Amino-terminal Acid Research Articles

Oxidation of Natural and Thermal Denatured Bovine Serum Albumin Hydrazyl Free Radicals in the Presence of Cyclodextrins

The influence of certain thermal treatments on the reactivity of bovine serum albumin (denaturation followed by renaturation in three different cooling conditions) was studied monitoring the kinetics of oxidation of BSA with two water-soluble stable hydrazyl radicals. The results showed that the addition to the reaction mixture of α-cyclodextrin and β-cyclodextrin decreases the oxidation rate, probably due to the encapsulation of terminal amino acid rests by the cyclodextrins cavity. β-Cyclodextrin protects more efficiently the albumin probes than α-cyclodextrin. The denatured albumin probes are more reactive than natural albumin as a consequence of the reorienting of the hydrophobic rests of albumin molecule to their surface.

Characterization of particulate proteins in Pacific surface waters

We investigated molecular characteristics of particulate proteins in Pacific surface waters using two‐dimensional electrophoresis (2DE). Most proteinaceous materials estimated by dye‐binding methods were characterized by the 2DE unresolved acidic materials with a broad range of molecular mass and the 2DE unresolved low molecular mass materials with a broad range of isoelectric point. The 2DE unresolved acidic and low molecular mass materials were considered to comprise peptides conjugated with acidic saccharides and degradation products (peptides) of proteins, respectively, which indicates that almost all proteins in living organisms failed to survive in detrital particulate organic matter (POM). Nevertheless, 23 discrete proteins were distinguished by the 2DE. Electrophoretic patterns of the discrete proteins indicated that they were a component of detrital POM. Three discrete proteins were subjected to N terminal amino acid sequence analysis. Two proteins out of three could not be determined because their N termini were blocked, and one protein was determined from the N terminus to the ninth amino acid residue. A homology search revealed that the N terminal amino acid sequence of the protein agreed completely with that of 70 kDa heat shock protein (HSP70) derived from photosynthetic organisms. HSP70 is a major member of the molecular chaperones that protect or repair proteins from damage under conditions of environmental stress. The occurrence of HSP70 in this study demonstrated that phytoplankton were able to induce the molecular chaperone(s). Clarification of factor(s) controlling induction of chaperones will enable us to assess the actual environmental stress on phytoplankton at the biomolecular level.

A structure-activity study of the neuropeptide PF1, SDPNFLRFamide, using the dorsal body wall muscle of the chicken nematode, Ascaridia galli.

The action of a range of N terminally modified peptides structurally related to the nematode peptide PF1, SDPNFLRFamide, has been investigated using a dorsal muscle strip preparation from the chicken nematode, Ascaridia galli. Acetylcholine contracts this muscle preparation in a concentration-dependent manner when applied in the range 1-100 microM with an EC50 value of 9 microM. These contractions are reduced in the presence of PF1 and its analogues, with a threshold effect of PF1 of around 1 nM and an IC50 value of 470 nM against 10 microM acetylcholine. All the PF1 analogues tested were less potent than PF1 in reducing the acetylcholine contractions, indicating the importance of the N terminal amino acids in the action of PF1 in this preparation.

Vesicle transport in oligodendrocytes: probable role of Rab40c protein.

Intracellular membrane trafficking plays an essential role in the structural and functional organization of oligodendrocytes, which synthesize a large amount of membrane to form myelin. Rab proteins are key components in intracellular vesicular transport. We cloned a novel Rab protein from an oligodendrocyte cDNA library, designating it Rab40c because of its homology with Rab40a and Rab40b. The DNA sequence of Rab40c shows an 843-base pair open reading frame. The deduced amino acid sequence is a protein with 281 amino acids, with a molecular weight of 31,466 Da and an isoelectric point of 9.83. Rab40c presents a number of distinct structural features including a carboxyl terminal extension and amino acid substitutions in the consensus sequence of the GTP-binding motifs. The carboxyl terminal region contains motifs that permit isoprenylation and palmitoylation. Binding studies indicate that Rab40c binds guanosine 5'-0-(3-thiotriphosphate) (GTP gamma S) with a K(d) of 21 microM and has a higher affinity for guanosine triphosphate (GTP) than for guanosine diphosphate (GDP). Rab40c is localized in the perinuclear recycling compartment, suggesting its involvement in endocytic events such as receptor recycling. The importance of this recycling in myelin formation is suggested by the increase in both Rab40c mRNA and Rab40c protein as oligodendrocytes differentiate.

