Nalbone G, Hostetler K Y
J Lipid Res. 1985 Jan;26(1):104-14.
During myocardial ischemia increased levels of lysoglycerophospholipids have been reported which may be deleterious to myocardial function. Phospholipases are presumed to be important in the regulation of this process. To further quantify and characterize the activity of heart phospholipases, we carried out a systematic analysis of phospholipase A activity in rat heart subcellular fractions isolated by the method of Palmer et al. (J. Biol. Chem. 1972. 262: 8731-8739). Neutral phospholipase A was recovered predominately in the cytosolic (soluble) fraction which represented 46% of recovered activity, while the microsomal and subsarcolemmal mitochondrial fractions represented 15% and 12% of the total recovered activity, respectively. Cytosolic phospholipase A differed from the two principal membrane-bound phospholipases A in its pH dependence and apparent Km for substrate. The cytosolic enzyme had a Km (apparent) for dioleoylphosphatidylcholine of 0.07 mM versus 0.28-0.33 mM for the membrane-associated phospholipases A. Acid phospholipase A activity had a subcellular distribution consistent with a lysosomal localization. Lysophospholipase was found principally in the cytosolic, microsomal, and the subsarcolemmal and interfibrillar mitochondrial fractions where it represented 46, 17, 6.3, and 6.9% of the recovered activity, respectively. The positional specificity of the respective phospholipases was assessed. This analysis was complicated by the fact that in heart, lysophospholipase has an observed Vmax 3.6- to 4.5-fold greater than that of phospholipase A in the various subcellular fractions. Equations were derived to obtain corrected values for the activity of phospholipases A1 and A2. Using this method we found that the cytosolic and lysosomal fractions contained phospholipase A1, while the mitochondrial fractions contained primarily phospholipase A2. In heart microsomes, the positional specificity of phospholipase A could not be determined because lysophospholipase activity was very high and lysophosphatidylcholine did not accumulate.
据报道，在心肌缺血期间，溶血甘油磷脂水平升高，这可能对心肌功能有害。磷脂酶被认为在这一过程的调节中起重要作用。为了进一步量化和表征心脏磷脂酶的活性，我们采用Palmer等人（《生物化学杂志》，1972年，262卷：8731 - 8739页）的方法对大鼠心脏亚细胞组分中的磷脂酶A活性进行了系统分析。中性磷脂酶A主要存在于胞质（可溶性）组分中，占回收活性的46%，而微粒体和肌膜下线粒体组分分别占总回收活性的15%和12%。胞质磷脂酶A在pH依赖性和底物的表观Km方面与两种主要的膜结合磷脂酶A不同。胞质酶对二油酰磷脂酰胆碱的Km（表观）为0.07 mM，而膜相关磷脂酶A的Km为0.28 - 0.33 mM。酸性磷脂酶A活性的亚细胞分布与溶酶体定位一致。溶血磷脂酶主要存在于胞质、微粒体、肌膜下和肌原纤维间线粒体组分中，分别占回收活性的46%、17%、6.3%和6.9%。评估了各磷脂酶的位置特异性。由于在心脏中观察到溶血磷脂酶的Vmax比各亚细胞组分中磷脂酶A的Vmax大3.6至4.5倍，这一分析变得复杂。推导了获得磷脂酶A1和A2活性校正值的方程。使用该方法我们发现胞质和溶酶体组分含有磷脂酶A1，而线粒体组分主要含有磷脂酶A2。在心脏微粒体中，由于溶血磷脂酶活性非常高且溶血磷脂酰胆碱不积累，无法确定磷脂酶A的位置特异性。
