Genetic alterations of ribonuclease-sensitive glycoprotein subunits of amino acid transport systems in Neurospora crassa conidia.

Neurospora crassa conidia have multiple and constitutive amino acid transport systems. Extraction by KCl releases amino acid-binding glycoproteins which have been purified by arginine affinity chromatography. Disappearance of certain fractions is coordinate with genetic lesions which reduce amino acid transport. Two such affinity fractions contain radioactivity when cells are grown on l-[¹⁴C]phenylalanine or on [¹⁴C]uridine, but not when cells are grown on [¹⁴C ]glucosamine. One purified arginine-binding fraction (B) contains 113 amino acid residues per minimum molecular weight. This glycoprotein also contains eight types of neutral sugar residues. No amino sugars were detected. Electrophoresis of crude extracts reveals five major Coomassie blue-staining species. The number of species is reduced, and the electrophoretic pattern is altered in extracts from transport-deficient strains. Tryptic “fingerprints” of these extracts indicate that mutations that reduce transport result in amino acid substitutions in the extractable glycoproteins. Nondialyzable material which absorbs light in the 260-nm region becomes dialyzable after digestion with RNase. Digestion of conidia with RNase reduces the amount of l-phenylalanine accumulated by the cells after 10 min of incubation with the amino acid.     

Isolation of an Arabidopsis cDNA sequence encoding a 22 kDa calcium-binding protein (CaBP-22) related to calmodulin

Complementary DNA sequences were isolated from a library of cloned Arabidopsis leaf mRNA sequences in gt10 that encoded a 21.7 kDa polypeptide (CaBP-22), which shared 66% amino acid sequence identity with Arabidopsis calmodulin. The putative Ca²⁺-binding domains of CaBP-22 and calmodulin, however, were more conserved and shared 79% sequence identity. Ca²⁺ binding by CaBP-22, which was inferred from its amino acid sequence similarity with calmodulin, was demonstrated indirectly by Ca²⁺-induced mobility shifting of in vitro translated CaBP-22 during SDS-polyacrylamide gel electrophoresis. CaBP-22 is encoded by a ca. 0.9 kb mRNA that was detected by northern blotting of leaf poly(A)⁺ RNA; this mRNA was slightly larger than the 809 bp CaBP-22 cDNA insert, indicating that the deduced amino acid sequence of CaBP-22 is near full-length. CaBP-22 mRNA was detected in RNA fractions isolated from leaves of both soil-grown and hydroponically grown Arabidopsis, but below the limits of detection in RNA isolated from roots, and developing siliques. Thus, CaBP-22 represents a new member of the EF-hand family of Ca²⁺-binding proteins with no known animal homologue and may participate in transducing Ca²⁺ signals to a specific subset of response elements.     

Amino acids,sugars and sterols of some Mediterranean brown algae

Free amino acids, amino sulfonic acids, sugars and sterols have been examined and quantitatively determined in 10 brown seaweeds. Acidic amino acids and their amides are the main components of the amino acid fraction. Cysteinolic acid, taurine and its N-methyl derivatives have been identified in most of the species examined. In all the algae, mannitol is present, sometimes in very large amounts. The sterol fractions of all the species contain fucosterol, cholesterol and 24-methylenecholesterol; minute amounts of 22-dehydrocholesterol and 24-methylcholesta-5, 22-dien-3β-ol have also been frequently detected.     

Metabolism of [2-14C] acetate to amino acids and proteins in segments of bean seedling roots

Summary ¹⁴C from [2-¹⁴C] acetate was found to be incorporated into soluble and protein amino acids in substantial amounts by bean root apices. The ¹⁴C was spread through a wide range of amino acids in both these fractions. Glutamic acid was found to be heavily labelled with ¹⁴C in both soluble and protein amino acid fractions. The data are discussed in relation to present ideas on transport and utilization of amino acids in root systems.     

