{"created":"2023-06-19T07:18:03.900781+00:00","id":3192,"links":{},"metadata":{"_buckets":{"deposit":"368b5b1f-75ac-4301-a62f-2cf8f1c2ccc0"},"_deposit":{"created_by":4,"id":"3192","owners":[4],"pid":{"revision_id":0,"type":"depid","value":"3192"},"status":"published"},"_oai":{"id":"oai:az.repo.nii.ac.jp:00003192","sets":["17:42:291","370:15:392"]},"author_link":["16221","16222"],"item_10006_date_granted_11":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"1986-01-16"}]},"item_10006_degree_grantor_9":{"attribute_name":"学位授与機関","attribute_value_mlt":[{"subitem_degreegrantor":[{"subitem_degreegrantor_name":"麻布大学"}]}]},"item_10006_degree_name_8":{"attribute_name":"学位名","attribute_value_mlt":[{"subitem_degreename":"獣医学博士"}]},"item_10006_description_22":{"attribute_name":"Abstract","attribute_value_mlt":[{"subitem_description":"The carbonic anhydrase catalyze the hydration of CO_2 and the dehydration of HCO_3 ( CO_2 + H_2 O  ⇔ HCO^-_3 + H^+) , a reaction which can occur at extremely high rates. Mammalian carbonic anhydrase is a zinc metalloenzyme which is widely distributed through the body. Two principal isozymes of carbonic anhydrase occur in mammalian erythrocytes ; a high-activity type designated as CA-II ( C ) and a low-activity type designated as CA-I ( B ). A basic protein noted by Blackburn et al. to be present in rabbit muscle extracts in relatively large amounts was later shown by the same laboratory to possess carbonic anhydrase activity. What appear to be similar enzymes have recently been discovered in the skeletal muscle of human, bovine, rat, sheep, chicken, mice and gorilla. This enzyme is the product of a distinct genetic locus and has been designated as carbonic anhydrase III ( CA-III ). It constitutes from 1 to 2 % of the soluble protein of muscle and has also been found in liver and lung extracts and in human erythrocytes. The sequence of the equine CA-I and CA-II has been determined by Deutsch et al. and it appeared of interest of initiate studies on the muscle type isozyme. In the present studies, equine CA-III has been isolated in crystalline form and describe its some properties.\nThe summarized as follows :\n\n1. Purification of CA-III from equine skeletal muscle\n The muscle tissue was excised from a fatally nembutalized pony were excised and placed in crushed ice, passed through a mechanical grinder with fine sieve. The minced muscle ( 1 Kg ) was homogenized with two volumes ( 2l ) of 0.01 M Tris-HCl ( pH 8.0 ) at 4℃ and centrifuged for 30 min at 8,000 xg. Then, iodoacetamide was added to give final concentration of 0.01 M, the pH of the solution was adjusted to 8.0 , and then , mixture was incubated for 30 min under non-denaturing condition. The sample was dialyzed against 0.01 M Tris- HCl ( pH 8.0 ) at 4 ℃. The sample was applied to a column (3.4 X 34 cm ) of CM-Sepharose CL-6B equilibrated with 0.01 M Tris-HCl ( pH 8.0 ). After washing the column extensively, the adsorbed proteins were eluted with a linear gradient of NaCl between 0 and 0.15 M in the same buffer. The various fractions eluted were tested for carbonic anhydrase activity. The enzymatically active fractions were pooled and concentrated by precipitation with saturated ammonium sulfate. The precipitate was dissolved in a small amount of pH 8.0, 0.01 M Tris-HCl containing 0.15 M NaCl and passed over a column of Sephadex G-75 ( Pharmacia ) equilibrated with this buffer. The enzymatically active fraction was dialyzed exhaustively against water, the small amount of precipitate formed was removed by centrifugation at 4℃ and the supernatant solution subjected to column electrofocusing in a pH 7-10.5 Ampholyte buffer ( Pharmacia ). The isoelectric point of CA-III was 8.9. About 300mg of enzyme are obtained per 1 Kg of muscle. A polymorphic form of the equine CA-III was designated as CA-IIIa and the isoelectric point of it was 8.1. Approximately 15 mg of CA-IIIa was obtained from 1 Kg