Yeast-based bioproduction of disulfide-rich peptides and their cyclization via asparaginyl endopeptidases.

Cyclic disulfide-rich peptides have attracted significant interest in drug development and biotechnology. Here, we describe a protocol for producing cyclic peptide precursors in Pichia pastoris that undergo in vitro enzymatic maturation into cyclic peptides using recombinant asparaginyl endopeptidases (AEPs). Peptide precursors are expressed with a C-terminal His tag and secreted into the media, enabling facile purification by immobilized metal affinity chromatography. After AEP-mediated cyclization, cyclic peptides are purified by reverse-phase high-performance liquid chromatography and characterized by mass spectrometry, peptide mass fingerprinting, NMR spectroscopy, and activity assays. We demonstrate the broad applicability of this protocol by generating cyclic peptides from three distinct classes that are either naturally occurring or synthetically backbone cyclized, and range in size from 14 amino acids with one disulfide bond, to 34 amino acids with a cystine knot comprising three disulfide bonds. The protocol requires 14 d to identify and optimize a high-expressing Pichia clone in small-scale cultures (24 well plates or 50 mL tubes), after which large-scale production in a bioreactor and peptide purification can be completed in 10 d. We use the cyclotide Momordica cochinchinensis trypsin inhibitor II as an example. We also include a protocol for recombinant AEP production in Escherichia coli as AEPs are emerging tools for orthogonal peptide and protein ligation. We focus on two AEPs that preferentially cyclize different peptide precursors, namely an engineered AEP with improved catalytic efficiency [C247A]OaAEP1b and the plant-derived MCoAEP2. Rudimentary proficiency and equipment in molecular biology, protein biochemistry and analytical chemistry are needed.

Milk whey from different animal species stimulates the in vitro release of CCK and GLP-1 through a whole simulated intestinal digestion.

Milk whey is effective in enhancing satiety mainly due to its protein composition. Peptides and amino acids derived from digestion of whey protein can act as suppressants of appetite by stimulation of receptors of satiety gut hormones. But, the protein fraction of whey can vary depending on species of animal, season, lactation period, etc. The aim of this study is to evaluate the satiety effect of milk whey from different species of ruminants (cow, sheep, goat and a mixture of them) through a simulated in vitro digestion, which performed the whole gastrointestinal process, from oral digestion to colonic fermentation. The satiety effect of each sample was measured by the production of satiating hormones (CCK and GLP-1) secreted by enteroendocrine cell line (STC-1) after 2 hours of incubation with non-digested, digested and fermented whey. Digested samples have shown to be potent CCK and GLP-1 secretagogues followed by fermented and non-digested samples, showing that the last one showed a weak hormone stimulation. Digested goat whey was the most efficient stimulator of GLP-1 (86.33 ± 4.55 pg mL-1) and fermented mixture whey produced the major release of CCK (80.78±1.81 pg mL-1). This study demonstrates that milk whey is a suitable ingredient to stimulate satiety through the effect of peptides, amino acids produced from digestion, and metabolites released by fermentation.

Synthesis and evaluation of biological activity of benzoxaborole derivatives of azithromycin.

Novel benzoxaborole derivatives of azithromycin in which benzoxaborole residue is attached to the 4″-hydroxy-group of azithromycin have been synthesized. Antibacterial activity of synthesized derivatives in comparison with azithromycin was tested on a panel of Gram-positive and Gram-negative bacterial strains. All the investigated compounds demonstrated broad spectrum of antibacterial activity being in general more active against Gram-positive strains. New benzoxaborole derivatives of azithromycin demonstrated high activity against Streptococcus pyogenes ATCC 19615 and Propionibacterium acnes ATCC 6919 strains. Some of the new compounds were more active than azithromycin against Streptococcus pneumoniae ATCC 49619 strain or Enterococcus faecium strains. Using a reporter construct created on the basis of the transcription attenuator region of the Escherichia coli tryptophan operon pRFPCER-TrpL2A it has been demonstrated that the mechanism of action of azithromycin analogs is blocking nascent peptide in ribosome tunnel.

