論文 - 田村　悦臣

Effects of gonadal hormones on the zonal expression of rat hepatic phenol sulfotransferases

Matsui M., Nakamura T., Tada M., Yamagata S., Tamura H., Homma H.

Biological and Pharmaceutical Bulletin （Biological and Pharmaceutical Bulletin）  21 （ 2 ） 97 - 101 1998年

研究論文（学術雑誌）,  ISSN  09186158

　概要を見る

Estradiol benzoate (EB) and testosterone propionate (TP) were administered to male and female Wistar rats, and their effects on the zonal expression of hepatic phenol sulfotransferase (P-ST) activities were determined at pH 5.5 and 7.4. Cytosolic fractions from periportal (PP) and perivenous (PV) hepatocytes were prepared by the dual-digitonin-pulse perfusion technique. In control rats, P-ST activities assayed at pH 5.5 and 7.4 were higher in males than in females, and were higher in the PV fraction than in the PP fraction in both sexes. P-ST activities were increased by the administration of TP in the PP and PV fractions of females, whereas the same treatment diminished the enzyme activities in both fractions of the males. EB administration gave reduced P-ST activity at pH 5.5 of both fractions, irrespective of sex, but not a marked difference at pH 7.4. Chromatofocusing of PP and PV fractions revealed the presence of P-ST isoforms eluted at approx. pH 8.0 (peak 1), 7.5 (peak 2), 7.0 (peak 3) and 6.0 (peak 4). In male rats, peak 3, which showed high enzyme activity at pH 5.5 in the PP and PV fractions, was markedly decreased by EB treatment, whereas in females, peak 3 was present only in the PV fraction and was not affected by EB administration. TP treatment did not show remarkable changes in P-ST peaks in the males, while peaks 2 and 3 were increased in the females. Immunoblot analysis revealed the presence of multiple P-ST isoforms which showed different immunoreactivity and electrochemical properties.

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Mg²⁺ binding and catalytic function of Sphingomyelinase from Bacillus cereus

Fujii S., Inoue B., Yamamoto H., Ogata K., Shinki T., Inoue S., Tomita M., Tamura H., Tsukamoto K., Ikezawa H., Ikeda K.

Journal of Biochemistry （Journal of Biochemistry）  124 （ 6 ） 1178 - 1187 1998年

研究論文（学術雑誌）,  ISSN  0021924X

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The modes of Mg2+ binding to SMase from Bacillus cereus were studied on the basis of the changes in the tryptophyl fluorescence intensity. This enzyme was shown to possess at least two binding sites for Mg2+ with low and high affinities. The effects of Mg2+ binding on the enzymatic activity and structural stability of the enzyme molecule were also studied. The results indicated that the binding of Mg2+ to the low-affinity site was essential for the catalysis, but was independent of the substrate binding to the enzyme. It was also indicated that the alkaline denaturation of the enzyme was partly prevented by the Mg2+ binding, whereas no significant protective effect was observed against the denaturation by urea. The pH dependence of the kinetic parameters for the hydrolysis of micellar HNP and mixed micellar SM with Triton X-100 (1:10), catalyzed by SMase from B. cereus, was studied in the presence of a large amount of Mg2+ to saturate both the low- and high-affinity sites. The pH dependence curves of the logarithm of 1/K(m) for these two kinds of substrates were similar in shape to each other, and showed a single transition. On the other hand, the shapes of the pH dependence curves of the logarithm of k(cat) for these two kinds of substrates were different from each other. The pH dependence curve for micellar HNP showed three transitions and, counting from the acidic end of the pH region, the first and third transitions having tangent lines with slopes of +1 and -1, respectively. On the other hand, the curve for mixed micellar SM with Triton X-100 showed one large transition with a slope of +1 (the first transition) and a very small transition (the third transition). On the basis of the present results and the three-dimensional structure of bovine pancreatic DNase I, which has a primary structure similar to that of B. cereus SMase, we proposed a catalytic mechanism for B. cereus SMase based on general-base catalysis.

