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Selective utilization of endogenous unsaturated phosphatidylcholines and diacylglycerols by cholinephosphotransferase of mouse lung microsomes
423
Biochimica et Biophysics Acta, 441 (1976) 423-432 0 Elsevier Scientific Publishing Company, Amsterdam
- Printed in The Netherlands
BBA 56851
SELECTIVE UTILIZATION OF ENDOGENOUS UNSATURATED PHOSPHATIDYLCHOLINES AND DIACYLGLYCEROLS BY CHOLINEPHOSPHOTRANSFERASE OF MOUSE LUNG MICROSOMES
M.G. SARZALA
* and L.M.G. VAN GOLDE **
Laboratory of Veterinary (The Netherlands) (Received
Biochemistry,
State University
of Utrechi,
Biltstraat
172, Utrecht
March 22nd, 1976)
1. In the presence of CMP, cholinephosphotransferase of mouse lung microsomes catalyzes the conversion of endogenous phosphatidylcholines into 1,2diacyl-sn-glycerols and CDPcholine. 2. In this conversion cholinephosphotransferase shows a distinct preference for those molecular species of phosphatidylcholine which contain an unsaturated fatty acid. The enzyme hardly utilizes endogenous dipalmitoylglycerophosphocholine as a substrate. 3. Membrane-bound 1,2-diacyl-sn-glycerols were also prepared by treatment of mouse lung microsomes with a pure phospholipase C from Bacillus cereus. These 1,2-diacyl-sn-glycerols were subsequently utilized as substrate by cholinephosphotransferase in the formation of phosphatidylcholine. In the latter reaction, cholinephosphotransferase exhibited a pronounced preference for unsaturated 1,2-diacyl-sn-glycerols and hardly utilized the endogenous 1,2-dipalmitoylsn-glycerol. 4. The low affinity of cholinephosphotransferase for either dipalmitoylglycerophosphocholine or 1,2-dipalmitoyl-sn-glycerol strongly endorses the concept that the CDPcholine pathway alone cannot be responsible for the production of pulmonary dipalmitoylglycerophosphocholine.
Introduction Dipalmitoylglycerophosphocholine choline) is known to be a principal * Present
(1,2-dipalmitoyl-sn-glycero-3-phosphophospholipid constituent of mammalian
address: Nencki Institute of Experimental Biology. Polish Academy of Sciences, Department of Biochemistry of Nervous System and Muscle, 3 Pasteur Street, Warszawa 22, Poland. ** To whom all correspondence should be addressed.
424
lung tissues [ 1,2]. It is also generally accepted as the major active component of the lung surfactant which prevents alveolar collapse during expiration [ 3-51. The known association between the “respiratory distress syndrome” of the newborn and a deficient surfactant production [6--81 has undoubtedly provoked interest in the mechanisms involved in the biosynthesis of pulmonary phosphatidylcholines. Based on our present knowledge two pathways can be considered to operate in the de novo formation of pulmonary phosphatidylcholines: (a) the ~DP~holine pathway established by Kennedy [9] and (b) a stepwise methylation of phosphatidylethanolamine f lo]. Accumulating evidence has been presented in the past few years that pathway b is of minor importance in the mammalian lung [ 11-17 1, including that or primates [ 18 J. Though all info~ation presently available (for reviews st efs. 5 and 19) points to an important role of the CDPcholine pathway in the biosynthesis of pulmonary phosphatidylcholines, it is unlikely that this pathway alone can be responsible for the formation of the dip~mitoylglycerophosphocholine species: both in vivo [ 14,201 and in vitro studies [ 171 have indicated that in the lung the CDPcholine pathway primarily functions in the production of unsaturated phosphatidylcholines. In addition, it has been demonstrated that a large portion of the palmitate at the 2-position of dipalmitoylgly~erophosphocholine is incorporated via a pathway bypassing CDPcholine [20,21]. So far, however, no explanation was available for the limited capacity of the CDPcholine pathway to produce pulmonary dipalmitoylglycerophosphocholine directly, i.e. without auxiliary mech~isms such as a deacylation-reacyl~tion cycle f 221 or a deacylation-transacylation process f 231. A possible explanation might be that cholinephosphotransferase (EC 2.7.8.2), which catalyzes the conversion of 1,2-diacyl-sn-glycerols into phosphatidylcholine, hardly utilizes 1,2-dipalmitoyl-sn-glycerol as substrate. As has been shown convincingly by Kanoh and Ohno [24], it is difficult to investigate the substrate specificity of cholinephosphotransferase with emulsions of exogenous 1,2-diacyl-sn-glycerols as substrates. These authors developed an elegant way to study the substrate specificity of choline- and ethanol~inepl~osphotr~sferase of rat liver microsomes. They made use of the known fact [11,25,26] that in the liver the reaction stimulated by cholinephosphotransferase is reversible : 1,2-diacyl-sn-glycerol +
CDPcholine
$ phosphatidylcholine
+ CMP
Kanoh and Ohno employed the back-reaction (b) as a tool to produce membrane-bound 1,2~iacyl-so-glycerols which were subsequently utilized as a substrate in the forward reaction (a) with a specificity that agreed very nicely with data from in vivo studies [ 241. In the present paper evidence will be provided that also in lung microsomes phosphatidylcholines can be utilized as substrate by cholinephosphotransfer~e in the back-reaction. Membrane-bound 1,2-diacyl-sn-glycerols were also prepared by treatment of lung microsomes with a pure phospholipase C from Bacillus cereus. From these two approaches it could be concluded that cholinephosphotransferase of lung microsomes exhibits a low affinity for 1,2-dipalmitoylsn-glycerol in the forward reaction and for dipalmitoylglycerophosphocholine in the back-reaction.
425
Materials and Methods Materials [9,10-3Hz]palmitic acid (spec. act. 500 Ci/mol) and [Me-14C]choline (spec. act. 52 Ci/mol) were purchased from the Radiochemical Centre, Amersham, Great Britain. CDP- [ Me- l 4 Clcholine (spec. act. 40 Ci/mol) was bought from N.E.N.-Chemicals, Dreieichenhain, Germany. Diisopropylfluorophosphate was supplied by EGA-Chemie KG, Steinheim, Germany. Unlabelled fatty acids were obtained from Sigma, St Louis, MO., U.S.A. and most biochemicals from Boehringer, Mannheim, Germany. All other chemicals and solvents were of analytical grade. Pure phospholipase C from B. cereus was a generous gift of Dr. R.A.F. Zwaal, Biochemistry Laboratory, State University of Utrecht, Utrecht, The Netherlands. The mice (Swiss random strain) were bought from the Central Institute for the breeding of Laboratory Animals, Zeist, The Netherlands. Methods Isolation of mouse lung microsomes. Groups of 10 mice were killed by cervical fracture. After removal and washing with 0.9% NaCl, the lungs were minced and homogenized in 4 ml of ice-cold 0.25 M sucrose using a Potter-Elvehjem homogenizer. After removal of the nuclear plus cell debris fraction by centrifugation at 1000 X g for 5 min, the supernatant was spun at 20 000 X g for 10 min in the SS-34 rotor of the Sorvall RC-2B centrifuge. The microsomes were then pelleted from the 20 000 X g supernatant by centrifugation at 105 000 X g for 90 min in the 50 rotor of a Beckman L-565 ultracentrifuge. The microsomal fraction obtained in this way was suspended in 2 ml of 0.125 M KCl/O.l M Tris (pH 7.4) and, if not used directly, stored at -80°C. Protein contents were estimated by the method of Lowry et al. [27]. Preparation of [Me-14C]choline- and [9,1 0-3Hz]palmitate-labelled microsomes. Groups of 10 mice received 0.1 ml. of saline containing 25 I.cCi[Me-14C]choline by injection into the tail vein. After 2.5 h the mice were killed and the microsomal fraction isolated from the lungs as described above. Under these conditions 95% of the labelled choline incorporated into the total lipids, was recovered in the phosphatidylcholine fraction. [9,10-3Hz]palmitic acid was complexed to albumin exactly as described by Wkesson et al. [28]. Aliquots of 100 E.tCi were injected into the tail vein of 10 mice and the lung microsomes isolated after 1 h. Phosphatidylcholine contained approx. 68% of the palmitate incorporated into the total lipid fraction. Incubation conditions for the back-reaction stimulated by cholinephosphotransferase. Varying amounts of microsomes labelled with either [Me-‘4C]choline or [9,10-3Hz]palmitate were incubated for the indicated periods of time (see Results) in a shaking water bath at 37°C. Unless specified otherwise, the incubation medium (1 ml) contaIned the following components: 100 mM Tris HCl (pH 8.5), 10 mM dithiothreitol, 10 mM MgClz, 5 mM EGTA * and 4 mM CMP. Parallel experiments were conducted in which the CMP was omitted. In incubations in which [Me- ’ 4 C] choline-labelled microsomes were used, the reactions were terminated by the addition of 1 ml of ice-cold 10% trichloroacetic acid. After centrifugation, aliquots were taken from the supematant to measure * EGTA:
ethyleneglycol-bis(&aminoethyl
etherkN,N-tetracetic
acid.
