Active Sulphydryl Groups Research Articles

Mechanism of inhibition of trypanothione reductase and glutathione reductase by trivalent organic arsenicals

The dithiol trypanothione, novel to trypanosomatids and analogous to glutathione in mammalian systems, has been shown to interact with anti-trypanocidal trivalent arsenical drugs forming a stable adduct, MelT. This adduct is a competitive inhibitor of the flavoprotein trypanothione reductase, responsible for maintaining intracellular trypanothione in the reduced form. Since trypanothione reductase and the analogous glutathione reductase both contain catalytically active sulphydryl groups we have examined the ability of several arsenicals to differentially inhibit these enzymes. Melarsen oxide [p-(4,6-diamino-s-triazin-2-yl)aminophenylarsenoxide] potently inhibits both enzymes in two stages, the first being essentially complete within 1 min, the second being time dependent, exhibiting saturable pseudo-first-order kinetics with kinact of 14.3 x 10(-4) s-1 and 1.06 x 10(-4) s-1 and Ki of 17.2 microM and 9.6 microM for trypanothione reductase and glutathione reductase, respectively. Inhibition requires prior reduction of the enzyme by NADPH and can be reversed by excess dithiols or prevented by MelT in the case of trypanothione reductase. In both cases a time-dependent loss of the characteristic charge-transfer absorbance band at 530 nm is observed upon addition of arsenical to pre-reduced enzyme, which with excess NADPH leads to a spectrum resembling the EH4 form and is accompanied by an increased ability to reduce molecular oxygen. A model for inhibition is proposed where, first, free arsenical and previously reduced enzyme immediately establish an equilibrium with an inactive monothioarsane enzyme-inhibitor complex involving the interchange cysteine distal to the FAD; second, a subsequent rearrangement about the sulphur-arsenic bond leads to the binding of the arsenical to the charge-transfer cysteine, proximal to the FAD, forming a more stable dithioarsane complex. Molecular modelling suggests that the differences in kinetic behaviour of the two enzymes can be attributed to structural features of their respective disulphide-binding sites. Incubation of reduced trypanothione reductase with excess dihydrotrypanothione and melarsen oxide prevents direct inhibition of the enzyme, suggesting that dihydrotrypanothione acts as a protectant in vivo, preventing the direct modification of trypanothione reductase by sequestering the arsenical as MelT.

Purification, characterization and origin of rat gastric peroxidase.

A membrane-bound peroxidase (EC 1.11.1.7) from rat stomach has been solubilized by 0.2% cetyltrimethylammonium bromide in the presence of 1.2 M NH4Cl. The enzyme was purified 3355-fold to apparent homogeneity as judged by acid polyacrylamide gel electrophoresis and appears to be a cationic protein. In sodium dodecyl sulfate gel electrophoresis, the enzyme shows single polypeptide band of Mr 45,000. In gel permeation, the Mr has been estimated as 47,000. Spectral properties indicate the presence of Soret band at 412 nm which shifts to 425 nm on complexation with CN- and to 430 nm on reduction with dithionite. The velocity constant, k1 for the reaction of the peroxidase with H2O2 is 1.38 X 10(7) M-1 s-1 and Km for H2O2 is 0.1 mM. The enzyme contains active sulphydryl groups and is inhibited by sulphydryl reagents of which p-hydroxymercuribenzoate is more reactive than mersalyl or N-ethylmaleimide. The enzyme is very resistant to thermal denaturation up to 65 degrees C and also to chaotropic reagents at least up to 2 M above which it is inactivated. The enzyme shows similarity with the intestinal eosinophil peroxidase as regards the molecular mass, spectral, kinetic and some of the catalytic properties. However, they differ significantly in terms of their interaction with fluoride ion, sulphydryl reagents, chaotropic reagent and also with the antiserum against the gastric peroxidase. Histochemically, the gastric peroxidase is shown to be localised in the gastric gland proper of the fundic stomach, rich in parietal and chief cells.

Proteases in germinating finger millet (Eleusine coracana) seeds

Proteolytic activity was estimated in germinated finger millet seedlings using the endogenous trypsin/amylase inhibitor as substrate and also with haemoglobin and albumin as substrates. The maximal proteolytic activity was observed on the third day of germination. With the inhibitor as substrate, the proteolytic activity was maximal at pH 2.5. The protease that acted on the inhibitor required sulphydryl groups for maximal activity and was suppressed by diazoacetyl norleucine methyl ester and Pepstatin. The protease that acted on haemoglobin with optimum pH of 5.0, was more stable on storage, did not depend on sulphydryl groups for activity and was unaffected by reagents that react with carboxyl groups.

