前号以来今日まで即ち平成9年7月から平成9年10月までの活動報告を行う。

I. TARA用実験ステーション

1. BL6B
TARA用実験ステーションBL6Bの大きな変更はない。

2. 大型IP読取装置

大型IP装置は完壁に作動するようになった。読取装置を制御する計算機INDY の0Sを6.2に上げ、NFSで接続したAlphaServer 4000のディスクに直接書き込 めるようにした。これにより読み取りながらデータをTARAハウスにあるサー バーに直接保存出来るようになり極めて便利になった。ユーザーが持ち帰るDA Tテープヘの書き込みエラーに備えて、データのシステムバックアップは1日l 回午前11時から開始される。パックアップに必要な時間はデータ量によるので はっきり言えないが、2時間程度と予想している。殆ど起こらないことではある が、書き込み途中のファイルがたまたまバックアップされると、不完全なファイ ルとしてDLTに保存されることになる。勿論、その時でもディスクヘの書き込 みは完全に実行される。通常、測定の途中で作られたファイルは測定後、各自の ポームディレクトリに移されるので、次のバックアップ時に再度バックアップさ れることになる。ディスク上のデータは１週間程度(ディスク使用量が70%に なるよう保存期間を決めたい)保存され、システムバックアップのDLTは、ビ ームタイム1期間の間保存される。しかし、サーバーがバックアップしたデータ を戻すことに頼らず、早めにDATに落として持ち帰るようにして頂きたい。

3. 計算機及びネットワーク等

TARAネットワークは、IPアドレス変換装置(PIX)を通してKEKネ ットワークに接続することによりセキュリティを強化した。これに伴いインター ネットワーク側からTARAのコンピュータに接続することは出来なくなったが、 TARAネットワーク内からインターネットに接続することは今まで通り可能で ある。このセキュリティの強化により、KEKで時々報告されるクラッカーによ る不法侵入の危険性が殆ど無くなったと考えている。また、大型IPデータの流 れを考慮して、ルータをBL6A/BL6BとBL18Bの2つに分離しネット ワークの混雑を緩和することにした。

各居室に10/100BASE-Tの情報コンセントを設置し、ユーザがコン ピュータを用意すれば、各居室でデータのバックアップ及びデータ処理が出来る 環境を用意した。

�U. 可搬型コンテナハウス(TARAプレハブ)
客員研究室を出資企業14社が使用する事が運営委員会で決定された。詳しく は運営委員会報告を参照されたい。

�V. 各種委員会報告

1. 編集員会

第7回編集委員会を平成9年9月3日(金)18時よりTARAハウスにて開 催した。構造生物Vol.3，No.3の原稿の最終チェックならびに印刷等のスケジュー ルの確認が行われた。続いて次号の内容についての検討が行われ、執筆依頼者及 び各委員の役割分担が決定された。

2. 運営委員会

第4回運営委員会がTARAハウスにおいて平成9年9月11l日(木)に開催 された。詳細は本号24頁に掲載された運営委員会報告を参照されたい。

IV.業績紹介

論文中にTARAのメンバー或いはTARAに謝意等を表明され、送付されて きた論文のリストを記載する。いずれ論文数が増えてきたら独立に欄を設けるベ きであるが、まだ始まったばかりで数も少ないのでここにまとめる。尚、本プロ ジェクトのメンパー名と所属を各論文の文頭に掲げた。

1.吾郷日出夫(JT)、稲垣栄二(JT)、宮野雅司(JT)

Cloning and Crystal Structure of Hematopoietic Prostaglandin D Synthase.
Cell, Vol. 90, 1085-1095 (1997).
Yoshihide Kanaoka¹, Hideo Ago², Eijj Inagaki², Toyomichi Nanayama², Masashi Miyano², Reiko Kikuno³, Yutaka Fujii⁴, Naomi Eguchi⁵, Hiroyuki Toh³, Yoshihiro Urade¹, and Osamu Hayaishi¹.
¹Department of Molecular Behavioral Biology, Osaka Bioscience Institute Osaka 565 Japan.
²Central Pharmaceutical Research Institute, Japan Tobacco, Inc. Osaka 569-11 Japan.
³Biomolecular Engineering Research Institute, Osaka 565 Japan.
⁴Department of Chemistry, Fukui Medical School, Fukui 910-11 Japan.
⁵PRESTO, Japan Science and Technology Corporation, Osaka 565 Japan.