Trace elements distribution and species fractionation in the wheat (Triticum aestivum) plant

Abstract

The status of 13 trace elements’ (both essential and toxic) was investigated in individual parts of the winter wheat plant(Triticum aestivum) taken during its whole cultivation period. The study includes the determination of total concentrations, portions soluble in 0.02 mol L^?1 Tris-HCI buffer solution (pH = 7.5), and the fractionation of soluble species of elements by SEC and ICP/MS. Ligands of trace elements from a low-molecular weight SEC fraction were isolated by affinity chromatography and characterised by MALDI/MS analyses and by amino acids composition. Inhomogeneous accumulation of trace elements was found in the analysed plant tissues. The concentrations of elements are also affected by the maturity of the plants. The distribution of the soluble species of the elements between chromatographic fractions exhibited some regularity in all the samples. Substantial amounts of trace elements are located in a low-molecular weight fraction (< 2 kDa). Only chromatograms of Zn (grain) and Cu (all samples) contain significant medium-molecular and high-molecular weight fractions. Compounds isolated from the low-molecular weight fractions are rich in cystein and dicarboxylic amino acids or their amides. MALDI/MS spectra of these compounds isolated from shoots, straw and grain confirmed the presence of the phytochelatin PC₅.     

Honeydew amino acids in relation to sugars and their role in the establishment of ant-attendance hierarchy in eight species of aphids feeding on tansy (Tanacetum vulgare)

Abstract. The ratio of the concentration of honeydew total amino acids to total sugars in the honeydew of eight species of aphids, all feeding on tansy, Tanacetum vulgare (L.), was determined and correlated with honeydew production and ant‐attendance. The honeydew of the five ant‐attended aphid species [Metopeurum fuscoviride (Stroyan), Trama troglodytes (v. Hayd), Aphis vandergooti (Börner), Brachycardus cardui (L.), Aphis fabae (Scopoli)] was rich in total amino acids, ranging from 12.9 to 20.8 nmol µL^?1 compared with the unattended aphid Macrosiphoniella tanacetaria (Kalt.) with only 3 nmol µL^?1. Asparagine, glutamine, glutamic acid and serine (all nonessential amino acids) were the predominant amino acids in the honeydew of all species. The total concentration of amino acids in the phloem sap of tansy was much higher (78.7 nmol µL^?1) then in the honeydew samples, and the predominant amino acids were glutamate (34.3%) and threonine (17.7%). A somewhat unexpected result was the finding that those aphid species with the highest total amino acid concentration in the honeydew always had the highest concentration of sugars. The lowest amino acid–sugar combined value was 104–28.8 nmol µL^?1 in the non ant‐attended species M. tanacetaria, and the highest value was an average of 270–89.9 nmol µL^?1 for the three most intensely attended aphid species M. fuscoviride, A. vandergooti and T. troglodytes. There is no evidence that any single amino acid or group of amino acids in the honeydew acted as an attractant for ant‐attendance in these eight aphid species. The richness of the honeydew (rate of secretion × total concentration of sugars), along with the presence of the attractant sugar melezitose, comprised the critical factors determining the extent of ant‐attendance of the aphids feeding on T. vulgare. The high total amino acid concentration in sugar‐rich honeydews can be explained by the high flow‐through of nutrients in aphids that are particularly well attended by ants.     

Distribution and biosynthesis of theanine in Theaceae plants

The theanine content of the leaves of 27 species or varieties of Theaceae plants was investigated. Theanine was present in 21 species or varieties, but in much lower amounts (<0.2 μmol/g fresh weight) than the quantity detected in Camellia sinensis var. sinensis. The major free amino acids in leaves of four species belonging to the genera Schima and Eurya, were glutamic acid, aspartic acid, glutamine, asparagine, alanine and proline and content of these amino acids is similar to or higher than theanine. Accumulation of free amino acids in these plants was generally lower than in C. sinensis var. sinensis. The biosynthetic activity of theanine, assessed by the incorporation of radioactivity from [¹⁴C]ethylamine, was detected in seedlings of two species of Schima. The theanine biosynthetic activity in roots was higher than that of leaves.     

