Preparation of functional acetyl-CoA carboxylase mRNA from rat mammary gland

ARCHIVES

OF E~IOCHEM~STRY

Vol. 257, No. 1, August

AND

Preparation FERNANDO

BIOPHYSICS

15, pp. 63-68,1987

of Functional Acetyl-CoA Carboxylase from Rat Mammary Gland’

mRNA

Li)PEZ-CASILLAS, MICHAEL E. PAPE, DONG-HOON DAVID N. KUHN, JACK E. DIXON, AND KI-HAN KIM2

Department

of Biochemistry, Received

Lkcember

Purdue l&1986,

University, and

in revised

West

Lafayette, form

April

Indiana

BAI,

47907

1, 1987

Poly(A)+ RNA from lactating rat mammary glands was fractionated according to size by isokinetic sucrose gradient centrifugation to obtain a fraction enriched for acetylCoA carboxylase. In vitro translation of this RNA preparation yielded apparent fulllength acetyl-CoA carboxylase with a molecular weight of 260,000. The synthesized protein was identified as acetyl-CoA carboxylase by specific immunoprecipitation. Tests with antiserum to fatty acid synthetase, revealed that the fractions containing acetylCoA carboxylase mRNA also contained mRNA for fatty acid synthetase; both of these mRNAs were approximately 10 kb. Fatty acid synthetase with a molecular weight of 250,000 was, synthesized. Using an in vitro rabbit reticulocyte lysate translation system, we have shown that the amount of translatable acetyl-CoA carboxylase mRNA increases during lactation. On the fifth day postpartum the level of translatable acetyl-CoA carboxylase mRNA increased to a peak level seven times that on the day of parturition. 0 1987 Academic

Press. Inc.

Mammalian acetyl-CoA carboxylase (ACC)” catalyzes the rate-limiting step in long-chain fatty acid synthesis. In this reaction, CO2is fixed into acetyl-CoA to form malonyl-CoNA (1). In contrast to the abundance of information on the short-term regulation of ACC (Z-5), our knowledge of its long-term regulation is limited. Long-term regulation of ACC has been studied in whole animals and in isolated cell culture systems under a variety of hormonal and nutritional states (1, 6-9). Collectively, these studies show that regulation of the amount of ACC

during certain nutritional and hormonal conditions can be accounted for, at least in part, by regulation of the rate of enzyme synthesis. Studies using inhibitors of RNA synthesis (S-10) as well as of the titration of polysomes involved in the synthesis of ACC (11) suggest that RNA synthesis is associated with changes in the rate of enzyme synthesis. However, the relationship between changes in RNA metabolism and control of enzyme synthesis is not clear. There are several apparent difficulties in the study of the transcriptional/translational control of mammalian ACC. (i) ACC is an unusually large enzyme, consisting of two identical subunits whose molecular weight is 260,000 (12). Correspondingly large mRNAs pose many difficulties in the preparation and in vitro translation of fulllength protein. (ii) The amount of mRNA for ACC in lipogenic tissues is extremely low; thus, it has previously been impossible to obtain RNA preparations containing translatable and identifiable amounts of this mRNA.

1 This research was supported by a grant from the National Institutes of Health (AM12865). This is Journal Paper No. K994 from the Purdue University Agricultural Experiment Station. ’ To whom correspondence should be addressed. a Abbreviations used: ACC, acetyl-CoA carboxylase; SDS, sodium dodecyl sulfate; FAS fatty acid synthetase; DTT, dithiothreitol; TCA, trichloroacetic acid; PAGE, polyacrylamide gel electrophoresis. 63

0003-9861/87 Copyright All rights

$3.00

Q 1987 hy Academic Press, Inc. of reproduction in any form reserved.

