- Email: [email protected]
Formation of γ1-A2, γ2-A2 AND γ3-A caseins by In vitro proteolysis of β-CASEIN A2 with bovine plasmin
FORMATION OF yl-A2, yz-A2 AND y3-A CASEINS BY IN VII-R0 PROTEOLYSIS OF fKASEIN A2 WITH BOVINE PLASMIN W. N. EIGEL Department
of Animal
Sciences,
Purdue
(Rec&&
University, 5 Octohrr
West Lafayette,
IN 47907,
1J.S.A
1976)
Abstract-l. In vitro proteolysis of I-casein A’ resulted in the formation of products identified as F~-A’ (y-A’), yz-A2 (TS-A’) and yLI-A(R-) caseins by polyacrylamide gel electrophoresis conducted under both acidic and alkaline conditions. 2: Additionally, “Stains-all” was used to identify the polypeptide bands corresponding to the N-tcrminal portions of &casein A’ released during proteolysis. 3. These observations are consistent with the hypothesis that plasmin is responsible for in t+r, production of these minor casein fractions.
INTRODL’CTION
The term casein is used to describe a group of heterogeneous phosphoproteins secreted in milk by mammary epithelial ceils. In the bovine, whole casein contains approx Xi”/, /I-casein, which exists as a single polypeptide chain of 209 amino acid residues (Ribadeau Dumas rl al., 1972). ,%Casein has a calculated mol. wt of 23,982 and contains 5 phosphate moieties covalently attached to serine at residues 15, 17, 18. 19 and 35. Eight genetic variants of @-casein (A’, A”, A3, B. B,, C, D and E), produced by the substitution of one or more amino acids, have been reported (Whitney et al., 1976). Bovine casein contains several minor components, chief among which is a group of related proteins designated T-, R-, S- and TS-caseins. Extensive comparisons of these minor casein fractions have been made with respect to their amino acid compositions, mol. wts. fingerprints following tryptic digestion and end-group amino acids (Gordon rt al., 1972). Numerous similarities led these investigators to determine the partial sequence of the N-terminal end of these proteins and to compare them with the primary structure of fi-casein. They concluded that ?-A’, TS-A2 and R-caseins are identical with amino acid residues 29-209, 106-209, and 108.--209, respectively, of fi-casein A2. Moreover, y-B, S- and TS-B caseins were reported to be identical to similar portions of /I-casein B. These findings have led the Protein Nomenclature Committee of the American Dairy Science Association (Whitney er al., 1976) to propose a new nomenclature for these minor proteins. Henceforth. they will be designated yl-(y-). y2 (X-A’ and S-) and y3-(R- and TS-B) caseins. Recently, Gordon and Groves (1975) have proposed that a limited, highly specific proteolysis of /?-casein might be responsible for the occurrence of these minor casein fractions in bovine milk. The presence of a natural-occurring protease in milk was first demonstrated by Babcock and Russell (1897). Kaminogdwa et al. (1971) have concentrated milk protease and have demonstrated that incubation with
@casein resulted in the formation of 1;JTS-) and y,-(R-) caseins (Kaminogawa & Yamauchi, 1972). Kaminogawa et al. (1972) compared the properties of concentrated milk protease with plasmin and found many similarities. However, the mol. wt which they obtained for milk protease was lower than published values for plasmin. Recently, Eigel et ul. (1976) obtained additional evidence for the presence of plasmin and its zymogen, plasminogen. in milk. In this report evidence indicating that if! r:itro proteolysis of /?-casein A’ by plasmin produces ?,-A” (y-). 7,-A” (TS-A2) and ?,-A (R-) caseins is presented.
Fresh milk was obtained from individual cows of the University herd. Cream was removed by centrifugation at 7000~ for 15 min. Casein was precipitated by adiusting the pH of the skim-milk to 4.6 ;sing 3 N HCi. cdllected by filtration through cheesecloth and washed 3 times with distilled water. x,,-Casein was prepared by the urea method of Zittle et al. (1959) and was further purified by ethanol treatj~ent (Zittle & Custer, 1963). ,%Casein was prepared by the urea method of Hipp rt al. (1952). The genetic variant of fl-casein was obtained by vertical polyacrvlamide gel elecirophoresis in 4.5 M uiea at pH’ 9.5 (Thompson c’t (II.. 1964). The A variant was determined bv disc gel electrophoresis in 8 M urea at pH 4.3 (Groves & Go&on, 1969). Blood was collected in the presence of heparin from a cow at slaughter. Insoluble material was removed bv centrifugation first at 5000 y at 4‘C and then at 27.000 g at 4’ C. The cold plasma was then filtered to remove insoluble lipid material,’ Bovine plasminogen was prepared from plasma by afinity chromatography (Chibber rf ~1.. 1974). Protrolysis
qf /I-cm&r
A” by plrrsmirl
Plasminogen was activated to plasmin by addition of urokinase (100 plough units/ml) to plasminogen (5 myml) dissolved in 0.05 M sodium tetraborate, pH X.4. Incubation was at 37’C for 30 min. The incubation mixture for proteolysis contained p-casein AZ (10 mg/ml) in 0.05 M sodium tetraborate, pH 8.4, and merthiolate (0.02”Gl) to inhibit microbial growth.
