Characterization of the tRNA and aminoacyl-tRNA synthetases of healthy and tumorous callus tissues from Nicotiana tabacum

Aminoacyl-tRNA synthetases extracted from healthy and crown gall tumor tissues (induced by Agrobacterium tumefaciens strain B6) from Nicotiana tabacum (strain Wisconsin 38) grown in vitro, showed the same ability to charge Phaseolus vulgaristRNA, for all the 15 amino acids tested. For each amino acid, optimal charging conditions (enzyme concentration, Mg²⁺/ATP ratios, K⁺ ion effects) have been determined with Phaseolus vulgaristRNA and were found to be the same whether aminoacyl-tRNA synthetases from healthy or tumor tissues were used. In each case, valyl- and glutamyl-tRNA synthetases were very sensitive to an excess of Mg²⁺ and K⁺ ions. Although tRNA's extracted from healthy and tumor tissues gave the same electrophoretic patterns, charging levels obtained with turner tRNAs were generally 45% higher than those obtained with tRNA's from healthy tissues.     

Stabilization of aminoacyl-tRNA synthetases by sephadex and polyacrylamide gels

Several aminoacyl-tRNA synthetases from the yellow lupin (Lupinus luteus) were stabilized against inactivation during storage at 0–4°, by entrapment in Sephadex or Biogel matrices and drying over P₂O₅. The degree of stabilization depended on the rate of drying of the gel and the pH of the medium and to a lesser extent on the ionic strength and protein concentration. With the exception of prolyl-tRNA synthetase, a greater stability was achieved with those enzymes which were relatively stable to thermal denaturation. Aminoacyl-tRNA synthetases for glutamic acid, glutamine, methionine and arginine, which become inactivated during purification, were considerably stabilized by this procedure.     

Amino acid incorporation on plant ribosomes: The transfer system

Pre-formed Vicia faba phenylalanyl-tRNA was active in a TYMV-RNA-directed Transfer System, whereas a similar tRNA preparation from yeast was not. Thus, lack of charging of yeast tRNA by enzymes from Phaseolus was not the only reason why yeast tRNA would not function in this Transfer System. In the poly U-directed Transfer System; where both types of tRNA were active, the pH and ionic parameters governing the reaction with yeast tRNA were more stringent.     

Purification and characterization of two tyrosyl-tRNA synthetase activities from soybean cotyledons

The tyrosyl-tRNA synthetases located in cytoplasm and chloroplasts of soybean cotyledons were purified to near homogeneity by ammonium sulfate precipitation, DEAE-cellulose chromatography, hydroxylapatite chromatography, and DEAE-Sephadex A-25 chromatography. Purified cytoplasmic tyrosyl-tRNA synthetase shows only a single band in acrylamide gel electrophoresis which corresponds to a MW of 126000. In SDS-acrylamide gel electrophoresis the enzyme again shows only a single band which corresponds to a MW of 61 000. Chloroplast tyrosyl-tRNA synthetase shows only one band in both acrylamide and SDS-acrylamide gel electrophoresis with MWs being 98 000 and 43 000, respectively. For cytoplasmic tyrosyl-tRNA synthetase the apparent K_ms determined are 6.8 μM L-tyrosine, 49 μM ATP, and 8.9 × 10^?8 M tRNA (as total tRNA). Apparent K_ms for chloroplast tyrosyl-tRNA synthetase are 4.9 μM L-tyrosine, 214 μM ATP and 2.2 × 10^?8 M tRNA (as BDC-ethanol fraction tRNA). Fractionation of soybean cotyledon-tRNA on RPC-5 columns gives 4 tyrosyl-tRNA species, the first two species (tRNA_{1 and 2}^Tyr) are acylated only by cytoplasmic tyrosyl-tRNA synthetase while the last two species (tRNA_{3 and 4}^Tyr) are acylated only by chloroplast tyrosyl-tRNA synthetase.     

Extraction of peroxidase from bitter gourd (Momordica charantia) by three phase partitioning with dimethyl carbonate (DMC) as organic phase

Three phase partitioning (TPP) is most renowned technique used for extraction and purification of natural products. In previous studies of TPP, t-butanol is mainly used as an organic phase. This is the first report that explores ability of dimethyl carbonate (DMC) in the field of TPP as an alternate solvent for t-butanol. In the present study TPP process with t-butanol and DMC as organic phase along with different salts was applied to waste bitter gourd powder to obtained peroxidase enzyme. DMC was found to be compatible with most of salts such as ammonium sulphate and sodium citrate and explored as more efficient solvent than t-butanol. This TPP system provides 4.84 fold purity of peroxidase enzyme at optimum source concentration of 0.15 g/mL, with a system comprising DMC as organic phase, sodium citrate (20%) as salt, agitation speed 120 rpm, pH 7, temperature 30 °C and extraction time of 3 h. Present study has aimed for extraction and separation of peroxidase from bitter gourd waste with TPP technique and ensures the scope of carbonated solvents in extraction and purification of proteins.     