Erythromycin leads to differential protein expression through differences in electrostatic and dispersion interactions with nascent proteins.

The antibiotic activity of erythromycin, which reversibly binds to a site within the bacterial ribosome exit tunnel, against many gram positive microorganisms indicates that it effectively inhibits the production of proteins. Similar to other macrolides, the activity of erythromycin is far from universal, as some peptides can bypass the macrolide-obstructed exit tunnel and become partially or fully synthesized. It is unclear why, at the molecular level, some proteins can be synthesized while others cannot. Here, we use steered molecular dynamics simulations to examine how erythromycin inhibits synthesis of the peptide ErmCL but not the peptide H-NS. By pulling these peptides through the exit tunnel of the E.coli ribosome with and without erythromycin present, we find that erythromycin directly interacts with both nascent peptides, but the force required for ErmCL to bypass erythromycin is greater than that of H-NS. The largest forces arise three to six residues from their N-terminus as they start to bypass Erythromycin. Decomposing the interaction energies between erythromycin and the peptides at this point, we find that there are stronger electrostatic and dispersion interactions with the more C-terminal residues of ErmCL than with H-NS. These results suggest that erythromycin slows or stalls synthesis of ErmCL compared to H-NS due to stronger interactions with particular residue positions along the nascent protein.

(R)-α-trifluoromethylalanine containing short peptide in the inhibition of amyloid peptide fibrillation.

The extracellular deposition of insoluble amyloid fibrils resulting from the aggregation of the amyloid-ß (Aß) is a pathological feature of neuronal loss in Alzheimer's disease (AD). Numerous small molecules have been reported to interfere with the process of Aß aggregation. Compounds containing aromatic structures, hydrophobic amino acids and/or the α-aminoisobutyric acid (Aib) as ß-sheet breaker elements have been reported to be effective inhibitors of Aß aggregation. We synthesized two peptides, one containing the Aib amino acid and the other including its trifluoromethylated analog (R)-α-Trifluoromethylalanine ((R)-Tfm-Alanine) and we evaluated the impact of these peptides on Aß amyloid formation. The compounds were tested by standard methods such as thioflavin-T fluorescence spectroscopy and transmission electron microscopy but also by circular dichroism, liquid state nuclear magnetic resonance (NMR) and NMR saturation transfer difference (STD) experiments to further characterize the effect of the two molecules on Aß structure and on the kinetics of depletion of monomeric, soluble Aß. Our results demonstrate that the peptide containing Aib reduces the quantity of aggregates containing ß-sheet structure but slightly inhibits Aß fibril formation, while the molecule including the trifluoromethyl (Tfm) group slows down the kinetics of Aß fibril formation, delays the random coil to ß-sheet structure transition and induces a change in the oligomerization pathway. These results suggest that the hydrophobic Tfm group has a better affinity with Aß than the methyl groups of the Aib and that this Tfm group is effective and important in preventing the Aß aggregation.

C-Abl inhibitor imatinib enhances insulin production by ß cells: c-Abl negatively regulates insulin production via interfering with the expression of NKx2.2 and GLUT-2.