426
by scintillation counting the amount of trichloroacetic acid-soluble radioactivity produced in the back-reaction. The lipids were extracted from the acidinsoluble fraction of the microsomes by the method of Bligh and Dyer [ 291. In experiments with unlabelled or [ 3Hlpalmitate-labelled microsomes, the microsomes were preincubated in the presence of 3 mM diisopropylfluorophosphate [ 241 at 37” C for 1 h to inhibit the possible action of microsomal lipase on the 1,2-diacyl-sn-glycerols formed during the back-reaction. After incubation of the microsomes in the presence and absence of CMP as described above, the reactions were stopped by the addition of 4 ml of methanol/chloroform (2 : 1, v/v) and the lipids extracted [ 291. Treatment of lung microsomes with phospholipase C. [9,10-3Hz]Palmitatelabelled microsomes (about 4 mg of protein) were suspended in 2 ml of a 0.1 M Tris . HCl (pH 8.3) buffer, containing 5 mM CaC&. After the addition of 20 (~1of phospholipase C from B. cereus (2.3 units), the microsomes were incubated at 37°C for 10 min. The reaction was terminated by the addition of 0.2 ml 0.1 M EGTA. The microsomes were then recovered from the incubation medium by layering the incubation mixture on 0.3 M sucrose and subsequent centrifugation at 30 000 X g for 90 min. The pellet was resuspended in 1 ml of 0.125 M KU/O.1 M Tris (pH 7.4). Aliquots of the microsomes were then taken for extraction [29] and lipid analysis. The remaining microsomes which contained membrane-bound 1,2-diacyl-sn-glycerols formed through the action of phospholipase C, were subsequently used as enzyme and substrate source in the forward reaction of cholinephosphotransferase. Incubation
conditions
for cholinephosphotransferase
in the forward
reaction.
Varying amounts of microsomes, containing membrane-bound 1,2-diacyl-snglycerols, were incubated at 37°C in a medium of the following composition: 100 ~1 0.1 M Tris - HCl (pH 8.5), 50 ~1 0.1 M MgCl?, 20 ,ul 0.1 M dithiothreitol and 10 ~120 mM CDP-[Me-14C]choline (spec. act. 0.5 Ci/mol) in a total volume of 0.2 ml. In parallel experiments untreated microsomes were used as enzyme and substrate source. After 10 min the reactions were stopped by the addition of 0.8 ml of methanol/chloroform (2 : 1, v/v) and the lipids extracted [ 291. Lipid analyses. Phosphatidylcholine was isolated from the total lipid mixtures by means of silica thin-layer chromatography using chloroform/methanol/ water (65 : 35 : 4, v/v) as developing solvent [30]. The phosphatidylcholine fraction was recovered quantitatively from the silica by extraction according to Bligh and Dyer [ 291. Aliquots were transferred into scintillation vials containing 10 ml of a scintillation fluid described by F’ricke [31]. The radioactivity measurements were carried out in a Packard Tricarb model 2425 B liquid scintillation counter. The remaining portions of the phosphatidylcholines were converted into 1,2-diacyl-sn-glycerols by treatment with phospholipase C and subsequently resolved by argentation thin-layer chromatography in disaturated, mono-, di-, tri-, tetra- and hexaenoic species. The details of this procedure have been published earlier [ 1,301. After extraction from the silica with chloroform/ methanol (2 : 1, v/v) [ 1,301, aliquots of the various species of 1,2-diacyl-snglycerols were taken for assay of the radioactivity. The amounts of the various species were estimated by means of gas-liquid chromatography using an internal standard as has been described in detail before [1,30]. 1,2-Diacyl-sn-glycerols present in the microsomal lipids or formed either by the back-reaction or by
427
the treatment of microsomes with phospholipase C, were isolated by silica thinlayer chromatography using benzene/chloroform/methanol (80 : 15 : 5, vlv) as developing solvent. After extraction from the silica with several portions of ether, the l,Z-diacyl-sn-glycerols were subsequently separated into the various molecular classes and analyzed for radioactivity content as described above. Results ~ccu~e~~e of the buck-reaction in mouse lung ~icroso~es The incubation conditions for investigation of the back-reaction in mouse lung microsomes were derived from preliminary experiments with microsomes containing phosphatidyl-[Me-’ 4 Clcholine. The decrease in radioactivity of phosphatidylcholine and the release of trichloroacetic acid-soluble radioactivity were used as parameters for the extent of the back-reaction [26]. The pH optimum for the action of cholinephosphot~sfer~e in the back-reaction was approx. 8.5 and a concentration of CMP of 4 mM was found to be required for maximal velocity of the reaction. EGTA was included in the reaction medium since Ca2* was found to be strongly inhibitory, as had been reported earlier for the back-reaction in rat liver microsomes [26]. Fig. 1 presents the distribution of radioactivity between phosphatidylcholine and the trichloroacetic acid-soluble fraction of the microsomes after different incubation times in the presence and absence of CMP. It is clear that addition of CMP induces a pronounced decrease in radioactivity content of phosphatidylcholine and a concomitant release of trichloroacetic acid-soluble radioactivity. By means of paper chromatography as described by Kanoh and Ohno [ 261 and using authentic CDPcholine and phospho~lchol~e as references, it could be established that the soluble radioactivity released from phosphatidylcholine in the presence of CMP consisted for more than 95% of CDPcholine. Fig. 2 presents the results obtained with [9,10-3H,]palmitate-labelled microsomes. It is clear that incubation in the presence of CMP causes a considerable formation of 1,2_diacyl-sn-glycerols. In strong contrast to the concomitant decrease in phosphatidylcholine (not shown), no conversion of phospha~dyleth~olamine was observed during the ineubation. The identity of the 1,2-diacyl-sn-glycerols formed by the back-reaction was established by cochromatography with an authentic synthetic 1,2-diacylsn-glycerol in various solvent systems. In addition they were effectively utilized as substrate by cholinephosphotransferase in the forward reaction. As had been found for rat liver microsomes f 241, it appeared to be necessary to preincubate the microsomes with d~sopropyl~uorophosphate to inhibit the action of a microsomal lipase on the 1,2-diacyl-sn-glycerols produced in the back-reaction. Omission of the preincubation led to a significantly smaller accumulation of 1,2diacyl-an-glycerols (Fig. 2) and to a production of radioactive monoacylglycerols and free fatty acids (data not shown). Apparently, a microsomal lipase is present, in contrast to an observation by Garcia et al. [32] who could not detect a lipase in lung microsomes. A possible explantion for this discrepancy might be that these authors only used exogenous triacylglycerols as substrate. Substrate specificity of cholinephosphotransferase in the utilization of phosphatidylcholines It was thought of great interest to investigate a possible specificity of cho-
428
10
20 lncubatlon
30 time
(man)
40
10
20 lncubaton
30
40
tome (man)
Fig. 1. Distribution of radioactivity between phosphatidylcholine and the trichloroacetic acid-soluble portion of mouse lung microsomes after incubation of [Me-l4 Clcholine-labelled microsomes in the presence and absence of CMP. The composition of the incubation medium has been described in detail in Materials and Methods. After the indicated periods of incubation. the reactions were terminated by the addition of 10% trichloroacetic acid and aliquots taken from the trichloroacetic acid-soluble portion and from phosphatidylcholine isolated from the insoluble portion of the microsomes. The radioactivity of phosphatidylcholine before the incubation (45 920 dpm) is taken as 100%. lL, phosphatidylcholine, in the presence of CMP; o----O, idem, in the absence of CMP; n-, trichloroacetic acid-soluble portion. in the presence of CMP and o-, idem, in the absence of CMP. Fig. 2. Radioactivity accumulating in 1.2-diacyl-sn-glycerols during incubation of [9.10-3H21palmitatelabelLed microsomes in the presence and absence of CMP. The details of the incubation procedures are described in Materials and Methods. The results are expressed as percentage of the radioactivity of phosphatidylcholine (66 000 dpm) before the incubation. 0~. in the presence of CMP, after preincubation with diisopropylfluorophosphate: A-. in the presence of CMP, without preincubation with diisopropylfluorophosphate and o-----U, in the absence of CMP, after preincubation.
