Influence of the nitrogen source and concentration on N fractions and free amino acid levels of grape vine explants

The influence of the source of inorganic nitrogen (KNO₃, (NH₄)₂SO₄ and NH₄NO₃) and its concentration (5, 10, 20 and 30 mM N) on total N incorporation, as well as on N distribution into different fractions (amminiacal, amino, amide and protein) and on free amino acid levels has been determined in grape vine explants cultured in vitro.Increasing concentrations of the nitrogen source resulted in increased total N content in tissues. This effect was small for KNO₃, higher for (NH₄)₂SO₄ and maximal for NH₄NO₃. In addition, nitrate promoted an increase in amino-N only, whereas ammonium increased both the ammoniacal-N and the amino-N fractions. Incorporation of N into amide-N and protein-N were not affected significantly by the N sources tested.The application of increasing quantities of N enhanced the accumulation of most free amino acids, especially arginine, alanine and proline, but to different extents, depending on both the N source and its concentration. The combination of ammonium and nitrate resulted in a higher accumulation of amino acids than that observed with either one of the two forms alone.     

Properties of albumins of milled rice

Extraction studies on IR36 milled rice showed that albumins solubilized by 0.1–0.15 M (NH₄)₂SO₄ consisted of about 20% high(～5%) lysine, fast-migrating proteins on electrophoresis at pH 8.3 and about 80% lower ～2%) lysine proteins of slower mobility. The 2%-lysine albumins were insoluble in 1.8 M (NH₄)₂SO₄ while the higher lysine albumins required 4 M (NH₄)₂SO₄ to precipitate. The 2%-lysine albumins were not fractionated by gel filtration and gave only one major fraction with MW 19 000. SDS-polyacrylamide gel electrophoresis confirmed the major subunit to be of MW 17 000. These albumins were separated by DEAE-Sephacel chromatography at pH 8.5 into three fractions of similar aminograms but differing in analytical get electrophoretic and isoelectric focusing patterns.     

Formation of delta-Aminolevulinic Acid from Glutamic Acid in Algal Extracts : Separation into an RNA and Three Required Enzyme Components by Serial Affinity Chromatography

Extracts from plant chloroplasts and algae catalyze the conversion of glutamate to δ-aminolevulinic acid (ALA) in the first committed step of the tetrapyrrole biosynthetic pathway leading to chlorophylls, hemes, and bilins. The conversion requires ATP, Mg²⁺, and NADPH as cofactors. Soluble extracts from Chlorella vulgaris have now been resolved into four macromolecular fractions, all of which are required to reconstitute activity. One fraction contains a low molecular weight RNA which can be separated from the protein components in an active high-speed supernatant by treatment with 1 molar NaCl followed by precipitation of the proteins with (NH₄)₂SO₄ at 70% saturation. The proteins recovered from the (NH₄)₂SO₄ precipitate are reactivated by addition of a fraction containing tRNAs isolated from Chlorella by phenol-chloroform extraction and DEAE cellulose chromatography. Three required protein fractions were resolved from the RNA-depleted (NH₄)₂SO₄ precipitate by serial affinity chromatography on Reactive Blue 2-Sepharose and 2′,5′-ADP-agarose. Glycerol was found to stabilize the enzyme activity during the separation process. The majority of the glutamate:tRNA ligase activity was associated with the fraction which was retained by Blue-Sepharose and not retained by ADP-agarose, in agreement with the reported properties of the affinity ligands. The active material in the fraction not retained by Blue-Sepharose eluted as a single component on gel filtration chromatography, with an apparent molecular weight of 67,000. The active component in the RNA fraction also eluted as a single component on gel filtration chromatography.     

