis for this sequence was provided by the arrangement of cyano- gen bromide-cleaved and trifluoroacetyl-blocked trypsin cleaved peptides reported earlier (13) and described in Fig. 1s and Table IS. Peptide CB-1 was digested with trypsin yielding two fragments, CB-1-T1 and CB-l-T2 (Table IS). The first peptide corresponded to residues 1-20 and the second, a larger fragment, to the remainder of CB-1, residues 24-84. The tripeptide 21-23 (Val-Glu-Lys) was not recovered from this digest. Although CB-1 contains five lysyl residues, primary cleavage occurred at the two Lys-Val bonds.

The amino-terminal third of the molecule is encompassed by tryptic fragments T-1A and T-lB, originally thought to have arisen from cleavage of an incompletely blocked lysyl residue (13). However, we can now deduce that despite the

Portions of this paper (including “Experimental Procedures,” Figs. IS and 2S, and Tables 1S-8S) are presented in miniprint at the end of this paper. The abbreviations used are: PTH, phenylthio- hydantoin; HPLC, high performance liquid chromatography. Mini- print is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chem- istry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 83M-2934, cite the authors, and include a check or money order for $7.20 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

32

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Sequence of Human &Casein 5133

I I_ I-P

FIG. 1. Polyacrylamide gel electrophoretic pattern (pH 9.5, 4 M urea) of human @-casein. 0- to 5-P indicates level of phos- phorylation.

use of L-1 -tosylamido-2-phenylethyl chloromethyl ketone- treated tr-ypsin in the digest, these peptides are the result of a chymotryptic-type split between leucine 49 and isoleucine 50. When the smallest cyanogen bromide peptide, CB-2, was subjected to automated Edman analysis, the first 14 of its 16 residues were identified and the terminal arginine of T-1B thereby located. The two remaining amino acids of CB-2 by composition, valine and methionine, serve as overlaps to the beginning of the next large fragment, T-2. Considering the accurate composition of CB-2, the fact that methionine must be the carboxyl-terminal residue and that arginine 98 marks the cleavage point between T-1B and T-2, it is most unlikely that any residues have been missed here.

Elucidation of the last third of the human @-casein molecule presented a somewhat unique problem when attempts at automated Edman degradation of CB-4 failed. It was obvious that the amino terminus was blocked, the most likely culprit being pyrrolidonecarboxylic acid (16) arising from the cycli- zation of glutamine 136. Treatment with pyrrolidonecarbox- ylyl peptidase as recommended by Doolittle (17) was unsuc-

1 5 10 15 NH2-ARG-GLU-THR-ILE-GLU-SER-LEU-SER-SER-S~R-GLU-GLU-SER-ILE-PRO-

, I I 4

P P

NH2-ARG-GLU-THR-ILE-GLU-SER-LEU-SER-SER-SER-GLU-GLU-SER-ILE-PRO- I I P P

NH2-ARG-GLU-THR-ILE-GLU-SER-LEU-SER-SER-SER-GLU-GLU-SER-ILE-PRO- I l l P P P

NH2-ARG-GLU-THR-ILE-GLU-SER-LEU-SER-SER-SER-GLU-GLU-SER-ILE-PRO- I I l l P P P P

NH2-ARG-GLU-THR-ILE-GLU-SER-LEU-SER-SER-SER-GLU-GLU-SER-ILE-PRO- I I I l l P P P P P

FIG. 2. Location of phosphate groups on the 1-P to 5-P forms of human &casein. See text for explanation of the dashed lines.

cessful; the blocked residue persisted. During the initial pu- rification of this cyanogen bromide fragment (13), fractions from the peak of interest were subjected to polyacrylamide gel electrophoresis at pH 4.3, 8 M urea. I t was observed that the beginning of the peak consisted of one gel band, the middle a mixture of two, and the descending area the second band of increased mobility. All these fractions had identical amino acid compositions and were designated CB-4 but not pooled. Since such multiple banding of purified peptides might be ascribed to amide differences (13), it seemed possible that the two bands represented a separation of a species with intact glutamine as the amino terminus and another with L- 2-pyrrolidonecarboxylic acid in that position. Confirming this speculation, sequence analysis of the two-banded fraction and that of the purified component of faster mobility proceeded without difficulty.

