309

Biochimica et Biophysica Acta, 667 (1981) 309--320 © Elsevier/North-Holland Biomedical Press

BBA 38617

PURIFICATION AND PROPERTIES OF A MAJOR CASEIN COMPONENT OF RAT MILK

MASAAKI HIROSE a, TOSHIO KATO b KENJI OMORI b MASATOSHI MAKI b MASAAKI YOSHIKAWA b, RYUZO SASAKI b and HIDEO CHIBA b

a Department of Food Science and Nutrition, Nara Women's University, Nara 630, and b Department of Food Science and Technology, Kyoto University, Kyoto 606 (Japan)

(Received June 17th, 1980)

Key words: Casein; Milk protein; Glycoprotein; (Rat milk)

Summary

A casein component (C2~asein) was purified by ion-exchange and gel filtra- t ion chromatography from rat milk, and the properties of this protein were examined. The molecular weight of C2-casein, as determined by Sepharose 4B gel filtration in 6 M guanidine hydrochloride, was 34 000 -+ 1000. The average hydrophobici ty calculated from the amino acid composition showed that C2- casein is a rather hydrophilic protein. The a-helix content obtained from optical rotatory dispersion experiments was about 12%. In ultracentrifugation analyses, monomer and polymer peaks of C2-casein were both seen, and the monomer-to-polymer ratio was not affected by changing temperature condi- tions. C 2 ~ s e i n was precipitated by the presence of 2.5 mM CaCI2, and the pre- eipitability was greatly decreased by the dephosphorylation of the protein. C2- casein was stabilized from Ca2+<lependent precipitation by the addition of another rat casein component (C3~asein) or of bovine K-casein.

Introduct ion

Milk protein synthesis and secretion in the mammary gland are regulated by the multiple interaction of several hormones. Previous reports have shown that a-lactalbumin [1], ~-lactoglobulin [2,3], and caseins [4,5] are synthesized as precursors with a signal peptide and then are processed by signal peptidase [6]. Other mechanisms are also involved in post-translational processing in the case of caseins, since they are secreted after phosphorylation, glycosylation, and subsequent molecular assemblage into colloidal particles called casein micelles [7]. To study the regulation mechanism of casein secretion, it is necessary that the molecular properties of casein components are elucidated in terms of casein miceUe formation.

310

Knowledge concerning the molecular structure of casein components from ruminants has been accumulated, and the primary structures of bovine casein components have been determined [8--11]. It has been shown that bovine casein micelles may be formed by a mechanism in which the Ca2*-dependent precipitation of ~sl- and fl-casein is stabilized by their interaction with K-casein [12,13]. In studies of the regulation of milk protein synthesis and secretion, rodents have been used as animal models [ 1,5,14--17 ]. Although casein compo- nents from rats [18--24], mice [25,26], guinea pigs [27], and rabbits [28] have been p~Lrtially characterized, the molecular mechanism of casein micelle formation remains to be solved in these cases. Previously, rat casein has been shown to be composed of three main components of different molecular weights [20,23~24]. In the present paper, we have purified a rat casein compo- nent, which is secreted most abundantly [29], the one designated C2-casein by McKenzie and Larson [23], and have compared the properties of this protein with bovine caseins. The molecular weight and the amino acid composition of C2-casein are significantly different from those of all the bovine casein compo- nents, but with respect to ultracentrifugation data analyses and Ca2*-dependent precipitability, the rat protein behaved similarly to bovine ~sl-casein.

