Cornejo J, Beale S I
Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912.
J Biol Chem. 1988 Aug 25;263(24):11915-21.
Enzymatic heme oxygenase activity has been partially purified from extracts of the unicellular red alga Cyanidium caldarium, and the macromolecular components have been separated into three protein fractions, referred to as Fractions I, II, and III, by serial column chromatography through DEAE-cellulose and Reactive Blue 2-Sepharose. Fraction I is retained by DEAE-cellulose at low salt concentration and eluted by 1 M NaCl. Fraction II is retained by Blue Sepharose at low salt concentration and eluted by 1 M NaCl. Fraction III is retained on 2',5'-ADP-agarose and eluted by 1 mM NADPH, while Fraction II is not retained on ADP-agarose. Fractions I-III, have Mr values of 22,000, 38,000, and 37,000, respectively (all +/- 2,000), as determined by Sephadex gel filtration chromatography. In vitro heme oxygenase activity requires the presence of all three fractions, plus substrate, O2, reduced pyridine nucleotide, and another reductant. Ascorbate, isoascorbate, and phenylenediamine serve equally well as the second reductant, but hydroquinone can also be used, with lower activity resulting. Fractions I-III are heat sensitive and inactive by Pronase digestion. Fraction I has a visible absorption spectrum similar to that of ferredoxin and is bleached by dithionite reduction or incubation with p-hydroxymercuribenzoate. Fraction I can be replaced by commercially available ferredoxin derived from the red alga Porphyra umbilicalis, and to a smaller extent, by spinach ferredoxin. Fraction III contains ferredoxin-linked cytochrome c reductase activity and can be partially replaced by spinach ferredoxin-NADP+ oxidoreductase. Reconstituted heme oxygenase and ferredoxin-linked cytochrome c reductase activities are both abolished if Fraction I or III is preincubated with 0.1 mM p-hydroxymercuribenzoate, but heme oxygenase activity is only slightly affected if Fraction II is preincubated with p-hydroxymercuribenzoate. Preincubation of Fraction II with 0.5 mM diethylpyrocarbonate inactivates heme oxygenase in the reconstituted system, and 10 microM mesohemin partially protects this Fraction against diethylpyrocarbonate inactivation. Algal heme oxygenase is inhibited 80% by 2 microM Sn-protoporphyrin even in the presence of 20 microM mesohemin. Fraction II is rate limiting in unfractionated and reconstituted incubation mixtures. None of the three cell fractions could be replaced by bovine spleen microsomal heme oxygenase or NADPH-cytochrome P450 reductase.
已从单细胞红藻嗜热栖热菌的提取物中部分纯化了酶促血红素加氧酶活性，并通过DEAE - 纤维素和活性蓝2 - 琼脂糖的连续柱色谱法将大分子成分分离为三个蛋白质组分，分别称为组分I、II和III。组分I在低盐浓度下被DEAE - 纤维素保留，并通过1 M NaCl洗脱。组分II在低盐浓度下被蓝色琼脂糖保留，并通过1 M NaCl洗脱。组分III保留在2',5'-ADP - 琼脂糖上，并通过1 mM NADPH洗脱，而组分II不保留在ADP - 琼脂糖上。通过葡聚糖凝胶过滤色谱法测定，组分I - III的Mr值分别为22,000、38,000和37,000（均±2,000）。体外血红素加氧酶活性需要所有三个组分、底物、O2、还原型吡啶核苷酸和另一种还原剂的存在。抗坏血酸、异抗坏血酸和苯二胺作为第二种还原剂同样有效，但对苯二酚也可使用，只是活性较低。组分I - III对热敏感，经链霉蛋白酶消化后无活性。组分I具有与铁氧化还原蛋白相似的可见吸收光谱，经连二亚硫酸盐还原或与对羟基汞苯甲酸孵育后会褪色。组分I可以被来源于紫菜的市售铁氧化还原蛋白替代，在较小程度上也可被菠菜铁氧化还原蛋白替代。组分III含有与铁氧化还原蛋白相关的细胞色素c还原酶活性，并且可以部分被菠菜铁氧化还原蛋白 - NADP +氧化还原酶替代。如果组分I或III与0.1 mM对羟基汞苯甲酸预孵育，则重组的血红素加氧酶和与铁氧化还原蛋白相关的细胞色素c还原酶活性均被消除，但如果组分II与对羟基汞苯甲酸预孵育，血红素加氧酶活性仅受到轻微影响。组分II与0.5 mM焦碳酸二乙酯预孵育会使重组系统中的血红素加氧酶失活，10 microM中血红素可部分保护该组分免受焦碳酸二乙酯失活的影响。即使存在20 microM中血红素，2 microM锡原卟啉也能抑制藻类血红素加氧酶80%。在未分级和重组的孵育混合物中，组分II是限速的。这三个细胞组分均不能被牛脾微粒体血红素加氧酶或NADPH - 细胞色素P450还原酶替代。
