Membrane Topology of Liver Microsomal Cytochrome P450 2B4 Determined via Monoclonal Antibodies Directed to the Halt-Transfer Signal

Abstract

The membrane topology of cytochrome P450 2B4 from the endoplasmic reticulum has been studied with highly-purified liver microsomes in a site-directed immunochemical approach. Microsomes were prepared from phenobarbital-induced rabbits, and the resulting microsomal fraction was washed 6 additional times with 0.1 M pyrophosphate buffer to effect removal of significant quantities of adventitiously-bound protein. Monoclonal antibodies were prepared against residues 18-29 of P450 2B4 (Leu18-Leu-Phe-Arg-Gly-His-Pro-Lys-Ala-His-Gly-Arg29), essentially corresponding to the halt-transfer signal. This region was chosen due to its mutually-exclusive location in the two alternative membrane topology models currently tenable [Black, S.D. (1992) FASEB J.6, 680-685]. Model "A" contains a single transmembrane anchor peptide with the amino terminus projecting into the lumen of the endoplasmic reticulum, while model "B" exhibits a hairpin loop of the first approximately 46 residues inserted into the membrane with the amino terminus located on the cytosolic side of the lipid bilayer; the halt-transfer signal peptide would be located at the cytosolic surface of the membrane in model "A" or as a loop on the lumenal side of the membrane in model "B". Nine antibodies, denoted as MmAbA, MmAbC, MmAbD, MmAbF, MmAbH, MmAbI, MmAbK, MmAbL, and MmAbP, were produced, and all were identified as IgM/kappa subtypes. Western blotting demonstrated that the antibodies could readily recognize P450 2B4 in microsomes. ELISA assays showed that all of the antibodies exhibited strong binding to intact microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigenic targets in tienilic acid hepatitis. Both cytochrome P450 2C11 and 2C11-tienilic acid adducts are transported to the plasma membrane of rat hepatocytes and recognized by human sera.

“…oriented as follows. No part of the molecule (except the N-terminal methionine) protrudes into the ER lumen (9). Except for the transmembrane segment, the molecule is located on the cytosolic side of the ER membrane (9).…”

“…No part of the molecule (except the N-terminal methionine) protrudes into the ER lumen (9). Except for the transmembrane segment, the molecule is located on the cytosolic side of the ER membrane (9). Vesicular migration of this cytosolic form to the PM should lead to a cytosolically oriented form in the PM (unless P450 switches to the opposite topology during its transport from the ER to the PM, which seems unlikely) (12).…”

“…P450s are cotranslationally inserted into the endoplasmic reticulum (ER) membrane, where they remain anchored by a noncleaved signal-anchor sequence (8,9). A cytosolic domain may serve as an ER retention signal (10).…”

Antibodies as a Probe in Cytochrome P450 Research

“…The other model suggested a hairpin loop of the first approx-imately 46 residues inserted into the membrane with the amino terminus located on the cytosolic side of the lipid bilayer. To distinguish between these two possibilities, Black et al (1994) used monoclonal antibodies prepared against residues 18 to 29 of CYP2B4, corresponding to the halt-transfer signal of the enzyme. Based on these two models, the cationic halt-transfer signal peptide of CYP2B4 (residues 21-29) should be located at the cytosolic surface of the membrane in the first model but at the luminal side of the membrane in the second model.…”

The 2002 Bernard B. Brodie Award Lecture

“…Microsomal drug-metabolizing enzymes generally exhibit a longer meander region and a longer polypeptide chain between helices F and G that includes helices FЈ and GЈ in CYP2C5/3LVdH. The figure was rendered using MOLSCRIPT (Kraulis, 1991) and RASTER3D (Merritt and Bacon, 1997). side of the endoplasmic reticulum when the CYP2B4 is bound to the microsomal membrane (Black et al, 1994).…”