Characterization of Cytosolic Glutathione S-Transferase in Spruce Needles

Active glutathione S-transferase (GST) has been purified from needles of Norway spruce (Picea abies L. Karst.). Two isoforms of the enzyme which exhibit different physico-chemical and catalytic properties were separated by (NH₄)₂SO₄ fractionation, affinity chromatography on epoxy-activated 4% cross-linked beaded agarose, using glutathione as the ligand, ion-exchange chromatography, and isoelectric focusing. The isozymes have pI values of 5.5 (GST I) and 4.3 (GST II). Both GST isozymes are homodimeric proteins with subunit sizes of 26 kD (GST I), and 23 kD (GST II). The kinetic properties of the enzymes are described and compared with other plants GSTs. Only GST II is able to conjugate the pesticides fluorodifen and alachlor.     

DNA-dependent RNA polymerases from murine testis. Properties of two activities.

Multiple forms of DNA-dependent RNA polymerase activities have been isolated from nuclei of mouse testis. Using highly purified nuclei, two activities can be solubilized and are separable by DEAE-Sephadex chromatography; peak I eluting at 0.11–0.14 M and peak II eluting at 0.24–0.27 M (NH₄)₂SO₄. A third form of RNA polymerase activity is observed eluting at 0.31–0.33 M (NH₄)₂SO₄ when an extract from a less highly purified nuclear preparation is analysed. At concentrations of 0.125 μg/ml, peak I is insensitive to the toxin α-amanitin, peak II is totally inhibited, and peak III is partially inhibited. Peak I activity is optimal at pH 8.4 in the presence of Mg²⁺ (2–6 mM) or Mn²⁺ (1 mM) and uses native and heat-denatured DNA template equally well. Peak II has optimal activity at pH 7.9 in the presence of Mn²⁺ (2 mM) and heat-denatured DNA. Mg²⁺ has little effect on the activity of peak II.     

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.     

Comparison of fractionation of groundnut proteins by two different methods

The salt-soluble proteins of groundnut meal were fractionated by precipitation with (NH₄)₂SO₄ by increasing the (NH₄)₂SO₄ saturation in steps of 10%. The sharp separation into arachin and conarachin claimed by earlier workers was not achieved, as protein was precipitated at each stage from 20 to 100% saturation with (NH₄)₂SO₄. The fractions so obtained were examined by disc electrophoresis on polyacrylamide gel and the amino acid compositions were determined by ion-exchange chromatography. Differences in both electrophoretic pattern and amino acid composition were found. The protein precipitated by CaCl₂ solution was similar in yield, nitrogen content, electrophoretic pattern, and amino acid composition to the fraction precipitating at 10–20% (NH₄)₂SO₄ saturation. The main differences in amino acid composition of the various fractions precipitated by (NH₄)₂SO₄ were found in the amino acids cystine, methionine, and lysine, which increased with increase in (NH₄)₂SO₄ saturation. The electrophoretic pattern and amino acid composition of “conarachin” varied according to the method of preparation.     

The transporting proteins of cholecalciferol and 25-hydroxycholecalciferol in serum of chicks and other species. Partial purification and characterization of the chick proteins

Chick serum contains two cholecalciferol-binding proteins, one of which binds mainly cholecalciferol (cholecalciferol-binding protein) and the other binds 25-hydroxycholecalciferol (25-hydroxycholecalciferol-binding protein). By means of Cohn fractionation, (NH₄)₂SO₄ precipitation, gel filtration on Sephadex G-200, ion-exchange chromatography on DEAE-Sephadex and an additional gel-filtration step on Sephadex G-100, these two binding proteins were purified. Both proteins possess β-globulin mobility on analytical polyacrylamide-disc-gel electrophoresis, a sedimentation coefficient of 3.5S and approximate molecular weights of 60000 for the cholecalciferol-binding protein and 54000 for the 25-hydroxycholecalciferol-binding protein. Sera obtained from rat, pig, human and monkey were shown to contain a single binding protein that is responsible for the transport of both cholecalciferol and 25-hydroxycholecalciferol. In the toad the lipoproteins are used for the transport of these two steroids.     

