Chromatographic analysis of lysosomal degradation of unlabeled native proteins in vitro by fluorescein isothiocyanate labeling.
Fluorescein isothiocyanate (FITC) has high affinity to both Toyopearl HW-40 and Sephadex G-25 gel. FITC-labeled amino acids showed no affinity for Sephadex G-25 whereas FITC-labeled proteins had no affinity. They were eluted in the order FITC-labeled proteins, FITC-labeled amino acids, and free FITC from a small combined column of Sephadex G-25/Toyopearl HW-40, which was prepared by layering a small volume of Toyopearl HW-40 gel on top of a larger volume of Sephadex G-25 gel. These findings were applied to analyzing the degradation of various unlabeled native proteins including L-lactate dehydrogenase (LDH) and rat serum albumin by total lysosomal enzymes in vitro. After degradation, proteins and amino acids released from a substrate protein were labeled with FITC and eluted through the small combined column. FITC-labeled amino acids were easily separated from FITC-labeled proteins and free FITC, and their production increased with degradation time and was markedly suppressed by the proteinase inhibitor leupeptin. The degradation of native LDH was also accompanied by inactivation and the disappearance of the 35-kDa subunit. This procedure is a simple and useful method for studying the proteolytic degradation of various unlabeled proteins by total lysosomal enzymes.
Ohshita
T
T
Division of Enzyme Chemistry, Institute for Enzyme Research, University of Tokushima, Japan.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D004229:Dithiothreitol; D005260:Female; D016650:Fluorescein-5-isothiocyanate; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D008247:Lysosomes / Q000378:metabolism*; D008297:Male; D008623:Mercaptoethanol; D011506:Proteins / Q000032:analysis*; D051381:Rats
D000596:Amino Acids; D011506:Proteins; D008623:Mercaptoethanol; D007770:L-Lactate Dehydrogenase; D016650:Fluorescein-5-isothiocyanate; D004229:Dithiothreitol