linephosphotransferase with respect to the various species of endogenous pulmonary phosphatidylcholines as substrates for the back-reaction. In these experiments [ 9,10-3 Hz lpalmitate-labelled microsomes were used in order to follow the formation of labelled molecular species of 1,2-diacyl-sn-glycerols. Table I presents the distribution of radioactive palmitate among the various molecular species of phosphatidylcholine before the incubation with CMP. In agreement with the findings of Moriya and Kanoh [20] in whole rat lung, the majority of the palmitate had been incorporated into the dipalmitoylglycerophosphocholine fraction of mouse lung microsomes. This fraction also possessed the highest relative specific activity (third column of Table I). Table II (column 1) presents the percentages of each molecular class of phosphatidylcholines utilized as substrate in the back-reaction. Very interesting is the observation that cholinephosphotransferase consumes preferentially the mono-, di-, and tetraenoic phosphatidylcholines whereas the disaturated species are hardly used as substrate by the enzyme. A possible selective utilization of the various species of 1,2-diacyl-sn-glycerols, produced in the microsomes by the back-reaction, as substrates for cholinephosphotransferase in the forward
429 TABLE I DISTRIBUTION OF RADIOACTIVITY AMONG THE VARIOUS CHOLINE OF MOUSE LUNG MICkOSOMES I h AFTER THE ~~,~~-~H~IPALM~TATE
CLASSES OF INTRAVENOUS
PHOSPHATIDYG INJECTION OF
For experimental details see Materials and Methods. The relative specific activity of a given class is defined as the percentage of radioactivity in that class (1st column) divided by the weight percentage (2nd column). The radioactivity of the total phosphatidylcholine fraction was 65 200 dpm. Chtsses of phosp~tidyIcho~ne
Diiaturated Monoenoic Dienoic Trienoic Tetraenoic Hexaenoic
Distribution
of
C9,19-3H21 pabnitate (%l
Weight distribution
66.3 16.6 13.0 3.8 5.8 5.3
Relative activity
specific
(%)
I.87
29.6 27.0 21.8 4.2 11.4 6.0
0.61 0.60 0.90 0.51 0.88
reaction was not pursued because of the very small amounts of 1,2-dipalmitoylsn-glycerol produced in the back-reaction. Therefore, phospholipase C treatment was chosen as an alternative method to generate endogenous l,Z-diacylsn-glycerols in [9,10-3Hz ~p~rni~te-labelled microsomes. Treatment of lung microsomes with phospholipase C resulted in an equal conversion of both phosphatidylcholine and phosphatidylethanolamine. The enzyme almost randomly attacked the various molecular specie8 of phosphatidylcholine including the disaturated class. The 1,2-diacyl-sn-glycerols, produced by phospholipase C, were subsequently utilized as substrates in the forward reaction catalyzed by cholinephospho~~sfer~e. Table II (column 2) shows the increase in radioacTABLE
II
UTILIZATION OF THE VARIOUS MOLECULAR CHOLINRS AND 1,2-DIACYL-sn-GLYCEROLS BY LUNG MICROSOMES
CLASSES OF ENDOGENOUS PHOSPHATIDYLCHOLINEPHOSPHOTRANSFERASE OF MOUSE
[9,10-3H21Palmitate-labeRed microsomes (activity of the total phosphatidylchollne fraction 65 200 dpm per mg protein) were incubated for 20 min in the presence of CMP in the standard medium for the backreaction (See Materials and Methods). Column 1 presents the percentage of each class of endogenous phosphatidylcholine utilized as substrate in the back-reaction. [9.10-3H2]Palmitate-labelIed microsomes were also treated with phospholipase Cl to produce labelled endogenous l,Zaiacyl-sn-glycerols. The selective utilization of the 1,2_diacyl_sn-glycerols by cholinephosphotransferase in the forward reaction is presented in column 2 as the percentage of increase in radioactivity of each molecular class of phosphatidylcholine. Molecular class of phosphatidylcholine
Utilization of each class in the back-reaction (98)
increase of radioactivity in each class due to utilization of endogenous 1,2&acyl-sn-glycerols 1%)
Disaturated Monoenoic Dienoic Trienoic Tetraenoic Hexaenoic
4 26 21 I 31 11
4 a2 88 38 134 a4
430
tivity of the various molecular classes of phosphatidylcholine due to the utilization of the labelled 1,2-diacyl-sn-glycerols produced by the phospholipase C treatment of [ 9,10-3Hz]palmitate-labelled microsomes. It is quite clear that there is a high preference of cholinephosphotransferase for the unsaturated species of 1,2-diacyl-sn-glycerols and a very limited utilization of 1,2-dipalmitoyl-sn-glycerol. Discussion Conclusive evidence has been presented by Kanoh and Ohno [ 241 that exogenous 1,2-diacyl-sn-glycerols should not be utilized as substrates to investigate the substrate specificity of choline- and ethanolaminephosphotransferase. In the present paper two methods were used to generate endogenous 1,2-diacyl-snglycerols in mouse lung microsomes : (1) the conversion of phosphatidylcholine into 1,2-diacyl-sn-glycerols by cholinephosphotransferase in the back-reaction and (2) treatment of lung microsomes with a pure phospholipase C from B. cereus. The first technique has been used succesfully by Kanoh and Ohno [ 241 in their studies on the substrate specificity of choline- and ethanolaminephosphotransferase of rat liver microsomes [ 241. Moriya and Kanoh [ 201 have suggested that also in lung microsomes phosphatidylcholines might be utilized as a substrate in the back-reaction. The actual capacity of lung ‘inicrosomes to catalyze the back-reaction was shown in this paper by the following observations: incubation of [Me-‘4C]choline-labelled microsomes in the presence of CMP resulted in the release of CDP-[Me-’ 4C]choline whereas a similar incubation of [9,10-3H2] palmitate-labelled microsomes yielded labelled 1,2diacyl-ssn-glycerols. In both experiments a concomitant decrease of phosphatidylcholine was noticed. In this study the back-reaction was used explicitly as a tool to investigate the substrate selectivity of cholinephosphotransferase towards endogenous phosphatidylcholines. Unlike cholinephosphotransferase from rat liver microsomes which utilized all molecular classes of phosphatidylcholine randomly [ 241, cholinephosphotransferase of lung microsomes showed a distinct preference for unsaturated classes of phosphatidylcholine and hardly converted dipalmitoylglycerophosphocholine. This does not necessarily imply a true substrate specificity of cholinephosphotransferase because the possibility cannot be completely excluded that the dipalmitoylglycerophosphocholine is inaccessible to the enzyme due to its spatial localization in the membrane fragments. On the other hand, phospholipase C which was used to generate endogenous 1,2-diacyl-sn-glycerols, did attack all phosphatidylcholines randomly, including the disaturated species. Using the endogenous 1,2-diacyl-sn-glycerols produced by phospholipase C as substrate for cholinephosphotransferase in the forward reaction it was clearly shown that the enzyme exhibits a low affinity for 1,2-dipalmitoyl-sn-glycerol. These observations concerning the preferential utilization of unsaturated 1,2diacyl-sn-glycerols are in contrast with the unpublished observation quoted by Hendry and Possmayer in ref. 33 that cholinephosphotransferase of lung microsomes exhibits a preference for exogenous 1,2-diacyl-sn-glycerols with a saturated fatty acid at the 2-position. However, our observations on the low affinity of cholinephosphotransferase for endogenous dipalmitoylglycerophosphocho-
