A peptide inhibitor for S-adenosyl-l-methionine-dependent transmethylation reactions: Purification and characterization

• 1.1. A proteinaceous inhibitor for S-adenosyl-l-methionine (AdoMet)-dependent transmethylation reactions has been purified to apparent homogeneity from rat liver cytosolic fraction.
• 2.2. The peptide was made up of 29 amino acid residues with a molecular weight of 2,584. Glycine accounted for 52% of the total amino acids.
• 3.3. Employing AdoMet: protein-carboxyl O-methyltransferase (Protein methylase II) and bovine serum γ-globulin as in vitro substrate, the mode of inhibition was found to be non-competitive with K_i value of 1.9 × 10⁻⁸ M.
• 4.4. When the inhibitor was present in the reaction mixture together with S-adenosyl-l-homocysteine (AdoHcy), which is a competitive inhibitor for AdoMet, the extent of inhibition exceeded that exerted by each individual inhibitor alone, suggesting that the sites of the inhibitors on the enzyme molecule are different.
• 5.5. Almost a stoichiometric relationship exists between the enzyme and the inhibitor molecule, the ratio being approx one.

     

Studies on germinal effects of quercetin, a naturally occurring flavonoid

Quercetin (3,3',4',5,7-pentahydroxyflavone) is one of the most widely occurring flavonoids ingested by man in food. It has been shown to be mutagenic in prokaryotes as well as in in vitro mammalian cell lines. In view of the unavoidability of ingesting it via a normal diet, there is a need to assess the potential genetic risk to man, due to flavonoid ingestion, using whole animal assays. Dominant lethal studies have been carried out in adult Swiss male mice and Wistar male rats to investigate the germinal effects of quercetin. Adult Swiss males were treated with 200, 300 or 400 mg/kg of quercetin dissolved in 60% dimethyl sulfoxide, intraperitoneally. In the rat study, 200 and 300 mg/kg of quercetin were used. Individually caged males were paired with untreated females for a week and six sequential matings were carried out. Two independent experiments in mice and a single experiment in rats constituted the study. Females were evaluated for inducted dominant lethality during the midterm of pregnancy. At 200 mg/kg dose of quercetin, there were no significant differences between control and the test group in pregnancy, total or live implantations in mice or rats during the whole test period that could be attributed to the flavonoid exposure. In mice at 300 and 400 mg/kg of quercetin, there was a profound reduction in fertility of the males during all six matings. The number of total and live implantations also decreased, particularly at the 400 mg/kg dose, although the sample size was too small to be statistically significant. Contrary to this, the rat study did not show any impairment of fertility, nor was there any substantial suppression of total and live implantations at the highest (300 mg/kg) dose tested. There were no significant differences between control and treated groups with regard to the number of dead implantations at any dosage level at any stage of the study in mice and rats. Thus, no post-implantation losses--a reliable measure of dominant lethal mutations--were induced by quercetin in mice or rats. The loss of fertility could be due to germinal cytotoxicity, oligospermia or impairment of fertilizing ability of the treated animals.(ABSTRACT TRUNCATED AT 400 WORDS)     

Acceleration of ionic reactions by naturally occurring glycosaminoglycans. II.

The reactions of halopentammine cobalt(III) complexes with both Hg(II) (aquation reaction) and Fe(II) (electron transfer reaction) have been studied in the presence of chondroitin-4-sulphate and heparin. The rates of both reactions are increased in the presence of the glycosaminoglycan polyeletrolytes, but by different mechanisms. Inhibition of the Hg(II) reaction at lower polymerto-reagent concentration ratios is consistent with specific polymer–Hg interactions which render a population of ‘bound’ ions unreactive. However, the activation energy of the Hg-induced aquation is lowered in the presence of the polyanion, consistent with stabilization of the expected trivalent cationic intermediate by electrostatic effects. Heparin, with the higher charge density, is especially effective in this regard. In contrast, the reduction of the Co(III) complexes by Fe(II) experiences an increased activation energy in the presence of polymer. The observed acceleration of this reaction may then be due largely to local concentration effects in the polymer domain, as implied by two-phase model. Investigation of the mechanisms of these rate accelerations will therefore provide more detailed information on the electrostatic fields and specific metal ion interactions of these important biological polymers.     

