Identification and characterization of three different subpopulations of the Drosophila multicatalytic proteinase (proteasome).
In Drosophila melanogaster the population of proteasome particles consists of three distinct subclasses. By fractionation of a 40,000 x g supernatant of Drosophila homogenate on a DEAE-Sephacel column, proteasome particles which elute at salt concentrations of 200, 300, and 500 mM KAc can be separated. The proteasomes of all three subfractions sediment at 19 S in sucrose gradients and are shown by two-dimensional gel electrophoretic analysis to possess the same protein content. They differ, however, with respect to their specific proteolytic activity against the substrates benzoyl-Val-Gly-Arg-4-methylcoumaryl-7-amide, succinyl-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide, and succinyl-Ala-Ala-Phe-4-methylcoumaryl-7-amide and the degree to which their hydrolytic activity can be enhanced by the addition of 30-110 microM sodium dodecyl sulfate (SDS). Our data show that the 200 mM proteasome fraction exhibits the lowest basal specific proteolytic activity but can be stimulated most by SDS. The 300 and 500 mM proteasome subfractions, on the other hand possess considerably higher but similar basal specific proteolytic activity. Of these only the proteolytic activity of the 300 mM subfraction against the substrates benzoyl-Val-Gly-Arg-4-methylcoumaryl-7-amide and succinyl-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide can be enhanced by SDS. Our data raise the possibility that the different subpopulations reflect structural differences between the proteasome particles, which in turn may result in different in vivo substrate specificities of the proteasome subpopulations.
Falkenburg
P E
PE
ZMBH/Molekulare Genetik, Heidelberg, Federal Republic of Germany.
Kloetzel
P M
PM
D000818:Animals; D002458:Cell Fractionation; D002845:Chromatography; D003546:Cysteine Endopeptidases; D003593:Cytoplasm / Q000201:enzymology; D004331:Drosophila melanogaster / Q000201:enzymology*; D015180:Electrophoresis, Gel, Two-Dimensional; D010450:Endopeptidases / Q000032:analysis*; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000032:analysis* / Q000037:antagonists & inhibitors; D011480:Protease Inhibitors; D046988:Proteasome Endopeptidase Complex; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013379:Substrate Specificity
D009097:Multienzyme Complexes; D011480:Protease Inhibitors; D012967:Sodium Dodecyl Sulfate; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex