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Function of NAD glycohydrolase in ADP-ribose uptake from NAD by human erythrocytes
Biochimica et Biophysica Acta, 1178 (1993) 121-126
121
© 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4889/93/$06.00
BBAMCR 13429
Function of NAD glycohydrolase in ADP-ribose uptake from NAD by human erythrocytes Uh-Hyun Kim a, Myung-Kwan Han a, Byung-Hyun Park a Hyung-Rho Kim a and Nyeon-Hyoung An b a Department of Biochemistry, Chonbuk National UniversityMedical School, Chonju (South Korea) and b Department of Biochemistry College of Pharmacy, Wonkwang University, Iri (South Korea)
(Received 6 April 1992)
Key words: NAD glycohydrolase;ADP-ribose uptake; ADP-ribose transport; Ectoenzyme; (Human); (Erythrocyte) The function of the ectoenzyme NAD glycohydrolase (NADase) in ADP-ribose uptake from extracellular NAD was studied in human erythrocytes that express relatively high NADase activity (adult erythrocytes) and erythrocytes expressing very low activity (newborn erythrocytes). The rates of ADP-ribose uptake from NAD in human erythrocytes were correlated with their NADase activities. In contrast, there was no significant difference in the rates of ADP-ribose uptake among these cells when incubated with ADP-ribose. These results indicate that ecto-NADase may have a role as supplier of ADP-ribose for its uptake into the cells and that the cleavage of NAD by NADase is necessary for the ADP-ribose uptake by human erythrocytes. From ADP-ribose uptake studies at 37°C a K m of 0.7 + 0.05/zM and a Vm~x of 2.04 + 0.1 pmol/min per/zl cell water was found for the uptake of [3H]ADP-ribose. The thiol-reactive reagents p-chloromercuribenzene sulfonic acid and N-ethylmaleimide inhibited the uptake ADP-ribose with IC50 values of 50 + 4 and 750 + 25 mM, respectively. Since efflux of [3H]ADP-ribose was negligible, the ADP-ribose transport system appears to be unidirectional. The unidirectionality was supported by the evidence that transported ADP-ribose was rapidly degraded to AMP which is impermeable to the membrane.
Introduction N A D glycohydrolase (NADase, EC 3.2.2.5) which catalyzes the hydrolysis of N A D into ADP-ribose and nicotinamide, has been reported to be ubiquitous in organisms from bacteria to mammals [1-5]. Most of eukaryotic NADases are membrane-associated enzymes [4,5]. In eukaryotes the activity of the enzyme was shown to be exclusively localized at the outer surface of the m e m b r a n e [6,7]. It was shown that a portion of ecto-NADase can be solubilized by treatment with a bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) and might be attached to the plasma m e m b r a n e through a phosphatidylinositol (PI)-glycan anchor [8]. Although the physiological significance of PI-anchor of N A D a s e is not elucidated,
the solubilization of PI-anchored N A D a s e by PI-PLC enabled us to purify this enzyme [9]. N A D a s e activity was shown to be low or absent in erythrocytes from newborn, but normal adult levels are reached within the first year of life [10]. The function of N A D a s e in vivo is not known, and the significance of the ecto-NADase in erythrocytes is uncertain. One possible role of ecto-NADase could be the hydrolysis of impermeable N A D in extracellular fluids into ADP-ribose and nicotinamide, which can be transported or diffused into the cell [11]. The availability of erythrocytes with high and low activities of NADase and reagents which inhibit N A D a s e activity or the uptake of ADP-ribose provide the opportunity to determine the role of this enzyme in the degradation of N A D for uptake of ADP-ribose.
Materials and Methods Correspondence to: U.-H. Kim, Department of Biochemistry, Chonbuk National University Medical School, Chonju 560-182, South Korea. Abbreviations; NADase, NAD glycohydrolase; ADP-ribose, adenosine diphosphoribose; PI-PLC, phosphatidylinositol-specific phospholipase C; NEM, N-ethylmaleimide; pCMBS, p-chloromercuribenzene sulfonic acid.
Materials. [4-3H]NAD was purchased from Amersham. [ adenine-2,8- 3H]NAD, 3-O-methyl-o-[ 1- 3H]glu. cose and o-[1-3H]sorbitol were obtained from New England Nuclear. N-ethymaleimide (NEM), p-chloromercuribenzene sulfonic acid (pCMBS) and silicone oil were purchased from Sigma. PI-PLC was prepared
122 from Bacillus cereus essentially as described by Ikezawa and Taguchi [12]. NADase of rabbit erythrocytes was solubilized by PI-PLC and purified with a Cibacron Blue column chromatography [9]. Erythrocyte preparation. Fresh erythrocytes from human donors were obtained by venipuncture and anticoagulated with heparin. Ceils were washed twice and the erythrocyte pellet was suspended in phosphatebuffered saline (PBS) containing 5 mM glucose (osmolarity 280 mosm/kg (pH 7.4)).
Preparation of [adenine-2,8-3H]ADP-ribose from [adenine-2,8-~H]NAD. [adenine-2,8- 3H]ADP-ribose was prepared by hydrolyzing [adenine-2,8-3H]NAD with purified NADase as described by Richter et al. [13]. The reaction mixture contained [adenine-2,83H]NAD (60 nmol, 3. l0 s cpm), 2.4 /zg purified NADase in 500 /~1 0.1 M phosphate buffer (pH 7.2). After incubation at 37°C for 20 min, the mixture was diluted 20-fold with distilled water and was then applied to a Dowex AG l-X8 (formate form) column. The ADP-ribose was eluted by applying a formic acid gradient from 1 to 10 M, lyophilized and adjusted to I mM with PBS. The recovery of [adenine-2,8-3H]ADPribose was 90-95% of the starting [adenine-2,83H]NAD.