Co-purification of Pea and Bean Leaf Soluble Auxin-binding Proteins with Ribulose-1,5-Bisphosphate Carboxylase

Soluble auxin-binding proteins (ABPs) were purified to constant specific activity from bean and pea leaves by a procedure involving (NH₄)₂SO₄ fractionation, anion exchange chromatography and gel filtration. Pea and bean ABPs exactly co-purify with ribulose-1,5-bisphosphate carboxylase (RuBPCase) in a variety of chromatographic separation procedures. The subunit compositions, electrophoretic purities and indole-3-acetic acid (IAA)-binding stoichiometries of the purified ABPs provide further evidence for the identity of RuBPCase and ABP. Pea ABP and bean ABP have dissociation constants for IAA of 0.8 and 1.3 micromolar, respectively, as determined by an (NH₄)₂SO₄ precipitation assay for IAA-binding to insolubilized ABP. IAA can bind to soluble bean and pea ABP (RuBPCase) as determined by equilibrium dialysis with affinities and stoichiometries similar to those determined for insolubilized ABP.     

Medium optimization for the feather-degradation by <Emphasis Type="Italic">Streptomyces fradiae Var</Emphasis><Emphasis Type="Italic">S-221</Emphasis> using the response surface methodology

In order to accelerate biodegradation of feather into more amino acids, the fermentation medium of feather-biodegrading Streptomyces fradiae Var S-221 was optimized in this paper. In the first optimization step, the effects of feather powder, beet molasses, (NH₄)₂SO₄ and KH₂PO₄ on amino acids formation were evaluated by using full factorial design. The results showed that feather powder and (NH₄)₂SO₄ had significant and positive effects on feather-biodegradation into amino acids. Then, the method of the steepest ascent was used to access the optimal region of the two significant factors. In the third step, the concentration of feather powder and (NH₄)₂SO₄ were further optimized with central composite design and response surface analysis. As a result, the composition of the optimal medium for S. fradiae Var S-221 fermentation were as follows (g/100 ml): feather powder, 19.504; beet molasses, 4.0; (NH₄)₂SO₄, 1.467; KH₂PO₄, 0.3; MgSO₄, 0.15; FeSO₄, 0.001; ZnSO₄, 0.0001; and MnSO₄, 0.0001. Using this optimal fermentation medium, the amino acids concentration was increased from 4.61 to 6.13 g/100 ml.     

A Method for Isolation of 2S, 7S and 11S Photeins of Soybean

A metnod is described for the isolation of 2S, 7S and 11S proteins of soybean. The unfractionated proteins are first precipitated with Hg(II) which yields a 11S-rich precipitate and this is further purified by (NH₄)₂SO₄precipitation. From Mg(II)supernatant which is rich in 7S and 2S proteins, they are separated by the use of (NH₄)₂SO₄ and cold ethanol. The 7S ana 11S protein are obtained in a highly purified form as indicated by ultracentrifugation and polyacrylamide gel electrophoresis.     

Peroxidase activity in mycelia of Aspergillus parasiticus that degrade aflatoxin

Summary Extracts of 9-day-old mycelia of Aspergillus parasiticus NRRL 2999 were assayed for peroxidase activity and for their ability to degrade aflatoxin. A positive relationship existed between rates of aflatoxin degradation and amount of peroxidase activity in these extracts. The supernatant fluid of homogenates from mycelia grown under similar conditions varied in amount of peroxidase present (170 to 2215 U/g). The fraction obtained, by precipitation with (NH₄)₂SO₄ at 45% of saturation, from six different homogenates prepared from three mycelial mats contained peroxidase and degraded aflatoxin. Rates of aflatoxin degradation by and amounts of peroxidase activity in each sample obtained from mycelial homogenates with (NH₄)₂SO₄ at 60% of saturation varied; however, when increased amounts of peroxidase activity were present, more aflatoxin was degraded and vice versa. Relatively little peroxidase activity was present in the fraction obtained with (NH₄)₂SO₄ at 30% of saturation and little or no aflatoxin was degraded by this precipitate. Trends for degradation of aflatoxin when more or less peroxidase activity was present in mycelial preparations suggest that the enzyme may be involved in degradation of aflatoxin by the Aspergillus.     

Physical properties of pectic polysaccharidases ofPseudomonas solanacearum from Nigeria

Pseudomonas solanacearum (obtained from Nigeria) produced certain pectic polysaccharidases when grown in aerobic batch cultures containing pectic substances. The pH optima of the enzymes were different. The optimum for polygalacturonase EC 3.2.2.15 was 5.5, and for pectate lyase EC 4.2.99.3 it was 8.5. The -1,4-glycosidic bonds between galacturonide units were cleaved at random, indicating the endo character of the enzymes. The pectic polysaccharidases were purified by (NH₄)₂SO₄ fractionation and by electrofocusing. Highest polygalacturonase activity and pectate lyase activity were obtained in the fractions at 41%–60% and 61%–80% (NH₄)₂SO₄ saturation, respectively. Polygalacturonase was resolved into two components with isoelectric points of 5.0 and 7.5; the isoelectric point of pectate lyase was 8.1.     

