Electrophoretic profiles and amino acid composition of rice endosperm proteins of a mutant with enhanced lysine and total protein after backcrosses for germplasm improvements

 Seed storage proteins from in vitro-derived rice mutants improved by several backcrosses to ‘Calrose 76’ and BC₂ and BC₃ were characterized for changes in five different solubility classes. Albumins, rsealb (water-soluble globulins), true salt-soluble globulins, prolamins and glutelins were SDS-PAGE separated in a single dimension, and some two-dimensionally, to identify protein modifications. The genetic transmission of the enhanced-lysine mutants in backcrosses and the linkage of lysine with grain chalkiness were confirmed. Advanced lines had altered globulin profiles similar to those of unimproved lines. Chalky/ enhanced-lysine phenotypes had similar prolamin and glutelin profiles in the mutant and controls at the same protein level. Mutants had increased levels of globulins at 50 kDa and 33 kDa but had substantially less protein at 25 kDa than the controls. High protein in the mutant contributed to an increase in prolamins and the major storage proteins in both the globulins and glutelins. A significant decrease in low-molecular-weight, 15- to 18-kDa albumins was associated with the chalky/enhanced-lysine mutant phenotype. Two proteins in the 15- to 18-kDa group were amino acid sequenced, and database comparisons identified these proteins as allergens. Advanced lines downregulated for allergens and with enhanced-lysine/protein but with normal fertility and seed weight should be useful in breeding programs for nutritional quality. Received: 14 October 1996 / Accepted: 8 November 1996     