188
W. N EIGEL
Prior to incubation the a-casein solution was heated at 80°C for 10 min. Freshly activated plasmin (0.28 mg/ml) _. was added and the solution incubated at 37°C. Aliquots (0.2 ml) were removed after 0.5. 1. 2, 4. 8. 15. 30 and 60 min and mixed with equal volumes of 8 M urea containing 0.2”:, 2-mercaptoethanol. Alkaline
phosphatase
treatment
A mixture
(0.5 ml) of p-casein A2 and plasmin, as described above, was incubated at 37’C. After 2 min an equal volume of 8 M urea containing 0.2”/, 2-mercaptoethanol was added. This solution was heated at 80°C for 10 min. cooled rapidly, and dialyzed against several changes of 0.005 M sodium acetate. pH 6.2, containing 0.001 M MgCl,. Incubation conditions with alkaline phosphatase Type VII from calf intestinal mucosa (Sigma) were similar to those of Green & Pastewka (1976) except that merthiolate (0.02”:,) was also included. Electrophorrsis
Proteolysis was monitored by disc gel electrophoresis in 4 M urea at pH 9.6 using the-technique of Davis (1964) as modified by Groves and Kiddy (1968). Gels were stained for protein with Amido Black IOB and for phosphoproteins with “Stains-all” (Green et al., 1973). Densitometric tracings of gels stained with Amido Black 10B and “Stainsall” w&e olbtained at 590 and 660 nm, respectively. Vertical polyacrylamide gel electrophoresis in 4.5 M urea at pH 3.0 (Peterson & Kopfler, 1966) was used for identification of y3- (R-) casein A. Mol.
wt determination
Sodium dodecyl sulfate (SDS) & Osborn. 1969) was used to /?-casein A2 and the products of Ovalbumin. chymotrypsinogen. chrome c were used as mol. wt
gel electrophoresis (Weber estimate the mol. wts of its proteolysis by plasmin. ribonuclease and cytostandards.
these electrophoretic conditions, yl-A2 (r-A2) and rs,caseins have similar electrophoretic mobilities (Groves, 1969). The 2 remaining bands have mobilities similar to y2-A2 (TS-A’) and y3-A (R-) caseins (Groves, 1969). SDS gel electrophoresis of p-casein A2 following incubation with plasmin for 4 min reveals the presence of 4 polypeptide bands (Fig. 3). The mol. wts calculated for 3 of these bands have been reported earlier (Eigel et al., 1976) and were used to identify these bands as 8-A’ , y1-A2 (r-A2) and y2-A2 (TX-A2) and Y~-A (R-) caseins (the latter 2 proteins differ in size by just 2 amino acid residues and thus cannot be differentiated by SDS polyacrylamide gel electrophoresis). The mol. wt estimates obtained for B-A’ and y1-A2 (y-A’) caseins are in agreement with the values obtained by Groves et al. (1973) using SDS gel electrophoresis. However, the mol. wt estimates for y2-A2 (TS-A2) and y3-A (R-) caseins are slightly high. If /j’-casein A2 can be converted directly to y2-AL (TS-A’) and yx-A (R-) caseins by plasmin, then the N-terminal portions of /I-casein A’ liberated in this process would be slightly larger than y2-A” (TS-A2) and y3-A (R-) caseins and may be partially responsible for the slightly higher mol. wt obtained for this polypeptide band. The mol. wt of the fastest-moving polypeptide band could not be determined with the standards used in this study. However, its mol. wt seems to be in the range of 9000-10,000 daltons and it probably represents the N-terminal end of Y~-(;J-) casein A2 released when y2-A2 (TS-A2) and y3-A (R-) caseins are formed by proteolysis of 7;1-(y-) casein A2 rather than b-casein A2 by plasmin. Disc gel electrophoretic patterns of the proteolytic products of p-casein A2 by plasmin also reveal the
RESULTS
Proteolysis of /?-casein A2 by plasmin was monitored by disc gel electrophoresis in 4 M urea at pH 9.6. These electrophoretic patterns (Fig. 1) reveal the gradual disappearance of the band corresponding to p-casein A2 along with the simultaneous appearance and increase in intensity of 2 slower-migrating bands with mobilities similar to y,-(1;-) and ‘/2-(TS-A2) caseins A2 (Groves, 1969). After 4 min of incubation with plasmin, the band corresponding to b-casein A2 has all but disappeared leaving the 2 major slowermigrating bands and a few faint faster-migrating bands (Fig. le). Further proteolysis by plasmin eventually results in the disappearance (after 30 min) of bands identified as yl-(y-) and y2-(TS-A’) caseins A2. Incubation of fi-casein A2 with urokinase under similar conditions had no effect on the electrophoretic pattern. Since y3- (R-) casein A has been reported to comigrate with yl-(y-) casein A2 in electrophoresis conducted under alkaline conditions (Groves, 1969), vertical gel electrophoresis in 4.5 M urea at pH 3.0 was used to further identify the products resulting from degradation of fl-casein A2 by plasmin. The electrophoretic pattern obtained after incubation of /j’-casein A’ with plasmin for 2 min revealed the presence of 4 polypeptide bands (Fig. 2a). The identifications of the p- and rl-(?;-) casein A2 bands were made by comparing their mobilities with samples of fl-casein A’ (Fig. 2b) and q,-casein (Fig. 2~). respectively. Under
P
II
(TS)I,
II
Fig. 4. Densitometric tracings of disc gel electrophoretic patterns (4M urea, pH 9.6) obtained for p-casein A’ (10 mg/ml) in 0.05 M sodium tetraborate. pH 8.4, incubated with bovine plasmin (0.28 mg/ml) at 37°C for 2 min. Duplicate samples were electrophoresed except that one gel was stained for protein with Amido black 10B (-) while the other gel was stained for phosphoproteins with “Stainsall” (---). Sample origin designated by arrow.
