Proteolytic hydrolysis and purification of the LRP/alfa-2-macroglobulin receptor domain from alpha-macroglobulins.
A new, easier and efficient purification method, using Sephacryl and DEAE-Sephacel, of the C-terminal fragment of two alpha-macroglobulins, alpha(2)-M and PZP, is presented. Two larger peptides were identified for each protein as the C-terminal fragment, with molecular weights of approximately 30 kDa and the N-terminal sequences were determined to be SSTQDTV for alpha(2)-M and VALHLS for PZP. The smaller peptides with molecular weights of 18 kDa correspond to a shorter C-terminal sequence of these proteins, and they were determined to be EEFPFA for alpha(2)-M and ALKVQTV for PZP, with no interfering sequences detected. The results confirmed the discriminatory capacity of the purification procedure and the purity of the fragments. This new methodology facilitates biological studies of alpha-macroglobulins, and will enable elucidation of the role the C-terminal region may exert to eliminate alpha-macroglobulin-proteinases complexes from the circulation by the LRP/receptor.
Barrera
Daniel Iván
DI
Grupo de Investigación en Biomoléculas, Universidad de Pamplona, Pamplona, Colombia.
Matheus
Luisa Marina
LM
Stigbrand
Torgny
T
Arbeláez
Luis Fernando
LF
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism; D015153:Blotting, Western; D002918:Chymotrypsin / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D006868:Hydrolysis; D026503:Low Density Lipoprotein Receptor-Related Protein-1 / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification*; D011247:Pregnancy; D011257:Pregnancy Proteins / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D020539:Sequence Analysis, Protein; D013997:Time Factors; D000511:alpha-Macroglobulins / Q000737:chemistry* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D026503:Low Density Lipoprotein Receptor-Related Protein-1; C472887:PZP protein, human; D010446:Peptide Fragments; D011257:Pregnancy Proteins; D000511:alpha-Macroglobulins; D010450:Endopeptidases; D002918:Chymotrypsin