Chronic myelogenous leukemia patients treated with tyrosine kinase inhibitor, Imatinib, were shown to have increased serum levels of C-peptide. Imatinib specifically inhibits the tyrosine kinase, c-Abl. However, the mechanism of how Imatinib treatment can lead to increased insulin level is unclear. Specifically, there is little investigation into whether Imatinib directly affects ß cells to promote insulin production. In this study, we showed that Imatinib significantly induced insulin expression in both glucose-stimulated and resting ß cells. In line with this finding, c-Abl knockdown by siRNA and overexpression of c-Abl markedly enhanced and inhibited insulin expression in ß cells, respectively. Unexpectedly, high concentrations of glucose significantly induced c-Abl expression, suggesting c-Abl may play a role in balancing insulin production during glucose stimulation. Further studies demonstrated that c-Abl inhibition did not affect the major insulin gene transcription factor, pancreatic and duodenal homeobox-1 (PDX-1) expression. Of interest, inhibition of c-Abl enhanced NKx2.2 and overexpression of c-Abl in ß cells markedly down-regulated NKx2.2, which is a positive regulator for insulin gene expression. Additionally, we found that c-Abl inhibition significantly enhanced the expression of glucose transporter GLUT2 on ß cells. Our study demonstrates a previously unrecognized mechanism that controls insulin expression through c-Abl-regulated NKx2.2 and GLUT2. Therapeutic targeting ß cell c-Abl could be employed in the treatment of diabetes or ß cell tumor, insulinoma.

MHC class I antigen presentation of DRiP-derived peptides from a model antigen is not dependent on the AAA ATPase p97.

CD8(+) T cells are responsible for killing cells of the body that have become infected or oncogenically transformed. In order to do so, effector CD8(+) T cells must recognize their cognate antigenic peptide bound to a MHC class I molecule that has been directly presented by the target cell. Due to the rapid nature of antigen presentation, it is believed that antigenic peptides are derived from a subset of newly synthesized proteins which are degraded almost immediately following synthesis and termed Defective Ribosomal Products or DRiPs. We have recently reported on a bioassay which can distinguish antigen presentation of DRiP substrates from other forms of rapidly degraded proteins and found that poly-ubiquitin chain disassembly may be necessary for efficient DRiP presentation. The AAA ATPase p97 protein is necessary for efficient cross-presentation of antigens on MHC class I molecules and plays an important role in extracting mis-folded proteins from the endoplasmic reticulum. Here, we find that genetic ablation or chemical inhibition of p97 does not diminish DRiP antigen presentation to any great extent nor does it alter the levels of MHC class I molecules on the cell surface, despite our observations that p97 inhibition increased the levels of poly-ubiquitinated proteins in the cell. These data demonstrate that inhibiting poly-ubiquitin chain disassembly alone is insufficient to abolish DRiP presentation.

Navß subunits modulate the inhibition of Nav1.8 by the analgesic gating modifier µO-conotoxin MrVIB.

Voltage-gated sodium channels (VGSCs) consist of a pore-forming α-subunit and regulatory ß-subunits. Several families of neuroactive peptides of Conus snails target VGSCs, including µO-conotoxins and µ-conotoxins. Unlike µ-conotoxins and the guanidinium alkaloid saxitoxin (STX), which are pore blockers, µO-conotoxins MrVIA and MrVIB inhibit VGSCs by modifying channel gating. µO-MrVIA/B can block Na(v)1.8 (a tetrodotoxin-resistant isoform of VGSCs) and have analgesic properties. The effect of Na(v)ß-subunit coexpression on susceptibility to block by µO-MrVIA/B and STX has, until now, not been reported. Here, we show that ß1-, ß2-, ß3-, and ß4-subunits, when individually coexpressed with Na(v)1.8 in Xenopus laevis oocytes, increased the k(on) of the block produced by µO-MrVIB (by 3-, 32-, 2-, and 7-fold, respectively) and modestly decreased the apparent k(off). Strong depolarizing prepulses markedly accelerated MrVIB washout with rates dependent on ß-subunit coexpression. Thus, coexpression of ß-subunits with Na(v)1.8 can strongly influence the affinity of the conopeptide for the channel. This observation is of particular interest because ß-subunit expression can be dynamic, e.g., ß2-expression is up-regulated after nerve injury (J Neurosci, 25:10970-10980, 2005); therefore, the effectiveness of a µO-conotoxin as a channel blocker could be enhanced by the conditions that may call for its use therapeutically. In contrast to MrVIB's action, the STX-induced block of Na(v)1.8 was only marginally, if at all, affected by coexpression of any of the ß-subunits. Our results raise the possibility that µO-conotoxins and perhaps other gating modifiers may provide a means to functionally assess the ß-subunit composition of VGSC complexes in neurons.