Antithrombotic and haemorrhagic effects of the naturally occurring thrombin inhibitor hirudin

Haemorrhagic effects of the naturally occurring thrombin inhibitor hirudin measured by the determination of bleeding time were compared with its antithrombotic actions in different models of experimental thrombosis. In mice and rats intravenous administration of hirudin caused a plasma concentration-dependent prolongation of bleeding time after transection of the tail tip and standardized incision of the tail, respectively. In rats intravenous infusion of hirudin prevented the formation of stasis-induced venous thrombosis of the jugular vein and the occurrence of thrombotic occlusion of the carotid artery after electrically induced damage of the vessel wall. Comparison of the haemorrhagic action of hirudin with its antithrombotic effectiveness showed that it caused haemorrhagic side effects only at plasma concentrations which are not required for the antithrombotic effects.     

Interaction of F1-ATPase, from ox heart mitochondria with its naturally occurring inhibitor protein. Studies using radio-iodinated inhibitor protein

The ox heart mitochondrial inhibitor protein may be iodinated with up to 0.8 mol 125I per mol inhibitor with no loss of inhibitory activity, with no change in binding affinity to submitochondrial particles, and without alteration in the response of membrane-bound inhibitor to energisation. Tryptic peptide maps reveal a single labelled peptide, consistent with modification of the single tyrosine residue of the protein. A single type of high-affinity binding site (Kd=96 . 10 (-9)M) for the inhibitor protein has been measured in submitochondrial particles. The concentration of this site is proportional to the amount of membrane-bound F1, and there appears to be one such site per F1 molecule. The ATp hydrolytic activity of submitochondrial particles is inversely proportional to the occupancy of the high-affinity binding site for the inhibitor protein. No evidence is found for a non-inhibitory binding site on the membrane or on other mitochondrial proteins. In intact mitochondria from bovine heart, the inhibitor protein is present in an approx. 1:1 ratio with F1. Submitochondrial particles prepared by sonication of these mitochondria with MgATP contain about 0.75 mol inhibitor protein per mol F1, and show about 25% of the ATPase activity of inhibitor-free submitochondrial particles. Additional inhibitor protein can be bound to these particles to a level of 0.2 mol/mol F1, with consequent loss of ATPase activity. If MgATP is omitted from the medium, or inhibitors of ATP hydrolysis are present, the rate of combination between F1 and its inhibitor protein is very much reduced. The equilibrium level of binding is, however, unaltered. These results suggest the presence of a single, high-affinity, inhibitory binding site for inhibitor protein on membrane-bound F1. The energisation of coupled submitochondrial particles by succinate oxidation or by ATP hydrolysis results in both the dissociation of inhibitor protein into solution, and the activation of ATP hydrolysis. At least 80% of the membrane-bound F1-inhibitor complex responds to this energisation by participating in a new equilibrium between bound and free inhibitor protein. This finding suggests that a delocalised energy pool is important in promoting inhibitor protein release from F1. Dissipation of the electrochemical gradient by uncouplers, or the binding of oligomycin or efrapetin effectively blocks energised release of the inhibitor protein. Conversely, the addition of aurovertin or adenosine 5'--[beta, lambda--imido]triphosphate enhances energy-driven release. The mode of action of various inhibitors on binding and energised release of the protein inhibitor is discussed.     

