トップページ - 横浜国立大学学術情報リポジトリ

全文

(1)ScL. Repts.,. Yokohama. NatZ.. Univ.. See.. Duodenum,. Changes. by. irrespective. AP. duodenum bone AP. and. An. while. or. Cbicken isozymes while,. S activity. plasma. alkaline. (Tamaki,. 1975).. all the birds B. (orS)and. 1976).. derives. enzyme. Cbicken. tissue. in small. phosphatase. (AP). individual. APes. intestine,. have. bone. and. been. isozymes. B. (or S) and be. can. AP. in. a. bird. Serum. AP. that. in bone isozymic. 30min. within. AP. bird, while. AP. B isozyme. serum. cycle,. lObr,. and. The. increase in. serum,. stages.. has either of isozyme. three F. or. urea. AP. may. by. found. S. types,. between. in. with. bone.. denaturation. (Tamaki. be tissue-specific,. because. heat. medullary. the association. High. dietary. of calcium is known. calcium. metabolism to. cause. ､ゝ. marked. *. decrease. Department Yokohama Yokohama,. of. in small. Agriculture,. National 240,. intestinalAP. Faculty. University,. JAPAN. activity. (Motzok,. of Education,. Tokiwadai,. Hodogaya-ku,. 1963;. F to. response. tissue.. investigated. B. and. isozyme. Furthermore,. been and. ど,. isozymes,. S isozyme. types.. have. tissue. chicken. two. isozymes. activity observed. in B isozymic activity. to. isozyme. considered laying. was. two. in resistance. specific. of. laying. differences. that. suggests. it. comprises. irrespective. and. F. serum. AP. to increase. between. significant. from. isozymes. had B only individual had. an. neuraminidase.. AP. serum. little change. isozyme. This. with. in egg. stages. in. Therefore, of. in plasma. as. in duodenum. to B isozyme. treated. of total. (Tamaki, 1975). Morichi,. plasma. B. have. (AP). phosphatase. APes. AP. S isozyme. corresponded. An. isozymes,. neuraminidase and. had. F or. when. activity. bone. duodenum. contribute. increse. alkaline. medullary. activities. at two. compared. after oviposition. AP in total serum F. bands. two. were. AP. 1996). B isozymes. respectively.. to either. bone. wasLSplitted. and Although. cent,. contribute. bone. F (or S) and. percentage. 3 per. meduuary. activities. types.. and. tissue specificity in laying. hen and of elucidating in Duodenum egg process. shell calcification activities. bone. while. and. AP. ll,. isozymic. relative. 97. abont. (白eceived,May medullary. Hens. Process. TAMAKI＋. and. Alkaline. Activities,during. Yoshinori. bird comprised. of isozyme. i?ozymes,the. in Laying. Caleifieation. for the purpose. types,. isozyme. were. bone,. 1996. Bone. lsozymic. Shell. DtlOdenum,. changes of serum AP inanindividual. 10, October,. -. Medullary. and. in Serum. investigated. pp. l. Isozymes. Egg. were. No.43,. Bone. Phosphatase. Abstraet.. H,. Daviesetal.,. 1970; McCuaig.

