Pulmonary phosphatidic acid phosphatase. Properties of membrane-bound phosphatidate-dependent phosphatidic acid phosphatase in rat lung

Pulmonary phosphatidic acid phosphatase. Properties of membrane-bound phosphatidate-dependent phosphatidic acid phosphatase in rat lung

212 Biochimica et Biophysics 0 Elsevier/North-Holland Acta, 574 Biomedical (1979) Press 212-225 BBA 57405 PULMONARY PHOSPHATIDIC ACID PHOSPH...

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Biochimica et Biophysics 0 Elsevier/North-Holland




(1979) Press


BBA 57405







a Department of Biochemistry, of Western Ontario, London, (Received November (Revised manuscript



and b Department Ontario, N6A 5A5

30th, 1978) February 16th,




a,b of Obstetrics (Canada)

and Gynaecology,



(Rat lung, Membrane-bound)

Summary 1. The membrane-bound phosphatidate-dependent phosphatidic acid phosphatase activity of rat lung has been investigated in cytosol and microsomal fractions using as a substrate [32P]phosphatidate bound to heat inactivated rat liver microsomes. Both activities demonstrated broad pH optima with a maximum of 7.4-8 for the cytosol and a maximum of 6.5-7.5 with the microsomal preparations. 2. At low concentrations (O-5 mM) Mg*’ produced a slight stimulation of the cytosol activity but at higher concentrations an inhibition was observed. Low concentrations (1.0-2.0 mM) of EDTA abolished the cytosol activity and reduced the microsomal activity to half. In both cases, the addition of Mg’+ in the presence of EDTA resulted in an activity which was more than 2-fold greater than that observed in the absence of chelator or divalent cation. 3. The cytosol activity was relatively resistant to the addition of ionic and nonionic detergents. In general, the addition of a number of phosphate esters increased rather than decreased the release of 32Pi, indicating a relative specificity for phosphate groups associated with a hydrophobic environment. The addition of aqueous dispersions of phosphatidate, lysophosphatidic acid or phosphatidylglycerophosphate markedly reduced the hydrolysis of membranebound [32P]phosphatidate. The cytosol activity was slightly inhibited by the addition of phosphatidylcholine. -Abbreviations: PA, phosphatidic acid; PAaq, aqueously dispersed phosphatidic acid; PAmb, membranebound phosphatidic acid: PAPase. phosphatidic acid phosphatase: EGTA, ethylene glycol-bis(fl-aminoethyl ether)-N.N’-tetraacetic acid: Tricine, N-tris(hydroxymethyl)methylglycine.


4. In an attempt to estimate the relative contributions of the cytosol and microsomal activities in vivo, these activities were assayed using [32P]phosphatidate endogenously generated on rat lung microsomes. With the 32P-labelled microsomes, the hydrolysis remained linear over the 45 min of the experiment. Addition of high speed supematant produced a rapid release of 32Pi during the first 10 min followed by a more gradual release similar to that observed with the microsomes alone. The cytosol activity remained greater than the microsomal activity at all times studied. 5. When [ “C]phosphatidate-labelled microsomes were incubated in the presence of nonradioactive CDPcholine, the addition of cytosol markedly stimulated the incorporation of radioactivity into phosphatidylcholine. This observation suggests that the phosphatidic acid phosphatase activity associated with the cytosol has a role in phosphatidylcholine (and presumably surfactant) biosynthesis in rat lung. __Introduction Phosphatidic acid phosphatase (PAPase) (EC, the enzyme which catalyzes the conversion of phosphatidic acid (PA) to 1,2-diacyl-sn-glycerol, occupies a key position in glycerolipid metabolism because its product serves as an immediate precursor for both phospholipids and neutral lipids. PAPase has been studied in a number of tissues including liver [ 1,2], adipose tissue [ 31, intestine [4] and lung [5,6]. Operationally at least two forms of the enzyme can be distinguished: that sedimenting with the particulate fractions including the mitochondria [ 7,8], microsomes [l-3,6,9], lysosomes [lo], plasma membrane [ll], and that associated with the soluble fraction [ 1,2,4,5,12]. Not only does the enzyme appear to exist in two separate physical states, but the properties of each vary, depending on the physical nature of the substrate used to measure the activity. A consistent observation in a number of tissues is that when aqueous dispersions of PA (PA,,) are used as substrate, the major activity is essentially particulate [ 1,3,4,6]. However, when the activities were measured using membrane-bound PA (PA,,,), the cytosol contains the major activity [ 1,2,4,5,12]. These observations have made it difficult to evaluate the role and/ or significance of PAPase activities from different subcellular fractions, with respect to glycerolipid synthesis in a number of tissues studied. The possible significance of PAPase to pulmonary glycerolipid synthesis has been emphasized by recent reports which have demonstrated that PAPase activity, measured with PA,,, increases both during fetal development [13,14] and after glucocorticoid administration to induce pulmonary maturation. However, the relation between these increases in hydrolase activity and the production of phosphatidylcholine by the endoplasmic reticulum [ 17,181 has not been established. In the present investigation, the PA,,, dependent PAPase activities in rat lung are investigated. This study, which represents the first comprehensive examination of the cytosol activity in this tissue, attempts to evaluate the importance of this activity in glycerolipid synthesis relative to the microsomal activity. The accompanying paper [ 191 describes the properties of PA,,-dependent PAPase in rat lung.