The heterogeneity of rat high density lipoproteins.

Ubiquitin was first found in nuclei in protein A24 where its carboxyl terminal is covalently bound to histone 2A by an isopeptide linkage (Goldknopf, I. L. and Busch, H. (1977) Proc. Natl. Acad. Sci. USA $\text{74}$, 864–868). Two-dimensional polyacrylamide gel electrophoresis of the 0.4 N H₂SO₄ soluble proteins from fractionated rat liver chromatin showed that protein A24 and histones H1, H2A, H2B, H3 and H4 were present in fractions P1 and P2 and markedly diminished in relative amounts in fraction S2. Conversely, a spot designated Ub was found in fraction S2 along with an increased amount and number of non-histone proteins. The Ub spot was not found in chromatin fractions P1 and P2. Ub was identified as ubiquitin by migration on two-dimensional gels and after purification by preparative polyacrylamide gel electrophoresis by its methionine NH₂-terminal amino acid and its amino acid composition.     

The Serine Hydroxymethyltransferase of Plasmodium lophurae*

SYNOPSIS. Plasmodium lophurae serine hydroxymethyltransferase (EC 2.1.2.1) was partially purified and characterized by (NH₄)₂SO₄ fractionation and chromatography on Sephadex G-100. The enzyme, precipitated by 3.0–3.3 m (NH₄)₂SO₄, had a molecular weight of 68,300 as estimated by exclusion chromatography on G-100. The pH optimum of the enzyme was 6.8–7.6 in sodium phosphate-citrate buffer. Citrate stabilized the enzyme during storage in phosphate buffer at 4 C. The K_m was 4.3 × 10^?3m for l -serine and 2.5 × 10^?4m for tetrahydrofolate.     

An Inhibitor of the δPKC Interaction with the d Subunit of F1Fo ATP Synthase Reduces Cardiac Troponin I Release from Ischemic Rat Hearts: Utility of a Novel Ammonium Sulfate Precipitation Technique

We have previously reported protection against hypoxic injury by a cell-permeable, mitochondrially-targeted δPKC-d subunit of F₁Fo ATPase (dF₁Fo) interaction inhibitor [NH₂-YGRKKRRQRRRMLA TRALSLIGKRAISTSVCAGRKLALKTIDWVSFDYKDDDDK-COOH] in neonatal cardiac myo-cytes. In the present work we demonstrate the partitioning of this peptide to the inner membrane and matrix of mitochondria when it is perfused into isolated rat hearts. We also used ammonium sulfate ((NH₄)₂SO₄) and chloroform/methanol precipitation of heart effluents to demonstrate reduced card-iac troponin I (cTnI) release from ischemic rat hearts perfused with this inhibitor. 50% (NH₄)₂SO₄ saturation of perfusates collected from Langendorff rat heart preparations optimally precipitated cTnI, allowing its detection in Western blots. In hearts receiving 20 min of ischemia followed by 30, or 60 min of reperfusion, the Mean±S.E. (n = 5) percentage of maximal cTnI release was 30±7 and 60±17, respectively, with additional cTnI release occurring after 150 min of reperfusion. Perfusion of hearts with the δPKC-dF₁Fo interaction inhibitor, prior to 20 min of ischemia and 60–150 min of reperfusion, reduced cTnI release by 80%. Additionally, we found that when soybean trypsin inhibitor (SBTI), was added to rat heart effluents, it could also be precipitated using (NH₄)₂SO₄ and detected in western blots. This provided a convenient method for normalizing protein recoveries between groups. Our results support the further development of the δPKC-dF₁Fo inhibitor as a potential therapeutic for combating cardiac ischemic injury. In addition, we have developed an improved method for the detection of cTnI release from perfused rat hearts.     