Syntheses, Structures, and Properties of Lanthanide Supramolecular Complexes with 1, 10-Phenanthroline and Terminal Amino Acids with Different Chain Lengths

Four lanthanide supramolecular coordination compounds, [Eu(gly)2(phen)2(H2O)2](ClO4)3(phen)4 · H2O (1), [Eu2(APA)6(phen)2](ClO4)6(phen)4 · 3H2O (2), [Tb2(ABA)4(phen)4](ClO4)6(phen)4 (3), and [Eu2(AHA)4(phen)4](ClO4)6(phen)2 · 2H2O · 2C2H5OH (4) (gly = glycine, APA = 3-aminopropionic acid, ABA = 4-aminobutanoic acid, AHA = 6-aminohexanoic acid, phen = 1, 10-phenanthroline), were synthesized and characterized by single crystal X-ray diffraction. Compound 1 has a 2-D supramolecular layered structure of mononuclear coordination cations and free phen molecules connected via hydrogen bonding and π-π stacking interactions. 2 forms a 3-D supramolecular network by hydrogen bonding between binuclear coordination cations and free phen molecules, between coordination cations and lattice water molecules, and π-π stacking interactions between free phen molecules. Compounds 3 and 4 form 2-D supramolecular structures with π-π stacking between coordinating phen molecules, and between free phen molecules hydrogen-bonded to the binuclear coordination cations. The high-resolution emission spectra show only one Eu3+ ion site in the title complexes. The aqueous solutions of the title complexes are all photochromic with the color of the solution changing from yellow to green when irradiated by mercury lamp. During the decoloration process, they return to yellow color. Synthesen, Strukturen und Eigenschaften von supramolekularen Lanthanid-Komplexen mit Phenanthrolin und endständigen Aminosäuren verschiedener Kettenlänge Vier supramolekulare Lanthanid-Koordinationsverbindungen [Eu(gly)2(phen)2(H2O)2](ClO4)3(phen)4 · H2O (1), [Eu2(APA)6(phen)2](ClO4)6(phen)4 · 3H2O (2), [Tb2(ABA)4(phen)4](ClO4)6(phen)4 (3) und [Eu2(AHA)4(phen)4](ClO4)6(phen)2 · 2H2O · 2C2H5OH (4) (gly = Glycin, APA = 3-Aminopropionsäure, ABA = 4-Aminobutansäure, AHA = 6-Aminohexansäure, phen = 1, 10-Phenanthrolin) wurden synthetisiert und durch Einkristall-Röntgenstrukturanalyse charakterisiert. 1 hat eine supramolekulare Schichtstruktur, die von einkernigen Kationen und freien Phenanthrolin-Molekülen durch Wasserstoffbrücken sowie π-π-Wechselwirkungen gebildet wird. 2 bildet ein dreidimensionales Netzwerk durch Wasserstoffbrückenbindungen zwischen zweikernigen Kationen und freien Phenanthrolinmolekülen, zwischen Kationen und Gitterwasser-Molekülen und durch π-π-Wechselwirkungen zwischen den freien Phenanthrolin-Molekülen. 3 and 4 bilden 2-D supramolekulare Strukturen durch π-π-Wechselwirkungen zwischen den koordinierten Phen-Molekülen und zwischen unkoordinierten Phen-Molekülen mit Wasserstoffbrückenbindungen zu den zweikernigen Kationen. Die hochaufgelösten Emissionsspektren zeigen, dass nur ein Eu3+-Ion im Titelkomplex vorliegt. Die wäßrigen Lösungen der Titelkomplexe ändern ihre Farbe von gelb nach grün bei Bestrahlung mit einer Quecksilberlampe und entfärben sich wieder nach gelb.

A new strategy for the preparation of peptide-targeted radiopharmaceuticals based on an fmoc-lysine-derived single amino acid chelate (SAAC). automated solid-phase synthesis, NMR characterization, and in vitro screening of fMLF(SAAC)G and fMLF[(SAAC-Re(CO)3)+]G.

