The Structure and Biosynthetic Mechanism of the Monomolecular Membrane made of Tetraether Type of Polar Lipids in Methan Menic Bacteria

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A pseudomurein-degrading enzyme from Methanobacterium wolfei autolyzate was partially puried to prepare protoplasts of Methanobacterium thermoautotrophicum. An improved assay method of the enzyme using purified cell walls of M. Thermoautotrophicum as a substrate made it possible to determine the enzyme activity quantitatively and to characterize its properties. The optimum pH and temperature were 6.8 - 7.4 and 75 ^ﾟC, respectively. Incubation of this enzyme in the absence of the substrate cell walls at 70 ^ﾟC for 10 min caused loss of half of the activity and heating at 85 ^ﾟC for 10 min inactivated it completely. However, the enzyme activity was recovered more or less by the incubation with the substrate at 62^ﾟC for14 hr even after heat treatment at 100^ﾟC for 30 min. Although the pseudomurein-degrading enzyme was oxygen-sensitive (active only under the anoxic condition), it was also active after storage in the presence of air without reducing agents if it was assayed under the anoxi … More c condition. On the basis of this property the enzyme was able to be purified under normal conditions in the presence of air. Intact cells, protoplasts, and vesicles prepared by passing of a French-pressure cell, of M. Thermoautotrophicum were reacted with trinitrobenzene sulfonic acid and total lipid was extracted. The trinitrophenylated amino lipids and the unreacted lipids were separated by use of thin-layer chromatography and percentage of trinitrophenylation was determined by phosphorus measurement. The vesicles prepared with a French-pressure cell is believed to be inside-out. Archaeitidylethanolamine was almost equally distributed between innerand outer leaflets of the membrane. Approximately 20 % of the amino group of gentiobiosyl caldarchaetidylethanolamine (tetraether type of phosphoglycolipid) oriented to the outside of the membrane. Most (90 %) of the amino groups of archaetidylserine and gentiobisyl caldarchaetidylserine were distributed inner leaflet. This gave another line of evidence for the heptad hypothesis which postulated that tetraether polar lipids were synthesized from diether polar lipids in the membrane without reorientation of those lipids. Less