- Email: [email protected]
[44] Preparation of microsomal β-glucuronidase and its membrane anchor protein, egasyn
[44]
EGASYN
AND fl-GLUCURONIDASE
MEMBRANE
BINDING
557
liver, but occurs to a large extent also in skeletal muscle. 87 Kidney and heart muscle also respond similarly. 82 Thyroid H o r m o n e s and M e m b r a n e Degradation
In contrast to what is observed after induction of the endoplasmic reticulum by phenobarbital (see above), there is a dissociation of membrane and enzyme degradation of induced skeletal muscle mitochondria, which speaks against an autophagic degradation mechanism in this case. 88 Transitions from hyperthyroid to normal and from normal to hypothyroid states cause a decrease in enzyme level, but no change, or even an increase, in the total mitochondrial membrane volume. Phthalate Ester Induction
As described in the section on peroxisomes, DEHP induces mitochondria also. 61-63 Table IV lists a few functions that are found to be induced in
isolated liver mitochondria after 2 weeks' treatment of rats with 2% DEHP in the diet. A detailed analysis of the enzyme pattern after such induction has not yet been performed, and, thus, the nature of the newly synthesized mitochondria is not known. Acknowledgments The work from the authors' laboratories cited here was supported by grants from the Swedish Medical and Natural Science Research Council and NIH Grant No. 1 RO1 CA 26261-02 awarded by the National Cancer Institute, DHEW, Bethesda, Maryland. 88L. Ernster, Fed. Proc., Fed. Am. Soc. Exp. Biol. 24, 1222(1965).
[44] P r e p a r a t i o n o f M i c r o s o m a l f l - G l u c u r o n i d a s e a n d Its Membrane Anchor Protein, Egasyn
By ALDONS J. LUSIS Acid hydrolases such as fl-glucuronidase are typically located in lysosomes, but in certain tissues of mammals substantial amounts of the enzyme are also present in the membranes of the endoplasmic reticulum. This has been demonstrated using both tissue fractionation and histochemical staining reactions. The microsomal enzyme is firmly bound to METHODS IN ENZYMOLOGY, VOL. 96
Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181996-5
558
BIOGENESIS AND ASSEMBLY OF MEMBRANE PROTEINS
8_
Eg
i~
[
S
EGASYN
~ M 3
5--
[44]
Gus
~ GLUCURONIDASE POLYPEPTIDE
LYSOSOMES
ENDOPLASMIC RETICULUM
Fro. l. Model for intracellular localization of mouse fl-glucuronidase. Six intracellular forms of/3-glucuronidase have been identified, The enzyme in lysosomes consists of a single form, termed L, that is a tetramer of identical subunits, each of molecular weight 71,000. The microsomal enzyme, on the other hand, occurs primarily as a series of higher molecular weight complexes, termed M1, M2, M3, and M4; these consist of a glucuronidase core of slightly different structure, X, complexed with one to four chains of a second polypeptide, egasyn. The X and L subunits are derived from a common structural gene, Gus, on chromosome 5, and differ by modification. Egasyn is an integral membrane glycoprotein of molecular weight 64,000. A mutation at the Eg gene on chromosome 8, present in strain YBR, results in the absence of immunoreactive egasyn and the inability to maintain/3-glucuronidase in endoplasmic reticulum membranes. This and other evidence suggest that egasyn stabilizes the membrane binding of/9-glucuronidase (see text). Reprinted from Lusis and Paigen. 4
membrane and detergents are required to extract it, while the enzyme in lysosomes is released upon rupture of the lysosomal membrane.l-3 A combination of biochemical and genetic approaches suggests a model for the localization of/3-glucuronidase in mouse tissues 4 (Fig. 1). Genetic studies indicate that the enzyme at both intracellular sites is derived from the same structural gene. The fl-glucuronidase in lysosomes occurs as a free tetramer of molecular weight about 280,000, whereas most of the enzyme in microsomes is complexed with an integral membrane protein, egasyn. The complexes can be extracted intact from membrane using Triton X-100, and the egasyn isolated from these complexes is a glycoprotein of molecular weight 64,000 that is structurally distinct from glucuronidase. The microsomal and lysosomal glucuronidase tetramers C. B. C. de Duve, R. Pressman, R. Gianetto, R. Wattiaux, and F. Appelmans, Biochem. J. 60, 604 (1955). 2 K. Paigen, Exp. Cell Res. 25, 286 (1961). 3 W. H. Fishman, S. S. Goldman, and R. DeLellis, Nature (London) 213, 457 (1967). 4 A. J. Lusis and K. Paigen, Isozymes: Curr. Top. Biol. Med. Res. 2, 63 (1977).
[44]
EGASYN AND fl-GLUCURONIDASE MEMBRANE BINDING
559
(termed X and L, respectively) differ by covalent modification, the microsomal form being slightly larger and less negatively charged than the lysosomal form, but the functional significance of these differences is not known. Several observations suggest that egasyn serves to stabilize the binding of fl-glucuronidase to membrane. Most convincing is the finding that a mouse strain that lacks egasyn is unable to maintain fl-glucuronidase in endoplasmic reticulum membrane. 4-7 In addition, among tissues and during development the levels of fl-glucuronidase in microsomes are correlated with the levels of egasyn. For example, liver and kidney contain 2050% microsomal glucuronidase and have relatively high levels of egasyn, whereas spleen contains only trace levels of microsomal glucuronidase and lacks detectable egasyn. Thus, egasyn appears to function as a membrane anchor for the highly polar glucuronidase tetramer. 4 Other mammals that have been examined, including rats, rabbits, and humans, also have substantial amounts of microsomal fl-glucuronidase in liver. 4 A membrane protein that may be analogous to egasyn has been isolated from rat liver microsomes. 8 The protein was extracted from microsomes with 2% Triton X-100, and it specifically binds B-glucuronidase. Unlike the mouse microsomal fl-glucuronidase complexes, the complex between rat glucuronidase and the binding protein is dissociated by relatively low concentrations of Triton X-100. Rabbit liver microsomal glucuronidase is extracted by Triton X-100 as a series of complexes with molecular weights of roughly 280,000, 330,000, 370,000, 420,000, and 500,000; thus, as in mice, the forms increase stepwise in molecular weight increments of roughly 55,000. The rabbit fl-glucuronidase complexes are considerably more stable than those of mice and rats, although they can be dissociated using 8 M urea. 9 fl-Glucuronidase Purification
Assay. Mammalian fl-glucuronidase is capable of hydrolyzing a wide variety of natural and synthetic fl-o-glucuronides. A variety of simple fluorometric and spectrophotometric assays of the enzyme, using artificial substrates such as the glucuronides of 4-methylumbelliferone and p-nitrophenol, have been developed. In terms of simplicity and sensitivity the fluorometric assay, employing 4-methylumbelliferyl-fl-o-glucuronide as the substrate, is the procedure of choice. R. 6 A. 7 A. 8 L. 9 R.
E. Ganschow and K. Paigen, Proc. Natl. Acad. Sci. U.S.A. 58, 938 (1967). J. Lusis, S. Tomino, and K. Paigen, Biochem. Genet. 15, 115 (1977). J. Lusis, S. Tomino, and K. Paigen, J. Biol. Chem. 251, 7753 (1976). D. Strawser and O. Touster, J. Biol. Chem. 254, 3716 (1979). T. Dean and M. Messer, Comp. Biochem. Physiol. 548, 107 (1976).
