Changes in protein fractions and leucine-[14C] incorporation during Sorghum grain development

Incorporation of leucine and changes in different protein fractions have been studied during Sorghum grain development. Most of the label from the injected leucine-[¹⁴C] was found in glutelin and residue fraction towards later stages of maturity. The label in albumin, globulin and prolamin decreased with a concomitant increase in label in glutelin and residue proteins. The concentration of lysine, aspartic acid and glycine decreased while that of leucine, proline, alanine, tyrosine, phenylalanine, and cystine increased during grain development. Increase in serine, methionine, valine and isoleucine was only marginal. The proportion of glutamic acid was high at all stages of grain development. Glutelin fraction resolved into two peaks on gel chromatography, only one of which with higher MW was labelled, while in albumin both the peaks were found to be labelled. Tannin content also increased during grain development.     

The Lack of Selective Reincorporation into Tissue Protein of the Amino Acids Derived from the Catabolism of Serum Protein

Homologous S³⁵-labeled albumin, gamma globulin, and alpha-beta globulin were transfused into rabbits and the specific activities of the electrophoretic fractions of the sera of the recipients were determined at various time intervals up to 12 days after injection. Detectable reincorporation into a fraction other than that transfused was found only in the gamma globulin fraction after albumin injection. This activity rose between 2 and 12 days and reached a level of 2 to 3 per cent of the extrapolated zero time activity of the albumin fraction. When homologous serum protein doubly labeled with I¹³¹ and S³⁵ was transfused into mice, marked drops in the ratios of I¹³¹ to S³⁵ in the serum and tissue proteins were observed between 1 and 48 hours after injection. On the basis of a determination of the absolute and relative amounts of I¹³¹ and S³⁵ found in the various tissue and serum proteins, the amount of reincorporation of S³⁵ into each protein was calculated. The relative amounts of reincorporation of S³⁵ among the various tissues were remarkably similar to the relative amounts of incorporation of S³⁵ after the injection of labeled free amino acids. It is concluded that serum protein does not form a major direct source of amino acids to the tissues but feeds them indirectly through the extracellular pool.     

Changeability of protein fractions and their amino acid composition during maturation of barley grain

The formation of the protein complex during barley grain maturation is characterized by unequal synthesis of different protein fractions. In ten day-old grain the dominating protein is glutelin, followed by albumin and globulin. The content of prolamin is at this stage negligible. Particularly after the eighteenth day of maturation intensive synthesis of prolamins begins, which continues during the entire maturing period in almost the same ratio as total N accumulation in the grain. From the twenty-fourth day the amount of prolamins exceeds that of glutelins, and at full maturity 50% of the proteins are prolamins. The glutelin content increases absolutely, but relatively it decreases from 41 to 26%. The amino acid composition of flour from total grain as well as the amino acid composition of the individual proteins is significantly changed during maturation. Generally the changes are manifested in an increasing content of Glu and Pro and a decreasing content of Asp, Ala and Lys. These changes seem to occur as a result of an increasing representation of prolamin, which is characterized by large content of Glu and Pro and low content of Asp, Ala and Lys compared with other protein fractions.     

Protein solubility, digestibility and fractionation after germination of sorghum varieties

The changes in crude protein, free amino acids, amino acid composition, protein solubility, protein fractionation and protein digestibility after germination of sorghum were investigated. Sorghum varieties (Dorado, Shandaweel-6, Giza-15) were soaked for 20 h followed by germination for 72 h; the results revealed that crude protein and free amino acids in raw sorghum varieties ranged from 10.62 to 12.46% and 0.66 to 1.03 mg/g, respectively. Shandaweel-6 was the highest variety in crude protein and free amino acids content. After germination, crude protein was decreased and free amino acids were increased. There was an increase in content of valine and phenylalanine amino acids after germination. On the other hand, there was a decrease in most of amino acids after germination. After germination protein solubility was significantly increased. Regarding protein fractions, there was an increase in albumin, globulin and kafirin proteins and a decrease in cross linked kafirin and cross linked glutelin after germination.     

