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Characterization of Mg2+- and (Ca2+ + Mg2+)-ATPase activity in adipocyte endoplasmic reticulum
ARCHIVES OF BIOCHEMISTRY Vol. 199, No. 1, January,
Characterization
BETTY Division
AND BIOPHYSICS pp. 92-102, 1980
of Mg2+- and (Ca2+ + Mg2+)-ATPase Endoplasmic Reticulum1 L.
BLACK,z
of Laboratory School
JAY
Medicine,
of Medicine,
M.
MCDONALD,3
Departments and Barnes Received
AND
qf Pathology Hospital, July
LEONARD
Activity
in Adipocyte
JARETT
and Medicine, Washington St. Louis, Missouri 63110
IJniversity
16, 1979
The presence of an energy-dependent calcium uptake system in adipocyte endoplasmic reticulum (D. E. Bruns, J. M. McDonald, and L. Jarett, 1976, J. Biol. Chem. 251, ‘71917197) suggested that this organelle might possess a calcium-stimulated transport ATPase. This report describes two types of ATPase activity in isolated microsomal vesicles: a nonspecific, divalent cation-stimulated ATPase (Mg*+-ATPase) of high specific activity, and a specific, calcium-dependent ATPase (Ca ‘+ + Mg*+-ATPase) of relatively low activity. Mg*+-ATPase activity was present in preparations of mitochondria and plasma membranes as well as microsomes, whereas the (Ca*+ + Mg’+)-ATPase activity appeared to be localized in the endoplasmic reticulum component of the microsomal fraction. Characterization of microsomal Mg2+-ATPase activity revealed apparent K, values of 115 PM for ATP, 333 yM for magnesium, and 200 PM for calcium. Maximum Mg*+-ATPase activity was obtained with no added calcium and 1 mM magnesium. Potassium was found to inhibit MgZ+-ATPase activity at concentrations greater than 100 mM. The energy of activation was calculated from Arrhenius plots to be 8.6 kcal/mol. Maximum activity of microsomal (Ca2+ + Mg2+)-ATPase was 13.7 nmol “*P/mgimin, which represented only 7% of the total ATPase activity. The enzyme was partially purified by treatment of the microsomes with 0.09% deoxycholic acid in 0.15 M KC1 which increased the specific activity to 37.7 nmol “‘Pimglmin. Characterization of (Ca2+ + Mg2+)-ATPase activity in this preparation revealed a biphasic dependence on ATP with a Hill coefficient of 0.80. The apparent K,s for magnesium and calcium were 125 and 0.6-1.2 FM, respectively. (Ca ?+ + Mg*+)-ATPase activity was stimulated by potassium with an apparent K, of 10 mM and maximum activity reached at 100 mM potassium. The energy of activation was 21.5 kcal/mol. The kinetics and ionic requirements of (Ca*+ + Mg2+)-ATPase are similar to those of the (Ca 2+ + Mg*+)-ATPase in sarcoplasmic reticulum. These results suggest that the (Ca*+ + Mg2+)-ATPase of adipocyte endoplasmic reticulum functions as a calcium transport enzyme.
Control of intracellular calcium levels appears to be crucial for the maintenance of normal cell function (1). Regulation of cytoplasmic calcium has been most extensively studied in muscle where the calcium concentration plays the major role in the contraction-relaxation cycle. In skeletal (2, 3),
cardiac (4), and smooth muscle (5, 6), cytoplasmic calcium concentrations are controlled primarily by the calcium transport system of sarcoplasmic reticulum. A similar calcium transport system has been described in plasma membranes of erythrocytes where calcium is extruded from the cells against a concentration gradient (7). Calcium transport in these systems is mediated by a calcium-stimulated Mg2+-ATPase with one to two calcium ions transported per ATP hydrolyzed, and the K, for calcium is in the micromolar range, consistent with estimated cytoplasmic concentrations of 10m7-
1 Presented in part at the 69th Annual Meeting of the American Society of Biological Chemists and supported by United States Public Health Service Research Grants AM 11892, AM 20097, AM 20579, and postdoctoral fellowship IF32 AM05666. 2 Present address: Department of Zoology, North Carolina State University, Raleigh, N. C. 27650. 3 To whom reprint requests should be sent. Telephone No. 314-454-2169. 0003.9861/80/010092-11$02.00/O Copyright 0 1980 by Academic Press, Inc. A11 rights of reproduction in any form reserved.
lo-6
M (1).