The purification of beta-galactosidase-specific polysomes by affinity chromatography.

Affinity chromatography on a β-galactosidase substrate analog-Sepharose column was used to purify β-galactosidase-specific polysomes from E. coli. The purification was monitored by hybridization of [³H]uridine pulse-labeled RNA extracted from polysomes to p lac 5 DNA. A purification of at least 12-fold was obtained. Binding of lac polysomes to the column required the presence of Sepharose-bound substrate analog; salt and pH conditions favorable to β-galactosidase binding; and intact polyribosomes. It was calculated that 40–50% of the labeled mRNA recovered was lac RNA.     

tRNA species in the developing grain of triticum aestivum

The level of lysyl- and prolyl-tRNA in various stages of the maturing wheat grain was measured by the aminoacylation procedure. The levels of these tRNAs changed only slightly during the maturation period. Several species of lysyl- and prolyl-tRNA were obtained from different parts of the developing grain by fractionation on benzoylated-DEAE cellulose (BD-cellulose). The embryo contained three discrete species of prolyl and at least three species of lysyl isoacceptor tRNA throughout development, whilst the tRNA obtained from the endosperm gave more complicated elution profiles on chromatography on BD-cellulose. Small changes were noted in the levels of aminoacylation of individual isoacceptor tRNA species for lysine or proline during seed maturation. However, these were insufficient to account for the changing pattern of lysine and proline in the storage protein during the development of the endosperm.     

Purification and characterization of an ovulation stimulating substance from the male accessory glands of Drosophila suzukii

An ovulation stimulating substance (OSS) was isolated from males of the fruit fly Drosophila suzukii, and purified to a homogeneous state by a 5-step purification procedure: extraction with 80% methanol, chloroform wash, heat treatment, ion-exchange chromatography, and reverse phase high performance liquid chromatography. Approximately 100-fold purification was obtained thereby yielding 39 μg of OSS from 1000 males for an overall yield of 34%. The OSS is a single peptide consisting of at least 35 amino acid residues and having a molecular weight of 3990. The purified OSS not only initiated ovulation in unmated females but also suppressed their receptivity towards males. The peptide of D. suzukii was found to be effective in the females of D. melanogaster, a species that belong to a different subgroup, but was less effective in a more closely related species, D. pulchrella.     

Efficient method for preparation of highly purified lipopolysaccharides by hydrophobic interaction chromatography

A method for the efficient preparation of highly purified lipopolysaccharides (LPSs) by hydrophobic interaction chromatography (HIC) has been developed. The procedure can be used for the purification of cell wall bound LPSs after hot phenol–water extraction and for the isolation of extracellular LPSs from the supernatant, respectively. The method described has been tested with artificial mixtures containing LPSs, polysaccharide, protein and RNA and subsequently employed for the preparative purification of two LPSs of different origin, namely the extracellular LPS secreted by Escherichia coli E49 into the culture medium, and the cell wall bound LPS from Pseudomonas aeruginosa VA11465/1. Compared to currently used methods for LPS purification such as enzymatic digestion and ultracentrifugation, the chromatographic separation reported here combines superior purity with minimal loss of LPS, high reproducibility and simple handling. The removal of contaminants such as protein, RNA and polysaccharides and the recovery of LPSs were monitored by appropriate assays.     

Leucine specific transfer ribonucleic acids and synthetases in the cotyledons of mature and germinating pea seeds

Changes in isoaccepting species of tRNA^Leu were determined in germinating pea seedlings and in developing pods. Leucine specific transfer ribonucleic acids of pea cotyledons can be fractionated into four isoaccepting species by reversed-phase chromatography (RPC-5) on a Plaskon column. In contrast, only two species of tRNA^Leu were observed in developing seed pods. Leucyl-tRNA synthetase purified by ammonium sulfate precipitation and DEAE cellulose column chromatography retained the full range of specificity towards all four tRNA^Leu species of pea cotyledons. This partially purified pea cotyledon enzyme could be further separated on a hydroxylapatite (HA) column into two peaks of leucyl-tRNA synthetase activity. Enzyme 1 is dominant in seed pods while 2 is predominant in cotyledons. Enzymes 1 and 2 from cotyledons were examined for the amino acid acceptor activity of twelve different amino acids. Both these fractions showed less than 3% acceptor activity for eleven other amino acids as compared to leucine-tRNA synthetase activity. Preliminary characterization of enzyme 2 from cotyledon, by isoelectric focusing and polyacrylamide gel electrophoresis indicates at least three subspecies.     

