Effects Of Competitive Inhibitors Research Articles

Biochemical study of the anti-diabetic action of the Egyptian plants Fenugreek and Balanites

Fenugreek and Balanites are two plants commonly used in Egyptian folk medicine as hypoglycemic agents. In the present study, the effects of 21 days oral administration of Fenugreek seed and Balanites fruit extracts (1.5 g/kg bw) on the liver and kidney glycogen content and on some key liver enzymes of carbohydrate metabolism in STZ-diabetic rats were studied. In addition, the effects of these two plant extracts on the intestinal alpha-amylase activity in vitro and starch digestion and absorption in vivo were also examined. Results indicated that single injection of STZ (50 mg/kg bw) caused 5-folds increase in the blood glucose level, 80% reduction in serum insulin level, 58% decrease in liver glycogen and 7-folds increase in kidney glycogen content as compared to the normal levels. The activity of glucose-6-phosphatase was markedly increased, whereas, the activities of both glucose-6-phosphate dehydrogenase and phospho-fructokinase were significantly decreased in the diabetic rat liver. Administration of Fenugreek extract to STZ-diabetic rats reduced blood glucose level by 58%, restored liver glycogen content and significantly decreased kidney glycogen as well as liver glucose-6-phosphatase activity. Meanwhile, Balanites extract reduced blood glucose level by 24% and significantly decreased liver glucose-6-phosphatase activity in diabetic rats. On the other hand, our results demonstrated that both the Fenugreek and Balanites extracts were able to in vitro inhibit alpha-amylase activity in dose-dependent manner. Fenugreek was more potent inhibitor than Balanites. This inhibition was reversed by increasing substrate concentration in a pattern which complies well with the effect of competitive inhibitors. Furthermore, this in vitro inhibition was confirmed by in vivo suppression of starch digestion and absorption induced by both plant extracts in normal rats. These findings suggest that the hypoglycemic effect of Fenugreek and Balanites is mediated through insulinomimetic effect as well as inhibition of intestinal alpha-amylase activity.

A Metabolic Enzyme That Rapidly Produces Superoxide, Fumarate Reductase of Escherichia coli

Aerobic organisms synthesize superoxide dismutases in order to escape injury from endogenous superoxide. An earlier study of Escherichia coli indicated that intracellular superoxide is formed primarily by autoxidation of components of the respiratory chain. In order to identify those components, inverted respiratory vesicles were incubated with five respiratory substrates. In most cases, essentially all of the superoxide was formed through autoxidation of fumarate reductase, despite the paucity of this anaerobic terminal oxidase in the aerobic cells from which the vesicles were prepared. In contrast, most dehydrogenases, the respiratory quinones, and the cytochrome oxidases did not produce any detectable superoxide. The propensity of fumarate reductase to generate superoxide could conceivably deluge cells with superoxide when anaerobic cells, which contain abundant fumarate reductase, enter an aerobic habitat. In fact, deletion or overexpression of the frd structural genes improved and retarded, respectively, the outgrowth of superoxide dismutase-attenuated cells when they were abruptly aerated, suggesting that fumarate reductase is a major source of superoxide in vivo. Steric inhibitors that bind adjacent to the flavin completely blocked superoxide production, indicating that the flavin, rather than an iron-sulfur cluster, is the direct electron donor to oxygen. Since the turnover numbers for superoxide formation by other flavoenzymes are orders of magnitude lower than that of fumarate reductase (1600 min-1), additional steric or electronic factors must accelerate its autoxidation.

13C-NMR studies of porcine kidney D-amino acid oxidase reconstituted with 13C-enriched flavin adenine dinucleotide. Effects of competitive inhibitors.

D-Amino acid oxidase (DAO) from porcine kidney was reconstituted with FAD's which were enriched with 13C at the 2-, 4-, 4a-, and 10a-positions of the isoalloxazine moiety, and 13C-NMR spectra of the reconstituted DAO were measured in the absence and presence of various competitive inhibitors. When compared to the corresponding chemical shifts of FMN at infinite dilution (Moonen, C.T.W. et al. (1984) Biochemistry 23, 4859-4867), the resonances of C(2), C(4), C(4a), and C(10a) of FAD bound to apoDAO were all shifted to higher field. However, when the reconstituted DAO was complexed with o-, m-, p-aminobenzoate, or benzoate, each of the four 13C-signals underwent a different change in chemical shift. In these changes we observed no characteristics which distinguish DAO-aminobenzoate complexes from DAO-benzoate complex, even though o-, m-, and p-aminobenzoates are known to form charge-transfer complexes with DAO. The signals due to 2- and 4-13C were more sensitive to the formation of the inhibitor-DAO complexes than those of the other carbon atoms. These findings suggest the modulation of the hydrogen bonds at the oxygen atoms of C(2) = O and C(4) = O with the protein moiety as the result of the inhibitor-binding.

