THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1984 by The American Society of Biological Chemists, Inc. Vol. 259, No. 4, Issue of February 25, pp. 2452-2456, 1984

Printed in U. S. A .

(Received for publication, September 6, 1983)

Mark C. BlaufussS, Jeffrey I. Gordon Q l l l l , Gustav SchonfeldQ**, Arnold W. StraussSllSS, and David H. AlpersQ From the Departments of $Pediatrics, §Medicine, TBiological Chemistry, and **Preventive Medicine, Washington University School of Medicine, St. Louis, Missouri 63220

We have examined the biosynthesis of rat apolipo- protein C-111 in the small intestine and liver. The pri- mary translation product of its mRNA was recovered from wheat germ and ascites cell-free systems. Com- parison of its NH2-terminal sequence with the NH2 terminus of plasma high density lipoprotein-associated apolipoprotein C-I11 showed that apo-(3-111 was ini- tially synthesized as a preprotein with a 20 amino acid long NH2-terminal extension: Met-X-X-X-Met-Leu- Leu-X-X-Ala-Leu-X-Ala-Leu-Leu-Ala-X-Ala-X-Ala. Co-translational cleavage of the cell-free translation product by signal peptidase generated a polypeptide with the same NH2 terminus as the mature protein (X-Glu-X-Glu-Gly-Ser-Leu-Leu-Leu-Gly-Ser-Met). Therefore, this apolipoprotein does not undergo post- translational proteolytic processing like two other high density lipoprotein-affiliated proteins, proapo-A-I and proapo-A-11.

The mRNA encoding apolipoprotein C-I11 comprises 0.4% of the translatable RNA species in adult rat liver and 0.14% of the translatable RNA species in small intestinal epithelium. Acute fat feeding with a triglyc- eride meal resulted in a 2-fold increase in intestinal preapo-C-I11 mRNA accumulation but no change in the levels of preproapo-A-I mRNA. Thus, the acute re- sponse of the apo-A-I and C-I11 genes to triacylglycerol absorption differs.

The C-apolipoproteins are a group of proteins which in plasma are associated with both the triglyceride-rich lipopro- teins, namely very low density lipoproteins and chylomicrons. They are less well represented in HDL’ (1-3). These low molecular weight polypeptides appear to be actively ex- changed between HDL and triglyceride-rich lipoproteins. During fat absorption, the C-apoproteins move from HDL to chylomicrons and the reverse process occurs as chylomicrons are catabolized (4, 5).

The most abundant C-apolipoprotein is apo-C-111 (3, 6-8). Human apo-C-I11 has M , = 8240 and contains 79 amino acids (9). Rat apo-C-I11 is larger with an estimated M , = 10,000 (10). Its sequence has not been determined. Rat and human

*This work was supported by Grants AM 14038, 07130, 07033, 30’292, and 20407 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

11 A Fellow of the John A. and George L. Hartford Foundation. $$ Established Investigator of the American Heart Association.

__ ~-

The abbreviations used are: HDL, high density lipoproteins; apo, apolipoprotein; SDS, sodium dodecyl sulfate.

apo-C-I11 have different NH2-terminal amino acids (aspartic acid uersus serine, respectively) as well as COOH-terminal amino acids (proline uersus alanine). Human apo-C-111 lacks cysteine and isoleucine, whereas rat apo-C-111 lacks cysteine and histidine (9, 10).

Human apo-C-111 is an 0-linked glycoprotein with carbo- hydrate attachment occurring at the threonine residue situ- ated a t position 74 (9). Apo-C-111 occurs as three isoforms. These differ according to whether they contain 0, 1, or 2 molecules of sialic acid at the terminal position of the carbo- hydrate chain, each chain consisting of galactose, mannose, and galactosamine (11). Rat apo-C-111 has been observed in two isoforms, one with no sialic acid or hexosamine (apo-C- 111-0) and the other major form (apo-C-111-3) with 1 residue of galactosamine and three of sialic acid/molecule (10).

