Presence Of Ethylenediaminetetraacetate Research Articles

Ion transport in heart mitochondria. XIII. The effect of ethylenediaminetetraacetate on monovalent ion uptake

1. Ethylenediaminetetraacetate (EDTA) markedly activates the accumulation of Na + and Li + and the swelling which accompanies the ion uptake by isolated heart mitochondria. This activation is reflected in the removal of limited amounts of endogenous Mg 2+ and extensive loss of K +. The removal of these cations requires the presence of Na +, a source of energy, and a permeant anion as well as EDTA. The effects of EDTA on the activation of Na + uptake and cation removal are duplicated by chelators with a high affinity for Mg 2+, but not by ethyleneglycol-bis-(β-aminoethylether)-N, N′-tetraacetic acid. Mg 2+ at concentrations 5 to 6 times less than EDTA prevents both activation of Na + uptake and cation removal. 2. EDTA does not appear to be bound by heart mitochondria. At neutral pH the chelator penetrates into the mitochondrial water volume to the same extent as sucrose and mannitol. At pH 8.1 where the removal of mitochondrial Mg 2+ by EDTA is more effective, EDTA penetrates virtually the entire water volume. This penetration requires the presence of a source of energy, a transported cation such as Na +, and a permeant anion. It appears possible that the oscillations in ion uptake and swelling observed in the presence of EDTA at pH 8.1 may be related to the presence of the chelator in the interior compartment under these conditions.

Studies on the Microbiological Degradation of Steroid Ring A

The synthesis of 2-oxo-cis-4-hexenoic acid from DL-2-amino-cis-4-hexenoic acid is described. The α-oxo acid appears to exist at neutral pH largely in the dienolic form. Ultracentrifuged extracts of steroid-induced Pseudomonas testosteroni rapidly converted 2-oxo-cis-4-hexenoic acid-1-14C to 14CO2. The same enzyme preparations, when supplemented with NAD and a NADPH-generating system, have been previously shown to convert Δ4-androstene-3,17-dione-4-14C to 14CO2 and to accumulate L-2-amino-cis-4-hexenoic acid-1-14C and DL-alanine-1-14C in the presence of ethylenediaminetetraacetate. It has now been shown that, under similar conditions, 2-oxo-amino-cis-4-hexenoic acid is efficiently converted to 2-amino-cis-4-hexenoic acid and alanine. The α-oxo acid also undergoes stereospecific enzymatic hydration to 2-oxo-4-hydroxyhexanoic acid, which is lactonized in the presence of acid to give a product that has been identified as one of the optically active isomers of 2-oxo-4-ethylbutyrolactone of unestablished configuration. These findings suggest that 2-oxo-cis-4-hexenoic acid is a key intermediate in the degradation of steroid ring A by microbial enzymes. The enzyme preparations also contain an enzyme which reduces the double bond of the α-oxo acid but does not appear to be involved in the degradative pathway of the steroids.

Mechanism and specifcity of L- and D-6-hydroxynicotine oxidase.

l‐6‐Hydroxynicotine oxidase, a FAD‐protein, coverts in the presence of oxygen l‐6‐hydroxynicotine to [6‐hydroxypyridyl(3)]‐(γ‐N‐methylaminopropyl)‐ketone; the overall process, whose stoichiometry has been established, consists of the enzyme‐catalyzed dehydrogenation of the substrate followed by spontaneous hydrolysis of the intermediate enamine. Titration of the enzyme anaerobically with substrate or dithionite spectrally does not indicate formation of a semiquinoid flavin, while upon irradiation in the presence of ethylenediaminetetraacetate, spectral changes take place which seem to relflect the existence of a transient radical stage. Molecular oxygen functions as electron acceptor in the oxidase reaction, other artificial or natural redox systems being inactive or even, as methylene blue, inhibitory to the l‐6‐hydroxynicotine oxidation. Previous evidence for the almost absolute structural and stereochemical specificity of both l‐and d‐6‐hydroxynicotine oxidases was extended.

