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Marked differences in the swainsonine inhibition of rat liver lysosomal α-d -mannosidase, rat liver golgi mannosidase II, and jack bean α-d -mannosidase
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 236, No. 1, January, pp. 427-434, 1985
Marked Differences in the Swainsonine Inhibition of Rat Liver Lysosomal a-D-Mannosidase, Rat Liver Golgi Mannosidase II, and Jack Bean a-D-Mannosidase D. R. P. TULSIANI, Departments
of Molecular
Biology
H. P. BROQUIST,
and Biochemistry,
Vanderbilt
Received August
AND
0. TOUSTER’
University,
NashviUe,
Tennessee
37235
13, 1984
Swainsonine, a plant toxin, strongly inhibits certain a-D-mannosidases but has no effect on others [D. R. P. Tulsiani, T. M. Harris, and 0. Touster (1982) J. BioZ. Chem. 257,7936-79391. The reversible inhibition of jack bean and lysosomal a-D-mannosidases has previously been suggested to be similar in nature but quite complex. Specific differences in the action of swainsonine on these two enzymes and on Golgi mannosidase II are reported. (a) The inhibition of the jack bean mannosidase, but not rat liver lysosomal a-D-mannosidase or Golgi mannosidase II, is increased by preincubation with the alkaloid. (b) The inhibition of the jack bean and lysosomal enzymes, but not mannosidase II, is competitive at inhibitor concentrations of ~0.5 PM. (c) The inhibition of jack bean cw-mannosidase is largely irreversible, its very limited reversibility being partially dependent upon the swainsonine concentration used and on the time of preincubation with the inhibitor. On the other hand, the inhibition of lysosomal a-mannosidase is largely reversible, as shown by dilution experiments and by the use of [3H]swainsonine. Golgi mannosidase II shows intermediate reversibility, the results indicating two modes of binding; one rapid and irreversible, the other much slower and reversible. o 19% Academic PESS, I~C.
Swainsonine, an indolizidine alkaloid, occurs in Swainsona canescens (1,2), spotted locoweed (Astragalus lentiginosus) (3), and the fungus Rhixoctonia leguminicola (4). It has been considered to be the agent in plants responsible for the neurological disease in livestock resembling the hereditary lysosomal disease a-mannosidosis (l-3), and recently this conclusion has been confirmed by a direct comparison of the effects of swainsonine and locoweed in the pig (5). Although swainsonine-containing plants are reported to induce a phenocopy of mannosidosis, and although swainsonine is a potent inhibitor of liver lysosomal, as well as jack bean, a-D-mannosidase (4,6-g), swainsonine administration to rats (10) and pigs (5) causes in1 To whom correspondence
crease in tissue levels of lysosomal mannosidase. Swainsonine is also a specific inhibitor of Golgi mannosidase II, but is without effect on Golgi mannosidase IA or IB or liver cytosolic a-mannosidase (9). As expected from in vitro experiments (9), swainsonine administration to animals lowers the level of liver Golgi mannosidase II (5, 10). Because of the inhibition of mannosidase II, the alkaloid causes cells to produce hybrid instead of complex glycoproteins (11-14). These findings raise questions regarding the mechanism by which swainsonine exerts its toxic effects, and suggests that further studies of its inhibitory action on enzymes should be of interest. Dorling et al. (6) studied the inhibition of jack bean a-D-mannosidase.
should be addressed. 427
and
mouse liver They reported
lysosomal that the
0003-9861185 $3.00 Copyright All rights
0 1985 by Academic Press, Inc. of reproduction in any form reserved.
428
TULSIANI,
BROQUIST,
inhibition of several mannosidases is reversible but “quite complex.” Subsequently, Kang and Elbein (8) studied the inhibition of the jack bean enzyme in more detail, and obtained results “suggesting that swainsonine may be a competitive inhibitor that binds tightly to the enzyme and is only slowly removed.” They were able to obtain only a limited degree of reversibility by various procedures. We report herein studies on the action of swainsonine on highly purified rat liver lysosomal a-D-mannosidase and Golgi mannosidase II and on jack bean a-Dmannosidase. Our results indicate that substantial differences exist in the effects of swainsonine on these enzymes. MATERIALS
AND
METHODS
Materials. Jack bean mannosidase (20 units/mg protein) and pnitrophenyl a-D-mannoside (PNPmannoside)’ were from Sigma; Bio-Gel P-10 (200400 mesh) was from Bio-Rad. Pure swainsonine was obtained from R leguminicola (4). [1-aH]Swainsonine (8.13 X lo6 cpm/mg) was prepared from R. leguminicola mycelium following growth on [l‘Hlhydroxyoctahydroindolizine (15) by procedures to be published. Rat liver lysosomal cY-D-mannosidase was purified through the Dowex 50 chromatography step as described (16). The purified enzyme had a specific activity of 9.0-9.2 units/mg protein, and was nearly 4500-fold purified over the crude liver extract. The enzyme contained N-acetyi-fl-D-ghrcosaminidase as the major contaminant (16). Homogeneous Golgi mannosidase II was prepared from purified, saltwashed Golgi membranes from rat liver (1’7). Bqfers. Buffer A, pH 6.0: 10 mM sodium acetate, 1 mM MgSO1, 1 mM 2-mercaptoethanol; Buffer B, pH 6.0: same as Buffer A containing 0.25 M NaCI. Buffer C, pH 6.0: 10 mM sodium acetate, 1 mM ZnCl,, and 0.25 M NaCl; Buffer D, pH 6.0: same as Buffer C containing 0.1% Triton X-100. Enzyme c~~says. Unless otherwise indicated, jack bean and rat liver lysosomal mannosidases were assayed in 100 mM sodium acetate buffer, pH 4.4. Golgi mannosidase II was assayed in the same buffer but at pH 5.5. The reaction mixtures contained buffer, 4 mM PNP-mannoside, and enzyme in a total volume of 0.5 ml. The incubations were carried out at 37°C for 15 to 30 min. The reaction was stopped
* Abbreviation oc-n-mannoside.