Materials and Methods

Preparation of casein Milk was collected from female Wistar rats at 15 days lactation using the

method of Woodward and Messer [21] and was defatted by centrifugation at 10 000 X g for 10 rain. 4 vol. distilled water were added to the skim milk, and the sample was adjusted to pH 4.0 by adding 3.4 M acetic acid. After the sam- pie was stirred for 2.5 h, it was centrifuged at 10 000 × g for 20 rain. The pre- cipitate was washed twice with cold distilled water and was dissolved in water by adjusting to pH 7.0 with the addition of 0.1 M NaOH. The whole casein sample was stored at --20°C until further processing. Purification of rat casein components was carried out as follows. The whole casein sample was dialyzed overnight against buffer A (10 mM imidazole, pH 7.1/1.0 mM EDTA/6.6 M urea) containing 1.0% ~-mercaptoethanol. The sample was applied to a DEAE- cellulose column equilibrated with buffer A containing 0.1%~-mercaptoethanol, and caseins were eluted with a 0--0.3 M NaCI linear gradient in 3 1 of the same buffer. Fractions containing casein components were collected, concentrated about five times on a Diaflo UM-10 membrane, and dialyzed against buffer A containing 1.0% ~-mercaptoethanol and 0.1 M NaC1. The casein solution was applied to a Sephacryl S-200 column equilibrated with buffer A containing 0.1% ~-mercaptoethanol and 0.1 M NaC1. The column was developed with the same buffer, and fractions of ~ ml each were collected. Bovine casein compo- nents were obtained as described previously [30]. Polyacrylamide gel electro- phoresis of the casein components was carried out in a buffer system contain- ing 0.1% sodium dodecyl sulfate as described by Laemmli [31]. Phosphopro- tein phosphatase was purified from bovine spleen according to the method of West and Dalgleish [32]. Caseins were dephosphorylated by phosphoprotein phosphatase in 0.1 M acetate buffer, pH 5.8, containing 5.0 mM ascorbic acid at 37°C as described previously [33].

311

Molecular weight determination After proteins (2 mg/ml, each protein) dissolved in 1.0 M Tris-HC1 buffer,

pH 8.5, containing 6.5 M guanidine hydrochloride were incubated with 0.05 M dithiothreitol for 2 h at room temperature, monoiodoacetate powder was added to the solution {final concentration, 0.1 M) and the samples were adjusted to pH 8--8.5 with 10 M NaOH. After a 1 h incubation, the modified proteins were subjected to gel filtration chromatography on a Sepharose 4B column (1.3 ×89 cm) equilibrated with 6.0M guanidine hydrochloride. Bovine serum albumin, ovalbumin, chymotrypsinogen, and myoglobin were used as molecular weight standards. Molecular weight was determined by plot- ting {distribution constant) 1/3 vs. (molecular weight) °'555 as described by Fish et al. [34].

Chemical analyses Casein was hydrolyzed at l l0°C for 24 or 72 h in 6.0 M HCI. Amino acid

analyses were carried out with a Hitachi amino acid analyser KLA-5. The values after 72 h hydrolysis were employed for valine and isoleucine content. The values of serine, threonine and tyrosine were obtained by extrapolation to 0 h. The tryptophan content was determined from the absorbance at 280 nm and 294.4 nm in 0.1 M NaOH [35]. The cysteine content was also determined by a chemical modification method using dithiobisnitrobenzoate after reduction with ~-mercaptoethanol [36]. Phosphorus contents were determined as described by Bartlett [37] after wet ashing in sulfuric acid.

Carbohydrate analyses were carried out by gas chromatography according to the method of Sweeley and Tao [38]. Casein or standard carbohydrates (man- nose, galactose, glucose, N~acetylgalactosamine, N~acetylglucosamine, and N~acetylneuraminic acid) were left to stand in methanolic HCI at 80°C for 22 h. Trimethylsilyl sugar was analyzed with a Shimadzu gas chromatograph GC-6M equipped with a 2% OV-17 column. Sialic acid was also analyzed by the color developing method described by Jourdian et al. [39].

Optical rotatory dispersion Optical rotatory properties of rat C2~casein and bovine asl~asein in the

300--600 nm region were examined with a JASCO optical rotatory dispersion recorder model J~5. Optical rotatory parameters were calculated as described by Moffitt and Yang [40].

