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Casein. IX. Carbohydrate Moiety of κ-Casein
Casein. IX. Carbohydrate Moiety of k-Casein V. D. TRAN and B. E. BAKER
Department of Agricultural Chemistry, Macdonald College of McGill University Macdonald College P.O., Quebec, Canada Abstract
Experimental Methods and Discussion
Glyeopeptides from pronase digests of K-casein had different molecular weights but contained equimolar amounts of galactose, ~¢-acetyl galactosamine, and sialic acid. The constitution of the carbohydrate moieties of the glycopeptides was investigated by enzymic hydrolysis, periodate oxidation, and alkaline degradation. The results indicate that the carbohydrate moiety of Kcasein consists of trisaecharide units which may be represented as a-~-acetyl neuraminyl (2->6)-fl-galactosyl-(1->3 or 6)-Nacetyl galactosamine, and that these units are attached to the peptide chain through the OH groups of serine or threonine, or both.
Sialyl glycopeptides. K-Casein (Variant A) prepared by the method of Zittle and Custer (20) was treated with rennin and the soluble macropeptide fraction was digested with pronase-P (Streptomyces grise~s protease). 1 The resultant product was subjected to column chromatography on Sephadex G-50 and then on Sephadex G-25. A glycopeptide fraction (GPA) was thereby isolated which contained no free amino acids or peptides of low molecular weight. The lyophilized fraction, Glycopeptide A, contained 18.1% galactose (16), 16.4% galactosamine (8), 28.2% sialie acid (19) (molar ratios, galaetose/galactosamine/sialie acid: 1.10/1.00/0.97). The fraction contained 51% of the bound hexose of the original K-casein. Glycopeptide A was dissolved in water and dialyzed against 3 changes of distilled water Introduction (20 volumes each; dialysis was 24 hours). The ~-Casein of a single genetic variant consists of monomeric units whose heterogeneity results diffusate and dialysate (fraction remaining in mostly from varying amounts (0 to 10%) of the dialyzing tube) were lyophilized to yield carbohydrates which the units contain. When fractions, Glycopeptides B and C. Each fraction the monomers are treated with rennin, each contained approximately 50% of the carbohyone produces water-insoluble paracasein and a drate of nndialyzed Glyeopeptide A and each water-soluble macropeptide which probably car- contained the three sugars in the same molar ties all of the carbohydrate o£ the original ratios. Neuraminidase (Vibrio choIerae) z released monomer. The macropeptide fraction derived approximately 95% of the sialic acid from Gly:~rom the carbohydrate-rich monomers of ~casein contains up to 30% carbohydrate and is eopeptide B, but glycosidases from almond seed (15) and rat epididymal extracts (13) did not soluble in 12% trichloroacetic acid (4). Alais and Joll~s (2) suggested that ~¢-acetyl release sugars from the glycopeptide. These neuraminic acid, galaetose, and galaetosamine results agree with those of others (2, 9) who are the only sugars in bovine K-macropeptide. have shown that sialic acid occupies a terminal ~tuang et al. (12) isolated from a papain digest position, but they also indicate that neither of K-casein, a glycopeptide fraction which con- galactose nor galactosamine occupies a terminal tained the three sugars aforementioned, in position. Periodate oxidation at 4 C (4 hours, in the nearly equivalent amounts. Baker and Hwang (5) isolated from a pronase digest of K-casein dark; periodate concentration 0.020 ~ ; molar a glyeopeptide fraction which contained about ratio, sialyl residue/periodate, 1/20) destroyed 64% carbohydrate. The authors observed a the neuraminie acid and the galactose but did stoichiometric relationship between the galactos- not destroy the galactosamine. When the oxidaamine and the threonine and serine contents of tion was performed at 25 C for 24 hours, sialic the glycopeptidie material and suggested that acid and galactose each yielded one mole of these amino acids are involved in the linkage titratable acid for each mole of sugar destroyed. between the carbohydrate moiety and the peptide I f one assumes that the acid produced is formic acid (14), then one might conclude that the chain. Our paper reports the results of further hydroxyl groups at C7, C8, and C9 of the sialie studies on the carbohydrate moiety of t:-casein. :Received for publication January 26, 1970.
