Abstract
The complete covalent structure of liver microsomal NADH-cytochrome b5 reductase from steer liver microsomes was determined. Cleavage at methionyl bonds gave 10 peptides accounting for all the residues of the protein. Acid cleavage of the reductase at the Asp-Pro bonds gave three peptides accounting for all the CNBr peptides in the molecule. Subfragmentation of these peptides by chemical and enzymatic cleavage provided overlaps which established all the fragments in an unambiguous sequence of 300 residues, corresponding to Mr 34,110. Limited tryptic digestion cleaved reductase at residues 28 and 119, yielding a preparation having two noncovalently linked peptides having a conformation which binds flavin and retains the structural features essential for NADH-cytochrome b5 activity. A model for the secondary structure of cytochrome b5 reductase is proposed that is based on computer-assisted analysis of the amino acid sequence. In this model the beta-turns are predominant and there is some 25% alpha and 30% beta structure.
Highlights
Bonds gave 10 peptides accounting forall the residues Chemical Cleavage of the Protein and Peptide Isolatwrwof the protein
Other in an unambiguous sequence of 300 residues, corre- CNBr peptides from the LH-60column were obtained by spondingto M, 34,110
A total of 10 CNBr peptides were cleaved reductase at residues 28 and 119, yielding a obtained. Their amino acid compositions are presented in preparation having two noncovalentlylinked peptides Table SI
Summary
Bonds gave 10 peptides accounting forall the residues Chemical Cleavage of the Protein and Peptide Isolatwrwof the protein. A total of 10 CNBr peptides were cleaved reductase at residues 28 and 119, yielding a obtained Their amino acid compositions are presented in preparation having two noncovalentlylinked peptides Table SI. The amino acid sequence of peptide CB-1 having a conformation which bindsflavin and retains and thenature of the NHz-terminalmasking group have been the structurafleaturesessentialfor NADH-cytochrome reported previously [2],its elution with respect to other CNBr bs activity. Peptide CB-8 cytochrome bs reductase is proposed that is based on eluted from reverse-phase HPLC as two peaks (Fig. S5) of computer-assistedanalysisof the amino acid sequencei.dentical amino aciod composition. Subfragmentation of peptide CB-7 at the tryptophanyl residues yielded three fragments resolvable by report here the primary structure of steer liver cytochromeb HPLC (Fig. S10).The emergence of such peptides as double reductase. Sodium dodecyl sulfate-polyacrylamide gel electropho- the sequence analysis of peptide DP-2,K-5 is shown in Table resis of the void volume fraction showed an absence of un- sv
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