Apo-C-I11 appears to have an important role in the metab- olism of triglyceride-rich lipoproteins. It inhibits apo-C-11’s activation of lipoprotein lipase and prevents the uptake of apo-E-containing lipoproteins (e.g. chylomicron remnants) by liver cells (12-14).

Interest in apo-(2-111 has been increased recently by a report of two sisters with severe, premature atherosclerosis, who were found to have no detectable plasma apo-A-I or apo-C- 111 (15). Antisera to normal apo-A-I absorbed a 45,000-Da polypeptide from the plasma of these patients (16). Southern blot analyses of their DNA disclosed a 7.5-kilobase insertion at the 3‘ end of the second intron of the A-I gene (17, 18). These same workers have shown that (a ) the A-I and C-111 genes are linked in normal individuals as well as these affected patients ( b ) that the C-111 gene which is situated approxi- mately 2.6-kilobases downstream from the apo-A-I gene is transcribed on the opposite strand as the A-I gene, and (c) that a restriction site polymorphism previously described at the 3’ end of the apo-A-I gene (19) and associated with hypertriglyceridemia is probably due to a single base pair change in the 3‘ noncoding portion of apo-C-I11 mRNA (20).

The structure of the primary translation product of apo-C- I11 RNA has not been described nor has its subsequent pro- teolytic processing. We have undertaken a study of rat apo- C-I11 biosynthesis in order to ascertain if the processing of its primary translation product could provide insights into its interactions with HDL and very low density lipoprotein. In addition, we have examined the effects of acute triacylglycerol feeding on apo-C-I11 gene expression to begin to understand those physiologic stimuli which modulate apo-C-111 gene expression.

EXPERIMENTAL PROCEDURES

Materials Adult Sprague-Dawley rats (150-200 g) and 14-day-old suckling

rats were purchased from Chappel Breeders, Bel Ridge, MO. Labeled

2452

by guest on May 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Rat Preapo-C-III Biosynthesis

amino acids of the highest specific activity available were obtained from Amersham Corp. ('H-amino-acids) and from New England Nuclear ([:%]methionine).

Methods

NH2-terminal Sequence Analysis of HDL-associated Apo-C-III- The procedures used for initial isolation of mature, rat apo-C-111-3 from serum lipoproteins have been described previously by our labo- ratories (21 ). For NH,-terminal sequence analyses, apo-C-111-3 was further purified by electrophoresis through a 20% polyacrylamide gel containing SDS (0.1%, Ref. 22), passively eluted in the presence of the serine protease inhibitors phenylmethylsulfonyl fluoride (1 mM) and benzamidine (1 mM), and subjected to Edman degradation using a 0.33 M Quadrol program and a Reckman 890C sequenator (23) . Phenylthiohydantoin-amino acid derivatives from each cycle of cleav- age were identified by high performance liquid chromatography (24).

Preparation of Total Cellular RNA from Rat Intestinal Mucosa and LiLw-Total cellular RNA was isolated from these tissues using guanidine HCI (24,25). Adult rats were maintained on a standard rat chow diet (Ralston Purina) for the steady state experiments hut were fasted overnight prior to acute fat feeding. Suckling rats were killed within 3 h after removal from their mother. After decapitation and exsanguination, the liver and entire length of small intestine were quickly removed from these suckling animals, rinsed with chilled NaCl(O.154 M), mechanically disrupted with a Polytron homogenizer, and extracted with 8 M guanidine HCI, 20 mM NaAc, pH 5.0 (25). RNA isolated in this fashion had an A2Wnm/Amnm of 1.7-1.8 and was shown to be intact he electrophoresis under denaturing conditions with methylmercuric hydroxide (26). The yield of RNA of small intestinal epithelium was 30-40A2~nm units/gand 40-50A2~.,units/ g of liver.