Isolation and characterization of a <Emphasis Type="Italic">Mastigocladus</Emphasis> species capable of growth,N<Subscript>2</Subscript>-fixation and N-assimilation at elevated temperature

A Mastigocladus species was isolated from the hot spring of Jakrem (Meghalaya) India. Uptake and utilization of nitrate, nitrite, ammonium and amino acids (glutamine, asparagine, arginine, alanine) were studied in this cyanobacterium grown at different temperatures (25°C, 45°C). There was 2–3 fold increase in the heterocyst formation and nitrogenase activity in N-free medium at higher temperature (45°C). Growth and uptake and assimilation of various nitrogen sources were also 2–3 fold higher at 45°C indicating that it is a thermophile. The extent of induction and repression of nitrate uptake by NO₃ ⁻ and NH₄ ⁺, respectively, differed from that of nitrite. It appeared that Mastigocladus had two independent nitrate/nitrite transport systems. Nitrate reductase and nitrite reductase activitiy was not NO₃ ⁻-inducible and ammonium or amino acids caused only partial repression. Presence of various amino acids in the media partially repressed glutamine synthetase activity. Ammonium (methylammonium) and amino acid uptake showed a biphasic pattern, was energy-dependent and the induction of uptake required de novo protein synthesis. Ammonium transport was substrate (NH₄ ⁺)-repressible, while the amino acid uptake was substrate inducible. When grown at 25°C, the cyanobacterium formed maximum akinetes that remained viable upto 5 years under dry conditions.     

Further observations on the production of amino acids by rat-liver mitochondria and other subcellular fractions

1. Rat-liver mitochondria showed a decrease in amino acid production after preparation in 0·25m-sucrose containing EDTA (1mm), but an increase in water content. When EDTA was replaced by Mn²⁺ (1mm) or succinate (1mm), both amino acid production and water content were lowered, whereas preparation in 0·9% potassium chloride caused an increase in both. 2. Amino acid production by rat-liver homogenates prepared in 0·9% potassium chloride or 0·25m-sucrose was similar (q_{amino acid} 0·047 and 0·042 respectively aerobically). After freezing-and-thawing q_{amino acid} values were approximately doubled, and approached that of a homogenate prepared in water. 3. All cations tested inhibited amino acid production by mitochondria, Hg²⁺ and Zn²⁺ being the most effective in tris–hydrochloric acid buffer. In phosphate buffer Mg²⁺ and Mn²⁺ had no effect. Of the anions tested only pyrophosphate and arsenate had any inhibitory effect at final concn. 1mm. 4. Iodosobenzoate (1mm) and p-chloromercuribenzenesulphonate (1mm) inhibited mitochondrial amino acid production by 70–80%, whereas soya-bean trypsin inhibitor, EDTA and di-isopropyl phosphorofluoridate inhibited by a maximum of 30%. Respiratory inhibitors had no effect. 5. Rat-liver homogenate and subcellular fractions each showed an individual pattern of inhibition when a series of inhibitors was tested. 6. Amino acid production by mitochondria was decreased by up to 50% in the presence of oxidizable substrate, apart from α-glycerophosphate and palmitate, which had no effect. CoA stimulated amino acid production in tris–hydrochloric acid but not in phosphate buffer, α-oxoglutarate abolishing the stimulation. 7. Cysteine and glutathione stimulated amino acid production by whole mitochondria by 30%, but only reduced glutathione stimulated production in broken mitochondria. 8. Adrenocorticotrophic hormone and growth hormone stimulated mitochondrial amino acid production by 21–24%, whereas insulin inhibited production by 25%. 9. Coupled oxidative phosphorylation increased amino acid production by up to 154% at 25° and 40°. The increase was abolished by 2,4-dinitrophenol. 10. Amino acid incorporation in mitochondria was accompanied by an increase in amino acid production, both being decreased by chloramphenicol. 11. Mitochondrial production of ninhydrin-positive material was increased in the presence of albumin. The biggest increase was noted for the soluble fraction of broken mitochondria. No increase was found in the presence of ¹⁴C-labelled algal protein or denatured mitochondrial protein.     

