Establishment of a Cell-Free System for the Biosynthesis of Nicotianamine

A cell-free system has been reported for the biosynthesis ofnicotianamine, a putative precursor of mugineic acid and itsderivatives (MAs). L-[l-¹⁴C]methionine was incubated with acrude homogenate of iron-deficient barley roots, and the reactionproducts were analyzed by HPLC and TLC. Radioactive nicotianaminewas formed enzymatically in a reaction that was dependent onATP. The effect of aminooxyacetic acid (AOA) was compared betweensystems for the synthesis of nicotianamine in vivo and in vitro. (Received December 28, 1988; Accepted April 12, 1989)     

Biosynthesis of T1 antigen in Salmonella: origin of D-galactofuranose and D-ribofuranose residues

The "T1 side chain" portion of cell wall lipopolysaccharide from T1 strains of Salmonella contains d-galactofuranose and d-ribofuranose residues. Isotope labeling studies, using intact cells of mutants each blocked at either of the two different steps of d-galactose metabolism (uridine diphosphate-glucose 4-epimerase and galactose-1-P uridylyl transferase) or at phosphoglucoisomerase, led to the following conclusions. (i) d-Galactofuranose residues are synthesized from d-galactopyranose or its derivatives, rather than by a direct conversion from other hexopyranoses or their derivatives. (ii) The pyranose-to-furanose conversion does not appear to take place at the level of the free d-galactose or d-galactose 1-phosphate. This result suggests that the conversion may occur at the stage of uridine diphosphate-d-galactose. (iii) In a mutant lacking phosphoglucoisomerase, d-ribofuranose residues in T1 side chains contained (14)C derived from exogenous d-fructose-U-(14)C, but little (3)H from exogenous d-glucose-1-(3)H. Thus, no evidence was found for a direct pathway of aldohexose-to-ribose conversion involving a loss of one of the carbons in the C2-C6 moiety of aldohexoses. This suggests, but does not prove, that the T1 ribofuranose residues are synthesized by conventional mechanisms involving hexose monophosphate shunt and transketolase-transaldolase reactions.     

Synthesis of Colicin E1 in a Cell-Free System

Colicin E1 was synthesized in a cell-free system. The in vitro synthesis was found to be dependent on the Col E1 DNA concentration and was not enhanced by the addition of mitomycin C.     

The Biosynthesis of Abscisic Acid from all-trans-{beta}-Carotene in a Cell-Free System from Citrus sinensis Exocarp

A cell-free system prepared from exocarp of Citrus sinensisfruits converted [¹⁴C]-all-trans-ß-carotene into zeaxanthin,antheraxanthin, violaxanthin and abscisic acid (ABA). ABA wasunequivocally characterized by combined capillary GC-MS. (Received August 3, 1993; Accepted August 6, 1993)     

Studies on the Cell-Free Biosynthesis of CNS Membrane Proteins

Abstract: The biosynthesis of CNS membrane proteins was studied in cell-free systems containing membrane-bound polysomes (rough endoplasmic reticulum; RER) or free polysomes from rat forebrain. In previous studies of CNS membrane proteins using two-dimensional gel electrophoretic analysis, five proteins (mol. wt.-pI: 75K 5.4, 68K 5.6, 61K 5.1, 58K 5.1, and 36K 5.6) were found in ceil membrane fractions including preparations enriched in RER, smooth endoplasmic reticulum, and plasma membranes. One of these proteins, 68K 5.6, was also present in cytosol and comigrated with a microtubule-associated protein. In our present study, cell-free systems containing RER were found to synthesize the 75K 5.4, 61K 5.1, and 58K 5.1 proteins. A protein, 34K 5.65, similar (but not identical) to the 36K 5.6 protein was also synthesized. After cell-free synthesis, the 75K 5.4 and 58K 5.1 proteins could be purified by concanavalin A affinity chromatography. Of the five common membrane proteins previously identified, only the 68K 5.6 protein was synthesized by the free polysome population. The free polysomes were also found to synthesize cyclic AMP binding proteins at 48K and 54K, known from previous studies to be present in both cytosol and plasma membrane fractions in mammalian brain tissue. In conclusion, RER synthesized proteins found exclusively in CNS membrane fractions, whereas free polysomes synthesized those proteins found in both soluble and membrane compartments.     