Energy-dependent 92
calcium transport
has
ATPase
ACTIVITY
OF ADIPOCYTE
recently been described in microsomal preparations from hepatocytes (8, 9>, fibroblasts (10, ll), kidney cells (12), brain tissue (13), and adipocytes (14). Although calcium-stimulated ATPase activity has been detected in some of these preparations, such studies have been complicated by the lability and low relative activity of the enzyme, by the presence of multiple ATPases, and by the heterogeneity of the microsomal preparations (13, 15, 16). Furthermore a relationship between enzyme activity and calcium transport has not been established. If these calcium transport systems are analogous to the well-characterized red cell and sarcoplasmic reticulum calcium pumps, then there must exist a specific calcium-stimulated ATPase which mediates the transport of calcium. The present study is part of a series of investigations designed to elucidate the mechanism and hormonal regulation of calcium transport in adipocyte endoplasmic reticulum. This paper characterizes the ATPase activity associated with this organelle and reveals the presence of two calciumstimulated enzymes: a nonspecific, divalent cation-stimulated ATPase (Mg2+-ATPase) of high specific activity and a specific, calcium-dependent ATPase ((Ca2+ + Mg’+)ATPase) of relatively low activity. Analysis of the kinetics and ionic requirements of both enzymes indicates that the Mg’+ATPase lacks the characteristics of a calcium transport ATPase, whereas all properties of the (Ca + Mg2+)-ATPase are strikingly similar to those of the transport (Ca + Mg2+)-ATPase of sarcoplasmic reticulum. EXPERIMENTAL
PROCEDURES
Materials. Male Wistar rats of 120 g were purchased from National Laboratory Animal Company (O’Fallon, MO.). Collagenase (Type I) from Clostridium histolyticum, bovine serum albumin (fraction V), and ATP (disodium salt) were purchased from Sigma Chemical Company (St. Louis, MO.). [Y-~~P]ATP was obtained from AmershamiSearle (Arlington Heights, Ill.), or was synthesized according to the method of Beutler and Guinto (17) from s2P, purchased from New England Nuclear (Boston, Mass.). All other reagents were of reagent grade quality and were purchased from standard sources. All reagents were prepared with water deionized by a double-chambered mixed bed ion
ENDOPLASMIC
RETICULUM
93
exchange resin system (Culligan, Inc., Northbrook, Ill.) which was filtered prior to use with a 0.25~pm pore size filter (Ultipor, PTM Corp., Cortland, N. Y.). Adipocyte fractionation. Adipocytes were isolated from rat epididymal fat pads as previously described (18). A modified method of obtaining microsomes was utilized since microsomes prepared by standard procedures did not transport calcium (14). Isolated adipocytes were homogenized in 0.25 M sucrose with 10 mM Tris-HCl, pH 7.4 at 4°C (Tris-sucrose). The homogenate was centrifuged at 20,OOOg for 15 min. ATP (0.5 mM) and MgCl, (0.25 mM) were added to the resulting supernatant (S,) which was centrifuged at 160,OOOg for 10 min to obtain a microsomal pellet. The pellet was resuspended in Tris-sucrose by brief homogenization. Previous characterization of microsomes isolated by a 60.min centrifugation of S, indicated that the preparation was highly enriched in endoplasmic reticulum (19. 20). The rapidly obtained microsomal preparation was found to be equally enriched in endoplasmic reticulum by assays of cytochrome c reductase activity which indicated an B.&fold enhancement of activity in the microsomes as compared to values in the homogenate. Based on the activity of this marker enzyme, 30% of the endoplasmic reticulum present in the homogenate was recovered in the rapidly prepared microsomes, similar to a 35% recovery in microsomes prepared by the standard 60-min centrifugation (N = 5). Microsomes were either immediately treated with DOCj or rapidly frozen in small aliquots with dry ice/ethanol and stored at -70°C until used. Mitochondrial and plasma membrane fractions were obtained as previously described (18. 