PURIFICATION AND CHARACTERIZATION OF A MOLLUSCAN EGG-SPECIFIC NADASE, A 2ND-MESSENGER ENZYME
PURIFICATION AND CHARACTERIZATION OF A MOLLUSCAN EGG-SPECIFIC NADASE, A 2ND-MESSENGER ENZYME
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DOI：
10.1091/mbc.2.3.193
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发表时间：
1991-03-01
期刊：
影响因子：
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通讯作者：
STRUMWASSER, F
中科院分区：
文献类型：
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作者：
HELLMICH, MR;STRUMWASSER, F
一种卵特异的NADase已经从海兔的卵巢中纯化为均一的。与其他NADase不同，Aplysia酶主要从NAD合成环状ADP-核糖(CADPR)，而不是ADP-核糖。研究表明，cADPR能刺激从海胆卵中制备的微粒体内钙离子的释放，并且当注入完整的海胆卵中时，可以激活皮质反应、多个核循环和DNA合成。该酶最初被鉴定为霍乱和百日咳毒素催化的ADP核糖化的抑制物。通过NADase分析，用顺序柱层析法从卵巢的水溶部分中纯化得到该蛋白。该酶的表观分子质量为29 kDa，NAD的K(M)为0.7 mM，转化率约为27000 molNAD。Min-1。30℃时的摩尔酶-1。制备抗NADase的单抗。二维凝胶免疫印迹显示该酶有多种异构体，pls在8.1到9.8之间。多种异构体用阳离子交换高压液相色谱柱分离，生成cADPR。对海兔卵巢冰冻切片的免疫组织化学分析表明，该酶是卵所特有的，仅限于5-10微米的大颗粒或小泡。到目前为止，已经在包括哺乳动物在内的各种生物中发现了cADPR生成酶的活性。Aplysia酶是第一个纯化产生cADPR的酶的例子。所有现有的证据表明，这个NADase是第二信使酶，这意味着其他NADase可能具有类似的功能。
An egg-specific NADase has been purified to homogeneity from the ovotestis of the opisthobranch mollusk Aplysia californica. Unlike other NADases, the Aplysia enzyme generates primarily cyclic-ADP-ribose (cADPR) rather than ADP-ribose from NAD. cADPR has been shown to stimulate the release of Ca2+ from microsomes prepared from sea urchin egg and, when injected into intact eggs, to activate the cortical reaction, multiple nuclear cycles, and DNA synthesis. The Aplysia enzyme was initially identified as an inhibitor of cholera and pertussis toxin-catalyzed ADP-ribosylation. By the use of an NADase assay, it was purified from the aqueous-soluble fraction of ovotestis by sequential column chromatography. The enzyme has an apparent molecular mass of 29 kDa, a K(m) for NAD of 0.7 mM, and a turnover rate of approximately 27 000 mol NAD. min-1.mol enzyme-1 at 30-degrees-C. Monoclonal antibodies were generated to the NADase. Immunoblots of two-dimensional gels revealed multiple isoforms of the enzyme, with pls ranging from 8.1 to 9.8. The multiple isoforms were resolved with a cation exchange high-pressure liquid chromatography column and shown to generate cADPR. Immunohistochemical analysis of cryostat sections of Aplysia ovotestis shows that the enzyme is specific to the eggs and restricted to large 5- to 10-mu-m granules or vesicles. To date the cADPR-generating enzyme activity has been identified in various organisms, including mammals. The Aplysia enzyme is the first example in which the enzyme that generates cADPR has been purified. All of the available evidence indicates that this NADase is a second-messenger enzyme, implying that other NADases may serve a similar function.