{"created":"2023-06-20T13:21:11.404096+00:00","id":1305,"links":{},"metadata":{"_buckets":{"deposit":"cccf9b35-7238-45a6-891b-033045cf08c4"},"_deposit":{"created_by":1,"id":"1305","owners":[1],"pid":{"revision_id":0,"type":"depid","value":"1305"},"status":"published"},"_oai":{"id":"oai:ir.soken.ac.jp:00001305","sets":["2:430:27"]},"author_link":["9556","9555","9557"],"item_1_creator_2":{"attribute_name":"著者名","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"徳元, 俊伸"}],"nameIdentifiers":[{}]}]},"item_1_creator_3":{"attribute_name":"フリガナ","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"トクモト, トシノブ"}],"nameIdentifiers":[{}]}]},"item_1_date_granted_11":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"1994-03-24"}]},"item_1_degree_grantor_5":{"attribute_name":"学位授与機関","attribute_value_mlt":[{"subitem_degreegrantor":[{"subitem_degreegrantor_name":"総合研究大学院大学"}]}]},"item_1_degree_name_6":{"attribute_name":"学位名","attribute_value_mlt":[{"subitem_degreename":"博士（理学）"}]},"item_1_description_12":{"attribute_name":"要旨","attribute_value_mlt":[{"subitem_description":"  It is generally accepted that proteolysis plays an important role in the regulation of the eukaryotic ce11 cycle.Eukaryotic cells contain a nonlysosomal large protease called the proteasome(of the multicatalytic protease) which is found in all eukaryotes,from yeast to man.Although it has been suggested that proteasomes are involved in the ubiquitin‐dependent proteolytic system,their exact role in intracellular protein breakdown is still uncertain.Recent studies using various protease inhibitors suggest that proteasomes are involved in meiotic maturation of animal oocytes.More recent studies have demonstrated that the termination of mitosis and meiosis,transition from metaphase to anaphase is induced by the degradation of cyclin B,a regulatory subunit of maturation‐promoting factor of M‐phase promoting factor(MPF).Furthermore,it has been suggested that cyclin B is degraded by an ubiquitin‐dependent proteolytic pathway,Since proteasomes are known to be a component of the ubiquitin‐dependent proteolytic system,it can be hypothesized that proteasomes play a role in cyclin degradation.However,there is no direct evidence for this hypothesis at present.<br />  Fish oocytes provide an appropriate experimental system to investigate above problems. In recent years,considerable progress has been made in identifying the factors responsible for the regulation of meiotic maturation of fish oocytes;theseinclude the isolation and characterization of a fish maturation‐inducing hormone(17α,20β‐dihydroxy‐4-pregnen‐3-one,17α,20β‐DP) and the components of MPF(p34cdc2,the catalytic subunit and cyclin,the regulatory subunit).<br />  The present study was designedto investigate(1) the structure of proteasomes present in goldfish oocytes,and(2) the possible role of the enzymes in the regulation of oocyte maturadtion and egg activation.A particular emphasis was placed on the role of proteasomes in cyclin B degradation,the event which is crucial in exiting metaphase and entering the next interphase during the ce11 cycle.To this end,a SDS‐dependent(laten) proteasome was purified and characterized from goldfish oocyte cytosol and raised monoc1onal antibodies against this enzyme.The cytosol fraction(150,000g supernatants) of goldfish ovary hydrolyzed a fluorogenic peptide,succiny1-Leu‐Leu‐Val一Tyr‐4-methylcoumary1-7‐amide,a we11‐known substrate for proteasome,regardless of the addition of SDS to the reaction mixtufe.Four steps of column chromatography resulted in a 135-fold purification of proteasome from this supernatant. Both SDS‐independent and SDS‐dependent hydrolyzing activities co‐migrated during purification.However,the SDS‐independent activity was markedly reduced during purification.The purified SDS‐dependent proteasome(latent proteasome)possessed weak hydrolyzing activity(chymotrypsin‐trypsin‐,and V8 protease‐like activities) even in the absence of SDS; the chymotrypsin‐and V8 protease‐1ike activities were significantly increased in the presence of SDS.Its molecular weight and sedimentation coefficient