Purification and Characterization of a Lectin from Chinese Quince (Chaenomeles sinensis) Fruit

Chaenomeles sinensis lectin (CSL) was isolated from Chinese quince fruit by affinity chromatography. The molecular weight of native CSL was estimated to be 16 kD by gel filtration chromatography. This lectin was found to contain approximately 57% carbohydrates. The molecular weight of deglycosylated CSL was estimated to be 3.6 kD by tricine-SIDS-PAGE under reduced conditions. Our results suggest that CSL may be a homodimer. The hemagglutinating activity of CSL was inhibited by N-acetyllactosamine and chicken ovalbumin, and it was drastically decreased at pH levels of 9.0 or greater. CSL may be a useful tool for the purification of glycoconjugates.     

Synthesis of diacyl and alkylacyl glycerophosphoserines

Diacyl and alkylacyl glycerophosphoserines were synthesized by condensation of the respective diradylglycerophosphates with N-t-butoxycarbonyl-L-serinebenzhydryl ester in the presence of 2,4,6-triisopropylbenzenesulfonyl chloride in pyridine at room temperature. The protective groups were simultaneously removed from the resultant N-t-butoxycarbonyl-phosphatidylserinebenzhydryl esters by treatment with HCl in chloroform at 0°C. Products were obtained in 60–65% yield after purification by medium pressure liquid chromatography (MPLC). Dipalmitoyl-N-methylphosphatidylserine was synthesized by an analogous procedure.     

Extraction and three-phase partitioning behavior of proteases from papaya peels

Dried papaya peels exhibited superior proteolytic activity to the fresh peels. An extraction with phosphate buffer pH 7.0 greatly maintained proteolytic activity when compared to water. SDS-PAGE verified that the extracted dried papaya peels held a wide range of proteins. To optimize the three-phase partitioning (TPP) for isolating the papaya peel proteases required a ratio of crude extract to t-butanol, the (NH₄)₂SO₄ concentration and the TPP cycles. The ratio of the crude extract to t-butanol of 1.0:0.5 with the presence of 20% (NH₄)₂SO₄ resulted in the highest proteolytic recovery at 253.5%, and 15.8-fold of purification in the bottom phase. The TPP was then optimized by adding up to 55% (NH₄)₂SO₄ to the bottom phase of the first step. A purification of 10.1-fold with about 89.4% recovery was obtained. This study showed the TPP can be effectively employed for the extraction of proteases from papaya peels.     

Substrate discrimination by prolyl-tRNA synthetase from various higher plants

Prolyl-tRNA synthetase from plants (e.g. Delonix regia) containing azetidine-2-carboxylic acid (A2C), activated imino acid analogues larger than proline (Pro) more efficiently than did the enzyme from plants lacking A2C. The reverse situation was observed for analogues, including A2C itself, that are smaller than Pro. The enzyme from A2C-producing species was quite labile and salt-sensitive, with a high pH optima for the ATP-³²PPi exchange reaction, whereas the enzyme from non-producer species was stable and insensitive to salts, with a lower pH optimum. Certain analogues of Pro, which failed to stimulate ATP-³²PPi in the presence of a particular type of Pro-tRNA synthetase, nevertheless could bind to the enzyme and inhibit the esterification of tRNA by Pro. In the absence of tRNA, no significant ATP-³²PPi exchange was catalyzed by the Delonix enzyme on addition of A2C; the addition of tRNA resulted in a low but real level of activation of the analogue relative to Pro. These findings are discussed in relation to the ability of the enzyme from A2C-producing plants to discriminate against the analogue.     

Rapid purification of bacterial plasmids and coliphage M13 RF without CsCl centrifugation

A simple and rapid procedure for the purification of plasmids from Escherichia coli K12 has been developed. Bacterial cells are subjected to the boiling procedure [D. S. Holmes, and M. Quigley Anal. Biochem.114, 193–197 (1981)] followed by removal of contaminating RNA by chromatography on Sepharose 2B and of genomic DNA by acid-phenol extraction. Plasmids are recovered with good yield. They can be restricted and ligated and will transform host cells. A simple modification of the procedure allows it to be used for the isolation of coliphage M13 RF DNA.     

