Redox and metal-regulated oligomeric state for human porphobilinogen synthase activation.

The oligomeric state of human porphobilinogen synthase (PBGS) [EC.4.2.1.24] is homooctamer, which consists of conformationally heterogenous subunits in the tertiary structure under air-saturated conditions. When PBGS is activated by reducing agent with zinc ion, a reservoir zinc ion coordinated by Cys(223) is transferred in the active center to be coordinated by Cys(122), Cys(124), and Cys(132) (Sawada et al. in J Biol Inorg Chem 10:199-207, 2005). The latter zinc ion serves as an electrophilic catalysis. In this study, we investigated a conformational change associated with the PBGS activation by reducing agent and zinc ion using analytical ultracentrifugation, negative staining electron microscopy, native PAGE, and enzyme activity staining. The results are in good agreement with our notion that the main component of PBGS is octamer with a few percent of hexamer and that the octamer changes spatial subunit arrangement upon reduction and further addition of zinc ion, accompanying decrease in f/f (0). It is concluded that redox-regulated PBGS activation via cleavage of disulfide bonds among Cys(122), Cys(124), and Cys(132) and coordination with zinc ion is closely linked to change in the oligomeric state.

Intermolecular disulfide bond to modulate protein function as a redox-sensing switch.

Recently, redox-regulated biological reactions have been elucidated. In the regulation of these reactions, redox-sensing molecular switches function as unique biological machineries that modulate the functional proteins present in enzymes, transcriptional factors, sensor proteins, and transcriptional factor modulators. The redox-sensing cysteine residues and the disulfide bond formed between these cysteine residues serve as redox-sensing molecular switches; these switches sense cellular oxidizing factors such as oxygen, reactive oxygen species, and cellular reducing factors such as thioredoxin (Trx), glutathione (GSH), and their family molecules. Depending on the redox status, the switch directly modulates the protein function via the "locking and unlocking" of the critically functional residue or indirectly modulates the protein function via "protein conformational changes," which affects the functioning of a distantly located critical residue in an allostery-like fashion or a topology change. Redox-sensing switches can be classified into two types-intramolecular (intrasubunit) and intermolecular (intersubunit) ones. Further, depending on the sensing specificity to reducing factors, the switch subtype is classified into Trx, GSH, or their family molecules-specific type. This review focused on the intermolecular redox-sensing switches found in various proteins.

Protein cysteine modifications: (1) medical chemistry for proteomics.

Protein cysteines (cysteinyl residues) play critical roles in biological processes. In the course of protein evolution under oxidizing atmosphere of the Earth, organisms have utilized highly reactive cysteines in many proteins essential for maintenance of life, i.e. enzymes, transcriptional factors, cytoskeletons, and receptors. In some enzymes, sophistical cysteine modification characterizes each catalytic mechanism. In vivo modification of protein cysteines with natural chemical compounds modulates protein functions as a molecular switch. Oxidation/reduction, thiol-disulfide exchange, nitrosylation, sulfuration, thiolation, acylation and prenylation are involved. Some protein cysteines coordinate metals or metal cofactors such as a heme or an iron sulfur cluster to form metalloproteins, serving as sensor proteins, metalloenzymes or transcriptional factors. Information on the in vitro chemical modifications and their reaction specificities of protein cysteines are essential for the investigation of the mechanisms and functions of in vivo protein cysteine modifications. In this review, we also mention historically important knowledge other than recent results on protein cysteine modification and modulation of protein function to fertilize medical proteomics.

New aspects in pathogenesis of konzo: neural cell damage directly caused by linamarin contained in cassava (Manihot esculenta Crantz).

Epidemic spastic paraparesis (konzo) found in tropical and subtropical countries is known to be caused by long-term intake of cassava (Manihot esculenta Crantz), which contains a cyanoglucoside linamarin (alpha-hydroxyisobutyronitrile-beta-d-glucopyranoside). It has been reported that linamarin is enzymatically converted to cyanide by bacteria in the intestine, and this is absorbed into the blood and then damages neural cells. However, unmetabolized linamarin was found in the urine after oral administration of cassava; thus, we hypothesized that konzo could be caused by direct toxicity of the unmetabolized linamarin that was transferred to the brain and could be transported into neural cells via a glucose transporter. In the present study it was confirmed that linamarin directly damaged neural culture pheochromocytoma cell (PC) 12 cells; 0.10 mm-linamarin caused cell death at 13.31 (SD 2.07) %, which was significantly different from that of control group (3.18 (SD 0.92) %, P=0.0004). Additional 10 microM-cytochalasin B, an inhibitor of a glucose transporter, prevented cell death: the percentage of dead cells significantly decreased to 6.06 (SD 1.98), P=0.0088). Furthermore, glucose also prevented cell death. These present results strongly suggest that linamarin competes with cytochalasin B and glucose for binding to a glucose transporter and enters into cells via glucose transporter.