Synthesis of novel acetylene-containing amino acids

Summary.  Novel synthetic procedures for the modification of non-proteinogenic acetylene-containing amino acids have been developed. The functionalization either proceeds via zinc/copper-mediated introduction of alkyl substituents, or via tungsten-catalyzed ring-closing alkyne metathesis reactions. Received March 28, 2002 Accepted October 3, 2002 Published online December 18, 2002 Acknowledgements These investigations are supported (in part) by the Netherlands Research Council for Chemical Sciences (CW) with financial aid from the Netherlands Technology Foundation (STW). Authors' address: Floris P. J. T. Rutjes, Prof. Dr., Department of Organic Chemistry, University of Nijmegen, Toernooiveld 1, NL-6525 ED Nijmegen, The Netherlands, E-mail: rutjes@sci.kun.nl  2, selected data: ¹H NMR (300 MHz, CDCl₃) δ 5.32 (d, J = 7.7 Hz, 1H), 4.44–4.40 (m, 1H), 3.76 (s, 3H), 2.75–2.73 (d, J = 5.0 Hz, 2H), 1.44 (s, 9H); ¹³C NMR (75 MHz, CDCl₃) δ 171.0, 155.0, 80.3, 74.6, 52.6, 51.9, 41.7, 28.3, 24.0; mp = 55°C.  Typical procedure for 5: zinc dust (116 mg, 1.408 mmol) was weighed into a 20 mL flask, which was repeatedly evacuated (with heating using a heat gun) and flushed with argon. Dry DMF (0.5 mL, distilled from CaH₂) and 1,2-dibromoethane (9.2 μL, 0.106 mmol) were added and the flask was heated at 80°C for 40 min. The reaction mixture was allowed to cool to room temperature, trimethylsilyl chloride (4 μL, 0.035 mmol) was added and the resulting mixture was stirred vigorously for a further 30 min under argon. Iodocyclohexane (69 μl, 0.528 mmol) was added and stirred at room temperature for 3 h more after which stirring was ceased to settle the zinc. CuCN (41 mg, 0.458 mmol) and LiCl (40 mg, 0.915 mmol) were heated to 150°C for 2 h and cooled to room temperature. Addition of DMF (1 mL) formed a soluble CuCN·2LiCl complex within 5 min. After cooling the Cu-complex to −15°C, the organozinc reagent was added dropwise followed by the bromoacetylene 2 (116 mg, 0.352 mmol). The mixture was allowed to stir overnight at room temperature. Water was added and the suspension was extracted using heptane, washed with brine, dried (MgSO₄) and concentrated. Purification using flash column chromatography (10% EtOAc in heptane) yielded 5 (100 mg, 81%) as a colorless oil. 5: IR ν 3355, 2929, 2852, 2359, 2337, 1749, 1717, 1498, 1447, 1365, 1251, 1181, 1060; ¹H NMR (300 MHz, CDCl₃) δ 5.28 (d, J = 7.7 Hz, 1H), 4.43–4.38 (m, 1H), 3.73 (s, 3H), 2.69–2.63 (m, 2H), 2.13 (m, 1H), 1.73–1.22 (m, 10H), 1.43 (s, 9H); ¹³C NMR (75 MHz, CDCl₃) δ 171.4, 155.0, 88.1, 79.9, 73.8, 52.3, 32.7, 32.7, 28.8, 28.2, 25.8, 24.6, 23.1; HRMS (EI): calculated for C₁₇H₂₇NO₄ 309.1940, found 309.1937.  A solution of the tungsten catalyst (7 mg, 10 mol%) in C₆H₅Cl (2 mL) was treated with a solution of 14 (49.0 mg, 0.120 mmol) in C₆H₅Cl (5.0 mL) under an argon atmosphere and the resulting mixture was heated at 80°C for 3 h. Evaporation followed by flash column chromatography (80% EtOAc in heptane) afforded 15 (21.0 mg, 50%; 64% after correction for starting material) and 14 (16 mg, 33%) as colorless oils. 15: [α]_D =–14.6 (c = 1, CH₂Cl₂); IR ν 3313, 2931, 2865, 2249, 1744, 1667, 1520, 1366, 1170; ¹H NMR (400 MHz, CDCl₃) δ 7.14 (d, J = 8.7 Hz, 1H), 6.08 (d, J = 8.3 Hz, 1H), 4.78 (q, J = 6.8 Hz, 1H), 4.27 (q, J = 7.9 Hz, 1H), 3.73 (s, 3H), 2.17–2.15 (m, 4H), 2.07–1.96 (m, 2H), 1.79–1.52 (m, 4H), 1.45 (s, 9H), 0.89–0.83 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 173.2, 171.8, 155.8, 80.4, 80.2, 79.3, 53.8, 52.5, 51.2, 32.8 (2×), 28.1, 24.6, 24.2, 18.3 (2×); HRMS (EI): calculated for C₁₈H₂₈N₂O₅     

Modification of amino acid composition of endosperm proteins from in-vitro-selected high lysine mutants in rice

Summary Endosperm protein mutants in rice may be recovered by biochemical selections with inhibitory levels of lysine and threonine. Among the phenotypes recovered from in vitro selections are lines with increased protein and percent lysine in the protein. This work was designed to identify changes in proteins of rice mutants and to further our understanding of the mechanisms of lysine plus threonine selections in rice. Among the most obvious amino acid changes in mutants was a higher lysine level in all protein solubility fractions and a decrease in tyrosine. Methionine and glutamate are reduced in some protein fractions. However, methionine is significantly higher in the mutant than the control in the glutelin fraction. Several other aspartate pathway amino acids are higher in the mutant than the unselected controls. Separation of proteins in SDS-PAGE gels showed shifts in the protein profiles in the mutants, including a decrease in the major 30 kDa low lysine globulin component, and an increase in several high-molecular-weight components, approximately 60–100 kDa. Increases in the lysine content of proteins of different solubility classes and different proteins within classes are detailed.     

Biosynthesis of lysine and other amino acids in the developing maize endosperm

Tracer studies with aspartic acid-[4-¹⁴C], alanine-[1-¹⁴C] acetate-[2-¹⁴C] and diaminopimelic acid-[1,(7)-¹⁴C] injected into the developing endosperm of maize revealed that the biosynthesis of lysine and other amino acids occurs in this organ. The data suggest that lysine is synthesized via the diaminopimelic acid pathway.     