560
B I O G E N E S IAND S ASSEMBLY OF MEMBRANE PROTEINS
[44]
I utilize the following assay protocol: 7 The sample is incubated at 37° with 0.4 mM 4-methylumbelliferyl-/3-o-glucuronide (Sigma Chemical Co.), 0.1% Triton X-100 (Sigma), and 0.1 M sodium acetate, pH 4.6, in a total volume of 0.1 ml. The reaction is stopped after 30 min by immersion in an ice bath followed by the addition of 1.0 ml of 0.1 M sodium carbonate. The fluorescence is measured with an Aminco fluorometer (catalog No. 4-7439) equipped with a Corning 7-60 excitation filter (peak wavelength 360 nm) and Kodak 2A emission filter (passing wavelengths above 415 nm), or an instrument with similar capability. One unit of activity is defined as that amount of enzyme that will hydrolyze 1/zmol of substrate per hr at 37°. At very low protein concentrations, the inclusion of 0.1% albumin during the incubation helps to stabilize the enzyme. If necessary, the sensitivity of the assay can be increased by prolonging the incubation period to 24 hr or more. Electrophoresis. The multiple forms of mouse/3-glucuronidase (L, X, M) are resolved using a nondenaturing polyacrylamide gel electrophoresis system containing Tris-glycine, pH 8.1, and 7% acrylamide. Glucuronidase activity is visualized by staining with naphthol-AS-BI-glucuronide (Sigma) as the substrate. ~°:1 The most sensitive technique for analyzing charge variation of the glucuronidase tetramer is isoelectric focusing. I have had best results using a gel system containing 6% acrylamide, 6 M urea, 1% pH 3.5-10 Ampholines and 1% pH 5-7 Ampholines. 11 Under these conditions the glucuronidase tetramer remains intact and the gel can be stained for enzyme activity, but the M form complexes between glucuronidase and egasyn are disrupted. Lysosomal (L Form) fl-Glucuronidase. Lysosomal fl-glucuronidase has been purified by conventional fractionation from several mammalian sources including bovine liver, human liver, rat liver and preputial gland, and mouse liver, kidney, and urine. 11,12Rat preputial gland 13 and mouse urine 11 are especially rich sources, permitting rapid and simple purification of milligram quantities of the enzyme. Antisera. High-titer, monospecific antibodies to/3-glucuronidase can be raised using rabbits or goats.14 Rabbit antiserum is prepared by inoculating outbred rabbits at multiple subcutaneous sites with 0.2 mg of purified L-form glucuronidase in Freund's complete adjuvant. Four weeks later, booster injections are given with 0.2 mg of enzyme in incomplete adjuvant. Serum is taken about 1 week after the boost. ~0R. T. Swank and K. Paigen, J. Mol. Biol. 77, 371 (1973). ll A. J. Lusis and K. Paigen, J. Biol. Chem. 253, 7336 (1978). 12 O. Touster, this series, Vol. 50, p. 488. 13R. K. Keller and O. Touster, J. Biol. Chem. 250, 2739 (1975). 14 S. Tomino, K, Paigen, D. Tulsiani, and O. Touster, J. Biol. Chem. 250, 8503 (1975).
[44]
EGASYN AND fl-GLUCURONIDASE MEMBRANE BINDING
561
Antibody Affinity Chromatography. Mouse glucuronidase can be readily purified from liver, kidney, and other tissues by antibody affinity chromatography. ~ Both microsomal (X) and lysosomal (L) forms of the enzyme can be purified, since immunologically they are not discernibly different. The protocol is as follows: Immunoglobin is purified from antiserum by repeated precipitation with ammonium sulfate at 45% saturation. The precipitate is dissolved in 0.02 M Tris, 0.15 M NaCI, pH 7.4 (TBS buffer), and dialyzed against the same buffer. The immunoglobin is coupled to cyanogen bromide-activated Sepharose 4B in 0.1 M NaHCO3, 0.5 M NaC1, and afterward the gel is washed extensively with coupling buffer and with 0.1 M sodium acetate, 0.5 M NaC1, pH 4.6. The gel is placed in a column and washed with 0.1 M sodium acetate, pH 5.2, containing 8 M urea to remove any loosely bound material. It is then equilibrated with TBS buffer, and the sample is applied in TBS buffer. Tissue homogenates, extracted with detergents such as Triton X-100 and centrifuged at high speed to remove particulate material (see below), are suitable for chromatography. For isolation of L-form enzyme, however, we have found it useful partially to purify the enzyme using heat treatment at acid pH prior to chromatography. ~ After application of the sample, the column is washed with at least 5 column volumes of (a) TBS buffer, (b) 0.02 M sodium acetate, pH 5.2, and (c) 0.02 M Tris, 0.3 M NaC1, pH 7.4. Glucuronidase is then eluted by applying 0.1 M sodium acetate, pH 5.2, containing 8 M urea to the column at a rate of about 4 column volumes per hour. The glucuronidase is normally eluted within about 3 column volumes, and the urea is removed by dialysis. Although glucuronidase is relatively resistant to inactivation by urea, dialysis is normally begun shortly after elution of the enzyme. The capacity of the gel can be estimated using a small column and increasing amounts of enzyme; it is possible to bind several hundred units of enzyme (2400 units per milligram of protein) per milliliter of gel. Normally, nearly all of the enzyme binds to the column, and recovery of activity averages 60-80%. After affinity chromatography, preparations contain only traces of contaminants, and these can be removed by chromatography on a 2.6 × 55 cm column of Ultrogel Ac-44 equilibrated with TBS. When stored at 4° in TBS containing 0.1% sodium azide, the affinity gel is stable for more than a year. Similar antibody affinity chromatography procedures have been reported for the isolation of glucuronidase from rat tissue. ~5,16 Purification of Microsomal Glucuronidase from Mouse Liver or Kidney. Tissues are homogenized in a Polytron homogenizer with 10 volumes 15 j. W. Owens, K. L. Gammon, and P. D. Stahl, Arch. Biochem. Biophys. 166, 258 (1975). ~6M. Himeno, Y. Nishimura, H. Tsuji, and K. Kato, Eur. J. Biochem. 70, 349 (1976).
562
BIOGENESIS AND ASSEMBLY OF MEMBRANE PROTEINS
[44]
of 0.02 M imidazole, pH 7.4. The homogenate is centrifuged at high speed (100,000 g, 60 min), and the supernatant solution (containing the bulk of the L-form enzyme) is removed. The pellet is suspended in 0.02 M imidazole, pH 7.4 (a volume equal to that of the original homogenate) using a Polytron homogenizer. The solution is recentrifuged at high speed, and the pellet (crude membrane) is suspended in an equal volume of 0.02 M imidazole, pH 7.4, containing 2% Triton X-100. This solution is centrifuged (100,000 g, 1 hr), and the supernatant solution, containing more than 90% of the activity, is carefully decanted. Glucuronidase is purified from this solution using antibody affinity chromatography as described above, except that after application of enzyme the column is first washed with several volumes of TBS containing 0.1% sodium deoxycholate (Sigma) to remove egasyn associated with glucuronidase. Enzyme yields average about 40% of the original membrane-bound activity. Kidneys from androgen-treated mice are the richest source of microsomal glucuronidase. The purified preparations contain about 40% L-form enzyme and about 60% X-form enzyme, judging from staining intensity after polyacrylamide gel electrophoresis. It is possible to obtain enzyme preparations with a greater proportion of X-form enzyme by fractionation and more extensive washing of membrane preparations; however, about 510% of the total L-form enzyme is tightly associated with membrane and can be extracted only with detergent. ~°,17 The X and L electrophoretic forms of glucuronidase can be separated on the basis of charge by DEAE-Sephadex chromatography at pH 6.9 (Fig. 2). Fractions obtained after chromatography are examined by electrophoresis at pH 8.1, and those containing enzyme approximately corresponding in mobility to the X or L forms observed in homogenates are pooled (these are termed the X and L fractions). When subjected to electrophoresis in the presence of sodium dodecyl sulfate, the X fraction migrates as a single band corresponding to an apparent molecular weight of about 74,000. The bulk of the L fraction migrates as a band of molecular weight 71,000, not discernibly different in mobility from L-form enzyme isolated from the lysosomal component of liver and kidney. When subjected to isoelectric focusing in polyacrylamide gels, the X fraction can be resolved into seven forms of about equal staining intensity, ranging from pI 6.0 to pI 6.6. The L fraction also contains several forms, ranging from pl 5.7 to pI 5.9, and it closely resembles the enzyme isolated from the lysosomal component of liver and kidney. The size difference between X and L/3-glucuronidase may result from differences in carbohydrate content, since after treatment with endoglycosidase H both forms comigrate on SDS gels with an apparent molecular weight of about 70,000. Endogly~7A. J. Lusis and K. Paigen, unpublished data (1977).