used: PNP-mannoside,
pnitrophenyl
AND
TOUSTER
by the addition of 1.0 ml of an alkaline buffer [0.133 M glycine, 0.06’7 M NaCl, and 0.083 M NazCOs adjusted to pH 10.7 (17)], and the absorbance at 400 nm was measured to determine the amount of pnitrophenol released. Enzyme and substrate controls were run with all assays. One unit of a-D-mannosidase is the amount of enzyme which catalyzed the release of 1 rmol pnitrophenol/min. Protein was assayed by the fluorometrie method (18), with bovine serum albumin as standard. RESULTS
Eflect of swainsmine concentration and on the extent of inhibition. Figure 1 shows that, whereas a concentration of 2 /IM swainsonine caused 50% inhibition of jack bean a-D-mannosidase, only 0.2 PM swainsonine was needed to cause 50% inhibition of the lysosomal enzyme and Golgi mannosidase II. The result with the jack bean enzyme was similar to that observed by Schneider et al. (4), but the concentration of inhibitor was 10 times higher than that reported by Dorling et al. (6). It was then observed that preincubation of jack bean mannosidase with the alkaloid at 3’7°C before the addition of substrate greatly increased the inhibition observed. Under these conditions, 50% inhibition was achieved at 0.4 PM swainsonine, a Concentration similar to that reported by Kang and Elbein (8), who have also shown that maximum inhibition requires preincubation with the alkaloid. Lysosomal a-D-mannosidase and Golgi mannosidase II, on the other hand, showed similar inhibition with or without preincubation with swainsonine (data not shown). Figure 1 also shows that the unlabeled and biosynthetically labeled swainsonine were essentially identical to each other in their inhibitory effects on the three enzymes. Competitive nature of the inhibition by swainsmine. The effect of PNP-mannoside concentration on the swainsonine inhibition of the three mannosidases was examined, and the results were plotted by the method of Lineweaver and Burk (Fig. 2). The alkaloid appeared to be a competitive inhibitor of jack bean and lysosomal a-mannosidases at concentrations of 0.5 /IM and below; at a higher concentration of preincubatim
SWAINSONINE
INHIBITION
429
OF a-D-MANNOSIDASES
iOLGl MANNOSIDASEII
0
2
4
6
8
IO SWANSONINE
(NM)
FIG. 1. Effect of swainsonine on cY-D-mannosidases. Jack bean ol-D-mannosidase and liver lysosomal Lu-D-mannosidase were assayed at pH 4.4 with 4 mM pnitrophenyl a-D-mannoside as substrate. Purified Golgi mannosidase II was assayed at pH 5.5 with 4 mM p-nitrophenyl cu-Dmannoside as described under Materials and Methods. The concentration of unlabeled (0) or labeled swainsonine (0) in the reaction mixture was varied as shown. The reaction was started by addition of jack bean mannosidase (0.2 pg protein), lysosomal mannosidase (0.4 pg protein), or Golgi mannosidase II (0.2 pg protein). In a different set of experiments (A), the jack bean mannosidase (0.2 fig protein) was first preincubated (10 min at 37°C) with varying concentrations of swainsonine in the above sodium acetate buffer before addition of the substrate. The reaction mixtures were incubated at 37°C for 15 min, and the released pnitrophenol was measured as described under Materials and Methods.
JACK
BEAN MANNOSIDASE
a0
LYSOSOMAL
MANNOSIDASE
16
GOLGI MANNOSIDASEII
1
0
0.2
0.4
0.6
0.8
1.0
0.2
0.4
ir 0;6
0.8
0
0.2
0.4
0.6
0.8
I.0
FIG. 2. Effect of substrate concentration on swainsonine inhibition of cY-D-mannosidases. The reaction mixture contained sodium acetate buffer (100 mM, pH 4.4 or 5.5), the indicated amount of swainsonine, and varying concentrations of pnitrophenyl a-D-mannoside. Following the addition of jack bean mannosidase (0.1 pg protein), purified rat liver lysosomal mannosidase (0.2 pg protein), or purified Golgi mannosidase II (0.3 pg protein), the mixture was incubated at 37’C for 30 min. The best curves obtained from the data by a linear regression program (Texas Instruments Calculator No. TI-55) are plotted by the method of Lineweaver and Burk. SW, swainsonine.
430
TULSIANI, 0 JACK
BEAN
BROQUIST,
MANNOSIDASE
DSDMAL
0 PREINCUBATION
TIME
AND
(rntn)
2
4
TOUSTER
MANNOSIDASE
6
PREINCUBATION
8
IO
24
!
I
I
I
I
I.,
r
0
2
4
6
8
IO
24
T1MElHr.l
PREINCUEATION
TIMEWr)
FIG. 3. Effect of time of preincubation with swainsonine on the reversibility of inhibition of (YD-mannosidases. Jack bean mannosidase (2 pg protein/O.1 ml Buffer A), purified rat liver lysosomal mannosidase (4 pg protein/O.1 ml Buffer A), or purified Golgi mannosidase II (4 pg/O.l ml Buffer D) was preincubated with different concentrations of swainsonine at 37°C. Aliquots containing 0.05 to 0.1 pg protein were withdrawn periodically and assayed for enzyme activity as described under Materials and Methods. 0, Control (no swainsonine); 0, 1 pM swainsonine; A, 5 pM swainsonine; A, 10 fire swainsonine.
(2 PM), the inhibition appeared to be noncompetitive. However, the inhibition of Golgi mannosidase II appeared to be noncompetitive even at low concentrations (0.2 PM) of swainsonine. Study of the reversibility of swainecmine inhibition by diluticm experiments. Figure 3 shows that the inhibition of jack bean mannosidase is essentially irreversible. In this experiment, the enzyme was preincubated with three different concentrations of swainsonine at 37°C for varying time periods. Aliquots were then diluted and assayed for enzymatic activity. It was apparent that the activity could not be recovered by dilution. However, the inhibition of the lysosomal mannosidase was partially reversible. Dilution of the mixture after preincubation led to recovery of 65-70% of the original enzymatic activity (Fig. 3). Figure 4 shows that the recovery of activity with the lysosomal enzyme is dependent upon the extent of dilution. The Golgi enzyme showed partial reversibility, the extent of which depended upon the concentration of swainsonine present during the preincubation (Fig. 3). The swainsonine concentration also influ-
enced the extent of the rapid, irreversible component of the inhibition (i.e., 0 time values in Fig. 3). Study of reversibility of inhibition using [‘H]swainsonine. The irreversibility of the
0
2
4 ENZYME
16
8 DILUTION
32
l-fold)
FIG. 4. Effect of dilution on the reversibility of the swainsonine inhibition of purified rat liver lysosomal ol-n-mannosidase. Purified enzyme (4 pg/50 ~1 Buffer A) was preincubated with (0) and without (0) 2 jiM swainsonine for 15 min at 37°C. The mixture was serially diluted in Buffer A, and the enzyme activity was measured as described under Materials and Methods.
