Development of glycerophosphate acytransferase in guinea pig lung mitochondria and microsomes

Biochimiea et Biophysica Acta, 802 (1984) 423-427 Elsevier

423

BBA 21930

DEVELOPMENT OF GLYCEROPHOSPHATE ACYLTRANSFERASE IN GUINEA PIG LUNG MITOCHONDRIA AND MICROSOMES SALIL K. DAS, PRATAPRAI B. KAKKAD and MELBA S. McCULLOUGH

Department of Biochemistry, Meharry Medical College, 1005 David Todd Boulevard, Nashville, TN 37208 (U.S.A.) (Received May 22nd, 1984) (Revised manuscript received September llth, 1984)

Key words: Glycerophosphate acyltransferase," Development," (Lung)

Development of mitochondrial and microsomal glycerophosphate acyltransferase in the fetal guinea pig lung was investigated. Mitochondrial and microsomal enzyme activity gradually increased from 45 days to 55 days of gestation. The specific activity in the microsomal fraction (8.2 nmol/min per mg protein) then declined until term, but increased again in the 24-h newborn from 2.5 to 6.1 nmol/min per mg protein. Glycerophosphate acyltransferase activity in the mitochondrial fraction declined after 55 days (3.5 nmol/min per mg) to a minimum level at 60 days (1.8 nmol/min per mg), but increased again in the 24-h newborn (4.0 nmol/min per mg). The specific activity of both mitochondrial and microsomal enzyme declined after 24 h after birth until adult levels were attained. Glycerophosphate acyltransferase activity in mitochondria and microsomes from adult lung was 0.8 and 2.0 nmol/min per mg, respectively. Microsomal enzyme activity was consistently inhibited (over 95%) throughout gestation and adulthood by exposure to any one of several proteinases: trypsin, chymotrypsin, papain, bromelain, pronase and nagarse. Although mitochondrial enzyme activity was also inhibited by these proteinases, there was a continuous increase in proteinase-resistant glycerophosphate acyltransferase activity between 45 days of gestation and term. In contrast, adult mitochondrial enzyme activity was stimulated by all the proteinases studied. These results suggest that early in gestation, glycerophosphate acyltransferase lies more exposed on the cytoplasmic side of the mitochondrial outer membrane and as gestation progresses it becomes embedded into the phospholipid bilayer.

Introduction

We have demonstrated earlier a bimodal subcellular distribution of glycerophosphate acyltransferase in fetal and adult guinea pig lung [1]. In comparison to the adult lung, the 55-day-old fetal lung had markedly higher mitochondrial and microsomal glycerophosphate acyltransferase activity. These results suggest that glycerophosphate acyltransferase may play an important role in lung development. It is generally agreed that the

Abbreviation: PC, phosphatidylcholine. 0304-4165/84/$03.00 © 1984 Elsevier Science Publishers B.V.

synthesis of lung surfactant phosphatidylcholine (PC) primarily occurs via the CDP-choline pathway [2]. The final step in the de novo synthesis of PC is catalyzed by cholinephosphotransferase with CDP-choline and diacylglycerol as substrates. We have shown that in fetal guinea pig lung, the subcellular cholinephosphotransferase activity is highest at approx. 81% (55 days) of the total gestational period [2]. Since the potential source of diacylglycerols is phosphatidic acid, a product formed by a pathway involving glycerophosphate acyltransferase, it is important that we study the subcellular developmental pattern of lung glycerophosphate acyltransferase. We have re-

