A quantitative assay to determine the amount of signal peptidase I in E. coli and the orientation of membrane vesicles.
The number of Signal Peptidase I (SPasel) molecules per E. coli cell was determined using western blot techniques. Different strains were found to contain approximately 1000 SPasel molecules per cell during exponential growth. Based on the activity of SPasel in vitro it could be estimated that this amount is sufficient to process all translocated precursors. SPasel did not appear to be under growth phase dependent control, but was constitutively expressed. The quantitative western blot technique was also used to establish the orientation and intactness of isolated inner membrane vesicles.
van Klompenburg
W
W
Department of Biochemistry of Membranes, Utrecht University, The Netherlands.
Whitley
P
P
Diemel
R
R
von Heijne
G
G
de Kruijff
B
B
D001426:Bacterial Proteins / Q000032:analysis* / Q000378:metabolism; D001692:Biological Transport; D015153:Blotting, Western / Q000379:methods*; D002462:Cell Membrane / Q000648:ultrastructure*; D019286:Endopeptidase K; D004926:Escherichia coli / Q000201:enzymology* / Q000254:growth & development; D008163:Luminescent Measurements; D008565:Membrane Proteins; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012697:Serine Endopeptidases / Q000032:analysis*
D001426:Bacterial Proteins; D008565:Membrane Proteins; D012697:Serine Endopeptidases; D019286:Endopeptidase K; C026515:type I signal peptidase