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Construction and expression of chimeric enzymes of rat liver hydroxysteroid sulfotransferase isozymes.

田村 悦臣

Arch Biochem Biophys 341 （ 2 ） 309 - 314 1997年05月

研究論文（学術雑誌）, 共著, 査読有り

Construction and expression of chimeric rat liver hydroxysteroid sulfotransferase isozymes

Tamura H., Morioka Y., Homma H., Matsui M.

Archives of Biochemistry and Biophysics （Archives of Biochemistry and Biophysics）  341 （ 2 ） 309 - 314 1997年05月

研究論文（学術雑誌）,  ISSN  00039861

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The ST-20 and ST-40 cDNAs encode rat liver hydroxysteroid sulfotransferases (HS-ST) that are 90% identical in amino acid sequence but exhibit different substrate preferences for dehydroepiandrosterone (DHEA), androsterone (AD), and cortisol (CS), ST-40 is active for all three substrates, whereas ST-20 is mainly active for cortisol. To determine the domain responsible for the substrate preferences of the HS-STs, 20 chimeric HS-STs were constructed by reciprocal exchanges of DNA fragments derived from the cDNAs and were expressed in Escherichia coli. Some chimeric enzymes were enzymatically active for all three substrates, and some displayed reduced or lost CS-ST activity, with retention of DHEA- and AD-ST activities. Others lost all HS-ST activity. Analysis revealed that a central region (region III spanning amino acids 102164 with five amino acid differences between ST-20 and ST-40) is essential for HS-ST activity, whereas regions II (amino acids 65-101) and IV (amino acids 165219) are unimportant with regard to substrate preference. It was also shown that the parental combination of regions I (amino acids 1-64) and V (amino acids 220-284) is essential for CS-ST activity. Photoaffinity labeling with [35S]3'-phosphoadenosine 5'- phosphosulfate (PAPS) revealed that some inactive chimeras lost affinity for PAPS. These results suggested that an ordered structure formed by regions I, III, and V is required for HS-ST activity, especially for substrate preference and PAPS binding.

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High sulfotransferase activity for phenolic aromatic odorants present in the mouse olfactory organ

Tamura H., Miyawaki A., Inoh N., Harada Y., Mikoshiba K., Matsui M.

Chemico-Biological Interactions （Chemico-Biological Interactions）  104 （ 1 ） 1 - 9 1997年04月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  00092797

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Mouse nasal cytosols show high sulfotransferase (ST) activities toward phenolic aromatic odorants, but have little activities for most alcoholic aromatic odorants. Most ST activities toward the phenolic odorants preferred slightly acidic pH (6.4) and were sensitive to 2,6-dichloro-4-nitrophenol, a specific inhibitor for phenol ST (P-ST) but were not inhibited by triethylamine and tetra-n-butylammonium chloride, which are specific inhibitors for hydroxysteroid ST (HS-ST). These results suggested that P-ST activities are responsible for sulfation of the phenolic odorants. The spectra of the ST activities for these odorants were similar in mouse nasal and liver cytosols, however, nasal cytosols showed much higher ST activity toward cinnamyl alcohol than liver cytosols. This activity preferred higher pH (7.4) compared to the phenolic odorant-ST activities and was inhibited by both types of inhibitors, specific for P-ST and HS-ST. These results appear to indicate the participation of multiple ST isoforms for the sulfation of odorants in mouse nasal cytosols. The existence of P-ST(s) active for the phenolic odorants in olfactory cytosols suggests a role in odorant perception, in particular, in the signal termination process.

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A distant evolutionary relationship between bacterial sphingomyelinase and mammalian DNase I

Matsuo Y., Yamada A., Tsukamoto K., Tamura H., Ikezawa H., Nakamura H., Nishikawa K.

