Amino Acid Selection in Protein Biosynthesis

Peterson, P. J.

doi:10.1111/j.1469-185x.1967.tb01530.x

Cited by 25 publications

(9 citation statements)

References 328 publications

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“…Because animals obtain selenium by direct or indirect consumption of vegetation, it becomes important to study the moleclar basis of selenium metabolism by plants in order to understand selenium toxicity. It has long been speculated that the inability of selenium sensitive plants to discriminate between selenoamino acids and their sulfur counterparts might be a mode of selenium toxicity (8,22,23). Such a lack of discrimination could result in the generation of malfunctioning selenium containing proteins which could lead to adverse metabolic consequences such as chlorosis and reduced growth rates, two of the classical symptoms of selenium toxicity (11).…”

Section: Discussionmentioning

confidence: 99%

In Vitro Incorporation of Selenomethionine into Protein by Vigna radiata Polysomes

Eustice

Foster²,

Kull³

et al. 1980

Plant Physiol.

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Vigna radiata polysomes efficiently incorporated 175Selselenomethio-nine, I"4Clmethionine, and I'4Clleucine in vitro. The optimal conditions for translation were determined to be 4.8 millimolar Mg2e, 182 milHimolar K+, and pH 7.4. The rates of incorporation of 175Selselenomethionine and 1'4CImethionine were similar when measured separately, but 175Selseleno-miethionine incorporation was 35% less than 14Clmethionine incorporation when both amino acids were present in equal molar concentrations. Polyacrylamide gel electrophoresis of the hot trichloroacetic acid precipitable translation products demonstrated synthesis of high molecular weight labeled proteins in the presence of 175Selselenomethionine or I35SImethi-onine. No major differences in molecular weights could be detected in the electrophoretic profiles. Utilization of selenomethionine during translation by Vigna radiata polysomes establishes a route for the assimilation of selenomethionine by plants susceptible to selenium toxicity. alteration of this type could give rise to abnormal properties such as altered tertiary structure or abnormal catalytic activity in newly synthesized proteins. As an example of such alterations, the Escherichia coli fi-galactosidase was found to withstand some substitution of methionine by selenomethionine, but an inactive 4S protein with high amounts of substitution was also generated (10). The 4S protein was interpreted to be an inactive subunit of ,Bgalactosidase, produced in excess.Though selenium toxicity to plant systems has been extensively studied with intact plants (23), an understanding at the subcellular level has been limited by the dearth of studies in this area of selenium research. To provide evidence for the in vitro incorporation of selenomethionine into protein, Vigna radiata polysomes and wheat germ supematants were prepared, and the selenium containing proteins synthesized in vitro were analyzed. Our results showed that the [75Se]selenomet2 labeling pattem differed in some respects from ["CJmet incorporation, yet [ 5Selselenomet was rapidly incorporated into high mol wt proteins.Selenium and sulfur constitute an isosteric pair based on chemical similarities and, therefore, have the potential to act as an analog pair in biological processes. Such an analog relationship was evinced from studies in which pasture plants, fed selenite, were able to synthesize the selenoanalogs of several typical sulfurcontaining metabolites (21). Other nonaccumulator plants, such as bromegrass (13) and duck week (21), also synthesize selenomethionine, an analog of methionine, when exposed to selenite. Selenomethionine synthesis in plants may protentiate a toxicity syndrome through indiscriminate utilization of the analog in biochemical reactions which normally use methionine.Methionine is a crucial compound in a multitude of metabolic processes. During the translation process, methionine participates in two key reactions, initiation of protein synthesis and internal insertion of methionine into proteins. In eukaryotes, th...

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Section: Discussionmentioning

confidence: 99%

In Vitro Incorporation of Selenomethionine into Protein by Vigna radiata Polysomes

Eustice

Foster²,

Kull³

et al. 1980

Plant Physiol.