Materials and Methods Materials High specific activity rat-glycerol 3-[32P]phosphate was prepared by a modification of previously described methods [20] as described by Kates [21] which essentially involved increasing the specific activity of the sodium [“*PIphosphate (Charles Frosst Pharmaceutical Division, Montreal) in the reaction mixture. In all cases the glycerol 3-[ 32P]phosphate was judged to be more than 90% pure by paper chromatography and autoradiography [22]. The phosphonate analogue of glycerol 3-phosphate, 3,4-dihydroxybutyl-1-phosphonate (dilithium salt), was a gift of Dr. Burton Tropp, Dept. of Chemistry, City University of New York. Phosphatidylglycerol phosphate (from Escherichia coli) was a gift from Peter McDonald, Dept. of Biochemistry, University of Western Ontario, London, Canada. Preparation of su bcellular fractions Microsomes from lung and liver tissue of male Sprague-Dawley rats (200300 g) were prepared essentially as described previously [ 231. The supernatant remaining after isolation of the lung microsomal fraction by centrifugation in a Beckman 60Ti rotor for 1 h at 104 000 X g, hereafter referred to as the ‘cytosol’ fraction, was quick-frozen with solid COJacetone and stored at -70°C. Cross-contamination of individual subcellular fractions was monitored by the use of appropriate marker enzymes [ 181. Preparation of PA,, [ 32P]PA was prepared enzymatically from rat-glycerol-3-[ 32P]phosphate with an esterification system described previously [5]. The high specific activity rat-glycerol 3-[32P]phosphate used in the preparation of the [32P]PAmb overcame the disadvantage of the short half-life of [““PI. The [32P]PAmb could be used as a sensitive indicator of PAPase activity fcr about 3 months. In a typical experiment 3.1 nmol [32P]PA/mg protein were formed on the liver microsomal membranes. Liver microsomes were used for convenience. Similar results were obtained in experiments with heat inactivated lung microsomes containing [32P]PA,b. Enzyme assays assayed in a standard PAPase activities toward [32P] PAmb were routinely incubation system (final volume 100 d) containing: 50 mM Tricine (pH 7.4), 0.4 mg cytosol protein, and 2.4-3 nmol [32P]PAmb. Unless otherwise indicated, the reaction was terminated by the addition of 200 ,ul cold 10% trichloroacetic acid, centrifuged, and an aliquot counted in Aquasol [ 151. In some cases, a Bligh and Dyer [24] biphasic system as described by Bleasdale et al. [25] was used to terminate the reaction and determine 32Pi released. There was good agreement between the two methods. PAPase activity towards PA,, was determined as described previously [5] using a pH of 7.4 for the incubations. The incorporation of [14C] PA into phosphatidylcholine was estimated by incubating lung microsomes labelled with sn-[ “C]glycerol 3-phosphate [ 51 as


described above for preparation of [ 32P]PAmb. The labelled microsomes were reisolated by centrifugation, resuspended to their original volume, and a 50 rJ aliquot incubated in the absence or presence of cytosol in a final volume of 300 ,~l containing (final concentration) 50 mM Tricine (pH 7.4), and 0.8 mM CDPcholine. The reaction was terminated by the addition of 10 ml chlorofrom/ methanol (1 : 1, v/v). The lipids were extracted by the method of Bligh and Dyer [ 241 and separated by thin-layer chromatography as described previously