of muscle. A 1 % solution of equine CA-III separated by electrofocusing was dialyzed at room temperature against repeated changes of 62 % saturated ammonium sulfate in pH 8.0, 0.05 M Tris buffer. After standing for several days at 4℃, crystals appeared. All three of the equine carbonic anhydrase isozymes have now been prepared in crystalline form. X-ray diffraction studies of them would be of interest to relate secondary-tertiary structural difference to their sequences and variant enzymatic activities and immunological properties. On the other hand equine CA-III was poorly retained by p-aminobenzenesulfonamide affinity columns. Koester et al. had previously reported the muscle enzyme to have a relative weak affinity for sulfonamides. Since considerable losses of activity were experienced in the early stages of fractionation author resorted to a somewhat different approach than that used by other investigators. It was based in parton the presence of what appear to be readily reactive cysteines in this enzyme.\n\n2. Physicochemical properties of equine CA-III\n The molecular weight of CA-III was estimated to be 26,500 by SDS-PAGE , 27,000 by gel filtration, respectively. The molecular weight of CA-IlIa was similar to those of CA-III. CA-IIIa was not a fragment of CA-Ill.\nEquine CA-III was dialyzed exhaustively against deionized, distilled water ( Milli-Q , Millipore Corp. ) and then centrifuged at 8,000 g for 10 min at 4 ℃ to remove a small amount of turbidity. Weighted aliquots of this solution were subject to measurements of dry weight at 105℃, O.D. at 280 nM. Equine CA-III was found to have an E ^1%_280 nm value of 15.5. From 40 to 60 % of the expected level of zinc was found in the crystallized enzyme after dialysis into pH 8.4, 0.033 M veronal buffer or distilled water. The relative low enzymatic activity of such a preparation could not be augmented when zinc was added to a level of 1 gm atom per mole of enzyme. The enzymatic activity of these preparations based on their zinc levels appeared to be in the range of that found for the muscle carbonic anhydrases of other species. It is not known whether the enzyme that has had 2 of its 4 cysteine alkylated tends to lose zinc more readily than the native form. Koester et al. have indicated that rabbit CA-III contains 1 gm atom of zinc and the metal is only slowly removed in pH 5.6, 100 mM sodium succinate buffer containing 5 mM o-phenanthroline and 1 mM dithiothreitol. Further studies of this problem which make use of the alkylated form of muscle carbonic anhydrases of other species will be required to define any effects of alkylation on the zinc binding activity of this enzyme.\n\n3. Chemical properties of equine CA-III\n The amino acid analyses were conducted on hydrolysates prepared in 5.7 N HCl at 110℃ for 20,40 and 100 hours, a Durrum D-500 apparatus being employed. The levels of labile components were extrapolated to zero time hydrolysis and maximum values for other amino acids were thaken. In cases where no significant level deviations were noted as a function of time , the values were averaged. A total of 257 residues was found when a molecular weightof 27,000 was assumed. The amino acid composition of CA-IIIa was similar to that of CA-III. When CA-III and CA-IIIa were alkylated with iodoacetamide under non- denaturing condition, 2 mol of S-carboxymethylcysteine ( CMC ) per mol of the enzyme were found , indicating that two cysteine residues may be located on the surface of the molecules and may readily reacts with the iodoacetamide. When CA-III and CA-IIIa were treated with 5 , 5-dithio-bis ( 2-nitrobenzoic acid ) ( DT NB ) in the absence of denaturing agent, no sulfhydryl groupwas detected, possibly because the sulghydryl groups of CA-III and CA-IIIa had already been alkylated with iodoacetamide before purification of the enzyme. However , titrations of the enzymes with DTNB in 6 M urea showed that CA-III