Isolation and properties of digestive carboxypeptidases from midguts of larvae of the black carpet beetle Attagenus megatoma

Two cationic carboxypeptidase fractions (CMS-G-1 and CMS-G-2) were isolated from the midgut homogenate of larvae of the black carpet beetle, Attagenus megatoma, through a series of ion exchange and affinity gel chromatographic steps. Fraction CMS-G-2 was highly active against the substrates hippuryl- dl-β-phenyllactate, N- carbobenzoxy-glycyl- l-phenylalanine , and N- carbobenzoxy-glycyl- l-tyrosine and was thus similar in specificity to bovine carboxypeptidase A. Fraction CMS-G-1 was most active against N- carbobenzoxy- l-valyl- l-leucine and N- carbobenzoxy-glycyl- l-methionine . Fraction CMS-G-1 also hydrolysed (in decreasing order) blocked peptides with carboxyl terminal l-isoleucine, l-leucine, l-alanine, and l-valine but was only slightly active against the ester and peptide substrates of carboxypeptidase A. Neither CMS-G-1 nor CMS-G-2 hydrolysed N- carbobenzoxy-glycyl- l-proline or glycyl-glycine. Between CMS-G-1 and CMS-G-2, at least four bands with peptidase activity were resolved on a cationic polyacrylamide gel system. The terminal and penultimate amino acids of the peptides were equally important in determining the specificity of individual peptidase bands. Fractions CMS-G-1 and CMS-G-2 each had a single, separate activity band with the substrate N- carbobenzoxy-glycyl- l-leucine . These two peptidases had molecular weights of about 35,500 and were most active at pH 7.5 to 8.5. Both enzymes were inhibited by the metal chelators EDTA and 1,10-phenanthroline and also by heavy metals such as Cu 2+, Hg 2+ and Cd 2+. The two enzymes thus appear to be metalloproteins with active sulphydryl groups.

Isolation and Characterization of a Membrane-associated Thiosulphate-oxidizing System of Thiobacillus novellus

Summary: A thiosulphate-oxidizing system of Thiobacillus novellus grown on thiosulphate was studied using intact cells, crude cell-free extracts and isolated membrane vesicles. All active preparations oxidized thiosulphate to sulphate, without intermediary accumulation of polythionates, consuming two moles of oxygen for every mole of thiosulphate oxidized. Less active cell-free preparations required reduced glutathione, NADH or sulphite for thiosulphate oxidation. An active membrane-associated thiosulphate-oxidizing system from crude cell-free extracts was isolated by either Sepharose 4B column chromatography or differential centrifugation. The isolated multi-enzyme complex system exhibited high specificity for thiosulphate with an apparent K m value of 0·12 mm. Enzyme activity was optimum at pH 7·5 and 25 °C. The system was sensitive to oxygen, storage at certain temperatures and freezing. Inhibition studies indicated that active sulphydryl groups and an electron transport chain were involved during thiosulphate oxidation. Electron micrographs of active crude extracts and the isolated membrane complex after negative staining showed the presence of spherical structures with a diameter of about 0·1 to 0·4 μm. An ultra-thin section of the membrane complex revealed that the large spherical particles observed in the negatively stained preparations were aggregated structures consisting of smaller vesicles 0·02 to 0·05 μm in diameter.

Toxic Effect and Action of Dead Sperm on Diluted Bovine Semen

The toxic effect of dead sperm on diluted bovine semen and the modifying effect of certain compounds on this toxicity were investigated.Toxicity is associated with an amino aeid oxidase which becomes active only after death of sperm. A metabolic product of this enzyme, peroxide, is responsible for the toxic effect of dead sperm. The enzyme is active against aromatic amino acids only. Its toxic effect is enhanced by increasing egg yolk, inhibited by ethylenediaminetetra-acetate (EDTA), and eliminated by catalase. Inhibition of toxicity by EDTA is by protection of sperm against deleterious effects of peroxide and not by inhibition of the enzyme. The enzyme is inhibited by ethyl maleimide indicating active sulphydryl groups. It is labile to heat being inactivated by heating to 90C for 2 minutes. It is also labile to pH 3 and pH 9. An active fraction was obtained from dead sperm by precipitation with 30% acetone. Activity of ejaculated dead sperm is only slightly less than freshly killed sperm. Practical implications are discussed.

ISOLATION, PROPERTIES, AND BIOLOGICAL EFFECTS OF PHOMIC ACID

Phomic acid, a toxic metabolic product of Phoma medicaginis, has been isolated from culture filtrates of the fungus. The toxin crystallizes as colorless rectangular plates having a refractive index of 0.56 and melting at 259–260 °C. It has absorption peaks at 208 and 260 mμ. The infrared spectrum of the toxin suggests that it may be an unsaturated aliphatic hydroxy acid.The toxic principle causes discoloration and drying of the leaf tip and leaf margin and a general stunting of the flax plant. It inhibits germination of seeds of flax, alfalfa, rape, and cress. The effect of phomic acid on seed germination can be reversed by glutathione and cysteine. Germination of the conidia of a few fungi is completely inhibited by the toxin, whereas it only retards the growth of the germ tube of Helminthosporium sativum. It inhibits the activity of triosephosphate dehydrogenase but not that of malic dehydrogenase. It is suggested that the toxin might act by inhibiting enzyme systems requiring sulphydryl groups for activity.

