studies on the synthesis of sterol carrier protein-2 in rat

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1987 by The American Society of Biological Chemists, Inc

Val. 262, No. 8, Isaue of March 15, pp. 3713-3717,1987 Printed in U.S.A.

Studies on the Synthesis of Sterol Carrier Protein-2 in Rat Adrenocortical Cells in Monolayer Culture REGULATION BY ACTH AND DIBUTYRYL CYCLIC 3‘,5’-AMP*

(Received for publication, September 9, 1986)

Wieslaw H. Trzeciak,O** Evan R. Simpson,“ Terrence J. Scallen,‘ George V. Vahouny? and Michael R. Waterman”.’ From the “Cecil H. and Ida Green Center for Reproductive Biology Sciences and the Department of Biochemistry, The University of Texas Health Science Center, Dallas, Texas 75235, the ‘Department of Biochemistry, School of Medicine, The University of New Mexico, Albuquerque, New Mexico 87131, and the dDepartment of Biochemistry, George Washington University, Washington, D. C. 20037

The effects of ACTH or dibutyryl cyclic AMP (Bt2 CAMP) on the synthesis of sterol carrier protein-2 (SCP2) have been studied in rat adrenocortical cells in monolayer culture. Radiolabeling of total cellular proteins with [3SS]methionine and immunoprecipitation with antibodies directed against rat liver SCP2, followed by polyacrylamide gel electrophoresis and fluorography, showed a 3-4-fold increase in the rate of synthesis of SCPz in cells treated for 48 h with ACTH (1 JLM) or BtzcAMP (0.1 mM). The induction of SCP2 synthesis depended upon the concentrations of ACTH or Bt2cAMP with an ED6o of 8 and 100 nM, respectively, and increased linearly with time between 12 and 48 h of treatment. Immunoprecipitation of SCP2 synthesized in a rabbit reticulocyte in vitro translation system programmed with RNA isolated from cells treated with ACTH or BtzcAMP revealed increased synthesis of SCP2 compared to RNA from control cells. The immunoprecipitable rat adrenal SCP2, synthesized in a cell-free translation system, showed mobility cor- responding to M, of 14,400 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was clearly larger than immunodetectable SCPz synthesized in cultured adrenal cells (Mr = 11,300). The electrophoretic mobilities of rat liver SCPz synthesized in cultured cells and in a cell-free translation system were the same as the respective forms from rat adrenal. It is concluded that the synthesis of SCP2 in rat adrenocortical cells is induced by ACTH and that the induction is mediated by CAMP and may involve increased levels of translatable mRNA encoding a higher molecular weight precursor form of SCP2, which presumably undergoes post-translational processing yielding the mature form.

Cholesterol and its precursors, as well as fatty acids, some phospholipids, and glycolipids, all poorly soluble in aqueous

~

Grants HD 13234, BNS 8318013, AM 28350, and AM 10628. The * This work was supported by United States Public Health Service

costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Permanent address: Dept. of Physiological Chemistry, Academy of Medicine, 6 Swiecickiego St., 60-781 Poznan, Poland.

‘It is with deep regret that we note the death of Dr. George Vahouny on August 1,1986.

’To whom reprint requests and correspondence should be ad- dressed.

media, require carrier proteins for intracellular transport and metabolism (for review see Ref. 1). Two major carriers of cholesterol precursors have been purified from bovine and rat liver and termed sterol carrier protein-1 (SCP,)’ and sterol carrier protein-2 (SCP2) (I), the latter also being called non- specific lipid transfer protein (2,3) or CM2 (4,5). While SCP, participates in the enzymatic conversion of squalene to lanosterol in liver microsomes (6, 7), SCP, enhances conversion of lanosterol to cholesterol (8), as well as cholesterol to 7a- hydroxycholesterol (8), and participates in cholesterol esteri- fication (9, 10) in liver microsomes.

