Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.17.3.2 (xanthine oxidase)
 8,383 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two types of complexes are formed during the interaction of xanthine oxidase with p-chloromercurybenzoate (pCMB). The reversible inactive complex (presumably of absorption nature) is formed practically instantaneously and competitively with regard to the substrate (Ki=6,2 . 10(-8) M) in 0,05 M phosphate buffer (pH 7,8, 25 degrees) and does not involve the fast-reacting SH-groups of the enzyme. Reactivation of xanthine oxidase is observed during prolonged incubation of the inactive complex at 0 degrees; it is associated with the interaction between pCMB and the fact-reacting SH-groups. This interaction results in a dissociation of the inactive complex. The blocking of the slow-reacting SH-groups is accompanied by an irreversible loss of the xanthine oxidase activity. The enzyme modification by blocking of 10 fast-reacting SH-groups does not involve the Fe-S clusters, but results in local changes in the enzyme conformation. This is manifested in a 2-fold increase of Km and the rate constants of proteolysis of the modified xanthine oxidase as compared to the native enzyme. The rate constants of proteolysis by trypsin for the native and modified enzymes in 0,05 M phosphate buffer (pH 7,8; 37 degrees) are 3,7 . 10(-3) min-1 and 7,0 . 10(-3) min-1, respectively; those for chymotrypsin in the same buffer (30 degrees) are 1,5 . 10(-2) min-1 and 6,0 . 10(-2) min-1, respectively.
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PMID:[Mechanism of interaction between milk xanthine oxidase and p-chloromercuribenzoate. Properties of the purified enzyme]. 49 85

A comprehensive study on enzyme ligand interactions by QSAR techniques is discussed. Thirteen correlation equations are presented which relate activity of 1086 ligands of isolated chloroplasts, chymotrypsin, dihydrofolate reductase, xanthine oxidase and guanine deaminase to their chemical structures. Two kinds of space within and on the surface of an enzyme are defined by means of pi and MR constants. Emphasis is put on the use of indicator variables as a means of rationalizing special enzyme-ligand interactions. The use of such studies for drug development is discussed.
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PMID:[Correlative analysis in the study of enzyme-ligand interactions]. 73 55

Bovine milk xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2) has been purified by a modified method without the use of proteases, and its structure has been analyzed by polyacrylamide gel electrophoresis. Native xanthine oxidase is found to consist of only two polypeptide chains A with molecular weights of 150 000 each. These chains have NH2-terminal methionine. Limited proteolysis with trypsin, chymotrypsin, or subtilisin at pH 8 did not affect molecular weight and activities of the enzyme while each of the A chains was cleaved under these conditions to three fragments C, E, and F with molecular weights of 92 00, 42 000 and 20 000, respectively. These fragments remained bound to each other and were relatively resistant to subsequent proteolysis. The isolation of xanthine oxidase in the presence of pancreatin as described by Hart et al. (1970, Biochem. J. 116, 851) gives partially digested enzyme composed mainly of chains C, E (Mr 35 000) and a small component (Mr approx. 15 0-0). The action of subtilisin on xanthine oxidase at pH 11 resulted in complete digestion of E chains, FAD separation, and total loss of xanthine:oxygen oxidoreductase activity while xanthine:indophenol oxidoreductase activity was relatively little affected. The residual enzyme has a molecular weight of about 200 000, is composed mainly of two C chains (and may probably contain F and/or proteolytic fragments of low molecular weight), contains molybdenum, and does not contain FAD.
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PMID:Subunit structure of bovine milk xanthine oxidase. Effect of limited cleavage by proteolytic enzymes on activity and structure. 126 10

Activated human neutrophils (PMN) degrade rTNF-alpha resulting in a loss of cytotoxic activity against murine L-929 cells (L cells). This inactivation is mediated through proteases released from activated PMN. Exposure of TNF to H2O2, glucose oxidase, xanthine oxidase, or myeloper-oxidase-H2O2-halide did not affect TNF cytotoxicity for L cells. Exposure to trypsin, chymotrypsin, pronase E, or elastase, however, did diminish TNF bioactivity. FMLP-stimulated PMN in the presence, but not in the absence, of cytochalasin B reduced TNF activity, whereas PMA-stimulated PMN did not affect TNF. Stimulation of PMN with opsonized bacteria also induced TNF inactivation as well as the supernatant of FMLP-stimulated cells. Addition of protease inhibitors to the FMLP-stimulated cytochalasin B-treated PMN abrogated the inactivation of TNF cytotoxicity for L cells, whereas scavengers were not protective. In addition, PMN from a chronic granulomatous disease patient also decreased TNF bioactivity. Inactivation of TNF by activated PMN correlated with granule release and not with superoxide production. Exposure of TNF to proteases and FMLP-activated PMN also resulted in a loss of reactivity with anti-TNF antibodies, as measured by ELISA, and in the formation of an approximately 10-kDa split product from the 17-kDa rTNF molecule. Partial degradation of TNF by proteases released from activated PMN may result in a diminished TNF bioactivity and thereby contribute to the regulation of local inflammatory reactions.
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PMID:Inactivation of recombinant human tumor necrosis factor-alpha by proteolytic enzymes released from stimulated human neutrophils. 194 Mar 72

