Calcium-activated neutral proteinase (CANP; calpain) activity in Schwann cells: immunofluorescence localization and compartmentation of mu- and mCANP.
Calcium-activated neutral proteinase (CANP; calpain) activity in Schwann cells: immunofluorescence localization and compartmentation of mu- and mCANP.
复制标题
雪旺细胞中钙激活的中性蛋白酶（CANP；钙蛋白酶）活性：免疫荧光定位和 mu- 和 mCANP 的划分。
DOI：
10.1002/jnr.490290310
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发表时间：
1991
影响因子：
4.2
通讯作者：
Hogan,EL
中科院分区：
文献类型：
--
作者：
Banik,NL;DeVries,GH;Neuberger,T;Russell,T;Chakrabarti,AK;Hogan,EL
在转化的雪旺细胞（tSc）的胞质和膜亚细胞组分中测定钙活化中性蛋白酶（CANP）的活性。通过DEAE和phenyl Sepharose柱层析分离μ m和mM Ca2+敏感（μ和mCANP）形式的CANP，后一步可以去除内源性抑制剂calpastatin。tSc比mM型含有更多的μCANP。超过75%的mCANP活性与膜相关，20%与细胞质相关。相比之下，大约80%的μCANP为细胞质，15%为膜质。Triton X‐100刺激了整个匀浆和膜颗粒的活性，但没有刺激细胞质部分的CANP活性。用胞浆抗体（抗cyt - mCANP）和髓磷脂抗体（抗myt - mCANP）研究了固定和通透性tSc中mM酶的免疫组织化学分布。anti - my - mCANP染色的活细胞（非通透性）有一个单一的丝状环包围单个细胞。经抗mCANP处理的通透性细胞在整个细胞内空间均有免疫无反应性沉积，但核周区域除外。当活细胞暴露于抗cyt - mCANP时，未检测到免疫组织化学染色，而渗透细胞在核周区域具有广泛的细胞内染色和最强烈的免疫反应性。结果表明，两种形式的CANP都存在于tSc中，并且μCANP的活性大部分是胞质性的，而mCANP则是颗粒性的。
Calcium‐activated neutral proteinase (CANP) activity was determined in cytosolic and membranous subcellular fractions of transformed Schwann cells (tSc). The μM and mM Ca2+‐sensitive (μ‐and mCANP) forms of CANP were separated by DEAE and phenyl Sepharose column chromatography, the latter step enabling removal of the endogenous inhibitor calpastatin. The tSc contained more μCANP than the mM isoform. More than 75% of mCANP activity was membrane‐associated and 20% was cytosolic. In contrast, approximately 80% of μCANP was cytosolic and 15% was membranous. Triton X‐100 stimulated activity of the whole homogenate and of the membrane pellet but did not stimulate CANP activity in the cytosolic fraction. Immunohistochemical distribution of mM enzyme was studied in both fixed and permeabilized tSc with cytosolic (anti‐cyt‐mCANP) and myelin (anti‐my‐mCANP) antibodies. Live cells (non‐permeabilized) stained with anti‐my‐mCANP had a single filamentous ring circumscribing individual cells. Permeabilized cells treated with anti‐my‐mCANP had immu‐noreactive deposits throughout the intracellular space but sparing the perinuclear region. No immunohistochemical staining was detected when live cells were exposed to anti‐cyt‐mCANP whereas permeabilized cells had extensive intracellular staining with the most intense immunoreactivity in the perinuclear region. Our results indicate that both forms of CANP are present in tSc and that the activity of most of the μCANP is cytosolic while mCANP is particulate.