Plasma amino acids concentration in amyotrophic lateral sclerosis patients

Summary.  Previous investigations showed an impairment of amino acids (AA) metabolism in amyotrophic lateral sclerosis (ALS). It was hypothesized that excitatory AA may play an important role in the etiopathogenesis of this disease. The aim of the study was to determine plasma AA concentrations in ALS patients, and to examine the relationship between AA and the clinical state of ALS patients, the type of ALS onset and the duration of the disease. The study involved 20 ALS patients and 30 control group people. The AA analysis was performed by ion – exchange chromatography on an automatic AA analyser. The results showed significantly decreased concentrations of valine, isoleucine, leucine, tyrosine and aspartate in the plasma of the whole group of ALS patients compared to the control group, and a significantly decreased concentration of arginine in the patients with a long duration of ALS compared to the patients with a short duration. The clinical state of ALS patients significantly influenced only plasma alanine concentration. Other plasma AA concentrations were not significantly associated with clinical parameters of the disease. Our study confirms that metabolic abnormalities concerning AA exist in ALS patients. However, the normal plasma glutamate concentration observed in this study in the whole group of ALS patients compared to the controls does not exclude that this excitatory AA may play a role in neurodegeneration in ALS. Received June 22, 2002 Accepted October 3, 2002 Published online January 23, 2003 Correspondence: Joanna Iłżecka M.D., Department of Neurology, Medical University, Jaczewskiego 8, 20-954 Lublin, Poland, Fax: +48 81 742 55 34, E-mail: Ilzecka@medscape.com     

Anaerobic accumulation of amino acids in rice roots: role of the glutamine synthetase/glutamate synthase cycle

Summary. Accumulation of amino acids was studied in rice roots of 3-day-old seedlings subjected for 48 h to anaerobic conditions. Alanine and Gaba were the main amino acids accumulated under anoxia. Their synthesis was strongly inhibited by MSX and AZA, inhibitors of glutamine synthetase and glutamate synthase. These activities increased after 8 h of anaerobic treatment and, by immunoprecipitation of ³⁵S-labeled proteins, it was shown that glutamine synthetase and ferredoxin-dependent glutamate synthase were synthesized during the treatment. These findings indicate that the glutamine synthetase/glutamate synthase cycle play an important role in anaerobic amino acid accumulation. Received April 5, 1999     

Lysine catabolism, an effective versatile regulator of lysine level in plants

Lysine is a nutritionally important essential amino acid, whose synthesis in plants is strongly regulated by the rate of its synthesis. Yet, lysine level in plants is also finely controlled by a super-regulated catabolic pathway that catabolizes lysine into glutamate and acetyl Co-A. The first two enzymes of lysine catabolism are synthesized from a single LKR/SDH gene. Expression of this gene is subject to compound developmental, hormonal and stress-associated regulation. Moreover, the LKR/SDH gene of different plant species encodes up to three distinct polypeptides: (i) a bifunctional enzyme containing the linked lysine-ketoglutarate (LKR) and saccharopine dehydrogenase (SDH) whose LKR activity is regulated by its linked SDH enzyme; (ii) a monofunctional SDH encoded by an internal promoter, which is a part of the coding DNA region of the LKR/SDH gene; and (iii) a monofunctional, highly potent LKR that is formed by polyadenylation within an intron. LKR activity in the bifunctional LKR/SDH polypeptide is also post-translationally regulated by phosphorylation by casein kinase-2 (CK2), but the consequence of this regulation is still unknown. Why is lysine metabolism super-regulated by synthesis and catabolism? A hypothesis addressing this important question is presented, suggesting that lysine may serve as a regulator of plant growth and interaction with the environment.     

