Effect of Overexpression of Truncated Calpain Small Subunit on Calpain System in L8 myoblast

• Main Authors: TSUN-CHIH CHUNG, 鍾尊智
• Other Authors: BOR-RUNG Ou
• Format: Others
• Language: en_US
• Published: 1999
• Online Access: http://ndltd.ncl.edu.tw/handle/58735383350385903530

碩士 === 東海大學 === 畜產學系 === 87 === The growth rate of skeletal muscle dependents on the rates of muscle protein synthesis and protein degradation. It was reported that calpains played an important role on the initiation of muscle protein degradation. The calpains depend on its calcium requirement can be grouped into - and m-calpain. Both and m-calpain contain two subunit, small subunit ( CS; 30KDa ) and large subunit ( CL; 80KDa ). The objectives of this study were to investigate the function of CS by expressing the autolysis form of CS in L8 myoblast. Rat post-autolysis small subunit ( 21KCS; 21KDa ) cDNA was amplified by polymerase chain reaction ( PCR ). The cDNA ( 577bp ) then was inserted into pMAMneo-SV40 ( pSV ) vector. The new plasmid is referred to pMAMneo-SV40-Small subunit ( pSV-SS ). The plasmid pSV and pSV-SS were transfected into L8 myoblasts, respectively. Then the single cloning was selected by G418 ( 300g/ ml ) containing medium. After selection, the transfected cell were analyzed by PCR using Neor primer against the genomic DNA and stable cloning cell line L8-Neo, SS1, SS2 and SS3 were established. CS mRNA were analyzed by Reverse transcription polymerase chain reaction ( RT-PCR ) in transfected and control cells using primer 21K-1 and oligo-dT. The data indicated that SS2 and SS3 had expression of exogenous CS mRNA ( 21KCS mRNA ), however, SS1 did not express the exogenous CS mRNA. In addition, and m-calpain were analyzed by Western blot in total and membrane protein. In analysis of total protein, the results showed that the protein concentration of - calpain in SS2 and SS3 were 15.7% and 17.3% higher than control ( L8-Neo ), respectively. The protein concentration of m-calpain in SS2 and SS3 were 23.3% and 16.6% higher than control ( L8-Neo ), respectively. In addition, SS3 and control ( L8-Neo ) were analyzed by immunoprecipitation to detect the stability of calpain. The data indicated that the stability of -calpain in SS3 was 36.3 hours higher than control ( 31.9hours ). The stability of -calpain was 40.5 hours higher than control ( 36.3hours ). In analysis of membrane calpain concentration, the transfected cells ( SS1、SS2 and SS3 ) and control cell ( L8-Neo ) have no significant difference by expression of exogenous CS. Also, there is no different in total protein degradation between transfected cells and control cells. These results imply that expression of exogenous 21KCS can increase the protein level of - and m-calpain and increase the stability of calpains. The result indicated that 21KCS effect on translation level but not on transcription level. On the pther hand, 21KCS can not improve the opportunity to translocte to the membrane or change the rate of protein degradation in myoblast of rat.