Cruse I, Maines M D
Department of Biophysics, University of Rochester School of Medicine, New York 14642.
J Biol Chem. 1988 Mar 5;263(7):3348-53.
Recently, we have reported on the presence of two forms of heme oxygenase in rat liver and testis microsomes, referred to as HO-1 and HO-2 (M. D. Maines, G. M. Trakshel, and R. K. Kutty (1986) J. Biol. Chem. 261, 411-419; G. M. Trakshel, R. K. Kutty, and M. D. Maines (1986) J. Biol. Chem. 261, 11131-11137). Although the two forms differed in several biochemical properties, we could not ascertain whether they represented two isozymes or whether they were isoforms of heme oxygenase. In the present study, we provide evidence suggesting that the two forms are isozymes and represent different gene products. We also provide data suggesting that HO-1 is the commonly known heme oxygenase form. The molecular weight and immunochemical properties of HO-1 and HO-2 did not vary depending on the tissue source examined, i.e. liver and testis. Major differences, however, were noted in the amino acid composition of the two forms including the presence of 3 cysteine/cystine residues in HO-2 only. Using antibody to HO-2, four testis clones and two liver clones were isolated, and one liver and one testis clone were sequenced. Both clones revealed a 274-base-pair insert, and the sequence of both inserts was the same. The validity of assignment was confirmed by matching a 14-amino-acid peptide obtained from purified HO-2 with the sequence. Approximately 43% amino acid homology was detected between the HO-2 insert and the published amino acid sequence of heme oxygenase. However, amino acid homology search revealed the presence of two regions of homology: one 22-mer sequence with only one unmatched amino acid, and one 10-mer sequence with one unmatched amino acid. Heme oxygenase appeared to be the HO-1 form, an assignment based on its amino acid sequence matching the sequence of 2 peptides obtained from purified HO-1 and the immunochemical properties of the cobalt-, hematin-, and bromobenzene-induced rat liver enzyme. The secondary structure prediction analysis revealed an area of 100% structural homology with only 72% sequence homology. We predict this region may represent the catalytic site of the enzyme.
最近，我们报道了大鼠肝脏和睾丸微粒体中存在两种形式的血红素加氧酶，分别称为HO - 1和HO - 2（M. D. 梅因斯、G. M. 特拉克斯赫尔和R. K. 库蒂（1986年）《生物化学杂志》261卷，411 - 419页；G. M. 特拉克斯赫尔、R. K. 库蒂和M. D. 梅因斯（1986年）《生物化学杂志》261卷，11131 - 11137页）。尽管这两种形式在若干生化特性上有所不同，但我们无法确定它们是代表两种同工酶还是血红素加氧酶的不同亚型。在本研究中，我们提供的证据表明这两种形式是同工酶，代表不同的基因产物。我们还提供数据表明HO - 1是常见的血红素加氧酶形式。HO - 1和HO - 2的分子量及免疫化学特性并不因所检测的组织来源（即肝脏和睾丸）而异。然而，在这两种形式的氨基酸组成上发现了主要差异，包括仅在HO - 2中存在3个半胱氨酸/胱氨酸残基。利用针对HO - 2的抗体，分离出4个睾丸克隆和2个肝脏克隆，并对1个肝脏克隆和1个睾丸克隆进行了测序。两个克隆均显示有一个274个碱基对的插入片段，且两个插入片段的序列相同。通过将从纯化的HO - 2获得的一个14氨基酸肽段与该序列匹配，证实了归属的有效性。在HO - 2插入片段与已发表的血红素加氧酶氨基酸序列之间检测到约43%的氨基酸同源性。然而，氨基酸同源性搜索显示存在两个同源区域：一个22聚体序列仅有一个不匹配的氨基酸，一个10聚体序列有一个不匹配的氨基酸。血红素加氧酶似乎是HO - 1形式，这一归属基于其氨基酸序列与从纯化的HO - 1获得的两个肽段的序列以及钴、血晶素和溴苯诱导的大鼠肝脏酶的免疫化学特性相匹配。二级结构预测分析显示存在一个结构同源性为100%但序列同源性仅为72%的区域。我们预测该区域可能代表该酶的催化位点。
