Esterification by Rat Liver Microsomes of Retinol Bound to Cellular ...

Vol. 263, No. 35, Issue of December 15, pp. 18693-18701,1988

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

Printed in U.S.A.

Esterification by Rat Liver Microsomes of Retinol Bound to Cellular Retinol-binding Protein* (Received for publication, April 25, 1988)

Robert W. Yost+, Earl H. Harrison, and A. Catharine Ross$ From the Division of Nutrition, Department of Physiology and Biochemistry, The Medical College of Pennsylvania, Philadelphia, Pennsylvania 19129

We haveinvestigatedtheesterification by liver Vitamin A is transported toliver as partof the chylomicron membranes of retinol bound to cellular retinol-binding remnant (1, 2) and is subsequently stored as fatty acid esters protein (CRBP). When CRBP carrying [‘Hlretinol as of retinol, largely in hepatic stellate cells (3,4). Itis becoming its ligand was purified from rat liver cytosol and in- clear that essentially all retinol, either in the extracellular cubated with rat livermicrosomes, a significant frac- circulation or the intracellular milieu, exists in protein-bound tion of the [‘Hlretinol was converted to [’Hlretinyl form. A number of retinoid-binding proteins have now been ester. Esterification of the CRBP-bound [‘Hlretinol, characterized (see Refs. 5 and 6 for reviews). Goodman and which was maximal at pH 6-7, did not require the co-workers (7, 8) first demonstrated that retinol circulates in addition of an exogenous fatty acylgroup.Indeed, plasma bound to retinol-binding protein (RBP),’ a protein of when additional palmitoyl-CoA or coenzyme A was 21 kDa that is secreted by liver parenchymal cells and is provided, the rate of esterification increased either thought to deliver retinol to its target organs. In a number of very slightly or not a t all. The esterification reaction had a K , for [’Hlretinol-CRBP of 4 f 0.6 PM and a organs, distinct intracellular binding proteins having ligand maximum velocity of 145 f 52 pmol/min/mg of micro- specificity for either retinol, retinal, or retinoic acid have also somal protein (n= 4). The major productswere retinyl been identified and characterized ( 5 , 6). Despite detailed palmitate/oleate and retinyl stearate in a ratio of ap- studies of the amino acid sequences of these proteins (9, lo), proximately 2 to 1 over a range of [‘Hlretinol-CRBP their tissue distribution (11-13), and their ligand specificity concentrations from1 to 8 PM. The addition of proges- (5, 6), the physiological functions of the retinoid-binding terone, a known inhibitorof the acyl-CoA:retinol acyl- proteins are notyet understood. Cellular retinol-binding protein (CRBP)was first identified transferase reaction,consistently increasedthe rate of retinyl ester formation when [’Hlretinol was delivered in 1973 by Bashor et al. (14) and subsequently purified from bound to CRBP. These experiments indicate that reti- rat liver (15), human liver (16), rat testis (17), and bovine nol presented to liver microsomal membranes by CRBP retina (18).CRBP has been demonstrated by immunochemican be converted to retinyl ester and that process, this cal methods in numerous organs (11-13) and is present in in contrast to the esterification of dispersed retinol, is relatively high concentrations in liver. Recently, we investiindependent of the addition of an activated fatty acid gated the subcellular distributions of esterified and unesteriand produces a pattern of retinyl ester species similar fied retinol, CRBP, and RBP in the livers of rats with widely A possible role of varying hepatic stores of vitamin A (19). At all levels of liver to that observedinintactliver. phospholipids as endogenous acyl donors in the esteri-retinol, there was sufficient retinol-binding protein in both fication of retinol bound to CRBP is supported by our the soluble fraction (as CRBP) and the microsomal fraction observations that depletion of microsomal phospholipid (as RBP) to account quantitatively for the amounts of free with phospholipase A% prior to addition of retinolCRBP decreasedthe retinol-esterifying activity almostretinol present in the two fractions. It thus seemed conceiv50%. Conversely, incubating microsomes with a lipid- able that CRBP may function as acytosolic transport protein generating system containing choline, CDP-choline, to direct retinol to various cellular sites of metabolism. We previously reported the presence in microsomes preglycerol 3-phosphate, and an acyl-CoA-generatingsystem prior to addition of retinol-CRBP increased retinol pared from liver (20), or the mammary gland (21), of an enzyme activity capable of esterifying solvent-dispersed retias compared tobufferesterificationsignificantly nol. Because the reaction rate was substantially increased by treated controls. inclusion of either preformed fatty acyl-CoA or ATP plus acylcoenzyme A, we inferred the presence of afatty CoAretinol acyltransferase in these organs. This enzymatic activity has now been described in intestine (22, 23) and a * This research was supported by National Institutes of Health number of other tissues (24, 25). Other evidence, however, Grant HL-22633 and funds from the Howard Heinz Endowment. The has supported an acyl-CoA-independent mechanism for reticosts of publication of thisarticle were defrayed in part by the nyl ester synthesis. Saari et al. (26, 27) have observed that payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 membranes from retinoblastoma cells and retinal pigment epithelium esterify retinol in the absence of exogenous actisolely to indicate this fact. $ Postdoctoral traineesupported by Training Grant HL07443 from vated fatty acids. In 1987,Ong et al. (28) presented the the Heart, Lung and Blood Institute, National Institutes of Health. 5 Recipient of a National Institutes of Health Research Career Development Award HD-00691. To whom correspondence and reprint requests should be addressed Dept. of Physiology and Biochemistry, Medical College of Pennsylvania, 3300 Henry St.,Philadelphia, PA 19129.

__

The abbreviations used are: RBP, retinol-binding protein; CRBP, cellular retinol-binding protein; ARAT, acyl-CoA:retinol acyltransferase; BSA,bovine serum albumin; DMSO, dimethyl sulfoxide; PMSF, phenyl methyl sulfonylfluoride; DTT, dithiothreitok HPLC, high pressure liquid chromatography; PLA2,phospholipase A*.