Uptake of [adenine-2,8-3H]ADP-ribose and [adenine2,8-3H]NAD by human erythrocytes. [adenine-2,83H]ADP-ribose uptake was measured by a rapid oilstop procedure [14], in which assay mixtures were layered on oil in 1.5-ml tubes and placed in a microfuge (Eppendorf model 5412). The uptake assay was initiated by rapid addition of 10 ~1 [adenine-2,8-3H]ADP ribose to 90/zl portions of cell suspension (3.106 cells) layered over 100 /zl of oil (silicone oil/paraffin oil, 80:20) and ended by pelleting the ceils at 16000 × g for 30 s. The tubes were frozen and cut through the oil layer, and the radioactivity associated with the cells was measured by liquid scintillation counting (Packard LS model A300C). The volume of extracellular medium trapped in the cell pellet and total water space of the cell pellet were determined using D-[1-3H]sorbitol and 3-O-methyl-i>[1-3H]glucose, respectively. The intracellular volume of the cell was taken as the difference between the total pellet water (determined with 3-0methyl-D-[1-3H]glucose) and the extracellular space water volume (determined with D-[1-3H]sorbitol. NADase assay. NADase activity was determined by measuring the formation of [4-3H]nicotinamide from [4-3H]NAD following elution from a Dowex-l-chloride column [1.5]. The assay was performed in PBS (pH 7.2), containing 25 nCi of [4-3H]NAD and 4. 10 6 cells in a total volume of 50/xl. After 1 h incubation at 37°C the reaction was terminated by the addition of 50/xl of ice cold 10 mM Tris-HCI and 5 mM MgCI 2 (pH 7.2) and the mixture was then applied to the Dowex column. [4-3H]nicotinamide was recovered by elution with 2.5
ml of 20 mM Tris-HC1 (pH 7.2) and radioactivity was determined by scintillation counting.
Analysis of the metabolites of ADP-ribose. [adenylate32p]ADP-ribose was adjusted to 60/zM with PBS. The uptake was initiated by addition of 5 ~1 of [adenylate32p]ADP-ribose or [adenylate-32P]NAD to 95 ~1 of cell suspention (1.107 RBC). The reaction mixtures were placed in a 1.5 ml microfuge tube containing 100/~I of oil (silicone oil/paraffin oil, 80:20) layered over 50/,~1 of 1 M perchloric acid. The cells were pelleted into the perchloric acid by centrifugation at 10000 × g for 1 min. After aspirating off the media and oil, the cell lysates were neutralized with 5 M KOH. To separate the metabolites of ADP-ribose, 10 ~1 aliquots of cell lysates were spotted gradually under a stream of cold air onto polyethyleneimine-cellulose thin-layer chromatography plates (J.T. Baker). As standard markers we spotted the mixtures of [adenylate-32p]NAD, [adenylate-32 P]ADP-ribose and [32p]AMP onto the origin of one lane as described [16]. The metabolites were separated by a two-step elution; (1), 5 cm in butanol/methanol/water (1 : 1:8) followed without drying by 15 cm in water. The plates were dried and a 2-cm strip from the bottom containing impurities was removed; (2), 12 cm in 0.3 M LiCI and approx. 6 cm in 1 M LiCI. The R f × 100 values of the separated metabolites were ADP-ribose, 45; AMP, 61 and NAD, 73. After elution, the plates were dried and autoradiographed.
Results
Relationship between NADase activity and [3H]ADPribose uptake from [3H]NAD We analyzed the rate of ADP-ribose uptake from NAD in adult erythrocytes and newborn infant erythrocytes, in which NADase activities are quite different. The rate of ADP-ribose uptake from NAD was much higher in adult erythrocytes than in newborn erythrocytes (Fig. 1A). Average uptake in adult erythrocytes was 1.28 + 0.2 pmol/h per /zl cell water, whereas in newborn erythrocytes the average was 0.11 + 0.01 pmol/h per/xl cell water. To determine whether or not the difference in ADP-ribose uptake from NAD between these two groups of erythrocytes was due to differences in NADase activity, we used erythrocytes exhibiting different levels of NADase activity. The finding that NADase activity were correlated well with the uptake of ADP-ribose from NAD (Fig. 2) indicates that differences of ADP-ribose uptake among the cells reflect differences of NADase activity among them. There was no significant difference in ADP-ribose uptake from [3H]ADP-ribose between newborn infant erythrocytes and adult erythrocytes (Fig. 1B). These
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Fig. 1. Time dependence of ADP-ribose uptake. Human erythrocytes were incubated as described in Materials and Methods. The NADase levels from adult erythrocytes (o) and neonate erythrocytes (e) were 3.08 pmol/h per 107 cells and 0.49 pmol/h per 107 cells, respectively. (A) Uptake from NAD. The uptake assay was started by adding 1 p,M [adenine-2,8-3H]NADin a final volume of 0.1 ml containing 3' 106 cells in PBS, 5 mM glucose. (B) Uptake from ADP-ribose. The uptake assay was started by adding 1 p.M [adenine-2,8-3H]ADP-ribosein a final volume of 0.1 ml containing 3.106 cells in PBS, 5 mM glucose. Data shown are from three experiments.
results suggest that ADP-ribose transporters are fully expressed in newborn erythrocytes, i.e., at the adult level, irrespective of the expression of NADase which may be delayed until first year of life [9].
Time and concentration dependence of ADP-ribose uptake in human erythrocytes Time-courses for the influx of 1 tzM [3H]ADP-ribose into cells revealed that cellular ADP-ribose reached steady-state levels of about 15/zM after 40 min (Fig. 1B). Fig. 3 shows the concentration dependence of ADP-ribose uptake into normal erythrocytes over the concentration range 0.25-10 gM. The results showed the saturable nature of ADP-ribose uptake by human erythrocytes. The g m for ADP-ribose uptake determined by double-reciprocal plots was 0.70 + 0.05 /~M and Vm~x was 2.04 + 0.1 pmol/min per ~1 cell water. 2.0 o
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Effects of inhibitors on NADase activity and ADP-ribose uptake To block ADP-ribose formation from NAD by NADase we used Cibacron Blue dye, a potent inhibitor for erythrocyte NADase. 0.3 /~M of Cibacron Blue, which significantly inhibited NADase activity of erythrocytes (76%), blocked the uptake of ADP-ribose from NAD at the similar extent (73%), but did not influence the uptake of ADP-ribose from [3H]ADPribose (Table I). NEM and pCMBS were shown to be inhibitors of ADP-ribose uptake with IC50 values of 750 + 25/zM and 50 + 4/zM, respectively (Fig. 4). In the presence of 1 mM NEM and 100 /zM pCMBS, which slightly inhibited NADase activity of erythrocytes, ADP-ribose uptake from both [3H]ADP-ribose and [3H]NAD was inhibited (Table I). To demonstrate that the low ADP-ribose uptake from NAD by the sulfhydryl-reagent-treated cells is not due to leakiness of cells, we measured the concentrations of ATP in the supernatants of the incubation mixtures of red cells: No change in ATP was observed before and after treatment with the sulfhydryl reagents (Table II). We
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NADaseactivity(pmol]h/107cells) Fig. 2. The relation of NADase activity and uptake of ADP-ribose formed from NAD by human erythrocytes. Erythrocytes were prepared from peripherial blood from newborn infants (e) and normal adults (o). 4-106 erythrocytes were used for measuring NADase activity and uptake of ADP-ribose from [adenine-2,8-3HlNAD. Cell suspensions were incubated at 37°C for 30 min. After two washes with PBS and dissolving in Aquasol, the radioactivity was determined in a liquid scintillation counter.
