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somes from pancreas that is not PMSF-sensitive and consequently is not LRAT or ARAT. It might be a reverse esterase reaction. At this time, sensitivity to both PMSF and NEM appears to be diagnostic for LRAT activity.

[52] B i l e S a l t - I n d e p e n d e n t R e t i n y l E s t e r H y d r o l a s e Activities Associated with Membranes of Rat Tissues B y E A R L H . HARRISON a n d JOSEPH L . NAPOLI

Introduction Hydrolysis of retinyl esters plays a major role in the overall metabolism of vitamin A in the body. In liver, retinyl ester hydrolysis occurs both during the uptake of chylomicron remnants and prior to the mobilization of retinyl esters stored in cellular lipid droplets. 1Although the majority of the endogenous retinoids in mammals consists of retinyl esters stored in the liver, liver is not the only site of retinyl ester storage. In kidney, lung, and testes esters can represent as much as 70% of the total neutral retinoids. 2-4 Being the enzymes required to release retinol accumulated and stored as esters, retinyl ester hydrolases are likely to play a key role in retinoid metabolism. Most studies of the hydrolysis of long-chain retinyl esters in liver and other tissues have focused on a neutral retinyl ester hydrolase activity that is markedly stimulated by millimolar concentrations of bile salts or their analogs. 3,5-1°This activity has an unusual distribution among subcellular fractions of rat liver, with most of the activity being distributed in the "nuclear" fraction and high-speed supernatant fraction of homogenates; D. S. Goodman and W. S. Blaner, in "The Retinoids" (M. B. Sporn, A. B. Roberts, and D. S. Goodman, eds.), Vol. 2, p. 1. Academic Press, New York, 1984. 2 j. B. Williams, B. C. Pramanik, and J. L. Napoli, J. Lipid Res. 25, 638 (1984). 3 j. L. Napoli, A. M. McCormick, B. O'Meara, and E. A. Dratz, Arch. Biochem. Biophys. 230, 194 (1984). 4 D. S. Goodman, H. S. Huang, and T. Shiratori, J. Lipid Res. 6, 390 (1965). 5 E. H. Harrison, J. E. Smith, and D. S. Goodman, J. Lipid Res. 20, 760 (1979). 6 j. H. Prystowsky, J. E. Smith, and D. S. Goodman, J. Biol. Chem. 256, 4498 (1981). 7 W. S. Blaner, J. H. Prystowsky, J. E. Smith, and D. S. Goodman, Biochim. Biophys. Acta 794, 419 (1984). 8 W. S. Blaner, G. Halperin, O. Stein, Y. Stein, and D. S. Goodman, Biochim. Biophys. Acta 794, 428 (1984). 9 D. A. Cooper and J. A. Olson, Biochim. Biophys. Acta 884, 251 (1986). i0 D. A. Cooper, H. C. Furr, and J. A. Olson, J. Nutr. 117, 2066 (1987).

METHODS 1N ENZYMOLOGY, VOL. 189

Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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very little activity is associated with the microsomal fraction. In addition, the absolute activity varies markedly among liver homogenates from individual rats. Also, when assayed in the presence of millimolar concentrations of bile salts (e.g., cholate) most preparations show higher apparent specific activity toward cholesteryl oleate and/or triolein than toward retinyl palmitate. We have recently detected bile salt-independent retinyl ester hydrolases in rat liver, kidney, lung, and testes.l 1,12These enzymes are clearly distinct from the previously described bile salt-dependent enzyme(s). This chapter describes the properties of these bile salt-independent retinyl ester hydrolases in liver and kidney, the two most studied tissues. Assay Retinyl ester hydrolase activity is determined using the sensitive, radiometric assay of Prystowsky et al.,6 as described in detail elsewhere in this volume. 13Briefly, reactions are carried out in a final volume of 0.2 ml by adding buffer and a source of enzyme in a final volume of 0.19 ml. Reactions are initiated by adding radioactive retinyl palmitate in 10/.d of ethanol and incubated at 37°. Reactions are terminated by adding 3.25 ml of methanol/chloroform/heptane (1.4: 1.25: 1) and 1 ml of potassium carbonate/borate buffer, pH 10, and the product, radioactive palmitic acid, is extracted into the alkaline aqueous upper phase. An aliquot of this phase is mixed with scintillation solvent and counted in a liquid scintillation counter. The amount of palmitic acid released is determined from the partition coefficient (-75%) of palmitic acid and the specific radioactivity of the substrate. We have used both [1-14C]palmitic acid and [9,10-3H]palmitic acid f o r the preparation of labeled retinyl palmitate. Retinyl [1-14C]palmitate is used at final specific activities of 2.5-10.0 /zCi//zmol. Retinyl [9,103H]palmitate is used at final specific activities of 17.0-23.0/~Ci/mol. For work with liver homogenates we routinely assay with 50 mM Trismaleate, pH 8.0 (the pH optimum), and I00/zM retinyl [1-14C]palmitate. Under these conditions the reaction is linear for 30 min and with up to approximately 15/xg of liver homogenate protein. For work with kidney homogenates routine assays are carded out using 10 mM HEPES, pH 8.0, and 100 ~M retinyl [9,10-3H]palmitate. Under these conditions the reaction is linear for 45 min and with up to 10/~g protein. 11 E. H. Harrison and M. Z. Gad, J. Biol. Chem. 264, 17142 (1989). 12 j. L. Napoli, E. B. Pacia, and G. J. Salerno, Arch. Biochem. Biophys. 274, 192 (1989). ~3D. A. Cooper, this volume [55].

