[45] Preparation and acidification activity of lysosomes and lysosomal membranes

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[45] P r e p a r a t i o n a n d Acidification Activity of L y s o s o m e s and Lysosomal M e m b r a n e s

By DONALD L. SCHNEIDERand JEAN CHIN Introduction Lysosomes are organelles of a membrane system which is known collectively as the vacuolar apparatus. This system includes lysosomes, coated vesicles, the Golgi apparatus, and endocytic vesicles, all of which participate in the essential cellular functions of endocytosis and exocytosis. The molecular events responsible for these functions are subjects of considerable current interest, especially now that proton pump activity, first found in lysosomes, is apparently present throughout the entire vacuolar apparatus. One would like to know whether the same molecular species of proton pump exists in all parts of the apparatus or whether each part has a distinct proton pump. The regulation of the proton pump(s) is another important area of research. In this chapter, we present methods for the isolation of lysosomes and lysosomal membranes from rat liver and from phagocytic cells and for the assay of acidification activity. In addition, the properties of the lysosomal ATP-driven proton pump will be considered.

Preparation of Lysosomes from Rat Liver This technique takes advantage of the buoyancy of lipoproteins to effect a very clean (and otherwise difficult) separation of Triton-lipoprotein-filled lysosomes (also known as tritosomes) from mitochondria. Rats are injected with the nonhemolytic agent Triton WR-1339 and, over the course of the next 3½ days, the 10-fold increase in plasma lipoproteins leads to accumulation of the Triton and lipoproteins in the liver lysosomes. A large granule fraction containing primarily mitochondria and lysosomes is prepared by differential centrifugation, and then, the lysosomes are floated up from the mitochondria by discontinuous sucrose gradient centrifugation.l-3 i A. Trouet, Arch. Int. Physiol. Biochem. 72, 698 (1964). 2 F. Leighton, B. Poole, H. Beaufay, P. Baudhuin, J. W. Coffey, S. Fowler, and C. de Duve, J. Cell Biol. 37, 482 (1968). 3 j. Burnside and D. L. Schneider, Biochern. J. 204, 525 (1982).

METHODS IN ENZYMOLOGY, VOL. 157

Copyright @ 1988by AcademicPress, Inc. All rights of reproduction in any form reserved.

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Reagents Triton WR-1339 (from Ruger Chemical, Irvington, NJ, or from Sigma, St. Louis, MO, as Tyloxapol): A 17% solution in saline is prepared by stirring at room temperature; complete dissolution may require stirring for about an hour. Although this stock solution of Triton WR-1339 is stable at ambient temperature, it should be stored frozen to avoid microbial growth that can be toxic to rats. Sucrose, 0.25 M, unbuffered. Prepare about 100 ml per rat and store in the refrigerator. Sucrose, 14.3% (w/w), density 1.06 g/ml, or 18% (w/v). Sucrose, 34.5% (w/w), density 1.155 g/ml, or 44% (w/v). Sucrose, 45% (w/w), density 1.21 g/ml, or 57% (w/v). Sucrose, 60% (w/w), density 1.295 g/ml, or 76.5% (w/v).

Procedure The preparation can easily be scaled up to a dozen rats or down to one; the desired number of rats are weighed individually and, under light ether anesthesia, each is injected intraperitoneally with 1 ml of Triton WR-1339 stock solution per I00 g body weight (dose: 850 mg of Triton WR-1339 per kilogram rat body weight). After 3½ days, decapitate the rats and rapidly excise and chill the livers by placing them in a tared beaker of ice-cold 0.25 M sucrose. Weigh the livers and, after mincing to 1-cm cubes, homogenize the livers one at a time in a size C Potter tissue grinder with motor-driven, serrated Teflon pestle in a volume of 25 ml of 0.25 M sucrose. A single stroke is sufficient. The homogenates are transferred to 50-ml centrifuge tubes and spun at 1500 g for 3 min. The supernatants are set aside on ice and the pellets are rehomogenized in the same volume and recentrifuged. The nuclear pellets are resuspended and a representative aliquot is saved. The combined supernatants are mixed well to give the extract (E) fraction; the volume is recorded. A l-ml aliquot is saved, and the remainder of the extract is centrifuged at 40,000 g (Sorvall SS34 rotor, 19,000 rpm) for 12 min. Decant the supernatants and set aside on ice. The pellets are resuspended in half the original volume of 0.25 M sucrose, using a hand-operated Potter tissue grinder with a smooth Teflon pestle. After recentrifugation, the supernatants are decanted, and the mitochondrial-lysosomal pellets are resuspended in 45% sucrose using a handoperated Potter tissue grinder. To assure that the density of the resuspended pellets is adequately high, a volume of 60% sucrose approximately equal to that of the pellets is added and, after very thorough mixing, a drop of the suspension is checked in a test tube containing a few milliliters of 34.5% sucrose in which it should sink. For large prepa-

