Membrane-associated phosphatidylglycerophosphate synthetase from Escherichia coli: purification by substrate affinity chromatography on cytidine 5'-diphospho-1,2-diacyl-sn-glycerol sepharose

Hirabayashi, Takashi; Larson, Timothy J.; Dowhan, William

doi:10.1021/bi00669a002

Cited by 101 publications

(92 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Triton X-100 has been used to solubilize the majority of these enzymes (8,10,12,(17)(18)(19)21) while digitonin (1,14), cholate (23), and octylglucoside (16) have been used to a less extent. PI synthase has been solubilized from rat brain (18) and rat liver (23) microsomes with Triton X-100 or cholate.…”

Section: Discussionmentioning

confidence: 99%

Solubilization of Microsomal-Associated Phosphatidylinositol Synthase from Germinating Soybeans

Robinson¹,

Carman²

1982

Plant Physiol.

View full text Add to dashboard Cite

CDP-1,2-diacysn-glycerol (CDP-diacylglycerol):myo-inositol phosphatidyltransferase (EC 2.7.8.11, phosphatidylinositol synthase) catalyzes the final step in the dcnovo synthesis ofphosphatidylinositol in the endoplasmic reticulum fraction of germinating soybeans (Glycine max L. var Cutler 71). A variety of solubilization agents were examined for their ability to release phosphatidylinositol synthase activity from the microsome fraction. The most effective agent to solubiize the enzyme was the nonionic detergent Brij W-1. A 2.1-fold increase in specific activity was achieved using 1% Brij W-1 with 69% activity solubilized.Maximal solubilization of phosphatidylinositol synthase was completely dependent on Brj W-1 (1%), potassium ions (0.3 M), and manganese ions (0.5 mM). Solubilization of the enzyme was not affected by the protein concentration of microsomes between 3 to 20 milligrams per milliliter. Solubilization was not affected by the pH of solubilization buffer between 6.5 to 8.5. To our knowledge, this is the first phospholipid biosynthetic enzyme solubilized from plant membranes. The Brij W-1-solubilized phosphatidylinositol synthase remained at the top of a glycerol gradient, whereas the membrane-associated enzyme sedimented to the bottom of the gradient. Maximal activity of the Brij W-1-solubilized phosphatidylinositol synthase was dependent on manganese (5 mM) or magnesium (30 mM) ions, and Triton X-100 (3.6 mm) at pH 8.0 with Tris-HCI buffer. The apparent K. values for CDP-diacylglycerol and myo-inositol for the solubilized enzyme was 0.1 mm and 46 ,UM, respectively. Solubilized phosphatidylinositol synthase activity was thermally inactivated at temperatures above 30°C.The enzyme responsible for the biosynthesis of PT3 is CDP-1,2-diacyl-sn-glycerol (CDP-diacylglycerol):myo-inositol phosphatidyltransferase (EC 2.7.8.11, PI synthase). PI synthase catalyzes the formation of PI and CMP from CDP-diacylglycerol and myoinositol (15). PI synthase has been identified from the membranes of a variety of plant tissues (5,20,22 membranes. The first step in the purification of a membraneassociate enzyme is the release of the enzyme in a soluble form from the membrane. Solubilization agents such as detergents and bile salts have been employed to release membrane-associated enzymes from cell membranes (11). In this communication, we report the conditions for the solubilization of PI synthase from germinating soybeans in reasonably high yield with good stability. PI synthase is localized in the endoplasmic reticulum of this seed (4). To our knowledge, this is the first report of the successful solubilization of a plant phospholipid biosynthetic enzyme. Therefore, we have paid particular attention to those factors required for the optimal release of PI synthase from soybean membranes. MATERIALS AND METHODSMaterials. All materials were reagent grade or better. PI, myoinositol, BSA, digitonin, sodium cholate, sodium deoxycholate, and Brij W-I (a mixture of 60-65% 20-cetyl ether and 30-35% 10-cetyl ether) were purch...

show abstract

Section: Discussionmentioning

confidence: 99%

Solubilization of Microsomal-Associated Phosphatidylinositol Synthase from Germinating Soybeans

Robinson¹,

Carman²

1982

Plant Physiol.

