- Email: [email protected]
Possible involvement of acyltransferase systems in the formation of pulmonary surfactant lipid in rat
ARCHIVES
OF BIOCHEMISTRY
AND BIOPHYSICS
Vol. 196, No. 1, August, pp. 209-219, 1979
Possible
Involvement of Acyltransferase Pulmonary Surfactant
Systems in the Formation Lipid in Rat
KAZUYO YAMADA AND HARUMI
of
OKUYAMA
The Faculty of Pharmaceutical Sciences, Nagoya City University, S-1 Tanabedori, Mizuhoku, Nagoya 467, Japan Received January 8, 1979; revised March 15, 1979 Dithiobis (2-nitrobenzoic acid)-resistant and -sensitive glycerophosphate acyltransferase systems were present in rat lung as in liver. The former was specific for palmitate while the latter could incorporate saturated and unsaturated acyl-CoAs comparably. The former has higher affinity for palmitate than the latter indicating that the l-position of glycerophosphate can be acylated selectively with palmitate under certain conditions. The specificities of 1-acylglycerophosphate and 1-acylglycerophosphocholine acyltransferase systems were similar in lung and liver; both systems showed higher specificities for unsaturated acyl-CoAs. However, the selectivities observed at lower concentrations of phospholipid acceptors in the presence of equimolar mixtures of saturated and unsaturated acyl-CoAs were much different; the lung systems showed relatively higher selectivities for palmitate than the liver systems in the formation of both diacylglycerophosphate and phosphatidylcholine. On the other hand, palmitate was excluded almost completely from the e-position in the 1-acylglycerophosphoethanolamine acyltransferase systems in lung and liver. These observations provide an enzymatic basis for describing the formation of pulmonary surfactant lipids in rat via acyltransferase systems.
Dipalmitoyl-sn-glycerol 3-phosphocholine is one of the major molecular species of phosphatidylcholine in mammalian lung tissue and is considered to play an important role in determining the surface properties of pulmonary surfactant (l-4). The processes which allow this particular molecular species to be formed in such abundance in lung in contrast to other tissues have been the subject of many investigators (2-4). Akino et al. (56) first proposed that an enzyme catalyzing a transesterification between two molecules of 1-acyl-sn-glycerol 3-phosphocholine (1-acyl-GPC)’ plays an important role in the synthesis of dipalmitoyl-GPC in rat lung. This idea is supported by the findings that this enzyme in lung can produce dipalmitoyl-GPC in vivo and in vitro, and that the enzyme activity increases at the latter stages of the development of embryo lung ’ Abbreviations used: -GP, -sn-glycerol3-phosphate; -GPC, -sn-glycerol3-phosphocholine; -GPE, -an-glycerol 3-phosphoethanolamine; DTNB, 5,5’-dithiobis (2-nitrobenzoic acid).
(7-ll), although other enzyme activities also increase (12- 14). However, the possible contributions of other biosynthetic routes to the formation of dipalmitoyl-GPC in lung have not been fully investigated and what data are available seem contradictory and confusing. For example, a significant proportion of the diacylglycerol and phosphatidylcholine formed from labeled glycerol or choline in vivo (15, 16) and in tissue slices (5) is the disaturated species, suggesting a potential role of the de novo synthetic pathway in the formation of disaturated species. Surprisingly, however, the CDP-choline:diacylglycerol cholinephosphotransferase system in lung tissue apparently does not utilize dipalmitoyl glycerol as a substrate in vitro (17, 18). Furthermore, the acyl-CoA:l-acylGPC acyltransferase system has been reported to exhibit higher specificity for unsaturated acyl-CoAs in vitro (19); and added radioactive 1-acyl-GPC is known to be acylated in viva and in slices (5, 6) preferentially with tetraene and diene fatty acids. In ap-
209
0003-9861/79/090209-11$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
210
YAMADA
AND OKUYAMA
parent contrast to this latter result, is the observation that radiolabeled palmitic acid, when added to lung slices, is esterified mainly at the e-position of phosphatidylcholine suggesting the involvement of l-acylGPC acyltransferase system in the formation of disaturated speciesin viva (5,6,20). When the synthesis of phosphatidylcholine from 1-acyl-GPC by microsomes was examined in the presence of fatty acids, ATP, and CoA, lung microsomes were found to incorporate relatively more palmitate into phosphatidylcholine than did liver microsomes (15). This indicates that through some specificity inherent in either the acyl-CoA synthetase system and/or acyltransferase systems in the lung, palmitate is more likely to be incorporated into the 2-position of lung phosphatidylcholine. One other difficulty in evaluating the contributions of the acyltransferase systems in dipalmitoyl-GPC synthesis is that the specificities reported for acyl-CoAs in lung microsomes by different workers vary considerably (15, 19, 2123). These variabilities may result from relatively high acyl-CoA hydrolase activities present in lung microsomes prepared by current methods. Even so, the specificities2 of the acyltransferase systems as measured by the maximal velocities for the individual acyl-CoAs may not be applicable in vivo. As we have shown previously (24-28), acyltransferase systems in many cases lack specificity for the acyl-CoA precursors and fail to produce an explanation for the highly selective positioning of various fatty acids at the l- and 2-positions of glycerophospholipids from rat liver or Escherichia coli. However, the selectivities2 of acyltransferase systems for acyl groups in mixtures of acyl donors vary in vitro depending on the relative concentrations of the substrates. The selectivities noted can be used to account for severalin viva phenomena (26-30). This paper compares and contrasts the p The term “specificity” is used to denote comparisons of acyltransfer rates measured in the presence of saturating concentrations of single acyl donors and single acyl acceptors. The term “selectivity” is used when the velocities of acyltransfer measured in the presence of mixtures of acyl donors or in the presence of suboptimal concentrations of substrates were compared.
properties of the acyltransferase systems of rat liver and lung with particular emphasis on variations in the selectivities observed in acyl group transfers as a function of substrate concentrations. We postulate these acyltransferase systems could contribute to the incorporation of palmitate into the 2-position of phosphatidylcholine in rat lung. MATERIALS
AND METHODS
[9,10-3H]Palmitic acid3 and [l-‘4C]linoleic acid were the products of the Amersham Radiochemical Centre (Amersham, England) and [lJ%]palmitic acid and [5,6,8,9,11,12,14,15-3H]arachidonicacid were theproducts of New England Nuclear (Boston, Mass.). CoA was obtained from Kyowa Hakko Company, Ltd. (Tokyo). Bovine serum albumin (fatty acid-poor) was obtained from Miles Laboratories, Inc.. 5,5’-Dithiobis (2-nitrobenzoic acid) (DTNB) and Crotalus adamanteus venom were purchased from Sigma. Acyl-CoA derivatives were prepared by a modification of Seubert’s procedure (31) as described previously (32). [3H]Glycerol 3-phosphate was prepared from [2-3H]glycerol (New England Nuclear) according to the method of Chang and Kennedy (33). I-Acyl-GPC was prepared from lecithin as described previously (34). 1-Acyl-GP was synthesized from 1-acyl-GPC according to the method of Long et al. (35). l-Acyl-GPE was prepared from eggphosphatidylethanolamine by treatment with phospholipase A, as described previously (34). These substrates contain palmitate and stearate as the major fatty acyl chain substituents. Rat Preparation of subcellular membranefractions. lung was minced and then homogenized in 5 vol of 0.25 M sucrose for 30 s with a Waring Blendor. The homogenate was filtered through three layers of gauze. The unfiltered residual material was rehomogenized with 5 vol of 0.25 M sucrose and again filtered. The filtrates were combined, and the pH was adjusted to 7.5 with 1 M Tris. The sample was centrifuged for 5 min at 6001~and the precipitate discarded. To the supernatant was added 0.11 vol of 1 M Tris-chloride, pH 9.0. Treatment of membranes at alkaline pH (8.0-8.5) eliminated acyl-CoA hydrolase activity which otherwise interfered with the DTNB-based spectrophotometric assay of acyltransferases. The supernatant obtained from centrifugation at SOOg for 5 min was centrifuged at 100,OOOgfor 90 min. The resulting pellet was suspended in 0.25 M sucrose-O.01 M Tris-chloride, pH 7.5, and designated as the whole membrane fraction. To obtain a mitochondrial fraction, the 6009 3 Specific radioactivities of labeled substrates were varied by diluting with nonlabeled substrates to obtain enough and comparable tritium and carbon-14 radioactivities for counting.
SURFACTANT
LIPID SYNTHESIS
supernatant was centrifuged at 78OOgfor 10 min, and the pellet homogenized in 0.1 M Tris-chloride, pH 9.0, centrifuged, and resuspended in 0.25 M sucrose-O.01 M Tris-chloride, pH 7.5. The intermediate fraction was prepared by centrifuging the 78OOgsupernatant at l5,OOOgfor 20 min. Then 0.11 vol of 1 M Tris-chloride, pH 9.0, was added to the resulting supernatant. Microsomes were then sedimented from this fraction by centrifugation at 100,OOOg for 90 min and suspended in 0.25 M sucrose-O.01 M Tris-chloride, pH 7.5. All membrane fractions were stored at -20°C until use. Subcellular fractions from liver were obtained as described previously (30). Assays. Acyl-CoA:glycerophosphate acyltransferase activities were assayed by measuring the rate of incorporation of [3H]glycerophosphate into the lipidsoluble fraction as described previously (28). Unless otherwise noted, the typical incubation mixture consisted of 2 mM [3H]glycerophosphate (2000 cpmnmol), 30 PM acyl-CoA, 2 mg/ml membrane protein, and 0.5 mg/ml bovine serum albumin in 1 ml of 0.1 M Trischloride, pH 7.5. When the effect of DTNB was to be examined, enzyme preparations were preincubated for 2 min in the presence of 4 mM DTNB, but in the absence of albumin, before starting the reactions by the addition of labeled substrate. When radiolabeled acylCoA was used, the fatty acids in the 1; and 2-positions of isolated diacyl-GP were analyzed for incorporation of radioactivity using phospholipase A, treatment as described previously (26). The activities of 1-acyl-GP, 1-acyl-GPC, and l-acylGPE acyltransferase systems were measured spectrophotometrically. Typically, the incubation mixture consisted of 10 to 20 PM acyl-CoA, phospholipid acceptor (75 PM 1-aeyl-GP, 150 PM 1-acyl-GPC, or 150 PM I-acyl-GPE), 0.15 to 0.2 mg/ml microsomal protein, and 1 mM DTNB in 1 ml of 0.08 M Tris-chloride, pH 7.5. Reactions were performed at 25°C and were initiated by adding acyl-CoA. Formation of thionitrobenzoate anion was monitored continuously at 413 nm with a Hitachi spectrometer (Model 181). Control rates were obtained from reactions performed without added acceptor and were subtracted from experimental values to give net acyltransfer rates. When radiolabeled acyl-CoA mixtures were used as acyl donors, the labeled fatty acids incorporated into phospholipids were determined after isolation of the products by thinlayer chromatography and hydrolysis by phospholipase A, as described previously (30). RESULTS
Glycerophosphute
Acyltransferase
Systems
It has previously been shown that two glycerophosphate acyltransferase systems exist in rat liver, one which is specific for palmitoyl-CoA and concentrated in mito-
211
IN RAT LUNG TABLE I
SPECIFICITIESOF GLYCEROPHOSPHATE ACYLTRANSFERASESYSTEMSIN LUNGANDLIVERO Acyltransfer rate (nmol/min/mg protein) Expt No.