[51. Results Properties of the PA,, -dependent activity in the cy tosol from rat lung PAPase activity in the cytosol fraction of rat lung was stable for at least two months at -70°C. Good proportionality existed between time of incubation or amount of cytosol protein added (Fig. 1). A broad pH optimum from pH 7.4 8 was observed with the cytosol and this tended to vary somewhat with different preparations. The pH optimum of the microsomal activity was somewhat lower (about pH 6.5-7.4). Kinetic studies demonstrated that if the concentration of cytosol protein was reduced, typical Michaelis-Menton profiles could be obtained (Fig. 2 inset). The double reciprocal plot of the velocity vs. concentration of PA,,., was linear with an apparent K, of 5.4 PM and V of 7.0 * 10m6 pmol/min per mg cytosol protein (Fig. 2). These kinetic data must however be interpreted with caution, since this system where the enzyme is soluble and the substrate particulate represents a case of heterogeneous catalysis [26,27]. In






.2 Protein


.3 (mg)









20 30 TIME (min)



I 50

Fig. 1. Release of “Pi from [32P]PA,b with respect to (A) amount of rat lung cytosol and (B) time. Incubations were carried out at 37’C as described under Materials and Methods. The incubation time in (A) was 15 min and the amount of protein used in (B) was 0.5 mg.

/ -O$

I -0.1





0.2 I/s







Fig. 2. Double reciprocal plot of 32Pi release as a function of increasing COncentratiOn Of [32P]PAmb. The incubation wm for 30 min in a mixture containing 0.125 mg cytosol protein and varying concentrations of [32P]PA,b. Other conditions are as described in the Materials and Methods. The reciprocal Plot was calculated by the least-squares method.

this type of system, the number of aggregates/unit volume could affect the kinetic features. However, although sonication of the PA,, containing vesicles resulted in smaller microsomal vesicles as seen by electron microscopy, the enzyme still exhibited hyperbolic Michaelis-Menton plots with similar K, and V values, regardless of the degree of sonication. The cytosol-associated PAPase activities of liver [ 121 and adipose tissue [ 31 demonstrate a dependency of Mg*+ provided that endogenous Mg*+ is depleted. Addition of the Mg*’ chelator, EDTA, to the standard incubation mixture (Fig. 3a) essentially abolished the PA,, -dependent activity at 1.2 mM. The addition of Mg*+ to the standard incubation mixture produced a slight stimulation followed by an inhibition (Fig. 3b). However, when various amounts of Mg*+ were added to the cytosol in the presence of 2.5 mM EDTA, the activity was not only restored but increased to a level more than 2-fold that observed in the absence of EDTA (Fig. 3b). The PAmb -dependent activity observed with lung microsomes in the presence of EDTA was also stimulated by the addition of Mg*’ to a level more than 2-fold the activity with untreated microsomes (Fig. 4). Cytosol PAmb dependent PAPase activity was not affected by the addition of low concentrations (O.Ol%, w/v) of the nonionic detergents Triton X-100, Nonidet P-40 or Lubrol WX. Some inhibition (20 -40%) was observed at a concentration of 0.1%. The ionic detergent deoxycholate had little effect even at 0.1% (w/v).



* 1.0 -

Fig. 3. The effect of varying the concentration of (A) EDTA and (B) MgClz in the presence of 2.5 mM EDTA on the hydrolysis of [ 32PIPAmb by rat lung cytosol. Incubations were conducted 89 described in the text.