431
line or endogenous 1,2-dipalmitoyl-sn-glycerols strongly support other in vitro [17] and in vivo [14,20] studies which showed that the CDPcholine pathway primarily functions in the formation of unsaturated phosphatidylcholines and that the dipalmitoylglycerophosphocholine is at least partially synthesized by means of auxiliary mechanisms which modify the unsaturated phosphatidylcholine into dipalmitoylglycerophosphocholine [ 20,211. In this respect two mechanisms have been proposed: a deacylation-reacylation cycle [ 14,34-361 and a deacylation-transacylation process [37,38]. Undoubtedly, research in the near future will be directed to the elucidation of the relative importance of these two processes. In this light it is important to realize that it is now almost generally accepted (for reviews see refs. 4, 5 and 19) that the Type II cells of the lung are the locus for surfactant production. Since methods are becoming available to obtain isolated Type II cells [ 21,39,40] these cells are undoubtedly the material to be chosen for tackling the question whether deacylation-reacylation or transacylation or both are the main contributors to the formation of pulmonary dipalmitoylglycerophosphocholine. Acknowledgements The investigations were supported in part by the Netherlands Foundation for Chemical Research (S.O.N.) with financial aid from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.). We are very much indebted to Mr. V. Oldenborg for his excellent technical assistance and advices in some of the experiments. References A., Van Golde, L.M.G. and Van Deenen, L.L.M. (1971) Biochim. Biophys. Acta 231. 1 Montfoort. 335-342 Mason, R.J. (1973) Am. Rev. Resp. Dis. 10’7.678-679 King, R.J. and Clements. J.A. (1972) Am. J. Physiol. 223. 707-714 Goerke, J. (1974) Biochim. Biophys. Acta 344, 241-261 Tierney. D.F. (1974) Annu. Rev. Physiol. 36. 209-231 Avery. M.E. and Mead, J. (1959) Am. J. Dis. Child. 97, 517-523 Chu. J., Clements, J.A.. Cotton, E.K., Klaus, M.H.. Sweet, A.Y. and Tooley, W.H. (1967) Pediatrics 40. 709-782 8 Adams, F.H. and EnthBming. G. (1966) Acta Physiol. Stand. 68. 23-27 9 Kennedy, E.P. (1961) Fed. Proc. 20.934-940 10 Bremer. J. and Greenberg, D.M. (1961) Biochim. Biophys. Acta 46, 205-216 11 BjGmstad, P. and Bremer. J. (1966) J. Lipid Res. 7, 3845 12 Spitzer, H.L. (1968) Biochim. Biophys. Acta 152, 562-558 13 Weinhold, P.A. (1968) J. Lipid Res. 9. 262-266 14 Vereyken. J.M., Montfoort. A. and Van Golde, L.M.G. (1971) Biochim. Biophys. Acta 260. 70-81 15 Di Augustine, R.P. (1971) Biochem. Biophys. Res. Commun. 43.311-317 16 Morgan, T.E. (1971) Arch. Int. Med. 127.401407 17 Akino, T., Abe, M. and Arai, T. (1971) Biochim. Biophys. Acta 248. 274-281 18 Epstein, M.F. and Farrell. P.M. (1975) Pediatr. Res. 9.658-665 19 Frosolono. M.F. (1976) Lipid Metabolism in Mammals (Snyder, F.. ed.) Plenum Press. New York, in the press 20 Moriya. T. and Kanoh, H. (1974) Tohoku J. EXP. Med. 112.241-256 21 Snyder, C.. Malone, B.. Nettesheim. P. and Snyder, F. (1973) Cancer Res. 33, 2437-2443 22 Lands. W.E.M. (1960) J. Biol. Chem. 236. 2233-2237 23 Erblsnd. J.F. and Madnetti. G.V. (1966) Biochim. Biophys. Acta 105. 128-138 24 Kanoh. H. and Ohno. K. (1975) Biochim. Biophys. Acta 380.199-207 25 Weiss, S.B., Smith, S.W. and Kennedy, E.P. (1958) J. Biol. Chem. 231. 53-64
432 26 27 28 29 30
Kanoh, H. and Ohno, K. (1973) Biochim. Biophys. Acta 306.203-217 Lowry, O.H., Rosebrough, N.J.. Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 Akesson, B.. Elovson, J. and Arvidson, G. (1970) Biochim. Biophys. Acta 380. 199-207 Bligh, E.G. and Dyer, W.J. (1959) Can. J. Biochem. Physiol. 37, Sll-Sl7 Van Golde, L.M.G., Pieterson, W.A. and Van Deenen, L.L.M. (1968) Biochim. Biophys. Acta 152, 84-95
31 32 33 34 35 36 37 38 39 40
Fricke. U. (1975) Anal. Biochem. 63, 555-558 Garcia, A., Newkirk, J.D. and Mavis. R.D. (1975) Biochem. Biophys. Res. Commun. 64. 128-135 Hendry. A.T. and Possmayer. F. (1974) Biochim. Biophys. Acta 369.156-172 Frosolono, M.F., Slivka, S. and Charms, B.L. (1971) J. Lipid Res. 12. 96-103 Snyder, F. and Malone, B. (1975) Biochem. Biophys. Res. Commun. 66, 914-919 Tansey, F.A.. and Frosolono, M.F. (1975) Biochem. Biophys. Res. Commun. 67.1560-1566 Akino. T., Yamazaki, I. and Abe, M. (1972) Tohoku J. Exp. Med. 108,133-139 Hallman, M. and Raivio. K. (1974) Pediatr. Res. 8. 874-879 Mason, R.. Williams, M.C. and Clements, J.A. (1975) Chest 67, 36%37s Kikkawa, Y., Yoneda, K., Smith, F., Packard, B. and Suzuki, M.D. (1975) Lab. Invest. 32, 295-302
Biochimica et Biophysics Acta, 441 (1976) 423-432 0 Elsevier Scientific Publishing Company, Amsterdam
- Printed in The Netherlands
BBA 56851
SELECTIVE UTILIZATION OF ENDOGENOUS UNSATURATED PHOSPHATIDYLCHOLINES AND DIACYLGLYCEROLS BY CHOLINEPHOSPHOTRANSFERASE OF MOUSE LUNG MICROSOMES
M.G. SARZALA
* and L.M.G. VAN GOLDE **
Laboratory of Veterinary (The Netherlands) (Received
Biochemistry,
State University
of Utrechi,
Biltstraat
172, Utrecht
March 22nd, 1976)
1. In the presence of CMP, cholinephosphotransferase of mouse lung microsomes catalyzes the conversion of endogenous phosphatidylcholines into 1,2diacyl-sn-glycerols and CDPcholine. 2. In this conversion cholinephosphotransferase shows a distinct preference for those molecular species of phosphatidylcholine which contain an unsaturated fatty acid. The enzyme hardly utilizes endogenous dipalmitoylglycerophosphocholine as a substrate. 3. Membrane-bound 1,2-diacyl-sn-glycerols were also prepared by treatment of mouse lung microsomes with a pure phospholipase C from Bacillus cereus. These 1,2-diacyl-sn-glycerols were subsequently utilized as substrate by cholinephosphotransferase in the formation of phosphatidylcholine. In the latter reaction, cholinephosphotransferase exhibited a pronounced preference for unsaturated 1,2-diacyl-sn-glycerols and hardly utilized the endogenous 1,2-dipalmitoylsn-glycerol. 4. The low affinity of cholinephosphotransferase for either dipalmitoylglycerophosphocholine or 1,2-dipalmitoyl-sn-glycerol strongly endorses the concept that the CDPcholine pathway alone cannot be responsible for the production of pulmonary dipalmitoylglycerophosphocholine.