424 ported [3] that incubation of adult guinea pig lung mitochondrial suspension in an isotonic lowionic-strength buffer containing various proteoiytic enzymes caused significant stimulation in glycerophosphate acyltransferase activity. The maximum stimulation ranged between 20 and 105%, and the order was as follows: bromelain > chymotrypsin > pronase > trypsin > papain > nagarse. Under hypotonic conditions, over 50% of glycerophosphate acyltransferase was destroyed by all the proteolytic enzymes. These results suggest that glycerophosphate acyltransferase in adult lung is located in the transverse plane of mitochondrial outer membrane. It is not known whether the submitochondrial location of glycerophosphate acyltransferase is similar in fetal and adult lung. This report presents the results of a study of the developmental pattern of glycerophosphate acyltransferase in both mitochondria and microsomes of fetal guinea pig lung. Materials and Methods Pregnant guinea pigs (Hartley strain) of timed gestation were purchased from C a m m Research Lab Animals, New Jersey. All animals were housed in stainless steel cages and fed ad libitum. Fresh cabbage and lettuce were given twice a week to provide vitamin C. The sources of chemicals were as follows: sn-[2-3H]glycerol 3-phosphate and Aquasol, New England Nuclear Corporation, Boston, MA; palmityl CoA and fatty acid free bovine serum albumin, Sigma, St. Louis, MO; asolectin, Associated Concentrations, Woodside, New York. Fetal guinea pigs were delivered by Caesarean section and the adult animals were killed by decapitation. The lungs were washed in ice-cold 0.9% saline, weighed and homogenized in 3 vol. of 0.25 M sucrose/1 mM EDTA (pH 7.4) with a Potter Elvehjem homogenizer. After the removal of nuclei and cell debris by centrifugation at 600 x g for 10 rain, mitochondria were sedimented at 10000 x g for 10 min in a Ti-50 rotor of a Beckman L5-50 ultracentrifuge. The pellet was resuspended in the isolation medium and resedimented at 6500 x g. The sediment was washed three times with 0.25 M sucrose/0.01 M Tris-HCl (pH 7.4) and was finally resuspended in the same buffer

and used as the mitochondrial preparation. The 10000 × g supernatant fluid was centrifuged at 105000 x g for 1 h to sediment the microsomes. The microsomal pellet was washed twice and resuspended in sucrose/Tris buffer. Protein concentration was measured by the procedure of Lowry et al. [4]. The mitochondrial and microsomal fractions were checked for cross contamination by assaying marker enzymes. Citrate synthase and N-ethylmaleimide-resistant glycerophosphate acyltransferase were used for the mitochondria, and NADPH-cytochrome c reductase and N-ethylmaleimide-sensitive glycerophosphate acyltransferase were used for microsomes, as described previously [1]. Mitochondrial and microsomal suspensions were incubated at 30°C for 5 min, and then a solution of proteolytic enzymes (bromelain, chymotrypsin, pronase, trypsin, papain and na.garse, 5 m g / m l in 1 mM HCI) was added to separate samples of suspension to give a p r o t e i n / proteolytic enzyme ratio of 1 : 2. After addition of a sample of proteolytic enzyme, the reaction mixture was incubated at 37°C for 10 min. The samples with or without proteolytic enzyme were immediately assayed for glycerophosphate acyltransferase. Glycerophosphate acyltransferase assays were performed in a total volume of 0.5 ml [5]. All assays contained an optimal concentration of palmityl CoA, 1.5 mM sn-[2-SH]glycerol 3-phosphate with a specific radioactivity of 10.103 cpm per nmol, 2 mM MgCI 2, 40 mM 2-(N-morpholino)ethanesulfonic acid, N-Tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid, glycylglycine buffer (pH 7.4), 2 mM KCN, 2 mg of bovine serum albumin, 200 #g asolectin and approx. 100 /*g of subcellular protein. Asolectin suspension (20 m g / m l in 10 mM Tris-HCl containing 1 mM EDTA (pH 7.4)) was prepared by sonicating the mixture for 5 min at 4 ° in a Model 185 Branson sonifier cell disruptor, centrifuging at 105 000 x g for 10 min and collecting the supernatant fluid. The assay was initiated by the addition of subcellular fraction and terminated after 3 rain by adding 1 ml of 1-butanol. A sample (0.7 ml) of the washed butanol extract was taken in a scintillation vial, mixed with 5 ml of Aquasol and counted in a Beckman LS-355 scintillation counter.