Outcome of hepatoblastoma treated with the JPLT-1 (Japanese Study Group for Pediatric Liver Tumor) Protocol-1: A report from the Japanese Study Group for Pediatric Liver Tumor.

PURPOSE: Hepatoblastoma is the most common malignant liver tumor in childhood. Multicenter studies elucidate the optimal pre- or postoperative chemotherapeutic regimens. This report reviews the results of the Japanese Study Group for Pediatric Liver Tumor Protocol-1 (JPLT-1) and compares its outcomes with published reports of other studies. METHODS: From March 1991 to December 1999, 154 patients with malignant liver tumor including 145 cases of hepatoblastomas were enrolled in the JPLT study. Data from 134 cases were analyzed in this study. JPLT-1 protocol 91A was used for patients with stage I or II hepatoblastoma. The chemotherapy regimen consisted of repeated courses of cisplatin (CDDP), 40 mg/m(2), and tetrahydropyranyl (THP)-Adriamycin, 30 mg/m(2). JPLT-1 protocol 91B was administered to patients with stage IIIA, IIIB, or IV hepatoblastoma. The chemotherapy regimen consisted of repeated courses of CDDP, 80 mg/m(2), and THP-Adriamycin, 30 mg/m(2)/day for 2 days. Courses were repeated every 4 weeks as tolerated. RESULTS: Seven patients died of chemotherapy-related side effects. Six of them died of sepsis caused by leukopenia and 1 case of liver failure. Overall survival rate (3-year/6-year) was 100%/100% for stage I (n = 9), 100%/95.7% for stage II (n = 32), 76.6%/73.8% for stage IIIA (n = 48), 50.3%/50.3% for stage IIIB (n = 25), 64.8%/38.9% for stage IV (n = 20), and 77.8%/73.4% overall. For stage IIIA and B disease, intravenous chemotherapy was better than intraarterial chemotherapy (66.4% v 38.1% for event-free survival and 69.3% v. 57.1% for overall survival). Patients less than 1 year of age had a better prognosis than older patients, but age was not a significant prognostic factor by multivariate analysis. CONCLUSIONS: The overall and event-free survival rates of the JPLT-1 study of hepatoblastoma were comparable with the results of other multicenter studies in Europe and the United States. The event-free survival rate at 3 years for stage IIIB and IV disease was under 50%. New treatment strategies are needed for patients with advanced hepatoblastoma.

Frequent beta-catenin mutation and cytoplasmic/nuclear accumulation in pancreatic solid-pseudopapillary neoplasm.

Significance of Wnt signaling with beta-catenin mutations on solid-pseudopapillary neoplasm (SPN) of the pancreas was studied by immunohistochemistry and molecular analysis. On immunohistochemistry, all 18 SPNs tested showed diffuse cytoplasmic/nuclear positivity for beta-catenin. Upon direct DNA sequencing of exon 3 of the beta-catenin gene, 15 (83%) of the 18 SPNs showed 1-bp missense mutation in codons 32 (5 cases), 33 (3 cases), 34 (3 cases), 37 (3 cases), and 41 (1 case). Immunoreactivity for cyclin D1, one of the intranuclear targets of beta-catenin complexes, was found in tumor cells of more than half the tumor cells of all of the 18 SPNs. The present study strongly suggested a significant role of Wnt signaling, mostly associated with beta-catenin mutations in the tumorigenesis of SPN.

Ewing's sarcoma family of tumor arising in the adrenal gland--possible diagnostic pitfall in pediatric pathology: histologic, immunohistochemical, ultrastructural, and molecular study.

