380
Biochrmicrr et B~ophysrca Am,
1042
(1990)380- 385 Elsevier
BBALIP
53330
Stimulation of CTP : phosphocholine cytidylyltransferase and phosphatidylcholine synthesis by incubation of rat hepatocytes with phospholipase A, Jasbinder
S. Sanghera
Lipid and Lipoprotein Research Group and The Deportment
(Revised
Key words:
Phosphocholine
cytidylyltransferase;
and Dennis E. Vance of Biochemistry,
(Received 17 July 1989) manuscript received 24 October
Enzyme translocation; (Rat hepatocyte)
University
of Alberta,Edmonton (Canada)
1989)
Phosphatidylcholine
synthesis;
Phospholipase
A *;
The effect of phospholipase A, treatment of rat hepatocytes on CTP: phosphocholine cytidylyltransferase and phosphatidylcholine synthesis was investigated. Cytidylyltransferase is recovered from the cytosol and in a membranebound form with the microsomes. Digitonin treatment of cells causes rapid release into the medium of the cytosolic, but not the microsomal form of the cytidylyltransferase. Incubation of hepatocytes for 10 min with phospholipase A, (0.9 units/dish) in the medium, resulted in a 33% decrease in the cytidylyltransferase activity released by digitonin treatment (2.5 f 0.15 nmol/min per mg compared to 3.9 f 0.10 nmol/min per mg in the control). In agreement with the digitonin experiments, incubation with 0.9 units/dish of phospholipase A, resulted in a decrease in the cytidylyltransferase activity in the cytosol (from 4.3 f 0.10 nmol/min per mg to 2.6 f 0.14 nmol/min per mg) and a corresponding increase in the microsomaf fraction (from 0.9 f 0.16 nmol/min per mg to 1.8 f 0.20 nmol/min per mg). The effect of phospholipase A 2 on cytidylyltransferase translocation was concentration- and time-dependent. Incubation of hepaotcytes in the presence of phospholipase A, (0.9 units/dish) for 10 min prior to pulse-chase experiments resulted in an increase in radiolabel incorporation into phosphatidylcholine (from 2.4 f 0.02 10 -s dpm/dish to 3.1 f 0.1 - 10 -5 dpm/dish) and a corresponding decrease in radiolabel associated with the choline (from 2.5 f 0.05 10 -’ to 1.4 f 0.03 * 10 -’ dpm) and phosphocholine fractions (from 8.5 f 0.07 * 10 -5 to 6.9 f 0.05 * 10 -5 dpm). We activity and conclude that phospholipase A 2 can cause a stimulation of CTP : phosphocholine cytidylyhransferase phosphatidylcholine synthesis in cultured rat hepatocytes. l
l
Introduction CTP : phosphocholine cytidylyltransferase (EC 2.7.7.15) catalyzes the conversion of CTP and phosphocholine to form CDP-choline. Studies involving pulsechase experiments with hepatocytes [l], isolated type II pneumocytes from adult [2] and fetal [3] lung, a myoblast cell line L, [4], and HeLa cells [5] indicated that this was the rate-limiting reaction in the incorporation of radioactive choline into PC. Cytidylyltransferase is
Abbreviations: DTT, dithiothreitol; GPC, glycerophosphocholine; LPC, lysophosphatidylcholine; PC, phosphatidylcholine; PMSF, CTP, phosphocholine cytidyphenylmethylsulfonyl fluoride; lyltransferase; DMEM, Dulbecco’s modified Eagle’s medium. Correspondence: and Department Alberta, Canada, 0005-2760/90/$03.50
D.E. Vance, Lipid and Lipoprotein Research Group of Biochemistry, University of Alberta, Edmonton, T6G 2S2. 4 1990 Elsevier Science Publishers
B.V. (Biomedical
