Conricode K M, Ochs R S
Department of Biochemistry, Kansas State University, Manhattan 66506.
Biochim Biophys Acta. 1989 May 15;1003(1):36-43. doi: 10.1016/0005-2760(89)90095-7.
The influence of proteins on phospholipase A2 was found to depend strongly on the enzyme assay system. We have used three different systems to measure phospholipase A2 which represent the different assay conditions used by a number of previous investigators. Two distinct stimulatory and two distinct inhibitory effects of proteins were observed. (1) A number of proteins - such as albumin, gamma-globulin and lysozyme - were found to inhibit phospholipase A2 activity only at very low substrate concentrations. This 'substrate depletion' was recently proposed as the mode of action for lipocortin. We therefore suggest that substrate depletion is not sufficiently specific to serve as a physiological regulatory mechanism and that the observed inhibition by lipocortin and other proteins more recently reported to mimic it are unlikely to be of physiological significance. (2) Use of liposomes at higher concentrations led to a nonlinear time-course. In this assay system, albumin (and other protein) stimulation can be accounted for as relief of product inhibition. (3) With high concentrations of phospholipids in the presence of cholate (mixed micelles), the behavior of proteins in the assay was complex. The assay time-course appeared linear in the absence of added protein, but at concentrations of added albumin up to 1 mg/ml, stimulation of phospholipase A2 activity was observed. Concentrations greater than this led to diminution of enzyme activity to the original activity. No effect whatever was observed when lysozyme was substituted for albumin. Since this biphasic result was not observed with liposomes, we suggest that the product whose inhibition is being relieved is the lysophosphatidylcholine, and not the free fatty acid. The inhibitory effect at high albumin concentrations is probably the result of removal of free fatty acids from the micelle: fatty acids are known to cause stimulation of phospholipase A2 by providing a negative charge to the lipid/water interface. (4) A different type of phospholipase A2 stimulation was apparent with melittin. This was found to be more specific than generally believed: we found no melittin stimulation of pancreatic phospholipase A2, yet confirmed a several-fold stimulation of bee venom phospholipase A2. We also found that high (millimolar) concentrations of calcium suppressed the melittin stimulation of bee venom phospholipase A2, and that a cationic detergent mimicked the stimulation by melittin. (5) We conclude that the effects of proteins on phospholipase A2 studied here can all be explained by proteins binding to substrate or product rather than enzyme-protein interactions.
人们发现蛋白质对磷脂酶A2的影响在很大程度上取决于酶的测定系统。我们使用了三种不同的系统来测量磷脂酶A2，它们代表了许多先前研究者所采用的不同测定条件。观察到蛋白质有两种明显的刺激作用和两种明显的抑制作用。（1）发现许多蛋白质，如白蛋白、γ球蛋白和溶菌酶，仅在非常低的底物浓度下抑制磷脂酶A2的活性。这种“底物耗竭”最近被认为是脂皮质素的作用方式。因此，我们认为底物耗竭作为一种生理调节机制的特异性不足，而且脂皮质素和最近报道的其他模仿它的蛋白质所观察到的抑制作用不太可能具有生理意义。（2）使用较高浓度的脂质体导致非线性的时间进程。在这个测定系统中，白蛋白（和其他蛋白质）的刺激作用可以解释为产物抑制的解除。（3）在胆酸盐（混合胶束）存在下，磷脂浓度较高时，测定中蛋白质的行为很复杂。在不添加蛋白质时，测定的时间进程呈线性，但添加白蛋白浓度高达1mg/ml时，观察到磷脂酶A2活性受到刺激。高于此浓度会导致酶活性降低至原始活性。用溶菌酶替代白蛋白时未观察到任何影响。由于用脂质体未观察到这种双相结果，我们认为被解除抑制的产物是溶血磷脂酰胆碱，而不是游离脂肪酸。高白蛋白浓度下的抑制作用可能是由于从胶束中去除了游离脂肪酸：已知脂肪酸通过向脂质/水界面提供负电荷来刺激磷脂酶A2。（4）蜂毒肽对磷脂酶A2有不同类型的刺激作用。发现这种作用比一般认为的更具特异性：我们发现蜂毒肽对胰磷脂酶A2没有刺激作用，但证实了对蜂毒磷脂酶A2有几倍的刺激作用。我们还发现高（毫摩尔）浓度的钙抑制了蜂毒肽对蜂毒磷脂酶A2的刺激作用，并且一种阳离子去污剂模仿了蜂毒肽的刺激作用。（5）我们得出结论，本文研究的蛋白质对磷脂酶A2的影响都可以通过蛋白质与底物或产物的结合来解释，而不是酶 - 蛋白质相互作用。