Correlations of amino acids in proteins

A correlation analysis among 20 amino acids is performed for four protein structural classes (, β, /β, and +β) in a total of 204 proteins. The correlation relationships among amino acids can be classified into the following four types: (1) strong positive correlation, (2) strong negative correlation, (3) weak correlation, and (4) no correlation. The correlation relationships are different for different proteins and are correlated with the features of their structural classes. The amino acids with the weak correlation relationship can be treated as the independent basis functions for the space where proteins are defined. The amino acids with large correlation coefficients are linear correlative with each other and they are not independent. The strong correlation among amino acids reflects their mutual constrained relationship, as exhibited by their relevant structural features. The information obtained through the correlation analysis is used for predicting protein structural classes and a better prediction quality is obtained than that by the simple geometry distance methods without taking into account the correlation effects.     

Amino acid requirement of growing pigs depending on amino acid efficiency and level of protein deposition 1st communication: lysine

In this study we investigated the influence of harvest date and genotype on the ruminai degradability of the organic matter of ensiled maize grains. Grains of the varieties Avenir, Byzance, CGS 5104 and CGS 5107 from six different harvest dates were available; they are classified as intermediate types between flint and dent com. The six harvest dates, during which time the dry matter content of the ensiled grains rose from 52% to 66%, extended from 1st September to 19th October. Assuming a passage rate of k=0.08, the effective ruminai degradability declined in this period on average from 93% to just under 79%; variety‐specific deviations also increased markedly during this period. The dry matter content (x, DM in %) of the ensiled grains had a profound influence on ruminai degradation: a highly significant curvilinear decline in ruminai degradability (y) was calculated at increasing DM levels (k = 0.08), which can be described by the equation y = ?0.072x² (±0.010)+7.417x(±1.186)?98.71(±34.58)(B=0.96;s_y,x[%]=1.36).

The ruminai degradability of ensiled maize grains is about 5–10% higher than that of fresh maize grains.     

Free amino acids in Crotalaria seeds

The free amino acids of seeds of 163 species of Crotalaria have been identified. Their pattern of distribution is compared with recent classifications of the genus and the possible ecological significance of their presence is discussed. Attention is drawn to the occurrence of toxic amino acids.     

Genetic engineering approaches to improve the bioavailability and the level of iron in rice grains

Iron deficiency is the most widespread micronutrient deficiency world-wide. A major cause is the poor absorption of iron from cereal and legume-based diets high in phytic acid. We have explored three approaches for increasing the amount of iron absorbed from rice-based meals. We first introduced a ferritin gene from Phaseolus vulgaris into rice grains, increasing their iron content up to two-fold. To increase iron bioavailability, we introduced a thermotolerant phytase from Aspergillus fumigatus into the rice endosperm. In addition, as cysteine peptides are considered a major enhancer of iron absorption, we overexpressed the endogenous cysteine-rich metallothionein-like protein. The content of cysteine residues increased about seven-fold and the phytase level in the grains about 130-fold, giving a phytase activity sufficient to completely degrade phytic acid in a simulated digestion experiment. High phytase rice, with an increased iron content and rich in cysteine-peptide, has the potential to greatly improve iron nutrition in rice-eating populations. Received: 15 April 2000 / Accepted: 12 May 2000     

Free amino acids and biogenic amines in red and white muscle of tuna stored in controlled atmospheres

Summary. This paper analyses the presence of and changes in free amino acids and biogenic amines in red and white muscle of bigeye tuna during storage in controlled atmospheres with 2 gas mixes containing different concentrations of CO₂ and O₂. Levels of amines were generally higher in white than in red muscle, with the exception of putrescine and spermidine. Levels of biogenic amines increased (p<0.05) throughout storage, commencing later in red than in white muscle. A correlation between the amino acid histidine and the biogenic amine histamine was observed, but only in white muscle. Only in the case of tryptophan did white and red muscle differ (p<0.05) in terms of essential free amino acid content. They also differed in anserine content. Concentrations of the non-essential FAAs glutamic acid, glycine and alanine were higher in red than in white muscle. The effectiveness of the atmospheres was reflected in the evolution of both biogenic amines and FAAs. Gas mix 1, containing a higher concentration of CO₂, was the more effective.     

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.     