We present an adrenal Ewing's sarcoma family of tumor (ESFT) arising in an 11-year-old Japanese boy. Although intensive chemoradiotherapy and radical surgery were performed, the patient died of obstinate disease 1 year and 3 months after the initial presentation. The primary site (adrenal gland) with radiologic findings (with foci of calcification), high titer of serum neuron specific enolase, and sheets of monotonous primitive rounded cells on histology mostly favored neuroblastoma. However, a diagnosis of ESFT was confirmed by immunohistochemical profile, including MIC2-positivity and molecular study disclosing EWS-FLI1 chimera gene verified by direct sequencing. Recognition of adrenal ESFT and use of newly developed diagnostic techniques are required for differential diagnosis of undifferentiated small round cell tumor of the adrenal gland.

Gastrectomized patients are in a state of chronic protein malnutrition analyses of 23 amino acids.

BACKGROUND/AIMS: Malnutrition is one of the major postoperative complications of radical subtotal or total gastrectomy for gastric cancer. This study was conducted to clarify the nutritional consequences of radical gastrectomy with respect to protein metabolism. METHODOLOGY: To evaluate the nutritional status and the abnormalities in protein metabolism in such cases, serum concentrations of 23 amino acids were measured by high performance liquid chromatography in 40 patients who had undergone either subtotal (n = 20) or total (n = 20) gastrectomy more than 6 months prior to this analysis. RESULTS: Serum concentrations of total amino acids and nonessential amino acids were the same between gastrectomized patients and healthy controls (n = 50). However, concentrations of essential amino acids, essential amino acid/nonessential amino acid and branched-chain amino acid/total amino acid ratios were significantly lower in patient groups than in normal controls. Each essential amino acid was decreased and concentrations of glutamate and citrulline were increased in both patient groups compared with controls. The major differences between patients with subtotal and total gastrectomies included an increased ornithine and a decreased arginine concentration in patients with subtotal gastrectomy. CONCLUSIONS: These changes suggest that malabsorption of protein from the intestinal tract causes persistent proteolysis in the skeletal muscle for long periods of time after surgery in these patients and that changes in ornithine and citrulline levels may reflect more severe alterations in those with total gastrectomy.

Mercaptopyruvate sulfurtransferase as a defense against cyanide toxication: molecular properties and mode of detoxification.

In cyanide poisoning, metalloproteins and carbonyl groups containing proteins are the main target molecules of nucleophilic attack by cyanide. To defend against this attack, cyanide is metabolized to less toxic thiocyanate via transsulfuration. This reaction is catalyzed by rhodanese and mercaptopyruvate sulfurtransferase (MST). Rhodanese is a well characterized mitochondrial enzyme. On the other hand, little was known about MST because it was unstable and difficult to purify. We first purified MST to homogeneity and cloned MST cDNA from rat liver to characterize MST. We also found that MST was an evolutionarily related enzyme of rhodanese. MST and rhodanese are widely distributed in rat tissues, and the kidney and liver prominently contain these enzymes. Immunohistochemical study revealed that MST is mainly distributed in proximal tubular epithelial cells in the kidney, pericentral hepatocytes in the liver, the perinuclear area of myocardial cells in the heart, and glial cells in the brain, and immunoelectron microscopical study concluded that MST was distributed in both cytoplasm and mitochondria, so that MST first detoxifies cyanide in cytoplasm and the cyanide which escapes from catalysis due to MST enters mitochondria. MST then detoxifies cyanide again in cooperation with rhodanese in mitochondria. Tissues other than the liver and kidney are more susceptible to cyanide toxicity because they contain less MST and rhodanese. Even in the same tissue, sensitivity to cyanide toxicity may differ according to the kind of cell. It is determined by a balance between the amount of proteins to be attacked and that of enzymes to defend.

Crystal structure of a multifunctional 2-Cys peroxiredoxin heme-binding protein 23 kDa/proliferation-associated gene product.

Heme-binding protein 23 kDa (HBP23), a rat isoform of human proliferation-associated gene product (PAG), is a member of the peroxiredoxin family of peroxidases, having two conserved cysteine residues. Recent biochemical studies have shown that HBP23/PAG is an oxidative stress-induced and proliferation-coupled multifunctional protein that exhibits specific bindings to c-Abl protein tyrosine kinase and heme, as well as a peroxidase activity. A 2.6-A resolution crystal structure of rat HBP23 in oxidized form revealed an unusual dimer structure in which the active residue Cys-52 forms a disulfide bond with conserved Cys-173 from another subunit by C-terminal tail swapping. The active site is largely hydrophobic with partially exposed Cys-173, suggesting a reduction mechanism of oxidized HBP23 by thioredoxin. Thus, the unusual cysteine disulfide bond is involved in peroxidation catalysis by using thioredoxin as the source of reducing equivalents. The structure also provides a clue to possible interaction surfaces for c-Abl and heme. Several significant structural differences have been found from a 1-Cys peroxiredoxin, ORF6, which lacks the C-terminal conserved cysteine corresponding to Cys-173 of HBP23.