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rations, the total volume should be at least 7 ml per rat. A 0.5-ml aliquot is set aside and the remainder of the resuspended mitochondrial-lysosomal fraction is applied to the gradient. Meanwhile, the supernatants are combined as the microsomal + soluble fraction (PS), and a 1-ml aliquot is saved. Sucrose Gradients. These are layered from the top down by adding, through a long, broken-tip Pasteur pipet, successive solutions to the bottom of the centrifuge tube. The Beckman SW 27 rotor with its capacity of 35 ml per tube provides a convenient compromise between capacity and centrifugation time, but other rotors may be used with appropriate adjustments. To avoid damage to organelles, excessive speeds are to be avoided. 4 Layer into each tube in the following order: (1) 5 ml of 14.3% sucrose, (2) 15 ml of 34.5% sucrose, (3) 13 ml of sample in 45% sucrose, and (4) 2 ml of 60% sucrose. When switching from one solution to the next, begin adding the next solution as the first enters the constricted part of the pipet so as to avoid (1) excessive mixing and (2) air bubbles that can disrupt the gradient. The gradients are centrifuged at 100,000 g (24,000 rpm) for 2 hr. Afterward the lysosomes at the 14.3-34.5% interface are apparent from the rich golden yellow-brown color (presumably due to aging pigment, lipofuscin). Since the lysosomes are above the mitochondria and other contaminants, it is important to unload the gradient from the top. This can be done in two ways. By using an inverted funnel (a handmade hollowed-out rubber stopper is perfectly adequate), one can pump heavy 60% sucrose into the bottom and float up the gradient. The lysosomes are collected in a volume of about 5 ml per gradient; all other parts of the gradient are combined as residue fraction. Alternatively, one can pierce the side of the tube with a syringe needle attached to a syringe and draw off the interface.

Analysis of the Preparation The lysosomal fraction will be enriched 40- to 60-fold relative to homogenate. If the rats are fasted overnight before sacrificing, the enrichment will be about 40; whereas without fasting, the enrichment will be about 60-fold. These differences presumably occur due to a greater protein content in lysosomes of fasted rats that are more active in protein degradation) Any number of marker enzymes are suitable, and a particu4 R. Wattiaux, S. Wattiaux-De Coninck, and M.-F. Ronveaux-Dupal, Eur. J. Biochem. 22, 31 (1971). 5 D. Ray, E. Cornell, and D. Schneider, Biochem. Biophys. Res. Commun. 71, 1246 (1976).

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lady convenient one is N-acetyl-fl-D-glucosaminidase (NA/3Gase) 6 assayed with p-nitrophenyl-N-acetyl-fl-o-glucosaminide, available from Sigma. In view of the sucrose content of gradient fractions and its interference in protein determinations, precipitation with trichloroacetic acid is advisable. The activity and protein values of the homogenate can be calculated from the values of the extract and nuclear fractions as recommended by de Dupe et al. 7 By assaying all of the fractions one can also make useful calculations concerning recoveries and yields, a detailed description of which is available) The overall yield of lysosomes is typically 30% or 10 mg of protein per rat. Preparation of Lysosomes from Phagocytic Cells Many cells take up inert, buoyant, latex beads and through endocytosis transfer the beads to their lysosomes. With latex, lysosomes from such diverse cell types as polymorphonuclear leukocytes, 9 amoebas, 1° and even fibroblastic L cells H can be isolated. Using again the principle of separation by flotation gradient centrifugation, a very clean separation of latex-filled lysosomes (more accurately referred to as phagolysosomes to denote the uptake of particulate material) from mitochondria is achieved. The preparation involves ingestion of the beads, homogenization, and flotation gradient centrifugation to isolate the lysosomes.

