JOURNAL OF CELLULAR PHYSIOLOGY 147:447454 (1991)
Characteristics of Taurine Transport in Rat Liver Lysosomes JAYDUTTV. VADGAMA,* KENT CHANG, JOEL D. KOPPLE, JOHN-MICHAELIDRISS, AND ADAM J , JONAS Division of Medical Genetics (j.V.V., K.C., I.-M./.,AJ.1.) and Division of h'ephrology it.D. KJ, Harbor-UCLA Medical Center, Torrance, California YO502 Taurine (2-aminoethanesulfonic acid) is a unique sulfur amino acid derivative that has putative nutritional, osmoregulatory, and neuroregulatory roles and is highly concentrated within a variety uf cells. The permeability of Percoll density gradient purified rat liver lysosomes to taurine was examined. lntralysosomal amino acid analysis showed trace levels of taurine compared to most other amino acids. Taurine uptake was Na+-independent, with an overshoot between 5-1 0 minutes. Trichloroacetic acid extraction studies and detergent lysis confirmed that free taurine accumulated in the lysosomal space. Kinetic studies revealed heterogeneous uptake with values for Km, = 31 z 1.82 and Km, > 198 ? 10.2 mM. The uptake had a pH optimal of 6.5 and was stimulated by the potassium specific ionophore valinomycin. The exodus rate was fairly rapid, with a tli2 of 5 minutes at 37°C. Analog inhibition studies indicated substrate specificity similar to the plasma membrane p-alanine carrier system, with inhibition by p-alanine, hypotaurine, and taurine. a-Alanine, 2-niethylaminoisobutyric acid (MeAIB), and threonine were poor inhibitors. No effects were observed with sucrose and the photoaffinity derivative of taurine NAP-tnurine (N-(il-azido-2-nitrophenyl)-2aminoethanesulfonatel. In summary, rat liver lysosomes possess a high Km system for taurine transport that is sensitive to changes in K' gradient and perhaps valinomycin induced diffusional membrane potential. These features may enable lysosomes to adapt to changing intracellular concentrations of this osmotic regulatory substance.
The metabolic role of taurine (2-aminoethanesulfonic acid) is a topic that has generated a great deal of interest. This molecule has the amino group on the beta carbon, not the alpha-carbon typical of most other amino acids, and has a sulfonic group in place of the more usual carboxylic group. Whereas the metabolism of taurine is limited, this compound has been implicated in a number of diverse processes. Besides its well-known function in bile salt synthesis (Hofmann and Small, 1967), taurine is involved in a variety of biochemical and physiological processes including osmoregulation (Thurston et al., 1980), cellular proliferation (Gaul1 et al., 1983), modulation of calcium and sodium fluxes (Sebring and Huxtable, 1985; Huxtable and Sebring, 19861, stimulation of glycolysis and glycogenesis (Kulakowski and Maturo, 1984),modulation of neuronal excitability (Oja and Kontro, 1983; Bernardi, 19851,detoxification (Emudianughe et al., 1983), membrane stabilization (Pasantes-Morales et al., 19851, antimutagenesis (Laidlaw et al., 19891, and retinal function in cats, premature or infant primates (Hayes et al., 1975; Sturman and Hayes, 19801, and possibly humans (Geggel et al., 1985; Sturman, 1986). Since most if not all cells accumulate intracellular taurine to levels much greater than plasma levels, it is conceivable that cellular organelles have developed mechanisms to regulate this substance in a manner 0 I991 WILEY-LISS. INC.
that would protect them from osmotic perturbations. The degradation of macromolecules within lysosomes results in the production of relatively large concentrations of smaller molecules such as amino acids and sugars. A number of specific transport systems (Pisoni et al., 1985, 1987; Bernar et al., 1986; Jonas, 1986; Collarini et al., 1989; Greene et al., 1990; Jonas and Jobe, 1990), are found in the lysosomal membrane, which allows egress of these materials, thus avoiding osmotic imbalances. Presumably, adaptive regulation of intracellular taurine concentration creates another environmental challenge for the lysosome. We have examined the permeability of lysosomes to taurine and have identified a system, although specific for p-amino acids, that is Na+-independent and with relatively high Km value(s). METHODS Materials All chemicals were obtained from Sigma Chemical Co. unless otherwise indicated. Female, 150-200 g, S3rague-Dawley rats were purchased from Harlan. [ HI-Taurine was obtained from Amersham. GFC filReceived November 9, 1990; accepted February 25, 1991 "To whom reprint requestdcorrespondence should be addressed.
448
VADGAMA ET AL.
ters (Fisher), Scintillation fluid 3a70B (RPI), and NAPtaurine lN-(4-azido-2-nitrophenyl)-2-aminoethane sulfonatel was purchased from Pierce Chemical Co. Lysosomal purification Rats were sacrificed by C02narcosis. The livers were excised, flushed with ice-cold buffer (0.25 M sucrose, 20mM Hepes, pH 7.0) to remove blood, and were ground in a Dounce homogenizer. An organelle pellet was prepared by differential centrifugation, and the lysosomes were further purified by Percoll density gradient centrifugation as previously described (Jonas, 1986). Following centrifugation, the high density fraction containing lysosomes was removed for use in subsequent studies. In a previous report, Jonas et al. (1986)had shown that lysosomes were purified approximately 90-fold by this method as indicated by the specific activity of p-hexosaminidase (Barret, 1972). One unit of enzyme activity is defined as hydrolysis of a pmole of substrateiminuteimg protein. Lysosomal latency. Lysosomal latency was assessed by the p-hexosaminidase assay as described by Pisoni et al. (1985). This procedure is a modification of the method of Hall et al. (1978). Essentially, 10-20 pl of lysosomal suspension with or without 1%triton X-100 were added t o 100 pl of 4 mM p-nitrophenyl(3-D-N-acetylglucosaminidein 0.36 M sucrose containing 0.043 M citric acid, 0.11 M Na2HP0, pH 4.9 buffer. Samples were incubated for 10 minutes at 37"C, and the reaction terminated by adding 1.0 ml of 0.4 M glycine! NaOH pH 10.4. The p-nitrophenol formed was read at 400 nM, and the amount was determined from a standard curve for p-nitrophenol prepared with the same reagents. One unit of p-hexosaminidase activity is defined as the amount of enzyme activity necessary to produce 1nmol of p-nitrophenolimin at 37°C. Using this procedure, the integrity of lysosomal preparation was determined to be between 90-95%. Taurine uptake In order to remove Percoll, lysosomes were diluted in a 10-fold excess of buffer and were collected by centrifugation at 13,000 g for 10 minutes at 4°C. The pellet was resuspended in ice-cold Krebs-Rin er choline phosphate buffer with 118 mM choline c loride, 5.9 mM KCL, 1.2mM MgS04 7H&, 0.5mM CaC1,-2H20, 2.0 mM KHCO', 5.5 mM D-glucose, and 25 mM choline phosphate. On certain occasions, this buffer was replaced with MOPS (50 mM) sucrose (0.25 M) EDTA (1mM) buffer adjusted to pH 7.0. Following incubation, lysosomes were collected on GFC glass fiber filters and were washed with 2X 10 ml aliquots of ice-cold phosphate-buffered saline (PBS). Filters were then dried and subjected to scintillation counting. Uptake was defined as pmoles taurine per mg lysosomal protein per minute. Total protein was determined b the method of Lowry et al. (1951). Note that we ha originally expressed our data in terms of mg of lysosomal protein. During the later studies, we compared uptake expressed in units of p-hexosaminidase activity. We found no difference in uptake profile, inhibition pattern, or on the kinetic parameters of Km. The primary data were applied to a basic program on an IBM-AT compatible computer.
