Glucosamine-labelled envelope proteins of Escherichia coli K-12. I. Electrophoretic studies and partial fractionation of phenol-soluble proteins.
Hydrophobic envelope proteins were extracted by phenol from a glucosamine- and leucine-requiring mutant of Escherichia coli K-12 (E-110). Three protein fractions labelled with D-[1-14C]glucosamine and L-[4,5-3H]leucine were obtained by electrophoretic separation. Envelopes were isolated from cells labelled with D-[1-14C]glucosamine--HCL and acid hydrolyzed. At least 68% of the radioactivity was recovered as glucosamine and glucose with no random distribution of label. Fingerprinting of pronase digests of glucosamine-labelled proteins showed four radioactive spots associated with peptides. The glycoproteins were pronase- and trypsin-sensitive and had apparent molecular weights of 11 000 (fast mobility), 35 000 (intermediate mobility) and 62 000 (slow mobility) as estimated by sodium dodecyl sulfate-polyacrylamide disc electrophoresis. The two heavier fractions were labelled with meso-diamino[1,7-14C2]pimelic acid, while orth[32P]phosphate was not incorporated into any fraction. The glucosamine radioactivity of the fast fraction underwent rapid changes upon a chase with non-radioactive glucosamine. Using a Sephadex LH-20 column, the radioactive proteins were separated from the phenol and subsequently fractionated on a DEAE-cellulose column. The DEAE-cellulose fractions were distinct from each other in the number and composition of protein bands, when analyzed by sodium dodecyl sulfate-polyacrylamide disc electrophoresis. Radioactive bands with intermediate and fast electrophoretic mobilities were found in separate DEAE-cellulose fractions.
D001426:Bacterial Proteins / Q000032:analysis* / Q000096:biosynthesis; D002455:Cell Division; D002462:Cell Membrane / Q000032:analysis / Q000378:metabolism; D004589:Electrophoresis, Disc; D004926:Escherichia coli / Q000032:analysis* / Q000378:metabolism; D005944:Glucosamine / Q000032:analysis*; D006023:Glycoproteins / Q000032:analysis* / Q000096:biosynthesis; D007930:Leucine / Q000032:analysis; D010636:Phenols; D011402:Pronase; D012995:Solubility; D014357:Trypsin
D001426:Bacterial Proteins; D006023:Glycoproteins; D010636:Phenols; D014357:Trypsin; D011402:Pronase; D007930:Leucine; D005944:Glucosamine