Use of [1 or 3-3H, U-14c]glucose to estimate the synthesis of glycerolipids via acyl dihydroxyacetone phosphate

Abstract

21-cI3 fibroblasts were incubated with [I-3H, U-14C]glucose or 14C]glucose to produce intracellular [3H]NADPH via the phosphogluconate pathway.3H and 14C were then determined at the th~ee positions of glycerol in glycerol phosphate, saponifiable glycerolipids, alkyl ether glycerolipids and plasmalogens.The 3H/14C ratio at C-2 in glycerol of saponifiable glycerolipids is 2-10 fold greater than in glycerol phosphate and approaches the ratio found in ether-containing glycerolipids.This suggests that a significant fraction of the glycerolipids in BHK-21-cI3 fibroblasts is synthesized via acyl dihydroxyacetone phosphate.There are at least two pathways through which lipid glycerol can be syni thesized from glycolytic intermediates -the glycerol phosphate (GP) and acyl dihydroxyacetone phosphate (DHAP) I pathways (1-3). The GP pathway preferentially consumes NADH (4) whereas the acyl DHAP pathway uses NADPH (5). This difference has previously been used to distinguish glycerolipid synthesis via the two pathways in disrupted cell preparations (1,2) but not as yet in intact cells. We report here the use of [1-3H, U-14C]glucose and [3-3H, U-14C] glucose to estimate the relative importance of the two pathways in intact BHK-21-c13 fibroblasts. Both [l-3H]glucose and [3-3H]glucose are expected to provide intracellular [3H]NADPH via the phosphogluconate oxidative pathway and both have been previously used to measure NADPH metabolism within cells (6-9). 3 Glycerolipid synthesized via acyl DHAP should contain H at C-2 of glycerol whereas that synthesized via GP should not. Ether-containing glycerolipids are thought to be synthesized only via acyl DHAP, so NADPH is an obligatory 1 Abbreviations: GP, sn glycerol-3-phosphate; DHAP, dihydroxyacetone phosphate. 658 Copyr~ht© 19~byAcademicPress,~ Allrightsofreproduction m any~rm rese~ed.

Glycerolipid biosynthesis in Saccharomyces cerevisiae: sn-glycerol-3-phosphate and dihydroxyacetone phosphate acyltransferase activities

“…Yeast membranous acyl-DHAP oxidoreductase activity. Acyl-DHAP is an important intermediate in the biosynthesis of ether lipids (6,25,27,38,40,49,57). Although we observed a significant DHAP acyltransferase activity in membranes from S. cerevisiae, other workers have been unable to detect any trace of neutral or ionic alkoxylipids in this organism (34).…”

“…Results are expressed as counts per minute of radioactive lipid product migrating similar to acyl-DHAP, lysophosphatidic acid (LPA), and phosphatidic acid (PA). controversial (9, 53, 5F, 57) despite the numerous investigations addressing this question (1, 18,35,37,[38][39][40]43). In previous studies of several tissues that prominently synthesize lipids (45, 46), we presented evidence strongly supporting the hypothesis that the microsomal glycerol-P and DHAP acyltransferase activities actually constitute dual catalytic functionm of a single enzyme.…”

“…Recent studies on the biosynthesis (12, 26, 49) and reduction (13,16,27,49) of acyl-dihydroxyacetone phosphate (acyl-DHAP) in liver particulate fractions have led to the proposal of a separate, independent route (the DHAP pathway) that allows DHAP to serve as a precursor for the backbone of glycerolipid. Despite extensive work (1, 18,35,37,[38][39][40]43), the relative contributions of the glycerol-P and DHAP pathways to phospholipid and triacylglycerol synthesis remain to be established in eucaryotic systems (53,55).…”

Συμβολη Στη Μελετη Της Βιοσυνθεσης Γλυκεριναιθερων Στο Πρωτοζωο Crithidia Fasciculata

“…Ή άποψη δμως πού επικρατεί σύμφωνα και με τα προη γούμενα δεδομένα, άλλα κυρίως με βάση νεώτερα πειρά ματα in vitro, είναι οτι οι άλκυλογλυκεριναιθέρες σχηματί ζονται από DL -3 -φωσφορική γλυκεριναλδεόδη (GAI -3Ρ) 121 (1) ή φωσφορική διυδροξυακετόνη (DHAP) 122 (2) ή άκυλιωμένη φωσφορική διυδροξυακετόνη 104 , 123 , σαν πρόδρο μες ενώσεις γλυκερινικής ρίζας και από λιπαρή αλκοόλη σαν πρόδρομη ένωση της άλειφατικής ρίζας. Ή συμπύκνω ση των δύο αυτών υποστρωμάτων γίνεται με μια πολύπλοκη αντίδραση ,πού απαιτεί ATP, Mg++, CoA, γλουταθειόνη ή άλλο ανάλογο αντιδραστήριο και NADPH σύμφωνα με τήν πορεία πού περιγράφεται στο Σχήμα 1 δπου φαίνεται οτι δεν έχει ακόμα ξεκαθαριστεί ό ρόλος των παραπάνω συμπαραγόντων στην αντίδραση.…”