Differential expression of the two methyl‐coenzyme M reductases in Methanobacterium thermoautotrophicum as determined immunochemically via isoenzyme‐specific antisera

Abstract

Methanobacterium thermoautotrophicum contains two isoenzymes of methyl-coenzyme M reductase (MCR), MCR I and MCR II, which catalyze the methane-forming step and which together represent more than 10% of the cellular protein. We describe here the preparation of isoenzyme-specific antisera against the two MCR isoenzymes and their use in the quantitative immunochemical determination of the two isoenzymes in the methanogen. The relative and absolute cellular concentration of the two proteins is shown to be strongly affected by growth conditions such as the temperature, pH, and substrate concentration. Conditions were found yielding cells which contained essentially only MCR I or MCR II. Using antisera against MCR I and MCR II, MCR from other methanogens were immunochemically compared. Evidence is presented that Methanobacterium wolfei also contains two isoenzymes of MCR.

Properties of the two isoenzymes of methyl‐coenzyme M reductase in Methanobacterium thermoautotrophicum

“…The availability of isoenzyme-specific antisera was exploited to permit the separation of the two isoenzymes (Bonacker et al, 1992). With these antisera, conjugated to Sepharose CL-4B beads, it was possible to achieve the quantitative adsorption of either one of the isoenzymes from the cell extract, resulting in a supernatant containing the active form of the non-adsorbed isoenzyme.…”

“…The respective non-adsorbed isoenzyme was purified from the supernatant (9 ml) at 0°C by fractionated ammonium sulfate precipitation (70-100% saturation) under an atmosphere of 5% H,/95% N,. The preparations thus obtained contained only one of the two isoenzymes, as revealed by SDS/PAGE and by analysis via the radial immunodiffusion technique (Mancini et al, 1965) as described by Bonacker et al (1992).…”

“…The antibodies specific for MCR I and MCR I1 were prepared from isoenzyme-specific rabbit antiserum obtained as described previously (Bonacker et al, 1992). 30 ml antiserum was applied to a DE 32 anion-exchange column (Whatman) which had previously been equilibrated with 30 mM sodium phosphate, pH 7.0.…”

“…Southern-blot analyses indicate the presence of two related but clearly separate MCR operons in the genomic DNA (Reeve, 1992). Evidence has been presented that MCR I is the predominant form when growth of the methanogen is limited by the rate of the H, and CO, supply and that MCR I1 predominates when the concentration of H2 and of CO, is not growth-rate limiting (Bonacker et al, 1992). It was not clear why this Archaeon uses different iso-…”

“…It was not clear why this Archaeon uses different iso-enzymes under different growth conditions. It was assumed that the two isoenzymes differ in their catalytic properties (Bonacker et al, 1992).…”