人肺腺癌细胞PI—PLC活性的变化和转移表型的关系

我们将高转移性肺腺癌细胞亚系Anip_(973)及其母系AGZY_(83-a)(无转移性)注入裸小鼠腹腔,待出现腹水后,分离癌细胞,测定其PI-PLC活性(并经转移试验证实Anip_(973)仍具有转移能力)。实验结果表明具有不同转移表型的Anip_(973)和AGZY_(83-a)细胞PI-PLC活性有明显差异,前者为后者的2.25—7.1倍。体外培养两种细胞,测定两者PI-PLC活性,两者比值随传代次数增加逐步缩小,甚至出现倒置。两种细胞培养液均显示PI-PLC活性,且Anip_(973)高于AGZY_(83-a)。不同转移表型肺腺癌细胞PI-PLC活性在不同条件下的变化截然不同,此变化可能和肺腺癌细胞的转移功能密切相关。推测PI-PLC可能将成为新的转移标记酶。     

不同转移表型肺腺癌细胞磷脂酰肌醇的合成

Fidler于七十年代从B_(16)癌细胞系分离出转移性高低不同的细胞亚群。又证实某些信息分子可使其互变。许多学者对高转移性和低(或不)转移性癌细胞的酶、脂类等进行了比较,但迄今为止,对两者生物学特性的差异尚无定论。本文通过同位素标记法,对高转移性及低(或不)转移性肺腺癌细胞磷脂酰肌醇(PI)的合成进行了研究。证明两者有明显差异。     

野生型P53、P16基因协同对肺腺癌细胞系生长抑制作用的研究

为了探讨野生型P53基因及P16基因在恶性肿瘤基因治疗中的作用,用腺病毒为载体将野生型P53基因转入高、低转移的肺腺癌细胞系Anip973、AGZY83-a和经野生型P16基因质粒转染的高、低转移肺腺癌细胞系Anip973（Anip973P16）、AGZY83-a（AGZY83-aP16）。对各组转染细胞进行生长曲线、MTT生长抑制率、原位末端标记、Western-blotting等技术检测分析。结果发现（1）野生型P53蛋白的过表达对上述肺腺癌细胞系均呈现出较强的生长抑制作用。（2）野生型P53蛋白的过表达对高转移肺癌细胞系Anip973的抑制作用明显高于低转移细胞系AGZY83-a。（3）野生型p53蛋白的过表达对经野生型P16基因转染的高、低转移的肺癌细胞Anip973、AGZY83-a抑制作用明显高于未经P16基因转染的细胞。野生型P53基因可以作为肺腺癌基因治疗的候选基因。肿瘤抑制基因P53、P16的联合转染可能是对肺腺癌进行基因治疗的有效手段。 Abstract：To investigate the suppression effect of tumor suppressor genes in lung adenocarcinoma cell lines,we transferred a pair of lung adenocarcinoma cell lines with different metastasis potential,Anip973(High-metastasis potential cell line) and AGZY83-a (Low-metastasis potential cell line)and this pair of cell lines transfected with P16 gene:AGZY83-a P16 and Anip973 P16 with wild type P53 gene with adenovirus vector.The suppression effects of P53 gene were evaluated by cell growth curve,MTT,western-blotting analysis and TUNEL technique.Overexpression of wild-type P53 gene in AGZY83-a,Anip973,Anip973 P16 and AGZY83-a P16 inhibited the growth of these four kinds of lung cancer cells and induced apoptosis of the cells.The suppression effect of P53 gene in Anip973 and Anip973 P16 was higher than AGZY83-a and AGZY83-a P16 while co-expression of P53 and　P16 in this pair of cell lines inhibited the cells more efficiently comparing with the expression of P53 alone.Wild-type P53 gene might act as a candidate gene in lung adenocarcinoma gene therapy while co-transfection of P53 and P16 genes was a more effective method.     

