Cysteinyl-tRNA Synthetase from Astragalus Species

The organ-specific accumulation, spatial distribution, and chemical speciation of selenium (Se) were previously unknown for any species of cactus. We investigated Se in Opuntia ficus-indica using inductively coupled plasma mass spectrometry, microfocused x-ray fluorescence elemental and chemical mapping (mXRF), Se K-edge x-ray absorption near-edge structure (XANES) spectroscopy, and liquid chromatography-mass spectrometry (LC-MS). mXRF showed Se concentrated inside small conic, vestigial leaves (cladode tips), the cladode vasculature, and the seed embryos. Se K-edge XANES demonstrated that approximately 96% of total Se in cladode, fruit juice, fruit pulp, and seed is carbon-Se-carbon (C-Se-C). Micro and bulk XANES analysis showed that cladode tips contained both selenate and C-Se-C forms. Inductively coupled plasma mass spectrometry quantification of Se in high-performance liquid chromatography fractions followed by LC-MS structural identification showed selenocystathionine-to-selenomethionine (SeMet) ratios of 75:25, 71:29, and 32:68, respectively in cladode, fruit, and seed. Enzymatic digestions and subsequent analysis confirmed that Se was mainly present in a "free" nonproteinaceous form inside cladode and fruit, while in the seed, Se was incorporated into proteins associated with lipids. mXRF chemical mapping illuminated the specific location of Se reduction and assimilation from selenate accumulated in the cladode tips into the two LC-MS-identified C-Se-C forms before they were transported into the cladode mesophyll. We conclude that Opuntia is a secondary Se-accumulating plant whose fruit and cladode contain mostly free selenocystathionine and SeMet, while seeds contain mainly SeMet in protein. When eaten, the organic Se forms in Opuntia fruit, cladode, and seed may improve health, increase Se mineral nutrition, and help prevent multiple human cancers.

Section: Discussionmentioning

confidence: 99%

Selenium Accumulation, Distribution, and Speciation in Spineless Prickly Pear Cactus: A Drought- and Salt-Tolerant, Selenium-Enriched Nutraceutical Fruit Crop for Biofortified Foods

Bañuelos

Fakra²,

Walse³

et al. 2010

“…The in vitro incorporation of selenomethionine into protein was also studied in E. coli (19), and it was shown to parallel methionine in both chain initiation and elongation. Most plant studies have examined the activation of selenoamino acids by aminoacyl-tRNA synthetase (4,5) and metabolic perturbations caused by selenomethionine (15,16) rather than the in vitro translational incorporation of selenoamino acids. The methionyl-tRNA synthetase from V. radiata does not discriminate effectively against selenomethionine and actually uses it quite efficiently (B. Beauchamp and A. Shrift, unpublished results).…”

Section: Discussionmentioning

confidence: 99%

“…Residual acetone was evaporated overnight at -20 C, and the pellet was resuspended in a buffer (pH 7.0) containing 0.5% 2-mercaptoethanol, 0.5% SDS, and 10 mm Na-phosphate. Solid urea was added to bring the concentration to 6.0 M, and the sample was incubated for 5 Figure 2. Integrity of polysome classes was high; the low ratio of monosomes to higher classes indicates excellent preservation (12).…”

Section: Methodsmentioning

confidence: 99%

In Vitro Incorporation of Selenomethionine into Protein by Vigna radiata Polysomes

Eustice

Foster²,

Kull³

et al. 1980

Self Cite

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...

“…A few comparable in vitro plant studies have focused on the following three aspects of selenium biochemistry: (a) aminoacylation of tRNA with selenoamino acids (2,3,21,24); (b) interactions of selenomethionine with methionine adenosyltransferase (12); and (c) the translational incorporation of selenomethionine into protein (5). Yet, in none of these studies in which selenium-sensitive plants were used, was a biochemical reaction correlated with selenium toxicity.…”

Section: Discussionmentioning

confidence: 99%

Selenium Toxicity: Aminoacylation and Peptide Bond Formation with Selenomethionine

Eustice¹,

Kull²,

Shrift³

1981

Self Cite

Selenomethionine and methionine were compared as substrates for in vitro aminoacylation, ribosome binding, and peptide bond formation with preparations from wheat germ. Selenomethionine paralleled methionine in all steps of the translation process except peptide bond formation. Peptide bond formation with the initiating species of tRNAMet demonstrated that selenomethionyl-tRNAM't was less effective as a substrate than was methionyl-tRNAf't. Participation of selenomethionine in the initiation process of translation could be expected to reduce the overall rate of protein synthesis and might aid in explaining selenium toxicity in selenium-sensitive plants.methionine, each step enroute to incorporation of this amino acid into protein should be studied independently of the other. For this purpose, ribosomes and supernatant fractors, prepared from wheat germ, were used. Selenomethionine and methionine functioned as equivalent substrates in the aminoacylation reaction and in ribosome binding of the aminoacyl-tRNA. Peptide bond formation between puromycin and selenomethionine, in contrast, was curtailed compared to bond formation between puromycin and methionine. As an explanation for selenium toxicity at the subcellular level, it would appear that selenomethionine exerts one of its effects in wheat by limiting the rate at which the first peptide bond is formed during protein synthesis. MATERIALS AND METHODSMethionine is a key intermediate in several crucial metabolic reactions. Since methionine contains a sulfur atom, substances which disrupt sulfur metabolism, such as those that contain selenium, in all likelihood will interfere with the metabolism of this sulfur amino acid. In selenium-sensitive plants, the assimilation of selenium usually parallels that of sulfur. As a consequence, selenoanalogs of several sulfur-containing metabolites can be synthesized (17). One of these analogs, selenomethionine, is known to be formed by a wide variety of plants (7,9,(16)(17)(18). Although the biochemical characteristics of selenomethionine resemble those of methionine in a great many ways, the metabolic activity of selenomethionine apparently differs in certain respects; one of these differences is, in general, reaction rate (4,5,11,12). Examination of those reactions in which selenomethionine functions at a rate different than methionine could reveal some of the factors that underlie selenium toxicity.One vital metabolic process with which these methionine analogs might interfere is the biosynthesis of protein. Methionine, required as the initiating amino acid during the translation process, is also incorporated at internal positions of most protein chains. Alteration of initiation process of translation or with the chain elongation process could drastically affect normal functions of the cell. Previous in vitro studies with polysomes from Vigna radiata root tips have established that methionine is a slightly better substrate than is selenomethionine during chain elongation, provided that both amino acids are supplied s...