Summary
Hematopoietic prostaglandin (PG) D synthase is the key enzyme for production of the D and J series of prostanoids in the immune system and mast cells. We isolated a CDNA for the rat enzyme, crystallized the recombinant enzynle, and determined the three-dimensional structure of the enzyme complexed with glutathione at 2.3 Å resolution. The enzyme is the first member of the sigma class glutathione S-transferase (GST) froln vertebrates and possesses a prominent cleft as the active site, which is never seen among other members of the GST family. The unique 3-D architecture of the cleft leads to the putative substrate binding mode and its catalytic mechanism, responsible for the specific isomerization from PGH₂ to PGD₂.

TARAに関する表現
Acknowledgerrlents; This study was supported in part by the Sakabe Project of the Tsukuba Advanced Research Alliance (TARA).

2. 祥雲弘文(筑波大)

Purification and characterization of a flavohemoglobin from the denitrifyjng fungus Fusarium oxysporum
FEBS Letters 414 (1997) 545-548
Naoki Takaya, Sawako Suzuki, Masaru Matsuo, Hirofumi Shoun, Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, lbaraki 305, Japan

Summary

A flavohemoprotein was purified to homogeneity from the denitrifyjng fungus Fusarium oxysporum. The purified protein existed as a monomer with a molecular weight of 44 kDa. It was purified in an oxidized form and exhibited the absorption maxima at 401, 540 and 643 nm in its resting form, and at 434 and 555 nm upon reduction with dithionite, respectively. The protein contained 0.5 mol protoheme/mol and 1. I mol FAD/mol, respectively. When the resting flavohemoprotein was aerobically incubated with NAD(P)H, it was converted to a spectral species that is spectrally very similar to oxyhemoglobins. These properties are characteristics of flavohemoglobins (FHb) of Alcaligenes eutrophus, Escherichia coli, and baker's yeast. Further the amino terminal amino acid sequence of the protein of F.oxysporum was similar to those of these FHbs. These results suggest that the isolated flavohemoprotein of F.oxysporum would be a counterpart of the proteins in the FHb family.

Key words: Flavohemoprotein; Hemoglobin; O₂ binding; NAD(P)H oxldase Denitrification; Fusarium oxysporum

TARAに関する表現
Acknowledgenlents; This study was supported by Sakabe Project TARA of University of Tsukuba

3.足立伸一(理研)、中川敦史(北大)、田中勲(北大)、祥雲弘文(筑波大)

Crystallization, preliminary diffraction and electron paramagnetic resonance studies of a single crystal of cytochrome P450nor
FEBS Letters 412 (1997) 346-350
Sam-Yong Park¹, Hideaki Shirrlizu², Shin-ichi Adachi¹, Yoshitsugu Shiro¹, Tetsutaro lizuka¹, Atsushi Nakagawa³, Isao Tanaka³, Hirofumi Shoun⁴, Hiroshi Hori⁵.
¹ The Institute of Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako, Saitama 351-01, Japan.
²Faculty of Science, Gakushuin University, Tokyo 170, Japan.
³ Division of Biologjcal Sciences, Graduate School of Science, Hokkaido University, Sapporo 060, Japan.
⁴ Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, lbaraki 305, Japan.
⁵Department of Biophysical Engineering, Facuity of Engineering Science, Osaka University, Toyonaka, Osaka 560, Japan.

Summary

Cytochrome P450nor (P450nor) is a heme-containing nitric oxide reductase from the denitrifying fungus, Fusariunl oxysporum. This enzyme catalyzes the reduction of NO to N₂O. In the present study, we report results from prel irrlinary crystal lographic and electron paramagnetic resonance (EPR) analysis of a single crystal of P450nor. The crystal was grown in 100 mM MES buffer at pH 5.6 using PEG 4000 as a precipitant. It belongs to the orthorhombic system with cell dimensions of a=54. 99Å, b=82.66Å,c= 87. 21Å, and the space group is P2₁2₁2₁. The crystal diffracts synchrotron radiation at higher than 2.0 Å resolutlon, and therefore it is suitable for X-ray crystal structure analysis at atomic resolution. Bijvoet and dispersive anonlalous difference Patterson maps show a clear peak corresponding to the henle iron. The structure solution is currently underway by means of MIR and MAD techniques. EPR analysis determined the orientation of the heme within the P450nor crystal.