Introduction Dipalmitoylglycerophosphocholine choline) is known to be a principal * Present
(1,2-dipalmitoyl-sn-glycero-3-phosphophospholipid constituent of mammalian
address: Nencki Institute of Experimental Biology. Polish Academy of Sciences, Department of Biochemistry of Nervous System and Muscle, 3 Pasteur Street, Warszawa 22, Poland. ** To whom all correspondence should be addressed.
424
lung tissues [ 1,2]. It is also generally accepted as the major active component of the lung surfactant which prevents alveolar collapse during expiration [ 3-51. The known association between the “respiratory distress syndrome” of the newborn and a deficient surfactant production [6--81 has undoubtedly provoked interest in the mechanisms involved in the biosynthesis of pulmonary phosphatidylcholines. Based on our present knowledge two pathways can be considered to operate in the de novo formation of pulmonary phosphatidylcholines: (a) the ~DP~holine pathway established by Kennedy [9] and (b) a stepwise methylation of phosphatidylethanolamine f lo]. Accumulating evidence has been presented in the past few years that pathway b is of minor importance in the mammalian lung [ 11-17 1, including that or primates [ 18 J. Though all info~ation presently available (for reviews st efs. 5 and 19) points to an important role of the CDPcholine pathway in the biosynthesis of pulmonary phosphatidylcholines, it is unlikely that this pathway alone can be responsible for the formation of the dip~mitoylglycerophosphocholine species: both in vivo [ 14,201 and in vitro studies [ 171 have indicated that in the lung the CDPcholine pathway primarily functions in the production of unsaturated phosphatidylcholines. In addition, it has been demonstrated that a large portion of the palmitate at the 2-position of dipalmitoylgly~erophosphocholine is incorporated via a pathway bypassing CDPcholine [20,21]. So far, however, no explanation was available for the limited capacity of the CDPcholine pathway to produce pulmonary dipalmitoylglycerophosphocholine directly, i.e. without auxiliary mech~isms such as a deacylation-reacyl~tion cycle f 221 or a deacylation-transacylation process f 231. A possible explanation might be that cholinephosphotransferase (EC 2.7.8.2), which catalyzes the conversion of 1,2-diacyl-sn-glycerols into phosphatidylcholine, hardly utilizes 1,2-dipalmitoyl-sn-glycerol as substrate. As has been shown convincingly by Kanoh and Ohno [24], it is difficult to investigate the substrate specificity of cholinephosphotransferase with emulsions of exogenous 1,2-diacyl-sn-glycerols as substrates. These authors developed an elegant way to study the substrate specificity of choline- and ethanol~inepl~osphotr~sferase of rat liver microsomes. They made use of the known fact [11,25,26] that in the liver the reaction stimulated by cholinephosphotransferase is reversible : 1,2-diacyl-sn-glycerol +
CDPcholine
$ phosphatidylcholine
+ CMP
Kanoh and Ohno employed the back-reaction (b) as a tool to produce membrane-bound 1,2~iacyl-so-glycerols which were subsequently utilized as a substrate in the forward reaction (a) with a specificity that agreed very nicely with data from in vivo studies [ 241. In the present paper evidence will be provided that also in lung microsomes phosphatidylcholines can be utilized as substrate by cholinephosphotransfer~e in the back-reaction. Membrane-bound 1,2-diacyl-sn-glycerols were also prepared by treatment of lung microsomes with a pure phospholipase C from Bacillus cereus. From these two approaches it could be concluded that cholinephosphotransferase of lung microsomes exhibits a low affinity for 1,2-dipalmitoylsn-glycerol in the forward reaction and for dipalmitoylglycerophosphocholine in the back-reaction.
425
Materials and Methods Materials [9,10-3Hz]palmitic acid (spec. act. 500 Ci/mol) and [Me-14C]choline (spec. act. 52 Ci/mol) were purchased from the Radiochemical Centre, Amersham, Great Britain. CDP- [ Me- l 4 Clcholine (spec. act. 40 Ci/mol) was bought from N.E.N.-Chemicals, Dreieichenhain, Germany. Diisopropylfluorophosphate was supplied by EGA-Chemie KG, Steinheim, Germany. Unlabelled fatty acids were obtained from Sigma, St Louis, MO., U.S.A. and most biochemicals from Boehringer, Mannheim, Germany. All other chemicals and solvents were of analytical grade. Pure phospholipase C from B. cereus was a generous gift of Dr. R.A.F. Zwaal, Biochemistry Laboratory, State University of Utrecht, Utrecht, The Netherlands. The mice (Swiss random strain) were bought from the Central Institute for the breeding of Laboratory Animals, Zeist, The Netherlands. Methods Isolation of mouse lung microsomes. Groups of 10 mice were killed by cervical fracture. After removal and washing with 0.9% NaCl, the lungs were minced and homogenized in 4 ml of ice-cold 0.25 M sucrose using a Potter-Elvehjem homogenizer. After removal of the nuclear plus cell debris fraction by centrifugation at 1000 X g for 5 min, the supernatant was spun at 20 000 X g for 10 min in the SS-34 rotor of the Sorvall RC-2B centrifuge. The microsomes were then pelleted from the 20 000 X g supernatant by centrifugation at 105 000 X g for 90 min in the 50 rotor of a Beckman L-565 ultracentrifuge. The microsomal fraction obtained in this way was suspended in 2 ml of 0.125 M KCl/O.l M Tris (pH 7.4) and, if not used directly, stored at -80°C. Protein contents were estimated by the method of Lowry et al. [27]. Preparation of [Me-14C]choline- and [9,1 0-3Hz]palmitate-labelled microsomes. Groups of 10 mice received 0.1 ml. of saline containing 25 I.cCi[Me-14C]choline by injection into the tail vein. After 2.5 h the mice were killed and the microsomal fraction isolated from the lungs as described above. Under these conditions 95% of the labelled choline incorporated into the total lipids, was recovered in the phosphatidylcholine fraction. [9,10-3Hz]palmitic acid was complexed to albumin exactly as described by Wkesson et al. [28]. Aliquots of 100 E.tCi were injected into the tail vein of 10 mice and the lung microsomes isolated after 1 h. Phosphatidylcholine contained approx. 68% of the palmitate incorporated into the total lipid fraction. Incubation conditions for the back-reaction stimulated by cholinephosphotransferase. Varying amounts of microsomes labelled with either [Me-‘4C]choline or [9,10-3Hz]palmitate were incubated for the indicated periods of time (see Results) in a shaking water bath at 37°C. Unless specified otherwise, the incubation medium (1 ml) contaIned the following components: 100 mM Tris HCl (pH 8.5), 10 mM dithiothreitol, 10 mM MgClz, 5 mM EGTA * and 4 mM CMP. Parallel experiments were conducted in which the CMP was omitted. In incubations in which [Me- ’ 4 C] choline-labelled microsomes were used, the reactions were terminated by the addition of 1 ml of ice-cold 10% trichloroacetic acid. After centrifugation, aliquots were taken from the supematant to measure * EGTA:
ethyleneglycol-bis(&aminoethyl
etherkN,N-tetracetic
acid.