Bovine liver SCP, is a basic protein of known primary structure (11, 12) while rat liver SCP, has a similar amino acid composition to that of the bovine protein, a PI of 8.6, and a molecular weight of 13,000 (11, 13). With the use of an antibody directed against this protein it was shown that SCP, is most abundant in liver and intestine (14, 15). SCPz is also found in the steroid hormone-synthesizing tissues such as adrenal cortex, ovary, and testis (15-17). In the adrenal cortex about 46% of this protein resides within the mitochondria whereas in the liver, SCP, is found predominantly in the microsomal fraction.* No appreciable diurnal variations in the SCP, content in the rat liver and adrenal gland were found, and no studies have been performed on hormonal regrhlation of the synthesis of this protein.

In the adrenal cortex SCP, facilitates the intracellular transfer of cholesterol from the lipid droplets to mitochondria (17, 18), and from the outer to the inner mitochondrial membranes (19), thereby stimulating pregnenolone formation (20) which is the rate-limiting step in steroid hormone biosyn- thesis (21). We have shown that in bovine adrenocortical cells in monolayer culture, ACTH or Bt,cAMP induces the synthesis of a number of steroidogenic enzymes, including components of the cholesterol side chain cleavage enzyme complex, and that this induction is accompanied by a rise in cortisol formation (21). These results suggest that an efficient supply of substrate cholesterol for the mitochondrial side chain cleavage enzyme is required and that the synthesis of SCP, which is the main carrier of cholesterol within adrenocortical cells (17, 18) may also be regulated by ACTH. The present studies were undertaken to investigate whether SCP, synthesis in rat adrenocortical cells in primary culture is

‘The abbreviations used are: SCP, sterol carrier prokin; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; ACTH, corticotropin; Bt,cAMP, dibutyryl cyclic AMP.

A. Khavroubi, R. Chanderbhan, T. J. Scallen, and G. V. Vahouny, unpublished observation.

3713

3714 Induction of Sterol Carrier Protein-2 Synthesis by ACTH

affected by ACTH. With the use of antibodies to SCP,, a specific induction of this protein by ACTH is shown in both cell-labeling and in vitro translation studies. The ACTH- induced synthesis of SCP, is directed by an mRNA species which encodes a higher molecular species of this protein, presumably a precursor form. A preliminary account of this work has been reported (22).

MATERIALS AND METHODS

Chemicals and Radiochemicals-Synthetic ACTHI_,, (Cortrosyn) was purchased from Organon (West Orange, NJ). Corticosterone and dibutyryl cyclic AMP were obtained from Sigma, and collagenase was from Boehringer Mannheim. Acrylamide, polyacrylamide, bisacryl- amide, N,N,N’,N’-tetramethylethylenediamine, and molecular weight standards for polyacrylamide gel electrophoresis were .purchased from Bio-Rad. [3H]Corticosterone (specific activity, 46.5 Ci/ mmol), [35S]methionine (specific activity, 1120 Ci/mmol), rabbit reticulocyte in uitro translation systems, and ‘*C-labeled molecular weight standards were obtained from New England Nuclear.

Antigens and Antibodies-SCP, was purified to homogeneity from rat liver (8), and SCP, antibodies were raised in rabbits, purified and characterized as described previously (22). Adrenodoxin, purified to homogeneity from bovine adrenals, was kindly provided by Dr. J. D. Lambeth (Emory University, Atlanta, GA), and antibodies to adrenodoxin were raised in rabbits (23). Antibodies to mitochondrial Fl- ATPase from bovine heart, raised in rabbits, were donated by Dr. G. A. Breen (University of Texas at Dallas).

The corticosterone antiserum was obtained from Endocrine Sci- ences (Tarzana, CA) and exhibited 3.3% cross-reactivity with deoxycorticosterone, 0.4% with pregnenolone, and 0.6% with progesterone.

Cell Culture and Radiolabeling of Proteins-Adrenal cell suspen- sions were prepared by collagenase digestion from 24-day-old female Sprague-Dawley rats (Holtzman Co., Madison, WI) as previously described (24). The cells were plated onto 60-mm tissue culture dishes and cultured in the medium comprising 4 ml of combined Ham’s F- 12 (KC Biological Inc., Lenexa, KS) and Dulbecco’s modified Eagle’s medium (Gibco Laboratories, Chagnin Falls, OH), with the additions of 2.5% fetal calf serum and 10% horse serum, penicillin (10 units/ ml), and streptomycin sulfate (0.1 mg/ml). The experiments were started with the addition of ACTH (1 p ~ ) or BhcAMP (0.1 mM), and the experiment continued for 48 h. Fetal rat hepatocytes were obtained and cultured as previously described (25). Prior to harvesting, cells were incubated for 2 h in combined Ham’s F12 and Dulbecco’s modified Eagle’s medium (methionine free), followed by 2-h incuba- tion in the same medium that contained [35S]methionine (50 pCi/ml) to radiolabel the newly synthesized proteins. The [35S]methionine- labeled protein extracts were prepared as previously described (26). Protein concentration in cell lysates was determined by the method of Lowry et al. (27).

