In vitro penicillin aminolysis: Application to a radioimmunoassay of trace amounts of penicillin

A specific and sensitive radioimmunoassay of trace amounts of penicillin G in biological fluids is proposed. It is based on the quantitative transformation of penicillin into penicilloyl by rupture of the β-lactam ring of the penicillin molecule, and then the determination of the penicilloyl groups thus formed. Using ?-aminocaproic acid as an acceptor which covalently binds with the penicilloyl groups formed, at alkaline pH, allows a complete and reproducible aminolysis of penicillin. The inhibition of the binding to antipenicilloyl antibodies and the association constant appear to be identical whether penicilloyl groups derive from penicillin or are present under standard penicilloyl ?-aminocaproate form. Besides this application to the radioimmunoassay of penicillin, the study of the in vitro aminolysis of penicillin should permit better understanding of the complex mechanism of the covalent binding of penicilloyl groups to proteins observed in vivo after penicillin therapy, binding which leads to the allergenic metabolite of penicillin.     

Cephalosporin-sensitive penicillin-binding proteins of Staphylococcus aureus and Bacillus subtilis active in the conversion of [14C]penicillin G to [14C]phenylacetylglycine.

Breakdown of the covalent complex formed between [14C]penicillin G and higher molecular weight, cephalosporin-sensitive penicillin-binding proteins was studied using mixtures of the purified proteins isolated from membranes of Staphylococcus aureus and Bacillus subtilis. These penicillin-binding proteins were found to release the bound 14C label in a first order process characterized by half-lives of 10 to 300 min at 37 degrees C. Denaturation of the penicilloyl.penicillin-binding proctein complex prevented this release, indicating that the process is enzyme-catalyzed. [14C]Phenylacetylglycine was identified as the major labeled fragmentation product, indicating that these cephalosporin-sensitive penicillin-binding proteins, for which no in vitro transpeptidase or carboxypeptidase activity has been found, catalyze the same fragmentation of the bound penicilloyl moiety previously described for several penicillin-sensitive D-alanine carboxypeptidases.     

Binding of benzyl penicilloyl to human serum albumin. Evidence for a highly reactive region at the junction of domains 1 and 2 of the albumin molecule

Tryptic digests of fragment C124-298 of penicilloylated serum albumin, obtained from a penicillin-treated patient or prepared by in vitro conjugation, were analyzed by HPLC. Determinations of benzyl penicilloyl groups (BPO) were performed on the different fractions. Three BPO-containing peptides were identified by their amino acid sequence and the bound BPO was located on lysines 190, 195 and 199 and serine 193. These four main BPO-binding sites are all located on a very short region (10 amino acid residues) of the albumin molecule at the junction of domains 1 and 2.     

Simultaneous release of penicilloic acid and phenylacetyl glycine by penicillin-binding proteins 5 and 6 of Escherichia coli.

Penicillin-binding proteins (PBPs) 5 and 6 of Escherichia coli released the bound penicilloyl moiety at an intermediate rate relative to, e.g., Staphylococcus aureus PBPs 4 (rapid) and 1 or 2 (slow). Each of these E. coli PBPs released the bound penicilloyl moiety as both penicilloic acid (hydrolysis) and phenylacetyl glycine (scission of the C-5--C-6 bond followed by hydrolysis).     

Sequence of active site peptides from the penicillin-sensitive D-alanine carboxypeptidase of Bacillus subtilis. Mechanism of penicillin action and sequence homology to beta-lactamases

