著者らは既に,牛乳カルシウムの利用性が高いことの理由のひとつに,小腸管内に生成するカゼインホスホペプチドが考えられることを指摘した.その機構を知る第一歩として本研究では,カゼイン摂取マウスの小腸管内容物からホスホペプチドを単離し,アミノ酸組成を調べた.

マウスには体重130～200gの雄ウイスター種を用い,β-カゼインもしくはHammarstenカゼインを含む飼料をspace-feedingし,その腸内容物からホスホペプチドを分離した.ホスホペプチドの単離並びに精製はゲル泳過法およびDowex50イナン交換クロマトグラフィーによった.またアミノ酸組成は,ホスホペプチドの加水分解物について日立アミノ酸自動分析器034型によって分析した.

単離したホスホペプチドは,グルタミン酸とホスホセリンが多く,これらがCa結合性残基となる可能性が考えられた.またN/P比は,トリプシン消化ペプチドについての文献値に近似していた

Imamura, Tsuneaki

Naito, Hiroshi

Suzuki, Hideo

Name not available

カゼイン摂取マウス腸管内で生成するホスホペプチドのアミノ酸組成

Hiroshima University Institutional Repository

]. Fac. Fish. Anim. Hush., Hiroshima Univ. (1974), 13:57-66 Amino Acid Composition of Phosphopeptide produced 1n the Small Intestinal Tract of Rat fed on Casein Tsuneaki IMAMURA, Hideo SuzuKI and Hiroshi NAITO Department of Food Chemistry and Technology, Faculty of Fisheries and Animal Husbandry, Hiroshima University, Fukuyama (Figs. 1-a - 5-b, Tables 1-6) Whether or not the nutritional value of protein or peptide is equal to that of the free amino acids mixture with an amino acid composition equivalent to that of protein or peptide is a significant problem in nutritional biochemistry. Although the presence of peptide in the digestive organ of mammalian has been reported, its nutrit"ional significance however has not been elucidated yet. Recently, MELLANDAR1J and NAITO et a/. 2) suggested that casein phos-phopeptide contributes to a high utilization of calcium in milk. But what kinds of amino acid residues or functional groups contribute to the calcium-tinding property of casein phosphopeptide is not yet evident. In view of this, casein phosphopeptide &'lmples were prepared from the intestinal contents of rats fed on casein and their amino acid compositions were determined. MATERIALS AND METHODS 1. Preparation of ,8-casein ,8-Casein was prepared by the urea method as modified by AscHAFFENBERG3J and about 20 mg of sample were obtained from 51 of fresh skim milk. According to the method of GRovEs et al.,4J the purity of the samples was checked by disc electrophoresis performed with a 7% polyacrylamide gel containing 4 M urea. The sample was found to be almost homogeneous with ,8-casein, though a. 5 - and "-casein could be detected slightly. 2. Brreeeding methods of rats Eight rats of one weak old were fed by way of a space feeding for eight days on the diet as shown in Table 1. After the preparative breeding period, they were divided into two equal groups and the names of H and ,8 were given under 58 Tsuneaki !MAMURA, Hideo SuzuKI and Hiroshi NAITO Table 1. The diet of rats for preparative breeding periods. Commercial first grade casein (20%) a·starch (38%) Vitamin Mineral Fibre ( 5.5%) soy bean oil ( 6.5%) sucrose (30%) sum Table 2. The diet for H-group. Hammarsten casein a-starch Vitamin Mineral Fibre soy bean oil sucrose choline chloride surn (20%) (38%) ( 5.5%) ( G.5%) (30%) Table 3. The diet for p-group. p-casein a-starch Vitamin Mineral Fibre soy bean oil sucrose (20%) (38%) ( 5.5%) ( 6.5%) (:10%) 200g 380g 55g 65g 300g 1000g 18 g 34.2g 4.95g 5.85g 27 g 0.18 g 90.18 g 18 g :H.2g <\. 95 g 5.85 g '27 g choline chloride 0.18 g sum 90.18 g Table 4. The growing records of each rat. ~~1~~";':,,.