Changes in salt-soluble proteins of rice during grain development

Salt-soluble proteins, albumin and globulin, were prepared from dehulled rice (Oryza sativa L., line IR1541-76-3) during grain development. Albumin and globulin progressively increased during grain development up to about 12 days after flowering (DAF) and then decreased slightly during grain desiccation. Free amino N was maximum at 10 DAF. Total protein and glutelin-prolamin (by difference) continued to increase up to 20 DAF. Aminogram of total protein and globulin showed a progressive decrease in lysine and threonine among the essential amino acids. Albumin showed a similar trend except for the lesser change in lysine content. Disc gel electrophoresis showed a maximum of four major and six minor protein bands for albumin and only one major and three minor bands for globulin. Sodium dodecyl sulfate-gel electrophoresis revealed three major polypeptide subunits for albumin with MW of 11 000, 8 500 and 16 000, and two for globulin with MW of 20 000 and 12 000.     

A genome-wide association analysis of quantitative trait loci for protein fraction content in Tibetan wild barley

The genetic associations and differences of four protein fractions were investigated in Tibetan wild barley. Albumin, globulin and hordein contents were under genetic control probably via multiple genes/quantitative trait loci. A correlation analysis showed that globulin was significantly associated with albumin, glutelin and hordein, while hordein was closely correlated with glutelin. Forty-nine diversity array technology (DArT) markers, which were distributed over seven chromosomes, were associated with the protein fraction contents. Those DArT markers associated with hordein were the same as those associated with globulin and glutelin. Only five markers associated with hordein, globulin and glutelin were also associated with albumin. Most of the protein fraction contents are therefore controlled by same genes which may contribute to total protein content. The discovery of new markers associated with specific protein fractions could be used to detect genes controlling protein content in the barley germplasm.     

Thein vitro activity of the microsomal fraction isolated from the spleen and the lymph nodes after immunization of the donor

The microsomal fraction from the spleen (after perfusion) of immunized rabbits incubated for 20 min at 37° C under usual conditions in the presence of energy sources incorporates¹⁴C-labelled amino acids both into the solubilized (by adding deoxycholate), and into the nonsolubilized part (15%). The cell supernatant incorporates under these conditions the¹⁴C-labelled amino acids into total proteins in the absence of microsomes but in a lower degree. The cell supernatant contains gamma globulin detectable by immunoelectrophoresis. Gamma globulin obtained by specific precipitation of the solubilized microsomal fraction with antigamma-globulin serum had an measurable radioactivity. The precipitate of gamma globulin obtained from the supernatant of the incubation medium in the same manner (after removing the microsomes) had a specific activity twice as high. On separating the microsomal fraction extract and the incubation medium supernatant on DEAE cellulose most fractions show on extinction maximum at 260 nm in the first case and at 280 nm in the second case. The microsomal fraction isolated from the spleen and lymph nodes of immunized pigs-48 and 72 h after revaccination, when incubatedin vitro, incorporated¹⁴C-labelled amino acids into total protein. After ultrasonic disintegration in 0.14m NaCl and filtration through a Sephadex G 25 column it is specifically precipitated with the antigammaglobulin serum. Gamma globulin isolated after incubation of the microsomal fraction had a measurable radioactivity. AntiHSA antibodies determined by adsorption on immunosorbent did not possess significant radioactivity. Only the concentrated supernatant of the incubation medium showed minute radioactivity of 75–94 counts/min /ml. The problem of investigating the formation of nascent specific antibodies on a subcellular levelin vitro during the early period of secondary response to the antigen is discussed, in particular the problem of their detection. An erratum to this article is available at .     