Enzyme source
1
Lung
2
Lung
3
Liver
DTNB
Pal-
+ + +
0.30 0.10 0.88 0.20 0.90 0.44
Ole-
Lin-
0.43 0.45 co.01 co.01 0.90 1.19 0.02 0.04 0.86 0.83 0.05 0.05
a The incubation mixtures consisted of 2 mM [3H]glycerophosphate (2000 cpm/nmol), 30 PM acyl-CoA, 2 mg/ml protein (whole membrane fraction, see Materials and Methods), and 0.5 mg/ml bovine serum albumin in 1 ml of 0.1 M Tris-chloride, pH 7.5, in the presence or absence of 4 mM DTNB. Incubations were performed for 2 min at 30°C. Palmitoyl (Pal-)-, oleoyl (Ole-)-, and lineoleoyl (Lin-)-CoAs were tested. A zero time control value (0.06 nmoYmin/mg protein) was subtracted to give net acyltransfer rates. Averages of two separate determinations (Expts 2 and 3) or three separate determinations (Expt 1) are presented. The maximal deviation from the mean was 15%. In Expt 2, an enzyme preparation suspended in 2 mM dithiothreitol0.25~ sucrose-O.01 MTris-chloride, pH 7.5, wasused, but 56fold excess DTNB was present in the assay mixture.
chondrial outer membrane preparation and another which is relatively nonspecific with respect to acyl-CoA donors and is found mainly in microsomal preparations (36-38). By using the sulfhydryl reagent, 5,5’-dithiobis (Znitrobenzoic acid) (DTNB) which selectively inhibits the microsomal acyltransferase, it has been possible for us to estimate the relative contributions of the mitochondrial and microsomal glycerophosphate acyltransferase systems to the synthesis of various molecular species of diacylGP in rat liver in vitro (30). We, therefore, applied this method to the lung system (Table I). It should be noted that saturating concentrations of a single acyl-CoA and a single acceptor were added to determine the maximal velocities for the individual acylCoAs and that use of the term “specificity” is confined to denote the comparisons of
212
YAMADA AND OKUYAMA
F 2 0.8. E 2 F _ 0.4 > .= .> 7l 4 0 0
20 Palmitoyl
40 60 CoA , pM
FIG. 1. Effects of palmitoyl-CoA concentration on glycerophosphate acyltransferase systems in lung. The incubation mixtures were as described in the legend to Table I, except that the concentrations of palmitoylCoA were varied. Whole membrane fraction (0) or mitochondrial fraction (0) was used as the enzyme source. The DTNB-sensitive activity (A) was calculated by subtracting the DTNB-resistant activity (A) from the total activity measured in the absence of DTNB. Each point represents the average of two separate determinations.
these maximal velocities (30).2 The DTNBresistant acyltransferase system in the whole membrane fraction prepared from lung was highly specific for palmitate, whereas the DTNB-sensitive system incorporated unsaturated and saturated acylCoAs at comparable rates. These properties of the two lung acyltransferase systems were quite similar to those observed in liver (30), both with respect to DTNB sensitivity and the lack of acyl group transfer specificity of the DTNB-sensitive system. However, the activity of the DTNB-resistant system was relatively lower in lung than in liver. The relatively low specificity for acyl-CoAs of the DTNB-sensitive system was qualitatively consistent with the results reported for the microsomal glycerophosphate acyltransferase system in rabbit lung (39). Dithiothreitol was found to protect the acyltransferase systems from a time-dependent inactivation (Expts 1 and 2 in Table I) and hence was added to the enzyme suspension in subsequent experiments on the glycerophosphate acyltransferase systems. The DTNB-resistant acyltransferase system in liver showed a much higher affinity for palmitoyl-CoA than did the DTNB-sen-
sitive system. This difference in affinity was found to be an important factor determining the molecular species of diacyl-GP formed in vitro (30). As shown in Fig. 1, the DTNBresistant system in mitochondria from lung also exhibited significantly higher affinity for palmitoyl-CoA than did the DTNB-sensitive system present in whole membrane fraction. These observations suggest that similar glycerophosphate acyltransferase systems are present in both liver and lung; the l-position of glycerophosphate can be acylated either with saturated acyl-CoAs via the DTNB-resistant system or with unsaturated acyl-CoAs in reactions catalyzed by the DTNB-sensitive system depending on the relative concentrations of acyl-CoAs. The apparent K, value for glycerophosphate was approximately 0.6 mM although concentrations above 3 mM did not completely saturate the system. The intracellular concentrations of glycerophosphate in liver is reported to be 0.11 to 0.17 pmol/g wet wt (40), but the concentration in lung has not been reported. The selectivity for acyl-CoAs in the formation of diacyl-GP by the microsomal glycerophosphate acyltransferase system was examined in the presence of an equimolar mixture of palmitoyl-CoA and linoleoyl-CoA and varying concentrations of glycerophosphate. Fatty acids incorporated into the l- and 2-positions of diacyl-GP, the major product, were analyzed after phospholipase A, hydrolysis. Linoleate and palmitate were incorporated into the l-position of diacyl-GP by microsomes from both lung and liver (Table II). This is consistent with the specificities of the DTNB-sensitive glycerophosphate acyltransferase systems shown in Table I. In preparations from liver, the 2-position of diacyl-GP was esterified preferentially with linoleate. In contrast, considerable amounts (35%) of palmitate were incorporated into the 2-position of diacyl-GP in lung microsomes. Changing the glycerophosphate concentration did not affect the ratio of the incorporated fatty acids in lung and affected the ratio only slightly in the liver microsomes. Thus, the capacity of lung microsomes to incorporate palmitate into the 2-position of diacyl-GP is much greater than that of liver microsomes.
SURFACTANT
LIPID SYNTHESIS
This difference in the selectivity with which the microsomal acyltransferase systems acylate the 2-position of the glycerophosphate moiety is compatible with the differences in the properties of microsomal 1-acyl-GP acyltransferase systems in lung and liver, which are described below.
TABLE III SPECIFICITIESOF MICROSOMALI-Acyl-GP ACYLTRANSFERASESYSTEMSIN LUNGANDLIVER" Relative acyltransfer rate Acyl-CoA
l-Acyl-GP Acyltransferase System Maximum velocities of microsomal lacyl-GP acyltransferase systems of lung and liver with various acyl-CoAs were determined in the presence of a single acylCoA and acceptor (Table III). The specifici-
213
IN RAT LUNG
PalSteOleLinAra-
Lung microsomes Liver microsomes l (12)b 0.83 1.3 1.6 0.80
1 (62)b 0.76 1.2 0.94 0.42
a The incubation mixtures consisted of 75 ~.LMl-acylGP, 10 PM acyl-CoA, 0.2 mg/ml microsomal protein, and 1 tiIM DTNB in 1 ml of 0.08 M Tris-chloride, pH 7.5. TABLE II Reactions at 25°C were monitored by continuously EFFECTOFTHEGLYCEROPHOSPHATECONCENTRATIONmeasuring the absorbance at 413 nm with a Hitachi ON THE SELECTIVITIESOFTHE MICROSOMAL spectrometer (Model 181)(37). Control values obtained GLYCEROPHOSPHATE ACYLTRANSFERASE from experiments performed in the absence of l-acylSYSTEMSIN LUNG AND LIVER* GP (below 1.1 nmollminimg protein for lung microsomes and 7 nmoYmin/mg protein for liver microsomes) Linoleate/palmitate molar ratio at were subtracted from values obtained in the presence of 1-acyl-GP to give net acyltransfer rates. Acyl-CoA Z-Position l-Position Glyceroconcentrations were examined in the range of 7.5 to phosphate 30 j&M. Acyl-CoA concentrations of lo-20 FM gave Liver Lung Liver Lung (mM) maximal velocities. Apparent K, values for l-palmitoyl-GP were 20 and 38 pM with palmitoyl-CoA and 0.73 2.0 2.0 8.4 1.5 arachidonoyl-CoA (Ara-), respectively. 12 1.7 0.85 0.5 1.8 * Activity expressed as nmol/min/mg of protein in 14 1.7 1.3 0.12 1.9 parentheses. Averages of two separate determinations (1.5) (1.8 (1.0) (1.0) (0) are presented. The maximal deviation from the mean was 10%. a The incubation mixtures consisted of 15 pM [3H]palmitoyl-CoA (7000 cpmnmol under dual-label counting conditions), 15 j.bM [14C]linoleoyl-CoA (8000 ties thus determined were similar and were cpminmol), varying concentration of glycerophosphate, relatively low in both the lung and liver and 2 mg/ml microsomal protein in 1 ml of 0.1 M Trissystems. In the case of rat liver microsomes, chloride, pH 7.5. The mixture was incubated at 30°C the apparent discrepancy between the refor 1 min and the reaction product, diacyl-GP, was latively low specificity of the 1-acyl-GP isolated using Silica Gel H thin-layer chromatography. The radioactive acids esterified to the l-and 2-positions acyltransferase system and the highly selecof diacyl-GP were determined after phospholipase A, tive incorporation of unsaturated fatty acids hydrolysis using methods described previously (28). into the S-position of diacyl-GP in vivo can Averages of three separate determinations for the lung be explained by the fact that the system can and two separate determinations for the liver are pre- be highly selective for unsaturated acylsented. The maximal deviations from means were 20 CoAs at lower concentrations of the acceptor and 15% for the lung and liver systems, respectively. in vitro possibly as a consequence of the Incorporation of radioactivity without added glycero- difference in affinities for the acceptor (26). phosphate was less than 29 and 7% of that obtained Bothin vivo and in the synthesis of diacyl-GP at 0.12 mM glycerophosphate for lung and liver systems, respectively. These values were subtracted from from glycerophosphate in vitro, the concenexperimental values in calculating the molar ratios of tration of the 1-acyl-GP intermediate is linoleate to palmitate incorporated into diacyl-GP. The maintained at a low level since the ratemolar ratios in the control experiments (no added ac- limiting step under these conditions is the glycerophosphate acyltransferase system. ceptor) are shown in parentheses.