Feeding pigs amino acids as protein-bound or in free form influences postprandial concentrations of amino acids,metabolites, and insulin

Dietary proteins need to be digested first while free amino acids (AAs) and small peptides are readily available for absorption and rapidly appear in the blood. The rapid postprandial appearance of dietary AA in the systemic circulation may result in inefficient AA utilisation for protein synthesis of peripheral tissues if other nutrients implicated in AA and protein metabolism are not available at the same time. The objective of this experiment was to compare the postprandial concentrations of plasma AA and other metabolites after the ingestion of a diet that provided AA either as proteins or as free AA and small peptides. Twenty-four male growing pigs (38.8 ± 2.67 kg) fitted with a jugular catheter were assigned to one of three diets that provided AA either in protein form (INT), free AA and small peptides (HYD), or as free AA (FAA). After an overnight fast and initial blood sampling, a small meal was given to each pig followed by serial blood collection for 360 min. Postprandial concentrations of plasma AA, glucose, insulin, and urea were then measured from the collected blood. Non-linear regression was used to summarise the postprandial plasma AA kinetics. Fasting concentrations of urea and some AA were higher (P < 0.05) while postprandial plasma insulin and glucose were lower (P < 0.01) for INT than for HYD and FAA. The area under the curve of plasma concentration after meal distribution was lower for INT for most AAs (P < 0.05), resulting in a flatter curve compared to HYD and FAA. This was the result of the slower appearance of dietary AA in the plasma when proteins are fed instead of free AA and small peptides. The flatter curve may also result from more AAs being metabolised by the intestine and liver when INT was fed. The metabolism of AA of the intestine and liver was higher for HYD than FAA. Providing AA as proteins or as free AA and small peptides affected the postprandial plasma kinetics of AA, urea, insulin, and glucose. Whether the flat kinetics when feeding proteins has a positive or negative effect on AA metabolism still needs to be explored.     

Changes in carbohydrates,amino acids and proteins in developing seed of chickpea

Developing seeds of chickpea cultivars G-130, L-550 and 850-3/27 grown under field conditions were sampled at different stages of maturity and analysed for soluble sugars, starch, soluble nitrogen, protein nitrogen and amino acids. Fr. wt of seeds of all three cultivars decreased after 28 days of flowering while the dry wt continued to increase. Rapid starch accumulation was observed between 14 and 28 days after flowering. Starch as per cent of seed dry wt started to decrease after 28 days, while starch per seed increased till maturity. The percentage of salt-soluble proteins decreased with maturation of seed. The electrophoretic pattern revealed that deposition of seed storage protein in cotyledons occurred 14 days after flowering. Most of the biochemical activity apparently occurred between 14 and 28 days after flowering.     

Changes in growth,proteins and free amino acids of developing seed and pod of fenugreek

The growth and development under field conditions of the pods and seeds of two cvs of Trigonella foenum graecum are described. Samples were harvested at different stages of ripeness for the determination of dry matter, protein and free amino acid content. During maturation, reserves of solutes are established in the pod wall before the seeds begin their exponential phase of growth. Later, these reserves disappear, providing about 20% of the seed's requirements for nitrogen. SDS-electrophoresis was used to follow the formation of proteins and it was shown that the synthesis of storage proteins takes place prior to dehydration of the seed. Production soluble nitrogenous compounds precedes protein accumulation. Free amino acids follow the same pattern. 4-Hydroxyisoleucine represents nearly 80% of free amino acid of dry seeds. The concentration does not decrease in the later stages of maturation of the seed but this unusual amino acid is absent in the storage proteins of the seeds.     