Acetyl Groups in Native Glucomannan from Easter Lily Bulbs

The in vitro digestibility of rice glutelin and wheat glutenin was investigated with a view to assessing their nutritional qualities, using casein and bovine serum albumin (BSA) as references. The following hydrolytic processes were adopted: pepsin-pancreation digestion (a model system before intestinal absorption) and aminopeptidase-prolidase hydrolysis [a model system for the intestinal mucosa (membrane digestion) and after intestinal absorption (intracellular hydrolysis)]. The pepsin-pancreatin digests were first examined. The degree of amino acid released from the proteins was 30% (glutelin), 23% (glutenin), 24% (casein) and 30% (BSA). A similar release pattern of individual amino acids was observed for all the proteins. The amounts of large peptide fractions increased in the order: glutelin < glutenin < casein < BSA. Glutelin was highly digestible. Apart from containing high amounts of glutamic acid (glutamine), cystine and proline, the large peptide fractions of glutelin were also rich in threonine, glycine and isoleucine while those of glutenin were only rich in glycine. The aminopeptidase-prolidase digests were examined next. Glutelin was almost completely hydrolyzed to amino acid, except for a low release of cystine, suggesting that the amino acid residues constituting glutelin could be easily utilized as nutrients in the living tissues. The degree of amino acid released from the proteins was 97% (glutelin), 93% (glutenin), 90% (casein) and 79% (BSA).

The convenient application of these model systems for the assessment of the in vitro digestibility of food proteins have been discussed.     

Distribution of N within Pea, Lupin, and Soybean Nodules

The ¹⁵N abundance of some, but not all, legume root nodules is significantly elevated compared to that of the whole plant. It seems probable that differences in ¹⁵N enrichment reflect differences in the assimilatory pathway of fixed N. In that context, we have determined the distribution of naturally occurring ¹⁵N in structural fractions of nodules from soybean (Glycine max L. Merr.), yellow lupin (Lupinus luteus), and pea (Pisum sativum) nodules and in chemical components from soybean nodules and to a lesser extent, pea and lupin nodules. None of the fractions of pea nodules (cortex, bacteriod, or host plant cytoplasm) was enriched in ¹⁵N. The differences among bacteriods, cortex, and plant cytoplasm were smaller in lupin than in soybean nodules, but in both, bacteriods had the highest ¹⁵N enrichment. In soybean nodules, the ¹⁵N abundance of bacteriods and cortex was higher than plant cytoplasm, but all three fractions were more enriched in ¹⁵N than the entire plant. Plant cytoplasm from soybean nodules was fractionated into protein-rich material, nonprotein alcohol precipitable material (NA), and a low molecular weight fraction. The N of the latter was further separated into N of ureides, nucleotides and free amino acids. Most of these components were either similar to or lower in ¹⁵N abundance than the plant cytoplasm as a whole, but the NA fraction showed unusual ¹⁵N enrichment. However, the percentage of nodule N in this fraction was small. NA fractions from yellow lupin and pea nodules and from soybean leaves were not enriched in ¹⁵N. Nor was the NA fraction in ruptured bacteriods and cortical tissue of soybean nodules. Variation among soybean nodule fractions in the preponderance in protein of different amino acids was not large enough to explain the differences in ¹⁵N abundances among them. A hypothesis, consistent with all known data, concerning the mechanism leading to the observed excess ¹⁵N of lupin and soybean bacteriods is offered.     

Assessing post-anthesis nitrogen uptake, distribution and utilisation in grain protein synthesis in barley (Hordeum vulgare L.) using 15N fertiliser and 15N proteinogenic and non-proteinogenic amino acids