Edman Degradation of the Intact and Co-translationally Cleaved Product of Apo-C-III mRNA Translation-Total cellular RNA from either liver or intestinal epithelium was translated in wheat germ lysates at a final concentration of 300 pg/ml in the presence of 1 radiolabeled and 19 unlabeled amino acids (25). Co-translational cleavage experiments were done with a fractionated ascites cell-free system that contained ascites microsomes possessing signal peptidase activity ( 2 7 ) . The intact or co-translationally cleaved apo-C-Ill poly- peptide was recovered from the cell-free systems by immunoprecipi- tation with monospecific antisera generated from apo-C-111-3 (21, 28). Procedures for immunoprecipitation have been described in detail by us in other publications (25,29). The only modification introduced for apo-C-Ill immunoadsorption was that the translated proteins were boiled in 1.5% SDS for 5 min prior to antibody addition. Following immunoprecipitation, the cell-free product was further purified by electrophoresis through discontinuous SDS-polyacrylam- ide gel, passively eluted in the presence of serine protease inhibitors (see above), combined with 100 nmol of carrier myoglobin, and subjected to automated sequential Edman degradation using the same

c-Ill. Quadrol program and sequenator that we used for plasma HDL-apo-

RESULTS

Since apo-C-I11 is a secreted protein, we assumed that it would be made as a larger precursor containing an NH,- terminal signal peptide (30). To test this assumption, we compared the NH2-terminal sequences of the primary trans- lation product and mature plasma HDL-associated protein. The primary translation product of apo-C-111 mRNA was identified by immunoprecipitation of the polypeptide from wheat germ lysates programmed with total cellular rat intes- tinal epithelial and liver RNA. Fig. 1 demonstrates that a single protein was immunoadsorbed by our monospecific anti- sera and that the mobility of the cell-free product was iden- tical whether encoded by liver or intestinal mRNA (compare Fig. 1, lanes 2 and 3) . This protein was not observed when purified plasma apo-C-111 was added to the immunoprecipi- tation mixture (Fig. 1, lane 5 ) . The estimated mass of this protein was 12,000 or 2,000 daltons greater than the reported mass of rat plasma apo-C-111 (10).

The NH,-terminal sequence of the intact liver primary translation product was determined by Edman degradation of

4 3 kDa

3OkDa

2lkDa 14kDa

1 2453

2 3 4 5

,pre Apo C I I I

FIG. 1. Wheat germ cell-free translation of rat intestinal and liver RNA. [R5S]Methionine-labeled polypeptides synthesized in uitro were separated by electrophoresis through a discontinuous 20% polyacrylamide slab gel containing SDS (22). A fluorograph of the gel is shown. Lane I , proteins encoded by total intestinal RNA. Lune 2, polypeptides obtained when the mixture in lane I was im- munoprecipitated with anti-apo-C-111 antiserum. Lane 3, polypep- tides obtained when the mixture in lane 4 was immunoprecipitated with anti-apo-C-I11 antiserum. Lane 4, proteins encoded by total liver RNA. Lane 5, immunoprecipitation of the products of lane 4 plus 10 pg of unlabeled plasma apo-C-111 with anti-apo-C-I11 antiserum. The position of migration of plasma HDL-associated apo-C-111-3 is indi- cated.

IO

(L, I *-IC., 51 a,

IO 20 30 CYCLE NUMBER

FIG. 2. NHz-terminal sequence analysis of preapo-C-111 synthesized in the wheat germ cell-free translation system. The primary translation product of apo-C-111 mRNA was labeled with ['Hlalanine, ["S]methionine, or ['HJleucine, purified by immu- noprecipitation and gel electrophoresis, and subjected to automated sequential Edman degradation. Loadings for sequencer runs included 6000 dpm of ['Hlleucine apo-C-Ill, 12,000 dpm of ["Hlalanine apo- C-111, and 6,000 dpm of ["S]methionine apo-C-Ill. The per cent recovery (-) is a hypothetical curve based on 96% repetitive yield and normalized to the first radioactive peak. The bottom panel in- cludes the sequence of plasma apo-C-111.

the radiolabeled protein. The results are shown in Fig. 2. Methionine residues were noted at positions 1,5, and 32. The yield of ["S]methionine at position 1 was 65% of the calcu- lated input disintegrations/min/residue which suggested that

by guest on May 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

2454 Rat Preapo-C-111 Biosynthesis

this residue was the initiator methionine and that we had recovered the intact primary translation product. The distri- bution of methionines in the NH, terminus of the cell-free product was identical whether this polypeptide was translated from liver or intestinal apo-C-I11 mRNA (data not shown). These sequence data were compared to those generated from the NH, terminus of plasma HDL-apo-C-111. No amino acids could be assigned to positions 1 and 3 of the plasma protein, in part because of the large amount of glycine coeluted from the final preparative SDS-polyacrylamide gel (Table I). How- ever, of the 16 positions given amino acid assignments, 9 were identical with those present in human plasma apo-C-I11 (Ta- ble 11). Table I1 also shows the overlap between the primary translation product and mature rat plasma protein. As an example, the leucine residues at positions 7 , 8, and 9 of the mature protein align perfectly with the leucine residues found

TABLE I NH2-terminal sequence analysis of rat plasma HDL-associated apo-

e-III ~ ~~ .. . ~-~ ~~~. . " . _ _ ~ _ ___"~

Cycle E G S L M Q Assigned number residues

"~ - ~- __ ~ _ _ ~ ~ ~ _ _ _ _ pmol phenylthiohydantoin amino acld recvoeredlcycle"

1 158 51,7906 -

2 27,895 3 105 2,085 4 1,445 5 324 (3,ooo1

7 (1352( 8 113351 9 rn

6 325

" lsi4) 14 b

15 / 9 0 8 / Y 16 m M I T E 18 imJ Q

Edman degradation of 5 nmol of apo-C-I11 was performed as described under "Exuerimental Procedures." Following conversion of

~- ~- __ _ _ ~ _ _ ~ __ ~

anilinothiazolinones.to phenylthiohydantoins, amino acids were iden- tified by high performance liquid chromatography. The recovery of each phenylthiohydantoin-amino acid was quantitated by comparing peak areas to that produced by known amounts of phenylthiohydan- toins-amino acid standards.

The large quantity of glycine recovered in these cycles is due to free glycine present in the gel purified sample.

Less than 100 pmol of phenylthiohydantoin amino acid was detected.

Amino acid assignments for each cycle or Edman degradation are indicated by a rectangle.

at positions 27, 28, and 29 of the polypeptide made in vitro. Similar overlap exists between the methionine at position 32 of the cell-free product and the methionine at position 12 of the plasma protein. From these areas of overlapping sequence we concluded that the primary translation product recovered by immunoprecipitation was a precursor of apo-C-I11 that contained a 20 amino acid long NH2-terminal extension.

The COOH-terminal amino acid of this 20 amino acid long sequence was alanine, an amino acid with a small aliphatic side chain that is frequently noted at the COOH terminus of signal segments (31). However, in order to prove that the entire 20 amino acid oligopeptide functioned as a signal se- quence, we performed a series of co-translational cleavage experiments. For these studies we used a fractionated ascites cell-free system that is known accurately and efficiently to remove the signal peptides from a wide variety of pre- and preproproteins (27,32). The results of these studies are shown in Figs. 3 and 4. Co-translational addition of ascites micro- somes containing signal peptidase activity resulted in a re- duction in the size of the apo-C-I11 primary translation prod- uct from 12,000 to 10,000 (Fig. 3). This cleavage was less efficient than that of preproapo-A-I (compare lunes 4 and 5 with 2 and 3 of the figure). A partial NHn-terminal sequence of the co-translationally cleaved, radiolabeled, C-I11 polypep- tide was determined by Edman degradation (Fig. 4). Residues 12 and 16 were identified as methionine and residues 6 and 11 as serine. These assignments correspond precisely to their respective positions in the mature molecule (see Table 11). We therefore concluded that apo-C-I11 is synthesized as a preprotein, does not contain a prosegment, and therefore does not undergo additional post-translational NHz-terminal pro- teolysis.

Having identified the primary translation product of apo- C-I11 mRNA, we were able to utilize the cell-free system to estimate the abundance of this mRNA in intestinal and liver RNA. To do this, the incorporation of three labeled amino acids (methionine, leucine, and alanine) into the cell-free product was expressed as a percentage of total protein syn- thesis in the wheat germ lysates (25). Apo-C-111 mRNA rep- resented 0.4% f 0.1 of the total translatable mRNA species in adult liver and 0.14% + 0.04 of the translatable mRNAs in total small intestinal epithelium. These values reflect the relative distribution of this mRNA sequence after long term feeding with a standard rat chow diet. Since apo-C-111 appears to be intimately involved in the metabolism of triglyceride- rich lipoproteins, we examined the effects of triacylglycerol absorption on intestinal apo-C-I11 accumulation. Changes occurring with acute feeding were analyzed. Total cellular RNA isolated from the entire length of small intestinal epi- t,helium was pooled from three animals a t each time point examined and used to program wheat germ lysates. Transla- tions were done on three separate occasions for each time point and the results evaluated using a pairwise comparison

TABLE I1 NH2-terminal sequence analysis of apo-C-III

~ . _ _ _ _ _ ~ -~

-20 . . . . - 1 5 . . . . - l o . . . .-5 . . . . 1 . . . 5 . . . . 1 0 . . . . 1 5 . . . Ratpreapo-C-111' Ra tHDLapo-C- I I Ib HumanHDLapo-C-111'

C o t r a n s l a t i o n a l c l e a v a g e s i t e I -Prepeptide Plasma protein-. . .

Determined by Edman degradation of the radiolabeled cell-free translation product. Determined by sequencing purified rat plasma HDL-associated apo-C-111.

~ - ~- .______

E As given in Ref. 9.

by guest on May 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Rat Preapo-C-III Biosynthesis 2455

1 4

43kDa-l I

30 kDa.

21 kDa

1 4 kDa,

2 3 4 5

,prepro A p o A I ' \p ro Apo A I

,preApo Clll " A p o C I I I

FIG. 3. Co-translational cleavage of preapo-C-111 and pre- proapo-A-I by ascites membranes. Total liver RNA was trans- lated in the mouse ascites tumor cell-free system in the presence and absence of ascites membranes (27). The ["S]methionine-labeled pep- tides were electrophoresed through a 20% polyacrylamide gel contain- ing SDS (0.1%). Lune I , translation of total liver RNA (15 pg/,iO p1 of reaction); lane 2, immunoprecipitate of lane 1 with rabbit antiserum to rat apo-C-111; lane 3 , immunoprecipitation with antiserum to rat apo-C-I11 after co-translational addition of ascites membranes; lanr 4 , immunoprecipitation of products shown in lane 1 with rabbit antiserum to rat apo-A-I; lane.5, immunoprecipitation with antiserum to rat apo-A-I after co-incubation with ascites membranes.

CYCLE NUMBER

FIG. 4. NH2-terminal amino acid sequence analysis of co- translationally cleaved apo-C-111. Total liver RNA was translated in the mouse ascites tumor cell-free system in the presence of ascites membranes, [:3iS]methionine and ["Hlserine. Immunoreactive apo-C- 111 was further purified by gel electrophoresis and subjected to auto- mated sequential Edman degradation. Top, [RsH]methionine-labeled co-translationally cleaved apo-C-I11 (2,000 dpm applied to the se- quenator cup). Bottom, [:3H]serine-labeled co-translationally cleaved apo-C-I11 (5,000 dpm). The bottom panel shows the sequence of rat plasma HDL-associated apo-C-111.

of least squares means (33). There was a 2-fold increase in the percentage of translatable apo-C-111 mRNA 4 h after corn oil feeding through a gastric tube (0.14-0.27%). This differ- ence was statistically significant ( p < 0.02) compared to the 0 and 1 h values. This response paralleled the increase pre- viously noted for apo-A-IV mRNA (Table I11 and Ref. 34). Translatable apo-A-I mRNA levels did not change in a statis- tically significant fashion during acute fat feeding (Table 111).

TABLE 111 Effect of triacylglycerol feeding on translatable intestinal preapo-C-

111, prrproapo-A-I, and prrapo-A-IV mRNA concentration Total cellular intestinal RNA was isolated at various times after

corn oil feeding groups of rats. Preapo-C-I11 and prepro-A-I mRNA levels were determined by purifying the [RsS]methionine-labeled pri- mary translation products from wheat germ lysates. The results were analyzed from three separate experiments using a pairwise compari- son of least squares means ( 3 3 ) .

Time after fat feeding Preapo-C-111 Preproapo-A-I Preapo-A-IV"

mRNA "

- ~ .

h % total, leavt squares mean

0 0.14 1.2 0.62 1 0.14 2

0.83 0.67 0.19 0.8 0.90

4 0.27' 1.13 1.3P 6 0.18

- . . ~ . ~~

1.1 1 .0

Preapo-A-IV data previously reported (34) using the same mRNA preparation. ' 2 Tail p value < 0.02 compared to 0 and 1 h.

These measurements average apolipoprotein mRNA accu- mulation throughout the entire length of small intestine. Greater alterations in mRNA concentration may occur in different segments of small bowel during acute triacylglycerol feeding.

Intestinal apo-C-I11 mRNA levels in suckling animals were compared to values observed in adults. The suckling rat is on a 13% fat diet (35) as opposed to the standard rat chow diet which contains 4% fat. mRNA encoding apo-C-I11 in suckling rat duodenum was increased 3-fold over steady state adult rat levels (0.17 uersus 0.06%).

DISCUSSION

We have analyzed the biosynthesis of apo-C-111 in rat small intestine and liver. Previous studies have shown that while very low density lipoprotein-apo-C incorporated radioactive amino acids during hepatic perfusion, chylomicron-apo-C iso- lated from intestinal lymph contained relatively little label (36). The role of the intestine in apo-C production was not certain from these studies since plasma proteins can be fil- tered into lymph. Apo-C-I11 has been detected within enter- ocytes by immune localization techniques (21). However, bio- synthesis of C-apoproteins has not been conclusively dem- onstrated in enterocytes. Our analysis of the products of intestinal epithelial mRNA translation provide evidence that apo-C-I11 is produced in the enterocyte.

Wu and Windmueller (36) used in vivo pulse labeling to show that approximately 5% of the C-apolipoproteins are synthesized in the intestine. The intestinal contribution to the plasma apo-C-111-0 was about 7% while at least 90% of apo-C-111-2 and 99% of apo-C-111-3 were derived from liver. Our in vitro estimate of the apo-C-I11 mRNA content of these tissues supports Wu and Windmueller's in vivo studies of their relative contribution to the total plasma apo-C-I11 pool. In adult rats, the liver weighs four times more than small intestinal mucosa (8 versus 2 g for 250-g animals) and yields one and one-half times the total RNA/g of tissue. The content of translatable preapo-C-111 mRNA in liver and gut after fat feeding can be calculated by multiplying concentration (0.4 and 0.17%, respectively) by a factor of 6 (4 for weight and 1.5 for yield of RNA). The ratio of liver to gut apo-C-I11 mRNA content becomes 14. Based on this calculation and assuming equal secretory rates, the intestine should supply about 7% of the total plasma apo-C-111.

The primary translation product of apo-C-111 mRNA is a preprotein that contains a 20 amino acid signal peptide.

by guest on May 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

2456 Rat Preapo-C-111 Biosynthesis

Although the sequence data are incomplete, the organization of the apo-C-I11 signal peptide is typical of other presegments. It possesses a high concentration of hydrophobic amino acids in its central domain (residues -15 to -3) and ends with a COOH-terminal alanine. Alanine is one of five amino acids that are commonly encountered at the signal peptidase cleav- age site, the others being glycine, cysteine, threonine, and serine (31). The presegment of apo-C-I11 shows less than 10% homology with the prepeptides of apo-A-I and A-IV which are a t least 59% homologous (37). The functional and evolu- tionary implications of these observations are not known.