The Role of Calcium in the Reactivation of Reduced Taka-amylase

Abstract Aspergillus oryzae α-amylase (Taka-amylase A) is a protein which contains four disulfide bonds, one free sulfhydryl group, and 1 essential atom of calcium per molecule. Full reduction in the presence of ethylenediaminetetraacetate and 8 m urea results in complete loss of enzymic activity. If Ca++ is present, reoxidation at pH 8.6 leads to full recovery of activity with a parallel decrease of free sulfhydryl groups to about one per molecule of protein. However, reoxidation in the absence of Ca++ leads to an inactive protein which contains about three sulfhydryl groups per molecule. Upon the addition of Ca++, this partially reoxidized product is fully reactivated, and the number of sulfhydryl groups again falls to about one per molecule. Reoxidation of unfolded Taka-amylase in the presence of dehydroascorbic acid also leads to rapid decrease in the number of free sulfhydryl groups to about one per molecule. The resulting material is inactive, but the addition of Ca++ results in almost complete restoration of activity. The disulfide bond-containing peptides formed during the reoxidation of reduced Taka-amylase under these various conditions have been located on two-dimensional tryptic peptide maps and on diagonal performic acid-treated peptic peptide patterns. Partially reoxidized, Ca++-free amylase forms three normally paired disulfide bonds, and the addition of Ca++ results in the appearance of the fourth disulfide bond. Dehydroascorbic acid causes all four normally paired disulfide bonds to form, and subsequent activation with Ca++ does not appear to involve disulfide interchange. The Ca++-induced formation of the one remaining disulfide bond in partially reoxidized Taka-amylase is accompanied by time-dependent changes in protein conformation, as detected by ultraviolet difference, solvent perturbation, and fluorescence spectroscopy. The development of a difference spectrum at 286 mµ following the addition of Ca++ closely parallels the recovery of enzymic activity and the loss of the two excess sulfhydryl groups. In addition, the decrease in the number of solvent-accessible chromophores and the loss of the tyrosine fluorescence peak indicate that Ca++ causes conformational transitions involving major portions of the molecule. We conclude that under normal conditions Ca++ is required for full reoxidation and proper folding of reoxidized Taka-amylase, but that Ca++ does not play a role in directing normal pairing of half-cystine residues.

Hormonal Control of Proteinase Activity in Squash Cotyledons

A crude proteolytic enzyme preparation which hydrolyzes casein was isolated from the cotyledons of squash seedlings. The presence of ethylene diamine tetraacetate or cysteine did not appreciably affect the activity of the preparation. During the course of germination, the level of proteolytic activity increased in the cotyledons of intact embryos through the third day and then decreased. The presence of the embryonic axis during the first 32 hours of germination was a prerequisite for the development of maximum proteolytic activity.The presence of a cytokinin, such as benzyladenine, kinetin, BTP [6-(benzylamino)-9-(2-tetrahydropyranyl)-9H-purine], or phenyladenine, in the culture solution could reproduce the effect of the embryonic axis. Other growth regulators did not produce this stimulation. High concentrations (1 mm) of all growth regulators examined were inhibitory. Various combinations of growth regulators failed to produce any synergistic effects. 6-Methylpurine inhibited the development of proteinase activity, and this inhibition was only partially restored by benzyladenine.A proteinase was partially purified from the cotyledons of 2-day-old squash seedlings and its synthesis was shown to be under the control of the embryonic axis.

Release of kallikrein from guinea pig lung during anaphylaxis.

An antigen-antibody reaction occurring in the perfused sensitized guinea pig lung, has been demonstrated to release kallikrein, a proteolytic enzyme related to the formation of kinins. This lung kallikrein is similar to plasma kallikrein in all properties studied, including susceptibility to the same inhibitors, electrophoretic mobility, and heterogeneity in molecular size. The release of kallikrein during anaphylaxis in the guinea pig lung occurs in the presence of ethylenediaminetetraacetate. Perfusion of ellagic acid into nonsensitized lungs will also release kallikrein, presumably through activation of Hageman factor. On the basis of these findings the hypothesis is suggested that the kallikrein in perfused lung activated by the antigen-antibody reaction is, in fact, plasma kallikrein. It is further suggested that activation of such kallikrein by the antigen-antibody reaction proceeds through Hageman factor.

Separation of the soluble proteins of bovine lenses on polyacrylamide gels

The soluble proteins of bovine lenses were separated by gel filtration on polyacrylamide gels. The presence of ethylenediaminetetra-acetate in the burfer was found to be essential for achieving a good separation of the lens homogenate into four fractions—A, B, C and D. Ultracentrifugal, electrophoretic and immunochemical analyses were used to compare these fractions with the soluble crystallin fractions of the lens. Fraction A was compared with α-crystallin, fractions B and C with β-crystallin and fraction D with γ-crystallin. The distribution of these four protein fractions in the nuclear and cortical parts of calf and cow lenses was determined. The oxidation-sensitive protein fraction of the calf lenses was separated from fraction B on an SE-Sephadex column.

Immunochemical studies on alpha-amylase. 3. Immunochemical relationships among amylases from various microorganisms.

Sirishinha, Stitaya (University of Rochester School of Medicine and Dentistry, Rochester, N.Y.), and Peter Z. Allen. Immunochemical studies on alpha-amylase. III. Immunochemical relationships among amylases from various microorganisms. J. Bacteriol. 90:1120-1128. 1965.-Immunochemical relationships among amylases obtained from a selected group of microorganisms were examined, and a cross-reaction was detected between the alpha-amylases of Bacillus stearothermophilus and B. subtilis. Immunodiffusion and quantitative precipitin studies, as well as cross-neutralization tests, indicate that B. stearothermophilus alpha-amylase reacts with a portion of antibody present in antisera to crystalline B. subtilis alpha-amylase. Amylases from these two species thus have some aspects of structure in common. Limited data obtained by immunodiffusion suggest that groupings which confer cross-reactivity to the B. stearothermophilus enzyme are lost after exposure to mercaptoethanol in the presence of ethylenediamine-tetraacetate, followed by treatment with iodoacetamide. With the antisera employed and within the concentration range examined, no immunochemical cross-reaction was observed among amylases from Aspergillus oryzae, B. subtilis, B. polymyxa, B. macerans, Pseudomonas saccharophila, and Euglena sanguinis. Immunoelectrophoresis of partially purified B. stearothermophilus alpha-amylase by use of antiserum to the crude enzyme, together with localization of amylase activity in immunoelectrophoretic plates, suggests that B. stearothermophilus alpha-amylase is antigenic in the rabbit.

Effect of ethylenediamine tetraacetate on the resistance of Pseudomonas aeruginosa to antibacterial agents.

Pseudomonas aeruginosa is a resistant pathogen which is dangerous in ophthalmology1, as a hospital cross-contaminant2, and as a plant pathogen3. We have used the spectrophotometric method of Brown and Richards4 to investigate the effect of disodium ethylenediamine tetraacetate (EDTA) on the action of several antibacterial agents against cultures of Ps. aeruginosa, NCTC 8203, in nutrient broth. It was found that the activity of polymyxin B sulphate, benzalkonium chloride and chlorhexidine diacetate against this organism was substantially increased in the presence of EDTA. Magnesium and calcium ions were found to block this potentiating action. EDTA was also found to potentiate the action of polymyxin against cells grown in undiluted serum.

THE PAPAIN-CATALYZED SYNTHESIS OF HIPPURYL ANILIDE. I. GENERAL PROPERTIES OF THE ENZYME SYSTEM.

1. 1. The ability of purified, crystalline, chymopapain-free papain (EC 3.4.4.10) to synthesize hippuryl anilide from hippuric acid and aniline has been investigated. The kinetic method of determining the reaction velocity by following aniline disappearance has been employed. 2. 2. The hydrolytic and synthetic activities of the enzyme were compared. Using 3. N-benzoyl-l-argininamide, and hippuric acid and aniline, as substrates for the estimation of hydrolytic and synthetic activities, respectively, papain was found to be over 1000 times more efficient in hydrolyzing N-benzoly-l-argininamide than in synthesizing hippuryl anilide. 4. 3. Chymopapain (EC 3.4.4.11), obtained in a partially purified state, was shown to be more active than crystalline papain in promoting the synthesis of hippuryl anilide. 5. 4. Papain is remarkably stable to heat in the absence of activator, but enzymic activity is readily lost in the presence of β-mercaptopropionic acid. Hippuric acid alters the heat stability of inactive papain and its presence enhances the rate at which papain loses its potential to undergo activation by sulfhydryl activator. 6. 5. 100 moles of sulfhydryl activator, β-mercaptopropionic acid, er mole of en enzyme are required for maximal activity. Diethyldisulfide-β,β′-dicarboxylic acid, the oxidation product of β-mercaptopropionic acid, inhibits enzymic synthesis at high concentrations. There is no synthesis in the absence of activator or in the presence of ethylenediaminetetra acetate alone. Ethylenediaminetetraacetate does not enhance the synthetic activity in the presence of sulfhydryl activator and citrate buffer. 7. 6. The velocity of hippuryl anilide synthesis is a linear function of papain concentration up to 6 mg/ml. 8. 7. Papain hydrolyzes the hippuryl anilide slowly due to the low solubility, 1.85 · 10−4 M, in citrate buffer of the anilide.