Fragmentation of the electron transport chain of Escherichia coli: Preparation of a soluble formate dehydrogenase-cytochrome b1 complex

The electron transport system of Escherichia coli has been studied with particular reference to the particulate enzymes formate dehydrogenase (formate: cytochrome b 1 oxidoreductase, EC 1.2.2.1) and cytochrome b 1. A soluble preparation of a formate dehydrogenase-cytochrome b 1 complex was obtained by extraction of suitably grown cells with deoxycholate and high concentrations of (NH 4) 2SO 4. The dehydrogenase, a flavoprotein requiring sulphydryl groups for activity, is quantitatively extracted with a 20-fold purification and is largely free of other particulate enzymes. A representative preparation contains about 0.5 mμmole flavin per mg protein, 1.5 mμmoles cytochrome b 1 per mg protein, 3% lipid and it oxidizes formate with ferricyanide as acceptor at a rate of 17 μmoles/min per mg protein. The cytochrome b 1 of the preparation is readily reducible by formate. The functional linkage between the dehydrogenase and cytochrome component is lost on mild aeration or aging of the preparations. The communication between the two components can be restored by the addition of vitamin K 3. However, neither vitamin K nor ubiquinone could be detected in fresh active preparations and it is therefore unlikely that a quinone is involved as a natural intermediate in this portion of the electron transport system of Escherichia coli.

The isolation and properties of a protease from proteus vulgaris

A method has been developed for the preparation, in a pure state, of a new proteolytic enzyme from cultures of Proteus vulgaris. The enzyme requires free sulphydryl groups for activity and can be inhibited by HgCl 2, PCMB and iodoacetate. The mercury-inhibited enzyme can be re-activated by cysteine or cyanide. The native enzyme, which has a more powerful action on denatured that on native proteins, is more active against a haemoglobin substrate that chymotrypsin, but has only one third the activity of trypsin. The pure protease is not resolved by electrophoresis in the Tiselius apparatus, between pH 6.6 and pH 9.6; it has a sedimentation constant S 20, w = 3.7 at 0.35% concentration in pH 8.0 buffer and has an optimum pH of activity between 9 and 10. The ultra-violet absorption spectrum between 240 mμ and 285 mμ is characteristic of a protein.

Intracellular changes in levels of polarographically active sulphydryl groups in rat liver during carcinogenesis.

Intracellular changes in levels of polarographically active sulphydryl groups in rat liver during carcinogenesis.

THE PHYSIOLOGICAL SIGNIFICANCE OF THE PHOSPHOMONOESTERASES

SummaryResearches of the past ten years bearing on the physiological significance of the common acid and alkaline phosphomonoesterases may be summarized in the following facts and concepts, (i) The alkaline phos‐phomonoesterase appears to be a salt‐soluble, the acid a water‐soluble, substance; both consist of a metallic coenzyme and a protein‐like carrier. Neither depends oh sulphydryl groups for activity, but the alkaline phosphatase seems to require amine and phenolic hydroxy! groups; it is affected only by strong oxidizing agents. There is not yet any incontestable evidence that alkaline phosphomonoesterases of different sources are not identical. (2) Phosphatases probably catalyse only hydrolyses, not syntheses. (3) The content of both phosphatases in the blood serum is characteristic of age and physiological state, and is used in the diagnosis of bone and liver disorders; biliary obstruction results in elevation of serum alkaline phosphatase probably because phosphatase is normally excreted by way of liver and gut, but other explanations are possible. Diet influences serum alkaline phosphatase level. (4) The study of phosphatase function has been advanced by the development of techniques for the histochemical demonstration of acid and alkaline phosphatases. (5) It is certain that alkaline phosphatase is indispensable in normal ossification; it is generally instrumental in abnormal calcifications, but in a few cases calcification occurs without it. (6) Glycolysis seems to accompany normal bone formation, but this fact does not clarify all the unsolved problems of ossification. (7) Hormones and vitamins influence both alkaline phosphatase activity and bone formation. (8) In kidney tubules alkaline phosphatase probably functions in dephosphorylating sugar molecules that are removed from the tubular fluid by phosphorylation. The enzyme is present at other sites where it might serve in a similar manner. (9) Phosphatases are commonly found in the cytoplasm of growing, regenerating, and secreting cells in which protein synthesis is being carried on; there appears to be a correlation in such cells between content of pentosenucleic acid and of phosphatase. In dividing nuclei, phosphatase parallels desoxyribonucleic acid. (10) Acid phosphatase is found in seminal fluid, and may help to nourish the sperm. (11) The occurrence of two common phosphomonoesterases with widely different pH optima may indicate a mechanism for allowing two dephosphorylating steps to go on simultaneously in a single cell. In tissues which carry on much traffic in glycogen, acid phosphatase is possibly provided to prevent bone formation. (12) The actual role which the phosphatases play in the physiology of the organism will be understood only with elucidation of the complete enzyme systems of which the phosphatases are a part.