Plasmid DNA delivery by arginine-rich cell-penetrating peptides containing unnatural amino acids

Cell-penetrating peptides (CPPs) have been developed as drug, protein, and gene delivery tools. In the present study, arginine (Arg)-rich CPPs containing unnatural amino acids were designed to deliver plasmid DNA (pDNA). The transfection ability of one of the Arg-rich CPPs examined here was more effective than that of the Arg nonapeptide, which is the most frequently used CPP. The transfection efficiencies of Arg-rich CPPs increased with longer post-incubation times and were significantly higher at 48-h and 72-h post-incubation than that of the commercially available transfection reagent TurboFect. These Arg-rich CPPs were complexed with pDNA for a long time in cells and effectively escaped from the late endosomes/lysosomes into the cytoplasm. These results will be helpful for designing novel CPPs for pDNA delivery.     

Identification of metal ion binding peptides containing unnatural amino acids by phage display

The bidentate metal binding amino acid bipyridylalanine (BpyAla) was incorporated into a disulfide linked cyclic peptide phage displayed library to identify metal ion binding peptides. Selection against Ni²⁺–nitrilotriacetic acid (NTA) enriched for sequences containing histidine and BpyAla. BpyAla predominated when selections were carried out at lower pH, consistent with the differential pK_a’s of histidine and BpyAla. Two peptides containing BpyAla were synthesized and found to bind Ni²⁺ with low micromolar dissociation constants. Incorporation of BpyAla and other metal binding amino acids into peptide and protein libraries should enable the evolution of novel binding and catalytic activities.     

Enhancing the incorporation of lysine derivatives into proteins with methylester forms of unnatural amino acids

We have improved the incorporation of l- and d-forms of unnatural amino acid (UAA) N^ε-thiaprolyl-l-lysine (ThzK) into ubiquitin (UB) and green fluorescent protein (GFP) by 2–6 folds with the use of the methylester forms of the UAAs in E coli cell culture. We also improved the yields of UAA-incorporated UB and GFP with the methylester forms of N^ε-Boc-l-Lysine (BocK) and N^ε-propargyl-l-Lysine (PrK) by 2–5 folds compared to their free acid forms. Our work demonstrated that using methylester-capped UAAs for protein expression is a useful strategy to enhance the yields of UAA-incorporated proteins.     

Utilization of aromatic amino acids as nitrogen sources in Brevibacterium linens: an inducible aromatic amino acid aminotransferase

The first step of the utilization of the aromatic amino acids as sole nitrogen sources by Brevibacterium linens strain 47 was found to be a transamination. The deaminated metabolites of the amino acids were detected in culture supernatants, and the enzyme activity was identified in cell free extracts. The cells contained increased aromatic amino acid aminotransferase activities on growth on the aromatic amino acids as sole nitrogen sources. Two aromatic aminotransferases (AT-I and AT-II) were separated upon diethylaminoethyl-Trisacryl M column chromatography of cell free extracts. Only AT-I was responsible for the increased level of aromatic amino acid aminotransferase activity of induced cells. The results suggested a catabolic role of AT-I in vivo.Abbreviations DNP dinitrophenyl - HPLC high performance liquid chromatography - PLP pyridoxal-5-phosphate     

Molecular characterization of ribonucleoproteic antigens containing repeated amino acid sequences from Trypanosoma cruzi

Trypanosoma cruzi expresses several proteins containing antigenic amino acid repeats. Here we characterized TcRpL7a and TcRBP28, which carry similar repeat motifs and share homology to the eukaryotic L7a ribosomal protein and to a Trypanosoma brucei RNA binding protein, respectively. Analyses of the full length and truncated recombinant TcRpL7a showed that the humoral response of patients with Chagas disease is directed towards its repetitive domain. Sequence analyses of distinct copies of TcRpL7a genes present in the genome of six T. cruzi strains indicate that the number of repeats is higher in proteins from T. cruzi II than T. cruzi I strains. A serum panel of 59 T. cruzi infected patients showed that 73% reacted with TcRpL7a, 71% reacted with TcRBP28 and 80% reacted with 1:1 mixture of both antigens. Synthetic peptides harboring the TcRpL7a repeat motif reacted with 46% of the serum samples. Antibodies raised against both antigens identified equivalent amounts of the native proteins in all three stages of the parasite life cycle. Analyses of subcellular fractions indicated that TcRBP28 is present in the cytoplasm whereas TcRpL7a co-fractionates with polysomes. Confirming their predicted cellular localization, GFP fusions showed that, whereas GFP::TcRBP28 localizes in the cytoplasm, GFP::TcRpL7a accumulates in the nucleus, where ribosome biogenesis occurs.     

