We propose to purify human thrombopoietin (Tpo) from the urine of patients with aplastic anemia in quantities sufficient for biochemical and biological studies. As shown in our preliminary studies, thrombopoietin has been identified as one of the glycoproteins which retains its in vivo activity after desialation. This specific property will also be pursued on the basis of its carbohydrate structure and affinity to several species of lectin. The main projects of this application are as follows: 1. Purification of human thrombopoietin. 2. Ascertain the levels of purity by a variety of procedures. 3. Determination of the carbohydrate structure and amino acid sequence. Purification of Tpo to molecular homogeneity will be accomplished with the continual in vivo and in vitro assessment of its biological activity. This process of purification will entail: 1. ethanol precipitation; 2. Sp-Sephadex cation exchange chromatography; 3. Sephadex G-100 column chromatography; 4. hydroxylapatite chromatography; 5. preparative gel electrophoresis. The biological mechanism(s) of the action of Tpo in conjunction with other known regulators of hematopoiesis [e.g., erythropoietin (Epo)] will then be pursued with these purified glycoprotein molecular probes.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL035411-02
Application #
3349257
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1985-02-01
Project End
1987-06-30
Budget Start
1985-07-01
Budget End
1986-06-30
Support Year
2
Fiscal Year
1985
Total Cost
Indirect Cost
Name
University of California Santa Barbara
Department
Type
Organized Research Units
DUNS #
City
Santa Barbara
State
CA
Country
United States
Zip Code
93106