Substrate specificity of the cytosol activity Competition experiments were conducted by the addition of a number of phosphate esters up to a final concentration of 1.0 mM. Although no inhibition was observed with sn-glycerol 3-phosphate or rut-glycerol 2-phosphate, a small decrease in activity (30%) was observed with the phosphonate analogue of glycerol 3-phosphate, 3,4dihydroxybutyl-1-phosphonate. With glucose 6-phosphate, AMP, NADPH and p-nitrophenylphosphate a concentration-independent stimulation (20-50%) of the control activity was observed. A similar phenomenon has been observed with the microsomal PA,,-dependent PAPase activity (see accompanying paper [ 191). Several workers- have reported that other phosphatase activities can be stimulated by the presence of chemically unrelated phosphate esters [28-301. These results suggested the cytosol activity was specific for phospholipids. In order to investigate the specificity of the cytosol for [32P]PA,b relative to various phospholipids, competition experiments were carried out with aqueous dispersions of various phospholipids (Table I). In con-

218 EDTA (mM)

4 I

8 I

12 1



t 1


















Mgt2 (mtvl) Fig. 4. The effect of varying the concentration of EDTA (0). MgClZ (A) and MgClZ in the presence of 2.5 mM EDTA (0) on the release of radioactivity from [ 32P]PAmb by rat lung microsomes. Incubations were conducted as indicated in the text.

trast to the water soluble phosphate esters tested, PA,,, lysophosphatidic acid and phosphatidylglycerol phosphate (E. co/i) do appear to compete for the same activity as [32P] PAmI,. Although the inhibition by lysophosphatidic acid could be a detergent effect, this is not likely in view of the fact that both ionic and TABLE






Rat lung cytosol [3_2P]PA,bdependent PAPase activity pholipids were sonicated in saline and added as indicated. 23.9 + 0.51 S.E. pmol/min per mg protein.

was assayed The specific


(% control)




Lysophosphatidic acid Phosphatidylglycerophosphate Phosphatidylcholine (egg yolk)




as described in the text. The phosactivity of the rat lung CytOSOl was







84.4 38.7 27.6 95.5

27.8 37.5 32.2 03.2

15.5 15.9 24.0 68.0


non-ionic detergents do not cause appreciable inhibition of the cytosol PAPase activity (Table I). The inhibition by PA,, was further investigated over a range of concen~ations (Fig. 5). At a concen~ation of 2.0 mN PA,,, the cytosol activity towards [3’P]PA,b was maximally inhibited to the extent of 85%. This concentration of PA,, is about ZO-fold in excess that of the [32P]PA,i, present in the incubation mixture. Higher concentrations of PA,, did not further inhibit 32Pi release. Half maximal inhibition of the cytosol activity occurred at 0.2 mM PA,,. ..


Utzhzatzon of PAmb and PA,, by the cytosol and microsomal fraction from rat lung (a) Comparison of the microsomal and cytosol activities towards exogenous PA,*. The use of PAmb in measuring PAPase activities initially suggested a role for the cytosol in ~ycerolipid synthesis [1,4]. However, the significance of these observations is still unclear. In liver [ 123 and intestine [4], the soluble activity is more active relative to the microsomal activity. The microsomal activity in adipose tissue, however, utilized PAmb more actively than that of the cytosol [ 3,311. The relative abilities of the cytosol and microsomal fractions of rat lung to hydrolyze [ 32P]PAmb on heat-inactivated liver microsomes was examined at different times (Fig. 6a). The microsomal activity demonstrated an increase in “Pi release after 20-30 min incubation. The reason for this effect which was noted in several experiments is not known. As a result of this increase and because the cytosol activity is not completely linear, the ratio of



Fig. 6. The effects of various concentrations of aqueoualy dispersed phosphatidate on the hydrolysis of C32PlPA,b by rat hzng c~tosol. The activity was detemhied as described in Table I.


the cytosol to microsomal activities decreased from 20-fold (10 min) to 3.5fold (60 min). A valid criticism of this experimental design is that the relative activity of the cytosol could be overestimated since the ability of an enzyme on one microsomal particle from lung is being examined for its ability to hydrolyze an exogenous substrate on a liver microsomal particle. However, if a similar experiment is conducted using PA,, (essentially particulate vesicles with a radius of about 200-300 8, [32]), the reverse relation is seen (Fig. 6b). In this case an activity on one microsomal particle more readily hydrolyses PA contained in a liposome relative to its cytosol counterpart. These results suggest that the inability of the rat lung microsomes to hydrolyse exogenous PAmi, relative to the cytosol, is not due to the exogenous nature of the substrate, but rather to the characteristics of the particle being presented. (b) Comparison of the cytosol and microsomal activities with [32P]PAmb endogenously generated on rat lung microsomes. The next experiments attempted to determine the relative importance of the cytosol and microsomal PAmb-dependent PAPase activities of rat lung in vivo. [32P]PAmb endogenously generated on lung microsomes was utilized as the substrate in order to eliminate the potential disadvantages associated with assaying the lung microsomal activ-