COMPONENTS OF GUINEA PIG COMPLEMENT. II. SEPARATION OF SERUM FRACTIONS ESSENTIAL FOR IMMUNE HEMOLYSIS.

The elution characteristics from DEAE cellulose are presented for four components of guinea pig serum, which are capable of interacting sequentially with sheep erythrocytes sensitized with antibody and the first, fourth, and second components of complement (EAC'1,4,2) to cause immune hemolysis, and information is given regarding some of the properties of these components, termed C'3c, C'3b, C'3a, and C'3d. All can react in the presence of ethylene-diaminetetraacetate, and are non-dialyzable. C'3c is quite stable at 56 degrees C, but is rapidly inactivated at low pH or by contact with hydrazine or ammonium hydroxide. C'3b is moderately heat-stable, quite susceptible to low pH, and less readily destroyed by hydrazine. C'3a is very heat-labile, but relatively stable at low pH, while C'3d is heat-labile, sensitive to low pH, and insensitive to bydrazine. EAC'1,4,2 reacts with C'3c to form EAC'1,4,2,3c, which reacts then with C'3b to give the intermediate, EAC'1,4,2,3cb. The following reaction with C'3a yields EAC'1,4,2,3cba, which reacts finally with C'3d to give EAC'1,4, 2,3cbad (E*). The first and last reactions proceed moderately well at 0 degrees C, but more rapidly at 30-37 degrees C. The reaction with C'3b is almost completely inhibited at 0 degrees C, while that involving C'3a proceeds almost as rapidly at 0 degrees C as at higher temperatures. EAC'1,4,2,3cba cells have an increased fragility as compared with the other intermediate forms. Depletion studies with purified fractions and appropriate intermediate complexes showed a high degree of depletion of C'3c, somewhat less of C'3b, and little or no depletion of C'3a from the fluid phase. Examination of a beta(1C) globulin prepared from fresh human serum revealed high C'3c and C'3b activity, and very little C'3a or C'3d.

Maturation, iron deficiency, and ligands in enteric radioiron transport in vitro.

The effects in rats, of age, iron-deficiency anemia, and ascorbic acid, citrate, fluoride, and ethylenediaminetetraacetate (EDTA) on enteric radioiron transport were studied in vitro by an everted gut-sac technique. Sacs from young animals transported more than those from older ones. Proximal jejunal sacs from anemic animals transported more than similar sacs from nonanemic rats, but the reverse effect appeared in sacs formed from proximal duodenum. When added to media containing ascorbic acid or citrate, fluoride depressed transport as did anaerobic incubation in the presence of ascorbic acid. Anaerobic incubation in the presence of EDTA appeared to permit elevated transport. Ascorbic acid, citrate, and EDTA all enhanced the level of Fe59 appearing in serosal media. These results appear to agree with previously established in vivo phenomena and tend to validate the in vitro method as one of promise for further studies of factors affecting iron absorption and of the mechanism of iron absorption.

