Membrane topology of bovine adrenocortical cytochrome P-450C21: structural studies by trypsin digestion in vesicle membranes.
Purified adrenocortical microsomal P-450C21 was incorporated into vesicle membranes composed of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine at a molar ratio of 5:3:1. Trypsinolysis of the incorporated P-450C21 resulted in the formation of 30-, 25-, and 20-kDa fragments. Similar fragment formation was observed by trypsinolysis of bovine adrenocortical microsomes with Western blotting using anti-P-450C21 IgG. In the detergent-solubilized state, trypsin cleaved P-450C21 into very small peptides. Washing of the trypsin-treated vesicles with 500 mM Na2CO3 failed to cause these fragments to separate from membranes. N-Terminal amino acid sequencing of these fragments showed that trypsin cleaved the 267 Arg-268Val and 332Arg-333Val bonds of P-450C21. The time course of fragment formation indicated that trypsin cleaved the 267Arg-268Val bond first to produce 30- and 25-kDa fragments and subsequently the 332Arg-333Val bond in the 25-kDa fragment to produce the 20-kDa fragment. Neither 21-hydroxylase activity, the reduced CO difference spectrum, nor the EPR spectrum of digested P-450C21 differed from those of undigested P-450C21. Heat treatment at 50 degrees C for 20 min did not cause any decrease in activity of digested P-450C21, when the substrate progesterone was present. This high stability toward heat treatment was not observed in the solubilized state. Rotational diffusion experiments on P-450C21 showed that the size of the molecule holding the heme was not changed significantly after digestion. On the basis of these results, P-450C21 is concluded to be deeply embedded in the vesicle membranes.
Kominami
S
S
Faculty of Integrated Arts and Sciences, Hiroshima University, Japan.
Tagashira
H
H
Ohta
Y
Y
Yamada
M
M
Kawato
S
S
Takemori
S
S
D000302:Adrenal Cortex / Q000201:enzymology*; D000595:Amino Acid Sequence; D000818:Animals; D016880:Anisotropy; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D004795:Enzyme Stability; D008081:Liposomes / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008861:Microsomes / Q000737:chemistry / Q000201:enzymology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D011510:Proteolipids / Q000737:chemistry; D013053:Spectrophotometry; D013255:Steroid 21-Hydroxylase / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism
D008081:Liposomes; D008565:Membrane Proteins; D010446:Peptide Fragments; D011510:Proteolipids; C020485:proteoliposomes; D013255:Steroid 21-Hydroxylase; D014357:Trypsin