e_. ,/, I




, , ,2 40

600 TIME (min)


/ Supernotont



A 20


I 60

and cytosol fractions of Fig. 6. cOmParisOn of the utilization of C32PlPAmb and PAaq by the microsomal rat lung. The microsomal (0) or cytosol(0) fractions recovered from 12 mg rat lung were incubated in the standard assay systems described in the Materials and Methods. The activities were corrected to nmollmin Per g lung by correcting for the recovery of protein in the microsomal (11.1 mg per g lung) and the cytosol (52.5 mg per g lung) fractions. The microsomal value was further corrected for the recovery of microsomes (48.6%) from the whole homogenate using the average recovery of three endoplasmic reticulum markers (NADPHxytochrome c reductase (EC; estrone sulphatase (EC and cholinephosphotransferase (EC and the cytosol value was also corrected for the recovery of cytosol (76.6%) by using the average recovery of the cytosol marker, lactate dehudrogenase (EC from three separate fractionations.


ity with substrate exogenously bound on heat-inactivated liver microsomes (see Materials and Methods). The experimental design permitted an examination of the competition between microsomal and cytosol activities for a common membrane-bound substrate. In this experiment rat lung microsomes biosynthetically labelled with [‘*PIPA which had been fractionated from 16 mg of lung tissue (wet weight), were incubated in the presence or absence of the cytosol obtained from an equivalent amount of tissue (Fig. 7a). Under these conditions, the release of 32Pi, catalyzed by the microsomal fraction over the 45 min examined, approached linearity. Addition of the cytosol resulted in a rapid release of 32Pi. The cytosol dependent activity (obtained by subtraction) exhibited a rapid initial phase followed by a more gradual hydrolysis with a slope similar to that observed with the microsomal fraction alone. In order to extrapolate the observed activities to the relative activities per g lung in vivo, the microsomal and cytosol activities were corrected for the recovery during the original fractionation and for the recovery of microsomal protein during the labelling with [“*PIPA (Fig. 7, legend). Since the cytosol activity uses membrane-bound substrate, the cytosol activity was expressed as the rate of hydrolysis catalysed by the cytosol of 1 g lung in the presence of the


TIME (mins) Fig. 7. Comparison of the utilization of endogenously generated [ 32P]PAmb by rat lung microsomes and by rat lung cytosol. Rat lung microsomes were biosynthetically labelled with f3*PlPA as described in the Materials and Methods for rat lung microsomes. The reisolated microsomes were resuspended with sucrose-EDTA to a volume corresponding to one-half of the original lung tissue. Aliquots of the 32Plabelled lung microsomal suspension (0.57 nmol [ 32PlPA) derived from 16 mg tissue were incubated in the absence (0) and presence (9) of the cytosol derived from 16 mg of lung (A). The supematant activity (A) was obtained by subtraction (A). In graph B, the supematant and microsomal activities are extrapolated to depict the activity per g lung by correcting for the recovery of microsomsl protein (40.5%) after labelling with 3 *P. The activities were also corrected for the recovery of protein in the initial isolation the microsomal and cytosol fractions and the recovery of endoplasmic reticulum and cytosol using marker enzymes as described in Fig. 6.




lung microsomes





with sn-[ 14Clglycerol




in the Methods


isolated by centrlfugation. The “C-labelled microsomes (0.11 mg protein) were incubated for 45 min at 37°C with CDPchollne (0.8 mM) in the presence or absence of rat lung cytosol (1.9 mg protein). The liquids were extracted and separated by thin-layer chromatography as described in the text. Condition

Microsomes Microsomes Microsomes


(zero time) (45 min) plus supernatant

(45 min)