Studies on peptide CB-4 extended the sequence data from residues 136 to 175 including the arginyl residue at 174 which marks the cleavage point for the final tryptic fragment, T-3. The latter peptide is devoid of lysine and even in the presence of Polybrene was not well retained in the spinning cup. The poor yields caused some uncertainty near the end of the sequence and amide or acid assignments could not be made for positions 205 and 207. The carboxyl-terminal three resi- dues were assigned by carboxypeptidase digestion of the whole molecule.

The total composition by sequence is 212 amino acids as compared to the reported value of 210 (5, 13). The changes involve one more serine (10 uersus 9), two additional proline residues (41 uersus 39), and one less leucine (25 uersus 26). Except for serine, these adjustments to the earlier composition involve amino acids present in high numbers, a not unex- pected finding.

Relevant to this composition, during Edman degradation position 142 (Table 7s ) yielded equal amounts of isoleucine and proline in direct contrast to the preceding and following residues where only one phenylthiohydantoin derivative was evident. This suggests genetic polymorphism at this site in- volving no change of charge-a so called “silent” genetic variant. A similar occurrence a t 169 where equal amounts of leucine and glutamine were found may be another uncharged substitution, but background accumulation at that stage of a sequence experiment prevents an unequivocal assertion. In-

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5134 Sequence of Human (3-Casein 1 5 10 15 20 25

FIG. 3. Strategy for determina- tion of the amino acid sequence of human ,&casein. The cyanogen bro- mide ( C B ) , trifluoroacetyl-blocked tryp- tic (T), and CB-1-T peptides are indi- cated below the line to which they refer. The forward arrows indicate those pep- tides sequenced; the reuerse arrows in- dicate the use of carboxypeptidases. Solid lines show residues placed by pep- tide composition only.

-----"""""""""" 4 - C E - 1 - 1 2

ARG-GLU-THR-ILE-GLU-SER-LEU-SER-SER-SER-GLU-GLU-SER-ILE-PRO-GLU-TYR-LYS-GLN-LYS-VAL-GLU-LYS-VAL-LYS-

C B - 1 - 7 1

1 - 1 A

I

I

30 40 50 HIS-GLU-ASP-GLN-GLN-GLN-GLY-THR-ASP-GLN-HIS-GLN-~P-LYS-J~-~R-PRO-SER-PHE-GLN-PRO-GLN~PRO~LEU~ILE~ """~"""""""""

,-A 1 - 1 E

""-.L

C B - 1 - 1 2

1 - 1 A

55 65 75 TYR-PRO-PHE-VAL-GLU-PRO-ILE-PRO-TYR-GLY-PHE-LTU-PRO-GLN-~N-ILE-~U-PRO-LEU-A~-G~-PRO-ALA-VAL-VAL- ""A""

80 90 100 LTU-PRO-VAL-PRO-GLN-PRO-GLU-ILE-~T-GLU-VAL-PRO-LYS-ALA-LYS-ASP-THR-VAL-TYR-THR-LYS-GLY-ARG-VAL-MET-

,-"""A""-"

""""A"""%2" 1 - 1 6

, A A

1 - 2

105 115 125 PRO-VAL-LEU-LYS-GLN-PRO-THR-ILE-PRO-PHE-PHE-ASP-P~-GLN-ILE-PRO-LYS-LEU-THR-ASP-LEU-GLU-ASN-LEU-HIS- t """-~"""""""""

1 - 2

130 140 150 LEU-PRa-LEU-PRO-LEU-LEU-GLN-PRO-SER-MET-GLN-GLN-VAL-PRO-GLN-PRO-ILE-PRO-GLN-THR-LEU-ALA-LEU-PRO-PRO-

1"""""""- ~~

""""4"- C B - 4

1 - 2

180 190 200

""""-~-4""""""---

VAL-PRO-VAL-GLN-ALA-LEU-LEU-LEU-ASN-GLN-GLU-LEU-LEU-LEU-ASN-PRO-PRO-H~S-GLN-ILE-TYR-PRO-VAL-PRO-GLU-

1-3

""A"""

volvement of these amino acids in such polymorphism would caseins (11, 15). According to Mercier et al. (18) (see the contribute to the uncertainty in totals discussed above. review (15) for details), the configuration ThrlSer-X-A, where