~Calbiochem, Los Angeles, California. 1009
1010
rRAN AND BAKER
acid residue and those at C2, C3, and C4 of the galactose residue are unsubstituted. The resistance of the galactosamine residue to periodate oxidation indicates that at least one of its hydroxyl groups at either C3 or C4 is substituted. Sialic acid-free glycopeptide. Glycopeptide B was treated with sulfuric acid (0.05 ~, 82 C, 2 hours) and after removing sulfate, the product was placed on a DEAE Sephadex (A-25) column (formate form). The column was washed with a gradient buffer (ammonium formate, 0.025 to 0.50 ~; p H 5.6) and the eluate fractions containing the glycopeptide material were freed of formate by gel filtration. One fraction, Glycopeptide B1, was eluted at low salt concentration and had the composition, expressed as molar ratios, of hexosamine, 1.00; hexose, 1.14; threonine, 1.63; serine, 1.23; alanine, 0.52; and proline, 0.38. The molecular weight of Glycopeptide B1, estimated by gel filtration (Sephadex G-15), was approximately 800. These results suggest that Glycopeptide B1 was a mixture of glycopeptides, each containing one galactose residue, one galactosamine residue, and three or four amino acid residues. The absence of acidic amino acids and the presence of threonine and serine in Glycopeptide B1 leave little doubt concerning the glycosidic nature of the linkage between the carbohydrate and the peptide moiety. Glycopeptide B1 was digested with glycosidases obtained from almond seeds and from rat epididymis, and the liberated monosaecharities were estimated by paper chromatogl~phy (18). The enzymes prepared from almond seed released approximately 90% of the galactose but no detectable quantity of ~acetyl galactosamine from the glycopeptide after digesting for several days. Rat epididymal extract released approximately 100% of the galactose and about 30% of the N-aeetyl galactosamine after digesting for 24 hours. This clearly indicates that galactose occupies a terminal position in the sialic acid-free glycopeptide fraction. This fact, combined with the results of periodate oxidation of Glycopeptide B1, constitutes indirect proof of the linkage of sialie acid to C6 of the galactose residues. Since epididymal extract did not hydrolyze a-nitropbenyl galactoside, it seems reasonable to assign a fl-configuration to the anomeric carbon of the galactose residue. Periodate oxidation of Glycopeptide B1 destroyed all the galactose and about 15% of the galactosamine. These results exclude the possibility that galactose and galactosamine are attached separately to the peptide chain and ~OURI~'AL OF I')AIRT SCIElqCE VOL. 53, ~O. 8
suggest that about 15% of the galactose is attached to galaetosamine by a 1-~6 linkage. Glycopeptide B1 was hydrolyzed with dilute NaOH (0.5 ~, 4 C, 24 hours) and the hydrolysate was de-ionized by passing it through a column of Dowex 50 (H form) and Dowex 1 (OH form). It was then placed on a Sephadex G-15 column. The column was washed with NaC1 solution (0.1 ~) and the eluate was analyzed for hexose, ~-acetyl hexosamine, and Morgan-Elson chromogens. Minor Peak A and two minor Peaks B and C were eluted from the column in the order A, B, C. Peak B gave a positive test for hexose and negative tests for hexosamine and Morgan-Elson chromogens. Peak C gave negative tests for hexose and hexosamine and a positive test for Morgan-Elson chromogens. Paper chromatographic analyses (Whatman no. 1; butanol/ethanol/water, 4/1/1 ; descending technique) showed that Peak B contained galactose (Rgluc~e, 0.92; AgNOa reagent) and possibly some talose, and that Peak C contained Morgan-Elson chromogens (Rgluc~e, 2.7; Ehrlich's reagent) (17). Minor Peak A gave positive tests for hexose and the Morgan-Elson chromogens and was negative for hexosamine. The solution was analyzed by paper chromatography and only one spot (Rgluc~e, 0.24) was detected either with the silver nitrate reagent or with Ehrlich's reagent. When the substance was treated with rat epididymal extract and the resultant solution was analyzed by the same paper chromatographic procedure, two spots were observed on the chromatogram; one spot had a Rglucosevalue the same as that of galaetose; and the other, the same as that of the Morgan-Elson chromogens. These results suggest that the substance in Peak A is galaetose linked to a Morgan-Elson chromogen. Previous workers (1, 3, 6, 7, 11) have shown that substances containing o-glycosidic linkages between carbohydrates and threonine or serine are degraded by mild alkali with the release of the carbohydrate moiety. The breakdown of the carbohydrate moiety of Glycopeptide B1 to galactose and the Morgan-Elson chromogens after release of the moiety by alkali treatment, indicates that galactose is attached to galactosamine by a 1-3 linkage (10). The detection of galactose linked to the Morgan-Elson chromogens among the alkali degradation products of Glycopeptide B1 indicates that some of the galactose is attached to galactosamine by a 1-6 linkage (10). The structure and the degradation of the carbohydrate moiety of Glycopeptide B are illustrated in Figure 1.
