大鼠细胞中5'-核苷酸酶的亚细胞分布与移动

Subcellular distribution and movement of 5'-nucleotidase in rat cells.

作者信息

Stanley K K, Edwards M R, Luzio J P

出版信息

Biochem J. 1980 Jan 15;186(1):59-69. doi: 10.1042/bj1860059.

DOI:10.1042/bj1860059

PMID:6245642

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1161503/

Abstract

Cell-surface 5'-nucleotidase was assayed by incubating whole-cell suspensions with 5'[3H]-AMP in iso-osmotic buffer and measuring [3H]adenosine production. The activity of cell-surface 5'-nucleotidase in hepatocytes, adipocytes and lymphocytes isolated from the rat was 15.0, 0.5 and 0.8pmol/min per cell at 37 degrees C respectively. 2. Disruption of the cells by vigorous mechanical homogenization or detergent treatment exposed additional 5'-nucleotidase activity, which represented 52%, 25% and 21% of the total activity in the three cell types respectively. This increase in 5'-nucleotidase activity which occurred when the cells were homogenized was due to a second pool of 5'-nucleotidase within the cell, rather than activation of the cell-surface enzyme. 3. In hepatocytes the intracellular 5'-nucleotidase activity was membrane-bound, indistinguishable from cell-surface 5'-nucleotidase in its inhibition by rabbit anti-(rat liver 5'-nucleotidase) serum and its kinetics with AMP, and was located on the extracytoplasmic face of vesicles within the cell. 4. The cell-surface 5'-nucleotidase of rat hepatocytes was rapidly inhibited when rabbit anti-(rat liver 5'-nucleotidase) serum or concanavalin A was added to the medium at 37 degrees C. Incubation with antiserum for 5 min at 37 degrees C inhibited 83 +/- 3% of the cell-surface enzyme. 5. Incubation of hepatocytes with exogenous antiserum or concanavalin A for 30 min at 37 degrees C resulted in over 50% inhibition of the intracellular enzyme. This inhibition was not prevented by disruption of the cytoskeleton or by ATP depletion. 6. Incubation of hepatocytes with exogenous antiserum or concanavalin A for up to 2h at 0 degrees C caused little or no inhibition of the intracellular enzyme, but over 75% inhibition of the cell-surface enzyme. 7. When surface-inhibited hepatocytes were washed and resuspended in buffer at 37 degrees C, 5'-nucleotidase was observed to redistribute from the intracellular pool to the cell surface.

摘要

通过将全细胞悬液与5'[³H]-AMP在等渗缓冲液中孵育并测量[³H]腺苷的生成，来测定细胞表面5'-核苷酸酶的活性。在37℃时，从大鼠分离的肝细胞、脂肪细胞和淋巴细胞中细胞表面5'-核苷酸酶的活性分别为每细胞15.0、0.5和0.8pmol/分钟。2. 通过剧烈机械匀浆或去污剂处理破坏细胞后，可暴露额外的5'-核苷酸酶活性，其分别占三种细胞类型总活性的52%、25%和21%。细胞匀浆时5'-核苷酸酶活性的这种增加是由于细胞内存在第二个5'-核苷酸酶池，而非细胞表面酶的激活。3. 在肝细胞中，细胞内5'-核苷酸酶活性与膜结合，在用兔抗（大鼠肝脏5'-核苷酸酶）血清抑制以及与AMP的动力学方面，与细胞表面5'-核苷酸酶无法区分，且位于细胞内囊泡的胞质外表面。4. 当在37℃将兔抗（大鼠肝脏5'-核苷酸酶）血清或伴刀豆球蛋白A添加到培养基中时，大鼠肝细胞的细胞表面5'-核苷酸酶会迅速受到抑制。在37℃与抗血清孵育5分钟可抑制83±3%的细胞表面酶。5. 在37℃将肝细胞与外源性抗血清或伴刀豆球蛋白A孵育30分钟，会导致细胞内酶受到超过50%的抑制。这种抑制不受细胞骨架破坏或ATP耗竭的影响。6. 在0℃将肝细胞与外源性抗血清或伴刀豆球蛋白A孵育长达2小时，对细胞内酶几乎没有抑制作用，但对细胞表面酶的抑制超过75%。7. 当表面受抑制的肝细胞被洗涤并重悬于37℃的缓冲液中时，观察到5'-核苷酸酶从细胞内池重新分布到细胞表面。