Ultracentrifugation analyses Sedimentation analyses were performed with a Beckman model E analytical

ultracentrifuge at controlled temperatures as described previously [ 30].

Ca 2 +-dependent precipitation After 1.0 mg/ml of C2~asein, C3~asein or as1 ~ s e i n was incubated at 37°C

for 1 h in btfffer B (10 raM imidazole, pH 7.1/70 mM KC1) with v~ious con- centrations of CaC12, the sample was centrifuged at 700 X g for 1 min. The supernatant was removed and was diluted with 0.05 M potassium citrate. The concentration of casein was estimated from the absorbance at 280 nm.

3 1 2

Stabilization from Ca2*-dependent precipitation Stabilization of C2-casein or asz-casein by other casein components was

tested under the same conditions as those of the precipitation experiments except that the reaction mixture contained several different concentrations of rat C3-casein or bovine K-casein.

lsoelectric point The isoelectric point of C2-casein was determined by 5% polyacrylamide gel

isoelectric focusing in the presence of 2% ampholine (pH 4--6) and 8.5 M urea. Proteins were detected by staining with 0.04% Coomassie Brilliant Blue G250 after fixing the gel in 15% trichloroacetic acid. Another gel was cut into pieces of 5 mm length and the pieces were suspended in 1 ml water. The pH values of the extracts were determined.

Results

Purification of rat casein components Fig. 1 shows an elution profile of rat casein components on DEAE-cellulose

chromatography. Rat casein had three main components. According to the nomenclature of McKenzie and Larson [23], the first, second and third peaks correspond to C3-, C2-, and Cl~asein, respectively. C2~casein fractions (frac- t ion Nos. 106--120) were collected, and concentrated about five times on a Diaflo UM-10 membrane. Gel electrophoretic analyses showed that the C2- casein fraction was still contaminated by other casein components. The con- tamination was able to be completely removed by gel filtration chromatog- raphy. As shown in Fig. 2A, the rat casein sample was found to be separated into two peaks of C1- and C2-casein. The fractions containing C2-casein (Nos. 80--91) and Cl-casein (Nos. 95--102) were collected, dialyzed against distilled water, and lyophilized. C3~asein was isolated using DEAE~cellulose chromatog-

1.0 .E

N

~0.5

20

C 2

1 I j~ ,c l . f - j 4o2 ~

C3 o !

~ ' w ~ "q "~ ' / o o 50 100 150 Fraction number

C2 o) A B

50 100 50 Fraction number

C3

A 100

Fig. 1. DEAE-cellulose co lumn chromatography of rat casein. Solut ion containing 360 mg whole casein was appUed to a DE-52 column (3 X 18 era) equil ibrated wi th buffer A (10 mM imidazole, pH 7.1/1.0 mM EDTA/6.6 M urea) containing 0.1% ~-mercaptoethanol. The column was developed w/th a ]/.near g~adlent of 0--~.3 M NaCI (each fraction, 20 m]) a t a flow rate of 30 m//h.

Fig. 2. Gel fa t ra t /on chromatography. The concentra ted C2-casein (A) or C3-casein sample (B) obtained from fractions in DE-52 chzomatography was applied to a Sephaeryl S-200 column (8 X 126 cm) equn/- brated wi th buffer A (10 mM im/dazole, pH 7.1/1.0 mM EDTA/6.6 M urea) containing 0.1% ~-mercapto- e thanol and 0.1 M N a t l . The co lumn was developed wi th the same buffer at a flow rate of 27 ml/h, and fractions (5 m] each) were collected.

313

raphy (fraction Nos. 63--72) and was subjected to gel filtration chromatogra- phy using Sephacryl S-200. As shown in Fig. 2B, C3~asein was eluted essenti- ally in a single peak. The fractions (Nos. 81--92) were collected, dialyzed against distilled water, and lyophilized. Yields from 360 mg acid casein were 8.4 mg for Cl~asein, 72.8 mg for C2~asein, and 2.1 mg for C3-casein. The preparations of these rat casein components were found to be homogeneous in polyacrylamide gel elect~ophoresis in the presence of sodium dodecyl sulfate (Fig. 3).

