研究者詳細 - 大橋　俊孝

論文 - 大橋　俊孝

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The primary structure of the Cl(-)-translocating ATPase, b subunit of Acetabularia acetabulum, which belongs to the F-type ATPase family. 査読国際誌

C Moritani, T Ohhashi, H Kadowaki, M Tagaya, T Fukui, F Lottspeich, D Oesterhelt, M Ikeda

Archives of biochemistry and biophysics 339 ( 1 ) 115 - 24 1997年3月

記述言語：英語掲載種別：研究論文（学術雑誌）

The genes possibly encoding the b subunit (50 kDa) of the Cl(-)-translocating ATPase of Acetabularia acetabulum were cloned from total RNA and from poly(A)+ RNA and sequenced. The deduced amino acid sequence of the open reading frame consisted of 478 amino acids and showed high similarity to the beta subunit of chloroplast F1-ATPase. Gene fragments encoding the putative beta subunit of chloroplast F1- (273 bp) and mitochondrial F1-ATPases (332 bp) were also cloned from A. acetabulum and sequenced, respectively. The deduced amino acid sequence of the chloroplast F1-ATPase showed 92.5% identity to be primary structure of the b subunit of the Cl(-)-translocating ATPase, while the nucleotide sequences were 79.9% identical. The deduced amino acid sequence of the latter was 77.3% identical to that of the b subunit of the Cl(-)-translocating ATPase and the nucleotide sequences were 67.5% identical. By Northern analysis, these three beta-like genes were demonstrated to be transcribed with different sizes of RNA species. A putative chloroplast F1-beta fragment also hybridized with chloroplast DNA isolated from the organism.

Absence of alpha 6(IV) collagen in kidney and skin of X-linked Alport syndrome patients. 査読国際誌

S Hino, T Takemura, Y Sado, M Kagawa, T Oohashi, Y Ninomiya, K Yoshioka

Pediatric nephrology (Berlin, Germany) 10 ( 6 ) 742 - 4 1996年12月

記述言語：英語掲載種別：研究論文（学術雑誌）

To identify the abnormalities of the type IV collagen alpha 6 chain, alpha 6(IV), in Alport syndrome, we examined renal and skin tissue using rat monoclonal antibodies against non-consensus amino acid sequences of alpha 6(IV). Immunofluorescence of normal human kidney and skin tissue revealed linear alpha 6(IV) staining in the basement membrane (BM) of Bowman's capsule, in some tubules, and also in the epidermal BM. Renal specimens from five male patients of four families with X-linked Alport syndrome showed no reactivity for alpha 6(IV) in Bowman's capsules and tubules. In these patients, alpha 1(IV) and alpha 2(IV) were normal, whereas alpha 3(IV), alpha 4(IV), and alpha 5(IV) were absent from the BMs of the kidney. In skin tissue of male patients, neither alpha 5(IV) nor alpha 6(IV) were detected. The epidermal BM of female heterozygotes with X-linked Alport syndrome showed a mosaic staining for alpha 5(IV) and alpha 6(IV). These findings indicate that, in addition to a disturbed alpha 3(IV)-alpha 4(IV)-alpha 5(IV) network, patients with X-linked Alport syndrome have abnormalities in alpha 6(IV) of the renal and epidermal BMs at the protein level.

DOI： 10.1007/s004670050206

Nephritogenicity and alpha-chain composition of NC1 fractions of type IV collagen from bovine renal basement membrane. 査読国際誌

S Rauf, M Kagawa, Y Kishiro, S Inoue, I Naito, T Oohashi, M Sugimoto, Y Ninomiya, Y Sado

Virchows Archiv : an international journal of pathology 428 ( 4-5 ) 281 - 8 1996年7月

