Research Description:

Purification of MARCKS from Bovine Brain: Development of a New Technique Based on Perchloric Acid

MARCKS, myristoylated alanine-rich C kinase substrate, is a rod-like protein with an acidic protein with a pI ranging from 4.12 (found in rats) and 4.42 (found in bovines). The protein does not contain significant regions of hydrophobicity, yet is largely membrane-associated in most cells and tissues. There are three main regions of the protein that are conserved/identical across five species- human, bovine, chicken, mouse and rat. One of these three of particular interest is the PSD, phosphorylation site domain, which consists of 25 amino acids and in contrast to the rest of the protein is quite basic. The PSD is thought to have many ligands, including calmodulin, inositol phospholipids, and actin. As the name suggests, this region is also a target for phosphorylation, with four serines that are potentially modified by protein kinase C.

In order to study the properties of MARCKS it must first be purified. There has been previous research that has purified protein kinase C substrates by taking advantage of the fact that they are often soluble in 2.5% perchloric acid. The major benefit of using perchloric acid is that it significantly reduces other proteins and therefore makes for a more rapid purification, and less steps are needed after treatment than with perchloric acid.

Currently in my work I am attempting to develop a purification scheme for obtaining native MARCKS from bovine brains using perchloric acid. At present, perchloric acid is being used to directly treat bovine brains. After treatment with perchloric acid, additional purification steps will be required to obtain pure protein. Detection by SDS-PAGE gels and western blots allows for identification of the protein at each purification step.