and CA-IIIa contained 1.7 and 0.9 mol of sulfhydryl group per mol of the enzyme, respectively. It is suggested that one of those cysteines reacted with DTNB whereas the other did not because it bound with some acidic components. The acidic peptide formed upon performic acid oxidation of the protein was isolated as a drop-through product on a Dowex 50-X2 column ( 0.5X 10 cm ) at pH 4.0 following removal of performic acid from the oxidation mixture. A part of this product was hydrolyzed for 20 hr in 6 N HCl at 110 ℃ and applied to the Durrum D-500 analyzer. The remaining part was directly applied to the Durrum D-500 analyzer without hydrolysis. Some acidic components were released from the purified CA-III a which eluted slightly before cysteic acid when applied to amino acid analyzer without hydrolysis. Amino acid composition of these acidic components was cysteic acid ( 0.63 nmol ) , glycine ( 0.85 nmol ), and glutamic acid ( 1.56 nmol ) suggesting that the components are glutathione. The activity of the equine CA-III was in the range of 350 to 400 Wilbur and Anderson units per mg of zinc containing protein. This is about 14 % , 1.4 % of the specific activity of CA-I , and CA-II, respectively, under identical assay conditions. CA-IIIa is 38 % of the activity of equine CA-III. Equine CA-III has a relatively low esterase activity ascompared to the CA-I and CA-II forms. Equine CA-III has been also found to possess weak alkaline and acid phospatase activities. These studies indicate that equine  CA-III is apparently a multi-site, multi-functional enyme. Aickin and Thomas reported that the red muscle fibers has 3 times higher CO_2 buffering capacity than the white muscle fibers and the membrane potential of the former is more sensitive to varying CO_2 levels than that of the latter, suggesting a greater need for CO_2 regulation in the red muscle. Thus, carbonic anhydrase may play a significant role in the regulation of CO_2 concentration and of pH in the red muscle. The physiologic meaning of the polymorphic forms of CA-IIIa remains obscure. The observation on these multiple forms probably provide support for the concept of nongenetic, interchangeable conformational modification of the CA-III molecule which has been termed as conformational isozymes.\n\n4. Immunochemical properties of equine CA-III\n Antibodies to equine CA-III were produced in all the seven rabbits employed. Each rabbit was injected initially with 1 mg of purified CA-III emulsified with an equal volume of Freund's complete adjuvant , followed by booster injection of the same amount of the enzyme every week for five times. Rabbits were bled via the auricular vein 10 days after the last injection. The specificity of the antiserum was evaluated by double immunodiffusion. Anti-equine CA-III serum reacted with equine CA-III, but not with equine CA-I and  CA-II. The antiserum to CA-III formed a single preciptin line with crude muscle extract, with purified CA-III, with liver extract and also with CA-IIIa , and all of the precipitin lines fused completely. On the other hand, anti-serum formed a single weak precipitin line with equine thymus. The cross-reactivity of CA-III of several animals using anti equine CA-III rabbit serum was investigated.  Carbonic anhydrase is a enzyme whose species specificity is rather low. Equine CA-III formed a spur over bovine, cat, dog, rat, and pig. The extract of muscle and liver from rabbitand chicken did not cross react with rabbit anti equine CA-III sera.\n\n5. Distribution of immunoreactive CA-III in various equine tissues and serum.\n A higly sensitive sandwich enzyme immunoassay ( EIA ) for equine muscle CA-III has been developed using microplate as a solidphase and peroxidase as a labelled enzyme. CA-III levels of tissue extracts were measured by EIA and calculated the values per gram wet tissue. The skeletal muscle, liver and thymus contained about 530 μg, 300 μg and 16.5 μg per gram wet tissue,respectively. But in other tissues examined, CA-III levels were only several ng per gram wet tissue. To find the normal serum CA-III level, sera from equine were assayed. The mean value was 0～27 ng /ml.