記述言語：英語掲載種別：研究論文（学術雑誌）

Nephritogenicity (anti-GBM-nephritis-inducing activity) and alpha-chain composition of globular-do-main (NCI) fractions of type IV collagen from bovine renal, pulmonary, and placental basement membranes (BMs) was examined by injecting these fractions with adjuvant into WKY/NCrj rats and by Western blotting using epitope-defined monoclonal antibodies to the six different alpha chains of type IV collagen. A purified nephritogenic fraction from renal BM contained alpha 1-alpha 6(IV)NCI, whereas a non-nephritogenic fraction contained only alpha 1-alpha 2(IV)NCI. Renal and pulmonary NCI had strong nephritogenic activity: placental NCI had weak activity. The renal and pulmonary fractions contained alpha 1-alpha 6(IV)NCI, and the placental fraction had a large amount of alpha 1-alpha 2(IV)NCI and a very small amount of alpha 3-alpha 6(IV)NCI. Immunohistochemical study of bovine renal BM with the monoclonal antibodies revealed that bovine glomerular BM contained alpha 1-alpha 5(IV) chains, but not the alpha 6(IV) chain. The absence of alpha 6(IV) chain in glomerular BM in bovine and in humans indicates that alpha 6(IV) chain is not a target antigen of anti-GBM nephritis. Nephritogenicity is apparently a property of alpha 3-alpha 5(IV)NCI.

Isolation and structure of the COL4A6 gene encoding the human alpha 6(IV) collagen chain and comparison with other type IV collagen genes. 査読国際誌

T Oohashi, Y Ueki, M Sugimoto, Y Ninomiya

The Journal of biological chemistry 270 ( 45 ) 26863 - 7 1995年11月

記述言語：英語掲載種別：研究論文（学術雑誌）

The genes COL4A5 and COL4A6, coding for the basement membrane collagen chains, alpha 5(IV) and alpha 6(IV), respectively, are located head-to-head in close proximity on human chromosome Xq22, and COL4A6 is transcribed from two alternative promoters in a tissue-specific fashion (Sugimoto M., Oohashi T., and Ninomiya Y. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 11679-11683). Immunofluorescence studies using alpha chain-specific antibodies demonstrated that the two genes are expressed in a tissue-specific manner (Ninomiya, Y., Kagawa, M., Iyama, K., Naito, L., Kishiro, Y., Seyer, J. M., Sugimoto, M., Oohashi, T., and Sado, Y. (1995) J. Cell Biol. 130, 1219-1229). We report here for the first time the isolation and the structural organization of the human COL4A6 gene. The entire gene presumably exceeds 200 kilobase pairs and contains 46 exons. Exons 1' and 1 encode the two different 5'-UTRs and the two amino-terminal parts of of the signal peptide. The carboxyl part of the signal peptide and the 7 S domain are coded for by the following 6 different exons, 2-7, whereas the exons 7-42 encode the central COL 1 domain, which contains the Gly-X-Y repeats. The last three exons, 43-45, encode the carboxyl-terminal NC1 domain. Sizes of more than a half of the exons of the gene are the same as those of Col4a2 but quite different from those of COL4A5. Within the COL4A6 gene we found three CA repeat markers that can be used for allele detection. The detailed structure of the COL4A6 gene and the high heterozygosity microsatellite markers located within the gene will be useful for linkage analysis and familial diagnosis of diseases caused by mutations of this gene.

ESTABLISHMENT BY THE RAT LYMPH-NODE METHOD OF EPITOPE-DEFINED MONOCLONAL-ANTIBODIES RECOGNIZING THE 6 DIFFERENT ALPHA-CHAINS OF HUMAN TYPE-IV COLLAGEN 査読

Y SADO, M KAGAWA, Y KISHIRO, K SUGIHARA, NAITO, I, JM SEYER, M SUGIMOTO, T OOHASHI, Y NINOMIYA

HISTOCHEMISTRY AND CELL BIOLOGY 104 ( 4 ) 267 - 275 1995年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SPRINGER VERLAG

