Early plant defense response is characterized by elevation of activity of peroxidases and enhanced insolubilization of hydroxyproline-rich glycoproteins, such as extensin, i n the cell wall.The insolubilization process (cross-linking between soluble extensin precursor molecules) is catalyzed by extensin peroxidases. We have ionically eluted extensin peroxidases from intact water-washed suspension-cultured tomato (hybrid of Lycopersicon esculenfum Mill.and Lycopersicon peruvianum L. [Mill.]) cells and purified them to homogeneity by molecular sieve and cation-exchange chromatography. Four ionic forms of peroxidase (PI, PII, EPIII, and EPIV) were resolved; only the latter two cross-linked tomato soluble extensin. The molecular weight (34,000-37,000), amino acid composition, and isoelectric point (9.0) of the extensin peroxidases were determined. Substrate specificities of the enzymes were investigated: soluble extensin and potato lectin (a hydroxyproline-rich glycoprotein with a domain that strongly resembles extensin) were crosslinked by only two forms of the enzyme, whereas bovine serum albumin, aldolase, insulin, a number of other marker proteins, and proteins eluted from tomato cells (except extensin) could not be cross-linked. We have also isolated a yeast elicitor that enhances total peroxidase activity and extensin insolubilization within 1 h of challenge i n cultured cells of tomato. A highly sensitive enzymelinked immunosorbent assay technique using polyclonal antiserum raised against soluble tomato extensin was used to demonstrate extensin insolubilization in vivo. A tomato cell-wall peroxidase that cross-links extensin has been purified and may have a role in plant defense.
Fibrolase, a fibrinolytic enzyme isolated from Agkistrodon c. contortrix (southern copperhead) venom, solubilizes fibrin primarily by rapid hydrolysis of the alpha and beta chains. Fibrolase is also an Aα, Bβ fibrinogenase. The breakdown products of fibrin and fibrinogen following incubation with fibrolase were different from those observed with plasmin. This enzyme is a metalloprotease that was inhibited by ethylenediaminetetraacetic acid. Fibrolase was inhibited by dithiothreitol, suggesting that disulfide bonds are important for catalytic activity. It was also inhibited by α2-macroglobulin, but not by the soybean or lima bean trypsin inhibitors, diisopropylfluorophosphate, or p-hydroxymercuribenzoate. Unlike thrombolytic agents such as streptokinase, fibrolase does not activate plasminogen as evidenced by the use of plasmin-specific chromogenic substrate S-2251 and by sodium do-decyl sulfate-polyacrylamide gel electrophoresis.
Egham, Surrey TW20 OEX.Plantperoxidases(EC 1.11.1.7)areinvolvedinamyriadofreactionssuch as lignin deposition. They may have anti-micmbii activity and are believed to Huysw (1) purifwd an anionic and a calionic peroxidase from peanut cells in suspensioo culture; they argued that anionic peroxidases are associated with lignin biosynthesis and cationic pxidases are responsible for IAA oxidation. despite the evidence that these anionic and cationic peroxidases have apparently identical catalytic properties with regard to oxidation of eugenol. aminoantipyrene , guaiacol and IAA. The. role of the microenvironment in vivo u p peroxidase activity is believed to be important in establishing evasive action of the plant to pathogenesis. The actual function of the maprity of peroxidases in the plant remains unclear. This is potentially a consequenwoftheabsenceof localization SUdiesconductedon specific peroxidase isozymes and the lack of informruion on substrate specifEities with respect to endogenous substrates. Pemxidases are thought to have a dual role during h e hypemsitive reaction. being implicated in the generation of free radicals and also smctural alterations in the plant cell wall.be. involved in the production and deloxificaIi0n of peroxides. chibbar and van . --j* Eiutloo volume (ml) Fig. 1. Mono S FPLC chromatogram of 'extensin peroxidase'Our pmtocol allows us the purify from suspensioncultured tomato cells both 'extensin peroxidase' and extensin, its perceived natural substrate.. Both extensin and extensin peroxidase are. ionicallyeluted from intxt water-washed m a t o cells (2). These were further purified by CMcellulose cationexchange and Sephacryl S-2OOgel filmtion chromatography (2). 'Ile extensin was found to be homogeneous. 'Extensin peroxidase' was further purified to homogeneity by CM-cellulose cationexchange chromatography and Mono S cationexchange FPLC (Fig. 1).In vuro extensin crosslinking was monitored by Superose-12 FPLC. Two ionic forms of p e r o x i h (Type 111 and IV) punfied by Mono S FPLC crosslinked extensin. The mokcular weights of these 'extensin peroxidases' were found to be approximately 32kDa and hey both have an isoeleceic point above pH 9.0. The amino acid profiles of the two ionic forms were similar. They lacked cysteine (no detectable cysteic acid was monitored after acid hydrolysis in 6N HCI at 110°C for 24 hour). Consequently. these two forms may be regarded as isoforms of 'extensin peroxidase'. Substrate. specificity of the two enzymes demonstrate that only soluble tomato extensin and potato lectin (an HRGP) can be crosslinked w h e w BSA, a l d o k , insulin and a number of other marker proteins and proteins eluted from tomato cells (except extensin) could nol be crosslinked. Other organic substrates were also tested relative to horseradish peroxidases ( Table 1).Hwseradish peroxidases were chosen because these are the only plant peroxidases that can be commercially-pmhased. Compared ID HRP isozymes, 'extensin peroxidase' IV appeared to have a strong aftiiity for syringaldaziie. ...
Fibrolase, a direct-acting fibrinolytic enzyme has been shown to cleave primarily the Aα and Bβ chains of human fibrin. We have previously reported that fibrolase also exhibits fibrinogenolytic activity and acts mainly as an α-chain fibrinogenase. In contrast to the action of streptokinase (plasminogen activator), fibrolase does not activate plasminogen. In vitro thrombolytic efficacy of fibrolase was determined by monitoring the release of radiolabel from iodinated fibrin and human blood clots. Fibrolase effectively digested the clots in a dose-dependent manner. The in vivo efficacy of fibrolase was evaluated in an animal model of arterial thrombosis. Fibrolase was found to be efficacious at dissolving femoral arterial clots following a single intravenous bolus administration. Time to reperfusion was dose dependent and similar to that observed with streptokinase. No adverse effects on blood pressure and heart rate were observed.
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