f l
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6
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ADPR concentration(gM) Fig. 3. Concentration dependence of ADP-ribose uptake. Human erythrocytes were incubated as described in Materials and Methods. ADP-ribose concentration range was 0.25-10/~M. Values shown are from three experiments.
124 TABLE i
Effect of inhibitors on the NADase activity and on the uptake of ADP-ribose from NAD and ADP-ribose in human erythrocytes Erythrocytes were preincubated with or without pCMBS (for 60 min) or NEM (for 30 min) and Cibacron Blue (for 30 min). Uptake and the NADase activity were then determined as described in Materials and Methods. Incubation time in uptake assay was 10 min. Values are means (+_ S.E.) of three experiments. The percent of control is given in parentheses.
Control pCMBS (100/xM) NEM (1 mM) Cibacron Blue (0.3/xM)
NADase activity (pmol/h per 107 cells)
Uptake from NAD (pmol/h per tzl cell water)
Uptake from ADP-ribose (pmol/h per/xl cell water)
4.12 +_0.8 3.91 +_0.6 (95.0) 3.74+-0.5 (90.1) 1.09 ± 0.1 (26.4)
3.36 + 0.7 1.37+_0.3 (40.9) 0.57+-0.1 (17.0) 0.78 +_0.09 (23.2)
56.4 +_1.2 21.4+- 1.5 (37.9) 7.6_+0.5 (13.5) 55.8 _+1.8 (98.9)
also considered the possibility that the low ADP-ribose uptake is due to retarded conversion of ADP-ribose to AMP by ADP-ribose pyrophosphatase, which might cause the accumulation and subsequent efflux of intracellular ADP-ribose. However, pCMBS did not inhibit ADP-ribose pyrophosphatase: ADP-ribose pyrophosphatase activity measured in the homogenate of human erythrocytes that had been incubated with 100 /xM pCMBS for 1 h at 37°C was 95% of that measured in control homogenate.
Effects of structural analogs on ADP-ribose uptake We analyzed the effect of structural analogs on ADP-ribose uptake. In this experiment, CibacronBlue-preincubated erythrocytes, in which NADase is completely blocked, were used to exclude the possible effects of cleavage products of NADase. 10/zM ADPribose, ADP-glucose, ADP-mannose, /3-NAD and aNAD were added to reaction mixtures containing 1 /xM [3H]ADP-ribose. Under these assay conditions, there was no inhibition of ADP-ribose uptake in the presence of any compounds, except ADP-ribose (Table III). It seems, therefore, that ADP-ribose transporter is specific for ADP-ribose.
1001 w 80-
~6040" 20"
10
20
30
40
50
Time (min) Fig. 5. Effux of transported [3HlADP-ribose as a function of time in erythrocytes. Erythrocytes were. loaded with 1 ~M [3H]ADP-ribose by incubation for 30 min at 37°C and then resuspended in ADPribose-free medium. After resuspension, the intracellular [3H]ADPribose content was determined as described in Materials and Methods. Control values (100%) represents accumulated [3H]ADP-ribose for 30 min at 37°C. Data shown are means of three determinations.
A 1
2
B 3
4
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2
3
4
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Fig. 4. Effects of pCMBS and NEM on ADP-ribose uptake. The percent inhibition of ADP-ribose uptake was determined by comparing the initial velocity of uptake in the presence of pCMBS (o) and NEM (e) to the control rate (0.94 pmol/min per /zl cell water) determined in the absence of inhibitors. Erythrocytes were preineubated with or without pCMBS and NEM for 60 rain at 37°C. The uptake was then determined as described in Materials and Methods. Values shown are from three experiments.
Fig. 6. Autoradiogram of thin-layer chromatography of ADP-ribose metabolites. The erythrocytes were incubated with [32P]ADP-ribose (A), or [32p]NAD (B) for 15 rain (lane 1), 30 min (lane 2), 45 min (lane 3), or 60 min (lane 4), were washed and lysed. Details of chromatography were described in Materials and Methods. The films were exposed for 3 days (A) and 6 days (B).
125 TABLE II
Discussion
Effect of sulfhydryl reagents on the hemolysis of human erythrocytes Erythrocytes (6-106 cells) were incubated with or without 1 /zM NEM or 100 /zM pCMBS in phosphate-buffered saline for 1 h at 37°C. Erythrocytes were fully lysed by incubation with 5 mM phosphate buffer (pH 7.2). ATP was then determined using the bioluminescent method. Values are means ( + S.E.) of three experiments. ATP (~M) Control NEM (1 mM) pCMBS (100/~M) Full lysis
0.224 :t: 0.09 0.226+0.08 0.239 + 0.07 10.45 5:0.2
Efflux of ADP-ribose from human erythrocytes To estimate the effiux of [3H]ADP-ribose transported into the cells, erythrocytes were preloaded in 1 /zM [3H]ADP-ribose for 30 min, the cells were collected and then incubated in PBS. The efflux was analyzed by measuring the remaining radioactivity in erythrocytes which were pelleted through oil layers. As shown in Fig. 5, the effiux of [3H]ADP-ribose was negligible during incubation in PBS. This suggests that the ADP-ribose uptake system is unidirectional.
Fate of transported ADP-ribose Thin-layer chromatography of perchloric acid extracts of erythrocytes following exposure to [a2p]ADPribose or [32p]NAD at 37°C for different times showed that almost all of the 32p in the ceils was present in the form of AMP (Fig. 6A). The results from the incubation of [32p]ADP-ribose or [32p]NAD were not much different, except for a slower rate of degradation to AMP due. to the delayed uptake of ADP-ribose in case of the incubation of [32p]NAD (Fig. 6A, B).