The regulation of aromatic amino acid biosynthesis in amino acid liberating mutant strains of Anabaena variabilis

Mutant strains of Anabaena variabilis which are resistant to the tryptophan analogue, 6-fluorotryptophan, liberated a wide range of amino acids although none liberated tryptophan in detectable quantities. Four strains (FT-7, FT-8, FT-9, FT-10) produced predominantly alanine together with small amounts of phenylalamine and tyrosine, strain FT-2 liberated mainly phenylalanine and tyrosine and strain FT-6 liberated mainly glutamate, NH ₄ ⁺ and several unidentified ninhydrin-positive compounds. Two forms of 3-deoxy-D-arbinoheptulosonate 7-phosphate (DAHP) synthase were identified in the parent strain, a tyrosine-sensitive form and a phenylalanine-sensitive form. In strains FT-2 and FT-6 the phenylalanine-sensitive enzyme was not detected and in strain FT-7 it was apparently deregulated with respect to inhibition by phenylalanine. No deregulation of anthranilate synthase was observed but mutant strains were found to have higher specific activities of this enzyme than the parent strain.Abbreviations chla chlorophyll a - 6-FT 6-fluorotryptophan - DAHP 3-deoxy-D-arabinoheptulosonate 7-phosphate - PEP phosphoenolpyruvate     

Purification and characterization of Plasmodium yoelii adenosine deaminase

Plasmodium lacks the de novo pathway for purine biosynthesis and relies exclusively on the salvage pathway. Adenosine deaminase (ADA), first enzyme of the pathway, was purified and characterized from Plasmodium yoelii, a rodent malarial species, using ion exchange and gel exclusion chromatography. The purified enzyme is a 41 kDa monomer. The enzyme showed K_m values of 41 μM and 34 μM for adenosine and 2′-deoxyadenosine, respectively. Erythro-9-(2-hydroxy-3-nonyl) adenine competitively inhibited P. yoelii ADA with K_i value of 0.5 μM. The enzyme was inhibited by DEPC and protein denaturing agents, urea and GdmCl. Purine analogues significantly inhibited ADA activity. Inhibition by p-chloromercuribenzoate (pCMB) and N-ethylmaleimide (NEM) indicated the presence of functional –SH groups. Tryptophan fluorescence maxima of ADA shifted from 339 nm to 357 nm in presence of GdmCl. Refolding studies showed that higher GdmCl concentration irreversibly denatured the purified ADA. Fluorescence quenchers (KI and acrylamide) quenched the ADA fluorescence intensity to the varied degree. The observed differences in kinetic properties of P. yoelii ADA as compared to the erythrocyte enzyme may facilitate in designing specific inhibitors against ADA.     

Permeability of membranes to amino acids and modified amino acids: Mechanisms involved in translocation

Summary The amino acid permeability of membranes is of interest because they are one of the key solutes involved in cell function. Membrane permeability coefficients (P) for amino acid classes, including neutral, polar, hydrophobic, and charged species, have been measured and compared using a variety of techniques. Decreasing lipid chain length increased permeability slightly (5-fold), while variations in pH had only minor effects on the permeability coefficients of the amino acids tested in liposomes. Increasing the membrane surface charge increased the permeability of amino acids of the opposite charge, while increasing the cholesterol content decreased membrane permeability. The permeability coefficients for most amino acids tested were surprisingly similar to those previously measured for monovalent cations such as sodium and potassium (approximately 10^–12–10^–13 cm · s^–1). This observation suggests that the permeation rates for the neutral, polar and charged amino acids are controlled by bilayer fluctuations and transient defects, rather than partition coefficients and Born energy barriers. Hydrophobic amino acids were 10² more permeable than the hydrophilic forms, reflecting their increased partition coefficient values.External pH had dramatic effects on the permeation rates for the modified amino acid lysine methyl ester in response to transmembrane pH gradients. It was established that lysine methyl ester and other modified short peptides permeate rapidly (P = 10^–2 cm · s^–1) as neutral (deprotonated) molecules. It was also shown that charge distributions dramatically alter permeation rates for modified di-peptides. These results may relate to the movement of peptides through membranes during protein translocation and to the origin of cellular membrane transport on the early Earth.Abbreviations DCP dicetylphosphate - DMPC dimyristoyl phosphatidylcholine - EPC egg phosphatidylcholine - LUV large unilamellar vesicle - MLV multilamellar vesicle - PLM planar lipid membrane - SUV small unilamellar vesicle - pH transmembrane pH gradient     