Key words: Nitric oxide reductase; Cytochrome P450; Crystallization; EPR; MAD

TARAに関する表現
Acknowledgements; This study was supported in part by the Sakabe Project at the TARA (Tsukuba Advanced Research Alliance) Center, University of Tsukuba Japan.

4. 祥雲弘文(筑波大)

Functional and structural comparison of nitric oxide reductases from denitrifyjng fungj Cylindrocarpon tonkinense and Fusarium oxysporum.
BBA 1338 (1997) 93-99.
Naoki Toritsuka¹, Hirofumi Shoun¹, Udai P. Singh², Sam-Yong Park², Tetsutaro lizuka², Yoshitsugu Shiro².
¹Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, lbaraki 305, Japan.
²The Institute of Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako, Saitama, 351-01, Japan.

Summary

Two isozymes of nitric oxide reductase (Nor) from the denitrifyjng fungus Cylindrocarpon tonkinense (c. Norl and c.Nor2) are the heme-enzyme cytochrome P-450's (Usuda et al. (1995) Appl. Environ. Microbiol. 61, 883-889). However, they catalyze the NO reduction to N.O, but not the monooxygenation reaction using O.. We kinetically and spectrophoto- metrically studied the reactions of the two Nor's with NO and electron donor, NAD(P)H, using flash-photolysis and stopped-flow rapjd scan methods. The enzyme in the Fe³⁺ state can bind NO to yield the Fe³⁺NO complex. When the resultant Fe³⁺NO complex reacted with the electron donor It was converted to the Fe³⁺ enzyme via a transient formation of the characteristic intermediate (1). The spectroscopic results were essentially the same as those of the Nor from another denitrifying fungus Fusarium oxysporum (f.Nor), which we previously reported (Shiro et al. (1995) J. Biol. Chem. 270, 1617-1623), suggesting that these fungal Nor's cataiyze the NO reduction by the same mechanism. Most probably, th Fe³⁺NO complex of the Nor is reduced with two-electrons dlrectly transferred from NAD(P)H to yield the Intermediate I, and then 39 the I reacts with another NO to generate N20 and the Fe3+ enzyme. However, the kinetic measurements showed that the reaction rate constant of each step was variable depending on the combination of the Nor and the electron donor; i.e., c.Norl + NADH, c.Nor2 + NADPH, c.Nor2 + NADH and f.Nor + NADH. In particular, the rate constant for the electron transfer step from the electron donor to the Fe³⁺ NO enzyme is dramatically different among these systems. On the other hand, we also measured paramagnetically shifted ¹H-NMR spectra of c.Nor2 and f.Nor in the ferrous (reduced) state where the iron-bound Cys β-CH₂, signal was observed at the same position ( about 270 ppm) for c.Nor2 and f.Nor, indicating that the Cys thiolate (S^-) coordinates to the heme iron in the same fashion in the Nor's. However, the heme peripheral proton signals were subtly but significantly different in their positions between the two enzymes. On the basis of these kinetic and spectroscopjc data, we suggested that the Fe-S^- binding character is not essential for the NO reduction reactivity, but that the subtie difference in interaction of their hemes with the surroundings is possibly responsible for the difference in th Nor reactivity, especially jn the electron transfer step from NAD(P)H to the Fe³⁺NO moiety.

Keywords: Fungal denitrification; Denitrification; Nitric oxide reductlon; Cytchrome P-450; (C. tonkinense); ( F. oxysporum)

TARAに関する表現
Acknowledgements; This work was supported by the Sakabe Project at the TARA (Tsukuba Advanced Research Alliance) Center, University of Tsukuba Japan.