Protein Science （Protein Science）  5 （ 12 ） 2459 - 2467 1996年12月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  09618368

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The three-dimensional structure of bacterial sphingomyelinase (SMase) was predicted using a protein fold recognition method: the search of a library of known structures showed that the SMase sequence is highly compatible with the mammalian DNase 1 structure, which suggested that SMase adopts a structure similar to that of DNase I. The amino acid sequence alignment based on the prediction revealed that, despite the lack of overall sequence similarity (less than 10% identity), those residues of DNase I that are involved in the hydrolysis of the phosphodiester bond, including two histidine residues (His 134 and His 252) of the active center; are conserved in SMase. In addition, a conserved pentapeptide sequence motif was found, which includes two catalytically critical residues. Asp 251 and His 252. A sequence database search showed that the motif is highly specific to mammalian DNase I and bacterial SMase. The functional roles of SMase residues identified by the sequence comparison were consistent with the results from mutant studies. Two Bacillus cereus SMase mutants (H134A and H252A) were constructed by site-directed mutagenesis. They completely abolished their catalytic activity. A model for the SMase-sphingomyelin complex structure was built to investigate how the SMase specifically recognizes its substrate. The model suggested that a set of residues conserved among bacterial SMases, including Trp 28 and Phe 55, might be important in the substrate recognition. The predicted structural similarity and the conservation of the functionally important residues strongly suggest a distant evolutionary relationship between bacterial SMase and mammalian DNase I. These two phosphodiesterases must have acquired the specificity for different substrates in the course of evolution.

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High-level expression of Bacillus thuringiensis phosphatidylinositol-specific phospholipase C by the Bacillus brevis host-vector system

Kobayashi T., Tamura H., Taguchi R., Udaka S., Ikezawa H.

Japanese Journal of Medical Science and Biology （Japanese Journal of Medical Science and Biology）  49 （ 3 ） 103 - 112 1996年06月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  00215112

　概要を見る

We succeeded in hyperproduction of Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PIPLC), using a Bacillus brevis 47 expression system. The recombinant B. thuringiensis PIPLC was expressed under the control of the middle wall protein gene promoter in B. brevis expression vector pNU211. A large amount of recombinant PIPLC (0.4 g per liter culture) was secreted into the medium as a mature enzyme, and the enzymatic properties of purified recombinant PIPLC were similar to those of the enzyme from wild-type B. thuringiensis. This system provides a useful approach to the three-dimensional structure-function relationship of PIPLC.

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Zonal distribution of sulfotransferase for phenol in olfactory sustentacular cells

Miyawaki A., Homma H., Tamura H., Matsui M., Mikoshiba K.

EMBO Journal （EMBO Journal）  15 （ 9 ） 2050 - 2055 1996年05月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  02614189

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We have immunolocalized phenol sulfotransferase (PST)G, an isoform of PST in sustentacular cells which reside in the dorso-medial portion of the nasal cavity of the mouse. The same topographical pattern of gene expression has been reported for some olfactory neuron-specific genes. When several established (phenol-containing) odorants were used as substrate, mouse nasal tissue cytosol showed a significant level of PST activity, as does mouse liver cytosol. This study is the first to demonstrate that gene expression in the olfactory sustentacular cells is also organized zonally, and indicates the involvement of sulfo-conjugation in olfactory perireceptor processes, such as odorant clearance and xenobiotic detoxification.

Studies on the active sites of Bacillus cereus sphingomyelinase substitution of some amino acids by site-directed mutagenesis

Ikezawa H., Tameishi K., Yamada A., Tamura H., Tsukamoto K., Matsuo Y., Nishikawa K.

Amino Acids （Amino Acids）  9 （ 3 ） 293 - 298 1995年09月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  09394451

　概要を見る

Chemical modifications suggested that acidic amino acids such as aspartic and glutamic acids are involved in the active sites of Bacillus cereus sphingomyelinase. Among aspartic acid residues in the conserved regions of this enzyme, Asp-126, Asp-156, Asp-233 and Asp-295 were converted to glycine by site-directed mutagenesis. According to prediction on structural similarity to pancreatic DNase I, His-151 and His-296 were also converted to alanine. The Asp and His mutants, D126G, D156G, D233G, D295G, H151A and H296A, were produced in Bacillus brevis 47, a protein-hyperproducing strain. The catalytic activities of D295G, H151A and H296A were completely abolished, and sphingomyelin-hydrolyzing activity of D126G or D156G was reduced by more than 50%. The activity of D126G toward p-NPPC was comparable to that of the wild-type, while D156G catalyzed the hydrolysis of HNP and p-NPPC more efficiently than the wild-type. Hemolytic activities of the mutants were parallel to their sphingomyelin-hydrolyzing activities. © 1995 Springer-Verlag.

