The subcellular distribution of transglutaminase was investigated by using the analytical approach of differential and isopycnic centrifugation as applied to three organs of the rat: liver, kidney and lung. After differential centrifugation by the method of de Duve, Pressman, Gianetto, Wattiaux & Appelmans [(1955) Biochem. J. 63, 604-617], transglutaminase is mostly recovered in the unsedimentable fraction S and the nuclear fraction N. After isopycnic centrifugation of the N fraction in a sucrose density gradient, a high proportion of the enzyme remains at the top of the gradient; a second but minor peak of activity is present in high-density regions, where a small proportion of 5'-nucleotidase, a plasma-membrane marker, is present together with a large proportion of collagen recovered in that fraction. 2. Fractions where a peak of transglutaminase was apparent in the sucrose gradient were examined by electron microscopy. The main components are large membrane sheets with extracellular matrix and free collagen fibers. 3. As these results seem to indicate that some correlation exists between particulate transglutaminase distribution and those of collagen and plasma membranes, the possible binding of transglutaminase by collagen (type I) and by purified rat liver plasma membrane was investigated. 4. The binding studies indicated that collagen is able to bind transglutaminase and to make complexes with plasma-membrane fragments whose density is higher than that of plasma-membrane fragments alone. Transglutaminase cannot be removed from such complexes by 1% Triton X-100, but can be to a relatively large extent by 0.5 M-KCl and by 50% (w/v) glycerol. 5. Such results suggest that the apparent association of transglutaminase with plasma membrane originates from binding in vitro of the cytosolic enzyme to plasma membrane bound to collagen, which takes place during homogenization of the tissue, when the soluble enzyme and extracellular components are brought together.
摘要
采用差速离心和等密度离心分析方法，对大鼠的肝脏、肾脏和肺这三个器官进行研究，以探究转谷氨酰胺酶的亚细胞分布。按照德迪夫、普雷斯曼、贾内托、瓦蒂奥克斯和阿佩尔曼斯（1955年，《生物化学杂志》63卷，604 - 617页）的方法进行差速离心后，转谷氨酰胺酶大多存在于未沉淀部分S和细胞核部分N中。将N部分在蔗糖密度梯度中进行等密度离心后，大部分酶保留在梯度顶部；在高密度区域出现第二个但较小的活性峰，该区域存在少量的5'-核苷酸酶（一种质膜标记物）以及大量在该部分回收的胶原蛋白。2. 对蔗糖梯度中出现转谷氨酰胺酶峰的部分进行电子显微镜检查。主要成分是带有细胞外基质的大膜片和游离的胶原纤维。3. 由于这些结果似乎表明颗粒状转谷氨酰胺酶的分布与胶原蛋白和质膜的分布之间存在某种关联，因此研究了转谷氨酰胺酶与I型胶原蛋白以及纯化的大鼠肝脏质膜的可能结合情况。4. 结合研究表明，胶原蛋白能够结合转谷氨酰胺酶，并与密度高于单独质膜片段的质膜片段形成复合物。1% Triton X - 100无法从这类复合物中去除转谷氨酰胺酶，但0.5 M - KCl和50%（w/v）甘油能在较大程度上做到这一点。5. 这些结果表明，转谷氨酰胺酶与质膜的明显关联源于细胞溶质酶在体外与结合了胶原蛋白的质膜的结合，这种结合发生在组织匀浆过程中，此时可溶性酶和细胞外成分聚集在一起。