TABLE III
Effect of structural analogs on ADP-ribose uptake The inhibition of [3H]ADP-ribose uptake was determined at 37°C, by comparing the rate of ADP-ribose uptake in the absence and presence of 10 /zM analog. The control rate for ADP-ribose (1 /zM) uptake was 0.94 pmol/min p e r / z l cell water. Human erythrocytes were preincubated with Cibacron Blue (1 /zM) for 30 min at 37°C. Values are means (5: S.E.) for three experiments. Addition
[3H]ADP-ribose uptake (percent of control)
None ADP-ribose ADP-glucose ADP-mannose /3-NAD a-NAD
100 0.1 + 0.03 99 -I-0.5 98 + 0.6 95 + 0.7 99 +0.4
NADase in most mammalian cells, including erythrocytes, is located on the outer surface of membrane. However, the role of NADase in vivo has remained an enigma. In this investigation, we obtained results indicating that the rate of ADP-ribose uptake from NAD correlates well with ecto-NADase activity in human erythrocytes (Figs. 1A and 2). Similar results were observed in mouse splenocytes (data not shown). In mouse splenocytes, which have higher NADase activity than human erythrocytes, the rate of ADP-ribose uptake from NAD was greater than that of human erythrocytes. These results also show that the two activities are correlated. In view of this situation, it is not surprising that a considerable amount of NAD is present in blood plasma [11]. This suggests that the breakdown of extracellular NAD by ecto-NADase may be a rate-limiting step for ADP-ribose uptake into the cells. Uptake of ADP-ribose in human erythrocytes follows Michaelis-Menten kinetics, and shows a saturable nature as a function of ADP-ribose concentration. ADP-ribose uptake was blocked by two kinds of sulfhydryl reagents [17], which suggests that the ADPribose transporter has essential sulfhydryl groups. pCMBS is known to work mainly at the surface of the erythrocyte membranes, whereas NEM can diffuse rapidly into the cytosol, reacting extensively with cytosolic proteins. The ADP-ribose transporter can discriminate among the structural analogs of ADP-ribose (Table III), and ADP-ribose uptake is not inhibited by nucleoside tranport inhibitors such as dipyridamol (data not shown). These data indicate that ADP-ribose transporter is quite specific for the ADP-ribose molecule. Since efflux of ADP-ribose trapped in the cell does not occur, the transporter apparently operates only unidirectionally. In contrast, [3H]nicotinamide, which is very diffusible and taken up into the cells rapidly, could be rapidly removed by incubating in PBS (data not shown). This is consistent with the findings reported by others [18]. Taken together, ADP-ribose uptake is strictly regulated via a unidirectional transport system, while nicotinamide diffuses freely through cell membranes. One possible explanation of this unidirectionality of ADP-ribose uptake is that ADP-ribose, once transported, can be converted into impermeable molecules such as AMP (Fig. 6). The degradation of transported ADP-ribose to AMP may be catalyzed by cytosolic ADP-ribose pyrophosphatase [19]. In fact, most of ADP-ribose pyrophosphatase was found to be in the cytosol of human erythrocytes (data not shown). Rapid conversion of ADP-ribose to AMP could explain the concentrative transport of ADP-ribose shown in Fig. lB. And also, the high rate of ADP-ribose uptake
126
in mouse splenocytes could be explained by the finding that mouse splenocytes had a much higher ADP-ribose pyrophosphatase activity than human erythrocytes (data not shown). References 1 Kaplan, N.O. (1955) Methods Enzymol. 2, 660-663. 2 Swislocki, N.I., Kalish, M.I., Chasalow, F.I. and Kaplan, N.O. (1967) J. Biol. Chem. 242, 1089-1094. 3 Srivastava. S.K., Maini, S.B. and Ramakrishnan, C.V. (1969) Phytochemistry 8, 1147-1154. 4 De Wolf, M.J.S., Van Dessel, G.A.F., Lagrou, A.R., Hilderson, H.J.J. and Dierick, W.S.H. (1985) Biochem. J. 226, 415-427. 5 Pekala, P.H. and Anderson, B.M. (1978) J. Biol. Chem. 253, 7453-7459. 6 Friedemann, H. and Rapopot, S.M. (1974) in Cellular and Molecular Biology of Erythrocytes (Yoshikawa, H. and Rapoport, S.M., eds.), pp. 181-259, University Park Press, Baltimore. 7 Alivisators, S.G.A. and Denstedt, O.F. (1951) Science 114, 281283.
8 Kim, U.-H., Rockwood S.F., Kim, H.R. and Daynes, R.A. (1988) Biochim. Biophys. Acta 965, 76-81. 9 Kim, V.-H., Kim, M.K., Kim, J.-S., Han, M.-K., Park, B.-H. and Kim, H.-R. (1993) Arch. Biochem. Biophys., in press. 10 Ng, W.G., Donnell, G.N. and Bergren, W.R. (1968) Nature 217, 64-65. 11 Dixon, M. and Webb, E.C. (1979) in Enzymes, 3rd Edn., pp. 465-518, Longman, London. 12 Ikezawa, H. and Taguchi, R. (1981) Methods Enzymol. 7l, 731741. 13 Richter, C., Winterhalter, K.H., Baumhuter, S., Lotscher, H.-R. and Moser, B. (1983) Proc. Natl. Acad. ScL USA 80, 3188-3192. 14 Dagnino, L., Bennet, L.L. and Paterson, A.R.P. (1991) J. Biol. Chem. 266, 6308-6311. 15 Moss, J., Manganiello, V.C. and Vaughan, M. (1976) Proc. Natl. Acad. Sci. USA 73, 4424-4427. 16 Suidan, H.S., Murrell, R.D. and Tolkovsky, A.M. (1991) Cell Regul. 2, 13-25. 17 May, J.M. (1985) J. Biol. Chem. 260, 462-467. 18 Lan, S.J. and Henderson, L.M. (1968) J. Biol. Chem. 243, 33883394. 19 Miro, A., Coatas, M.J., Garcia-Diaz, M. Hernandez, M.T. and Cameselle, J.C. (1989) FEBS Lett. 244, 123-126.