64

LGPEZ-CASILLAS

In this communication, we report a detailed procedure for obtaining an mRNA preparation that is enriched in both ACC mRNA and fatty acid synthetase mRNA. This preparation of mRNA can be translated into apparent full-length protein in vitro and we have used this in vitro translation system to quantitate the amount of translatable ACC mRNA during lactation. MATERIALS

AND

METHODS

Chemicals. Guanidine thiocyanate was from Fluka A. G.; RNAse-free sucrose from BRL; oligo(dT)-cellulose type 7 from P-L Biochemicals; [3SS]-methionine (800 Ci/mmol) and EnaHance from New England Nuclear. Formaldehyde-fixed Staphylococcus aUreu.s cells (Pansorbin) were purchased from Calbiochem. Before use, Pansorbin was boiled in 3% SDS containing 10% @-mercaptoethanol, as described by Richert et al. (13) and suspended as a 10% solution (v/v) in NET buffer (50 mM Tris-Cl, pH 7.5, 0.15 M NaCI, 5 mM EDTA, 0.05% Nonidet-P40, 1 mg/ml ovalbumin, and 0.02% NaN3). Nuclease-treated rabbit reticulocyte lysate was prepared as described (14) and stored at -70°C. Animals. Wistar female rats from our department animal facility weighing 300 ? 10 g were used. Animals were kept on a fat-free diet for 24 h before they were killed. Antibodies. Preparation of rabbit antibody against rat liver ACC has been previously described (15). Fatty acid synthetase (FAS)-specific antiserum was a generous gift of Dr. A. Goodridge at Case Western Reserve University. Poly(A)+ RNA preparation. Total RNA was prepared from the mammary glands of lactating animals as described by Cathala (16) with the following modifications. The concentration of SDS was 1% in the solubilization buffer and solubilization of the initial precipitate was carried out at 37°C using a Dounce homogenizer for a period of 30-60 min. Poly(A)+ RNA was separated from the total RNA using oligo(dT)-cellulose as follows. Approximately 12 mg total RNA in 2 ml of 10 mM Tris-Cl, pH 7.5, containing 1 mM EDTA, was denatured by heating for 5 min in a boiling water bath, quickly chilled in an ice water bath, adjusted to 0.5 M KCl, and loaded onto an oligo(dT)-cellulose column (0.5 g dry wt) which had been equilibrated in the same buffer. After extensive washing with the same buffer, poly(A)+ RNA was eluted with 10 mM Tris-Cl, pH 7.5, containing 1 mM EDTA. Isokinetic mcrose gradient centrifugatim of p&y(A)+ RNA. Isokinetic sucrose gradients (5-20%)were prepared according to No11 (17). Buffers used and denaturation of RNA samples follow the methods described by MacLeod et al. (18).

ET

AL.

Fractionation of poly(A)+ RNA by agarose gel electrophoresis. Poly(A)+ RNA was subjected to agarose gel electrophoresis in the presence of methyl mercury hydroxide (19). The separated RNAs were then eluted from the gels using the IBI unidirectional electroeluter (International Biotechnologies, Inc.). The gels were cut into 5-mm segments and the gel pieces were soaked in 1X EEB (20 mM Tris-Cl, pH 8.0, 0.2 mM EDTA, 5 mM NaCI) containing 10 mM DTT, for 2 h at room temperature, before RNAs were eluted by the procedure described by the manufacturer. Elution by means of electrophoresis was performed at room temperature for 45 min at a constant voltage of 130 V. Eluted material was mixed with carrier tRNAs (40 fig) before phenol/chloroform extraction and precipitation by ethanol. In vitro translation of mRNA. In vitro translation mixtures contained the following in a final volume of 28 ~1: 20 pl of reticulocyte lysate, 10 &i of [?S]methionine, 2.5 nmol of each one of the other 19 amino acids, 0.25 pmol creatine phosphate, 2.5 Fmol KCl, 12.5 nmol MgCl,, 31.2 nmol DTT, and l-2 ~1 of the RNA (l-2 fig). Incubation was carried out at 30°C for 1.5 h. The reaction was stopped by the addition of 7 ~1 of 0.1 M methionine to the reaction mixture in an ice bath. Typically, 3 ~1 of the reaction mixture was assayed for TCA-precipitable radioactivity (total incorporation); 10 ~1 was used to examine the pattern of total translation products by SDS-polyacrylamide gel electrophoresis. The remaining reaction mixture was used in specific immunoprecipitation analyses. Immunoprecipitation and SDS-gel electrophoresis. Translation mixtures to be immunoprecipitated were incubated for 15 min at room temperature with 200 pl of NET buffer and 30 ~1 of Pansorbin in order to remove nonspecific binding materials. Antisera diluted in NET buffer were then mixed with the Pansorbintreated reaction mixture as indicated in the figure legends. This mixture was kept at room temperature for 2-4 h. After the addition of 50 ~1 of Pansorbin, the incubation was continued for 15 min. Pansorbin-antibody-antigen complexes were collected by centrifugation and washed three times with 500 ~1 of a solution containing 15 mM sodium phosphate, pH 7.4, 1 mM EDTA, 0.5 M NaCl, 0.5% Triton X-100, and 0.05% SDS. The precipitates were resuspended in 100 ~1 of SDS-PAGE sample buffer, (25 mM Tris-Cl, pH 8.0,2.5 ITIM EDTA, 2.5% SDS, 25% sucrose, 10% P-mercaptoethanol, and 25 pg/ml pyronin Y) and heated for 5 min in a boiling water bath. The supernatants containing the solubilized immunoprecipitates were obtained by centrifugation and subjected to SDSpolyacrylamide gel electrophoresis (20). For fluorography, gels were fixed in solution containing 10% isopropranol and 5% acetic acid, stained with Coomassie blue, destained, and treated with EnaHance as recommended by the manufacturer.