Branched-Chain Amino Acid Biosynthesis in Salmonella typhimurium: a Quantitative Analysis

We report here the first quantitative study of the branched-chain amino acid biosynthetic pathway in Salmonella typhimurium LT2. The intracellular levels of the enzymes of the pathway and of the 2-keto acid intermediates were determined under various physiological conditions and used for estimation of several of the fluxes in the cells. The results led to a revision of previous ideas concerning the way in which multiple acetohydroxy acid synthase (AHAS) isozymes contribute to the fitness of enterobacteria. In wild-type LT2, AHAS isozyme I provides most of the flux to valine, leucine, and pantothenate, while isozyme II provides most of the flux to isoleucine. With acetate as a carbon source, a strain expressing AHAS II only is limited in growth because of the low enzyme activity in the presence of elevated levels of the inhibitor glyoxylate. A strain with AHAS I only is limited during growth on glucose by the low tendency of this enzyme to utilize 2-ketobutyrate as a substrate; isoleucine limitation then leads to elevated threonine deaminase activity and an increased 2-ketobutyrate/2-ketoisovalerate ratio, which in turn interferes with the synthesis of coenzyme A and methionine. The regulation of threonine deaminase is also crucial in this regard. It is conceivable that, because of fundamental limitations on the specificity of enzymes, no single AHAS could possibly be adequate for the varied conditions that enterobacteria successfully encounter.     

Presynaptic Effect of Ethosuximide: a Study on a Cell-Free Model System

The effect of an antiepileptic drug, ethosuximide, on fusion of synaptic vesicles with the synaptosomal plasma membranes was studied. It was shown that ethosuximide increases the rate of the Ca²⁺-dependent fusion reaction. We found that ethosuximide-induced fusion of synaptic vesicles with the plasma membrane in a Ca²⁺-free medium is much lower than the Ca²⁺-induced effect under the same conditions. Thus, the fusion-inducing effect of ethosuximide is mostly Ca²⁺-dependent. Ethosuximide-evoked fusion was suppressed by pentylenetetrazole.     

Biosynthesis of Xyloglucan in Suspension-Cultured Soybean Cells. Synthesis of Xyloglucan from UDP-Glucose and UDP-Xylose in the Cell-Free System

When UDP-[¹⁴C]glucose or UDP-[¹⁴C]xylose was incubated witha particulate fraction from soybean cells, radioactive polymerswere synthesized. On digestion with Aspergillus oryzae enzymes,these polymers gave ¹⁴C-monosaccharides and a ¹⁴C-disaccharidewith chromatographic and electrophoretic mobilities indistinguishablefrom those of authentic isoprimeverose (6-O--D-xylopyranosyl-D-glucopyranose).The disaccharide consisted of xylose and glucose, and the latterwas located at the reducing end. Evidence that the disaccharideis isoprimeverose was provided by methylation analysis. Hydrolysisof the methylated disaccharide yielded 2,3,4-tri-O-methyl-D-xyloseand 2,3,4-tri-O-methyl-D-glucose. Thus, incorporation of radioactivityinto isoprimeverose, the smallest structural unit of xyloglucan,suggests that xyloglucan is synthesized in vitro from UDP-glucoseand UDP-xylose. (Received November 20, 1980; Accepted February 14, 1981)     

Phosphorylation of Simian Virus 40 Proteins in a Cell-Free System

We have shown previously that all the structural proteins of simian virus 40 (SV40) are phosphoproteins. Virus phosphorylated in vivo could be further phosphorylated with exogenous cellular protein kinases in a cell-free system containing gamma-(32)P-ATP as phosphate donor. In intact infectious virus only polypeptides 1 and 2 (mol wt 49,000 and 40,800, respectively) were further phosphorylated in vitro. However, when infectious SV40 was partially disrupted, treated with nucleases, and then phosphorylated in vitro, all five structural polypeptides accepted additional phosphate groups. Similarly, all polypeptides of intact empty capsids, derived from infected cells, were further phosphorylated in vitro. Phosphorylation of empty capsids and infectious SV40 in vitro was enhanced from 4- to 11-fold after prior treatment of virus with alkali. The phosphate group was linked only to serine residues of the viral polypeptides phosphorylated both in vitro and in vivo.     