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Mutation in aspartic acids modifies catalytic and hemolytic activities of Bacillus cereus sphingomyelinase.

H. Tamura*, K. Tameishi, A. Yamada, M. Tomita, Y. Matsuo, K. Nishikawa and H. Ikezawa

Biochem J 309 （ 3 ） 757 - 764 1995年08月

研究論文（学術雑誌）, 共著, 査読有り

Studies on catalytic sites of glycosylphosphatidylinositol-anchoring aminopeptidase N from larval midgut of the silkworm, Bombyx mori and its expression in virus-infected silkworm.

田村 悦臣

Protein Engng 8   966 1995年

研究論文（学術雑誌）, 共著, 査読有り

Mutation in aspartic acid residues modifies catalytic and haemolytic activities of Bacillus cereus sphingomyelinase

Tamura H., Tameishi K., Yamada A., Tomita M., Matsuo Y., Nishikawa K., Ikezawa H.

Biochemical Journal （Biochemical Journal）  309 （ 3 ） 757 - 764 1995年

研究論文（学術雑誌）,  ISSN  02646021

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Four aspartic acid residues (Asp126, Asp156, Asp233 and Asp295) of Bacillus cereus sphingomyelinase (SMase) in the conservative regions were changed to glycine by in vitro mutagenesis, and the mutant SMases [D126G (Asp126 → Gly etc.), D156G, D233G and D295G] were produced in Bacillus brevis 47, a protein-producing strain. The sphingomyelin (SM)-hydrolysing activity of D295G was completely abolished and those of D126G and D156G were reduced by more than 80%, whereas that of D233G was not so profoundly affected, Two mutant enzymes (D126G and D156G) were purified and characterized further. The hydrolytic activities of D126G and D156G toward four phosphocholine-containing substrates with different hydrophobicities, SM, 2-hexadecanoylamino-4-nitrophenylphosphocholine (HNP), lysophosphatidylcholine (lysoPC) and p-nitrophenylphosphocholine (p-NPPC), were compared with those of the wild-type. The activity of D126G toward water-soluble p-NPPC was comparable with that of the wild-type. On the other hand, D156G catalysed the hydrolysis of hydrophilic substrates such as HNP and p-NPPC more efficiently (> 4-fold) than the wild-type. These results suggested that Asp126 and Asp156, located in the highly conserved region, may well be involved in a substrate recognition process rather than catalytic action. Haemolytic activities of the mutant enzymes were found to be parallel with their SM-hydrolysing activities. Two regions, including the C-terminal region containing Asp295, were found to show considerable sequence identity with the corresponding regions of bovine pancreatic DNase I. Structural predictions indicated structural similarity between SMase and DNase I. An evolutionary relationship based on the catalytic function was suggested between the structures of these two phosphodiesterases.

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Purification of glycosylphophatidylinositol-anchoring aminopeptidase N from the plasma membrane of larval midgut epithelial cells of the silkworm, Bombyx mori.

M. Tomita*, H. Obara, Y. Takesue, H. Tamura, S. Miyajima, R. Taguchi and H. Ikezawa

Int J Biochem 26 （ 8 ） 977 - 986 1994年08月

研究論文（学術雑誌）, 共著, 査読有り

Purification of glycosylphosphatidylinositol-anchoring aminopeptidase in from the plasma membrane of larval midgut epithelial cells of the silkworm, Bombyx mori

Masahiro T., Hiroshi O., Yoshiki T., Hiro-Omi T., Shigetoshi M., Ryo T., Hiroh I.