ACETYL-CoA

CARBOXYLASE

Dried gels were exposed to Kodak XAR5 at -70°C. Densitometric tracings were done in an E-C paratus Corp. densitometer.

mRNA

FROM

RAT

MAMMARY

GLAND

65

film Ap15

RESULTS

Preparaticna and in Vitro Translation of ACC m>RNA Lactating mammary glands were chosen for the purification of ACC mRNA because at the peak of lactation ACC constitutes about 0.4% of total soluble proteins in mammary gland tissues (21). This is about two-fold higher than the carboxylase content in livers of rats that have been starved and refed a fat-free diet; a nutritional scheme intended to stimulate hepatic lipogenie activities (22). Although we selected an experimental system that is known to be actively involved in fatty acid synthesis, experiments in which we attempted to identify AC!C mRNA by translating total and poly(A)+ RNA (1 to 3 pg RNA) followed by immunoprecipitation of the synthesized products did not reveal any ACC synthesis. Therefore, we proceeded to fractionate total poly(A)‘- RNA by size using isokinetic sucrose gradient centrifugation with the goal of obtaining a pool of poly(A)+ RNA that was enriched for ACC mRNA. In vitro translation of the fractions and immunoprecipitation of the synthesized proteins demonstrated that this process allows detection of the rare ACC mRNA (Figs. 1 and 2). In Fig 1, the relative sedimentation position of ACC mRNA in relation to the ribosomal RNAs is shown. Those fractions containing .ACC mRNA still contained a considerable amount of mRNA encoding smaller proteins, as evidenced by the total translation products of each fraction (lane T, Fig. 2). Immunoprecipitates obtained using anti-ACC serum revealed newly synthesized ACC bands (lane A, fractions 2-5, Fig. 2) with an estimated molecular weight of 260,000. When the mRNA for FAS was ex,amined using the specific antiFAS serum:, we found that the fractions containing ACC mRNA (lane A) also contained FAS mRNA (lane F). We further substantiated that the two mRNA species are similar in size by fractionating the

05

FRACTION

FIG. 1. Isokinetic sucrose gradient fractionation of rat mammary gland poly(A)+ RNA. Poly(A)+ RNA (250 rg) prepared from total RNA of lactating rat mammary gland (Day 6 postpartum) was separated in a 5-20s isokinetic sucrose gradient as described under Materials and Methods. Spectrophotometric readings at a wavelength of 260 nm were taken for each of the 0.5-ml fractions collected and corrected with readings obtained from a similarly fractionated control sample. Bars indicate fractions containing translatable ACC and FAS mRNAs.