189
a
b
t
Fig. I. Disc gel electrophoresis at pH 9.6 in 4 M urea of /3-casein AZ (IO mg/ml) in 0.05 M sodium tctraborate, pH 8.4. incubated at WC with bovine plasmin (0.28 mg/ml) for (a) 0. (b) 0.5, (c) I, (d) 2. (c) 4. (I] 8, (g) 15, (h) 30 and (i) 60 min, respectively. Merthiolate was included to inhibit microbial growth.
190
Fig. 2. Vertical gel electrophoresis (4.5 M urea. pH 3.0) of (a) p-casein A’ (10 mg/ml) in 0.05 M sodium tetraborate, pH 8.4. incubated at 37°C with bovine plasmin (0.28 mg/ml) for 2 min, (b) p-casein A’ and (c) qcasein. Fig. 3. Sodium dodecyl sulfate gel electrophoresis ate, pH 8.4, incubated with bovine
of j-casein A* (10 mg/ml) in 0.05 M sodium plasmin (0.28 mg/ml) at 37°C for 4 min.
tetrabor-
&-A’,
191
j(*-AZ and y3-A caseins
of 2 faint, fast-migrating bands (Figs. 1 tracing of the disc gel pattern obtained after incubation of j-casein A2 with plasmin for 2 min is shown in Fig. 4. Superimposed on this tracing is the pattern obtained when a duplicate gel was stained with “Stains-all” instead of Amido black 1OB. Green rt al. (1973) reported that phosphoproteins produce a blue band with “Stains-all” while nonphosphorylated proteins give either a red band or do not stain at all. /$ And y,-(y-) caseins A2 contain 5 and 1 phosphate moieties, respectively, covalently attached to serine residues near their N-terminal ends (Ribadeau Dumas et a/., 1972). yz-A2 FS-A’) and y3-A (R-) caseins are devoid of covalently attached phosphate. Comparison of the 2 densitometric patterns (Fig. 4) indicates that b- and rl-(r-) caseins A2 stain as expected for phosphoproteins while yZ- (TSA’) casein A2 did not produce a band with “Stainsall”. The 2 fast-migrating bands observed in gels stained with Amido black IOB produced blue bands with “Stains-all”. Treatment of the sample with alkaline phosphatase prior to electrophoresis resulted in the disappearance of all blue bands when the gel was stained with “Stains-all”. Thus, the 2 fast-migrating bands are most likely the phosphorylated N-terminal ends of /j’- and/or y,-(y-) caseins A2 produced by plasmin during formation of y2-AZ (TS-A’) and Y~-A (R-) caseins.
appearance
ee). A densitometric
DlSCUSSIOlV
In this work, disc gel electrophoresis, conducted under both acidic and alkaline conditions, and SDS gel electrophoresis have been used to identify yl-A2 (‘J-A’) , y2-A2 (TS-A’) and y3-A (R-) caseins as products of the proteolytic cleavage of /&casein A2 by bovine plasmin. An increasing amount of indirect evidence strongly suggests the presence of plasmin and plasminogen in bovine milk. Kaminogawa et al. (1972) first suggested that milk protease may be plasmin, however, when concentrated milk protease was incubated with p-casein, only y2-(TS-) and y3-(R-) caseins were identified as products (Kaminogawa & Yamauchi, 1972). These workers did not report the formation of yl-(y-) casein as a result of this proteolysis. Magee et al. (1976) have demonstrated that conversion of galactosyltransferase from its high mol. wt form (58,000) to a low mol. wt from (44,000) is catalyzed ii7 t&o by plasmin and have suggested that the same process ~JI vim is responsible for differences in mol. wts reported for galactosyltransferase. Eigel et al. (1976) observed the electrophoretic changes produced in casein by the natural-occurring protease in milk and demonstrated that these changes could be reproduced by treatment of casein with isolated bovine plasmin. Moreover, they reported that activity of the naturally-occurring protease in milk was stimulated by urokinase, drastically inhibited by low levels ( lO-4 M) of E-aminccaproic acid (EACA) and completely inhibited by EACA and diisopropylfluorophosphate (DFP). The localization in the mammary gland of the site of proteolysis of /%casein by plasmin remains to be determined. Incubation of fi-casein B with trypsin results in the formation of several electrophoretic bands (Gordon & Groves. 1975). Three of these have been reported
to have mobilities in alkaline gel electrophoresis similar to ?/,-(y-), “J*-(S-) and yJ-(TS-) caseins B. Plasmin is very similar to trypsin in specificity in that both hydrolyze lysinyl and arginyl bonds. However, plasmin has been shown to hydrolyze lysine methyl esters more rapidly than arginine methyl esters while trypsin hydrolyzes both types equally well (Sherry et al., 1966). Initial formation of ?,-A2 (?-A’), yz-A2 FS-A’) and >a3-A(R-) caseins involves proteolysis of b-casein A2 at lysine 28, 105 and 107 (Ribadeau Dumas et al., 1972).