Partial purification and characterization of dipeptidyl peptidase II (DPP II) from guinea pig testes

Depeptidyl peptidase (DPP II) was partially purified from guinea pig testes by (NH₄)₂SO₄ precipitation, Con A-Sepharose 4B chromatography, and Sephadex G-200 chromatography to a specific activity of 27.4 μmol Ala₃ hydrolyzed min^?1 mg^?1 protein. Chromatography on a calibrated G-200 column yielded a molecular weight of 135,000 daltons for the enzyme. Sodium dodecyl sulfate polyacrylamide electrophoresis showed an enrichment of a broad doublet at 64–66,000 daltons. The enzyme had optimal activity toward hydrolysis of L-alanyl-alanyl-alanine at pH 4.5 and showed sensitivity to cations of increasing size with Tris producing the most inhibition of those tested. The enzyme was moderately inhibited by serine proteinase inhibitors. Thin-layer chromatography revealed the dipeptidase nature of the enzyme's activity on tripeptides and dipeptidyl arylamides. A doublet of activity occurred when nitrocellulose electroblots of nondenaturing gel electrophoresis of the (NH₄)₂SO₄ fraction were reacted with the specific DPP II substrate, lysyl-alanyl-4-methoxy-2-napthylamide. Analytical isoelectric focusing of the G-200 fraction followed by fluorescent enzyme activity detection that used cellulose triacetate overlay membranes impregnated with the specific DPP II substrate, lysyl-alanyl-7-amino-4-trifluoromethylcou-marin, revealed multiple isoforms focusing at pI = 4.8–5.6. Two prominent bands focused at pI = 4.9 and pI = 5.1. The properties of guinea pig testicular DPP II are compared and contrasted with similar dipeptidyl peptidases from other sources.     

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.     

Biochemical and immunological characterization of a histone variant associated with spermatogenesis in the mouse

A chromosomal histone, H2S, specific to the mouse testis has been purified. Amino acid analysis indicated lack of cysteine and a high basic amino acid content typical of histones. Specific antibodies against histones H2S have been generated in rabbits and partially purified using (NH₄)₂SO₄ precipitation and ion-exchange chromatography. Protein transfer experiments indicate presence of antigenically similar histones in the rat and rabbit testes but not in the guinea pig and dog testes. In addition, histone complement of somatic tissues such as lung, kidney, liver and spleen lacked antigenically similar proteins. Immunocytochemical studies using peroxidase-antiperoxidase complex indicated presence of immunoreactive cells in the seminiferous epithelium which were lacking in the interstitium. These data demonstrate histone H2S to be a unique histone associated with spermatogenesis in the mouse.     

Allosterische Regulation der Aktivität der Glutamatdehydrogenase aus Erbsenkeimlingen durch das Substrat α-Ketoglutarsäure

Summary Several investigations on the properties of glutamate dehydrogenase from plant sources indicate that the enzymatic activity (reductive amination) follows a Michaelis-Menten-type kinetic when velocity is plotted versus rising concentrations of the substrate -ketoglutarate. In the course of our investigations on the effect of SO₂ on pea plant enzymes we found that SO ₄ ^2- , added as (NH₄)₂SO₄ to the assay system, causes this type of activity response because of its ability to function as an activator. When (NH₄)₂SO₄ is replaced by NH₄Cl in the in vitro system however, activity response is sigmoidal. Addition of competitive inhibitor to the latter system again gives rise to a sigmoidal kinetic with reduced initial velocities. Identical kinetic behaviour is observed when either 120 fold enriched enzyme from shoots or highly purified glutamate dehydrogenase from pea roots is used, a fact which justifies the assumption that the enzyme from pea plants belongs to the MIC-type of regulatory proteins (modulator independent cooperativity). The activity response caused by other effectors, especially purine nucleotides, is discussed with regard to the above findings.     

Physicochemical properties of salt-soluble,unsheared chromatin

Salt-dependent structural changes of rat liver chromatin isolated by an extraction procedure not involving shear and exogeneous nucleases were investigated by sedimentation and light scattering methods. The effects observed are complex involving changes of the molecular weight and expansion. Between 0.1 M and 0.2 M (NH₄)₂SO₄ where histone H1 is released, a fragmentation into molecules of half molecular weight is found which is accompanied by an expansion into a more extended conformation gradually increasing to 0.4 M (NH₄)₂SO₄. The H1-free chromatin does not exhibit the reduction in molecular weight but undergoes this expansion. The original conformation is not reversible on re-decreasing the salt concentration to 0.1 M (NH₄)₂SO₄.     