异搏定对小鼠肝癌腹水瘤细胞系PI-PLC活性的影响

本文采用615近交系小鼠肝癌腹水瘤H_(ca)-F_(25)/CL-A_2细胞,测定钙拮抗剂异搏定作用前后磷脂酰肌醇特异的磷脂酶C(PI-PLC)活性的变化,并与同样处理的瘤株内钙恒稳的有关指标相比较。结果表明经异搏定处理后的A_2细胞PI-PLC活性显著降低,并与钙恒稳的指标基本呈现相应的平行变化趋势。提示PI-PLC可能参与肿瘤细胞内钙恒稳的变化过程,异搏定的作用可能与磷脂酰肌醇信号传导系统相关。     

脂肪酸对人肺腺癌细胞膜流动性的影响

脂肪酸是细胞膜正常流动性的主要调节因素之一。本文报导了二种不同转移表型人肺腺细胞与九种不同脂肪酸共孵育后,对其细胞膜流动性的影响。结果表明,不同转移一夫肺腺癌细胞对各种脂肪酸有不同的敏感性,高转移癌细胞Ａｎｉｐ对棕榈酸和花生酸较敏感,而低转移癌细胞ＡＧＺＹ对棕榈烯酸和亚油酸较敏感。     

胸水中肺腺癌细胞与血清中PTN多效蛋白分子生物学的研究

通过检测PTN蛋白在肺腺癌患者术前血清标本及相对应的恶性胸水肺腺癌细胞2种不同标本中的表达及对比其表达的差异,探讨其诊断意义.利用Western-blot免疫印迹方法检测50例恶性胸水及相对应的术前血清,并对肺腺癌细胞进行石蜡包埋、免疫细胞化学检查.同时分别以10例正常献血者血清、20例胸水良性增生细胞作为对照.肺腺癌患者血清和恶性胸水细胞中PTN蛋白的表达分别高于对照组PTN蛋白的表达,恶性胸水中PTN蛋白的表达59.0％ (49/83)高于肺腺癌患者血清中PTN蛋白的表达32.5％ (27/83).差异均具有统计学意义(P＜0.05),恶性胸水肺腺癌细胞中的PTN蛋白表达和波形蛋白Vimentin呈正相关关系(P ＜0.01,r =0.728),而与钙粘连蛋白E-ca呈负相关.PTN蛋白在肺腺癌患者血清和恶性胸水细胞标本中高表达,恶性胸水肺腺癌细胞中PTN蛋白的表达高于血清中PTN蛋白的表达,肺腺癌细胞中PTN蛋白的表达与波形蛋白Vimentin表达相一致,肺腺癌细胞在转移过程中已发生了向间质细胞转化EMT的过程,同时增强了肺腺癌细胞的高侵袭性,而恶性胸水肺腺癌细胞PTN蛋白的高表达更促进了肺腺癌细胞的转移.提示对未发生胸水转移的肺腺癌患者进行血清中PTN蛋白的检测,对已发生胸水转移的肺腺癌患者同样要检测PTN蛋白,以期提高肺腺癌患者的诊断率.     

了哥王中西瑞香素的提取分离及抗肿瘤作用研究

研究西瑞香素(daphnoretin)的体外抗肿瘤作用.采用溶剂提取法、硅胶柱层析、重结晶法从中药了哥王中分离西瑞香素并用薄层色谱法和高效液相色谱法测定纯度.应用MTT法观察不同浓度的西瑞香素对体外培养的人肺腺癌细胞AGZY-83-a、人喉癌细胞Hep2和人肝癌细胞HepG2的抑制作用.采用激光扫描共聚焦显微镜(LSCM)观察细胞形态的变化并检测细胞内钙离子浓度.经紫外光谱、质谱和核磁共振光谱确定提取分离西瑞香素的结构,其纯度为99.75%.西瑞香素对体外培养的人肺腺癌细胞AGZY-83-a、人喉癌细胞Hep2和人肝癌细胞HepG2均有明显抑制作用,且呈浓度依赖性.其对上述三种肿瘤细胞的半数抑制浓度IC50值分别为8.73、9.71和31.34 μg/mL.激光扫描共聚焦显微镜(LSCM)检测结果表明应用西瑞香素处理肿瘤细胞72 h后,细胞内钙离子浓度显著升高(P<0.05).西瑞香素体外对人肺腺癌细胞AGZY-83-a、人喉癌细胞Hep2和人肝癌细胞HepG2均有明显抑制作用,机制可能与细胞内钙超载有关.     