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Heptatic Retinyl Ester Hydrolases We present below information on the properties of the bile salt-independent retinyl ester hydrolase (REH) activity of rat liver, n In particular, three areas are discussed which serve to distinguish the bile salt-independent hydrolase(s) from the bile salt-dependent enzyme(s).

Variability i~ Activity When homogenates of the livers of individual rats are assayed for retinyl palmitate hydrolase activity in the presence of 20 mM cholate, there is marked variation in absolute activity. 5,6 Table I shows the neutral retinyl palmitate hydrolase (RPH) activity for eight individual animals assayed in the presence or the absence of cholate. As expected the activities in the presence of cholate varied extensively. In marked contrast, the activities in the absence of bile salt showed little variation. Indeed, in this series of animals the addition of cholate inhibited the neutral RPH activity in seven of the homogenates. These results are consistent with there being a neutral, bile salt-independent RPH activity that is inhibited by bile salt and a separate bile salt-dependent activity present to various extents in the livers of individual animals.

TABLE I BILE SALT-DEPENDENT AND BILE SALT-INDEPENDENT RETINYL ESTER HYDROLASE ACTIVITIES IN INDIVIDUAL RAT LIVERS a

Retinyl ester hydrolase activity (nmol/h/g liver) Rat

No cholate

20 m M cholate

1 2 3 4 5 6 7 8

1244 1143 1044 881 1268 1567 1224 1151

0 0 0 135 278 350 876 2339

a Whole homogenates of rat livers were assayed in 30-min incubations containing 50 m M T r i s maleate buffer (pH 8.0) and 100 /.~M retinyl palrnitate, with and without 20 m M cholate. Reproduced with permission. 11

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Subcellular Distribution

Table II provides information on the distribution of the neutral, bile salt-independent RPH activity among subcellular fractions prepared by differential centrifugation of rat liver homogenates. Most of the enzyme activity is recovered in the microsomal fraction of the homogenate, and its distribution is very similar to those of marker enzymes for the endoplasmic reticulum (glucose-6-phosphatase and o-nitrophenyl acetate esterase) and plasma membrane (alkaline phosphodiesterase). Thus, in terms of subcellular distribution, the bile salt-independent RPH activity is entirely unlike the previously studied, cholate-stimulated RPH that is nearly absent from microsomes and, rather, is found associated with the "nuclear" and high-speed supernatant fractions of the liver homogenate. T A B L E II DISTRIBUTIONS OF BILE SALT-INDEPENDENT RETINYL ESTER HYDROLASE AND MARKER ENZYMES IN SUBCELLULAR FRACTIONS OF RAT LIVER a Percentage of recovered a m o u n t b Constituent

N

ML

P

S

Protein (4) N-Acetyl-fl glucosaminidase (lysosomes) (4) Glucose-6phosphatase (ER) (3) Esterase a (ER) (1) Alkaline phosphodiesterase (plasmalemma) (4) Retinyl ester hydrolase (4)