SWAINSONINE
INHIBITION
reaction of swainsonine with jack bean mannosidase was investigated by incubating the enzyme at 37°C with [3H]swainsonine for varying lengths of time. Each mixture was then applied to a Bio-Gel P10 column to remove free inhibitor, the void volume fractions presumably containing enzyme and enzyme-inhibitor complex. Recovery of enzymatic activity was inversely proportional to the amount of inhibitor bound to the enzyme (Figs. 5A and B). This observation held for varying incubation times and for varying [3H]swainsonine concentrations. When the jack bean mannosidase was preincubated with 1 PM inhibitor for 15 min at 3’7°C and applied to the Bio-Gel P-10 column, the void volume fraction contained 40% of the original enzymatic activity and 0.013 nmol labeled inhibitor (Fig. 5A).
P 5
0
5
IO
However, when 5 PM inhibitor was used (Fig. 5B), the recovered enzyme was essentially inactive and contained more than twice as much (0.03 nmol) bound swainsonine. Attempts to dissociate the enzyme-inhibitor complex by incubation with 10 mM mannose, 10 mM PNP-mannoside, or 10 mM cY-methylmannoside at pH 4.4 or 6.0 were unsuccessful, results suggesting that the swainsonine was tightly bound to the active site of the enzyme. That the alkaloid was not covalently bound was indicated by the fact that treatment of the enzyme-[3H]swainsonine complex in Buffer B at 100°C for 5 min dissociated 87% of this complex. The presence of sodium dodecyl sulfate (2.5%, w/v) in this reaction mixture resulted in 100% dissociation of the complex. This was shown by applying the treated mix-
15
b
cl
I
3
0
5
MIN. 0,lO
0
2
I
431
OF a-D-MANNOSIDASES
lb MIN.
IS DI
I
i!
z 100
z 5
i
HR.
HR. PREINCUBATION
T I ME
FIG. 5. Relationship of irreversibly bound swainsonine to the recovery of a-D-mannosidase activity. Jack bean mannosidase (5 pg protein/O.1 ml buffer A) was preincubated at 37’C with two different concentrations of [‘Hlswainsonine (A, 1 HIM; B, 5 PM). At the indicated times, the mixture was applied to a Bio-Gel P-10 column (1 X 26 cm, -400 mesh) equilibrated with Buffer B to separate the enzyme-swainsonine complex from free [aH]swainsonine. The column was eluted with the buffer at a flow rate of 3 ml/h. The radioactivity and the enzyme activity in fractions (1 ml) were measured in aliquots as described under Materials and Methods. The column void volume fractions (8-13) contained enzyme and bound [3I]swainsonine, whereas free pH]swainsonine eluted in fractions 19-25. In (C), the same procedure was used with purified lysosomal a-Dmannosidase (4 fig/50 ~1 buffer A) incubated with 5 pM [aH]swainsonine. In (D), purified Golgi mannosidase II (5 pg/50 pl Buffer D) was preincubated at 37°C with 5 pM [3H]swainsonine. With the latter enzyme, the Bio-Gel P-4 was equilibrated with Buffer D.
432
TULSIANI,
BROQUIST,
ture to a Bio-Gel P-10 column and measuring the radioactivity eluted from the column in the position of free swainsonine. The labeled swainsonine was released only under severe denaturing conditions without recovery of any enzymatic activity. Differing extents of reversibility of inhibition of lysosomal mannosidase and mannosidase II were found. When a mixture of the lysosomal enzyme and 5 PM [3H]swainsonine was preincubated at 37°C for varying lengths of time and then applied to a Bio-Gel P-10 column, more than 60% of the activity, but very little radioactivity, was recovered in the void volume fractions (Fig. 5C). This result agreed with the dilution experiments shown in Fig. 3B. However, when mannosidase II was used in the same type of gel-filtration experiment, only 30% of the enzymatic activity was present in the void volume fractions, which also contained some radioactivity (Fig. 5D). In another experiment involving mannosidase II, the binding of [3H]swainsonine to salt-washed Golgi membranes was investigated with (Table I, tube B) and without (Table I, tube C) prior incubation with swainsonine. After extensive washing of the membranes, each sample was incubated with
AND
10 pM [3H]swainsonine, a concentration that gave essentially complete inhibition of mannosidase II (Fig. 1). After the membranes were exhaustively washed with Buffer C, assay for mannosidase II activity showed that both samples had regained about 30% of the activity (cf. control; Table I, tube A). Radioactivity in membranes from tube C was nearly five times greater than from tube B. These results indicated that (a) some of mannosidase II activity was regained when excess free swainsonine was removed, and (b) when swainsonine-binding sites on mannosidase II were saturated, little exchange or further addition of the alkaloid occurred. In viva studies in the rat have also yielded evidence suggesting the presence of tightly bound swainsonine in liver Golgi membranes (10). DISCUSSION
The pioneering work of Dorling and his associates led to the identification of swainsonine as a potent inhibitor of jack bean and lysosomal a-D-mannosidases (1, 6). Subsequently, we (9) showed that the alkaloid also strongly inhibits Golgi mannosidase II but not mannosidases IA or
TABLE THE BINDING
Membrane suspension” (sample)
Preincubatior? (additions)
A
None
B
SW (lo fiM)
C
None
OF SWAINSONINE
TOUSTER
I
TO RAT LIVER
Incubation’ (additions) None [3H]SW (10 ELM) [3H]SW (10 /.tM)
GOLGI MEMBRANES
Membrane-bound [3H]swainsonined (nmol/mg protein) None 0.005 0.033
Mannosidase II activityd (%I) 100
28 33
“The salt-washed rat liver Golgi membranes were suspended in Buffer C (13 mg protein/ml). Aliquots containing 2 mg protein were pipetted into each of three tubes (A, B, and C). * Swainsonine (SW) solution (tube B), or water (tubes A and C) was added in each tube. After preincubation at 3’7°C for 15 min, the mixture was diluted with 2 ml Buffer C and centrifuged at 105,OOOgfor 30 min. The pellet was washed four times in 2 ml of the same buffer, each time homogenizing and centrifuging as above. “The washed pellet (see b above) was suspended in Buffer C. [3H]Swainsonine was added only to tubes B and C, the same volume of water being added to tube A. The reaction mixtures (0.2 ml) were incubated at 37°C for 15 min, and the free [3H]swainsonine was removed by extensive washing in Buffer C (see above). d The washed pellet (see b above) from each tube was suspended in Buffer C (0.4 ml). Aliquots were dissolved in 2 N NHIOH, neutralized with glacial acetic acid, and used for the determination of radioactivity (11). Aliquots of the suspension were also assayed for Golgi mannosidase II activity as described under Materials and Methods.
SWAINSONINE
INHIBITION
IB or cytosolic a-D-mannosidase. That the reversible inhibition of jack bean mannosidase is not explainable by a straightforward reversible, competitive mechanism has been evident from the work of Dorling et al. (6) and Kang and Elbein (8). The latter authors first showed that preincubation of swainsonine with jack bean mannosidase increased the observed inhibition of the enzyme, an observation confirmed in the present report. However, we find that such preincubation does not increase the swainsonine inhibition of rat liver lysosomal a-D-mannosidase or Golgi mannosidase II. It is not evident why Dorling et aL (6) found that swainsonine was inhibitory at considerably lower concentrations than employed by Kang and Elbein (8), Schneider et al. (4), or by us. We have directly compared swainsonine obtained from S. caneseem with that isolated from R. leguminicola, and found that both preparations behaved identically in all enzymatic tests employed. In regard to the finding (9) that cytosolic cY-D-mannosidase, with 4 mM PNP-mannoside as substrate, is not inhibited by swainsonine, the recent report of Bischoff and Kornfeld (19) is pertinent. In their paper reporting the occurrence of a-Dmannosidase activity in rat liver endoplasmic reticulum, they state that swainsonine was not inhibitory to this activity unless the p-nitrophenyl cY-mannoside concentration was considerably lower than 4 mM. They therefore suggested that mannosidases with low affinity for PNP-mannoside are inhibited by swainsonine. In support of this suggestion, it is relevant that rat liver cytosolic a-D-mannosidase has a lower K, for PNP-mannoside (20) than lysosomal mannosidase or Golgi mannosidase II (21). Further investigation of this suggestion would be of interest. The results reported herein point to further differences among the enzymes which are very susceptible to inhibition by swainsonine. Reversibility of inhibition is minimal with jack bean a-mannosidase, intermediate with Golgi mannosidase II, and greatest with the lysosomal enzyme. There appears to be two modes of binding
OF a-D-MANNOSIDASES
433
to the liver enzymes (Fig. 3). With mannosidase II, one mode of binding is very rapid and irreversible. The other is slower, with the extent of reversibility dependent upon the original swainsonine concentration and the time of exposure of the enzyme to the inhibitor. With lysosomal a-mannosidase the irreversible mode is very small, and the reversible mode is essentially independent of swainsonine concentration and time of exposure. Although the explanation for the two modes is not apparent at this time, it is possible that multiple forms of the liver enzymes are present in the purified enzymes used. For example, since they are glycoproteins, differently processed forms may be present. The fact that the swainsonine inhibitions of liver lysosomal a-mannosidase and Golgi mannosidase II are partially reversible has relevance to studies of enzyme levels in animals fed swainsonine or plants containing this toxin. Swainsonine administration to rats lowers Golgi mannosidase II to 27% of control activity (lo), an amount very similar to that found in Table I. Since homogenization of liver and preparation of Golgi membranes for assay of enzymatic activity may have been accompanied by the release of some inhibitor, the activity of the enzyme in the intact liver may have in fact been less than 27%. Similarly, while swainsonine administration to rats leads to a considerable elevation of assayable lysosomal CYmannosidase activity, the reversibility of inhibition of this enzyme precludes estimating the actual activity of this enzyme in intact liver by measuring the activity in tissue extracts. Experiments in progress are designed to determine the functional enzyme in intact animals given swainsonine. ACKNOWLEDGMENTS A sample of swainsonine isolated from Swainsona canescens and obtained from Dr. P. R. Dorling was kindly furnished to us by Dr. Philip Stahl, Washington University School of Medicine. We are grateful to Mrs. Vera Coleman for excellent technical assistance. This investigation was supported by Grant GM 26430 to O.T., Grant AM 16019 to H.P.B., and
434
TULSIANI,
Biomedical Research Support from The National Institutes States Public Health Service.
Grant S07-RR07201 of Health, United
BROQUIST,
REFERENCES 1. COLEGATE, S. M., DORLING, P. R., AND HUXTABLE, C. R. (1979) Au&. J. Chem, 32, 2257-2264. 2. DORLING, P. R., HUXTAELE, C. R., AND VOGEL, P. (1978) Neuropathol. Appl Neurobid 4, 285295. 3. MOLYNEUX, R. J., AND JAMES, L. F. (1982) Science (Washington, D. C.) 216, 190-191. 4. SCHNEIDER, M. J., UNGEMACH, F. S., BROQUIST, H. P., AND HARRIS, T. M. (1983) Tetruhedmn 39, 29-32. 5. TIJLSIANI, D. R. P., BROQUIST, H. P., JAMES, L. F., AND TOUSTER, 0. (1984) Arch Biochem. Biophys. 232, 76-85. 6. DORLING, P. R., HUXTABLE, C. R., AND COLEGATE, S. M. (1980) Biochem J 191, 649-651. 7. CHOTAI, K., JENNINGS, C., WINCHESTER, B., AND DORLING, P. (1983) J. CeU. Biochem. 21, 107117. 8. KANG, M. S., AND ELBEIN, A. D. (1983) Plant Physiol 71, 551-554.
AND
TOUSTER
9. TULSIANI, D. R. P., HARRIS, T. M., AND TOUSTER, 0. (1982) J. Biol. Chem 257, 7936-7939. 10. TULSIANI, D. R. P., AND TOUSTER, 0. (1983) Arch. B&hem Biophys. 242,594-600. 11. TULSIANI, D. R. P., AND TOUSTER, 0. (1983) J. Biol. Chem 258, 7578-7585. 12. GROSS, V., TRAN-THI, T.-A., VOSBECK, K., AND HEINRICH, P. A. (1983) J. Biol Chem 258, 4032-4036. 13. KANG, M. S., AND ELBEIN, A. D. (1983) J. Vird 46, 60-69. 14. ARUMUGHAM, R. G., AND TANZER, M. L. (1983) J. Biol Chem 258,11883-11889. 15. GUENGERICH, F. P., SNYDER, J. J., AND BROQUIST, H. P. (1973) Biochemistry 12,4264-4269. 16. OPHEIM, D. J., AND TOUSTER, 0. (1978) J. Biol. Chem 253, 1017-1023. 17. TULSIANI, D. R. P., BUSCHIAZZO, H. O., TOLBERT, B., AND TOUSTER, 0. (1977) Arch B&hem. Biophys. 181, 216-227. 18. ANDERSON, P. M., AND DESNICK, R. J. (1979) .I BioL Chem. 254.6924-6930. 19. BISCHOFF, J., AND KORNFELD, R. (1983) .I. Biol Chem 258, 7907-7910. 20. SHOUP, V. A., AND TOUSTER, 0. (1976) J. Biol Chem 251, 3845-3852. 21. DEWALD, B., AND TOUSTER, 0. (1973) J. Biol Chem. 248, 7223-7233.