Interaction between F1-ATPase and its naturally occurring inhibitor protein. Studies using a specific anti-inhibitor antibody

An antibody was raised to cross-linked ox-heart mitochondrial inhibitor protein, which cross-reacts with the free inhibitor but with no other mitochondrial membrane protein. This antibody yields an immunoprecipitate with the cross-linked inhibitor protein, but a soluble antibody-antigen complex with free inhibitor. The antibody binds well to inhibitor protein whether the latter is complexed with F1-ATPase or not. Antibody binding has no effect on the ability of the inhibitor protein to inhibit the ATPase activity of F1. These findings suggest that the antibody does not block the site of interaction between the inhibitor and F1. The inhibitor protein content of submitochondrial membrane preparations was determined by radioimmunoassay, activity measurements and an immunochemical 'back titration' technique. The inhibitor content of the membranes is shown to decrease after energisation, suggesting a loss of inhibitor from the membranes into solution. Binding antibody to the inhibitor protein on submitochondrial particles has no effect on the steady-state rate of phosphorylation, but it increases the lag phase preceding phosphorylation from 30 to 54 s. The rate constant for the approach to the steady state drops from 0.078 to 0.052 s-1. This effect confirms that the lag phase is due to inhibition of phosphorylation by the inhibitor protein. The increase in ATPase activity following energisation takes place by a fast phase (80% maximal activity reached within 90 s) and a slower phase (lasting about 10 min.). The rate constant of the rapid phase (0.017 s-1) is of the same order as that for the activation of phosphorylation. It is concluded that the rapid phase of ATPase induction is fast enough for this process to occur simultaneously with the activation of phosphorylation.     

Isolation of a naturally occurring inhibitor for dark Pfr reversion from etiolated Pisum epicotyls

The in vitro reversion of Pfr to Pr in the dark was preventedby adding the low molecular weight fraction obtained from aSephadex G-50 column chromatogram to the crude homogenate ofetiolated pea epicotyl tissues, and by the methanolic and boilingaquous extracts of the tissue. The degree of inhibition of darkPfr reversion was related to the logarithmic concentrationsof the methanol-extractable material. The active substance washighly soluble in water-saturated n-butanol; but not in chloroform,ethyl acetate, n-hexane, benzene and pure n-butanol. Thus, alow molecular substance(s) which can inhibit dark Pfr reversionwas indicated to be contained in pea epicotyl tissues. (Received January 28, 1975; )     

The identification and properties of a naturally occurring inhibitor of malic enzyme

The inhibitor of malic enzyme present in potato tubers has been identified as oxalic acid. Oxalic acid proves to be a particularly potent inhibitor with a K_I = 50 μM. A kinetic analysis indicates that inhibition is not due to chelation of Mg²⁺ and suggests that oxalate binds tightly to malic enzyme after NADPH has been bound.     

Tetraoxygenated naturally occurring xanthones

This review, with 350 references, gives information on the chemical study of 234 naturally occurring tetraoxygenated xanthones in 12 families, 53 genus and 182 species of higher plants, and two which are described as fungal and lichen metabolites. The value of these groups of substances in connection with pharmacological activity and therapeutic use of some species is described. The structural formulas of 135 isolated compounds, and their distribution, are also given.     

Separation procedures for naturally occurring antioxidant phytochemicals

Phytochemicals in fruits, vegetables, spices and traditional herbal medicinal plants have been found to play protective roles against many human chronic diseases including cancer and cardiovascular diseases (CVD). These diseases are associated with oxidative stresses caused by excess free radicals and other reactive oxygen species. Antioxidant phytochemicals exert their effect by neutralizing these highly reactive radicals. Among the tens of thousands of phytochemicals found in our diets or traditional medicines, polyphenols and carotenoids stand out as the two most important groups of natural antioxidants. However, although collectively these phytochemicals are good antioxidants, the roles and effect of individual compounds are often not well known. Hundreds of carotenoids and thousands of polyphenols have been identified so far from various plants. A single plant could contain highly complex profiles of these compounds, which sometimes are labile to heat, air and light, and they may exist at very low concentrations in the plants. This makes the separation and detection of these antioxidant phytochemicals a challenging task. The present review focuses on the antioxidant activity, chemical types, sampling and sample processing procedures, and separation using various chromatographic and electrophoretic techniques. Detection and quantification using ultraviolet-visible-diode array and mass spectrometry will be discussed.     