Acknorvlrdgenlents-Journal paper No. 6383 of the Purdue Agricultural Experiment Station. Lafayette, Indiana. This research was supported in part by a grant from the Purdue Cancer Committee (Indiana Elks). REFERENCES
BABCOCK S. M. & RUSSELL H. L. (1897) Unorganized ferments of milk: a new factor in the ripening of cheese. Wis. Agr. rxp. Sta. 22. 161-193. CHIBBER B. A. K.. D~UTSCH D. G. & MERTZ E. T. (1974) Plasminogen. In Methods in En~ymolmg,v Vol. 34. pp. 424-432. Academic Press, New York. DAVIS B. J. (1964) Disc electrophoresis-II. Method and application to human serum proteins. A~I. N.Y. Acud. Sci. 121. 4Ok427. EIGEL W. N., CHIBBER B. A. K. & TOMICH J. M. (1977) Plasminogen and plasmin: evidence for their presence in bovine milk. Science, submitted for publication. GREEN M. R. & PASTEWKA J. V. (1976) Characterization of major milk proteins from BALB/c and C3H mice. J. Dairy Sci. 59. 207-215. GREEN M. R.. PASTEWKA J. V. & PEACOCK A. C. (1973) Differential staining of phosphoproteins on polyacrylamide gels with a cationic carbocyanine dye. Analog. Biochzm. 56. 43-51. GORDON W. G. & GROVES M. L. (1975) Primary sequence of beta, gamma, and minor caseins. J. Dairy Sci. 58, 574-582. GORDON W. G., GROVES M. L., GREENBERGR.. JONES S. B., KALAN E. B., PETERSON R. F. & TO~NEND R. E. (1972) Probable identification of y-. TS-, R- and S-caseins as fragments of B-casein. J. Dairy Sci. 55. 261-263. GROVES M. L. (1969) Some minor components of casein and other phosphoproteins in milk. A review. J. Dairy Sci. 52. 1155-1165. GROVES M. L. & GORDON W. G. (1969) Evidence from amino acid analysis for a relationship in the biosynthesis of y- and /I-caseins. Biochim. hiophys. Acta 194, 421-432. GROVES M. L. & KIDDY C. A. (1968) Polymorphism of r-casein in cow’s milk. Archs Biochem. BiophJs. 126, 18% 193. GROVES M. L., GORDON W. G., KALAN E. B. & JONES S. B. (1973) TS-A’. TS-B. R- and S-caseins: their isolation. composition. and relationship to the b-y-casein polvmorphs AZ and B. J. Dairy Sci. 56. 558%568. HI&-N. J.: GROVES M. L., CUSTER J. H. & MCMEEKIN T. L. (1952) Separation of r-. /& and y-casein. J. Dairy Sci. 35. 272-281. KAMIN~CAWA S., MIZOBIJCHI H. & YAMAUCHI K. (1972) Comparison of bovine milk protease with plasmin. Aur. hiol. &WI. 12. 2163-2167. _ KAMIN~GAWA S. & YAMAUCHI K. (1972) Decomposition of b-casein by milk protease. Similarity of the’decomposed products to temperature-sensitive and R-caseins. Agr. hiol. Chetn. 36. 255-260. KAMIN~CAWA S., SATO F. & YAMAUCHI K. (1971) Purification and some properties of milk protease. Agr. hiol. Chwi. 35. 1465-1467.
192
W. N. EIGEL
MAGEE S. C., GREEN C. R. & EBNER K. E. (1976) Plasmin and the conversion of the molecular forms of bovine milk galactosyltransferase. Biochim. biophys. Acfa 420. 187- 194. PETERSON R. F. & KOPFLER F. C. (1966) Detection of new types of /%casein by polyacrylamide gel electrophoresis at acid pH. A proposed nomenclature. Biochrm. biophys. Res. Commun 22. 388-392. RI~ADEAU DuhlAS B., BRIGNON G., GROSCLAUDE F. & MERTIER J. C. (1972) Structure Primaire de la caseine /I bovine. Sequence complete. Eur. J. Biochem. 25, 505-514. SHERRY S.. ALKJAERSIG N. & FLETCHER A. P. (1966) Activity of plasmin and streptokinase-activator on substituted arginine and lysine esters. Thromh. Diath. Haemorrhag. 16. 18-3 1. THOMPSON M. P., KIDDY C. A., JOHNSTON J. 0. & WEIN-
BERG R. M. (1964) Genetic polymorphism in caseins of cows’ milk--II. Confirmation of the genetic control of fl-casein variation. J. Dairy Sci. 47. 378-381. WEBER K. & OSBORN M. (1969) The reliability of molecular weight determinations by dodecyl sulfate-iolyacrylamide gel electrophoresis. J. hiol. Chem. 244, 44064412. W&TNEY R.-M., BRUNNER J. R.. EBNER K. E.. FARRELL H. M., JR., JOSEPHXIN R. V., MORR C. V. & SWAI~GCI~U H. E. (1976) Nomenclature of the proteins of cow’s milk: fourth revision. J. Dairy Sci. 59, 795-815. ZITTLE C. A., CERBULIS J., PEPPER L. & DELLA-MONICA E. S. (1959) Preparation of calcium-sensitive r-caesin. J. Dairy Sci. 42. 1897-1902. ZITTLE C. A. & CUSTER J. H. (1963) Purification and some of the properties of c(,, and K-casein. J. Dniry Sci. 46. 1183-1188.