~~=~~~~hc--,~,___H-f'wcih' ,-booA 4 we~h< :"' ,,, . w;;~:· r':t;e_t ~' _:7;~~t__ 0:~ ~et weigl1! • of iet __ i w1~:;t~l 0:~ ~~ 3 0 138 0 168 4 5 6 7 8 9 10 --jUst-before _1-illing~----128 5 128 7 124 (j 121 9 122 7 119 10 119 10 -~--· I 134 I (0.7)* ' -~-~--,-~-~~~--'~-'-•-oo--164 162 162 162 164 163 160 17:\ 10 10 10 10 9 10 'I ( 1. 2)* 188g 20g 20 196 4 194 4 186 10 188 8 182 10 186 8 178 9 183 7 17:i 10 180 8 173 9 178 9 173 7 180 6 1W 9 176 5 189 (0.2)* Amino Acid Composition of Phosphopeptide produced in the Small Intestinal 59 Tract of Rat fed on Casein {i-1 {i-2 (3-3 {i-4 body weight body weight body weight body I weight weight of diet weight of d1et weight of diet weight of diet 1st day 172 g 20g 180g 20g 184g 20g 142g I 20g 2 20 20 20 20 3 186 4 194 4 192 8 162 4 4 176 8 182 12 186 12 152 10 5 173 8 182 14 184 14 156 9 6 172 8 181 9 185 12 158 9 7 172 11 176 12 184 13 158 12 8 172 12 175 13 188 12 162 12 9 173 10 17G 10 187 10 164 9 10 171 10 173 10 Hl3 9 164 10 Lustbefore --- --------------~-- ----~---~-------------- ----' ----------------illing I 186 (0.2)* 192 I (0.3)* ; 196 I (0.3)* i 182 ' (0.1)* -----~---··-----~---------·--------·--( )* the lost weight of diet their respective diets. H- and ,8-group were space-fed for two days on the diets as shown in Table 2 and 3, respectively. The growmg records of each rat are shown in Table 4. 3. Isolation of casein phosphopeptide On the lOth day, that is the last day of the breeding period, each individ~l rat was given its diet with a time lag of three minutes and then killed under ether anaethesia at exactly one hour after the meal. The intestinal contents of each rats were washed out with cold water. The washings were frozen in dry ice-acetone and stored at - 20°C. To the frozen samples, after thawing to such an extent that small amounts of ice crystall remained, a few drops of 50% tri-chloroacetic acid (TCA) solution were added first and then ·larger quantities of an ice-cold TCA solution in order to bring the final concentration up to about 10%. Themixturewas homogenized and centrifuged (lO,OOOxg, 20min, lOOC). A 5 ml portion of the supernatant was filtered with a Sephadex G-25 column previously washed with 0. 05 N acetic acid and a NjP ratio of the fraction eluted at the void volume was measured. Each of the remaining supernatants of H- and ,8-group was eluted by successive gel filtrations with Sephadex G-25 and G-50 columns previously washed with 0. 05 N acetic acid. The fractions eluted at the void volume were chromatographed on Dowex 50 x 2 columns equilibrated previ-ously with 0. 2 N pyridine-acetate buffer, pH 3. 0,51 and finally desalted by the Sephadex G-25 filtration. In the above gel filtrations and chromatography, the effluents were fractionated to 5 ml and 10 ml portion, respectively. 60 Tsuneaki IMAMURA, Hideo SuzuKr and Hiroshi NArTo 4. Determination of peptide and organic phosphorus The amount of peptide was determined by the ninhydrin colorimetry,Sl and that of orgaritc phosphorous by the modified ALLEN's method.7l 5. Amino acid analysis Amino acid analysis of the casein phosphopeptide sample was done by the method of SPACKMAN et al.8) using an automatic amino acid analyzer (Hitachi Model 034). The sample of l.lmg was hydrolyzed with 3.3ml of 6 N HCl in a sealed tube for 24 hrs at l10°C. After removing humin and HCl by filtration and evaporation, respectively, the hydrolyzate was kept in 'l/acuo overnight and dissolved in 1. 