20), except that EDTA was omitted throughout the procedure (14). DOG’-treated microsornes. Freshly isolated microsomes at a concentration of l-2 mg protein/ml were added to an equal volume of DOC in KC1 giving a final concentration of 0.09% DOC-0.15 M KCl. The DOCmicrosomes solution was stirred for 5 min at 24°C. diluted 5-10x with Tris-sucrose plus 0.5 mM ATP and 0.25 mM MgCl,, and centrifuged at 160,000~ for 10 min. The resulting pellet was resuspended m Trissucrose, rapidly frozen, and stored at ~ 70°C until used, ATPnae assays. ATP hydrolysis by microsomal, mitochondrial, and plasma membrane fractions was measured by an isotopic assay method (21). Standard incubations were performed in duplicate in conical glass tubes with constant shaking at 37°C. The Assam was initiated by addition of lo-20 wg of protein to a standard incubation medium containing 50 mM TrisHCl, pH 6.8 at 37”C, 100 mM KCl, 1 mM MgCl,, 5 rnM K+-oxalate or Tris-oxalate, 0.5 mM Na,ATP with tracer [y-“*P]ATP, 0.2 mM EGTA, and 0.18 mifl CaCl, 4 Abbreviations used: DOC. deoxycholic acid; EGTA, ethylene glycol bis (P-aminoethyl ether) N,N ‘-tetraacetic acid; PCMBS, p-chloromercuribenzene sulfonate; S, supernatant.
94
BLACK,
MCDONALD, TABLE
ATPase Subcellular fraction Microsomes
Mitochondria Plasma membranes
ACTIVITYINADIPOCYTE
Assay Standard Standard Standard Standard Standard
medium medium medium medium medium
I
RESULTS
Subcellular
The Mg2+-ATPase activity (207 2 18.9 nmol 32P/mg/min) of the rapidly obtained microsomal fraction (Table I) was comparable to that found in the original adipocyte microsomal preparation of Jarett and McKee1 (24). Addition of 10 FM free calcium to the standard incubation medium increased total ATPase activity by approximately ‘7%, yielding an additional 13.7 nmol
FRACTIONLP
Mg*+-ATPase (nmol 32P/mg/min)
(12)* plus 50 PM PCMBS (5) minus potassium (5) (8) (10)
(approximately 10 PM free calcium) in a total of 500 ~1. The oxalate was included to make the buffer identical to that used in calcium transport studies (22). The reaction was terminated after 2-20 min by addition of 1% sodium lauryl sulfate. The hydrolyzed 32P was extracted in xylene:isobutanol and quantitated by liquid scintillation counting in toluene:omnifluor: ethanol. In all assays, appropriate control tubes without protein were included to correct for nonenzymatic ATP hydrolysis. To measure (Ca2+ + Mg2+)ATPase activity, assays were performed with and without calcium. Values obtained in the absence of calcium (Mg*+-ATPase activity) were subtracted from the total ATPase activity in the presence of calcium to give a value for (Ca*+ + Mg2+)-ATPase activity. Other assays. Protein was measured by the method of Lowry et al. (23) using bovine serum albumin as a standard. NADH-cytochrome c reductase and succinic dehydrogenase activity were measured as previously described (20). Calculations. Free calcium concentrations in the incubation buffer were calculated from the EGTAI calcium ratios as described by Schatzmann (7). Data are expressed as means with standard errors given in cases where the number of preparations is greater than 4.
in Adipocyte
JARETT
SUBCELLULAR
conditions
n Mg*+-ATPase and (CaZ+ + MgZ+)-ATPase activities described under Experimental Procedures. b Values represent the mean (-tSEM) of the number
ATPase Activity Fractions
AND
were
207 183 260 613 1094 assayed
of preparations
+ k +? *
(Ca*+ + Mg2+)-ATPase (nmol 3*P/mg/min)
18.9 20.0 20.0 92.0 149
13.7 -0.4 0.6 13.9 -1.6
in the standard listed
incubation
5 t t + 2
1.8 0.2 3.6 12.5 14.5 medium
as
in parentheses.