X represents any amino acid, and A, an acidic residue, is required for casein phosphorylation. Sites where A is a dicar-

Phosphorylation-The sequence cluster a t residues 5-12 of boxylic amino acid are termed primary sites; and those with human @-casein corresponding to the site of phosphorylation phosphoserine in that position, secondary sites. From Fig. 2, is also found a t varying locations in bovine p-, asl-, and a,*- note that human @-casein contains primary phosphorylation

DISCUSSION

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Sequence of Human @-Casein 5135

1 10 20 30 1(0 50

HUMN R E T I E S L S S S E E S I P E Y K Q K V E K V K H E D Q Q Q G S D ~ H O D K I Y P S F Q P Q P L I

BOVINE R E L E E L N V P G E I V T R I N K - I - F O S - E - E - E L - H - F A - T - S - V

OVINE R E Q E E L N V V G-V T H I N K - 1 ° F O S-E - € " E I-" H-F A--I-S-V

1 10 20 30 40 M

FIG. 4. Sequences of &caseins aligned for homologies. Numbering a t the top refers to the human sequence and at the bottom, the bovine homologue. Deletion in the alignment is indicated by o and the presence of an extra residue by r. The standard one-letter symbols for amino acids are used (28). A solid line indicates density with the human se- quence. The bovine BA2 and ovine B' sequences were determined by other in- vestigators (11, 12).

51 60 70 80 90 1W Y P F V E P I P Y G F L P Q N I L P L A Q P A V V L P V P Q P E I M E V P K A K D T V Y T K G R V R

PG-N S - P - T - T P - V - P F - V - G - S " V " E A ~ A P - - H K E -

P G-A N S 1-1 P-V-P F- G-V-E--I( V P -H K E-

- [I -

60 70 so 1 9 0 100

101 110 1M 130 140 150 P V L K O P T I P F F D P O I P K L T D L f N L H L P L P L L Q P S U Q Q V P O P l P O T L A L P P

-F P-V-V 0-T E S-S L 1-V-K- P L-S Y-H-P H-L-P-V R F - -F P-Y-V E-T E S-S L 1-V V-S Y-H-P-L-P-V U F -

110 1 m 1 9 140 150

151 160 1 70 180 190 2w O P L Y S V P E P K V L P I P ~ E V L P Y ~ V ~ A ~ P V ~ ~ L L L N O E L L ~ N P P H Q ~ Y P ~ P ~

"s v L-t s 0 s- V-E K A V-0-D M-I-F-Y-Q P V-G-V R G P F - I I V

"s V L-L S 8 - V-K A V O - - 0 - D -I(-l"F--Y"P V-G-V R G P F--I L V

160 170 180 190 200 209

201 212

P S T T Z A B H P I S V

sites at positions 3 ,9 , and 10 and secondary sites at positions 6 and 8. As mentioned earlier, the finding of phosphorus only on specific seryl and threonyl residues in forms phosphoryl- ated a t different levels indicates that the components are indeed homogeneous with respect to phosphorylated sites and not merely charge class mixtures containingphosphate groups randomly distributed. The 1-P form alone is a mixture, with half the molelcules phosphorylated at position 9 and half at position 10. Therefore, in this case only, these two sites are equivalent; each is followed N + 2 by glutamic acid and the casein kinase cannot distinguish between serines 9 and 10. After this, for the 2-, 3- , 4-, and 5-P forms, phosphate addition appears to be stepwise and there are no intermediate forms. Possible phosphate migration after incorporation seems to be ruled out by its absence on the neighboring seryl residue 8 in the hiphosphorylated molecule.