121
© 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4889/93/$06.00
BBAMCR 13429
Function of NAD glycohydrolase in ADP-ribose uptake from NAD by human erythrocytes Uh-Hyun Kim a, Myung-Kwan Han a, Byung-Hyun Park a Hyung-Rho Kim a and Nyeon-Hyoung An b a Department of Biochemistry, Chonbuk National UniversityMedical School, Chonju (South Korea) and b Department of Biochemistry College of Pharmacy, Wonkwang University, Iri (South Korea)
(Received 6 April 1992)
Key words: NAD glycohydrolase;ADP-ribose uptake; ADP-ribose transport; Ectoenzyme; (Human); (Erythrocyte) The function of the ectoenzyme NAD glycohydrolase (NADase) in ADP-ribose uptake from extracellular NAD was studied in human erythrocytes that express relatively high NADase activity (adult erythrocytes) and erythrocytes expressing very low activity (newborn erythrocytes). The rates of ADP-ribose uptake from NAD in human erythrocytes were correlated with their NADase activities. In contrast, there was no significant difference in the rates of ADP-ribose uptake among these cells when incubated with ADP-ribose. These results indicate that ecto-NADase may have a role as supplier of ADP-ribose for its uptake into the cells and that the cleavage of NAD by NADase is necessary for the ADP-ribose uptake by human erythrocytes. From ADP-ribose uptake studies at 37°C a K m of 0.7 + 0.05/zM and a Vm~x of 2.04 + 0.1 pmol/min per/zl cell water was found for the uptake of [3H]ADP-ribose. The thiol-reactive reagents p-chloromercuribenzene sulfonic acid and N-ethylmaleimide inhibited the uptake ADP-ribose with IC50 values of 50 + 4 and 750 + 25 mM, respectively. Since efflux of [3H]ADP-ribose was negligible, the ADP-ribose transport system appears to be unidirectional. The unidirectionality was supported by the evidence that transported ADP-ribose was rapidly degraded to AMP which is impermeable to the membrane.
Introduction N A D glycohydrolase (NADase, EC 3.2.2.5) which catalyzes the hydrolysis of N A D into ADP-ribose and nicotinamide, has been reported to be ubiquitous in organisms from bacteria to mammals [1-5]. Most of eukaryotic NADases are membrane-associated enzymes [4,5]. In eukaryotes the activity of the enzyme was shown to be exclusively localized at the outer surface of the m e m b r a n e [6,7]. It was shown that a portion of ecto-NADase can be solubilized by treatment with a bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) and might be attached to the plasma m e m b r a n e through a phosphatidylinositol (PI)-glycan anchor [8]. Although the physiological significance of PI-anchor of N A D a s e is not elucidated,
the solubilization of PI-anchored N A D a s e by PI-PLC enabled us to purify this enzyme [9]. N A D a s e activity was shown to be low or absent in erythrocytes from newborn, but normal adult levels are reached within the first year of life [10]. The function of N A D a s e in vivo is not known, and the significance of the ecto-NADase in erythrocytes is uncertain. One possible role of ecto-NADase could be the hydrolysis of impermeable N A D in extracellular fluids into ADP-ribose and nicotinamide, which can be transported or diffused into the cell [11]. The availability of erythrocytes with high and low activities of NADase and reagents which inhibit N A D a s e activity or the uptake of ADP-ribose provide the opportunity to determine the role of this enzyme in the degradation of N A D for uptake of ADP-ribose.
Materials and Methods Correspondence to: U.-H. Kim, Department of Biochemistry, Chonbuk National University Medical School, Chonju 560-182, South Korea. Abbreviations; NADase, NAD glycohydrolase; ADP-ribose, adenosine diphosphoribose; PI-PLC, phosphatidylinositol-specific phospholipase C; NEM, N-ethylmaleimide; pCMBS, p-chloromercuribenzene sulfonic acid.
Materials. [4-3H]NAD was purchased from Amersham. [ adenine-2,8- 3H]NAD, 3-O-methyl-o-[ 1- 3H]glu. cose and o-[1-3H]sorbitol were obtained from New England Nuclear. N-ethymaleimide (NEM), p-chloromercuribenzene sulfonic acid (pCMBS) and silicone oil were purchased from Sigma. PI-PLC was prepared
122 from Bacillus cereus essentially as described by Ikezawa and Taguchi [12]. NADase of rabbit erythrocytes was solubilized by PI-PLC and purified with a Cibacron Blue column chromatography [9]. Erythrocyte preparation. Fresh erythrocytes from human donors were obtained by venipuncture and anticoagulated with heparin. Ceils were washed twice and the erythrocyte pellet was suspended in phosphatebuffered saline (PBS) containing 5 mM glucose (osmolarity 280 mosm/kg (pH 7.4)).
Preparation of [adenine-2,8-3H]ADP-ribose from [adenine-2,8-~H]NAD. [adenine-2,8- 3H]ADP-ribose was prepared by hydrolyzing [adenine-2,8-3H]NAD with purified NADase as described by Richter et al. [13]. The reaction mixture contained [adenine-2,83H]NAD (60 nmol, 3. l0 s cpm), 2.4 /zg purified NADase in 500 /~1 0.1 M phosphate buffer (pH 7.2). After incubation at 37°C for 20 min, the mixture was diluted 20-fold with distilled water and was then applied to a Dowex AG l-X8 (formate form) column. The ADP-ribose was eluted by applying a formic acid gradient from 1 to 10 M, lyophilized and adjusted to I mM with PBS. The recovery of [adenine-2,8-3H]ADPribose was 90-95% of the starting [adenine-2,83H]NAD.
