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S Ichihara

Publications and source records attributed to S Ichihara.

At least 91 records · Page 5Linked to original sources
PubMed1986

Purification and characterization of human hepatic cysteine-conjugate beta-lyase.

Cysteine-conjugate beta-lyase (EC 4.4.1.13) was purified about 880-fold from human liver obtained post mortem. The purification procedure included (NH4)2SO4 precipitation, chromatography on DEAE-cellulose and hydroxyapatite, gel filtration on Sephadex G-200, and chromatofocusing. The purified enzyme cleaves the C-S bond of several S-aryl-L-cysteines to yield equimolar amounts of thiols, pyruvic acid and ammonia via an alpha beta-elimination reaction. The Mr of the enzyme was estimated to be 88,000 by gel filtration. The enzyme is thermolabile, has a pH optimum of 8.5, and an apparent Km of 0.7 mM towards S-(p-bromophenyl)-L-cysteine. The enzyme requires pyridoxal 5'-phosphate as a cofactor, and hence the enzyme activity was completely abolished by hydroxylamine. No effect of EDTA or thiol-blocking reagents was observed on the activity of the enzyme.

Carbon-Sulfur Lyases↗
PubMed1985

Accessibility of lysyl residues of Escherichia coli B/r porin (OmpF) to covalent labeling reagents of different sizes. An approach for a three-dimensional structure of a channel-forming protein.

The three-dimensional structure of Escherichia coli B/r porin (OmpF) was studied by chemical modification using activated sugars of different size. Galactose and galactosides of different penetration properties through the porin channel were oxidized by galactose oxidase, and the 6-aldehydes formed were linked to amino groups in porin by reduction with NaBH3CN. Tryptic fragments of modified and unmodified porin were separated by reversed-phase high pressure liquid chromatography and identified by amino acid and amino-terminal analysis from the known primary structure of OmpF. Modification of purified native porin trimers in beta-octylglucoside revealed three classes of amino groups: (i) those not modified by any sugars; (ii) those modified only by small sugars that diffuse rapidly through the pore, such as galactose or melibiose; and (iii) those modified by either small or large sugars, the latter including pore-impermeant sugars such as stachyose. The results suggest that the three classes of amino groups correspond, respectively, to groups buried in the trimeric molecule, those in the interior of the pore and those exposed on the surface of porin. In addition modification experiments performed on whole cells suggested that all the reactive groups modified by the pore-impermeant sugars (class iii) are located on the surface of porin exposed on the outside of the outer membrane.

Bacterial Outer Membrane Proteins↗
PubMed1984

Protease IV, a cytoplasmic membrane protein of Escherichia coli, has signal peptide peptidase activity.

During export of the outer membrane lipoprotein across the cytoplasmic membrane, the signal peptide of the lipoprotein undergoes two successive proteolytic attacks, cleavage of the signal peptide by signal peptidase and digestion of the cleaved signal peptide by an enzyme called signal peptide peptidase(s) (Hussain, M., Ichihara, S., and Mizushima, S. (1982) J. Biol. Chem. 257, 5177-5182; Hussain, M., Ozawa, Y., Ichihara, S., and Mizushima, S. (1982) Eur. J. Biochem. 129, 233-239). Here we report that protease IV, a cytoplasmic membrane protease, exhibits the signal peptide peptidase activity. The signal peptide peptidase activity was cofractionated with protease IV throughout the entire process of purification of the latter enzyme. Only the signal peptide was digested by the peptidase among membrane proteins. Both the signal peptide peptidase activity and the protease IV activity were inhibited to similar degrees by antipain, leupeptin, chymostatin, and elastatinal that are known to inhibit the signal peptide peptidase activity in the cell envelope. From these results we conclude that protease IV is the signal peptide peptidase that is responsible for signal peptide digestion in the cytoplasmic membrane. The peptidase attacked the signal peptide only after its release from the precursor protein.

Chromatography, DEAE-Cellulose↗
PubMed1984

Purification and characterization of C-S lyase from Fusobacterium varium. A C-S cleavage enzyme of cysteine conjugates and some S-containing amino acids.

An enzyme responsible for the carbon-sulfur bond cleavage of various S-aryl, S-aralkyl, and S-alkyl cysteines has been purified about 270-fold from Fusobacterium varium. Incubation of a cysteine conjugate of p-bromobenzene with the enzyme yielded equimolar amounts of p-bromobenzenethiol, pyruvic acid, and ammonia, indicating that the carbon-sulfur bond cleavage proceeds via an alpha, beta-elimination reaction. The enzyme activity was inhibited either by hydroxylamine or KCN and stabilized by pyridoxal phosphate, which probably acted as cofactor. The broad substrate spectrum of this enzyme suggested an important role of the intestinal microflora in the in vivo formation of methylthio-containing metabolites of various xenobiotics.

Chromatography, DEAE-Cellulose↗
PubMed1984

Metabolism of tenoxicam in rats.

