Incorporation of paratose residue into Salmonella O-specific polysaccharides using enzymatic reactions]

The block mechanism of O-specific polysaccharides biosynthesis was demonstrated for Salmonella nitra (serogroup A) and S. haifa (serogroup B). Due to the moderate specificity of glycosyl transferases from S. nitra, S. typhimurium, S. haifa and S. kentucky (serogroup C3) towards the 3,6-dideoxyhexose structure a paratose residue can be incorporated into the polysaccharide chain instead of an abequose residue, and vice versa.     

Acceptor specificity of mannosyl transferases from Salmonella of serotypes C2 and C3

Synthetic mono- and disaccharide derivatives of moraprenyl pyrophosphate were studied as mannose acceptors during the assembly of the repeating unit Rha-Man-Man-Gal of the Salmonella newport (serogroup C2) and S. kentucky (serogroup C3) O-antigens. Mannosyl transferases revealed strict specificity towards the configuration of terminal monosaccharide residue at C1 as well as to the type of linkage between monosaccharide residues in the disaccharide acceptor. The specificity of mannosyl transferases towards the structure of subterminal monosaccharide was not absolute. Alpha-D-Glucose and alpha-D-mannose derivatives were found not to serve as mannosyl residue acceptors, whereas those of alpha-D-talose, alpha-D-fucose, 4-deoxy-D-xylo-hexose and Man (alpha 1-3) glucose were substrates in enzymatic mannosylation with formation of polyprenyl pyrophosphate trisaccharides. These derivatives could serve as substrates for two subsequent enzymatic reactions: rhamnosylation and polymerization of the repeating units, yielding 40-60% of the polysaccharides.     

A Survey of Argentine and Lebanese Bone for Salmonellas with Particular Reference to the Isolation of Salmonella typhimurium

The incidence of Salmonella typhimurium in imported bone meal may be underestimated by conventional isolation techniques because of the presence of other salmonella serotypes. A method was developed with a serological bias towards recovery of serotypes with 'i'as H phase 1 antigen. By this technique, 44 strains of S. typhimurium were isolated from Argentinian bone meal compared with nine strains cultured by the unbiased method. The technique was less successful with Lebanese bone. The serological technique was most effective when several salmonella serotypes not possessing the 'i'antigen were present in a sample. S. kentucky (8, 20:i:z₆), like S. typhimurium was also efficiently isolated by the serological method. In 12 specimens both S. typhimurium and S. kentucky were present. The two serotypes were easily separated by examining four 25 g sub-samples of the specimen.     

Incorporation of abequose residues into O-specific polysaccharides of Salmonella serotypes B, C2 and C3

The possibility of chemical-enzymatic synthesis of O-specific polysaccharide and its modified derivatives was demonstrated with a cell envelope preparation from Salmonella typhimurium using synthetic polyprenyl pyrophosphate oligosaccharides and CDP-[14C]Abe. It was shown that during biosynthesis of O-specific polysaccharides from S. newport and S. kentucky abequosylation reaction occurs prior to polymerization of oligosaccharide repeating units.     

The structure of O-antigenic polysaccharide of gram negative bacteria Salmonella kentucky strain 98/39(0:8, H:i,Z6)

The computerized calculation of the 13C NMR spectra of polysaccharide and oligosaccharides (ANMROL), together with chemical analysis (methylation and Smith degradation) showed that the polysaccharide has the following structure: (formula; see text) which differs from the previous data published for Salmonella kentucky strain I. S. 98 (8.20).     

The mosaic of proteins synthesized by Salmonella typhimurium

A profusion of proteins with heterologous serological specificities was synthesized by S. typhimurium grown on artificial media; accordingly, sera prepared in rabbits with these proteins displayed an abundance of antibodies reacting, in agar gel, against numerous heterologous proteins. the absorption of the sera with different Enterobacterial proteins proved that the S. typhimurium proteins are a mixture of specific proteins, and common E. coli and Salmonellae determinants; in addition, a group of strongly cross-precipitating proteins common to S. typhimurium and S. choleraesuis and to S. typhimurium and S. kentucky were identified that were not present in the proteins common to S. enteritidis, S. typhi and E. coli, or in the S. paratyphi A proteins used absorptions. The specific proteins of S. typhimurium were synthesized on artificial media in, apparently, smaller amounts than the common proteins; their role in the protection of mice against infection with their natural pathogen was, however, proof of their specificity and contrasted with the ineffectiveness, in protecting the mice, of the common proteins.     