Partitioning and separation of α-lactalbumin and β-lactoglobulin in polyethylene glycol/ammonium sulphate aqueous two-phase systems

The partition behaviour of -lactalbumin (la) and -lactoglobulin (lg) on PEG/(NH₄)₂SO₄ system was studied. For purified proteins, a partition coefficient of 12.8 for la and 0.34 for lg, with mass recovery yields of 96.7% for la in the upper phase and 83.8% for lg in the lower phase was obtained, in 18% (w/w) PEG 900/14% (w/w) (NH₄)₂SO₄ system, at pH 7. PEG/(NH₄)₂SO₄ system was an economical alternative for the recovery and separation of the two proteins in cheese whey, allowing a 50% reduction in costs. An efficient and inexpensive separation of both proteins in cheese whey could be achieved, by using 16% (w/w) PEG 900/15% (w/w) (NH₄)₂SO₄, at pH 7.5.     

Development of high-molar-mass cellobiase complex by spontaneous protein-protein interaction in the culture filtrate ofTermitomyces clypeatus

The 450 kDa cellobiase fromTermitomyces clypeatus which migrates as a single band on IEF, PAGE and SDS-PAGE, was found to possess appreciable sucrase activity. The fungus produced sucrase and cellobiase constitutively in different media but with different activity ratios. The kinetics of secretion of the two enzymes was similar underin vivo andin vitro conditions. HPGPLC analysis of the culture filtrates indicated the presence of both sucrase and cellobiase in the same protein fractions of different molar mass, even in the 30-kDa protein fraction. No free sucrase or cellobiase could be detected in the culture filtrates. It was also observed that fractionation of cellobiase by (NH₄)₂SO₄ precipitation was different with different amounts of associated sucrase activity present in the culture filtrate. The (NH₄)₂SO₄-precipitated cellobiase fraction also contained cellobiases in proteins of widely varied molar mass ranges. However, none of the low-molar mass proteins other than the 450-kDa enzyme could be purified, as all low-molar-mass fractions spontaneously aggregated to the 450-kDa enzyme. Hydrophobic chromatography of the (NH₄)₂SO₄-precipitated fractions followed by HPGPLC of the eluted active fraction yielded both cellobiase-free sucrase and a very low sucrase-containing cellobiase fraction. The cellobiase fraction, homogeneous in PAGE, was also a high-molar-mass protein complex dissociating into a number of protein bands on SDS-PAGE. It was suggested that the 450-kDa cellobiase was not liberated by the fungus as a preformed enzyme complex but that the complex developed through interaction of cellobiase with sucrase underin vitro conditions and the possibility of the involvement of other proteins in the aggregation cannot be excluded.     

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     

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.     

Characterization of the Ca2+-regulatory complex of chick embryonic muscles: Polymorphism of tropomyosin in adult and embryonic fibers

The Ca²⁺-regulatory tropomyosin-troponin complex was purified from chick embryonic muscles by a combination of DEAE-cellulose chromatography and (NH₄)₂SO₄ fractionation. The embryonic complex was very similar to that obtained from adult chicken muscles with respect to stoichiometry of components and biological activity. Tropomyosin of embryonic skeletal muscles contains both α and β subunits, the β form being the major species. In the adult stage the β form is decreased with a concomitant increase in the α form. These results indicate that i) the Ca²⁺-regulatory proteins are not deficient in early embryonic muscles as previously thought (Hitchcock, S.E., Develop. Biol. $\text{23}$, 399, 1970), and ii) different structural genes coding for tropomyosin subunits are expressed differentially in embryonic and adult muscle fibers.     

Alkaline inorganic pyrophosphatase from guar (Cyamopsis tetragonoloba) cotyledons

Alkaline inorganic pyrophosphatase from guar cotyledons was purified x 110 with about 34% recovery by (NH₄)₂SO₄ fractionation, acetone prec     

A PHASE RULE STUDY OF THE PROTEINS OF BLOOD SERUM: THE EFFECT OF CHANGES IN CERTAIN VARIABLES

Changes were studied in the standard solubility curve of fresh serum proteins by alterations in pH, temperature, concentration of protein, and nature of the salt used for precipitation. The principal factor affecting the precipitation of protein fractions was a change in temperature. In order to investigate the proteins in their original states low temperatures are necessary. Protein fraction A is altered by a change in pH and with the use of (NH₄)₂SO₄ as a precipitant, fraction B by a change in pH and temperature, and use of (NH₄)₂SO₄, C by a change in temperature and concentration of the protein, and D by a change in temperature and pH. The solubility of D is independent of the amount of protein in solution in high concentrations of salt.