CASEI~ IX
1011
Sialyl Glycopeptides (Fraction Glycopeptide-B} ¢r-Slalyl-(Z--6)-~-Gal-( I ~ 3) -N -AcGal -O-Peptide a-Sialyl-(Z--~ ) -~ -GILl -( I--6)-N-AcGal -O-Peptide
1
Neur aminidas • o¢ dilute H~O 4 Siali< a c l d - f r e e RIycopeptides (Fraction [Iyc
o!e pride - S l ;
85%
cyp, I,
15% type 2)
~-Gal-~ ~3]-bl-AcGat-O*Pept~de ~t~/~e I} (;old alkali
Glycosidase ( r a t epididy~nis)
Periodate
. .
dGeal;c:;:;
F .....
to s e
Ao..
and N-acet~lSelect os arnlne
[
Glycosidase ( r a t epididyrnis)
[
Glycosidase
Joa.to
Cold alkali
Pe "
r-L
......
and N - a c e t y l galactol avs~ne
[l}-Gal -( I~3} -N -AcGal)
I
Galactose and N - a c e t y l slalacto0 arnlne destroyed Galactose linked t~ Morgan-Elson chromogens (peak A)
(~ala~ctose (Peak B] ÷ Morgan*Elson T~le~;. ch . . . . sen. (Peak C)
t
Glycosldas •
(rat epididymis) Galactose ÷ Morgan -El~a~ss chromosena
Fro. 1. Sehematic representation of proposed structure and degradation of the carbohydrate moiety of glycopeptides isolated from pronase digests of K-casein.
Acknowledgments The authors t h a n k the Agricultural Research Council of Quebec, Canada, and the National Research Council of Canada for financial support which defrayed p a r t of the cost of this investigation.
(8)
References (1) Adams, J. B. 1965. Studies on the muein derived from human colloid breast carcinoma. Bioehem. J., 94: 368. (2) Alais, C., and P. Joll~s. 1961. ~tude compar~e des Caseinoglycopeptides form,s p a r Action de la Presure sur les Caseines de Vache et de Ch~vre. I I . ~.tude de la P a r t i e non-peptidique. Biochim. Biophys. Acta, 51 : 315. (3) Anderson, B., P. Hoffman, and K. Meyer. 1963. Serine-linked peptide of chondroitin sulfate. Biochim. Biophys. Acta, 74: 309. (4) Armstrong, C. E., A. G. Mackinlay, R. J. Hill, and R. G. Wake. 1967. The action of rennin on x-casein: the heterogeneity and origin of the soluble product. Biochim. Biophys. Acts, 140: 123. (5) Baker, B. E., and P. C. Hwang. 1967. Casein. V I I I . Isolation of a glyeopeptide from an enzymic hydrolysate of K-casein. J. Dairy Sci., 50: 1206. (6) Bhavanandan, V. P., E. Buddeeke, R. Carubelli, and A. Gottschalk. 1964. Complete enzymic degradation of glycopeptides conraining o-seryl and o-threonyl linked carbohydrate. Biochim. Biophys. Res. Commun., 16 : 333. (7) Carubelli, R., V. P. Bhavanandan, and A.