425

Results and Discussions

The change in the activity of lung mitochondrial and microsomal glycerophosphate acyltransferase with gestational age is shown in Fig. 1. The activity of both subcellular fractions varied with gesta-. tional age: the activity was greatest (8.2 n m o l / m i n per mg for microsomes and 3.5 n m o l / m i n per mg for mitochondria) at approx. 81% of the total gestation period (55 days). The microsomal activity was higher than that of the mitochondria at all stages of development. The difference in the enzyme activity between these two subcellular fractions was highest at 45 days of gestation (5-fold) and lowest at term (only 25%). After 55 days of gestation, the specific activity of the microsomal fraction declined until term, but increased again in the 24-h newborn from 2.5 to 6.1 n m o l / m i n per mg protein. The activity in the mitochondrial fraction declined after 55 days (3.5 n m o l / m i n per mg) to a minimum level at 60 days (1.8 n m o l / m i n per mg), but increased again in the 24-h newborn (4.0 n m o l / m i n per mg). The specific activity of both mitochondrial and microsomal glycerophosphate acyltransferase declined after 24 h postpartum until adult levels were atained. The activity in the mitochondrial and microsomal fractions of adult lung was 0.8 n m o l / m i n per mg and 2.0 n m o l / m i n per mg, respectively. Measurement of marker enzymes suggested that there was little cross-con-

9o[ c 80

~5

//

~L 70~

//

D~ 6 0 ~

x

~o i

\ \ ,\

~

'~

• 30

@ ~o I

}~ \~\

\

/

D/ 45 50 55 Gestatloncll Age (days)

60

I6'h [ ldcly ' "Adurt Term 12hr's Post nqta[

Fig. I. The activity of glycerophosphate acyltransferase as a function of gestational age. Each assay employed 100 ~g of protein. PalmityI-CoA at concentrations of 72 /LM for mitochondria and 144/~M for microsomes was used as the acyl donor. Each point represents mean nmol glycerol 3-phosphate incorporated/min per mg protein+S.E, of five samples. Mitochondria, 0 O ; Microsomes, • O.

tamination of mitochondrial matrix and endoplasmic reticulum marker enzymes. The mitochondrial fraction showed no detectable N-ethylmaleimide sensitive glycerophosphate acyltransferase and little NADPH-cytochrome c reductase, while containing high citrate synthase activity. The microsomai fraction, similarly showed no detectable citrate synthase, but did contain glycerophosphate acyltransferase, which was completely inhibited by N-ethylmaleimide and had high NADPH-cytochrome c reductase (results not shown). To our knowledge, no such developmental study has been undertaken with subcellular glycerophosphate acyltransferase of any tissue, particularly lung. In a recent limited study with microsomal glycerophosphate acyltransferase in rat liver microsomes, Coleman and Haynes [6] have observed a sharp rise in glycerophosphate acyltransferase activity after birth. However, unlike guinea pig lung, glycerophosphate acyltransferase activity in rat liver continually increased until adult levels were attained at 5-days after birth. Furthermore, these investigators limited their study to the late gestational period and did not detect any appreciable microsomal glycerophosphate acyltransferase activity. In our study, we also observed low levels of glycerophosphate acyltransferase activity in late gestation. Glycerophosphate acyltransferase being the first enzyme in the acylation of glycerophosphate, is an important regulatory enzyme in the formation of phosphoglycerides. The microsomal, but not the mitochondrial, glycerophosphate acyltransferase can be inhibited in vitro by various agents, such as sulfydryl group reagents, proteolytic enzymes, polymyxin B and acetone [1,3,5,7]. In vitro, there are no known inhibitors selective for mitochondrial glycerophosphate acyltransferase. However, in vivo dietary and hormonal deficiencies are known to affect the mitochondrial, but not microsomal glycerophosphate acyltransferase [8-11]. For example, m i t o c h o n d r i a l , but not m i c r o s o m a l glycero~hosphate acyltransferase activity is inhilS.ited in fasting [8] or in diabetic animal [9], regenerating rat liver [10] and cells maintained in tissue culture [11]. In our system, both mitochondrial and microsomal glycerophosphate acyltransferase varied substantially during gestation. It is not known how this variation is regulated. It is