\n\n6. CA-III immunohistochemical localization in equine skeletal muscle\n Conjugation of peroxidase to the specific antibody was done by the periodate method. The tissue was embedded in paraffin and sectioned at 5μm. For the immunohistochemical localization of CA-III direct immuno peroxidase method was used. The sections were pretreated with 0.3% H_2 O_2 - methanol to block endogeneous peroxidase activity and with normal rabbit sera to block Fc receptors. The sections were then examined by the direct immunoperoxidase method using peroxidase conjugated specific anti CA-III antibody. As a control, peroxidase labelled normal rabbit Ig-G was used insted. By the direct immuno-histochemical method , the characteristic dark brown peroxidase reaction product was localized mainly inside the muscle fibers but not all of them, consequently producing a mosaic appearance. Moynihan ( 1977 ) found the presence of extravascular carbonic anhydrase activity biochemically in rat skeletal muscles. He noted that the red soleus muscle contained a significantly higher concentration of the enzyme compared with the white muscles. To confirm these data, Ridderstrale ( 1979 ) examined the rat skeletal muscles histochemically using a modified Hansson's cobalt phosphate method but could not find any significant difference. Lonnerholm ( 1980 ) examined sections of resin-embedded rabbit skeletal muscle with the same method and found that the staining of the cytoplasm of the muscle fiber varied in its intensity, but did not clarify whether carbonic anhydrase-rich fibers were of the so-called red or white type. However, he noticed that the sarcoplasmic staining was at least 1,000 times less sensitive to acetazolamide inhibition than the staining of structures, such as walls of capillaries situated within the muscle bundles. According to the recent studies on CA-III, this isozyme shows relatively poor CO_2 hydration activity and is less sensitive to acetazolamide inhibition compared with red cell isozymes. Using the indirect immunoperoxidase method, the localization of CA-III in frozen sections of human skeletal muscle was investigated by Shima et al. Human CA-III was found to be localized in Type-I muscle fibers when compared with serial sections stained with myosin ATP ase and other reactions. This fact along with other investigators result suggested that cytoplasmic staining could reflect CA-III activity. In this study, auther suggested that CA-III was indeed localized in  Type-I fibers ( red muscle type ) in accordance with the above mentioned data, but detailed localization of CA-III inside the muscle fiber was difficult to ascertain by this method.\n 7. Peptide map of CA-III\n CA-III was reduced with 0.05 M dithiothreitol in pH 8.5 , 0.05 M Tris-HCl containing 6 M urea and then alkylated with 0.125 M iodoacetamid. For citraconylation , 10 mg of reduced and alkylated  CA-III were dissolved in 1 ml of 2 % sodium bicarbonate ( pH 8.2 ), which 0.01 ml citraconic anhydride were added three times at intervals of 20 min with stirring. During this process, the pH of the solution was adjusted to 8.2 with 2 N NaOH. The mixture was then allowed to react at room temperature for 3 hours and dialyzed against 0.01 M ammonium bicarbonate ( pH 8.0 ) to remove excess reagents. Citraconylated CA-III was removed from dialysis tube and lyophilized. 