(9)
(10)
(11) (12)
(13)
(14)
(15)
(16)
Gottechalk. 1965. Studies on glyeoproteins. XI. The o-glycosidlc linkage of •-aeetyl galactosamine to seryl and threonyl residues in ovine s u b m ~ l ] a r y gland glycoproteins. Bioehim. Biophys. Acts, 161: 67. Cessi, C., and F. Piliego. 1959. Determination of amino sugars in the presence of amino acids and glucose. Biochem. J., 77: 508. Gibbons, R. A., and G. C. Cheesemann. 1962. Action of rennin on casein: the function of the neuraminic acid residues. Biochim. Biophys. Acts, 5 6 : 3 5 4 . Gottsch-]k, A. 1960. The Chemistry and Biology of Sialie Acid and Related Substances. University Press, Cambridge. Gottschalk, A., ed. 1966. Glyeoprotein. Elsevier Publishing Co., Amsterdam. Huang, F. Y. -Y., G. O. Henneberry, and B. E. Baker. 1964. Studies on casein. VII. The carbohydrate constitution of glycopeptidle material isolated from enzymic hydrolysates of ~-casien. Biochim. Biophys. Acta, 83: 333. Levvy, G. A., and J. Conchie. 1966. Mammalian glycosidases and their inhibition by aldonolaetones. Methods in Enzymology, 8 : 571. Malaprade, L. 1928. Action of polyaleohol on periodic acid. Analytical application. Bull. Soc. Chim. 1~rance, 43:683. Malhotra, O. P., and Mohan Dey Prakash. 1967. Purification and physical properties of sweet almond a-galactosidase. Biochem. J., 103 : 508. Montgomery, R. 1961. F u r t h e r studies of JOURNAL OF DAIRY SCIENCE VOL. 53, NO. 8
1012
TRAN
AND
the phenol-sulfuric acid reagent for carbohydrates. Bioehim. Biophys. Acta, 48: 591. (17) Salton, M. R. 1959. An improved method for the detection of l~-acetylamino sugars on paper ehromatograms. Biochim. Biophys. Aeta, 34: 308. (18) Spiro, R. G. 1960. Studies of fetuin, a
JOURNAL OF DAIRY SCIINCE VOL. 53, NO. 8
BAKER
glyeoprotein of fetal serum. I. Isolations, chemical composition and physiochemieal properties. J. Biol. Chem., 235: 2860. (19) Warren, L. 1959. The thiobarbiturie acid assay of sialie ae/d. J. Biol. Chem., 234: 705. (20> Zittle, C. A., and J. H. Custer. 1963. Purification and some of the properties of as-casein and K-casein. J. Dairy Sei., 46:1183.
Department of Agricultural Chemistry, Macdonald College of McGill University Macdonald College P.O., Quebec, Canada Abstract
Experimental Methods and Discussion
Glyeopeptides from pronase digests of K-casein had different molecular weights but contained equimolar amounts of galactose, ~¢-acetyl galactosamine, and sialic acid. The constitution of the carbohydrate moieties of the glycopeptides was investigated by enzymic hydrolysis, periodate oxidation, and alkaline degradation. The results indicate that the carbohydrate moiety of Kcasein consists of trisaecharide units which may be represented as a-~-acetyl neuraminyl (2->6)-fl-galactosyl-(1->3 or 6)-Nacetyl galactosamine, and that these units are attached to the peptide chain through the OH groups of serine or threonine, or both.
Sialyl glycopeptides. K-Casein (Variant A) prepared by the method of Zittle and Custer (20) was treated with rennin and the soluble macropeptide fraction was digested with pronase-P (Streptomyces grise~s protease). 1 The resultant product was subjected to column chromatography on Sephadex G-50 and then on Sephadex G-25. A glycopeptide fraction (GPA) was thereby isolated which contained no free amino acids or peptides of low molecular weight. The lyophilized fraction, Glycopeptide A, contained 18.1% galactose (16), 16.4% galactosamine (8), 28.2% sialie acid (19) (molar ratios, galaetose/galactosamine/sialie acid: 1.10/1.00/0.97). The fraction contained 51% of the bound hexose of the original K-casein. Glycopeptide A was dissolved in water and dialyzed against 3 changes of distilled water Introduction (20 volumes each; dialysis was 24 hours). The ~-Casein of a single genetic variant consists of monomeric units whose heterogeneity results diffusate and dialysate (fraction remaining in mostly from varying amounts (0 to 10%) of the dialyzing tube) were lyophilized to yield carbohydrates which the units contain. When fractions, Glycopeptides B and C. Each fraction the monomers are treated with rennin, each contained approximately 50% of the carbohyone