5. 足立伸一(理研)、中川敦史(北大)、田中勲(北大)、祥雲弘文(筑波大)

Crystal structure of nitric oxide reductase from denitrifying fungus Fusarium oxysporum.
Nature Structural Biology, 4, 827-832 (1997).
Sam-Yong Park¹, Hideaki Shimizu², Shin-ichi Adachi¹, Atsushi Nakagawa³, Isao Tanaka³, Kazuhiko Nakahara⁴, Hirofunli Shoun⁴, Eiji Obayashi⁵, Hiro Nakamura¹, Tetsutaro lizuka¹ and Yoshitsugu Shiro¹.
¹The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-01, Japan.
² Gakushuin University, Mejjro, Toshima-ku, Tokyo 170, Japan.
³ Division of Biologjcal Sciences, Graduate School of Science, Hokkaido University, Sapporo 060, Japan.
⁴Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, lbaraki 305, Japan.
⁵Department of Applied Chenlistry, Chuo Universityn Bunkyo-ku, Tokyo 112, Japan.

Summary

Structures of nitric oxide reductase (NOR) in the ferric resting and the ferrous CO states have been solved at 2.0 A resolution, These structures provide significant new insights into how NO is reduced in biological systems. The haem distal pocket is open to solvent, implicating this regjon as a possible NADH binding site. In combination with mutagenesis results, a hydrogen-bonding network from the water molecule adjacent to the iron ligand to the protein surface of the distal pocket through the hydroxyl group of Ser 286 and the carboxyl group of Asp 393 can be assigned to a pathway for proton delivery during the NO reduction reaction.

TARAに関する表現
Acknowledgements; Thls work was supported in part by the Sakabe Project at the TARA (Tsukuba Advanced Research Alliance) Center, University of Tsukuba Japan.

6. 田中可昌(筑波大)

A site-specific DNA endonuclease specified by one of two ORFS encoded by a group I intron in Dictyostelium discoideum mitochondrial DNA.
Gene 191, 15-121 (1997)
Shinjj Ogawa¹, Kayo Naito¹, Kiyohiko Angata¹, Takahiro Morio¹, Hideko Urushihara¹, Yoshimasa Tanaka^1,2
1 Institute of Biologjcal Sciences, Unlverslty of Tsukuba Tsukuba
²Center for TARA*, University of Tsukuba, Tsukuba, Ibaraki 305, Japan

Summary

The second intron (Dd0X1/2. 2) of Dictyostelium discoideum cytochrome oxidase subunit 1/2 fused gene has two free-standing ORF genes (Dd ai2a and Dd ai2b) in a loop, which have similar amino acid sequences and are homologous to al4 DNA endonuclease (1-Scell) of Saccharomyces cerevisiae. To elucidate the functions of these ORFs, we cloned the ORFS into an expression vector and introduced the conlposite vectors into E. coli. The expression of Dd ai2a in E. coli caused growth inhibition and degradation of the E. coli genomic DNA. To determine whether Dd ai2a protein is a homing type DNA endonuclease, the ability to cleave the homing site of its intron in vivo was exanlined. Dd a2ia cleaved only one strand of intronless DNA sequence at the site which coincides with the I-Scell cleavage recognition site. We suppose that Dd ai2a functions actually as a homing type DNA endonuclease in D. discoideum mito- chondria by virtue of other factors. To obtaln further information about the relationship between the existence of introns and the mating system, we carried out in vitro self-spjicing assay and polymerase chain reaction analysis using 13 strains of the cellular slime mold.

Keywords: Intronic ORF; Self-spl icing; Celluiar slime mold; Mating type; cox1/2 fused gene.

TARAに関する表現
所属の欄:² 及び
footnote:*Tsukuba Advanced Research Al liance (TARA researcher for Sakabe Project)
Acknowledgement: -----This study was partly supported by ----and by Sakabe project of TARA (Tsukuba Advanced Research Alliance) of this University.