426
by scintillation counting the amount of trichloroacetic acid-soluble radioactivity produced in the back-reaction. The lipids were extracted from the acidinsoluble fraction of the microsomes by the method of Bligh and Dyer [ 291. In experiments with unlabelled or [ 3Hlpalmitate-labelled microsomes, the microsomes were preincubated in the presence of 3 mM diisopropylfluorophosphate [ 241 at 37” C for 1 h to inhibit the possible action of microsomal lipase on the 1,2-diacyl-sn-glycerols formed during the back-reaction. After incubation of the microsomes in the presence and absence of CMP as described above, the reactions were stopped by the addition of 4 ml of methanol/chloroform (2 : 1, v/v) and the lipids extracted [ 291. Treatment of lung microsomes with phospholipase C. [9,10-3Hz]Palmitatelabelled microsomes (about 4 mg of protein) were suspended in 2 ml of a 0.1 M Tris . HCl (pH 8.3) buffer, containing 5 mM CaC&. After the addition of 20 (~1of phospholipase C from B. cereus (2.3 units), the microsomes were incubated at 37°C for 10 min. The reaction was terminated by the addition of 0.2 ml 0.1 M EGTA. The microsomes were then recovered from the incubation medium by layering the incubation mixture on 0.3 M sucrose and subsequent centrifugation at 30 000 X g for 90 min. The pellet was resuspended in 1 ml of 0.125 M KU/O.1 M Tris (pH 7.4). Aliquots of the microsomes were then taken for extraction [29] and lipid analysis. The remaining microsomes which contained membrane-bound 1,2-diacyl-sn-glycerols formed through the action of phospholipase C, were subsequently used as enzyme and substrate source in the forward reaction of cholinephosphotransferase. Incubation
conditions
for cholinephosphotransferase
in the forward
reaction.
Varying amounts of microsomes, containing membrane-bound 1,2-diacyl-snglycerols, were incubated at 37°C in a medium of the following composition: 100 ~1 0.1 M Tris - HCl (pH 8.5), 50 ~1 0.1 M MgCl?, 20 ,ul 0.1 M dithiothreitol and 10 ~120 mM CDP-[Me-14C]choline (spec. act. 0.5 Ci/mol) in a total volume of 0.2 ml. In parallel experiments untreated microsomes were used as enzyme and substrate source. After 10 min the reactions were stopped by the addition of 0.8 ml of methanol/chloroform (2 : 1, v/v) and the lipids extracted [ 291. Lipid analyses. Phosphatidylcholine was isolated from the total lipid mixtures by means of silica thin-layer chromatography using chloroform/methanol/ water (65 : 35 : 4, v/v) as developing solvent [30]. The phosphatidylcholine fraction was recovered quantitatively from the silica by extraction according to Bligh and Dyer [ 291. Aliquots were transferred into scintillation vials containing 10 ml of a scintillation fluid described by F’ricke [31]. The radioactivity measurements were carried out in a Packard Tricarb model 2425 B liquid scintillation counter. The remaining portions of the phosphatidylcholines were converted into 1,2-diacyl-sn-glycerols by treatment with phospholipase C and subsequently resolved by argentation thin-layer chromatography in disaturated, mono-, di-, tri-, tetra- and hexaenoic species. The details of this procedure have been published earlier [ 1,301. After extraction from the silica with chloroform/ methanol (2 : 1, v/v) [ 1,301, aliquots of the various species of 1,2-diacyl-snglycerols were taken for assay of the radioactivity. The amounts of the various species were estimated by means of gas-liquid chromatography using an internal standard as has been described in detail before [1,30]. 1,2-Diacyl-sn-glycerols present in the microsomal lipids or formed either by the back-reaction or by
427
the treatment of microsomes with phospholipase C, were isolated by silica thinlayer chromatography using benzene/chloroform/methanol (80 : 15 : 5, vlv) as developing solvent. After extraction from the silica with several portions of ether, the l,Z-diacyl-sn-glycerols were subsequently separated into the various molecular classes and analyzed for radioactivity content as described above. Results ~ccu~e~~e of the buck-reaction in mouse lung ~icroso~es The incubation conditions for investigation of the back-reaction in mouse lung microsomes were derived from preliminary experiments with microsomes containing phosphatidyl-[Me-’ 4 Clcholine. The decrease in radioactivity of phosphatidylcholine and the release of trichloroacetic acid-soluble radioactivity were used as parameters for the extent of the back-reaction [26]. The pH optimum for the action of cholinephosphot~sfer~e in the back-reaction was approx. 8.5 and a concentration of CMP of 4 mM was found to be required for maximal velocity of the reaction. EGTA was included in the reaction medium since Ca2* was found to be strongly inhibitory, as had been reported earlier for the back-reaction in rat liver microsomes [26]. Fig. 1 presents the distribution of radioactivity between phosphatidylcholine and the trichloroacetic acid-soluble fraction of the microsomes after different incubation times in the presence and absence of CMP. It is clear that addition of CMP induces a pronounced decrease in radioactivity content of phosphatidylcholine and a concomitant release of trichloroacetic acid-soluble radioactivity. By means of paper chromatography as described by Kanoh and Ohno [ 261 and using authentic CDPcholine and phospho~lchol~e as references, it could be established that the soluble radioactivity released from phosphatidylcholine in the presence of CMP consisted for more than 95% of CDPcholine. Fig. 2 presents the results obtained with [9,10-3H,]palmitate-labelled microsomes. It is clear that incubation in the presence of CMP causes a considerable formation of 1,2_diacyl-sn-glycerols. In strong contrast to the concomitant decrease in phosphatidylcholine (not shown), no conversion of phospha~dyleth~olamine was observed during the ineubation. The identity of the 1,2-diacyl-sn-glycerols formed by the back-reaction was established by cochromatography with an authentic synthetic 1,2-diacylsn-glycerol in various solvent systems. In addition they were effectively utilized as substrate by cholinephosphotransferase in the forward reaction. As had been found for rat liver microsomes f 241, it appeared to be necessary to preincubate the microsomes with d~sopropyl~uorophosphate to inhibit the action of a microsomal lipase on the 1,2-diacyl-sn-glycerols produced in the back-reaction. Omission of the preincubation led to a significantly smaller accumulation of 1,2diacyl-an-glycerols (Fig. 2) and to a production of radioactive monoacylglycerols and free fatty acids (data not shown). Apparently, a microsomal lipase is present, in contrast to an observation by Garcia et al. [32] who could not detect a lipase in lung microsomes. A possible explantion for this discrepancy might be that these authors only used exogenous triacylglycerols as substrate. Substrate specificity of cholinephosphotransferase in the utilization of phosphatidylcholines It was thought of great interest to investigate a possible specificity of cho-
428
10
20 lncubatlon
30 time
(man)
40
10
20 lncubaton
30
40
tome (man)
Fig. 1. Distribution of radioactivity between phosphatidylcholine and the trichloroacetic acid-soluble portion of mouse lung microsomes after incubation of [Me-l4 Clcholine-labelled microsomes in the presence and absence of CMP. The composition of the incubation medium has been described in detail in Materials and Methods. After the indicated periods of incubation. the reactions were terminated by the addition of 10% trichloroacetic acid and aliquots taken from the trichloroacetic acid-soluble portion and from phosphatidylcholine isolated from the insoluble portion of the microsomes. The radioactivity of phosphatidylcholine before the incubation (45 920 dpm) is taken as 100%. lL, phosphatidylcholine, in the presence of CMP; o----O, idem, in the absence of CMP; n-, trichloroacetic acid-soluble portion. in the presence of CMP and o-, idem, in the absence of CMP. Fig. 2. Radioactivity accumulating in 1.2-diacyl-sn-glycerols during incubation of [9.10-3H21palmitatelabelLed microsomes in the presence and absence of CMP. The details of the incubation procedures are described in Materials and Methods. The results are expressed as percentage of the radioactivity of phosphatidylcholine (66 000 dpm) before the incubation. 0~. in the presence of CMP, after preincubation with diisopropylfluorophosphate: A-. in the presence of CMP, without preincubation with diisopropylfluorophosphate and o-----U, in the absence of CMP, after preincubation.