Reagents Latex beads: These are available from Sigma or Difco as aqueous suspensions. It is preferable to use beads about 1/zm in diameter, about the same size as bacteria (whence "fake bacteria"), because small beads (0.2 /zm) are not ingested efficiently and large ones (> 1.5/zm) promote the secretion of lysosomal enzymes and render the lysosomes fragile to manipulations. It is also to be noted that the beads are shipped in detergent solution to promote dispersion. Before using the latex, detergent is removed by diluting with l0 volumes of saline and centrifuging at 20,000 g (SorvaU SS34 rotor, 15,000 rpm) for 20 min. The pelleted beads are gently resuspended in a small volume of saline using a hand-operated Potter tissue 6 j. Findlay, G. A. Levvy, and C. A. Marsch, Biochem. J. 69, 467 (1958). 7 C. de Dupe, B. C. Pressman, R. Gianetto, R. Wattiaux, and F. Appelmans, Biochem. J. 60, 604 (1955). s C. de Dupe, J. Cell Biol. 50, 20D (1971). 9 D. R. Crawford and D. L. Schneider, J. Biol. Chem. 258, 5363 (1983). to E. D. Korn, this series, Vol. 31, p. 686. tt A. L. Hubbard and Z. A. Cohn, J. Cell Biol. 64, 461 (1975).

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grinder. To effect monodispersion, the washed beads are briefly subjected to gentle bath sonication just prior to use. Ingestion media: This can be either the growth media or phosphatebuffered saline if the cells will tolerate the lack of serum proteins and nutrients. Since uptake depends on the adsorptive properties of the beads, the presence of serum proteins may decrease ingestion and the ultimate yield of lysosomes. Phosphate-buffered saline (PBS): 150 mM sodium chloride, 10 mM sodium phosphate, pH 7.4. Albumin-PBS: Dissolve 1 g of bovine serum albumin in 100 ml of PBS and readjust the pH to 7.4. Sucrose solutions (w/v): 10%, 20%, and 30%, 100 ml each, chilled to 0_4 °. Sucrose solution (w/w), 60%, chilled. Sucrose solutions, 0.25 and 2 M.

Procedure Ingestion of latex is effectively carried out with quite concentrated solutions of beads and cells. One gram wet weight of packed cells is dispersed in 100 ml of ingestion media containing 0.1 g wet weight of packed latex. After incubation, at either 30 or 37° for 20 min with gentle shaking, the mixture is thoroughly chilled. The cells are collected by lowspeed centrifugation, washed twice with 30 ml of cold albumin-PBS, washed twice with chilled PBS, and resuspended in a small volume of 0.25 M sucrose to give 2 ml. Homogenization. The resuspended cells are transferred to a Dounce tissue grinder (14 ml size, type A, tight) and diluted to 50 mM sucrose by addition of 8 ml of ice-cold water. Cell disruption is achieved by 10 strokes of a tight pestle and isoosmolarity is restored by adding 1.2 ml of 2 M sucrose. Sucrose Gradient. Layer in order from top to bottom by adding through a long, broken-tip Pasteur pipet in an SW 27 tube the following: (1) 3 ml of 10% sucrose, (2) 10 ml of 20% sucrose, (3) 10 ml of 30% sucrose, (4) 10 ml of a 50 : 50 mixture of homogenate and 60% sucrose, and (5) 2 ml of 60% sucrose. Add successive solutions when the previous one enters the constricted part of the pipet. The gradients are centrifuged at 60,000 g (18,000 rpm) for 45 min. The lysosomes are collected from the 10-20% interface by pumping 60% sucrose into the bottom and floating the gradient up through an inverted funnel.