A group of rat monoclonal antibodies recognizing the six different alpha chains of human type IV collagen have been established by our novel method. The method is designated the rat lymph node method in which enlarged medial iliac lymph nodes of a rat injected with an antigen emulsion via hind footpads are used as a source of B cells for cell fusion to produce hybridomas. The immunogens used were synthetic peptides having non-consensus amino acid sequences near the carboxyl termini of type IV collagen alpha chains. Hybridomas were screened both by ELISA with synthetic peptides and by indirect immunofluorescence with cryostat sections of human kidneys. Because the epitopes of all antibodies were determined by multipin-peptide scanning, they were confirmed to be isoform-specific. They are useful for identification of alpha chains of type IV collagen at the protein level in normal and abnormal conditions. The combined use of synthetic peptides as immunogens, the rat lymph node method as making monoclonal antibodies, and the multipin-peptide scanning as epitope mapping is found to be a strong tool for identification of peptides and proteins whose amino acid sequences are known or have been deduced.

Establishment by the rat lymph node method of epitope-defined monoclonal antibodies recognizing the six different alpha chains of human type IV collagen. 査読国際誌

Y Sado, M Kagawa, Y Kishiro, K Sugihara, I Naito, J M Seyer, M Sugimoto, T Oohashi, Y Ninomiya

Histochemistry and cell biology 104 ( 4 ) 267 - 75 1995年10月

記述言語：英語掲載種別：研究論文（学術雑誌）

A group of rat monoclonal antibodies recognizing the six different alpha chains of human type IV collagen have been established by our novel method. The method is designated the rat lymph node method in which enlarged medial iliac lymph nodes of a rat injected with an antigen emulsion via hind footpads are used as a source of B cells for cell fusion to produce hybridomas. The immunogens used were synthetic peptides having non-consensus amino acid sequences near the carboxyl termini of type IV collagen alpha chains. Hybridomas were screened both by ELISA with synthetic peptides and by indirect immunofluorescence with cryostat sections of human kidneys. Because the epitopes of all antibodies were determined by multipin-peptide scanning, they were confirmed to be isoform-specific. They are useful for identification of alpha chains of type IV collagen at the protein level in normal and abnormal conditions. The combined use of synthetic peptides as immunogens, the rat lymph node method as making monoclonal antibodies, and the multipin-peptide scanning as epitope mapping is found to be a strong tool for identification of peptides and proteins whose amino acid sequences are known or have been deduced.

Differential expression of two basement membrane collagen genes, COL4A6 and COL4A5, demonstrated by immunofluorescence staining using peptide-specific monoclonal antibodies. 査読国際誌

Y Ninomiya, M Kagawa, K Iyama, I Naito, Y Kishiro, J M Seyer, M Sugimoto, T Oohashi, Y Sado

The Journal of cell biology 130 ( 5 ) 1219 - 29 1995年9月

記述言語：英語掲載種別：研究論文（学術雑誌）

Genes for the human alpha 5(IV) and alpha 6(IV) collagen chains have a unique arrangement in that they are colocalized on chromosome Xq22 in a head-to-head fashion and appear to share a common bidirectional promoter. In addition we reported a novel observation that the COL4A6 gene is transcribed from two alternative promoters in a tissue-specific manner (Sugimoto, M., T. Oohashi, and Y. Ninomiya. 1994. Proc. Natl. Acad. Sci. USA. 91:11679-11683). To know whether the translation products of both genes are colocalized in various tissues, we raised alpha 5(IV) and alpha 6(IV) chain-specific rat monoclonal antibodies against synthetic peptides reflecting sequences near the carboxy terminus of each noncollagenous (NC)1 domain. By Western blotting alpha 6(IV) chain-specific antibody recognized 27-kD monomers and associated dimers of the human type IV collagen NC1 domain, which is the first demonstration of the presence in tissues of the alpha 6(IV) polypeptide as predicted from its cDNA sequence. Immunofluorescence studies using anti-alpha 6(IV) antibody demonstrated that in human adult kidney the alpha 6(IV) chain was never detected in the glomerular basement membrane, whereas the basement membranes of the Bowman's capsules and distal tubules were positive. The staining pattern of the glomerular basement membrane was quite different from that obtained with the anti-alpha 5(IV) peptide antibody. The alpha 5(IV) and alpha 6(IV) chains were colocalized in the basement membrane in the skin, smooth muscle cells, and adipocytes; however, little if any reaction was seen in basement membranes of cardiac muscles and hepatic sinusoidal endothelial cells. Thus, both genes are expressed in a tissue-specific manner, perhaps due to the unique function of the bidirectional promoter for both genes, which is presumably different from that for COL4A1 and COL4A2.