磷脂酰胆碱特异性和磷脂酰肌醇特异性磷脂酶C酶学性质及其在油脂脱胶中的应用

通过基因工程方法将具有磷脂酰胆碱(PC)、磷脂酰乙醇胺(PE)特异性磷脂酶C(PC-PLC)和磷脂酰肌醇(PI)特异性磷脂酶C(PI-PLC)基因分别在枯草芽孢杆菌WB800中进行胞外表达。对2种重组蛋白粗酶的酶学性质进行研究,并探究其用于油脂脱胶中的效果。结果表明:PC-PLC和PI-PLC均在30～45℃和中性偏酸的条件下具有较好的稳定性,2 h后残余活性仍在60%以上。Zn~(2+)、Mg~(2+)对PC-PLC的活性有促进作用,而Ca~(2+)对PI-PLC的活性有促进作用。油脂脱胶研究结果表明,PC-PLC的最适脱胶条件为50℃、pH 6.0、反应2 h、酶添加量为80 mg/kg;PI-PLC的最适脱胶条件为45℃、pH 7.0、反应2 h、酶添加量为60 mg/kg。PC-PLC和PI-PLC联合脱胶实验中大豆毛油中磷含量从531 mg/kg降至4.1 mg/kg,此时甘油二酯(DAG)含量增加0.95%。     

一个与非小细胞肺癌转移相关的基因--RAB5A基因

采用mRNA差异展示技术(mRNA DD)研究具有相同细胞来源,但转移能力高低不同的人肺腺癌细胞系AGZY83-a(低转移)和Anip973(高转移),分析在两个细胞系中基因差异表达的情况,发现在高转移细胞系中有RAB5A基因的表达.该基因为蛋白质入胞信号的调控者,为RAS超家族成员.为进一步证实其转录表达的调控改变情况,以及RAB5A高表达的临床意义,进一步采用RT-PCR和免疫组织化学的方法检测了50例临床非小细胞肺癌的手术标本,结果表明,RAB5A的表达有随转移发生而增强的趋势,而RAB5A的蛋白表达程度在有转移的病例中明显增强(P<0.05).     

龙脑液导致癌细胞凋亡的实验研究

目的:在细胞培养下检验加入龙脑液诱导癌细胞凋亡的作用.方法:采用人鼻咽癌、肺鳞癌、肺腺癌,乳腺癌细胞和正常细胞株,保持细胞正常培养基浓度下,用不同浓度稀释的龙脑液培养细胞,培养不同时间用四唑盐(MTT)比色试验观察龙脑液对癌细胞增殖的影响,采用细胞涂片染色观察细胞死亡性质,在流式细胞仪分析其凋亡率和性质.结果:二倍稀释浓度的龙脑液能快速导致癌细胞凋亡,四倍稀释浓度的龙脑液培养,均有细胞凋亡发生,凋亡程度从高到底依次为肺鳞癌、肺上皮癌和鼻咽癌,正常细胞293T则只有基础水平的凋亡发生;在撤出龙脑液回到正常培养环境下正常细胞能较快恢复到正常生长状态,而癌细胞则在一定时间内继续发生凋亡.在形态学上出现胞膜起泡或出芽,染色质凝聚分裂、细胞质减少,细胞碎片化等.早期细胞膜磷脂酰丝氨酸外翻明显导致细胞凋亡,也通过Caspases3凋亡通路变化而引起癌细胞凋亡.结论:龙脑对癌细胞有促凋亡作用.     

Upregulation of BNIP3 promotes apoptosis of lung cancer cells that were induced by p53

It has been shown that p53 induces cell apoptosis and the Bcl-2 family plays key roles in this process. However, the molecular mechanism of p53 apoptotic pathway is still unclear. Here, we show that overexpression of exogenous wild-type p53 induced apoptosis in lung cancer cells and high metastasis potential cells had a faster rate of apoptosis than low metastasis potential cells. The expression of pro-apoptotic gene BNIP3 was increased significantly both in Anip973 and 95D cell lines which have high metastasis ability, but not AGZY83-a or little increased in 95C cell lines which possess low metastasis ability. Overexpression of BNIP3 increases apoptotic rate induced by p53 in AGZY83-a cells. Blocking the expression of BNIP3 by siRNA in Anip973 cells decreased apoptotic rate mediated by p53. Taken together, these data suggest that high level expression of BNIP3 mediated rapid apoptosis that was triggered by p53 in lung cancer cells.     