5 ml of 0. 2 N citrate tuffer (pH 2. 2). A 0. 5 ml portion of the hydrolyzate solution was used for the analysis of amino acid composition. RESULTS 1. Phosphopeptide obtained from the H-group The mean value of the NJP ratios of the supernatants in the TCA treatment was found to be about 100. The remaining supernatants from individnal rats 1.0 0.8 e c 0 00 N 0.6 ... oS ~ ·;;; c ~ 0.4 Qj .S:l ... "' 0 0.2 Tube number Fig. 1-a Gel filtration pattern of H-group on a Sephadex G-25 column. Column: 2.5x40cm Eluent : 0. 25 N acetic acid s " 0 00 N :0 ::-... " ~ Oi ·~ "' 0 0. 6 0.5 0.4 0.3 0.2 0.1 Tube number Fig. 2-a Gel filtration pattern of H-group on a Sephadex G-50 column. Column: 2.5x40cm Eluent : 0.05 N acetic acid Amino Acid Composition of Phosphopeptide produced in the Small Intestinal Tract of Rat fed on Casein 61 were combined and concentrated. The concentrate was filtered with a Sephadex G-25 column. The elution curve is shown in Fig. 1-a. After concentration, the effiuent (Tube Nos. 14-25) was filtered again with a Sephadex G-50 column. The elution curve is shown in Fig. 2-a. Among the three peaks, the fraction of the second peak was concentrated and filtered with a Sephadex G-25 column, because the second one contained phosphopeptide. The result obtained is shown 0. 8 E c: 0.6 0 CO "' ~ $ ·;;; 0.4 c: " "" <a <.) 8 0.2 0 10 40 Tube number Fig. 3-a Gel filtration pattern of H-group on a Sephadex G-25 column. 0.4 § 0 0.3 CO "' :6 '§ 0.2 ;;:; "" <a .So! c. 0 0.1 Column : 2. 5 x 40 cm Eluent : 0.05 N acetic acid Tube number Fig. 4-a Chromatography of H-group with a Dowex 50 x 2 column. Column: 1.8x50cm Eluent: 0.2 N pyridine-acetate buffer (pH 3.0) 0-0 peptide (0. D. at 570 nm) 6-6 organic phosphorus (0. D. at 660 nm) 50 m Fig. 3-a. The NjP ratio of the fraction eluted at the void volume was 17.6; the value IS one-fifth of that of the original superna tan t. To the fraction, after concentrated to 10 ml volume, 2.5 ml of 1 N pyridine-acetate buffer was added so as get 0. 2 N pyridine m the final concentration. After the pH was .adjusted to 2.5 with 6 N ·HCl, the sample was inserted on the upper bed of a column of Dowex 50 x 2 .. : The elution curve 0.08 E 8 0.06 00 N .... ., ~ ·;;; 0. 04 ~ ~ <a -~ ~ 0.02 00~--~~l~OoX~~~----~30~­Tube number Fig. 5-a Desaltation of H-group Sephadex G-25 colum. Column: 2.5x40cm Eluent : water with a 62 Tsuneaki lMAMVRA, Hideo SvztrKI and Hiroshi NAITO Table 5. The amino acid compositions of isolated phosphopeptides from the intestinal tract. Amino Acid H·group ~·group Lys His NHa 26 7.5 Arg Asp 4 1.75 Thr 5.5 4.75 Ser 10 5.5 Glu 9.5 5.25 Pro + + Gly 2.5 1.75 Ala 2.5 1.25 Cys Val 2.5 1. 75 Met + ± lieu 3.0 1.0 Leu 1.0 1.0 Tyr Phe + + Trp N. D.* N. D.* (Leu=l.O) *N. D.znot determined shown in Fig. 4-a was obtained. The fraction (Tube Nos. 10-15) containing organic phosphorus was desalted with a Sephadex G-!}.5 column (Fig. 5-a). The fraction eluted at the void volume was lyophilized and about 6. 3 mg of phosphopeptide was obtained as a purified sample. This sample of 1 . 1 mg was used for amino acid analysis and found to have the composition as shown in Table 5. 