18693

Microsomal Esterification of CRBP-bound Retinol

18694

4 ELMand a V,, of 66 pmol/min/mg of microsomal protein. In similar studies using partially purified CRBP-bound retinol, the same I(,was determined, 4.1 rt 0.9 PM. The V, had a greater range, 171 f 89 pmol/min/mg of microsomal protein ( n = 3). The variability in the observed V,, values is most likely the result of using variouspreparations of microsomes from individualrat livers. Using aliquots of the [3H]retinyl ester extracts from the experiment shown in Fig. 1, individual 13H]retinylester species were isolated by HPLC (20,32), Two major peaks of 3H were eluted in a ratio of approximately2 to 1(Fig. 2). Although we observed no obvious shoulder on the descending limb of EXPERIMENTAL PROCEDWRES~ the major peak of ['Hlretinyl palmitate, the retinyl oleate standard eluted just after the retinyl palmitate standardand, RESULTS therefore, these data do not exclude the possibility that a ~ r e l i Studies ~ i ~using ~ Partially Purified pW/Retiml- small amount of [3H]retinyloleate was also formed. The data CRBP-Using partially purified ['Hlretinol-CRBP, we inves- from Figs. 1 and 2 were used to construct the saturation plot tigated whether this protein-ligand complex is capable of shown in Fig. 3. Over a range of con~entrati~ns of added [3H] acting as a source of retinol for esterification by rat liver retinol-CRBP, the ratio of retinyl palmitatejoleate to retinyl microsomes. At a concentration of 5 ELM[3H]retinol-CRBP, stearate formed by rat liver microsomes was quite constant. in the absence of exogenous cofactors, 11%of the CRBP- Similar results were obtained for the esterification of I3H] bound t3H]retinol was esterified after 30 min by 0.5 mgof retinol presented by partially purified CRBP (datanot microsomal protein. To determine whether an even greater shown). portion of [3H]retinolbound to CRBP is available for esteriCharacterizationof the Basal Reaction Conditions-Using a fication, incubations were conducted for 30 min with 6 mg of concentration of 5 ~ L CRBP-bound M retinol, the optimel conmicrosomal protein. These incubations demonstra~d that ditions for the basal reaction (without additional cofactors) liver microsomes werecapable of converting 38% of the [3H] were characterized. The effect of protein concentration, time retinol bound to CRBP to ["Hjretinyi ester. After incubation, of incubation, buffer pH, and incubation temperature on the over 90% of the [3H~retinyl ester was recovered in the reiso- esterification reaction were investigated.The effect of increaslated microsomal fraction, whereas93% of the remaining ing microsomal protein on retinol esterification is shown in unesterified retinol remained in the supernatant. Having established that CRBP-bound retinol is available I RP for esterification by rat liver microsomes, we examined the parameters of this reaction in more detail. These incubations were conducted in the absence of added cofactors (palmitoylCoA or coenzyme A) since studies (described below) showed little effect of these agents on the esterification of CRBPbound retinol. Kinetic S ~ ~ i e s - I n i t i a lstudies were conducted to determine the K,,, and VmeLfor the esterification of CRBP-bound retinol (Fig. 1).These data were obtained using CRBP-bound retinol that was purified by HPLC. The reaction had a K, of

provocative observations that rat intestinal mucosal microsomes were able to synthesize retinyl esters from retinol presented to themembranes bound to CRBP, type If, a form of cellular retinoid-binding protein found in high concentration in the intestinal mucosa. Further,this esterification reaction did not require exogenous acyl-CoA,had a relatively low K,, and produced a mixture of fatty acid esters like those found in intestinal lymph and isolated chylomicrons. Wethus havebeen prompted to reexamine the process of hepatic retinyl ester synthesis and to investigate the possible role of liver CRBP in directing this esterification reaction.

i

TIME, rnin

FXG.2. An aliquot of the total ['Hlretinylestersformed with 7.9 &M CRBP-bound ['H]retinol from Fig. 1 was separated using HPLC (see "Methods") andfractions ofcolumn effluent were counted for 'H, The elution position of known standards (retinyl palmitate, RP; retinyl oleate, RO; and retinyt stearate, R S ) are indicated.

,r' -.2

0

I 0

J

I

.2

.4

.6

.8

lis, UM

FIG. 1. Lineweaver-Burkplot for the esterification of HPLC-purified CRBP-bound ['H]retinol. The reaction tube contained 100 fig of microsomal protein, 2 mM D m , and 0.15 M phosphate buffer, pH 7.0, in a final volume of 250 pi. The concentration of CRBP-bound retinol ranged from 1.2 to 7.9 #M. The line obtained by linear regression analysis of the data is: Y = 60.2X 15.1; 7 = 0.971. From these data, theK, is 4 pM, and theVmexis 66 pmol/min/ mg of microsomal protein.

20

+

0

2

4

6

8

RETINOL, pM

FIG. 3. By using datafrom Fig. 2, asaturationPlotfor "Experimental Procedures" are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard esterification of CRBP-bound['Hlretinol W&S constructed. magnifying glass. Full size photocopies are included in the microfilm The rate of formation of total retinyl esters (01, retiny1 palmitate/ and retinyl stearate (Dl are shown. oleate (a), edition of the Journal that is available from Waverly Press.