Chemico-enzymatic synthesis of branched O-specific polysaccharides of Salmonella serotype C2 and C3]

The possibility of chemical-enzymatic synthesis of branched polysaccharides was demonstrated with the enzymes from Salmonella newport and S. kentucky using synthetic polyprenyl pyrophosphate oligosaccharides. Formation of polymers with alpha 1-2-, beta 1-2-alpha 1-4- and beta 1-4-linkages between glucose residues in the branch and galactose residues of the main chain was shown.     

Thermal inactivation of eight Salmonella serotypes on dry corn flour.

Dry heat was used to inactivate Salmonella newington, Salmonella typhimurium, Salmonella anatum, Salmonella kentucky, Salmonella cubana, Salmonella seftenberg, Salmonella thompson, and Salmonella tennessee in corn flour at 10 and 15% moisture. The flour was spray inoculated at 10(5) Salmonella cells per g and then stored at 49 degrees C (120 degrees F); viable Salmonella cells were counted on Trypticase (BBL Microbiology Systems) soy agar plates every 30 min for the first 4 h and then at 4-h intervals for 20 additional h of storage. After 24 h, 99.9% of all Salmonella cells were killed. S. thompson and S. tennessee were more resistant to heat inactivation than the other serotypes. Naturally occurring contamination by Salmonella spp. in dry food products could be significantly reduced with this treatment.     

昆虫谷胱甘肽S-转移酶分离纯化的新方法

谷胱甘肽Ｓ－转移酶（ｇｌｕｔａｔｈｉｏｎｅＳ－ｔｒａｎｓｆｅｒａｓｅｓ,ＧＳＴ）是一类具有多种生理功能的同功酶．从蜡螟幼虫（Ｇａｌｅｒｉａｍｅｌｏｎｅｌａ）的提取液中分离纯化谷胱甘肽Ｓ－转移酶的基本方法如下：首先将冷冻的蜡螟幼虫在磷酸缓冲液中匀桨,经１００００ｇ和１０００００ｇ分级离心;取上清液通过ＱＡＥ－ＳｅｐｈａｄｅｘＡ－２５离子交换柱层析除去部分色素和杂蛋白;然后采用谷胱甘肽－琼脂糖凝胶亲和层析（ＧＳＨ－ＱＴ４）,四溴酚酞二磺酸盐－琼脂糖凝胶亲和层析（ＢＳＰ－ＱＴ４）,铜离子－琼脂糖凝胶螯合层析（Ｃｕ２＋－ＱＴ４）及ＰＢＥ９４－Ｓｅｐｈａｒｏｓｅ（ＰＢＥ９４）聚焦层析等层析技术进一步分离纯化．将上述方法获得的色谱峰以ＣＤＮＢ和ＤＣＮＢ为底物检测生物活性．具有生物活性部分的蛋白质,通过ＳＤＳ－ＰＡＧＥ测定其分子量．实验结果表明,采用ＧＳＨ－ＱＴ４亲和层析法获得的活性峰,在ＳＤＳ－ＰＡＧＥ图谱上呈现出两条带,分子量为２４ｋＤ,２４．５ｋＤ左右;Ｃｕ２＋－ＱＴ６螯合层析法分离的活性峰,呈现出一条带,分子量为２４ｋＤ左右;ＰＢＥ９４－聚焦层析法分离获得三个活性峰：第一色谱峰,呈现出一条带,分子量为２３ｋＤ左右     

Identity of malonyl and palmitoyl transferase of fatty acid synthetase from yeast. Functional interrelationships between the acyl transferases.

Functional interrelationships between the acyl transferases of yeast fatty acid synthetase were investigated. In binding assays with synthetase modified by 5,5'-dithiobis(2-nitrobenzoic acid), 4--5 malonyl transferase entities per multienzyme complex molecule could be titrated. In the presence of palmitoyl-CoA these malonyl transferases were found inaccessible to malonyl-CoA, whereas the acetyl transferases were reactive towards acetyl-CoA. Between four and five palmitoyl transferase entities per synthetase equivalent were found reactive towards palmitoyl-CoA, the palmitoyl binding being inhibited by malonyl-CoA. Following palmitoyl binding the acetyl transferases were found towards acetyl-CoA. Substrate model assays were consistent with these data. It is concluded that malonyl and palmitoyl transferases are closely coupled enzyme components of the multienzyme complex which are fairly independent of the acetyl transferase entities. The molecular basis for the observed coupling will be given in the following paper.     

