Sutyak J, Austen K F, Soberman R J
Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115.
J Biol Chem. 1989 Sep 5;264(25):14818-23.
We have previously reported that cytochrome P-450LTB in the microsomes of human polymorphonuclear leukocytes (PMN) catalyzes three omega-oxidations of leukotriene B4 (LTB4), leading to the sequential formation of 20-OH-LTB4, 20-CHO-LTB4, and 20-COOH-LTB4 (Soberman, R.J., Sutyak, J.P., Okita, R.T., Wendelborn, D.F., Roberts, L.J., II, and Austen, K. F. (1988) J. Biol. Chem. 263, 7996-8002). The identification of the novel final intermediate, 20-CHO-LTB4, allowed direct analysis of its metabolism by PMN microsomes in the presence of adenine nucleotide cofactors. Microsomes in the presence of 100 microM NAD+ or 100 microM NADP+ converted 1.0 microM 20-CHO-LTB4 to 20-COOH-LTB4 with a Km of 2.4 +/- 0.8 microM (mean +/- S.E., n = 4) and a Vmax of 813.9 +/- 136.6 pmol.min-1.mg-1, for NAD+, as compared to 0.12 microM and 5.0 pmol.min-1.mg-1 (n = 2) for NADPH as a cofactor. The conversion of 1.0 microM of 20-CHO-LTB4 to 20-COOH-LTB4 in the presence of saturating concentrations (1.0 mM) of both NAD+ and NADP+ was not greater than the reaction in the presence of 1.0 mM of each cofactor separately, indicating that NAD+ and NADP+ were cofactors for the same enzyme. Antibody to cytochrome P-450 reductase did not inhibit the conversion of 20-CHO-LTB4 to 20-COOH-LTB4. When 1.0 microM 20-OH-LTB4 was added to microsomes in the presence of NADPH, approximately three-fourths of the product formed (63.7 +/- 5.1 pmol; mean +/- S.E., n = 3) was 20-CHO-LTB4 and approximately one-fourth (21.3 +/- 3.9 pmol; mean +/- S.E., n = 3) was 20-COOH-LTB4. In the presence of both NADPH and NAD+, only 20-COOH-LTB4 (85.5 +/- 9.9 pmol; mean +/- S.E., n = 3) was formed. PMN microsomes also contain an NADH-dependent aldehyde reductase which converts 20-CHO-LTB4 to 20-OH-LTB4, a member of the LTB4 family of molecules with biological activity. Based upon kinetic, cofactor and inhibition data, microsomal aldehyde dehydrogenase preferentially regulates the final and irreversible inactivation step in the LTB4 metabolic sequence.
我们之前报道过，人多形核白细胞（PMN）微粒体中的细胞色素P - 450LTB催化白三烯B4（LTB4）的三次ω-氧化，导致依次形成20 - OH - LTB4、20 - CHO - LTB4和20 - COOH - LTB4（索伯曼，R.J.，苏蒂亚克，J.P.，奥基塔，R.T.，温德尔伯恩，D.F.，罗伯茨，L.J.，二世，以及奥斯汀，K.F.（1988年）《生物化学杂志》263卷，7996 - 8002页）。新型最终中间体20 - CHO - LTB4的鉴定使得在腺嘌呤核苷酸辅因子存在的情况下，能够直接分析其被PMN微粒体代谢的情况。在100微摩尔/升NAD⁺或100微摩尔/升NADP⁺存在下，微粒体将1.0微摩尔20 - CHO - LTB4转化为20 - COOH - LTB4，对于NAD⁺，Km为2.4±0.8微摩尔（平均值±标准误，n = 4），Vmax为813.9±136.6皮摩尔·分钟⁻¹·毫克⁻¹，而对于作为辅因子的NADPH，Km为0.12微摩尔，Vmax为5.0皮摩尔·分钟⁻¹·毫克⁻¹（n = 2）。在饱和浓度（1.0毫摩尔/升）的NAD⁺和NADP⁺同时存在的情况下，将1.0微摩尔20 - CHO - LTB4转化为20 - COOH - LTB4的反应并不比单独存在1.0毫摩尔/升每种辅因子时的反应更大，这表明NAD⁺和NADP⁺是同一种酶的辅因子。细胞色素P - 450还原酶抗体并不抑制20 - CHO - LTB4向20 - COOH - LTB4的转化。当在NADPH存在的情况下向微粒体中加入1.0微摩尔20 - OH - LTB4时，形成的产物中大约四分之三（63.7±5.1皮摩尔；平均值±标准误，n = 3）是20 - CHO - LTB4，大约四分之一（21.3±3.9皮摩尔；平均值±标准误，n = 3）是20 - COOH - LTB4。在NADPH和NAD⁺同时存在的情况下，仅形成了20 - COOH - LTB4（85.5±9.9皮摩尔；平均值±标准误，n = 3）。PMN微粒体还含有一种NADH依赖性醛还原酶，它将20 - CHO - LTB4转化为20 - OH - LTB4，20 - OH - LTB4是具有生物活性的LTB4分子家族的一员。基于动力学、辅因子和抑制数据，微粒体醛脱氢酶优先调节LTB4代谢序列中的最终且不可逆的失活步骤。
