Enzymes of Phospholipid Metabolism in the Endoplasmic Reticulum of Castor Bean Endosperm

Moore, Thomas; Lord, J. Michael; Kagawa, T.; Beevers, Harry

doi:10.1104/pp.52.1.50

Cited by 105 publications

(52 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Phosphatidic acid phosphatase has been reported to be located predominantly in ER but may also be found in all other fractions (26). It is usually not discussed as a potential contributor to misleading phospholipid data of plant membranes although a sequential action of phospholipase D and phosphatidic acid phosphatase is well known (19).…”

Section: Methodsmentioning

confidence: 99%

“…The concomitant increase in the phosphatidic acid containing fractions of silica gel matches the decrease in intact phospholipids only in membrane fractions C and D. Thus, a second activity is suggested to be present in addition to that of phospholipase D. To test this possibility, we monitored phosphatidic acid phosphatase which has been described to be most active in the ER but is active also in other membrane compartments (26). Table II shows that phosphatidic acid phosphatase activity is mainly located in our fractions A and B to account for the additional degradative changes in these fractions.…”

Section: Methodsmentioning

confidence: 99%

“…The phosphatidic acid phosphatase assay was as described (26) (31,38). The airdried plates were stained with iodine vapor.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Action and Inhibition of Endogenous Phospholipases during Isolation of Plant Membranes

Scherer

Morré²

1978

Plant Physiol.

View full text Add to dashboard Cite

Endogenous phospholipase D and phosphatidic acid phosphatase activities were demonstrated in membrane fractions isolated from soybean (Glycine max L.) hypocotyls. Phospholipase D activity was distributed widely among different membrane fractions while phosphatidic acid phosphatase was found predominantly in membranes equilibrating in lower sucrose densities. Phospholipase D action was unaffected by ethylenediaminetetraacetic acid, sodium salt or ethylene glycol-bis(fl-aminoethyl ether)-N,N'-tetraacetic acid but was prevented by a mixture of 4% choline and 4% ethanolamine. Phosphatidic acid phosphatase was inhibited by 10 millimolar glycerol 1-phosphate or by homogenization media prepared with coconut milk as a solvent instead of water. Under fuily protected conditions the phospholipid composition of soybean membrane fractions remained unchanged for at least 1 hour at 20 C. Membranes prepared under protected conditions had low phosphatidic acid contents and the phosphoipid compositions closely resembled those of corresponding animal membranes.Quantitative comparisons of the phospholipid compositions of different membrane fractions have been complicated by poor recoveries of individual phospholipids and degradative alterations (22,31). As a part of ongoing investigations of the in vitro interactions of plant membranes with plant growth hormones, we were prompted to undertake a detailed investigation of the stability of isolated plant membranes in cell homogenates. We found that phospholipids of membranes from soybean hypocotyls under investigation were surprisingly susceptible to degradation. Both phospholipase D and phosphatidic acid phosphatase appeared to contribute. Subsequently, a procedure was developed to inhibit phospholipid degradation by these enzymes. The procedure kept membranes stable even at room temperature so that physiological effects of hormones could be studied with less drastically altered membrane preparations.The phenomenon of rapid phospholipid degradation we describe here is probably common for isolated plant membranes with some clear exceptions (9,10 the hypocotyls (about 1 cm long) were used for membrane isolations.Membrane Isolations. The isolation medium contained 0.1 M K-phosphate (pH 6.5), 20 mM EDTA2 or EGTA, and 0.5 M sucrose in freshly prepared and filtered coconut milk. Hypocotyls were homogenized for 45 sec with a Polytron 20 ST (Kinematica, Lucerne, Switzerland) operating at about 5,000 rpm. Membranes from about 80-g bean hypocotyls were isolated as described (40). Four membrane fractions were collected finally from a discontinuous coconut milk-sucrose gradient and designated A, B, C, and D. (40). For protection against lipid degradation 4% choline (w/v), 4% ethanolamine (v/v), and 10 mm glycerol-1-P were added to all solutions.Assays. Membrane pellets of the individual gradient fractions were resuspended with an all-glass homogenizer of the PotterElvehjem type in either 6 ml (experiment in Fig. 1) or 18 ml (all other experiments) ofassay buffer. The standard incubation ...