a
IK
Taurine efflux Lysosomes were equilibrated with 10 pM L3H1-taurine for 10 minutes at 37°C. The taurine-loaded lysosomes were washed with 10 volumes of cold choline KRP buffer and were collected by centrifugation. The pellet was resuspended in buffer and used for efflux experiments. Lysosomes were collected as described for uptake studies. Membrane potential Membrane potential was measured fluorometrically using the cyanine dye di-OC6 (Seliymann and Gallin, 1983). Excitation wavelength was 460 nM and emission wavelength 510 nM. Transport kinetics Data obtained from concentration dependent transport studies were subjected to kinetic analysis using Cleland's computer program (Cleland, 1979) for biphasic uptake called Twoonl, where Log v = log
Vmax~.[S] Kml [S]
+
+ Vmaxp[S] Kma + [S]
Note that in this kinetic program, the data are processed after correcting for the nonsaturable component obtained from another equation called Toorgl, where Log v = log
where Kd
=
Vmax.[S] Km [S]
+
+ Kdo[S]
the nonsaturable component.
RESULTS Time course of uptake and cation selectivity Figure 1 shows the uptake of 10 FM 'H-taurine into rat liver lysosomes in sodium containing or sodium-free choline KRP, pH 7.4 at 37°C. Although the data appear to show no significant (p < 0.1) difference between uptake in sodium and sodium-free conditions, the overshoot in Na+-media was less compared to that observed in choline media. Following an initial overshoot between 5 to 10 minutes, the uptake reached equilibrium by 20 minutes. Next, in order to be certain that the uptake measurements were not reflecting 3H-taurine binding onto lysosomal membranes, we subjected incubated lysosomes to TCA precipitation (Fig. 1B). Aliquots of lysosomal suspensions (200 pl) were removed at intervals, placed into ice-cold PBS, and centrifuged immediately for 2 minutes in Eppendorf microfuge. The pellet was rinsed once more with 1ml of ice-cold PBS and was suspended into 220 pl of 5%TCA. Virtually all of the label was removed in the TCA soluble fraction, consistent with uptake of free taurine. Additional tests were performed to confirm that the labeled accumulation with time represented uptake and not simple binding. These were as follows: (1)the uptake was measured in the presence of 1%detergent Triton X-100, and the results (Fig lr) showed no increase in accumulation with respect to time, and (2) at the end of the normal uptake procedure, lysosomes were washed with ice-coldd-H20instead of PBS. In this
449
LYSOSOMAL TAURINE TRANSPORT 3.0
T
2.54
35 T
a P
0
04 10
20
30
40
50
60
0
Time. minutes
Fig. 1. Time course of 10 pM [3H-taurine uptake into rat liver lysosomes. A. The uptake of 10 ELMr3H taurine was measured up to 60 minutes in N a ' containing or Na+-free(choline) Krebs Ringer phosphate (KRP) buffer at pH 7.4 at 37°C. The data are mean i- SEM of 3 determinations. The composition of the buffer was as follows: NaCL 118mM, KCL 5.9mM, Na,HPO, 25mM, MgSO, 7H,O 1.2mM, KHCO, 2.0 mM, CaCl, 2H20 0.51 mM, and D-glucose 5.5 mM. For sodium-free buffer, sodium chloride and sodium hydro en phosphate were replaced by equimolar choline chloride and cho7ine hydrogen phosphate. The data in ( A ) indicate uptake in the presence of detergent. triton X-100 at 1%. B. The uptake of 10 pM [jHI-taurine was measured a s in A, except that it was terminated by a rapid
I
10
20
30 40 Time, minutes
50
60
centrifugation assay; 200 p1 aliquots of the lysosomal suspension were removed at specific time intervals and placed into 1.0 ml of ice-cold choline KRP. The suspension was immediately centrifuged for 2 minutes in Eppendorf microcentrifuge at 15,OOOxg.The supernatant was aspirated and the lysosomes were rinsed once more under the same conditions. The pellet was then suspended into 120 p1 of 5% trichloracetic acid (TCA) for 1hour, and the suspension centrifuged for 5 minutes in the Eppendorf centrifuge; 100 111 of the supernatant was removed for isotope counting. The residual TCA was removed, the pellet dissolved in 1N NaOH, and total protein was determined by the method of Lowry et al., 1951.
TABLE 1. Concentration of amino acids in rat liver lysosomes' Amino acid Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine Cystine Tyrosine Alanine
pmoles/mg protein
LIM
Amino acid
0.103 0.187 0.372 0.287 0.124 0.208
23 42 83 64 28 39 46
ND
ND
Arginine Asparagine Aspartic acid Glutamic acid Glutamine Glycine Ornithine Proline Serine Taurine Citrulline H ydroxyproline
0.175 0.253
56
ND
ND
0.159 0.294
35 66
pmoles/mg protein
pM
0.052 0.165 0.091 0.136 0.181 0.223 0.165 0.189 0.282 0.005 0.011
15 37 20 30 40 50 37 42 63 1.1 -~ 2.4
ND
ND
'Percoll purified lysosomes were suspended in Mops/Tris pH 7.0 buffer containing 0.25M sucrose, 1 mM Na.2EDTA, and 1.0% Triton X-100. This suspension was kept on ice for 10 minutes to facilitate lysosomal lysis. Soon after, total protein was precipitated with 5%TCA (trichloroacetic acid) followed by a 10-minute centrifugation at 16,000 xg. The TCA soluble supernatant was then processed for amino acid analysis on a Beckman amino acid analyzer (model 121 MB). The protein pellet was dissolved in 1 N NaOH, and its content estimated by the method of Lowry. The values are mean of 3 determinations and were expressed at pmoles per mg of total protein or as concentration in pM. In the later case, intralysosomal water space is assumed to be 4.5 pL per mg of protein (Schneider, 1983).