一个与非小细胞肺癌转移相关的基因――RAB5A基因

采用mRNA差异展示技术（mRNA DD）研究具有相同细胞来源,但转移能力高低不同的人肺腺癌细胞系AGZY83-a（低转移）和Anip973(高转移),分析在两个细胞系中基因差异表达的情况,发现在高转移细胞系中有RAB5A基因的表达。该基因为蛋白质入胞信号的调控者,为RAS超家族成员。为进一步证实其转录表达的调控改变情况,以及RAB5A高表达的临床意义,进一步采用RT-PCR和免疫组织化学的方法检测了50例临床非小细胞肺癌的手术标本,结果表明,RAB5A的表达有随转移发生而增强的趋势,而RAB5A的蛋白表达程度在有转移的病例中明显增强(P<0.05)。 Abstract：　Using mRNA differential display (mRNA DD)techniques, we analyzed the differences of gene expression between two human lung adenocarcinoma cell lines,AGZY83-a and Anip973. Anip973 was isolated from AGZY83-a, but manifested much higher metastatic potential than the parent line. The results showed that there were significant differences on gene expression between the two cell lines and that there was over-expression of RAB5Agene in the Anip973 cell line. The product of RAB5Agene was recognized as signal regulators of endocytotic pathway and protein trafficking at the cell surface, and belong to a member of the RAS superfamily. Furthemore, we compared to the expression of RAB5Agene and RAB5Aprotein in clinical samples of 50 cases non-small lung carcinoma and nearby lymph node by means of RT-PCR and immunohistochemistry method. The results indicated that the high expression of RAB5Ain metastatic tumor and the enhancement level of RAB5Ain metastatic tumor and the enhancement level of expression were corresponded with the increase of metastatic degree. And there were significance of difference on the expression degree of RAB5Aprotein between non-small lung carcinoma with metastasis and non- metastasis (P<0.05).     

Purification and characterization of a phosphoinositide-specific phospholipase C from guinea pig uterus. Phosphorylation by protein kinase C in vivo

Two peaks of phosphoinositide-specific phospholipase C (PI-PLC) activity were resolved when guinea pig uterus cytosolic proteins were chromatographed on a DEAE-Sepharose column. The first peak of enzyme activity eluting from the DEAE-Sepharose column (PI-PLC I) was further purified to homogeneity, whereas the second peak of enzyme activity was enriched 300-fold. PI-PLC I migrated as a 62-kDa protein on sodium dodecyl sulfate-polyacrylamide gels. Antibodies prepared against PI-PLC I failed to react with PI-PLC II. PI-PLC I hydrolyzed all three phosphoinositides, exhibiting a greater Vmax for phosphatidylinositol 4,5-bisphosphate greater than phosphatidylinositol 4-phosphate greater than phosphatidylinositol. Hydrolysis of phosphatidylinositol was calcium-dependent, whereas significant hydrolysis of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate occurred in the presence of 2.5 mM EGTA. At physiological concentrations of calcium, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were the preferred substrates. Antibodies specific for PI-PLC I reacted with a 62-kDa protein in both the cytosol and membrane fractions from guinea pig uterus. Quantitation of the immunoblots revealed that 25% of the 62-kDa protein was membrane-associated, whereas only 5% of the total enzyme activity was membrane-associated. Approximately 20% of the membrane-bound phospholipase C activity and immunoreactive material were loosely bound, whereas the remainder required detergent extraction for complete solubilization. The 62-kDa protein associated with the membrane fractions did not bind lectin affinity columns, suggesting that it was not glycosylated. PI-PLC I was identified as a phosphoprotein in [32P]orthophosphate-labeled rat basophilic leukemia (RBL-1) cells by two-dimensional gel electrophoresis followed by immunoblotting. In untreated cells, 32P-labeled PI-PLC I was found in the cytosolic fraction. Treatment of RBL-1 cells with those phorbol esters which are known to activate the Ca2+/phospholipid-dependent enzyme protein kinase C, resulted in a time-dependent increase in the phosphorylation of both membrane-bound and cytosolic PI-PLC I. Thus, in RBL-1 cells, protein kinase C may play an important role in the regulation of phospholipase C through protein phosphorylation.     