[44]
563
EGASYN AND fl-GLUCURONIDASE MEMBRANE BINDING 76+88~ 76 80 84 88 ~76~8 {--)FRACTIONNUMBER
g B
100
T ORIGIN IiBI ~-~ =-a ~..., ,__, i==1
(+)
B 15
J
f
--
__ Z so 80
g,
8
z c~
70 z
5
0 rr
60
5
z
;¢ ~
I 0
--
7O
80
90
100
50 110
FRACTION NUMBER FIG. 2. Separation of X and L mouse/3-glucuronidase using DEAE-Sephadex chromatography. Glucuronidase was isolated from crude microsomes of pooled livers and kidneys of 100 male C57BL/6J mice using antibody affinity chromatography. The enzyme (13.6 ml, containing 260 units of enzyme activity) was then dialyzed against 0.02 M imidazole, pH 6.9, and applied to a 0.6 x 10 cm column of DEAE-Sephadex A-25, equilibrated with 0.02 M imidazole, pH 6.9. Essentially all activity bound to the column. The column was washed with several volumes of equilibration buffer, and the activity was eluted using a linear salt gradient, from 0 to 0.1 M NaC1, in equilibration buffer (total volume 200 ml) at a rate of about 20 ml/hr. Fractions of 2.4 ml were collected, glucuronidase activity was determined, and aliquots from peak fractions were subjected to polyacrylamide gel electrophoresis at pH 8.1, followed by staining for glucuronidase activity. The inset shows the pattern of glucuronidase activity after electrophoresis of fractions 76, 80, 84, 88, and mixtures of 76 and 88 (30-tzl aliquots were used). Fractions 74-81, corresponding approximately to X enzyme in electrophoretic mobility, and fractions 88-90, corresponding approximately to L enzyme in mobility, were pooled for further characterization (see text).
cosidase treatment has little effect on the charge of glucuronidase.17 The charge difference, on the other hand, may be due in part to a C-terminal peptide that can be removed from X-form enzyme by protease treatment (Fig. 3). The proteases tested did not discernibly affect the apparent molecular weight of glucuronidase as judged by electrophoresis in sodium dodecyl sulfate, suggesting that the peptide removed from X by protease treatment is short.17 Rat Microsomal fl-Glucuronidase. Rat liver microsomal fl-glucuronidase can be isolated by similar procedures. As in the mouse, the rat microsomal enzyme appears to be slightly larger and less acidic than the lysosomal enzyme.~5,16 Chemical analyses suggest that the two forms differ in carbohydrate and amino acid composition? 8J9 i8 D. R. P. Tulsiani, H. Six, and O. Touster, Proc. Natl. Acad. Sci. U.S.A. 75, 3080 (1978). ~9 M. Himeno, Y. Nishimura, K. Takahashi, and K. Kato, J. Biochem. (Tokyo) 83, 511 (1978).
564
BIOGENESIS AND ASSEMBLY OF MEMBRANE PROTEINS pH
5.5
6.0
/[EIIrl iJ/[~lJll ./ [ !~ll EI f //" ill, ill IJ I I~lllll II I I'~,11
[44]
6.5
I I f I I I
i FIG. 3. Effect of protease treatment on the isoelectric focusing pattern of X- and L-form glucuronidase. Isolated glucuronidase preparations (Fig. 2) were treated with proteases and subjected to isoelectric focusing as described. HThe gels were stained for glucuronidase activity and scanned at 550 nm. The pH gradient along the gel is shown at the top. About 0.5 unit of enzyme activity was applied to each slot, and protease treatment had no effect on total activity. Shown are absorbance tracings for control X form glucuronidase (X), X form treated with aminopeptidase M (X + AP), X form treated with carboxypeptidase Y (X + CP), control L form (L), and L form treated with carboxypeptidase Y (L + CP). Trypsin and chymotrypsin treatment gave the same results as carboxypeptidase Y.
Isolation and A s s a y of E g a s y n
Isolation from Mouse Tissues. I n the m o u s e , m i c r o s o m a l glucuronid a s e - e g a s y n c o m p l e x e s c a n be e x t r a c t e d intact f r o m m e m b r a n e s with Triton X-100. l° T h e m i c r o s o m a l g l u c u r o n i d a s e can then be specifically precipitated using a n t i - g l u c u r o n i d a s e a n t i b o d y , 2° and e g a s y n can be selectively solubilized f r o m s u c h i m m u n o p r e c i p i t a t e s b y disrupting the glucu r o n i d a s e - e g a s y n c o m p l e x with heating at 50 ° o r with 0.1% s o d i u m d e o x y c h o l a t e . 7 It also is feasible to isolate e g a s y n b y binding m i c r o s o m a l g l u c u r o n i d a s e c o m p l e x e s to a n t i - g l u c u r o n i d a s e affinity c o l u m n s and selectively eluting e g a s y n b y passing 0.1% s o d i u m d e o x y c h o l a t e in T B S t h r o u g h the columns.17 2o S. Tomino and K. Paigen, J. Biol. Chem. 250, 116 (1975).
[44]
EGASYN A N D f l - G L U C U R O N I D A S E M E M B R A N E B I N D I N G
565
Homogenate Centrifuge 2500 g, i0 min
Pellet
1
Supernatant Centrifuge i00,000 g, 60 min
1
I Pellet
Supernatant
Wash Suspend in T r i t o n X-100 C e n t r i f u g e i00,000 g, 60 min
Supernatant Add e q u i v a l e n t a n t i - g l u c u r o n i d a s e I n c u b a t e a t 0 °, 12 hr C e n t r i f u g e 10,000 g, 10 min
Pellet
1 Pellet
Supernatant
Wash
Heat at 50 °, 5 min, or treat with 0.1% deoxycholate Centrifuge i0,000 g, i0 min
Pellet
Supernatant (containing egasyn)
FIG. 4. Isolation of egasyn by release from glucuronidase immunoprecipitates.
The purification of egasyn by specific release from immunoprecipitated microsomal/3-glucuronidase is summarized in Fig. 4. All steps are carried out at 0 - 4 ° to minimize disruption of egasyn-glucuronidase complexes. The protocol is as follows: 1. The tissues are removed and homogenized in 10 volumes of icecold 0.02 M imidazole, 0.25 M sucrose, pH 7.4, using a Waring blender (4 min at low speed). I normally use as a source the pooled livers and kidneys from 50-100 mice, representing 75-150 g wet weight of tissue. Livers and kidneys are used, since they are relatively rich sources of microsomal glucuronidase.m Most of the common strains of mice have similar levels of microsomal enzyme, but C3H/HeJ, AKR/J, and CBA/J have very low levels of fl-glucuronidase and YBR/J lacks egasyn. Male or testosterone-induced female mice have elevated levels of kidney enzyme and, thus, are preferable to untreated female mice for egasyn purification.4
566
B I O G E N E S I S A N D ASSEMBLY OF M E M B R A N E PROTEINS
[44]
2. The debris is removed by low-speed centrifugation (2500 g, 10 min). The supernatant is centrifuged at high speed (100,000 g, 60 min), and the pellet is washed by suspension in the original volume of 0.02 M imidazole, 0.25 M sucrose, pH 7.4, in a Waring blender, followed by recentrifugation at high speed. 3. The washed high-speed pellet, containing the crude membrane fraction, is suspended in the original volume of 0.02 M imidazole, 0.25 M sucrose, pH 7.4. Triton X-100 is added to a final concentration of 2%. This suspension is allowed to stand on ice for about 1 hr, and then the solution is centrifuged at 100,000 g for 60 min. The clear supernatant, containing over 80% of the microsomal /3-glucuronidase activity, is carefully decanted. 4. The high-speed Triton X-100 extract is diluted by addition of an equal volume of 0.02 M Tris-HC1, 0.3 M NaCI, pH 7.4, and its glucuronidase activity is determined. An equivalent amount of anti-glucuronidase antibody is added, and the mixture is allowed to stand overnight on ice or at 4°. Our original procedure 7 utilized F(ab)2 fragment of anti-glucuronidase IgG, but similar results can be obtained using intact antibody. 5. The immunoprecipitate is collected by centrifugation (10,000 g, 10 min) and washed three times with several milliliters of TBS buffer. The immunoprecipitate is then suspended in a small volume of TBS (about 0.2 ml per 100 g of tissue), and the egasyn is quantitatively released by heating at 50° for 5 min or by the addition of sodium deoxycholate (0.1% final concentration). The egasyn is separated from the remaining immunoprecipitate by centrifugation (10,000 g, 10 min). When mouse liver is used, the final yield of egasyn is 1-2/xg per gram of tissue. 6. The purity of isolated egasyn should be examined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. 7 Most preparations exhibit a single protein band corresponding to molecular weight 64,000. Glucuronidase Binding Protein from Rat Tissues. A protein that specifically binds rat fl-glucuronidase is extracted from rat liver microsomes using 2% Triton X-100. Unlike the mouse glucuronidase-egasyn complex, the rat glucuronidase-binding protein complex is sensitive to relatively low concentrations of detergent (0.25% Triton X-100), and thus, it is not possible to recover intact complexes after extraction of tissues with detergents. The binding protein extracted from rat microsomes can be isolated on the basis of its ability to adhere to columns containing immobilized/~-glucuronidase, and it resembles mouse egasyn in certain respects.8 Antiserum against Egasyn. Rabbits are inoculated intradermally at multiple sites on the back with 50/~g of purified mouse egasyn emulsified in Freund's complete adjuvant. Booster injections containing the same