18695

curve from 4 to 24 "C is 26 kcal/mol, and the Q ~ ois 4.9. Above 24 "C, the activation energy is considerably reduced (11.7 kcal/mol), and theQl0 is 1.9. Wehave not yet determined the cause of this change in activation energy; however, possible explanations suggested by the work of others include change in membrane fluidity, or intrinsic temperature dependence of the catalytic efficiency of an enzyme (38,391. To determine whether the increased activity observed a t higher temperatures was the result of breakdown of the [3H] retinol-CRBP complex, the protein-Iigand complex was incubated alone, under similar conditions, for 20 min at 52 "C. The ultraviolet absorption spectrum of the treated complex was essentially identical to thatof the untreated complex (not shown). This indicates that breakdown of the ligand-protein complex, releasing free retinol, was not likely to be a contributing factor inthe increased esterification of retinol observed with increasing temperature. In contrast to the stability of the retinol-CRBP complex, the microsomal enzyme activity was somewhat unstable. Incubation of microsomes a t 37 "C for 15 min prior to addition of [3H]retinol-CRBP resulted in a 30% decrease in retinol esterification compared to microsomes held on ice for the same time. At higher temperatures (45 or 55 "C for 15 min followed by incubation with [3H]retinol-CRBP at 37 "C) the esterifying activity decreased even more rapidly, with 54% of activity remaining after preincubation at 45 "Cand essentially no activity remaining after incubation at 55 "C. Therefore, thermal instability of the microsomes may contribute to the failure to observe greater product formation at 37 "C with longer incubation times (Fig. 4B). In contrast, acylCoAretinol acyltransferase activity was not reduced as a result of pretreatment of microsomes at 37 "C for up to 90 min. Acyl-CoAretinol acyltransferase activity was reduced 60% by pretreatment at 52 "C for 15 min, and a complete loss of activity was observed after pretreatment at 55 "C for 15 min. The microsomal activities were, however,quite stableto 0 1 2 3 0 4 8 12 16 20 freezing; preparations retained activity for at least 4 weeks TIME, min MICROSOMAL PROTEIN, mg and all experiments were conducted within this time. The esterification of [3H]retinol bound to CRBP, like that of dispersed retinol (20, 21), was inhibited completely by the sulfhydryl reagent, p-chloromercuribenzoate (approximately 5 mM). 58-035, an acylamide shown to inhibit the esterification of microsomal cholesterol (29), did not inhibit the esterification of either CRBP-bound retinol, or dispersed retinol. Effect of ExogenousCofactors-Because previous studies had demonstrated that the addition of fatty acyl-CoA to rat 5 6 7 8 910 3.3 3.2 3.4 3.5 3.6 3.7 liver microsomes increased the esterification of dispersed pH [ 1/T°K]x lo3 retinol upto 5-6-fold, it was of interest to determine whether 45 24 i the esterification of [3H]retinol bound to CRBP couldbe OC stimulated by exogenous fatty acyl-CoA. In several experiFIG.4. Esterification of partially purified CRBP-['H]reti) ments, the esterification of CRBP-bound retinol (5 p ~ was no1 as a function of microsomal protein concentration ( A ) , either only slightly enhanced or was unaffected by palmitoyllength of incubation ( B ) , pH (C), and temperature ( D ) .All reaction tubes contained 5 g M CRBP-[3H]retinol, 2 mM DTT,and CoA over the concentration range of2.5-10 p~ (Fig. 5A). ) 0.15 M buffer in a total volume of 200 p l . Reactions shown in panels Indeed, higher concentrations of palmitoyLCoh (50 p ~ inA, B, and D were conducted in phosphate buffer a t 37 "C for 4 min hibited the esterification process. The addition of 100p~ BSA and, for panels B, C, and D,included 100 pg of microsomal protein. relieved this inhibition but did not result in a stimulation of All values are for at least duplicate assays. Panel A , the reaction was retinyl ester synthesis. Even a t a much greater concentration conducted a t pH 7.4 in the presence of 0.025-3 mg of microsomal protein. Panel B , the reaction was conducted at pH7.0. Panel C, the of [3H]retinol-CRBP (90 p M ) , 10 pM palmitoyl-CoA stimureaction was conducted over a pH range of 5-10. Phosphate buffer lated esterification by only 30%, and the addition of 50 p~ palmitoyl-CoA was still inhibitory (data not shown). In some (0)was used from pH 5 to 7.84, Tris-HCI (0)was used for pH 8.69.5 and glycine-NaOH (A) was used for pH 10.3. Panel D,Arrhenius experiments the possible involvement of coenzyme A in the plot for the esterification reaction. The microsomes were warmed to esterification of [3H]retinol-CRBP was determined, The adthe respective temperatures for 1 min prior to theaddition of CRBP[3H]retinol. Using linear regression analysis, for the points from 4 to dition of CoA over the range of 0.1-5 mM had no effect on 24 "C the line had a slope of -5.71 and an r value of 0.999. For points the rate of esterification of [3H]retinolbound to CRBP (data from 24 to 46 "C the values were -2.58 and 0.982. The calculated not shown). activation energies are 26 and 11.7 kcal/mol, respectively. In contrast, and as reported previously (ZO), the esterificaFig. 4A. Retinyl ester synthesis was directly proportional to the amount of microsomal protein up to approximately 1mg. The effect of the length of incubation on retinyl ester formation is shown in Fig. 4B. The formation of retinyl esters was approximately linear for 4 min. After 15-20 min, little additional retinyl ester was formed. An additional hour of incubation, a total of 86 min, resulted in only a slight increase (23%) in esterified retinol compared to the amount of ester formed after 20 min. The effect of the reaction buffer pH on the esterification of CRBP-bound [3H]retinol is presented in Fig. 4C. Retinol esterification was maximal at pH6-7. At pH 7.4, the pH used in some of our preliminary studies, the amount of ester formed was still 88% of the maximum observed at pH6.7-7. Esterification was demonstrable, albeit at lesser rates, into thebasic pH range of 8-10. Thus we selected to use 50 pg of microsomal protein, 5 pM [3H]retinol-CRBP, pH 7.0, and an incubation time of 4 min in most of our subsequent studies. for CRBP-bound retinol (5 p ~ and ) Working near the K,,, at a constantmicrosomal enzyme to substrate ratio,the effect of the incubation temperature on retinol esterification was investigated. Esterification activity increased with the temperature of the incubation between 4 and 46 "C, the highest temperature we examined. When presented as an Arrhenius plot (Fig. 4D), these data indicate a change of slope a t about 24 "C. The calculated activation energy for the portion of the


18696 -

250

-

200

-

150

-

500

A

L

0

0.5 1

5 10

50 100

PALMITOYL - COA (vM)