The effect of C/N ratio on lipid production byRhodosporidium toruloides ATCC 10788

Summary An optimum initial molar C/N ratio of about 77 was found for maximum lipid production from glucose or fructose with a nitrogen source from either (NH₄)₂SO₄, NH₄NO₃ or urea. The fatty acid composition of the lipids was affected by the initial C/N ratio and by the type of sugar but not significantly by the type of nitrogen.     

Purification,Characterization, and Quantitation of the Antigen Employed in the Immunodiffusion Test for Diagnosis of Equine Infectious Anemia

Equine infectious anemia (EIA) antigen extracted from the spleen of horses infected with EIA virus was purified by pH treatment, (NH₄)2SO₄fractionation and affinity chromatography. The homogeneity of the antigen was indicated by sedimentation rate and sedimentation equilibrium experiments. A S_{20, w} of 0.51 was determined and a molecular weight of 7600 was calculated from sedimentation equilibrium analysis. The amino acid composition of the pure antigen indicated the antigen is an acidic protein. Employing radical immunodiffusion (RID) and pure antigen a method for quantitating antigen content of antigen containing preparations was developed.     

Effect of ammonium sulfate on the phytotoxicity, foliar uptake, and translocation of imazamethabenz in wild oat

Experiments were conducted in greenhouse, growth chamber, and laboratory conditions to determine the effect of ammonium sulfate [(NH₄)₂SO₄] on the phytotoxicity, foliar uptake, and translocation of imazamethabenz on wild oat. Rates of (NH₄)₂SO₄ up to 5% (w/v) applied with a greenhouse sprayer did not affect the phytotoxicity of the herbicide when the mix was applied at the one- to two-leaf stage. However, inclusion of 1 and 2% (NH₄)₂SO₄ increased the phytotoxicity of the herbicide when the mix was sprayed at the two- to three-leaf, or the three- to four-leaf stage. At 10%, (NH₄)₂SO₄ decreased the phytotoxicity of the sublethal dosage of the herbicide. When the herbicide was applied as individual drops to the growth chamber-grown plants, inclusion of (NH₄)₂SO₄ at 1% did not affect phytotoxicity as measured by shoot growth. The presence of (NH₄)₂SO₄ did not affect the amount of imazamethabenz retained by wild oat foliage, but it decreased [¹⁴C]imazamethabenz absorption, slightly antagonized acropetal translocation, and increased the basipetal translocation of [¹⁴C]imazamethabenz. It was concluded that application methods greatly modify the effect of (NH₄)₂SO₄ on imazamethabenz phytotoxicity. Herbicide absorption and translocation as determined by one method do not necessarily represent the absorption and translocation patterns when different application methods are used. Absorption and translocation were not the factors that were responsible for the observed effect of (NH₄)₂SO₄ on the herbicide phytotoxicity.Abbreviations SC suspension concentrate     

Gibberelic acid-mediated activation of monophenolase in de-embryonated half-seeds of wheat (Triticum Aestivum)

Stimulation of monophenolase activity was observed when de-embryonated prewashed half-seeds of wheat were imbibed in a solution of gibberellic acid (GA₃, 10^t?5 M). Crude extracts, prepared from GA₃-treated half-seeds, showed ca a two-fold increase in monophenolase activity over the controls at pH 6.6, while a dramatic rise in enzyme activity (seven- to nine-fold) was observed at pH 9.0. The partially purified (NH₄)₂SO₄ fraction precipitate (30–50% saturation) also showed enhancement of enzyme activity at pH 9.0 in GA₃-treated half-seeds, while in controls, there was negligible activity at this pH. Administration of five amino acid analogues (1 mM each) to half-seeds showed no significant inhibition of GA₃-stimulated monophenolase activity, but proved very effective in decreasing (86% inhibition) the GA₃-induced amylase activity. This indicated that the hormone-regulated monophenolase activity was not dependent on de novo protein synthesis. Treatment of half-seeds with GA₃ modified the monophenolase and altered the electrophoretic pattern. The enzyme was relatively thermostable at 55° and the pH optimum was shifted from pH 7.0 to 9.0. In addition, the GA₃-treated half-seeds showed relatively high stability of enzyme activity in the presence of (NH₄)₂S0₄ ions. These alterations in the GA₃-stimulated nomophenolase suggest there is activation of preformed enzyme molecules. The appearance of slow-migrating multiple forms on acrylamide gels, in response to GA₃ treatment, is probably due to the association of fast-migrating forms. Such oligomerization could result in a conformational change leading to enzyme activation. This may be an adaptive mechanism so that the enzyme can function with varying temperature, pH and ionic strength during early stages of seed germination.     