We investigated the effects of nitrogen (N) availability during the vegetative phase on (a) post‐anthesis N uptake and (b) its translocation into ears in barley plants grown in a greenhouse at two levels of N: low (50 mg N kg^?1 sand) and optimal N supply (150 mg N kg^?1 sand). Plants in the two N treatments were fertilised with the same amount of labelled ¹⁵N [50 mg ¹⁵N kg^?1 sand at 10% ¹⁵N_exc (N_excess, i.e. N_exc, is defined as the abundance of enriched stable isotope minus the natural abundance of the isotope) applied as ¹⁵NH₄¹⁵NO₃] 10 days after anthesis (daa). In a separate experiment, the uptake and transport into ears of proteinogenic and non‐proteinogenic amino acids were studied to determine whether a relationship exists between amino acid transport into ears and their proteinogenic nature. Plants were fed with either ¹⁵N‐α‐alanine, a proteinogenic amino acid, or ¹⁵N‐α‐aminoisobutyric acid, a non‐proteinogenic amino acid. Both these amino acids were labelled at 95.6% ¹⁵N_exc. Results showed that N accumulations in stems, leaves and especially in ears were correlated with their dry matter (dm) weights. The application of 150 mg N kg^?1 sand significantly increased plant dm weight and total N accumulation in plants. During their filling period, ears absorbed N from both external (growth substrate) and internal (stored N in plants) sources. Nitrogen concentration in ears was higher in optimal N‐fed plants than in low N‐fed plants until 10 daa, but from 21 to 35 daa, differences were not detected. Conversely, ¹⁵N_exc in ears, leaves and stems was higher in low N‐fed plants than in optimal N‐fed plants. Ears acted as strong sink organ for the post‐anthesis N taken up from the soil independently of pre‐anthesis N nutrition: on average, 87% of the N taken up from the soil after anthesis was translocated and accumulated in ears. Low N‐fed plants continued to take up N from the post‐anthesis N fertiliser during the later grain‐filling period. The increase of pre‐anthesis N supply rate led to a decrease in the contribution of nitrogen derived from post‐anthesis ¹⁵N‐labelled fertiliser (N_dff) to total N in all aboveground organs, especially in ears where 44% and 22% of total N originated from post‐anthesis N uptake in low N‐fed and optimal N‐fed plants, respectively. The experiment with labelled amino acids showed that there was greater transport of proteinogenic amino acid into the ear (50% of total ¹⁵N) than non‐proteinogenic amino acid (39%). However, this transport of the non‐proteinogenic amino acids into ear suggested that the transport of N compounds from source (leaves) to sink organs (ear) might not be intrinsically regulated by their ability to be incorporated into storage protein of ears.     

Endosperm Protein Synthesis and l-[S]Methionine Incorporation in Maize Kernels Cultured In Vitro

This study was conducted to examine protein synthesis and l-[³⁵S] methionine incorporation into the endosperm of Zea mays L. kernels developing in vitro. Two-day-old kernels of the inbred line W64A were placed in culture on a defined medium containing 10 microCuries l-[³⁵S] methionine per milliliter (13 milliCuries per millimole) and harvested at 10, 15, 20, 25, 30, 35, and 40 days after pollination. Cultured kernels attained a final endosperm mass of 120 milligrams compared to 175 milligrams for field-grown controls. Field and cultured kernels had similar concentrations (microgram per milligram endospern) for total protein, albumin plus globulin, zein, and glutelin fractions at most kernel ages.     

The Isolation and Characterization of the Major Polypeptides of the Seed Globulin of Cowpea (Vigna unguiculata L. Walp) and their Sequential Synthesis in Developing Seeds

Globulin protein formed the major protein fraction of matureseeds of cowpea; it was found to be heterogeneous when examinedby using chromatography and zonal isoelectric precipitation.Both 7S and US globulin were present and the fraction was dissociatedby SDS treatment into three major subunits with apparent molecularweights 56 000, 54 000, and 52 000 as determined in SDS-acrylamidegels. The individual major subunits were purified by using ion-exchangechromatography in 8 M urea and further characterized by chemicalanalysis. Two of the major subunits had low but different contentsof S-containing amino acid residues and were probably subunitsof 7S glycoproteins. The pattern of seed development was investigated and four phaseswere identified. The protein profile and the amino acid compositionof the seeds changed during development; most of the seed globulinwas synthesized in the third and fourth phases and the net synthesisof the three major sub-units of the globulin differed. The profileof the amino acid composition of the pod walls did not changeduring the period of synthesis of the seed globulin fraction.The essential amino acid content of the meal is determined bythat of the globulin fraction except for cysteine/cystine whichis mostly supplied by the albumin fraction, the latter probablycontaining some proteins rich in S-containing amino acids.     