Synthesis of D-lyxitol and D-ribitol analogues of the naturally occurring glycosidase inhibitor salacinol

The synthesis of analogues of the naturally occurring glycosidase inhibitor, salacinol, in which the D-arabinitol ring has been replaced by D-lyxitol or D-ribitol, is described. Salacinol is one of the active principles in the aqueous extracts of Salacia reticulata, which are traditionally used in India and Sri Lanka for the treatment of Type II diabetes. The synthetic strategy relies on the nucleophilic attack of 1,4-anhydro-2,3,5-tri-O-p-methoxybenzyl-4-thio-D-lyxitol or 1,4-anhydro-2,3,5-tri-O-p-methoxybenzyl-4-thio-D-ribitol at the least hindered carbon of the benzylidene-protected L-cyclic sulfate derived from L-erythritol. Screening of these compounds against recombinant human maltase glucoamylase (MGA), a critical intestinal glucosidase involved in the processing of oligosaccharides of glucose into glucose itself, shows that they are not effective inhibitors of MGA and demonstrates the importance of the d-arabinitol configuration in the heterocyclic ring for effective inhibition.     

Parathyroid hormone-related peptide is a naturally occurring,protein kinase A-dependent angiogenesis inhibitor

Angiogenesis is a highly regulated process that results from the sequential actions of naturally occurring stimulators and inhibitors. Here, we show that parathyroid hormone-related peptide, a peptide hormone derived from normal and tumor cells that regulates bone metabolism and vascular tone, is a naturally occurring angiogenesis inhibitor. Parathyroid hormone-related peptide or a ten-amino-acid peptide from its N terminus inhibits endothelial cell migration in vitro and angiogenesis in vivo by activating endothelial cell protein kinase A. Activation of protein kinase A inhibits cell migration and angiogenesis by inhibiting the small GTPase Rac. In contrast, inhibition of protein kinase A reverses the anti-migratory and anti-angiogenic properties of parathyroid hormone-related peptide. These studies show that parathyroid hormone-related peptide is a naturally occurring angiogenesis inhibitor that functions by activation of protein kinase A.     

Actinonin, a naturally occurring antibacterial agent, is a potent deformylase inhibitor

Peptide deformylase (PDF) is essential in prokaryotes and absent in mammalian cells, thus making it an attractive target for the discovery of novel antibiotics. We have identified actinonin, a naturally occurring antibacterial agent, as a potent PDF inhibitor. The dissociation constant for this compound was 0.3 x 10(-)(9) M against Ni-PDF from Escherichia coli; the PDF from Staphylococcus aureus gave a similar value. Microbiological evaluation revealed that actinonin is a bacteriostatic agent with activity against Gram-positive and fastidious Gram-negative microorganisms. The PDF gene, def, was placed under control of P(BAD) in E. coli tolC, permitting regulation of PDF expression levels in the cell by varying the external arabinose concentration. The susceptibility of this strain to actinonin increases with decreased levels of PDF expression, indicating that actinonin inhibits bacterial growth by targeting this enzyme. Actinonin provides an excellent starting point from which to derive a more potent PDF inhibitor that has a broader spectrum of antibacterial activity.     

Social constraints on naturally occurring preschool altruism

Social interaction among 26 children ranging in age from three to five years was videotaped for 30 hours during free play at a University preschool. Altruistic exchanges were identified from these tapes and coded into one of 16 categories. Approximately 1200 exchanges were observed. Nearly 60% of observed altruism was directed toward peers and 40% toward teachers. The degree of generality in children's altruistic behavior depended strongly on whether such activity was directed toward peers or teachers, and on whether the peer was a friend. A strong relationship was evident between individual rates of initiated and received altruistic behavior. These findings were related to previous experimental research on young children's “prosocial” behavior, and discussed with reference to both psychological and sociobiological conceptions of human altruism.