Purification and characterization of a methionine-specific aminopeptidase from Salmonella typhimurium.
An aminopeptidase specific for methionine (peptidase M) has been purified from wild-type and mutant Salmonella typhimurium strains. Recombinant peptidase M was also purified from Escherichia coli. These preparations were characterized with respect to their physicochemical properties using analytical ultracentrifugation, SDS/PAGE, isoelectric focusing, titration curve analysis, amino acid analysis, N-and C-terminal sequencing and various spectroscopic methods. Peptidase M activity is stimulated by Co2+, in agreement with previous studies using crude extracts of Salmonella. The purified preparations did not contain significant amounts of any metal. Enzymically important metal is loosely associated and lost during enzyme purification. Peptidase M was shown to contain seven free sulphydryl residues none of which are involved in either intra-or inter-molecular disulphide bonds. Most appear solvent-accessible as evidenced by their reactivity under native conditions. Limited modification of the sulphydryl residues with either iodoacetamide or 5,5'-dithiobis(2-nitrobenzoic acid) led to inactivation. Several cysteines were shown to be labelled to various degrees by peptide mapping of inactivated S-[14C]carboxymethylated protein. Whether cysteine modification affects enzymic activity directly (blocking an active site) or indirectly (by causing conformational change) remains to be established.
Wingfield
P
P
Biogen S.A., Geneva, Switzerland.
Graber
P
P
Turcatti
G
G
Movva
N R
NR
Pelletier
M
M
Craig
S
S
Rose
K
K
Miller
C G
CG
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000626:Aminopeptidases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005285:Fermentation; D007525:Isoelectric Focusing; D013058:Mass Spectrometry; D063208:Methionyl Aminopeptidases; D008969:Molecular Sequence Data; D009154:Mutation; D010449:Peptide Mapping; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification; D012486:Salmonella typhimurium / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013438:Sulfhydryl Compounds / Q000032:analysis; D014357:Trypsin; D014461:Ultracentrifugation
D000596:Amino Acids; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D000626:Aminopeptidases; D063208:Methionyl Aminopeptidases; D014357:Trypsin