Regulation of Homocysteine Biosynthesis in Salmonella typhimurium

The regulation of the homocysteine branch of the methionine biosynthetic pathway in Salmonella typhimurium has been reexamined with the aid of a new assay for the first enzyme. The activity of this enzyme is subject to synergistic feedback inhibition by methionine plus S-adenosylmethionine. The synthesis of all three enzymes of the pathway is regulated by noncoordinate repression. The enzymes are derepressed in metJ and metK regulatory mutants, suggesting the existence of regulatory elements common to all three. Experiments with a methionine/vitamin B(12) auxotroph (metE) grown in a chemostat on methionine or vitamin B(12) suggested that the first enzyme is more sensitive to repression by methionine derived from exogenous than from endogenous sources. metB and metC mutants grown on methionine in the chemostat did not show hypersensitivity to repression by exogenous methionine. Therefore, it appears that the metE chemostat findings are peculiar to the phenotype of this mutant; such evidence suggests a possible role for a functional methyltetrahydrofolate-homocysteine transmethylase in regulating the synthesis of the first enzyme. Thus there appear to be regulatory elements which are common to the repression of all three enzymes, as well as some that are unique to the first enzyme. The nature of the corepressor is not known, but it may be a derivative of S-adenosylmethionine. metJ and metK mutants of Salmonella have a normal capacity for S-adenosylmethionine synthesis but may be blocked in synthesis or utilization of a corepressor derived from it.     

ent-Kaurene Biosynthesis in Cell-Free Extracts of Excised Parts of Tall and Dwarf Pea Seedlings

Investigations on the sites of ent-kaur-16-ene (ent-kaurene) biosynthesis were conducted with cell-free extracts from several excised parts of 10-, 13-, and 16-d-old tall and dwarf pea (Pisum sativum L.) seedlings. [¹⁴C]Mevalonic acid was incorporated into ent-kaurene in cell-free extracts from young developing leaves and elongating internodes of tall (`Alaska') and dwarf (`Progress No.9') pea seedlings at all three stages of development. ent-Kaurene biosynthesis also occurred readily in cell-free extracts from shoot tips, petioles, and stipules near the young elongating internodes. The ent-kaurene-synthesizing activity found in young developing tissues declined as tissues matured. Little or no activity was detectable in enzyme extracts from cotyledons and root tips at different stages. In light grown tall pea internodes ent-kaurene-synthesizing activity was low as they began to elongate, reached a maximum when the internodes reached about 2 cm in length and declined as they matured. Activity in extracts of dwarf shoot tips and internodes was generally lower than in equivalent tall plants, but the activity in dwarf leaves and stipules was somewhat higher than in tall plants. With the exception of root tips, there is a strong correlation between growth potential of a tissue and the rate of ent-kaurene biosynthesis in extracts from that tissue.     

Preparation of a Cell-Free, Isobutene-Forming System from Rhodotorula minuta

The cell-free isobutene-forming system of Rhodotorula minuta IFO 1102 was prepared. This system consisted of isovaleric acid, NADPH, and a cell extract. The maximal activity obtained by using the described cell-free system was 17 nl/mg of protein per h. It appears that this cell-free isobutene-forming system operates in living cells of R. minuta.     

Biosynthesis of the O Antigen from Citrobacter 139

The biosynthesis of the O antigen of Citrobacter 139 (Escherichia coli 3 Zurich 4,5,12:z(20)) was shown to proceed through a series of lipid-linked intermediates, similar to those involved in O-antigen synthesis in Salmonella. Galactose was the first sugar incorporated, followed by rhamnose and mannose. Abequose was incorporated from cytidine diphosphate (CDP)-abequose only when all three of the other nucleotide sugars (uridine diphosphate galactose, guanosine diphosphate mannose, and thymidine diphosphate rhamnose) were present. Rhamnosyl-galactosyl 1-phosphate and mannosyl-rhamnosyl-galactosyl 1-phosphate were identified as the products of mild alkaline hydrolysis of the lipid-linked intermediates.     