produces water-insoluble paracasein and a drate of nndialyzed Glyeopeptide A and each water-soluble macropeptide which probably car- contained the three sugars in the same molar ties all of the carbohydrate o£ the original ratios. Neuraminidase (Vibrio choIerae) z released monomer. The macropeptide fraction derived approximately 95% of the sialic acid from Gly:~rom the carbohydrate-rich monomers of ~casein contains up to 30% carbohydrate and is eopeptide B, but glycosidases from almond seed (15) and rat epididymal extracts (13) did not soluble in 12% trichloroacetic acid (4). Alais and Joll~s (2) suggested that ~¢-acetyl release sugars from the glycopeptide. These neuraminic acid, galaetose, and galaetosamine results agree with those of others (2, 9) who are the only sugars in bovine K-macropeptide. have shown that sialic acid occupies a terminal ~tuang et al. (12) isolated from a papain digest position, but they also indicate that neither of K-casein, a glycopeptide fraction which con- galactose nor galactosamine occupies a terminal tained the three sugars aforementioned, in position. Periodate oxidation at 4 C (4 hours, in the nearly equivalent amounts. Baker and Hwang (5) isolated from a pronase digest of K-casein dark; periodate concentration 0.020 ~ ; molar a glyeopeptide fraction which contained about ratio, sialyl residue/periodate, 1/20) destroyed 64% carbohydrate. The authors observed a the neuraminie acid and the galactose but did stoichiometric relationship between the galactos- not destroy the galactosamine. When the oxidaamine and the threonine and serine contents of tion was performed at 25 C for 24 hours, sialic the glycopeptidie material and suggested that acid and galactose each yielded one mole of these amino acids are involved in the linkage titratable acid for each mole of sugar destroyed. between the carbohydrate moiety and the peptide I f one assumes that the acid produced is formic acid (14), then one might conclude that the chain. Our paper reports the results of further hydroxyl groups at C7, C8, and C9 of the sialie studies on the carbohydrate moiety of t:-casein. :Received for publication January 26, 1970.
~Calbiochem, Los Angeles, California. 1009
1010
rRAN AND BAKER
acid residue and those at C2, C3, and C4 of the galactose residue are unsubstituted. The resistance of the galactosamine residue to periodate oxidation indicates that at least one of its hydroxyl groups at either C3 or C4 is substituted. Sialic acid-free glycopeptide. Glycopeptide B was treated with sulfuric acid (0.05 ~, 82 C, 2 hours) and after removing sulfate, the product was placed on a DEAE Sephadex (A-25) column (formate form). The column was washed with a gradient buffer (ammonium formate, 0.025 to 0.50 ~; p H 5.6) and the eluate fractions containing the glycopeptide material were freed of formate by gel filtration. One fraction, Glycopeptide B1, was eluted at low salt concentration and had the composition, expressed as molar ratios, of hexosamine, 1.00; hexose, 1.14; threonine, 1.63; serine, 1.23; alanine, 0.52; and proline, 0.38. The molecular weight of Glycopeptide B1, estimated by gel filtration (Sephadex G-15), was approximately 800. These results suggest that Glycopeptide B1 was a mixture of glycopeptides, each containing one galactose residue, one galactosamine residue, and three or four amino acid residues. The absence of acidic amino acids and the presence of threonine and serine in Glycopeptide B1 leave little doubt concerning the glycosidic nature of the linkage between the carbohydrate and the peptide moiety. Glycopeptide B1 was digested with glycosidases obtained from almond seeds and from rat epididymis, and the liberated monosaecharities were estimated by paper chromatogl~phy (18). The enzymes prepared from almond seed released approximately 90% of the galactose but no detectable quantity of ~acetyl galactosamine from the glycopeptide after digesting for several days. Rat epididymal extract released approximately 100% of the galactose and about 30% of the N-aeetyl galactosamine after digesting for 24 hours. This clearly indicates that galactose occupies a terminal