The structures of six metabolites of tenoxicam in rats (2 mg/kg, orally), elucidated by physicochemical analyses or the reverse-isotope dilution method, were 5'-hydroxytenoxicam (5% dose), 3-(methylsulphamoyl)-2-thiophenecarboxylic acid (9% dose), and the C-7 or C-8 O-glucuronide of tenoxicam (30% dose). The mechanism of formation of N-methylthiophenesulphimide, a possible precursor of 3-(methylsulphamoyl)-2-thiophenecarboxylic acid from tenoxicam, is discussed.

Animals↗
PubMed1984

[Pelvic recurrence of bladder carcinoma following total cystectomy].

The patients who undergo total cystectomy for high stage bladder carcinoma have poor prognosis because of local extension or distant metastases. In the case of recurrent lesions after total cystectomy, radiotherapy and chemotherapy are generally selected. In some cases of recurrent tumors localized within the pelvic cavity and with no distant metastases, however, surgical resection of the tumor is effective in local disease control combined with radio- and chemotherapy. Surgical treatment is not only effective in reducing the cell number in a bulky tumor, but also improves clinical symptoms and conditions such as pain, bleeding, rectal irritability, intestinal obstruction and so on. We present here three patients who underwent extensive operation for pelvic recurrence of bladder carcinoma following total cystectomy. Two of these cases died of progressive disease, nine months and five months after the extensive operations. The third patient died of acute pericarditis thirteen months after the second operation. Autopsy revealed that the surgical treatment was effective in controlling the local recurrence in the third case. Furthermore, some problems regarding pelvic recurrence and surgical treatment are discussed.

Aged↗
PubMed1982

Mechanism of signal peptide cleavage in the biosynthesis of the major lipoprotein of the Escherichia coli outer membrane.

On treatment of Escherichia coli cells with globomycin, a glyceride-containing precursor of the major outer membrane lipoprotein accumulates in the cytoplasmic membrane (Hussain, M., Ichihara, S., and Mizushima, S. (1980) J. Biol. Chem. 255, 3707-3712). When the envelope fraction from such cells was incubated in a suitable buffer, this precursor could be processed to the mature lipoprotein. The processing involved removal of the signal peptide and subsequent acylation of the NH2 terminus thus bared. Two types of peptidase and an acylation enzyme(s) were found to be involved in these processes. The enzyme that cleaves the signal peptide, called signal peptidase in this paper, had many unique properties: being highly resistant to high temperature, having a wide optimum pH range, and being highly sensitive to detergents. The other peptidase(s), called signal peptide peptidase in this paper, was assumed to be responsible for the digestion of the signal peptide that had been cleaved from the precursor lipoprotein. This enzyme was rather heat-sensitive. Thus the processing from the precursor to the mature lipoprotein at a high temperature resulted in accumulation of a peptide that was most probably the intact signal peptide. The third enzyme(s) involved in the processing was the one that is responsible for acylation of the newly bared NH2 terminus of the lipoprotein. The enzyme activity was also lost at 80 degrees C. In the light of these findings, the biosynthetic pathway of the lipoprotein is discussed.

Cell Membrane↗
PubMed1982

Mechanism of export of outer membrane lipoproteins through the cytoplasmic membrane in Escherichia coli. Binding of lipoprotein precursors to the peptidoglycan layer.

Upon treatment of Escherichia coli cells with globomycin, precursors of Braun's lipoprotein, a peptidoglycan-associated lipoprotein (PAL) and several new species of lipoproteins accumulated in the cell envelope (Hussain, M. Ichihara, S., and Mizushima, S. (1980) J. Biol. Chem. 255, 3707-3012; and Ichihara, S., Hussain, M., and Mizushima, S. (1981) J. Biol. Chem. 256, 3125-3129). The precursors of the Braun's lipoprotein and PAL thus accumulated were able to interact with the peptidoglycan layer. A considerable fraction of the precursor of Braun's lipoprotein was covalently bound to the peptidoglycan layer through its COOH-terminal lysine residue in the same manner as in the mature form. The manner of interaction of the precursor of PAL with the peptidoglycan layer was also the same as that of its mature form in which the central to COOH-terminal region of the lipoprotein is important for the interaction. Both precursors were localized in the cytoplasmic membrane when the outer and cytoplasmic membranes were separated after digestion by lysozyme of the peptidoglycan layer. When the cell envelope fraction was incubated, these precursors were chased to the corresponding mature forms. These results indicate that these proteins can be exported through the cytoplasmic membrane while they still retain the signal peptide that is most probably held in the cytoplasmic membrane.

Anti-Bacterial Agents↗
PubMed1982

Signal peptide digestion in Escherichia coli. Effect of protease inhibitors on hydrolysis of the cleaved signal peptide of the major outer-membrane lipoprotein.

