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

Journal Article · Thu Apr 01 00:00:00 EST 1982 · J. Bacteriol.; (United States)

OSTI ID:6933710

Kranz, R G; Gennis, R B

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-phenylenediamine (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, a/sub 1/, 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.

• Research Organization: Univ. of Illinois, Urbana
• DOE Contract Number: AC02-80ER10682
• OSTI ID: 6933710
• Journal Information: J. Bacteriol.; (United States), Vol. 150:1
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
ESCHERICHIA COLI
CELL MEMBRANES
HEME
STRUCTURAL CHEMICAL ANALYSIS
PROTEINS
CELL CYCLE
CYTOPLASM
ELECTROPHORESIS
IMMUNE REACTIONS
RESPIRATION
BACTERIA
CARBOXYLIC ACIDS
CELL CONSTITUENTS
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
MEMBRANES
MICROORGANISMS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PIGMENTS
PORPHYRINS
550200* - Biochemistry