FIG. 5. The effect of increasing concentrationsofpalmitoylCoA on (A) the esterification of partially purified CRBPbound ['Hlretinol and ( B ) dispersed retinol. Panel A, the reaction tube contained 50 pg of microsomal protein, 2 mM DTT, 0.15 M phosphate buffer, pH 7.0 or 7.4, 5 p~ CRBP-bound retinol, and an

increasing concentration of palmitoyl-CoA in the absence (a)and presence (0)of 100 p~ BSA in a total volume of 100 pl. The average basal activity in the absence of BSA was 100 f 9 (S.E.) pmol/min/ mg of microsomal protein for six experiments. The activity in the presence of BSA was 128 pmol/min/mg of protein. Points shown represent average values of duplicates from one to four experiments. Panel B , the reaction tube contained 100 pg of microsomal protein, 5 mM DTT, 20 p~ BSA, 0.15 M phosphate buffer, pH 7.4, and either 5 p~ (0)or 110 p~ (0)retinol dispersed in dimethyl sulfoxide in a total volume of 250 pl. The basal activity in the absence of palmitoylCoA and BSA was 90 and 172 pmol/min/mg of microsomal protein, respectively. tion of t3H]retinol presented asa dispersion torat liver microsomes was significantly increased when exogenous palmitoyl-CoA was added (Fig. 5 B ) . At a low concentration of dispersed retinol (5 PM, comparable to theK,,, for t3H]retinol bound to CRBP), theaddition of 5 to 100 p~ palmitoyl-CoA in the presence of 20 p~ BSA significantly increased retinol esterification. When a higher concentration of dispersed retinol (110 PM) was added as substrate, the stimulatory effect of palmitoyl-CoA was even greater and had not reached saturation at a concentration of 100 pM palmitoyl-CoA. In some experiments we pretreated microsomes with hydroxylamine, a compound which reacts with endogenous acylCoAs to form hydroxamic acids (40),prior to assaying retinolesterifying activity. As previously reported (20) the amount of retinyl esters formed was significantly reduced when a high concentration of retinol dispersed in dimethyl sulfoxide was presented to hydroxylamine-treated microsomes. Retinyl ester formation was restored to control levels with the addition of palmitoyl-CoA and BSA. In contrast when hydroxylaminepretreated microsomes were used to study the esterification of [3H]retinol bound to CRBP, the amount of retinyl esters formed was reduced to 17 f 5% (n = 4) of the buffer controls, but the addition of palmitoyl-CoA did not restore the esterifying activity. Effect of Phospholipase Az Treatment on RetinolEsterification-Phosphatidylcholine has recently been postulated to be the acyl donor for the esterification of CRBP-bound retinol by microsomes from the small intestine (41). To investigate this possibility for liver membranes, we added exogenous egg

phosphatidylcholine, chosen because the fatty acyl species resemble those of retinyl esters, to microsomes before the addition of CRBP-bound retinol. Other experiments were conducted using dilauroylphosphatidylcholine. Although other investigators have demonstrated that medium-length fatty acyl chains from phosphatidylcholine can be transferred to retinol (41), we did not observe an increase in the rate of retinol esterification after addition of these phospholipids from 0.08 to 4 mM. In fact, at thehigher concentrations this addition inhibited retinol esterification (datanot shown). Considering that sufficient microsomal phospholipid might be present endogenously, we then asked whether decreasing the endogenous phospholipid contents by pretreating microsomes with phospholipase AP would also reduce the rate of retinol esterification. Table I shows the results of a representative experiment. After treating microsomes with phospholipase Az,with or without addition of 5% BSA to facilitate removal of the products of lipolysis, retinol esterification was substantially reduced phospholipase A2 treatment alone resulted in a 25% decrease in esterifying activity, with a concomitant decrease in phosphatidylcholine and an increase in lysophosphatidylcholine. When BSA was added, the esterifying activity was reduced 44% compared to the respective control with a greater reduction in the totalphospholipid and lysophosphatidylcholine pools. Retinol esterification was not affected by addition of BSA to microsomes incubated in the absence of phospholipase AP,nor did addition of BSA affect the mass of total phospholipid or phosphatidylcholine. The lysophosphatidylcholine pool was less than 1 pg/mg microsomal protein in both groups. These values are in good agreement with previous measurements in liver microsomes (4244). We also asked whether the esterification of retinol could be stimulated when phospholipid is generated by liver microsomes from exogenous precursors. A lipid-generating system previously used to investigate the synthesis of phosphatidylcholine fromdiacylglycerolby lung microsomes (34) was employed. Microsomes from the experiment described in Table I were incubated in thepresence and absence of this lipidgenerating system, or buffer alone. The results are shown in Table 11. We observed similar increases (about 50%) in the esterification of retinol by microsomes pretreated with the lipid-generating system regardless of prior treatment with phospholipase. The addition of a lipid-generating system resulted in a small but consistentincrease in the total phospholipid pool of all samples. There is also a slight but consistent increase in the amount of phosphatidylcholine, with the exception of the 41% increase observed in the phospholipase Az-treated group with no added BSA. This large change in phosphatidylcholine poolsize is probably the result of a reacylation of the lysophosphatidylcholine present in these microsomes. These dataindicate that theability of the microsomes to esterify retinol bound to CRBPis not lost as a result of phospholipase Az treatment and suggest that it is possible for treated microsomes to resynthesize an endogenous acyl donor with an associated stimulation of retinol-esterifying activity. Effect of Progesterone onRetinol Esterification-Retinol esterification was also studied in the presence of progesterone, a steroid hormone which has been shown to inhibit acylCoAcholesterol acyltransferase (45,46) and acyl-CoAretinol acyltransferase (20) activity in microsomes. The effect of treating microsomes with increasing concentrations of progesterone for 6 min before the subsequent addition of either CRBP-bound retinol or dispersed retinol is shown in Fig. 6. At final concentrations of 50 PM and above, progesterone


18697

TABLEI The effect of phospholipase A2 pretreatment of microsomes Microsomes were incubated with phospholipase A, (PLAP)with or without the subsequent addition of 5% BSA prior to reisolation and determination of retinol-esterifying activity (see “Methods” in the Miniprint Section). Additional aliquots were taken for lipid analysis (see “Methods”) or for further studies shown in Table 11. All values are corrected for volume and are expressed per milligram of starting microsomal protein. The values in parentheses are percent of controls incubated without phospholipase AP.The data are shown for a representative experiment. Esterifying activity

Total phospholipid/ mg protein

pmol/min/mg microsomal protein

Amount phosphatidylcholine/ mg protein

Amount lysophosphatidyl choline/mg protein

P&?