P2-purinergic receptors activate a guanine nucleotide-dependent phospholipase C in membranes from HL-60 cells

We have previously determined that human neutrophils and monocytes, as well as neutrophil/monocyte progenitor cells, express a subtype of P2-purinergic receptors (for ATP) which activate the inositol phospholipid signalling system. In the present study, membranes prepared from HL-60 promyelocytic leukemia cells were used to examine the mechanism by which these ATP receptors activate phosphatidylinositol-specific phospholipase C (PI-PLC) under defined in vitro conditions. Micromolar concentrations of the receptor agonists ATP, UTP, and ATP gamma S stimulated the GTP-dependent formation of inositol bisphosphate (IP2) and inositol trisphosphate (IP3) in washed membranes prepared from undifferentiated HL-60 cells prelabeled with [3H]inositol. The stimulatory effects of these nucleotides on PI-PLC appeared to be mediated through a GTP binding protein since minimal inositol polyphosphate accumulation was observed in the absence of guanine nucleotides. The increased inositol polyphosphate formation triggered by these nucleotide receptor agonists did not result from inhibition of GTP breakdown. Neither was it a consequence of increased [3H]polyphosphatidylinositol levels resulting from enhanced activity of membrane-associated PI- or PIP-kinases. Instead, the stimulated phospholipase activity was apparently receptor-mediated. The rank order of potency observed in these in vitro membrane assays (ATP = UTP greater than ATP gamma S much greater than TTP greater than CTP much greater than beta, gamma-CH-ATP) was similar to that observed with intact HL-60 cells. This order of potency appears to distinguish the P2-purinergic receptors expressed by human phagocytic leukocytes from the P2 gamma-purinergic receptors which activate PI-PLC in turkey erythrocyte membranes.     

Activation of protease-constitutive recA proteins of Escherichia coli by all of the common nucleoside triphosphates.

To understand why the RecA proteins of the protease-constitutive recA1202 and recA1211 mutants show very high protease activities in vivo without the usual need for DNA damage (E. S. Tessman and P. Peterson, J. Bacteriol. 163:677-687, 1985), we examined the activation of the mutant proteins by nucleoside triphosphates (NTPs) in vitro. In vivo, the mutant protease activities are resistant to inhibition by cytidine plus guanosine (C + G) in the growth medium, in contrast to the activities of weaker mutants, such as recA441, which are sensitive to C + G inhibition. We found that RecA1202 and RecA1211 proteins, in contrast to RecA+, can use natural NTPs other than ATP and dATP as cofactors in the cleavage of LexA repressor. The effectiveness of NTPs in promoting LexA cleavage by RecA1202 and RecA1211 proteins decreased in roughly the following order: dATP greater than ATP greater than UTP greater than ATP-gamma S greater than dCTP greater than CTP greater than dGTP greater than GTP greater than TTP. These mutant proteins showed higher affinities for ATP and single-stranded DNA and higher repressor cleavage activities than RecA+ protein. With the various effectors (single-stranded DNA or NTPs), the RecA1202 protein always showed more activity than RecA1211 in the cleavage of LexA repressor in vitro, which is consistent with the greater activity of the recA1202 mutant in vivo. The results explain, in part, why some recA mutants have unusually high constitutive RecA protease activity and why that activity is more or less resistant to C + G inhibition.     

DJ-1 may contribute to metastasis of non-small cell lung cancer

Lung cancer is a leading cause of cancer-related death, about 40% human non-small cell lung cancer (NSCLC) patients showed lymph node involvements. However, the precise mechanism for the metastasis is still not fully understood. This study was to analyze the potential molecular mechanism for lung cancer metastasis. In the current study, proteomics analysis by two-dimensional electrophoresis (2-DE) was performed first to identify the differentially expressed protein between the higher metastasis lung adenocarcinoma cell line Anip973 and the lower metastasis lung adenocarcinoma cell line AGZY83-a. We confirmed the result by RT-PCR, immunoblotting and immunocytochemistry analyses in these two cell lines. Then we examined the expression of the differentially expressed protein in tumor tissues of NSCLC patients by immunoblotting and immunohistochemistry analyses. Using 2-DE analysis, we have identified DJ-1 was expressed higher in the higher metastasis Anip973 compared to the parental cell line AGZY83-a, that was confirmed by RT-PCR, immunoblotting and immunocytochemistry analyses. In NSCLC patients?? tumor tissues study, immunoblotting data showed that, DJ-1 expression level was significantly higher in 72.2% (13/18) of NSCLC tissue samples compared to that in paired normal lung tissues (P?=?0.044). Immunohistochemistry analysis demonstrated increased DJ-1 expression in 85 NSCLC tumor tissue samples compared with 7 normal lung tissue samples (P?=?0.044). DJ-1 expression was also found to be significantly correlated with cancer lymphatic metastasis (P?=?0.039). DJ-1 might contribute to the metastasis of NSCLC.     