Characterization and partial purification of three glycosidases from castor bean endosperm

α-Mannosidase and β- N-acetylhexosaminidase, which could function in the cleavage of glycosidic linkages in the native Ricinus communis lectins, and β-galactosidase were purified some 100-fold from the endosperm tissue of castor bean seedlings. The procedure used ammonium sulphate precipitation followed by chromatography on CM-cellulose, hydroxyapatite and Sephacryl S-300 to separate the three activities. All three glycosidases were present, with the lectins, in the protein bodies of dry seed and increased in activity during the time that lectins are broken down in the vacuoles. The enzymes show optimal activity in the range pH 3–5.5. The α-mannosidase had a K m of 0.77 mM for p- nitrophenyl-α- D-mannopyranoside. The β-galactosidase showed a K m of 1.39 mM for p-nitrophenyl-β- D-galactopyranoside. The β- N-acetylhexasominidase had a K m of 0.47 mM for p-nitrophenyl- N-acetyl-β- N-glucosamide and a K m of 0.33 mM for p-nitrophenyl- N-acetyl-β- D-galactosamide. Effects of competitive inhibitors and cations were described.

Kinetic study on the initial stage of the fibrinogen-fibrin conversion by thrombin (III) effects of competitive inhibitors

By using the turbidimetrical procedure, the inhibition constants, K ip , of competitive inhibitors of thrombin were estimated in systems of fibrinogen-thrombin-inhibitor. In the presence of a competitive inhibitor such as benzamidine, p-aminobenzamidine, tosylarginine methyl ester, MD-805, and antithrombin III-heparin, the turbidity change of reaction mixture was traced on the initial stage of the fibrinogen-fibrin conversion catalyzed by thrombin. The formation rate of the smallest polymer as detectable by turbidimetry was measured from evaluating the induction period of turbidity-time curves. The K ip values thus obtained were a little less than or agreed well with the inhibition constants, K i , reported on systems of synthetic substrate-thrombin-inhibitor.

Affinity chromatography on immobilised adenosine 5'-monophosphate. Some kinetic parameters involved in the binding of group-specific enzymes.

The interaction of several enzymes with N6−(6-aminohexyl)-5′-AMP-Sepharose was examined under column chromatographic and batchwise conditions. The dissociation constant (KL) of lactate dehydrogenase and glycerokinase for immobilised 5′-AMP was 1.1 μM when determined under batchwise conditions; similar values were recorded for lactate dehydrogenase in the presence of a competitive inhibitor (NADH). The effect of competitive inhibitors on the binding of alcohol dehydrogenase, glycerokinase and lactate dehydrogenase to N6-(6-aminohexyl)-5′-AMP-Sepharose under column chromatographic conditions was studied. Under these conditions the dissociation constant (KL) of lactate dehydrogenase for the immobilised 5′-AMP was about 0.27 μM, the strength of the interaction (β) decreased with increasing NADH concentrations and a relationship was apparent between the elution volume and inhibitor concentration. These results have been discussed in relation to the principles of affinity chromatography, and are correlated with the known kinetic and molecular properties of the enzymes in free solution.

Kinetics of beta-lactamase inactivation of penicillins. II. Effect of competitive inhibitors.

Experiments were conducted that showed Staphylococcusaureusβ-lactamase activity to be inhibited the most by dicloxacillin, cephaloglycin, and 7-ACA, to a lesser degree by cloxacillin, quinacillin, oxacillin, 6-APA, cephalexin, nafcillin, and ancillin and least of all by methicillin. Bacillus cereus β-lactamase activity was inhibited most by nafcillin and dicloxicillin and only moderately by all the other inhibitors cited, including methicillin, which was least effective. The degree of inhibition was shown to depend on the structural characteristics of the inhibitor, the time of exposure of enzyme to inhibitor, and the ratio of amount of inhibitor to amount of substrate. The inhibition was of the competitive type. The compounds having the highest affinity for the enzyme or the lowest Ki value were the strongest inhibitors and were most effective in protecting the susceptible substrate from hydrolysis.

Effect of competitive inhibitors on the inactivation of d-amino acid oxidase and its protein component by oxygen

Competitive inhibitors of d-amino acid oxidase decrease inactivation of the oxidase by oxygen, but do not protect the protein component of the oxidase against the inactivation. This implies interaction between the inhibitors and oxidase that does not occur between the inhibitors and catalytic protein. The order of effect of the inhibitors on the inactivation of the oxidase and oxidase activity is the same. The results are compatible with the hypothesis that in the formation of the ternary complex of catalytic protein, flavine nucleotide, and inhibitor, the initial, obligatory step is the formation of the oxidase.

The Effect of Competitive Inhibitors on the Milk Clotting Activity of α-Chymotrypsin1

The Effect of Competitive Inhibitors on the Milk Clotting Activity of α-Chymotrypsin¹