The aim of this project is to isolate and characterize a protein from adult rat liver that produces a reversible inhibition of the proliferation of liver-derived cells. The purification procedure has recently been extensively modified in order to obtain high recovery and greater purification of the growth inhibitor. Following the initial anion exchange and gel filtration chromatographic procedures, two high resolution ion exchange chromatographic steps are utilized, anion exchange on a Mono Q column followed by cation exchange on a Mono S column. The resulting preparation which has an ID50 of about 1-5 ng/ml is then subjected to high-resolution hydrophobic interaction chromatography using an propyl aspartamide HPLC. This produces a preparation with an ID50 of 150-500 pg/ml and two-dimensional polyacrylamide electrophoresis has revealed the presence of about 10-15 polypeptides. A further microscale purification procedure utilizing microbore reverse phase HPLC is currently being investigated. Chromatography of the propyl aspartamide purified samples indicates the presence of three major protein peaks. Although the conditions employed in this procedure cause a major loss of protein activity, it has been possible to tentatively narrow down the growth inhibitory activity to either one of these major proteins or a minor component-of the preparation which coelutes with it at a 43.5% acetonitrile concentration. Current work is focused on determining which of these proteins is responsible for the inhibitory activity. This newly developed procedure should allow the purification of this inhibitor to the purity required for sequence analysis. Concomitantly the large-scale purification of this liver-derived growth inhibitor is being pursued with the aim of immunization in order to obtain neutralizing antibodies to its activity.

Agency
National Institute of Health (NIH)
Institute
Division of Cancer Epidemiology And Genetics (NCI)
Type
Intramural Research (Z01)
Project #
1Z01CP005373-07
Application #
3896358
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
7
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Cancer Epidemiology and Genetics
Department
Type
DUNS #
City
State
Country
United States
Zip Code