Regulation of apo-C-I11 biosynthesis would appear to be important to the metabolism of triglyceride-rich lipoproteins since it serves both as regulator of lipoprotein lipase and chylomicron uptake (12, 13). In this light, it is intriguing that triacylglycerol feeding acutely increased apo-C-I11 mRNA ac- cumulation in the small intestinal epithelium by 2-fold within 4 h.

There have been no studies published which compare the rate of synthesis of apo-A-I and apo-C-111. Net apolipoprotein production (tissue and medium) by jejunal explants in vitro has been determined (28). Apo-C-I11 production increased by 2-fold 4 h after addition to oleic acid. These increases observed in vitro correspond exactly with increases in translatable apo- C-I11 mRNA observed in oivo using fat fed animals. However, the apo-A-I content of explant tissue and medium rose by 2.8- fold after oleic acid addition. No increase in translatable preproapo-A-I was found after acute fat feeding (Table 111). Studies using explants showed that apo-A-I secretion was increased 2-fold after fat addition while apo-C-I11 secretion did not change. Taken altogether these data suggest that regulation of apo-C-I11 and apo-A-I synthesis is not always coordinate.

It is not known if the rat apo-A-I and C-I11 genes are closely linked and convergently transcribed as in humans (20). How- ever, our observations imply that there is a difference in the acute response of the rat apo-C-I11 and A-I genes to triacyl- glycerol absorption. The effects of longer term dietary manip- ulations on apo-A-I, A-IV, and C-I11 accumulation are cur- rently under investigation.

Acknowledgments-We wish to thank Drs. I. Boime and S. Lentz for their assistance in setting up the ascites co-translational cleavage system, Phillip Miller for statistical analyses, and S. Winkler, S. Silverman, and C. Camp for typing the manuscript.

REFERENCES

1. Alaupovic, R. (1971) Atherosclerosis 13, 141-146 2. Kashyap, M. L., Srivastava, L. S., Chen, C. Y., Perisutti, G.,

Campbell, M., Lutmer, R. F., and Glueck, C. J. (1977) J . Clin. Inuest. 60, 171-180

3 . Schonfeld, G., George, P. K., Miller, J., Reilly, P., and Witztum, J. (1979) Metab. Clin. Exp. 28, 1001-1010

4. Havel, R. J., Kane, J. P., and Kashyap, M. L. (1973) J. Clin. Inuest. 52, 32-38

5. Kashyap, M. L., Barnart, R. L., Srivastava, L. S., Perisutti, G., Alden, C., Hogg, E., Glueck, C. J., and Jackson, R. L. (1983) Am. J . Clin. Nutr. 37 , 233-243

6. Catapano, A. L. (1980) Atherosclerosis 35, 419424 7. Kane, J. P., Sata, T., Hamilton, R. L., and Havel, R. J . (1975) J.

8. Brown, W. V., Levy, R. I., and Frederickson, D. S. (1970) J. Bwl.

9. Brewer, H. B., Jr., Shulman, R., Herbert, P., Roman, R., and

10. Herbert, P. N., Windmueller, H. G., Bersot, T. P., and Shulman,

11. Vaith, P., Assmann, G., and Uhlenbnick, G. (1978) Biochim.

12. Brown, W. V., and Baginsky, M. L. (1972) Biochem. Biophys.

13. Windler, E., Choo, Y., and Havel, R. J. (1980) J . Biol. Chem.

14. Quarfordt, S. H., Michalopoulos, G., and Schirmer, B. (1982) J. BWL. Chem. 257,14642-14647

15. Norum, R. A., Lakier, J. B., Goldstein, S., Angel, A., Goldberg, R., Block, W., Nottze, 0. K., Dolphin, P. J., Edelglass, J., Bogorad, D. D., and Alaupovic, P. (1982) N. Engl. J . Med. 306,