2 mg of enzyme were dissolved in 1 ml of pH 8.0, 0.01 M ammonium bicarbonate and heated at 100℃ for 1 min. Digestions of heated denaturing enzymes by trypsin were carried out in pH 8.0,  0.01 M ammonium bicarbonate. Proteolysis was initiated by the addition of an amount of TPCK-treated trypsin equal to 1% of the protein weight. These digeation were allowed to proceed for 6 hours at 37 ℃ with shaking. The digests were then centrifuged to remove any in- soluble, \" core peptides \", and the clear supernatant of digestswere lyophilized. Peptide mapping of tryptic digests was carried out on Whatman 3 M chromatograph paper ( 46 X 57 cm ). The sample was completely dried on the chromatograph paper before proceeding. The paper was eqilibrated with the chromatographic solvent by hanging it in the chromatocase. Descending chromatography with a n-butanol-acetic. acid - water mixture ( 40 : 10 : 50 ) was conducted for 16 hours at room temperature. After chromatography the paper was removed and dried at 60 ℃ for 1 hour in the chromatogram drying oven. The electrophoretic dimension of the peptide map is developed vertically to the chromatographic dimmention at pH 6.5, pyridineacetatebuffer ( 10 % pyridine, 0.5 % acetic acid ). Electrophoresis was performed at 2,500 V, for 1 hour using the high voltage electrophorator. After electrophores is the paper was dried completely at room temperature. And then, the chromatogram was dipped in a 3 % pyridine solution in acetone in order to adjust the pH before fluorescamine staining. Peptide were visualized by spraying with a 0.0005 % solution of fluorescamine in aceton. After spraying the fluorescamine, the peptides were distinctly visible under ultra violet illumination( 365 nm ). The peptide maps prepared by trypsin digestion of equine CA-III disclosed the presence of 26 peptides. The spots of peptides were removed by cut off the chromatograph paper and peptides were eluted from paper with 25% acetic acid. The eluted peptides were lyophilized and hydrolyzed in 6 N HCl for 20 hour at 110  ℃ in evacuated and sealed tubes. By amino acid analysis of the eluate from each peptide spot, there were 12 peptides that contained arginine, 13 peptides that contained lysine and 1 peptide that contained both arginine and lysine. The map prepared by trypsin digestion of citraconylated CA-III disclosed the presence of 13 peptides. Amino acid analysis of each peptide identified 8 peptides with arginine, 1 peptide with lysine and 4 peptides with both lysine and arginine. Peptide No.21 contained 2 cysteines when CA-III was alkylated under nondenaturing condition. This seems to indicate that they were located on the protein surface. Two more cysteines were detected in peptide No.25 when CA-III was alkylated with iodoacetamide in 6 M urea. The peptide map of CA-IIIa corresponded to that of CA-III with the exception of disappearance of peptide No.25. If CA-IIIa is associated with the loss of one amide group, a peptide containing either asparagine or glutamine should migrate further to the anode side on the peptide map. Binding of glutathione to CA-IIIa , has been suggested. It is,therefore, presumed that in CA-IIIa, binding of glutathione to either of the 2 cysteines moieties of peptide No.25 made the peptide insoluble, because that it contains 50 % hydrophobic amino acid, and thus it was precluded from the detection.\n\n8. Sequence of equine CA-III\n Equine CA-III was reduced with a 10-fold molar excess of 2-mercaptoethanol or dithiothreitol in pH 8.5, 0.5 M Tris-HCl buffer containing 6 M guanidine-HCl or urea and 0.01 M EDTA at 50 ℃ for 4 hrs and then alkylated with a one- fold excess of iodoacetamide. Reduce and Alkylation ( RA ) protein was used in all sequence determinations. Some of the RA-enzyme employed had been also citraconylated. Peptides were isolated from tryptic, chymotryptic digests of enzyme so derivatized. Clevage of the protein was also effected with CNBr. The proteolytic digests were fractionated over columns of Sephadex G-25 equilibrated with pH 8.0, 0.15 M AmHCO_3. Peptide fractions resolved by gel filtration were further separated by high pressure liquid chromatography ( HPLC ). Aliquots of purified peptides were hydrolyzed for 20 hrs. at 110 ℃ in 5.7 N HCl and analyzed for their