Effects of hypoxia on plasma amino acids of fetal sheep

Summary. Secondary amino acid disturbances from circulatory responses during hypoxia may cause problems in interpreting plasma amino acid profiles of sick babies investigated for possible inherited defects. Systematic studies to characterise them are difficult in man. We investigated the effects of hypoxia on plasma amino acids by studying 9 late gestation fetal sheep in utero during 11 one hour episodes of moderately severe isocapnic hypoxia. In 6 experiments, maternal plasma amino acids were also monitored. Fourteen fetal plasma amino acids increased significantly, with the largest proportionate changes in alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, ornithine and lysine. Maternal amino acids did not increase. Probable explanations were reflex peripheral vasoconstriction in skeletal muscle beds and decreased hepatic blood flow. The findings extend our knowledge of the fetal response to hypoxic stress, demonstrate the importance of skeletal muscle in branched-chain amino acid metabolism, and should help with interpretation of postnatal plasma amino acid disturbances. Received January 29, 1999, Accepted February 22, 1999     

Free radical-mediated oxidation of free amino acids and amino acid residues in proteins

Summary. We summarize here results of studies designed to elucidate basic mechanisms of reactive oxygen (ROS)-mediated oxidation of proteins and free amino acids. These studies have shown that oxidation of proteins can lead to hydroxylation of aromatic groups and aliphatic amino acid side chains, nitration of aromatic amino acid residues, nitrosylation of sulfhydryl groups, sulfoxidation of methionine residues, chlorination of aromatic groups and primary amino groups, and to conversion of some amino acid residues to carbonyl derivatives. Oxidation can lead also to cleavage of the polypeptide chain and to formation of cross-linked protein aggregates. Furthermore, functional groups of proteins can react with oxidation products of polyunsaturated fatty acids and with carbohydrate derivatives (glycation/glycoxidation) to produce inactive derivatives. Highly specific methods have been developed for the detection and assay of the various kinds of protein modifications. Because the generation of carbonyl derivatives occurs by many different mechanisms, the level of carbonyl groups in proteins is widely used as a marker of oxidative protein damage. The level of oxidized proteins increases with aging and in a number of age-related diseases. However, the accumulation of oxidized protein is a complex function of the rates of ROS formation, antioxidant levels, and the ability to proteolytically eliminate oxidized forms of proteins. Thus, the accumulation of oxidized proteins is also dependent upon genetic factors and individual life styles. It is noteworthy that surface-exposed methionine and cysteine residues of proteins are particularly sensitive to oxidation by almost all forms of ROS; however, unlike other kinds of oxidation the oxidation of these sulfur-containing amino acid residues is reversible. It is thus evident that the cyclic oxidation and reduction of the sulfur-containing amino acids may serve as an important antioxidant mechanism, and also that these reversible oxidations may provide an important mechanism for the regulation of some enzyme functions.     

Free amino acids in the haemolymph of honey bee queens (Apis mellifera L.)

In queen honey bees the free amino acid content in the haemolymph clearly depends on the physiological function and social environment of the individual. While in drones and workers the content of free amino acids increases after emergence until it reaches a peak in 5-day-old animals and decreases afterwards, the amino acid content in queens reaches its highest level (>60 nmol/ microl haemolymph) with the onset of egg laying (10 d of age). This level is about 2.5 times more than the highest level found in workers. Queens maintain this high level also when they are older (>30 d) and continue to lay eggs in average colonies. As in drones and workers, in queens the predominant amino acid is proline, which accounts for more than 50% of the total content of free amino acids in egg-laying individuals. When 10-day-old queens are prevented from mating and do not lay eggs, their amino acid content is significantly lower compared to laying queens of the same age. Also the social environment influences the contents of free amino acids in queens. When virgin queens were kept for 6 days with 20 worker bees and sufficient honey and pollen in an incubator, they had significantly lower concentrations of amino acids than virgin queens living for the same period with about 8000 workers in a colony. Most probably, the high amino acid concentration in the haemolymph is the basis for the high protein synthesis activity of laying queens.     

EPR study of anion radicals of various N-quinonyl amino acids

Summary. Since peptide quinones possess great clinical potential in targeted chemotherapy, several series of novel N-quinonyl amino acids have been synthesized and their first products of reduction were studied by EPR spectroscopy. EPR spectra of the corresponding radical adducts were identified by computer simulation. The dependence between the splitting constants and the chemical structure of the N-quinonyl amino acids anion radicals was examined. Received January 4, 2000; Accepted March 14, 2000