Reduced phospholipase activity, peptide storage and the pathogenesis of canine neuronal ceroid-lipofuscinosis.
Reduced phospholipase activity, peptide storage and the pathogenesis of canine neuronal ceroid-lipofuscinosis.
复制标题
磷脂酶活性降低、肽储存和犬神经元蜡质脂褐质沉积症的发病机制。
DOI：
10.1007/bf00710276
复制
发表时间：
1993
影响因子：
4.2
通讯作者：
Siakotos,AN
中科院分区：
文献类型：
--
作者：
Dawson,G;Kilkus,J;Siakotos,AN
METHODSPreparation of lysosomes from normal and NCL dogs: Dogs were anaesthetized and injected intraperitoneally with 850 mg of WR-1339 per kg (Leighton et al 1968). They were anaesthetized 48 h later and sacrificed; the aortas were transected and the liver was removed. Liver (100 g) was homogenized in 0.25 mol/L sucrose (electric blender then Potter-Elvejem pestle). Nuclei and debris were removed (600g for 10 min) and a lysosome/mitochondrial fraction was obtained by centrifugation at 230 000g. rain for 1 min. This fraction was resuspended in 45% sucrose and centrifuged in a swinging bucket rotor on a 14.3% to 60.7% sucrose gradient. The WR-1339-engorged lysosomes banded between 14.3% and 34.5% sucrose (Leighton et al 1968) and were removed by aspiration for PLA1 assay and DCCD-binding mitochondrial ATP synthase isolation.Labelling with DCCD: Membrane preparations or isolated lyosomes (0.3-1.0 mg) in water were added to an Eppendorf tube containing 1/~ Ci (20 nmol; 2.05 GBq/mmol) of N, N"-dicyclohexyl [l* C] carbodiimide ([14C] DCCD) and brought to 0.4 ml in 10mmol/L Hepes, pH 7.3. Samples were rotated overnight at 4 C, centrifuged for 10rain, washed twice with Hepes, resuspended in LiDS and run on a 3-phase polyacrylamide gel (4% loading, 10% spacer and 16.5% main gel containing 0.1% LiDS and 6 mol/L urea) designed to resolve low-molecular-weight proteins (Schagger and von Jagow 1987). Gels were dried and subjected to autoradiography.