214
YAMADA AND OKUYAMA TABLE IV
EFFECT OF THE I-ACYLGP CONCENTRATION ON THE SELECTIVITIES OF MICROSOMAL l-ACYL-GP ACYLTRANSFERASE SYSTEMSIN LUNG AND LIVER”
the 2-position of diacyl-GP at low phospholipid acceptor concentrations than is the liver system. 1-Acyl-GPC
Acyltransferase
System
The specificities for acyl-CoAs of microsomal 1-acyl-GPC acyltransferase systems 1-Acyl-GP in lung and liver are shown in Table V. AlLiver Lung (PM) though the specific activities of the two microsomal preparations were different, 50 2.0 k 0.1 (6) 2.1 + 0.1 (7) the specificities for the acyl-CoAs were 10 2.1 + 0.1 (2) 2.7 5 0.4 (3) quite similar; both systems showed higher 2.3 + 0.1 (4) 3.9 2 1.3 (3) 5 specificities for unsaturated acyl-CoAs than 3 2.9 2 0.4 (4) 7.9 * 2.4 (6) saturated acyl-CoAs. However, the differ1.5 3.2 2 0.8 (2) 13.7 k 1.9 (3) (2.3 + 0.2) (8) (2.4 k 0.5) (7) ences in the properties of the lung and (0) liver systems were noted when the acceptor (2The incubation mixtures consisted of 15 ELM[3H]- concentrations were varied in the presence palmitoyl-CoA (12,000cpm/nmol), 15 PM [W]linoleoylof saturating concentrations of equimolar CoA (8000cpm/nmol), varying concentrations of l-acylmixtures of saturated and unsaturated acylGP, 0.2 mg/ml microsomal protein, and 1 mM DTNB CoAs. As shown in Table VI, a pronounced in 1 ml of 0.08 M Tris-chloride, pH 7.5. The mixture selectivity for linoleate was observed at was incubated for 1 min at 30°C and the product, dilower concentrations of 1-acyl-GPC in liver acyl-GP, was analyzed as described in the legend to microsomal system with palmitate being alTable II. The numbers of experiments are shown in most excluded from the 2-position of phosparentheses. Total incorporation of radioactivity withphatidylcholine. In contrast, the selectivity for out added 1-acyl-GP was less than 42% of that observed linoleate decreased at lower concentrations with 1.5 PM l-acyl-GP for both the lung and liver. This value was subtracted from the experimental val- of the acceptor in the lung system, allowing ues in calculating the molar ratios of linoleate to palmitate to be incorporated into the 2-posipalmitate incorporated into the 2-position. tion of phosphatidylcholine. These features were even more noticeable with the palmitate/arachidonate combination. The liver Thus, the selectivity for acyl-CoAs observed system was highly selective for arachidoat lower concentrations of 1-acyl-GP was considered to reflect the selectivity in the acylation at the 2-position of diacyl-GP in TABLE V the livers of rats fed normal diets. In conSPECIFICITIES OF MICROSOMAL l-AcYLGPC trast to the results obtained with liver ACYLTRANSFERASE SYSTEMS IN microsomes, the microsomal 1-acyl-GP acylLUNG AND LIVERY transferase system in lung incorporated palmitate and linoleate into the 2-position Relative acyltransfer rate of diacyl-GP with comparable rates at all concentrations of the acceptor, indicating Acyl-CoA Lung microsomes Liver microsomes that the affinities for 1-acyl-GP for palmiPal1 (21)” toy1 transfer and linoleoyl transfer reactions 1 W’ Ste0.50 0.66 are similar (Table IV). Thus, the specificiOle2.5 2.5 ties for acyl-CoAs as measured by the Lin4.4 3.5 respective maximal velocities were not much Ara4.3 4.0 different between the 1-acyl-GP acyltransferase systems in lung and liver. However, ’ The incubation mixtures were as described in the the selectivities for palmitate and linoleate legend to Table III, except that 150 PM 1-acyl-GPC observed at lower concentrations of l-acyl-GP was used as the acceptor. were significantly different; the lung system b Specific activities expressed as nmoYminimg of prois more likely to incorporate palmitate into tein are shown in parentheses. Linoleate/palmitate molar ratio 2 SE
SURFACTANT TABLE VI
LIPID SYNTHESIS
215
IN RAT LUNG
of 1-acyl-GPC acyltransferase system was examined with equimolar mixtures of arachidonoyl-CoA and palmitoyl-CoA. In both lung and liver systems, more arachidonate than palmitate was incorporated into the 2-position of phosphatidylcholine at a satuArachidonatel rating concentration of acyl-CoAs, but the palmitate selectivity for arachidonate decreased when molar ratio the levels of acyl-CoA mixtures were lowered. These results are consistent with an Lung Liver observation by Hasegawa-Sasaki and Ohno 34 (19) that palmitate incorporation is inhibited 4.8 by arachidonoyl-CoA but arachidonate in>lOO 2.2 corporation is not inhibited by palmitoyl1.2 >lOO (1.0) (17) CoA in the rat lung 1-acyl-GPC acyltransferase system (Table VII).
EFFECT OF THE l-ACYL-GPC CONCENTRATIONON SELECTIVITIES OF MICROSOMALl-ACYL-GPC ACYLTRANSFERASESYSTEMSIN LUNG AND LIVER”
l-Acyl-GPC concentration (PM(I) 75 15 6 (0)
Lineoleatei palmitate molar ratio Lung
Liver
22 17.5 80 9.1 5.1 >lOO (3.6) (>lOO)
(2The incubation mixtures consisted of 10 PM [3H]palmitoyl-CoA (7400 cpm/nmol) plus 10 PM [W]linoleoyl-CoA (820 cpm/nmol) or 10 PM [‘*C]palmitoyl-CoA (12,500 cpmnmol) plus 10 PM [3H]arachidonoyl-CoA (2500cpm/nmol), varying concentration of 1-acyl-GPC, 1 mM DTNB, and 0.2 mg/mI microsomal protein in 1 ml of 0.1 M T&-chloride, pH 7.5. The mixtures were incubated for 1 min at 30°C. The fatty acids incorporated into the 2-position of the product, phosphatidylcholine, were analyzed after phospholipase Al hydrolysis. Averages of two separate determinations are presented. The maximal deviation from the mean was 30%. The molar ratios in the control experiments are shown in parentheses.
nate, while the lung system became more selective for palmitate at lower concentrations of the acceptor; in lung the rates of palmitate and arachidonate incorporation at a 1-acyl-GPC concentration of 6 PM were comparable whereas about fivefold more arachidonate than palmitate was incorporated at 75 PM 1-acyl-GPC under these conditions. Thus, while the specificities for acylCoAs of the 1-acyl-GPC acyltransferase systems from liver and lung microsomes were not significantly different when measured at saturating substrate concentrations, the lung system exhibited a selectivity for the transfer of palmitate to 1-acyl-GPC at lower concentrations of the phospholipid acceptor significantly higher than that in the liver system. In contrast, the liver l-acylGPC acyltransferase system showed a marked selectivity toward arachidonoyl transfer. In the next experiments, the effect of varying acyl-CoA levels on the selectivity
I-Acyl-GPE
Acyltransferase System
The specificities for acyl-CoAs of l-acylGPE acyltransferase systems in lung and liver microsomes are shown in Table VIII. Both systems showed higher specificities for unsaturated acyl-CoAs examined, except oleoyl-CoA. The effect of the acceptor concentration on the selectivity of this enzyme TABLE VII EFFECT OF ACYL-CoA CONCENTRATIONON SELECTIVITY OF MICROSOMALl-ACYLGPC ACYLTRANSFERASESYSTEMS IN LUNG AND LIVERY Arachidonatei palmitate molar ratio
Acyl-CoA concentration (PM)
Lung
Liver
2+2 5+5 10 + 10 25 + 25 50 + 50
3.7 6.1 9.4 12.2 19.3
2.1 5.3 25 >lOO >lOO
a The incubation mixtures consisted of varying concentration of [W]palmitoyl-CoA (12,500 cpmnmol) plus [3H]arachidonoyl-CoA (2200 cpm/nmol), 75 PM I-acyl-GPC, 1 mM DTNB, and 0.1 mg of microsomal protein in 1 ml of 0.1 M Tris-chloride, pH 7.5. The mixtures were incubated for 1 min at 25°C. The fatty acids incorporated into the e-position of phosphatidylcholine were analyzed after phospholipase A, hydrolysis. Averages of two separate determinations are presented. The maximal deviation from mean was 30%.
216
YAMADA
AND OKUYAMA
TABLE IX system is shown in Table IX. In both lung and liver systems, palmitate tended to be EFFECTOFTHE l-ACYL-GPE CONCENTRATION ONTHE excluded from the 2-position, a character- SELECTIVITIES OF l-ACYL-GPE ACYLTRANSFERASE istic which was even more pronounced when SYSTEMSIN LUNGANDLIVER" the 1-acyl-GPE acceptor concentration was Arachidonatel lower. These observations are compatible palmitate with the absence of palmitate at the 2-posil-Acyl-GPE molar ratio tion of phosphatidylethanolamine isolated concentration from rat lung and liver (41). DISCUSSION
Enzyme systems acylating the l-position of glycerophosphate have abilities to incorporate saturated and unsaturated acyl-CoAs comparably as revealed by the maximal velocities (Table I). However, this acylation step can be selective for palmitate in the presence of two glycerophosphate acyltransferase systems at lower concentrations of saturated and unsaturated acyl-CoAs, since the DTNB-resistant system has much higher affinity for palmitoyl-Coil than the DTNBsensitive system (Fig. 1). The localization of the former in the mitochondrial fraction is not easily understandable. Whether l-palmitoyl-GP produced in mitochondria is used as substrate for microsomal enzyme or the DTNB-resistant system was artificially associated with mitochondria during subcelTABLE
VIII
SPECIFICITIESOF MICROSOMALl-ACYL-GPE ACYLTRANSFERASESYSTEMSIN LUNGANDLIVER" Acyltransfer rate (nmoYmin/mg protein) Acyl-CoA
Lung
Liver
PalOleLinAra-
1.8 1.9 6.4 8.0
2.5 1.0 14.6 14.2
’ The incubation mixtures consisted of 20 PM acylCoA, 150 pM 1-acyl-GPE, 1 mM DTNB, and 0.15 mgiml microsomal protein in 1 ml of 0.08 M T&-chloride, pH 7.5. The reactions were followed spectrophotometrically. Averages of two separate determinations are presented. The maximum deviation from the mean was 5%. I-Acyl-GPE concentrations of 150-250 PM gave maximal velocities.
(PM)
Lung
Liver
300 150 50
9.8 14 25
>lOO >lOO
(0)
6-j
>lOO G-)
L1The incubation mixtures consisted of 10 PM [3H]arachidonoyl-CoA (2500 cpmnmol), 10 PM [“‘Clpalmitoyl-CoA (12,500 cpm/nmol), varying concentration of I-acyl-GPE, and 0.2 mg/ml microsomal protein in 1 ml of 0.08 M Tris-chloride, pH 7.5. The mixtures were incubated for 1 min at 30°C. The fatty acids incorporated into phosphatidylethanolamine were analyzed after phospholipase A, hydrolysis (30). Incorporation of radioactivity in the absence of 1-acyl-GPE was negligible.
lular fractionation (30) is to be clarified. Although little unsaturated fatty acid is found esterified to the l-position of glycerophospholipids purified from lungs or livers of rats fed normal laboratory diets (41,42), the in vivo situations under which unsaturated fatty acids are found at the l-position are known in liver but not in lung as noted earlier (30). These results suggested that the concentration and composition of the acyl donor pool are important in determining the fatty acid pattern at the l-position of diacyl-GP in lung. In this sense, it seems interesting that the compositions of acyl-CoA pools in the lung and liver are quite different.4 The contribution made to diacyl-GP synthesis by the relatively low 2-acyl-GP acyltransferase activity found in liver (24) is not clear at present in either the liver or lung systems. Recently, the dihydroxyacetone phosphate pathway was reported to play a quantitatively more important role in glycerolipid synthesis in the lung than the glycerophosphate pathway (43). Although the specificity for acyl-CoAs of this pathway 4 Y. Tabata and Y. Suzuki (19’78) Abstract of the 6th meeting of Jap. Med. Sot. Biol. Interface, p. 8.
SURFACTANT
LIPID
SYNTHESIS
in lung has not been determined, the specificities of dihydroxyacetonephosphate pathways present in liver mitochondrial (peroxisomal) and microsomal fractions are known to be similar to those of glycerophosphate acyltransferase systems in these subcellular fractions (44, 45). The fatty acids at the 2-position of diacyl-GP formed in vivo are quite different between lung and liver tissues (5,6,46,47). Although an enzyme activity which acylates the 2-position of glycerophosphate to form 2-unsaturated acyl-GP as an intermediate in liver was reported by Lamb and Fallon (48), we have been unable to reproduce their results (24). Hence, we focused our attention on the 1-acyl-GP acyltransferase system present in lung microsomes. A striking difference between the 1-acyl-GP acyltransferase systems in the lung and liver was noted. Although both systems showed similar specificities at saturating substrate concentrations, the selectivities for acyl donors differed markedly at lower concentrations of the 1-acyl-GP acceptor (Tables II and IV). l-Acyl-GPC acyltransferase systems are believed to be involved in determining the fatty acid composition of the 2-position of phosphatidylcholine. These systems showed very similar specificities in both lung and liver (Table IV) but again showed quite different selectivities for various acyl-CoAs at lower concentrations of acceptor (Table VI). At present, it is unclear whether substrate concentrations vary substantially in vivo. However, one can calculate that the phospholipid acceptor concentration in vivo is considerably lower than the saturating concentrations used to determine specificities in vitro (- 150 nmol/0.2 mg microsomal protein). Thus, it seems reasonable to assume that the selectivities of acyltransferase systems at lower concentrations of acceptors may closely mimic the reaction conditions existing in vivo. The present experiments have clearly shown that the 1-acyl-GP and 1-acyl-GPC acyltransferase systems in lung can incorporate palmitate into the 2-position of diacyl-GP and phosphatidylcholine at rates comparable to those observed with unsaturated fatty acids. Such is not the case with the liver acyltransferase systems which essentially exclude palmitate. The selectivi-
IN RAT LUNG
217
ties observed with lung and liver in vitro are consistent with the in vivo distribution of acyl groups (6, 15, 16, 41, 42). Variations in the selectivities of acyltransferase systems in response to changes in substrate concentrations might also help explain the apparently inconsistent results noted in tracer experiments performed with lung tissue slices; radiolabeled 1-acyl-GPC added to the incubation mixture was found to be acylated mainly with unsaturated fatty acids by the acyltransferase system whereas acylations of endogenous lipid precursor with added palmitate yielded phosphatidylcholine with palmitate located mainly at the 2-position (5). On the one hand, esterification of labeled 1-acyl-GPC in these experiments may have resulted from acyltransfer reactions involving low levels of endogenous fatty acyl donors and relatively high concentrations of exogenous 1-acyl-GPC while on the other hand, selective incorporation of labeled palmitate occurred through acylation of low endogenous levels of 1-acyl-GPC. One could thus rationalize the results obtained in the tissue slice experiments based on the selectivities observed in acyl group transfers at high and low 1-acyl-GPC concentrations (Table VI). It should be noted, however, that the relative concentrations of acylCoAs could affect the selectivities of acyltransferase systems (26, 28, 30), although the selectivities were determined in the current experiments in the presence of equimolar mixtures of acyl-CoAs. Some kinetic parameters of the l-acylGPC acyltransferase system in lung have been reported by other workers (15,19,2123,49) and are summarized in Table X. The specificities, shown as relative maximal velocities for acyl-CoAs, as well as the K, values for the substrates differ significantly from each other. We could prepare lung microsomes practically free of acyl-CoA hydrolase activities, which enabled us to use a spectrophotometric assay with DTNB (37) to follow the acyltransfer reactions continuously. Our data are most similar to those reported by Hasegawa-Sasaki and Ohno (19) except the K, value for archidonoyl-CoA. The kinetic parameters determined with lung microsomes were not significantly different from those obtained with liver mi-
218
YAMADAANDOKUYAMA TABLE X KINETIC CONSTANTS FOR l-ACYL-GPC ACYLTRANSFERASESYSTEMSIN LUNG
K, for Acyl-CoA (PM)
K, for l-acyl-GPC (PM)
Relative V max
PalOle-
-4
-
1 1.1
(15)
PalOleAra-
6 6 44
18 25 30
1 1.8 4.8
(19)
PalOle-
-
-
1 0.24
(23)
PalOleAra-
<5 <5
19 29
1 2.5 4.3
Rabbit lung
PalOle-
36 8
34 37
1 1.5
(21)
Rabbit cultured cell
PalOle-
15 16
-
1 0.73
(21)
Enzyme source Rat lung
Acyl-CoA
Reference
Present experiments
n Not reported.
crosomes and thus do not provide a simple explanation for the fatty acid composition of phosphatidylcholine isolated from lung and liver. However, differences in properties of 1-acyl-GP and 1-acyl-GPC acyltransferase systems in the lung and liver became quite apparent when the selectivities of acyltransfer were measured as a function of phospholipid acceptor concentration. We have previously presented evidence that the observed variations in the selectivity of the acyltransferase system are not caused by changes in the physicochemical properties of substrate micelles but are an inherent property of the enzyme system itself (2426, 30). Additional support for this concept comes from the observations (a) that lowering 1-acyl-GPC concentrations had opposite effects on the selectivities in lung and liver systems (Table VI) and (b) that lowering the 1-acyl-GPC and 1-acyl-GPE concentrations had opposite effects on the incorporation of palmitate and arachidonate in the lung (Tables VI and IX). The specificities of 1-acyl-GPC and l-acylGPE acyltransferase systems were not the same. For example, the rate of transfer for
oleoyl-CoA was much less with the l-acylGPE acyltransferase systems than with the l-acyl-GPC acyltransferase systems in both lung and liver microsomes. Furthermore, lowering the concentrations of 1-acyl-GPC and 1-acyl-GPE had opposite effects on the selectivity for palmitate and arachidonate in lung. These results are consistent with the concept (32) that the acylations of l-acylGPC and 1-acyl-GPE are catalyzed by different microsomal enzyme systems although it is unknown how many different acyltransferase enzymes are actually present. It is also unclear with which cell type of the lung the various acyltransferases are associated. In this context, it would be of particular interest to determine the properties of the acyltransferase systems in the alveolar Type II cells (16, 50), those cells which are known to be involved in the formation of dipalmitoyl-GPC in lung. ACKNOWLEDGMENT The authors are indebted to Professor H. Ikezawa, Nagoya City University, Nagoya, for his helpful discussions.
SURFACTANT
LIPID
SYNTHESIS
REFERENCES 1. GOERKE, J. (19’74) Biochim. Biophys. Acta 344, 241-261. 2. OHNO, K., AKINO, T., AND FUJIWARA, T. (1978) Rev. Perinat. Med. 2, 227-318. 3. AKINO, T., AND ABE, M. (1977) J. Japan. Med. Sot. Biol. Interjace 8, 2-18. 4. VAN GOLDE, L. M. G. (1976) Amer. Rev. Resp. Dis. 114, 977-1000. 5. AKINO, T., ABE, M., AND ARAI, T. (1971) Biochim. Biophys. Acta 248, 274-281. 6. AKINO, T., YAMAZAKI, I., AND ABE, M. (1972) Tohoku J. Exp. Med. 108, 133-139. 7. VAN DEN BOSCH, H., BONTE, H. A., AND VAN DEENEN, L. L. M. (1965) Biochim. Biophys. Acta 98, 648-651. 8. ABE, M., OHNO, K., AND SATO, R. (1974) Biochim. Biophys. Acta 369, 361-370. 9. HALLMAN, M., AND RAIVIO, K. (1975) Biol. Neonate 27, 329-338. 10. OLDENBORG, V., AND VAN GOLDE, L. M. G. (1976) Biochim. Biophys. Acta 441, 433-442. 11. OKANO, G., AND AKINO, T. (1978) Biochim. Biophys. Acta 528, 3’73-384. 12. TSAO, F. H. C., AND ZACHMAN, R. Z. (1977) Pediat. Res. 11, 858-861. 13. FARRELL, P. M., LUNDGREN, D. W., AND ADAMS, A. J. (1974) Biochem. Biophys. Res. Commun. 57, 696-701. 14. ROONEY, S. A., WAI-LEE, T. S., GOBRAN, L., AND MOTOYAMA, E. K. (1976) Biochim. Biophys. Acta 431, 447-458. 15. VEREYKIN, J. M., MONTFOORT, A., AND VAN GOLDE, L. M. G. (1972) Biochim. Biophys. Acta 260, 70-81. 16. KIKKAWA, Y. (1978) J. Japan. Med. Sot. Biol. Interface 9, 1-13. 17. SARZALA, M. G., AND VAN GOLDE, L. M. G. (1976) Biochim. Biophys. Acta 441, 423-432. 18. POSSMAYER, F., DUWE, G., AND HAHN, M. (1977) Canad. J. Biochem. 55, 609-617. 19. HASEGAWA-SASAKI, H., AND OHNO, K. (1975) Biochim. Biophys. Acta 380, 486-495. 20. MORIYA, K., AND KANOH, H. (1974) Tohoku J. Exp. Med. 112, 241-256. 21. TANSEY, F. A., AND FROSOLONO, M. F. (1975) Biochem. Biophys. Res. Commun. 67, 15601566. 22. FROSOXINO, M. F., SLIVKA, S., AND CHARM, B. L. (1971) J. Lipid Res. 12, 96-102. 23. ENGLE, M. J., SANDERS, R. L., AND LONGMORE, W. J. (1976) Arch. Biochem. Biophys. 173, 586-595. 24. OKUYAMA, H., EIBL, H., AND LANDS, W. E. M. (1971) Biochim. Biophys. Acta 248, 263-273.
IN RAT LUNG
219
25. OKUYAMA, H., AND WAKIL, S. J. (1973) J. Biol. Chem. 248, 5197-5205. 26. OKUYAMA, H., AND LANDS, W. E. M. (1972) J. Biol. Chem. 247, 1414-1423. 27. OKUYAMA, H., YAMADA, K., AND IKEZAWA, H. (1975) J. Biol. Chem. 250, 1710-1713. 28. OKUYAMA, H., YAMADA, K., IKEZAWA, H., AND WAKIL, S. J. (1976) J. Biol. Chem. 251, 24872492. 29. OKUYAMA, H., YAMADA, K., KAMEYAMA, Y., IKEZAWA, H., AKAMATSU, Y., AND NOJIMA, S. (1977) Biochemistry 16, 2668-2673. 30. YAMADA, K., AND OKUYAMA, H. (1978)Arch. Biothem. Biophys. 190, 409-420. 31. SEUBERT, W. (1960) Biochem. Prep. 7, 80-83. 32. OKUYAMA, H., LANDS, W. E. M., CHRISTIE, W. W., AND GUNSTONE, F. D. (1969) J. Biol. Chem. 244, 263-273. 33. CHANG, Y. Y., AND KENNEDY, E. P. (1967) J. Lipid Res. 8, 447-455. 34. OKUYAMA, H., LANDS, W. E. M., GUNSTONE, F. D., AND BARVE, J. A. (1972) Biochemistry 11, 4392-4398. 35. LONG, C., ODAVI~, R., AND SARGENT, E. J. (1967) Biochem. J. 102, 211-229. 36. MONROY, G., KELKER, H. C., AND PULLMAN, M. E. (1973) J. Biol. Chem. 247, 6884-6894. 37. LANDS, W. E. M., AND HART, P. (1965) J. Biol. Chem. 240, 1905-1911. 38. DAAE, L. N. W., ANDBREMER, J. (1970)Biochim. Biophys. Acta 210, 92-104. 39. HENDRY, A. T., AND POSSMAYER, F. (1974) Biochim. Biophys. Acta 369, 156-172. 40. GUYNN, R. W., VELOSO, D., AND VEECH, R. L. (1972) J. Biol. Chem. 247, 7325-7331. 41. OKANO, G., KAWAMOTO, T., AND AKINO, T. (1978) Biochim. Biophys. Acta 528, 385-393. 42. MONTFOORT, A., VAN GOLDE, L. M. G., AND VAN DEENEN, L. L. M. (1971) Biochim. Biophys. Acta 231, 335-342. 43. MASON, R. J. (1978) J. Biol. Chem. 253, 33673370. 44. HAJRA, A. K. (1968) J. Biol. Chem. 243, 34583465. 45. JONES, C. L., AND HAJRA, A. K. (1977) Biochem. Biophys. Res. Commun. 76, 1138-1143. 46. HILL, E. E., HUSBANDS, D. H., AND LANDS, W. E. M. (1968) J. Biol. Chem. 243,4440-4451. 47. AKESSON, B., ELOVSON, J., AND ARVIDSON, G. (1970) Biochim. Biophys. Acta 210, 15-27. 48. LAMB, R. G., AND FALLON, H. J. (1970) J. Biol. Chem. 245, 3075-3083. 49. SNYDER, F., AND MALONE, B. (1975) Biochem. Biophys. Res. Commun. 66, 914-919. 50. BATENBURG, J. J., LONGMORE, W. J., AND VAN GOLDE, L. M. G. (1978) Biochim. Biophys. Acta 529, 160-170.