[44]
EGASYN AND fl-GLUCURONIDASEMEMBRANE BINDING
567
mixture are given at 2-week intervals. Antibody is detected by Ouchterlony double diffusion (with a few micrograms of purified egasyn as the antigen) or by the ability to bind microsomal glucuronidase in Triton X100 extracts (see below). Rabbits are bled after maximal titer is obtained (this required about 2 months in my experience). The immunoglobin fraction can be partially purified from serum by precipitation with ammonium sulfate at 40% saturation. The antibody preparations that I obtained are specific for egasyn and show no cross-reactivity with either L- or X-form mouse glucuronidase; however, they do bind the M-form complexes containing both glucuronidase and egasyn. 7 A sensitive method for detecting antibody to egasyn is to expose microsomal glucuronidase to antibody and examine the subsequent ability of glucuronidase-egasyn complexes to migrate in polyacrylamide gels: Fresh homogenates of mouse liver are made 2% in Triton X-100 and centrifuged at high speed. Electrophoresis of the supernate on 7% polyacrylamide gels at pH 8.1, followed by staining for fl-glucuronidase with naphthol-AS-BI-glucuronide substrate, gives a band corresponding to lysosomal (L form) glucuronidase and 4 bands corresponding to the microsomal (M form) complexes containing glucuronidase and egasyn. Incubation with anti-egasyn antibody (overnight at 4°) results in the removal of M-form, but not L-form, enzyme upon electrophoresis. 7 Labeling with usI. To a test tube are added, in order, 1 mCi of carrierfree Na125I (in 10/zl); 0.5 M sodium phosphate, pH 7.5 (10/zl); 10/xg of egasyn isolated using the heat-release procedure described above (20/zl); and 50/xg of chloramine-T in water (10 tzl). The mixture is shaken briefly, and 250/xg of sodium metabisulfite (in 0.5 ml) and 100/zg of KI (in 100/xl) are added. Unreacted iodine is separated from the labeled egasyn by passing the mixture through a column of Sephadex G-25 (0.7 cm x 25 cm) equilibrated with 0.15 M NaCI, 0.02 M Tris, 0.1% Triton X-100, and 0.1% bovine serum albumin, pH 7.5. Fractions of 0.6 ml are collected, and the peak fractions eluting in the void volume, which contain about 30% of the total counts, are pooled. When examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the labeled product contains a significant amount of activity that migrates slightly behind the tracking dye in addition to a peak that migrates at the position of an egasyn marker. This low molecular contamination, possibly a breakdown product resulting from the iodination procedure, is only partially removed by exhaustive dialysis; however, the contamination can be removed by chromatography on a 0.5-ml concanavalin A-Sepharose column equilibrated with 0.15 M NaCI, 0.02 M Tris, 0.1% Triton X- 100, 0.1% bovine serum albumin, pH 7.5. The sample is applied slowly to the column, and the column is then washed
568
B I O G E N E S I S A N D ASSEMBLY OF M E M B R A N E PROTEINS
[44]
with several volumes of equilibration buffer. This removes the low molecular weight contamination, which does not bind to the concanavalin A. The labeled egasyn is then eluted by percolating 5 ml of equilibration buffer containing 0.2 M methyl-a-mannoside through the column. The lzSI-labeled egasyn is stored frozen until use in the radioimmunoassay. As judged by electrophoresis and immunoreactivity, it is stable for at least 6 weeks. Titration of Labeled Egasyn with Anti-Egasyn Antibody. Titrations and radioimmunoassays can be performed in small glass tubes (0.6 cm x 5 cm). For titrations, antibody to egasyn is added at varying dilutions to about 10,000 cmp of 125I-labeled egasyn, in a final volume of 100/zl. All dilutions are made in TBS containing 0.1% Triton X-100 and 0.1% bovine serum albumin. The mixtures are incubated for 1 hr at 37° and then 24 hr at 4 °. Carrier control rabbit serum (40/xl of 4% serum) and antibody to rabbit IgG (30/zl) are added, and the mixtures are incubated for 1 hr at 37° and 4 hr at 4 °. The immunoprecipitate pellets are collected by centrifugation (10,000 g, 10 min) and washed twice with dilution buffer. Using our antibody preparations, 80-90% of the label is precipitated at high levels of antibody (10-fold dilution) and less than 3% of the label is precipitated in the absence of anti-egasyn antibody. 7 Radioimmunoassay of Egasyn. For the radioimmunoassay, a concentration of antibody that corresponds to about 50% binding of the 125Ilabeled egasyn is used. The antibody is first incubated with sample, 125Ilabeled egasyn is then added, and after further incubation the immune complexes are precipitated by the addition of goat antiserum raised against rabbit IgG. The egasyn content of samples is estimated by their ability to displace radioactivity from the precipitate to the supernatant fraction. Standard curves are obtained by the addition of unlabeled isolated egasyn. Most of the antigenic sites on egasyn in homogenates of liver are masked after extraction with Triton X-100 and become immunoreactive only after treatment with 1% sodium deoxycholate (Fig. 5). The reason for this is unclear but it is, therefore, necessary to carry out the radioimmunoassay in the presence of deoxycholate. 7 The assay described here is a liquid-phase procedure with double-antibody precipitation; other variations have not been tested. Thus, it may be possible to improve the sensitivity or simplicity of the assay, for example, with solid-phase immunoassay. The protocol for the assay is as follows: 1. Anti-egasyn IgG and the sample are incubated in TBS containing 1% sodium deoxycholate (100/xl final volume) for 1 hr at 37° and 24 hr at 4°. The amount of antibody used is sufficient to precipitate about 50% of the labeled egasyn (see below) in the absence of competing antigen and
[44]
EGASYN AND fl-GLUCURONIDASE MEMBRANE BINDING EGASYN
CONCENTRATION
(ng 0.1 i
PER
TUBE)
1 i
569
10
I
i
i
100 i
i
0.5
- ~'~^t .~-~o~ 0.4 LL
0.3
0.2 0.1 0 I
0.1
I
I
I
1
HOMOGENATE (p.I
I
I
10
I
100
CONCENTRATION PER
TUBE)
FIG. 5. Radioimmunoassay of mouse egasyn: effect of deoxycholate. Egasyn was assayed in the absence (©, A) or the presence (0, &) of 1% sodium deoxycholate as described in the text. The standard ( 0 , 0 ) was a preparation of egasyn purified from mouse liver and kidney by release from immunoprecipitates of microsomal glucuronidase. The sample (A, &) was a 10% homogenate of strain C57BL/6J liver extracted with Triton X-100 and centrifuged at high speed. Plotted on the ordinate is the ratio of antibody-bound nsI (B) to free n~I (F).
represents a 500-fold dilution of the antiserum. Samples are diluted in TBS containing 0.1% bovine serum albumin and 1% Triton X-100. The particulate matter in homogenates results in some interference with the assay. For this reason, assays are generally performed on high speed supernatant solutions of samples extracted with 2% Triton X-100, which solubilizes both egasyn and the microsomal glucuronidase complexes. 7 2. To the mixture is then added 5000 cpm for n5I-labeled egasyn (in 50 /~1 of sample buffer). This is incubated for 1 hr at 37° and 72 hr at 4°. 3. Egasyn bound to antibody is separated from free egasyn by the addition of goat antibody to rabbit IgG (30/xl) and 4% control rabbit serum as carrier (40/~1). This is incubated for 1 hr at 37° and 4 hr at 4°. The mixture is centrifuged at 10,000 g for 10 min, and the immunoprecipitate pellet is washed twice with 0.5 ml of sample buffer. The radioactivity in the pellet is measured with a gamma counter. A purified preparation of egasyn (from 1 to 50 ng per tube) is used as the standard. Typical radioimmunoassay results are shown in Fig. 5.