Marked Differences in the Swainsonine Inhibition of Rat Liver Lysosomal a-D-Mannosidase, Rat Liver Golgi Mannosidase II, and Jack Bean a-D-Mannosidase D. R. P. TULSIANI, Departments
of Molecular
Biology
H. P. BROQUIST,
and Biochemistry,
Vanderbilt
Received August
AND
0. TOUSTER’
University,
NashviUe,
Tennessee
37235
13, 1984
Swainsonine, a plant toxin, strongly inhibits certain a-D-mannosidases but has no effect on others [D. R. P. Tulsiani, T. M. Harris, and 0. Touster (1982) J. BioZ. Chem. 257,7936-79391. The reversible inhibition of jack bean and lysosomal a-D-mannosidases has previously been suggested to be similar in nature but quite complex. Specific differences in the action of swainsonine on these two enzymes and on Golgi mannosidase II are reported. (a) The inhibition of the jack bean mannosidase, but not rat liver lysosomal a-D-mannosidase or Golgi mannosidase II, is increased by preincubation with the alkaloid. (b) The inhibition of the jack bean and lysosomal enzymes, but not mannosidase II, is competitive at inhibitor concentrations of ~0.5 PM. (c) The inhibition of jack bean cw-mannosidase is largely irreversible, its very limited reversibility being partially dependent upon the swainsonine concentration used and on the time of preincubation with the inhibitor. On the other hand, the inhibition of lysosomal a-mannosidase is largely reversible, as shown by dilution experiments and by the use of [3H]swainsonine. Golgi mannosidase II shows intermediate reversibility, the results indicating two modes of binding; one rapid and irreversible, the other much slower and reversible. o 19% Academic PESS, I~C.
Swainsonine, an indolizidine alkaloid, occurs in Swainsona canescens (1,2), spotted locoweed (Astragalus lentiginosus) (3), and the fungus Rhixoctonia leguminicola (4). It has been considered to be the agent in plants responsible for the neurological disease in livestock resembling the hereditary lysosomal disease a-mannosidosis (l-3), and recently this conclusion has been confirmed by a direct comparison of the effects of swainsonine and locoweed in the pig (5). Although swainsonine-containing plants are reported to induce a phenocopy of mannosidosis, and although swainsonine is a potent inhibitor of liver lysosomal, as well as jack bean, a-D-mannosidase (4,6-g), swainsonine administration to rats (10) and pigs (5) causes in1 To whom correspondence
crease in tissue levels of lysosomal mannosidase. Swainsonine is also a specific inhibitor of Golgi mannosidase II, but is without effect on Golgi mannosidase IA or IB or liver cytosolic a-mannosidase (9). As expected from in vitro experiments (9), swainsonine administration to animals lowers the level of liver Golgi mannosidase II (5, 10). Because of the inhibition of mannosidase II, the alkaloid causes cells to produce hybrid instead of complex glycoproteins (11-14). These findings raise questions regarding the mechanism by which swainsonine exerts its toxic effects, and suggests that further studies of its inhibitory action on enzymes should be of interest. Dorling et al. (6) studied the inhibition of jack bean a-D-mannosidase.
should be addressed. 427
and
mouse liver They reported
lysosomal that the
0003-9861185 $3.00 Copyright All rights
0 1985 by Academic Press, Inc. of reproduction in any form reserved.
428
TULSIANI,
BROQUIST,
inhibition of several mannosidases is reversible but “quite complex.” Subsequently, Kang and Elbein (8) studied the inhibition of the jack bean enzyme in more detail, and obtained results “suggesting that swainsonine may be a competitive inhibitor that binds tightly to the enzyme and is only slowly removed.” They were able to obtain only a limited degree of reversibility by various procedures. We report herein studies on the action of swainsonine on highly purified rat liver lysosomal a-D-mannosidase and Golgi mannosidase II and on jack bean a-Dmannosidase. Our results indicate that substantial differences exist in the effects of swainsonine on these enzymes. MATERIALS
AND
METHODS
Materials. Jack bean mannosidase (20 units/mg protein) and pnitrophenyl a-D-mannoside (PNPmannoside)’ were from Sigma; Bio-Gel P-10 (200400 mesh) was from Bio-Rad. Pure swainsonine was obtained from R leguminicola (4). [1-aH]Swainsonine (8.13 X lo6 cpm/mg) was prepared from R. leguminicola mycelium following growth on [l‘Hlhydroxyoctahydroindolizine (15) by procedures to be published. Rat liver lysosomal cY-D-mannosidase was purified through the Dowex 50 chromatography step as described (16). The purified enzyme had a specific activity of 9.0-9.2 units/mg protein, and was nearly 4500-fold purified over the crude liver extract. The enzyme contained N-acetyi-fl-D-ghrcosaminidase as the major contaminant (16). Homogeneous Golgi mannosidase II was prepared from purified, saltwashed Golgi membranes from rat liver (1’7). Bqfers. Buffer A, pH 6.0: 10 mM sodium acetate, 1 mM MgSO1, 1 mM 2-mercaptoethanol; Buffer B, pH 6.0: same as Buffer A containing 0.25 M NaCI. Buffer C, pH 6.0: 10 mM sodium acetate, 1 mM ZnCl,, and 0.25 M NaCl; Buffer D, pH 6.0: same as Buffer C containing 0.1% Triton X-100. Enzyme c~~says. Unless otherwise indicated, jack bean and rat liver lysosomal mannosidases were assayed in 100 mM sodium acetate buffer, pH 4.4. Golgi mannosidase II was assayed in the same buffer but at pH 5.5. The reaction mixtures contained buffer, 4 mM PNP-mannoside, and enzyme in a total volume of 0.5 ml. The incubations were carried out at 37°C for 15 to 30 min. The reaction was stopped
* Abbreviation oc-n-mannoside.