A tridentate single amino acid chelate (SAAC) derived from N-alpha-Fmoc-l-lysine was incorporated within a short peptide sequence using an automated peptide synthesizer. Novel derivatives of the chemotactic peptide fMLF were prepared such that the SAAC and its Re complex were selectively placed between a terminal glycine amino acid and the targeting fMLF sequence. The products, which were synthesized in parallel, were characterized by mass spectrometry and multi-NMR spectroscopy. The latter technique demonstrated that the structures of the targeting portions of the peptides are the same in the SAAC and Re-SAAC derivatives. The affinities of the reported compounds for the formyl peptide receptor were subsequently determined using flow cytometry and were found to be comparable to that of the parent peptide. The results of this work demonstrate the feasibility and numerous benefits of using the SAAC system to prepare peptide-targeted Tc(I) and Re(I) radiopharmaceuticals.

N-terminal amino acid sequences of troponin T fragments, including 30 kDa one, produced during postmortem aging of bovine longissimus muscle

We have determined the amino (N)-terminal amino acid (AA) sequences of five troponin T (TnT) fragments produced during postmortem aging of bovine longissimus muscle. Western blot analysis showed that 32.1, 28.8, 27, and 25.8 kDa anti-fast-type TnT (fTnT)-positive fragments and a 31 kDa anti-slow-type TnT (sTnT)-positive fragment were present at 14 d postmortem. The N-terminal AA sequences of the 32.1, 28.8 (conventional 30 kDa), 27, and 25.8 kDa fragments were APPPPAEV, EVHEPEEK, EKPRPRLT, and APKIPEGE, respectively, and they were mapped to the N-terminal region of bovine fTnT isoforms. The N-terminal sequences of the 31 kDa fragment, EAPEEPEP, were mapped to the sTnT isoforms. These findings indicate that the two isoform types of fTnT predominantly expressed in the longissimus muscle are cleaved specifically at Glu 21–Ala 22 and Glu 15–Ala 16, His 37–Glu 38 and His 31–Glu 32, Glu 43–Glu 44 and Glu 37–Glu 38, and/or Thr 51–Ala 52 and Thr 45–Ala 46, respectively, and that a sTnT isoform is cleaved specifically at Glu 23–Glu 24.

Gene organization of human transporter associated with antigen processing-like (TAPL, ABCB9): analysis of alternative splicing variants and promoter activity

The gene organization of human TAPL (TAP-like, ABCB9) was determined. The TAPL gene consists of 12 exons including the first non-coding exon on human chromosome 12q23.34. Three alternative splicing variants of the 12th exon have been identified by 3 ′RACE using RNA from human cell lines and isolated lymphocytes. As expected from the similarity of the amino acid sequences of TAP1, TAP2, and TAPL, the intron insertion points in these three genes are essentially the same. However, the TAP2 and TAPL genes are closely related, since each has common non-coding exon and splicing isoforms. The novel splicing variants of TAPL termed 12B and 12C have shorter carboxyl terminal amino acid sequences than 12A, reportedly a conserved isoform in rodents and human. The proximal promoter region of the TAPL gene lacks a canonical TATA-box but contains several GC-box elements. The 60 bp upstream sequence containing two GC-boxes from the human TAPL transcriptional start site confers basal promoter activity.

Characters of dendritic poly( l-lysine) analogues with the terminal lysines replaced with arginines and histidines as gene carriers in vitro

The development of a non-viral gene delivery system into cells is an important key to realize the safe delivery of therapeutic genes without the side effects often pointed out for viral vectors. We have shown that dendritic poly( l-lysine) of the 6th generation (KG6) shows high transfection efficiency into several cultivated cells with low cytotoxicity. Here, to investigate the effect of substituting terminal cationic groups on the gene delivery into cells, we synthesized KGR6 and KGH6, in which terminal amino acids were replaced by arginines and histidines, respectively. DNA-binding analysis showed that KGR6 could bind to the plasmid DNA as strongly as KG6, whereas KGH6 showed decreased binding ability. KGR6 showed 3- to 12-fold higher transfection efficiency into several cultivated cells than KG6. In contrast, KGH6 showed no transfection efficiency. However, once KGH6 was mixed with the DNA under acidic conditions (pH 5.0), DNA-complexes were formed and they showed high transfection efficiency compared to that in KG6-mediated transfection. DNA-complexes of KGH6 formed under acidic conditions were 1–2 μm and spherical, and relatively stable under neutral conditions. The size and spherical shape of the complexes were the same as those of KG6. The unique character of KGH6 will be one of the basic and valuable tools which will enable us to construct a functional gene transfection system in vitro and in vivo.