Involvement of nuclear phosphatidylinositol-dependent phospholipases C in cell cycle progression during rat liver regeneration

Nuclear lipid metabolism is involved in the regulation of cell proliferation. Modulation of the expression and activity of nuclear PI-phospholipase C (PI-PLC) has been reported during liver regeneration after partial hepatectomy, although it has not been determined whether different PLC isoforms play specific roles in the regulation of cell cycle progression. Here, we report evidence that the increased activity of nuclear PLCs in regenerating rat liver occurs before the peak of DNA replication and involves the enzyme activity associated to the chromatin and not that associated to the nuclear membrane. Immunocytochemical analyses indicate that PI-PLC beta(1) isoform is exclusively localized at the chromatin level, PI-PLC beta(1) co-localizes with DNA replication sites much more than PI-PLC gamma(1), which is also present at the nuclear envelope. These findings and the increased amount of PI-PLC gamma(1) occurring after the peak of DNA replication suggest that PI-PLC beta(1) and gamma(1) play different roles in cell cycle progression during regenerating liver. The increased activity of PI-PLC beta(1) constitutively present within the hepatocyte nucleus, should trigger DNA replication, whereas PI-PLC gamma(1) should be involved in G2/M phase transition through lamin phosphorylation.     

Hyperosmotic stress induces a rapid and transient increase in inositol 1,4,5-trisphosphate independent of abscisic acid in Arabidopsis cell culture

Phospholipid metabolism is involved in hyperosmotic-stress responses in plants. To investigate the role of phosphoinositide-specific phospholipase C (PI-PLC)-a key enzyme in phosphoinositide turnover-in hyperosmotic-stress signaling, we analyzed changes in inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) content in response to hyperosmotic shock or salinity in Arabidopsis thaliana T87 cultured cells. Within a few s, a hyperosmotic shock, caused by mannitol, NaCl, or dehydration, induced a rapid and transient increase in Ins(1,4,5)P3. However, no transient increase was detected in cells treated with ABA. Neomycin and U73122, inhibitors of PI-PLC, inhibited the increase in Ins(1,4,5)P3 caused by the hyperosmotic shock. A rapid increase in phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in response to the hyperosmotic shock also occurred, but the rate of increase was much slower than that of Ins(1,4,5)P3. These findings indicate that the transient Ins(1,4,5)P3 production was due to the activation of PI-PLC in response to hyperosmotic stress. PI-PLC inhibitors also inhibited hyperosmotic stress-responsive expression of some dehydration-inducible genes, such as rd29A (lti78/cor78) and rd17 (cor47), that are controlled by the DRE/CRT cis-acting element but did not inhibit hyperosmotic stress-responsive expression of ABA-inducible genes, such as rd20. Taken together, these results suggest the involvement of PI-PLC and Ins(1,4,5)P3 in an ABA-independent hyperosmotic-stress signal transduction pathway in higher plants.     

Characterization of phosphoinositide-specific phospholipase C from human platelets.

Phosphoinositide-specific phospholipase C (PI-PLC) from human platelet cytosol was purified 190-fold to a specific activity of 0.68 mumol of phosphatidylinositol (PI) cleaved/min per mg of protein. It hydrolyses PI and phosphatidylinositol 4,5-bisphosphate (PIP2), but not phosphatidylcholine, phosphatidylserine or phosphatidylethanolamine. The enzyme exhibits an acid pH optimum of 5.5 and has a molecular mass of 98 kDa as determined by Sephacryl S-200 gel filtration. It required millimolar concentrations of Ca2+ for PI hydrolysis, whereas micromolar concentrations are optimal for PIP2 hydrolysis. Mg2+ could substitute for Ca2+ when PIP2, but not PI, was used as the substrate. EDTA was more effective than EGTA in inhibiting the basal PI-PLC activity towards PIP2. Sodium deoxycholate strongly inhibits the purified PI-PLC activity with either PI or PIP2 as substrate. Ras proteins, either alone or in the form of liposomes, have no effect on PI-PLC activity.