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1.
Diamine oxidase follows bi-ter ping-pong kinetics, with an intermediate, "reduced" free-enzyme form being generated after the anaerobic conversion of amine to aldehyde. Visible spectra of diamine oxidase reacting at subzero temperatures provide evidence that this intermediate enzyme form is obtained via several other intermediates and that the environment of the Cu(II) changes dramatically during the course of the reaction [even though it is not reduced to Cu(I) during the catalytic cycle]. The spectrum of this form of diamine oxidase, which is obtained 0.5--2 h after the addition of amine at -5 to -15 degrees C, is independent of substrate, is identical with that obtained by anaerobic addition of substrate at room temperature, and provides evidence for a direct interaction of Cu(II) with the organic cofactor of the enzyme. This interaction is apparently charge transfer in nature. Upon removal of Cu(II) from the native enzyme, one obtains spectral evidence that the organic cofactor is still present. However, removal of the Cu(II) from the reduced (intermediate) enzyme form yields a featureless enzyme spectrum and a Cu(II)--chelate complex which contains a new ligand, which is presumably the second prosthetic group.  相似文献   

2.
1,4-Diamino-2-butyne is a mechanism-based inhibitor of diamine oxidase (EC 1.4.3.6) from pea cotyledons. It shows saturation kinetics Km = 1 mM like a substrate, but its interaction leads to time-dependent loss of enzyme activity which is not restored by gel filtration. The substrate 1,4-diaminobutane and the competitive inhibitor 1,4-diamino-2-butanone protect the enzyme against inactivation. Changes in the enzyme electronic spectra with 1,4-diamino-2-butyne were found. The mechanism of the interaction involves an intermediate aminoallenic compound, which is formed with covalent bound pyrrole in the reaction of the nucleophile with the enzyme. The presence of a pyrrole in the inactivated enzyme was confirmed by reaction with Ehrlich's reagent. The kinetic data obtained in this study indicate that 1,4-diamino-2-butyne is a mechanism-based inactivator with number of turnovers, r = 17 and characteristic constants K' = 0.32 mM and k(in) = 4.89 min-1.  相似文献   

3.
Occurrence of diamine oxidase in the apoplast of pea epicotyls   总被引:4,自引:0,他引:4  
R. Federico  R. Angelini 《Planta》1986,167(2):300-302
Most of the diamine oxidase (EC 1.4.3.6) present in pea (Pisum sativum L. cv. Rondo) epicotyls is found in the fluid obtained by centrifuging pea epicotyl sections previously infiltrated under vacuum with a buffer solution. No detectable amount of the cytoplasmic enzyme glucose-6-phosphate dehydrogenase is present in this fluid, showing that there is very little contamination by cell contents. Polyacrylamide-gel electrophoresis and specific-activity data indicate that diamine oxidase is the most plentiful protein in the extracellular solution obtained from pea epicotyl sections and that an active process is involved in the selective transfer of the enzyme outside the cell. The possible involvement of diamine oxidase in the supply of H2O2 to peroxidase-catalyzed reactions occurring inside the cell wall is discussed.Abbreviations DAO diamine oxidase - Glc6P glucose-6-phosphate  相似文献   

4.
In the oxidation of a homologous series of monodansyldiamines by pea seedling diamine oxidase, monodansylcadaverine was the best substrate. Monodansyldiaminohexane was oxidized at 74% of the rate with monodansylcadaverine, and monodansylputrescine and monodansyldiaminopropane were oxidized only very slowly. The optimum pH for the oxidation of monodansylcadaverine was 8.5, and the Km 2.4 × 10?4 M. Under optimum conditions, putrescine was oxidized eleven times faster than monodansylcadaverine. Oxidation of monodansylcadaverine by diamine oxidase, and the exhaustive dansylation of lysine in equivalent amounts ultimately showed equal fluorescence in the dansyl-5-aminovaleraldehyde formed, indicating stoichiometric conversion to this product in both reactions.  相似文献   

5.
Diamine oxidase (DAO) (EC 1.4.3.6) was purified from pea epicotyls to homogeneity by the criterion of polyacrylamide gel electrophoresis (PAGE). The pu  相似文献   

6.
The level of diamine oxidase in pea seedling stems has been determined as a function of time after germination in both etiolated and non-etiolated plants. The maximum amount of enzyme per plant is obtained between 11 and 13 days. The amount of activity per gram of tissue appears to be proportional to the rate of growth. We describe an efficient method of isolation of pea seedling stem diamine oxidase from 12-day-old etiolated seedlings, a procedure that brings the enzyme to purity after a 97-fold purification. A new assay procedure for pea seedling diamine oxidase is detailed and compared to previously used methods. The kinetic parameters for three common substrates have also been determined. SDS-acrylamide gel electrophoresis, gel filtration chromatography and copper analyses have been used to determine that pea seedling diamine oxidase exists as a dimer of two apparently identical subunits, the dimer molecular weight being about 190,000. The isoelectric point of this enzyme was determined to be 6.5.  相似文献   

7.
Diamine oxidase present in the cotyledons of germinating pea seeds is induced by putrescine, spermidine and ornithine. Auxins inhibit enzyme synthesis in cotyledons only in the presence of embryo. Cycloheximide inhibits the synthesis of the cotyledon enzyme but has no effect on the embryo enzyme. 5-Fluorouracil inhibits the synthesis of both cotyledon and embryo enzymes.  相似文献   

8.
The activity of cotyledon and embryo diamine oxidase was reduced by feeding ethrel and chloroethanol to the seedlings. The inhibitory effect of 2,4-D on the activity of enzyme in the cotyledon which may be mediated through ethylene was reversed by exposure of seeds to red light.  相似文献   

9.
Diamine oxidase was purified separately from cotyledon and embryo of pea seedlings germinated for 6 days. The Km of the cotyledon enzyme for putrescine was 1.6 × 10?4M while that for the embryo enzyme was 9 × 10?5M. On heating for 15 min at 70° the embryo enzyme retained about 90% activity whereas the cotyledon enzyme retained only 20% activity. The electrophoretic mobility of the cotyledon enzyme was ca twice that of the enzyme from embryo.  相似文献   

10.
11.
The effect of the removal of cotyledons on the growth of etiolated pea plants and on the changes in diamine oxidase activity were followed. Plants with removed cotyledons showed higher diamine oxidase activity both in the shoots and in the roots when expressed per fresh weight unit. Higher diamine oxidase activity can be found also in the remaining cotyledon when one of the two cotyledons is removed (when expressed per fresh weight unit and per cotyledon). The plants belonging to those experimental variants in which a higher diamine oxidase activity was established (plants without cotyledons) had simultaneously a high content of its substrates. These plants at the same time contained in the shoots and in the roots smaller amounts of potassium. On the other hand, these findings cannot be applied when comparing individual organs (shoots and roots), as higher diamine oxidase activity can be found in the shoots than in the roots, but the content of its substrates is in the shoots lower. These data indicate that the relationship between the activity of diamine oxidase and the content of its substrates is probably rather complicated. In the connection with the fact that a high diamine oxidase activity can occur in the plant simultaneously with a high content of its substrates, it is possible to assume that (a) changes in the pattern of substrates may occur after the removing of the cotyledons, (b) the synthesis of diamine oxidase may be induced owing to a higher accumulation of substrates, (c) the enzyme may be spacially separated from the substrates in the cells.  相似文献   

12.
As a first step in investigations of the properties of crystalline enzymes, the binding of indole, N-formyl-l-phenylalanine, and N-formyl-l-p-iodophenylalanine to α-chymotrypsin crystals, and the binding of indole to tosyl-α-chymotrypsin crystals, has been studied. The methods used were spectrophotometric measurements of the concentration of indole in the supernatant, or measurements of the concentration of radioactively labeled indole in both the supernatant and the crystal. The dissociation constants of the specific binding site of the crystalline enzyme have been determined for indole and N-formyl-l-phenylalanine. It was found that indole does not bind to tosyl-α-chymotrypsin crystals and that N-formyl-p-iodophenylalanine does not bind to the substrate binding site of the crystalline enzyme.The information obtained from these simple equilibrium measurements is in agreement with X-ray diffraction studies. The approach is, therefore, capable of determining whether or not compounds bind to the active site of a crystalline enzyme, and whether the occupancy of this site is sufficient for structure determinations using X-ray diffraction methods.  相似文献   

13.
Hirasawa E 《Plant physiology》1983,73(4):1062-1064
A reexamination has been made for the supposed regulation of pea (Pisum sativum cv Alaska) cotyledonary diamine oxidase (EC 1.4.3.6) activity by the embryonic axis. When dry cotyledons from which the embryo and testa have been removed surgically are imbibed by soaking in water, there is little increase of the enzyme activity during subsequent incubation on filter paper. However, if the dry cotyledons are imbibed and maintained on filter paper from the first, the increase of the enzyme activity is similar to that in the intact seedling. Thus, rapid imbibition of the isolated dry cotyledons is responsible for repression of enzyme development, and a role for the axis need not be invoked.  相似文献   