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Analysis of the Preparation The NAflGase activity, discussed above, is a convenient marker. Lysosomelike granules or phagolysosomes are relatively abundant in amoebae and neutrophils, and the phagolysosomal fraction will be at most 10fold enriched in marker activity relative to homogenate. Fibroblasts are less rich in lysosomes, and an enrichment of more than 20 is expected. With the latex procedure, the yields are commonly low, usually about 10% of total. Other comments are in order regarding analysis of phagolysosomal preparations. First, the neutrophils, known to contain different kinds of granules, carry out a sequence of fusion events between the forming phagolysosome and tertiary granules, specific granules, and azurophilic granules. When incubating latex beads with cells for short times, 6 min or less, it is important to be mindful that the azurophilic granules, marked by NA/3Gase, may not yet have fused with the forming phagolysosomes. Under such conditions, gelatinase for the tertiary granules or lactoferrin for the specific granules is a better marker. However, especially for these granules, fusion may precede closure of the forming phagolysosome from the outside, and considerable secretion occurs that selectively depletes phagolysosomes and may lead to an underestimation of enrichment. Second, protein determination of latex-laden phagolysosomes is facilitated by centrifugation of the assay solutions, after addition of all reagents (Lowry C and Folin) but prior to reading the absorbance, at 15,000 g for 10 min to eliminate the turbidity of the latex beads. Lysosomes from Other Cells At the present time no single technique is applicable in every instance. Ones that have a wide spectrum of uses and merit consideration are centrifugation in metrizamide, ~2centrifugation in Percoll, 13 and free flow electrophoresis. 14 Assay of Acidification Activity

In our laboratory, we routinely use the radioactive methylamine assay with rapid gel filtration ~5because it is very sensitive, easy to do batchwise t2 R. Wattiaux, S. Wattiaux-De Coninck, M.-F. Ronveaux-Dupal, and F. Dubois, J. Cell Biol. 78, 349 (1978). 13 L. H. Rome, A. J. Garvin, M. M. Allientta, and E. F. Neufeld, Cell (Cambridge, Mass.) 17, 143 (1979). ~4R. Stahn, K. P. Maier, and K. Hannig, J. Cell Biol. 46, 576 (1970). 15 D. L. Schneider, J. Biol. Chem. 256, 3858 (1981).

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with large numbers of samples (we routinely do 48 assays at once, about 2 hr from start to finish), and the activity value obtained is ApH that is more obviously related to acidity than a rate value such as nmol H ÷ per min. mg. The theory of using permeant weak bases to probe intravesicle pH is well founded as discussed in the literature. 16,17

Reagents Methylamine, 14C-labeled, is commercially available. The hydrochloride is not volatile; a stock aqueous solution, 0. I mCi per milliliter, is convenient and stable when stored frozen at -20 ° . The chemical concentration of methylamine in the assay is low, 1-10/~M, and one should be mindful that millimolar levels of permeant weak bases, including methylamine, ammonium sulfate, etc., are inhibitory and must be avoided when measuring acidification with any assay. ATP, 45 mM, pH 7.0, stock solution stored at - 2 0 °. MOPS [3-(N-morpholino)propanesulfonic acid], 0.2 M, pH 7. DTT (dithiothreitol), 1 M, aqueous solution, stored at - 2 0 °. EDTA (ethylenediaminetetraacetic acid) and EGTA (ethylene glycol bis(fl-aminoethyl ether)-N,N,N',N'-tetraacetic acid) solutions, 200 and 50 mM, respectively, pH 7. Magnesium chloride, 0.2 M. Potassium chloride and sodium chloride solutions, I M each. BSA (bovine serum albumin), 20 mg/ml aqueous solution, pH 7, sterilized by membrane filtration, stored at 4° . Phospholipid solutions, crude soybean (a mixture consisting primarily of phosphatidylcholine and phosphatidylethanolamine) or brain phosphatidylserine, 20 mg/ml aqueous dispersions obtained by homogenizing in a Potter tissue grinder and bath sonicating, dialyzing against 40 volumes of 1 mM Tris, 4 changes, 4 hr each. These are stored at - 2 0 °. Sephadex G-50:6 g swollen overnight at 4° in 100 ml of 20 mM potassium MOPS, pH 7, 60 mM sucrose, 60 mM potassium chloride, 1 mM EDTA, 2 mM magnesium chloride, 0.5 mM EGTA, is enough for 60 assays.

