| Author/Editor | Cirman, Tina | |
| Title | Vloga lizosomskih proteaz v apoptozi | |
| Type | monografija | |
| Place | Ljubljana | |
| Publisher | Medicinska fakulteta | |
| Publication year | 2001 | |
| Volume | str. 69 | |
| Language | slo | |
| Abstract | Because of impact of apoptosis on the normal development of the organism and its role in the aetiology of different diseases, it has been one of the most intensively studied areas in the last decade. Despite this, there are a lot of issues that remain to be solved. Cysteine proteases, termed caspases, play an important role in the initiation and execution of apoptosis. More recently, lysosomal proteases have been shown to play an important role in regulating certain apoptotic pathways. Their precise role in triggering and executing apoptosis and their interactions with caspases remain to be elucidated. In attempt to understand the latter, we have isolated cell components in order to model their interactions. Lysosomes, uncontaminated with cytochrome c, were prepared by subcellular fractionation of mouse liver. Liver homogenate was centrifuged on a gradient of 30% Percoll. Fractions that contained only lysosomes (and not mitochondria) were pooled and either used immediately or frozen at -70 C. Mitochondria were prepared by simple centrifugation of mouse heart homogenate. Using this technique almost pure intact mitochondria were obtained. Samples of cytosol were preepared from Jurkat-T cells that were swelled in hypotonic buffer. Passing them through a needle disrupted only the cell membrane while organelles remained intact and were removed from the sample by centrifugation. Bradford assay was used to determine total protein concentration and extracts with sufficient protein concentration were stored at -70 C. To study the effect of caspases and mitochondria on lysosomal stability, the components were mixed in a lysosomal buffer (0,25M sucrose and 0,2mM Hepes, pH = 7,2) which was shown to ensure the stability of all the components for the duration of the experiment. After a certain period of incubation, the lysosomes were checked for leakage by assaying for a specific lysosomal enzyme, B-hexosaminidase. (Abstract truncated at 2000 characters). | |
| Descriptors | LYSOSOMES BETA-N-ACETYLHEXOSAMINIDASE CYTOCHROME C MITOCHONDRIA, HEART APOPTOSIS MICE CYTOSOL |