EGASYN
AND fl-GLUCURONIDASE
MEMBRANE
BINDING
557
liver, but occurs to a large extent also in skeletal muscle. 87 Kidney and heart muscle also respond similarly. 82 Thyroid H o r m o n e s and M e m b r a n e Degradation
In contrast to what is observed after induction of the endoplasmic reticulum by phenobarbital (see above), there is a dissociation of membrane and enzyme degradation of induced skeletal muscle mitochondria, which speaks against an autophagic degradation mechanism in this case. 88 Transitions from hyperthyroid to normal and from normal to hypothyroid states cause a decrease in enzyme level, but no change, or even an increase, in the total mitochondrial membrane volume. Phthalate Ester Induction
As described in the section on peroxisomes, DEHP induces mitochondria also. 61-63 Table IV lists a few functions that are found to be induced in
isolated liver mitochondria after 2 weeks' treatment of rats with 2% DEHP in the diet. A detailed analysis of the enzyme pattern after such induction has not yet been performed, and, thus, the nature of the newly synthesized mitochondria is not known. Acknowledgments The work from the authors' laboratories cited here was supported by grants from the Swedish Medical and Natural Science Research Council and NIH Grant No. 1 RO1 CA 26261-02 awarded by the National Cancer Institute, DHEW, Bethesda, Maryland. 88L. Ernster, Fed. Proc., Fed. Am. Soc. Exp. Biol. 24, 1222(1965).
[44] P r e p a r a t i o n o f M i c r o s o m a l f l - G l u c u r o n i d a s e a n d Its Membrane Anchor Protein, Egasyn
By ALDONS J. LUSIS Acid hydrolases such as fl-glucuronidase are typically located in lysosomes, but in certain tissues of mammals substantial amounts of the enzyme are also present in the membranes of the endoplasmic reticulum. This has been demonstrated using both tissue fractionation and histochemical staining reactions. The microsomal enzyme is firmly bound to METHODS IN ENZYMOLOGY, VOL. 96
Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181996-5
558
BIOGENESIS AND ASSEMBLY OF MEMBRANE PROTEINS
8_
Eg
i~
[
S
EGASYN
~ M 3
5--
[44]
Gus
~ GLUCURONIDASE POLYPEPTIDE
LYSOSOMES
ENDOPLASMIC RETICULUM
Fro. l. Model for intracellular localization of mouse fl-glucuronidase. Six intracellular forms of/3-glucuronidase have been identified, The enzyme in lysosomes consists of a single form, termed L, that is a tetramer of identical subunits, each of molecular weight 71,000. The microsomal enzyme, on the other hand, occurs primarily as a series of higher molecular weight complexes, termed M1, M2, M3, and M4; these consist of a glucuronidase core of slightly different structure, X, complexed with one to four chains of a second polypeptide, egasyn. The X and L subunits are derived from a common structural gene, Gus, on chromosome 5, and differ by modification. Egasyn is an integral membrane glycoprotein of molecular weight 64,000. A mutation at the Eg gene on chromosome 8, present in strain YBR, results in the absence of immunoreactive egasyn and the inability to maintain/3-glucuronidase in endoplasmic reticulum membranes. This and other evidence suggest that egasyn stabilizes the membrane binding of/9-glucuronidase (see text). Reprinted from Lusis and Paigen. 4
membrane and detergents are required to extract it, while the enzyme in lysosomes is released upon rupture of the lysosomal membrane.l-3 A combination of biochemical and genetic approaches suggests a model for the localization of/3-glucuronidase in mouse tissues 4 (Fig. 1). Genetic studies indicate that the enzyme at both intracellular sites is derived from the same structural gene. The fl-glucuronidase in lysosomes occurs as a free tetramer of molecular weight about 280,000, whereas most of the enzyme in microsomes is complexed with an integral membrane protein, egasyn. The complexes can be extracted intact from membrane using Triton X-100, and the egasyn isolated from these complexes is a glycoprotein of molecular weight 64,000 that is structurally distinct from glucuronidase. The microsomal and lysosomal glucuronidase tetramers C. B. C. de Duve, R. Pressman, R. Gianetto, R. Wattiaux, and F. Appelmans, Biochem. J. 60, 604 (1955). 2 K. Paigen, Exp. Cell Res. 25, 286 (1961). 3 W. H. Fishman, S. S. Goldman, and R. DeLellis, Nature (London) 213, 457 (1967). 4 A. J. Lusis and K. Paigen, Isozymes: Curr. Top. Biol. Med. Res. 2, 63 (1977).
[44]
EGASYN AND fl-GLUCURONIDASE MEMBRANE BINDING
559
(termed X and L, respectively) differ by covalent modification, the microsomal form being slightly larger and less negatively charged than the lysosomal form, but the functional significance of these differences is not known. Several observations suggest that egasyn serves to stabilize the binding of fl-glucuronidase to membrane. Most convincing is the finding that a mouse strain that lacks egasyn is unable to maintain fl-glucuronidase in endoplasmic reticulum membrane. 4-7 In addition, among tissues and during development the levels of fl-glucuronidase in microsomes are correlated with the levels of egasyn. For example, liver and kidney contain 2050% microsomal glucuronidase and have relatively high levels of egasyn, whereas spleen contains only trace levels of microsomal glucuronidase and lacks detectable egasyn. Thus, egasyn appears to function as a membrane anchor for the highly polar glucuronidase tetramer. 4 Other mammals that have been examined, including rats, rabbits, and humans, also have substantial amounts of microsomal fl-glucuronidase in liver. 4 A membrane protein that may be analogous to egasyn has been isolated from rat liver microsomes. 8 The protein was extracted from microsomes with 2% Triton X-100, and it specifically binds B-glucuronidase. Unlike the mouse microsomal fl-glucuronidase complexes, the complex between rat glucuronidase and the binding protein is dissociated by relatively low concentrations of Triton X-100. Rabbit liver microsomal glucuronidase is extracted by Triton X-100 as a series of complexes with molecular weights of roughly 280,000, 330,000, 370,000, 420,000, and 500,000; thus, as in mice, the forms increase stepwise in molecular weight increments of roughly 55,000. The rabbit fl-glucuronidase complexes are considerably more stable than those of mice and rats, although they can be dissociated using 8 M urea. 9 fl-Glucuronidase Purification
Assay. Mammalian fl-glucuronidase is capable of hydrolyzing a wide variety of natural and synthetic fl-o-glucuronides. A variety of simple fluorometric and spectrophotometric assays of the enzyme, using artificial substrates such as the glucuronides of 4-methylumbelliferone and p-nitrophenol, have been developed. In terms of simplicity and sensitivity the fluorometric assay, employing 4-methylumbelliferyl-fl-o-glucuronide as the substrate, is the procedure of choice. R. 6 A. 7 A. 8 L. 9 R.
E. Ganschow and K. Paigen, Proc. Natl. Acad. Sci. U.S.A. 58, 938 (1967). J. Lusis, S. Tomino, and K. Paigen, Biochem. Genet. 15, 115 (1977). J. Lusis, S. Tomino, and K. Paigen, J. Biol. Chem. 251, 7753 (1976). D. Strawser and O. Touster, J. Biol. Chem. 254, 3716 (1979). T. Dean and M. Messer, Comp. Biochem. Physiol. 548, 107 (1976).