Molecular weight determination The molecular weight of C2~asein was determined by the gel filtration

method described by Fish et al. [34]. The plot of (distribution constant) 1/8 vs. (molecular weight) °'555 showed a linear relation with the four marker proteins of bovine serum albumin, ovalbumin, chymotrypsinogen and myoglobin. With this plot, the molecular weight of C2~asein was estimated to be 34 000 ± 1000. The molecular weight, estimated in the same way, of bovine asl~asein was 22 500, which is close to the value obtained from the primary structure (23 615) [8].

Chemical composition In Table I, the data from amino acid analyses of C2-casein are compared

with bovine casein components [8--11]. The amount of glutamic acid plus glutamine was much greater in rat C2~asein than in bovine as1, as2-, ~- or ~ a s e i n (molar percentages were: C2, 27.7; a,1, 19.6; a82, 19.4; ~, 18.7; ~,

Fig. S. Gel electrophoras/s of ra t casein components . Purified ra t casein componen ts were analyzed by 0.1% sodium dodecyl sulfate-12% polyacrylamide gel e leo t rophoru / s as described by Laemmli [ S l ] . Gels were stained in 0.1% Cooma .~e b lue R250/50% tr lchloroaeet lc a d d for 1 h, and destained in T% acet/c acid. A, 20 #S ra t whole casein; B, 6.0/zg C3-easein; C, 6.0 #g C2-vasein; D, 6.0 #g Cl-casein.

Fig. 4. Sedimenta t ion analyses of C2-easein. 5.0 rng/ml C2-casein solut ion in buffer B cont..in!rig 2.0 mM di th io thre i to l was centr i fuged a t 60 000 rev./mln. Photographs were taken S0 rain af ter reaching maxi- mum speed at 5°C (A) and 14 mln af ter reaehlng ma~dmum speed a t 85°C (B).

314

T A B L E I

C H E M I C A L C O M P O S I T I O N O F R A T C 2 - C A S E I N

Values f o r a m i n o ac id r e s i d u e s a n d N H 3 r e p r e s e n t m o l a r p e r c e n t a g e s . All t he d a t a o f bovine casein c o m - p o n e n t s a r e f r o m r e p o r t s b y D u m a s and his c o - w o r k e r s [ 8 - - 1 1 ] un less speci f ied.

R a t Bov ine C2-case in

a s l - C a s e i n B ~s2-Caseln E-casein A 2 ~-case in B

A s p Jl' 8 .1 3 .5 1 .9 1 .9 2 .4 A s n 4 .0 6 .8 2 .4 4 .1 T h r 2 .6 2 .5 7 .3 4 .3 8 .3 Se t 1 0 . 7 8 .0 8 .2 7 .7 7 .7 Glu 1 2 7 . 7 12 .1 12 .1 8 .6 7 .7 Gin J 7 .5 -7.3 I 0 . I 8 .3 P ro 6 .2 8 .5 4 .8 1 6 . 8 I I . 8 G ly 0 .6 4 . 5 1 .0 2 .4 1 .2 Ala 1 2 . 0 4 . 5 3 .9 2 .4 8 .9 Cys 0 .3 0 1 .0 0 1 .2 Val 3 .4 5 .5 6 . 8 9 .1 6 .5 Met 2.3 2 .5 1 .9 2 .9 1 .2 n e 2 .7 5 .5 5 .3 4 .8 7 .7 L e u 9 .9 8 .5 6 .3 1 0 . 5 4 .7 T y r 3.1 5 .0 5 .8 1 .9 5 .8 Phe 1.7 4 . 0 2 .9 4 .3 2 .4 L y s 3 .3 7 .0 1 1 . 6 5 .3 5 .3

