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Purification and partial characterization of lipoxygenase with dual catalytic activities from human term placenta.

ABSTRACT: Lipoxygenase possessing dual catalytic activities, i.e. dioxygenase and hydroperoxidase, was purified from the cytosols of term placentas from non-smoking women. Concanavalin A affinity chromatography followed by phenyl-Sepharose CL-4B chromatography resulted in the separation of one hydrophobic and one non-hydrophobic isoenzyme. The concanavalin A-purified enzyme was used in all subsequent experiments. The dioxygenase activity of the enzyme exhibited a Vmax. of 204.37 +/- 17.66 nmol/min per mg of protein and a Km of 0.79 mM for linoleic acid. The involvement of dioxygen in enzymic linoleic acid oxidation was confirmed by O2 uptake studies. Arachidonic acid and linolenic acid also served as substrates for the dioxygenase activity. The placental lipoxygenase co-oxidized benzidine in the presence of linoleic acid (hydroperoxidase activity). Both the dioxygenase and hydroperoxidase activities were significantly stimulated by Ca2+ (1-100 microM), ATP (10-400 nM) and H2O2 (1-10 nM). Similarly, these two activities were inhibited by nordihydroguaiaretic acid, 5,8,11-eicosatriynoic acid, gossypol, esculetin, butylated hydroxyanisole and butylated hydroxytoluene. Boiled enzyme was without significant dioxygenase and hydroperoxidase activities. Pyrogallol, 3,3'-dimethoxybenzidine, 3,3',5,5'-tetramethylbenzidine, tetramethylphenylenediamine and 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) were also co-oxidized by the placental lipoxygenase. These results suggest that: (i) lipoxygenase from human term placenta exhibits both dioxygenase and hydroperoxidase activities, and (ii) this enzyme represents an important pathway for chemical oxidation in the placentas of non-smoking women.

SUBMITTER: Joseph P

PROVIDER: S-EPMC1134323 | biostudies-other | 1993 Jul

REPOSITORIES: biostudies-other

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Project description:Heparan sulphate (HS) is an important component of the extracellular matrix (ECM) and the vasculature basal lamina (BL) which functions as a barrier to the extravasation of metastatic and inflammatory cells. Platelet-tumour cell aggregation at the capillary endothelium results in activation and degranulation of platelets. Cleavage of HS by endoglycosidase or heparanase activity produced in relatively large amounts by the platelets and the invading cells may assist in the disassembly of the ECM and BL, and thereby facilitate cell migration. Using a recently published rapid, quantitative assay for heparanase activity towards HS [Freeman, C. and Parish, C.R. (1997), Biochem. J., 325, 229-237], human platelet heparanase has now been purified 1700-fold to homogeneity in 19% yield by a five column procedure, which consists of concanavalin A-Sepharose, Zn2+-chelating-Sepharose, Blue A-agarose, octyl-agarose and gel filtration chromatography. The enzyme, which was shown to be an endoglucuronidase that degrades both heparin and HS, has a native molecular mass of 50 kDa when analysed by gel filtration chromatography and by SDS/PAGE. Platelet heparanase degraded porcine mucosal HS in a stepwise fashion from a number average molecular mass of 18.5 to 13, to 8 and finally to 4.5 kDa fragments as determined by gel filtration analysis. Bovine lung heparin was degraded from 8.9 to 4.8 kDa while porcine mucosal heparin was degraded from 8.1 kDa to 3.8 and finally to 2.9 kDa fragments. Studies of the enzyme's substrate specificity using modified heparin analogues showed that substrate cleavage required the presence of carboxyl groups, but O- and N-sulphation were not essential. Inhibition studies demonstrated an absolute requirement for the presence of O-sulphate groups. Platelet heparanase was inhibited by heparin analogues which also inhibited tumour heparanase, suggesting that sulphated polysaccharides which inhibit tumour metastasis may act to prevent both tumour cell and platelet heparanase degradation of endothelial cell surface HS and the basal laminar.

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Purification and partial characterization of lipoxygenase with dual catalytic activities from human term placenta.

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