It has been proposed that penicillin and other beta-lactam antibiotics are substrate analogs which inactivate certain essential enzymes of bacterial cell wall biosynthesis by acylating a catalytic site amino acid residue (Tipper, D.J., and Strominger, J.L. (1965) Proc. Natl. Acad. Sci. U.S.A. 54, 1133-1141). A key prediction of this hypothesis, that the penicilloyl moiety and an acyl moiety derived from substrate both bind to the same active site residue, has been examined. D-Alanine carboxypeptidase, a penicillin-sensitive membrane enzyme, was purified from Bacillus subtilis and labeled covalently at the antibiotic binding site with [14C]penicillin G or with the cephalosporin [14C]cefoxitin. Alternatively, an acyl moiety derived from the depsipeptide substrate [14C]diacetyl L-Lys-D-Ala-D-lactate was trapped at the catalytic site in near-stoichiometric amounts by rapid denaturation of an acyl-enzyme intermediate. Radiolabeled peptides were purified from a pepsin digest of each of the 14C-labeled D-alanine carboxypeptidases and their amino acid sequences determined. Antibiotic- and substrate-labeled peptic peptides had the same sequence: Tyr-Ser-Lys-Asn-Ala-Asp-Lys-Arg-Leu-Pro-Ile-Ala-Ser-Met. Acyl moieties derived from antibiotic and from substrate were shown to be bound covalently in ester linkage to the identical amino acid residue, a serine at the penultimate position of the peptic peptide. These studies establish that beta-lactam antibiotics are indeed active site-directed acylating agents. Additional amino acid sequence data were obtained by isolating and sequencing [14C]penicilloyl peptides after digestion of [14C]penicilloyl D-alanine carboxypeptidase with either trypsin or cyanogen bromide and by NH2-terminal sequencing of the uncleaved protein. The sequence of the NH2-terminal 64 amino acids was thus determined and the active site serine then identified as residue 36. A computer search for homologous proteins indicated significant sequence homology between the active site of D-alanine carboxypeptidase and the NH2-terminal portion of beta-lactamases. Maximum homology was obtained when the active site serine of D-alanine carboxypeptidase was aligned correctly with a serine likely to be involved in beta-lactamase catalysis. These findings provide strong evidence that penicillin-sensitive D-alanine carboxypeptidases and penicillin-inactivating beta-lactamases are related evolutionarily.     

Hydroxylaminolysis of penicillin binding componenets is enzymatically catalyzed.

The hydroxylaminolysis of the penicilloyl moiety from [14C]penicillin G binding component (PBC) complexes of the Bacillus subtilis D-alanine carboxypeptidase and of the mixture of PBC's of Staphylococcus aureus was inhibited by denaturation of the complexes by heat (55 degrees), detergent (1% sodium dodecyl sulfate), or trichloroacetic acid. The kinetics of inhibition by denaturation were comparable to those of the inhibition of [14C]penicillin G binding to the PBC's and of carboxypeptidase activity of the B. subtilis enzyme under identical denaturing conditions. These data establish that the hydroxylaminolysis is an enzymatically catalyzed process suggesting that penicillin G is bound to an enzymatically active site. Treatment of the denatured [14C]penicillin G-carboxypeptidase complex with sodium borohydride or at pH 12 resulted in the release of the penicilloyl moiety. These results are consistent with a carboxylic ester bond for the penicilloyl-PBC instead of a thiolester linkage as was initially presumed.     

New temperate Pseudomonas aeruginosa phage, phi297: Specific features of genome structure

The genome structure and some specific features of temperate Pseudomonas aeruginosa phage phi297 are considered. Analysis of sequencing data and genome annotation suggest that the phi297 genome displays a mosaic structure, which has formed through combining gene blocks from bacteria of taxonomically remote groups and/or their phages. The results of a comparison of the phi297 DNA homology level and pattern with the genome sequences of the currently known related P. aeruginosa bacteriophages are interpreted from the perspective of assumed active migration of these phages between different bacterial species.     

不同形式蛋氨酸对建鲤生长性能及血清游离氨基酸含量的影响

为考察不同形式蛋氨酸对建鲤生长的作用效果, 实验以豆粕、鱼粉、棉粕为蛋白源, 配制缺乏蛋氨酸的基础饲料(对照组, 蛋氨酸含量为0.48%), 在基础饲料中分别添加晶体蛋氨酸、微囊蛋氨酸、蛋氨酸羟基类似物(MHA)及蛋氨酸羟基类似物钙盐(MHA-Ca), 使蛋氨酸含量达到0.58%, 获得5个饲料处理组, 饲养平均体重为(8.61.0) g的建鲤(Cyprinus carpio var Jian)8周。结果显示: 各组鱼体增重率分别为343.51%、350.77%、382.80%、384.02%和385.59%; 饲料系数分别为1.58、1.55、1.42、1.42和1.41; 晶体蛋氨酸组鱼体增重率、饲料系数与对照组无显著差异(P0.05), 微囊蛋氨酸组、MHA组、MHA-Ca组增重率较对照组提高11.4%、11.8%、12.2% (P0.05), 饲料系数降低10.1%、10.1%、10.8% (P0.05)。各处理组在肌肉水分、脂肪含量间无显著差异(P0.05), MHA组肌肉粗蛋白含量较晶体蛋氨酸组显著下降, 其他各组间无显著差异(P0.05)。对摄食后不同时间的血清游离氨基酸浓度变化的分析表明, 对照组在摄食后2h或3h达到峰值, 晶体蛋氨酸组、MHA组在摄食后1h达到吸收峰值, 微囊蛋氨酸组在摄食后1h或2h达到峰值, 而MHA-Ca组则在摄食后3h达到峰值。上述结果表明, 在蛋氨酸缺乏的颗粒饲料中补充晶体蛋氨酸, 对建鲤生长性能无改善作用, 而添加微囊蛋氨酸、蛋氨酸羟基类似物、蛋氨酸羟基类似物钙盐则显著提高了鱼体生长性能, 降低饲料系数。       