Functional Importance of the Carboxyl-terminal Region of Striated Muscle Tropomyosin

Striated muscle tropomyosin (TM) interacts with actin and the troponin complex to regulate calcium-mediated muscle contraction. Previous work by our laboratory established that alpha- and beta-TM isoforms elicit physiological differences in sarcomeric performance. Heart myofilaments containing beta-TM exhibit an increased sensitivity to calcium that is associated with a decrease in the rate of relaxation and a prolonged time of relaxation. To address whether the carboxyl-terminal, troponin T binding domain of beta-TM is responsible for these physiological alterations, we exchanged the 27 terminal amino acids of alpha-TM (amino acids 258 -284) for the corresponding region in beta-TM. Hearts of transgenic mice that express this chimeric TM protein exhibit significant decreases in their rates of contraction and relaxation when assessed by ex vivo work-performing cardiac analyses. There are increases in the time to peak pressure and a dramatic increase in end diastolic pressure. In myofilaments, this chimeric protein induces depression of maximum tension and ATPase rate, together with a significant decrease in sensitivity to calcium. Our data are the first to demonstrate that the TM isoform-specific carboxyl terminus is a critical determinant of sarcomere performance and calcium sensitivity in both the whole heart and in isolated myofilaments.

Neoepitopes as biomarkers of cartilage catabolism.

Progressive degradation of articular cartilage is a central feature of arthritis and a major determinant of long term joint dysfunction. There are no treatments able to halt the progression of cartilage destruction presently available, and monitoring the benefit of potential therapies is hampered by our inability to measure the "health" of articular cartilage. Serial radiographic assessment of joint space narrowing, the current gold standard, requires measurements over a prolonged time (1-5 years) and is prone to technical difficulties. Other strategies for evaluating cartilage degradation are needed to enable both short and long term monitoring of disease progression and response to therapy. One avenue that holds promise is the use of biomarkers that accurately reflect the degradative state of the articular cartilage. Antibodies that recognise terminal amino acid sequences generated by proteolysis at specific sites in the core protein of both aggrecan and type II collagen (neoepitope antibodies) have become available in recent years. These antibodies have been invaluable for identifying the proteinases responsible for cartilage breakdown both in vitro and in vivo. The presence of neoepitope sequences generated by specific metalloenzyme cleavage of aggrecan and type II collagen correlates well with the progression of cartilage degeneration, both in vitro and in mouse models of arthritis. Preliminary results with quantitative assays of type II collagen neoepitopes suggest that they may be useful markers of joint disease in humans. Long term studies correlating neoepitope concentration with clinical and radiographic disease are now required to validate the utility of neoepitopes as surrogate markers of cartilage degeneration and joint disease.

Purification of Her-2 extracellular domain and identification of its cleavage site

The EGF family of receptors belongs to the tyrosine kinase receptor (TKR) family and plays an important role during embryonic and postnatal development and also in the progression of tumors. Her-2/neu/c-erbB-2, a member of the epidermal growth factor receptor family, can be cleaved into a soluble extra cellular domain (ECD) and a membrane-bound stub fragment. Her-2 ECD from a breast cancer cell line SKBR3 was immunopurified and analyzed with matrix-assisted laser desorption ionization (MALDI) and carboxyl terminal amino acid sequencing. A sequence within the juxtamembrane region (only 11 amino acid residues) P AEQR↓AS P was identified most likely as a primary site of cleavage, P A↓EQRAS P as a minor site, that generate the ECD. The sites of cleavage are within the signature motif P/GX 5–7P/G highly conserved in the EGF receptor family.

Determinants of proteasome recognition of ornithine decarboxylase, a ubiquitin-independent substrate.

Ornithine decarboxylase (ODC) is regulated by its metabolic products through a feedback loop that employs a second protein, antizyme 1 (AZ1). AZ1 accelerates the degradation of ODC by the proteasome. We used purified components to study the structural elements required for proteasomal recognition of this ubiquitin-independent substrate. Our results demonstrate that AZ1 acts on ODC to enhance the association of ODC with the proteasome, not the rate of its processing. Substrate-linked or free polyubiquitin chains compete for AZ1-stimulated degradation of ODC. ODC-AZ1 is therefore recognized by the same element(s) in the proteasome that mediate recognition of polyubiquitin chains. The 37 C-terminal amino acids of ODC harbor an AZ1-modulated recognition determinant. Within the ODC C terminus, three subsites are functionally distinguishable. The five terminal amino acids (ARINV, residues 457-461) collaborate with residue C441 to constitute one recognition element, and AZ1 collaborates with additional constituents of the ODC C terminus to generate a second recognition element.