Upon incubation of the envelope fraction of Escherichia coli a precursor of the major outer membrane lipoprotein that accumulates in the cytoplasmic membrane of the globomycin-treated cell is processed to the mature form [Hussain, M., Ichihara, S., and Mizushima, S. (1980) J. Biol. Chem. 255, 3707-3712; (1982) J. Biol. Chem. 257, 5177-5182]. When this precursor-containing envelope fraction was incubated in the presence of protease inhibitors such as antipain, leupeptin, chymostatin and elastatinal, a new peptide appeared on a polyacrylamide gel at the position where the signal peptide was expected to appear. This was proved to be the signal peptide of the lipoprotein from the following facts: (a) its appearance is in proportion to the appearance of the lipoprotein and disappearance of the precursor; (b) when the cleavage of the signal peptide from the precursor was inhibited by globomycin, the peptide did not appear on the gel; and (c) the results of labeling of the peptide with [3H]leucine, [35S]methionine and [3H]arginine were consistent with the amino acid composition of the signal peptide. The signal peptide thus accumulated in the envelope fraction was hydrolyzed by an enzyme named 'signal peptide peptidase' when the envelope fraction was washed to remove the inhibitors. The hydrolysis was inhibited by re-addition of these inhibitors. The signal peptide peptidase hydrolyzed the signal peptide only after its cleavage from the lipoprotein precursor.

Bacterial Proteins↗
PubMed1981

Characterization of new membrane lipoproteins and their precursors of Escherichia coli.

By labeling cells heavily with [3H]glycerol or [3H]-palmitic acid several new species of lipoproteins, in addition to Braun's lipoprotein and a peptidoglycan-associated lipoprotein called PAL, were found in the envelope of Escherichia coli. The new lipoproteins were immunochemically different from both Braun's lipoprotein and PAL. A strain lacking the structural gene for Braun's lipoprotein contained new lipoproteins and PAL. In addition to Braun's lipoprotein and PAL, four new lipoproteins were found to be localized in the outer membrane, while other two species were found in the cytoplasmic membrane. The localization of one species is unknown. We previously reported that, on treatment of cells with globomycin, a precursor of Braun's lipoprotein accumulated in the cell envelope (Hussain, M., Ichihara, S., and Mizushima, S. (1980) J. Biol. Chem. 255, 3707-3712). Similarly, the putative precursors of new lipoproteins and that of PAL accumulated in globomycin-treated cells. These precursors contained glycerol and fatty acid(s) as that of Braun's lipoprotein did. It is suggested that the structures of the "signal" region and the mechanisms of processing of all the lipoproteins of E. coli are similar.

Cell Membrane↗
PubMed1980

Accumulation of glyceride-containing precursor of the outer membrane lipoprotein in the cytoplasmic membrane of Escherichia coli treated with globomycin.

The protein accumulated in the cell envelope of Escherichia coli treated with globomycin was identified as the precursor of the outer membrane lipoprotein. The prolipoprotein was almost exclusively localized in the cytoplasmic membrane. The prolipoprotein could be immunoprecipitated with antilipoprotein immunoglobulin and could be chased to the lipoprotein in both in vivo and in vitro. Globomycin inhibited the chase. The prolipoprotein contained glycerol and fatty acid residues, whereas no free sulfhydryl group was detected in it. From these results, it is concluded that the prolipoprotein possesses a glyceride which is covalently bound to the cysteine residue in the peptide as the lipoprotein does and that the removal of signal peptide takes place after the modification. The inhibition of bacterial growth with increasing concentrations of globomycin was accompanied by a gradual increase in the accumulation of the prolipoprotein. Furthermore, growth of the lipoprotein-negative mutant was highly resistant to globomycin. These results strongly indicate that the accumulation of the prolipoprotein in the cytoplasmic membrane causes the death of cells.

Anti-Bacterial Agents↗
PubMed1980

Tissue distribution of 5'-deoxy-5-fluorouridine and derived 5-fluorouracil in tumor-bearing mice and rats.

Distribution of 5-fluorouracil (5-FU) in various tissues including transplanted tumors was examined 1, 3 (or 4), and 24 hr after a single oral dose of 5'-deoxy-5-fluorouridine (5'-DFUR), Ftorafur (FT-207), or 5-FU itself to groups of mice bearing sarcoma-180 and of rats bearing Walker carcinoma-256. The levels of 5-FU derived from 5'-DFUR were highest in the tumors among the tissues examined 3 (or 4) and 24 hr after administration. At the 1st hr after dosing the 5-FU level in the small intestine was close to, but that in other tissues was much lower than, the 5-FU level in the tumor of both types. Such a selective distribution of 5-FU in tumor tissues was not observed after dosing of either FT-207 or 5-FU itself. Since the anticancer effect of 5'-DFUR is likely to be manifested after its conversion to 5-FU, the 5'-deoxyribose moiety of 5'-DFUR may be deemed as an efficient carrier by which the 5-FU moiety is conveyed selectively to cancer tissues.

Animals↗