Cupp M, Bensadoun A, Melford K
J Biol Chem. 1987 May 5;262(13):6383-8.
The mechanism responsible for the stimulation of secretion of lipoprotein lipase by heparin in cultured cells was studied with avian adipocytes in culture. Immunoprecipitation followed by electrophoresis and fluorography were used to isolate and quantitate the radiolabeled enzyme, whereas total lipoprotein lipase was quantitated by radioimmunoassay. Rates of synthesis of lipoprotein lipase were not different for control or heparin treatments as judged by incorporation of L-[35S]methionine counts into lipoprotein lipase during a 20-min pulse. This observation was corroborated in pulse-chase experiments where the calculation of total lipoprotein lipase synthesis, based on the rate of change in enzyme-specific activity during the chase, showed no difference between control (8.13 +/- 3.1) and heparin treatments (9.1 +/- 5.3 ng/h/60-mm dish). Secretion rates of enzyme were calculated from measurements of the radioactivity of the secreted enzyme and the cellular enzyme-specific activity. Degradation rates were calculated by difference between synthesis and secretion rates of enzyme. In control cells 76% of the synthesized enzyme was degraded. Addition of heparin to the culture medium reduced the degradation rate to 21% of the synthetic rate. The presence of heparin in cell media resulted in a decrease in apparent intracellular retention half-time for secreted enzyme from 160 +/- 44 min to 25 +/- 1 min. The above data demonstrate that the increase in lipoprotein lipase protein secretion, observed upon addition of heparin to cultured adipocytes, is due to a decreased degradation rate with no change in synthetic rate. Finally, newly synthesized lipoprotein lipase in cultured adipocytes is secreted constitutively and there is no evidence that it is stored in an intracellular pool.
利用培养的禽脂肪细胞研究了肝素刺激培养细胞中脂蛋白脂肪酶分泌的机制。采用免疫沉淀法，随后进行电泳和荧光自显影来分离和定量放射性标记的酶，而总脂蛋白脂肪酶则通过放射免疫测定法定量。根据在20分钟脉冲期间L-[35S]甲硫氨酸计数掺入脂蛋白脂肪酶的情况判断，对照或肝素处理的脂蛋白脂肪酶合成速率没有差异。在脉冲追踪实验中证实了这一观察结果，在该实验中，根据追踪期间酶比活性的变化率计算总脂蛋白脂肪酶合成，结果显示对照（8.13±3.1）和肝素处理（9.1±5.3 ng/h/60-mm培养皿）之间没有差异。酶的分泌速率通过测量分泌酶的放射性和细胞酶比活性来计算。降解速率通过酶的合成速率与分泌速率之差来计算。在对照细胞中，76%的合成酶被降解。向培养基中添加肝素将降解速率降低至合成速率的21%。细胞培养基中存在肝素导致分泌酶的表观细胞内保留半衰期从160±44分钟降至25±1分钟。上述数据表明，向培养的脂肪细胞中添加肝素后观察到的脂蛋白脂肪酶蛋白分泌增加是由于降解速率降低，而合成速率没有变化。最后，培养的脂肪细胞中新合成的脂蛋白脂肪酶是组成性分泌的，没有证据表明它储存在细胞内池。