(2) Y.. 2. Motzok,. and stage. of egg AP. serum. and. present. bone. medullary in egg. in. 9-month-old. to elucidate. investigated isozymes. AP. in laying. duodenum. serum,. Serum. within. to have. an. laid. Duodenum laying. hen. at. lObr. 8.0, containing. (Janke medullary. glass beaker Thereafter,. remove. electrophoresis. substrate,. by. samples. and. examine. incubated urea,. to examine. obtained. for 15min. of the. to. examine. not. stage. studied. with. intestine, borle. of small. AP. serum. Hiratsuka. an. egg. laying. bone. medullary. isozymic. from. taken. were. Farm. in. Yokohama. were. The. by. 37℃ with. the effect of. urea. urea,. for lOmin. for 30min. were. (1970).. sodium. salt. (Sigma. Chemical. Co.,U.S.A.)as. After. for. 1. Samples. at. the incubation,. gel AP. to. (5%). isozyme. was. Co.,U.S.A.) as. lmg. with Samples. 37℃.. heated. were. remove. a. dye-coupler.. 1 physiological. :. temperature.. at room. lhr. to. - 20℃.. incubated. were. ml. dilnting. a. in. placed. temperature. at. Tanabe. treatment.. 4℃. at. and. and. temperature･. same. stored. bone. and. at room. for 1 hr at the. of uterus.. Equipment. The. crusher. for 2days. from. buffer, pH. horizontalpolyacrylamide. Co.,U.S.A.) per 4M. Tris-HCl. Laboratory. with. at 6,000rpm. samples. same. recognized. section. Tamaki. Chemical. the. sampled. slaughter.. pieces. supernatants. neuraminidase,. Chemical. Turrax. solution. to. were. of 0.1M. after. small. subjected. were. in the upper. egg. at 38,000rpm. phosphate. of. an. equalvolume. centrifuged. taken. (femur). bone. buffer. from. - 20℃ until used.. at. immediately. same. cycle. All the birds. stored. into very. contained.. acid. at 3,000rpm. in. l℃ by IKA-Ultra. the effect of heat treatment.. centrifuging. at. with at. were. described. effect. at. kept. birds had. crushed. B salt (Sigma. (Sigma. hen. The. Germny). above. previously. the. are. of. and. medullary. ultracentrifuged. particles. fast blue. changes. bone. and. for lmin. volume. and. and. Methods. sample. homogenized. were. equal. hen. stages. serum. (femur). Co.,West. β ･napbthyl. neuraminidase. by. MgCl2. tissue. samples. detected. The. all the tissue samples. any. The. 8M. 0.05M. cellular debris. gross. To. were. with. two. after oviposition,. mucosa. bone. tissue specificity. lOhr, after ovIPOSition.. day.. bone. Kunkel. and. in the starting. they. and. laying. at. and. next. mucosa,. duodenum. The. 30min. egg. Rock. taken. were. samples. in the starting. activity. 1980.. since. individuals,. mucosa,. Plymouth. Nationalロniversity. AP. process.. shell calcification. White. higher. et al., 1965). However,. Materials. Samples. have. activity. was. study. (Taylor. too. bone. (Stringer, 1962). Simillary,. process. increases,. of isozymic. activities. in medullary. shell calcification. activity. associaton The. Osteoblasts. 1974).. TAMAKI. saline at. of were. containing. 60℃ for lOmin were. supernatants. All the samples. obtained. subjected. to. electrophoresis. TotalAP. activity. in. serum. was. measured. by. the method. of Kindand. King. (1954).. One.

(3) AP. unit Tbe with. the activity. shows. (Kayagaki-Irika activity. lmg. activities. were. neuraminidase. total AP. liberating. percentage. relative. Isozymes. Co.,. and. relative. Hens. band. in. 3. lOOml. per. of phenol. of each. measured. Kogyo. in Laying. serum. AP. activity. (500nm). percentage. band. of each. duodenum. and. spectrophotometrically. Japan).. 15min.. per. in. serum. treated. samples by was. a. densitometer. calculated. from. activity.. ResuIts. 1. Duodenum, Fig. bone. 1 shows. Duodenum anodic. side and. bands. in laying. AP. between. bone. medulla7y. the comparison. bone. medullary. and. isozymes of AP. of the zymograms. hen. between two. compared. types. isozyme. bands,. the. band. toward. the tailing minor isozyme. AP. was. observed;. isozymes. bone. in duodenum,. and. types.. intensely. more. cathodic S type. had. stained. side. a. band. major. Mobility. little slower. toward. diference. in major rate. migration. than. Ftype. Bone. the. and. same.. bone. medullary But. APes. the migration. had. rates. a. single. intensely. of their bands. were. band.. stained slower. than. Their. mobilities. that in duodenum. was. AP.. A Medullary. Bone. Duodenum. bone Fig.1.. Electrophoregram. of. isozymesin. hen.. bottom,. laying direction. duodenum, Samples. of migration. bone A, C and upwards.. and. bone. medullary. E: Ftype;. B, D. and. alkaline F:S. type.. phospatase Origin. at.

(4) Y.. F Fig.2.. Alteration treated. of. with. treatment.. mobility. S. type of alkaline. neuraminidase.. Origin. TAMAI(Ⅰ. at bottom,. type. pbospbatase. Samples. A and. direction. isozymes. C: untreated;. of migration. Non-treatment Fig.3. Schematic. 1 1. l. l. I. I. I. I. l. l. I I. l l. I. I. I. I. neuraminidase. _____J. F type. 一■■￣一-■■■■￣▼. l l. l. L_____J. S. treatment.. of duodenum. alkaline. phosphatase. type treatment. Neuraminidase. illustration. neuraminidase. 1. +. ■S type. D:. B and. duodenum. upwards.. r--. ドtype. in chicken. bands. with. and. without.

