Sarcosine reductase of Tissierella creatinophila: purification and characterization of its components.
Sarcosine reductase is the only reductase system present in Tissierella creatinophila when grown on creatinine plus formate. The acetyl-phosphate-forming component protein C was purified to homogeneity. SDS-PAGE of the purified protein revealed two protein bands with apparent mol. masses of 62 and 50 kDa. The N-terminal amino acid sequence of the two subunits was determined. Antibodies raised against each of the subunits of protein C from Eubacterium acidaminophilum cross-reacted with the corresponding protein present in T. creatinophila, Clostridium litorale and Clostridium sporogenes. The arsenate-dependent hydrolysis of acetyl phosphate catalyzed by protein C was partly inhibited by antibodies directed against the large subunit. Antibodies raised against the small subunit were twice as effective, which indicates that this subunit is the primary site of acetyl transfer from acetyl phosphate. The protein A component of the sarcosine reductase of T. creatinophila was purified to homogeneity by cochromatography with thioredoxin reductase on DEAE-Sephacel, hydroxylapatite, Q-Sepharose, and Sephacryl 100-HR. Protein A had an apparent mol. mass of 21 kDa. Its N-terminal amino acid sequence showed high similarities to that of other proteins A. Initial steps for the purification and preliminary characterization of the sarcosine-specific, substrate-binding protein Bsarcosine component of T. creatinophila indicated the involvement of a 50-kDa protein.
Harms
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C
Institut für Mikrobiologie, Georg-August-Universität, Grisebachstrasse 8, D-37077 Göttingen, Germany.
Ludwig
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U
Andreesen
J R
JR
D000594:Amino Acid Oxidoreductases / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial; D000918:Antibody Specificity; D002847:Chromatography, Agarose; D003013:Clostridium / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D010755:Organophosphates / Q000378:metabolism; D011486:Protein C / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D011817:Rabbits; D011961:Receptors, Fc / Q000737:chemistry / Q000302:isolation & purification; D017421:Sequence Analysis; D016622:Silver Staining; D013053:Spectrophotometry; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000302:isolation & purification
D000907:Antibodies, Bacterial; C045281:IgA receptor; D010755:Organophosphates; D011486:Protein C; D011961:Receptors, Fc; D013205:Staphylococcal Protein A; C011632:acetyl phosphate; D000594:Amino Acid Oxidoreductases; C097251:sarcosine reductase