Treatment results of advanced neuroblastoma with the first Japanese study group protocol. Study Group of Japan for Treatment of Advanced Neuroblastoma.

PURPOSE: To elucidate the efficacy of intensive induction and consolidation chemotherapy regimens (Study Group of Japan for Advanced Neuroblastoma [JANB] 85) for patients with advanced neuroblastoma aged 1 year or older. PATIENT AND METHODS: One hundred fifty-seven patients with newly diagnosed advanced neuroblastoma were entered into this study between January 1985 and December 1990. Eligible patients were 12 months old or older with stage III or IV disease. The patients first received six cyclic courses of intensive induction chemotherapy (designated regimen A1) consisting of cyclophosphamide (1,200 mg/m2), vincristine (1.5 mg/m2), tetrahydro-pyranyl Adriamycin (pirarubicin; 40 mg/m2), and cisplatin (90 mg/m2). The patients were further treated with three different consolidation protocols: 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-1-(2-chloroethyl)-1-nitrosour ea, dacarbazine, and bone marrow transplantation. RESULTS: Overall survival rates for patients with stage III disease without reference to the consolidation protocols were 80.8%, 76.9%, and 66.3% at 2, 5, and 10 years, respectively. The overall survival rates for patients with stage IV disease were 58.8%, 34.4%, and 28.9% at 2, 5, and 10 years, respectively. There were no statistically significant differences between the three consolidation treatment groups. Patients who did not achieve complete remission (CR) with induction chemotherapy and surgery all died, suggesting that CR is essential for the cure of advanced neuroblastoma. The overall 5-year survival rate of the 24 patients with N-myc amplified stage III and IV disease was 33.3%, and the longest survival time of a relapse-free patient was 103 months. CONCLUSION: The intensive induction chemotherapy regimen used in this study may be of significant value in increasing the CR rate and survival for patients with N-myc amplified and nonamplified advanced neuroblastoma.

Crystallographic characterization of a stress-induced multifunctional protein, rat HBP-23.

HBP-23 is a stress-induced multifunctional rat protein that belongs to a novel family of antioxidant proteins, referred to as peroxiredoxins, and exhibits heme-binding and inhibition of c-Abl protein tyrosine kinase. Recombinant HBP-23 was crystallized by a hanging-drop vapor-diffusion method. The crystals belong to space group P41212 or P43212 with unit-cell dimensions of a = b = 73.47 A, c = 210.37 A and contain two protein molecules in the asymmetric unit. A data set at 2.7-A resolution was collected with a cryo-crystallographic technique. Crystals of selenomethionyl HBP-23 were also obtained under the same conditions.

Mucoadhesive microspheres containing amoxicillin for clearance of Helicobacter pylori.

In an effort to augment the anti-Helicobacter pylori effect of amoxicillin, mucoadhesive microspheres, which have the ability to reside in the gastrointestinal tract for an extended period, were prepared. The microspheres contained the antimicrobial agent and an adhesive polymer (carboxyvinyl polymer) powder dispersed in waxy hydrogenated castor oil. The percentage of amoxicillin remaining in the stomach both 2 and 4 h after oral administration of the mucoadhesive microspheres to Mongolian gerbils under fed conditions was about three times higher than that after administration in the form of a 0.5% methylcellulose suspension. The in vivo clearance of H. pylori following oral administration of the mucoadhesive microspheres and the 0.5% methylcellulose suspension to infected Mongolian gerbils was examined under fed conditions. The mucoadhesive microspheres and the 0.5% methylcellulose suspension both showed anti-H. pylori effects in this experimental model of infection, but the required dose of amoxicillin was effectively reduced by a factor of 10 when the mucoadhesive microspheres were used. In conclusion, the mucoadhesive microspheres more effectively cleared H. pylori from the gastrointestinal tract than the 0.5% methylcellulose suspension due to the prolonged gastrointestinal residence time resulting from mucoadhesion. A dosage form consisting of mucoadhesive microspheres containing an appropriate antimicrobial agent should be useful for the eradication of H. pylori.

Tissue and subcellular distribution of mercaptopyruvate sulfurtransferase in the rat: confocal laser fluorescence and immunoelectron microscopic studies combined with biochemical analysis.

