Isoelectric focusing and crossed immunoelectrophoresis of heme proteins in the Escherichia coli cytoplasmic membrane

Abstract

Isoelectric focusing (IEF), agarose electrophoresis, and crossed immunoelectrophoresis (CIE) were used to resolve the heme-containing proteins of the Escherichia coli cytoplasmic membrane after solubilization by Triton X-100. Two bands in IEF stained for heme with pI values of 4.7 and 5.3. One of the bands, with an isoelectric point of pH 5.3, was present only when the cells were grown to late log or stationary phase and possessed N,N,N,'N'-tetramethyl-p-phenylene-diamine (TMPD) oxidase activity. The pI 4.7 band was present in cells harvested in both mid-log and stationary phases. Agarose electrophoresis, using larger samples, revealed the same two components apparent by IEF, and, in addition, a third component. The heme-containing fractions were extracted after agarose electrophoresis and subjected to further study. The component which was present in cells grown to stationary phase contained hemes b, a1, and d. The other two fractions contained only b heme. One of these corresponded to the component with pI 4.7 in IEF and had catalase activity. Antisera were raised against Triton X-100-solubilized cytoplasmic membranes and against the focused TMPD oxidase complex. With these anti-sera, CIE in the presence of Triton X-100 revealed four precipitin complexes containing heme. Three of these corresponded to the components identified by IEF and agarose electrophoresis. We demonstrate that the combined use of IEF and CIE is valuable for analysis of membrane proteins. In particular, this work represents a substantial initial step toward a structural elucidation of the E. coli aerobic respiratory chain.

SENSITIVITY OF HemA MUTANT Escherichia coli CELLS TO INACTIVATION BY NEAR‐UV LIGHT DEPENDS ON THE LEVEL OF SUPPLEMENTATION WITH δ‐AMINOLEVULINIC ACID

“…The spectra observed with the extracts from cells grown in the presence of 6-ALA are consistent with those reported by others for stationary E . coli exhibiting peaks at about 520, 560 and 620 nm (Green and Gennis, 1983;Kranz and Gennis, 1982). Although the protein concentrations for the cellular extracts from cells grown without 6-ALA supplementation were about one-fifth of those from cells grown with such supplementation, the spectra give no indication of peaks characteristic of difference spectra (reduced-minus-oxidized) for strains synthesizing cytochromes.…”

Isolation and characterization of an Escherichia coli mutant lacking cytochrome d terminal oxidase

“…With Paracoccus dentrificans, Azotobacter vinelandii, and Rhodopseudomonas capsulata, the use of artificial electron donors specific for one branch of multibranched electron transport systems made possible the isolation of cytochrome-deficient mutants (16,25,42). In this report, a similar strategy was used to obtain a mutant of E. coli K-12 missing cytochrome d. This mutant was detected by its failure to oxidize the artificial electron donor N,N,N',N'tetramethyl-p-phenylenediamine (TMPD), lending support to the proposal that TMPD donates electrons primarily to the cytochrome d branch (22). In addition to cytochrome d, cytochromes a1 and probably b558 are missing in the mutant.…”

“…Mutant isolation. Previous studies suggested that high TMPD oxidase activity in the membranes from E. coli grown under conditions of low aeration is due to the presence of cytochrome d (22). Since intact E. coli cells do not react with TMPD (39), an agar plate test was devised that disrupts cells in situ, thus permitting the reaction of TMPD with the electron transport system.…”

“…Membrane preparation and protein determination. Membrane preparation and protein determination procedures have been described previously (22). Difference spectra.…”

Succinate dehydrogenase in Rhodopseudomonas sphaeroides: subunit composition and immunocross-reactivity with other related bacteria

“…Both the anti-inner membrane serum and the anti-SDH serum were purified by the procedure described by Kranz and Gennis (29).…”