&

No added BSA +PLAz -PLAz

44 (71%) 62

408 (90%) 453

175 (74%) 238

35 0.8

5% BSA +PLAP -PLA,

33 (56%) 59

343 (72%) 473

170 (70%) 243

2.5 0.3

TABLEI1 The effect of lipid regeneration on retinol esterification Microsomes from the experiment shown in Table I were incubated with a lipid-regenerating system (see “Methods” in the Miniprint Section) prior to reisolation and determination of retinol-esterifying activity. The data are expressed as a percent of control incubations without the lipid-generating system. Esterifying activity Prior treatment (pmol/min/mg microsomal of controls protein) after Total Phosphatidylincubation with phospholipid choline without lipidgenerating system lipid-generating system

No added BSA +PLAz -PLA, 5% BSA +PLAz -PLAz

%

%

%

141 167

108 109

141 107

154 158

102 107

103 109

consistently increased the initial rate of retinol esterification when CRBP-bound retinol was the substrate (Fig. 6A). In parallel experiments, the effect of progesterone on the esterification of dispersed retinol was determined using both alow concentration (5 WM,Fig. 6B) and ahigher concentration (100 p ~ Fig. , 6C) of dispersed retinol, either in the absence of exogenous palmitoyl-CoA and BSA or in their presence. In contrast to our results with CRBP-bound retinol, progesterone had little effect on retinol esterification in theabsence of palmitoyl-CoA and BSA, but inhibited esterification in their presence. The latter effect is most notable when the esterification reaction is driven at higher rates by addition of 110 p~ dispersed retinol andexogenous palmitoyl-CoA and BSA. The effects of progesterone both in increasing the esterification of [3H]retinol bound toCRBPand in inhibiting the acylCoAxetinol acyltransferase reaction were almost immediate; maximum changes were observed after 1 min of pretreatment in both cases (data not shown).

this interpretation. First, a significant portion of the CRBPbound retinol we added to rat liver microsomes was available for esterification. Second, the apparent K , for retinol esterification by rat liver microsomes, approximately 4 p M , agrees very wellwith our estimates of the physiological concentration of CRBP in rat liver. Based on a value of 73 pg of CRBP/g of liver from rats with adequate vitamin A stores (19) and the molecular weight of CRBP (14,600), we estimate a concentration of approximately 5 pmol of CRBP/kgof liver, or approximately 6-7 pmol/“liter” of cytoplasmic ~ompartment.~ In comparison, we have reported an approximate K , of 30-40 p~ for solvent-dispersed retinol presented to either rat liver or mammary gland microsomes (20, 21). Thus, the K,,, measured in vitro is appropriate for the physiological concentration of CRBP in the intracellular milieu. Similarly, recent studies on the esterification of retinol associated with CRBP, type 11, by intestinal microsomes, indicated a K, which is substantially lower (28) than that estimated from studies on the esterification of dispersed retinol by rat or human intestinal membranes (22,23). Thus, inboth tissues, the affinity of the substrate for the esterifying enzyme(s) is enhanced in the presence of the appropriate binding protein. Additionally, the maximum initial velocity we have determined for the esterification of CRBP-bound retinol in uitro appears to be more than adequate to account for the hepatic esterification in vivo of newly assimilated vitamin A? A thirdargument for the physiological involvement of CRBPin directing retinol esterification derives from the pattern of retinyl esters formed by liver microsomes presented with CRBP-bound retinol. It is well known from the early work of Goodman and co-workers (1, 48) that the fatty acid composition of retinyl esters is quite organ-specific and, in contrast to glyceryl esters, varies only slightly with differences

Based on morphometric determinations that cytoplasm comprises nearly 75% of the nonnuclear volume of rat liver (47), the concentration of CRBP, which is nearly entirely soluble (19),in the cytoplasmic compartment is approximately 6-7 ,UM. T o estimate a reasonable rate of retinol esterification in uivo, we have assumed that, on a vitamin A-adequate diet, a rat may consume DISCUSSION 100 fig of retinol in a daily meal. Since nearly all vitamin A that is The present study demonstratesthat CRBP is an effective newly delivered to the liver has been shown to undergo hydrolysis vehicle for delivering retinol to liver microsomal enzyme(s) and complete reesterification in about 3 h (I), this would represent that esterify retinol and thussuggests that one possible phys- an average rate of 12 X lo3 pmol/lO g of liver/min. Assuming that cellular membranes yielding the microsomal fraction comprise 50 mg iological rolefor CRBP is to direct retinol to appropriate sites of protein/g of liver, an in uivo rate of 0.4 pmol/min/mg of microsomal of metabolism, and/or to act as a substrate for theacylation protein is estimated. In comparison, the average Vmaxwe have measof retinol. At least three lines of evidence are supportive of ured in vitro is 145 pmol/min/mg of microsomal protein.