The ability of various reactive oxygen species and serine proteases to activate latent collagenase (matrix metalloproteinase-1) purified from human neutrophils was examined. Latent 70-75 kD human neutrophil collagenase (HNC) was efficiently activated by known non-proteolytic activators phenylmercuric chloride (an organomercurial compound) and gold thioglucose (Au(I)-salt). Corresponding degree of activation was achieved by reactive oxygen species including hypochlorous acid (HOCl), hydrogen peroxide (H2O2) and hydroxyl radical generated by hypoxanthine/xanthine oxidase (HX/XAO). The presence of trace amounts of iron and EDTA were necessary and even enhanced H2O2 induced activation of latent HNC. This activation could be abolished by an iron chelator desferrioxamine and a hydroxyl radical scavenger mannitol. HOCl induced activation of latent HNC was not affected by desferrioxamine and mannitol. Thus, these compounds do not inhibit the active/activated form of HNC. Latent HNC could also be activated by trypsin and chymotrypsin but not by plasmin and plasma kallikrein. The ability of mannitol and desferrioxamine to inhibit the H2O2-induced activation of HNC suggests the transition metal dependent Fenton reaction to be responsible for localized and/or site-specific generation of hydroxyl radical/hydroxyl radical -like oxidants to act as the activating oxygen species. Our results support the ability of myeloperoxidase derived HOCl to act as a direct oxidative activator of HNC and further suggest the existence of a new/alternative oxidative activation pathway of HNC involving hydroxyl radical.
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PMID:Activation of latent human neutrophil collagenase by reactive oxygen species and serine proteases. 217 13

Superoxide anion (O2-) generated from xanthine oxidase/xanthine has been used to decrease the half life of endothelium derived relaxing factor (EDRF). However, by itself, xanthine oxidase causes endothelium dependent relaxation. This relaxation is unrelated to the oxidative property of the enzyme since it is not inhibited by allopurinol. In addition, the relaxation is not inhibited by the cyclooxygenase inhibitor, indomethacin, or the phospholipase A2 inhibitor, p-bromophenacyl bromide. On the other hand the relaxation is inhibited by the trypsin inhibitor (TI) from chicken egg white. A similar endothelium dependent relaxation elicited by pancreatin and trypsin is also inhibited by TI. Pancreatin used in the preparation of xanthine oxidase contains trypsin, chymotrypsin and carboxypeptidase. When compared to trypsin both chymotrypsin and carboxypeptidase elicit little relaxation. Thus the endothelium dependent relaxation elicited by xanthine oxidase is likely due to contamination with trypsin. Our results emphasize that when the superoxide generating system, xanthine oxidase/xanthine is used to study the effect of oxygen radicals on EDRF, it is advantageous to ensure that only purified preparations of xanthine oxidase are used.
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PMID:Xanthine oxidase and endothelium dependent relaxation. 282 Apr 11

The cationic proteins from neutrophil lysosomes have been shown to modulate phagocytic activity of granulocytes. The present study reports the effects of the cationic protein fractions on the generation of O2- by human PMNs during phagocytosis. Human PMNs were reacted with different phagocytic stimuli in the presence and absence of lysosomal cationic proteins and the amount of O2- generated was determined by superoxide dismutase inhibitable reduction of cytochrome c. Total cationic protein extract from neutrophil lysosomes enhanced O2- generated by PMNs during the phagocytosis of IgG-coated latex beads and opsonized zymosan particles. The analysis of the fractions of cationic proteins obtained from a Sephadex G-75 column showed that the O2- generation-enhancing activity was associated with the proteins eluted in fractions III and IV. A protein fraction mainly eluted in void volume inhibited the cytochrome c reduction by O2- formed during phagocytosis. This was due to the presence of superoxide dismutase-like activity since O2- generated by the xanthine-xanthine oxidase system was also inhibited by this fraction. The cationic protein fractions III and IV from the Sephadex G-75 column were further subfractionated. Although the O2(-)-enhancing activity was eluted in the same fractions as chymotrypsin activity, there was no quantitative correlation between the amount of O2- generation and chymotrypsin activity. Moreover, commercial chymotrypsin did not enhance O2- generation. Electrophoretic analysis of the isolated protein fractions suggests that O2- generation enhancing protein (SGEP) is different from lysozyme or chymotrypsin and probably represents previously undescribed protein.
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PMID:Influence of neutrophil cationic proteins on generation of superoxide by human polymorphonuclear cells during phagocytosis. 303 79