560
B I O G E N E S IAND S ASSEMBLY OF MEMBRANE PROTEINS
[44]
I utilize the following assay protocol: 7 The sample is incubated at 37° with 0.4 mM 4-methylumbelliferyl-/3-o-glucuronide (Sigma Chemical Co.), 0.1% Triton X-100 (Sigma), and 0.1 M sodium acetate, pH 4.6, in a total volume of 0.1 ml. The reaction is stopped after 30 min by immersion in an ice bath followed by the addition of 1.0 ml of 0.1 M sodium carbonate. The fluorescence is measured with an Aminco fluorometer (catalog No. 4-7439) equipped with a Corning 7-60 excitation filter (peak wavelength 360 nm) and Kodak 2A emission filter (passing wavelengths above 415 nm), or an instrument with similar capability. One unit of activity is defined as that amount of enzyme that will hydrolyze 1/zmol of substrate per hr at 37°. At very low protein concentrations, the inclusion of 0.1% albumin during the incubation helps to stabilize the enzyme. If necessary, the sensitivity of the assay can be increased by prolonging the incubation period to 24 hr or more. Electrophoresis. The multiple forms of mouse/3-glucuronidase (L, X, M) are resolved using a nondenaturing polyacrylamide gel electrophoresis system containing Tris-glycine, pH 8.1, and 7% acrylamide. Glucuronidase activity is visualized by staining with naphthol-AS-BI-glucuronide (Sigma) as the substrate. ~°:1 The most sensitive technique for analyzing charge variation of the glucuronidase tetramer is isoelectric focusing. I have had best results using a gel system containing 6% acrylamide, 6 M urea, 1% pH 3.5-10 Ampholines and 1% pH 5-7 Ampholines. 11 Under these conditions the glucuronidase tetramer remains intact and the gel can be stained for enzyme activity, but the M form complexes between glucuronidase and egasyn are disrupted. Lysosomal (L Form) fl-Glucuronidase. Lysosomal fl-glucuronidase has been purified by conventional fractionation from several mammalian sources including bovine liver, human liver, rat liver and preputial gland, and mouse liver, kidney, and urine. 11,12Rat preputial gland 13 and mouse urine 11 are especially rich sources, permitting rapid and simple purification of milligram quantities of the enzyme. Antisera. High-titer, monospecific antibodies to/3-glucuronidase can be raised using rabbits or goats.14 Rabbit antiserum is prepared by inoculating outbred rabbits at multiple subcutaneous sites with 0.2 mg of purified L-form glucuronidase in Freund's complete adjuvant. Four weeks later, booster injections are given with 0.2 mg of enzyme in incomplete adjuvant. Serum is taken about 1 week after the boost. ~0R. T. Swank and K. Paigen, J. Mol. Biol. 77, 371 (1973). ll A. J. Lusis and K. Paigen, J. Biol. Chem. 253, 7336 (1978). 12 O. Touster, this series, Vol. 50, p. 488. 13R. K. Keller and O. Touster, J. Biol. Chem. 250, 2739 (1975). 14 S. Tomino, K, Paigen, D. Tulsiani, and O. Touster, J. Biol. Chem. 250, 8503 (1975).
[44]
EGASYN AND fl-GLUCURONIDASE MEMBRANE BINDING
561
Antibody Affinity Chromatography. Mouse glucuronidase can be readily purified from liver, kidney, and other tissues by antibody affinity chromatography. ~ Both microsomal (X) and lysosomal (L) forms of the enzyme can be purified, since immunologically they are not discernibly different. The protocol is as follows: Immunoglobin is purified from antiserum by repeated precipitation with ammonium sulfate at 45% saturation. The precipitate is dissolved in 0.02 M Tris, 0.15 M NaCI, pH 7.4 (TBS buffer), and dialyzed against the same buffer. The immunoglobin is coupled to cyanogen bromide-activated Sepharose 4B in 0.1 M NaHCO3, 0.5 M NaC1, and afterward the gel is washed extensively with coupling buffer and with 0.1 M sodium acetate, 0.5 M NaC1, pH 4.6. The gel is placed in a column and washed with 0.1 M sodium acetate, pH 5.2, containing 8 M urea to remove any loosely bound material. It is then equilibrated with TBS buffer, and the sample is applied in TBS buffer. Tissue homogenates, extracted with detergents such as Triton X-100 and centrifuged at high speed to remove particulate material (see below), are suitable for chromatography. For isolation of L-form enzyme, however, we have found it useful partially to purify the enzyme using heat treatment at acid pH prior to chromatography. ~ After application of the sample, the column is washed with at least 5 column volumes of (a) TBS buffer, (b) 0.02 M sodium acetate, pH 5.2, and (c) 0.02 M Tris, 0.3 M NaC1, pH 7.4. Glucuronidase is then eluted by applying 0.1 M sodium acetate, pH 5.2, containing 8 M urea to the column at a rate of about 4 column volumes per hour. The glucuronidase is normally eluted within about 3 column volumes, and the urea is removed by dialysis. Although glucuronidase is relatively resistant to inactivation by urea, dialysis is normally begun shortly after elution of the enzyme. The capacity of the gel can be estimated using a small column and increasing amounts of enzyme; it is possible to bind several hundred units of enzyme (2400 units per milligram of protein) per milliliter of gel. Normally, nearly all of the enzyme binds to the column, and recovery of activity averages 60-80%. After affinity chromatography, preparations contain only traces of contaminants, and these can be removed by chromatography on a 2.6 × 55 cm column of Ultrogel Ac-44 equilibrated with TBS. When stored at 4° in TBS containing 0.1% sodium azide, the affinity gel is stable for more than a year. Similar antibody affinity chromatography procedures have been reported for the isolation of glucuronidase from rat tissue. ~5,16 Purification of Microsomal Glucuronidase from Mouse Liver or Kidney. Tissues are homogenized in a Polytron homogenizer with 10 volumes 15 j. W. Owens, K. L. Gammon, and P. D. Stahl, Arch. Biochem. Biophys. 166, 258 (1975). ~6M. Himeno, Y. Nishimura, H. Tsuji, and K. Kato, Eur. J. Biochem. 70, 349 (1976).
562
BIOGENESIS AND ASSEMBLY OF MEMBRANE PROTEINS
[44]
of 0.02 M imidazole, pH 7.4. The homogenate is centrifuged at high speed (100,000 g, 60 min), and the supernatant solution (containing the bulk of the L-form enzyme) is removed. The pellet is suspended in 0.02 M imidazole, pH 7.4 (a volume equal to that of the original homogenate) using a Polytron homogenizer. The solution is recentrifuged at high speed, and the pellet (crude membrane) is suspended in an equal volume of 0.02 M imidazole, pH 7.4, containing 2% Triton X-100. This solution is centrifuged (100,000 g, 1 hr), and the supernatant solution, containing more than 90% of the activity, is carefully decanted. Glucuronidase is purified from this solution using antibody affinity chromatography as described above, except that after application of enzyme the column is first washed with several volumes of TBS containing 0.1% sodium deoxycholate (Sigma) to remove egasyn associated with glucuronidase. Enzyme yields average about 40% of the original membrane-bound activity. Kidneys from androgen-treated mice are the richest source of microsomal glucuronidase. The purified preparations contain about 40% L-form enzyme and about 60% X-form enzyme, judging from staining intensity after polyacrylamide gel electrophoresis. It is possible to obtain enzyme preparations with a greater proportion of X-form enzyme by fractionation and more extensive washing of membrane preparations; however, about 510% of the total L-form enzyme is tightly associated with membrane and can be extracted only with detergent. ~°,17 The X and L electrophoretic forms of glucuronidase can be separated on the basis of charge by DEAE-Sephadex chromatography at pH 6.9 (Fig. 2). Fractions obtained after chromatography are examined by electrophoresis at pH 8.1, and those containing enzyme approximately corresponding in mobility to the X or L forms observed in homogenates are pooled (these are termed the X and L fractions). When subjected to electrophoresis in the presence of sodium dodecyl sulfate, the X fraction migrates as a single band corresponding to an apparent molecular weight of about 74,000. The bulk of the L fraction migrates as a band of molecular weight 71,000, not discernibly different in mobility from L-form enzyme isolated from the lysosomal component of liver and kidney. When subjected to isoelectric focusing in polyacrylamide gels, the X fraction can be resolved into seven forms of about equal staining intensity, ranging from pI 6.0 to pI 6.6. The L fraction also contains several forms, ranging from pl 5.7 to pI 5.9, and it closely resembles the enzyme isolated from the lysosomal component of liver and kidney. The size difference between X and L/3-glucuronidase may result from differences in carbohydrate content, since after treatment with endoglycosidase H both forms comigrate on SDS gels with an apparent molecular weight of about 70,000. Endogly~7A. J. Lusis and K. Paigen, unpublished data (1977).