His 1 .2 2 .5 1 .5 2 . 4 1 .8 Azg 3.3 3 .0 2 .9 1 .9 3 .0 Trp 0 .9 1 .0 1 .0 0 . 5 0 .6 N H 3 26 .1 ( 1 1 . 6 ) ( 1 4 . 0 ) ( 1 2 . 4 ) ( 1 2 . 4 )

M o l e c u l a r w e i g h t 3 4 0 0 0 2 3 6 1 5 2 5 1 5 0 - - 2 5 390"" 23 9 8 3 19 0 2 3 a

Average h y d r o p h o b i c i t y ( ca l / r e s idue ) 9 0 7 1 1 7 0 1 1 1 0 1 3 3 0 1 2 2 4

P h o s p h o r u s ( m o l / m o l ) 1 1 . 5 8 1 0 - - I 3 5 1

C a r b o h y d r a t e Glc , 0 .6 n o n e - - n o n e O a l N A c , 1 - - 2 ; ( m o l / m o l ) Gal, 1 - - 2 ;

Sial ie a c i d , 1 - - 2

E11c~m a t 2 8 0 n m 6 .8 b 1 0 . 0 5 e - - 4 .6 e 1 2 . 2 d

I soe lee t r ic p o i n t 4 .6 4 . 8 2 - - 5 . 1 6 w 5 . 3 5 5 .37

a V a l u e fo r c a r b o h y d r a t e - f r e e p r o t e i n . b V a l u e in b u f f e r B. c D a t a f r o m T h o m p s o n [ ~ ] . d D a t a f r o m Zitf le and C u s t e r [ 54 ] . e D a t a f r o m W h i t n e y e t :a l , [ 5 5 ] .

16.0). The proline content of C2-casein was slightly higher than ~,z~asein, but lower than the other bovine casein components (molar percentages were: C2, 6.2; ~,1, 8.5; ~,2, 4.8; ~, 16.8; ~, 11.8). The presence of about 1 mol cysteine was detected in C2-casein: the same value was obtained by a chemical modifi- cation technique using dithiobisnitrobenzoate after reduction with ~-mercapto- ethanol [36]. The average hydrophobicity, as defined by Bigelow [41], of C2- casein, calculated from the amino acid composition, was only 907 cal per resi- due. This is in marked contrast to the bovine casein components, typical

3 1 5

examples having high average hydrophobicity (a~l, 1170; a,2, 1110; ~, 1330; ~, 1224).

Table I also shows that about 12 mol phosphorus were detected in C2-casein. This is similar to bovine asl~asein when phosphorus contents are normalized by the molecular weights.

The data from carbohydrate analyses by gas chromatography revealed that 0.6 tool glucose is present in i tool C2~asein. No other carbohydrate moiety, including mannose, galactose, N~cetylgalactosamine, N-acetylglucosamine, and sialic acid was detected by this technique. Sialic acid was also not detected by the color developing method described by Jourdian et al. [39].

Optical rotatory properties The secondary structure of C2-casein was analysed by the optical rotatory

dispersion method, and was compared with that of bovine ~,l~asein. As shown in Table II, a-helix contents were 11.6% for C2-casein and 5.9% for ~,l- casein. The latter value is consistent with the previous data given by Herskovits (6.0%) [42].

Ultracentrifugation analyses It has been shown that bovine casein components are easily polymerized

under physiological conditions. The degree of self~ssociation of ~ a s e i n is greater at higher temperature [43], while that of ~sl-casein is not affected by changing temperature conditions [44]. To compare sedimentation properties of C2-casein and bovine caseins, the rat protein was analyzed by ultracentrifu- gation at two different temperatures. Fig. 4 shows that C2~asein sedhnented mainly at 10.8 S, which would correspond to the sedimentation coefficient of C2~asein polymer. A minor peak of 2.2 S is probably the monomer form of the protein. There was no effect of temperature on the sedimentation patterns of the rat casein. Therefore, C2-casein is dissimilar to bovine ~-casein in terms of sedimentation properties.