Uptake of [adenine-2,8-3H]ADP-ribose and [adenine2,8-3H]NAD by human erythrocytes. [adenine-2,83H]ADP-ribose uptake was measured by a rapid oilstop procedure [14], in which assay mixtures were layered on oil in 1.5-ml tubes and placed in a microfuge (Eppendorf model 5412). The uptake assay was initiated by rapid addition of 10 ~1 [adenine-2,8-3H]ADP ribose to 90/zl portions of cell suspension (3.106 cells) layered over 100 /zl of oil (silicone oil/paraffin oil, 80:20) and ended by pelleting the ceils at 16000 × g for 30 s. The tubes were frozen and cut through the oil layer, and the radioactivity associated with the cells was measured by liquid scintillation counting (Packard LS model A300C). The volume of extracellular medium trapped in the cell pellet and total water space of the cell pellet were determined using D-[1-3H]sorbitol and 3-O-methyl-i>[1-3H]glucose, respectively. The intracellular volume of the cell was taken as the difference between the total pellet water (determined with 3-0methyl-D-[1-3H]glucose) and the extracellular space water volume (determined with D-[1-3H]sorbitol. NADase assay. NADase activity was determined by measuring the formation of [4-3H]nicotinamide from [4-3H]NAD following elution from a Dowex-l-chloride column [1.5]. The assay was performed in PBS (pH 7.2), containing 25 nCi of [4-3H]NAD and 4. 10 6 cells in a total volume of 50/xl. After 1 h incubation at 37°C the reaction was terminated by the addition of 50/xl of ice cold 10 mM Tris-HCI and 5 mM MgCI 2 (pH 7.2) and the mixture was then applied to the Dowex column. [4-3H]nicotinamide was recovered by elution with 2.5
ml of 20 mM Tris-HC1 (pH 7.2) and radioactivity was determined by scintillation counting.
Analysis of the metabolites of ADP-ribose. [adenylate32p]ADP-ribose was adjusted to 60/zM with PBS. The uptake was initiated by addition of 5 ~1 of [adenylate32p]ADP-ribose or [adenylate-32P]NAD to 95 ~1 of cell suspention (1.107 RBC). The reaction mixtures were placed in a 1.5 ml microfuge tube containing 100/~I of oil (silicone oil/paraffin oil, 80:20) layered over 50/,~1 of 1 M perchloric acid. The cells were pelleted into the perchloric acid by centrifugation at 10000 × g for 1 min. After aspirating off the media and oil, the cell lysates were neutralized with 5 M KOH. To separate the metabolites of ADP-ribose, 10 ~1 aliquots of cell lysates were spotted gradually under a stream of cold air onto polyethyleneimine-cellulose thin-layer chromatography plates (J.T. Baker). As standard markers we spotted the mixtures of [adenylate-32p]NAD, [adenylate-32 P]ADP-ribose and [32p]AMP onto the origin of one lane as described [16]. The metabolites were separated by a two-step elution; (1), 5 cm in butanol/methanol/water (1 : 1:8) followed without drying by 15 cm in water. The plates were dried and a 2-cm strip from the bottom containing impurities was removed; (2), 12 cm in 0.3 M LiCI and approx. 6 cm in 1 M LiCI. The R f × 100 values of the separated metabolites were ADP-ribose, 45; AMP, 61 and NAD, 73. After elution, the plates were dried and autoradiographed.
Results
Relationship between NADase activity and [3H]ADPribose uptake from [3H]NAD We analyzed the rate of ADP-ribose uptake from NAD in adult erythrocytes and newborn infant erythrocytes, in which NADase activities are quite different. The rate of ADP-ribose uptake from NAD was much higher in adult erythrocytes than in newborn erythrocytes (Fig. 1A). Average uptake in adult erythrocytes was 1.28 + 0.2 pmol/h per /zl cell water, whereas in newborn erythrocytes the average was 0.11 + 0.01 pmol/h per/xl cell water. To determine whether or not the difference in ADP-ribose uptake from NAD between these two groups of erythrocytes was due to differences in NADase activity, we used erythrocytes exhibiting different levels of NADase activity. The finding that NADase activity were correlated well with the uptake of ADP-ribose from NAD (Fig. 2) indicates that differences of ADP-ribose uptake among the cells reflect differences of NADase activity among them. There was no significant difference in ADP-ribose uptake from [3H]ADP-ribose between newborn infant erythrocytes and adult erythrocytes (Fig. 1B). These
123 ~
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10
20
~0
40
Time (min)
~-
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/ l0
20
30
40
50
Time (min)
Fig. 1. Time dependence of ADP-ribose uptake. Human erythrocytes were incubated as described in Materials and Methods. The NADase levels from adult erythrocytes (o) and neonate erythrocytes (e) were 3.08 pmol/h per 107 cells and 0.49 pmol/h per 107 cells, respectively. (A) Uptake from NAD. The uptake assay was started by adding 1 p,M [adenine-2,8-3H]NADin a final volume of 0.1 ml containing 3' 106 cells in PBS, 5 mM glucose. (B) Uptake from ADP-ribose. The uptake assay was started by adding 1 p.M [adenine-2,8-3H]ADP-ribosein a final volume of 0.1 ml containing 3.106 cells in PBS, 5 mM glucose. Data shown are from three experiments.
results suggest that ADP-ribose transporters are fully expressed in newborn erythrocytes, i.e., at the adult level, irrespective of the expression of NADase which may be delayed until first year of life [9].
Time and concentration dependence of ADP-ribose uptake in human erythrocytes Time-courses for the influx of 1 tzM [3H]ADP-ribose into cells revealed that cellular ADP-ribose reached steady-state levels of about 15/zM after 40 min (Fig. 1B). Fig. 3 shows the concentration dependence of ADP-ribose uptake into normal erythrocytes over the concentration range 0.25-10 gM. The results showed the saturable nature of ADP-ribose uptake by human erythrocytes. The g m for ADP-ribose uptake determined by double-reciprocal plots was 0.70 + 0.05 /~M and Vm~x was 2.04 + 0.1 pmol/min per ~1 cell water. 2.0 o
o o
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o
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o
Effects of inhibitors on NADase activity and ADP-ribose uptake To block ADP-ribose formation from NAD by NADase we used Cibacron Blue dye, a potent inhibitor for erythrocyte NADase. 0.3 /~M of Cibacron Blue, which significantly inhibited NADase activity of erythrocytes (76%), blocked the uptake of ADP-ribose from NAD at the similar extent (73%), but did not influence the uptake of ADP-ribose from [3H]ADPribose (Table I). NEM and pCMBS were shown to be inhibitors of ADP-ribose uptake with IC50 values of 750 + 25/zM and 50 + 4/zM, respectively (Fig. 4). In the presence of 1 mM NEM and 100 /zM pCMBS, which slightly inhibited NADase activity of erythrocytes, ADP-ribose uptake from both [3H]ADP-ribose and [3H]NAD was inhibited (Table I). To demonstrate that the low ADP-ribose uptake from NAD by the sulfhydryl-reagent-treated cells is not due to leakiness of cells, we measured the concentrations of ATP in the supernatants of the incubation mixtures of red cells: No change in ATP was observed before and after treatment with the sulfhydryl reagents (Table II). We
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o
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2
3
4
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6
7
NADaseactivity(pmol]h/107cells) Fig. 2. The relation of NADase activity and uptake of ADP-ribose formed from NAD by human erythrocytes. Erythrocytes were prepared from peripherial blood from newborn infants (e) and normal adults (o). 4-106 erythrocytes were used for measuring NADase activity and uptake of ADP-ribose from [adenine-2,8-3HlNAD. Cell suspensions were incubated at 37°C for 30 min. After two washes with PBS and dissolving in Aquasol, the radioactivity was determined in a liquid scintillation counter.