(5) AP. When. duodenum. of F type. became. equal. affected. (Fig. was. not. duodenum the mobility. of the. irrespective. of isozyme. two. peaks. bands. were. 5 show Both. respectively･ closer to origin the. one. toward. 6. duodenum,. was. bone. and. intensely. bone AP. in duodenum. AP treatment･. and. the. up. and. In case. side. effects. of heat. (60oC,. stable. the single major the. same. heat. band. result. 10min). and. was. and. minor. types.. bone. AP. up. to the. retarded it was. and. isozymes, position. two. splitted stained. 4M. bands, faintly. more. and. medullary. treatments. urea. The. respectively.. treatments,. urea. in bone. origin. in mobility.. of F type,. isozymes. the. of the major. the other. and. to. densitometrically,. meduuary. AP,. chick. treatment. closer. of isozyme. of bone. differences. to. activities. were. in. observed. drawn. band. of S type. band. neuraminidase. was. isozymes. catbodic. AP. position. boneand. small. was. Also,. AP. treatment･. bone. the. curve. on. bone. toward. the. irrespective. respectively,. included. medullary. isozymes. to. major. been. By. percentage. relative. medullary. of the has. 1977).. etal.,. of the major. mobility. phenomenon. the distribution. The. 5. the mobility. retarded. When. 3%,. side, which. in duodenum. to either. types･. 7 show. and. band. minor. same. the effect of neuraminidase. more. stained. The. Hens. neuraminidase,. whereas. (Tamaki. by neuraminidase. anodic. Figs. 3).. of age. 97 and. about. Figs 4 and. 2 and. in Laying. with. to that of S type. distinguished.. were. treated. were. samples. at 4 weeks. AP. Isozymes. while bone. major. the minor APes. were. on. band band labile. in the S type.. obtained. D F Fig･4･ Effect B. and. of neuraminidase C: untreated;. migration. upwards.. A and. type treatment. S on. bone. D: neuraminidase. type. alkaline treatment.. phospbatase Origin. isozymes. at bottom,. Samples. direction. of.

(6) Y.. F. Fig.5.. Effect isozymes. bottom,. of. direction. S. type. neuraminidase Samples. TAMAKI. B and. treatment. C: untreated;. of migration. upwards.. on. type. medullary. A and. bone. D: neuraminidase. alkaline treatment1. phosphatase. Origin. at.

(7) AP. Isozymes. Duodenum. Fig･7･. of 4M. Effect. 2.. Changes. To. in. reconfirm. Origin. activity in. the. change. in. AP. laying. cycle･. observed. between. two. to about. 8 units. isozymes. between. Taylaor. in activity. 1). On. two. activity, changes. of 8 units. in egg. Change. in the F. (or S). slightly. reduced. activity. in. AP. serum. was. small. level between. two. very. level. were. percentage. activities was. was. corresponded. were. corresponded. few. a. stages. cent. in. activities. irrespective and. per. the total AP. isozymic. process. 50. of of about. activity. activity. at two. compared. (Table 2). From. calcification. activity. were. the relative. stages. urea. process. shell calcification. the increase. in B isozymic. Change shell. isozyme. hand,. egg. activities. the increased. F:. and. cycle. during. AP. a, D. alkaline. upwards.. laying. egg. study,. the other. between. change. an. bone. medullary. E: untreated;. of migration. serum. (Table 1) and. stages. (Table 3).. stages. total. and. A, C and. activity. Also, in the present. percentage. two. serum. (1965),. et all. (Table. did not. the relative. direction. isozymic. in. AP. bottom,. bone. Samples. AP. by. egg. duodenum,. on. in F type.. at. Medullary bone. ser7Lm. described an. treatment. isozymes. treatment.. Hens. Bone. urea. phosphatase. in Laying. serum. activity. and. caluculated to the. of isozyme. individuals. one. types.. had. even. stages.. Diseussion. Major be. band. identical. identically Tamaki. and. in duodenum to. when. F. or. S. treated. Morichi,. AP isozyme with. 1976).. By. in F in. or. S isozyme. chick. plasma,. neuraminidase, the. same. type. reason,. urea. in laylng. because and. the minor. hen. the. heat. can. AP. be considered. isozyme. treatments. band. in duodenum. to. responded. (Tamaki, AP. 1975;. and. the.