of Animal
Sciences,
Purdue
(Rec&&
University, 5 Octohrr
West Lafayette,
IN 47907,
1J.S.A
1976)
Abstract-l. In vitro proteolysis of I-casein A’ resulted in the formation of products identified as F~-A’ (y-A’), yz-A2 (TS-A’) and yLI-A(R-) caseins by polyacrylamide gel electrophoresis conducted under both acidic and alkaline conditions. 2: Additionally, “Stains-all” was used to identify the polypeptide bands corresponding to the N-tcrminal portions of &casein A’ released during proteolysis. 3. These observations are consistent with the hypothesis that plasmin is responsible for in t+r, production of these minor casein fractions.
INTRODL’CTION
The term casein is used to describe a group of heterogeneous phosphoproteins secreted in milk by mammary epithelial ceils. In the bovine, whole casein contains approx Xi”/, /I-casein, which exists as a single polypeptide chain of 209 amino acid residues (Ribadeau Dumas rl al., 1972). ,%Casein has a calculated mol. wt of 23,982 and contains 5 phosphate moieties covalently attached to serine at residues 15, 17, 18. 19 and 35. Eight genetic variants of @-casein (A’, A”, A3, B. B,, C, D and E), produced by the substitution of one or more amino acids, have been reported (Whitney et al., 1976). Bovine casein contains several minor components, chief among which is a group of related proteins designated T-, R-, S- and TS-caseins. Extensive comparisons of these minor casein fractions have been made with respect to their amino acid compositions, mol. wts. fingerprints following tryptic digestion and end-group amino acids (Gordon rt al., 1972). Numerous similarities led these investigators to determine the partial sequence of the N-terminal end of these proteins and to compare them with the primary structure of fi-casein. They concluded that ?-A’, TS-A2 and R-caseins are identical with amino acid residues 29-209, 106-209, and 108.--209, respectively, of fi-casein A2. Moreover, y-B, S- and TS-B caseins were reported to be identical to similar portions of /I-casein B. These findings have led the Protein Nomenclature Committee of the American Dairy Science Association (Whitney er al., 1976) to propose a new nomenclature for these minor proteins. Henceforth. they will be designated yl-(y-). y2 (X-A’ and S-) and y3-(R- and TS-B) caseins. Recently, Gordon and Groves (1975) have proposed that a limited, highly specific proteolysis of /?-casein might be responsible for the occurrence of these minor casein fractions in bovine milk. The presence of a natural-occurring protease in milk was first demonstrated by Babcock and Russell (1897). Kaminogdwa et al. (1971) have concentrated milk protease and have demonstrated that incubation with
@casein resulted in the formation of 1;JTS-) and y,-(R-) caseins (Kaminogawa & Yamauchi, 1972). Kaminogawa et al. (1972) compared the properties of concentrated milk protease with plasmin and found many similarities. However, the mol. wt which they obtained for milk protease was lower than published values for plasmin. Recently, Eigel et ul. (1976) obtained additional evidence for the presence of plasmin and its zymogen, plasminogen. in milk. In this report evidence indicating that if! r:itro proteolysis of /?-casein A’ by plasmin produces ?,-A” (y-). 7,-A” (TS-A2) and ?,-A (R-) caseins is presented.
Fresh milk was obtained from individual cows of the University herd. Cream was removed by centrifugation at 7000~ for 15 min. Casein was precipitated by adiusting the pH of the skim-milk to 4.6 ;sing 3 N HCi. cdllected by filtration through cheesecloth and washed 3 times with distilled water. x,,-Casein was prepared by the urea method of Zittle et al. (1959) and was further purified by ethanol treatj~ent (Zittle & Custer, 1963). ,%Casein was prepared by the urea method of Hipp rt al. (1952). The genetic variant of fl-casein was obtained by vertical polyacrvlamide gel elecirophoresis in 4.5 M uiea at pH’ 9.5 (Thompson c’t (II.. 1964). The A variant was determined bv disc gel electrophoresis in 8 M urea at pH 4.3 (Groves & Go&on, 1969). Blood was collected in the presence of heparin from a cow at slaughter. Insoluble material was removed bv centrifugation first at 5000 y at 4‘C and then at 27.000 g at 4’ C. The cold plasma was then filtered to remove insoluble lipid material,’ Bovine plasminogen was prepared from plasma by afinity chromatography (Chibber rf ~1.. 1974). Protrolysis
qf /I-cm&r
A” by plrrsmirl
Plasminogen was activated to plasmin by addition of urokinase (100 plough units/ml) to plasminogen (5 myml) dissolved in 0.05 M sodium tetraborate, pH X.4. Incubation was at 37’C for 30 min. The incubation mixture for proteolysis contained p-casein AZ (10 mg/ml) in 0.05 M sodium tetraborate, pH 8.4, and merthiolate (0.02”Gl) to inhibit microbial growth.