Growth of the Peritrich Opercularia coarctata in Axenic Culture*

A synthetic medium for Opercularia coarctata was developed that contains 20 amino acids, 10 vitamins, an 8-component balanced salt solution, Fe₂(SO₄)₃·(NH₄)₂SO₄·24H₂O, Tween 80, stigmasterol, a 7-component nucleic acid mixture, phenol red as an indicator, and 2,500 U.S.P. units/ml penicillin to maintain sterility. This medium supported axenic survival for 96 hr. Multiple supplements of thioctic acid, niacin, niacinamide, inositol, PABA, oleic acid, and Fe(NO₃)₂·9H₂O instead of Fe₂(SO₄)₃·(NH₄)₂SO₄·24H₂O coverted the survival medium into a growth medium, which permitted 36–45 days continuous cultivation of populations in excess of 4 × 10³ cells/3.0 ml final volume. Five generations were produced during the 48 hr logarithmic growth period. Serial transfers at 72 hr and during periods of greatest cell density produced a maximum of 8 generations 96 hr after initiation but the medium failed to sustain growth through more than 6 serial transfers. Extension of this investigation to formulating a minimal axenic medium is discussed.     

Nitrogen regulation of gibberellin biosynthesis in Gibberella fujikuroi

Summary In Gibberella fujikuroi, ammonium (NH₄ ⁺) interfered with the production of gibberellic acid (GA₃). Optimal production occurred at 19 mm (NH₄)₂SO₄ and the synthesis of GA₃ was reduced threefold in a medium with 38 mm (NH₄)₂SO₄. Using a resting cell system with mycelia previously grown on two concentrations (19 mm and 38 mm) of (NH₄)₂SO₄, it was found that NH₄ ⁺ depressed synthesis of the gibberellin-synthesizing enzymes. Furthermore, addition of NH₄ ⁺ to a producing system shut off gibberellin formation, indicating that the negative effect of NH₄ ⁺ ions is also due to inhibition of one or more enzymes in the gibberellin biosynthesis pathway. The onset of gibberellin biosynthesis in media with high (38 mm) and low (19 mm) concentrations of (NH₄)₂SO₄ was studied by addition of cycloheximide to batch cultures of various ages. Offprint requests to: B. Brückner     

In Vitro Fatty Acid Synthesis and Complex Lipid Metabolism in the Cyanobacterium Anabaena variabilis: I. Some Characteristics of Fatty Acid Synthesis

In vitro fatty acid synthesis was examined in crude cell extracts, soluble fractions, and 80% (NH₄)₂SO₄ fractions from Anabaena variabilis M3. Fatty acid synthesis was absolutely dependent upon acyl carrier protein and required NADPH and NADH. Moreover, fatty acid synthesis and elongation occurred in the cytoplasm of the cell. The major fatty acid products were palmitic acid (16:0) and stearic acid (18:0). Of considerable interest, both stearoyl-acyl carrier protein and stearoyl-coenzyme A desaturases were not detected in any of the fractions from A. variabilis. The similarities and differences in fatty acid synthesis between A. variabilis and higher plant tissues are discussed with respect to the endosymbiotic theory of chloroplast evolution.     

Rapid purification of chicken gizzard alpha-actinin

A method of purification has been developed which yields highly purified α-actinin and requires less than one day to complete. The α-actinin is extracted from washed chicken gizzard muscle with water at 37°. Actin and a 55,000 dalton protein are quantitatively precipitated from the extract with 20 mM MgCl₂. The α-actinin is subsequently precipitated from the extract by 30% (NH₄)₂SO₄ and fractionated on DEAE cellulose. Spontaneous protein aggregation is prevented by adding 10% glycerol.