RNA Isolation and in Vitro Translation-Total RNA was vrevared from the cultured rat adrenal cells and from rat liver as desdribed by Chirgwin et al. (28). Prior to RNA extraction the cell cultures were incubated for 48 h without additions or with additions of ACTH (1 p ~ ) or Bt2cAMP (0.1 mM). The isolated RNA was translated in vitro using a rabbit reticulocyte lysate translation system and [35S]methionine (1 mCi/ml) to radiolabel the newly synthesized proteins.

Immunoisolation and SDS-Polyacrylamide Gel Electrophmesis- The newly synthesized radiolabeled SCP,, adrenodoxin, and F1-ATP- ase were immunoisolated from cell lysates and from in vitro translation products by the method of Ivarie and Jones (29) modified as described earlier (30). Staphylococcus aureus cell membranes (Pan- sorbin, Behring Diagnostics) were used as an immunosorbent. Im- munoisolated proteins were subjected to polyacrylamide gel electrophoresis (31) followed by treatment of the gels with EN3HANCE (New England Nuclear), drying, and exposure to X-Omat AR films (Kodak). The intensity of the bands on the autoradiograms was evaluated by densitometric scanning. Molecular weights of the separated proteins were calculated by the least square regression analysis method using I4C-labeled standards.

Corticosterone Assay-The concentration of corticosterone was determined by radioimmunoassay as described by Rainey et al. (32). Concentrations of steroids in the media were also evaluated by reverse-phase high performance liquid chromatography (33). The combined samples were extracted with chloroform and separated using a commercially available CI8 pBondapak column (25 X 2.1 mm) (Waters Associates, Milford, MA) in 6040 (v/v) methanokwater,

using a high pressure liquid chromatograph ”45 (Waters Associ- ates). The steroids were detected at 240 nm and quantified on the basis of peak heights which were directly proportional to the mass of steroid injected. Cortisone was used as an internal standard. The precision of individual measurements was *3%.

RESULTS

Effect of ACTH and Bt,cAMP on Corticosterone Forma- tion-Radioimmunoassay revealed that corticosterone formation in cultured cells increased exponentially with increasing concentration of ACTH and Bt,cAMP with EDs0 values of 8 and 100 nM, respectively. Corticosterone formation in response to the maximally effective dose of either ACTH or Bt,cAMP increased in a linear fashion between 12 and 48 h of culture and declined thereafter. High performance liquid chromatography demonstrated that corticosterone and 18- hydroxydeoxycorticosterone comprised about 90% of total steroids detected i n the culture media with a molar ratio of the two compounds of 3:l. Deoxycorticosterone comprised less than 10% of total Czl steroids, and only trace amounts of progesterone and no 17a-hydroxy derivatives of Cpl steroids were detected (results not shown).

The Effects of ACTH and BtzcAMP on the Synthesis of SCP, and Adrenodoxin in Cultured Cells-The protein immunoprecipitated from rat adrenocortical cell lysates with antibodies to rat liver SCP, co-migrated with purified liver SCP, and with adrenodoxin immunoprecipitated with antibodies directed against bovine adrenodoxin (Fig. 1). Judging from the electrophoretic mobilities of both liver SCP, and t h e protein immunoprecipitated with the liver SCP, antibody, the M, of both proteins was 12,300 and was very close to the M, of bovine adrenodoxin (not shown). The radiolabeled protein immunoprecipitated with antibody to rat liver SCP, was almost completely displaced from the antigen-antibody complex with 5 pg of purified SCP, (lane 4, left-hand side), but i t was not affected by 5 pg of purified bovine adrenodoxin (lane 5, left-hand side), which completely displaced the radiolabeled rat adrenodoxin from its complex with the adrenodoxin antibody (lane 4, right-hand side). Also, the SCP, purified from rat liver did not displace rat liver adrenodoxin from antigen-