case, we found little or no counts remaining on the GFC filter. Intralysosomal concentration of taurine. Table 1 shows that, in comparison to most amino acids, the endogenous content of taurine is significantly low.
were observed when data (up to concentration range of 250 mM) in Figure 2A were transformed into a Hofstee plot (Fig. 2B) to obtain values for the kinetic parameters Km and Vmax. The estimated values are Kml = 30.9 ? 1.82 mM; Km2= 198 k 10.2mM; Vmaxl,= 3.4 2 0.29 and Vmax2 = 19.0 ? 0.81 pmollmg protein/ min. These values were determined from the kinetic Concentration dependent uptake and kinetic equation suitable for estimating biphasic uptake. This analysis of taurine uptake program is called Twoonl, and the equation is described Figure 2A shows the concentration-dependent up- in Methods. Note that the nonsaturable component take of taurine into rat liver lysosomes. The concentra- (0.076nmol/mg proteinimin) was corrected before protion range varied from 0.01 to 250 mM with a range of cessing the data for this equation. In addition, this 30 different concentration points. The data show ap- kinetic study was repeated 3 times, and uptake exparent saturation at substrate concentrations over pressed in terms of pmols/unit of P-hexaseimin. The 50 mM. However, the uptake was linear up to 5 mM values for Km were identical to those expressed here. taurine concentration (Fig. 2A inset). Biphasic kinetics The Vmax values were 1.2 and 12.5 pmolslheximin.
450
VADGAMA ET AL. 1210.0 -'\
\
\ \
*\\
o.1c5 0.0
I
0
25
50
75
150 [Tourine], rnM
100
125
175 200
225
250
0.05
Fig. 2. A. Concentration-dependent uptake of taurine into rat liver lysosomes. The uptake of 0.01 to 250 mM r3H1-taurine was measured for 1 minute in choline KRP, pH 7.4, and at 37°C. A shows the concentration-dependent uptake up to 250 mM taurine. Osmotic corrections were made by reducing choline chloride to appropriate concentrations. The inset shows that uptake was linear up to substrate concentrations of 5 mM. The uptake and termination procedures are as described in the methods section. Note that lysosomal latency was examined with each taurine concentration and was found to be
8 5 9 0 % B. Kinetics of taurine uptake into rat liver lysosomes. The data from the Michaelis-Menten plot in A were transformed into a Hofstee plot to determine kinetic parameters Km and Vmax. The plot revealed a biphasic uptake with Km values of 30.9 ? 1.82 mM and 198 ? 10.2 mM. Vmax values were 3.4 i 0.29 and 19 i 0.81 pmolimg proteinimin. These studies were re-examined and later expressed in units of P-hex activity. The Km values remained identical, whereas the Vmax values were 1.2 and 12.5 pmolslheximin.
2.5 7
"T
T
1
D3ontrol .5
E
-
P
c c
1.5.-
10% in potential generated by the proton pump. In summary, marked changes in cystoplasmic osmoeach case. larity due to influx of taurine could have profound Fig. 10. Effect of KCL, valinomycin, taurine, and cccp in di-0-C, effects on lysosomal volume and possibly function. fluorescence in the presence of lysosomes. Lysosomes were added at Bidirectional permeability of lysosomes to taurine althe first arrow to a cuvette containing 2 mL of 25 nM di-0-C, in choline KRP, pH 7.4 at room temperature. Dye was allowed to lows these structures t o adapt to such intracellular equilibrate with lysosomes (chart speed 1mm/min). A. Vertical changes. This ability may be of great importance in arrows indicate the addition of 5 pM valinomycin (Val), aliquots of tissues with marked accumulation of taurine and at 1M KCL, (up to a final concentration of 31 mM). B. Same as A, but different stages of development. Currently, taurine has followed by 0.1 mM taurine (Tau). C. 9.1 mM taurine was added prior no known function in lysosomes. Studies with intact to the addition of valinomycin and KCL. Excitation wavelength was 460 nM, emission wavelength was 510 nM. These results are repre- cells have suggested that taurine may help stabilize sentative of 5 separate determinations. membranes (Pasantes-Morales et al., 1985). Additional
B
B
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Jonas, A.J., and Jobe, H. (1990) Sulfate transport by rat liver lysosomes. J . Biol. Chem., 265:17545-17549. Jonas, A.J., Greene, A.A., Smith, M.L., and Schneider, J.A. (1982) Cystine accumulation and loss in normal heterozygous and cystiACKNOWLEDGMENTS notic fibroblasts. Proc. Natl. Acad. Sci. U.S.A., 79t4442-4445. D.P., Miller, L.A., and Chesney, R.W. (1990) Adaptive regulaWe thank Carol Rivera for her aid in preparation of Jones, tion of taurine transport in two continuous renal epithelial cell the manuscript. This work was supported by NIH lines. Kidney Int., 38.219-226. awards DK39147 and DK37403. Knauf, P.A., Breuer, W., McCulloch, L., and Rothstein, A. (1978) N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-taurinej as a photoaffinity probe for identifying membrane components conLITERATURE CITED taining the modifier site of the human red blood cell anion exchange system. J. Gen. Physiol., 72:631-649. Barrett, A.J. (1972) In Lysosomes, A Laboratory Handbook. J.T. Kulakowski, E.C., and Maturo, J. (1984) Hypoglycemic properties of Dingle, ed. Elsevier, New York, pp. 46-216. taurine not mediated by enhanced insulin release. Biochem. PharBashford. C.L.. and Smith, J.C. (1979) Methods of Enzymol., 55:569macol., 33:2835-2838. 