DOI： 10.1083/jcb.130.5.1219

The genes COL4A5 and COL4A6, coding for basement membrane collagen chains alpha 5(IV) and alpha 6(IV), are located head-to-head in close proximity on human chromosome Xq22 and COL4A6 is transcribed from two alternative promoters. 査読国際誌

M Sugimoto, T Oohashi, Y Ninomiya

Proceedings of the National Academy of Sciences of the United States of America 91 ( 24 ) 11679 - 83 1994年11月

記述言語：英語掲載種別：研究論文（学術雑誌）

The genes for the alpha 5(IV) and alpha 6(IV) chains of human basement membrane collagen type IV have been found together on chromosome X at segment q22 and have been reported to be arranged in a head-to-head fashion. Here we report the 5' flanking sequences of COL4A5 and COL4A6 and that COL4A6 is transcribed from two alternative promoters in a tissue-specific fashion. Analysis of the sequence immediately upstream of the transcription start sites revealed some features of housekeeping genes--i.e., the lack of a TATA motif and the presence of CCAAT and CTC boxes. Further analysis revealed that COL4A6 contains two alternative promoters that control the generation of two different transcripts. One transcription start site (from exon 1') is 442 bp away from the transcription start site of COL4A5, while an alternative transcription start site (from exon 1) is located 1050 bp from the first one and drives the expression of a second transcript that encodes an alpha 6(IV) chain with a different signal peptide. Reverse transcription-PCR experiments revealed that the transcript from exon 1' is abundant in placenta, whereas the transcript from exon 1 is more frequently found in kidney and lung. These results provide additional clues to answering the general question of what mechanisms are used to generate unique basement membrane structures in different tissues.

DOI： 10.1073/pnas.91.24.11679

Purification and characterization of a membrane-bound ATPase from Acetabularia cliftonii that corresponds to a Cl(-)-translocating ATPase in Acetabularia acetabulum. 査読国際誌

C Moritani, T Ohhashi, S Satoh, D Oesterhelt, M Ikeda

Bioscience, biotechnology, and biochemistry 58 ( 11 ) 2087 - 9 1994年11月

記述言語：英語掲載種別：研究論文（学術雑誌）

A Mg(2+)-ATPase was solubilized from membranes of Acetabularia cliftonii using nonanoyl-N-methylgluconamide and purified by ion-exchange and gel permeation chromatography. One active ATPase fraction after Mono Q chromatography had a specific activity of 10 units/mg of protein. Judged from subunit composition [54 (a), 50 (b) with a fainter band around 40 kDa], catalytic properties, and N-terminal amino acid sequence of the b subunit, the isolated enzyme was comparable to the Cl(-)-ATPase of Acetabularia acetabulum. Immunological characterization of both subunits showed significant similarity to the F type of ATPase. Cl(-)-transport activity was observed by reconstitution studies into liposomes.

Identification of a new collagen IV chain, alpha 6(IV), by cDNA isolation and assignment of the gene to chromosome Xq22, which is the same locus for COL4A5. 査読国際誌