Procedure Tuberculin syringes, 1-ml size, are outfitted with flitted disks, and filled with the swollen Sephadex G-50 previously equilibrated at room 16 C. de Duve, T. De Barsy, A. Trouet, P. Tulkens, and F. Van Hoff, Biochem. Pharmacol. 23, 2495 (1974). 17 D. J. Reijngoud and J. M. Tager, Biochem. Biophys. Acta 472, 419 (1977).

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temperature. Immediately prior to use, these are centrifuged at 200 g for 3 min. The sample is preincubated with buffer and [14C]methylamine (500,000 cpm, about 10 nmol or 5/xM) at room temperature for I0 min (volume 180 /xl), and the assay is initiated by addition of 20/zl of either ATP (+ATP) or EDTA ( - A T P control). The final concentrations are 20 mM MOPS, pH 7, 60 mM sucrose, 50 m M potassium chloride, 12.5 mM sodium chloride, 0.5 mM EGTA, 0.1 mg/ml BSA, 75/zg/ml phospholipid, 9 mM magnesium chloride, and 4.5 mM of either ATP or EDTA. After incubation (usually 10 min), 150 /xl of each mixture is transferred to a tuberculin syringe filled with Sephadex (prespun) and centrifuged at 200 g for 3 min. The eluate, collected in a clean 13 × 100 mm tube during centrifugation, is diluted to 250/zl with water (add about 125/zl), and protein is precipitated by addition of 40/zl of 40% trichloroacetic acid. After centrifugation, 250/~1 of the supernatant is removed and counted for radioactivity, and the residue is analyzed for protein by the Lowry method.18 Calculations. The counts are first corrected for methylamine flowthrough, then the ATP-dependent proton gradient is calculated by dividing the cpm/mg value of the +ATP sample by that of the - A T P one as shown in the tabulation below. The cpm corrected value is obtained by subtracting an appropriate amount based on protein, e.g., the 0.0243 value for lysos-ATP is in part due to bovine serum albumin (7.25/0.0091 = 797 cpm/mg flow-through, 76.6/zg sample + 20 ~g BSA or 20.7% BSA); therefore, the correction is (0.0243)(797)(0.207) = 4.01 cpm. The ATPdependent ApH is obtained by taking the log, log 12.99 = 1.11 = ApH. Since the outside pH is 7.0, the pH inside is 5.89. It should be noted that this method assumes insignificant volume changes, an assumption that has been verified for lysosomes, 4/zl/mg _ ATP) 5 Sample

Net cpm

mg protein

cpm corrected

cpm/mg

Proton gradient

Flow-through Lysos-ATP Lysos+ATP

7.25 141.6 1739.6

0.0091 0.0243 0.0236

-137.6 1735.7

5,662.5 73,547

12.99×

A number of other methods for measuring acidification activity exist. 19,20 t80. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). ~9 X.-S. Xie, D. K. Stone, and E. Racker, this volume [49]. 20 N. Nelson, S. Cidon, and Y. Moriyama, this volume [48].