Mutants of Salmonella typhimurium responding to cysteine or methionine: their nature and possible role in the regulation of cysteine biosynthesis.

Nineteen mutants of Salmonella typhimurium responding to either cysteine or methionine (cym) have been identified amongst cysteine (cys) and methionine (met) auxotrophs. Their growth responses to known intermediates in the related pathways of cysteine and methionine biosynthesis and complementation patterns in abortive transduction tests divided the mutants into six groups. Results of conjugation, cotransduction and deletion mapping experiments substantiated these groups, each of which carried a lesion within known cys genes. Enzyme assays on cym mutants from five of the six groups confirmed their cys gene deficiencies. Growth response and enzyme assay data were not consistent with mutants being leaky cys mutants (spared by methionine). None of eight cym mutants tested were able to convert [35S]methionine into [35S]cysteine. Selenate specifically inhibits the early enzymes of cysteine synthesis. In cym mutants this inhibition was relieved by cysteine but not by methionine, indicating that cym mutants require active cys enzymes for growth on methionine. There was evidence that methionine stimulated in vivo activity of cys enzymes in a cym mutant. Resistance to inhibition by 1,2,4-triazole results in reduced levels of the O-acetyl serine sulphydrylase. In cym mutants triazole resistance gave unstable suppression of the cym phenotype. Cym mutants may result from mutation in regulatory regions common to each of the cys genes, with the precise role of methionine as yet unknown.     

Assignment of a single disulfide bridge in rat liver methionine adenosyltransferase.

Rat liver methionine adenosyltransferase incorporated 8 mol of N-ethylmaleimide per mol of subunit upon denaturation in the presence of 8 M urea, whereas 10 such groups were labelled when dithiothreitol was also included. This observation prompted a re-examination of the state of the thiol groups, which was carried out using peptide mapping, amino acid analysis and N-terminal sequencing. The results obtained revealed a disulfide bridge between Cys35 and Cys61. This disulfide did not appear to be conserved because cysteines homologous to residue 61 do not exist in methionine adenosyltransferases of other origins, therefore suggesting its importance for the differential aspects of the liver-specific enzyme.     

Reevaluation of dietary methionine requirement by plasma methionine and ammonia concentrations in surgically modified rainbow trout,Oncorhynchus mykiss

This study aimed to reevaluate the dietary methionine requirement by means of the plasma methionine and ammonia concentrations in surgically modified rainbow trout, Oncorhynchus mykiss. A total of 35 rainbow trout averaging 505 ± 6.5 g (initial body weight, mean ± SD) were randomly distributed into seven groups with five fish in each group. After 48 h of food deprivation, each group was fed one of seven L‐amino acid‐based diets containing graded levels of methionine (0.25, 0.40, 0.50, 0.60, 0.70, 0.80 or 0.95% of diet, dry matter bases) by intubation at 1% bodyweight. Blood samples were taken at 0, 5 and 24 h after intubation. Post‐prandial plasma free methionine concentrations (PPmet, 5 h after intubation) and post‐absorptive plasma free methionine concentrations (PAmet, 24 h after intubation) of fish fed diets containing 0.60% or more methionine were significantly (P < 0.05) higher than those of fish fed diets containing 0.50% or less methionine. PPmet and PAmet in fish fed diets containing 0.60% or higher methionine were not significantly different except the PPmet of fish fed a diet containing 0.95% methionine. Post‐prandial plasma ammonia concentrations (PPA, 5 h after intubation) of fish fed diets containing 0.70% or more methionine were significantly higher than those of fish fed diets containing 0.60% or less methionine, and PPA of fish fed diets containing 0.25% and up to 0.60% methionine were not significantly different from each other. Broken‐line model analyses on PPmet, PAmet, and PPA indicated that the dietary methionine requirement of rainbow trout was between 0.59% (1.69) and 0.67% (1.91) of diets (% dietary protein bases) when the diets contained 0.5% cystine.     