linephosphotransferase with respect to the various species of endogenous pulmonary phosphatidylcholines as substrates for the back-reaction. In these experiments [ 9,10-3 Hz lpalmitate-labelled microsomes were used in order to follow the formation of labelled molecular species of 1,2-diacyl-sn-glycerols. Table I presents the distribution of radioactive palmitate among the various molecular species of phosphatidylcholine before the incubation with CMP. In agreement with the findings of Moriya and Kanoh [20] in whole rat lung, the majority of the palmitate had been incorporated into the dipalmitoylglycerophosphocholine fraction of mouse lung microsomes. This fraction also possessed the highest relative specific activity (third column of Table I). Table II (column 1) presents the percentages of each molecular class of phosphatidylcholines utilized as substrate in the back-reaction. Very interesting is the observation that cholinephosphotransferase consumes preferentially the mono-, di-, and tetraenoic phosphatidylcholines whereas the disaturated species are hardly used as substrate by the enzyme. A possible selective utilization of the various species of 1,2-diacyl-sn-glycerols, produced in the microsomes by the back-reaction, as substrates for cholinephosphotransferase in the forward
429 TABLE I DISTRIBUTION OF RADIOACTIVITY AMONG THE VARIOUS CHOLINE OF MOUSE LUNG MICkOSOMES I h AFTER THE ~~,~~-~H~IPALM~TATE
CLASSES OF INTRAVENOUS
PHOSPHATIDYG INJECTION OF
For experimental details see Materials and Methods. The relative specific activity of a given class is defined as the percentage of radioactivity in that class (1st column) divided by the weight percentage (2nd column). The radioactivity of the total phosphatidylcholine fraction was 65 200 dpm. Chtsses of phosp~tidyIcho~ne
Diiaturated Monoenoic Dienoic Trienoic Tetraenoic Hexaenoic
Distribution
of
C9,19-3H21 pabnitate (%l
Weight distribution
66.3 16.6 13.0 3.8 5.8 5.3
Relative activity
specific
(%)
I.87
29.6 27.0 21.8 4.2 11.4 6.0
0.61 0.60 0.90 0.51 0.88
reaction was not pursued because of the very small amounts of 1,2-dipalmitoylsn-glycerol produced in the back-reaction. Therefore, phospholipase C treatment was chosen as an alternative method to generate endogenous l,Z-diacylsn-glycerols in [9,10-3Hz ~p~rni~te-labelled microsomes. Treatment of lung microsomes with phospholipase C resulted in an equal conversion of both phosphatidylcholine and phosphatidylethanolamine. The enzyme almost randomly attacked the various molecular specie8 of phosphatidylcholine including the disaturated class. The 1,2-diacyl-sn-glycerols, produced by phospholipase C, were subsequently utilized as substrates in the forward reaction catalyzed by cholinephospho~~sfer~e. Table II (column 2) shows the increase in radioacTABLE
II
UTILIZATION OF THE VARIOUS MOLECULAR CHOLINRS AND 1,2-DIACYL-sn-GLYCEROLS BY LUNG MICROSOMES
CLASSES OF ENDOGENOUS PHOSPHATIDYLCHOLINEPHOSPHOTRANSFERASE OF MOUSE
[9,10-3H21Palmitate-labeRed microsomes (activity of the total phosphatidylchollne fraction 65 200 dpm per mg protein) were incubated for 20 min in the presence of CMP in the standard medium for the backreaction (See Materials and Methods). Column 1 presents the percentage of each class of endogenous phosphatidylcholine utilized as substrate in the back-reaction. [9.10-3H2]Palmitate-labelIed microsomes were also treated with phospholipase Cl to produce labelled endogenous l,Zaiacyl-sn-glycerols. The selective utilization of the 1,2_diacyl_sn-glycerols by cholinephosphotransferase in the forward reaction is presented in column 2 as the percentage of increase in radioactivity of each molecular class of phosphatidylcholine. Molecular class of phosphatidylcholine
Utilization of each class in the back-reaction (98)
increase of radioactivity in each class due to utilization of endogenous 1,2&acyl-sn-glycerols 1%)
Disaturated Monoenoic Dienoic Trienoic Tetraenoic Hexaenoic
4 26 21 I 31 11
4 a2 88 38 134 a4
430
tivity of the various molecular classes of phosphatidylcholine due to the utilization of the labelled 1,2-diacyl-sn-glycerols produced by the phospholipase C treatment of [ 9,10-3Hz]palmitate-labelled microsomes. It is quite clear that there is a high preference of cholinephosphotransferase for the unsaturated species of 1,2-diacyl-sn-glycerols and a very limited utilization of 1,2-dipalmitoyl-sn-glycerol. Discussion Conclusive evidence has been presented by Kanoh and Ohno [ 241 that exogenous 1,2-diacyl-sn-glycerols should not be utilized as substrates to investigate the substrate specificity of choline- and ethanolaminephosphotransferase. In the present paper two methods were used to generate endogenous 1,2-diacyl-snglycerols in mouse lung microsomes : (1) the conversion of phosphatidylcholine into 1,2-diacyl-sn-glycerols by cholinephosphotransferase in the back-reaction and (2) treatment of lung microsomes with a pure phospholipase C from B. cereus. The first technique has been used succesfully by Kanoh and Ohno [ 241 in their studies on the substrate specificity of choline- and ethanolaminephosphotransferase of rat liver microsomes [ 241. Moriya and Kanoh [ 201 have suggested that also in lung microsomes phosphatidylcholines might be utilized as a substrate in the back-reaction. The actual capacity of lung ‘inicrosomes to catalyze the back-reaction was shown in this paper by the following observations: incubation of [Me-‘4C]choline-labelled microsomes in the presence of CMP resulted in the release of CDP-[Me-’ 4C]choline whereas a similar incubation of [9,10-3H2] palmitate-labelled microsomes yielded labelled 1,2diacyl-ssn-glycerols. In both experiments a concomitant decrease of phosphatidylcholine was noticed. In this study the back-reaction was used explicitly as a tool to investigate the substrate selectivity of cholinephosphotransferase towards endogenous phosphatidylcholines. Unlike cholinephosphotransferase from rat liver microsomes which utilized