Membrane glycoprotein transferases: Time courses of activity changes in a synchronized cell population and enzyme activity half-lives in an asynchronous population

The time course of activity changes of five membrane glycoprotein transferases: the glycoprotein:galactosyl, the fetuin:fucosyl, the PSM:fucosyl, the fetuin:N-acetylglucosaminyl, and the polypeptide: N-acetylgalactosaminyl was determined using defined acceptors and conditions in synchronized L5178Y cells. In addition, time course of activity changes of these transferases was determined for the endogenous acceptors present in the cell extract. Each of the membrane glycoprotein transferases was an S peak enzyme in the L5178Y cell. Activity with endogenous acceptors, in general, was constant throughout the cell cycle indicating that acceptor availability may, in part, control glycoprotein synthesis in vivo.     

Cloning of a human tRNA isopentenyl transferase

A cDNA of human origin is shown to encode a tRNA isopentenyl transferase (E.C. 2.5.1.8). Expression of the gene in a Saccharomyces cerevisiae mutant lacking the endogenous tRNA isopentenyl transferase MOD5 resulted in functional complementation and reintroduction of isopentenyladenosine into tRNA. The deduced amino acid sequence contains a number of regions conserved in known tRNA isopentenyl transferases. The similarity to the S. cerevisiae MOD5 protein is 53%, and to the Escherichia coli MiaA protein 47%. The human sequence was found to contain a single C2H2 Zn-finger-like motif, which was detected also in the MOD5 protein, and several putative tRNA transferases located by BLAST searches, but not in prokaryotic homologues.     

Cytosolic glutathione transferases from rat liver. Primary structure of class alpha glutathione transferase 8-8 and characterization of low-abundance class Mu glutathione transferases.

Six GSH transferases with neutral/acidic isoelectric points were purified from the cytosol fraction of rat liver. Four transferases are class Mu enzymes related to the previously characterized GSH transferases 3-3, 4-4 and 6-6, as judged by structural and enzymic properties. Two additional GSH transferases are distinguished by high specific activities with 4-hydroxyalk-2-enals, toxic products of lipid peroxidation. The most abundant of these two enzymes, GSH transferase 8-8, a class Alpha enzyme, has earlier been identified in rat lung and kidney. The amino acid sequence of subunit 8 was determined and showed a typical class Alpha GSH transferase structure including an N-acetylated N-terminal methionine residue.     

Synthesis of oligosaccharide fragments of the repeating unit of Salmonella kentucky O-specific polysaccharide and conversion of the oligosaccharides into the glycosyl phosphates

alpha-D-Man-(1----2)-alpha-D-Man-(1----3)-D-Gal, a structural fragment of the main chain of Salmonella serogroups C2 and C3 O-specific polysaccharides, and the isomer with the central residue beta have been synthesised, as have some oligosaccharides related to the structure of the O-specific polysaccharide of S. kentucky (serogroup C3), namely, alpha-D-Glc-(1----4)-D-Gal, alpha-D-Man-(1----3)-[alpha-D-Glc-(1----4)]-D-Gal, and alpha-D-Man-(1----2)-alpha-D-Man-(1----3)-[alpha-D-Glc-(1----4)]-D-Gal, and the isomers with the D-Glc unit beta. Each oligosaccharide was converted into the alpha-glycosyl phosphate.     

Mutagenicity of fractions of cigarette smoke condensate in Neurospora crassa and Salmonella typhimurium

The mutagenicities of selected fractions of cigarette smoke condensate (CSC) were studied in Neurospora crassa for the presence of direct-acting mutagens. CSCs from the University of kentucky Reference Cigarette 1R1 were assayed in a forward-mutation test at the adenine-3 (ad-3) region in resting conidia of a 2-component heterokaryon. Direct-acting mutagenic activity was found in an enriched polycyclic aromatic hydrocarbons (EPAH) fraction and in a basic fraction (Swain 5). No direct-acting mutagenic activity was detected in an acidic fraction (Swain 8), although it was highly toxic to resting conidia. The EPAH fraction also was tested in the presence of S9 mix prepared from Aroclor-1254-induced rat liver. It was found to be mutagenic, but higher doses were required than in the absence of S9 mix. In addition, the mutagenicities of CSC and 10 fractions of CSC were investigated in Salmonella typhimurium TA1538 by the incorporation and preincubation methods. In general, preincubation did not enhance the mutagenicities of the fractions, and the two rankings of mutagenic potency of the condensates that were obtained by the two methods were not significantly different. This is the first report of the presence of potent direct-acting mutagenicity in the EPAH and Swain 5 fractions of CSC.     