partially purified mRNA preparation by electrophoresis in an agarose gel under denaturing conditions. After isolating the mRNA from various slices of the gel and translating the mRNA, we determined that both mRNA species comigrated (data not shown). As indicated by the migration patterns of the translation products (Fig. 2) the molecular weights of the in vitro synthesized ACC and FAS are very similar. Therefore, we wanted to verify that both antiserum preparations were specific for their respective enzymes The newly synthesized ACC that was labeled in the translation reaction was displaced from the immunoprecipitates by the addition of authentic ACC that was purified from rat liver (data not shown). In addition, we demonstrated that prior treatment of the translation mixture with anti-FAS serum did not affect

66

LGPEZ-CASILLAS

FIG. 2. Analysis of cell free translation products by SDS-gel electrophoresis. Poly(A)+ RNA (300 pg) obtained from lactating rat mammary gland (6th day postpartum) was separated as described in Fig. 1. Aliquots (l/25) of the first six bottom fractions collected were translated in vitro as described under Materials and Methods. After an aliquot from each fraction was taken for measurement of total incorporation, the translation reaction mixtures were divided into three identical portions. One portion was mixed with an appropriate amount of SDS-PAGE loading buffer for the examination of total translation product (T). The other two portions were pretreated with Pansorbin to remove nonspecific binding materials, and then immunoprecipitated with 1 ~1 of anti-ACC (enough to precipitate 0.45 pg of rat liver ACC) and with 7.5 ~1 of anti-FAS (titer unknown) as described under Materials and Methods. The anti-ACC and anti-FAS immunoprecipitates were run in lanes A and F, respectively, of the SDS-PAGE. The fluorograms, obtained after 3 days of exposure at -70°C are shown. Numbers at the top of the figure refer to sucrose gradient fractions as numbered in Fig. 1. Numbers in the left margin of the figure indicate the sizes of molecular weight standards in kilodaltons.

the amount of labeled ACC that was immunoprecipitated with anti-ACC serum (data not shown). Estimation Lactating

of ACC mRNA Content in Mammary Glands

Without knowing the factors that are involved in determining the stability and the differential translational efficiency of mRNAs, it is difficult to estimate the amount of recovery of ACC mRNA at different stages of the preparation. However, assuming that all the mRNAs are translated at the same efficiency, one can make a rough estimation of the amount of ACC mRNA in the preparation.

ET

AL.

The amount of RNA in fractions 2-5 obtained by sucrose gradient centrifugation corresponds to 1.3% of the total poly(A)+ RNA. Densitometric scanning analyses of the fluorograms indicate that radioactivity incorporated into the proteins larger than 200 kDa accounts for only 6% of the total de novo protein synthesis directed by mRNAs in these fractions. The scarcity of ACC and FAS mRNA is further demonstrated when the relative amounts of ACC and FAS synthesized were compared with the total translation products larger than 200 kDa. Taking sucrose gradient fraction No. 4 as an example, only 12% of the radioactivity present in the above 200-kDa band in lane 4T (Fig. 2) corresponds to ACC (lane 4A, Fig. 2). From these figures, we can estimate that the amount of translatable ACC mRNAs is 2.6 ng/mg of total RNA in the mammary gland at Day 6 of lactation. Through the procedure just outlined, we estimate that there is about 100 ng of translatable mRNA for ACC in 8 g of lactating rat mammary gland (Table 1).

TABLE

I

RECOVERY OF RNA AT DIFFERENT STAGES OF ENRICHMENT % of total Total RNA Poly(A)+ RNA Poly(A)+ RNA in fractions of sucrose gradient centrifugation Translatable acetyl-CoA carboxylase mRNA Translatable fatty acid synthetase mRNA

37.8 mg 1.2 mg

100 3.2

15.3 /lg

0.04

100 ng

0.0003

70 ng

0.0002

(2-5)

Note. Data represent the recovery of RNA during the enrichment procedure, as described under Materials and Methods, using the inguinal mammary glands (8 g) from a rat at Day 6 postpartum. Total RNA, poly(A)+ RNA, and RNA in sucrose gradient fractions were estimated by spectrophotometry at 260 nm (1 absorbance unit = 40 pg RNA/ml). Translatable mRNA levels were estimated from densitometric tracings of fluorograms of in vitro translation products and immunoprecipitates as discussed in the text.