Amino acid incorporation on 80s plant ribosomes: The complete system

A cell-free system directed by poly U or turnip yellow mosaic virus (TYMV)-RNA was obtained from imbibed seeds of Phaseolus aureus; this in vitro system was dependent upon exogenous tRNA. The poly U-directed system functioned in the presence of tRNAs from P. aureus, Vicia faba and yeast, whereas TYMV-RNA was translated only in the presence of tRNAs from P. aureus or V. faba. The pH and Mg²⁺ optima for aminoacylation of tRNAs of P. aureus, V. faba and yeast by leucine and phenylalanine were related to the overall pH and ionic concentration optima for the complete system.     

Large-Scale Purification,Characterization, and Spore Outgrowth Inhibitory Effect of Thurincin H,a Bacteriocin Produced by Bacillus thuringiensis SF361

Large-scale purification of the highly hydrophobic bacteriocin thurincin H was accomplished via a novel and simple two-step method: ammonia sulfate precipitation and C18 solid-phase extraction. The inhibition spectrum and stability of thurincin H as well as its antagonistic activity against Bacillus cereus F4552 spores were further characterized. In the purification method, secreted proteins contained in the supernatant of a 40 h incubated culture of B. thuringiensis SF361 were precipitated by 68 % ammonia sulfate and purified by reverse-phase chromatography, with a yield of 18.53 mg/l of pure thurincin H. Silver-stained SDS–PAGE, high-performance liquid chromatography, and liquid chromatography–mass spectrometry confirmed the high purity of the prepared sample. Thurincin H exhibited a broad antimicrobial activity against 22 tested bacterial strains among six different genera including Bacillus, Carnobacterium, Geobacillus, Enterococcus, Listeria, and Staphylococcus. There was no detectable activity against any of the selected yeast or fungi. The bacteriocin activity was stable for 30 min at 50 °C and decreased to undetectable levels within 10 min at temperatures above 80 °C. Thurincin H is also stable from pH 2–7 for at least 24 h at room temperature. Thurincin H is germicidal against B. cereus spores in brain heart infusion broth, but not in Tris–NaCl buffer. The efficient purification method enables the large-scale production of pure thurincin H. The broad inhibitory spectrum of this bacteriocin may be of interest as a potential natural biopreservative in the food industry, particularly in post-processed and ready-to-eat food.     

Extraction and purification of a lectin from small black kidney bean (Phaseolus vulgaris) using a reversed micellar system

Lectin from crude extract of small black kidney bean (Phaseolus vulgaris) was successfully extracted using the reversed micellar extraction (RME). The effects of water content of organic phase (W_o), ionic strength, pH, Aerosol-OT (AOT) concentration and extraction time on the forward extraction and the pH and ionic strength in the backward extraction were studied to optimize the extraction efficiency and purification factor. Forward extraction of lectin was found to be maximum after 15 min of contact using 50 mM AOT in organic phase with W_o 27 and 10 mM citrate-phosphate buffer at pH 5.5 containing 100 mM NaCl in the aqueous phase. Lectin was backward extracted into a fresh aqueous phase using sodium-phosphate buffer (10 mM, pH 7.0) containing 500 mM KCl. The overall yield of the process was 53.28% for protein recovery and 8.2-fold for purification factor. The efficiency of the process was confirmed by gel electrophoresis analysis.     

Efficient two-step chromatographic purification of penicillin acylase from clarified Escherichia coli ultrasonic homogenate

A two-step chromatographic purification procedure from clarified Escherichia coli ultrasonic homogenate was evaluated. The capture step included immobilized metal affinity chromatography with Cu²⁺ as metal ion. Two elution methods were performed: 1 M NH₄Cl and 0.01 M imidazole. Respectively, we obtained a different purification fold (16.5 to 3.15) and a similar result for the recovery of activity (90–99%). The best elution method was chosen for the procedure. The second step, hydrophobic interaction chromatography, gave a 3.8-fold purification with 77.7% of activity. The total procedure gave a 66-fold purification in relation to the initial crude extract with 70% for the recovery of activity and was performed without any conditioning step and at the same pH value.     

Isolation and purification of peptide and alkaloid toxins from nabaena flos-aquae using high-performance thin-layer chromatography

A procedure has been developed for the sequential extraction and purification of the peptide and alkaloid toxins from a single batch of Anabaena flos-aquae cells. After solvent extraction and adsorption onto C₁₈ Sep-Pak cartridges, the cyanobacterial toxins were purified by high-performance thin-layer chromatography and identified by scanning with a densitometer. The purified components were tested separately by mouse bioassay. Liver damage and nervous disorder were induced by the two peptide and single alkaloid toxins, respectively.