Cloning and characterization of an aromatic amino acid and leucine permease of Penicillium chrysogenum

The gene encoding the amino acid permease ArlP (Aromatic and leucine Permease) was isolated from the filamentous fungus Penicillium chrysogenum after PCR using degenerated oligonucleotides based on conserved regions of fungal amino acid permeases. The cDNA clone was used for expression of the permease in Saccharomyces cerevisiae M4054, which is defective in the general amino acid permease Gap1. Upon overexpression, an increase in the uptake of l-tyrosine, l-phenylalanine, l-tryptophan and l-leucine was observed. Further competition experiments indicate that ArlP recognizes neutral and aromatic amino acids with an unbranched β-carbon atom.     

Purification and characterization of secreted acid phosphatase under phosphate-deficient condition inPholiota nameko

Activity of acid phosphatase secreted by mycelia ofPholiota nameko on cultivation for 30d in Pi-depleted medium was 88-fold higher than the corresponding activity in the Pi-supplied medium. One isozyme of the secreted acid phosphatases was purified from the culture filtrate of Pi-depleted medium by ammonium sulfate fractionation and cation exchange chromatography. The purified enzyme was homogeneous on electrophoresis. Gel filtration analysis showed change chromatography. The purified enzyme was homogeneous on electrophoresis. Gel filtration analysis showed that the native molecule had a molecular weight of 117,000. The molecular weight on gel electrophoresis with SDS was 52,000, indicating that the native form of the enzyme was a homodimer. The optimum pH and temperature of the enzyme were, 5.5 and 45°C, respectively, and the isoelectric point of the enzyme was pH 6.9. Adsorption on Con A-Sepharose and periodic-Schiff stain suggested that the enzyme is a glycoprotein. The enzyme hydrolyzed a wide variety of phosphate esters, nucleoside phosphates, sugar phosphates, and phosphorylated amino acids. Cu²⁺, Fe²⁺, Hg²⁺, iodoacetate, molybdate, tartaric acid, and SDS inhibited the enzyme activity. Fe³⁺ (1 mM), Triton X-100, methanol, and ethanol activated it. Fifteen residues of the N-terminal amino acid sequence were determined.     

Isolation and characterization of an unusual 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase gene from Enterobacter cloacae UW4

A genomic library of the 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-containing plant growth-promoting bacterium Enterobacter cloacae UW4 in pUC19 in Escherichia coli was screened for the ability to utilize ACC as a sole source of nitrogen. One of the clones that was isolated contained a plasmid with an insert of approximately 0.8 kb that conferred ACC deaminase activity. Sequence analysis revealed that this DNA fragment contains an open-reading frame of 696 nucleotides predicted to encode a protein of 232 amino acids, a member of the amidohydrolase protein superfamily, i.e., a deaminase that contains a mononuclear or binuclear metal center as compared to the canonical ACC deaminase which contains pyridoxal phosphate as a co-factor.     

Branched chain amino acid aminotransferase isoenzymes of Pseudomonas cepacia

Pseudomonas cepacia grew rapidly using a mixture of all three branched chain amino acids as carbon source, but failed to use individual branched chain amino acids as sole carbon source. Extracts of bacteria grown on branched chain amino acids had between 2- and 3-fold higher levels of -ketoglutarate-dependent branched chain amino acid aminotransferase activity than extracts of glucose-grown bacteria. The increase in enzyme activity was due to the presence of a second aminotransferase not detected in extracts of glucose-grown bacteria. The enzyme, which presumably plays a role in branched chain amino acid degradation, had an apparent molecular weight (mol. wt.) of 75,000. The other aminotransferase was formed constitutively and apparently functions in synthesis of branched chain amino acids. It was more stable than the 75,000 mol.wt. enzyme, and was purified to homogeneity and found to be a 180,000 mol.wt. oligomer containing 6 subunits of approximately 30,000 mol.wt. Antiserum prepared against the purified enzyme inhibited its activity but failed to influence the activity of the 75,000 mol.wt. aminotransferase, suggesting that the two isoenzymes are encoded by different genes.     