CHARACTERISTICS OF AMINO ACID ACCUMULATION BY SYNAPTOSOMAL PARTICLES ISOLATED FROM RAT BRAIN

—The characteristics of the accumulation of 14 L-amino acids (Leu, Ileu, Val, His, Tyr, Phe, Gly, Ala, Ser, Thr, Asp, Pro, Arg and Lys) by synaptosomal fractions prepared from rat brains were studied. Distinct differences were observed in the ion requirements for the accumulation of these amino acids. The accumulation of Asp and Pro alone showed a total requirement for Na⁺; uptakes of the other amino acids were either maximal in Na⁺-free media or only partially dependent on the presence of external Na⁺. With brain maturation, two types of developmental alterations could be distinguished: (1) changes in rates of influx, and (2) changes in the effects of ions. Synaptosomal fractions prepared from brains of immature rats accumulated Leu, Arg and Lys to a greater extent and Val, Tyr, Pro and Asp to a lesser extent than did the fractions prepared from brains of mature animals. The accumulation of Ser and Thr by immature fractions was partially dependent on external Na⁺, whereas their accumulation by adult fractions was Na⁺-independent. These alterations in Na⁺ requirements coincided with developmental changes in mutual inhibitions of amino acid transport.     

Characteristics of amino acid accumulation by isolated intestinal epithelial cells

Summary Accumulation of neutral amino acids by isolated chick epithelial cells has been studied and the results discussed in terms of the ion gradient model, and a model invoking a direct input of metabolic energy. The cells establish four- to eightfold concentration gradients of amino acids at an extracellular concentration of 1mm. The accumulation is sodium-dependent, inhibited by high extracellular potassium concentrations, and is sensitive to a variety of metabolic inhibitors. Also, amino acid uptake is depressed by actively transported sugars, and certain other amino acids, and is stimulated by phloridzin.Cells equilibrated with valine and loaded with 30 to 40mm intracellular sodium begin immediately to actively accumulate valine when suddenly introduced to media containing 20mm sodium. The cells establish a threefold gradient of amino acid during the interval when intracellular sodium is higher than extracellular sodium.Amino acid accumulation and²²Na efflux were monitored simultaneously in cells treated with phloridzin. While phloridzin causes a 30% stimulation of amino acid uptake, no variation in the rate of²²Na efflux or the steady-state level of²²Na maintained by the cells can be detected. Similarly, either 2.5mm glucose or 2.5mm 3-O-methylglucose cause approximately a 50% inhibition of 1mm valine uptake, but no detectable change in steady-state cellular²²Na content. Several aspects of the data seem inconsistent with concepts embodied in the ion gradient hypothesis, and it is suggested that a directly energized transport mechanism can better accommodate all of the data.     

Amino acid sequences of ovomucoid third domain from 25 additional species of birds

Ovomucoids were isolated from 25 avian species other than the 101 studied in Laskowskiet al. (1987,Biochemistry 26, 202–221). These were subjected to limited proteolysis with an appropriate enzyme, and connecting peptide extended ovomucoid third domains were isolated and sequenced to the end in a protein sequencer. Of the 25 new sequences, 13 duplicate ones were already known, and 12 are unique. Probably the most striking findings are a Pro¹⁴ Ser¹⁴ replacement in weka, an Ala¹⁴Thr¹⁵ replacement in Bulwer's pheasant, the discovery of two additional amino acid residues Ile¹⁸ and Gly¹⁸ at the P₁ reactive site position in Kalij pheasant and tawny frogmouth, respectively, and the first finding of a negative (Glu³⁴) rather than positive (Lys³⁴ or Arg³⁴) amino acid residue at the NH₂ terminus of the helix in caracara ovomucoid third domain. These results complete the determination of all the sequences of ovomucoid third domains in the four species genusGallus, in the five species genusSyrmaticus, and in the two species generaAix andPavo.     

Effect of EDTA on transport forms of manganese in maize xylem exudate

Xylem exudates were collected from 20-day-old maize seedlings grown for 24 h in labelled manganese chloride solution (10 ppm) with or without EDTA (18 mgl^-1). The distribution of radioactivity on paper ohromatograms of exudates did not show the existence of free Mn. Fractionation of exudate constituents by gel filtration on Sephadex G-26 showed that⁵⁴Mn is associated with three UV (254 nm) -absorbing fractions. Two fractions contain amino acids (M. wt.:≧ 10 000 and ∼ 900). The third fraction (M. wt. ∼ 600) however, appears to be a carrier of a different type, which is practically amino acid free, although it contains 10.23% N (by elementary analysis). The properties of this carrier are discussed. Application of EDTA has favoured the; transport of Mn in xylem sap with fractions of relatively lower molecular weights.     