[44]
563
EGASYN AND fl-GLUCURONIDASE MEMBRANE BINDING 76+88~ 76 80 84 88 ~76~8 {--)FRACTIONNUMBER
g B
100
T ORIGIN IiBI ~-~ =-a ~..., ,__, i==1
(+)
B 15
J
f
--
__ Z so 80
g,
8
z c~
70 z
5
0 rr
60
5
z
;¢ ~
I 0
--
7O
80
90
100
50 110
FRACTION NUMBER FIG. 2. Separation of X and L mouse/3-glucuronidase using DEAE-Sephadex chromatography. Glucuronidase was isolated from crude microsomes of pooled livers and kidneys of 100 male C57BL/6J mice using antibody affinity chromatography. The enzyme (13.6 ml, containing 260 units of enzyme activity) was then dialyzed against 0.02 M imidazole, pH 6.9, and applied to a 0.6 x 10 cm column of DEAE-Sephadex A-25, equilibrated with 0.02 M imidazole, pH 6.9. Essentially all activity bound to the column. The column was washed with several volumes of equilibration buffer, and the activity was eluted using a linear salt gradient, from 0 to 0.1 M NaC1, in equilibration buffer (total volume 200 ml) at a rate of about 20 ml/hr. Fractions of 2.4 ml were collected, glucuronidase activity was determined, and aliquots from peak fractions were subjected to polyacrylamide gel electrophoresis at pH 8.1, followed by staining for glucuronidase activity. The inset shows the pattern of glucuronidase activity after electrophoresis of fractions 76, 80, 84, 88, and mixtures of 76 and 88 (30-tzl aliquots were used). Fractions 74-81, corresponding approximately to X enzyme in electrophoretic mobility, and fractions 88-90, corresponding approximately to L enzyme in mobility, were pooled for further characterization (see text).
cosidase treatment has little effect on the charge of glucuronidase.17 The charge difference, on the other hand, may be due in part to a C-terminal peptide that can be removed from X-form enzyme by protease treatment (Fig. 3). The proteases tested did not discernibly affect the apparent molecular weight of glucuronidase as judged by electrophoresis in sodium dodecyl sulfate, suggesting that the peptide removed from X by protease treatment is short.17 Rat Microsomal fl-Glucuronidase. Rat liver microsomal fl-glucuronidase can be isolated by similar procedures. As in the mouse, the rat microsomal enzyme appears to be slightly larger and less acidic than the lysosomal enzyme.~5,16 Chemical analyses suggest that the two forms differ in carbohydrate and amino acid composition? 8J9 i8 D. R. P. Tulsiani, H. Six, and O. Touster, Proc. Natl. Acad. Sci. U.S.A. 75, 3080 (1978). ~9 M. Himeno, Y. Nishimura, K. Takahashi, and K. Kato, J. Biochem. (Tokyo) 83, 511 (1978).
564
BIOGENESIS AND ASSEMBLY OF MEMBRANE PROTEINS pH
5.5
6.0
/[EIIrl iJ/[~lJll ./ [ !~ll EI f //" ill, ill IJ I I~lllll II I I'~,11
[44]
6.5
I I f I I I
i FIG. 3. Effect of protease treatment on the isoelectric focusing pattern of X- and L-form glucuronidase. Isolated glucuronidase preparations (Fig. 2) were treated with proteases and subjected to isoelectric focusing as described. HThe gels were stained for glucuronidase activity and scanned at 550 nm. The pH gradient along the gel is shown at the top. About 0.5 unit of enzyme activity was applied to each slot, and protease treatment had no effect on total activity. Shown are absorbance tracings for control X form glucuronidase (X), X form treated with aminopeptidase M (X + AP), X form treated with carboxypeptidase Y (X + CP), control L form (L), and L form treated with carboxypeptidase Y (L + CP). Trypsin and chymotrypsin treatment gave the same results as carboxypeptidase Y.
Isolation and A s s a y of E g a s y n
Isolation from Mouse Tissues. I n the m o u s e , m i c r o s o m a l glucuronid a s e - e g a s y n c o m p l e x e s c a n be e x t r a c t e d intact f r o m m e m b r a n e s with Triton X-100. l° T h e m i c r o s o m a l g l u c u r o n i d a s e can then be specifically precipitated using a n t i - g l u c u r o n i d a s e a n t i b o d y , 2° and e g a s y n can be selectively solubilized f r o m s u c h i m m u n o p r e c i p i t a t e s b y disrupting the glucu r o n i d a s e - e g a s y n c o m p l e x with heating at 50 ° o r with 0.1% s o d i u m d e o x y c h o l a t e . 7 It also is feasible to isolate e g a s y n b y binding m i c r o s o m a l g l u c u r o n i d a s e c o m p l e x e s to a n t i - g l u c u r o n i d a s e affinity c o l u m n s and selectively eluting e g a s y n b y passing 0.1% s o d i u m d e o x y c h o l a t e in T B S t h r o u g h the columns.17 2o S. Tomino and K. Paigen, J. Biol. Chem. 250, 116 (1975).
[44]
EGASYN A N D f l - G L U C U R O N I D A S E M E M B R A N E B I N D I N G
565
Homogenate Centrifuge 2500 g, i0 min
Pellet
1
Supernatant Centrifuge i00,000 g, 60 min
1
I Pellet
Supernatant
Wash Suspend in T r i t o n X-100 C e n t r i f u g e i00,000 g, 60 min
Supernatant Add e q u i v a l e n t a n t i - g l u c u r o n i d a s e I n c u b a t e a t 0 °, 12 hr C e n t r i f u g e 10,000 g, 10 min
Pellet
1 Pellet
Supernatant
Wash
Heat at 50 °, 5 min, or treat with 0.1% deoxycholate Centrifuge i0,000 g, i0 min
Pellet
Supernatant (containing egasyn)
FIG. 4. Isolation of egasyn by release from glucuronidase immunoprecipitates.
The purification of egasyn by specific release from immunoprecipitated microsomal/3-glucuronidase is summarized in Fig. 4. All steps are carried out at 0 - 4 ° to minimize disruption of egasyn-glucuronidase complexes. The protocol is as follows: 1. The tissues are removed and homogenized in 10 volumes of icecold 0.02 M imidazole, 0.25 M sucrose, pH 7.4, using a Waring blender (4 min at low speed). I normally use as a source the pooled livers and kidneys from 50-100 mice, representing 75-150 g wet weight of tissue. Livers and kidneys are used, since they are relatively rich sources of microsomal glucuronidase.m Most of the common strains of mice have similar levels of microsomal enzyme, but C3H/HeJ, AKR/J, and CBA/J have very low levels of fl-glucuronidase and YBR/J lacks egasyn. Male or testosterone-induced female mice have elevated levels of kidney enzyme and, thus, are preferable to untreated female mice for egasyn purification.4
566
B I O G E N E S I S A N D ASSEMBLY OF M E M B R A N E PROTEINS
[44]
2. The debris is removed by low-speed centrifugation (2500 g, 10 min). The supernatant is centrifuged at high speed (100,000 g, 60 min), and the pellet is washed by suspension in the original volume of 0.02 M imidazole, 0.25 M sucrose, pH 7.4, in a Waring blender, followed by recentrifugation at high speed. 3. The washed high-speed pellet, containing the crude membrane fraction, is suspended in the original volume of 0.02 M imidazole, 0.25 M sucrose, pH 7.4. Triton X-100 is added to a final concentration of 2%. This suspension is allowed to stand on ice for about 1 hr, and then the solution is centrifuged at 100,000 g for 60 min. The clear supernatant, containing over 80% of the microsomal /3-glucuronidase activity, is carefully decanted. 4. The high-speed Triton X-100 extract is diluted by addition of an equal volume of 0.02 M Tris-HC1, 0.3 M NaCI, pH 7.4, and its glucuronidase activity is determined. An equivalent amount of anti-glucuronidase antibody is added, and the mixture is allowed to stand overnight on ice or at 4°. Our original procedure 7 utilized F(ab)2 fragment of anti-glucuronidase IgG, but similar results can be obtained using intact antibody. 5. The immunoprecipitate is collected by centrifugation (10,000 g, 10 min) and washed three times with several milliliters of TBS buffer. The immunoprecipitate is then suspended in a small volume of TBS (about 0.2 ml per 100 g of tissue), and the egasyn is quantitatively released by heating at 50° for 5 min or by the addition of sodium deoxycholate (0.1% final concentration). The egasyn is separated from the remaining immunoprecipitate by centrifugation (10,000 g, 10 min). When mouse liver is used, the final yield of egasyn is 1-2/xg per gram of tissue. 6. The purity of isolated egasyn should be examined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. 7 Most preparations exhibit a single protein band corresponding to molecular weight 64,000. Glucuronidase Binding Protein from Rat Tissues. A protein that specifically binds rat fl-glucuronidase is extracted from rat liver microsomes using 2% Triton X-100. Unlike the mouse glucuronidase-egasyn complex, the rat glucuronidase-binding protein complex is sensitive to relatively low concentrations of detergent (0.25% Triton X-100), and thus, it is not possible to recover intact complexes after extraction of tissues with detergents. The binding protein extracted from rat microsomes can be isolated on the basis of its ability to adhere to columns containing immobilized/~-glucuronidase, and it resembles mouse egasyn in certain respects.8 Antiserum against Egasyn. Rabbits are inoculated intradermally at multiple sites on the back with 50/~g of purified mouse egasyn emulsified in Freund's complete adjuvant. Booster injections containing the same
