A new method for the detection of small amounts of amino acid oxidase activity is described. The method is based on the inactivation of catalase by 3-amino-1,2,4-triazole in the presence of H20z generated by the amino acid oxidase reaction. The oxidation of 2-5 mpmoles of amino acid is easily detectable. The application of the method to the study of enzyme specificity is discussed. It is shown that D-lySine is a substrate of D-amino acid oxidase and that L-serine and L-threonine are substrates of L-amino acid oxidase.The classic method for the determination of amino acid oxidase activity is the manometric measurement of oxygen consumption (Krebs, 1935). Although more sensitive methods exist for specific substrates such as phenylalanine (Knox and Pitt, 1957;Wellner and Meister, 1960) and hydroxyproline (Corrigan et al., 1963), these are not applicable to all amino acids. The recent observation of Neims and Hellerman (1962) that glycine is slowly oxidized by amino acid oxidase indicated the need for a re-examination of the specificity of amino acid oxidases by the use of an assay procedure capable of detecting very low rates of enzymatic oxidation and applicable to a large number of amino acids. Such a procedure is described. It is shown that some amino acids which were previously thought not to be susceptible to the action of D-and L-amino acid oxidases are oxidized by these enzymes.The procedure is based on the finding of Margoliash and Novogrodsky (1958) that catalase is irreversibly inhibited by hydrogen peroxide in the presence of 3-amino-1,2,4-triazole. Since hydrogen peroxide is a product of the amino acid oxidase reaction, if the reaction is carried out in the presence of catalase and aminotriazole, the amount of amino acid oxidized may be estimated by measuring the inhibition of catalase. It is possible in this way to measure the oxidation of as little as 2-5 mpmoles of amino acid. The great sensitivity attainable is owing to the catalytic amplification of the effect of the product being measured. Since each molecule of catalase is capable of decomposing several million molecules of hydrogen peroxide, the inactivation of a few catalase molecules produces a greatly amplified and easily measurable effect. The principle of catalytic amplification has been utilized previously by Berger et al. (1959) in a sensitive assay for pepsin, using ribonuclease as substrate, in which the inactivation of ribonuclease was used as a measure of pepsin activity. and Meister, 1960). 3-Amino-1,2,4-triazole was obtained from Calbiochem. The amino acids were bought from Mann Research Laboratories, Nutritional Biochemicals Corp., and Schwarz BioResearch. ~-3 , 4 -Dehydroproline was synthesized by the method of Robertson and Witkop (1962). DL-a-Methylphenylalanine was obtained from Merck, Sharp and Dohme.Enzyme Units.-One unit of catalase is defined as the amount of enzyme required to decompose 1 pmole of H202 per minute at p H 7.0 and 25 O when the H202 concentration is 0.01 M. (The catalase activity was determined according to th...
Highly purified preparations of D-amino acid oxidase catalyze the oxidation of L-proline to 1-pyrroline-2-carboxylic acid and of l-3 ,4-dehydroproline to pyrrole-2-carboxylic acid. These reactions are strongly inhibited by benzoate, a known inhibitor of D-amino acid oxidase. The oxidation of l-3 ,4-dehydroproline is inhibited by D-alanine but not by L-alanine. The apparent Km for L-proline is about 0.15 m. L-Proline is oxidized about 3000-4000 times more slowly than D-proline.
The haemostatic components of venom from the genus Porthidium has been poorly studied, although it is known that severe manifestations occur when humans are envenomed, which include invasive oedema and disseminated ecchymosis. The effects of venom on blood platelets are commonly studied and are normally carried out with platelet-rich plasma (PRP). A series of crude venom dilutions was used to determine the effects of adenosine diphosphate (2 microM) and adrenaline (11 microM) induced platelet aggregation. Venom of Porthidium lansbergii hutmanni was fractioned by anionic exchange chromatography, and the fractions were also used to determine the 50% inhibition of adenosine diphosphate (ADP) and adrenaline-induced platelet aggregating dose (AD50). Crude venom has more effect in inhibiting adrenaline-induced aggregation (AD50 = 0.0043 microg) followed by the adenosine diphosphate (AD50 = 17 microg). Peaks I and II obtained by chromatography also inhibited adrenaline-induced platelet aggregation with an AD50 of 3.2 and 0.013 microg, respectively, and both peaks inhibited ADP-induced platelet aggregation with an AD50 of 10 microg. The main purpose of this work was to characterise the in vitro effects caused by P. lansbergii hutmanni crude venom and its fractions on the platelet aggregation mediated by adrenaline and ADP agonists.
The Porthidium genus is represented by the P. lansbergii rozei and P. lansbergii hutmanni (Plh) subspecies in Venezuela. The venom components of these have been little studied, probably due to the low incidence of reported accidents, although acute and serious local effects such as invasive edema and disseminated ecchymosis are present during human envenonation. The aim of this work was to characterize the in vitro effects of crude P. l. hutmanni venom, and its fractions, on platelet aggregation triggered by two physiologic agonists: thrombin and collagen. The effects of thrombin and collagen were observed on a platelet-rich plasma (PRP) solution (3 x 10(5) platelets/microL) using serial dilutions of P. l. hutmanni venom (0.625-40 microg). The crude venom was fractionated by anionic exchange chromatography and two peaks obtained. Crude venom and both fractions were highly inhibitory on platelet aggregation mediated by the two agonists. The anti-aggregating dose (AD(50)) for both agonists was determined. PRP collagen-triggered aggregation was most inhibited by the crude venom (AD(50) = 0.67 microg) when compared with PRP thrombin-triggered aggregation (AD(50) = 4.92 microg). Collagen-induced aggregation was more intensely inhibited by venom than thrombin-induced aggregation. In conclusion, to specify the inhibition mechanisms involved for each of the active components in the venom from these subspecies, we must characterize and purify the inhibitors of aggregation from P. l. hutmanni venom, with the purpose of suggesting new pharmacological substances to be incorporated into the therapeutic arsenal to treat hemostatic pathologies related to high levels of platelet aggregation.
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