An LKB1-interacting protein negatively regulates TNFalpha-induced NF-kappaB activation.

The Peutz-Jeghers syndrome (PJS) is a hereditary disorder that predisposes an individual to benign and malignant tumors in multiple organ systems. Recently, the locus responsible for PJS was mapped genetically to the LKB1 gene, with a subsequent investigation proving that it is responsible for most cases of PJS. LKB1 encodes a nuclear serine/threonine protein kinase, and potential tumor-suppressing activity has been attributed to LKB1 kinase. However, how LKB1 exerts its tumor-suppressing function remains to be determined. In this report, we describe the identification of a putative human LKB1-interacting protein, FLIP1, using the yeast two-hybrid system. Two regions of the LKB1 sequence have been determined to be crucial for the interaction with FLIP1. FLIP1 encodes a protein of 429 amino acids with a predicted molecular weight of 47 kd. In contrast to LKB1, which is mainly nuclear, FLIP1 is a cytoplasmic protein, and its expression is ubiquitous in all human tissues examined to date. Interestingly, deletion of the 195 N- terminal amino acids allows FLIP1 to enter the nucleus, suggesting the presence of a regulatory mechanism through its N-terminus for nuclear entry. In addition, we found that ectopic expression of FLIP1 selectively blocks cytokine-induced NF-kappaB activation. The involvement of FLIP1 in the regulation of NF-kappaB activity may shed new light on the role of LKB1 in tumor suppression.

An LKB1-Interacting Protein Negatively Regulates TNFα-Induced NF-κB Activation

The Peutz-Jeghers syndrome (PJS) is a hereditary disorder that predisposes an individual to benign and malignant tumors in multiple organ systems. Recently, the locus responsible for PJS was mapped genetically to the LKB1 gene, with a subsequent investigation proving that it is responsible for most cases of PJS. LKB1 encodes a nuclear serine/threonine protein kinase, and potential tumor-suppressing activity has been attributed to LKB1 kinase. However, how LKB1 exerts its tumor-suppressing function remains to be determined. In this report, we describe the identification of a putative human LKB1-interacting protein, FLIP1, using the yeast two-hybrid system. Two regions of the LKB1 sequence have been determined to be crucial for the interaction with FLIP1. FLIP1 encodes a protein of 429 amino acids with a predicted molecular weight of 47 kd. In contrast to LKB1, which is mainly nuclear, FLIP1 is a cytoplasmic protein, and its expression is ubiquitous in all human tissues examined to date. Interestingly, deletion of the 195 N- terminal amino acids allows FLIP1 to enter the nucleus, suggesting the presence of a regulatory mechanism through its N-terminus for nuclear entry. In addition, we found that ectopic expression of FLIP1 selectively blocks cytokine-induced NF-ĸB activation. The involvement of FLIP1 in the regulation of NF-ĸB activity may shed new light on the role of LKB1 in tumor suppression.

Identification, purification and partial characterization of tissue inhibitor of matrix metalloproteinase-2 in bovine pulmonary artery smooth muscle.

Bovine pulmonary artery smooth muscle possesses the tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) as revealed by Western immunoblot study of its cytosol fraction with bovine polyclonal TIMP-2 antibody. This potent polypeptide inhibitor of matrix metalloproteinases (MMPs) was purified to homogeneity from cytosol fraction of bovine pulmonary artery smooth muscle. This inhibitor was purified by ammonium sulfate precipitation followed by gelatin sepharose and lentil lectin sepharose affinity chromatography and continuous elution electrophoresis by Prep Cell Model 491 (Bio-Rad, USA). SDS-PAGE revealed that the inhibitor has an apparent molecular mass of 21 kDa and was confirmed as TIMP-2 by (i) Western immunoblot assay using bovine polyclonal TIMP-2 antibody; and also by (ii) amino terminal amino acid sequence analysis of the purified inhibitor is found to be identical with TIMP-2 obtained from other sources. The purified 21 kDa inhibitor was found to be active against matrix metalloproteinase-2 (MMP-2, 72 kDa gelatinase) and matrix metalloproteinase-9 (MMP-9, 92 kDa gelatinase), the ambient MMPs in the pulmonary artery smooth muscle. The inhibitor was also found to be sensitive to the activated 72 kDa gelatinase-TIMP-2 complex and also active human interstitial collagenase. By contrast, it was found to be insensitive to the serine proteases: trypsin and plasmin. The inhibitor was heat and acid resistant and it had the sensitivity to trypsin degradation and reduction-alkylation. Treatment of the inhibitor with hydrogen peroxide, superoxide generating system (hypoxanthine plus xanthine oxidase) and peroxynitrite inactivated the inhibitor.