16. Zannis, V. L., Karathanasis, S. K., and Breslow, J. L. (1983) Fed.

17. Karathanasis, S. K., Norum, R. A., Zannis, V. L., and Breslow,

18. Karathanasis, S. K., Zannis, V. I., and Breslow, J. L. (1983) Fed

19. Rees, A., Shoulders, C. C., Stocks, J., Galton, D. J., and Baralle,

20. Karathanasis, S. K., McPherson, J., Zannis, V. I., and Breslow,

21. Schonfeld, G., Grimme, N., and Alpers, D. (1980) J. Cell. Bid.

22. Laemmli, U. K. (1970) Nature (Lond.) 227,680-685 23. Thomas, K. A., Silverman, R. E., Jeng, I., Baglan, N. C., and

Bradshaw, R. A. (1981) J. Bid. Chem. 256,9147-9155 24. Deeley, R. G., Gordon, J . I., Burns, A. T. H., Mullinix, K. P.,

Bina-Stein, M., and Goldberger, R. F. (1977) J. Bwl. Chem.

25. Gordon, J . I., Smith, D. P., Andy, R., Alpers, D. H., Schonfeld,

26. Bailey, J. M., and Davidson, N. (1976) Anal. Biochem. 70,75-85 27. Hortin, G., and Boime, I. (1980) Proc. Natl. Acad. Sci. U. S. A.

77,1356-1360 28. Alpers, D. H., Lancaster, N., and Schonfeld, G. (1982) Metab.

Clin. Exp. 31 , 784-790 29. Gordon, J . I., Sims, H. F., Lentz, S. R., Edelstein, C., Scanu, A.

M., and Strauss, A. W. (1983) J. Biol. Chem. 258, 4037-4044 30. Blobel, G., and Dobberstein, B. (1975) J. Cell Bid. 67, 835-862 31. Austen, B. M. (1979) FEBS Lett. 103,308-313 32. Szczesna, C., and Boime, I. (1978) Proc. Natl. Acad. Sci. U. S. A.

33. Helwig, C. J. T., and Council, K. A. (eds) (1979) SAS Users Guide,

34. Gordon, J. I., Smith, D. P., Alpers, D. H., and Strauss, A. W.

35. Altman, P. (1961) in Blood and Other Bodily Fluids (Dittmer, D.

36. Wu, A.-L., and Windmueller, H. G. (1979) J. BWl. Chem. 2 5 4 ,

37. Gordon, J. I., Smith, D. P., Alpers, D. H., and Strauss, A. W.

C h . Invest. 56, 1622-1634

Chem. 245,6588-6594

Wehrly, R. (1974) J. Biol. Chem. 249,4975-4984

R. S. (1974) J. Bwl. Chem. 249,5718-5724

Biophys. Acta 541,234-240

Res. Commun. 46,375-381

255,5475-5480

1513-1518

Proc. 42, 1816

J. L. (1983) Nature (Lond.) 303, 718-720

Proc. 42, 1817

F. (1983) Lancet 1, 444-446

J. L. (1983) Nature (Lond.) 304, 371-373

86,562-567

252,8310-8319

G., and Strauss, A. W. (1982) J. Biol. Chem. 257, 971-978

73,1179-1183

SAS Institute, Raleigh, NC

(1982) Biochemistry 2 1 , 5424-5431

S., ed) FASEB, p. 416

7316-7322

(1982) J. Biol. Chem. 257,8418-8423

by guest on May 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

M C Blaufuss, J I Gordon, G Schonfeld, A W Strauss and D H AlpersBiosynthesis of apolipoprotein C-III in rat liver and small intestinal mucosa.

1984, 259:2452-2456.J. Biol. Chem. 

  http://www.jbc.org/content/259/4/2452Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/259/4/2452.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on May 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from