Toxicity and glutathione implication in the effects observed by exposure of the liver fish cell line PLHC-1 to pure cylindrospermopsin.

Cylindrospermopsin (CYN), a cyanotoxin produced by several freshwater cyanobacteria species, has been reported to cause human and animal intoxications. CYN is a potent inhibitor of protein and glutathione synthesis. In order to study these effects, various in vitro models have been used, which are representative of the organs targeted by the toxin. However, studies concerning CYN toxicity to fish species, both in vivo and in vitro, are still very scarce. To our knowledge, this is the first work dealing with the effects of CYN in a fish cell line. In the present work, we tried to test the hypothesis that CYN could be hepatotoxic to fish causing cell damage and oxidative stress, which may lead to pathogenicity. To deal this purpose, PLCH-1 cells, derived from fish liver, were exposed to concentrations that ranged from 0.3 to 40 µg/mL CYN during 24 and 48 h for the cytotoxicity study, and 2, 4 and 8 µg/mL CYN for the oxidative stress assays. The basal cytotoxicity endpoints studied were protein content, neutral red uptake and the tetrazolium salt, MTS, reduction. The biomarkers used for the oxidative stress study were reactive oxygen species (ROS) content, reduced glutathione content and γ-glutamylcysteine synthetase activity. The cytotoxicity endpoints revealed a decrease in the cellular viability in a time and concentration-dependent way. Moreover, when cells were exposed to pure CYN, an increase in the ROS content was observed, being more marked at the higher concentrations used. Finally, the present work shows alterations in GSH content and synthesis due to CYN. Moreover, a relationship between cytotoxic effects and ROS production has been evidenced. The results obtained confirm the alteration on fish liver cells, which should be considered relevant to what it may happen in real scenarios since fish are frequently in contact with this cyanotoxin.

Distinct pathways generate peptides from defective ribosomal products for CD8+ T cell immunosurveillance.

To understand better the endogenous sources of MHC class I peptide ligands, we generated an antigenic reporter protein whose degradation is rapidly and reversibly controlled with Shield-1, a cell-permeant drug. Using this system, we demonstrate that defective ribosomal products (DRiPs) represent a major and highly efficient source of peptides and are completely resistant to our attempts to stabilize the protein. Although peptides also derive from nascent Shield-1-sensitive proteins and "retirees" created by Shield-1 withdrawal, these are much less efficient sources on a molar basis. We use this system to identify two drugs--each known to inhibit polyubiquitin chain disassembly--that selectively inhibit presentation of Shield-1-resistant DRiPs. These findings provide the initial evidence for distinct biochemical pathways for presentation of DRiPs versus retirees and implicate polyubiquitin chain disassembly or the actions of deubiquitylating enzymes as playing an important role in DRiP presentation.

RNA polymerase II inhibitors dissociate antigenic peptide generation from normal viral protein synthesis: a role for nuclear translation in defective ribosomal product synthesis?

Following viral infection, cells rapidly present peptides from newly synthesized viral proteins on MHC class I molecules, likely from rapidly degraded forms of nascent proteins. The nature of these defective ribosomal products (DRiPs) remains largely undefined. Using inhibitors of RNA polymerase II that block influenza A virus neuraminidase (NA) mRNA export from the nucleus and inhibit cytoplasmic NA translation, we demonstrate a surprising disconnect between levels of NA translation and generation of SIINFEKL peptide genetically inserted into the NA stalk. A 33-fold reduction in NA expression is accompanied by only a 5-fold reduction in K(b)-SIINFEKL complex cell-surface expression, resulting in a net 6-fold increase in the overall efficiency of Ag presentation. Although the proteasome inhibitor MG132 completely blocked K(b)-SIINFEKL complex generation, we were unable to biochemically detect a MG132-dependent cohort of NA DRiPs relevant for Ag processing, suggesting that a minute population of DRiPs is a highly efficient source of antigenic peptides. These data support the idea that Ag processing uses compartmentalized translation, perhaps even in the nucleus itself, to increase the efficiency of the generation of class I peptide ligands.