32P/mg/min. This additional ATPase activity was specific for calcium ((Ca2+ + Mg2+)ATPase) since increasing the magnesium concentration from 1 mM to 2 mM in the absence of calcium did not increase the total ATPase activity (data not shown). Addition of 50 j..&M PCMBS to the standard incubation buffer or omission of potassium slightly altered Mg2+-ATPase activity to 88 and 126% of control values, respectively (Table I). In contrast, (Ca2+ + Mg2+)-ATPase activity was virtually eliminated under these conditions. ATPase activity was assayed in highly enriched mitochondrial and plasma membrane preparations to ascertain whether the Mg2+-ATPase or (Ca2+ + Mg2+)-ATPase activity in the microsomal fraction could be accounted for by small amounts of mitochondrial or plasma membrane contaminants of the microsomal preparations. Mitochondrial Mg2+-ATPase activity was three-fold higher than that of the microsomes (Table I). The addition of 10 PM free calcium stimulated mitochondrial ATPase by 13.9 nmol 32P/mg/min. The maximum mitochondrial contamination of the microsomal fraction is 6%, based on assays of succinic dehydrogenase activity which gave values of 116.8 + 22 and 7.2 k 1.4 nmol succinate oxidizedlmglmin (N = 8) for mitochondrial and microsomal fractions, respectively. Thus mitochondrial (Ca”+ + Mg2+)-ATPase could account for a maximum of 5.8% (approximately 0.8 nmol/mg/ min) of the microsomal (Ca2+ + Mg2+)ATPase activity. The Mg2+-ATPase specific
ATPase
ACTIVITY
OF ADIPOCYTE
FIG. 1. Dependence of Mg2+-ATPase activity on concentration of ATP. Mg2+-ATPase activity was determined using an assay buffer containing 50 mM Tris-HCl, pH 6.8 at 37”C, 1 mM MgCl,, and 0.05-1.0 mM ATP. Each point represents the mean value obtained from four microsomal preparations, each assayed in duplicate. The inset is a double-reciprocal plot of the data.
activity of plasma membranes was five-fold higher than that of the microsomal fraction, but calcium-stimulated ATPase activity was not detectable under the standard incubation conditions. Therefore, plasma membrane contaminants could not contribute to the (Ca2+ + Mg2+)-ATPase activity in the microsomal fraction. On the other hand, because of the extremely high specific activity of Mg2+-ATPase in the mitochondrial and plasma membrane fractions, even a small amount of contamination of the microsomal preparation by these organelles could account for a substantial portion of the microsomal Mg2+-ATPase activity.
ENDOPLASMIC
RETICULUM
95
concentration is shown in Fig. 1. Analysis by double-reciprocal plots yields an apparent K, of 115 pM. Mg2+ATPase activity was dependent upon the presence of divalent cations. In the absence of any added divalent cations, ATPase activity was approximately 2% of that found under optimal conditions (data not shown). This residual activity could be eliminated by the addition of 150 pM EDTA and thus was presumed to reflect the presence of divalent cations in the microsomal membranes or incubation medium. ATP hydrolysis as a function of either calcium or magnesium concentration was dose dependent and saturable (Fig. 2). Analysis of doublereciprocal plots yielded an apparent K, for magnesium of 333 pM and a V of 556 nmol “ZPlmglmin. The addition of 200 pM EGTA to the incubation buffer did not alter these parameters, indicating that magnesium is effective in the complete absence of calcium. Calcium proved to be a fair substitute for magnesium. In the absence of added magnesium, the apparent K, for calcium was 200 PM and V was 370 nmol 32Plmglmin. As nonsaturating concentrations of magnesium were increased from 10 pM to ZOO pM, stimulation of activity by 50 pM calcium was reduced from 400 to 21% (data not shown), indicating that calcium stimulation was inhibited by magnesium. No calcium stimulation occurred at magnesium concentrations above 500 PM. Thus, high rates of ATP hydrolysis could be supported by
Characterization of Mg2+-ATPase Activity in Microsomes
The Mg2+-ATPase activity of the microsomal fraction was further characterized in an incubation buffer containing 50 mM TrisHCl, pH 6.8 at 37”C, 0.5 mM ATP, and 1 mM MgCl,. Potassium was omitted from the buffer to eliminate (Ca2+ + Mg2+)-ATPase activity (Table I). Omission of EGTA under these assay conditions had no significant effect on Mg2+-ATPase activity. The Mg2+ATPase activity was linear for 2-3 min at protein concentrations between 10 and 40 Fg/ml (data not shown). ATP, magnesium, and calcium dependency of Mg2+-ATPase activity. Mg2+-
ATPase
activity
as a function
of ATP
FIG. 2. Dependence of Mg2+-ATPase activity on concentrations of magnesium and calcium. M$+ATPase activity was determined using an assay buffer containing 50 mM Tris-HCl, pH 6.8 at 37”C, 0.5 mM ATP, and 0.05-2.0 mM magnesium (e) or calcium (0). Each point represents the mean value obtained from four microsomal preparations, each assayed in duplicate.
