Limited proteolysis of alpha 1-proteinase inhibitor (alpha 1-PI) in acute leukemia: studies on the resulting fragments and implication for the structure of the inactivated inhibitor.
In patients with acute myeloid leukemia, a 41 kDa glycoprotein appears in the urine during remission induction chemotherapy. We have recently reported on the isolation and preliminary characterization of this protein and the generation of specific monoclonal antibodies which showed that it is a proteolytically modified form of alpha 1-proteinase inhibitor (Dengler et al., 1992, Biol. Chem. Hoppe-Seyler 373, 581-588). In the paper presented here, results from further characterization experiments as well as from studies on the effects of proteolysis on the conformation and the resulting functional properties of the truncated inhibitor are reported. N-terminal amino acid sequence analysis showed that proteolysis has occurred in the N-terminal part as well as in the reactive site loop of alpha 1-PI. The resulting core protein of 41 kDa is composed of approximately 324 amino acid residues with the C-terminus located close to Lys343 of alpha 1-PI.A 4 kDa peptide remaining bound to this fragment throughout the entire purification procedure could be separated by SDS treatment. N- and C-terminal sequence analysis of this peptide after isolation by gel filtration showed that it is comprised of residues Ile359 up to Lys394, thus representing the peptide located C-terminal to the reactive site loop of alpha 1-PI. Transverse urea gradient gel electrophoresis indicates that the proteolyzed inhibitor is in the thermodynamically stable, relaxed (R)-conformation known for proteinase-complexed and cleaved serpins. The truncated inhibitor exhibits no chemotactic activity towards neutrophils when tested in a standard assay.
Dengler
R
R
Medizinische Klinik im Klinikum Innenstadt, Universität München, Germany.
Lottspeich
F
F
Oberthür
W
W
Mast
A E
AE
Emmerich
B
B
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002634:Chemotaxis, Leukocyte / Q000187:drug effects; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D007525:Isoelectric Focusing; D015470:Leukemia, Myeloid, Acute / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011489:Protein Denaturation; D014508:Urea / Q000737:chemistry; D000515:alpha 1-Antitrypsin / Q000737:chemistry / Q000378:metabolism*
D000596:Amino Acids; D007202:Indicators and Reagents; D010446:Peptide Fragments; D000515:alpha 1-Antitrypsin; D014508:Urea