586. Bernar, J., Tietze, F., Kohn, L.D., Bernardini, I., Harper, G.S., Laidlaw, S.A., Dietrich, M.F., Lamtenzan, M.P., Block, J.B., and Komle. J.D. 119891Antimutagenic effects of taurine in a bacterial Grollman, E.F., and Gahl, W.A. (1986) Characterization of a lysoa&y system. Cancer Res., 49766004604. soma1 membrane transport system for tyrosine and other neutral Lowry, O.H., Rosebrough, K.J., Farr, A.L., and Randall, R.J. (1951) amino acids in rat thyroid cells. J. Biol. Chem., 261:17107-17112. Protein measurement with the folin phenol reagent. J . Biol. Chem., Bernardi, N. (1985) On the role of taurine in the cerebellar cortex: A 193:265-275. reappraisal. Acta Physiol. Pharmacol. Latinoamer., 35:153-164. Chesney, R.W. (1985) Taurine: Its biological role and clinical impli- Oja, S.S., and Kontro, P. (1983) Taurine, In Handbook of Neurochemistry, 2nd ed. A. Lajtha, ed. 3:501. Plenum, New York, pp. 501. cations. Adv. Pediatr., 32:l-42. Christensen, H.N. (1988) Amino acid transport systems of lysosomes: Pasantes-Morales, H., and Cruz, C. (1985) Taurine: A physiological stabilizer of photoreceptor membranes, In Taurine: Biological AcPossible substitute utility of a surviving transport system for one tions and Clinical Perspectives. S.S. Oja. L. Ahtee, and P. Kontro, et congenitally defective or absent. Bioscience Rep., 8:121-129. al., eds. Alan R. Liss, New York, pp. 371-381. Christensen, H.N., Hess, B., and Riggs, T.R. (1954) Concentration of taurine, p-alanine, and tri-iodothyronine by ascites carcinoma cells. Pasantes-Morales, H., Wright, C.E., and Gaull, G.E. (1985) Taurine protection of lymphoblastoid cells from iron-ascorbate-induced damCancer Res., 14,124-127. age. Biochem. Pharmacol., 34:2205-2207. Cleland, W.W.(1979) Methods Enzymol., 63:103-138. Collarini, E.J., Pisoni, R.L., and Christensen, H.N. (1989) Character- Pisoni, R.L., Thoene, J.G., and Christensen, H.N. (1985) Detection and characterization of carrier-mediated cationic amino acid transport ization of a transport system for anionic amino acids in human in lysosomes of normal and cystinotic human fibroblasts. J. Biol. fibroblast lysosomes. Biochem. Biophys. Acta, 987:139-144. Chem., 260:47914798. Emudianughe, T.S., Caldwell, J., and Smith, R.L. (1983) The utilization of exogenous taurine for the conjugation of xenobiotic acids in Pisoni, R.L., Flickinger, K.S., Thoene, J.G., and Christensen, H.N. (1987) Characterization of carrier-mediated transport systems for the ferret. Xenobiotica, 13:133-138. small neutral amino acids in human fibroblast lysosomes. J. Biol. Gahl, W.A., Bashan, N., Tietze, F., Bernardini, I., and Schulman, J.D. Chem., 262:6010-6017. (1982) Cystine transport is defective in isolated leukocyte lysosomes from patients with cystinosis. Science, 21 7:1263-1265. Pisoni, R.L., Acker, T.L., Lisowski, K., Lemons, R.M., and Thoene, Gahl, W.A., Tietze, F., Bashan, N., Bernardini, I., Radford, D., and J.G. (1990) A cysteine specific lysosomal transport system provides Schulman, J.D. (1983) Characteristics of cystine countertransport a major route for the delivery of thiol to human fibroblast lysosomes: in normal and cystinotic lysosome-rich leucocyte granular fractions. possible role in supporting lysosomal proteolysis. J. Cell Biol., Biochem. J., 216t393-400. 110:327-335. Gaull, G.E., Wright, C.E., and Tallan, H.H. (1983) Taurine in human Reeves, J.P. (1983)In: Lysosomes in Biology and Pathology, vol. 7. J.T. lymphoblastoid cells: Uptake and role in proliferation. In: Sulfur Dingle, R.T. Dean, and W. Sly, eds. Elsevier Biomedical Press, Amino Acids: Biochemical and Clinical Aspects. Liss. New York, pp. Amsterdam. 297-303. Schneider, D.L. (1983) ATP-dependent acidification of membrane Geggel, H.S., Ament, M.E., Heckenlively, J.R., Martin, D.A., and vesicles isolated from purified rat liver lysosomes. J . Biol. Chem., Kopple, J.D. (1985) Nutritional requirement for taurine in patients 258:1833-1838. receiving long-term parenteral nutrition. New Engl. J. Med., Sebring, L.A., and Huxtable, R.J. (1985) Taurine modulation of 312:142-146. calcium binding to cardiac sarcolemma. J. Pharmacol. Exp. Therap., Greene, A.A., Marcusson, E.G., Morell, G.P., and Schneider, J.A. 232:445-45 1. (1990) Characterization of lysosomal cystine transport system in Seliymann, B.E., and Gallin, J.I. (1983)J . Cell Physiol., 115:105-115. mouse L-929 fibroblasts. J . Biol. Chem., 265:9888-9895. Stewart, B.H., Collarini, E.J., Pisoni, R.L., and Christensen, H.N. Hall, C.W., Liebraers, I., DiNatale, P., and Neufeld, E.F. (1978) 11989) Separate and shared lysosomal transport of branched and Methods Enzymol., 50:439-456. aromatic dipolar amino acids. Biochim. Biophys. Acta, 987145-153. Harikumar, P., and Reeves, J.P. (1983) The lysosomal proton pump is Sturman, J.A. (1986) Nutritional taurine and central nervous system electrogenic. J. Biol. Chem., 258:10403-10410. development. Ann. N.Y. Acad. Sci. 477r196-213. Hayes, K.C., Carey, R.E., and Schmidt, S.Y. (1975) Retinal degener- Sturman, J.A., and Hayes, K.C. (1980) The biology of taurine in ation associated with taurine deficiency in the cat. Science> nutrition and development. Adv. Nutr. Res., 353-299. 188r949-95 1. Thurston, J.H., Hauhart, R.E., and Dirgo, J.A. (1980) Taurine: A role Hofmann, A.F., and Small, D.M. 11967) Detergent properties of bile in osmotic regulation of mammalian brain and possible clinical salts: Correlation with physiological function. Annu. Rev. Med., significance. Life Sci., 26:1561-1568. 18:333-367. Vadgama, J.V., and Kopple, J.D. (1989) Regulation of taurine transHuxtable, R.J., and Sebring, L.A. (1986)Towards a unifying theory for port into renal tubular cells in culture. FASEB J., 3:#4846, A1060. the actions of tauriue. Trends Pharmacol. Sci., 9:481485. Wright, C.E., Tallan, H.H., Lin, Y.Y., and Gaull, G.E. (1986) Taurine: Jonas, A.J. (1986) Cystine transport in purified rat liver lysosomes. Biological update. Ann. Rev. Biochem., 55:427453. Biochem. J., 236:671-677.
studies will be required to determine whether this is the case for lysosomes.
