The biosynthesis of mannosyl-1-phosphoryl-polyisoprenol in Micrococcus lysodeikticus and its role in mannan synthesis.

as a-D-mannosyl-(l -# 3')-diglyceride and a-D-mannosyl-(l 3)-a-D-mannosyl(1 3')-diglyceride.1 In this communication, the results of studies on the structure of the third mannose-containing lipid are presented. All the available evidence indicates that the structure of this compound is mannosyl-1-phosphorylpolyisoprenol (MPP). Preliminary experiments suggest that IMPP serves as an intermediate in the enzymatic transfer of mannosyl groups from GDP-mannose to mannan. Methods.-Enzyme preparation: A particulate preparation that catalyzes the synthesis of MPP, mannan, and mannosyldiglyceride was obtained by centrifugation of a crude, cell-free extract of M. lysodeikticus' at 100,000 X g for 1 hr. The MPP synthetase was prepared from the crude extract as follows: Crude extract (60 ml) was heated at 40° for 20 min. The pH of the solution (at 00) was adjusted to 5.5 with 1 N acetic acid, and 15.7 gm of (NH4)2SO4 was added with stirring. After 30 min, the precipitate was collected by centrifugation, dissolved in 0.02 M Tris-HCI, pH 7.6 (buffer A), and dialyzed overnight against 2 liters of buffer A. The dialyzed preparation (64 ml) was added with rapid stirring to 550 ml of acetone at -25°. The resultant precipitate was washed successively with 300 ml each of acetone and diethyl ether at -25° and then dried in vacuo for 2 hr (1.35 gm dry wt). Prior to use, 300 mg of acetone powder was suspended in 8.0 ml of buffer A and stirred at 0° for 45 min. Insoluble material was removed by centrifugation at 6000 X g for 5 min. Preparation and purification of MPP: To 12.5 mmoles of Tris-maleate, pH 8.5; 10 mmoles of MgCl2; and 0.5 mmole of Tris-HCl, pH 7.6, in 60 ml of H20 were added 60 ml of a solution of acetone powder enzyme, 75 ml of a sonicated suspension of 1.45 gm of total lipids from M. lysodeikticus,l and 2 ml of GDP-mannose-U-C"4 (14.72 ,umoles, specific activity 2.45 X 105 cpm/,umole). After incubation at 370 for 2 hr, the reaction was terminated and the lipids were extracted as previousyl described.' The lipid (2.0 X 106 cpm, 8.15 ,umoles of mannose) was applied to a silicic acid column (60 gm) and eluted successively with 1000 ml of CHCl3, 600 ml of acetone, and 600 ml CHCls-CH30H, 1:1. Crude MPP, quantitatively recovered in the last fraction, was dissolved in CHCl-CH30H, 2:1 (CM) and applied to a DEAE-cellulose column (4.5 X 30 cm) prepared in CM. The column was eluted with 2400 ml of CM, 700 ml of CH30H, and 700 ml of CM containing 84 ml of concentrated NH40H. All of the radioactive lipid was recovered in the acidic lipid fraction eluted with the last solvent. This fraction was evaporated to dryness and dissolved in 16 ml of CHC13-CH30H, 1:4. After addition of 1.5 ml of 1 N NaOH, the solution was incubated at 370 for 15 min. Then 1.5 ml of 1 N acetic acid, 30 ml of CHCl3-CH30H, 9:1, 15 ml of isobutanol, and 30 ml of H20 were added and the solution was mixed vigorously. The aqueous layer was discarded and the CHCl3 layer was washed with 15 ml of H20-CH30H, 2:1, and then evaporated to dryness. The C14-MPP (1.72 X 106 cpm, 7.0 ,umoles) was applied to a DEAE-cellulose column and