Ca2+-dependent precipitation of C2-casein Bovine casein miceUes have been shown to be formed by a mechanism in

which calcium-sensitive components (as1- and ~<asein) are stabilized from pre- cipitation by calcium-insensitive ~<asein [12,13]. Therefore, the interactions of casein components with Ca 2+ appear to be necessary steps for micelle forma- tion. Deph0sphorylated C2-casein as well as the native protein were examined for their precipitability in the presence of Ca 2+, and these properties were corn-

T A B L E II

O P T I C A L R O T A T O R Y P A R A M E T E R S O F C 2 - C A S E I N

Optical rotatory dispersion of C2-case in ( 1 . 9 3 m g / m l ) a n d a s l - C a s e i n ( 2 . 7 7 m g l m l ) w a s examined in buffer B a t r o o m t e m p e r a t u r e . The percentage of a-hel ix content w a s c a l c u l a t e d f r o m t h e b 0 va lue .

~'c ( n m ) a 0 b 0 % H e l i x

C2-casein 230 --359 --73 11.6 asl-Casein 221 --552 ~37 5.9

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pared with ~sl~asein. Another rat casein component, C3~asein, was also exa- mined for its Ca2÷-sensitivity.

As shown in Fig. 5, native C2-casein was more sensitive to Ca 2÷ than native ~sz~asein. The Ca2÷~iependent precipitability of C2-casein was decreased markedly by dephosphorylation. The necessary Ca 2÷ concentration for 50% protein precipitation was about 2.5 mM for native C2-casein and about 7.0 mM for dephosphorylated C2~asein. C3-casein was found to be almost completely insensitive to Ca 2 *.

Stabilization of C2-casein from precipitation Bovine casein micelles have been demonstrated to be formed in vitro by add-

ing Ca 2* to a mixture of K~asein and Ca2*-sensitive components [12,13]. C3- casein was quite insensitive to Ca 2* (Fig. 5). A previous report by Woodward [22] showed that protein in one peak in DEAE~ellulose chromatography of rat caseins, which probably corresponds to our C3-casein, exerts effects to pro- tect other caseins against Ca2*~lependent precipitation. The stabilizing ability of C3-casein was examined in more detail and was compared with that of bovine ~ a s e i n . Fig. 6 shows that C3~asein was able to stabilize both C2- and esl~asein from Ca2*~lependent precipitation. Both esZ" and C2-casein were completely precipitated in the presence of 20 mM CaC12 (see Fig. 5), whereas these proteins were stabilized almost completely by the presence of C3~asein in a weight ratio of 0.6 and 1.0, respectively. However, the stabilizing ability of C3~asein was lower than that of bovine ~ a s e i n . C2~asein was less stabi- lized than bovine ~,z~asein by either K~asein or CS~asein, possibly because C2-casein is more sensitive to Ca 2. than is ~sz ~asein.

100

_c

0

" '~" ~ 1 0 0

5 10 15 20 "50 CaCI 2 ( rnM )

oI~ o!, oI~ o18 ,io C 3 or "K /C 2 or ctsl ( Wt rQtio )

Fig. 5 . C a l c i u m - d e p e n d e n t p r e c i p i t a t i o n . 1 . 0 m g / m l na t ive C2-case in (o) , d e p h o s p h o r y l a t e d C2-case in (A), n a t i v e ~s l -Case in (o) , o r na t i ve C3-case in ( 4 ) w a s i n c u b a t e d a t 3 7 ° C f o r 1 h w i t h d i f f e r e n t concentrat ions o f CaCl 2 , t h e n t h e s a m p l e w a s c e n t l d f u g e d a t 7 0 0 × g f o r 1 ra in . I n t h e o r d i n a t e , a b s o r b a n c e a t 2 8 0 run in t he s u p e ] m a t a n t w i t h o u t a d d i t i o n o f CaC12 is e x p r e s s e d as 1 0 0 % .