Effects of medium osmolarity on the release of amino acids from isolated cotyledons of developing pea seeds

The release of endogenous amino acids from isolated, immature pea (Pisum sativum L. cv. Marzia) cotyledons was investigated in relation to their developmental stage and the osmolarity of the bathing medium. The water potential of the cotyledons was about-1.1 MPa from which it could be inferred that the osmolarity of their apoplastic fluids will be approximately 450 mosmol·l^?1. The time course of amino-acid release conformed to an exponential function. Rate constants of the release were in the range 0.3 to 0.9 · h ^?1. No indication was found for increased permeability of the plasmamembrane for amino acids at low medium osmolarity. Rate constants were even 1.5-fold lower in 0 mM mannitol than in medium with 400 mM mannitol. This effect could be ascribed to reduced protein synthesis in hypotonic media. In the presence of 400 mM mannitol the release was nearly proportional to the total amino-acid pool of the cotyledons and ranged from 12% to 8% for the various developmental stages. Amino-acid release was stimulated by incubation in a hypotonic medium (< 400 mM mannitol), up to fourfold in a medium without mannitol where as much as 45% of the cotyledonary amino-acid content could be released. The extra aminoacid release induced by the hypotonic condition declined during development and eventually vanished completely. Release of amino acids into a medium with 400 mM mannitol was more selective than into a medium without mannitol. For instance, arginine was one of the main constituents of the cotyledonary amino-acid pool (19%) as well as of the released amino-acid mixture when the medium contained no mannitol (10%), whereas it was virtually absent when the medium contained 400 mM mannitol. As an overall interpretation of these results, it is proposed that the hypotonic condition greatly enhances the permeability of the tonoplast (not that of the plasmalemma) for amino acids so that the otherwise well-sequestered amino acids in the vacuole become available for release into the bathing medium.     

Novel biosynthetic routes to non-proteinogenic amino acids as chiral pharmaceutical intermediates

Transaminases catalyse the reversible transfer of amino and keto groups between an amino acid and keto acid substrate pair. Many bacterial transaminases accept a wide array of keto acids as amino acceptors and are useful as commercial biocatalysts in the preparation of amino acids. Since the reaction equilibrium typically lies close to unity, several approaches have been described to improve upon the 50% product yield, using additional enzymes. The present work describes an efficient means to significantly increase product yield in transamination using the aromatic transaminase of Escherichia coli encoded by the tyrB gene, with -aspartate as the amino donor. This is achieved by the introduction of the alsS gene encoding the acetolactate synthase of Bacillus subtilis, which eliminates pyruvate and alanine produced as a by-product of aspartate transamination. The biosynthesis of the non-proteinogenic amino acid -2-aminobutyrate is described using a recombinant strain of E. coli containing the cloned tyrB and alsS genes. The strain additionally carries the cloned ilvA gene of E. coli encoding threonine deaminase to produce the substrate 2-ketobutyrate from -threonine. An alternate coupled process uses lysine -aminotransferase in concert with a transaminase using -glutamate as the amino donor.     

Production of -amino acids by N-acyl- -amino acid amidohydrolase and its structure and function

-Amino acids have been widely used as synthetic materials for various compounds such as pharmaceuticals and agrochemicals. The manufacture of -amino acids by fermentation is difficult, and enzymatic methods are mainly employed. At present, the optical resolution method using N-acyl- -amino acid amidohydrolase is the most useful and convenient. In this review, the application of N-acyl- -amino acid amidohydrolase to the production of -amino acids and recent progress in the study of structure–function relationships from the standpoint of improving this enzyme for industrial application are discussed.     