96
BLACK,
MCDONALD,
AND
JARETT
(Table I). At concentrations of 150 and 200 mM, Mg2+-ATPase activity was inhibited by 22 and 33%, respectively (data not shown). Temperature dependency of Mg2+-ATPase activity. The Mg2+-ATPase activity in-
creased with temperature between 15 and 35°C. Analysis of the data by Arrhenius plots revealed a Q1,, of 1.64 and an activation energy of 8.65 kcal/mol (Fig. 3). Characterization of the (Ca2+ + Mg2+)ATPase Activity FIG. 3. Arrhenius plots of ATPase activity. Mg*+ATPase activity (0) in microsomal preparations was determined at temperatures of 15-35°C using an incubation buffer containing 50 mM Tris-HCI, adjusted to pH 6.8 at each temperature, 1 mM MgCl,, and 0.5 mM ATP. (Ca2+ + My+)-ATPase activity (0) in DOC-treated microsomal preparations was determined at temperatures of ZO-40°C using the standard incubation buffer with the pH adjusted to 6.8 at each temperature. Each point represents the mean value obtained from three preparations, each assayed in duplicate.
either magnesium or calcium, but magnesium in the absence of calcium elicited maximum ATPase activity. Effect activity.
of potassium
on Mg2+-ATPase
The Mg2+-ATPase activity was slightly inhibited (20%) by 100 mM potassium, in contrast to (Ca’+ + Mg2+)-ATPase which required this cation for full activity TABLE COMPARISON
OF ATPase
ACTIVITY
ATPase after
microsomes” microsomes’
activity recovered DOC treatmentd
Optimal
conditions
for DOC treatment.
The effect of DOC treatment (see Experimental Procedures) on the ATPase activity of subsequently resuspended vesicles was tested for DOC concentrations between 0.06 and 0.12%. The Mg2+-ATPase activity was lost much more rapidly than (Ca2+ + Mg2+)-ATPase activity at DOC concentrations between 0.06 and 0.09%. The (Ca2+ + Mg2+)-ATPase to total ATPase ratio was highest with 0.09% DOC. Lowering the temperature during DOG treatment II
IN UNTREATED
Mg*+-ATPase (nmol 3ZP/mg/min) Untreated DOC-treated
It was necessary to reduce Mg2+-ATPase activity relative to (Ca2+ + Mg2+)-ATPase activity to further characterize the microsomal (Ca2+ + Mg*+)-ATPase, since the (Ca2+ + Mg2+)-ATPase activity represented only 7% of the total ATPase activity present. This was accomplished by a brief treatment of the microsomal vesicles with DOC.
vs DOGTREATED
(Ca’+ + Mg’+)ATPase (nmol “*P/mg/min)
207 t 18.9 60.9 ? 6.5
13.7 -t 1.8 37.7 -+ 2.1
7.8%
72.6%
MICROSOMES” (Ca2’
+ Mg*+)-ATPasei total ATPase (%I 6.7 t 1.0 40.9 2 1.0
a Mg*+-ATPase and (Ca2+ + Mg2+)-ATPase activities were assayed in the standard incubation medium as described under Experimental Procedures. * Values represent the mean (t SEM) of 12 preparations. c Values represent the mean (2 SEM) of 27 preparations. d The protein recovered in the DOC-treated microsomes was 26.4 + 1.3% (n = 12) of the protein in the original, untreated microsomal preparation.