Fig . 6 . S t a b i l i z a t i o n o f C2-casa ln f r o m c a l c i u m - d e p e n d e n t p r e c i p i t a t i o n . 1 .0 m g / m l C2-casc in o r ~s l -Case in w a s i n c u b a t e d in t h e p r e s e n c e o f 2 0 m M CaC12 w i t h v a r i o u s c o n c e n t r a t i o n s o f ca l c ium- insena l t i ve case in (C8° o r K-casein) in t h e c o m b i n a t i o n s o f o~1- p lu s K-casein (o) , C2- p lus ~-case in (e ) , ~ s l " p lu s CS-ca l e in (A), a n d C 2 - p lu s CS-case in (A). T h e s a m p l e w a s c e n t r i f u g e d a n d a b s o r b a n c e in t h e s u p e r n a t a n t w a s de te r ° m i n e d i n t h e s a m e w a y as in t h e c a l c i u m - d e p e n d e n t p r e c i p i t a t i o n e x p e r i m e n t s .

317

Discussion

In previous reports, the molecular weight of C2~asein has been estimated to be 38 000--42 000 by gel electrophoresis in sodium dodecyl sulfate [20,22-- 24]. However, caseins have been shown to behave abnormally in the electro- phoresis [26]. We found that the apparent molecular weight of bovine asl- casein estimated by gel filtration analysis in 6.0 M guanidine hydrochloride is almost exactly the same as the value calculated from the amino acid sequence of this protein. Therefore, the molecular weight of C2~asein (34 000 -+ 1000) obtained by the gel filtration method in this paper is probably more accurate than the values reported previously [20,22--24]. C2~asein and a casein com- ponent from mouse milk [26] are unique examples of high molecular weight caseins.

The chemical composition of C2-casein was significantly different from bovine casein components. Bovine caseins have been shown to be examples of proteins of high hydrophobicity. Hydrophobic residues are in clusters in the primary structures of the bovine caseins and are at least in part on the surfaces of the molecules [8--10]. It is believed that in bovine caseins hydrophobic bonds are involved in micelle formation as well as in self-association of each component [43--45]. The average hydrophobicity of C2~asein was only 907 cal per residue. This value is extremely low, considering that most self-associ- ating proteins have high average hydrophobicity values [41], and that all the casein components from other animals have values of more than 1100 cal per residue [8--11,27~28]. The bonding involved in molecular interactions of C2- casein is an interesting subject.

a-Helix content of C2~asein (11.6%) was higher than those of bovine casein components (as1, 6%; ~, 5%; ~, 5%) [42]. This may be related to the fact that the proline content of C2-casein is lower than these bovine casein components (Table I).

The existence of glucose in C2-casein as carbohydrate moiety in a glycopro- tein is unusual. The single example of a glycopeptide containing glucose is the low molecular weight glycopeptide from human erythrocyte membranes [46]. In this glycopeptide, glucose has been shown to be covalently attached S-glyco- sidically to a cysteine residue. However, this may be unlikely in C2~asein, since about 1 tool sulfhydryl group, a value close to data from amino acid analyses, was detected by a chemical modification method using dithiobisnitrobenzoate. These suggest that in C2-casein glucose may be attached to the peptide by a novel type of chemical linkage. During the preparation of this manuscript, a report by Pelissier et al. [47] which describes the chemical compositions of rat caseins has appeared. They have shown that proteins eluted from DEAE-cellu- lose, which probably correspond to our C2¢asein, can be separated into two components (B1 and B2 proteins), and that these proteins may possess the same peptide chain with a variable quantity of sialic acid. The contents of amino acid and phosphorus in their B1 and B2 proteins are similar to our C2- casein except in the case of glycine, isoleucine and cysteine. The contents of glycine and isoleucine in C2-casein were significantly lower than B1 and B2 proteins (glycine, 50%; isoleucine, 66%). About I mol cysteine was detected in C2~asein, but no cysteine residue was detected in their B1 and B2 proteins. In

318

addition, sialic acid was not detected in our C2-casein by either gas chromato- graphy or the color developing method. The reason for the inconsistency in the chemical compositions is not clear. The quanti ty or the type of the carbohy- drate moiety might be dependent on physiological conditions of the animals.