OF BIOCHEMISTRY
AND BIOPHYSICS
Vol. 196, No. 1, August, pp. 209-219, 1979
Possible
Involvement of Acyltransferase Pulmonary Surfactant
Systems in the Formation Lipid in Rat
KAZUYO YAMADA AND HARUMI
of
OKUYAMA
The Faculty of Pharmaceutical Sciences, Nagoya City University, S-1 Tanabedori, Mizuhoku, Nagoya 467, Japan Received January 8, 1979; revised March 15, 1979 Dithiobis (2-nitrobenzoic acid)-resistant and -sensitive glycerophosphate acyltransferase systems were present in rat lung as in liver. The former was specific for palmitate while the latter could incorporate saturated and unsaturated acyl-CoAs comparably. The former has higher affinity for palmitate than the latter indicating that the l-position of glycerophosphate can be acylated selectively with palmitate under certain conditions. The specificities of 1-acylglycerophosphate and 1-acylglycerophosphocholine acyltransferase systems were similar in lung and liver; both systems showed higher specificities for unsaturated acyl-CoAs. However, the selectivities observed at lower concentrations of phospholipid acceptors in the presence of equimolar mixtures of saturated and unsaturated acyl-CoAs were much different; the lung systems showed relatively higher selectivities for palmitate than the liver systems in the formation of both diacylglycerophosphate and phosphatidylcholine. On the other hand, palmitate was excluded almost completely from the e-position in the 1-acylglycerophosphoethanolamine acyltransferase systems in lung and liver. These observations provide an enzymatic basis for describing the formation of pulmonary surfactant lipids in rat via acyltransferase systems.
Dipalmitoyl-sn-glycerol 3-phosphocholine is one of the major molecular species of phosphatidylcholine in mammalian lung tissue and is considered to play an important role in determining the surface properties of pulmonary surfactant (l-4). The processes which allow this particular molecular species to be formed in such abundance in lung in contrast to other tissues have been the subject of many investigators (2-4). Akino et al. (56) first proposed that an enzyme catalyzing a transesterification between two molecules of 1-acyl-sn-glycerol 3-phosphocholine (1-acyl-GPC)’ plays an important role in the synthesis of dipalmitoyl-GPC in rat lung. This idea is supported by the findings that this enzyme in lung can produce dipalmitoyl-GPC in vivo and in vitro, and that the enzyme activity increases at the latter stages of the development of embryo lung ’ Abbreviations used: -GP, -sn-glycerol3-phosphate; -GPC, -sn-glycerol3-phosphocholine; -GPE, -an-glycerol 3-phosphoethanolamine; DTNB, 5,5’-dithiobis (2-nitrobenzoic acid).
(7-ll), although other enzyme activities also increase (12- 14). However, the possible contributions of other biosynthetic routes to the formation of dipalmitoyl-GPC in lung have not been fully investigated and what data are available seem contradictory and confusing. For example, a significant proportion of the diacylglycerol and phosphatidylcholine formed from labeled glycerol or choline in vivo (15, 16) and in tissue slices (5) is the disaturated species, suggesting a potential role of the de novo synthetic pathway in the formation of disaturated species. Surprisingly, however, the CDP-choline:diacylglycerol cholinephosphotransferase system in lung tissue apparently does not utilize dipalmitoyl glycerol as a substrate in vitro (17, 18). Furthermore, the acyl-CoA:l-acylGPC acyltransferase system has been reported to exhibit higher specificity for unsaturated acyl-CoAs in vitro (19); and added radioactive 1-acyl-GPC is known to be acylated in viva and in slices (5, 6) preferentially with tetraene and diene fatty acids. In ap-
209
0003-9861/79/090209-11$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
210
YAMADA
AND OKUYAMA
parent contrast to this latter result, is the observation that radiolabeled palmitic acid, when added to lung slices, is esterified mainly at the e-position of phosphatidylcholine suggesting the involvement of l-acylGPC acyltransferase system in the formation of disaturated speciesin viva (5,6,20). When the synthesis of phosphatidylcholine from 1-acyl-GPC by microsomes was examined in the presence of fatty acids, ATP, and CoA, lung microsomes were found to incorporate relatively more palmitate into phosphatidylcholine than did liver microsomes (15). This indicates that through some specificity inherent in either the acyl-CoA synthetase system and/or acyltransferase systems in the lung, palmitate is more likely to be incorporated into the 2-position of lung phosphatidylcholine. One other difficulty in evaluating the contributions of the acyltransferase systems in dipalmitoyl-GPC synthesis is that the specificities reported for acyl-CoAs in lung microsomes by different workers vary considerably (15, 19, 2123). These variabilities may result from relatively high acyl-CoA hydrolase activities present in lung microsomes prepared by current methods. Even so, the specificities2 of the acyltransferase systems as measured by the maximal velocities for the individual acyl-CoAs may not be applicable in vivo. As we have shown previously (24-28), acyltransferase systems in many cases lack specificity for the acyl-CoA precursors and fail to produce an explanation for the highly selective positioning of various fatty acids at the l- and 2-positions of glycerophospholipids from rat liver or Escherichia coli. However, the selectivities2 of acyltransferase systems for acyl groups in mixtures of acyl donors vary in vitro depending on the relative concentrations of the substrates. The selectivities noted can be used to account for severalin viva phenomena (26-30). This paper compares and contrasts the p The term “specificity” is used to denote comparisons of acyltransfer rates measured in the presence of saturating concentrations of single acyl donors and single acyl acceptors. The term “selectivity” is used when the velocities of acyltransfer measured in the presence of mixtures of acyl donors or in the presence of suboptimal concentrations of substrates were compared.
properties of the acyltransferase systems of rat liver and lung with particular emphasis on variations in the selectivities observed in acyl group transfers as a function of substrate concentrations. We postulate these acyltransferase systems could contribute to the incorporation of palmitate into the 2-position of phosphatidylcholine in rat lung. MATERIALS
AND METHODS
[9,10-3H]Palmitic acid3 and [l-‘4C]linoleic acid were the products of the Amersham Radiochemical Centre (Amersham, England) and [lJ%]palmitic acid and [5,6,8,9,11,12,14,15-3H]arachidonicacid were theproducts of New England Nuclear (Boston, Mass.). CoA was obtained from Kyowa Hakko Company, Ltd. (Tokyo). Bovine serum albumin (fatty acid-poor) was obtained from Miles Laboratories, Inc.. 5,5’-Dithiobis (2-nitrobenzoic acid) (DTNB) and Crotalus adamanteus venom were purchased from Sigma. Acyl-CoA derivatives were prepared by a modification of Seubert’s procedure (31) as described previously (32). [3H]Glycerol 3-phosphate was prepared from [2-3H]glycerol (New England Nuclear) according to the method of Chang and Kennedy (33). I-Acyl-GPC was prepared from lecithin as described previously (34). 1-Acyl-GP was synthesized from 1-acyl-GPC according to the method of Long et al. (35). l-Acyl-GPE was prepared from eggphosphatidylethanolamine by treatment with phospholipase A, as described previously (34). These substrates contain palmitate and stearate as the major fatty acyl chain substituents. Rat Preparation of subcellular membranefractions. lung was minced and then homogenized in 5 vol of 0.25 M sucrose for 30 s with a Waring Blendor. The homogenate was filtered through three layers of gauze. The unfiltered residual material was rehomogenized with 5 vol of 0.25 M sucrose and again filtered. The filtrates were combined, and the pH was adjusted to 7.5 with 1 M Tris. The sample was centrifuged for 5 min at 6001~and the precipitate discarded. To the supernatant was added 0.11 vol of 1 M Tris-chloride, pH 9.0. Treatment of membranes at alkaline pH (8.0-8.5) eliminated acyl-CoA hydrolase activity which otherwise interfered with the DTNB-based spectrophotometric assay of acyltransferases. The supernatant obtained from centrifugation at SOOg for 5 min was centrifuged at 100,OOOgfor 90 min. The resulting pellet was suspended in 0.25 M sucrose-O.01 M Tris-chloride, pH 7.5, and designated as the whole membrane fraction. To obtain a mitochondrial fraction, the 6009 3 Specific radioactivities of labeled substrates were varied by diluting with nonlabeled substrates to obtain enough and comparable tritium and carbon-14 radioactivities for counting.
SURFACTANT
LIPID SYNTHESIS
supernatant was centrifuged at 78OOgfor 10 min, and the pellet homogenized in 0.1 M Tris-chloride, pH 9.0, centrifuged, and resuspended in 0.25 M sucrose-O.01 M Tris-chloride, pH 7.5. The intermediate fraction was prepared by centrifuging the 78OOgsupernatant at l5,OOOgfor 20 min. Then 0.11 vol of 1 M Tris-chloride, pH 9.0, was added to the resulting supernatant. Microsomes were then sedimented from this fraction by centrifugation at 100,OOOg for 90 min and suspended in 0.25 M sucrose-O.01 M Tris-chloride, pH 7.5. All membrane fractions were stored at -20°C until use. Subcellular fractions from liver were obtained as described previously (30). Assays. Acyl-CoA:glycerophosphate acyltransferase activities were assayed by measuring the rate of incorporation of [3H]glycerophosphate into the lipidsoluble fraction as described previously (28). Unless otherwise noted, the typical incubation mixture consisted of 2 mM [3H]glycerophosphate (2000 cpmnmol), 30 PM acyl-CoA, 2 mg/ml membrane protein, and 0.5 mg/ml bovine serum albumin in 1 ml of 0.1 M Trischloride, pH 7.5. When the effect of DTNB was to be examined, enzyme preparations were preincubated for 2 min in the presence of 4 mM DTNB, but in the absence of albumin, before starting the reactions by the addition of labeled substrate. When radiolabeled acylCoA was used, the fatty acids in the 1; and 2-positions of isolated diacyl-GP were analyzed for incorporation of radioactivity using phospholipase A, treatment as described previously (26). The activities of 1-acyl-GP, 1-acyl-GPC, and l-acylGPE acyltransferase systems were measured spectrophotometrically. Typically, the incubation mixture consisted of 10 to 20 PM acyl-CoA, phospholipid acceptor (75 PM 1-aeyl-GP, 150 PM 1-acyl-GPC, or 150 PM I-acyl-GPE), 0.15 to 0.2 mg/ml microsomal protein, and 1 mM DTNB in 1 ml of 0.08 M Tris-chloride, pH 7.5. Reactions were performed at 25°C and were initiated by adding acyl-CoA. Formation of thionitrobenzoate anion was monitored continuously at 413 nm with a Hitachi spectrometer (Model 181). Control rates were obtained from reactions performed without added acceptor and were subtracted from experimental values to give net acyltransfer rates. When radiolabeled acyl-CoA mixtures were used as acyl donors, the labeled fatty acids incorporated into phospholipids were determined after isolation of the products by thinlayer chromatography and hydrolysis by phospholipase A, as described previously (30). RESULTS
Glycerophosphute
Acyltransferase
Systems
It has previously been shown that two glycerophosphate acyltransferase systems exist in rat liver, one which is specific for palmitoyl-CoA and concentrated in mito-
211
IN RAT LUNG TABLE I
SPECIFICITIESOF GLYCEROPHOSPHATE ACYLTRANSFERASESYSTEMSIN LUNGANDLIVERO Acyltransfer rate (nmol/min/mg protein) Expt No.