compositions on a Durrum D-500 analyzer. Peptides were subjected to automated Edman degredatian with the Beckman Model 890 C Sequencer using a 1.0 M Quadrol program with the addition of 3 mg Polybrene before the firstcycle was initiated. Cleavage was effected with anhydrous heptafluorobutyric acid ( HFBA ) and the phenylthiohydantoin ( PTZ ) derivatives were extracted with butyl chloride. The phenylthiohydatioin ( PTH ) derivitives were formed by heating the dried extracts with 1 N HCl at 50℃ for 15 mins. Identification of the later utilized the IBM Model LC/ 9533 Liquid Chromatograph in conjunction with a Waters Wisp 710 B automated sampler. The sequence of equine CA-III has been determined. To provide for homology with the erythrocyte carbonic anhydrases, the first residueis numbered 2 and gap is introduced at residue 126. The extent of homology with the bovine muscle enzymeand with CA-I ( low activity ) and CA-II ( high activity ) equine erythrocyte forms is caluculated. A strong sequence homology to other mammalian carbonic anhydrase exists, and 86.6% of the residues in the equine and bovine  CA-III are identical. Although CA-III shows strong sequence homologies to the higher activity erythrocyte forms, they vary in many properties. The latter enzymes usually contain a single cysteine, although bovine CA-II has none and one minor form of equine carbonic anhydrase and one of equine carbonic anhydrase II has 2 such residues. In contrast, equine CA-III has 4 cysteines, rabbit has 6, chicken has 7,and bovine has 3.\n\n9. Chemical modification of equine CA-III\n Equine CA-III shows a much weaker esterase activity for p-nitrophenyl acetate than the type I and II erythrocyte isozymes. The components formed by reaction with the aromatic esters and carbamoyl phosphate possessed more anodic charges at alkaline pH than the starting enzyme. Equine CA-III behaves in undergoing extensive acylation of N∈ -lysine residues upon reacting with p-nitrophenyl esters. To determine the extent of such derivatization , native enzyme and the different charged components isolated by electrofocusing were dinitrophenylated with an excess of fluorodinitrobenzene ( FDNB ) at pH 9.0. After removal of reagents, the protein was hydrolyzed for 20 hrs. at 110 ℃ in 5.7 N HCl and the amino acid compositions determined with a Durrum D-500 Amino Acid Analyzer. An acylated amino acid, i.e. N∈ -lysine would not react with FDNB. The level of free lysine in the samples reacted with the ester , i. e. acylated protein, over that in the enzyme controls, would be a measure of the number of lysines that had been derivatized since the acyl groups but not the FDNB derivatives would be removed during hydrolysis. The experiments with carbamoyl phosphate, which appeared to result in the carbamylation of N∈ -lysine to form homocitrulline, were more difficult to quantitate due to the back hydrolysis of some of the homocitrulline to lysine. The modification of from 6 to  7 lysine residues results in the production of a series of more a-nodic electrophoretic components. The derivatization of lysine residues leads to a marked decrease in the enzyme's ability to hydrate  CO_2. No such modification was noted for equine CA-II. A small amount , i.e. less than 10 % , of equine CA-I was converted into a more anodic form by its reaction with PNPA. Thus, the extensive alkylation of CA-III during its hydrolysis of aromatic esters appears to be a unique property of the CA-III. The ability of the enzyme to auto-acylate some of its X∈ -lysine residues is inttriguing but also seeks a physiological meaning. The reactions of CA-III with carbamoyl phosphate are also of interest, particulary so since this compound is fairly rapidly converted to cyanate. Carbamoyl phosphate at a level of 3mM has been reported to almost completely inhibit all three isozymes of carbonic anhydrase ( Carter et al ). The experiments of these investigators would