were estimated to be 620kDa and 19.2 S,respectively‐Three kinds of monoclonal antibodies were raised against the purified latent proteasome.Western blot analyses revealed that these antibodies recognized a single species of protein on native PAGE,but recognized several subunits ranging in molecular mass from 23.5 to 31.5kDa on SDS‐PAGE.Cytosol fractions containing the SDS‐independent activity had a band which migrated slower than that of purified latent proteasome.The SDS‐independent protease activity was depleted when the cytosol fraction was immunoprecipitated with the anti‐latent proteasome antibody‐From these structural and enzymatical properties it is concluded that the purified proteasome corresponds to 20S proteasomes reported in other eukaryotic cells.<br />  The preceding studies demonstrated that although goldfish oocyte cytosols contain an active form of proteasome which can hydrolyze its substrates in the absence of activators,this enzymatic activity markedly reduced during further purification.A new procedure involving the use of a step elution and  ATP was developed to prevent the loss of activity during purification.Using this method,active proteasome was purified to homogeneity from ovarian cytosol using five steps of chromatography.The purified active proteasome had chymotrypsin‐,trypsin‐,and V8 protease‐like activities even in the absence of SDS.The enzyme exhibited two bands on native PAGE.Electrophoresis and Western blot analyses showed that the enzyme consists of at 1east 15 protein components ranging in molecular mass from 35.5 to 140kDa,as we11 as the multiple subunits of the latent proteasome(20Sproteasome)ranging in molecular mass from 23.5 to 31.5kDa.The molecular weight and sedimentation coefficient of the active proteasome were estimated to be 1,200kDa and 29.4 S,respectively,both of which are larger than those of the 1atent proteasome of the same species.In electron micrographs,the active proteasome appeared as a dumbbe11-1ike image.It is concluded that the active proteasome purified from goldfish oocyte cytosol is identical to the 26S proteolytic complex reported in human and rabbit.<br />  As a first step to understand the possible function of proteasomes during meiotic maturation of goldfish oocytes,changes in the protein levels and activity of protesomes during oocyte maturation and egg activation were examined.Proteasome activity was measured using the fluorogenic peptide.During oocyte maturation,the activity and protein levels of proteasome changed synchronously with two peaks,one prior to the migration of germinal vesicle and the other just after the completion of germinal vesicle breakdown.Upon egg activation,there was a two-fold increase in the activity of proteasome,followed by a sharp drop soon after cyclin B degradation.This drop in the enzyme activity was coincident with asignincant decrease in the 1evels of immunoreactive proteasome components,except for one component which was weakly stained with one of the anti‐proteasome monoc1onal antibodies.These results suggest that active proteasome is involved in the regulation  of oocyte maturadon and egg activation,and further support the hypothesis that active proteasome may play an important role in the regulation of MPF inactivation,namely cyclin degradation,which occurs immediately after egg activation.<br />  Next,the possible role of active proteasome in the regulation of cyclin B degradation was investigated,for the first time,using E.coli produced goldfish cyclin B and purined goldfish active proteasome.It was found that active proteasome can digest the wild cyclin B,producing an intermediate cyclin B protein(42kDa).In contrast,cyclin B mutants lacking the first 42,68,and 96N‐terminal amino acids were not digested with active proteasome,suggesting that the N‐terminal amino acids are necessary for cyclin B degradation.Amino acid sequence analysis of the 42kDa intermediate protein revealed that active proteasome cuts the C‐terminal peptide bond of lysine 57.Experiments using various protease inhibitors suggest that trypsin‐like activity may be responsible