A dominant interfering mutation in RAS1 of Saccharomyces cerevisiae

A mutant allele of RAS1 that dominantly interferes with the wild-type Ras function in the yeast Saccharomyces cerevisiae was discovered during screening of mutants that suppress an ira2 disruption mutation. A single amino acid substitution, serine for glycine at position 22, was found to cause the mutant phenotype. The inhibitory effect of the RAS1 ^Ser22 gene could be overcome either by overexpression of CDC25 or by the ira2 disruption mutation. These results suggest that the RAS1^Ser22 gene product interferes with the normal interaction of Ras with Cdc25 by forming a dead-end complex between Ras1^Ser22 and Cdc25 proteins.     

A dominant interfering mutation in RAS1 of Saccharomyces cerevisiae

A mutant allele of RAS1 that dominantly interferes with the wild-type Ras function in the yeast Saccharomyces cerevisiae was discovered during screening of mutants that suppress an ira2 disruption mutation. A single amino acid substitution, serine for glycine at position 22, was found to cause the mutant phenotype. The inhibitory effect of the RAS1 ^Ser22 gene could be overcome either by overexpression of CDC25 or by the ira2 disruption mutation. These results suggest that the RAS1^Ser22 gene product interferes with the normal interaction of Ras with Cdc25 by forming a dead-end complex between Ras1^Ser22 and Cdc25 proteins.     

Concerning the presence and formation of ascorbic acid in yeasts

The selective, sensitive method of analysis of ascorbic acid by high performance liquid chromatography with electrochemical detection (HPLC/EC) has been used to determine the ascorbic acid content of cell extracts from yeasts grown in glucose-free medium, 0.3 M D-glucose, and 0.112 M L-galactono-1,4-lactone. Saccharomyces cerevisiae (strain G-25 and its tetraploid) and a commercial baker's yeast contained less than 2 μg ascorbic acid g^?1 wet wt. of cells when grown for 22 h in glucose-free medium. In 0.3 M D-glucose, only the commercial baker's yeast gave a slight increase (2–50 μg g^?1 wet wt. in 22 h). In 0.112 M L-galactono-1,4-lactone, all three strains produced ascorbic acid (372–587 μg g^?1 wet wt. in 22 h). Lypomyces starkeyi, a species previously reported to contain a significant amount of ascorbic acid (Heick et al., Can. J. Biochem., 47 (1972) 752), was essentially devoid of ascorbic acid under all three conditions of incubation although it did contain an HPLC/EC reactive peak (R_T = 0.87 relative to ascorbic acid) that was readily oxidized by charcoal in the presence of oxygen. The identity of this new compound remains to be determined.     

Properties of spinach chloroplast fructose-1,6-diphosphatase

Photosynthetic fructose-1,6-diphosphatase (FDPase) fractions I and II, earlier purified from spinach leaves, show a similar amino acid composition, with the exception of a higher glutamic acid content in the latter. In both fractions glutamic and aspartic acids are the main amino acids. pH activity profiles of fractions I and II are similar, with optima at 8·65–8·70, both showing a high specificity for fructose- 1,6-diphosphate. These two fractions are Mg²⁺-dependent for activity, with an Optimum Mg²⁺ concentration of 10 mM in standard conditions, which shifts to 5 mM when the MG²⁺/EDTA ratio is increased to 10; Mn²⁺ and Co²⁺ are slightly active. EDTA enhances FDPase activity slightly, with an optimum at 0·4–0·8 mM. Cysteine has no activating effect, and acts as an inhibitor above 10 mM. Both I and II have an optimum substrate concentration of 4 mM, and the substrate inhibits at concns above this value. Kinetic velocity curves are sigmoidal, with the concave zone located in the range of physiological substrate concns. (Hill coefficient 1·75 for both). This suggests a strong regulatory role of fructose-1,6-diphosphate. K_m values are 1·4 × 10⁻³ M (fraction I) and 1·1 × 10⁻³ M (fraction II). The highest activity rate occurs at 60°, in accordance with the high thermostability of both fractions; the activation energies are 14·3 kcal/mol (fraction I) and 13·0 kcal/mol (fraction II).     