A Radioimmunoassay-Based Method for Measuring the True Affinity of a Monoclonal Antibody with Trace Amounts of Radioactive Antigen: Illustration with the Products of a Cell-Free Protein Synthesis System

This communication describes a novel highly sensitive method for measuring the affinity of a monoclonal antibody for its antigen. It is based on a radioimmunoassay in which the antigen is labeled with radioactivity. It is therefore particularly well adapted to the study of trace amounts of radiolabeled polypeptide chains produced either in vivo, or in vitro by a cell free protein synthesis system or by chemical radiolabeling. It offers several advantages over previously described methods. Though making use of insolubilized antibody, it does measure the true affinity constant of the monoclonal antibody in solution for the antigen. It can be used even when the antigen is present at concentrations far below the dissociation constant of the antibody/antigen complex. It does not require the antigen or the antibody to be purified. In most cases, it requires no sophisticated equipment. This method could be easily adapted to the determination of the equilibrium constant of any type of protein/ligand system.     

Biosynthesis During Recovery of Heat-Injured Salmonella typhimurium

Salmonella typhimurium 7136 incorporated label from glucose-U-(14)C into nucleic acids, lipids, and pool material during recovery from heat injury. There was very little incorporation of label into protein during recovery.     

Glucosinolate Biosynthesis: Sulfation of Desulfobenzylglucosinolate by Cell-Free Extracts of Cress (Lepidium sativum L.) Seedlings

After removal of myrosinase activity by concanavalin A-Sepharose 4B chromatography, cell-free extracts of light-grown cress (Lepidium sativum L.) seedlings, catalyzed the sulfation of desulfobenzylglucosinolate (K_m, 0.23 millimolar) to benzylglucosinolate using PAPS (K_m, 1 millimolar) as sulfur donor. Sulfotransferase activity, which was optimal at pH 9.0, was stimulated by MgCl₂, MnCl₂, β-mercaptoethanol, and dithiothreitol and was inhibited by ZnSO₄ and SH-reagents. The enzyme also sulfated desulfoallyglucosinolate to allylglucosinolate (sinigrin) but was inactive towards all phenylpropanoids and flavonoids tested.     

Flavanone Glycoside Biosynthesis in Citrus: Chalcone Synthase, UDP-Glucose:Flavanone-7-O-Glucosyl-Transferase and -Rhamnosyl-Transferase Activities in Cell-Free Extracts

Previous indirect evidence suggested that the biosynthesis of flavonoids in Citrus may not proceed via the usual chalcone synthase reaction and that glycosylation occurs during chalcone formation and not afterward, as has been reported in other species. We detected chalcone-synthase and UDP-glucose:flavanone-7-O-glucosyl-transferase activities in cell-free extracts of Citrus. The glucosylated flavanone was further rhamnosylated when exogenous UDP-glucose and NADPH were added to the extract. Chalcone-synthase activity was detected in cell-free extracts derived from young leaves and fruits. Young fruits (2 millimeter diameter) had the highest chalcone synthase activity. UDP-glucose:flavanone-7-O-glucosyl-transferase activity was measured in cell-free extracts derived from young leaves and fruits of Citrus mitis and Citrus maxima. The highest UDP-glucose:flavanone-7-O-glucosyl-transferase activity was found in young C. maxima leaves. These data indicate that Citrus contains a flavonoid pathway similar to that studied in other species.     

Inhibition of T-Cell Proliferation Induced by a Cell-Free Salmonella typhimurium Extract Does Not Involve a Nitric Oxide-Mediated Mechanism

In a previous study, we observed that a cell-free Salmonella typhimurium extract induced suppression of mitogen-induced T-cell proliferation and that this suppression involved non-responsiveness of T-cells to interleukin-2 (IL-2) and augmentation of IL-2 receptor (IL-2R) expression. In this study, we found that inhibition of phytohemagglutinin (PHA)-stimulated murine spleen cell proliferation induced by a cell-free S. typhimurium extract was reversed by treatment with an anti-interferon-γ monoclonal antibody (anti-IFN-γ Ab), but not by interleukin-4 or N^G-monomethyl-l -arginine, which is known to inhibit nitric oxide (NO)-secretion from spleen cells in culture. However, IL-2R expression was augmented by treatment with the extract, although this was independent of an NO-mediated mechanism. Only anti-IFN-γ Ab treatment reduced the augmented IL-2R expression to a normal level. These results suggest that the suppression of T-cell proliferation induced by the Salmonella cell-free extract is associated with augmentation of IL-2R expression in an NO production-independent manner.