In our previous study, we found that mercaptopyruvate sulfurtransferase (MST) was evolutionarily related to mitochondrial rhodanese. To elucidate the difference between MST and rhodanese, the tissue, cellular, and subcellular distribution of rat MST was determined biochemically and immunohistochemically by using anti-MST antibody raised in rabbit. In an immunohistochemical study, tetramethyl rhodamine isothiocyanate-conjugated phalloidin against F-actin and fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin as a secondary antibody to the anti-MST antibody were used for double fluorescent staining. They were detected by confocal laser fluorescence microscopy. In the immunoelectron microscopic study of hepatocyte and renal tubular epithelium, a postembedding immunogold method was used. Biochemical studies including western blot analyses of various tissues and subcellular fractions of the liver were also performed. MST was widely distributed in rat tissues but the cellular distribution was found to be different in each tissue. MST was predominantly localized in proximal tubular epithelium in the kidney, pericentral hepatocytes in the liver, cardiac cells in the heart, and neuroglial cells in the brain. This immunocytochemical study also found that MST was localized in both mitochondria and cytoplasm.

Evaluation of oral mucoadhesive microspheres in man on the basis of the pharmacokinetics of furosemide and riboflavin, compounds with limited gastrointestinal absorption sites.

When sustained-release adhesive and non-adhesive microspheres which release the same drugs at similar rates are administered orally, drug absorption after administration of adhesive microspheres should, if the gastrointestinal residence of adhesive microspheres is prolonged as a result of mucoadhesion, be higher than that after administration of non-adhesive microspheres. The gastrointestinal transit of oral adhesive microspheres in man has been evaluated pharmacokinetically using furosemide and riboflavin, compounds with limited absorption sites in the upper small intestine. In a preliminary experiment with fasted rats it was confirmed that a higher percentage of the drug remained in the stomach and that plasma drug levels were higher when furosemide was administered in the form of adhesive rather than non-adhesive microspheres. Two kinds of sustained-release microsphere, adhesive and non-adhesive, containing furosemide and riboflavin in hard gelatin capsules were prepared and orally administered to 10 healthy fasted volunteers in a cross-over design. Areas under the plasma concentration-time curves (AUC) were 1.8 times larger for furosemide and urinary recovery was 2.4 times higher for riboflavin when adhesive microspheres rather than when non-adhesive microspheres were used. When adhesive microspheres containing riboflavin were administered to fed volunteers, urinary recovery was 2.1 times higher and mean residence time (MRT) was more prolonged than when the microspheres were administered to fasted volunteers. Adhesive microspheres were found to adhere to the gastric or intestinal mucosa with high affinity in man and rats, resulting in prolonged gastrointestinal residence.

Evaluation of patients with advanced neuroblastoma surviving more than 5 years after initiation of an intensive Japanese protocol: a report from the Study Group of Japan for Treatment of Advanced Neuroblastoma.

In January 1985, a single protocol consisting of cyclophosphamide, vincristine, tetrahydropyranyl adriamycin, and cis-platinum for the treatment of advanced neuroblastoma was begun nationwide in Japan and was found to improve clinical results significantly in terms of 2- or 3-year survival rate. Between January 1985 and December 1988, 113 eligible patients (7 infants younger than 12 months of age with stage IVA disease and 106 patients aged 12 months or older with stage III or IV disease) were enrolled and followed up for 5 years or more after initiation of treatment, as of March 1994. In this study, the usefulness of the protocol for the treatment of advanced neuroblastoma was evaluated with survival rates in relation to age, tumor site, stage, and N-myc amplification for patients surviving more than 5 years after initiation of the protocol. Fifty of the 113 patients were alive 5 years or more after initiation of the treatment, 39 without any episodes of disease recurrence. Fourteen (70%) of 20 patients with stage III, 6 (50%) of 12 with stage IVB, and 24 (30%) of 81 with stage IVA disease were alive and disease-free 5 years after initiation of the protocol. Twenty (56%) of 36 patients without N-myc amplification were alive at 5 years after initiation of the protocol. Only one patient who was alive without evidence of the disease at 5 years had recurrence afterward.

Role of amino acid residues in the active site of rat liver mercaptopyruvate sulfurtransferase. CDNA cloning, overexpression, and site-directed mutagenesis.

