Ravid K, Rosenthal R A, Doctrow S R, Lowenstein J M
Graduate Department of Biochemistry, Brandeis University, Waltham, MA 02254.
Biochem J. 1989 Mar 15;258(3):653-61. doi: 10.1042/bj2580653.
Membranes prepared from calf brain were solubilized and chromatographed on a column containing 5'-amino-5'-deoxyadenosine covalently linked to agarose through the 5'-amino group. When the column was eluted with adenosine, a pure protein emerged with subunit molecular mass of 28 kDa. The protein was extracted from the membranes with sodium cholate, but not with 100 microM-adenosine or 0.5 M-NaCl. A similar 28 kDa protein was isolated from the soluble fraction of calf brain. The yield of membrane-bound and soluble 28 kDa protein per gram of tissue was about the same. The 28 kDa protein was also found in membrane and soluble fractions of rabbit heart, rat liver and vascular smooth muscle from calf aorta. The yield per gram of tissue fell into the order brain greater than heart approximately vascular smooth muscle greater than liver for the 28 kDa protein from the membrane fraction, and brain approximately heart greater than vascular smooth muscle greater than liver for the 28 kDa protein from the soluble fraction. Polyclonal antibodies to pure 28 kDa protein from calf brain membranes cross-reacted with the 28 kDa protein from calf brain soluble fraction and with 28 kDa proteins isolated from other tissues. The 28 kDa protein from calf brain membranes was also eluted from the affinity column by AMP and 2',5'-dideoxyadenosine, but at a concentration higher than that at which adenosine eluted the protein, but N6-(R-phenylisopropyl)adenosine, 5'-N-ethylcarboxamidoadenosine, ADP, ATP, GTP, NAD+, cyclic AMP and inosine failed to elute the protein at concentrations up to 1 mM. The 28 kDa protein from the soluble fraction was not eluted by 3 mM-AMP or 1 mM-N6-(R-phenylisopropyl)adenosine,-5'-N-ethylcarboxamidoadenosine or -cyclic AMP. Unexpectedly, the soluble 28 kDa protein was eluted by AMP in the presence of sodium cholate. Soluble 28 kDa protein from calf brain had a KD for adenosine of 12 microM. Membrane 28 kDa protein from calf brain had a KD of 14 microM in the presence of 0.1% sodium cholate. Amino acid compositions of the 28 kDa proteins were similar, but not identical.
从小牛脑制备的膜被溶解，并在一个含有通过5'-氨基与琼脂糖共价连接的5'-氨基-5'-脱氧腺苷的柱上进行色谱分析。当用腺苷洗脱该柱时，出现了一种亚基分子量为28 kDa的纯蛋白。该蛋白用胆酸钠从膜中提取，但不能用100 microM-腺苷或0.5 M-氯化钠提取。从牛脑的可溶性部分分离出了一种类似的28 kDa蛋白。每克组织中膜结合和可溶性28 kDa蛋白的产量大致相同。在兔心脏、大鼠肝脏和小牛主动脉的血管平滑肌的膜和可溶性部分中也发现了28 kDa蛋白。对于来自膜部分的28 kDa蛋白，每克组织的产量顺序为脑＞心脏＞血管平滑肌＞肝脏，对于来自可溶性部分的28 kDa蛋白，顺序为脑＞心脏＞血管平滑肌＞肝脏。针对从小牛脑膜中提取的纯28 kDa蛋白的多克隆抗体与从小牛脑可溶性部分提取的28 kDa蛋白以及从其他组织中分离的28 kDa蛋白发生交叉反应。从小牛脑膜中提取的28 kDa蛋白也被AMP和2',5'-二脱氧腺苷从亲和柱上洗脱下来，但洗脱浓度高于腺苷洗脱该蛋白的浓度，而N6-(R-苯异丙基)腺苷、5'-N-乙基羧酰胺腺苷、ADP、ATP、GTP、NAD+、环AMP和肌苷在浓度高达1 mM时未能洗脱该蛋白。来自可溶性部分的28 kDa蛋白未被3 mM-AMP或1 mM-N6-(R-苯异丙基)腺苷、-5'-N-乙基羧酰胺腺苷或-环AMP洗脱。出乎意料的是，在胆酸钠存在下，可溶性28 kDa蛋白被AMP洗脱。从小牛脑中提取的可溶性28 kDa蛋白对腺苷的KD为12 microM。在0.1%胆酸钠存在下，从小牛脑膜中提取的28 kDa蛋白的KD为14 microM。28 kDa蛋白的氨基酸组成相似，但不完全相同。