‘


18698

whole lymph or isolated chylomicrons (32, 48). Thus, in both organs, delivery of retinol by the major cellular binding protein results in a retinyl ester pattern that closely resembles that found in vivo. It is also of interest that retinol associated with CRBP was esterified by microsomal membranes in the absence of an exogenous donor of the fatty acyl group, and that no significant increase in the rateof esterification resulted when reaction mixtures were supplemented with palmitoyl-CoA. This finding is in contrast to our previous reports that the esterification of dispersed retinol is increased considerably when performed fatty acyl-CoA or an acyl-CoA-generating system 100 is provided (20, 21), but is consistent with observations of Saari et al. (27) that membranes of the bovine retinal pigment epithelium esterify CRBP-bound retinol in the absence of an exogenous acyldonor and of Ong et al. (28) that retinol bound to CRBP, type 11, is esterified by microsomal membranes of 40 1 the rat intestine in the absence of exogenous acyl-CoA. The *o} presence of an endogenous acyl donor has been postulated (27, 28) and, based on the similarity between the fatty acid composition of retinyl esters in lymph and the n-1 fatty acid IC of phosphatidylcholine, Huang and Goodman (48) proposed lecithin as a potential precursor of the fatty acyl group of intestinal retinyl esters. Very recently, MacDonald and Ong loo 80 (41) have reported that fatty acid from exogenous phosphatidylcholine can be transferred to retinol presented to intestinal microsomes by CRBP, type 11, and have thus proposed the existence of a 1ecithin:retinol acyltransferase in rat intestinal membranes. A difficulty in reconciling the acylCoAretinol acyltransferase reaction with liver physiology is that this enzymatic process is able to utilize a variety of 10 50 100 200 300 species of fatty acyl-CoA, including odd-chain acyl-CoAs not typical of the retinyl ester composition of cells (50). Thus, PROGESTERONE pM FIG. 6. The effect of progesterone pretreatment of micro- presentation of retinol to microsomal membranes from three somes on the esterification of [SH]retinol. Microsomes were organs (eye, intestine, and liver) by an appropriate binding pretreated with progesterone dissolved in 2 pl of acetone (20) for 6 protein appears to make available an endogenous second min at 37 "C before the subsequent addition of CRBP-bound t3H] substrate, leading mainly to formation of the specific saturetinol or dispersed [3H]retinol. Panel A, to investigate the esterifi- rated fatty acid esters of retinol found in vivo. cation of CRBP-bound retinol, the reaction tube contained 50 pg of To extend the observations that phosphatidylcholine is treated microsomes, 5 1M CRBP-bound [3HJretinoI,2 mM DTT, and likely to be important in the esterification of retinol by liver 0.15 M phosphate buffer, pH 7.0, in a total volume of 100 pl. The effect of progesterone on esterification of 5 pM [3H]retinol dispersed membranes, we conducted experiments with phospholipase in dimethyl sulfoxide in the absence of palmitoyl-Coh and BSA (0) A2 to perturb the endogenous pool of membrane phosphoand in their presence (H) is shown in panel B. The effect on esterifi- lipids. We observed a decrease in retinol-esterifying activity cation of 110 p~ dispersed retinol in the absence of palmitoyl-CoA as a result of phospholipase Az treatment, the magnitude of and BSA (A) and in their presence (A)is shown in panel C. The whichwas greatest (43%) when phospholipase A2-treated reaction tubes contained 100 pg of treated microsomes, 5 mM DTT, and phosphate buffer pH 7.4 rt 100 FM palmitoyl-CoA and 20 FM samples were incubated with BSA to remove the products of BSA (20). The total volume was250 rl. Inall studies the incubations lipolysis. We have compared the retinol-esterifying activity were for 4 min at 37 "C. The esterification rates for acetone-treated versus the microsomal mass of total phospholipid, phosphacontrol microsomes were 118 5 (n = 3) pmol/min/mg (panel A ) , 85 tidylcholine, or phosphatidylcholine plus lysophosphatidyland 117 pmol/min/mg in the absence and presence of palmitoyl-CoA choline after each of the four treatments shown in Table I, and BSA (panel B ) , and 102 and 572 pmol/min/mg in the absence and have found the best linear relationship ( r = 0.99) between and presence of palmitoyl-CoA and BSA (panel C ) . esterifying activity and the sum of phosphatidylcholine plus in the type of dietary fat. Analysis of the retinyl ester com- lysophosphatidylcholine. Thesedata suggest that, while a decreased availability of phosphatidylcholine does result in a position of liver from numerous species has demonstrated that more than 95%of the total retinol is esterified with long- decrease in retinyl ester synthesis, the esterifying enzyme chainsaturatedfatty acids (48). Specifically inrat liver, may also be able to utilize lysophosphatidylcholine acylated retinyl palmitate andretinyl stearate comprise approximately at thecarbon-1 position. Although addition of exogenous egg phosphatidylcholine or 70 and 15%, respectively, of total retinyl esters (1, 49). In good agreement with this steady-state composition, we have dilauroylphosphatidylcholine did not increase the rate of esobserved that newly synthesized esters of retinol presented to terification of retinol bound to CRBP, it was possible to rat liver microsomes by CRBP are also principally retinyl demonstrate an increase under conditions in which phosphopalmitate/oleate and retinyl stearate in a ratio close to 2 to 1. lipids were synthesized in situ. In all cases, incubation with Our results are similar to the important finding of Ong et al. the lipid-generating system resulted in a significant increase (28) that the esterification of retinol presented by CRBP, in retinol-esterifying activity and a slight but consistent intype 11, to intestinal microsomes leads in vitro to a pattern of crease in phospholipid content. Although these changes in retinyl esters very much like that previously determined in lipid content are relatively small on a percentage basis, they

p-"

*

Microsomal Esterificationof CRBP-bound Retinol represent a relatively large change in mass. For example, a 1%change in phosphatidylcholine (average molecular weight of 770) content in a typical incubation represents a mass change of 2000pmol/mg of original microsomal protein. Since the maximal rate of retinol esterification is on the order of 145 pmol/min/mg of protein, the totalchange in phospholipid appears to be in great excess of that required for this reaction. Further detailed studies on the requirements for regeneration of retinol-esterifying activity willbe necessary to identify more precisely the specific endogous donor of the acyl groups to retinol. A question of interest is whether or not a single enzyme esterifies retinol presented in either dispersed form or bound to CRBP. A definitive answer most likely must await studies showing the physical separation, or copurification, of these enzymatic activities. Although several properties or characteristics are shared, others differ significantly. Both enzymatic reactions are inhibited by mercurial reagents (20, 211, both show rather similar instability at elevated temperature, and neither are inhibited by the drug, 58-035 (29). Although we found no evidence that the esterification of CRBP-bound retinol requires the presence of an activated fatty acid, we have found both in our previous studies (20, 21) and this investigation that some esterification of dispersed retinol also takes place in unsupplemented microsomes. Thus, dependence or independence of fatty acyl-CoA alone does not completely distinguish these activities. It is quite clear, however, that kinetic distinctionsdo exist; as notedabove, the apparent K , for retinol is lower when it is presented by its cellular binding proteins. Additionally, using progesterone as a modulator, our data indicate two very different patterns of response. Brief treatment of the microsomes with progesterone inhibits the esterification of dispersed retinol (20), an effect that was greatest at high concentrations of retinol and in the presence of palmitoyl-CoA when esterification is driven at maximal rates. In contrast, when retinol was delivered by CRBP, therewas a consistent,modest enhancement of retinyl ester synthesis afterprogesterone addition. Progesterone has been demonstrated to inhibit acyl-CoAcholesterol acyltransferase (45, 46), and it has been argued that this steroid acts directly on the membrane to inhibit the delivery of membraneassociated cholesterol to its esterifying enzyme (51). Our studies demonstrate a similar reduction in the esterification of dispersed retinol, but suggest that retinol associated with CRBP is specifically “targeted to its site of esterification even when progesterone is present. Membranes treated with hydroxylamine to deplete endogenous acyl-CoA nonetheless retain the ability to catalyze the acyl-CoAretinol acyltransferase reaction from dispersed retinol and exogenously added acyl-CoA (20). In contrast, these membranes lose the ability to catalyze the esterification of retinol associated with CRBP, even when exogenous acyl-CoA is available (27; and text). In addition to theability of neutral hydroxylamine to react with fatty acyl thioesters (40), Jauhiainen and Dolphin (52) have recently demonstrated its direct interaction with the active site of 1ecithin:cholesterol acyltransferase; thus, direct inhibition of the transacylation of retinol presented by CRBP is quite plausible. Based on these and otherstudies, we believe that itis most likely that, under physiological conditions, CRBP directs the retinol to an acyl-CoA-independent esterifying enzyme. It is also plausible that the acyl-CoA:retinol acyltransferase reaction provides a second system for conversion of the membrane-seeking and potentially damaging retinol molecule (53) to more hydrophobic retinyl esters when, as may be the case