Production of peptide antifungal antibiotic and biocontrol activity of Bacillus subtilis.

Among different bacterial cultures, a potent Bacillus subtilis MTCC-8114 was isolated from garden soil samples which showed 16 and 14 mm inhibition zones by spot inoculation method and 24 and 22 mm inhibition zones by well agar diffusion method against test fungi i.e. Microsporum fulvum and Trichophyton species. Among four media tested, the maximum growth and antibiotic production was found in trypticase soya broth (TSB) medium at 37 degrees C, pH-7 and 48 h of incubation. The Rf value (0.64) by Thin Layer Chromatography (TLC) technique and UV and FTIR spectral data of the active antifungal compound, indicated that the isolated compound belongs to peptide antifungal antibiotic group. MIC value of antifungal antibiotic was 135 and 145 microg/ml.

Tight linkage between translation and MHC class I peptide ligand generation implies specialized antigen processing for defective ribosomal products.

There is mounting evidence that MHC class I peptide ligands are predominantly generated from defective ribosomal products and other classes of polypeptides degraded rapidly (t1/2 < 10 min) following their synthesis. The most direct evidence supporting this conclusion is the rapid inhibition of peptide ligand generation following cycloheximide-mediated inhibition of protein synthesis. In this study, we show that this linkage is due to depleting the pool of rapidly degraded proteins, and not to interference with other protein synthesis-dependent processes. Our findings indicate that in the model systems used in this study, MHC class I peptides are preferentially generated from rapidly degraded polypeptides relative to slowly degraded proteins. This conclusion is supported by the properties of peptide presentation from slowly degraded (t1/2 = 4 h) defective ribosomal products generated artificially by incorporation of the amino acid analog canavanine into a model viral Ag. We propose that specialized machinery exists to link protein synthesis with class I peptide ligand generation to enable the rapid detection of viral gene expression.

Corticosterone impairs MHC class I antigen presentation by dendritic cells via reduction of peptide generation.

The presentation of viral peptide-MHC class I complexes by antigen presenting cells, such as dendritic cells (DCs), is obligatory for the generation of antiviral effector and memory CD8(+) cytotoxic T lymphocyte (CTL) responses. Prolonged psychological stress is immunosuppressive and undermines primary and memory CTL-mediated antiviral immunity; however, the mechanisms involved are unknown. Using a panel of novel reagents and techniques, we quantitatively measured the effect of the stress-induced hormone corticosterone (CORT) on the efficiency of DCs to process and present virally expressed antigen, characterized the conditions for this CORT-mediated effect, and delineated the components of the MHC class I pathway that were affected. We found that physiologically relevant levels of CORT, prior to infection and acting via the glucocorticoid receptor, suppressed the formation of peptide-MHC class I complexes on the surface of infected DCs. We further showed that this suppression of peptide-MHC class I complexes is via the action of CORT on elements of the class I pathway upstream from TAP that are involved in the generation of antigenic peptides. This CORT-mediated suppression of peptide-class I complexes on DCs also resulted in a marked reduction of their ability to activate a specific T cell hybridoma. These findings offer a mechanism contributing to the stress-induced suppression of host defenses against viral diseases and have implications for the efficacy of antiviral vaccines. At the most fundamental cellular level, this impairment of antigen processing has implications for the regulation of protein degradation in all cells, which is critical to many aspects of immune function.

Expression of adrenomedullin and proadrenomedullin N-terminal 20 peptide in PC12 cells after exposure to nerve growth factor.

Adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) are multi-functional peptides derived from the same precursor, proadrenomedullin. We have studied the regulatory mechanism of expression of these peptides during neuronal differentiation of rat pheochromocytoma PC12 cells by nerve growth factor (NGF). The cellular levels of the peptides increased slightly, and then progressively decreased below the control by NGF. Immunoreactive (ir)-AM in the medium was transiently increased by NGF. Cytochemical staining showed that ir-AM and ir-PAMP were abundantly present in cytoplasm in the undifferentiated cells, and were decreased during culture with NGF. There was no preferential localization of ir-AM or ir-PAMP in neurites in comparison with in cytoplasm in the differentiated cells. Northern blot analysis showed that mRNA encoding these peptides, as detected as a band of 1.6 kb, increased more than three-fold at 1 h after the addition of NGF and then progressively decreased to one fifth of the control during 72 h. Degradation rate of the mRNA was slowed by NGF even when mRNA level is decreased after 72 h of NGF treatment. The transcription rate of their gene increased transiently and then decreased by the long-term treatment with NGF. These results demonstrate that expression of AM and PAMP is regulated by NGF along with time-dependent differentiation: AM gene transcription is transiently activated by NGF, whereas it was suppressed during neuronal differentiation of the cells.

Mononucleotide derivatives as ribosomal P-site substrates reveal an important contribution of the 2'-OH to activity.

The chemical synthesis of various acylaminoacylated mononucleotides is described and their activities as donor substrates for the ribosomal peptide synthesis were investigated using PhetRNA(Phe) as an acceptor. This minimal reaction was characterized in detail and was shown to be stimulated by CMP, cytidine and cytosine. By using several cytidine and cytosine analogs evidence is provided that this enhancement is rather caused by base pairing to rRNA, followed by a structural change, than by a base mediated general acid/base catalysis. Only derivatives of AMP proved active as P-site substrates. Further, a significant contribution of the 2'-OH to activity was indicated by the finding that AcLeu-dAMP was inactive as donor substrate, although it is a good inhibitor of peptide bond formation and thus, is presumably bound to the P-site. However, Di(AcLeu)-2'-OCH(3)-Ade and DiAcLeu-AMP were moderately active in this assay suggesting that the reactivity of the 3'-acylaminoacid ester is stimulated by the presence of the 2'-oxygen group. A model is discussed how further interactions of the 2'-OH in the transition state might influence peptidyl transferase activity.

Ginkgo biloba extract inhibits tumor necrosis factor-alpha-induced reactive oxygen species generation, transcription factor activation, and cell adhesion molecule expression in human aortic endothelial cells.

OBJECTIVE: This study was conducted to examination whether Ginkgo biloba extract (GBE), a Chinese herb with antioxidant activity, could reduce cytokine-induced monocyte/human aortic endothelial cell (HAEC) interaction, a pivotal early event in atherogenesis. METHODS AND RESULTS: Pretreatment of HAECs with GBE (50 and 100 microg/mL for 18 hours) significantly suppressed cellular binding between the human monocytic cell line U937 and tumor necrosis factor-alpha (TNF-alpha)-stimulated HAECs by using in vitro binding assay (68.7% and 60.1% inhibitions, respectively). Cell enzyme-linked immunosorbent assay and immunoblot analysis showed that GBE (50 microg/mL for 18 hours) significantly attenuated TNF-alpha-induced cell surface and total protein expression of vascular cellular adhesion molecule-1 and intracellular adhesion molecule-1 (63.5% and 69.2%, respectively; P<0.05). However, pretreatment with probucol (5 micromol/L for 18 hours) reduced the expression of vascular cellular adhesion molecule-1 but not intracellular adhesion molecule-1. Preincubation of HAECs with GBE or probucol significantly reduced intracellular reactive oxygen species formation induced by TNF-alpha (76.8% and 68.2% inhibitions, respectively; P<0.05). Electrophoretic mobility shift assay demonstrated that both GBE and probucol inhibited transcription factor nuclear factor-kappaB activation in TNF-alpha-stimulated HAECs (55.2% and 65.6% inhibitions, respectively) but only GBE could inhibit the TNF-alpha-stimulated activator protein 1 activation (45.1% inhibition, P<0.05). CONCLUSIONS: GBE could reduce cytokine-stimulated endothelial adhesiveness by downregulating intracellular reactive oxygen species formation, nuclear factor-kappaB and activator protein 1 activation, and adhesion molecule expression in HAECs, supporting the notion that the natural compound Ginkgo biloba may have potential implications in clinical atherosclerosis disease.