SCP, Adx I 1-

92.5- 69 .O - 46 .O -

c)

30.0- X L

I

12.3-

S T 1 2 3 4 5 1 2 3 4

FIG. 1. Identification of immunoreactive SCP2. Rat adrenocortical cells were grown, treated, and cell proteins labeled as described under “Materials and Methods.” The radiolabeled SCP, was immunoprecipitated from samples of cell lysates each containing 2 X lo6 cpm of protein-bound radioactivity with antibody directed against rat liver SCP,. The radiolabeled adrenodoxin (Adz) was immunoprecipitated with bovine adrenodoxin antibody. The immunoisolated proteins were separated by 15% SDS-PAGE followed by fluorography. ST, purified SCP,, stained with Coomassie Brilliant Blue. Lanes: I, no treatment; 2, ACTH (1 p ~ ) ; 3, Bt2cAMP (0.1 mM); 4, Bt2cAMP (immunoprecipitation in the presence of 5 pg of purified SCP,); 5, Bt2cAMP (immunoisolation in the presence of 5 pg of purified adrenodoxin). The positions of l4C-labeled molecular weight standards are shown on the left-hand side of the autoradiogram.

Induction of Sterol Carrier Protein-2 Synthesis by ACTH 3715

antibody complex (not shown). Thus, it is concluded that the radiolabeled protein immunoprecipitated with the antibodies to rat liver SCP, is rat adrenal SCP,.

The incorporation of [35S]methionine into immunoprecipitable SCP, and adrenodoxin during 2-h incubations of cells treated for 48 h with ACTH or Bt,cAMP at concentrations which maximally stimulate corticosterone formation revealed higher incorporation of [35S]methionine into these proteins than in nontreated cells. As shown in Fig. 1, the synthesis of SCP, was about 3-fold higher in ACTH- or Bt,cAMP-treated cells as compared with untreated cells, whereas about a 10- fold increase in the synthesis of adrenodoxin in response to ACTH or BtzcAMP treatment was observed. Under these conditions the synthesis of the subunits of the constitutive mitochondrial enzyme F,-ATPase was not affected by ACTH or BtzcAMP (results not shown).

Dose Dependence and Time Course of ACTH- or Bt2cAMP- stimulated SCP, Synthesis-Upon addition of ACTH a specific dose-related increase in the synthesis of SCPz with an ED,, of 10 nM was observed (Fig. 2). At 1 p M concentration of ACTH a 4-fold increase in the synthesis of SCP, was noted after 48 h of culture. Likewise, the synthesis of SCP, was stimulated 4-fold by BtzcAMP with an EDso of 100 nM, the maximally effective dose being 0.1 mM (not shown). Synthesis of SCP, increased with time, reaching a maximum (3.6-fold) between 36 and 48 h of treatment with ACTH (1 p ~ ) (Fig. 3). Similar results were obtained when the rates of Bt,cAMP- stimulated synthesis of SCP, were measured (not shown). The half-life of newly synthesized SCP, in the presence of ACTH was measured by a procedure previously described by this laboratory (34) and found to be about 30 h. Thus the incorporation of [35S]methionine during the 2-h pulse time used in the synthesis studies is not greatly influenced by SCP, turnover.

Identification of the Higher Molecular Weight Forms of SCP, Synthesized in an in Vitro Translation System Programmed with RNA Isolated from Rat Adrenocortical Cells and Rat Liver-Electrophoretic analysis of SCP, synthesized in a cell- free translation system programmed with RNA isolated from cultured rat adrenocortical cells and also from rat liver dem-

92.5 - 69.0 - 46.0 -

92.5 - 69.0 - 46.0 -

Time (h) FIG. 3. Electrophoretic analysis of the time course of

ACTH-stimulated synthesis of SCP2. The conditions of cell culture, protein labeling, immunoisolation, and separation by SDS- PAGE were the same as described in the legend to Fig. 1 except for variable times of treatment. The bars show integrated areas of the peaks obtained from densitometric scanning. A second cell culture gave a profile similar to that shown here. Con, control.