Sequential Comparison of Peptides Containing Half-cystine Residues from Ovalbumins of Six Avian Species

Hen ovalbumin contains one cystine disulfide (Cys⁷³-Cys¹²⁰) and four cysteine sulfhydryl groups (Cys¹¹,Cys³⁰,Cys³⁶⁷, and Cys³⁸²) in a single polypeptide chain of 385 amino acid residues. To investigate whether or not such a structure is shared by related avian species, the contents of disulfide-involved half-cystine residues and their positions in the primary structure of ovalbumins from five species were compared with those of hen ovalbumin. Ovalbumins were alkylated with a fluorescent dye, IAEDANS, under disulfide-reduced and disulfide-intact conditions and digested with a number of proteolytic enzymes. The sequences were deduced from peptides containing half-cystine residues labeled with the fluorescent dye. The results showed that the number of free cysteine sulfhydryl groups of ovalbumins was different among the species, three for guinea fowl and turkey (Cys¹¹, Cys³⁶⁷, and Cys³⁸²); and two for Pekin duck, mallard duck, and Emden goose (Cys¹¹ and Cys³³¹). On the other hand, a single intrachain disulfide bond could be identified from ovalbumins of five species using a combination of peptide mapping and N-terminal amino acid sequencing analysis under reduced and non-reduced conditions, in which the intrachain disulfide bond was like that of hen ovalbumin (Cys⁷³-Cys¹²⁰). The results also indicated that the variations in amino acid sequences on these peptides containing half-cystine residues bear a close relationship with the phylogeny of the six species.     

Erratic Evolution of Glycerol-3-Phosphate Dehydrogenase in Drosophila,Chymomyza, and Ceratitis

We have studied the evolution of Gpdh in 18 fruitfly species by sequencing 1,077 nucleotides per species on average. The region sequenced includes four exons coding for 277 amino acids and three variable-length introns. Phylogenies derived by a variety of methods confirm that the nominal genus Zaprionus belongs within the genus Drosophila, whereas Scaptodrosophila and Chymomyza are outside. The rate of GPDH evolution is erratic. The rate of amino acid replacements in a lineage appears to be 1.0 × 10⁻¹⁰/site/year when Drosophila species are considered (diverged up to 55 million years ago), but becomes 2.3 × 10⁻¹⁰ when they are compared to Chymomyza species (divergence around 60 My ago), and 4.6 × 10⁻¹⁰ when species of those two genera are compared with the medfly Ceratitis capitata (divergence around 100 My ago). In order to account for these observations, the rate of amino acid replacement must have been 15 or more times greater in some lineages and at some times than in others. At the nucleotide level, however, Gpdh evolves in a fairly clockwise fashion. Received: 13 June 1996 / Accepted: 16 August 1996     

Mutagenicity of the Pyrolysis Products of Ammonium Salts

One molecular species of prothoracicotropic hormone with a molecular weight of about 22, 000 (22K-PTTH) of the silkworm, Bombyx mori, was isolated from 5 × 10⁵ adult heads. The purification procedure consisted of 16 steps including defatting, salt-extraction, fractional precipitations, conventional column chromatographies, and high performance liquid chromatographies. An approximately 5 × 10⁶-fold purification was attained to yield 5.4 μg (0.25 nmol) of the pure hormone with a recovery of 3.3%. Injection of 0.11 ng of the purified 22K-PTTH could elicit adult development in a brainless Bombyx pupa. 22K-PTTH is a basic protein (pI 7.7 ~ 8.7) containing disulfide bond(s). The amino-terminal amino acid sequence of 22K-PTTH was determined to be Gly-Asn-Ile-Gln-Val-Glu-Asn-Gln-Ile-Pro-Asp-Pro-.