Prabhakar P, Laboy J I, Wang J, Budker T, Din Z Z, Chobanian M, Fahien L A
Department of Pharmacology, Department of Pediatrics, University of Wisconsin Medical School, 1330 University Avenue, Madison, Wisconsin, 53706, USA.
Arch Biochem Biophys. 1998 Dec 15;360(2):195-205. doi: 10.1006/abbi.1998.0939.
At pH 7.05 NADH-X prepared by incubating NADH with glyceraldehyde-3-phosphate dehydrogenase (E.C. 1.2.1.12) was a potent noncompetitive inhibitor, with respect to coenzyme, of NADPH oxidation by pure rabbit muscle cytosolic glycerol-3-phosphate dehydrogenase (E.C. 1.1.1.8) and also a potent inhibitor of NADPH oxidation catalyzed by this enzyme in a rat pancreatic islet cytosolic fraction. It was a much less potent inhibitor of NADPH oxidation catalyzed by this enzyme in a rat liver cytosolic fraction and of NADH oxidation catalyzed by this enzyme from all three sources. Glycerol-3-phosphate dehydrogenase purified from muscle cytosol contains tightly bound NADH-X, NAD, and ADP-ribose, each in amounts of about 0.1 mol per mole of enzyme polypeptide chain. A deproteinized supernatant of this enzyme contained these three ligands and produced the same type of inhibition of the enzyme described above for prepared NADH-X with a Ki, in the reaction with NADPH at pH 7.05, in the range of 0.2 microM with respect to the total concentration of ligands ([ADP-ribose] + [NAD] + [NADH-X] = 0. 2 microM). However, only the NADH-X component could account for the potent inhibition because NAD, ADP-ribose, and the primary acid product (which can be produced from NADH-X) each had a Ki considerably higher than 0.2 microM. Although at pH 7.05 NADH-X inhibited NADPH oxidation considerably more than NADH oxidation, the reverse was the case at pH 7.38. Since the enzyme purified from muscle contains tightly bound NADH-X, NADH-X might become attached to the enzyme in vivo where it could play a role in regulating the ratio of NADH to NADPH oxidation of the enzyme.
在pH 7.05条件下，通过将NADH与甘油醛-3-磷酸脱氢酶（E.C. 1.2.1.12）温育制备的NADH-X，对于辅酶而言，是纯兔肌肉胞质甘油醛-3-磷酸脱氢酶（E.C. 1.1.1.8）催化的NADPH氧化的强效非竞争性抑制剂，也是该酶在大鼠胰岛胞质部分中催化的NADPH氧化的强效抑制剂。它对该酶在大鼠肝脏胞质部分中催化的NADPH氧化以及对来自所有三种来源的该酶催化的NADH氧化的抑制作用要弱得多。从肌肉胞质溶胶中纯化的甘油醛-3-磷酸脱氢酶含有紧密结合的NADH-X、NAD和ADP-核糖，每摩尔酶多肽链中每种的含量约为0.1摩尔。该酶的脱蛋白上清液含有这三种配体，并对上述酶产生与制备的NADH-X相同类型的抑制作用，在pH 7.05与NADPH的反应中，相对于配体的总浓度（[ADP-核糖]+[NAD]+[NADH-X]=0.2 microM），其抑制常数（Ki）在0.2 microM范围内。然而，只有NADH-X成分能够解释这种强效抑制作用，因为NAD、ADP-核糖和初级酸性产物（可由NADH-X产生）的抑制常数均远高于0.2 microM。尽管在pH 7.05时NADH-X对NADPH氧化的抑制作用比对NADH氧化强得多，但在pH 7.38时情况则相反。由于从肌肉中纯化的酶含有紧密结合的NADH-X，NADH-X可能在体内附着于该酶，在那里它可能在调节该酶的NADH与NADPH氧化比例中发挥作用。
