Characterization and purification of a protease in serum that cleaves proatrial natriuretic factor (ProANF) to its circulating forms.
Characterization and purification of a protease in serum that cleaves proatrial natriuretic factor (ProANF) to its circulating forms.
复制标题
血清中一种蛋白酶的表征和纯化，该蛋白酶可将心房钠尿因子 (ProANF) 裂解为其循环形式。
DOI：
10.1021/bi00400a030
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发表时间：
1987
期刊：
影响因子：
2.9
通讯作者：
Homcy,CJ
中科院分区：
文献类型：
--
作者：
Zisfein,JB;Graham,RM;Dreskin,SV;Wildey,GM;Fischman,AJ;Homcy,CJ
细胞和分子研究实验室，心脏科，马萨诸塞州总医院，波士顿，马萨诸塞州02114接收日期：1987年4月15日;修订版手册接收日期：1987年6月30日摘要：心房利钠因子（ANF）是一种17千道尔顿（kDa）、126个氨基酸的多肽，在心房细胞中合成和储存，但在循环中较小，前ANF羧基末端的24和28个氨基酸肽片段。以前的研究表明，proANF是从培养的心房心肌细胞中完整分泌的，并且可以被血清蛋白酶切割成较小的3-kDa肽，被认为是循环形式。本报告描述了从大鼠血清中纯化和鉴定这种proANF切割蛋白酶。裂解两个35 S-标记的proANF来自大鼠心房细胞培养物，作为评估的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）/放射自显影，和合成的对硝基苯胺含底物被用作检测酶活性的测定。在大鼠血清中发现ProANF裂解活性，在大鼠血浆中未检测到此类活性。血清中的裂解不依赖于血小板或其他细胞成分的存在。用蛋白酶抑制剂苯甲脒、亮抑酶肽、苯甲磺酰氟和氟磷酸二异丙酯（DFP）可完全抑制前心钠素裂解，但用抑肽酶、大豆胰蛋白酶抑制剂、胃蛋白酶抑制剂或水蛭素则不能。与维生素K依赖性血浆蛋白不同，proANF裂解蛋白酶不吸附硫酸钡。依次应用离子交换、羟基磷灰石、凝集素亲和层析和凝胶过滤层析，从大鼠血清中获得5000-6000倍纯化的酶。通过凝胶过滤色谱法对全血清或纯化的酶进行分级，显示出对应于Stokes半径为45的蛋白质的单一活性峰。发现酶的p/为约5.6。用[3 H] DFP孵育纯化的酶，然后进行SDS-PAGE和放射自显影，显示出特异性标记的38-kDa肽，底物结合亚基。如前所述，通过纯化的酶裂解35 S-标记的proANF产生的3-kDa产物的高效液相色谱分析揭示了两种放射性标记的肽，其与28和24个氨基酸的C-末端肽共洗脱。这些观察结果暗示了一种前体-产物关系，即proANF最初裂解为28个氨基酸的肽，然后裂解为24个氨基酸的肽。这些研究表明，在大鼠血清中发现的大多数proANF裂解活性是由一种独特的丝氨酸蛋白酶所代表的，该丝氨酸蛋白酶的性质不同于多种充分表征的蛋白酶，如激肽释放酶、纤溶酶和维生素K依赖性血浆蛋白。这种蛋白酶在proANF体内加工中的作用仍有待确定。A.试验利钠因子（ANF）在心房心肌细胞中合成为152个氨基酸的“前激素原”（Seidman等，1984; Yamanaka等人，1984; Maki等人，1984）并作为126个氨基酸的多肽proANF储存在心房颗粒中（Kangawa等人，1984; Glembotski等人，1985; Vuolteenaho等人，1985; Zisfein等人，1986 a; Miyota等人，1985年）。血液中免疫反应性和生物活性ANF的分析揭示了主要的循环形式是24和28个氨基酸的肽，对应于proANF的羧基末端片段（Sugarawa et al.，1985 …
Cellular and Molecular Research Laboratory, Cardiac Unit, Massachusetts General Hospital, Boston, Massachusetts02114 Received April 15, 1987; Revised Manuscript Received June 30, 1987 abstract: Atrial natriureticfactor (ANF) is synthesized and stored in atrial cardiocytes as a 17-kilodalton (kDa), 126 amino acid polypeptide, proANF, butcirculates as smaller, 24 and 28 amino acid peptide fragments of the carboxy terminus of proANF. It has previously been shown that proANF is secreted intact from cultured atrial cardiocytesand can be cleaved by a serum protease to smaller, 3-kDa peptides believed to be thecirculating forms. This report describes the purification and characterization of this proANF-cleaving protease from rat serum. The cleavages both of 35S-labeled proANF derived from rat atrial cell cultures, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/autoradiography, and of a synthetic p-nitroanilide-containing substrate were used as assays for the detection of enzyme activity. ProANF-cleaving activity was found in rat serum, with no such activity detectable in rat plasma. Cleavage in serum was not dependent on the presence of platelets or other cellular elements. Complete inhibition of proANF cleavage was obtained with the protease inhibitors benzamidine, leupeptin, phenylmethanesulfonyl fluoride, and diisopropyl fluorophosphate (DFP) but not with aprotinin, soybean trypsin inhibitor, pepstatin, or hirudin. Unlike the vitamin K dependent plasma proteins, the proANF-cleaving protease did not adsorb to barium sulfate. With the sequential application of ion-exchange, hydroxylapatite, lectin affinity, and gel filtration chromatography, a 5000-6000-fold purification of the enzyme from rat serum was achieved. Fractionation of either whole serum or the purified enzyme by gel filtration chromatography revealed a single peak of activity corresponding to a protein with a Stokes radius of 45 A. The p/of the enzyme was found to be approximately 5.6. Incubation of the purified enzyme with [3H] DFP followed by SDS-PAGE and autoradiography revealed a specifically labeled 38-kDa peptide, the substrate binding subunit. Analysis by high-performance liquid chromatography of the 3-kDa products resulting from the cleavage of 35S-labeled proANF by the purified enzyme revealed, as previously described with whole serum, two radiolabeled peptides which coeluted with the 28 and 24 amino acid C-terminal peptides. Moreover, a time-dependent increase in the abundance of the latter peptide was found. These observations imply a precursor-product relationship, with the initial cleavage of proANF to the 28 amino acid peptide, which is then cleaved to the 24 amino acid peptide. These studies indicate that the majority of proANF cleavage activity found in rat serum is represented by that of a distinct serine protease whose properties are different from a variety of wellcharacterized proteases, such as kallikrein, plasmin, and the vitamin K dependent plasma proteins. The role of this protease in the in vivoprocessing of proANF remains to be defined.. A. trial natriuretic factor (ANF) is synthesized in atrial cardiocytes as a 152 amino acid “preprohormone”(Seidman et al., 1984; Yamanaka et al., 1984; Maki et al., 1984) and is stored in atrial granules as a 126 amino acid polypeptide, proANF (Kangawa et al., 1984; Glembotski et al., 1985; Vuolteenaho et al., 1985; Zisfein et al., 1986a; Miyota et al., 1985). Analysis of immunoreactive and bioactive ANF in blood has revealed that the major circulating forms are the 24 and 28 amino acid peptides, corresponding to the car-boxy-terminal fragments ofproANF (Sugarawa et al., 1985 …