188
W. N EIGEL
Prior to incubation the a-casein solution was heated at 80°C for 10 min. Freshly activated plasmin (0.28 mg/ml) _. was added and the solution incubated at 37°C. Aliquots (0.2 ml) were removed after 0.5. 1. 2, 4. 8. 15. 30 and 60 min and mixed with equal volumes of 8 M urea containing 0.2”:, 2-mercaptoethanol. Alkaline
phosphatase
treatment
A mixture
(0.5 ml) of p-casein A2 and plasmin, as described above, was incubated at 37’C. After 2 min an equal volume of 8 M urea containing 0.2”/, 2-mercaptoethanol was added. This solution was heated at 80°C for 10 min. cooled rapidly, and dialyzed against several changes of 0.005 M sodium acetate. pH 6.2, containing 0.001 M MgCl,. Incubation conditions with alkaline phosphatase Type VII from calf intestinal mucosa (Sigma) were similar to those of Green & Pastewka (1976) except that merthiolate (0.02”:,) was also included. Electrophorrsis
Proteolysis was monitored by disc gel electrophoresis in 4 M urea at pH 9.6 using the-technique of Davis (1964) as modified by Groves and Kiddy (1968). Gels were stained for protein with Amido Black IOB and for phosphoproteins with “Stains-all” (Green et al., 1973). Densitometric tracings of gels stained with Amido Black 10B and “Stainsall” w&e olbtained at 590 and 660 nm, respectively. Vertical polyacrylamide gel electrophoresis in 4.5 M urea at pH 3.0 (Peterson & Kopfler, 1966) was used for identification of y3- (R-) casein A. Mol.
wt determination
Sodium dodecyl sulfate (SDS) & Osborn. 1969) was used to /?-casein A2 and the products of Ovalbumin. chymotrypsinogen. chrome c were used as mol. wt
gel electrophoresis (Weber estimate the mol. wts of its proteolysis by plasmin. ribonuclease and cytostandards.
these electrophoretic conditions, yl-A2 (r-A2) and rs,caseins have similar electrophoretic mobilities (Groves, 1969). The 2 remaining bands have mobilities similar to y2-A2 (TS-A’) and y3-A (R-) caseins (Groves, 1969). SDS gel electrophoresis of p-casein A2 following incubation with plasmin for 4 min reveals the presence of 4 polypeptide bands (Fig. 3). The mol. wts calculated for 3 of these bands have been reported earlier (Eigel et al., 1976) and were used to identify these bands as 8-A’ , y1-A2 (r-A2) and y2-A2 (TX-A2) and Y~-A (R-) caseins (the latter 2 proteins differ in size by just 2 amino acid residues and thus cannot be differentiated by SDS polyacrylamide gel electrophoresis). The mol. wt estimates obtained for B-A’ and y1-A2 (y-A’) caseins are in agreement with the values obtained by Groves et al. (1973) using SDS gel electrophoresis. However, the mol. wt estimates for y2-A2 (TS-A2) and y3-A (R-) caseins are slightly high. If /j’-casein A2 can be converted directly to y2-AL (TS-A’) and yx-A (R-) caseins by plasmin, then the N-terminal portions of /I-casein A’ liberated in this process would be slightly larger than y2-A” (TS-A2) and y3-A (R-) caseins and may be partially responsible for the slightly higher mol. wt obtained for this polypeptide band. The mol. wt of the fastest-moving polypeptide band could not be determined with the standards used in this study. However, its mol. wt seems to be in the range of 9000-10,000 daltons and it probably represents the N-terminal end of Y~-(;J-) casein A2 released when y2-A2 (TS-A2) and y3-A (R-) caseins are formed by proteolysis of 7;1-(y-) casein A2 rather than b-casein A2 by plasmin. Disc gel electrophoretic patterns of the proteolytic products of p-casein A2 by plasmin also reveal the
RESULTS
Proteolysis of /?-casein A2 by plasmin was monitored by disc gel electrophoresis in 4 M urea at pH 9.6. These electrophoretic patterns (Fig. 1) reveal the gradual disappearance of the band corresponding to p-casein A2 along with the simultaneous appearance and increase in intensity of 2 slower-migrating bands with mobilities similar to y,-(1;-) and ‘/2-(TS-A2) caseins A2 (Groves, 1969). After 4 min of incubation with plasmin, the band corresponding to b-casein A2 has all but disappeared leaving the 2 major slowermigrating bands and a few faint faster-migrating bands (Fig. le). Further proteolysis by plasmin eventually results in the disappearance (after 30 min) of bands identified as yl-(y-) and y2-(TS-A’) caseins A2. Incubation of fi-casein A2 with urokinase under similar conditions had no effect on the electrophoretic pattern. Since y3- (R-) casein A has been reported to comigrate with yl-(y-) casein A2 in electrophoresis conducted under alkaline conditions (Groves, 1969), vertical gel electrophoresis in 4.5 M urea at pH 3.0 was used to further identify the products resulting from degradation of fl-casein A2 by plasmin. The electrophoretic pattern obtained after incubation of /j’-casein A’ with plasmin for 2 min revealed the presence of 4 polypeptide bands (Fig. 2a). The identifications of the p- and rl-(?;-) casein A2 bands were made by comparing their mobilities with samples of fl-casein A’ (Fig. 2b) and q,-casein (Fig. 2~). respectively. Under
P
II
(TS)I,
II
Fig. 4. Densitometric tracings of disc gel electrophoretic patterns (4M urea, pH 9.6) obtained for p-casein A’ (10 mg/ml) in 0.05 M sodium tetraborate. pH 8.4, incubated with bovine plasmin (0.28 mg/ml) at 37°C for 2 min. Duplicate samples were electrophoresed except that one gel was stained for protein with Amido black 10B (-) while the other gel was stained for phosphoproteins with “Stainsall” (---). Sample origin designated by arrow.
