PHOSPHOLIPASE-C IN DICTYOSTELIUM-DISCOIDEUM CYCLIC-AMP SURFACE-RECEPTOR AND G-PROTEIN-REGULATED ACTIVITY IN-VITRO
PHOSPHOLIPASE-C IN DICTYOSTELIUM-DISCOIDEUM CYCLIC-AMP SURFACE-RECEPTOR AND G-PROTEIN-REGULATED ACTIVITY IN-VITRO
复制标题
DOI：
10.1042/bj2970181
复制
发表时间：
1994-01-01
影响因子：
4.1
通讯作者：
VANHAASTERT, PJM
中科院分区：
文献类型：
--
作者：
BOMINAAR, AA;KESBEKE, F;VANHAASTERT, PJM
细胞黏菌Dictyosteelium discoideum在用化学引诱物cAMP刺激后显示出几种反应，包括环AMP（cAMP）、cGMP和Ins（1，4，5）P-3的瞬时升高。本文介绍了磷脂酶C的体外调控。在我们的实验条件下，商业PtdIns（4，5）P-2不能用于分析在Dictyosteoblasts裂解物中的磷脂酶C活性，因为它主要水解成甘油磷酸肌醇而不是Ins（1，4，5）P-3。用内源性未标记的PtdInsP（2）作为底物测定酶活性。产物经同位素稀释法测定，鉴定为真正的Ins（1，4，5）P-3。由于磷脂酶C是严格依赖于Ca 2+的，最佳浓度范围为1-100 μ M，因此在EGTA中制备细胞裂解物，并通过加入10 μ M游离Ca 2+开始酶反应。磷脂酶C活性增加了2倍，在Dictyoslalphae的发展，直到8小时的饥饿，之后的活性下降到营养水平的10%以下。体外酶活性增加到2倍后，刺激细胞与激动剂cAMP在体内。在裂解过程中加入10 μ M鸟苷5 '-[γ-硫代]三磷酸以相同的程度激活酶，这种作用被鸟苷5'-[β-硫代]二磷酸拮抗。这些结果有力地表明，表面cAMP受体和G-蛋白调节磷脂酶C在Dictyosteroids的发展。
The cellular slime mould Dictyostelium discoideum shows several responses after stimulation with the chemoattractant cAMP, including a transient rise in cyclic AMP (cAMP), cGMP and Ins(1,4,5)P-3. In this paper the regulation of phospholipase C in vitro is described. Under our experimental conditions commercial PtdIns(4,5)P-2 cannot be used to analyse phospholipase C activity in Dictyostelium lysates, because it is hydrolysed mainly to glycerophosphoinositol instead of Ins(l,4,5)P-3. Enzyme activity was determined with endogenous unlabelled PtdInsP(2) as a substrate. The product was measured by isotope-dilution assay and identified as authentic Ins(1,4,5)P-3. Since phospholipase C is strictly Ca2+-dependent, with an optimal concentration range of 1-100 mu M, cell lysates were prepared in EGTA and the enzyme reaction was started by adding 10 mu M free Ca2+. Phospholipase C activity increased 2-fold during Dictyoslelium development up to 8 h of starvation, after which the activity declined to less than 10 % of the vegetative level. Enzyme activity in vitro increased up to 2-fold after stimulation of cells with the agonist cAMP in vivo. Addition of 10 mu M guanosine 5'-[gamma-thio]triphosphate during lysis activated the enzyme to the same extent, and this effect was antagonized by guanosine 5'-[beta-thio]diphosphate. These results strongly suggest that surface cAMP receptors and G-proteins regulate phospholipase C during Dictyostelium development.