f l
1
2
3
4
5
l
6
l
7
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8
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ADPR concentration(gM) Fig. 3. Concentration dependence of ADP-ribose uptake. Human erythrocytes were incubated as described in Materials and Methods. ADP-ribose concentration range was 0.25-10/~M. Values shown are from three experiments.
124 TABLE i
Effect of inhibitors on the NADase activity and on the uptake of ADP-ribose from NAD and ADP-ribose in human erythrocytes Erythrocytes were preincubated with or without pCMBS (for 60 min) or NEM (for 30 min) and Cibacron Blue (for 30 min). Uptake and the NADase activity were then determined as described in Materials and Methods. Incubation time in uptake assay was 10 min. Values are means (+_ S.E.) of three experiments. The percent of control is given in parentheses.
Control pCMBS (100/xM) NEM (1 mM) Cibacron Blue (0.3/xM)
NADase activity (pmol/h per 107 cells)
Uptake from NAD (pmol/h per tzl cell water)
Uptake from ADP-ribose (pmol/h per/xl cell water)
4.12 +_0.8 3.91 +_0.6 (95.0) 3.74+-0.5 (90.1) 1.09 ± 0.1 (26.4)
3.36 + 0.7 1.37+_0.3 (40.9) 0.57+-0.1 (17.0) 0.78 +_0.09 (23.2)
56.4 +_1.2 21.4+- 1.5 (37.9) 7.6_+0.5 (13.5) 55.8 _+1.8 (98.9)
also considered the possibility that the low ADP-ribose uptake is due to retarded conversion of ADP-ribose to AMP by ADP-ribose pyrophosphatase, which might cause the accumulation and subsequent efflux of intracellular ADP-ribose. However, pCMBS did not inhibit ADP-ribose pyrophosphatase: ADP-ribose pyrophosphatase activity measured in the homogenate of human erythrocytes that had been incubated with 100 /xM pCMBS for 1 h at 37°C was 95% of that measured in control homogenate.
Effects of structural analogs on ADP-ribose uptake We analyzed the effect of structural analogs on ADP-ribose uptake. In this experiment, CibacronBlue-preincubated erythrocytes, in which NADase is completely blocked, were used to exclude the possible effects of cleavage products of NADase. 10/zM ADPribose, ADP-glucose, ADP-mannose, /3-NAD and aNAD were added to reaction mixtures containing 1 /xM [3H]ADP-ribose. Under these assay conditions, there was no inhibition of ADP-ribose uptake in the presence of any compounds, except ADP-ribose (Table III). It seems, therefore, that ADP-ribose transporter is specific for ADP-ribose.
1001 w 80-
~6040" 20"
10
20
30
40
50
Time (min) Fig. 5. Effux of transported [3HlADP-ribose as a function of time in erythrocytes. Erythrocytes were. loaded with 1 ~M [3H]ADP-ribose by incubation for 30 min at 37°C and then resuspended in ADPribose-free medium. After resuspension, the intracellular [3H]ADPribose content was determined as described in Materials and Methods. Control values (100%) represents accumulated [3H]ADP-ribose for 30 min at 37°C. Data shown are means of three determinations.
A 1
2
B 3
4
1
2
3
4
I~D--
100~
AMP-80 6O
ADP-dbOm--
40 20 0 0
4 Inhibitors, log[raM]
Fig. 4. Effects of pCMBS and NEM on ADP-ribose uptake. The percent inhibition of ADP-ribose uptake was determined by comparing the initial velocity of uptake in the presence of pCMBS (o) and NEM (e) to the control rate (0.94 pmol/min per /zl cell water) determined in the absence of inhibitors. Erythrocytes were preineubated with or without pCMBS and NEM for 60 rain at 37°C. The uptake was then determined as described in Materials and Methods. Values shown are from three experiments.
Fig. 6. Autoradiogram of thin-layer chromatography of ADP-ribose metabolites. The erythrocytes were incubated with [32P]ADP-ribose (A), or [32p]NAD (B) for 15 rain (lane 1), 30 min (lane 2), 45 min (lane 3), or 60 min (lane 4), were washed and lysed. Details of chromatography were described in Materials and Methods. The films were exposed for 3 days (A) and 6 days (B).
125 TABLE II
Discussion
Effect of sulfhydryl reagents on the hemolysis of human erythrocytes Erythrocytes (6-106 cells) were incubated with or without 1 /zM NEM or 100 /zM pCMBS in phosphate-buffered saline for 1 h at 37°C. Erythrocytes were fully lysed by incubation with 5 mM phosphate buffer (pH 7.2). ATP was then determined using the bioluminescent method. Values are means ( + S.E.) of three experiments. ATP (~M) Control NEM (1 mM) pCMBS (100/~M) Full lysis
0.224 :t: 0.09 0.226+0.08 0.239 + 0.07 10.45 5:0.2
Efflux of ADP-ribose from human erythrocytes To estimate the effiux of [3H]ADP-ribose transported into the cells, erythrocytes were preloaded in 1 /zM [3H]ADP-ribose for 30 min, the cells were collected and then incubated in PBS. The efflux was analyzed by measuring the remaining radioactivity in erythrocytes which were pelleted through oil layers. As shown in Fig. 5, the effiux of [3H]ADP-ribose was negligible during incubation in PBS. This suggests that the ADP-ribose uptake system is unidirectional.
Fate of transported ADP-ribose Thin-layer chromatography of perchloric acid extracts of erythrocytes following exposure to [a2p]ADPribose or [32p]NAD at 37°C for different times showed that almost all of the 32p in the ceils was present in the form of AMP (Fig. 6A). The results from the incubation of [32p]ADP-ribose or [32p]NAD were not much different, except for a slower rate of degradation to AMP due. to the delayed uptake of ADP-ribose in case of the incubation of [32p]NAD (Fig. 6A, B).
