Molecular cloning of the type 5, iron-containing, tartrate-resistant acid phosphatase from human placenta.
Molecular cloning of the type 5, iron-containing, tartrate-resistant acid phosphatase from human placenta.
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发表时间：
1989-01
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通讯作者：
C. Ketcham;R. Roberts;R. Simmen;Harry S. Nick
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作者：
C. Ketcham;R. Roberts;R. Simmen;Harry S. Nick
5型含铁抗酒石酸酸性磷酸酶（TR-AP）是人类酸性磷酸酶的一种相对较小的细胞内同工酶，与子宫铁蛋白（猪子宫分泌的黄体酮诱导蛋白）有免疫关系。本文描述了从人胎盘中纯化少量TR-AP的方法。利用子宫铁蛋白编码区两个32p标记的短cDNA克隆筛选胎盘lambda gt11 cDNA文库，鉴定出一个1412碱基对的cDNA，可编码整个人类TR-AP同工酶。该cDNA包含969个碱基对的开放阅读框，对应323个氨基酸的蛋白质。存在一个由19个氨基酸和两个潜在糖基化位点组成的假定信号序列。推导出的人TR-AP的氨基酸序列与猪子宫铁蛋白的氨基酸序列有85%相同（本文也首次完整地报道了猪子宫铁蛋白的氨基酸序列），与牛脾脏磷蛋白磷酸酶的部分氨基酸序列的相应区域有82%相同。采用标记的TR-AP cDNA探针的Northern印迹技术显示，在毛细胞白血病患者的白细胞、人K562红白血病细胞和爱泼斯坦-巴尔病毒转化的人b细胞中存在1.5千碱基的转录物，但在人t细胞系中不存在。K562细胞在10(-8)M phorbol 12-肉豆蔻酸13-乙酸酯的作用下培养48-72 h，每个细胞TR-AP活性提高约30倍，TR-AP mRNA水平相应升高。
The type 5, iron-containing, tartrate-resistant acid phosphatase (TR-AP) constitutes a relatively minor intracellular isozyme of acid phosphatase in the human that is immunologically related to uteroferrin, a secreted progesterone-induced protein of the porcine uterus. Here, the purification of small amounts of TR-AP from human placenta is described. When a placental lambda gt11 cDNA library was screened with two short 32P-labeled cDNA clones from within the coding region of uteroferrin, a 1412-base pair cDNA was identified that encodes the entire human TR-AP isozyme. This cDNA contains an open reading frame of 969 base pairs, corresponding to a protein of 323 amino acids. A putative signal sequence of 19 amino acids and two potential glycosylation sites are present. The deduced amino acid sequence of the human TR-AP is 85% identical to that of porcine uteroferrin (whose sequence is also reported here in complete form for the first time) and 82% identical to the corresponding regions of a partial amino acid sequence of a bovine spleen phosphoprotein phosphatase. Northern blotting techniques employing a labeled TR-AP cDNA probe revealed the presence of a 1.5-kilobase transcript in white cells from a patient with hairy cell leukemia, in human K562 erythroleukemic cells, and in Epstein-Barr virus-transformed human B-cells, but not in a human T-cell line. Culture of K562 cells in presence of 10(-8) M phorbol 12-myristate 13-acetate ester for 48-72 h enhanced TR-AP activity per cell about 30-fold and led to a corresponding increase in TR-AP mRNA levels.