Properties of glutelin from mature and developing rice grain

Aminograms and SDS-polyacrylamide electrophoresis of milled rice glutelin of 12 Oryza sativa samples showed similar composition and ratio of 1 : 1 : 1 for subunits with MW 38 000:25 000: 16 000, indicating little possibility of finding variants of rice glutelins. Fractionation of S-cyanoethyl glutelin of 3 rices on polyacrylamide-agarose gels gave MW subunits differing in amino acid analysis of which the subunits with MW > 38 000 had the highest lysine content. Of the solubility fractions of endosperm glutelin, the fraction extracted by 0.5 M NaCl-0.6 % β-mercapto-ethanol-0.5% SDS was closest to glutelin in properties. In the developing grain of two varieties, appearance of protein bodies and rapid synthesis of glutelin from 7 days after flowering onward coincided with a drop in lysine content and appearance of MW 38 000 and 25 000 of crude glutelin. The MW 38 000 subunit is thus unique to endo-sperm glutelin.     

Differential ammonia metabolism in Aedes aegypti fat body and midgut tissues

In order to understand at the tissue level how Aedes aegypti copes with toxic ammonia concentrations that result from the rapid metabolism of blood meal proteins, we investigated the incorporation of ¹⁵N from ¹⁵NH₄Cl into amino acids using an in vitro tissue culture system. Fat body or midgut tissues from female mosquitoes were incubated in an Aedes saline solution supplemented with glucose and ¹⁵NH₄Cl for 10-40 min. The media were then mixed with deuterium-labeled amino acids, dried and derivatized. The ¹⁵N-labeled and unlabeled amino acids in each sample were quantified by mass spectrometry techniques. The results demonstrate that both tissues efficiently incorporate ammonia into amino acids, however, the specific metabolic pathways are distinct. In the fat body, the ¹⁵N from ¹⁵NH₄Cl is first incorporated into the amide side chain of Gln and then into the amino group of Gln, Glu, Ala and Pro. This process mainly occurs via the glutamine synthetase (GS) and glutamate synthase (GltS) pathway. In contrast, ¹⁵N in midgut is first incorporated into the amino group of Glu and Ala, and then into the amide side chain of Gln. Interestingly, our data show that the GS/GltS pathway is not functional in the midgut. Instead, midgut cells detoxify ammonia by glutamate dehydrogenase, alanine aminotransferase and GS. These data provide new insights into ammonia metabolism in A. aegypti mosquitoes.     

Growth and composition of maize kernels cultured in vitro with varying supplies of carbon and nitrogen

This study employed in vitro seed culture to determine how C and N supply influence the growth (i.e. starch accumulation) and protein composition of maize (Zea mays L.) endosperm. Immature kernels were grown to maturity on liquid medium containing various concentrations of C (sucrose at 234 millimolar [low] and 468 millimolar [high]) and N (amino acid mixture ranging in N from 0 to 144 millimolar). Low C supply limited starch, but not N, accumulation in the endosperm. With high C, endosperm starch and protein content increased concomitantly as N supply increased from 0 to 13.4 millimolar. Endosperm growth was unaffected by additional N until concentrations exceeding approximately 72 millimolar reduced starch accumulation. A similar inhibition of starch deposition occurred with lower N concentrations when kernels were grown with low C. Endosperm total N content reached a point of saturation with approximately 36 millimolar N in the medium, regardless of C supply. Zein synthesis in the endosperm responded positively across all N levels, while glutelin content remained static and albumin/globulin proteins were reduced in amount when N supply was greater than 36 millimolar. A reciprocal, inverse relationship was observed in mature endosperm tissue between the concentrations of free amino acids and soluble sugars. Our data suggest that under N stress starch and protein accumulation in the endosperm are interdependent, at least in appearance, but are independent otherwise.     

Dynamics of fertilizer-derived organic nitrogen fractions in an arable soil during a growing season

Background

Inorganic fertilizer is one of the most important anthropogenic inputs which influences soil nutrient turnover in agricultural ecosystems. However, as the key process involved in the maintenance, transformation and stability of soil nitrogen (N), the incorporation and allocation of fertilizer N between different soil organic N (SON) fractions in a growing season remains largely unknown.

Methods

In this study, a field experiment was conducted in triplicate of micro-plots and a total of 200 kg N ha^?1 (¹⁵?N-labeled (NH₄)₂SO₄, 98 atom %) was applied as a basal dressing and two top dressings, at jointing and filling stages, respectively, to a maize crop during one growing season. The distribution and seasonal dynamics of fertilizer N in different SON fractions (i.e., amino acids, amino sugars, hydrolyzable ammonium N and acid insoluble-N) were measured by liquid/gas chromatography–mass spectrometry (LC/GC-MS) and element analysis-combustion-isotope ratio mass spectrometry (EA-C-IRMS) techniques. Path analysis was used to evaluate the transformation processes between organic N fractions derived from fertilizer and N supply strategy in soil-plant system.