recovered from cells in two interconvertible forms; an inactive cytosolic form and an active microsomal form [6-91. The cytosolic form and the pure enzyme can be fully activated by anionic phospholipids and by PC-fatty acid vesicles [lo-121. It is now well established that the activity of the cytidylyltransferase is regulated by several different mechanisms [13]. PC synthesis and cytidylyltransferase activity are inhibited by treatment of hepatocytes with CAMP analogues [14] and evidence for regulation of cytidylyltransferase by reversible protein phosphorylation was obtained [15]. More recently, pure cytidylyltransferase has been demonstrated to be regulated in vitro by reversible phosphorylation and serves as a substrate for CAMP-dependent protein kinase [16]. Phosphate (32P) was incorporated into serine residue(s) on the cytidylyltransferase and this led to inhibition of the cytidylyltransferase activity by translocation of the enzyme from a lipid membrane fraction to the superDivision)
381 natant fraction. Treatment of the enzyme with alkaline phosphatase caused cytidylyltransferase to bind to membranes [16]. Thus, regulation of cytidylyltransferase and PC synthesis by phosphorylation/dephosphorylation seems well established. Another factor which has been reported to regulate cytidylyltransferase activity is free fatty acids [8,17,18]. Addition of long-chain unsaturated fatty acids such as oleic acid to hepatocytes stimulates cytidylyltransferase activity by translocation of the enzyme from the cytosolic fraction to the microsomal fraction. A third mechanism for regulation of cytidylyltransferase translocation is via diacylglycerol [18,19], which has been shown to cause binding of cytidylyltransferase to membranes. A possibly related observation is that treatment of cells with phospholipase C causes cytidylyltransferase translocation to membranes and stimulates PC synthesis [20,21]. It has recently been shown that even though the phospholipase C is on the exterior of the cells, cytidylyltransferase is translocated to the endoplasmic reticulum [22]. The mechanism by which phospholipase C causes cytidylyltransferase translocation is not understood. One possibility is due to generation of diacylglycerol in the membranes. Alternatively, a decrease in the concentration of PC would somehow mediate this effect or the fatty acid levels might be increased in the phospholipase-treated cells. In the present study, the effects of phospholipase A, on cytidylyltransferase activity and PC syntesis in cultured rat hepatocytes was investigated. The results show that degradation of PC causes an activation of PC synthesis.
dish. After this time interval, the medium was removed and the cells washed with fresh serum-free medium prior to the start of the experiment. For labelling studies, the hepatocytes were initially resuspended in cholineand methionine-free medium during the pulse period and then transferred to choline and methionine containing medium during the chase. To study the effect of phospholipase A, on choline incorporation into PC, the hepatocytes were incubated with phospholipase A, for various time intervals (O-20 mm). The medium was removed and the cells pulsed for 30 min with 15 pCi/dish of [methyl- 3H]choline chloride in serum-free medium. After this interval, the medium was removed and the cells washed with unlabelled medium. The prelabelled cells were subsequently incubated for 20 min with 2 ml/dish of serum-free medium. At the end of the incubations, the medium was removed for lipid analysis and the cells washed with 2 ml/dish of fresh serum-free medium. The cells were scraped from the dishes in 3 x 1 ml of 50% methanol with a rubber policeman. The cell suspension was sonicated for 30 s with an Ultrasonic processor 85 (Heat Systems Ultrasonic, Farmingdale, NY, U.S.A.) at an output setting of 4. A portion of the sonicated cell suspension (30 ~1) or medium was taken for protein determination by the Bio-Rad assay, and the remainder used for extraction and analysis of lipids and cholinecontaining water-soluble compounds. Trypan blue stain exclusion and constant cellular protein content indicated that the hepatocytes were viable throughout the incubation period.