Phosphatidic acid



31.2 16.6 11.1

0.0 0.0 9.9

44.5 21.6 26.6

[ 32P]PA-labelled microsomes from 1 g lung. The cytosol dependent activity was greater than the microsomal activity at each incubation period examined (Fig. 7b). However, as can be seen from the profile of the cytosol curve, the ratio of the cytosol to microsomal activities decreased from 8.9 (10 min) to 3.6 (30 min). The reason for the two phases in the cytosol dependent release of 32P. is not known, but may be related to the amount of [32P]PA bound to the lung microsomes. When cytosol was present, 61% of the membrane-bound radioactivity was released during the incubation. The role of the cytosol fraction in the biosynthesis of 3-sn-phosphatidylcholine in rat lung The relative importance of pulmonary cytosol and microsomal PAPase activities in the generation of 1,2-sn-diacylglycerol for phosphatidylcholine synthesis was examined in an experiment in which [‘4C]PAmb-labelled rat lung microsomes were incubated with nonradioactive CDPcholine in the presence and absence of cytosol. The addition of cytosol markedly enhanced the incorporation of radioactivity into phosphatidylcholine (Table II). Surprisingly, under both conditions there was a marked decrease in the recovery of 14C-labelled lipid. This decrease is presumably due to the degradation of diacylglycerol ny microsomal lipase [ 331. Mitchell et al. [ 121 and Fallon et al. [34] previously reported that the addition of rat liver cytosol stimulated the incorporation of radioactivity from [ 14C]PAmb into phosphatidylcholine in rat liver microsomes. The results presented in Table II clearly demonstrate that the PAmb-dependent PAPase activity in rat lung cytosol can play a role in the biosynthesis of lung phosphatidylcholine and presumably in surfactant production. Discussion The precise mechanisms which control pulmonary phospholipid metabolism have not been elucidated. The observation that PAPase activity increase late in gestation [ 13,141 and during the glucocorticoid induction of pulmonary maturity [ 15,161 has led to the suggestion that this enzyme could play an important


role in fetal surfactant synthesis [13,14,16,45]. If the de novo synthesis of phosphatidylcholine in lung were limited by the availability of diacylglycerol, an increase in PAPase activity could result in an overall stimulation of this uiosynthetic pathway. However, although an increase in PAPase activity has been demonstrated in fetal rabbit lung just prior to the rapid accumulation of phosphatidylcholine [ 131, a more detailed understanding of the properties of this enzyme in lung is required before this activity can be implicated in the control of surfactant synthesis. For example, since present evidence indicates that the bulk of surfactant phosphatidylcholine is synthesized on the endoplasmic reticulum [17,18,35,36], it is apparent that the PAPase activities in the microsomal or cytosol fractions could most reasonably be associated with phosphatidylcholine synthesis. Fetal pulmonary maturation is characterized by the appearance of numerous lamellar bodies [ 35,361, lysosomal related organelles which reputedly contain PAPase activity [ 37-391, However, since the lamellar bodies do not contain a number of the enzymes required for phosphatidylcholine synthesis [17,18], an increase in lamellar body PAPase activity would not necessarily explain the accumulation of phosphatidylcholine. Secondly, if a particular PAPase activity is important for glycerolipid synthesis, it should interutilize PA,,, , which presumably represents the intracellular biosynthetic mediate. With the exception of a preliminary communication from this laboratory [ 53, all other studies have measured PAPase activity with PA,,. Considerable evidence has accumulated in other tissues which indicates that information obtained with membrane-bound substrates more closely describes the situation regarding glycerolipid production in vivo than that derived with aqueous dispersions of lipids [ 12,411. Thus, although Schulz et al. [ 131 and more recently Mavis et al. [6] have emphasized the importance of the PA,, dependent microsomal activity in lung, it is important to establish whether this activity can produce diacylglycerol which can be utilized for phosphatidylcholine synthesis. In this respect, it is interesting to note that although in intact cells PA is present in trace amounts, the esterification of glycerol 3-phosphate by microsomal preparations from a number of tissues [ 1,4,31,40] including lung [ 5,231 leads to an accumulation of PAmb. The cytosol PAPase activity described in this paper fulfills the criteria for an activity which could act on the PA formed by the endoplasmic reticulum and function in glycerolipid synthesis in lung. This suggestion is consistent with the present state of knowledge concerning glycerolipid synthesis in liver, intestine and adipose tissue [ 1,2,4,12]. The PAPase activity from lung cytosol was similar to the cytosol activities with this substrate in other tissues in that the activity demonstrated a broad neutral pH optimum and was susceptible to higher concentration of divalent cations and fluoride [42,43]. The kinetic constants are comparable to those reported for other tissues, although there is considerable variation in the literature presumably due to the insoluble nature of the substrate. In addition, some deviation from Michaelis-Menten type kinetics was observed with brain cortex cytosol when PA,,, was the substrate [42]. Lung cytosol PAPase exhibited classic kinetic profiles which were not affected by sonication of the PA-containing vesicles. None of the detergents tested greatly affected the cytosol PAPase activity. With rat brain both the nonionic detergent Triton X-100 and the ionic detergent deoxycholate greatly enhanced the cytosolic activity at high