ACETYL-CoA

CARBOXYLASE

mRNA

FROM

RAT

MAMMARY

GLAND

67

Effect of Lactation on the Level of Translatable ACC mRNA The analysis of in vitro translation products by immunoprecipitation with specific antiserum has provided, for the first time, an assay system to examine the level of functional mRNA for ACC. Therefore, we investigated the effect of lactation on the amount of functional ACC mRNA. Functional acetyl-CoA carboxylase mRNA increases about sevenfold at Day 5 postpartum and remains at high levels (Fig. 3). Accompany:ing studies into changes in total RNA revealed that the amount of extractable RNA per gram of tissue during the lactation period remains relatively constant at about 2 mg, except on Day 7 postpartum when the amount of total RNA increases significantly. However, the percentage of poly(A)+ and total RNA that is translatable in vitro in these RNA preparations is essentially the same (data not shown). DISCUSSION

It has been impossible for many investigators, including us, to obtain ACC mRNA preparations from any tissues that could be translated in vitro into full-length ACC. In the caseof mammary glands, these difficulties may have several causes: (a) rat mammary glands contain relatively high levels of ribonuclease (23,24) and high molecular weight mRNA is a prime target for such enzymes; (b) rat mammary glands contain high levels of mRNA for smaller milk proteins such as casein (25) and, in terms of protein synthesis capacity, a large mRNA such as that for ACC cannot compete efficiently with smaller mRNAs; (c) the relative amount of ACC mRNA is simply too smal.1. Our procedure for enriching ACC mRNA neither involves any new techniques nor does it explain the causes of past difficulties. However, it produces functional mRNA which can be translated into apparent full-length ACC in vitro. Using this translation system, we have been able to correlate the role of translatable mRNA accumulation in the synthesis of ACC during the lactation process. Pre-

01234567 CAYS POST-PARTUM

FIG. 3. Changes in the level of ACC mRNA during lactation. Lactating rats were sacrificed at the indicated number of days postpartum. Day 0 refers to full-term pregnancy. Total RNA and poly(A)+ RNA from inguinal mammary gland were prepared as described under Materials and Methods. All samples were processed at the same time under identical conditions. Poly(A)+ RNAs (275 pg each) were fractionated by sucrose gradient centrifugation as described in the legend to Fig. 1, and the fractions were translated in vitro and immunoprecipitated with anti-ACC as described in Fig. 2. The relative amount of ACC mRNA in each sample was determined by scanning the Auorograms of the translation products with a densitometer. The amount of translatable ACC mRNA is given in arbitrary units, using Day 0 as the unit of comparison.

viously, Numa and co-workers have shown that the amounts of nascent ACC polypeptide chains detected in rat liver polysomes correlated well with the increased level of ACC found in re-fed animals (11). Such results now could be interpreted as the increased capacity for acetyl-CoA carboxylase synthesis was due to the increased amount of specific ribosome population as a result of increased amount of the specific mRNA. Our results show directly for the first time that lactation-induced lipogenesis (21) actually involves an increase in the amount of translatable ACC mRNAs and therefore, seemingly reveals the importance of transcriptional control in this case. The data presented in Table I may seriously underestimate the amount of mRNA because of the lengthy process in-

68

LOPEZ-CASILLAS

volved in the final determination of the amount of functional mRNA versus the actual amount of mRNA. However, the relative increase in the mRNA population during the lactation period (Fig. 3) should provide a reasonable estimation as to the time course of active ACC mRNA accumulation as well as the relative degree of the mRNA increase. The molecular weight of ACC from rat liver (12) and mammary gland (26) is 260,000, whereas the molecular weight of FAS has been reported to be 230,000 in the rat mammary gland (27) and 250,000 in the rabbit mammary gland. The results from our in vitro translations also demonstrate that the molecular size of FAS is slightly smaller than ACC. However, our data demonstrate that the size of both mRNAs is about the same and this is an agreement with data obtained by Northern blot analysis using cDNA for each enzyme as a probe (28-30). The estimated size of each is approximately 10 kb. REFERENCES 1. NUMA,

S., AND

YAMASHITA,

C’urr.

S. (1974)

Top.

ET

VAGELOS,

M. D., Moss,

11.

12. 13. 14.

NAKANISHI, NUMA,

J., AND

POLAKIS,

S. E. (1974)

16.