T Oohashi, M Sugimoto, M G Mattei, Y Ninomiya

The Journal of biological chemistry 269 ( 10 ) 7520 - 6 1994年3月

記述言語：英語掲載種別：研究論文（学術雑誌）

To date, five distinct alpha chains have been identified in basement membrane collagen IV. We have cloned a gene encoding a new alpha chain belonging to basement membrane collagen IV by cDNA isolation under low stringency conditions. Isolation of overlapping clones and the technique of 5' rapid amplification of cDNA ends enabled us to derive the primary structure of the entire polypeptide. The deduced collagen polypeptide contained 1678 amino acid residues, including a 21-residue signal peptide, a 24-residue amino-terminal NC domain, a central 1405-residue collagenous (COL1) domain, and a 228-residue carboxyl-terminal NC1 domain. We have designated this newly found alpha chain as the alpha 6(IV) chain. The gene was mapped to chromosome Xq22 by in situ hybridization of metaphase lymphocytes. This is the same region of chromosome X where the gene coding for the alpha 5(IV) collagen chain (COL4A5) resides. Mutations in COL4A5 have been characterized in more than 50 patients with Alport syndrome. However, some of the X-linked cases of Alport syndrome are not apparently caused by COL4A5 mutations. The gene we describe in this paper therefore seems to be a candidate for mutations in this group of Alport syndrome patients.

Chloroplast ATPase in Acetabularia acetabulum: purification and characterization of chloroplast F1-ATPase. 査読国際誌

S Satoh, C Moritani, T Ohhashi, K Konishi, M Ikeda

Bioscience, biotechnology, and biochemistry 58 ( 3 ) 521 - 5 1994年3月

記述言語：英語掲載種別：研究論文（学術雑誌）

ATPases were isolated from chloroplasts of the unicellular marine alga Acetabularia acetabulum. Two preparations of ATPase, a chloroplast-enriched fraction and an alpha beta gamma-complex were compared. The alpha beta gamma-complex was released into an EDTA solution and purified by anion-exchange chromatography, hydrophobic chromatography, and gel permeation chromatography. The subunit composition of this enzyme appeared to be 52-53 (alpha), 51 (beta), and 40 (gamma) kDa from SDS-PAGE. ATPase activity was enriched about 260-fold to a specific activity of approximate 4.1 U.mg protein-1. The catalytic properties of the alpha beta gamma-complex were as follows: pH optimum at 7.5; substrate specificity, ATP > ITP, GTP > UTP = CTP (Km for ATP 0.2 mM); divalent cation requirement, Mg2+ = Mn2+ = Co2+ > Zn2+ > Ni2+ > Ca2+; ATPase activity was inhibited by monovalent anions (NO3-, SCN-), while monovalent cations had neither inhibitory nor stimulatory effect. Orthovanadate had no inhibitory effect on the enzyme activity of alpha beta gamma-complex. Azide was the most effective inhibitor of the alpha beta gamma-complex. N-Terminal amino acid sequences of the alpha and beta subunits were not obtained and appeared to be blocked. The gamma subunit gave a sequence of AGLKEMKD-XIGSVXNTKKI, which showed 60% similarity to the gamma subunits of spinach and Chlamydomonas reinhardtii CF1-ATPase and EF1-ATPase.

Isolation and characterization of human cDNAs and genomic DNAs encoding alpha-4(IV) and alpha-6(IV) chains reveal the presence of a distinct subclass of collagen IV genes. 査読国際誌

M Sugimoto, T Oohashi, M G Mattei, A Fukutomi, N Kashihara, N Matsuo, H Yoshioka, Y Ninomiya

Contributions to nephrology 107 29 - 35 1994年

cDNA isolation and partial gene structure of the human alpha 4(IV) collagen chain. 査読国際誌

M Sugimoto, T Oohashi, H Yoshioka, N Matsuo, Y Ninomiya

FEBS letters 330 ( 2 ) 122 - 8 1993年9月

記述言語：英語掲載種別：研究論文（学術雑誌）

A novel collagen IV chain, alpha 4(IV), has recently been identified in basement membranes. We describe part of the primary structure of the human alpha 4(IV) polypeptide for the first time, which has been determined by cloning and sequencing of cDNAs encoding 241 amino acid residues of the COL domain and 231 residues of the NC1 domain. We also characterized a genomic DNA fragment containing 4 exons coding for the entire NC1 domain. Among five known alpha chains of collagen IV, the alpha 4(IV) chain is distinct from the other four chains. However, it is more similar to the alpha 2(IV) chain than to the alpha 1(IV), alpha 3(IV) and alpha 5(IV) chains in terms of amino acid sequence homology, domain structure of polypeptides and exon/intron structure of the genes, suggesting the presence of two phylogenetically distinct subclasses of collagen IV alpha chains; one composed of alpha 2 and alpha 4 chains and the other of alpha 1, alpha 3 and alpha 5 chains.

