碘化人肾α-D-甘露糖苷酶在大鼠肝脏中的摄取——与膜成分的关联以及体内外稳定性

Uptake of iodinated human kidney alpha-D-mannosidase by rat liver- Association with membrane elements and stability in vivo and in vitro.

作者信息

Marinkovic D V, Petrovic S L, Martinovic J V, Marinkovic J N

出版信息

Biochem J. 1976 Dec 1;159(3):729-36. doi: 10.1042/bj1590729.

DOI:10.1042/bj1590729

PMID:1008830

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1164175/

Abstract

Human kidney alpha-D-mannosidase (form A) was labelled with 125I to a specific radio-activity of approx. 2250muCi/mg of protein, essentially without loss of enzymic activity. The enzymic activity and radioactivity of the iodinated material also co-migrated in gel filtration and gel electrophoresis. 2. The binding of 125I-labelled mannosidase in vitro to particulate material in liver and kidney homogenates was of the other of 2 pg/mg of particulate material in liver and kidney homogenates was of the order of 2pg/mg of particulate protein withing 16h at 37 degrees C, and essentially zero in intervals of up to 60 min. The degradation in vitro of labelled exogenous mannosidase was of the order of 10-20pg/ 16th per mg of protein in postnuclear supernatant, and it was saturated entirely within 1h at 37 degrees C. 3. The binding of labelled mannosidase in vivo to particulate elements of liver homogenates 60 min after intravenous injection was at least 10 times higher in terms of specific radioactivity than the highest value attainable in vitro. Virtually all exogenous enzyme bound to liver particulate material could be recovered in macromolecular form after disruption of membranes by detergents. 4. The radioactive enzyme bound to liver particulate material could be detached almost completely by shearing, repeated freezing and thawing, and exposure to strong detergents under conditions that do not eliminate rough-endoplasmic-membrane structure. It could bot be released, however, by high salt concentration (0.5M-KC1) or by exposure to weak detergents such as Tween 80. The particle-bound enzyme should thus be associated with plasma membranes and lysosome-like elements. 5. Of the rat tissues studied, only liver could approach, within 60 min after the injection, the concentration of exogenous mannosidase found in the blood serum. The activity per g tissue weight fell progressively from liver (60% of serum value) to kidney (16% of serum value), lung (8% of serum vlaue), spleen (6% of serum value) and brain (0.9% of serum value). Most of the radioactive enzyme found in tissues other than liver appeared to be present in a free form, whereas in liver more than 50% of the labelled enzyme was associated with membrane elements.

摘要

人肾α-D-甘露糖苷酶（A 型）用¹²⁵I 标记，比放射性约为 2250μCi/mg 蛋白质，酶活性基本未损失。碘化材料的酶活性和放射性在凝胶过滤和凝胶电泳中也共同迁移。2. 在 37℃下，¹²⁵I 标记的甘露糖苷酶在体外与肝和肾匀浆中的颗粒物质结合，肝和肾匀浆中颗粒物质的结合量约为 2pg/mg 颗粒蛋白，16 小时内达到该水平，在长达 60 分钟的时间段内基本为零。标记的外源性甘露糖苷酶在体外的降解量约为核后上清液中每毫克蛋白质每 16 小时 10 - 20pg，在 37℃下 1 小时内完全饱和。3. 静脉注射 60 分钟后，标记的甘露糖苷酶在体内与肝匀浆颗粒成分的结合，就比放射性而言，至少比体外可达到的最高值高 10 倍。在用去污剂破坏膜后，几乎所有与肝颗粒物质结合的外源性酶都能以大分子形式回收。4. 与肝颗粒物质结合的放射性酶几乎可以通过剪切、反复冻融以及在不破坏粗面内质网结构的条件下暴露于强去污剂而完全解离。然而，它不能通过高盐浓度（0.5M - KCl）或暴露于弱去污剂如吐温 80 而释放。因此，颗粒结合的酶应该与质膜和溶酶体样成分相关。5. 在研究的大鼠组织中，只有肝脏在注射后 60 分钟内能够接近血清中外源性甘露糖苷酶的浓度。每克组织重量的活性从肝脏（血清值的 60%）逐渐下降到肾脏（血清值的 16%）、肺（血清值的 8%）、脾脏（血清值的 6%）和大脑（血清值的 0.9%）。在肝脏以外的组织中发现的大多数放射性酶似乎以游离形式存在，而在肝脏中，超过 50%的标记酶与膜成分相关。