14.
The determination of diamine oxidase activity with the ninhydrin reagent was used for monitoring of simultaneous oxidation of two homologous substrates, putrescine and cadaverine, which give different colour products (519 and 417 nm). We measured the reaction rates of oxidation of both substrates in different proportion and compared them with the total reaction rate determined by the guaiacol method. The substrates show competition with inhibition constants of putrescine against cadaverine of 0.14 mmol.l-1 and cadaverine against putrescine of 6.4 mumol.l-1.  相似文献   

15.
16.
Diamine oxidase (histaminase) is produced at very high levels by the decidual cells of the placenta. The presence of diamine oxidase has been demonstrated in human neutrophils. Purification of human placenta diamine oxidase was performed by four subfractionation steps and led to the isolation of one polypeptide whose molecular weight was 84,000, as assessed by SDS PAGE. Using polyclonal antibodies raised against the purified enzyme, we have demonstrated that the neutrophil diamine oxidase is immunochemically identical to the placental diamine oxidase. Development of immunological methods will be useful for detection and quantitation of diamine oxidase in neutrophils during the inflammation process.  相似文献   

17.
A simple colorimetric method for estimation of DAO activity with 4-nitrobenzylamine as a substrate (9,10) was developed. Sensitivity of this method, based on conversion of the aldehyde formed in course of the enzymatic reaction into its 4-nitrophenylhydrazone with subsequent measuring of optical density at 590 nm in strongly alkaline medium, exceeded about 25-fold that of the conventional colorimetric procedure for estimation of DAO activity (14).Sensitivity of the spectrophotometric method for estimation of DAO activity with 4-dimethylaminomethylbenzylamine as a substrate (4) was increased about fivefold by conversion of the aldehyde formed in course of the enzymatic reaction into its 4-nitrophenylhydrazone with subsequent measuring of optical density at 530 nm in strongly alkaline medium.  相似文献   

18.
The association rate constant for the binding of bilirubin to bovine serum albumin has been determined in a continuous-flow experiment. The value obtained is 0.9 x 106m?1S?1. Furthermore the dissociation rate constant is determined from the rate of the peroxidase-catalyzed oxidation of bilirubin in a bilirubin-albumin solution. This figure is 3.1 × 10?2s 1. Calculation of the apparent binding equilibrium constant from the two rate constants gives 2.9 x 107m?1. The above mentioned peroxidase oxidation has also been used for a direct estimation of the binding equilibrium constant giving 2.7 × 107m?1. All experiments are carried out at 36 °C and pH 7.4.  相似文献   

19.
A method for determining individual rate constants for nucleotide binding to and dissociation from membrane bound pig kidney Na,K-ATPase is presented. The method involves determination of the rate of relaxation when Na,K-ATPase in the presence of eosin is mixed with ADP or ATP in a stopped-flow fluorescence apparatus. It is shown that the nucleotide dependence of this rate of relaxation--taken together with measured equilibrium binding values for eosin and ADP--makes possible a reasonably reliable determination of the rate constant for dissociation of nucleotide, i.e., determination of the rate constant k-1 in the following model (where E denotes Na,K-ATPase): [formula: see text] All experiments are carried out at about 4 degrees C in a buffer containing 200 mM sucrose, 10 mM EDTA, 25 mM Tris and 73 mM NaCl (pH 7.4). Values obtained for the rate constants for dissociation are about 6 s-1 for ADP and 2-3 s-1 for ATP.  相似文献   

20.
Agarose crossed affinity electrophoresis (aff-EP) was employed for the determination of lectin-sugar dissociation constants (Ki). In the first dimension of the aff-EP increasing amounts of sugar (alpha-methyl-D-mannoside) were added to a given concentration of lectin (concanavalin A). Then the electrophoresis was run with alpha 1-acid glycoprotein, alpha 1-antitrypsin and alpha-fetoprotein as markers of lectin-sugar interactions. Mathematical equations for determination of the mechanisms and constants of lectin-sugar-glycoprotein interactions were developed. The mean value of the concanavalin A-alpha-methyl-D-mannoside dissociation constant calculated according to the introduced equations was 0.28 mM. In this system it was also possible to determine lectin-glycoprotein dissociation constants (K). The observed influence of the sugar on lectin-glycoprotein binding might be due to hydrophobic interactions since the addition of nonionic detergent caused reversal of this phenomenon.  相似文献   

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