Abstract
Rat liver microsomes catalyzed the formation of A,E,E-geranylgeranyl diphosphate from farnesyl diphosphate and isopentenyl diphosphate in the presence of Triton X-100. Studies on product specificity using various primers such as Z,E-farnesyl diphosphate, E,E-farnesyl diphosphate, Z,E,E-geranylgeranyl diphosphate, E,E,E-geranylgeranyl diphosphate, Z,E,E,E-geranylfarnesyl diphosphate, and E,E,E,E-geranylfarnesyl diphosphate suggested that the microsomal dehydrodolichyl diphosphate synthase has such properties that it releases Z,E,E-geranylgeranyl diphosphate, the first intermediate, in the reactions with farnesyl diphosphate as the starting primer. Metabolic labeling of rat liver slices with [2-3H]mevalonic acid revealed the accumulation of E,E,E-geranylgeranyl (di)phosphates as well as dolichyl (di)phosphate (C85 and C90) and dehydrodolichol (C85 and C90), but no accumulation of Z,E,E-geranylgeranyl (di)phosphate or E,E-farnesyl (di)phosphate was detected. Microsomal enzyme preparations from mouse liver and hamster liver also produced Z,E,E-geranylgeranyl diphosphate from farnesyl diphosphate and isopentenyl diphosphate.
Highlights
Rat liver microsomes catalyzed the formation of 2, to c80 and
Dehydrodolichyl diphosphate synthase hasbeen found in animal diphosphate,E,E,E-geranylgeranyldiphosphate,Z,E,E-geranylgerany1 diphosphate, Z,E,E,E-geranylfarnesyl diphosphate, and E,E,E, E-geranylfarnesyl diphosphate were prepared as described in a pretissuessuchas chickenliver [5],hen oviduct [6], Ehrlich vious paper [3] or according to Poulter’s method [11]M. ale Spragueascitestumor cells [7], rat liver [8], andtestes [9]
E,E,E,E-Geranylfarnesyl-PP 13,250 3,12652 somes from Rat, Mouse, and Hamster Livers-To better understand whether or not the formationof Z,E,E-geranylgeranyl diphosphate in thepresence of Triton X-100 is specific for rat liver microsomes, we compared the microsomal enzymes prepared from rat,mouse, and hamsterlivers
Summary
To thecorresponding monophosphate, which is responsible for lipopolysaccharide or peptidoglycanbiosynthesis. Chloromicrosomes in the presence of Triton X-100 were subjected form:Methanol(2:1) Extracts-We compared butanol soluble to reversed-phase TLC (Fig, lA), two radioactivity spots were products with chloroform:methanol(21s)oluble products deobserved as described previously[10].One spot corresponding rived from the same incubation mixture. When diphosphate was used as anallylic substrate instead of E,E- a fraction containing products not bound toDEAE-cellulose farnesyl diphosphate in the presence of Triton X-100, two was subjected to silica gel TLCinsolvent I, three major radioactivity spots were observed, withtheshorterchain radioactivity peaks were observed corresponding to squalene, length prenols having slightly larger mobility than reference coenzyme Qlo and cholesterol. In the coafsZe,E-farnesyl diphos- solvent IV, no radioactivity peaks corresponding to CIS and phate or Z,E,E-geranylgeranyl diphosphate, a radioactivity spot corresponding to a shorter prenol was observed but it was smaller than thadterived from all-Eseries of substrates. When radioactive hydrolysates formed by acipdhosphatase treatment of charged products eluted from DEAE-cellulose by 6 M CH,COONH, saturated butano1:methanol (1O:l)were
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