View full text Add to dashboard Cite

show abstract

“…Not wanting to repeat the difficulties of time and material I experienced with the PS decarboxylase, we capitalized on new approaches to protein purification by developing substrate affinity chromatography methods. For the PGP synthase, we covalently attached the lipid substrate CDP-diacylglycerol to a Sepharose column and eluted the enzyme with its substrate CDP-diacylglycerol (25). PS synthase tightly binds to phosphocellulose, which we specifically eluted using CDP-diacylglycerol (26).…”

Section: Time To Find Gainful Employmentmentioning

confidence: 99%

Understanding phospholipid function: Why are there so many lipids?

Dowhan¹

2017

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

In the 1970s, phospholipids were still considered mere building blocks of the membrane lipid bilayer, but the subsequent realization that phospholipids could also serve as second messengers brought new interest to the field. My own passion for the unique amphipathic properties of lipids led me to seek other, non-signaling functions for phospholipids, particularly in their interactions with membrane proteins. This seemed to be the last frontier in protein chemistry and enzymology to be conquered. I was fortunate to find my way to Eugene Kennedy's laboratory, where both membrane proteins and phospholipids were the foci of study, thus providing a jumping-off point for advancing our fundamental understanding of lipid synthesis, membrane protein biosynthesis, phospholipid and membrane protein trafficking, and the cellular roles of phospholipids. After purifying and characterizing enzymes of phospholipid biosynthesis in Escherichia coli and cloning of several of the genes encoding these enzymes in E. coli and Saccharomyces cerevisiae, I was in a position to alter phospholipid composition in a systematic manner during the cell cycle in these microorganisms. My group was able to establish, contrary to common assumption (derived from the fact that membrane proteins retain activity in detergent extracts) that phospholipid environment is a strong determining factor in the function of membrane proteins. We showed that molecular genetic alterations in membrane lipid composition result in many phenotypes, and uncovered direct lipid-protein interactions that govern dynamic structural and functional properties of membrane proteins. Here I present my personal "reflections" on how our understanding of phospholipid functions has evolved.

show abstract

“…In B. subtilis, a sequential bi-bi mechanism (in a reaction involving two products and two reactants, the presence of one of the products can stimulate an exchange reaction between a product and one of the reactants when the other reactant is present) appears to be followed by the PSS reaction (7). The same mechanism was also proposed for S. cerevisiae PSS and E. coli phosphatidylglycerophosphate synthase (1,14). In contrast, the E. coli PSS reaction has characteristics of ping-pong mechanisms in which one or more products are released before all substrates have been added (22,34).…”

Section: Discussionmentioning

confidence: 73%

The Helicobacter pylori gene encoding phosphatidylserine synthase: sequence, expression, and insertional mutagenesis

Taylor

1997

J Bacteriol

View full text Add to dashboard Cite

The Helicobacter pylori pss gene, coding for phosphatidylserine synthase (PSS), was cloned and sequenced in this study. A polypeptide of 237 amino acids was deduced from the PSS sequence. H. pylori PSS exhibits significant amino acid sequence identity with the PSS proteins found in the archaebacterium Methanococcus jannaschii, the gram-positive bacterium Bacillus subtilis, and the yeast Saccharomyces cerevisiae but none with its Escherichia coli counterpart. Expression of the putative pss gene in maxicells gave rise to a product of ϳ26 kDa, which is in agreement with the predicted molecular mass of 26,617 Da. A manganese-dependent PSS activity was found in the membrane fractions of the E. coli cells overexpressing the H. pylori pss gene product. This result indicates that this enzyme is a membrane-bound protein, a conclusion which is supported by the fact that the PSS protein contains several local hydrophobic segments which could form transmembrane helices. The pss gene was inactivated with a chloramphenicol acetyltransferase cassette on the plasmid. However, an isogenic pss gene-disrupted mutant of H. pylori UA802 could not be obtained, suggesting that this enzyme plays an essential role in the growth of this organism.

show abstract

Membrane-associated phosphatidylglycerophosphate synthetase from Escherichia coli: purification by substrate affinity chromatography on cytidine 5'-diphospho-1,2-diacyl-sn-glycerol sepharose

Cited by 101 publications

References 36 publications

Solubilization of Microsomal-Associated Phosphatidylinositol Synthase from Germinating Soybeans

Solubilization of Microsomal-Associated Phosphatidylinositol Synthase from Germinating Soybeans

Understanding phospholipid function: Why are there so many lipids?

The Helicobacter pylori gene encoding phosphatidylserine synthase: sequence, expression, and insertional mutagenesis

Contact Info

Product

Resources

About