I
,
1
Characteristics of Taurine Transport in Rat Liver Lysosomes JAYDUTTV. VADGAMA,* KENT CHANG, JOEL D. KOPPLE, JOHN-MICHAELIDRISS, AND ADAM J , JONAS Division of Medical Genetics (j.V.V., K.C., I.-M./.,AJ.1.) and Division of h'ephrology it.D. KJ, Harbor-UCLA Medical Center, Torrance, California YO502 Taurine (2-aminoethanesulfonic acid) is a unique sulfur amino acid derivative that has putative nutritional, osmoregulatory, and neuroregulatory roles and is highly concentrated within a variety uf cells. The permeability of Percoll density gradient purified rat liver lysosomes to taurine was examined. lntralysosomal amino acid analysis showed trace levels of taurine compared to most other amino acids. Taurine uptake was Na+-independent, with an overshoot between 5-1 0 minutes. Trichloroacetic acid extraction studies and detergent lysis confirmed that free taurine accumulated in the lysosomal space. Kinetic studies revealed heterogeneous uptake with values for Km, = 31 z 1.82 and Km, > 198 ? 10.2 mM. The uptake had a pH optimal of 6.5 and was stimulated by the potassium specific ionophore valinomycin. The exodus rate was fairly rapid, with a tli2 of 5 minutes at 37°C. Analog inhibition studies indicated substrate specificity similar to the plasma membrane p-alanine carrier system, with inhibition by p-alanine, hypotaurine, and taurine. a-Alanine, 2-niethylaminoisobutyric acid (MeAIB), and threonine were poor inhibitors. No effects were observed with sucrose and the photoaffinity derivative of taurine NAP-tnurine (N-(il-azido-2-nitrophenyl)-2aminoethanesulfonatel. In summary, rat liver lysosomes possess a high Km system for taurine transport that is sensitive to changes in K' gradient and perhaps valinomycin induced diffusional membrane potential. These features may enable lysosomes to adapt to changing intracellular concentrations of this osmotic regulatory substance.
The metabolic role of taurine (2-aminoethanesulfonic acid) is a topic that has generated a great deal of interest. This molecule has the amino group on the beta carbon, not the alpha-carbon typical of most other amino acids, and has a sulfonic group in place of the more usual carboxylic group. Whereas the metabolism of taurine is limited, this compound has been implicated in a number of diverse processes. Besides its well-known function in bile salt synthesis (Hofmann and Small, 1967), taurine is involved in a variety of biochemical and physiological processes including osmoregulation (Thurston et al., 1980), cellular proliferation (Gaul1 et al., 1983), modulation of calcium and sodium fluxes (Sebring and Huxtable, 1985; Huxtable and Sebring, 19861, stimulation of glycolysis and glycogenesis (Kulakowski and Maturo, 1984),modulation of neuronal excitability (Oja and Kontro, 1983; Bernardi, 19851,detoxification (Emudianughe et al., 1983), membrane stabilization (Pasantes-Morales et al., 19851, antimutagenesis (Laidlaw et al., 19891, and retinal function in cats, premature or infant primates (Hayes et al., 1975; Sturman and Hayes, 19801, and possibly humans (Geggel et al., 1985; Sturman, 1986). Since most if not all cells accumulate intracellular taurine to levels much greater than plasma levels, it is conceivable that cellular organelles have developed mechanisms to regulate this substance in a manner 0 I991 WILEY-LISS. INC.
that would protect them from osmotic perturbations. The degradation of macromolecules within lysosomes results in the production of relatively large concentrations of smaller molecules such as amino acids and sugars. A number of specific transport systems (Pisoni et al., 1985, 1987; Bernar et al., 1986; Jonas, 1986; Collarini et al., 1989; Greene et al., 1990; Jonas and Jobe, 1990), are found in the lysosomal membrane, which allows egress of these materials, thus avoiding osmotic imbalances. Presumably, adaptive regulation of intracellular taurine concentration creates another environmental challenge for the lysosome. We have examined the permeability of lysosomes to taurine and have identified a system, although specific for p-amino acids, that is Na+-independent and with relatively high Km value(s). METHODS Materials All chemicals were obtained from Sigma Chemical Co. unless otherwise indicated. Female, 150-200 g, S3rague-Dawley rats were purchased from Harlan. [ HI-Taurine was obtained from Amersham. GFC filReceived November 9, 1990; accepted February 25, 1991 "To whom reprint requestdcorrespondence should be addressed.
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VADGAMA ET AL.
ters (Fisher), Scintillation fluid 3a70B (RPI), and NAPtaurine lN-(4-azido-2-nitrophenyl)-2-aminoethane sulfonatel was purchased from Pierce Chemical Co. Lysosomal purification Rats were sacrificed by C02narcosis. The livers were excised, flushed with ice-cold buffer (0.25 M sucrose, 20mM Hepes, pH 7.0) to remove blood, and were ground in a Dounce homogenizer. An organelle pellet was prepared by differential centrifugation, and the lysosomes were further purified by Percoll density gradient centrifugation as previously described (Jonas, 1986). Following centrifugation, the high density fraction containing lysosomes was removed for use in subsequent studies. In a previous report, Jonas et al. (1986)had shown that lysosomes were purified approximately 90-fold by this method as indicated by the specific activity of p-hexosaminidase (Barret, 1972). One unit of enzyme activity is defined as hydrolysis of a pmole of substrateiminuteimg protein. Lysosomal latency. Lysosomal latency was assessed by the p-hexosaminidase assay as described by Pisoni et al. (1985). This procedure is a modification of the method of Hall et al. (1978). Essentially, 10-20 pl of lysosomal suspension with or without 1%triton X-100 were added t o 100 pl of 4 mM p-nitrophenyl(3-D-N-acetylglucosaminidein 0.36 M sucrose containing 0.043 M citric acid, 0.11 M Na2HP0, pH 4.9 buffer. Samples were incubated for 10 minutes at 37"C, and the reaction terminated by adding 1.0 ml of 0.4 M glycine! NaOH pH 10.4. The p-nitrophenol formed was read at 400 nM, and the amount was determined from a standard curve for p-nitrophenol prepared with the same reagents. One unit of p-hexosaminidase activity is defined as the amount of enzyme activity necessary to produce 1nmol of p-nitrophenolimin at 37°C. Using this procedure, the integrity of lysosomal preparation was determined to be between 90-95%. Taurine uptake In order to remove Percoll, lysosomes were diluted in a 10-fold excess of buffer and were collected by centrifugation at 13,000 g for 10 minutes at 4°C. The pellet was resuspended in ice-cold Krebs-Rin er choline phosphate buffer with 118 mM choline c loride, 5.9 mM KCL, 1.2mM MgS04 7H&, 0.5mM CaC1,-2H20, 2.0 mM KHCO', 5.5 mM D-glucose, and 25 mM choline phosphate. On certain occasions, this buffer was replaced with MOPS (50 mM) sucrose (0.25 M) EDTA (1mM) buffer adjusted to pH 7.0. Following incubation, lysosomes were collected on GFC glass fiber filters and were washed with 2X 10 ml aliquots of ice-cold phosphate-buffered saline (PBS). Filters were then dried and subjected to scintillation counting. Uptake was defined as pmoles taurine per mg lysosomal protein per minute. Total protein was determined b the method of Lowry et al. (1951). Note that we ha originally expressed our data in terms of mg of lysosomal protein. During the later studies, we compared uptake expressed in units of p-hexosaminidase activity. We found no difference in uptake profile, inhibition pattern, or on the kinetic parameters of Km. The primary data were applied to a basic program on an IBM-AT compatible computer.