position in the sialic acid-free glycopeptide fraction. This fact, combined with the results of periodate oxidation of Glycopeptide B1, constitutes indirect proof of the linkage of sialie acid to C6 of the galactose residues. Since epididymal extract did not hydrolyze a-nitropbenyl galactoside, it seems reasonable to assign a fl-configuration to the anomeric carbon of the galactose residue. Periodate oxidation of Glycopeptide B1 destroyed all the galactose and about 15% of the galactosamine. These results exclude the possibility that galactose and galactosamine are attached separately to the peptide chain and ~OURI~'AL OF I')AIRT SCIElqCE VOL. 53, ~O. 8
suggest that about 15% of the galactose is attached to galaetosamine by a 1-~6 linkage. Glycopeptide B1 was hydrolyzed with dilute NaOH (0.5 ~, 4 C, 24 hours) and the hydrolysate was de-ionized by passing it through a column of Dowex 50 (H form) and Dowex 1 (OH form). It was then placed on a Sephadex G-15 column. The column was washed with NaC1 solution (0.1 ~) and the eluate was analyzed for hexose, ~-acetyl hexosamine, and Morgan-Elson chromogens. Minor Peak A and two minor Peaks B and C were eluted from the column in the order A, B, C. Peak B gave a positive test for hexose and negative tests for hexosamine and Morgan-Elson chromogens. Peak C gave negative tests for hexose and hexosamine and a positive test for Morgan-Elson chromogens. Paper chromatographic analyses (Whatman no. 1; butanol/ethanol/water, 4/1/1 ; descending technique) showed that Peak B contained galactose (Rgluc~e, 0.92; AgNOa reagent) and possibly some talose, and that Peak C contained Morgan-Elson chromogens (Rgluc~e, 2.7; Ehrlich's reagent) (17). Minor Peak A gave positive tests for hexose and the Morgan-Elson chromogens and was negative for hexosamine. The solution was analyzed by paper chromatography and only one spot (Rgluc~e, 0.24) was detected either with the silver nitrate reagent or with Ehrlich's reagent. When the substance was treated with rat epididymal extract and the resultant solution was analyzed by the same paper chromatographic procedure, two spots were observed on the chromatogram; one spot had a Rglucosevalue the same as that of galaetose; and the other, the same as that of the Morgan-Elson chromogens. These results suggest that the substance in Peak A is galaetose linked to a Morgan-Elson chromogen. Previous workers (1, 3, 6, 7, 11) have shown that substances containing o-glycosidic linkages between carbohydrates and threonine or serine are degraded by mild alkali with the release of the carbohydrate moiety. The breakdown of the carbohydrate moiety of Glycopeptide B1 to galactose and the Morgan-Elson chromogens after release of the moiety by alkali treatment, indicates that galactose is attached to galactosamine by a 1-3 linkage (10). The detection of galactose linked to the Morgan-Elson chromogens among the alkali degradation products of Glycopeptide B1 indicates that some of the galactose is attached to galactosamine by a 1-6 linkage (10). The structure and the degradation of the carbohydrate moiety of Glycopeptide B are illustrated in Figure 1.
CASEI~ IX
1011
Sialyl Glycopeptides (Fraction Glycopeptide-B} ¢r-Slalyl-(Z--6)-~-Gal-( I ~ 3) -N -AcGal -O-Peptide a-Sialyl-(Z--~ ) -~ -GILl -( I--6)-N-AcGal -O-Peptide
1
Neur aminidas • o¢ dilute H~O 4 Siali< a c l d - f r e e RIycopeptides (Fraction [Iyc
o!e pride - S l ;
85%
cyp, I,
15% type 2)
~-Gal-~ ~3]-bl-AcGat-O*Pept~de ~t~/~e I} (;old alkali
Glycosidase ( r a t epididy~nis)
Periodate
. .
dGeal;c:;:;
F .....
to s e
Ao..
and N-acet~lSelect os arnlne
[
Glycosidase ( r a t epididyrnis)
[
Glycosidase
Joa.to
Cold alkali
Pe "
r-L
......
and N - a c e t y l galactol avs~ne
[l}-Gal -( I~3} -N -AcGal)
I
Galactose and N - a c e t y l slalacto0 arnlne destroyed Galactose linked t~ Morgan-Elson chromogens (peak A)
(~ala~ctose (Peak B] ÷ Morgan*Elson T~le~;. ch . . . . sen. (Peak C)
t
Glycosldas •
(rat epididymis) Galactose ÷ Morgan -El~a~ss chromosena
Fro. 1. Sehematic representation of proposed structure and degradation of the carbohydrate moiety of glycopeptides isolated from pronase digests of K-casein.