A complete amino acid structure of rat liver mercaptopyruvate sulfurtransferase (MST, EC 2.8.1.2) was determined by sequence analysis of cDNA and purified enzyme. The enzyme consists of 296 amino acid residues with a calculated molecular mass of 32,808 Da. Sequence identity in cDNA and the deduced amino acid sequence are 65 and 60% respectively, between rat MST and rhodanese. By their entire sequence similarity MST and rhodanese are confirmed to be evolutionarily related enzymes (Nagahara, N., Okazaki, T., and Nishino, T. (1995) J. Biol. Chem. 270, 16230-16235). The conversion of MST to rhodanese was attempted, and the role of amino acid residues was studied by site-directed mutagenesis with the isolated cDNA of rat liver MST. There is a strong possibility that Cys247 is a catalytic site of MST. Arg187 is suggested to be a binding site of both mercaptopyruvate and thiosulfate in MST. Arg196, which is missed in rhodanese, is important for catalysis in MST. On the other hand, the substitution of Arg for Gly248 or Lys for Ser249 facilitates catalysis of thiosulfate in MST. It is concluded that Arg187 and Arg196 of rat MST are critical residues in determining substrate specificity for mercaptopyruvate. On the other hand, Arg185, Arg247, and Lys248 of rat rhodanese are critical residues in determining substrate specificity for thiosulfate.

Unfavorable DNA ploidy and Ha-ras p21 findings in neuroblastomas detected through mass screening.

BACKGROUND: Urinary mass screening has been available for 6-month-old infants throughout Japan since 1985. It is still controversial as to whether the program contributes to the detection of unfavorable neuroblastomas destined to present clinically when a patient reaches an older age. DNA diploidy and tetraploidy, low expression of Ha-ras p21, and an amplified N-myc gene status relate to an unfavorable prognosis. The authors examined these biologic indicators in neuroblastomas detected by urinary mass screening. PATIENTS AND METHODS: Seventy-eight neuroblastomas detected by mass screening were studied for DNA ploidy using DNA flow cytometry, Ha-ras p21 expression using immunostaining, and N-myc gene copy number using slot-blot or Southern blot hybridization methods. RESULTS: Of 73 tumors with analyzable DNA flow cytometric results, 18 (24.7%) had diploidy (n = 7) or tetraploidy (n = 11). Twenty-eight (40.0%) of 70 tumors examined showed low-to-absent expression of Ha-ras p21. DNA diploid and tetraploid status correlated significantly with the low-to-absent expression of Ha-ras p21 (P = 0.00021). Fourteen (20.0%) of the 70 patients had both of these two unfavorable prognostic markers. N-myc amplification was not detected in 41 of 41 tumors studied. All 78 patients were alive 8-92 months after completion of treatment. CONCLUSIONS: At least 20.0% of neuroblastomas detected by mass screening have unfavorable biologic prognostic markers. These patients may benefit from early detection and immediate treatment. However, the biologic features associated with a poor prognosis are not predictive of poor outcome in individual patients, and, therefore, should not be used to justify more intensive therapies.

Cytosolic mercaptopyruvate sulfurtransferase is evolutionarily related to mitochondrial rhodanese. Striking similarity in active site amino acid sequence and the increase in the mercaptopyruvate sulfurtransferase activity of rhodanese by site-directed mutagenesis.

Rat liver mercaptopyruvate sulfurtransferase (MST) was purified to homogeneity. MST is very similar to rhodanese in physicochemical properties. Further, rhodanese cross-reacts with anti-MST antibody. Both purified authentic MST and expressed rhodanese possess MST and rhodanese activities, although the ratio of rhodanese to MST activity is low in MST and high in rhodanese. In order to compare the active site regions of MST and rhodanese, the primary structure of a possible active site region of MST was determined. The sequence showed 66% homology with that of rat liver rhodanese. An active site cysteine residue (Cys246; site of formation of persulfide in catalysis) and an arginine residue (Arg185; substrate binding site) in rhodanese were also conserved in MST. On the other hand, two other active site residues (Arg247 and Lys248) were replaced by Gly and Ser, respectively. Conversion of rhodanese to MST was tried by site-directed mutagenesis. After cloning of rat liver rhodanese, recombinant wild type and three mutants (Arg247-->Gly and/or Lys248-->Ser) were constructed. The enzymes were expressed in Escherichia coli strain BL21 (DE3) with a T7 promoter system. The mutation of these residues decreases rhodanese activity and increases MST activity.