International Journal of Biochemistry （International Journal of Biochemistry）  26 （ 8 ） 977 - 986 1994年08月

研究論文（学術雑誌）,  ISSN  0020711X

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1. 1. Aminopeptidase N was selectively released from larval midgut of silkworm, Bombyx mori, by phosphatidylinositol-specific phospholipase C, and purified to a homogeneous state by ion exchange, gel filtration. Con A-Sepharose and 4-aminobenzyl phosphonic acid-agarose column chromatographies. 2. 2. The purified aminopeptidase N preparation showed 190.8 U/mg of specific activity. Its molecular weight was estimated to be around 100 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 3. 3. Purified aminopeptidase N molecule preferentially hydrolyzed Leu-, Ala- and Met-p-nitroanilide as substrates. Especially, Leu-p-nitroanilide proved to be the best substrate for aminopeptidase N from larval midgut of silkworm. 4. 4. By treatment with phosphatidylinositol-specific phospholipase C, two other hydrolases, alkaline phosphatase and alkaline phosphodiesterase I, were also solubilized from silkworm midgut. © 1994.

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Inhibition of NGF-induced neurite outgrowth of PC12 cells by Bacillus cereus sphingomyelinase, a bacterial hemolysin

Tamura H., Noto M., Kinoshita K., Ohkuma S., Ikezawa H.

Toxicon （Toxicon）  32 （ 5 ） 629 - 633 1994年05月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  00410101

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H-o. Tamura, M. Noto, K. Kinoshita, S. Ohkuma and H. Ikezawa. Inhibition of NGF-induced neurite outgrowth of PC12 cells by Bacillus cereus sphingomyelinase, a bacterial hemolysin. Toxicon 32, 629-633, 1994.-Sphingomyelinase of Bacillus cereus, a bacterial hemolysin, reduced nerve growth factor (NGF)-induced neurite outgrowth of PC12 cells in a dose-dependent manner. At 200 mU/ml, sphingomyelinase repressed half the neurite outgrowth of the cells at 250 ng/ml NGF. The c-fos superinduction, one of the early responses induced by NGF, was not influenced by this treatment, suggesting that the repression by sphingomyelinase occurred via a protein kinase C-independent pathway. © 1994.

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Mutational analysis of the COOH-terminal hydrophobic domain of 5'-nucleotidase as a signal for the glycosylphosphatidylinositol (GPI)-anchor modification.

Y. Furukawa, H. Tamura* and H. Ikezawa

Biochim Biophys Acta 1190 （ 2 ） 273 - 278 1994年03月

研究論文（学術雑誌）, 共著, 査読有り

Mutational analysis of the COOH-terminal hydrophobic domain of bovine liver 5′-nucleotidase as a signal for glycosylphosphatidylinositol (GPI) anchor attachment

Furukawa Y., Tamura H., Ikezawa H.

BBA - Biomembranes （BBA - Biomembranes）  1190 （ 2 ） 273 - 278 1994年03月

研究論文（学術雑誌）,  ISSN  00052736

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In order to address the minimum domain of the COOH-terminal hydrophobic region responsible for GPI modification of bovine liver 5′-nucleotidase, we constructed a series of the deletion mutants of the COOH-terminus and expressed them in COS cells. Cells transfected by the deletion mutant of 6 amino acids (-IIILYQ) from the hydrophobic domain (-FSLIFLSVLAVIIILYQ) did not show any elevation of cell surface-associated 5′-nucleotidase activity, whereas the 2 (-YQ) or 4 (-ILYQ) amino acid deletion mutant retained the bovine liver-derived activity on the cell surface as a GPI-anchored protein. Loss of half the hydrophobic domain (6 or 8 amino acids) resulted in accumulation of the activity in the cell. On the other hand, deletion of the whole hydrophobic domain (17 amino acids) or the entire cleaved-off domain (25 amino acids) made the product secreted into the medium. In conclusion, the hydrophobicity of 13 amino acids in length was enough for the GPI modification of the bovine liver 5′-nucleotidase. © 1994.

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Studies on the GPI-anchored enzyme, hepatic 5'-nucleotidase. Microheterogeneity of the anchor, processing and localization.