MANIATIS, (1982)

D. G. (1980)

in Molecular

Aspects

17. 18. 19.

P. W.,

Chem.

21.

KILBURN,

E. (1969)

S.,

Biochem. 8. GIFFHORN, 221,343-350.

AND

NUMA,

J. T., AND PITOT, 12&X,15-20. A. W., FERGUSON,

S.

N. R. (1984)

C. (1982)

Eur.

ALBERTS,

K., HENNESSY,

MANTIAL,

J. A., AND

BAXTER,

2.329-335. Nature (London)

NOLL, H. (1967) 215,360-363. MACLEOD, A. R., KARN, J., AND BRENNER, S. (1981) Nature (l&,&m) 291,386-390. BAILEY, J. M., AND DAVIDSON, N. (1976) Anal

70,75-85.

LAEMMLI, 685. MACKALL,

U. K. (1970) J. C., AND

Nature

(LmLdon)

23.

LIN,

S., AND

FRITZ,

D. K.,

LIAO,

W.

227, 680-

M. D. (1977)

LANE,

S.

25.

26. 27.

B&hem. Eur.

(1970)

J.

P. J. (1977)

16,3361-3368.

LIU,

D. K., KULICK, D., AND WILLIAM, G. H. (1979) J. 178,241-244. HOBBS, A. A., RICHARD, D. A., KESSLER, D. J., AND

J.

ROSEN, J. M. (1982) J. Biol. Chem. 251, 35963605. AHMAD, F., ADMAD, P. M., PIERETTI, L., AND WATTERS, G. T. (1978) J. BioZ. Chem. 253,1733-1737. HARDIE, D. G., AND COHEN, P. (1978) Eur. J.

B&hem.

J.

J.

Biochem. 10.

Labora-

LEE, K-H., AND KIM, K-H. (1977) J. Biol. Chem, 252.1748-1751. CATHALA, G., SAVOURET, J-F., MENDEZ, B., WEST,

29.

ZEHNER WAKIL,

S., AND

30.

92,25-34. Z. E., MATTICK, J. S., STUART, R. AND S. J. (1980) J. Biol. Chem. 255, 9519-

9522. NEPOKROEFF,

Biol. H.

Cold Spring Harbor Harbor, NY.

NUMA,

J. Biol.

B&hem.

S., AND

Sci. USA 73,

22.

of Cel-

Eur.

(1970)

Acad

J. 162,635-642. NAKANISHI, S., AND B&hem. 16,161-173.

28.

16,161-173. S., AND KATZ,

T., HARIKAWA,

NatL

B&hem.

244,6254-6262.

7. NAKANISHI,

9. SPENCE,

AND

pp. 344-347, Cold Spring

B&hem. 20.

24.

lular Regulation (Cohen, P., Ed.), Vol. 1, pp. 3362, Elsevier, Amsterdam. 5. WITPERS, L., MORIARITY, D., AND MARTIN, D. B. (1979) J. Biol Chem. 254,6644-6649. 6. MAJERUS,

249,5241-

T., FRITSCH, E. F., AND SAINBROOK, J. Molecular Cloning. A Laboratory Man-

B. L., KARIN, M., J. D. (1983) DNA

Cum: 3. KIM, 4. HARDIE,

Chem.

2304-2307. SONG, S. C., AND KIM, K.-H. (1981) J. Biol. Chem. 256,7786-7788. RICHERT, N. D., DAVIES, P. J. A., JAY, G., AND PASTAN, I. H. (1979) J. Viral 31, 695-706.

ual, tory, 15.

S., TANABE, S. (1976) Proc.

Biochemistry

Top. Cell. Reg. 8,139-187. K.-H. (1983) Curr. Top. Cell Reg. 22,143-176.

J. Biol.

P. R. (1974)

5249.

Cell. Reg. 8, 197-246. 2. LANE,

AL.

BACK, R.

C. M.,

Chem.

AND

PORTER,

J. W.

(1978)

J.

253,2279-2283.

D. W., GOLDMAN, M. J., FISCH, J. E., OCHS, S., AND GOODRIDGE, A. G. (1986) J. Biol.

Chem.

261,4190-4197.