24 -+ 4 19 -+ 7

20 -+ 4 48 -+ 6

19 -+ 2 ' 28 -+ 4

37 -+ 6 4 -+ 4

26 -+ 2

I 1 -+ 3

60 -+ 5

3 -+ 0

105 --+-8

29

7

60

4

91

23 --- 5

17 --+ 9

58 -+ 13

2 - 1

110 + 13

56 --- 9

13 --- 6

99 --- 11

23 --- 5

8 + 5

R e c o v e r y (%)c 97 -+ 5 94 -+ 10

R e p r o d u c e d with permission. H b R e s u l t s are given as m e a n s --- 1 S.D. T h e n u m b e r of e x p e r i m e n t s is given in parentheses. Relative values are p r e s e n t e d for the distribution of each constituent a m o n g the four fractions: nuclear (N), m i t o c h o n d r i a l - l y s o s o m a l (ML), microsomal (P), and supernatant (S). Values are percentages o f the constituent recovered in each fraction relative to the a m o u n t s r e c o v e r e d in all four fractions (taken as 100%). c R e c o v e r y r e p r e s e n t s the total a m o u n t recovered in all four fractions relative to the a m o u n t in the whole h o m o g e n a t e . d " N o n s p e c i f i c " esterase w a s a s s a y e d using o-nitrophenyl acetate as the substrate.

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BILE SALT-INDEPENDENT RETINYL ESTER HYDROLASE T A B L E III RELATIVE SPECIFIC ACTIVITIES OF BILE SALT-INDEPENDENT RETINYL ESTER HYDROLASE AND MARKER ENZYMES IN PLASMA MEMBRANE FRACTIONS OF RAT LIVER HOMOGENATES a'b Exp. 1

Exp. 2

Constituent

RSA C

Recovery d

RSA c

Recovery d

Protein N-Acetyl-/3-glucosaminidase Glucose-6-phosphatase Esterase e Alkaline phosphodiesterase Retinyl ester hydrolase

1 1 2 -29 35

96 82 103 -89 115

1 2 1 1 15 19

89 91 96 89

101 124

a Reproduced with permission. N b Plasma membrane-rich fractions were isolated from the microsomal fraction by the method of O. Touster, N. N. Aronson, J. T. Dulaney, and H. Hendrickson, J. Cell Biol. 47, 604 (1970). c Relative specific activity (RSA) is the percentage of activity recovered in the plasma

membrane fraction divided by the percentage of total homogenate protein recovered in the fraction. The isolated plasma membrane fractions contained 0.5-1.0% of the homogenate protein. d Recoveries represent the sum of the constituent in the plasma membrane-rich fraction and all other fractions relative to the amount observed in the unfractionated homogenate. e " N o n s p e c i f i c " esterase was assayed using o-nitrophenyl acetate as the substrate.

In order to further resolve the membrane components of the microsomal fraction, fractions rich in plasma membranes are prepared from it. The analysis of these preparations is presented in Table III. The results agree well with those presented in the original description of the method. Thus, the isolated fraction is highly enriched in plasma membrane fragments as assessed by the enrichment of alkaline phosphodiesterase activity. Significantly, the preparation shows no enrichment of endoplasmic reticulum markers such as glucose-6-phosphatase and o-nitrophenyl acetate esterase. The bile salt-independent REH activity is enriched in the isolated plasma membrane fraction to the same extent as alkaline phosphodiesterase. This result suggests that most or all of the bile salt-independent REH activity is localized in the plasma membrane. In this regard it is entirely different from the cholate-dependent activity, which has been shown to be absent in highly enriched plasma membrane fractions of rat liver.

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Differential Responses to Pancreatic Cholesteryl Ester Hydrolase Antibodies A final demonstration that the bile salt-independent RPH activity is due to an enzyme(s) different than that (those) responsible for the bile salt-stimulated hydrolysis is indicated by the differential inhibition of the activities by antibodies to pancreatic cholesteryl ester hydrolase (EC 3.1.1.13). In order to make this comparison, whole homogenates of rat liver are prescreened for retinyl palmitate hydrolase activity in the presence and absence of 20 mM cholate. A homogenate is then chosen that shows substantial bile salt-dependent REH activity. As shown in Fig. 1, incubation of this homogenate with anti-pancreatic cholesteryl ester hydrolase IgG led to marked inhibition (70%) of the bile salt-dependent RPH activity without affecting the bile salt-independent activity. In a separate experiment (data not shown), 15/xg of IgG led to 80% inhibition of the bile salt-dependent activity without affecting the bile salt-independent activity. In both experiments, the remaining activity may be due to the hydrolysis of the substrate by the bile salt-independent hydrolase(s) also present in these whole homogenates.

~Q. 2000 H-I

Bile salt-independent retinyl ester hydrolase activities associated with membranes of rat tissues.

[52] BILE SALT-INDEPENDENT R E T I N Y L ESTER HYDROLASE 459 somes from pancreas that is not PMSF-sensitive and consequently is not LRAT or ARAT...
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