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Section: Methodsmentioning

confidence: 99%

“…Altered aminoacyl-tRNA synthetases have been described in other plants which synthesize large amounts of nonprotein amino acids. Modified prolyl-tRNA synthetases, for example, can be isolated from Polygonatum and Convallaria species that synthesize azetidine-2-carboxylic acid; this nonprotein amino acid analog of proline is excluded from the proteins of these plants but not from the proteins of organisms poisoned by the analog (7,8,22,25 Buffer B, for dissolving the 45 to 65% (NH4)2SO4 fraction, and for equilibration of Sephadex and DE-52 columns, contained 0.05 M tris and the other components of buffer A; the pH was brought to 7.7 with HCl.Buffer C, pH 7.7, for hydroxylapatite and cellulose phosphate columns, contained 50 ml of 0.02 M KH2PO4, 500 ml of 0.02 K2HPO4, 25 mm 2-mercaptoethanol, and 10% (w/v) glycerol.Buffer D, pH 7.7, for gradient elution of hydroxylapatite and cellulose phosphate columns, contained 70 ml of 0.4 M KH2PO4 and 500 ml of 0.4 M K2HPO4, 25 mm 2-mercaptoethanol, andPreparation of Crude Enzyme Extract and Preliminary Fractionation. All operations were carried out at 3 to 4 C. Phaseolus aureus seed, milled to a powder, was homogenized for 1 min in a blender with the ratio of tissue to buffer at 1 g to 10 ml.…”

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confidence: 99%

Utilization of Selenocysteine by a Cysteinyl-tRNA Synthetase from Phaseolus aureus

et al. 1976

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An L-cysteinyl-tRNA synthetase (EC 6.1.1.16) from Phaseolus aureus has been purified approximately 200-fold. The enzyme uses selenocysteine as substrate in the ATP-PPi exchange assay; other cysteine analogs were inactive. The molecular weight as determined by Sephadex G-200 column chromatography is about 61,000; sodium dodecyl sulfate and 8 Murea acrylamide gel electrophoresis indicate that the enzyme is a dimer conssting of two identical monomers of molecular weight 30,000.A method for the preparation of selenocysteine from selenocystine is described.digests from several organisms (2, 10, 15, 23), but it was reported absent, despite the presence of selenomethionine, from proteins of other organisms grown with radioactive selenite or selenate (13, 21). Such findings suggest that different organisms vary in their ability to activate selenocysteine and incorporate it into their proteins. Therefore, to examine the activation of selenocysteine, a method for its preparation from selenocystine and its use as substrate by a purified cysteinyl-tRNA synthetase from Phaseolus aureus was undertaken and will be described here. MATERIALS AND METHODSThe ability of methionyl-and cysteinyl-tRNA synthetases to use selenium isologs as substrates has been suggested as the cause of selenium toxicity. According to this hypothesis, both the sulfur and selenium amino acids are activated and incorporated into proteins; these altered proteins, with an excess of selenoamino acids, function abnormally (7,29). On the other hand, tolerance of selenium, notably in selenium-accumulator plants, is thought to have arisen from evolutionary modifications to these aminoacyl-tRNA synthetases so that the selenium isologs, no longer activated, are excluded from proteins; instead, the selenium isologs are converted to and accumulate as nonprotein selenoamino acids (7,24,28,31). Altered aminoacyl-tRNA synthetases have been described in other plants which synthesize large amounts of nonprotein amino acids. Modified prolyl-tRNA synthetases, for example, can be isolated from Polygonatum and Convallaria species that synthesize azetidine-2-carboxylic acid; this nonprotein amino acid analog of proline is excluded from the proteins of these plants but not from the proteins of organisms poisoned by the analog (7,8,22,25 Buffer B, for dissolving the 45 to 65% (NH4)2SO4 fraction, and for equilibration of Sephadex and DE-52 columns, contained 0.05 M tris and the other components of buffer A; the pH was brought to 7.7 with HCl.Buffer C, pH 7.7, for hydroxylapatite and cellulose phosphate columns, contained 50 ml of 0.02 M KH2PO4, 500 ml of 0.02 K2HPO4, 25 mm 2-mercaptoethanol, and 10% (w/v) glycerol.Buffer D, pH 7.7, for gradient elution of hydroxylapatite and cellulose phosphate columns, contained 70 ml of 0.4 M KH2PO4 and 500 ml of 0.4 M K2HPO4, 25 mm 2-mercaptoethanol, andPreparation of Crude Enzyme Extract and Preliminary Fractionation. All operations were carried out at 3 to 4 C. Phaseolus aureus seed, milled to a powder, was homogenized for 1 min in ...