Enzyme source
1
Lung
2
Lung
3
Liver
DTNB
Pal-
+ + +
0.30 0.10 0.88 0.20 0.90 0.44
Ole-
Lin-
0.43 0.45 co.01 co.01 0.90 1.19 0.02 0.04 0.86 0.83 0.05 0.05
a The incubation mixtures consisted of 2 mM [3H]glycerophosphate (2000 cpm/nmol), 30 PM acyl-CoA, 2 mg/ml protein (whole membrane fraction, see Materials and Methods), and 0.5 mg/ml bovine serum albumin in 1 ml of 0.1 M Tris-chloride, pH 7.5, in the presence or absence of 4 mM DTNB. Incubations were performed for 2 min at 30°C. Palmitoyl (Pal-)-, oleoyl (Ole-)-, and lineoleoyl (Lin-)-CoAs were tested. A zero time control value (0.06 nmoYmin/mg protein) was subtracted to give net acyltransfer rates. Averages of two separate determinations (Expts 2 and 3) or three separate determinations (Expt 1) are presented. The maximal deviation from the mean was 15%. In Expt 2, an enzyme preparation suspended in 2 mM dithiothreitol0.25~ sucrose-O.01 MTris-chloride, pH 7.5, wasused, but 56fold excess DTNB was present in the assay mixture.
chondrial outer membrane preparation and another which is relatively nonspecific with respect to acyl-CoA donors and is found mainly in microsomal preparations (36-38). By using the sulfhydryl reagent, 5,5’-dithiobis (Znitrobenzoic acid) (DTNB) which selectively inhibits the microsomal acyltransferase, it has been possible for us to estimate the relative contributions of the mitochondrial and microsomal glycerophosphate acyltransferase systems to the synthesis of various molecular species of diacylGP in rat liver in vitro (30). We, therefore, applied this method to the lung system (Table I). It should be noted that saturating concentrations of a single acyl-CoA and a single acceptor were added to determine the maximal velocities for the individual acylCoAs and that use of the term “specificity” is confined to denote the comparisons of
212
YAMADA AND OKUYAMA
F 2 0.8. E 2 F _ 0.4 > .= .> 7l 4 0 0
20 Palmitoyl
40 60 CoA , pM
FIG. 1. Effects of palmitoyl-CoA concentration on glycerophosphate acyltransferase systems in lung. The incubation mixtures were as described in the legend to Table I, except that the concentrations of palmitoylCoA were varied. Whole membrane fraction (0) or mitochondrial fraction (0) was used as the enzyme source. The DTNB-sensitive activity (A) was calculated by subtracting the DTNB-resistant activity (A) from the total activity measured in the absence of DTNB. Each point represents the average of two separate determinations.
these maximal velocities (30).2 The DTNBresistant acyltransferase system in the whole membrane fraction prepared from lung was highly specific for palmitate, whereas the DTNB-sensitive system incorporated unsaturated and saturated acylCoAs at comparable rates. These properties of the two lung acyltransferase systems were quite similar to those observed in liver (30), both with respect to DTNB sensitivity and the lack of acyl group transfer specificity of the DTNB-sensitive system. However, the activity of the DTNB-resistant system was relatively lower in lung than in liver. The relatively low specificity for acyl-CoAs of the DTNB-sensitive system was qualitatively consistent with the results reported for the microsomal glycerophosphate acyltransferase system in rabbit lung (39). Dithiothreitol was found to protect the acyltransferase systems from a time-dependent inactivation (Expts 1 and 2 in Table I) and hence was added to the enzyme suspension in subsequent experiments on the glycerophosphate acyltransferase systems. The DTNB-resistant acyltransferase system in liver showed a much higher affinity for palmitoyl-CoA than did the DTNB-sen-
sitive system. This difference in affinity was found to be an important factor determining the molecular species of diacyl-GP formed in vitro (30). As shown in Fig. 1, the DTNBresistant system in mitochondria from lung also exhibited significantly higher affinity for palmitoyl-CoA than did the DTNB-sensitive system present in whole membrane fraction. These observations suggest that similar glycerophosphate acyltransferase systems are present in both liver and lung; the l-position of glycerophosphate can be acylated either with saturated acyl-CoAs via the DTNB-resistant system or with unsaturated acyl-CoAs in reactions catalyzed by the DTNB-sensitive system depending on the relative concentrations of acyl-CoAs. The apparent K, value for glycerophosphate was approximately 0.6 mM although concentrations above 3 mM did not completely saturate the system. The intracellular concentrations of glycerophosphate in liver is reported to be 0.11 to 0.17 pmol/g wet wt (40), but the concentration in lung has not been reported. The selectivity for acyl-CoAs in the formation of diacyl-GP by the microsomal glycerophosphate acyltransferase system was examined in the presence of an equimolar mixture of palmitoyl-CoA and linoleoyl-CoA and varying concentrations of glycerophosphate. Fatty acids incorporated into the l- and 2-positions of diacyl-GP, the major product, were analyzed after phospholipase A, hydrolysis. Linoleate and palmitate were incorporated into the l-position of diacyl-GP by microsomes from both lung and liver (Table II). This is consistent with the specificities of the DTNB-sensitive glycerophosphate acyltransferase systems shown in Table I. In preparations from liver, the 2-position of diacyl-GP was esterified preferentially with linoleate. In contrast, considerable amounts (35%) of palmitate were incorporated into the 2-position of diacyl-GP in lung microsomes. Changing the glycerophosphate concentration did not affect the ratio of the incorporated fatty acids in lung and affected the ratio only slightly in the liver microsomes. Thus, the capacity of lung microsomes to incorporate palmitate into the 2-position of diacyl-GP is much greater than that of liver microsomes.
SURFACTANT
LIPID SYNTHESIS
This difference in the selectivity with which the microsomal acyltransferase systems acylate the 2-position of the glycerophosphate moiety is compatible with the differences in the properties of microsomal 1-acyl-GP acyltransferase systems in lung and liver, which are described below.
TABLE III SPECIFICITIESOF MICROSOMALI-Acyl-GP ACYLTRANSFERASESYSTEMSIN LUNGANDLIVER" Relative acyltransfer rate Acyl-CoA
l-Acyl-GP Acyltransferase System Maximum velocities of microsomal lacyl-GP acyltransferase systems of lung and liver with various acyl-CoAs were determined in the presence of a single acylCoA and acceptor (Table III). The specifici-
213
IN RAT LUNG
PalSteOleLinAra-
Lung microsomes Liver microsomes l (12)b 0.83 1.3 1.6 0.80
1 (62)b 0.76 1.2 0.94 0.42
a The incubation mixtures consisted of 75 ~.LMl-acylGP, 10 PM acyl-CoA, 0.2 mg/ml microsomal protein, and 1 tiIM DTNB in 1 ml of 0.08 M Tris-chloride, pH 7.5. TABLE II Reactions at 25°C were monitored by continuously EFFECTOFTHEGLYCEROPHOSPHATECONCENTRATIONmeasuring the absorbance at 413 nm with a Hitachi ON THE SELECTIVITIESOFTHE MICROSOMAL spectrometer (Model 181)(37). Control values obtained GLYCEROPHOSPHATE ACYLTRANSFERASE from experiments performed in the absence of l-acylSYSTEMSIN LUNG AND LIVER* GP (below 1.1 nmollminimg protein for lung microsomes and 7 nmoYmin/mg protein for liver microsomes) Linoleate/palmitate molar ratio at were subtracted from values obtained in the presence of 1-acyl-GP to give net acyltransfer rates. Acyl-CoA Z-Position l-Position Glyceroconcentrations were examined in the range of 7.5 to phosphate 30 j&M. Acyl-CoA concentrations of lo-20 FM gave Liver Lung Liver Lung (mM) maximal velocities. Apparent K, values for l-palmitoyl-GP were 20 and 38 pM with palmitoyl-CoA and 0.73 2.0 2.0 8.4 1.5 arachidonoyl-CoA (Ara-), respectively. 12 1.7 0.85 0.5 1.8 * Activity expressed as nmol/min/mg of protein in 14 1.7 1.3 0.12 1.9 parentheses. Averages of two separate determinations (1.5) (1.8 (1.0) (1.0) (0) are presented. The maximal deviation from the mean was 10%. a The incubation mixtures consisted of 15 pM [3H]palmitoyl-CoA (7000 cpmnmol under dual-label counting conditions), 15 j.bM [14C]linoleoyl-CoA (8000 ties thus determined were similar and were cpminmol), varying concentration of glycerophosphate, relatively low in both the lung and liver and 2 mg/ml microsomal protein in 1 ml of 0.1 M Trissystems. In the case of rat liver microsomes, chloride, pH 7.5. The mixture was incubated at 30°C the apparent discrepancy between the refor 1 min and the reaction product, diacyl-GP, was latively low specificity of the 1-acyl-GP isolated using Silica Gel H thin-layer chromatography. The radioactive acids esterified to the l-and 2-positions acyltransferase system and the highly selecof diacyl-GP were determined after phospholipase A, tive incorporation of unsaturated fatty acids hydrolysis using methods described previously (28). into the S-position of diacyl-GP in vivo can Averages of three separate determinations for the lung be explained by the fact that the system can and two separate determinations for the liver are pre- be highly selective for unsaturated acylsented. The maximal deviations from means were 20 CoAs at lower concentrations of the acceptor and 15% for the lung and liver systems, respectively. in vitro possibly as a consequence of the Incorporation of radioactivity without added glycero- difference in affinities for the acceptor (26). phosphate was less than 29 and 7% of that obtained Bothin vivo and in the synthesis of diacyl-GP at 0.12 mM glycerophosphate for lung and liver systems, respectively. These values were subtracted from from glycerophosphate in vitro, the concenexperimental values in calculating the molar ratios of tration of the 1-acyl-GP intermediate is linoleate to palmitate incorporated into diacyl-GP. The maintained at a low level since the ratemolar ratios in the control experiments (no added ac- limiting step under these conditions is the glycerophosphate acyltransferase system. ceptor) are shown in parentheses.