a
IK
Taurine efflux Lysosomes were equilibrated with 10 pM L3H1-taurine for 10 minutes at 37°C. The taurine-loaded lysosomes were washed with 10 volumes of cold choline KRP buffer and were collected by centrifugation. The pellet was resuspended in buffer and used for efflux experiments. Lysosomes were collected as described for uptake studies. Membrane potential Membrane potential was measured fluorometrically using the cyanine dye di-OC6 (Seliymann and Gallin, 1983). Excitation wavelength was 460 nM and emission wavelength 510 nM. Transport kinetics Data obtained from concentration dependent transport studies were subjected to kinetic analysis using Cleland's computer program (Cleland, 1979) for biphasic uptake called Twoonl, where Log v = log
Vmax~.[S] Kml [S]
+
+ Vmaxp[S] Kma + [S]
Note that in this kinetic program, the data are processed after correcting for the nonsaturable component obtained from another equation called Toorgl, where Log v = log
where Kd
=
Vmax.[S] Km [S]
+
+ Kdo[S]
the nonsaturable component.
RESULTS Time course of uptake and cation selectivity Figure 1 shows the uptake of 10 FM 'H-taurine into rat liver lysosomes in sodium containing or sodium-free choline KRP, pH 7.4 at 37°C. Although the data appear to show no significant (p < 0.1) difference between uptake in sodium and sodium-free conditions, the overshoot in Na+-media was less compared to that observed in choline media. Following an initial overshoot between 5 to 10 minutes, the uptake reached equilibrium by 20 minutes. Next, in order to be certain that the uptake measurements were not reflecting 3H-taurine binding onto lysosomal membranes, we subjected incubated lysosomes to TCA precipitation (Fig. 1B). Aliquots of lysosomal suspensions (200 pl) were removed at intervals, placed into ice-cold PBS, and centrifuged immediately for 2 minutes in Eppendorf microfuge. The pellet was rinsed once more with 1ml of ice-cold PBS and was suspended into 220 pl of 5%TCA. Virtually all of the label was removed in the TCA soluble fraction, consistent with uptake of free taurine. Additional tests were performed to confirm that the labeled accumulation with time represented uptake and not simple binding. These were as follows: (1)the uptake was measured in the presence of 1%detergent Triton X-100, and the results (Fig lr) showed no increase in accumulation with respect to time, and (2) at the end of the normal uptake procedure, lysosomes were washed with ice-coldd-H20instead of PBS. In this
449
LYSOSOMAL TAURINE TRANSPORT 3.0
T
2.54
35 T
a P
0
04 10
20
30
40
50
60
0
Time. minutes
Fig. 1. Time course of 10 pM [3H-taurine uptake into rat liver lysosomes. A. The uptake of 10 ELMr3H taurine was measured up to 60 minutes in N a ' containing or Na+-free(choline) Krebs Ringer phosphate (KRP) buffer at pH 7.4 at 37°C. The data are mean i- SEM of 3 determinations. The composition of the buffer was as follows: NaCL 118mM, KCL 5.9mM, Na,HPO, 25mM, MgSO, 7H,O 1.2mM, KHCO, 2.0 mM, CaCl, 2H20 0.51 mM, and D-glucose 5.5 mM. For sodium-free buffer, sodium chloride and sodium hydro en phosphate were replaced by equimolar choline chloride and cho7ine hydrogen phosphate. The data in ( A ) indicate uptake in the presence of detergent. triton X-100 at 1%. B. The uptake of 10 pM [jHI-taurine was measured a s in A, except that it was terminated by a rapid
I
10
20
30 40 Time, minutes
50
60
centrifugation assay; 200 p1 aliquots of the lysosomal suspension were removed at specific time intervals and placed into 1.0 ml of ice-cold choline KRP. The suspension was immediately centrifuged for 2 minutes in Eppendorf microcentrifuge at 15,OOOxg.The supernatant was aspirated and the lysosomes were rinsed once more under the same conditions. The pellet was then suspended into 120 p1 of 5% trichloracetic acid (TCA) for 1hour, and the suspension centrifuged for 5 minutes in the Eppendorf centrifuge; 100 111 of the supernatant was removed for isotope counting. The residual TCA was removed, the pellet dissolved in 1N NaOH, and total protein was determined by the method of Lowry et al., 1951.
TABLE 1. Concentration of amino acids in rat liver lysosomes' Amino acid Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine Cystine Tyrosine Alanine
pmoles/mg protein
LIM
Amino acid
0.103 0.187 0.372 0.287 0.124 0.208
23 42 83 64 28 39 46
ND
ND
Arginine Asparagine Aspartic acid Glutamic acid Glutamine Glycine Ornithine Proline Serine Taurine Citrulline H ydroxyproline
0.175 0.253
56
ND
ND
0.159 0.294
35 66
pmoles/mg protein
pM
0.052 0.165 0.091 0.136 0.181 0.223 0.165 0.189 0.282 0.005 0.011
15 37 20 30 40 50 37 42 63 1.1 -~ 2.4
ND
ND
'Percoll purified lysosomes were suspended in Mops/Tris pH 7.0 buffer containing 0.25M sucrose, 1 mM Na.2EDTA, and 1.0% Triton X-100. This suspension was kept on ice for 10 minutes to facilitate lysosomal lysis. Soon after, total protein was precipitated with 5%TCA (trichloroacetic acid) followed by a 10-minute centrifugation at 16,000 xg. The TCA soluble supernatant was then processed for amino acid analysis on a Beckman amino acid analyzer (model 121 MB). The protein pellet was dissolved in 1 N NaOH, and its content estimated by the method of Lowry. The values are mean of 3 determinations and were expressed at pmoles per mg of total protein or as concentration in pM. In the later case, intralysosomal water space is assumed to be 4.5 pL per mg of protein (Schneider, 1983).