Production of Two Types of Phytase from Aspergillus oryzae during Industrial Koji Making

In our previous study, it was determined that phytase produced by Aspergillus oryzae plays an important role in supplying phosphate to yeast in the process of making sake. During koji making, two types of phytase (Phy-I and Phy-II) are produced. The purified phytases have high thermal and pH stability, in comparison to phytase purified from a submerged culture (ACP-II). In the present study, Phy-I and Phy-II retained their activities for 45 h. The NH 2 -terminal sequence of Phy-1, which is eight amino acids in length, was identical to that of ACP-II, but the molecular weights of these two forms, as estimated by SDS-PAGE, were quite different from each other (Phy-I, 120 kDa; ACP-II, 58 kDa). From the NH 2 -terminal amino acid sequence analysis of the predominant phytase (Phy-II), a molecular weight of 116 kDa was expected to reflect a new type of phytase produced only in koji culture. The substrate specificity of Phy-II was sufficiently broad that it hydrolyzed not only phytic acid and p -nitro phenyl phosphate, but also glucose 6-phosphate and glycerol 1-phosphate. In the process of making koji, Phy-I was produced at an early stage, followed by Phy-II; with both phytases being thought to function to hydrolyze phytic acid cooperatively.

Fusion of GFP to the carboxyl terminus of connexin43 increases gap junction size in HeLa cells.

The pattern of gap junctional coupling between cells is thought to be important for the proper function of many types of tissues. At present, little is known about the molecular mechanisms that control the size and distribution of gap junctions. We addressed this issue by expressing connexin43 (Cx43) constructs in HeLa cells, a connexin-deficient cell line. HeLa cells expressing exogenously introduced wild-type Cx43 formed small, punctate gap junctions. By contrast, cells expressing Cx43-GFP formed large, sheet-like gap junctions. These results suggest that the GFP tag, which is fused to the carboxyl terminus of Cx43, alters gap junction size by masking the carboxyl terminal amino acids of Cx43 that comprise a zonula occludins-1 (ZO-1) binding site. We are currently testing this hypothesis using deletion and dominant-negative constructs that directly target the interaction between Cx43 and ZO-1.

Transaldolase/glucose-6-phosphate isomerase bifunctional enzyme and ribulokinase as factors to increase xylitol production from D-arabitol in Gluconobacter oxydans.

Xylitol production from D-arabitol by the membrane and soluble fractions of Gluconobacter oxydans was investigated. Two proteins in the soluble fraction were found to have the ability to increase xylitol production. Both of these xylitol-increasing factors were purified, and on the basis of their NH(2)-terminal amino acid sequences the genes encoding both of the factors were cloned. Expression of the cloned genes in Escherichia coli showed that one of the xylitol-increasing factors is the bifunctional enzyme transaldolase/glucose-6-phosphate isomerase, and the other is ribulokinase. Using membrane and soluble fractions of G. oxydans, 3.8 g/l of xylitol were produced from 10 g/l D-arabitol after incubation for 40 h, and addition of purified recombinant transaldolase/glucose-6-phosphate isomerase or ribulokinase increased xylitol to 5.4 g/l respectively, confirming the identity of the xylitol-increasing factors.

Purification and Characterization of Recombinant Bovine Cathepsin C.

Purification of recombinant cathepsin C (CTC) from the fermentation broth of methylotrophic yeast, Candida boidinii was conducted. After centrifugation, fermentation supernatant was processed with four consecutive chromatography columns (cation exchange, hydrophobic, anion exchange, and gel filtration). As a result, the recombinant CTC of 4.7 U/mg was purified from the fermentation broth containing 169 U/l of CTC and the yield was 35%. From the gel filtration analysis, molecular weight of the recombinant CTC was determined to be about 200 kDa, that was almost the same as the native one. From SDS-PAGE analysis and N- terminal amino acid analysis, the monomer bands of the recombinant CTC were discussed. In order to compare the enzymatic property of the recombinant and native CTC, (1) pH profile of the activities, (2) temperature profile of the activities, and (3) enzymatic specificity for several substrates were investigated. From these studies, it was revealed that the enzymatic property of the recombinant CTC was almost the same as that of the native one.