Kleczkowski L A, Edwards G E
Department of Botany, Washington State University, Pullman, Washington 99164-4230.
Plant Physiol. 1989 Sep;91(1):278-86. doi: 10.1104/pp.91.1.278.
At least two hydroxypyruvate reductases (HPRs), differing in specificity for NAD(P)H and (presumably) utilizing glyoxylate as a secondary substrate, were identified by fractionation of crude maize leaf extracts with ammonium sulfate. The NADH-preferring enzyme, which most probably represented peroxisomal HPR, was precipitated by 30 to 45% saturated ammonium sulfate, while most of the NADPH-dependent activity was found in a 45 to 60% precipitate. The HPRs had similar low K(m)s for hydroxypyruvate (about 0.1 millimolar), regardless of cofactor, while affinities of glyoxylate reductase (GR) reactions for glyoxylate varied widely (K(m)s of 0.4-12 millimolar) depending on cofactor. At high hydroxypyruvate concentrations, the NADPH-HPR from the 30 to 45% precipitate showed negative cooperativity with respect to this reactant, having a second K(m) of 6 millimolar. In contrast, NADPH-HPR from the 45 to 60% precipitate was inhibited at high hydroxypyruvate concentrations (K(1) of 3 millimolar) and, together with NADPH-GR, had only few, if any, common antigenic determinants with NADH-HPR from the 30 to 45% fraction. Both NADPH-HPR and NADPH-GR activities from the 45 to 60% precipitate were probably carried out by the same enzyme(s), as found by kinetic studies. Following preincubation with NADPH, there was a marked increase (up to sixfold) in activity of NADPH-HPR from either crude or fractionated extracts. Most of this increase could be attributed to an artefact resulting from an interference by endogeneous NADPH-phosphatase, which hydrolyzed NADPH to NADH, the latter being utilized by the NADH-dependent HPR. However, in the presence of 15 millimolar fluoride (phosphatase inhibitor), preincubation with NADPH still resulted in over 60% activation of NADPH-HPR. The NADPH treatment stimulated the V(max) of the reductase but had no effect on its K(m) for hydroxypyruvate. Enzyme distribution studies revealed that both NADH and NADPH-dependent HPR and GR activities were predominantly localized in the bundle sheath compartment. Rates of NADPH-HPR and NADPH-GR in this tissue (over 100 micromoles per hour per milligram of chlorophyll each) are in the upper range of values reported for leaves of C(3) species.
通过用硫酸铵分级分离玉米叶片粗提物，鉴定出至少两种对NAD(P)H特异性不同且（可能）以乙醛酸作为第二底物的羟基丙酮酸还原酶（HPR）。偏好NADH的酶很可能代表过氧化物酶体HPR，在硫酸铵饱和度为30%至45%时沉淀，而大部分依赖NADPH的活性则存在于45%至60%的沉淀中。无论辅酶如何，HPR对羟基丙酮酸的K(m)值都很低（约0.1毫摩尔），而乙醛酸还原酶（GR）反应对乙醛酸的亲和力则因辅酶不同而有很大差异（K(m)值为0.4 - 12毫摩尔）。在高浓度羟基丙酮酸条件下，30%至45%沉淀中的NADPH - HPR对该反应物表现出负协同性，其第二个K(m)值为6毫摩尔。相比之下，45%至60%沉淀中的NADPH - HPR在高浓度羟基丙酮酸时受到抑制（K(i)为3毫摩尔），并且与NADPH - GR一起，与30%至45%组分中的NADH - HPR几乎没有共同的抗原决定簇（如果有的话）。动力学研究发现，45%至60%沉淀中的NADPH - HPR和NADPH - GR活性可能由同一种酶催化。用NADPH预孵育后，粗提物或分级分离提取物中的NADPH - HPR活性显著增加（高达六倍）。这种增加大部分可归因于内源性NADPH磷酸酶干扰导致的假象，该酶将NADPH水解为NADH，后者被依赖NADH的HPR利用。然而，在存在15毫摩尔氟化物（磷酸酶抑制剂）的情况下，用NADPH预孵育仍导致NADPH - HPR激活超过60%。NADPH处理刺激了还原酶的V(max)，但对其对羟基丙酮酸的K(m)没有影响。酶分布研究表明，依赖NADH和NADPH的HPR及GR活性主要定位于维管束鞘区室。该组织中NADPH - HPR和NADPH - GR的活性（每毫克叶绿素每小时超过100微摩尔）处于C3植物叶片报道值的上限范围。
