Sodium ion translocation by N5-methyltetrahydromethanopterin: coenzyme M methyltransferase from Methanosarcina mazei Gö1 reconstituted in ether lipid liposomes.
The N5-methyltetrahydromethanopterin (H,MPT):coenzyme M methyltransferase is a membrane associated, corrinoid-containing protein that uses the methylation of coenzyme M (HS-CoM) by methyl-tetrahydromethanopterin to drive an energy-conserving sodium ion pump. The enzyme was purified from acetate-grown Methanosarcina mazei Gö1 by a two-step solubilization with n-octyl-beta-glucoside, chromatography on hydroxyapatite, and by gel filtration on Superdex 200 or Sepharose CL-6B. The highly purified protein was apparently composed of six different subunits of 34, 28, 20, 13, 12, and 9 kDa. The N-terminal amino acid sequences of these polypeptides were determined. The native enzyme exhibited an apparent molecular mass of about 380 kDa. During purification, the enzyme was stabilized with 10 microM hydroxocobalamin. The highest specific activity reached during purification was 10.4 U/mg. The purified enzyme was reconstituted in monolayer liposomes prepared from ether lipids of M. mazei Gö1. In experiments with radioactive sodium ions, it was shown that the methyltransferase catalyzes the vectorial translocation of sodium ions across the membrane. Methyltransferase activity was stimulated by sodium ions. 1.7 mol Na-/mol methyl groups transferred were translocated. Methyltetrahydrofolate and methyl-cobalamin could substitute for methyl-H,MPT.
Lienard
T
T
Institut für Mikrobiologie der Georg August Universität, Göttingen, Germany.
Becher
B
B
Marschall
M
M
Bowien
S
S
Gottschalk
G
G
D000595:Amino Acid Sequence; D001692:Biological Transport; D004795:Enzyme Stability; D004987:Ethers; D008055:Lipids; D008081:Liposomes; D017020:Methanosarcina / Q000201:enzymology*; D008780:Methyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017421:Sequence Analysis; D012964:Sodium / Q000378:metabolism*
D004987:Ethers; D008055:Lipids; D008081:Liposomes; D012964:Sodium; D008780:Methyltransferases; C048755:tetrahydromethanopterin S-methyltransferase