all molecular classes of phosphatidylcholine randomly [ 241, cholinephosphotransferase of lung microsomes showed a distinct preference for unsaturated classes of phosphatidylcholine and hardly converted dipalmitoylglycerophosphocholine. This does not necessarily imply a true substrate specificity of cholinephosphotransferase because the possibility cannot be completely excluded that the dipalmitoylglycerophosphocholine is inaccessible to the enzyme due to its spatial localization in the membrane fragments. On the other hand, phospholipase C which was used to generate endogenous 1,2-diacyl-sn-glycerols, did attack all phosphatidylcholines randomly, including the disaturated species. Using the endogenous 1,2-diacyl-sn-glycerols produced by phospholipase C as substrate for cholinephosphotransferase in the forward reaction it was clearly shown that the enzyme exhibits a low affinity for 1,2-dipalmitoyl-sn-glycerol. These observations concerning the preferential utilization of unsaturated 1,2diacyl-sn-glycerols are in contrast with the unpublished observation quoted by Hendry and Possmayer in ref. 33 that cholinephosphotransferase of lung microsomes exhibits a preference for exogenous 1,2-diacyl-sn-glycerols with a saturated fatty acid at the 2-position. However, our observations on the low affinity of cholinephosphotransferase for endogenous dipalmitoylglycerophosphocho-
431
line or endogenous 1,2-dipalmitoyl-sn-glycerols strongly support other in vitro [17] and in vivo [14,20] studies which showed that the CDPcholine pathway primarily functions in the formation of unsaturated phosphatidylcholines and that the dipalmitoylglycerophosphocholine is at least partially synthesized by means of auxiliary mechanisms which modify the unsaturated phosphatidylcholine into dipalmitoylglycerophosphocholine [ 20,211. In this respect two mechanisms have been proposed: a deacylation-reacylation cycle [ 14,34-361 and a deacylation-transacylation process [37,38]. Undoubtedly, research in the near future will be directed to the elucidation of the relative importance of these two processes. In this light it is important to realize that it is now almost generally accepted (for reviews see refs. 4, 5 and 19) that the Type II cells of the lung are the locus for surfactant production. Since methods are becoming available to obtain isolated Type II cells [ 21,39,40] these cells are undoubtedly the material to be chosen for tackling the question whether deacylation-reacylation or transacylation or both are the main contributors to the formation of pulmonary dipalmitoylglycerophosphocholine. Acknowledgements The investigations were supported in part by the Netherlands Foundation for Chemical Research (S.O.N.) with financial aid from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.). We are very much indebted to Mr. V. Oldenborg for his excellent technical assistance and advices in some of the experiments. References A., Van Golde, L.M.G. and Van Deenen, L.L.M. (1971) Biochim. Biophys. Acta 231. 1 Montfoort. 335-342 Mason, R.J. (1973) Am. Rev. Resp. Dis. 10’7.678-679 King, R.J. and Clements. J.A. (1972) Am. J. Physiol. 223. 707-714 Goerke, J. (1974) Biochim. Biophys. Acta 344, 241-261 Tierney. D.F. (1974) Annu. Rev. Physiol. 36. 209-231 Avery. M.E. and Mead, J. (1959) Am. J. Dis. Child. 97, 517-523 Chu. J., Clements, J.A.. Cotton, E.K., Klaus, M.H.. Sweet, A.Y. and Tooley, W.H. (1967) Pediatrics 40. 709-782 8 Adams, F.H. and EnthBming. G. (1966) Acta Physiol. Stand. 68. 23-27 9 Kennedy, E.P. (1961) Fed. Proc. 20.934-940 10 Bremer. J. and Greenberg, D.M. (1961) Biochim. Biophys. Acta 46, 205-216 11 BjGmstad, P. and Bremer. J. (1966) J. Lipid Res. 7, 3845 12 Spitzer, H.L. (1968) Biochim. Biophys. Acta 152, 562-558 13 Weinhold, P.A. (1968) J. Lipid Res. 9. 262-266 14 Vereyken. J.M., Montfoort. A. and Van Golde, L.M.G. (1971) Biochim. Biophys. Acta 260. 70-81 15 Di Augustine, R.P. (1971) Biochem. Biophys. Res. Commun. 43.311-317 16 Morgan, T.E. (1971) Arch. Int. Med. 127.401407 17 Akino, T., Abe, M. and Arai, T. (1971) Biochim. Biophys. Acta 248. 274-281 18 Epstein, M.F. and Farrell. P.M. (1975) Pediatr. Res. 9.658-665 19 Frosolono. M.F. (1976) Lipid Metabolism in Mammals (Snyder, F.. ed.) Plenum Press. New York, in the press 20 Moriya. T. and Kanoh, H. (1974) Tohoku J. EXP. Med. 112.241-256 21 Snyder, C.. Malone, B.. Nettesheim. P. and Snyder, F. (1973) Cancer Res. 33, 2437-2443 22 Lands. W.E.M. (1960) J. Biol. Chem. 236. 2233-2237 23 Erblsnd. J.F. and Madnetti. G.V. (1966) Biochim. Biophys. Acta 105. 128-138 24 Kanoh. H. and Ohno. K. (1975) Biochim. Biophys. Acta 380.199-207 25 Weiss, S.B., Smith, S.W. and Kennedy, E.P. (1958) J. Biol. Chem. 231. 53-64
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Kanoh, H. and Ohno, K. (1973) Biochim. Biophys. Acta 306.203-217 Lowry, O.H., Rosebrough, N.J.. Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 Akesson, B.. Elovson, J. and Arvidson, G. (1970) Biochim. Biophys. Acta 380. 199-207 Bligh, E.G. and Dyer, W.J. (1959) Can. J. Biochem. Physiol. 37, Sll-Sl7 Van Golde, L.M.G., Pieterson, W.A. and Van Deenen, L.L.M. (1968) Biochim. Biophys. Acta 152, 84-95
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Fricke. U. (1975) Anal. Biochem. 63, 555-558 Garcia, A., Newkirk, J.D. and Mavis. R.D. (1975) Biochem. Biophys. Res. Commun. 64. 128-135 Hendry. A.T. and Possmayer. F. (1974) Biochim. Biophys. Acta 369.156-172 Frosolono, M.F., Slivka, S. and Charms, B.L. (1971) J. Lipid Res. 12. 96-103 Snyder, F. and Malone, B. (1975) Biochem. Biophys. Res. Commun. 66, 914-919 Tansey, F.A.. and Frosolono, M.F. (1975) Biochem. Biophys. Res. Commun. 67.1560-1566 Akino. T., Yamazaki, I. and Abe, M. (1972) Tohoku J. Exp. Med. 108,133-139 Hallman, M. and Raivio. K. (1974) Pediatr. Res. 8. 874-879 Mason, R.. Williams, M.C. and Clements, J.A. (1975) Chest 67, 36%37s Kikkawa, Y., Yoneda, K., Smith, F., Packard, B. and Suzuki, M.D. (1975) Lab. Invest. 32, 295-302