Vesterqvist O, Sargent C A, Grover G J, Warrack B M, DiDonato G C, Ogletree M L
Department of Pharmacology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000.
Anal Biochem. 1994 Mar;217(2):210-9. doi: 10.1006/abio.1994.1111.
We have developed an assay for studying myocardial phospholipase A2 activity by measuring accumulation of lysophospholipids resulting from hydrolysis of the endogenous choline glycerophospholipid pool. This assay was used to characterize phospholipase A2 activity in rabbit myocardium. Lyophilized rabbit myocardium was incubated at 37 degrees C in Tris-HCl buffer containing either ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA)/EDTA or calcium, and palmitoyl-lysophosphatidylcholine (P-LPC), oleoyl-LPC, stearoyl-LPC, and 16:0-lysoplasmenylcholine were measured using a recently developed HPLC method. The identity of the individual species was confirmed by ion-spray LC-MS-MS. In the presence of EGTA/EDTA, incubation for up to 30 min caused a linear increase in all lysophospholipids. The main increases were found in P-LPC and 16:0-lysoplasmenylcholine, which increased by 37 +/- 3 (mean +/- SE, N = 8) and 48 +/- 3 nmol/g dry wt x min, respectively. The apparent phospholipase A2 activity was found to be calcium, temperature, and pH sensitive. The pH optimum was between 6.5 and 8.0, and incubation at room temperature and 45 degrees C decreased the activity by 80 and 40%, respectively. Studies of the metabolism of the formed lysophospholipids showed a substantial metabolism of the lysophospholipids that accounted for about 40% of the total phospholipase A2 activity. This method offers a novel approach to study phospholipase A2 activities by measuring accumulation of products resulting from hydrolysis of endogenous phospholipid pools.
我们开发了一种检测方法，通过测量内源性胆碱甘油磷脂池水解产生的溶血磷脂积累来研究心肌磷脂酶A2活性。该检测方法用于表征兔心肌中的磷脂酶A2活性。将冻干的兔心肌在含有乙二醇双（β-氨基乙醚）N，N'-四乙酸（EGTA）/乙二胺四乙酸（EDTA）或钙的Tris-HCl缓冲液中于37℃孵育，使用最近开发的高效液相色谱（HPLC）方法测量棕榈酰溶血磷脂酰胆碱（P-LPC）、油酰-LPC、硬脂酰-LPC和16:0-溶血缩醛磷脂酰胆碱。通过离子喷雾液相色谱-质谱-质谱联用（LC-MS-MS）确认了各个种类的身份。在EGTA/EDTA存在的情况下，孵育长达30分钟会导致所有溶血磷脂呈线性增加。主要增加的是P-LPC和16:0-溶血缩醛磷脂酰胆碱，分别以37±3（平均值±标准误，N = 8）和48±3 nmol/g干重×分钟的速度增加。发现表观磷脂酶A2活性对钙、温度和pH敏感。最适pH在6.5至8.0之间，在室温及45℃孵育分别使活性降低80%和40%。对形成的溶血磷脂代谢的研究表明，溶血磷脂有大量代谢，约占总磷脂酶A2活性的40%。该方法通过测量内源性磷脂池水解产物的积累，为研究磷脂酶A2活性提供了一种新方法。