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Preparation of Membranes with Acidification Activity Freshly prepared lysosomes are mixed with proteinase inhibitors 21 and disrupted by freezing. Proteinase action is minimized by adding 1 mM Tris free base, 50 mM potassium phosphate, pH 7, 50/zM chymostatin, 25 /xM pepstatin, 3 /zM leupeptin, 5 mg/ml BSA, 2 mM p-aminobenzamidine, and 0.5/xM aprotinin. It is important to use a complete mixture of inhibitors because a limited mixture will stabilize the residual proteinase activities 22 and thereby labilize the acidification activity. Freezing at -20 ° is satisfactory for disruption and one overnight storage. When stored at - 8 0 ° with the proteinase inhibitors, the acidification activity is stable for at least 6 months. To isolate the membranes, the thawed lysosomes are diluted and either collected by discontinuous sucrose gradient centrifugation 21 or pelleted by centrifuging at 100,000 g for 35 min. The pelleted membranes are resuspended by hand in a small volume of 0.25 M sucrose using a Potter tissue grinder. Significantly, the pelleted membranes are active in acidification only if the proteinase inhibitor mixture is used. Also, it should be pointed out that the dilution is required: in 20% sucrose as isolated, the membranes are quite slow sedimenting as their equilibrium position is at a sucrose density of 1.12 (25% sucrose). Furthermore, excessive dilution, to less than density 1.03 (8% sucrose), will lead not only to pelleting of lysosomal membranes but also some of the lysosomal contents. For example, a significant part of the total lysosomal cholesterol (assayed enzymatically23), more than half in the case of tritosomes, appears on linear sucrose gradients at density 1.05 with about one third of the phospholipid (assayed as described24). The cholesterol is presumably from endocytosed lipoprotein cholesterol inasmuch as these gradient fractions contain no ATPase, acidification, nor acid 5'-nucleotidase activities. One might also point out that the Triton WR-1339 does not sediment but fractionates with the soluble proteins. Additional modifications are useful for the removal of latex beads from phagolysosomal membranes. The disrupted preparation is diluted to less than 15% sucrose (w/w) and layered over a cushion of 15% sucrose; by centrifugation at I00,000 g for 35 min, the membranes are pelleted while the latex beads accumulate above the 15% sucrose cushion. These are completely removed by inverting the tubes and wiping the walls with a swab before righting the tubes. 21 D. L. Schneider, J. Biol. Chem. 258, 1833 (1983). ,_2 D. L. Schneider, lntracell. Protein Catabolism, Proc. Int. Syrup., 5th, p. 291 0985). 23 C. A. Allain, L. S. Poon, C. S. G. Chan, W. Richmond, and P. C. Fu, Clin. Chem. 20, 470 (1974). 24 j. Chin and K. Bloch, J. Biol. Chem. 259, 11735 (1984).

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Concerning the isolation of lysosomal membranes, some miscellaneous points should be made. The addition of millimolar amounts of magnesium is often beneficial for the pelleting of membranes active in acidification. The effect is not an aggregative one, but may be related to a suppression of proteinase activity 22or perhaps due to the stabilization of a nonsedimentable factor. In addition, unless the ionic strength is at least 0.2 M, significant amounts of the soluble hydrolases like NA/3Gase will cofractionate with the membranes. 2~