Xanthine oxidase (EC 1.2.3.2) was purified from fresh cows' milk by differential centrifugation and hydroxylapatite chromatography in the absence of reducing agents and proteases. The purified isolate possessed an absorbance at 280 nm:absorbance at 450 nm ratio of 4.84; an absorbance (1 cm at 280 nm 1%) of 11.9; an activity:absorbance at 450 nm of 141, a specific activity of 3.59 units/mg; and detectable dehydrogenase activity. The enzyme preparation was obtained in a reversible oxidase form that could be partially converted to xanthine dehydrogenase in the presence of 10mM dithiothreitol or 1% mercaptoethanol. Amino acid analyses revealed that the enzyme was hydrophobic in nature and that lysine constituted its N-terminal residue. The protein contained 22 disulfide and 38 sulfhydryl groups, four of which were detectable in the undenatured protein complex. Discontinuous PAGE in the presence of selected dissociation agents did not result in further resolution. Sodium dodecyl sulfate-PAGE of the purified enzyme revealed a sharp zone with a molecular weight of 151,000 +/- 4000 (i.e., monomer). The purified enzyme exhibited oxidase activity in the presence of 6 M urea and following limited proteolysis by trypsin, chymotrypsin, plasmin, pancreatin, pepsin, and papain. Proteolyzed xanthine oxidase migrated as a single zone in polyacrylamide gels in the presence and absence of dissociating agents such as 1% mercaptoethanol and 6 M urea. Restricted digestion of xanthine oxidase by proteases was indicated by the presence of three major zones with molecular weights ranging from 85,000 to 100,000, 30,000 to 35,000, and 18,000 to 20,000 commonly observed in SDS gels. Amino acid profiles of the principal peptidyl fragments of trypsin-cleaved xanthine oxidase indicated their hydrophobic nature and lysine as the N-terminal residue for all fragments.
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PMID:Characteristics of purified cows' milk xanthine oxidase and its submolecular characteristics. 339 6

1. The xanthine oxidase of cow's milk, crude or purified, appears as an oxidase (type O), and can be converted almost completely into a NAD(+)-dependent dehydrogenase (type D) by treatment with dithioerythritol or dihydrolipoic acid, but only to a small extent by other thiols. 2. The D form of the enzyme is inhibited by NADH, which competes with NAD(+). 3. The kinetic constants of the two forms of the enzyme are similar to those of the corresponding forms of rat liver xanthine oxidase. 4. Milk xanthine oxidase is converted into an irreversible O form by pretreatment with chymotrypsin, papain or subtilisin, but only partially with trypsin. 5. The enzyme as purified shows a major faster band and a minor slower band on gel electrophoresis. The slower band is greatly reinforced after xanthine oxidase is converted into the irreversible O form by chymotrypsin.
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PMID:Milk xanthine oxidase type D (dehydrogenase) and type O (oxidase). Purification, interconversion and some properties. 435 4

1. Two major forms of xanthine oxidase are demonstrated for the mouse. On polyacrylamide-gel electrophoresis the duodenal form migrates faster towards the anode than that of the liver. Both forms also differ in their (NH(4))(2)SO(4) precipitation patterns and sucrose-density-gradient molecular-weight determinations. 2. The liver form is fully converted into the duodenal form by incubation at 37 degrees C with 2.5mg of crude trypsin/ml for 1(1/2)h, without loss of activity. The trypsin-treated liver form behaves like the normal duodenal form as characterized by electrophoresis, (NH(4))(2)SO(4) precipitation patterns, and sucrose-density-gradient molecular-weight determinations. 3. Partial conversion is also brought about by purified trypsin and chymotrypsin, but not with beta-carboxypeptidase or lipase. The conversion is inhibited by soya-bean trypsin inhibitor. 4. In embryo mice the duodenal form is similar to the liver form on electrophoresis. 5. These studies indicate, as might be expected, that the duodenal form is a modified version of the liver enzyme, probably caused by proteolytic alteration.
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PMID:Characterization of alternative molecular forms of xanthine oxidase in the mouse. 472 35


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