The reason for the existence of multiphosphorylated forms in human B-casein as compared with the fully phosphorylated cow homologue remains unclear. Possible explanations in- volve single or multiple kinase or phosphatase activity during biosynthesis or a variability in transport mechanism between the two species. An additional point to be considered is the appearance of quantitative variation among the six p-casein components. The pattern in Fig. 1 where the 2-P and 4-P forms show greater intensities than the others is common for many individual and pooled milk samples. Differing band intensities have been ascribed to genetic control (19). During our screening studies of individual samples, variations in band intensity patterns have been encountered. Other factors, such as stage of lactation, maternal health, or altered chemistry of calcium and phosphate, may also play a role in governing the quantitative variation. Correlation of this observation with the nutritional adequacy for the preterm infant in terms of calcium and phosphate availability may lead to the elucidation of some cause/effect relationship.

Sequence Comparison-Fig. 4 shows a comparison of the human P-casein sequence with the ruminant homologues, bovine (11) and ovine (121, the only other p-caseins for which such data are available. To align the structures it is necessary to place the amino terminus of the human molecule at position 10 of the other two sequences. A compensating carboxyl- terminal extension of the human protein provides for the approximate size equivalency, 212 uersus 209 residues. This "frame shift" relationship is rather unusual, and considering that several residues at the amino and carboxyl termini of the three proteins are identical, the mutation may exist in the genome and could reflect a difference in the number and location of exons. There is an insert of an extra residue at human position 61 and a deletion between human I 9 and 80 (bovine residue 88). In terms of amino acid identit,ies between the homologues, the value is 47% for the human-bovine comparison, 51% for human-ovine, and 90% for the more closely related species, bovine-ovine. Most of the differences between the three proteins represent single base changes. All three molecules, characteristic of caseins in general, contain large amounts of proline (39-41 residues/mol). Over 60% of these prolyl residues are identically situated, showing conser- vation of the teleological requirement for openness and there- fore, maximum digestibility. If the human and bovine P-casein sequences are aligned as in Fig. 4 and their secondary struc- tures compared, the human homologue exhibits even more aperiodicity than the bovine protein (11, 20), previously re- ported to contain a low proportion of its residues in periodic conformation. Comparison of the hydrophobicity profiles of the molecules reveals no striking differences. Details of these comparisons will be presented elsewhere. Charge distribution along the three sequences is again quite similar-a perhaps predictable observation considering that each of these pro- teins is packaged by protein interaction for delivery via the "casein micelle." Several explanations are extant for the bo-

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5136 Sequence of Human &Casein

vine micelle structure (21, 22), but the dearth of knowledge of the components of the human casein system does not allow straightforward extrapolations.

Seven genetic variants of bovine @-casein have been re- ported (22), all demonstrable by acid or alkaline polyacryl- amide gel electrophoresis. These variants, containing one or more charged residue differences, have been confirmed as truly genetic by gel pattern analysis of dam, sire, daughter relationships. For substitutions of uncharged residues, as seems to be the case in human @-casein, there is no easy way to demonstrate this phenomenon. Careful sequence analysis with good quantitation should suffice to confirm the existence of heterozygosity in the human protein. Voglino et al. (19), from an analysis of gel band patterns and intensities (similar to Fig. 1), propose the existence of a charged residue substi- tution in human @-casein but they have not isolated or char- acterized any material to support this contention. Since milk for the present sequence study was obtained from a single individual (5) and the multibanded pattern of Fig. 1 is due only to differences in phosphorylation, we conclude that we are dealing with heterozygous @-casein, i.e. genetic poly- morphism involving uncharged amino acids.

Recently Monti and Jolles (23) reported on a temperature- sensitive human milk whey protein, referred to as galacto- thermin, which they concluded was intact 0-P @-casein. We suggest that solubility a t pH 4.6 allows distribution of this form between the whey and casein fractions.

Peptides with opioid activity have been isolated from bovine caseins by several investigators (24-27). One of these, (3- casomorphin (25), is equivalent to residues 60-66 of bovine @-casein, and has been prepared from an enzyme digest of whole casein. The human homologue contains several se- quences characteristic of such peptides and studies are under- way to test these fragments for activity.