case, we found little or no counts remaining on the GFC filter. Intralysosomal concentration of taurine. Table 1 shows that, in comparison to most amino acids, the endogenous content of taurine is significantly low.
were observed when data (up to concentration range of 250 mM) in Figure 2A were transformed into a Hofstee plot (Fig. 2B) to obtain values for the kinetic parameters Km and Vmax. The estimated values are Kml = 30.9 ? 1.82 mM; Km2= 198 k 10.2mM; Vmaxl,= 3.4 2 0.29 and Vmax2 = 19.0 ? 0.81 pmollmg protein/ min. These values were determined from the kinetic Concentration dependent uptake and kinetic equation suitable for estimating biphasic uptake. This analysis of taurine uptake program is called Twoonl, and the equation is described Figure 2A shows the concentration-dependent up- in Methods. Note that the nonsaturable component take of taurine into rat liver lysosomes. The concentra- (0.076nmol/mg proteinimin) was corrected before protion range varied from 0.01 to 250 mM with a range of cessing the data for this equation. In addition, this 30 different concentration points. The data show ap- kinetic study was repeated 3 times, and uptake exparent saturation at substrate concentrations over pressed in terms of pmols/unit of P-hexaseimin. The 50 mM. However, the uptake was linear up to 5 mM values for Km were identical to those expressed here. taurine concentration (Fig. 2A inset). Biphasic kinetics The Vmax values were 1.2 and 12.5 pmolslheximin.
450
VADGAMA ET AL. 1210.0 -'\
\
\ \
*\\
o.1c5 0.0
I
0
25
50
75
150 [Tourine], rnM
100
125
175 200
225
250
0.05
Fig. 2. A. Concentration-dependent uptake of taurine into rat liver lysosomes. The uptake of 0.01 to 250 mM r3H1-taurine was measured for 1 minute in choline KRP, pH 7.4, and at 37°C. A shows the concentration-dependent uptake up to 250 mM taurine. Osmotic corrections were made by reducing choline chloride to appropriate concentrations. The inset shows that uptake was linear up to substrate concentrations of 5 mM. The uptake and termination procedures are as described in the methods section. Note that lysosomal latency was examined with each taurine concentration and was found to be
8 5 9 0 % B. Kinetics of taurine uptake into rat liver lysosomes. The data from the Michaelis-Menten plot in A were transformed into a Hofstee plot to determine kinetic parameters Km and Vmax. The plot revealed a biphasic uptake with Km values of 30.9 ? 1.82 mM and 198 ? 10.2 mM. Vmax values were 3.4 i 0.29 and 19 i 0.81 pmolimg proteinimin. These studies were re-examined and later expressed in units of P-hex activity. The Km values remained identical, whereas the Vmax values were 1.2 and 12.5 pmolslheximin.
2.5 7
"T
T
1
D3ontrol .5
E
-
P
c c
1.5.-
10% in potential generated by the proton pump. In summary, marked changes in cystoplasmic osmoeach case. larity due to influx of taurine could have profound Fig. 10. Effect of KCL, valinomycin, taurine, and cccp in di-0-C, effects on lysosomal volume and possibly function. fluorescence in the presence of lysosomes. Lysosomes were added at Bidirectional permeability of lysosomes to taurine althe first arrow to a cuvette containing 2 mL of 25 nM di-0-C, in choline KRP, pH 7.4 at room temperature. Dye was allowed to lows these structures t o adapt to such intracellular equilibrate with lysosomes (chart speed 1mm/min). A. Vertical changes. This ability may be of great importance in arrows indicate the addition of 5 pM valinomycin (Val), aliquots of tissues with marked accumulation of taurine and at 1M KCL, (up to a final concentration of 31 mM). B. Same as A, but different stages of development. Currently, taurine has followed by 0.1 mM taurine (Tau). C. 9.1 mM taurine was added prior no known function in lysosomes. Studies with intact to the addition of valinomycin and KCL. Excitation wavelength was 460 nM, emission wavelength was 510 nM. These results are repre- cells have suggested that taurine may help stabilize sentative of 5 separate determinations. membranes (Pasantes-Morales et al., 1985). Additional
B
B
454
VADGAMA ET AL
Jonas, A.J., and Jobe, H. (1990) Sulfate transport by rat liver lysosomes. J . Biol. Chem., 265:17545-17549. Jonas, A.J., Greene, A.A., Smith, M.L., and Schneider, J.A. (1982) Cystine accumulation and loss in normal heterozygous and cystiACKNOWLEDGMENTS notic fibroblasts. Proc. Natl. Acad. Sci. U.S.A., 79t4442-4445. D.P., Miller, L.A., and Chesney, R.W. (1990) Adaptive regulaWe thank Carol Rivera for her aid in preparation of Jones, tion of taurine transport in two continuous renal epithelial cell the manuscript. This work was supported by NIH lines. Kidney Int., 38.219-226. awards DK39147 and DK37403. Knauf, P.A., Breuer, W., McCulloch, L., and Rothstein, A. (1978) N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-taurinej as a photoaffinity probe for identifying membrane components conLITERATURE CITED taining the modifier site of the human red blood cell anion exchange system. J. Gen. Physiol., 72:631-649. Barrett, A.J. (1972) In Lysosomes, A Laboratory Handbook. J.T. Kulakowski, E.C., and Maturo, J. (1984) Hypoglycemic properties of Dingle, ed. Elsevier, New York, pp. 46-216. taurine not mediated by enhanced insulin release. Biochem. PharBashford. C.L.. and Smith, J.C. (1979) Methods of Enzymol., 55:569macol., 33:2835-2838. 