used: PNP-mannoside,
pnitrophenyl
AND
TOUSTER
by the addition of 1.0 ml of an alkaline buffer [0.133 M glycine, 0.06’7 M NaCl, and 0.083 M NazCOs adjusted to pH 10.7 (17)], and the absorbance at 400 nm was measured to determine the amount of pnitrophenol released. Enzyme and substrate controls were run with all assays. One unit of a-D-mannosidase is the amount of enzyme which catalyzed the release of 1 rmol pnitrophenol/min. Protein was assayed by the fluorometrie method (18), with bovine serum albumin as standard. RESULTS
Eflect of swainsmine concentration and on the extent of inhibition. Figure 1 shows that, whereas a concentration of 2 /IM swainsonine caused 50% inhibition of jack bean a-D-mannosidase, only 0.2 PM swainsonine was needed to cause 50% inhibition of the lysosomal enzyme and Golgi mannosidase II. The result with the jack bean enzyme was similar to that observed by Schneider et al. (4), but the concentration of inhibitor was 10 times higher than that reported by Dorling et al. (6). It was then observed that preincubation of jack bean mannosidase with the alkaloid at 3’7°C before the addition of substrate greatly increased the inhibition observed. Under these conditions, 50% inhibition was achieved at 0.4 PM swainsonine, a Concentration similar to that reported by Kang and Elbein (8), who have also shown that maximum inhibition requires preincubation with the alkaloid. Lysosomal a-D-mannosidase and Golgi mannosidase II, on the other hand, showed similar inhibition with or without preincubation with swainsonine (data not shown). Figure 1 also shows that the unlabeled and biosynthetically labeled swainsonine were essentially identical to each other in their inhibitory effects on the three enzymes. Competitive nature of the inhibition by swainsmine. The effect of PNP-mannoside concentration on the swainsonine inhibition of the three mannosidases was examined, and the results were plotted by the method of Lineweaver and Burk (Fig. 2). The alkaloid appeared to be a competitive inhibitor of jack bean and lysosomal a-mannosidases at concentrations of 0.5 /IM and below; at a higher concentration of preincubatim
SWAINSONINE
INHIBITION
429
OF a-D-MANNOSIDASES
iOLGl MANNOSIDASEII
0
2
4
6
8
IO SWANSONINE
(NM)
FIG. 1. Effect of swainsonine on cY-D-mannosidases. Jack bean ol-D-mannosidase and liver lysosomal Lu-D-mannosidase were assayed at pH 4.4 with 4 mM pnitrophenyl a-D-mannoside as substrate. Purified Golgi mannosidase II was assayed at pH 5.5 with 4 mM p-nitrophenyl cu-Dmannoside as described under Materials and Methods. The concentration of unlabeled (0) or labeled swainsonine (0) in the reaction mixture was varied as shown. The reaction was started by addition of jack bean mannosidase (0.2 pg protein), lysosomal mannosidase (0.4 pg protein), or Golgi mannosidase II (0.2 pg protein). In a different set of experiments (A), the jack bean mannosidase (0.2 fig protein) was first preincubated (10 min at 37°C) with varying concentrations of swainsonine in the above sodium acetate buffer before addition of the substrate. The reaction mixtures were incubated at 37°C for 15 min, and the released pnitrophenol was measured as described under Materials and Methods.
JACK
BEAN MANNOSIDASE
a0
LYSOSOMAL
MANNOSIDASE
16
GOLGI MANNOSIDASEII
1
0
0.2
0.4
0.6
0.8
1.0
0.2
0.4
ir 0;6
0.8
0
0.2
0.4
0.6
0.8
I.0
FIG. 2. Effect of substrate concentration on swainsonine inhibition of cY-D-mannosidases. The reaction mixture contained sodium acetate buffer (100 mM, pH 4.4 or 5.5), the indicated amount of swainsonine, and varying concentrations of pnitrophenyl a-D-mannoside. Following the addition of jack bean mannosidase (0.1 pg protein), purified rat liver lysosomal mannosidase (0.2 pg protein), or purified Golgi mannosidase II (0.3 pg protein), the mixture was incubated at 37’C for 30 min. The best curves obtained from the data by a linear regression program (Texas Instruments Calculator No. TI-55) are plotted by the method of Lineweaver and Burk. SW, swainsonine.
430
TULSIANI, 0 JACK
BEAN
BROQUIST,
MANNOSIDASE
DSDMAL
0 PREINCUBATION
TIME
AND
(rntn)
2
4
TOUSTER
MANNOSIDASE
6
PREINCUBATION
8
IO
24
!
I
I
I
I
I.,
r
0
2
4
6
8
IO
24
T1MElHr.l
PREINCUEATION
TIMEWr)
FIG. 3. Effect of time of preincubation with swainsonine on the reversibility of inhibition of (YD-mannosidases. Jack bean mannosidase (2 pg protein/O.1 ml Buffer A), purified rat liver lysosomal mannosidase (4 pg protein/O.1 ml Buffer A), or purified Golgi mannosidase II (4 pg/O.l ml Buffer D) was preincubated with different concentrations of swainsonine at 37°C. Aliquots containing 0.05 to 0.1 pg protein were withdrawn periodically and assayed for enzyme activity as described under Materials and Methods. 0, Control (no swainsonine); 0, 1 pM swainsonine; A, 5 pM swainsonine; A, 10 fire swainsonine.
(2 PM), the inhibition appeared to be noncompetitive. However, the inhibition of Golgi mannosidase II appeared to be noncompetitive even at low concentrations (0.2 PM) of swainsonine. Study of the reversibility of swainecmine inhibition by diluticm experiments. Figure 3 shows that the inhibition of jack bean mannosidase is essentially irreversible. In this experiment, the enzyme was preincubated with three different concentrations of swainsonine at 37°C for varying time periods. Aliquots were then diluted and assayed for enzymatic activity. It was apparent that the activity could not be recovered by dilution. However, the inhibition of the lysosomal mannosidase was partially reversible. Dilution of the mixture after preincubation led to recovery of 65-70% of the original enzymatic activity (Fig. 3). Figure 4 shows that the recovery of activity with the lysosomal enzyme is dependent upon the extent of dilution. The Golgi enzyme showed partial reversibility, the extent of which depended upon the concentration of swainsonine present during the preincubation (Fig. 3). The swainsonine concentration also influ-
enced the extent of the rapid, irreversible component of the inhibition (i.e., 0 time values in Fig. 3). Study of reversibility of inhibition using [‘H]swainsonine. The irreversibility of the
0
2
4 ENZYME
16
8 DILUTION
32
l-fold)
FIG. 4. Effect of dilution on the reversibility of the swainsonine inhibition of purified rat liver lysosomal ol-n-mannosidase. Purified enzyme (4 pg/50 ~1 Buffer A) was preincubated with (0) and without (0) 2 jiM swainsonine for 15 min at 37°C. The mixture was serially diluted in Buffer A, and the enzyme activity was measured as described under Materials and Methods.