189
a
b
t
Fig. I. Disc gel electrophoresis at pH 9.6 in 4 M urea of /3-casein AZ (IO mg/ml) in 0.05 M sodium tctraborate, pH 8.4. incubated at WC with bovine plasmin (0.28 mg/ml) for (a) 0. (b) 0.5, (c) I, (d) 2. (c) 4. (I] 8, (g) 15, (h) 30 and (i) 60 min, respectively. Merthiolate was included to inhibit microbial growth.
190
Fig. 2. Vertical gel electrophoresis (4.5 M urea. pH 3.0) of (a) p-casein A’ (10 mg/ml) in 0.05 M sodium tetraborate, pH 8.4. incubated at 37°C with bovine plasmin (0.28 mg/ml) for 2 min, (b) p-casein A’ and (c) qcasein. Fig. 3. Sodium dodecyl sulfate gel electrophoresis ate, pH 8.4, incubated with bovine
of j-casein A* (10 mg/ml) in 0.05 M sodium plasmin (0.28 mg/ml) at 37°C for 4 min.
tetrabor-
&-A’,
191
j(*-AZ and y3-A caseins
of 2 faint, fast-migrating bands (Figs. 1 tracing of the disc gel pattern obtained after incubation of j-casein A2 with plasmin for 2 min is shown in Fig. 4. Superimposed on this tracing is the pattern obtained when a duplicate gel was stained with “Stains-all” instead of Amido black 1OB. Green rt al. (1973) reported that phosphoproteins produce a blue band with “Stains-all” while nonphosphorylated proteins give either a red band or do not stain at all. /$ And y,-(y-) caseins A2 contain 5 and 1 phosphate moieties, respectively, covalently attached to serine residues near their N-terminal ends (Ribadeau Dumas et a/., 1972). yz-A2 FS-A’) and y3-A (R-) caseins are devoid of covalently attached phosphate. Comparison of the 2 densitometric patterns (Fig. 4) indicates that b- and rl-(r-) caseins A2 stain as expected for phosphoproteins while yZ- (TSA’) casein A2 did not produce a band with “Stainsall”. The 2 fast-migrating bands observed in gels stained with Amido black IOB produced blue bands with “Stains-all”. Treatment of the sample with alkaline phosphatase prior to electrophoresis resulted in the disappearance of all blue bands when the gel was stained with “Stains-all”. Thus, the 2 fast-migrating bands are most likely the phosphorylated N-terminal ends of /j’- and/or y,-(y-) caseins A2 produced by plasmin during formation of y2-AZ (TS-A’) and Y~-A (R-) caseins.
appearance
ee). A densitometric
DlSCUSSIOlV
In this work, disc gel electrophoresis, conducted under both acidic and alkaline conditions, and SDS gel electrophoresis have been used to identify yl-A2 (‘J-A’) , y2-A2 (TS-A’) and y3-A (R-) caseins as products of the proteolytic cleavage of /&casein A2 by bovine plasmin. An increasing amount of indirect evidence strongly suggests the presence of plasmin and plasminogen in bovine milk. Kaminogawa et al. (1972) first suggested that milk protease may be plasmin, however, when concentrated milk protease was incubated with p-casein, only y2-(TS-) and y3-(R-) caseins were identified as products (Kaminogawa & Yamauchi, 1972). These workers did not report the formation of yl-(y-) casein as a result of this proteolysis. Magee et al. (1976) have demonstrated that conversion of galactosyltransferase from its high mol. wt form (58,000) to a low mol. wt from (44,000) is catalyzed ii7 t&o by plasmin and have suggested that the same process ~JI vim is responsible for differences in mol. wts reported for galactosyltransferase. Eigel et al. (1976) observed the electrophoretic changes produced in casein by the natural-occurring protease in milk and demonstrated that these changes could be reproduced by treatment of casein with isolated bovine plasmin. Moreover, they reported that activity of the naturally-occurring protease in milk was stimulated by urokinase, drastically inhibited by low levels ( lO-4 M) of E-aminccaproic acid (EACA) and completely inhibited by EACA and diisopropylfluorophosphate (DFP). The localization in the mammary gland of the site of proteolysis of /%casein by plasmin remains to be determined. Incubation of fi-casein B with trypsin results in the formation of several electrophoretic bands (Gordon & Groves. 1975). Three of these have been reported
to have mobilities in alkaline gel electrophoresis similar to ?/,-(y-), “J*-(S-) and yJ-(TS-) caseins B. Plasmin is very similar to trypsin in specificity in that both hydrolyze lysinyl and arginyl bonds. However, plasmin has been shown to hydrolyze lysine methyl esters more rapidly than arginine methyl esters while trypsin hydrolyzes both types equally well (Sherry et al., 1966). Initial formation of ?,-A2 (?-A’), yz-A2 FS-A’) and >a3-A(R-) caseins involves proteolysis of b-casein A2 at lysine 28, 105 and 107 (Ribadeau Dumas et al., 1972).