TABLE III
Effect of structural analogs on ADP-ribose uptake The inhibition of [3H]ADP-ribose uptake was determined at 37°C, by comparing the rate of ADP-ribose uptake in the absence and presence of 10 /zM analog. The control rate for ADP-ribose (1 /zM) uptake was 0.94 pmol/min p e r / z l cell water. Human erythrocytes were preincubated with Cibacron Blue (1 /zM) for 30 min at 37°C. Values are means (5: S.E.) for three experiments. Addition
[3H]ADP-ribose uptake (percent of control)
None ADP-ribose ADP-glucose ADP-mannose /3-NAD a-NAD
100 0.1 + 0.03 99 -I-0.5 98 + 0.6 95 + 0.7 99 +0.4
NADase in most mammalian cells, including erythrocytes, is located on the outer surface of membrane. However, the role of NADase in vivo has remained an enigma. In this investigation, we obtained results indicating that the rate of ADP-ribose uptake from NAD correlates well with ecto-NADase activity in human erythrocytes (Figs. 1A and 2). Similar results were observed in mouse splenocytes (data not shown). In mouse splenocytes, which have higher NADase activity than human erythrocytes, the rate of ADP-ribose uptake from NAD was greater than that of human erythrocytes. These results also show that the two activities are correlated. In view of this situation, it is not surprising that a considerable amount of NAD is present in blood plasma [11]. This suggests that the breakdown of extracellular NAD by ecto-NADase may be a rate-limiting step for ADP-ribose uptake into the cells. Uptake of ADP-ribose in human erythrocytes follows Michaelis-Menten kinetics, and shows a saturable nature as a function of ADP-ribose concentration. ADP-ribose uptake was blocked by two kinds of sulfhydryl reagents [17], which suggests that the ADPribose transporter has essential sulfhydryl groups. pCMBS is known to work mainly at the surface of the erythrocyte membranes, whereas NEM can diffuse rapidly into the cytosol, reacting extensively with cytosolic proteins. The ADP-ribose transporter can discriminate among the structural analogs of ADP-ribose (Table III), and ADP-ribose uptake is not inhibited by nucleoside tranport inhibitors such as dipyridamol (data not shown). These data indicate that ADP-ribose transporter is quite specific for the ADP-ribose molecule. Since efflux of ADP-ribose trapped in the cell does not occur, the transporter apparently operates only unidirectionally. In contrast, [3H]nicotinamide, which is very diffusible and taken up into the cells rapidly, could be rapidly removed by incubating in PBS (data not shown). This is consistent with the findings reported by others [18]. Taken together, ADP-ribose uptake is strictly regulated via a unidirectional transport system, while nicotinamide diffuses freely through cell membranes. One possible explanation of this unidirectionality of ADP-ribose uptake is that ADP-ribose, once transported, can be converted into impermeable molecules such as AMP (Fig. 6). The degradation of transported ADP-ribose to AMP may be catalyzed by cytosolic ADP-ribose pyrophosphatase [19]. In fact, most of ADP-ribose pyrophosphatase was found to be in the cytosol of human erythrocytes (data not shown). Rapid conversion of ADP-ribose to AMP could explain the concentrative transport of ADP-ribose shown in Fig. lB. And also, the high rate of ADP-ribose uptake
126
in mouse splenocytes could be explained by the finding that mouse splenocytes had a much higher ADP-ribose pyrophosphatase activity than human erythrocytes (data not shown). References 1 Kaplan, N.O. (1955) Methods Enzymol. 2, 660-663. 2 Swislocki, N.I., Kalish, M.I., Chasalow, F.I. and Kaplan, N.O. (1967) J. Biol. Chem. 242, 1089-1094. 3 Srivastava. S.K., Maini, S.B. and Ramakrishnan, C.V. (1969) Phytochemistry 8, 1147-1154. 4 De Wolf, M.J.S., Van Dessel, G.A.F., Lagrou, A.R., Hilderson, H.J.J. and Dierick, W.S.H. (1985) Biochem. J. 226, 415-427. 5 Pekala, P.H. and Anderson, B.M. (1978) J. Biol. Chem. 253, 7453-7459. 6 Friedemann, H. and Rapopot, S.M. (1974) in Cellular and Molecular Biology of Erythrocytes (Yoshikawa, H. and Rapoport, S.M., eds.), pp. 181-259, University Park Press, Baltimore. 7 Alivisators, S.G.A. and Denstedt, O.F. (1951) Science 114, 281283.
8 Kim, U.-H., Rockwood S.F., Kim, H.R. and Daynes, R.A. (1988) Biochim. Biophys. Acta 965, 76-81. 9 Kim, V.-H., Kim, M.K., Kim, J.-S., Han, M.-K., Park, B.-H. and Kim, H.-R. (1993) Arch. Biochem. Biophys., in press. 10 Ng, W.G., Donnell, G.N. and Bergren, W.R. (1968) Nature 217, 64-65. 11 Dixon, M. and Webb, E.C. (1979) in Enzymes, 3rd Edn., pp. 465-518, Longman, London. 12 Ikezawa, H. and Taguchi, R. (1981) Methods Enzymol. 7l, 731741. 13 Richter, C., Winterhalter, K.H., Baumhuter, S., Lotscher, H.-R. and Moser, B. (1983) Proc. Natl. Acad. ScL USA 80, 3188-3192. 14 Dagnino, L., Bennet, L.L. and Paterson, A.R.P. (1991) J. Biol. Chem. 266, 6308-6311. 15 Moss, J., Manganiello, V.C. and Vaughan, M. (1976) Proc. Natl. Acad. Sci. USA 73, 4424-4427. 16 Suidan, H.S., Murrell, R.D. and Tolkovsky, A.M. (1991) Cell Regul. 2, 13-25. 17 May, J.M. (1985) J. Biol. Chem. 260, 462-467. 18 Lan, S.J. and Henderson, L.M. (1968) J. Biol. Chem. 243, 33883394. 19 Miro, A., Coatas, M.J., Garcia-Diaz, M. Hernandez, M.T. and Cameselle, J.C. (1989) FEBS Lett. 244, 123-126.