SWAINSONINE
INHIBITION
reaction of swainsonine with jack bean mannosidase was investigated by incubating the enzyme at 37°C with [3H]swainsonine for varying lengths of time. Each mixture was then applied to a Bio-Gel P10 column to remove free inhibitor, the void volume fractions presumably containing enzyme and enzyme-inhibitor complex. Recovery of enzymatic activity was inversely proportional to the amount of inhibitor bound to the enzyme (Figs. 5A and B). This observation held for varying incubation times and for varying [3H]swainsonine concentrations. When the jack bean mannosidase was preincubated with 1 PM inhibitor for 15 min at 3’7°C and applied to the Bio-Gel P-10 column, the void volume fraction contained 40% of the original enzymatic activity and 0.013 nmol labeled inhibitor (Fig. 5A).
P 5
0
5
IO
However, when 5 PM inhibitor was used (Fig. 5B), the recovered enzyme was essentially inactive and contained more than twice as much (0.03 nmol) bound swainsonine. Attempts to dissociate the enzyme-inhibitor complex by incubation with 10 mM mannose, 10 mM PNP-mannoside, or 10 mM cY-methylmannoside at pH 4.4 or 6.0 were unsuccessful, results suggesting that the swainsonine was tightly bound to the active site of the enzyme. That the alkaloid was not covalently bound was indicated by the fact that treatment of the enzyme-[3H]swainsonine complex in Buffer B at 100°C for 5 min dissociated 87% of this complex. The presence of sodium dodecyl sulfate (2.5%, w/v) in this reaction mixture resulted in 100% dissociation of the complex. This was shown by applying the treated mix-
15
b
cl
I
3
0
5
MIN. 0,lO
0
2
I
431
OF a-D-MANNOSIDASES
lb MIN.
IS DI
I
i!
z 100
z 5
i
HR.
HR. PREINCUBATION
T I ME
FIG. 5. Relationship of irreversibly bound swainsonine to the recovery of a-D-mannosidase activity. Jack bean mannosidase (5 pg protein/O.1 ml buffer A) was preincubated at 37’C with two different concentrations of [‘Hlswainsonine (A, 1 HIM; B, 5 PM). At the indicated times, the mixture was applied to a Bio-Gel P-10 column (1 X 26 cm, -400 mesh) equilibrated with Buffer B to separate the enzyme-swainsonine complex from free [aH]swainsonine. The column was eluted with the buffer at a flow rate of 3 ml/h. The radioactivity and the enzyme activity in fractions (1 ml) were measured in aliquots as described under Materials and Methods. The column void volume fractions (8-13) contained enzyme and bound [3I]swainsonine, whereas free pH]swainsonine eluted in fractions 19-25. In (C), the same procedure was used with purified lysosomal a-Dmannosidase (4 fig/50 ~1 buffer A) incubated with 5 pM [aH]swainsonine. In (D), purified Golgi mannosidase II (5 pg/50 pl Buffer D) was preincubated at 37°C with 5 pM [3H]swainsonine. With the latter enzyme, the Bio-Gel P-4 was equilibrated with Buffer D.
432
TULSIANI,
BROQUIST,
ture to a Bio-Gel P-10 column and measuring the radioactivity eluted from the column in the position of free swainsonine. The labeled swainsonine was released only under severe denaturing conditions without recovery of any enzymatic activity. Differing extents of reversibility of inhibition of lysosomal mannosidase and mannosidase II were found. When a mixture of the lysosomal enzyme and 5 PM [3H]swainsonine was preincubated at 37°C for varying lengths of time and then applied to a Bio-Gel P-10 column, more than 60% of the activity, but very little radioactivity, was recovered in the void volume fractions (Fig. 5C). This result agreed with the dilution experiments shown in Fig. 3B. However, when mannosidase II was used in the same type of gel-filtration experiment, only 30% of the enzymatic activity was present in the void volume fractions, which also contained some radioactivity (Fig. 5D). In another experiment involving mannosidase II, the binding of [3H]swainsonine to salt-washed Golgi membranes was investigated with (Table I, tube B) and without (Table I, tube C) prior incubation with swainsonine. After extensive washing of the membranes, each sample was incubated with
AND
10 pM [3H]swainsonine, a concentration that gave essentially complete inhibition of mannosidase II (Fig. 1). After the membranes were exhaustively washed with Buffer C, assay for mannosidase II activity showed that both samples had regained about 30% of the activity (cf. control; Table I, tube A). Radioactivity in membranes from tube C was nearly five times greater than from tube B. These results indicated that (a) some of mannosidase II activity was regained when excess free swainsonine was removed, and (b) when swainsonine-binding sites on mannosidase II were saturated, little exchange or further addition of the alkaloid occurred. In viva studies in the rat have also yielded evidence suggesting the presence of tightly bound swainsonine in liver Golgi membranes (10). DISCUSSION
The pioneering work of Dorling and his associates led to the identification of swainsonine as a potent inhibitor of jack bean and lysosomal a-D-mannosidases (1, 6). Subsequently, we (9) showed that the alkaloid also strongly inhibits Golgi mannosidase II but not mannosidases IA or
TABLE THE BINDING
Membrane suspension” (sample)
Preincubatior? (additions)
A
None
B
SW (lo fiM)
C
None
OF SWAINSONINE
TOUSTER
I
TO RAT LIVER
Incubation’ (additions) None [3H]SW (10 ELM) [3H]SW (10 /.tM)
GOLGI MEMBRANES
Membrane-bound [3H]swainsonined (nmol/mg protein) None 0.005 0.033
Mannosidase II activityd (%I) 100
28 33
“The salt-washed rat liver Golgi membranes were suspended in Buffer C (13 mg protein/ml). Aliquots containing 2 mg protein were pipetted into each of three tubes (A, B, and C). * Swainsonine (SW) solution (tube B), or water (tubes A and C) was added in each tube. After preincubation at 3’7°C for 15 min, the mixture was diluted with 2 ml Buffer C and centrifuged at 105,OOOgfor 30 min. The pellet was washed four times in 2 ml of the same buffer, each time homogenizing and centrifuging as above. “The washed pellet (see b above) was suspended in Buffer C. [3H]Swainsonine was added only to tubes B and C, the same volume of water being added to tube A. The reaction mixtures (0.2 ml) were incubated at 37°C for 15 min, and the free [3H]swainsonine was removed by extensive washing in Buffer C (see above). d The washed pellet (see b above) from each tube was suspended in Buffer C (0.4 ml). Aliquots were dissolved in 2 N NHIOH, neutralized with glacial acetic acid, and used for the determination of radioactivity (11). Aliquots of the suspension were also assayed for Golgi mannosidase II activity as described under Materials and Methods.