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“…The enzymes catalyzing this step, the amino acid:tRNA ligases (amino acid-activating enzymes, aminoacyl tRNA synthetases, EC 6.1.1. ), have been shown to change in activity during germination of pea (13), bean (1), and wheat (21), during differentiation of pea (7) and bean (14) roots, and during senescence of soybean cotyledons (5) and tobacco leaves (19), and several workers have suggested that changes in the activity of these enzymes, coupled with changes in isoaccepting species of tRNA, may regulate the temporal development of specific enzymes (2,4,23,27). However, although these developmental changes in ligase activity are well characterized and may play an important role in cellular differentiation nothing is known about how these changes are brought about.…”

Section: Introductionmentioning

confidence: 99%

Leucine: tRNA Ligase from Cultured Cells of Nicotiana tabacum var. Xanthi

Gore¹,

Wray²

1978

Plant Physiol.

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Leudne:tRNA 1gmn was asayed in extracts from cultured tobacco (coVatiana tabacwn) XD Of the component steps in the de novo synthesis of proteins from amino acids in higher plants, the first step-that is the activation and attachment of amino acid to tRNA -has received the most attention (see review by Lea and Norris [17]). The enzymes catalyzing this step, the amino acid:tRNA ligases (amino acid-activating enzymes, aminoacyl tRNA synthetases, EC 6.1.1.), have been shown to change in activity during germination of pea (13), bean (1), and wheat (21), during differentiation of pea (7) and bean (14) roots, and during senescence of soybean cotyledons (5) and tobacco leaves (19), and several workers have suggested that changes in the activity of these enzymes, coupled with changes in isoaccepting species of tRNA, may regulate the temporal development of specific enzymes (2,4,23,27). However, although these developmental changes in ligase activity are well characterized and may play an important role in cellular differentiation nothing is known about how these changes are brought about. 3To whom requests for reprints should be addressed.Many of the recent advances in our understanding of the basic control mechanisms involved in plant nitrogen metabolism have been realized due to the utilization of cell suspension cultures (e.g. 9, 29, 30). Such cultures possess several obvious advantages over the intact plant especially the investigators' ability to manipulate and control the environment of the cells. In view of this, and the fact that specific temporal changes in the activity levels of most amino acid:tRNA ligases occur during growth of batch suspension cultures of tobacco XD cells (33), cell suspension cultures seem to be ideally suited to an investigation of the regulatory mechanisms underlying the development of these enzymes. Here, we present density labeling experiments which indicate that at least one of these enzymes, leucine:tRNA ligase, is synthesized de novo and that its cellular activity is controlled by a balance between the synthesis and loss of functional enzyme molecules. A preliminary account of some of this work has been presented (11).MATERIALS AND METHODS Chemicals. Ninety-nine mol % deuterium oxide and 99 mol % [15Nlsodium nitrate were obtained from Bio-Rad Laboratories Ltd., Bromley, Kent. L-[4,53Hlleucine (53 Ci/mol) was obtained from the Radiochemical Centre, Amersham, Bucks; 2,5-diphenyloxazole (PPO) and 1,4-di-2-(5-diphenyloxazolyl)benzene (POPOP) and toluene were obtained from Koch-

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Amino Acid Selection in Protein Biosynthesis

Cited by 25 publications

References 328 publications

In Vitro Incorporation of Selenomethionine into Protein by Vigna radiata Polysomes

In Vitro Incorporation of Selenomethionine into Protein by Vigna radiata Polysomes

Utilization of Selenocysteine by a Cysteinyl-tRNA Synthetase from Phaseolus aureus

Leucine: tRNA Ligase from Cultured Cells of Nicotiana tabacum var. Xanthi

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