Acknorvlrdgenlents-Journal paper No. 6383 of the Purdue Agricultural Experiment Station. Lafayette, Indiana. This research was supported in part by a grant from the Purdue Cancer Committee (Indiana Elks). REFERENCES
BABCOCK S. M. & RUSSELL H. L. (1897) Unorganized ferments of milk: a new factor in the ripening of cheese. Wis. Agr. rxp. Sta. 22. 161-193. CHIBBER B. A. K.. D~UTSCH D. G. & MERTZ E. T. (1974) Plasminogen. In Methods in En~ymolmg,v Vol. 34. pp. 424-432. Academic Press, New York. DAVIS B. J. (1964) Disc electrophoresis-II. Method and application to human serum proteins. A~I. N.Y. Acud. Sci. 121. 4Ok427. EIGEL W. N., CHIBBER B. A. K. & TOMICH J. M. (1977) Plasminogen and plasmin: evidence for their presence in bovine milk. Science, submitted for publication. GREEN M. R. & PASTEWKA J. V. (1976) Characterization of major milk proteins from BALB/c and C3H mice. J. Dairy Sci. 59. 207-215. GREEN M. R.. PASTEWKA J. V. & PEACOCK A. C. (1973) Differential staining of phosphoproteins on polyacrylamide gels with a cationic carbocyanine dye. Analog. Biochzm. 56. 43-51. GORDON W. G. & GROVES M. L. (1975) Primary sequence of beta, gamma, and minor caseins. J. Dairy Sci. 58, 574-582. GORDON W. G., GROVES M. L., GREENBERGR.. JONES S. B., KALAN E. B., PETERSON R. F. & TO~NEND R. E. (1972) Probable identification of y-. TS-, R- and S-caseins as fragments of B-casein. J. Dairy Sci. 55. 261-263. GROVES M. L. (1969) Some minor components of casein and other phosphoproteins in milk. A review. J. Dairy Sci. 52. 1155-1165. GROVES M. L. & GORDON W. G. (1969) Evidence from amino acid analysis for a relationship in the biosynthesis of y- and /I-caseins. Biochim. hiophys. Acta 194, 421-432. GROVES M. L. & KIDDY C. A. (1968) Polymorphism of r-casein in cow’s milk. Archs Biochem. BiophJs. 126, 18% 193. GROVES M. L., GORDON W. G., KALAN E. B. & JONES S. B. (1973) TS-A’. TS-B. R- and S-caseins: their isolation. composition. and relationship to the b-y-casein polvmorphs AZ and B. J. Dairy Sci. 56. 558%568. HI&-N. J.: GROVES M. L., CUSTER J. H. & MCMEEKIN T. L. (1952) Separation of r-. /& and y-casein. J. Dairy Sci. 35. 272-281. KAMIN~CAWA S., MIZOBIJCHI H. & YAMAUCHI K. (1972) Comparison of bovine milk protease with plasmin. Aur. hiol. &WI. 12. 2163-2167. _ KAMIN~GAWA S. & YAMAUCHI K. (1972) Decomposition of b-casein by milk protease. Similarity of the’decomposed products to temperature-sensitive and R-caseins. Agr. hiol. Chetn. 36. 255-260. KAMIN~CAWA S., SATO F. & YAMAUCHI K. (1971) Purification and some properties of milk protease. Agr. hiol. Chwi. 35. 1465-1467.
192
W. N. EIGEL
MAGEE S. C., GREEN C. R. & EBNER K. E. (1976) Plasmin and the conversion of the molecular forms of bovine milk galactosyltransferase. Biochim. biophys. Acfa 420. 187- 194. PETERSON R. F. & KOPFLER F. C. (1966) Detection of new types of /%casein by polyacrylamide gel electrophoresis at acid pH. A proposed nomenclature. Biochrm. biophys. Res. Commun 22. 388-392. RI~ADEAU DuhlAS B., BRIGNON G., GROSCLAUDE F. & MERTIER J. C. (1972) Structure Primaire de la caseine /I bovine. Sequence complete. Eur. J. Biochem. 25, 505-514. SHERRY S.. ALKJAERSIG N. & FLETCHER A. P. (1966) Activity of plasmin and streptokinase-activator on substituted arginine and lysine esters. Thromh. Diath. Haemorrhag. 16. 18-3 1. THOMPSON M. P., KIDDY C. A., JOHNSTON J. 0. & WEIN-
BERG R. M. (1964) Genetic polymorphism in caseins of cows’ milk--II. Confirmation of the genetic control of fl-casein variation. J. Dairy Sci. 47. 378-381. WEBER K. & OSBORN M. (1969) The reliability of molecular weight determinations by dodecyl sulfate-iolyacrylamide gel electrophoresis. J. hiol. Chem. 244, 44064412. W&TNEY R.-M., BRUNNER J. R.. EBNER K. E.. FARRELL H. M., JR., JOSEPHXIN R. V., MORR C. V. & SWAI~GCI~U H. E. (1976) Nomenclature of the proteins of cow’s milk: fourth revision. J. Dairy Sci. 59, 795-815. ZITTLE C. A., CERBULIS J., PEPPER L. & DELLA-MONICA E. S. (1959) Preparation of calcium-sensitive r-caesin. J. Dairy Sci. 42. 1897-1902. ZITTLE C. A. & CUSTER J. H. (1963) Purification and some of the properties of c(,, and K-casein. J. Dniry Sci. 46. 1183-1188.