The use of glyoxal bis-(2-hydroxyanil) in determining microgram amounts of uranium

In alkaline solution, uranium VI readily forms with glyoxal bis-(2- hydroxyanil) a violet complex containing two molecules of reagent to one of uranium, this reaction was made the basis of a method for determining uranium. The complex, soluble in aqueous methanolic solution, has maximum absorption at 575 m mu and is stable in presence of ethylenediaminetetraacetate, which suppresses formation of other metallic complexes. Phosphate and carbonate reduce the sensitivity of the method. (auth)

Studies of the Specificity of Deoxyribonuclease I: III. HYDROLYSIS OF CHAINS CARRYING A MONOESTERIFIED PHOSPHATE ON CARBON 5'

Kunitz (3) crystallized pancreatic deoxyribonucleasel and studied its kinetics and the products of its action on deoxyribonucleic acid. With a ratio of enzyme to substrate not higher than 1: 500, the products obtained after exhaustive digestion had an average length of a tetranucleotide. Studies from this and Sinsheimer’s laboratory (for details see the recent review (4)), showed that the digest thus obtained represented a complex mixture of fragments varying in length from mononucleotides to at least o&a-, possibly dodecanucleotides. The attempts to redigest larger oligonucleotides (by the use of a low enzyme-substrate ratio) into smaller fragments failed (5). It was therefore assumed that the digest thus obtained is a “limit digest” and that it is composed of fragments containing only internucleotide linkages which are totally resistant to the action of DNase I. Hurst (6) and Hurst and Fmdlay (7) observed that with a very large amount of enzyme (ratio of enzyme to substrate of the order 1: lo), at pH 8.0, in the presence of ethylenediaminetetraacetate and Mn++, the hydrolysis proceeds beyond the “limit digest” stage. To explain their findings, Hurst and Findlay (7) postulated that crystalline DNase I contains still another enzyme, an oligonucleotidase, which is capable of hydrolyzing oligonucleotides present in the “liiit digest” into smaller fragments, as measured by solubility in a uranyl acetate reagent. Independently, in the previous paper of this series (2), it was observed that when oligonucleotides carrying monoesterified phosphate on carbon 3’ were digested with very high amounts of DNase I, the predominant products were derivatives of dinucleotides. The hypothesis was advanced (2) that dinucleotides represent an ultimate product of the action of DNase I on deoxyribonucleic acid and that an apparent end point (“limit digest”) is caused by an insufficient amount of enzyme and by product inhibition. Thus, an alternative explanation was suggested, without postulating

THE EFFECT OF INGESTION OF DISODIUM ETHYLENEDIAMINETETRAACETATE ON THE ABSORPTION AND METABOLISM OF RADIOACTIVE IRON BY THE RAT

Disodium ethylenediaminetetraacetate (EDTA) was fed to young adult rats at a level of 0.1% of the diet. The effect on absorption, metabolism, and excretion of dietary iron was studied after administration of radioactive ferric chloride by stomach tube. The presence of EDTA decreased the absorption of iron and increased the excretion in urine. When EDTA was not fed until after the Fe59had been absorbed, the iron appeared to be increased in the blood and the spleen, and to be removed from the liver.

Copper salicylaldoxime and its use in the heterometric microdeterminatton of copper in the presence of foreign metals

1. The initial precipitation of copper-salicylaldoxime occurs at pH~1.5.Between pH 1.5 and 3.0, the precipitation is incomplete. A complete precipitation occurs in the pH-region of 3–10. Between pH 10.1 and 10.7 the precipitate redissolves At higher pH's a clear solution is obtained. 2. The solubility of copper salicylaldoximate is about ten times greater in 0.01M sulfuric acid (~0.5·10 -4 mol) than in 0.1 M acetic acid. 3. In 10 ml of 0.1 M acetic acid solution in the presence of ~99% foreign bivalent metals, one mg copper could be determined heterometrically with an error of 1–2%. 4. In the presence of ~95% alumin um or chromium, either citrate or tartrate (0.2 M) were used as masking agents. In the presence of iron (~ 95%), clear end-points were obtained only with citrate. One mg of copper could be determined with an error of 0–3%. 5. Whichever pH is used, copper is not precipitated in the presence of ethylenediaminetetraacetate.

Oxidation of glutathione in extracts of lens in the presence of ethylenediaminetetraacetate.

CHELATING agents are being increasingly used for removing traces of metals. We therefore think it worth while to report a case where a chelating agent accelerated the oxidation of a substance which it was intended to preserve in the reduced state.