7. 田中可昌(筑波大)

Cloning and characterization of the gene encoding a mitochondrially localized DNA topoisomerase II in Dictyostel ium discoideum Western blot analysis
Biochimica et Biophysica Acta 1352, 63-72 (1997)
Kayoko Komori¹, Kenjj Kuroe¹ Kaichiro Yanagisawa¹, Yoshimasa Tanaka^1,2,*
1Institute of Biological Sciences, University of Tsukuba, Tsukuba, lbaraki 305, Japan
²Center for TARA*, University of Tsukuba, Tsukuba, Ibaraki 305, Japan

Summary

We cloned a gene (topA) encoding DNA topoisomerase 11 from Dictyostelium discoideum nuclear DNA using oligo probes corresponding to the consensus amino acld sequences found in the gene in other eukaryotes. The gene encoding a predicted polypeptide of 1282 amino acids with M. of about 146 kDa, is a single copy that is expressed as a polyadenylated 4.5 kb RNA. The predicted amino acid sequence shares similarity with those of other eukaryotes with identity between 32 and 46%. The protein is 260-300 amino acids shorter in the C-terminal regjon and 50-80 Ionger in the N-terminal regjon than those of other eukaryotes. In TOpA of D. discoideum, the N-terminal regjon with stretches of charged and hydrophilic amino acids is predicted to fold into an amphiphilic α-helix which is characteristic of a mitochondriai targeting signal presequence. Four independent polyclonal antibodies against bacterial ly expressed GST fusion proteins containing four portions of the polypeptide detected a single band on Western blots at about 135 kDa. Western blots analysis of subceliular fractions revealed that this protein is locaiized in mitochondria. The protein and the mRNA are present in growth phase and during development, although levels of both declined as development proceeded.

Keywords: Cellular slime mold; Mitochondrial localization; Mitochondrial targeting sequence; Molecular clonin ;DNA topoisomerase II;

TARAに関する表現
所属の欄:² 及び
footnote:*Tsukuba Advanced Research Alliance (TARA researcher for Sakabe Project)
Acknowledgement: -----This study was partly supported by ----and by Sakabe project of TARA (Tsukuba Advanced Research Alliance) of this University.

8. 岡村直道(筑波大)

Characterization and Identification of Protelns Secreted In the Varlous Regions of the Adult Boar Epjdidymis*.
BIOLOGY OF REPRODUCTION 55, 956-974 (1996)
Patrick Syntin¹, Franpoise Dacheux^1,2, Xavier Druart¹, Jean Luc Gatti¹ Naomichi Okamura³, and Jean-Louis Dacheux¹.
¹Laboratoire de Physiologie de la Reproduction, URA INRA-CNRS 1291, 373 80 Nouzilly France.
²Laboratoire de Biologje de la Reproduction, Faculte des Sciences et Techniques, 37200 Tours, France.
³Institute of Basic Medical Sciences and Center for TARA, University of Tsukuba, Tsukuba, Ibaraki 305, Japan.
^*N.O. is TARA researcher for the Sakabe Project.

Summary

The synthesis and secretion of proteins by the boar genital tract were studied in vitro by jncubating epjdidymal tissues with [³⁵S]methionine and cysteine. Characterization of the major neosynthesized proteins was performed electrophoretical ly by one and two-dimensional PAGE analysis, and an epididymal protein cartography was established. Some of the proteins secreted were found to be unregjonalized. Polarization studies of the secretions in the epididymal tubule were carried out by jn vitro incubation of isolated tubules, and most of these unregjonalized proteins were found not to be secreted in the epjdidymal lunen. Inside the epididymal lumen, protein secretion was highly regionalized, and electrophoresis analysis detected few proteins secreted at all points along the organ. A total of 146 epjdidymal proteins, covering 220 spots, were found to be secreted by the epididymis. The distal caput showed the highest number of spots, the lowest number of proteins secreted being found in the proximal caput and cauda. Most of the epjdidymal proteins analyzed are highly polymorphic in terms of both isoelectric point and molecular mass.

The presence and importance of the different compounds in the various regions of the epididymis were established. Several distinct secretory regjons of the epjdidymis can be deternlined by the presence of major characteristic proteins. The concentrations of a gjven protein in the fluids of various regjons were not reiated to the respective secretion intensity of that protein. Identification of some major epididymal proteins was accomplished by N-terminal amino microsequencing and by the use of specific antisera. Of the various major proteins, clusterin, glutathione peroxidase, retinol-binding protein, Iactoferrin, EP4, β-N- acetyl-hexosaminidase, α-mannosidase, and procathepsin L were identified and localized along the organ. Several polypeptides found in this study remain unidentified.