Adenosine deaminase from Azotobacter vinelandii. Purification and properties

Adenosine deaminase (EC 3.5.4.4) was found to occur in the extract of Azotobacter vinelandii, strain 0, and purified by heating at 65°C, fractionation with ammonium sulfate, DEAE-cellulose chromatography and gel filtration on Sephadex G-150. Purified adenosine deaminase was effectively stabilized by the addition of ethylene glycol. The molecular weight of the enzyme was estimated to be 66,000 by gel filtration on Sephadex G-150. The enzyme specifically attacked adenosine and 2-deoxyadenosine to the same extent, and formycin A to a lesser extent. The pH optimum of the enzyme was observed at pH 7.2. Double reciprocal plot of initial velocity versus adenosine concentration was concave upward, and Hill interaction coefficient was calculated to be 1.5, suggesting the allosteric binding of the substrate. ATP inhibited adenosine deaminase in an allosteric manner, whereas other nucleotides were without effect. The physiological significance of the enzyme was discussed in relation to salvage pathway of purine nucleotides.     

Site-specific solvent exposure analysis of a membrane protein using unnatural amino acids and F nuclear magnetic resonance

Membrane proteins play an essential role in cellular metabolism, transportation and signal transduction across cell membranes. The scarcity of membrane protein structures has thus far prevented a full understanding of their molecular mechanisms. Preliminary topology studies and residue solvent exposure analysis have the potential to provide valuable information on membrane proteins of unknown structure. Here, a ¹⁹F-containing unnatural amino acid (trimethylfluoro-phenylalanine, tfmF) was applied to accomplish site-specific ¹⁹F spin incorporation at different sites in diacylglycerol kinase (DAGK, an Escherichia coli membrane protein) for site-specific solvent exposure analysis. Due to isotope effect on ¹⁹F spins, a standard curve for ¹⁹F-tfmF chemical shifts was drawn for varying solvent H₂O/D₂O ratios. Further site-specific ¹⁹F solvent isotope shift analysis was conducted for DAGK to distinguish residues in water-soluble loops, interfacial areas or hydrophobic membrane regions. This site-specific solvent exposure analysis method could be applied for further topological analysis of other membrane proteins.     

锁阳中氨基酸类化合物研究进展

锁阳（Cynomorium songaricum）是寄生于白刺属（Nitraria L.）植物根部的肉质草本植物,含有多种生物活性成分,如黄酮类、萜类、氨基酸等。氨基酸是锁阳中的一类重要化合物,与锁阳的品质密切相关,具有较大的研究价值。本文通过查阅近几年的相关文献,从锁阳中含有的氨基酸种类、含量、生物活性、检测方法及人工培育前景等方面对锁阳中氨基酸类成分的研究现状进行整理和归纳,旨在为锁阳中氨基酸类成分的进一步研究、开发和利用提供一定的支持与帮助。     

Purification and characterization of an acid phosphatase from the commercial mushroom Agaricus bisporus

Acid phosphatase [AP; EC 3.1.3.2], a key enzyme involved in the synthesis of mannitol in Agaricus bisporus, was purified to homogeneity and characterized. The native enzyme appeared to be a high molecular weight type glycoprotein. It has a molecular weight of 145 kDa and consists of four identical 39-kDa subunits. The isoelectric point of the enzyme was found at 4.7. Maximum activity occurred at 65°C. The optimum pH range was between 3.5 and 5.5, with maximum activity at pH 4.75. The enzyme was unaffected by EDTA, and inhibited by tartrate and inorganic phosphate. The enzyme exhibits a K _m for p-nitrophenylphosphate and fructose-6-phosphate of 370 M and 3.1 mM, respectively. A broad substrate specificity was observed with significant activities for fructose-6-phosphate, glucose-6-phosphate, mannitol-1-phosphate, AMP and -glycerol phosphate. Only phosphomonoesters were dephosphorylated. Antibodies raised against the purified enzyme could precipitate AP activity from a cell-free extract in an anticatalytic immunoprecipitation test.     

Isolation and characterization of amino acid-analogue-resistant mutants of Spirulina platensis

Amino acid-analogue-resistant mutants of the cyanobacterium Spirulina platensis were isolated using amino acid analogues -2-thienylalanine, p-fluorophenylalanine, ethionine and azetidine-2-carboxylic acid. The growth and other cellular contents in these mutants were less than in the parent. The internal free amino acid pool showed varying amounts. Maximal overproduction occurred of proline whereas overproduction of aspartic acid, alanine and lysine was much less.