214
YAMADA AND OKUYAMA TABLE IV
EFFECT OF THE I-ACYLGP CONCENTRATION ON THE SELECTIVITIES OF MICROSOMAL l-ACYL-GP ACYLTRANSFERASE SYSTEMSIN LUNG AND LIVER”
the 2-position of diacyl-GP at low phospholipid acceptor concentrations than is the liver system. 1-Acyl-GPC
Acyltransferase
System
The specificities for acyl-CoAs of microsomal 1-acyl-GPC acyltransferase systems 1-Acyl-GP in lung and liver are shown in Table V. AlLiver Lung (PM) though the specific activities of the two microsomal preparations were different, 50 2.0 k 0.1 (6) 2.1 + 0.1 (7) the specificities for the acyl-CoAs were 10 2.1 + 0.1 (2) 2.7 5 0.4 (3) quite similar; both systems showed higher 2.3 + 0.1 (4) 3.9 2 1.3 (3) 5 specificities for unsaturated acyl-CoAs than 3 2.9 2 0.4 (4) 7.9 * 2.4 (6) saturated acyl-CoAs. However, the differ1.5 3.2 2 0.8 (2) 13.7 k 1.9 (3) (2.3 + 0.2) (8) (2.4 k 0.5) (7) ences in the properties of the lung and (0) liver systems were noted when the acceptor (2The incubation mixtures consisted of 15 ELM[3H]- concentrations were varied in the presence palmitoyl-CoA (12,000cpm/nmol), 15 PM [W]linoleoylof saturating concentrations of equimolar CoA (8000cpm/nmol), varying concentrations of l-acylmixtures of saturated and unsaturated acylGP, 0.2 mg/ml microsomal protein, and 1 mM DTNB CoAs. As shown in Table VI, a pronounced in 1 ml of 0.08 M Tris-chloride, pH 7.5. The mixture selectivity for linoleate was observed at was incubated for 1 min at 30°C and the product, dilower concentrations of 1-acyl-GPC in liver acyl-GP, was analyzed as described in the legend to microsomal system with palmitate being alTable II. The numbers of experiments are shown in most excluded from the 2-position of phosparentheses. Total incorporation of radioactivity withphatidylcholine. In contrast, the selectivity for out added 1-acyl-GP was less than 42% of that observed linoleate decreased at lower concentrations with 1.5 PM l-acyl-GP for both the lung and liver. This value was subtracted from the experimental val- of the acceptor in the lung system, allowing ues in calculating the molar ratios of linoleate to palmitate to be incorporated into the 2-posipalmitate incorporated into the 2-position. tion of phosphatidylcholine. These features were even more noticeable with the palmitate/arachidonate combination. The liver Thus, the selectivity for acyl-CoAs observed system was highly selective for arachidoat lower concentrations of 1-acyl-GP was considered to reflect the selectivity in the acylation at the 2-position of diacyl-GP in TABLE V the livers of rats fed normal diets. In conSPECIFICITIES OF MICROSOMAL l-AcYLGPC trast to the results obtained with liver ACYLTRANSFERASE SYSTEMS IN microsomes, the microsomal 1-acyl-GP acylLUNG AND LIVERY transferase system in lung incorporated palmitate and linoleate into the 2-position Relative acyltransfer rate of diacyl-GP with comparable rates at all concentrations of the acceptor, indicating Acyl-CoA Lung microsomes Liver microsomes that the affinities for 1-acyl-GP for palmiPal1 (21)” toy1 transfer and linoleoyl transfer reactions 1 W’ Ste0.50 0.66 are similar (Table IV). Thus, the specificiOle2.5 2.5 ties for acyl-CoAs as measured by the Lin4.4 3.5 respective maximal velocities were not much Ara4.3 4.0 different between the 1-acyl-GP acyltransferase systems in lung and liver. However, ’ The incubation mixtures were as described in the the selectivities for palmitate and linoleate legend to Table III, except that 150 PM 1-acyl-GPC observed at lower concentrations of l-acyl-GP was used as the acceptor. were significantly different; the lung system b Specific activities expressed as nmoYminimg of prois more likely to incorporate palmitate into tein are shown in parentheses. Linoleate/palmitate molar ratio 2 SE
SURFACTANT TABLE VI
LIPID SYNTHESIS
215
IN RAT LUNG
of 1-acyl-GPC acyltransferase system was examined with equimolar mixtures of arachidonoyl-CoA and palmitoyl-CoA. In both lung and liver systems, more arachidonate than palmitate was incorporated into the 2-position of phosphatidylcholine at a satuArachidonatel rating concentration of acyl-CoAs, but the palmitate selectivity for arachidonate decreased when molar ratio the levels of acyl-CoA mixtures were lowered. These results are consistent with an Lung Liver observation by Hasegawa-Sasaki and Ohno 34 (19) that palmitate incorporation is inhibited 4.8 by arachidonoyl-CoA but arachidonate in>lOO 2.2 corporation is not inhibited by palmitoyl1.2 >lOO (1.0) (17) CoA in the rat lung 1-acyl-GPC acyltransferase system (Table VII).
EFFECT OF THE l-ACYL-GPC CONCENTRATIONON SELECTIVITIES OF MICROSOMALl-ACYL-GPC ACYLTRANSFERASESYSTEMSIN LUNG AND LIVER”
l-Acyl-GPC concentration (PM(I) 75 15 6 (0)
Lineoleatei palmitate molar ratio Lung
Liver
22 17.5 80 9.1 5.1 >lOO (3.6) (>lOO)
(2The incubation mixtures consisted of 10 PM [3H]palmitoyl-CoA (7400 cpm/nmol) plus 10 PM [W]linoleoyl-CoA (820 cpm/nmol) or 10 PM [‘*C]palmitoyl-CoA (12,500 cpmnmol) plus 10 PM [3H]arachidonoyl-CoA (2500cpm/nmol), varying concentration of 1-acyl-GPC, 1 mM DTNB, and 0.2 mg/mI microsomal protein in 1 ml of 0.1 M T&-chloride, pH 7.5. The mixtures were incubated for 1 min at 30°C. The fatty acids incorporated into the 2-position of the product, phosphatidylcholine, were analyzed after phospholipase Al hydrolysis. Averages of two separate determinations are presented. The maximal deviation from the mean was 30%. The molar ratios in the control experiments are shown in parentheses.
nate, while the lung system became more selective for palmitate at lower concentrations of the acceptor; in lung the rates of palmitate and arachidonate incorporation at a 1-acyl-GPC concentration of 6 PM were comparable whereas about fivefold more arachidonate than palmitate was incorporated at 75 PM 1-acyl-GPC under these conditions. Thus, while the specificities for acylCoAs of the 1-acyl-GPC acyltransferase systems from liver and lung microsomes were not significantly different when measured at saturating substrate concentrations, the lung system exhibited a selectivity for the transfer of palmitate to 1-acyl-GPC at lower concentrations of the phospholipid acceptor significantly higher than that in the liver system. In contrast, the liver l-acylGPC acyltransferase system showed a marked selectivity toward arachidonoyl transfer. In the next experiments, the effect of varying acyl-CoA levels on the selectivity
I-Acyl-GPE
Acyltransferase System
The specificities for acyl-CoAs of l-acylGPE acyltransferase systems in lung and liver microsomes are shown in Table VIII. Both systems showed higher specificities for unsaturated acyl-CoAs examined, except oleoyl-CoA. The effect of the acceptor concentration on the selectivity of this enzyme TABLE VII EFFECT OF ACYL-CoA CONCENTRATIONON SELECTIVITY OF MICROSOMALl-ACYLGPC ACYLTRANSFERASESYSTEMS IN LUNG AND LIVERY Arachidonatei palmitate molar ratio
Acyl-CoA concentration (PM)
Lung
Liver
2+2 5+5 10 + 10 25 + 25 50 + 50
3.7 6.1 9.4 12.2 19.3
2.1 5.3 25 >lOO >lOO
a The incubation mixtures consisted of varying concentration of [W]palmitoyl-CoA (12,500 cpmnmol) plus [3H]arachidonoyl-CoA (2200 cpm/nmol), 75 PM I-acyl-GPC, 1 mM DTNB, and 0.1 mg of microsomal protein in 1 ml of 0.1 M Tris-chloride, pH 7.5. The mixtures were incubated for 1 min at 25°C. The fatty acids incorporated into the e-position of phosphatidylcholine were analyzed after phospholipase A, hydrolysis. Averages of two separate determinations are presented. The maximal deviation from mean was 30%.
216
YAMADA
AND OKUYAMA
TABLE IX system is shown in Table IX. In both lung and liver systems, palmitate tended to be EFFECTOFTHE l-ACYL-GPE CONCENTRATION ONTHE excluded from the 2-position, a character- SELECTIVITIES OF l-ACYL-GPE ACYLTRANSFERASE istic which was even more pronounced when SYSTEMSIN LUNGANDLIVER" the 1-acyl-GPE acceptor concentration was Arachidonatel lower. These observations are compatible palmitate with the absence of palmitate at the 2-posil-Acyl-GPE molar ratio tion of phosphatidylethanolamine isolated concentration from rat lung and liver (41). DISCUSSION
Enzyme systems acylating the l-position of glycerophosphate have abilities to incorporate saturated and unsaturated acyl-CoAs comparably as revealed by the maximal velocities (Table I). However, this acylation step can be selective for palmitate in the presence of two glycerophosphate acyltransferase systems at lower concentrations of saturated and unsaturated acyl-CoAs, since the DTNB-resistant system has much higher affinity for palmitoyl-Coil than the DTNBsensitive system (Fig. 1). The localization of the former in the mitochondrial fraction is not easily understandable. Whether l-palmitoyl-GP produced in mitochondria is used as substrate for microsomal enzyme or the DTNB-resistant system was artificially associated with mitochondria during subcelTABLE
VIII
SPECIFICITIESOF MICROSOMALl-ACYL-GPE ACYLTRANSFERASESYSTEMSIN LUNGANDLIVER" Acyltransfer rate (nmoYmin/mg protein) Acyl-CoA
Lung
Liver
PalOleLinAra-
1.8 1.9 6.4 8.0
2.5 1.0 14.6 14.2
’ The incubation mixtures consisted of 20 PM acylCoA, 150 pM 1-acyl-GPE, 1 mM DTNB, and 0.15 mgiml microsomal protein in 1 ml of 0.08 M T&-chloride, pH 7.5. The reactions were followed spectrophotometrically. Averages of two separate determinations are presented. The maximum deviation from the mean was 5%. I-Acyl-GPE concentrations of 150-250 PM gave maximal velocities.
(PM)
Lung
Liver
300 150 50
9.8 14 25
>lOO >lOO
(0)
6-j
>lOO G-)
L1The incubation mixtures consisted of 10 PM [3H]arachidonoyl-CoA (2500 cpmnmol), 10 PM [“‘Clpalmitoyl-CoA (12,500 cpm/nmol), varying concentration of I-acyl-GPE, and 0.2 mg/ml microsomal protein in 1 ml of 0.08 M Tris-chloride, pH 7.5. The mixtures were incubated for 1 min at 30°C. The fatty acids incorporated into phosphatidylethanolamine were analyzed after phospholipase A, hydrolysis (30). Incorporation of radioactivity in the absence of 1-acyl-GPE was negligible.
lular fractionation (30) is to be clarified. Although little unsaturated fatty acid is found esterified to the l-position of glycerophospholipids purified from lungs or livers of rats fed normal laboratory diets (41,42), the in vivo situations under which unsaturated fatty acids are found at the l-position are known in liver but not in lung as noted earlier (30). These results suggested that the concentration and composition of the acyl donor pool are important in determining the fatty acid pattern at the l-position of diacyl-GP in lung. In this sense, it seems interesting that the compositions of acyl-CoA pools in the lung and liver are quite different.4 The contribution made to diacyl-GP synthesis by the relatively low 2-acyl-GP acyltransferase activity found in liver (24) is not clear at present in either the liver or lung systems. Recently, the dihydroxyacetone phosphate pathway was reported to play a quantitatively more important role in glycerolipid synthesis in the lung than the glycerophosphate pathway (43). Although the specificity for acyl-CoAs of this pathway 4 Y. Tabata and Y. Suzuki (19’78) Abstract of the 6th meeting of Jap. Med. Sot. Biol. Interface, p. 8.