TARAに関する表現
所属の欄;³ Institute of Basic Medical Sciences and Center for TARA, University of Tsukuba, Tsukuba, Ibaraki 305, Japan.
Foot note; N.O. is TARA researcher for the Sakabe Project

9. 岡村直道(筑波大)

Direct Evidence for the Secretion of Lactoferrin and Its Binding to Sperm in the Porcine Epididymis.
Molecular Reproduction and Development 47, 490-496 (1997)
Yin-zhe Jjn¹, Shiro Bannai¹, Francoise Dacheux², Jean-Louis Dacheux² and Naomichi Okamura^1,3
1Institute of Basic Medical Sciences. University of Tsukuba, Japan.
²Laboratoire de Physiologje de la Reproduction, Institut National de la Recherche Agronomique. Monnaie, France.
³Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba, Ibaraki, Japan.

Summary

Lactoferrin has been for the first time purified from the porcine cauda epididynlal fluid as a 70 kDa protein. Both Western and Northern blot analyses show that lactoferrin is synthesized in the regions from the distal caput to the cauda epididymis and secreted into the luminal fiuid. Lactoferrin is first secreted as a 75 kDa glycoprotein and its carbo hydrate moieties are gradually digested to form 70 kDa protein in the cauda epjdidymis.

Lactoferrin has already bound to the surface of the epididymal sperm because the anti-lactoferrin antiserum induces the mature sperm tail-to- tail agglutination. These results strongly suggest new physiological functions of lactoferrin on the sperm maturation in the epididymis.

Key Words: Sperm maturation; estrogen; retinoic acid.

TARAに関する表現
所属の欄;³ Center for TARA Tsukuba Advanced Alllance Unlverslty of Tsukuba, Tsukuba, Ibaraki, Japan.
Foot note; Contact Grant sponsor:Tsukuba Advanced Research Alliance (TARA Sakabe Project).

10. 岡村直道(筑波大)

Molecular cloning and characterization of the epididymis-specific glutathione peroxidase-like protein secreted in the porcine epjdidymal fluid.
BBA 1336 (1997) 99-109.
Naomichi Okamura^1,2, Yuka lwaki¹, Shinsuke Hiramoto¹, Michiko Tamba¹ Shiro Bannai¹, Yoshiki Sugita², Patrick Syntin⁴, Francoise Dacheux⁴, Jean-Louis Dacheux⁴.
¹Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba Ibaraki 305, Japan.
²Center for TARA, Universlty of Tsukuba, Tsukuba lbaraki 305, Japan.
³Ibaraki Prefectural University of Health Sciences, Ami-machi, Inashiki-gun, Ibaraki 300-03, Japan.
⁴Laboratoire de Physiologje de la Reproduction, INRA, 37380 Monnaie, France.

Summary

The epjdidymis-specific glutathione peroxidase was purified from the porcine cauda epididymal fluid in order to analyze its enzymatic activity and roles in the epididymls. The purified protein was found to consist of four identical 23 kDa subunits. The complementary DNA encoding the 23 kDa subunit was cloned from the cDNA Iibrary of the porcine proximal caput epididymis, only where the 23 kDa subunit is expressed. Although the selenocysteine codon (TGA) is contained in the CDNA of the other cytosolic type of glutathione peroxidases, it is replaced by cysteine codon (TGT) in the 23 kDa subunit CDNA, similarly to the results previously obtained for CDNAS encoding the epididymis- specific form of the secreted glutathione peroxidases of mouse, rat and monkey. By the direct analysis of the selenium, the purified protein was proved to contain no selenium atom in the molecule. The activities of the purified epididymis-specific glutathione peroxidase toward hydrogen peroxide or organic hydroperoxides were by far lower than the activity of cytosolic selenium-dependent glutathione peroxidase (less than 0.1%). In addition, the concentration of glutathione in the porcine epididymal fluids was about 20μM, which is much lower than the optimal concentration for the glutathione peroxidase activity of the purified protein. These results strongly suggest that this protein is enzymatically quiescent at least in the porcine epididymal fluid. An immunocytochemical study showed that this protein was found to bind to the acrosonlal region of the epididymal sperm and to disappear during the acrosome reaction. Furthermore, this protein significantiy retarded the acrosome reaction induced in vitro. The possibilities have been discussed that it protects sperm from the premature acrosome reaction and maintains sperm fertilizing ability in the epididymis.

