Meeting Abstract

P1.124  Thursday, Jan. 3  Identifying Novel Protein Stablizers by Co-immunoprecipitation in Porcelain Crabs, Genus Petrolisthes CAYENNE, Andrea P.*; STILLMAN, Jonathon H.; Romberg Tiburon Center, SFSU andrealilpenny@yahoo.com

Biochemical adaptation of enzymes, specifically those involving sequence variation among orthologous homologues, allows organisms to conserve metabolic kinetic properties in a wide range of environmental conditions. However, the role(s) of protein-protein interactions in enzyme adaptation are less well understood. Previous examination of the glycolytic enzyme lactate dehydrogenase (LDH) in porcelain crabs showed that interspecific differences in thermal stability were related to both intrinsic and extrinsic protein stabilizers. Here, we attempted to identify the extrinsic stabilizing proteins in porcelain crab muscle tissue using co-immunoprecipitation. To do this, we homogenized muscle tissue from Petrolisthes elongatus from New Zealand in RIPA buffer and incubated the homogenate supernatant with purified rabbit polyclonal IgG anti-Petrolisthes cinctipes LDH. To separate LDH and its associates proteins, we used protein-A sepharose beads and low speed cintrifugation. The samples were then run on a 12% SDS-PAGE and Coomassie Blue stained. We found a 45 kDa band that was not present in either the purified LDH, IgG, or Protein-A beads. To identify this unknown protein, we are in the process of extracting the protein from the gel and preparing the sample for peptide mass fingerprinting (MS/MS) using MALDI-TOF/TOF mass spectrometry. Additonal species will be examined using the same methods including Petrolisthes cinctipes. Funded by: NIH MBRS-SCORE to JHS, and NIH-RISE to APC