SURFACTANT
LIPID
SYNTHESIS
in lung has not been determined, the specificities of dihydroxyacetonephosphate pathways present in liver mitochondrial (peroxisomal) and microsomal fractions are known to be similar to those of glycerophosphate acyltransferase systems in these subcellular fractions (44, 45). The fatty acids at the 2-position of diacyl-GP formed in vivo are quite different between lung and liver tissues (5,6,46,47). Although an enzyme activity which acylates the 2-position of glycerophosphate to form 2-unsaturated acyl-GP as an intermediate in liver was reported by Lamb and Fallon (48), we have been unable to reproduce their results (24). Hence, we focused our attention on the 1-acyl-GP acyltransferase system present in lung microsomes. A striking difference between the 1-acyl-GP acyltransferase systems in the lung and liver was noted. Although both systems showed similar specificities at saturating substrate concentrations, the selectivities for acyl donors differed markedly at lower concentrations of the 1-acyl-GP acceptor (Tables II and IV). l-Acyl-GPC acyltransferase systems are believed to be involved in determining the fatty acid composition of the 2-position of phosphatidylcholine. These systems showed very similar specificities in both lung and liver (Table IV) but again showed quite different selectivities for various acyl-CoAs at lower concentrations of acceptor (Table VI). At present, it is unclear whether substrate concentrations vary substantially in vivo. However, one can calculate that the phospholipid acceptor concentration in vivo is considerably lower than the saturating concentrations used to determine specificities in vitro (- 150 nmol/0.2 mg microsomal protein). Thus, it seems reasonable to assume that the selectivities of acyltransferase systems at lower concentrations of acceptors may closely mimic the reaction conditions existing in vivo. The present experiments have clearly shown that the 1-acyl-GP and 1-acyl-GPC acyltransferase systems in lung can incorporate palmitate into the 2-position of diacyl-GP and phosphatidylcholine at rates comparable to those observed with unsaturated fatty acids. Such is not the case with the liver acyltransferase systems which essentially exclude palmitate. The selectivi-
IN RAT LUNG
217
ties observed with lung and liver in vitro are consistent with the in vivo distribution of acyl groups (6, 15, 16, 41, 42). Variations in the selectivities of acyltransferase systems in response to changes in substrate concentrations might also help explain the apparently inconsistent results noted in tracer experiments performed with lung tissue slices; radiolabeled 1-acyl-GPC added to the incubation mixture was found to be acylated mainly with unsaturated fatty acids by the acyltransferase system whereas acylations of endogenous lipid precursor with added palmitate yielded phosphatidylcholine with palmitate located mainly at the 2-position (5). On the one hand, esterification of labeled 1-acyl-GPC in these experiments may have resulted from acyltransfer reactions involving low levels of endogenous fatty acyl donors and relatively high concentrations of exogenous 1-acyl-GPC while on the other hand, selective incorporation of labeled palmitate occurred through acylation of low endogenous levels of 1-acyl-GPC. One could thus rationalize the results obtained in the tissue slice experiments based on the selectivities observed in acyl group transfers at high and low 1-acyl-GPC concentrations (Table VI). It should be noted, however, that the relative concentrations of acylCoAs could affect the selectivities of acyltransferase systems (26, 28, 30), although the selectivities were determined in the current experiments in the presence of equimolar mixtures of acyl-CoAs. Some kinetic parameters of the l-acylGPC acyltransferase system in lung have been reported by other workers (15,19,2123,49) and are summarized in Table X. The specificities, shown as relative maximal velocities for acyl-CoAs, as well as the K, values for the substrates differ significantly from each other. We could prepare lung microsomes practically free of acyl-CoA hydrolase activities, which enabled us to use a spectrophotometric assay with DTNB (37) to follow the acyltransfer reactions continuously. Our data are most similar to those reported by Hasegawa-Sasaki and Ohno (19) except the K, value for archidonoyl-CoA. The kinetic parameters determined with lung microsomes were not significantly different from those obtained with liver mi-
218
YAMADAANDOKUYAMA TABLE X KINETIC CONSTANTS FOR l-ACYL-GPC ACYLTRANSFERASESYSTEMSIN LUNG
K, for Acyl-CoA (PM)
K, for l-acyl-GPC (PM)
Relative V max
PalOle-
-4
-
1 1.1
(15)
PalOleAra-
6 6 44
18 25 30
1 1.8 4.8
(19)
PalOle-
-
-
1 0.24
(23)
PalOleAra-
<5 <5
19 29
1 2.5 4.3
Rabbit lung
PalOle-
36 8
34 37
1 1.5
(21)
Rabbit cultured cell
PalOle-
15 16
-
1 0.73
(21)
Enzyme source Rat lung
Acyl-CoA
Reference
Present experiments
n Not reported.
crosomes and thus do not provide a simple explanation for the fatty acid composition of phosphatidylcholine isolated from lung and liver. However, differences in properties of 1-acyl-GP and 1-acyl-GPC acyltransferase systems in the lung and liver became quite apparent when the selectivities of acyltransfer were measured as a function of phospholipid acceptor concentration. We have previously presented evidence that the observed variations in the selectivity of the acyltransferase system are not caused by changes in the physicochemical properties of substrate micelles but are an inherent property of the enzyme system itself (2426, 30). Additional support for this concept comes from the observations (a) that lowering 1-acyl-GPC concentrations had opposite effects on the selectivities in lung and liver systems (Table VI) and (b) that lowering the 1-acyl-GPC and 1-acyl-GPE concentrations had opposite effects on the incorporation of palmitate and arachidonate in the lung (Tables VI and IX). The specificities of 1-acyl-GPC and l-acylGPE acyltransferase systems were not the same. For example, the rate of transfer for
oleoyl-CoA was much less with the l-acylGPE acyltransferase systems than with the l-acyl-GPC acyltransferase systems in both lung and liver microsomes. Furthermore, lowering the concentrations of 1-acyl-GPC and 1-acyl-GPE had opposite effects on the selectivity for palmitate and arachidonate in lung. These results are consistent with the concept (32) that the acylations of l-acylGPC and 1-acyl-GPE are catalyzed by different microsomal enzyme systems although it is unknown how many different acyltransferase enzymes are actually present. It is also unclear with which cell type of the lung the various acyltransferases are associated. In this context, it would be of particular interest to determine the properties of the acyltransferase systems in the alveolar Type II cells (16, 50), those cells which are known to be involved in the formation of dipalmitoyl-GPC in lung. ACKNOWLEDGMENT The authors are indebted to Professor H. Ikezawa, Nagoya City University, Nagoya, for his helpful discussions.
SURFACTANT
LIPID
SYNTHESIS
REFERENCES 1. GOERKE, J. (19’74) Biochim. Biophys. Acta 344, 241-261. 2. OHNO, K., AKINO, T., AND FUJIWARA, T. (1978) Rev. Perinat. Med. 2, 227-318. 3. AKINO, T., AND ABE, M. (1977) J. Japan. Med. Sot. Biol. Interjace 8, 2-18. 4. VAN GOLDE, L. M. G. (1976) Amer. Rev. Resp. Dis. 114, 977-1000. 5. AKINO, T., ABE, M., AND ARAI, T. (1971) Biochim. Biophys. Acta 248, 274-281. 6. AKINO, T., YAMAZAKI, I., AND ABE, M. (1972) Tohoku J. Exp. Med. 108, 133-139. 7. VAN DEN BOSCH, H., BONTE, H. A., AND VAN DEENEN, L. L. M. (1965) Biochim. Biophys. Acta 98, 648-651. 8. ABE, M., OHNO, K., AND SATO, R. (1974) Biochim. Biophys. Acta 369, 361-370. 9. HALLMAN, M., AND RAIVIO, K. (1975) Biol. Neonate 27, 329-338. 10. OLDENBORG, V., AND VAN GOLDE, L. M. G. (1976) Biochim. Biophys. Acta 441, 433-442. 11. OKANO, G., AND AKINO, T. (1978) Biochim. Biophys. Acta 528, 3’73-384. 12. TSAO, F. H. C., AND ZACHMAN, R. Z. (1977) Pediat. Res. 11, 858-861. 13. FARRELL, P. M., LUNDGREN, D. W., AND ADAMS, A. J. (1974) Biochem. Biophys. Res. Commun. 57, 696-701. 14. ROONEY, S. A., WAI-LEE, T. S., GOBRAN, L., AND MOTOYAMA, E. K. (1976) Biochim. Biophys. Acta 431, 447-458. 15. VEREYKIN, J. M., MONTFOORT, A., AND VAN GOLDE, L. M. G. (1972) Biochim. Biophys. Acta 260, 70-81. 16. KIKKAWA, Y. (1978) J. Japan. Med. Sot. Biol. Interface 9, 1-13. 17. SARZALA, M. G., AND VAN GOLDE, L. M. G. (1976) Biochim. Biophys. Acta 441, 423-432. 18. POSSMAYER, F., DUWE, G., AND HAHN, M. (1977) Canad. J. Biochem. 55, 609-617. 19. HASEGAWA-SASAKI, H., AND OHNO, K. (1975) Biochim. Biophys. Acta 380, 486-495. 20. MORIYA, K., AND KANOH, H. (1974) Tohoku J. Exp. Med. 112, 241-256. 21. TANSEY, F. A., AND FROSOLONO, M. F. (1975) Biochem. Biophys. Res. Commun. 67, 15601566. 22. FROSOXINO, M. F., SLIVKA, S., AND CHARM, B. L. (1971) J. Lipid Res. 12, 96-102. 23. ENGLE, M. J., SANDERS, R. L., AND LONGMORE, W. J. (1976) Arch. Biochem. Biophys. 173, 586-595. 24. OKUYAMA, H., EIBL, H., AND LANDS, W. E. M. (1971) Biochim. Biophys. Acta 248, 263-273.
IN RAT LUNG
219
25. OKUYAMA, H., AND WAKIL, S. J. (1973) J. Biol. Chem. 248, 5197-5205. 26. OKUYAMA, H., AND LANDS, W. E. M. (1972) J. Biol. Chem. 247, 1414-1423. 27. OKUYAMA, H., YAMADA, K., AND IKEZAWA, H. (1975) J. Biol. Chem. 250, 1710-1713. 28. OKUYAMA, H., YAMADA, K., IKEZAWA, H., AND WAKIL, S. J. (1976) J. Biol. Chem. 251, 24872492. 29. OKUYAMA, H., YAMADA, K., KAMEYAMA, Y., IKEZAWA, H., AKAMATSU, Y., AND NOJIMA, S. (1977) Biochemistry 16, 2668-2673. 30. YAMADA, K., AND OKUYAMA, H. (1978)Arch. Biothem. Biophys. 190, 409-420. 31. SEUBERT, W. (1960) Biochem. Prep. 7, 80-83. 32. OKUYAMA, H., LANDS, W. E. M., CHRISTIE, W. W., AND GUNSTONE, F. D. (1969) J. Biol. Chem. 244, 263-273. 33. CHANG, Y. Y., AND KENNEDY, E. P. (1967) J. Lipid Res. 8, 447-455. 34. OKUYAMA, H., LANDS, W. E. M., GUNSTONE, F. D., AND BARVE, J. A. (1972) Biochemistry 11, 4392-4398. 35. LONG, C., ODAVI~, R., AND SARGENT, E. J. (1967) Biochem. J. 102, 211-229. 36. MONROY, G., KELKER, H. C., AND PULLMAN, M. E. (1973) J. Biol. Chem. 247, 6884-6894. 37. LANDS, W. E. M., AND HART, P. (1965) J. Biol. Chem. 240, 1905-1911. 38. DAAE, L. N. W., ANDBREMER, J. (1970)Biochim. Biophys. Acta 210, 92-104. 39. HENDRY, A. T., AND POSSMAYER, F. (1974) Biochim. Biophys. Acta 369, 156-172. 40. GUYNN, R. W., VELOSO, D., AND VEECH, R. L. (1972) J. Biol. Chem. 247, 7325-7331. 41. OKANO, G., KAWAMOTO, T., AND AKINO, T. (1978) Biochim. Biophys. Acta 528, 385-393. 42. MONTFOORT, A., VAN GOLDE, L. M. G., AND VAN DEENEN, L. L. M. (1971) Biochim. Biophys. Acta 231, 335-342. 43. MASON, R. J. (1978) J. Biol. Chem. 253, 33673370. 44. HAJRA, A. K. (1968) J. Biol. Chem. 243, 34583465. 45. JONES, C. L., AND HAJRA, A. K. (1977) Biochem. Biophys. Res. Commun. 76, 1138-1143. 46. HILL, E. E., HUSBANDS, D. H., AND LANDS, W. E. M. (1968) J. Biol. Chem. 243,4440-4451. 47. AKESSON, B., ELOVSON, J., AND ARVIDSON, G. (1970) Biochim. Biophys. Acta 210, 15-27. 48. LAMB, R. G., AND FALLON, H. J. (1970) J. Biol. Chem. 245, 3075-3083. 49. SNYDER, F., AND MALONE, B. (1975) Biochem. Biophys. Res. Commun. 66, 914-919. 50. BATENBURG, J. J., LONGMORE, W. J., AND VAN GOLDE, L. M. G. (1978) Biochim. Biophys. Acta 529, 160-170.