Liu G Q, Olivecrona T
Department of Medical Biochemistry and Biophysics, University of Umeå, Sweden.
Am J Physiol. 1991 Dec;261(6 Pt 2):H2044-50. doi: 10.1152/ajpheart.1991.261.6.H2044.
Lipoprotein lipase (LPL) in guinea pig hearts was labeled by perfusion for 15 min with [35S] methionine, followed by chase perfusion with unlabeled medium for less than or equal to 90 min. Suitable conditions for pulse and chase and for immunoprecipitation of LPL were worked out. Differences in labeling between individual hearts were found to be substantial. A technique to take serial biopsies (approximately 20 mg) from the heart ventricle was therefore worked out and validated. Regional differences of LPL labeling within the ventricle were small. There was no evidence for enhanced release of LPL caused by biopsy. When heparin was present throughout the chase, labeled LPL began to appear in the medium after 30 min. Thus LPL can move rapidly from synthesis in parenchymal cells to the endothelial surface and into the medium. After 90 min chase with heparin, approximately equal amounts of LPL (approximately 32%) had been released to the medium, remained in the heart, or had been degraded, respectively. In contrast, no detectable amount of labeled LPL appeared in the medium during 90 min chase without heparin, and a heparin flush at this time brought out only approximately 9% of the pulse-labeled LPL.
用[35S]甲硫氨酸对豚鼠心脏中的脂蛋白脂肪酶(LPL)进行15分钟的灌注标记,随后用未标记的培养基进行90分钟或更短时间的追踪灌注。确定了LPL脉冲标记和追踪标记以及免疫沉淀的合适条件。发现不同个体心脏之间的标记差异很大。因此,研究并验证了一种从心室进行连续活检(约20毫克)的技术。心室内LPL标记的区域差异很小。没有证据表明活检会导致LPL释放增加。在整个追踪过程中加入肝素后,标记的LPL在30分钟后开始出现在培养基中。因此,LPL可以从实质细胞中的合成迅速转移到内皮表面并进入培养基。用肝素追踪90分钟后,分别约有等量的LPL(约32%)释放到培养基中、保留在心脏中或被降解。相比之下,在无肝素的90分钟追踪过程中,培养基中未检测到标记的LPL,此时用肝素冲洗仅带出约9%脉冲标记的LPL。