Ikezawa H., Taguchi R., Tamura H., Kobayashi T.

Brazilian journal of medical and biological research = Revista brasileira de pesquisas médicas e biológicas / Sociedade Brasileira de Biofísica ... [et al.] （Brazilian journal of medical and biological research = Revista brasileira de pesquisas médicas e biológicas / Sociedade Brasileira de Biofísica ... [et al.]）  27 （ 2 ） 395 - 399 1994年02月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0100879X

　概要を見る

Hepatic 5'-nucleotidases of vertebrates were investigated for localization in the lysosomes and the plasma membrane, microheterogeneity of the glycosylphosphatidylinositol (GPI)-anchor moiety and minimal requirement of the C-terminal signal peptide for GPI attachment. Using PIPLC of Bacillus thuringiensis and subcellular fractionation by Percoll gradient centrifugation, we found that chicken liver 5'-nucleotidase can be transferred from plasma membrane to lysosomes in the GPI-anchored or soluble form. Bovine liver ecto 5'-nucleotidase was solubilized by PIPLC, purified to a homogeneous state, and analyzed for the structures of GPI-anchor isoforms by HPLC and ESI-MS in combination with glycosidase treatments, after peptide-bond cleavage by CNBr or trypsin. Several isomers of the GPI anchor were thus characterized; major components contained two phosphorylethanolamine residues, whereas the component containing three phosphorylethanolamine residues was present only as a small percentage of the total. The cleavage/attachment site of the GPI anchor in the C-terminal of 5'-nucleotidase was shown to be Ser523. The peptide region cleaved off at the posttranslational processing has a length of 25 amino acid residues which contains a hydrophobic stretch of 17 amino acids. By site-directed mutagenesis, we determined the minimal length of the hydrophobic peptide to be 13 amino acids for expression of 5'-nucleotidase as a GPI-anchored form on the COS cell surface. When peptide length was shortened to less than 13 amino acids, the expressed enzyme was not sorted to the cell surface but present within, or secreted out of the cells.

The role of acidic amino acid residues in catalytic and adsorptive sites of Bacillus cereus sphingomyelinase.

M. Tomita*, Y. Ueda, H. Tamura, R. Taguchi and H. Ikezawa

Biochim Biophys Acta 1203 （ 1 ） 85 - 92 1993年11月

研究論文（学術雑誌）, 共著, 査読有り

The role of acidic amino-acid residues in catalytic and adsorptive sites of Bacillus cereus spingomyelinase

Tomita M., Ueda Y., Tamura H., Taguchi R., Ikezawa H.

Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular （Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular）  1203 （ 1 ） 85 - 92 1993年11月

研究論文（学術雑誌）,  ISSN  01674838

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By the modification of acidic amino-acid residues with Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3′-sulfonate), the activity of sphingomyelinase of Bacillus cereus was decreased by 80-90%. Also, the reduction of Cys residues in the sphingomyelinase molecule by dithiothreitol cause a drasic decrease in enzymatic activity, whereas the spingomyelinase activity was not affected by treatment with p-chloromercuribenzenesulfonic acid. Actually, no inactivation of sphingomyelinase activity was observed after selective modification of basic amino-acid residues such as Lys, His and Arg, and of the unchanged amino-acid residues Ser and Thr. The treatment of the sphingomyelinase molecule with Woodward's reagent K or dithiothreitol also brought about the inhibition of the specific adsorption of sphingomyelinase toward intact erythrocyte membranes. However, the extent of inhibition in the enzyme adsorption, 20-50%, was less than that observed in the sphingomyelinase activity. These results suggest that acidic amino-acid residues, such as Asp and Glu, in the sphingomyelinase molecule are involved in the catalytic sites and the adsorptive sites. Apparently, the disruption of disulfide linkage in the sphingomyelinase molecule by dithiothreitol destabilized its structure, resulting in a drastic decrease in sphingomyelin-hydrolyzing activity and specific adsorption of sphingomyelinase towards crythrocyte membranes. © 1993.

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