Production and fate of methylated sulfur compounds from methionine and dimethylsulfoniopropionate in anoxic salt marsh sediments

Anoxic salt marsh sediments were amended with dl-methionine and dimethylsulfoniopropionate (DMSP). Microbial metabolism of methionine yielded methane thiol (MSH) as the major volatile organosulfur product, with the formation of lesser amounts of dimethylsulfide (DMS). Biological transformation of DMSP resulted in the rapid release of DMS and only small amounts of MSH. Experiments with microbial inhibitors indicated that production of MSH from methionine was carried out by procaryotic organisms, probably sulfate-reducing bacteria. Methane-producing bacteria did not metabolize methionine. The involvement of specific groups of organisms in DMSP hydrolysis could not be determined with the inhibitors used, because DMSP was hydrolyzed in all samples except those which were autoclaved. Unamended sediment slurries, prepared from Spartina alterniflora sediments, contained significant (1 to 10 muM) concentrations of DMS. Endogenous methylated sulfur compounds and those produced from added methionine and DMSP were consumed by sediment microbes. Both sulfate-reducing and methane-producing bacteria were involved in DMS and MSH consumption. Methanogenesis was stimulated by the volatile organosulfur compounds released from methionine and DMSP. However, apparent competition for these compounds exists between methanogens and sulfate reducers. At low (1 muM) concentrations of methionine, the terminal S-methyl group was metabolized almost exclusively to CO(2) and only small amounts of CH(4). At higher (>100 muM) concentrations of methionine, the proportion of the methyl-sulfur group converted to CH(4) increased. The results of this study demonstrate that methionine and DMSP are potential precursors of methylated sulfur compounds in anoxic sediments and that the microbial community is capable of metabolizing volatile methylated sulfur compounds.     

Cysteine and methionine-precursors of methyl sulphone metabolites of 2,4',5-trichlorobiphenyl in mice

In order to account for the origin of the sulphur atom in methyl sulphones derived from polychlorinated biphenyls (PCB), groups of mice were treated with 2,4'-5-trichlorobiphenyl and [35S]cysteine or [35S]methionine, respectively. Control animals received the labelled amino acids only. The radioactive substances extracted from lung tissue were characterized by partition between hexane and sulphuric acid, thin-layer radiochromatography (TLRC), gas chromatography (GC) and mass fragmentography (MF). In lung extracts of both experimental groups sulphuric acid soluble metabolites were present: Their Rf-values on TLRC were identifical with that of 4-methylsulphonyl-2,4'-5-trichlorobiphenyl and their identity was confirmed by GC and GC-MF, which also indicated the presence of a minor sulphone isomer. The study shows that both cysteine and methionine could function as donors of the sulphur atom in the methyl sulphone metabolites derived from PCB.     

甲硫氨酸脑啡肽对猴免疫缺陷病毒早期感染引起CEM x1 74细胞凋亡的可能作用(英文)

探讨短期甲硫氨酸脑啡肽作用对SIV感染CEMx174细胞凋亡的可能作用 .用MTS法测定甲硫氨酸脑啡肽对感染CEMx174细胞存活率的影响 ,流式细胞仪分析SIV诱导细胞凋亡及其甲硫氨酸脑啡肽的作用 ,并测定了cAMP含量、PKA活性和组蛋白磷酸化的水平 .SIV能够显著减少CEMx174细胞数 ,甲硫氨酸脑啡肽可以提高感染细胞的存活率 .AnnexinⅤ结合实验显示 ,1μmol L甲硫氨酸脑啡肽可以增加存活细胞的比率 ,减少凋亡细胞数 .甲硫氨酸脑啡肽降低正常细胞和感染细胞cAMP的含量和PKA活性 ,但是在感染组较为明显 .在感染的情况下 ,磷酸化的组蛋白增加 ,甲硫氨酸脑啡肽可以减少其含量 .甲硫氨酸脑啡肽的作用可以被纳酪酮所拮抗 .研究结果提示 ,甲硫氨酸脑啡肽对SIV感染引起早期细胞凋亡的作用涉及cAMP PKA信号传递过程     

