Screening for beta-poly(L-malate) binding proteins by affinity chromatography.
Poly(beta-L-malic acid) is a cell type-specific polymer of myxomycetes (true slime molds) with the physiological role to organize mobility of certain proteins over the giant multinucleated plasmodia. We have developed an affinity chromatography employing 1,6-diamino-n-hexane-Sepharose-coupled poly(malic acid) to identify such proteins in cellular extracts of Physarum polycephalum. Molecular masses were measured by SDS-PAGE and non-denaturing PAGE after silver staining and/or Western blotting. Protein complexes/subunits were detected by 2-dimensional non-denaturing PAGE/SDS-PAGE. A simplified gel shift experiment displayed binding to fragmented calf thymus DNA. Nuclei were richest in poly(malate) binding proteins followed by cytoplasm and membranes. A protein of 370 kDa dissociated into 11 subunits of 11-29 kDa, indicative of a highly complex protein. This and other proteins displayed binding to nucleic acid in gel shift experiments. Poly(malate) is considered a structural and functional equivalent of long contiguous aspartate repeats in proteins of eukaryotes.
Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, D-93040 Regensburg, Germany.
Thomas
T
Göttler
Eggehard
E
Holler
D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel; D003959:Diamines / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D008293:Malates / Q000737:chemistry / Q000378:metabolism*; D008970:Molecular Weight; D016839:Physarum polycephalum / Q000737:chemistry* / Q000254:growth & development; D011108:Polymers / Q000737:chemistry / Q000378:metabolism*; D012685:Sepharose / Q000737:chemistry
D002352:Carrier Proteins; D003959:Diamines; D008293:Malates; D011108:Polymers; C074640:poly(malate); D012685:Sepharose; C007297:1,6-diaminohexane