Improvement of reconstitution of the Cl--translocating ATPase isolated from Acetabularia acetabulum into liposomes and several anion pump characteristics 査読

Toshitaka Ohhashi, Takashi Katsu, Mikiko Ikeda

BBA - Biomembranes 1106 ( 1 ) 165 - 170 1992年4月

記述言語：英語掲載種別：研究論文（学術雑誌）

The improved reconstitution of the Mono Q-III fraction, a Cl--translocating ATPase, isolated from Acetabularia acetabulum (Ikeda et al. (1990) Biochemistry 29, 2057-2065) into liposomes rendered transport properties of this enzyme clear. The liposomes were prepared by the reversed-phase method using egg lecithin and cholesterol in a molar ration of 2:1 and the purified ATPase was incorporated into the liposomes by a dialysis for 3 h. About 80% of the ATPase was incorporated into the liposomes. The weight ratio of the enzyme to lipid was 1:400-600. A sigmoid curve was obtained when the Cl--transport activity of the enzyme was plotted against Cl- concentration. Hill's plot afforded a half-subsrate concentration [S]0.5 of 45 mM and a Hill's coefficient n of 2.33. Effects of Br- and F- on the Cl--transport were also examined in the reconstituted system, both halide ions decrease the 36Cl- efflux significantly. These kinetic data are in good agreement with the electrophysiological data presented by Tittor et al. (1983) J. Membr. Biol. 75, 129-139). © 1992.

DOI： 10.1016/0005-2736(92)90235-E

Improvement of reconstitution of the Cl(-)-translocating ATPase isolated from Acetabularia acetabulum into liposomes and several anion pump characteristics. 査読国際誌

T Ohhashi, T Katsu, M Ikeda

Biochimica et biophysica acta 1106 ( 1 ) 165 - 70 1992年4月

記述言語：英語掲載種別：研究論文（学術雑誌）

The improved reconstitution of the Mono Q-III fraction, a Cl(-)-translocating ATPase, isolated from Acetabularia acetabulum (Ikeda et al. (1990) Biochemistry 29, 2057-2065) into liposomes rendered transport properties of this enzyme clear. The liposomes were prepared by the reversed-phase method using egg lecithin and cholesterol in a molar ratio of 2:1 and the purified ATPase was incorporated into the liposomes by a dialysis for 3 h. About 80% of the ATPase was incorporated into the liposomes. The weight ratio of the enzyme to lipid was 1:400-600. A sigmoid curve was obtained when the Cl(-)-transport activity of the enzyme was plotted against Cl- concentration. Hill's plot afforded a half-substrate concentration [S]0.5 of 45 mM and a Hill's coefficient n of 2.33. Effects of Br- and F- on the Cl(-)-transport were also examined in the reconstituted system, both halide ions decreased the 36Cl- efflux significantly. These kinetic data are in good agreement with the electrophysiological data presented by Tittor et al. ((1983) J. Membr. Biol. 75, 129-139).

Preparative high-yield electroelution of proteins after separation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and its application to analysis of amino acid sequences and to raise antibodies. 査読国際誌

T Ohhashi, C Moritani, H Andoh, S Satoh, S Ohmori, F Lottspeich, M Ikeda

Journal of chromatography 585 ( 1 ) 153 - 9 1991年10月

記述言語：英語掲載種別：研究論文（学術雑誌）

A method for the preparative high-yield electroelution of proteins from sodium dodecyl sulphate (SDS) polyacrylamide gel strips was established. The method consisted of SDS-polyacrylamide gel electrophoresis, detection of proteins with sodium acetate and electrophoretic elution at 200 V for 3 h by utilizing a horizontal flat-bed gel electrophoresis apparatus. Standard proteins with molecular masses of 14-66 kilodalton (cytochrome c, aldolase, ovalbumin and bovine serum albumin) were recovered with an average yield of 73.6 +/- 2.3%. A membrane-bound protein, rat skeletal muscle Ca(2+)-ATPase (100 kilodalton) was also well recovered (over 60%). This method was applicable to the purification of proteins required for N-terminal amino acid sequencing and to raise antibodies.