Experimental
Extraction and analysis of lipid and water-soluble compounds The sonicated cell preparation or medium (3 ml) was transferred to screw-cap tubes and a further 3 ml of 50% methanol added. 6 ml of chloroform-containing antioxidant 2,6 di-t-butyl-4-methylphenol(50 mg/l) was added and the lipid- and water-soluble compounds extracted as described by the method of Folch et al. [24]. After centrifugation of the extract at 1000 X g for 15 min, the upper aqueous phase was removed and the lower chloroform phase was washed twice with 3 ml of theoretical upper phase (methanol/water/chloroform 48 : 47 : 3, v/v). The lower chloroform phase containing the lipids was evaporated to dryness under N,. The dried lipid was resuspended in 500 ~1 of chloroform and 25 ~1 of this was spotted onto a silica-gel 60 thin-layer chromatography plate (E. Merck, Darmstadt, F.R.G.). The plates were developed in chloroform/ methanol/ acetic acid/ formic acid/water solvent (70 : 30 : 12 : 4 : 2, v/v) containing antioxidant 2,6 di-t-butyl-Cmethylphenol. After the plates had been air-dried, the LPC. and PC bands were visualized with I, vapour and scraped into scintillation vials. Water (0.5 ml) and 5 ml
procedures
Materials Female Sprague-Dawley rats (SO-100 g) were obtained from animal services, University of Alberta. [methyl- 3HICholine chloride (15 Ci/mmol) was obtained from Amersham. Phospholipase A, (from Naja mocambique), phospholipase C (from Clostridium perfringens), BSA, digitonin, and substrates for enzyme assays were purchased from Sigma. Dulbecco’s modified Ealge’s medium (DMEM), was from Gibco. Cell culture and labelling studies Hepatocytes were isolated by a collagenase-perfusion technique similar to that described previously [23]. The isolated hepatocytes were washed and resuspended in DMEM containing 17% fetal calf serum (1 . lo6 cells/ml). The hepatocytes were dispersed into plastic culture dishes (Falcon 3802 Primaria; 60 mm X 15 mm) (3 ml/dish) and incubated at 37 o C under an atmosphere of air/CO, (95 : 5) for 3-4 h to allow attachment to the
382 of aqueous counting scintillant (ACS) was added and the radioactivity was measured after 24 h. Radioactivity associated with the choline-containing water-soluble compounds was determined by spotting 100 ~1 of the aqueous phase on silica-gel 60 thin-layer chromatography plates. 10 ~1 of standard, containing choline (15 mg/ml), phosphocholine (60 mg/ml), CDP-choline (10 mg/ml) and betaine (15 mg/ml) was applied to each lane. 100 pg of GPC was also applied to each lane to examine incorporation of label into GPC. The plate was developed in methanol/0.6% sodium chloride/ammonium hydroxide solvent (10 : 10 : 0.9, v/v). After air drying the plate, the choline containing compounds were visualized by exposure to I, vapour. Bands of choline containing compounds were scraped into scintillation vials and 0.5 ml water added. The radioactivity was measured by adding 5 ml of ACS and counting after 24 h.
C_~tidyij&ransferase release by digitonin treatment Cytidylyltransferase was released from hepatocytes by digitonin treatment as described by Mackall et al. [25]. Digitonin (0.5 mg/ml) was dissolved in 10 mM Tris-HCl (pH 7.4) 0.15 M NaCl, 0.25 M sucrose and 0.5 mM PMSF. Hepatocytes were initially cultured for 4 h in 60 mm x 15 mm dishes. After a subsequent incubation of cells with phospholipase A,, the medium was removed and the cells washed with 2 ml/dish of ice-cold phosphate-buffered saline. This was subsequently removed and the dishes placed on an ice-cold glass plate in an ice bath. 1 ml of digitonin solution was added to each dish and after 2, 4, and 8 min, the digitonin solution was removed with a pipette. 40 ~1 of the sample was used for enzyme assay, while 10 ~1 of sample was used for protein estimation.
Subcellular fractionation After treatment of hepatocytes with phospholipase AZ, the medium was removed and the cells washed with 2 ml/dish of ice-cold phosphate-buffered saline. The cells were scraped using a rubber policeman in ice-cold buffer A (20 mM Tris-HCl (pH 7.4), 0.15 M NaCl,l mM PMSF, and 2 mM DTT) (2 X 1 ml). The cells were pelleted by centrifugation at 2000 x g for 5 min at 4” C. The pellet was resuspended in buffer A and homogenized with 40 strokes of a tight-fitting Dounce homogenizer. The extract was centrifuged at 10000 X g for 10 min at 4” C. The supernatant was removed and the pellet rehomogenized and centrifuged as before. The combined 10000 x g supernatant was centrifuged at 100000 X g for 1 h at 4” C. The microsomal pellet was resuspended in 2 ml of buffer A with 20 strokes of a glass-Teflon homogenizer. 20 ~1 of cytosolic fractions and microsomal fractions were used for enzyme assay.