SWAINSONINE
INHIBITION
IB or cytosolic a-D-mannosidase. That the reversible inhibition of jack bean mannosidase is not explainable by a straightforward reversible, competitive mechanism has been evident from the work of Dorling et al. (6) and Kang and Elbein (8). The latter authors first showed that preincubation of swainsonine with jack bean mannosidase increased the observed inhibition of the enzyme, an observation confirmed in the present report. However, we find that such preincubation does not increase the swainsonine inhibition of rat liver lysosomal a-D-mannosidase or Golgi mannosidase II. It is not evident why Dorling et aL (6) found that swainsonine was inhibitory at considerably lower concentrations than employed by Kang and Elbein (8), Schneider et al. (4), or by us. We have directly compared swainsonine obtained from S. caneseem with that isolated from R. leguminicola, and found that both preparations behaved identically in all enzymatic tests employed. In regard to the finding (9) that cytosolic cY-D-mannosidase, with 4 mM PNP-mannoside as substrate, is not inhibited by swainsonine, the recent report of Bischoff and Kornfeld (19) is pertinent. In their paper reporting the occurrence of a-Dmannosidase activity in rat liver endoplasmic reticulum, they state that swainsonine was not inhibitory to this activity unless the p-nitrophenyl cY-mannoside concentration was considerably lower than 4 mM. They therefore suggested that mannosidases with low affinity for PNP-mannoside are inhibited by swainsonine. In support of this suggestion, it is relevant that rat liver cytosolic a-D-mannosidase has a lower K, for PNP-mannoside (20) than lysosomal mannosidase or Golgi mannosidase II (21). Further investigation of this suggestion would be of interest. The results reported herein point to further differences among the enzymes which are very susceptible to inhibition by swainsonine. Reversibility of inhibition is minimal with jack bean a-mannosidase, intermediate with Golgi mannosidase II, and greatest with the lysosomal enzyme. There appears to be two modes of binding
OF a-D-MANNOSIDASES
433
to the liver enzymes (Fig. 3). With mannosidase II, one mode of binding is very rapid and irreversible. The other is slower, with the extent of reversibility dependent upon the original swainsonine concentration and the time of exposure of the enzyme to the inhibitor. With lysosomal a-mannosidase the irreversible mode is very small, and the reversible mode is essentially independent of swainsonine concentration and time of exposure. Although the explanation for the two modes is not apparent at this time, it is possible that multiple forms of the liver enzymes are present in the purified enzymes used. For example, since they are glycoproteins, differently processed forms may be present. The fact that the swainsonine inhibitions of liver lysosomal a-mannosidase and Golgi mannosidase II are partially reversible has relevance to studies of enzyme levels in animals fed swainsonine or plants containing this toxin. Swainsonine administration to rats lowers Golgi mannosidase II to 27% of control activity (lo), an amount very similar to that found in Table I. Since homogenization of liver and preparation of Golgi membranes for assay of enzymatic activity may have been accompanied by the release of some inhibitor, the activity of the enzyme in the intact liver may have in fact been less than 27%. Similarly, while swainsonine administration to rats leads to a considerable elevation of assayable lysosomal CYmannosidase activity, the reversibility of inhibition of this enzyme precludes estimating the actual activity of this enzyme in intact liver by measuring the activity in tissue extracts. Experiments in progress are designed to determine the functional enzyme in intact animals given swainsonine. ACKNOWLEDGMENTS A sample of swainsonine isolated from Swainsona canescens and obtained from Dr. P. R. Dorling was kindly furnished to us by Dr. Philip Stahl, Washington University School of Medicine. We are grateful to Mrs. Vera Coleman for excellent technical assistance. This investigation was supported by Grant GM 26430 to O.T., Grant AM 16019 to H.P.B., and
434
TULSIANI,
Biomedical Research Support from The National Institutes States Public Health Service.
Grant S07-RR07201 of Health, United
BROQUIST,
REFERENCES 1. COLEGATE, S. M., DORLING, P. R., AND HUXTABLE, C. R. (1979) Au&. J. Chem, 32, 2257-2264. 2. DORLING, P. R., HUXTAELE, C. R., AND VOGEL, P. (1978) Neuropathol. Appl Neurobid 4, 285295. 3. MOLYNEUX, R. J., AND JAMES, L. F. (1982) Science (Washington, D. C.) 216, 190-191. 4. SCHNEIDER, M. J., UNGEMACH, F. S., BROQUIST, H. P., AND HARRIS, T. M. (1983) Tetruhedmn 39, 29-32. 5. TIJLSIANI, D. R. P., BROQUIST, H. P., JAMES, L. F., AND TOUSTER, 0. (1984) Arch Biochem. Biophys. 232, 76-85. 6. DORLING, P. R., HUXTABLE, C. R., AND COLEGATE, S. M. (1980) Biochem J 191, 649-651. 7. CHOTAI, K., JENNINGS, C., WINCHESTER, B., AND DORLING, P. (1983) J. CeU. Biochem. 21, 107117. 8. KANG, M. S., AND ELBEIN, A. D. (1983) Plant Physiol 71, 551-554.
AND
TOUSTER
9. TULSIANI, D. R. P., HARRIS, T. M., AND TOUSTER, 0. (1982) J. Biol. Chem 257, 7936-7939. 10. TULSIANI, D. R. P., AND TOUSTER, 0. (1983) Arch. B&hem Biophys. 242,594-600. 11. TULSIANI, D. R. P., AND TOUSTER, 0. (1983) J. Biol. Chem 258, 7578-7585. 12. GROSS, V., TRAN-THI, T.-A., VOSBECK, K., AND HEINRICH, P. A. (1983) J. Biol Chem 258, 4032-4036. 13. KANG, M. S., AND ELBEIN, A. D. (1983) J. Vird 46, 60-69. 14. ARUMUGHAM, R. G., AND TANZER, M. L. (1983) J. Biol Chem 258,11883-11889. 15. GUENGERICH, F. P., SNYDER, J. J., AND BROQUIST, H. P. (1973) Biochemistry 12,4264-4269. 16. OPHEIM, D. J., AND TOUSTER, 0. (1978) J. Biol. Chem 253, 1017-1023. 17. TULSIANI, D. R. P., BUSCHIAZZO, H. O., TOLBERT, B., AND TOUSTER, 0. (1977) Arch B&hem. Biophys. 181, 216-227. 18. ANDERSON, P. M., AND DESNICK, R. J. (1979) .I BioL Chem. 254.6924-6930. 19. BISCHOFF, J., AND KORNFELD, R. (1983) .I. Biol Chem 258, 7907-7910. 20. SHOUP, V. A., AND TOUSTER, 0. (1976) J. Biol Chem 251, 3845-3852. 21. DEWALD, B., AND TOUSTER, 0. (1973) J. Biol Chem. 248, 7223-7233.