2. Phosphopeptide obtained from the ,8-group The N/P rl;ltios of the supernatants by the TCA treatment from indivic,lual rats of the ,8-group (,8-1, ,8-2, ,8-3 and ,8-4) were 58. 4, 54. 0, 56.7 and 94. 3, 1.0 0.8 E <: ~ "' 0.6 ;; ~ ·u; "' "' 0. 4 "" -.; .!:! Q. 0 0.2 00 10 20 30 40 T .. be number Fig. 1-b Gel filtration pattern of fil·group on a Sephadex G-25 column. Column : 2. 5 x 40 cm Eluent : 0.05 N acetic ecid 1.5 E <: ~ ~ 1.0 ~ 'iii ~ -.; .sa o. s Q. 0 40 Tube number Fig. 2-b Gel filtration pattern of j3-group on a Sephadex G-50 column. Column: 2.5x40cm Eluent : 0.05 N acetic acid Amino Acid Composition of Phosphopeptide produced in the Small Intestinal 63 Tract of Rat fed on Casein 2.0 1.5 s = 0 CO N ~ ~ 1.0 ·;;; = ~ 'Oj ·3 c. 0 0.5 00 30 Tube number Fig. 3-b Gel filtration pattern of fi·group on a Sephadex G-25 column. Column: 2.5x40cm Eluent : 0.05 N acetic acid 0.6 E ~ 0.5 ;; 0.4 :;. ·;;; 0.3 = ~ 0.2 '" ·" c. 0. 1 0 Tub" rmmh<'r Fig. 5-b Desaltation of fi·group Sephadex G-25 column. with a 0 '; E " 0 0.3 C() "' ;;; ~ ·;;. ~ 0.2 "" -;;; -~ c.. c 0.1 Tube number Fig. 4-b Chromatography of .S·group with a Dowex 50 x 2 column. Column : 1. 8 x 50 cm Eluent : 0. 2 N pyridine-acetate buffer (pH 3.0) 0-0 peptide (0. D. at 570 nm) f:::.-1:::. organic phosphopus (0. D. at 660 nm) respectively. Among them. the three supematants with a s1milar N/P ratio were combined and concen-trated. The concentrate was filtered with Sephadex G-25 and G-50 columns successively and chromato-graphed with a Dowex 50 x 2 column in the same way as used on the above H-group. The elution Column: 2.5x40cm curves are shown in Figs. 1-b. Eluent : water 2-b. 3-b. 4-b and 5-b. The yield of the purified phosphopeptide was 4. 1 mg. The amino acid composition is shown in Table 5. CONSIDERATIONS AND CONCLUSIONS 1) Two casein phosphopeptide preparations. i.e .• H. C. P. and (3. C. P. were 64 Tsuneaki IMAMURA, Hideo SuzuKI and Hiroshi NAITO prepared from the contents of small intestinal tracts of rats by successive gel filtrations. In the case of the H. C. P. which was prepared from the 4 rats fed on Hammarsten casein, the NfP ratio of the phosphopeptide fraction obtained through the fir!St gel filtration with a Sephadex G-25 column was found to be 10 times higher than that of the one reported by NAITo et a/.2l Although the ration used in this experiment contained both sucrose and starch as appetizers for the ;rats, the one used by NAITO et al.2l contained starch only. The difference in ration seems to influence the digestive conditions of rats, but the cause for the above difference of N/P ration is still not clear. 2) Another phosphopeptide preparation was the {3. C. P., plepared from the 3 rats fed on {3-casein. Borh the two preparations, H. C. P. and {3. C. P., had a similar amino acid composition, and contained considerable amounts of glutamic acid and phosphoserine. Accordingly, they were acidic peptides. The calculated N/P ratios of H. C. P. and {3. C. P. on the basis of their phosphoserine contents were 6.5 and 5.7, respectively. These values are similar to those of NAITo et al.2l and of others9H 3l (Table 6). Considering their amino acid compositions, Table 6. The amino acid composition of phosphopeptide from casein (by literature). Am A .d I M<cu•·l (),.,.. SCHORMtlLLERlO) I DuMAsllll2> MANSON13l I mo Cl DAR1> BERG9l a-casein I a-casein I . I . I fo-casein fo-casein as 1-casem ias 1 -casem Lys 2 1 1 1 1 I His NH3 I 2 2 Arg 2.8 4 1 1 1 3 1 1 Asp 1.7 1 1 1 1 Thr 9.5 5 5 4 4 2 4 5 Ser 10.3 10 7 5 7 4-5 7 7 Glu 1.0 1 1 1 1 1 Pro 1.2 1 1 1 1 Gly 1.0 2 1 1 1 1 Ala I Cys 2.0 2 2 1 2 2 2 Val 2 1 I 1 1 1 2 Met 3.7 4 2 1 2 2 2 lieu 1.0 3 3 Leu (Leu=l.O) Tyr Phe Trp p I 18.6 6 5 4 5 3 5 4 Ser/P 0.5 0.8 1 1 0.9 0.7 0.8 1.25 N/P I 2.6 5.9 4.45 Amino-N/P . 