Acknowledgments The authors t h a n k the Agricultural Research Council of Quebec, Canada, and the National Research Council of Canada for financial support which defrayed p a r t of the cost of this investigation.
(8)
References (1) Adams, J. B. 1965. Studies on the muein derived from human colloid breast carcinoma. Bioehem. J., 94: 368. (2) Alais, C., and P. Joll~s. 1961. ~tude compar~e des Caseinoglycopeptides form,s p a r Action de la Presure sur les Caseines de Vache et de Ch~vre. I I . ~.tude de la P a r t i e non-peptidique. Biochim. Biophys. Acta, 51 : 315. (3) Anderson, B., P. Hoffman, and K. Meyer. 1963. Serine-linked peptide of chondroitin sulfate. Biochim. Biophys. Acta, 74: 309. (4) Armstrong, C. E., A. G. Mackinlay, R. J. Hill, and R. G. Wake. 1967. The action of rennin on x-casein: the heterogeneity and origin of the soluble product. Biochim. Biophys. Acts, 140: 123. (5) Baker, B. E., and P. C. Hwang. 1967. Casein. V I I I . Isolation of a glyeopeptide from an enzymic hydrolysate of K-casein. J. Dairy Sci., 50: 1206. (6) Bhavanandan, V. P., E. Buddeeke, R. Carubelli, and A. Gottschalk. 1964. Complete enzymic degradation of glycopeptides conraining o-seryl and o-threonyl linked carbohydrate. Biochim. Biophys. Res. Commun., 16 : 333. (7) Carubelli, R., V. P. Bhavanandan, and A.
(9)
(10)
(11) (12)
(13)
(14)
(15)
(16)
Gottechalk. 1965. Studies on glyeoproteins. XI. The o-glycosidlc linkage of •-aeetyl galactosamine to seryl and threonyl residues in ovine s u b m ~ l ] a r y gland glycoproteins. Bioehim. Biophys. Acts, 161: 67. Cessi, C., and F. Piliego. 1959. Determination of amino sugars in the presence of amino acids and glucose. Biochem. J., 77: 508. Gibbons, R. A., and G. C. Cheesemann. 1962. Action of rennin on casein: the function of the neuraminic acid residues. Biochim. Biophys. Acts, 5 6 : 3 5 4 . Gottsch-]k, A. 1960. The Chemistry and Biology of Sialie Acid and Related Substances. University Press, Cambridge. Gottschalk, A., ed. 1966. Glyeoprotein. Elsevier Publishing Co., Amsterdam. Huang, F. Y. -Y., G. O. Henneberry, and B. E. Baker. 1964. Studies on casein. VII. The carbohydrate constitution of glycopeptidle material isolated from enzymic hydrolysates of ~-casien. Biochim. Biophys. Acta, 83: 333. Levvy, G. A., and J. Conchie. 1966. Mammalian glycosidases and their inhibition by aldonolaetones. Methods in Enzymology, 8 : 571. Malaprade, L. 1928. Action of polyaleohol on periodic acid. Analytical application. Bull. Soc. Chim. 1~rance, 43:683. Malhotra, O. P., and Mohan Dey Prakash. 1967. Purification and physical properties of sweet almond a-galactosidase. Biochem. J., 103 : 508. Montgomery, R. 1961. F u r t h e r studies of JOURNAL OF DAIRY SCIENCE VOL. 53, NO. 8
1012
TRAN
AND
the phenol-sulfuric acid reagent for carbohydrates. Bioehim. Biophys. Acta, 48: 591. (17) Salton, M. R. 1959. An improved method for the detection of l~-acetylamino sugars on paper ehromatograms. Biochim. Biophys. Aeta, 34: 308. (18) Spiro, R. G. 1960. Studies of fetuin, a
JOURNAL OF DAIRY SCIINCE VOL. 53, NO. 8
BAKER
glyeoprotein of fetal serum. I. Isolations, chemical composition and physiochemieal properties. J. Biol. Chem., 235: 2860. (19) Warren, L. 1959. The thiobarbiturie acid assay of sialie ae/d. J. Biol. Chem., 234: 705. (20> Zittle, C. A., and J. H. Custer. 1963. Purification and some of the properties of as-casein and K-casein. J. Dairy Sei., 46:1183.