Cytidylyltransferase assay Assay of the cytidylyltransferase was done as described previously [15]. PC: fatty acid vesicles (1 : 1 molar ratio, 0.2 mM) were used as the lipid activators of the inactive cytosolic enzyme. Protein determination Protein was determined with the Bio-Rad protein assay based on the method of Bradford [26]. Bovine serum albumin was used as the standard. Standards were prepared with the same concentration of the enzyme buffer ingradients that were in the sample. Results Digitonin-mediated release of cytidylyltransferase by phospholipase C and A, in rat hepatocytes Hepatocytes were treated with various concentrations of phospholipase C for 10 min and the release of cytidylyltransferase into the medium examined after digitonin treatment. As the phospholipase C concentration increased, there was a substantial decrease in the amount of cytidylyltransferase activity released by digitonin treatment compared to control (Fig. 1). With 0.9 units/dish of phospholipase C in the medium, there was only 3.1 &-0.13 nmol/min per mg of cytidylyltransferase activity released (P < 0.05) compared to 4.0 f 0.11 nmol/min per mg released after 8 min of digitonin treatment in the control. Since digitonin permeabilized cells have been shown to release rapidly cytosolic enzymes, such as lactate dehydrogenase, into the medium but not membrane-bound enzymes [18]. this indicated that phospholipase C caused a decrease in the cytosolic cytidylyltransferase activity resulting in less enzyme being released by digitonin. Similarly, hepatocytes were treated with various concentrations of phospholipase A, for 5 or 10 min and
8
4.0
z
3.o
E P
g 2.0 5 za .= 10 .e
t; Q
0.0 0
2
4
8
10
Time (nk)
Fig. 1. Effect of phospholipase C on digitonin mediated release of cytidylyltransferase. Hepatocytes were incubated with 3 ml/dish of modified Eagle’s medium containing 0 units (0) 0.3 units (A), or 0.9 units (m) phospholipase C (from Clostridium perfrrn~ens) for 10 min. The medium was subsequently removed. The cells were washed with 2 ml/dish of phosphate-buffered saline and then 1 ml of digitonin solution was added to each dish. After 2, 4. or 8 min, the digitonin solution was removed and assayed for cytidylyltransferase activity. The results are the averages, k SD.. of three separate experiments ( * P c 0.05 using the students f-test).
383
4 O.OY 0 2
Ti4me @tin)
B
10
2 ,,,Y 0
2
Ti4me (iin)
8
10
release of Fig. 2. Effect of phosphohpase A, on digitonin-mediated cytidylyltransferase. Hepatocytes were incubated with 3 ml/dish of modified Eagle’s_medium containing 0 units (0). 0.3 units (A), or 0.9 units (m) phospholipase A, (from Naja mocumhique) for 5 min (A). or 10 min (9). The medium was subsequently removed, the cells washed with 2 ml/dish of phosphate-buffered saline and then 1 ml of digitonin solution added to each dish. After 2, 4, or 8 min, the digitonin solution was removed and assayed for cytidylyltransferase activity. The results are the averages. *SD., of three separate determinations (* P < 0.05 using student’s r-test).
the release of cytidylyltransferase activity examined by digitonin treatment. After 5 min incubation, the amount of cytidylyltransferase activity released from phospholipase A, treated cells was less than the release from control cells. However, the effect of phospholipase A, treatment was more pronounced after a 10 min incubation (Fig. 2). Incubation with 0.9 units/dish of phospholipase A, for 10 min resulted in 2.5 + 0.15 nmol/min per mg of cytidylyltransferase activity released (P < 0.01) compared to 3.9 & 0.10 nmol/min per mg in the control after 8 min of digitonin treatment. Thus, both phospholipase A, and C can decrease the amount of cytidylyltransferase activity released by digitonin treatment compared to control. The viability of the hepatocytes was not affected by treatment with phospholipase C or A, as determined by exclusion of Trypan blue. Addition of phospholipase A, and C to the assay buffer had no significant effect on the cytidylyltransferase activity (data not shown).
1.8 _t 0.20 nmol/min per mg. Thus, translocation of the cytidylyltransferase from the cytosol to the microsomes could account for over 75% of the decrease in cytidylyltransferase activity released by digitonin treatment. Hepatocytes were incubated with 0.6 units/dish of phospholipase A, for various time intervlas and then homogenized and fractionated into the cytosolic and microsomal fraction (Fig. 3B). As the time of incubation of cells with phospholipase A, increased, this resulted in a decrease in cytidylyltransferase activity in the cytosolic fraction and an increase in the microsomal fraction. After 20 min incubation, the cytidylyltransferase activity in the cytosol decreased from 4.1 f 0.10 to 2.7 &-0.20 nmol/min per mg, while it increased in the microsomal fraction from 0.9 + 0.05 to 2.0 10.10 nmol/min per mg. Effect of phospholipase A2 on PC synthesis Initially, hepatocytes were pulsed with [methyl‘HIcholine chloride for 30 min and then chased with unlabelled medium, containing various concentrations of phospholipase Az for 10 min. When we extracted the cellular lipids and analyzed incorporation of the label into PC, we found a significant amount of label incorporated into the LPC fraction with very little incorporation into PC (data not shown). This suggested that the label was initially being incorporated into PC but this was quickly degraded by the phospholipase A, to form LPC. To overcome this problem, we incubated hepatocytes with phospholipase A, for various time intervals prior to pulsing the cells. The medium was removed and the cells pulsed with [methyl-3H]choline chloride for 30 min and chased with unlabelled medium. As the concentration of phospholipase A, was increased, this resulted in an increase in label associated with PC (Fig.
4.0
Subcellular fractionation after treatment with phosphoiipase A, Since phospholipase A, was decreasing the release of cytidylyltransferase by digitonin treatment, we wanted to examine whether or not this decrease was due to translocation of the cytosolic form to the microsomes or whether the decrease was due to inhibition of the cytosolic activity by some other mechanisms. Hepatocytes treated with phospholipase A,, were homogenized and fractionated into the cytosolic and microsomal components. With increasing phospholipase A, concentrations, there was a decrease in the cytidylyltransferase activity in the cytosol and an increase in the microsomal fraction (Fig. 3A). With 0.9 units/dish of phospholipase A, and a 10 min incubation, the cytidylyltransferase decreased in the cytosolic fraction from 4.3 f 0.10 to 2.6 f 0.14 nmol/min per mg, while it increased in the microsomal fraction from 0.9 & 0.16 to
*
c*oro, Himsome
3.0
2.0 1.0
0.0
0.2
0.4
0.6
Units PLA 2
0.8
1.0
0.0_::::I 0
5
IO Time
15
20
(min)
Fig. 3. Effect of phospholipase AZ on cytidylyltransferase translocation. Hepatocytes were incubated with 3 ml/dish of modified Eagle’s medium containing various concentrations of phospholipase A> for 10 min (A) or 0.6 units phosphohpase Az for various time intervals (B). The medium was subsequently removed. and the cells washed with 2 ml/dish of phosphate-buffered saline. The cells were scraped and homogenized with 40 strokes of a tight-fitting Dounce homogenier in 20 mM Tris-HCI (pH 7.4) 0.15 M NaCI, 1 mM PMSF. and 2 mM DTT. The extract was centrifuged at 10000~ g for 10 min at 4OC. The resulting supernatant was centrifuged at 100000~ g for 1 h at 4°C. The cytosolic (0) and the microsomal (m) fractions were assayed for cytidylyltransferase activity. The results are the averages. k S.E., of three separate determinations.
384
2.2’ 0.0
’ 0.2
0.4
’ 0.6
0.8
1
Units PLA 2
Fig. 4. Effect of phospholipase A, on PC synthesis. Hepatocytes were incubated with various concentrations of phospholipase A, for 10 min (A) or 0.6 units phospholipase A, for various time intervals (B). The medium was removed and the cells pulsed with 15 pCi/dish of [ merh?;l- 3 HIcholine chloride for 30 min in choline- and methionine-free modified Eagle’s medium. After this interval, the medium was removed, the cells washed with 2 ml/dish of unlabelled medium prior to incubation with 3 ml/dish of unlabelled medium containing 28 PLM choline chloride for 20 mm. The medium and the cells were removed and the total radioactivity in PC determined. The results are the average of three separate determinations* SD. ( * P < 0.01: P i 0.01 using student’s r-test).
4A). Incubation with 0.9 units/dish of phospholipase A, resulted in an increase in label into PC from (2.4 * 0.02) . 10e5 dpm/dish to (3.1 f 0.1). lop5 dpm/dish (P < 0.01). In a similar fashion, increasing phospholipase A, concentrations resulted in a corresponding decrease in the label associated with both the choline (from (2.5 + 0.05). lop5 to (1.4 f 0.03). 10M5 dpm) and phosphocholine (from (8.5 t- 0.07). lo-’ to (6.9 * 0.05). lop5 dpm) (P < 0.05) fractions. The results are the average + SD. of three separate experiments. There was no effect of phospholipase A, on label associated with other choline containing compounds such as CDPcholine, and GPC. Therefore, phospholipase A, can stimulate PC synthesis by activating the cytidylyltransferase. A time-course of the effects of 0.6 units/dish of phospholipase A, on PC synthesis was studied. With increasing time intervals, the label into PC increased (Fig. 4B). After 20 min incubation, the label into PC increased from 2.6 + 0.03. lop5 dpm/dish to (3.4 + 0.05) . 10e5 dpm/dish. Thus, phospholipase A, treatment of hepatocytes can increase incorporation of label into PC. Discussion
Several conclusions can be derived from the above results regarding the effects of phospholipase A, on cytidylyltransferase activity and PC synthesis: (A) Phospholipase A, and phospholipase C can cause a significant decrease, within 10 min, in the amount of cytidylyltransferase activity released by digitonin treatment. Since digitonin treatment causes rapid release of soluble cytoplasmic components, this suggested that the amount of soluble cytidylyltransferase activity was decreased
upon phospholipase treatment; (B) The decrease in the cytidylyltransferase activity release by phospholipase A, upon digitonin treatment is due largely to the translocation of the enzyme from the cytosolic compartment to the microsomal compartment. Since the microsomal form of the enzyme is the active form of the enzyme. this indicated that phospholipase A, can activate the cytidylyltransferase; and (C) Phospholipase A, caused an increase in PC synthesis both in a concentrationand time-dependent manner. This was to be expected since the cytidylyltransferase was stimulated by phospholipase A,. Our results indicate that treatment of hepatocytes with phospholipase A z can activate the cytidylyltransferase and ultimately PC synthesis. Aeberhard et al. [27] also found stimulation of cytidylyltransferase in fetal Type II cells upon addition of the phospholipase A, activator mellitin. The product of phospholipase A, reaction is the generation of free fatty acid and lysophospholipid. Previous work [8,17,18], has already demonstrated activation of the cytidylyltransferase and PC synthesis by exogenously added free fatty acid. One possible explanation for the increased binding of cytidylyltransferase to membranes would be an increase in fatty acids due to the action of phospholipase A,. Our results also support the nation that a decrease in the level of PC via phospholipase action, can modulate the rate of PC synthesis. This concept was initially proposed based on the results of phospholipase C on the cytidylyltransferase and PC synthesis [6,20,21]. It was established that degradation of phospholipids via phospholipase C action generated diacylglycerol [6] which could directly or indirectly activate the cytidylyltransferase and ultimately PC synthesis. Alternatively, Sleight and Kent have proposed that translocation of cytidylyltransferase could be due to a decrease in the concentration of PC in the membranes [21,28]. Since we now find indications for translocation via phospholipase A, treatment of hepatocytes, a decrease in PC levels might explain this translocation of cytidylyltransferase. In this context it is noteworthy that the translocation of cytidylyltransferase in choline-deficient hepatocytes has been shown to be due to a change in the level of PC in these cells (Jamil, H., Yao, Z. and Vance, D.E., unpublished results). If one or more of these three possible mechanisms (increase in fatty acids or diacylglycerol, or decrease in PC) is found to be responsible for translocation of cytidylyltransferase, it would demonstrate a rather sensitive mechanism for regulation of PC biosynthesis. Future studies should distinguish among these and other possible mechanisms. That the effect of phospholipase A, on cytidylyltransferase and PC synthesis is rapid indicates that this mode of regulation may be important in the short-term regulation of PC synthesis. It also demonstrates that the degradation of the lipid content
385 of the cell is quickly compensated synthesis. This feature is essential viable.