426 possible that this variation is associated with either a change in the lipid microenvironment in the subcellular m e m b r a n e a n d / o r change associated with maternal nutritional status and hormonal levels. We do not know at this time what is the relative distribution of glycerophosphate acyltransferase in different cell types. These questions must be addressed before firm conclusions can be reached concerning the role of glycerophosphate acyltransferase in fetal lung development. Nevertheless, the present results strengthen our previous hypothesis that glycerophosphate acyltransferase plays an important role in lung surfactant synthesis. Results on the effects of proteolytic enzymes on the mitochondrial glycerophosphate acyltransferase activity of fetal lung of different gestational age are shown in Fig. 2. Although the enzyme activity was inhibited by all the proteinases studied, there was a gradual increase in the protease-resistant mitochondrial glycerophosphate acyltransferase activity between 45 days of gestation and

40

term. Adult mitochondrial enzyme activity was stimulated by all the proteinases studied. Microsomal glycerophosphate acyltransferase activity was consistently inhibited (over 95%) throughout gestation and adulthood to a m i n i m u m level (0.2 n m o l / m i n per mg) by all the proteinases (results not shown). This suggests that at early gestational age, glycerophosphate acyltransferase is relatively more exposed to the cytoplasmic side of the mitochondrial outer m e m b r a n e and becomes progressively more embedded into the phospholipid bilayer as gestational age increases. However, before firm conclusions can be reached on the proteinase experiments, we must have evidence that the subcellular fractions contain vesicles which are impermeable to the digestive enzymes. In order to accomplish this goal, we are currently studying the effect of proteolytic enzymes on the adenylate kinase activity of fetal lung mitochondria of varied gestational age. We have observed [3] that incubation of guinea pig lung mitochondrial suspension in an isotonic low ionic strength buffer containing various proteolytic enzymes causes no alteration in the activity of adenylate kinase, a marker for inner surface of outer mitochondrial m e m b r a n e of lung [12].

35

Acknowledgements 30

This work was supported by N I H G r a n t R R 08037 funded by the Division of Research Resources and the National Heart, Lung and Blood Institutes. We thank Dr. Dipak Haldar for his excellent comments, and Mrs. Anita Frierson for typing this manuscript.

~25

8~x 2.0

5

E c

©

References 05

Term

Adult

GestQtlonal Age (doys)

Fig. 2. The effects of proteolytic enzymes on the activity of fetal and adult lung mitochondrial glycerophosphate acyltransferase. Each assay contained 100 ~tg of protein and 200/~g of proteolytic enzyme. Palmityl-CoA at concentration of 72 #M was used as the acyl donor. Each point represents mean nmol glycerol 3-phosphate incorporated/min per mg protein + S.E. of five samples. Control, • 0; nagarse, 0 - - 0 ; papain, © O; trypsin, • •; pronase, • •; chymotrypsin, [] •; bromelain, A ZX.

1 Das, S.K., McCullough, M.S. and Halder, D. (1981) Biochem. Biophys. Res. Commun. 101,237-242 2 Stith, I.E. and Das, S.K. (1982) Biochim. Biophys. Acta 714, 250-256 3 Das, S.K. and Haldar, D. (1984) Biochem. Biophys. Res. Commun. 123, 569-573 4 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 5 Haldar, D., Wung-W. Tso and Pullman, M.E. (1979) J. Biot Chem. 254, 4502-4509 6 Coleman, R.A. and Haynes, E.B. (1983) J. Biol. Chem. 258, 450-456 7 Fitzpatrick, S.M., Sorresso, G. and Halder, D. (1982) J. Neurochem. 39, 286-289

427 8 Bates, E.J. and Saggerson, E.D. (1979) Biochem. J. 182, 751-762 9 Bates, E.J. and Saggerson, E.D. (1977) FEBS Lett. 84, 229 232 10 Haldar D., Magot, S., Fitzpatrick, S., Lee, I. and Lin, Y. (1978) J. Cell Biol. 79, 317a

11 Stern, W. and Pullman, M.E. (1978) J. Biol. Chem. 253, 8047-8055 12 Das, S.K. (1981) Biochem. Biophys. Res. Commun. 103, 1145-1148