18699

in hypervitaminosis A, an excess of unbound retinol is present in cells. Acknowledgments-We acknowledge the expert technical assistance of Elizabeth M. Gardner. REFERENCES 1. Goodman, D. S., Huang, H. S., and Shiratori, T. (1965) J. Lipid Res. 6,390-396 2. Ross, A.C., and Zilversmit, D. B. (1977) J. Lipid Res. 1 8 , 169181 3. Blaner, W . S., Hendriks, H. F. J., Brouwer, A., de Leeuw, A. M., Knook, D. L., and Goodman, D. S. (1985) J. Lipid Res. 2 6 , 1241-1251 4. Blomhoff, R., Rasmussen, M., Nilsson, A., Norum, K. R., Berg,

T., Blaner, W . S., Kato, M., Mertz, J. R., Goodman, D. S., Eriksson, U., and Peterson, P. A. (1985) J. Bioi. Chem. 2 6 0 , 13560-13565 5. Chytil, F., and Ong, D. E. (1987) Annu. Reu. Nutr. 7 , 321-335 6. Chytil, F., and Ong, D. E. (1984) in The Retinoids (Sporn, A. B., Roberts, M. B., and Goodman, D. S., eds) Vol. 2, pp. 90-123,

Academic Press Inc., New York 7. Kanai, M., Raz, M., and Goodman, D. S. (1968) J. Clin. Invest. 47,2025-2044 8. Goodman, D. S. (1984) in The Retinoids (Sporn, A. B., Roberts, M. B., and Goodman, D. S., eds) Vol. 2, pp. 42-89, Academic

Press Inc., New York 9. Sundelin, J., Busch, C., Das, K., Das, S., Eriksson, U., Jonsson,

K.H., Kampe, O., Laurent, B., Liljas, A., Newcomer, M., Nilsson, M., Norlinder, H., Rask, L., Ronne, H., and Peterson, P. A. (1983) J. Invest. Dermatol. 8 1 , 5 9 ~ - 6 3 s 10. Colantuoni, V., Cortese, R., Nilsson, M., Lundvall, J., Bavik, C. O., Eriksson, U., Peterson, P. A., and Sundelin, J. (1985) Biochern. Biophys. Res. Commun. 1 3 0 , 431-439 11. Adachi, N., Smith, J. E., Sklan, D., and Goodman, D. S. (1981) J. Biol. Chem. 256,9471-9476 12. Ong, D. E., Crow,J. A., and Chytil, F. (1982) J. Bwl. Chem. 2 5 7 , 13385-13389 13. Eriksson, U., Das, K., Busch, C., Nordlinder, H., Rask, L., Sundelin, J., Sallstrom, J., and Peterson,P. A. (1984) J. Biol. Chem. 259,13464-13470 14. Bashor, M. M., Toft, D. O., and Chytil, F. (1973) Proc. Natl. Acad. Sci. U. S. A . 70,3483-3487 15. Ong, D. E., and Chytil, F. (1978) J. Bwl. Chem. 253,828-832 16. Ong, D. E. (1982) Cancer Res. 4 2 , 1033-1037 17. Ross, A. C., Takahashi, Y. I., and Goodman, D. S. (1978) J. B i d . Chem. 253,6591-6598 18. Saari, J. C., Futterman, S., and Bredberg, L. (1978) J. Biol. Chem. 253,6432-6436 19. Harrison, E. H., Blaner, W . S., Goodman, D. S., and Ross, A. C. (1987) J . Lipid Res. 28,973-981 20. Ross, A.C. (1982) J. Biol. Chen. 2 5 7 , 2453-2459 21. Ross, A. C. (1982) J. Lipid Res. 2 3 , 133-144 22. Helgerud, P., Petersen, L. B., and Norum, K. R. (1982) J. Lipid Res. 23,609-618 23. Helgerud, P., Petersen, L. B., and Norum, K. R. (1983) J. Clin. Invest. 7 1,747-753 24. Donoghue, S., Johnson, K., Donawick, W . J., and Sklan,D. (1981) J. Dairy Sci. 6 4 , 2419-2421 25. Torma, H., and Vahlquist, A. (1987) J. Inuest. Derrnatol. 88, 398-402 26. Saari, J. C., Bredberg, L., and Futterman, S. (1980) Invest. Ophthalmol. Visual Sci. 19,1301-1308 27. Saari, J. C., Bunt-Milan, A. H., Bredberg, D. L., and Gamin, G. G. (1984) Vision Res. 2 4 , 1595-1603 28. Ong,D. E., Kakkad, B., and MacDonald, P. N. (1987) J. Biol. Chem. 262,2729-2736 29. Ross, A. C., Go, K. J., Heider, J. G., and Rothblat, G. H. (1984) J. Biol. Chem. 2 5 9 , 815-819 30. Ong, D. E., and Chytil, F. (1980) Methods Enzymol. 6 7 , 288-296 31. Markwell, M. A. K., Haas, S. M., Bieber, L. L., and Tolbert, N. E. (1978) Anal. Biochem. 8 7 , 206-210 32. Ross, A.C. (1981) Anal. Biochem. 115,324-330 33. Bamberger, M., Glick, J. M., and Rothblat, G. R. (1983) J. Lipid Res. 2 4 , 869-876 34. Rustow, B., and Kunze, D. (1985) Biochim. Biophys. Acta 8 3 5 , 273-278

