Mustonen P, Lehtonen J, Kõiv A, Kinnunen P K
Department of Medical Chemistry, University of Helsinki, Finland.
Biochemistry. 1993 May 25;32(20):5373-80. doi: 10.1021/bi00071a012.
As revealed by resonance energy transfer utilizing pyrene-labeled phosphatidylcholine donor, the mainly electrostatically controlled binding of adriamycin (Adr) and cytochrome c (cyt c) to mixed egg yolk phosphatidic acid/phosphatidylcholine (eggPA/eggPC, 15:85 molar ratio) liposomes was reversed upon the inclusion of increasing contents of sphingosine. At a [sphingosine]/[eggPA] molar ratio of approximately 2:1, the degree of fluorescence quenching by cyt c and Adr was approximately the same as when using liposomes lacking eggPA. Similarly, the increase in the surface pressure of sphingosine/eggPA monolayers on an air/water interface due to the membrane penetration of either cyt c or Adr was progressively reduced by increasing the content of sphingosine in the monolayers. The above critical [sphingosine]/[acidic phospholipid] stoichiometry yielding dissociation of the positively charged ligands Adr or cyt c from membrane acidic phospholipids was shifted from 2:1 to 1:1 upon substituting egg phosphatidylglycerol (eggPG) for eggPA. Accordingly, charge neutralization of the acidic phospholipids by sphingosine could be involved. One eggPA (having maximally two negative charges) appears to require two molecules of sphingosine whereas the maximally singly charged eggPG is neutralized by one sphingosine. For comparison we also studied the effects of sphingosine on the phospholipase A2 catalyzed hydrolysis of the pyrene-labeled acidic alkylacyl phospholipid analog 1-octacosanyl-2-[6-(pyren-1-yl)]hexanoyl-sn-glycero-3- phosphatidylmethanol (C28-O-PHPM) and the corresponding phosphatidylcholine (C28-O-PHPC). In the presence of low Ca2+ concentrations (approximately 50 nM) limiting the rate of the enzymatic reaction, sphingosine gradually inhibited the hydrolysis of phosphatidylcholine, and at 1:6 sphingosine/C28-O-PHPC a nearly complete lack of hydrolysis was evident.(ABSTRACT TRUNCATED AT 250 WORDS)
利用芘标记的磷脂酰胆碱供体进行共振能量转移实验发现，阿霉素（Adr）和细胞色素c（cyt c）与混合的蛋黄磷脂酸/磷脂酰胆碱（eggPA/eggPC，摩尔比15:85）脂质体之间主要受静电控制的结合，会随着鞘氨醇含量的增加而逆转。当[鞘氨醇]/[eggPA]摩尔比约为2:1时，cyt c和Adr引起的荧光猝灭程度与使用不含eggPA的脂质体时大致相同。同样，由于cyt c或Adr穿透膜导致鞘氨醇/eggPA单层在空气/水界面上表面压力的增加，会随着单层中鞘氨醇含量的增加而逐渐降低。上述导致带正电配体Adr或cyt c从膜酸性磷脂上解离的关键[鞘氨醇]/[酸性磷脂]化学计量比，在将蛋黄磷脂酰甘油（eggPG）替代eggPA后从2:1变为1:1。因此，可能涉及鞘氨醇对酸性磷脂的电荷中和。一个eggPA（最多带有两个负电荷）似乎需要两个鞘氨醇分子，而最多带一个负电荷的eggPG则被一个鞘氨醇中和。为作比较，我们还研究了鞘氨醇对磷脂酶A2催化芘标记的酸性烷基酰基磷脂类似物1 - 二十八烷基 - 2 - [6 - （芘 - 1 - 基）]己酰基 - sn - 甘油 - 3 - 磷脂酰甲醇（C28 - O - PHPM）和相应磷脂酰胆碱（C28 - O - PHPC）水解的影响。在低Ca2 +浓度（约50 nM）限制酶促反应速率的情况下，鞘氨醇逐渐抑制磷脂酰胆碱的水解，当鞘氨醇/C28 - O - PHPC为1:6时，几乎完全没有水解现象明显。（摘要截短于250字）