1.8 6.0 I 4.4 4 4 5-6 5.8 7.75 Amino Acid Composition. of Phosphopeptide produced in the Small Intestinal Tract of Rat fed on Casein some acidic peptides seem to be present in the samples as impurities. 65 When tryptic digestion occured, lysine and arginine must have been present in the peptide preparations. As shown in Table 5, however, they could not be observed in the amino acid compositions of the two phosphopeptide preparations. 3) The localized presence of a phosphoric acid group on the casein phos-phopeptide chain has been reported by DuMAS11)12) and MANSON13l and the formation of the Ca-complex or the Ca-binding property of the casein phosphopeptide by MELLANDAR1) and '0STERBERG.9l A Ca-binding protein contaming much glutamic acid had been isolated from chick intestinal mucosa. 14l On the basis of these findings, the casein phosphopeptide preparations in this study must have a significant physiological activity. This study was carried out under the guidance of Dr. H. NAITo previously assistant professor in our laboratory. REFERENCES 1) MELLANDAR, 0.: Acta Soc. Med. Upsal., 52, 107 (1947). 2) NAITO, H., KAwAKAMI, A. and lMAMURA, T.: f. Agr. Biol. Chem., 36, 409 (1972). 3) AscHAFFENBERG, R.: f. Dairy Res., 30, 259 (1963). 4) GRovEs, M. L. and Kmov, C. A.: Arch. Biochem. Biophys., 1.26, 188 (1968). 5) ScHOEDER, W.A., }oNES, R.L., CoRMICK, ]. and MeCALLA, K.: Anal. Chem., 34, 1570 (1962). 6) YEMM, E. W. and CocKING, E. C.: Analyst, 80, 209 (1955). 7) ALLEN, R. ]. L.: Biochem. f., 34, 858 (1940). 8) SPACKMAN, D. H., STEIN, W. H. and MooRE, S.: Anal Chem·, 30, 1190 (1958). 9) OsTERBERG, R.: Acta Chem. Scand., 18, 795 (196~). 10) ScHORMOLLER, }., HANS, R. und BELITZ, H. D.: Z. Le bens. Unters. Fors h., 131, 65 (1966). 11) GRoscLAUDE, F., MERCIER, J·C. and DuMAS, B. R.: Eur. f. Biochem., 14, 98 (1970). 12) DuMAS, B. R., GRosCLAUDE, F. and MERCIER, J·C.: Eur. f. Biochem., 14, 451 (1970). 13) MANSON, W. and ANNAN, W.D.: Arch. Biochem. Biophys., 145, 16 (1971). 14) WAssERMAN, R. H., CARRADINO, R. A. and TAYLOR, A.: f. Biol. Chem., 243, 3978 (1968). 66 Tsuneaki IM..MuR.. Hideo SUZUKl and Hiroshi N"'ITO カゼイン摂取マウス腸管内で生成するホスホベプチドのアミノ酸組成今村経明・鈴木秀男・内藤博著者らは既に，牛乳カルシウムの利用性が高いことの理由のひとつに，小腸管内に生成するカゼインホスホベプチドが考えられることを指摘した.その機構を知る第一歩として本研究では，カゼイン摂取マウスの小腸管内容物からホスホベプチドを単離し，アミノ酸組成を調べた.マウスには体重 130-2∞gの雄ウイスター種を用い.s-カゼインもしくは Hammarstenカゼインを含む飼料を space-feedingしその腸内容物からホスホベプチドを分離した.ホスホベプチドの単離並びに精製はゲル泳過法および Dowex50 イオン交換クロマトグラフィーによった.またアミノ酸組成は，ホスホベプチドの加水分解物について日立アミノ酸自動分析器034型によって分析した.単離したホスホベプチドは，グルタミン酸とホスホセリンが多く，これらが Ca結合性残基となる可能性が考えられた.また N/P比は， トリプシン消化ベプチドについての文献値に近似していた.

Amino Acid Composition of Phosphopeptide produced in the Small Intestinal Tract of Rat fed on Casein

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カゼイン摂取マウス腸管内で生成するホスホペプチドのアミノ酸組成

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