The effect of organic diets on the performance of pullets maintained under semi-organic conditions

The effects of organic diets, with or without supplements of betaine, saponin, fructo-oligosaccharide and methionine, on the health, performance and gut flora of pullets were investigated. A comparison was also made between birds fed organic diets and those fed a non-organic diet. Day-old Lohmann Tradition pullets were reared in 24 groups of 64 chicks indoors until 11 weeks, and then in 48 groups of 24 to 27 chicks with access to range until 17 weeks of age. Groups of birds were fed one of eight diets, a conventional rearing diet with supplementary amino acids, an organic basal diet, organic basal plus methionine and organic basal supplemented with one of the test ingredients. At most stages of growth the birds fed the conventional diet and those fed the basal diet with methionine performed better than those that had no supplemental methionine. Other dietary treatments had no consistently significant effect on growth, the microbial populations within the gastro-intestinal tract of the birds or the number of parasite eggs excreted. After 5 weeks with access to range, the birds that were fed three out of five diets regarded as deficient in sulphur amino acids achieved similar weights (P > 0.05) to birds that received diets adequate in sulphur amino acids. Health and welfare of birds fed organic diets was not adversely affected; however, an investigation of birds housed in larger flocks and taken into the laying phase, when physical demands on birds are greatest, is required.     

Methionine and cysteine metabolism in Fasciola hepatica

Radiolabel from the methyl groups of serine and methyltetrahydrofolate was readily incorporated into methionine in adult Fasciola hepatica, and a substantial proportion of the label from [³⁵S]methionine appeared in cysteine. The data suggest that methionine synthesis is via methyltetrahydrofolate-homocysteine methyltransferase and that there is cysteine synthesis from methionine. Cystathionine-β-synthase and γ-cystathionase activities were demonstrated in homogenates.     

Toward an in vitro test for the diagnosis of allergy to penicillins. Synthesis, characterization, and use of beta-lactam and beta-lactam metabolite poly-L-lysines which recognize human IgE antibodies.

Conjugates of poly-L-lysine (PLL) containing a penicillin or a penicilloyl residue were prepared and characterized by 1H NMR and by size-exclusion (SE) HPLC. These conjugates were used in a radio allergo sorbent tests (RAST) test for the determination of allergy toward beta-lactams. The chemiometric evaluation of the data indicates that allergy to amoxycillin is different from allergy to the other beta-lactams tested. Furthermore, careful chemical characterization of the conjugates appears to be crucial to obtain meaningful information from the RAST data.     

Preparation of multifunctional and multireactive polypeptides via methionine alkylation

We report the development of a new "click"-type reaction for polypeptide modification based on the chemoselective alkylation of thioether groups in methionine residues. The controlled synthesis of methionine polymers and their alkylation by a broad range of functional reagents to yield stable sulfonium derivatives are described. These "methionine click" functionalizations are compatible with deprotection of other functional groups, use an inexpensive, natural amino acid that is readily polymerized and requires no protecting groups, and allow the introduction of a diverse range of functionality and reactive groups onto polypeptides.     

槲皮素对甲硫氨酸负载大鼠氨基酸代谢的影响

为探讨槲皮素对甲硫氨酸(Met)负载大鼠氨基酸代谢的影响,将Wistar大鼠24只随机分为3组,即对照组、1％甲硫氨酸组以及1％甲硫氨酸和0.5％槲皮素组,喂养6周后,采用高压液相色谱法测定血清中半胱氨酸含量,全自动氨基酸分析仪测定血清中其他氨基酸含量.结果显示,1％甲硫氨酸干预后除对牛磺酸产生显著影响外,对其他氨基酸没有明显影响.0.5％槲皮素干预后,血清必需氨基酸苏氨酸、缬氨酸含量较1％ Met组显著升高(p＜0.05),牛磺酸、缬氨酸、亮氨酸和异亮氨酸较对照组亦显著升高(p＜0.05),而血清丝氨酸和脯氨酸含量较对照组显著降低(p＜0.05).结果表明,槲皮素可能加强甲硫氨酸转硫化代谢途径.