586. Bernar, J., Tietze, F., Kohn, L.D., Bernardini, I., Harper, G.S., Laidlaw, S.A., Dietrich, M.F., Lamtenzan, M.P., Block, J.B., and Komle. J.D. 119891Antimutagenic effects of taurine in a bacterial Grollman, E.F., and Gahl, W.A. (1986) Characterization of a lysoa&y system. Cancer Res., 49766004604. soma1 membrane transport system for tyrosine and other neutral Lowry, O.H., Rosebrough, K.J., Farr, A.L., and Randall, R.J. (1951) amino acids in rat thyroid cells. J. Biol. Chem., 261:17107-17112. Protein measurement with the folin phenol reagent. J . Biol. Chem., Bernardi, N. (1985) On the role of taurine in the cerebellar cortex: A 193:265-275. reappraisal. Acta Physiol. Pharmacol. Latinoamer., 35:153-164. Chesney, R.W. (1985) Taurine: Its biological role and clinical impli- Oja, S.S., and Kontro, P. (1983) Taurine, In Handbook of Neurochemistry, 2nd ed. A. Lajtha, ed. 3:501. Plenum, New York, pp. 501. cations. Adv. Pediatr., 32:l-42. Christensen, H.N. (1988) Amino acid transport systems of lysosomes: Pasantes-Morales, H., and Cruz, C. (1985) Taurine: A physiological stabilizer of photoreceptor membranes, In Taurine: Biological AcPossible substitute utility of a surviving transport system for one tions and Clinical Perspectives. S.S. Oja. L. Ahtee, and P. Kontro, et congenitally defective or absent. Bioscience Rep., 8:121-129. al., eds. Alan R. Liss, New York, pp. 371-381. Christensen, H.N., Hess, B., and Riggs, T.R. (1954) Concentration of taurine, p-alanine, and tri-iodothyronine by ascites carcinoma cells. Pasantes-Morales, H., Wright, C.E., and Gaull, G.E. (1985) Taurine protection of lymphoblastoid cells from iron-ascorbate-induced damCancer Res., 14,124-127. age. Biochem. Pharmacol., 34:2205-2207. Cleland, W.W.(1979) Methods Enzymol., 63:103-138. Collarini, E.J., Pisoni, R.L., and Christensen, H.N. (1989) Character- Pisoni, R.L., Thoene, J.G., and Christensen, H.N. (1985) Detection and characterization of carrier-mediated cationic amino acid transport ization of a transport system for anionic amino acids in human in lysosomes of normal and cystinotic human fibroblasts. J. Biol. fibroblast lysosomes. Biochem. Biophys. Acta, 987:139-144. Chem., 260:47914798. Emudianughe, T.S., Caldwell, J., and Smith, R.L. (1983) The utilization of exogenous taurine for the conjugation of xenobiotic acids in Pisoni, R.L., Flickinger, K.S., Thoene, J.G., and Christensen, H.N. (1987) Characterization of carrier-mediated transport systems for the ferret. Xenobiotica, 13:133-138. small neutral amino acids in human fibroblast lysosomes. J. Biol. Gahl, W.A., Bashan, N., Tietze, F., Bernardini, I., and Schulman, J.D. Chem., 262:6010-6017. (1982) Cystine transport is defective in isolated leukocyte lysosomes from patients with cystinosis. Science, 21 7:1263-1265. Pisoni, R.L., Acker, T.L., Lisowski, K., Lemons, R.M., and Thoene, Gahl, W.A., Tietze, F., Bashan, N., Bernardini, I., Radford, D., and J.G. (1990) A cysteine specific lysosomal transport system provides Schulman, J.D. (1983) Characteristics of cystine countertransport a major route for the delivery of thiol to human fibroblast lysosomes: in normal and cystinotic lysosome-rich leucocyte granular fractions. possible role in supporting lysosomal proteolysis. J. Cell Biol., Biochem. J., 216t393-400. 110:327-335. Gaull, G.E., Wright, C.E., and Tallan, H.H. (1983) Taurine in human Reeves, J.P. (1983)In: Lysosomes in Biology and Pathology, vol. 7. J.T. lymphoblastoid cells: Uptake and role in proliferation. In: Sulfur Dingle, R.T. Dean, and W. Sly, eds. Elsevier Biomedical Press, Amino Acids: Biochemical and Clinical Aspects. Liss. New York, pp. Amsterdam. 297-303. Schneider, D.L. (1983) ATP-dependent acidification of membrane Geggel, H.S., Ament, M.E., Heckenlively, J.R., Martin, D.A., and vesicles isolated from purified rat liver lysosomes. J . Biol. Chem., Kopple, J.D. (1985) Nutritional requirement for taurine in patients 258:1833-1838. receiving long-term parenteral nutrition. New Engl. J. Med., Sebring, L.A., and Huxtable, R.J. (1985) Taurine modulation of 312:142-146. calcium binding to cardiac sarcolemma. J. Pharmacol. Exp. Therap., Greene, A.A., Marcusson, E.G., Morell, G.P., and Schneider, J.A. 232:445-45 1. (1990) Characterization of lysosomal cystine transport system in Seliymann, B.E., and Gallin, J.I. (1983)J . Cell Physiol., 115:105-115. mouse L-929 fibroblasts. J . Biol. Chem., 265:9888-9895. Stewart, B.H., Collarini, E.J., Pisoni, R.L., and Christensen, H.N. Hall, C.W., Liebraers, I., DiNatale, P., and Neufeld, E.F. (1978) 11989) Separate and shared lysosomal transport of branched and Methods Enzymol., 50:439-456. aromatic dipolar amino acids. Biochim. Biophys. Acta, 987145-153. Harikumar, P., and Reeves, J.P. (1983) The lysosomal proton pump is Sturman, J.A. (1986) Nutritional taurine and central nervous system electrogenic. J. Biol. Chem., 258:10403-10410. development. Ann. N.Y. Acad. Sci. 477r196-213. Hayes, K.C., Carey, R.E., and Schmidt, S.Y. (1975) Retinal degener- Sturman, J.A., and Hayes, K.C. (1980) The biology of taurine in ation associated with taurine deficiency in the cat. Science> nutrition and development. Adv. Nutr. Res., 353-299. 188r949-95 1. Thurston, J.H., Hauhart, R.E., and Dirgo, J.A. (1980) Taurine: A role Hofmann, A.F., and Small, D.M. 11967) Detergent properties of bile in osmotic regulation of mammalian brain and possible clinical salts: Correlation with physiological function. Annu. Rev. Med., significance. Life Sci., 26:1561-1568. 18:333-367. Vadgama, J.V., and Kopple, J.D. (1989) Regulation of taurine transHuxtable, R.J., and Sebring, L.A. (1986)Towards a unifying theory for port into renal tubular cells in culture. FASEB J., 3:#4846, A1060. the actions of tauriue. Trends Pharmacol. Sci., 9:481485. Wright, C.E., Tallan, H.H., Lin, Y.Y., and Gaull, G.E. (1986) Taurine: Jonas, A.J. (1986) Cystine transport in purified rat liver lysosomes. Biological update. Ann. Rev. Biochem., 55:427453. Biochem. J., 236:671-677.
studies will be required to determine whether this is the case for lysosomes.
I
,
1