Keywords: Sperm maturation; Glutathione peroxidase; Acrosome reactlon Epididymis.

TARAに関する表現
所属の欄;² Center for TARA Unlverslty of Tsukuba Tsukuba lbarakl 305, Japan.
Acknowlegements; This work was supported in part by Tsukuba Advanced Research A1liance (TARA Sakabe project).

11. 岡村直道(筑波大)

Assignnlent of a -mannosidase gene (MAN2B2) to swine Chrolnosome 8p23- pter by fluorescence in situ hybridization.
Mammalian Genome Vol.8, N0.2, 158-159 (1997).
Kousuke Ohata^1,4, Naomichi Okamura², Misaki Kojlma⁴, Hiroshi Yasue^3,4
1Faculty of Agriculture, University of Tsukuba, Tsukuba. Ibaraki 305, Japan.
²Institute of Basic Medical Sciences and Center for TARA (TARA researcher for Sakabe project) University of Tsukuba, Ts.ukuba, Ibaraki 305, Japan.
³Institute of Biologjcs, University of Tsukuba, Tsukuba, Ibaraki 305, Japan.
⁴Aninlal Genome Research Group, National Institute of Animal Industry, P.O. Box 5, Norin Kenkyu Danchi, Tsukuba, Ibaraki 305, Japan.

Summary

Recently, we have cloned a cDNA encoding a novel α-D-mannosidase (NAN2B2). MAN2B2 is specifically secreted from the border region between the porcine caput and corpus epididymis, and then binds to the equatorial segment of the immature sperm, suggesting that it is involved in the sperm-egg interaction. In the present study, its gene was found to reside on swine Chr8p23-pter by fluorescence in situ hybridization.

TARAに関する表現
所属の欄;² Instltute of Baslc Medlcal Sclences and Center for TARA (TARA researcher for Sakabe project) University of Tsukuba, Tsukuba, lbaraki 305, Japan.

12. 水野　洋(農業生物資源研、筑波大)

Structure of coagulation factors IX/X-binding protein, a heterodimer of C-type lectin domains.
Nature Structural Biology Vol.4,N0.6, 438-441 (1997)
Hiroshi Mizuno¹, Zui Fujimoto¹, Mika Koizumi¹, Hideko Atoda² and Takashi Morita².
¹National Institute of Agrobiological Resources, Tsukuba, Ibaraki 305, Japan
²Biochemistry, Meiji College of Pharmacy, Tanashi, Tokyo 188, Japan.

Summary

Coagulation factors IX/X-binding protein is an intertwined dimer with a central loop projecting into the adjoining subunit. Excluding this loop, each subunit has a fold similar to rat Hlannose-binding protein.

TARAに関する表現
Method: A complete data set was collected with a Weisenberg camera for macromolecules at the Photon Factory in Tsukuba on BL-6A2 and BL-6B (TARA data col lection system).

13. 水野　洋(農業生物資源研、筑波大)

Crystallization and preliminary X-ray diffraction studies of tulip aryl acylamidase: a key enzyme in plant herbicide detoxification.
Acta Cryst. D53, 342-344 (1997).
Kouichi Fukuda¹, Takashi Matsumoto², Kiyoshi Hagiwara², Zui Fujimoto², and Hiroshi Mizuno².
¹Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, lbaraki 305, Japan.
²Department of Molecular Biology, National Institute of Agrobiological Resources. Tsukuba, Ibaraki 305, Japan.

Summary

Crystals of aryl acylamidase (E.C. 3.5. 1. 13) from tulip bulbs have been obtained by the hangjng-drop vapor-diffusion method using polyethylene glycol (PEG) 8000 as a precipjtant. The crystals belong to space group P2₁2₁2₁ with unit-cell dimensions a=68.7, b=80.1 and c=112.9 Å. Assuming two molecules of molecular weight of 34 kDa in the asymmetric unit, Vm is 2.28A³ Da^-1 , indicating a solvent content of approximately 46%. The intensity data have been collected to 2.5 Å resolution with an R_merge of 0.067.

TARAに関する表現
HM is a member of the TARA (Tsukuba Advanced Research A1liance) project of University of Tsukuba, Japan.