Properties of the Lysosomal Proton Pump Acidification activity is inhibited by treatment of preparations with dicyclohexylcarbodiimide, diisothiocyanostilbenedisulfonic acid, and Nethylmaleimide but not by oligomycin, ouabain, or vanadate. Thus, it is clearly different from mitochondria126 and fungal plasma membrane 27 proton pumps. In addition, acidification activity is inhibited by the ionophores carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) 21 and monensin. 28 The relationship of the proton pumps in the various regions of the vacuolar apparatus is less clear, inasmuch as they all have similar sensitivities to dicyclohexylcarbodiimide and N-ethylmaleimide and insensitivities to oligomycin, ouabain, and vanadate. 15,29-33 Suggestion that the lysosomal pump may be unique is based on (1) a less strict specificity for ATP (GTP supports acidification activity nearly as w e l l ) , 15'34 and (2) an insensitivity to duramycin. 35 However, there are alternative explanations for the observed data, and protein purification studies are necessary. Concerning the studies on reconstitution of acidification carried out in Racker's laboratory, 36 which indicate that the 25 D. L. Schneider, J. Burnside, F. R. Gorga, and C. J. Nettleton, Biochem. J. 176, 75 (1978). 26 E. Racker, " A New Look at Mechanisms in Bioenergetics." Academic Press, New York, 1976. 27 B. J. Bowman, F. Blasco, and C. W. Slayman, J. Biol. Chem. 256, 17343 (1981). 28 j. A. Fink, M. J. Cahilly, and D. L. Schneider, Biochem. Arch. 1, 37 (1985). 29 S. Ohkuma, Y. Moriyama, and T. Takano, Proc. Natl. Acad. Sci. U.S.A. 79, 2758 (1982). 30 M. Forgac, L. Cantley, B. Wiedenmann, L. Altstiel, and D. Branton, Proc. Natl. Acad. Sci. U.S.A. 80, 1300 (1983). 31 D. K. Stone, X.-S. Xie, and E. Racker, J. Biol. Chem. 258, 4059 (1983). ~2 F. Zhang and D. L. Schneider, Biochem. Biophys. Res. Commun. 114, 620 (1983). 33 j. Glickman, K. Croen, S. Kelley, and Q. AI-Awgati, J. Cell Biol. 97, 1303 (1983). 34 C. J. Galloway, G. E. Dean, M. Marsh, G. Rudnick, and I. Mellman, Proc. Natl. Acad. Sci. U.S.A. 80, 3334 (1983). 35 D. K. Stone, X.-S. Xie, and E. Racker, J. Biol. Chem. 259, 2701 (1984). 36 X.-S. Xie, D. K. Stone, and E. Racker, J. Biol. Chem. 259, 11676 (1984).

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coated-vesicle pump consists of a unique set of polypeptides that fit the F~F0 paradigm in which the ATPase (F~ portion) is more readily solubilized than the proton channel (F0 portion), it is worth noting that both the coated vesicle and the lysosomal ATPases are stimulated by dithiothreitol and phosphatidylserine. To date, there are no known inhibitors specific for the lysosomal proton pump. Although micromolar concentrations of trifluoperazine inhibit acidification and ATPase activities (IC50 = 50/~M), the involvement of calmodulin is unlikely inasmuch as 50/zM levamisole, bromotetramisole, and calmidazolium are without effect, and, therefore, the inhibitory effect of trifluoperazine is presumably nonspecific. The N-ethylmaleimide sensitivity of the lysosomal proton pump requires comment. Although the majority of the ATPase activity in rat liver lysosomes is membranous, only 5.5% of the membranous ATPase is inhibited by N-ethylmaleimide. Moreover, the majority of the N-ethylmaleimide-sensitive ATPase activity in lysosomes is soluble (greater than 80%) and not membrane bound. Thus, we are faced with the awesome probability that the proton pump is a very minor protein component of lysosomes.

[46] A n a l y s i s of E n d o s o m e a n d L y s o s o m e Acidification in Vitro By CYNTHIA J. GALLOWAY, GARY E. DEAN, RENATAFUCHS, and IRA MELLMAN The low internal pH of endocytic organelles plays a critical role in maintaining the orderly traffic of receptors and receptor-bound ligands during endocytosis. 1,2In endosomes, acidic pH promotes the dissociation of many ligands from their receptors, allowing the receptor to recycle to the cell surface and the ligand to be transported to lysosomes. In lysosomes, acidic pH facilitates degradation of internalized macromolecules by lysosomal hydrolases, many of which have acidic pH optima. In addition to these beneficial functions, low intravesicular pH is used by a variety of pathogenic agents to enter the cell. For example, many enveloped viruses and bacterial toxins penetrate endosomal or lysosomal memi A. Helenius, I. Mellman, D. Wall, and A. Hubbard, Trends Biochem. Sci. 8, 245 (1983). 2 M. S. Brown, R. G. W. Anderson, and J. L. Goldstein, Cell (Cambridge, Mass.) 32, 663 (1983).

METHODS IN ENZYMOLOGY, VOL. 157

Copyright © 1988by Academic Press, Inc, All rights of reproduction in any form reserved,