Cell I n vitro Labeling Translation "

92.5- 1 ' 69 .O - 46 .O -

P 30.0- 9 x, 5

44.4- 12.3 -

L A A L L D A D FIG. 4. Identification of the higher molecular weight form

of SCP2. Adrenocortical cells and fetal hepatocytes were cultured as described under "Materials and Methods." At 48 h of culture cell proteins were radiolabeled with [35S]methionine. Total RNA was isolated from adrenocortical cells cultured for 48 h or from rat liver and translated using a rabbit reticulocyte in vitro translation system. SCP, was immunoisolated from synthesizedproteins (2 X IO6 protein- bound cpm) as described in legend to Fig. 1. L, liver; A, adrenal. The subscript D in the last two lanes indicates displacement of radiolabeled SCPp by addition of 10 pg of purified rat liver SCPp. The bars indicate positions of unlabeled molecular weight standards.

Lo,

ACTH (log M) FIG. 2. Electrophoretic analysis of the effects of increasing

doses of ACTH on SCPz synthesis. The conditions of cell culture, radiolabeling, protein immunoisolation, and separation by SDS- PAGE were the same as described in the legend to Fig. 1 except for variable concentrations of ACTH. The bars show integrated areas of the peaks obtained from densitometric scanning. A second cell culture gave a profile similar to that shown here. Con, control.

onstrated the synthesis of higher molecular weight forms of rat adrenal SCP, and rat liver SCP, (Fig. 4). A major protein band was present with an electrophoretic mobility corre- sponding to an M , of 14,400, which was larger by about 2,000 than the M , of SCPz immunoisolated from intact adrenal or liver cells. This major band was accompanied by a less pro- nounced band of somewhat larger molecular weight. Both bands were displaced by addition of 10 pg of purified liver SCP, to either sample.

Effects of ACTH or BtzcAMP on SCP, Synthesis as Deter- mined by in Vitro Translation-Both ACTH and Bt,cAMP at concentrations maximally effective in inducing the synthe-

3716 Induction of Sterol Carrier Protein-2 Synthesis by ACTH

sis of SCP, as determined by cell labeling also increased the synthesis of the putative SCP, precursor as determined by in vitro translation (Fig. 5 ) . Immunoprecipitation with the antibody directed against adrenodoxin revealed the synthesis of a precursor form of rat adrenodoxin with an M , of 26,500. The synthesis of the adrenodoxin precursor was highly induced by the maximally effective doses of ACTH or Bt,cAMP, a result similar to that observed in cell labeling studies shown in Fig. 1. Under these conditions the synthesis of the precursor forms of the subunits of mitochondrial F,-ATPase was not affected by ACTH or Bt2cAMP treatment (results not shown).

DISCUSSION

We have previously described the coordinate induction of the synthesis of the components of the side chain cleavage enzyme complex by ACTH (35,36) or Bt,cAMP (37) and the effect of serum low-density lipoprotein cholesterol to induce the synthesis of P-450,, (38). To investigate factors that might affect intracellular transport of cholesterol, we studied the effect of ACTH on the synthesis of SCP, in rat adrenocortical cells in monolayer culture.

As shown in Fig. 1, the synthesis of SCP, could be readily detected in cultured cells and was enhanced by ACTH or Bt2cAMP in a time- (Fig. 3) and dose-dependent (Fig. 2) fashion with an ED50 equal to that producing a half-maximal increase in corticosterone formation in these cells. The synthesis of the subunits of a constitutive mitochondrial F,- ATPase enzyme which was used as an internal control was not affected by ACTH or Bt2cAMP treatment, indicative that SCP, is a member of the small group of proteins, including steroid hydroxylases and related enzymes, whose synthesis is increased by ACTH or Bt2cAMP. The constitutive synthesis of SCP, was higher than that of adrenodoxin and was enhanced by ACTH to a lesser extent (Fig. 1). The M , of 12,300 found for the rat adrenal SCP, was close to the values reported in the literature for purified SCP, from rat and bovine liver (8, 15) and identical with the M , of high pressure liquid chromatography-purified rat liver SCP, (13). Immunoisola- tion of SCP, from cell-free translation systems programmed with RNA isolated from cultured adrenal or liver cells revealed

