Characterization of human serum N-acetylmuramyl-L-alanine amidase purified by affinity chromatography.
Human serum N-acetylmuramyl-L-alanine amidase was purified to homogeneity by a relatively short procedure including affinity chromatography. For this purpose, a specific adsorbent was prepared by coupling the main substrate of the enzyme, N-acetyl-muramyl-L-alanine-gamma-D-glutamyl-L-meso-2,6[3,4,5-3H] diaminopimelic acid, to a divinylsulfone agarose gel. The enzyme is unable to hydrolyze this muramylpeptide when it is attached as a ligand to the gel, whereas a high affinity is conserved. In addition to affinity chromatography, the presented purification scheme includes ion exchange chromatography on DEAE-Sepharose and molecular sieving on Superdex 200. The enzyme was purified 739-fold with a yield of 22.5%. One single band at 135 kDa was obtained on native gradient PAGE. Gradient PAGE in denaturing conditions gave one single band at 74 kDa, which was lowered to 64 kDa when the enzyme was denatured in nonreducing conditions. This suggests that the native enzyme is a dimer consisting of two subunits of identical molecular weight with only intramolecular disulfide bonds. Isoelectric focusing gave one single band at pI 5.0. Glycan detection before and after treatment with N-glycosidase F showed that the enzyme is a glycoprotein. Further analysis by lectin immuno detection on dot blots confirmed that the enzyme is an N-glycosylated protein of complex type with sialic acid, terminally linked alpha (2-->6) to galactose or N-acetylgalactosamine. The 15 amino acid N-terminal sequence was determined by microsequence analysis.
De Pauw
P
P
Research Institute, C.E.R.I.A.-C.O.O.V.I., Brussels, Belgium.
Neyt
C
C
Vanderwinkel
E
E
Wattiez
R
R
Falmagne
P
P
D000119:Acetylmuramyl-Alanyl-Isoglutamine / Q000031:analogs & derivatives / Q000378:metabolism; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000097:blood* / Q000302:isolation & purification; D006801:Humans; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000097:blood* / Q000302:isolation & purification; D011485:Protein Binding; D011487:Protein Conformation; D017421:Sequence Analysis
D006023:Glycoproteins; D000119:Acetylmuramyl-Alanyl-Isoglutamine; C035104:muramylNAc-Ala-isoGln-Lys-tripeptide; D009238:N-Acetylmuramoyl-L-alanine Amidase