Lee S B, Rhee S G
Laboratory of Cell Signaling, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Biol Chem. 1996 Jan 5;271(1):25-31. doi: 10.1074/jbc.271.1.25.
Complementary DNAs encoding a previously unidentified phosphoinositide-specific phospholipase C (PLC) isozyme were cloned from a rat brain cDNA library by the polymerase chain reaction with degenerate oligonucleotide primers based on sequences common to three known delta-type PLC isozymes. The encoded polypeptide contains 772 amino acids (calculated molecular mass, 88,966 daltons) and is similar in primary structure to delta-type PLC isozymes, with overall sequence identities of 45% to PLC-delta 1, 72% to PLC-delta 2, and 47% to PLC-delta 3. Thus, the new PLC isozyme was named PLC-delta 4. Recombinant PLC-delta 4 was purified from extracts of HeLa cells that had been infected with vaccinia virus containing the corresponding cDNA. The purified protein exhibited an apparent molecular mass of 90 kDa on SDS-polyacrylamide gels. The specific activity of PLC-delta 4 and its dependence on Ca2+ were similar to those of PLC-delta 1. The distribution of PLC-delta 4 in 16 different rat tissues was studied by immunoblot analysis with PLC-delta 4-specific antibodies of fractions obtained after an enzyme-enrichment procedure. The 90-kDa immunoreactive protein was detected unambiguously in only eight tissues and was present at concentrations that were low compared to those of other major PLC isozymes. A 93-kDa immunoreactive protein was also prominent in testis but was not detected in the other seven positive tissues. The 93-kDa enzyme appears to be derived from a splice variant of the mRNA that encodes the 90-kDa PLC-delta 4 and contains an additional 32 amino acids between the X and Y catalytic domains. Splice variants have not previously been detected for delta-type PLC isozymes.
利用基于三种已知δ型磷脂酶C（PLC）同工酶共有序列的简并寡核苷酸引物，通过聚合酶链反应从大鼠脑cDNA文库中克隆出编码一种先前未鉴定的磷酸肌醇特异性磷脂酶C同工酶的互补DNA。编码的多肽含有772个氨基酸（计算分子量为88,966道尔顿），其一级结构与δ型PLC同工酶相似，与PLC-δ1的总体序列同一性为45%，与PLC-δ2为72%，与PLC-δ3为47%。因此，这种新的PLC同工酶被命名为PLC-δ4。重组PLC-δ4是从感染了含有相应cDNA的痘苗病毒的HeLa细胞提取物中纯化得到的。纯化后的蛋白在SDS-聚丙烯酰胺凝胶上的表观分子量为90 kDa。PLC-δ4的比活性及其对Ca2+的依赖性与PLC-δ1相似。通过用PLC-δ4特异性抗体对酶富集程序后获得的组分进行免疫印迹分析，研究了PLC-δ4在16种不同大鼠组织中的分布。仅在8种组织中明确检测到了90 kDa的免疫反应性蛋白，其浓度与其他主要PLC同工酶相比很低。一种93 kDa的免疫反应性蛋白在睾丸中也很突出，但在其他7种阳性组织中未检测到。93 kDa的酶似乎来源于编码90 kDa PLC-δ4的mRNA的剪接变体，在X和Y催化结构域之间含有另外32个氨基酸。此前尚未在δ型PLC同工酶中检测到剪接变体。