(8) Y.. a. Table. 1. Changes. in. total. in egg. stages. serum. laying. No.. of bi rds. type. within. 10 hours. 30 min. later. two. 5. s. 5. 16.4 ± 2.0. activity. (B-A). ち-A. A. (B). F. in AP. fromoviposition. afteroviposition. 16.2±4,9'. XIOO. (Unit). (%). 25.0±7.4. 8.8. 54.3. 24.3 ± 2.3. 7.7. 47.0. ±SE.. *Mean. 2. A. Table. comparison. isozymes. of. percentage. relative two. between. in egg. stages. laylng. Isozyme. No.. of. Within. bi rds. type. 30 min. F. 5. 21.5±3.2*. s. 5. 15.9±4.3. by. Measured. phosphatase. percentage. (%)I. activity 10 hours. B isozyme. F. or. later from. oviposition B isozyme. S isozyme. 78.6±3.2. 23.3±4.2. 76.8±4.2. 84.2±4.3. 13.0±4.8. 86.9±4.8. I. (500nm).. densitometer. ±SE.. *Mean. Table. alkaline. CyCle･. after oviposition. S isozyme. F or. serum. of. activity. Relative. *. between. activity. Changes. (mg pbenol/100ml). activity. (A). *. (AP). phosphatase. alkaline. Cycle.. Total AP Isozyme. TAMAKI. 3. Changes. in in egg. stages. calculated Isozyme. alkaline. laying AP 30. Within. type. serum. (mg phenol/100ml)''. isozymeactivity min. 10. after. hours. two. between. BI. F2. /100ml) between. S2. or. (mg. activity. later from. ovlpOSition. SI. or. activities. cycle.. ovlpOSition Fl. isozyme. (AP). phosphatase. B2. pbenol. activity twostages. B2･Bl. (S2･Sl). F2･Fl. F. 3.3'. 1 2.9. 5.0. 20.0. 1.7. 7.1. s. 2.7. 1 3.7. 3.0. 2 1.3. 0.3. 7.6. *Mean. *. *. single. major. the B isozyme Duodenum, can. bone. from. caluculated. band. in bone. and. 1). and. relative. medullary. AP. percentage. bone. APes. activities. (Table. be considered. can. to. 2).. be. to. identical. in plasma. bone. be considered or. total (Table. medullary. or. medullary. that duodenum bone. AP. AP. bone AP. isozymes. were. tissue-specific. contributes. to the F. to. B isozyme. contribute. that. (or S) in. (Fig 1). isozyme serum. in AP. serm. and. thus. it. AP, while. in laying. hen･.

(9) AP. Therefore,. the present. is due. cycle. hypothesis. of Taylor. associated isozyme. B. 6).. This. having due. (1965), of AP. isozyme. B. The. 1972).. 2, replaced. duodenum. AP. B. isozyme･. composed. of F. only. isozyme,. In. Tamaki. (or S). and. further. function. AP. B. laying. different. moving. which. cycle. the. suports. AP. serum. be medullary. might. bone.. (Fig. closer to the origin. bands. kinds. of two. of glico･proteins. in bone. quantitatively. S). to peak. isozyme,. in laying. study. while. AP. be. may. AP. tissue. bone. and. 1 defined. 97%. them, to. AP. of total bone. medullary. isozymes. had were. be considered. can. the biochemical. elucidate. by. corresponded. about. liver. isozymes. ontogeny. with. which hen,. that. reported. As chicken. laylng. cell.. the F (or. to. tissue. corresponded. (1979). will need. origin of. change. S isozyme,. or. the. in王∋ isozyme. in bone. in egg. activity partially. is composed. the present. the F. isozymes.. studies. in egg. and. Watanabe. and. be tissue-specific, in three. as. that. to. AP. (Table 3). isozyme. found. were. 9. serum. heterogeneity. embryo. existed. B. of existence. it after hatching. activity. in total. AP. The. isozymes. the majorityin20-day-chick. peak. two. in bone. in the place. Ⅲens. suggested. activity. weight.. AP. Moog,. who. splitted. molecular. duodenum and. in the. was. B. the difference. (Change had. AP. that. means. Cbicken. in activity. et al.. in bone. different. to. increase. the increase. with. in Laying. the. that. result. the increase. to. Isozymes. and. to. physiological. isozymes.. 摘. 要. 産卵鶏のアルカリ性フォスファタ-ゼ(AP)アイソザイムの臓器特異性および卵殻形成時の血清AP活性の増加についてアイソザイム活性の観点からその変化を明らかにした｡産卵鶏の十二指腸APアイソザイムは血清APと同様に2つのアイソザイムからなっていた｡すなわち,. FタイプはFとB,. SタイプはSとBアイソザイムの2本のバンドを持っていた｡. 骨(大腿骨)および骨髄骨細胞のAPアイソザイムは血清中のBバンドに相当する1本のバンドのみ検出された｡これら臓器中APアイソザイムをノイラミニダーゼや尿素および熱処理したところ,血清APアイソザイムと同一の反応を示した｡卵殻形成時の血清AP活性の増加はBアイソザイムの増加によっていた｡. Referenees Chang,. C. E., and F. Moog,. partial. characterization. post-hatching Davies･. Kind･. M･. Ⅰ･･G･ M･. chicks:. Influence. Poultry. S°i.49. P･ R･. 1972.. N･･ and. stages･. :. the. of Biochim･. Ritcey, and of. Alkaline. dietary. of the. phosphatses foms. multiple Biophys.. I･ Motzok,. Acta. 1970･. calcium,. of. chicken. duodenum.. duodenal. Ⅰ.∫solation in. phospbatase. and. pre-and. 258:154･165.. Intestinal. inorganic. and. phytase phytate. and. alkaline. phosphorus. pbospbatase. and. vitamin. of D.. 1280-1286. E･ J･ Ring,. 1954･. Estimation. of. plasma. phosphatase. by. determination. of.

(10) Y.. 10. hydrolysed McCuaig,. phenol. L. W.,. Motzok, and stringer,. Ⅰ.,1 963.. Studies. reaction. velocity. in the. Tamaki,. isozymes. Tamaki,. Blood Tanabe,. in the. Y., and. isozymes Tamaki,. Y., and. isozymes Tamaki,. Y.,. isozymes Taylor,. :. D. Thesis,. University. studies Grps. 1976.. S. Watanabe, in chicken. S.. phosphatase. 45ト457.. S･. Moriuchi,. J. Kirrley,. during. egg. shell. Britain.. Great. alkaline. plasma. phosphatase. :. and. of. the. alkaline. 798-804･ chicken. Blood. forms. plasma. alkaline. biochem･. Grps genetic. Genet･. control. 7. of alkaline. phosphatase :. 179･184･ phosphatase. S°i.. Blood. 1965.. optima. to medullary. reference. molecular. multiple. 1977･. Anim･. duodenum･. chicken. of. Characterization. liver. 丁ap.∫.Zootecb.. and. of. Anim･. special. Reading,. of. pH. 172-180･. :. with. on. minerals. 195-200･. Sci･ 49. Poultry. treatments･. and. :. Characterization. 1979.. Watanabe,. in chicken. control. plasma.. heat. and. Genet･. Genetic. 6. 90-95.. of dietary. of bone,. control. :. in the. phospbatase. alkaline 52. ∫.87. of Reading,. genetic. biochem.. 1970･. Moricbi,. urea. of. and. Pbamacol.. Biochem.. physiology. 322-326･. :. P, Zn,. 4･ Influence. of fowl.. and. chicken. T. G., A. Williams,. alkaline 43. by. T.. of Ca,. phospbatase･. chemistry. Further. Y.. phosphatase Tamaki,. Ph.. Anim.. Y., and. alkaline. of phosphatase. The. fowl.. Y., 1975.. on. Interaction. Ca level. Can. ∫.Pbysiol.. of dietary. D. A., 1962.. bone. 1974･. 7. J. Clin. Path･. aminoantypyrine.. I. Motzok,. and. ⅠⅠ. Effect. cbick.. with. TAMAKI. Genetic Grps. Cyclic. calcification. control. biocbem･. in. Genet･. in the. changes the. of. alkaline. 8. :. activities. domestic. fowl･. phosphatase. 33-38･ of plasma Can･. acid. J･ Biochem･. and.

(11)