14. 坂田誠(名大)

Structure of Endohedral Dimetallofullerene Sc₂@ C₈₄
Physical Review Letters Vol.78, N0.17, 3330-3333 (1997)
Masaki Takata¹, Eiji Nishibori¹, Buntaro Umeda¹, Makoto Sakata¹, Etsuji Yamamoto², and Hisanori Shinohara².
¹Department of Applied Physics, Nagoya University, Nagoya 464-01, Japan
²Department of Chemistry, Nagoya University, Nagoya 464-01, Japan.

Summary

The endohedral nature of dimetallofullerene Sc2@c84 is determined for the first time by a method which is a combination of the maximum entropy method (MEM) and the Rietveld refinement from synchrotron powder diffractlon data. The obtained MEM charge density clearly shows the D2d- symmetry cage structure, indicating the rotation of Sc.eC84 molecules in solid state is almost quenched even at room temperature. From the MEM charge density, the encapsulated Sc-Sc distance and the nearest Sc-C distance are 3.9(1) and 2.4(1) A, respectively.

TARAに関する表現
This work was supported by ----- , the TARA Sakabe Project,

15. 福山恵一(阪大)

Binding mode of benzhydroxamic acid to Arthromyces ramosus peroxidase shown by X-ray crystaliographic analysis of the complex at 1.6 A resolution.
FEBS Letters 412, 107-110 (1997)
Hiroyuki Itakura, Yutaka Oda, Keiichl Fukuyama
Department of Biology, Graduate School of Science. Osaka University, Toyonaka, Osaka 560, Japan

Summary

The crystal structure of Arthromyces ramosus peroxidase (ARP) in complex with benzhydroxamic acid (BHA) as determined by X-ray analysis at 1.6 Å shows unambiguously how BHA binds to ARP. BHA is located in the distal heme pocket. Its functional groups are held by three hydrogen bonds to His⁵⁶N, Arg⁵²N_ε, and Pro¹⁵⁴O, but are too far away to interact with the heme iron. The aromatic ring of BHA is positioned at the entrance of the channel to the heme pocket, approximately parallel to the heme group. Most water molecules at the active site of the native enzyme are replaced by BHA, leaving a ligand, probably a water molecule, at the sixth position of the heme. Results are compared with spectro- scopic data.

Key words:Peroxldase; Heme enzyme; Benzhydroxamic acid ; X-ray crystal lography; Arthromyces ramosus

TARAに関する表現
Acknowledgements; This work was supported in part by... . . . . . and by the Sakabe project of TARA (Tsukuba Advaced Research A1liance), of the University of Tsukuba.

16. 甲斐泰(阪大)、原田繁春(阪大、現在東大)

Structure of the Zinc Endoprotease froln Streptomyces caespjtosus
J. Biochem. 121, 304-308 (1997)
Genjj Kurisu, Takayoshi Kinoshita, Akiko Sugjmoto, Akinobu Nagara, Yasushi Kal, Nobutami Kasai, and Shigeharu Harada
Department of Applied Chemistry, Faculy of Engjneering, Osaka Uniuersi'ty, Suita, Osaka 565.

Summary

A zinc endoprotease produced by Streptomyces caespitosus (ScNp) specifically hydrolyzes the peptide bond at the imino side of aromatic residues and is the smallest protease found to date. Although SCNP carries the zinc-binding sequence HEXXH, its primary structure of 132 amino acid residues differs from those of other known zinc metalloendo- proteases. X-ray structural analysis of SCNP at 1.6 Å resolution revealed that despjte a lack of sequence honlology, the common topolo- gjcal feature of main-chain folding and a β-turn containing methionine, which is a feature of the zinc metalloendoprotease superfamily of metzincins, is conserved in ScNP. The zinc atom of SCNP is tetrahedrally ligated by the two histidines in the HEXXH sequence, an aspartate residue and a water molecule. Thus, SCNP represents a novel subfamily of metzincins with a HEXXHXXGXXD zinc-binding sequence. A plausible substrate recognition pocket to which arontatic residues bind is located near the catalytic zinc ion. Key words: crystal structure, novel subfamily, substrate recognition pocket, zinc metallo-protease.

TARAに関する表現
footnote; S.Harada and Y.Kai are members of the TARA Sakabe project of University of Tsukuba, Japan.