appear to be complicated by the formation of cyanate which has been shown by Maren and Canto (1979) and Maren and Sanyal ( 1983 ) to be a strong inhibitor of both the hydration of CO_2 and dehydration of bicarbonate. In this experiments which employed carbamoyl phosphate at about 3 mM levels for a long period of time led to extensive carbamoylation of CA-III. However, the individual components isolated still showed considerable hydration activity. The components after extensive dialysis and isolation by electrofocusing for 24 hrs. would not be expected to contain cyanate and the inhibition noted must be due to the carbamylation. The inhibitory results obtained by Carter et al. ( 1984 ) would be anticipated to be due to a specific effect of carbamoyl phosphate. The variable and extensive acetylation of the lysine residues of CA-III is of interest but no significant biological meaning is apparent as yet. Further studies of the correlation of the effects of derivatization of muscle carbonic anhydrase on various of its biological activities may eventually lead to a better understanding of its true physiologic function.\n\n"}]},"item_10006_description_7":{"attribute_name":"抄録","attribute_value_mlt":[{"subitem_description":"炭酸脱水酵素(以下CAと略す)は1933年，Meldrum及びRoughtonによって発見された，亜鉛を含みCO_2 + H_2 0 ⇔ HCO^-_3 + H^+の可逆反応を触媒する金属酵素である。CAはCO_2の運搬や酸塩基平衡に役立っている酵素であり，ウマにおいては，Deutschらにより赤血球中からCA-IとCA-IIの二種類のアイソザイムが精製され報告されている。CAの第3番目のアイソザイムであるCA-IIIに関する研究は，Koesterらが1977年にウサギの筋肉から精製したpeak XがCA活性を持つことを報告したのに端を発した。現在までにウマのCA-IIIに関する報告はなされておらず，本論文ではウマの筋肉からのCA-IIIの精製方法と結晶化方法について報告した。そして，精製したCA-IIIの生化学的，物理化学的，免疫化学的，免疫組織化学的性状の分析結果に加え，一次構造の解析，化学修飾による影響について検討を行いCA-IIIの性状を明確にした。更に，CA-IIIの微量測定法を開発し獣医臨床領域への応用を試みた。それらの結果について要約すると下記のごとくである。\n1. CA-IIIの精製と結晶化\n　精製にはポニーの中臀筋を用い，トリス塩酸緩衝液(pH8.0)を加えてホモジネートを作成した。遠心分離後，上清をヨードアセトアミドを用いてアルキル化を行った。次に，CMセファローズCL-6Bを用いて筋肉抽出液の陽イオン交換クロマトグラフィーを行った。吸着した蛋白質は塩濃度を直線的に上昇させることにより溶出した。そして，CA活性の存在する溶出分画をセファデックスG-75でゲル濾過を行い，つづいてカラム等電点電気泳動を行なった。その結果CA-IIIは等電点8.9の分画で精製された。精製したCA-IIIは澱粉ゲル電気泳動，SDS-ポリアクリルアミドゲル電気泳動において単一の蛋白質であることを証明した。精製したCA-IIIをウサギへ免疫することによりCA-IIIに対する抗体のみを作成することができた。上記の方法で精製したCA-IIIは60%硫安を含む0.05Mトリス緩衝液(pH8.6)で平衡化し，硫安濃度を62%に上昇させ温度を室温から4℃へ徐々に下げることにより結晶を得た。CA-IIIの多形であるCA-IIIaは，同じ方法で精製することができた。1kgの筋肉を用いてCA-IIIは約300mg，CA-IIIaは約15mgが精製された。CA-IIIはCA-I，CA-IIと異なり，サルファマイドをリガンドとしたアフィニティークロマトでは精製されなかった。\n2. CA-IIIの物理化学的性状\n　CA-III，CA-IIIaの分子量は共にゲル濾過法で27,000，SDS-PAGEで26,500と推定され，単量体の酵素蛋白質であった。\n　CA-IIIの極大吸収は280nmにあり，1%溶液の吸光度は280nmの波長において15.5と算出された。等電点は，CA-IIIが8.9，CA-IIIaは8.1であった。亜鉛の含有量を測定した結果，およそ1分子のCA-III中に1原子の亜鉛が存在した。\n3. CA-IIIの生化学的性状\n　ウマのCA-I，CA-II, CA-III，CA-IIIaのCO_2水和活性を測定し比較した結果CA-IIIの値はCA-Iの1/7，CA-IIの1/74であった。CA-IIIaはCA-IIIの1/2.6であった。エステラーゼ活性については，活性の高い順にCA-II，CA-I, CA-IIIであった。酸性フォスファターゼ活性は，3種のアイソザイムにおいてはほぼ等しく，アルカリフォスファターゼ活性はCA-IIIのみ存在した。CA-IIIのCO_2水和活性は60℃，5分間の加熱で活性を失ったが，エステラーゼ活性については100℃，5分間の加熱でも失活しなかった。\n　アミノ酸分析の結果，CA-IIIのアミノ酸総数は257残基であった。システインは4残基含まれており，その内の2残基は分子表面に存在し残りの2残基は分子内に埋もれた状態で在在していた。CA-IIIaに関してはアミノ酸組成はCA-IIIに非常に類似しており，4残基のシステインのうち内部に埋もれた2つのシステイン残基の一方にグルタチオンが結合していることを示唆した。CA-IIIのアミン酸組成の特徴は，システインとアルギニンの数においてCA-Iは1個と6個，CA-IIは1個と9個であるのに対し，CA-IIIでは4個と14個とそれぞれの数が多いことであった。\n4. CA-IIIの免疫化学的性状\n　抗ウマCA-III血清を用いたオクタロニー法では，ウマのCA-I，CA-IIとは沈降線を形成せずCA-IIIのみと一本の沈降線を形成した。更に，CA-IIIaとも一本の沈降線を形成した。ウマの各種臓器抽出液を用いてオクタロニー法を行った結果，CA-IIIと同一の明瞭な沈降線を形成したのは筋肉と肝臓の抽出液であった。更にウマの胸腺とは弱い沈降線を形成した。\n　CA-IIIの種特異性を検索した結果，ウシ，イヌ，ネコ，ラットの筋肉抽出液と抗ウマCA-III血清との間にスパ一を形成する沈降線が観察された。\n　一方，肝臓ではウシ，ブタ，ネコ，ラット(雄)との間にもスパーが出現する沈降線を認めた。ウサギ，ニワトリの筋肉と肝臓とは沈降線を形成しなかった。\n5. ウマの各種臓器のCA-III含有量\n　サンドイッチ法による酵素免疫測定法を確立しCA-IIIの定量を行った結果，臓器湿重量1g当りの筋肉では530μg，肝臓では300μg，胸腺で16.5μgであった。その他の臓器では，平均57.3ngと極めて低値であった。赤血球中には，ヘモグロビン1g当り319.2ng存在した。この値は，赤血球中に存在するCA-Iの1/41,692, CA-IIの1/5,549であった。\n6. CA-IIIの組織局在性\n　ウマ筋肉の組織切片を作製し，ペルオキシダーゼ標識抗ウマCA-III血清を用いる直接法で筋線維中のCA-IIIを染色した。その結果，強く染色される線維と淡く染色される線維，そして染色されない線維が確認された。強く染色される線維の割合が少ないことからCA-IIIは赤筋線維に局在すると考察した。\n7. CA-IIIのペプチドマップ\n　CA-IIIをトリプシンで分解し，ペプチドマップを作製すると26個のペプチドが検出された。