Rapid assay and purification of a unique signal peptidase that processes the prolipoprotein from Escherichia coli B.
A simple and accurate assay for prolipoprotein signal peptidase activity has been described that is based on the solubility of the signal peptide in 80% acetone. The unprocessed precursor and the mature form of the lipoprotein are quantitatively recovered in the precipitate. The signal peptide, from the acetone supernatant utilizing the purified signal peptidase, contains labeled methionine at its NH2 terminus and has Mr = 2200 (S.E. = 69). A specific signal peptidase that processes the modified form of Braun's prolipoprotein to its correct mature form has been purified. This enzyme is globomycin sensitive and has been purified 35,000-fold from the membranes of Escherichia coli by extraction at pH 4.0 with 2% Triton X-100 and heating, followed by conventional column chromatography at room temperature. This prolipoprotein signal peptidase has a pH optimum at 6.0, is not inhibited by EDTA, and requires 1 mM dithiothreitol for stability. The monomer molecular weight of this specific signal peptidase is 17,800 (S.E. = 900) as determined by sodium dodecyl sulfate-gel electrophoresis.
D001425:Bacterial Outer Membrane Proteins; D002462:Cell Membrane / Q000201:enzymology; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D007700:Kinetics; D008074:Lipoproteins / Q000235:genetics*; D046911:Macromolecular Substances; D008565:Membrane Proteins; D008970:Molecular Weight; D011498:Protein Precursors / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D012697:Serine Endopeptidases; D013104:Spheroplasts / Q000201:enzymology
D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D008074:Lipoproteins; C099932:Lpp protein, E coli; D046911:Macromolecular Substances; D008565:Membrane Proteins; D011498:Protein Precursors; D010450:Endopeptidases; D012697:Serine Endopeptidases; C026515:type I signal peptidase