for by increased lipid if the cell is to remain
Acknowledgments This work was supported by a grant from cal Research Council of Canada. J.S.S. was by a Studentship Award from the Alberta Foundation for Medical Research. D.E.V. is Scientist of the Alberta Heritage Foundation cal Research.
the Medisupported Heritage a Medical for Medi-
References 1 Pritchard, P.H. and Vance, D.E. (1981) Biochem. J. 196, 261-267. 2 Post, M., Batenburg, J.J., Schuurmans, E. and Van Golde, L.M.G. (1982) Biochim. Biophys. Acta 712, 390-394. 3 Post, M., Batenburg, J.J., Van Golde, L.M.G. and Smith, B.T. (1984) Biochim. Biophys. Acta 795, 558-563. 4 Cornell, R.B. and Goldfine, H. (1983) B&him. Biophys. Acta 750. 504-520. 5 Vance, D.E., Trip, E.M. and Paddon, H.B. (1980) J. Biol. Chem. 255, 1064-1069. 6 Sleight, R. and Kent, C. (1980) J. Biol. Chem. 255, 10644-10650. 7 Weinhold, P.A., Feldman, D.A., Quade, M.M., Miller, J.C. and Brooks, R.L. (1981) Biochim. Biophys. Acta 665, 134-144. 8 Pelech, S.L., Pritchard, P.H., Brindley, D.N. and Vance, D.E. (1983) J. Biol. Chem. 258, 6782-6788. 9 Sleight, R. and Kent, C. (1983) J. Biol. Chem. 258, 831-835. 10 Feldman, D.A., Kovac, C.R., Dranginis, P.L. and Weinhold, P.A. (1978) J. Biol. Chem. 253, 4980-4986.
11 Feldman, D.A. and Weinhold, P.A. (1987) J. Biol. Chem. 262, 9075-9081. 12 Feldman, D.A., Rounsifer, W.E. and Weinhold, P.A. (1985) Biochim. Biophys. Acta 429, 429-437. 13 Pelech, S.L. and Vance, D.E. (1984) Biochim. Biophys. Acta 779. 217-251. 14 Pelech, S.L., Pritchard, P.H. and Vance, D.E. (1981) J. Biol. Chem. 256, 8283-8286. 15 Pelech, S.L. and Vance, D.E. (1982) J. Biol. Chem. 257. 14198-14202. 16 Sanghera, J.S. and Vance, D.E. (1989) J. Biol. Chem. 264. 1215-1223. 17 Weinhold, P.A., Rounsifer. W.E., Williams, S.E., Brubaker, P.G. and Feldman, D.A. (1984) J. Biol. Chem. 259, 10315-10321. 18 Cornell, R. and Vance, D.E. (1987) Biochim. Biophys. Acta 919, 26-36. 19 Choy, P.C., Farren, S.B. and Vance. D.E. (1979) Can. J. Biochem. 57, 605-612. 20 Sleight, R. and Kent, C. (1983) J. Biol. Chem. 258, 824-830. 21 Sleight, R. and Kent, C. (1983) J. Biol. Chem. 258, 836-839. 22 Terc’e. F., Record, N., Ribbes. G., Chap, H. and Douste-Blazy. L. (1988) J. Biol. Chem. 263, 3142-3149. 23 Davis, R.A., Engelhorn, SC., Pangburn, S.H., Weinstein, D.B. and Steinberg. D. (1979) J. Biol. Chem. 254. 2010-2016. 24 Folch, J., Lees, M. and Sloane-Stanley, G.H. (1957) J. Biol. Chem. 226, 497-509. 25 Mackall, J., Meredith, M. and Lane, M.D. (1979) Anal. Biochem. 95, 270-274. 26 Bradford, M. (1976) Anal. B&hem. 72. 248-254. 27 Aeberhard, E.E., Barrett, C.T., Kaplan. S.A. and Scott, M.L. (1986) Biochim. Biophys. Acta 875, 6-11. 28 Sleight, R. and Kent, C. (1983) J. Biol. Chem. 258, 836-839.