SCP, Ad x ”

92.5- 66.2- 45.0 -

? 31.0- 8 cL 21.5-

14.4-

Con ACTH Bt, Con ACTH Bt, cAMP cAMP

FIG. 5. Electrophoretic analysis of the effect of ACTH or BtzcAMP on the synthesis of SCPz in an in vitro translation system programmed with RNA isolated from cultured cells. The conditions of cell culture, treatments, and concentrations of added compounds were the same as in the legend to Fig. 1. A t 48 h of culture total RNA was isolated and translated using a rabbit reticulocyte in vitro translation system. The SCP2 and adrenodoxin (Adx) were immunoisolated from synthesized proteins (2 X lo6 cpm of protein-bound radioactivity) as described in the legend to Fig. 1. Con, no treatment. The bars indicate positions of molecular weight mark- ers. A second cell culture led to results similar to those shown here.

that the immunodetectable SCP, co-migrated on SDS-PAGE with a molecular weight standard of M , 14,400 (Fig. 4). This suggests that both the adrenal and liver SCPZ are originally synthesized as higher molecular weight precursor forms, which presumably undergo post-translational processing. Work is in progress to elucidate the nature of this post- translation modification. The additional immunoprecipitable protein of M , about 50,000 present in liver cells (Fig. 4) was also detected by others who used the immunoblotting tech- nique (16, 39).

Immunoisolation from in vitro translation products showed a moderate increase in the amount of immunodetectable SCP, in samples programmed with RNA from ACTH- or Bt2cAMP- treated cells as compared with samples from nontreated cells. This indicated that the effect of ACTH to induce the synthesis of SCP, in rat adrenal cells involves increased levels of translatable mRNA encoding SCP,. Under the same conditions the amount of immunodetectable adrenodoxin precursor was increased to a much greater degree. In previous studies we have shown that the induction of synthesis of steroid hydroxylases and adrenodoxin is associated with a specific increase in the content of mRNAs encoding these enzymes and results primarily from increased gene transcription (35, 36,40-45). Thus, we might predict that the increased level of translatable RNA for SCP, in response to ACTH treatment arises from increased transcription of the SCP, gene. How- ever, further studies are necessary to prove this point. We have also observed that the molecular weight of the precursor form of adrenodoxin was about 26,500 which is close to the value of 24,000 recently reported for the human adrenodoxin precursor (46) but is quite different from the M, of adrenodoxin precursor from bovine adrenocortical cells (23). The reason for these apparent size differences among species is currently under investigation.

It is concluded that the synthesis of SCP, in rat adrenocortical cells in culture is induced by ACTH in a dose- and time- related fashion and that this induction is mediated by CAMP. However, the constitutive level of SCP, synthesis is main- tained relatively high in adrenocortical cells cultured for several days in the absence of ACTH, unlike adrenodoxin whose synthesis decreased profoundly during this period of culture. This comparison suggests that while cAMP may contribute to the regulation of SCP, synthesis, other factors, perhaps nonpituitary in origin, also play an important role in regulation of SCP, synthesis. The effect of ACTH to induce the synthesis of SCP, involves increased levels of translatable mRNA encoding a higher molecular weight form of SCP,. This putative higher molecular weight precursor presumably undergoes post-translational processing yielding the mature form.

Thus, the chronic action of ACTH to regulate optimal steroidogenic capacity in the adrenal cortex is not limited to steroid hydroxylases and closely related enzymes but also includes proteins involved in determining the availability of substrate cholesterol for the cholesterol side-chain cleavage enzyme complex such as 3-hydroxy-3-methylglutaryl-coen- zyme A reductase (32) and SCP,.

Acknowledgments-We thank Leticia Cortez for skillful technical assistance in preparing rat adrenocortical cells and Ruby Lu in estimating corticosterone concentrations. The expert editorial assistance of Jo Ann Killebrew is gratefully acknowledged.

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Biophys. Acta 812,387-392

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Biophys. Res. Commun. 136,348-355

studies on the synthesis of sterol carrier protein-2 in rat

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