concentrations (0.5%). The slight inhibition (20%) by Triton X-100 (0.1%) observed with lung cytosol may indicate a difference from the brain enzyme. However, it is not possible to distinguish whether the detergents exert their effects on the enzyme or the substrate. Although the cytosol activity was not susceptible to inhibition by water soluble phosphate esters, it was reduced by the presence of PA,,, lysophosphatidic acid and phosphatidylglycerol phosphate. The PAPase activity associated with human amniotic fluid can hydrolyse lysophosphatidic acid [44], l-alkylglycerol phosphate [ 251 and phosphatidylglycerol phosphate [44,45]. It has been suggested that PAPase functions in phosphatidylglycerol synthesis in lung [ 451. The present results with rat lung suggest that cytosol may be able to hydrolyse a number of the primary phosphate esters of hydrophobic molecules: but more direct experiments are required to substantiate this possibility. However, it should be noted that the ability to hydrolyse these lipids in vitro is an indication but not a definitive proof of an in vivo function. It was of interest to note that although a Mg” requirement was not evident under the standard incubation conditions, a Mg” dependency could be demonstrated with the cytosol and the microsomal PA,, dependent activities in the presence of EDTA (2.5 mM). Furthermore, the activity observed under these conditions was more than 2-fold greater than with the standard conditions. A similar phenomenon has been observed with EDTA-treated microsomes from rat liver [ 21. In contrast, the PA,, dependent microsomal and cytosol activities of rat lung do not demonstrate this activation (see accompanying paper), suggesting that separate proteins may be responsible for the hydrolase activity observed with the aqueously dispersed substrate. The studies described in this paper suggest that although the microsomal and cytosol fractions both contain PA,,, -dependent PAPase activity, the hydrolase in the cytosol is more significant on a per g lung basis (Fig. 7). This cytosol activity can play a role in the de novo pathway for phosphatidylcholine synthesis (Table II). However, it is not yet known whether this activity functions in the control of phosphatidylcholine or is of importance in the induction of surfactant synthesis in fetal development.

Acknowledgement These investigations Council of Canada.

were supported

by grants from

the Medical


References 1












Sedgwick. and



J.M.. P.G.,


Fallon, Fallon. Rao,



H.J. H.J.










J. Lipid

P.A. G.F.

Hiibsher, Res.


and Schwarz. and

G. (1967)




J. Biochem.

517-524 G.E.


(1967) F.