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Action and Inhibition of Endogenous Phospholipases during Isolation of Plant Membranes

Scherer

Morré²

1978

Plant Physiol.

View full text Add to dashboard Cite

show abstract

“…Phosphatidylcholine [ 11, phophatidylinositol [ 21, phosphatidylserine [2] and phosphatidylglycerol [3] are all synthesized by this cellular fraction, and as important structural components of mitochondrial and glyoxysomal membranes [4], establish a role for the endoplasmic reticulum during the biogenesis of these organelles.…”

Section: Introductionmentioning

confidence: 99%

Development of phospholipid synthesizing enzymes in castor bean endosperm

Bowden¹,

Lord²

1975

FEBS Letters

View full text Add to dashboard Cite

“…Although this enzyme was first demonstrated in plants by Kates (19) in 1955, since then it has been characterized extensively with animal tissues (2). Nevertheless, since the 1970s plant phosphatidate phosphatases have been studied with seedlings of castor bean (25), broad bean (20), and mung bean (11). Douce and coworkers (4,18) characterized the chloroplastic phosphatidate phosphatase of spinach leaves, and demonstrated that it is localized in the inner envelope.…”

mentioning

confidence: 99%

Microsomal Phosphatidate Phosphatase in Maturing Safflower Seeds

Ichihara¹,

Norikura²,

Fujii³

1989

Plant Physiol.

View full text Add to dashboard Cite

An assay system comprising sodium phosphatidate, phosphatidylcholine, and bovine serum albumin has been developed for the reproducible determination of phosphatidate phosphatase activity in maturing seeds of safflower (Carthamus tinctorius L.). The activity was detected in both membrane and soluble fractions, and the microsomal phosphatidate phosphatase was characterized. The optimum pH for Pi release was 6.7, and the activity depended on the concentration of Mg2 . Phosphatidylcholine and bovine serum albumin stimulated the phosphatase reaction. This phosphatase was highly specific for phosphatidate; lysophosphatidate, and water-soluble phosphate esters did not serve as substrate. The specific activity was approximately 20 nanomoles per minute per milligram of protein, which was close to that of glycerol-phosphate acyltransferase and higher than that of diacylglycerol acyltransferase. Furthermore, the activity per seed was enough to account for the rate of triacylglycerol accumulation in vivo. The step of diacylglycerol formation by phosphatidate phosphatase does not appear to be rate-limiting for triacylglycerol synthesis during seed maturation.Phosphatidate phosphatase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) is a key enzyme of glycerolipid biosynthesis, and catalyzes the hydrolysis of phosphatidate to 1,2-diacylglycerol and Pi. The diacylglycerol product is not only the direct precursor of triacylglycerol but also a substrate for the synthesis of membrane glycerolipids. Although this enzyme was first demonstrated in plants by Kates (19) In mammalian tissues, the phosphatidate phosphatase reaction is considered to be the rate-limiting step of triacylglycerol synthesis (2). We will discuss whether this is also the case for maturing safflower seeds.To assay the activity of phosphatidate phosphatase with good reproducibility, we have developed an effective phosphatidate substrate, the preparative procedure for which is also described. MATERIALS AND METHODS Plant Material and Preparation of MicrosomesMaturing seeds of safflower (Carthamus tinctorius L.) were harvested 14 to 15 d after flowering when '4C-labeled acetate was most rapidly incorporated into diacylglycerol (15), and cotyledons were homogenized in 10 mm Tris-HCl (pH 7.0) containing 0.4 M sorbitol. The homogenate was centrifuged at 5000g for 10 min, and then the supernatant obtained was centrifuged at 100,000g for 1 h. The precipitate was suspended in the same buffer and stored at -20°C. Protein was determined by the method of Lowry et al. (21)

show abstract

Enzymes of Phospholipid Metabolism in the Endoplasmic Reticulum of Castor Bean Endosperm

Cited by 105 publications

References 29 publications

Action and Inhibition of Endogenous Phospholipases during Isolation of Plant Membranes

Action and Inhibition of Endogenous Phospholipases during Isolation of Plant Membranes

Development of phospholipid synthesizing enzymes in castor bean endosperm

Microsomal Phosphatidate Phosphatase in Maturing Safflower Seeds

Contact Info

Product

Resources

About