Abstract
Detergent-solubilized liver microsomal NADPH-cytochrome P-450 reductase is known to retain the ability to transfer electrons to cytochrome P-450, whereas the trypsin-solubilized reductase transfers electrons only to artificial acceptors. Due to the loss of a hydrophobic fragment by the action of trypsin, the altered reductase is no longer capable of binding cytochrome P-450. In the present study the primary tryptic attack on the rabbit reductase was shown to be at the Lys 44-Ile 45 bond to liberate the hydrophilic domain (molecular weight, 71,000) from the intact enzyme (molecular weight, 77,000). The other fragment (molecular weight, 4,800) undergoes tryptic attack at the Lys 34-Lys 35-Lys 36 sequence to yield a polypeptide representing the hydrophobic domain of the reductase and a nona- or decapeptide (Lys 35 or Lys 36 through Lys 44) which serves as the connecting region. The hydrophobic peptide, which is derived from the NH2-terminal end of the reductase, has an acetylated NH2 terminus and a region (Val 16 through Phe 32) which is exceptionally hydrophobic, with a predicted beta-sheet structure, and is believed to be involved in the binding of cytochrome P-450 and phospholipid. The site of attack on the reductase by various proteases is different, but the cleavage points are localized within a short segment of the polypeptide chain. A comparison of the tryptic forms (representing the hydrophilic domains) of the rabbit and rat reductases by terminal sequence analysis showed a high degree of similarity, with about 80% of the residues in exact correspondence and only a short variable region near the Ile NH2 terminus.
Highlights
Detergent-solubilized liver microsomal NADPH-cy- as a protease-treated form, “NADPH-cytochrome c reductochrome P-450 reductase is known to retain thaebility tase’’, which is monomeric in aqueous solution(Mr to transferelectrons to cytochrome P-450, whereas the = 71,000) and has high activitnythe reductionof cytochrome trypsin-solubilized reductase transfers electrons only c as an artificial acceptor, but this shortened forcmompletely to artificial acceptors
NH2-terminal Blocking Grouinp Native Reductase-Since a free NH2 terminus in t h e detergent-solubilized reductase could not be detected by dansylation,aminopeptidase M digestion, and automated and manual sequence analysis in preliminary experiments, the presence of a blocking group seemed likely
The sequence of the NHz-terminal region of rabbit hepatic NADPH-cytochrome P-450 reductase has been established with the aid of methods designed for hydrophobic peptides
Summary
Converted to this smaller molecular weight form This rabbit reduc- Amino Acid SequenceDetermination-Manual sequence analysis tase preparation is referred to as “adventitious.” Purification of the of small and large peptides was performed essentially according to trypsin-solubilizedreductase from liver microsomesof phenobarbital- the methods of Tam (33), with the following modifications to the treatedrats (12,27) was carried outas described for the rabbit method for large polypeptides. Large proteolytic fragments added in gradient back to initial conditions was run in 4 min Another hexafluoroacetone solution were analyzed without carrier, whereas sample was injected when the base-line was stabilized, typically aft.er small hydrophobic peptide samples in formic acid (88%)were applied an elapsed time of 110 min. T h e source of other materials is given elsewhere (12, 21, 38)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