REFERENCES

1. Committee on Nutrition, American Academy of Pediatrics (1980)

2. Ogra, P. L., and Greene, H. L. (1982) Pediatr. Res. 16, 266-271 3. Lonnerdal, B., Forsum, E., and Hambraeus, L. (1976) Nutr. Rep.

4. Hambraeus, L., Lonnerdal, B., Forsum, E., and Gebre-Medhin,

5. Groves, M. L., and Gordon, W. G. (1970) Arch. Biochem. Biophys.

Pediatrics 65, 854-857

Znt. 13, 125-134

M. (1978) Acta. Pediatr. Scand. 67, 561-565

140,47-51

6. Nagasawa, T., Kiyosawa, I., and Kuwahara, K. (1970) J. Dairy

7. Gross, S. J. (1983) N . Engl. J. Med. 308, 237-241 8. Chessex, P., Reicbman, B., Verellen, G., Putet, G., Smith, J. M.,

9. Lemons, J. A., Moye, L., Hall, D., and Simmons, M. (1982)

10. Greer, F. R., Steichen, J. J., and Tsang, R. C. (1982) Am. J . Dis.

11. Ribadeau Dumas, B., Brignon, G., Grosclaude, F., and Mercier,

12. Richardson, B. C., and Mercier, J.-C. (1979) Eur. J. Biochem.

13. Groves, M. L., and Greenberg, R. (1978) Biachem. J. 169, 337-

14. Greenberg, R., Groves, M. L., and Peterson, R. F. (1976) J. Dairy

15. Mercier, J.-C. (1981) Biochimie 63, 1-17 16. Doolittle, R. F., and Armentrout, R. W. (1968) Biochemistry 7,

17. Doolittle, R. F. (1970) Methods Enzymol. 19, 555-569 18. Mercier, J.-C., Grosclaude, F., and Ribadeau Dumas, B. (1971)

19. Voglino, G. F., Caone, M., and Ponzone, A. (1975) Biomedicine

20. Creamer, L. K., Richardson, T., and Parry, D. A. D. (1981) Arch.

21. Farrell, H. M., Jr. (1973) J. Dairy Sci. 56, 1195-1206 22. Whitney, R. McL., Brunner, J . R., Ebner, K. E., Farrell, H. M.,

Jr., Josephson, R. V., Morr, C. B., and Swaisgood, H. E. (1976)

23. Monti, J. C., and Jolles, P. (1982) Experientia (Basel) 38, 1211- 1213

24. Brantl, V., Teschemacher, H., Henschen, A., and Lottspeich, F. (1979) Hoppe-Seyler’s 2. Physiol. Chem. 360, 1211-1216

25. Henschen, A., Lottspeich, F., Brantl, V., and Teschemacher, H. (1979) Hoppe-SeylerS Z. Physiol. Chem. 360, 1217-1224

26. Zioudrou, C., Streaty, R. A., and Klee, W. A. (1979) J . Biol. Chem. 254,2446-2449

27. Brantl, V., and Teschemacher, H. (1982) Milchwissenschuit 37,

28. IUPAC-IUB Commission on Biochemical Nomenclature (1968)

29. Davis, B. J. (1964) Ann. N. Y. Acad. Sci. 121,404-427 30. Greenberg, R., and Groves, M. L. (1979) J. Dairy Res. 46, 235-

239 31. Pisano, J. J., Bronzert, T. J., and Brewer, H. B., Jr. (1972) Anal.

Biochem. 45,43-59 32. Jeppson, J. O., and Sjoquist, J. (1967) Anal. Biochem. 18, 264-

269 33. Smithies, O., Gibson, D., Fanning, E. M., Goodfliesh, R. M.,

Gilman, J. G., and Ballantyne, D. C. (1971) Biochemistry 10,

SC~. 53, 136-145

Heim, T., and Swyer, P. R. (1983) J. Pediatr. 102, 107-112

Pediatr. Res. 16, 113-117

Child. 136, 581-583

J . 4 . (1972) Eur. J . Biochem. 25, 505-514

99,285-297

342

S C ~ . 59, 1016-1018

516-521

Eur. J. Biochem. 23,41-51

(Paris) 23, 344-345

Biochem. Biophys. 2 11,689-696

J. Dairy Sei. 59, 795-815

581-583

J. Biol. Chem. 243,3557-3559

4912-4921

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Sequence of Human /%Casein 5137