82,627-633 6





Fink&t&n. B. and





G. (1965)






J. Lipid




19, 106,

467-477 63-77





8 Johnston, J.M. and Bearden, J.H. (1962) Biochim. Biophys. Acta 56, 365--367 9 Tzur. R. and Shapiro, B. (1976) Eur. J. Biochem. 64, 301-305 G.F. and Kennedy, E.P. (1963) J. Biol. Chem. 238.2615-2619 10 w&am, Kent. C. and Vagelos. P.R. (1976) Biochim. Biophys. Acta 436. 377-386 Mitchell, M.P., Brindley, D.N. and Hiihscher, G. (1971) Eur. J. Biochem. 18.214-220 Schultz, F.M., Jimenez, J.M., MacDonald, P.C. and Johnston, J.M. (1974) Gynecol. Invest. 5. 222229 14 Ravinuthala, H.R.. Miller, J. C. and Weinhold, P.A. (1978) Biochim. Biophys. Acta 530, 347-356 F., Duwe. G., Metcalfe, R., Stewart-DeHaan, P.J.. Wong, C., Las Heras, J. and Harding, 15 Possmayer, P.G.R. (1977) Biochem. J. 166.485-494 Biophys. 16 Brehier, A., Benson. B.J., Williams, M.C.. Mason, R.J. and Ballard, P.L. (1977) Biochem. Res. Comm. 77,883-890 17 Tsao, F.H.C. and Zachman, R.D. (1977) Pediat. Res. 11.849-857 18 Baraliska, J. and Van Golde, L.M.G. (1977) Biochim. Biophw. Acta 488. 285-293 19 Yeung, A.. Casola, P.. Wong, C., Fellows, C.F. and Possmayer. F. (1979) Biochim. Biophys. Acta 574,

11 12 13

226-239 20 McMurray. W.C.. Strickland, K.P.. Berry, J.F. and Rossiter, R.J. (1957) Biochem. J. 66. 634-644 20 Sastry. P.S. and Kates, M. (1966) Can. J. Biochem. 44, 459.-467 Techniques of Lipidology, PP. 475-476. North Holland/American Elsevier, 21 Kates, M. (1975) 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Amsterdam Chang, Y.Y. and Kennedy, E.P. (1967) J. Lipid Res. 8. 447-455 Hendry, A.T. and Possmayer, F. (1974) Biochim. Biophys. Acta 369,156-172 Bligh. E.G. and Dyer, N.J. (1959) Can. J. Biochem. Physiol. 37,911--917 Bleasdaie, J.E., Davis, C.S. and Agranoff, B.W. (1978) Biochim. Biophys. Acta 528, 331-343 Gatt, S. and Bartfai. T. (1977) Biochim. Biophys. Acta 488, l-12 Gatt, S. and Bartfsi, T. (1977) Biochim. Biophys. Acta 488, 13-24 Egi. Y. and Kawasaki, T. (1977) J. Biochem. 82.307-309 Khandelwal. R.L. (1977) Biochim. Biophys. Acta 485, 379-390 Nakai, C. and Thomas, J.A. (1974) J. Biol. Chem. 249,6459-6467 Jamdar. S.C. and Fallon, H.J. (1973) J. Lipid Res. 14, 509-516 Mille, M. and Vanderkooi. G. (1977) J. Colloid Interface Sci. 61, 455-474 Samala, M.G. and van Golde, L.M.G. (1976) Biochim. Biophys. Acta 441,423-432 Fallon, H.J., Barwick, J., Lamb, R.G. and van den Bosch, H. (1976) in Lipids (Paoletti, R.. Porcellati, G. and Jancini, G., eds.), Vol. 1. PP. 67-74, Raven Press, New York, NY Chevalier. G. and Collet, A.J. (1972) Anat. Rec. 174, 289-310 Rooney, S.A., Page-Roberts, B.A. and Motoyama, E.K. (1975) J. Lipid Res. 16. 418-425 Meban. C. (1972) J. CeIi Biol. 53, 249-252 Spitzer, H.L., Rice, J.M., MacDonald, P.C. and Johnston, J.M. (1975) Biochem. Biophys. Res. Comm. 66.17-23 Jimenez, J.M. and Johnston, J.M. (1975) Pediatr. Res. 10, 767-769 Shapiro, B. and Tzur. R. (1978) Ann. N.Y. Acad. Sci. 149,784-790 Fallon, H.J.. Barwick. J., Lamb, R.G. and van den Bosch, H. (1975) J. Lipid Res. 16. 107-115 Davis, C.S. (1976) Ph.D. Thesis. University of Michigan, Chicago, MI Hosaka, K., Yamashita, S. and Numa. S. (1975) J. Biol. Chem. 77. 501-509 Benson, B.J. and Clements, J.A. (1978) Fed. Proc. 37.1493 Johnston, J.M.. Reynolds, G.. Wylie. M.B. and MacDonald, P.C. (1978) Biochim. Biophys. Acta 531, 63-71 Kako, K.J. and Patterson, S.D. (1975) Biochem. J. 152, 313-323