[44]
EGASYN AND fl-GLUCURONIDASEMEMBRANE BINDING
567
mixture are given at 2-week intervals. Antibody is detected by Ouchterlony double diffusion (with a few micrograms of purified egasyn as the antigen) or by the ability to bind microsomal glucuronidase in Triton X100 extracts (see below). Rabbits are bled after maximal titer is obtained (this required about 2 months in my experience). The immunoglobin fraction can be partially purified from serum by precipitation with ammonium sulfate at 40% saturation. The antibody preparations that I obtained are specific for egasyn and show no cross-reactivity with either L- or X-form mouse glucuronidase; however, they do bind the M-form complexes containing both glucuronidase and egasyn. 7 A sensitive method for detecting antibody to egasyn is to expose microsomal glucuronidase to antibody and examine the subsequent ability of glucuronidase-egasyn complexes to migrate in polyacrylamide gels: Fresh homogenates of mouse liver are made 2% in Triton X-100 and centrifuged at high speed. Electrophoresis of the supernate on 7% polyacrylamide gels at pH 8.1, followed by staining for fl-glucuronidase with naphthol-AS-BI-glucuronide substrate, gives a band corresponding to lysosomal (L form) glucuronidase and 4 bands corresponding to the microsomal (M form) complexes containing glucuronidase and egasyn. Incubation with anti-egasyn antibody (overnight at 4°) results in the removal of M-form, but not L-form, enzyme upon electrophoresis. 7 Labeling with usI. To a test tube are added, in order, 1 mCi of carrierfree Na125I (in 10/zl); 0.5 M sodium phosphate, pH 7.5 (10/zl); 10/xg of egasyn isolated using the heat-release procedure described above (20/zl); and 50/xg of chloramine-T in water (10 tzl). The mixture is shaken briefly, and 250/xg of sodium metabisulfite (in 0.5 ml) and 100/zg of KI (in 100/xl) are added. Unreacted iodine is separated from the labeled egasyn by passing the mixture through a column of Sephadex G-25 (0.7 cm x 25 cm) equilibrated with 0.15 M NaCI, 0.02 M Tris, 0.1% Triton X-100, and 0.1% bovine serum albumin, pH 7.5. Fractions of 0.6 ml are collected, and the peak fractions eluting in the void volume, which contain about 30% of the total counts, are pooled. When examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the labeled product contains a significant amount of activity that migrates slightly behind the tracking dye in addition to a peak that migrates at the position of an egasyn marker. This low molecular contamination, possibly a breakdown product resulting from the iodination procedure, is only partially removed by exhaustive dialysis; however, the contamination can be removed by chromatography on a 0.5-ml concanavalin A-Sepharose column equilibrated with 0.15 M NaCI, 0.02 M Tris, 0.1% Triton X- 100, 0.1% bovine serum albumin, pH 7.5. The sample is applied slowly to the column, and the column is then washed
568
B I O G E N E S I S A N D ASSEMBLY OF M E M B R A N E PROTEINS
[44]
with several volumes of equilibration buffer. This removes the low molecular weight contamination, which does not bind to the concanavalin A. The labeled egasyn is then eluted by percolating 5 ml of equilibration buffer containing 0.2 M methyl-a-mannoside through the column. The lzSI-labeled egasyn is stored frozen until use in the radioimmunoassay. As judged by electrophoresis and immunoreactivity, it is stable for at least 6 weeks. Titration of Labeled Egasyn with Anti-Egasyn Antibody. Titrations and radioimmunoassays can be performed in small glass tubes (0.6 cm x 5 cm). For titrations, antibody to egasyn is added at varying dilutions to about 10,000 cmp of 125I-labeled egasyn, in a final volume of 100/zl. All dilutions are made in TBS containing 0.1% Triton X-100 and 0.1% bovine serum albumin. The mixtures are incubated for 1 hr at 37° and then 24 hr at 4 °. Carrier control rabbit serum (40/xl of 4% serum) and antibody to rabbit IgG (30/zl) are added, and the mixtures are incubated for 1 hr at 37° and 4 hr at 4 °. The immunoprecipitate pellets are collected by centrifugation (10,000 g, 10 min) and washed twice with dilution buffer. Using our antibody preparations, 80-90% of the label is precipitated at high levels of antibody (10-fold dilution) and less than 3% of the label is precipitated in the absence of anti-egasyn antibody. 7 Radioimmunoassay of Egasyn. For the radioimmunoassay, a concentration of antibody that corresponds to about 50% binding of the 125Ilabeled egasyn is used. The antibody is first incubated with sample, 125Ilabeled egasyn is then added, and after further incubation the immune complexes are precipitated by the addition of goat antiserum raised against rabbit IgG. The egasyn content of samples is estimated by their ability to displace radioactivity from the precipitate to the supernatant fraction. Standard curves are obtained by the addition of unlabeled isolated egasyn. Most of the antigenic sites on egasyn in homogenates of liver are masked after extraction with Triton X-100 and become immunoreactive only after treatment with 1% sodium deoxycholate (Fig. 5). The reason for this is unclear but it is, therefore, necessary to carry out the radioimmunoassay in the presence of deoxycholate. 7 The assay described here is a liquid-phase procedure with double-antibody precipitation; other variations have not been tested. Thus, it may be possible to improve the sensitivity or simplicity of the assay, for example, with solid-phase immunoassay. The protocol for the assay is as follows: 1. Anti-egasyn IgG and the sample are incubated in TBS containing 1% sodium deoxycholate (100/xl final volume) for 1 hr at 37° and 24 hr at 4°. The amount of antibody used is sufficient to precipitate about 50% of the labeled egasyn (see below) in the absence of competing antigen and
[44]
EGASYN AND fl-GLUCURONIDASE MEMBRANE BINDING EGASYN
CONCENTRATION
(ng 0.1 i
PER
TUBE)
1 i
569
10
I
i
i
100 i
i
0.5
- ~'~^t .~-~o~ 0.4 LL
0.3
0.2 0.1 0 I
0.1
I
I
I
1
HOMOGENATE (p.I
I
I
10
I
100
CONCENTRATION PER
TUBE)
FIG. 5. Radioimmunoassay of mouse egasyn: effect of deoxycholate. Egasyn was assayed in the absence (©, A) or the presence (0, &) of 1% sodium deoxycholate as described in the text. The standard ( 0 , 0 ) was a preparation of egasyn purified from mouse liver and kidney by release from immunoprecipitates of microsomal glucuronidase. The sample (A, &) was a 10% homogenate of strain C57BL/6J liver extracted with Triton X-100 and centrifuged at high speed. Plotted on the ordinate is the ratio of antibody-bound nsI (B) to free n~I (F).
represents a 500-fold dilution of the antiserum. Samples are diluted in TBS containing 0.1% bovine serum albumin and 1% Triton X-100. The particulate matter in homogenates results in some interference with the assay. For this reason, assays are generally performed on high speed supernatant solutions of samples extracted with 2% Triton X-100, which solubilizes both egasyn and the microsomal glucuronidase complexes. 7 2. To the mixture is then added 5000 cpm for n5I-labeled egasyn (in 50 /~1 of sample buffer). This is incubated for 1 hr at 37° and 72 hr at 4°. 3. Egasyn bound to antibody is separated from free egasyn by the addition of goat antibody to rabbit IgG (30/xl) and 4% control rabbit serum as carrier (40/~1). This is incubated for 1 hr at 37° and 4 hr at 4°. The mixture is centrifuged at 10,000 g for 10 min, and the immunoprecipitate pellet is washed twice with 0.5 ml of sample buffer. The radioactivity in the pellet is measured with a gamma counter. A purified preparation of egasyn (from 1 to 50 ng per tube) is used as the standard. Typical radioimmunoassay results are shown in Fig. 5.