Purification and characterization of recombinant histidine‐tagged human platelet 12‐lipoxygenase expressed in a baculovirus/insect cell system

Abstract

A baculoviral expression vector consisting of a sequence encoding a six-histidine tag apposed to the human platelet 12-lipoxygenase cDNA, under control of the polyhedrin promoter, was constructed. Recombinant 12-lipoxygenase baculoviruses were used to infect Spodoptera frugiperda insect cells (Sf9). At 54 h post-infection, maximal 1Zlipoxygenase activity and protein levels were achieved ; the enzyme was purified to apparent homogeneity in a single step by nickel-ion-chelation chromatography in which the (His),-tagged 12-lipoxygenase was eluted with 100 mM imidazole. The purified enzyme metabolized arachidonic acid almost exclusively to 12-hydroperoxyeicosatetraenoic acid with little, if any, epoxyalcohol or reduction products and had a V,,, of 2-4 pmol min-' mg protein-', K , of 10 pM and k,,, of =250 min-I. Linoleic acid, on the other hand, was converted to (13S)-13-hydroperoxy-octadecadienoic acid at a rate which was about 2% of that obtained with arachidonic acid as substrate, but displayed the same K,. The enzyme was most active between pH 7.5 -8 and activity was stimulated significantly in the presence of 0.006% Tween-20. A polyclonal antibody to the recombinant enzyme was generated and found to recognize a single 75-kDa band in platelets, human erythroleukemia cells and 12-lipoxygenase baculoviral-infected Sf9 cells by immunoblot and immunoprecipitation methods. 12-Lipoxygenase protein represented 0.1 % of the total soluble protein in platelet preparations. In immunofluorescence experiments 12-lipoxygenase was observed in the cytoplasm of infected insect cells and in the human megakaryoblastic DAMI cell line. The isolation of large quantities of pure human platelet 12-lipoxygenase should facilitate detailed biochemical structure/function studies.Arachidonate 12-lipoxygenase catalyzes the insertion of molecular oxygen stereospecifically onto C-12 of arachidonic acid to yield (1 2S)-12-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE) [l-31. This was the first described mammalian lipoxygenase, found in platelets of different species [l, 31. More recently, the cDNA encoding human platelet 12-lipoxygenase has been isolated [4-61. The enzyme consists of 663 amino acids with a molecular mass of 75 kDa. Human and bovine platelet 12-lipoxygenases differ distinctly from the 12-lipoxygenases found in porcine and bovine leukocytes by several biochemical and immunological criteria [7, 81. The porcine leukocyte enzyme has been purified and characterized extensively [9]. This enzyme is 65% identical to the human platelet enzyme based on the deduced primary structure [4][5][6] 101. Although the human Correspondence to C.

Oxidative metabolism of lipoamino acids and vanilloids by lipoxygenases and cyclooxygenases

“…The kinetic parameters determined for each enzyme with arachidonic acid were in agreement with values reported in the literature [2,[35][36][37]. Notably pl12-LO exhibited very similar kinetic parameters for the lipaomino acids and arachidonate.…”

Differential characteristics of human 15‐lipoxygenase isozymes and a novel splice variant of 15S‐lipoxygenase

“…For example, 12-HETE and 15-HETE have been implicated in tumour metastases and the formation of atherosclerotic plaques, respectively [11,12]. The important biological roles of these LO metabolites and the complexity of substrate utilization by members of the LO family emphasize the importance of understanding factors which affect the activity and regulation of these enzymes.Overexpression and characterization of recombinant 5-LO, 12-LO and 15-LOa have been carried out allowing kinetic analysis of these enzymes [9,13,14] and the hypotheses as to the mechanism by which LOs oxygenate fatty acids have been developed [15]. In vitro, both 5-LO and 15-LOa undergo a burst phase of activity before reaching a steady rate of product formation and then undergoing suicide inactivation by their respective enzyme products [9,14].…”

“…Overexpression and characterization of recombinant 5-LO, 12-LO and 15-LOa have been carried out allowing kinetic analysis of these enzymes [9,13,14] and the hypotheses as to the mechanism by which LOs oxygenate fatty acids have been developed [15]. In vitro, both 5-LO and 15-LOa undergo a burst phase of activity before reaching a steady rate of product formation and then undergoing suicide inactivation by their respective enzyme products [9,14].…”

Lipoxygenase activity in heart cells

“…An optimal pH of 6.8 was found for the showed a pH optimum of 6.8-7.3, which is similar to the microsomal activity, 7.0-7.2 for the AS pellet, and 7.3 for optimum of 6.8-7.2 obtained for the enzyme in human leuko-LOX from cultured rat cardiomyocytes, cytes [21]. In human platelets, the pH optimum was in the In order to further characterize LOX activity, several range of 7.5-8.0 [22], and at the range of 7.0-8.0 in porcine known inhibitors of eicosanoid metabolism were investigated, epidermis [23]. The results are summarized in Table 2.…”