Cylindrospermopsin-induced protein synthesis inhibition and its dissociation from acute toxicity in mouse hepatocytes.

The toxicology of the cyanobacterial alkaloid cylindrospermopsin (CYN), a potent inhibitor of protein synthesis, appears complex and is not well understood. In exposed mice the liver is the main target for the toxic effects of CYN. In this study primary mouse hepatocyte cultures were used to investigate the mechanisms involved in CYN toxicity. The results show that 1-5 microM CYN caused significant concentration-dependent cytotoxicity (52%-82% cell death) at 18 h. Protein synthesis inhibition was a sensitive, early indicator of cellular responses to CYN. Following removal of the toxin, the inhibition of protein synthesis could not be reversed, showing behavior similar to that of the irreversible inhibitor emetine. In contrast to the LDH leakage, protein synthesis was maximally inhibited by 0.5 microM CYN. No protein synthesis occurred over 4-18 h at or above this concentration. Inhibition of cytochrome P450 (CYP450) activity with 50 microM proadifen or 50 microM ketoconazole diminished the toxicity of CYN but not the effects on protein synthesis. These findings imply a dissociation of the two events and implicate the involvement of CYP450-derived metabolites in the toxicity process, but not in the impairment of protein synthesis. Thus, the total abolition of protein synthesis may exaggerate the metabolite effects but cannot be considered a primary cause of cell death in hepatocytes over an acute time frame. In cell types deficient in CYP450 enzymes, protein synthesis inhibition may play a more crucial role in the development of cytotoxicity.

Effect of polyamines on the inhibition of peptidyltransferase by antibiotics: revisiting the mechanism of chloramphenicol action.

Chloramphenicol is thought to interfere competitively with the binding of the aminoacyl-tRNA 3'-terminus to ribosomal A-site. However, noncompetitive or mixed-noncompetitive inhibition, often observed to be dependent on chloramphenicol concentration and ionic conditions, leaves some doubt about the precise mode of action. Here, we examine further the inhibition effect of chloramphenicol, using a model system derived from Escherichia coli in which a peptide bond is formed between puromycin and AcPhe-tRNA bound at the P-site of poly(U)-programmed ribosomes, under ionic conditions (6 mM Mg2+, 100 mM NH4+, 100 microM spermine) more closely resembling the physiological status. Kinetics reveal that chloramphenicol (I) reacts rapidly with AcPhe-tRNA.poly(U).70S ribosomal complex (C) to form the encounter complex CI which is then isomerized slowly to a more tight complex, C*I. A similar inhibition pattern is observed, if complex C modified by a photoreactive analogue of spermine, reacts in buffer free of spermine. Spermine, either reversibly interacting with or covalently attached to ribosomes, enhances the peptidyltransferase activity and increases the chloramphenicol potency, without affecting the isomerization step. As indicated by photoaffinity labeling, the peptidyltransferase center at which chloramphenicol binds, is one of the preferred cross-linking sites for polyamines. This fact may explain the effect of spermine on chloramphenicol binding to ribosomes.

Continuous cell-free protein synthesis directed by messenger DNA and catalyzed by extract of Thermus thermophilus HB27.

Polypeptide synthesis directed by DNA as the messenger in a cell-free system of Thermus thermophilus was investigated. Polypeptides were synthesized with the addition of neomycin in the presence of DNA catalyzed by the cell extract. The stability of messenger DNA was greater than that of messenger RNA. Continuous cell-free translation with messenger DNA produced polypeptides at the rate of more than 8 microg/h in the presence of spermine.