各ペプチドを抽出してアミノ酸分析を行った結果，リジンを含むペプチドは13個，アルギニンを含むペプチドは12個であった。1個のペプチドは両者を含んでいた。CA-IIIをシトラコニール化しトリプシンで分解した場合，リジンのC末端が切断されないため15個のペプチドが検出された。CA-IIIaのペプチドマップは，グルタチオンが結合していると推定ざれるペプチドの1個が消失した以外はCA-IIIと同じであった。\n8. CA-IIIのアミノ酸配列\n　アミノ酸配列の決定は，CA-IIIをペプチドに分解し，精製したペプチドをシークエンサーにより自動Edman分解を行い，N末端からのアミノ酸の同定はHPLCで行った。CA-IIIの分解は，プロムシアン，トリプシン，キモトリプシンを用いて行った。その結果ウマのCA-IIIの一次構造は259残基中の92%を決定することができた。ウマCA-IIIアミノ酸配列は，CA-Iと55.3%，CA-IIとは57.3%の類似性があり，ウシCA-IIIとは86.6%が等しかった。CA-IIIの活性中心は，他のCAアイソザイムと同様に，94番，96番，119番に位置するヒスチヂンであることを推察した。さらに，64番目の活性に関与するアミノ酸がアルギニンであることがCA-IIIの活性の低い原因と考察した。\n9. CA-IIIの化学修飾\n　p-ニトロフェノールブチレートを用いてCA-IIIをアセチル化した結果リジン残基のみが修飾された。アセチル化されるリジンが増えるにつれ等電点は低下し，酵素活性も低下することが認められた。CAの阻害剤として報告されたカルバミルリン酸を用いるとCA-IIIのリジンがカルバミル化されることを認めた。カルバミル化されるリジンが増加するにつれ，等電点が低下し酵素活性も低下した。\n10. ウマ血清中のCA-III値\n　酵素抗体測定法でウマ血清中のCA-IIIを測定した。今回用いたウマ血清中の値は，安静時においては0～27ng/mlであった。競走用サラブレッドにおいては，トレーニング後には最高で84ng/mlに上昇したものも認めた。一頭のウマについては，安静時においても1,380ng/mlと非常に高い値を示したことから，なんらかの筋疾患の存在を推察した。これらのことから，CA-IIIの測定は運動生理学の領域や筋疾患の診断に興味ある成績を与えるものと考察した。\n"}]},"item_10006_dissertation_number_12":{"attribute_name":"学位授与番号","attribute_value_mlt":[{"subitem_dissertationnumber":"乙第236号"}]},"item_10006_version_type_18":{"attribute_name":"著者版フラグ","attribute_value_mlt":[{"subitem_version_resource":"http://purl.org/coar/version/c_ab4af688f83e57aa","subitem_version_type":"AM"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"西田, 利穂"}],"nameIdentifiers":[{}]},{"creatorNames":[{"creatorName":"Nishita, Toshiho","creatorNameLang":"en"}],"nameIdentifiers":[{}]}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2013-02-08"}],"displaytype":"detail","filename":"diss_dv_otsu0236.pdf","filesize":[{"value":"33.0 MB"}],"format":"application/pdf","licensetype":"license_note","mimetype":"application/pdf","url":{"label":"diss_dv_otsu0236","url":"https://az.repo.nii.ac.jp/record/3192/files/diss_dv_otsu0236.pdf"},"version_id":"a928f96a-8280-49f6-b17c-1732841cd877"},{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2014-08-18"}],"displaytype":"detail","filename":"diss_dv_otsu0236_jab&rev.pdf","filesize":[{"value":"329.1 kB"}],"format":"application/pdf","licensetype":"license_note","mimetype":"application/pdf","url":{"label":"diss_dv_otsu0236_jab&rev","url":"https://az.repo.nii.ac.jp/record/3192/files/diss_dv_otsu0236_jab&rev.pdf"},"version_id":"837f97a4-97c6-4df6-87f4-79f8fb531c4d"},{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2013-02-08"}],"displaytype":"detail","filename":"diss_dv_otsu0236_jab.pdf","filesize":[{"value":"169.3 kB"}],"format":"application/pdf","licensetype":"license_note","mimetype":"application/pdf","url":{"label":"diss_dv_otsu0236_jab","url":"https://az.repo.nii.ac.jp/record/3192/files/diss_dv_otsu0236_jab.pdf"},"version_id":"fb58685d-7b27-4a13-b46c-cf76a312f080"},{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2013-02-08"}],"displaytype":"detail","filename":"diss_dv_otsu0236_eab.pdf","filesize":[{"value":"543.7 kB"}],"format":"application/pdf","licensetype":"license_note","mimetype":"application/pdf","url":{"label":"diss_dv_otsu0236_eab","url":"https://az.repo.nii.ac.jp/record/3192/files/diss_dv_otsu0236_eab.pdf"},"version_id":"4f90f8ac-d38a-4dde-acba-cee2fa3019ea"}]},"item_language":{"attribute_name":"言語","attribute_value_mlt":[{"subitem_language":"jpn"}]},"item_resource_type":{"attribute_name":"資源タイプ","attribute_value_mlt":[{"resourcetype":"thesis"}]},"item_title":"ウマの筋肉型炭酸脱水酵素(CA-III)に関する研究","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"ウマの筋肉型炭酸脱水酵素(CA-III)に関する研究"},{"subitem_title":"Studies of equine muscle carbonic anhydrase (CA-III)","subitem_title_language":"en"}]},"item_type_id":"10006","owner":"4","path":["291","392"],"pubdate":{"attribute_name":"公開日","attribute_value":"2013-01-29"},"publish_date":"2013-01-29","publish_status":"0","recid":"3192","relation_version_is_last":true,"title":["ウマの筋肉型炭酸脱水酵素(CA-III)に関する研究"],"weko_creator_id":"4","weko_shared_id":4},"updated":"2023-06-19T08:19:44.932484+00:00"}