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35. Bligh, E. G., and Dyer, W. J. (1959) Can. J. Biochern. Physiol. 37,911-917 36. Touchstone, J. C., Chen, J. C., and Beaver, K. M. (1979) Lipids 15,61-62 37. Marinetti, C.V. (1962) J. Lipid Res. 3,1-20 38. Houslay, M. D., and Stanley, K. K. (eds) Dynamics of Biological Membranes, pp. 106-116, John Wiley and Sons, New York 39. Bador, H., Morelis, R., and Louisot, P. (1984) Biochirn. Biophys. Acta 800, 75-86 40. Stadtman, E. R. (1957) Methods Enzymol. 3 , 931-941 41. MacDonald, P. N., and Ong, D. E. (1988) FASEB J. 2 , Abstr. 3182 42. Davidson, S. C., and Wills, E. D. (1974) Biochem J. 1 4 0 , 461468 43. Suzuki, Y., Depierre, J. W., and Ernster, L. (1980) Biochim. Biophys. Acta 601, 532-543 44. Waskell, L.,Koblin, D., and Canova-Davis, L. (1982) Lipids 17, 317-320

45. Goldstein, J. L., Faust, F. R., Dygos, J. H., Chorvat, R. J., and Brown, M. S. (1978) Proc. Nutl. Acad. Sci. U. S. A. 75, 18771881 46. Simpson, E. R., and Burkhart,M. F. (1980) Arch. Biochem. Biophys. 2 0 0 , 77-85 47. Bouin, A., Bolender, R. P., and Weibel, E. W. (1977) J. Cell Biol. 72,441-455 48.Huang, H. S., andGoodman, D. S. (1965) J. Biol. Chen. 240, 2839-2844 49. Futterman, S., and Andrews, J. S. (1964) J. Biol. Chem. 2 3 9 , 4077-4080 50. Ball, M. D., and Olson, J. A. (1988) FASEB J. 2 , Abstr. 3183 51. Middelton, B. (1987) Biochem. Biophys. Res. Cornmun. 145,350356 52. Jauhiainen, M., and Dolphin, P. J. (1986) J. Biol. Chem. 261, 7032-7043 53. Dingle, J. T., and Lucy, J. A. (1962) Biochem. J. 84,611-621

Microsomal Esterification of CRBP-bound Retinol 0.28 M sucrose 0 . 1 M Tr15 - HC1, pH 7 . 4 were lncublted with 6 mM C a C l , 150 m N a C 1 , 1% BSA. with or without the addltlon o € 4 0 u l of PLA2 (1.9 units) I D a flnal volume of 6.9 m l . The IncubatLon was for 5 minutes at 37' C followed the TO some tubes w e added BSA to a flnal addltlon Of 165 p l of 0 . 3 H EDTA ~

concentration O f 5 % and to other tubes an equal volume Of buffer. The tubes were lncubated another 5 mlnutes at 37O c before a d d m g 10 ml of ~ c ecold An buffer. The m~crosomes were isolated by ultrd~en~rlfugatlona s above allquot of resuspended microsomes was used to lnvestlgate the esterlflcatlon of CRBP-bound retlnol following PLA: treatment. and the Iemalolng mlCrosOmes were dlvlded m t o two groups to lnvestugate the effect Of llpld regeneratLon on the esterlficatlon Of CRBP-bound retinol. Micmsomes from the pLA2 expeiment were Incubated wlth a llpld generating System a s descrlbed by R Y S t O Y and K u n z e ( 3 4 1 to effect the synthesis o f I n addltlon to the resuspended phosphatldylcholine from dlacylqlycerol. mrcrosomes, the lncubarion tube contamed, at a f m a l concentratron i n 50 m HEPES. pH 7.8: 0 . 1 ml4 EDTA, 10 mH cysteine-HCl. 0 2 Ru( COR, 150 mM K C 1 . 3mM MqC12, 3.5 mM ATP, 0.5 mM glyceral-3~phosphate. 1 mH chollne chloride. 1 mM CDP-chollne and 20 pM palrnltlc acrd complexed to BSR (molar ratlo 5 : l ) . The control incubations contalned microsomes treated vlth cysteine-HC1. A11 tubes w e ~ e Incubated for 20 minutes at 31° c after removlnq them from l e e . then dlluted with rce Cold buffer and >Solated as descrrbed above The treated mlciosornes were resuspended l n 0 . 1 5 M potassium phosphate buffer, pH 7.0. One allquot was assayed for esterlflcatlan Of CRBP-bound retlnai a s outlined above. and another dllquot was used for lipld analysis.

Isolation and ~. analysls of m i ~ r o ~ o m allplds l

" "

H1CrOSomal llpkds were extracted followlnq the procedure of Bllqh and Dyer 05). The llplds were brought to a known Volume in chloroform plus BHT and stored at -20" C Untll they were analyzed. Phospholipids were separated by ID thin l a y e r chromatography u s i n g s l l i c a g e l G plates developed c h l o ~ o f o ~ : e t h a n o l : t ~ ~ ~ t h y l( 3~0 ~: 3~4 :"3 =0 :~B ~ ~v /tv ~ ) ~ (36). Liplds were vrsuallzed by spraylng the plates wlth 0.025% rhodamlne6G I" ethanol and ldentifled by comparison to known standards run I" a parallel lane. The gel cantalnlnq each l i p l d band was scraped Into a test tube, and the llpld was quantltated u s l n q the method of Marlnett1 (371. After color development and before reading the Optical density of the sample. the tubes were centrifuged to sedment the s111ca g e l .