HUMAN B-CASEIN' AMINO ACID SEQUENCE AND lDENTlF iCATlON OF PHOSPHORYLATION SITES

- Cycle

1 2 3 4 5 6 7 8 9

10 11 12 1 3 14 15 16 17 18

20 19

21 22 23 24

Amlno acld n mole. Vleld

__

214

113

96

81

MefhW o f defectmn

a . b . d 2,b.c.d

a.d a,b a . c a.b a .c a , c a r c a.b a.b a ,c a . b

a.b d.b a.b

a.b.d, a.b,d

a.b a.b

a.b a , b

a,b d.e

b,C,d a. b

b b

31 Gln b 32 Glv b

Table 35

Amlno Acld Sequence Of CB- I -TZ

c y c i e -

1 2 3 4 5 6 1 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22

24 23

Amlno acld

Val

n moles Yle ld

__ 300 269

260

1 00 83

81

87

Method of detectson

a .d a . d d .e

a.c,d d .d a.d 3.d

c . d a.c.d a.d d.e d d d

a.d a . d a.d. c . d a.d d

a . d d

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5138 Sequence of Human P-Casein Tab le 6s

Amino Actd Sequence of T - 2

Table 45

A m n o Acld Sequence of T- I0

Cycle - 1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18

20 19

21 22 23 24 25 26 2 1 28 29 30 31 32 33 34 35 36 37

39 38

40 41

43 42

44

Method of defeCf8on

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 2 1

29 28

30 31 3 2 33

35 34

36 37 38 39 40

328

235 267

191

204

1 13 146

107 145

17

85

62 62 60

44

2 1 48

25 29

24 2 1

20

l o

9

1 2 3 4 5 6 1 8 9

10 11 12 13 14 15 16 17 18 19 20 21

23 22

24 25 26 2 1

29 28

30 31

33 32

35 34

36

495

321 487

277

142 126

65 12 77

31 31

21 12 12 10

a.d a.d a.d a.d C . d

a.cl a.d a.d d d

d d d d d d d

d d d d d d d d

E.d d d d d d

d d

c . d

d.e

d.e

Ile Le" PTO 139 Le" A11

129 121

Gln

Ala PW 101

Val 99

Val 99

LC" 70 P W Val

65

P W 83 Gln

74

Glu Ile 42 Met G,"

39

Val PW Val 27 24 LYS Ala L Y S ASP Thr Val 17

37

26 660 n moles applied; repemwe y w d val

4 * 24. 85%

See footnote - Table 2 s 5 w n mler applied; reperltive yleld

Met 2 * 31, 91%

See fmfnore - Table 2 s

Table 75

Amno A a d Sequence of CB-4

Table 55

Amino ACld Sequence Of '0-2 Method ol detection ___

d d d d d d d d d d d d d d d d d d d

C.d d d d d d d d d d d d d d d d d d d

d d.e

154 138 130 121

108 78

42 43 8 1

35 25 39

59 54

22 43

20 31

34 16

17 9

25

18

13

1 Gln 2 Gln 3 Val 4 P W 5 Gln 6 Prn 7 I h Pro 8 P W 9

10 Thr t i "

11 Le" 12 Ala 13 14

Le"

15 Pro PW

16 1 7 P W

Gln

18 19 Trp

Le"

20 5er 21 Val 22 23

P W Glu

24 P W 25

2 1 26

L Y S Val

28 29

Le" Pro

30 Pro I le

31 Gln 32 GI" 33 Val

35 34 Leu Gln

36 37

Tv r P W

38 P W Val

39 40

Arg Ala

6 7 8 9

10 11 12 13

160 d d d

a.d d d d

97

48 12

L Y S Clv

Yleld Leu 11 i 18, 94%

See footnote - Table 25

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R Greenberg, M L Groves and H J Dowersites.

Human beta-casein. Amino acid sequence and identification of phosphorylation

1984, 259:5132-5138.J. Biol. Chem. 

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