mesna and methylamine

Methanogenesis from methylamines requires the intermediate methylation of 2-mercaptoethanesulfonate (CoM). In vitro reconstitution of CoM methylation with monomethylamine was achieved with three purified proteins: a monomethylamine corrinoid protein (MMCP), the "A" isozyme of methylcobamide:CoM methyltransferase (MT2-A), and a newly isolated protein termed monomethylamine methyltransferase (MMAMT).MMAMT is a 170-kDa protein with 52-kDa subunits. The MMAMT polypeptide was rate-limiting for methyl transfer until at a 2-fold molar excess over MMCP. MMAMT is a monomethylamine:MMCP methyltransferase, since methylation of MMCP required MMAMT but not MT2-A. MMCP and MMAMT formed a complex detectable by size exclusion high pressure liquid chromatography. Methyl group transfer from methyl-MMCP to CoM was mediated by MT2-A, since methyl iodide:CoM methyl transfer by MMCP and MT2-A did not require MMAMT. MT2-M, an isozyme of MT2-A, was inactive in MMCP-dependent methyl transfer. Immunodepletion of MMCP from the extract inhibited CoM methylation with monomethylamine but not dimethylamine. Purified MMCP reconstituted activity in immunodepleted extracts. These results show that MMCP is the major corrinoid protein for methanogenesis from monomethylamine detectable in extracts and that it interacts with two methyltransferases. MMAMT functions as a MMA:MMCP methyltransferase, while MT2-A functions as a methyl-MMCP:CoM methyltransferase.

Topics: Bacterial Proteins; Mesna; Methanosarcina; Methylamines; Methylation; Methyltransferases

An assay which allowed detection of proteins involved in the trimethylamine- or monomethylamine (MMA)-dependent methylation of coenzyme M (CoM) was developed. The two activities could be separated by anion-exchange chromatography. The unresolved activity responsible for MMA:CoM methyl transfer eluted from a gel permeation column in the molecular mass range of 32 kDa. The activity was purified to two monomeric proteins of 40 and 29 kDa. The preparation contained protein-bound corrinoid in a mixture of Co(II) and Co(III) states, as well as methyl-B12:CoM methyltransferase (MT2) activity. N-terminal sequence analysis demonstrated that the polypeptides were two previously identified proteins of undefined physiological function. The smaller polypeptide was the monomeric 29-kDa corrinoid protein. The larger polypeptide was the "A" isozyme of MT2. Individually purified preparations of both proteins increased the rate of MMA-dependent CoM methylation by approximately 1.7 mumol/min/mg of purified protein above background activity in the extract of methanol-grown cells. These results indicate that the 29-kDa corrinoid protein and the "A" isozyme of MT2 function in methanogenesis from MMA. A likely mechanism is that the 29-kDa corrinoid is methylated by MMA and the methyl group is then transferred by the "A" isozyme of MT2 to CoM.

Topics: Amino Acid Sequence; Archaeal Proteins; Bacterial Proteins; Chromatography, Ion Exchange; Isoenzymes; Kinetics; Mesna; Methanosarcina barkeri; Methylamines; Methylation; Methyltransferases; Molecular Sequence Data; Spectrophotometry, Ultraviolet

The disulfide bridge pattern of human alpha 2-macroglobulin (alpha 2M) given earlier (Sottrup-Jensen, L., Stepanik, T. M., Kristensen, T., Wierzbicki, D. M., Jones, C. M., Lønblad, P. B., Magnusson, S., and Petersen, T. E. (1984) J. Biol. Chem. 259, 8318-8327) has been revised by showing that Cys255 and Cys408 in one subunit are bridged to Cys408 and Cys255, respectively, in the adjacent subunit of the proteinase binding dimer. Thus, the alpha 2M-dimer contains two interchain disulfide bridges, and the individual subunits are arranged in an antiparallel fashion. These results are the outcome of partial reduction experiments, where reduction of methylamine-treated alpha 2M with 1-8 mM mercaptoethanesulfonate at pH 8.0 resulted in the appearance of 2.6 mol of SH-groups per mol of free subunit. Apart from reduction of the two interchain bridges, the intrachain bridges Cys228-Cys276, Cys572-Cys748, Cys798-Cys826, and Cys824-Cys860 are reduced to a minor extent under these conditions. The disulfide bridge pattern of alpha 2M has been completed by showing that the alpha 2M subunit contains 11 intrachain bridges, including a bridge connecting Cys447 with Cys540.

Topics: alpha-Macroglobulins; Amino Acid Sequence; Amino Acids; Disulfides; Endopeptidases; Humans; Mesna; Methylamines; Oxidation-Reduction