The 3-ketodihydrosphingosine synthetase of Bacteroides melaninogenicus: Partial purification and properties

Lev, M.; Milford, Albert F.

doi:10.1016/0003-9861(81)90384-2

Cited by 28 publications

(18 citation statements)

References 11 publications

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“…3). This is comparable to the values reported for the enzyme from other sources, which are in the range of 0.3 to 1.1 m (Lev and Milford, 1981;Williams et al, 1984;Merrill et al, 1988;Holleran et al, 1990). To allow sufficient recovery of radioactivity in the assay extract for accurate measurement of enzyme activity while minimizing the amount of [3H]Ser used, assays were routinely performed using a Ser concentration of 2 rrw and containing 1 pCi of [3H]Ser.…”

Section: Substrate Requirements and Kinetic Analysissupporting

confidence: 89%

“…The activity of Ser palmitoyltransferase from squash is the highest reported to date for any microsomal preparation from a complex tissue (Holleran et al, 1990) and is higher than the activity reported for a crude preparation from B. melaninogenicus (Lev and Milford, 1981) and preparations from yeast (based on our calculations from results reported by DiMari et al, 1971). Presumably, this level of activity is required to maintain the sphingolipid content of the cell membranes and to meet the biosynthetic demands of growth.…”

Section: Discussionmentioning

confidence: 45%

“…The squash enzyme appears similar to Ser palmitoyltransferase from other sources with respect to temperature and pH optima, kinetics, substrate specificity, and subcellular distribution (DiMari et al, 1971;Lev and Milford, 1981;Williams et al, 1984;Merrill et al, 1985;Holleran et al, 1990).…”

Section: Discussionmentioning

confidence: 83%

“…Early studies of Ser palmitoyltransferase using preparations from the yeast Hansenula cifferi (Braun and Snell, 1968;Stoffel et al, 1968a;Snell et al, 1970;DiMari et al, 1971), rat liver (Stoffel et al, 1968a;Stoffel, 1970), and brain (Braun et al, 1970;Kanfer and Bates, 1970) demonstrated that Ser and palmitoyl-COA are substrates for the reaction, J-ketosphinganine is the immediate product in the absence of NADPH, and pyridoxal 5'-P is required for activity. Ser palmitoyltransferase partially purified from the anaerobic bacterium Bacteroides melaninogenicus was found to have properties similar to the eukaryotic enzyme (Lev and Milford, 1981).…”

mentioning

confidence: 99%

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Sphingolipid Long-Chain Base Synthesis in Plants (Characterization of Serine Palmitoyltransferase Activity in Squash Fruit Microsomes)

Lynch

Fairfield

1993

Plant Physiol.

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Section: Substrate Requirements and Kinetic Analysissupporting

confidence: 89%

Section: Discussionmentioning

confidence: 45%

Section: Discussionmentioning

confidence: 83%

mentioning

confidence: 99%

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Sphingolipid Long-Chain Base Synthesis in Plants (Characterization of Serine Palmitoyltransferase Activity in Squash Fruit Microsomes)

Lynch

Fairfield

1993

Plant Physiol.

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“…Subsequent work has indicated that SPT may be rate limiting in LCB synthesis in various animal tissues (10,16). SPT has not been obtained in highly pure form from any source, although partial purification from the obligate anaerobe Bacteroides melaninogenicus has been achieved (14). Little is known about the regulation of LCB synthesis in any tissue, a question that will receive increased attention since exogenous sphingolipid LCBs exhibit a variety of profound effects on animal cells (9,11).…”

Section: Discussionmentioning

confidence: 99%

Characterization of enzymatic synthesis of sphingolipid long-chain bases in Saccharomyces cerevisiae: mutant strains exhibiting long-chain-base auxotrophy are deficient in serine palmitoyltransferase activity

1992

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We have begun a biochemical-genetic analysis of the synthesis of sphingolipid long-chain bases in Saccharomyces cerevisiae and found evidence for the occurrence of serine palmitoyltransferase (SPT) and 3-ketosphinganine reductase, enzymes that catalyze the initial steps of the pathway in other organisms. SPT activity was demonstrated in vitro with crude membrane preparations from S. cerevisiae as judged by the formation of radiolabeled 3-ketosphinganine from the condensation of palmitoyl-coenzyme A (CoA) with radiolabeled serine. Shorter (C12 and C14) and longer (C18) acyl-CoAs sustain significant SPT activity, a result consistent with the finding of both C18 and C20 long-chain bases in the organism. Three products of the long-chain-base synthetic pathway, 3-ketosphinganine, erythrosphinganine, and phytosphingosine, neither directly inhibited the reaction in vitro nor affected the specific activity of the enzyme when these bases were included in the culture medium ofwild-type cells. Thus, no evidence for either feedback inhibition or repression of enzyme synthesis could be found with these putative effectors. Mutant strains of S. cerevisiae that require a sphingolipid long-chain base for growth fall into two genetic complementation groups, LCB1 and LCB2. Membrane preparations from both kcbl and -b2 mutant strains exhibited negligible SPT activity when tested in vitro.Step 2 of the long-chain-base synthetic pathway was demonstrated by the stereospecific NADPHdependent reduction of 3-ketosphinganine to erythrosphinganine. Membranes isolated from wild-type cells and from an kcbl mutant exhibited substantial 3-ketosphinganine reductase activity. We conclude that the Lcbphenotype of these mutants results from a missing or defective SPT, an activity controlled by both the LCBI and LCB2 genes. These results and earlier work from this laboratory establish that SPT plays an essential role in sphingolipid synthesis in S. cerevisiae.Fungi and plants contain sphingolipids that are distinguished from animal sphingolipids; they contain phosphoinositol as part of their polar head groups with phytosphingosine (PHS) as the major long-chain-base (LCB) component (1,8). Such sphingolipids have been shown to be highly localized in the plasma membrane of Saccharomyces cerevisiae (17). To study the metabolism and function of sphingolipids in S. cerevisiae, we isolated mutant strains, termed Lcb-, auxotrophic for sphingolipid LCBs. The Lcb-strains fell into two genetic complementation groups, termed LCBI and LCB2 (18). Without an appropriate LCB, these strains were unable to grow and synthesize sphingolipid (18, 27) and rapidly lost viability (18); these data suggested one or more vital roles for the yeast sphingolipids.No information is available concerning the initial steps of sphingolipid LCB synthesis in S. cerevisiae. We have characterized the first two steps of LCB synthesis (Fig. 1) as catalyzed by isolated membranes from S. cerevisiae. These in vitro enzymatic studies indicate that step 1, carried out by serine palmitoyltransfe...

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Serine C-palmitoyltransferase

Springer Handbook of Enzymes

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The 3-ketodihydrosphingosine synthetase of Bacteroides melaninogenicus: Partial purification and properties

Cited by 28 publications

References 11 publications

Sphingolipid Long-Chain Base Synthesis in Plants (Characterization of Serine Palmitoyltransferase Activity in Squash Fruit Microsomes)

Sphingolipid Long-Chain Base Synthesis in Plants (Characterization of Serine Palmitoyltransferase Activity in Squash Fruit Microsomes)

Characterization of enzymatic synthesis of sphingolipid long-chain bases in Saccharomyces cerevisiae: mutant strains exhibiting long-chain-base auxotrophy are deficient in serine palmitoyltransferase activity

Serine C-palmitoyltransferase

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