Zelewski M, Swierczyński J
Department of Biochemistry, Academic Medical School, Gdańsk, Poland.
Eur J Biochem. 1991 Oct 15;201(2):339-45. doi: 10.1111/j.1432-1033.1991.tb16291.x.
In human liver, almost 90% of malic enzyme activity is located within the extramitochondrial compartment, and only approximately 10% in the mitochondrial fraction. Extramitochondrial malic enzyme has been isolated from the post-mitochondrial supernatant of human liver by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose, ADP-Sepharose-4B and Sephacryl S-300 to apparent homogeneity, as judged from polyacrylamide gel electrophoresis. The specific activity of the purified enzyme was 56 mumol.min-1.mg protein-1, which corresponds to about 10,000-fold purification. The molecular mass of the native enzyme determined by gel filtration is 251 kDa. SDS/polyacrylamide gel electrophoresis showed one polypeptide band of molecular mass 63 kDa. Thus, it appears that the native protein is a tetramer composed of identical-molecular-mass subunits. The isoelectric point of the isolated enzyme was 5.65. The enzyme was shown to carboxylate pyruvate with at least the same rate as the forward reaction. The optimum pH for the carboxylation reaction was at pH 7.25 and that for the NADP-linked decarboxylation reaction varied with malate concentration. The Km values determined at pH 7.2 for malate and NADP were 120 microM and 9.2 microM, respectively. The Km values for pyruvate, NADPH and bicarbonate were 5.9 mM, 5.3 microM and 27.9 mM, respectively. The enzyme converted malate to pyruvate (at optimum pH 6.4) in the presence of 10 mM NAD at approximately 40% of the maximum rate with NADP. The Km values for malate and NAD were 0.96 mM and 4.6 mM, respectively. NAD-dependent decarboxylation reaction was not reversible. The purified human liver malic enzyme catalyzed decarboxylation of oxaloacetate and NADPH-linked reduction of pyruvate at about 1.3% and 5.4% of the maximum rate of NADP-linked oxidative decarboxylation of malate, respectively. The results indicate that malic enzyme from human liver exhibits similar properties to the enzyme from animal liver.
在人类肝脏中，苹果酸酶活性的近90%位于线粒体外部分，而线粒体部分中仅约占10%。通过硫酸铵分级分离、DEAE-纤维素柱层析、ADP-琼脂糖-4B柱层析和Sephacryl S-300柱层析，从人肝脏线粒体后上清液中分离出线粒体外苹果酸酶，直至通过聚丙烯酰胺凝胶电泳判断达到表观均一性。纯化酶的比活性为56 μmol·min⁻¹·mg蛋白⁻¹，这相当于约10000倍的纯化倍数。通过凝胶过滤测定的天然酶分子量为251 kDa。SDS/聚丙烯酰胺凝胶电泳显示一条分子量为63 kDa的多肽带。因此，天然蛋白似乎是由相同分子量亚基组成的四聚体。分离出的酶的等电点为5.65。该酶催化丙酮酸羧化的速率至少与正向反应相同。羧化反应的最适pH为7.25，而与NADP相连的脱羧反应的最适pH随苹果酸浓度而变化。在pH 7.2时测定的苹果酸和NADP的Km值分别为120 μM和9.2 μM。丙酮酸、NADPH和碳酸氢盐的Km值分别为5.9 mM、5.3 μM和27.9 mM。在10 mM NAD存在下，该酶在最适pH 6.4时将苹果酸转化为丙酮酸的速率约为以NADP时最大速率的40%。苹果酸和NAD的Km值分别为0.96 mM和4.6 mM。NAD依赖性脱羧反应不可逆。纯化的人肝脏苹果酸酶催化草酰乙酸脱羧以及丙酮酸的NADPH依赖性还原，其速率分别约为苹果酸的NADP依赖性氧化脱羧最大速率的1.3%和5.4%。结果表明，人肝脏中的苹果酸酶与动物肝脏中的酶具有相似的性质。