Glutathione transferases. Catalysis of nucleophilic reactions of glutathione

Homogeneous preparations of the glutathione transferases from rat liver have been tested for their ability to catalyze a number of diverse nucleophilic reactions of GSH. Although disulfide interchange with GSSG or L-cystine, and cis-trans isomerization of maleic acid, are clearly promoted by thiols in solution, the reactions were not catalyzed by the glutathione transferases. In contrast, certain more hydrophobic analogs of these compounds were found to serve as substrates. The transferases also catalyze the glutathione-dependent release of p-nitrophenol from p-nitrophenyl acetate and p-nitrophenyl trimethylacetate. These observations are consistent with the formulation that catalysis may result from close juxtaposition of sufficiently electrophilic, nonpolar compounds with GSH on the enzyme surface.     

Membrane glycoprotein biosynthesis: Changes in levels of glycosyl transferases in fibroblasts transformed by oncogenic viruses

Fibroblasts transformed by oncogenic viruses were found to have greater enzymic activities of four membrane glycoprotein:glycosyl transferases on a cell or protein basis then two non-transformed fibroblastic lines. These enzymes are responsible for the synthesis of membrane glycoproteins; each of the four transferases studied, the polypeptide:N-acetylgalactosaminyl, glycoprotein:galactosyl, fetuin:fucosyl and PSM:fucosyl transferases, was more than twice as active in the transformed cell lines using both endogenous and added receptor. The most pronounced differences occurred with the doubly (SV-PY-3T3) transformed fibroblasts in all cases; with the N-acetylgalactosaminyl and galactosyl transferases the increase was 8–16 fold over the non-transformed cells. It was demonstrated that these results do not arise from a changed level of glycosidase activities.     

Alteration of oligomeric state and domain architecture is essential for functional transformation between transferase and hydrolase with the same scaffold

Transferases and hydrolases catalyze different chemical reactions and express different dynamic responses upon ligand binding. To insulate the ligand molecule from the surrounding water, transferases bury it inside the protein by closing the cleft, while hydrolases undergo a small conformational change and leave the ligand molecule exposed to the solvent. Despite these distinct ligand‐binding modes, some transferases and hydrolases are homologous. To clarify how such different catalytic modes are possible with the same scaffold, we examined the solvent accessibility of ligand molecules for 15 SCOP superfamilies, each containing both transferase and hydrolase catalytic domains. In contrast to hydrolases, we found that nine superfamilies of transferases use two major strategies, oligomerization and domain fusion, to insulate the ligand molecules. The subunits and domains that were recruited by the transferases often act as a cover for the ligand molecule. The other strategies adopted by transferases to insulate the ligand molecule are the relocation of catalytic sites, the rearrangement of secondary structure elements, and the insertion of peripheral regions. These findings provide insights into how proteins have evolved and acquired distinct functions with a limited number of scaffolds.     

Identification of a basic hybrid glutathione S-transferase from human liver. Glutathione S-transferase delta is composed of two distinct subunits (B1 and B2).

The purification of a hybrid glutathione S-transferase (B1 B2) from human liver is described. This enzyme has an isoelectric point of 8.75 and the B1 and B2 subunits are distinguishable immunologically and are ionically distinct. Hybridization experiments demonstrated that B1 B1 and B2 B2 could be resolved by CM-cellulose chromatography and have pI values of 8.9 and 8.4 respectively. Transferase B1 B2, and the two homodimers from which it is formed, are electrophoretically and immunochemically distinct from the neutral enzyme (transferase mu) and two acidic enzymes (transferases rho and lambda). Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis demonstrated that B1 and B2 both have an Mr of 26 000, whereas, in contrast, transferase mu comprises subunits of Mr 27 000 and transferases rho and lambda both comprise subunits of Mr 24 500. Antisera raised against B1 or B2 monomers did not cross-react with the neutral or acidic glutathione S-transferases. The identity of transferase B1 B2 with glutathione S-transferase delta prepared by the method of Kamisaka, Habig, Ketley, Arias & Jakoby [(1975) Eur. J. Biochem. 60, 153-161] has been demonstrated, as well as its relationship to other previously described transferases.     

Purification and characterization of three distinct glutathione transferases from mouse liver

Three distinct glutathione transferases in the liver cytosol fraction of male NMRI mice have been purified by affinity chromatography and fast protein liquid chromatofocusing. These enzymes account for approximately 95% of the activity detectable with 1-chloro-2,4-dinitrobenzene as electrophilic substrate. Differences between the three forms are manifested in isoelectric points, apparent subunit molecular mass values, amino acid compositions, N-terminal structures, substrate specificities, and sensitivities to inhibitors, as well as in reactions with specific antibodies raised against glutathione transferases from rat and human tissues. The results indicate strongly that the three mouse enzymes are products of different genes. A comparison of the mouse glutathione transferases with rat and human enzymes revealed similarities between the transferases from different species. Mouse glutathione transferases have been named on the basis of their respective subunit compositions.