Results

The accumulation of fertilizer-derived N in different organic fractions was season-specific. At jointing stage, preferential enrichment of ¹⁵?N was found in soil amino acids plus amino sugars, indicating the active biological immobilization of basal dressing fertilizer N. Nevertheless, there is still a small proportion of fertilizer N stabilized in the acid insoluble fraction. The accumulation of the residual fertilizer N in hydrolyzable ammonium N reached a maximum at filling stage and then declined significantly, implying the rapid release of the fertilizer N remained in mineral forms. The contents of amino acids changed slightly, but they played a very important role in mediating SON transformation.

Conclusion

The hydrolyzable ammonium N was a temporary pool for rapid fertilizer N retention and simultaneously was apt to release N for crop uptake in the current season. In contrast, the amino acids could serve as a transitional pool of available N in the soil-crop system, while the acid insoluble fraction was as a stable pool of fertilizer N. Importantly, there is an interim shift among different pools to maintain soil N turnover; hence N in the amino acid fraction mediates N supply and the depolymerization of SON constituents controls the proceeding of fertilizer N cycling in the soil-plant system.     

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.     

Biochemical and molecular characterization of cowpea landraces using seed storage proteins and SRAP marker patterns

Seven landraces of cowpea [Vigna unguiculata (L.) Walp.] were assessed for genetic variability in total proteins, protein fractions viz. albumins, globulins, prolamins, and glutelins by SDS-polyacrylamide gel electrophoresis and DNA polymorphism using sequence-related amplified polymorphisms (SRAP) markers. The solubility-based protein fractionation data indicated that the salt soluble fraction (globulin) and water-soluble fraction (albumin) proteins were the predominant fractions in cowpea seeds comprising 45–50.3% and 31.2–35.5% of total soluble proteins, respectively. The electrophoretic pattern revealed the molecular heterogeneity among total proteins as well as different protein fractions. The molecular weights of protein bands obtained by SDS-PAGE varied between 10 to 250, 15 to 110, 15 to 150, and 15 to 130?kDa for total proteins, albumins, globulins, and glutelins, respectively. A large number of bands were found common to the various landraces, indicative of their close relationship with one another. However, a few bands distinctive to some specific landraces were also detected, indicating varietal differences. A 34 SRAP primer pair combination generated a total of 1003 amplicons (loci) showed 100% polymorphism with an average of 0.93 polymorphism information content (PIC) value. Landraces displayed an average 0.50 similarity coefficient which clustered the landraces corresponding to their growth habit in main clusters and to their geographical origin in subcultures. Molecular and biochemical analysis were correlated with a medium level (Mantel test, r?=?0.56, P?<?0.02). These findings revealed that seed proteins and DNA polymorphism provide valuable information regarding the variability among landraces and this information could be utilized for breeding purposes in the enhancement of protein quality and quantity in grain legumes.     

Molecular characterization of oat seed globulins

We have isolated full-length cDNA clones that encode oat (Avena sativa) seed storage globulin mRNAs from a cDNA library in the expression vector lambda gtll. The longest of these clones, pOG2, has an 1840-base pair insert that encodes a complete precursor subunit with a signal peptide of 24 amino acids followed by an acidic polypeptide of 293 amino acids and a basic polypeptide of 201 amino acids. Near the C terminus of the acidic polypeptide are four repeats of a highly conserved, glutamine-rich octapeptide. Other oat globulin cDNA clones contain five of these repeats. Nucleotide sequence comparisons between these clones indicate that the genes encoding these proteins are highly conserved. We estimate there to be 7 to 10 genes for the oat globulin per haploid genome. Comparisons of amino acid sequences show that the oat globulin is 30 to 40% homologous with storage globulins of legumes and about 70% homologous with the rice seed storage globulin (glutelin).