DOI： 10.1016/0021-9673(91)85069-R

Characterization of fibroblast-derived prolidase. The presence of two forms of prolidase. 査読国際誌

T Oono, H Yasutomi, T Ohhashi, H Kodama, J Arata

Journal of dermatological science 1 ( 5 ) 319 - 23 1990年9月

記述言語：英語掲載種別：研究論文（学術雑誌）

Crude enzyme solutions of prolidase were extracted from cultured human skin fibroblasts derived from control and prolidase-deficient sisters. Two forms of prolidases (prolidase-I and II) were partially purified by high performance liquid chromatography equipped with an ion exchange column. On gel filtration, the relative molecular weights of prolidase-I and II were estimated to be MW = 105,000 and 151,000, respectively. The substrate specificity of partially purified prolidase-I and II in control fibroblasts was estimated against Gly-Pro, Ala-Pro, Met-Pro. Each form of prolidase differed in its substrate specificity. In prolidase-deficient sisters, the elder with typical clinical manifestations and the younger with only slight clinical manifestations, the activity of prolidase-I was absent. However, the activity of prolidase-II was sufficiently present in both sisters. The substrate specificity of prolidase-II in the patients was similar to that of control. No difference in substrate specificity was found between these two patients.

Characterization of prolidase I and II from erythrocytes of a control, a patient with prolidase deficiency and her mother. 査読国際誌

T Ohhashi, T Ohno, J Arata, K Sugahara, H Kodama

Clinica chimica acta; international journal of clinical chemistry 187 ( 1 ) 1 - 9 1990年1月

記述言語：英語掲載種別：研究論文（学術雑誌）

Prolidase I (EC 3.4.13.9) was purified to homogeneity from the erythrocytes of a normal human (control) and the patient's mother, and prolidase II from erythrocytes of a control and the patient's mother, and prolidase from the patient's erythrocytes was also highly purified. The various properties of the patient's prolidase were compared to those of prolidase from a control and the patient's mother. Prolidase I from a control and the patient's mother had a molecular weight of about 112,000, and was composed of two subunits with an identical molecular weight of 56,000. The Km values for Gly-Pro of the control's and the patient's mother's prolidase I were 2.90 +/- 0.22 and 2.88 +/- 0.27 mM, but the Vmax values for Gly-Pro of the mother's enzyme was reduced about 30% compared to that of control enzymes (mother: 6.02 units/mg protein, control: 22.21 units/mg protein). Isoionic points of these enzymes by chromatofocusing were pH 4.6 approximately 4.7. Prolidase II from the control and the patient's mother, and the patient's prolidase had a molecular weight of about 185,000, and was composed of two subunits with an identical molecular weight of 95,000. The Km and Vmax values for various substrates of prolidase II from a control and the patient's mother, and the patient's prolidase were almost the same.

Characteristics and partial purification of prolidase and prolinase from leukocytes of a normal human and a patient with prolidase deficiency. 査読国際誌

H Kodama, T Ohhashi, C Ohba, T Ohno, J Arata, I Kubonishi, I Miyoshi

Clinica chimica acta; international journal of clinical chemistry 180 ( 1 ) 65 - 72 1989年3月

CHARACTERISTICS OF PARTIALLY PURIFIED PROLIDASE FROM ERYTHROCYTES OF NORMAL INDIVIDUALS, OF 2 PATIENTS WITH PROLIDASE DEFICIENCY, AND OF THE PATIENTS MOTHER 査読

T OHHASHI, T OHNO, J ARATA, H KODAMA

JOURNAL OF CLINICAL BIOCHEMISTRY AND NUTRITION 5 ( 3 ) 183 - 192 1988年11月

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