for cyclin B degradation.Full-length goldfish cyclin B was also degraded in Xenopus egg extracts after activation by the addition of Ca2+.Taken together,these results provide the first evidence to indicate that proteasomes are involved in the regulation of cyclin Bdegradation. <br />  Ubiquitin was purified from the cytosol fraction of goldfish ovaries containing full-grown postvitellogenic oocytes using four steps of column chromatagraphy. It was also shown that a large amount of ubiquitin occurs as a free polypeptide in immature oocytes of goldfish. Furthermore, approximately the same amount of proteasome as ubiquitin was present in immature oocytes. These findings are consistent with the notion that both proteasome and ubiquitin are involved in cyclin B degradation during the meiotic cell cycle of goldfish oocytes and eggs. <br />  Based on the data presented in this study, together with those of other studies, a hypothetical model for the possible participation of proteasomes in the regulation of cyclin B degradation is presented as follows. Upon fertilization (egg activation), an increase in Ca2+ activates a Ca2+/calmodulin dependent protein kinase. This serine/threonine kinase then either directly activates proteasome or makes cyclin B a better substrate for the proteasome through an unknown mechanism. Proteasome then cuts the N-terminus (Lys 57) of cyclin B and expose an ubiquitinating site of cyclin B, thus enabling cyclin B to interact with ubiquitinating enzymes. After ubiquitination, cyclin B-ubiquitin complexes are degraded by active proteasome to small peptides, leading to the inactivation of MPF.","subitem_description_type":"Other"}]},"item_1_description_18":{"attribute_name":"フォーマット","attribute_value_mlt":[{"subitem_description":"application/pdf","subitem_description_type":"Other"}]},"item_1_description_7":{"attribute_name":"学位記番号","attribute_value_mlt":[{"subitem_description":"総研大甲第93号","subitem_description_type":"Other"}]},"item_1_select_14":{"attribute_name":"所蔵","attribute_value_mlt":[{"subitem_select_item":"有"}]},"item_1_select_8":{"attribute_name":"研究科","attribute_value_mlt":[{"subitem_select_item":"生命科学研究科"}]},"item_1_select_9":{"attribute_name":"専攻","attribute_value_mlt":[{"subitem_select_item":"X2 分子生物機構論専攻"}]},"item_1_text_10":{"attribute_name":"学位授与年度","attribute_value_mlt":[{"subitem_text_value":"1993"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"TOKUMOTO, Toshinobu","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":"2016-02-17"}],"displaytype":"simple","filename":"甲93_要旨.pdf","filesize":[{"value":"461.7 kB"}],"format":"application/pdf","licensetype":"license_11","mimetype":"application/pdf","url":{"label":"要旨・審査要旨 / Abstract, Screening Result","url":"https://ir.soken.ac.jp/record/1305/files/甲93_要旨.pdf"},"version_id":"44ef6288-687c-43da-8cc0-d917fbf2b5ed"},{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2016-02-17"}],"displaytype":"simple","filename":"甲93_本文.pdf","filesize":[{"value":"5.6 MB"}],"format":"application/pdf","licensetype":"license_11","mimetype":"application/pdf","url":{"label":"本文","url":"https://ir.soken.ac.jp/record/1305/files/甲93_本文.pdf"},"version_id":"4ae638e8-4c54-439d-9ab4-ff20b5ce6f51"}]},"item_language":{"attribute_name":"言語","attribute_value_mlt":[{"subitem_language":"eng"}]},"item_resource_type":{"attribute_name":"資源タイプ","attribute_value_mlt":[{"resourcetype":"thesis","resourceuri":"http://purl.org/coar/resource_type/c_46ec"}]},"item_title":"キンギョの卵内プロテアソーム：その構造と卵成熟．受精における機能","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"キンギョの卵内プロテアソーム：その構造と卵成熟．受精における機能"},{"subitem_title":"Oocyte Proteasomes: The Structure and Role in the Regulation of Meiotic Maturation and Fertilization in the Goldfish, Carassius auratus","subitem_title_language":"en"}]},"item_type_id":"1","owner":"1","path":["27"],"pubdate":{"attribute_name":"公開日","attribute_value":"2010-02-22"},"publish_date":"2010-02-22","publish_status":"0","recid":"1305","relation_version_is_last":true,"title":["キンギョの卵内プロテアソーム：その構造と卵成熟．受精における機能"],"weko_creator_id":"1","weko_shared_id":1},"updated":"2023-06-20T14:46:07.333856+00:00"}