It has been demonstrated that both intermolecular associations of as1-, fl- and K~asein and their interactions with Ca 2+ are implicated in the formation of bovine casein miceUe [12,13,45]. Experiments were carried out to assign the role of C2-casein in miceUe formation in rat milk. C2-casein was found to be precipitated by the addition of 2.5 mM Ca 2+ (Fig. 5). In this respect, C2~asein seems more sensitive to Ca 2+ than asl- and fl~.asein, which require 5--10 mM Ca 2+ for their precipitation. A minor bovine casein component, designated as2- casein, has been shown to be more sensitive than asl- and fl-casein [48,49]. Rat C2-casein appears similar to bovine as2-Casein in Ca2÷-sensitivity. At present, however, we are not able to evaluate in more detail the analogy between rat C2~casein and bovine as2~Casein, since data to compare these proteins directly are not sufficient. Bovine asl- and fl-casein have been shown to be distinguish- able by ultracentrifugation analyses: self-association of fl-casein is greatly dependent on temperature [43], but that of asl-Casein is not [44]. As shown in Fig. 4, no temperature dependency was observed in the self-association of C2- casein. In a previous report we demonstrated that fl-casein completely loses its Ca2+<lependent precipitability after dephosphorylation by spleen phospho- protein phosphatase [33], while the precipitability of asl-casein still remained after the enzymatic t rea tment [50 ]. Although the Ca 2+<lependent precipitabfl- ity of C2-casein was lessened by dephosphorylation, it remained significant (Fig. 5). Furthermore, C2-casein was precipitable under low temperature condi- tions which almost completely inhibited the Ca2+-dependent precipitability of fl-casein but not asl-casein (unpublished data). These observations show that C2~asein resembles ~sl~asein rather than fl~asein. Ca2+-binding to carboxyl groups may have an essential role in the precipitation of C2-casein, as in the case of bovine asl-casein [51].

As shown in Fig. 6, rat C2~asein was efficiently stabilized from Ca2+<lepen - dent precipitation by the addition of bovine K-casein, which is known to be insensitive to Ca 2÷ and to stabilize a~l- and fl-casein from precipitation by form- ing stable casein micelles [12,13]. C3-casein was found to be insensitive to Ca 2+ (Fig. 5) and stabilized C2-casein (Fig. 6), suggesting that the former protein may function like bovine K-casein for casein micelle formation in rat milk. However, the stabilization of C2-casein was less efficient by C3-casein than by K-casein (Fig. 6). Therefore, it appears still inconclusive that C3-casein is the sole component to stabilize C2-casein in rat milk. Pelissier et al. [47] have shown similarity in the N-terminal sequence of rat A1 and A2 proteins, which probably correspond to our C3-casein, with bovine fl-casein but not with bovine K-casein. We have shown previously that dephosphorylated bovine fl-casein can stabilize a~rcasein from Ca~+-dependent precipitation in an about 1 : 1 weight ratio [52]. In this respect, rat C3-casein might be related to dephosphorylated bovine fl-casein. Green and Pastewka [26] have reported that in mouse casein a fourth minor component has K-casein-like characteristics. In the present paper, a fourth minor component , which was seen in the gel electrophoresis of whole casein (Fig. 3), and Cl-casein were not characterized. To study the

319

mechanism of casein miceUe formation in rat milk in more detail, it may be important to examine all combinations of the rat casein components.

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