ATPase
ACTIVITY
OF ADIPOCYTE
ENDOPLASMIC
RETICULUM
97
ATP dependency of (Ca2+ + Mg2+)ATPase activity. The (Ca’+ + Me+)-ATPase
FIG. 4. Dependence of (Ca” + Mg2+)-ATPase activity on concentration of ATP. (Ca*’ + Mg*-)ATPase activity was determined using the standard incubation buffer with 0.01-1.0 mM ATP. Each point represents the mean value of three DOC-treated microsomal preparations, each assayed in duplicate. The inset is a double-reciprocal plot of the data.
from 24°C to 4°C elicited a 10% increase in (Ca2+ + Mg2+)-ATPase activity, but Mg2+ATPase specific activity increased 6000/c, giving a low (Ca2+ + Mg’+)-ATPase to total ATPase ratio. The (Ca2+ + Mg2+)ATPase activity in preparations treated with 0.09% DOC at 24°C remained constant during 3-6 min of treatment, but dropped after longer exposure to the detergent. Therefore, subsequent studies of (Ca2+ + Me+)-ATPase utilized microsomes treated with 0.09% DOC at 24°C for 5 min. The specific activity of (Ca’+ + Mg2+)ATPase of microsomes prepared in this manner was almost threefold higher than that in untreated microsomes (Table II). The (Ca’+ + Mg’+)-ATPase as a percentage of total ATPase activity was increased from 7% to 41%. Recovery studies indicated that 75% of the original (Ca2+ + Mg2+)-ATPase activity present in the untreated microsomes was recovered, as compared to only 7-8% of the Mg*+-ATPase activity. Stability, linearity, and protein ency of (Ca2+ + Mg2i)-ATPase
activity was dose dependent and saturable as a function of ATP concentration (Fig. 4). Analysis of the data by double-reciprocal plots yielded a broken line with two apparent components. An apparent K, of 36 PM was found in the ATP concentration range of lo-50 PM, whereas the apparent K,, at higher concentrations was 118 FM. A Hill plot of the data yielded a straight line with a Hill coefficient of 0.80. Calcium and magnesium dependency of (Ca2+ + Mg2+)-ATPase activity. Calcium-
stimulated ATP hydrolysis in DOG-treated microsomes required the presence of both calcium and magnesium, in contrast to the requirements of the Mg’+-ATPase activity. In the presence of 1 mM magnesium, the addition of as little as 0.2 PM free calcium stimulated ATPase activity by lo-15%. Maximum stimulation by calcium occurred at 3-5 FM (Fig. 5). Double reciprocal plots revealed a K, for free calcium of 0.6 PM and V of 30.3 nmol ~“P/mg/min. The free calcium concentrations used in these calculations were obtained from the calcium/EGTA ratios in the incubation buffer as described by Schatzman (7). A similar K, for free calcium of 1.2 PM is calculated by estimating free calcium concentrations by the method of Katz (25) which includes a correction for the calcium complexed by ATP. The magnesium dependency of (Ca” I
.’
rl
dependactivity.
The (Ca2+ + Mg2+)-ATPase activity in DOC-treated microsomes was stable at 4°C for 25-30 min, but decreased to 82% by 60 min. Activity remained constant for at least 6 weeks when the preparation was rapidly frozen and stored at -70°C. In the standard incubation buffer at 37”C, (Ca2+ + Mg2+)-ATPase activity was linear with time for 20-40 min using protein concentrations from lo-40 pgiml (data not shown).
FIG. 5. Dependence tivity on concentration ATPase activity was incubation buffer with point represents the from 13 DOC-treated assayed in duplicate. plot of the data.
of (Ca2+ + Mg*+)-ATPase acof calcium. (Ca*+ + Mg2’)determined using the standard 0.5-10.0 pM free calcium. Each mean value (?SE) obtained microsomal preparations, each The inset is a double-reciprocal
98
BLACK,
MCDONALD.
+ Mg2+)-ATPase activity is illustrated in Fig. 6. The low activity present in the absence of added magnesium presumably reflects the presence of contaminating magnesium ions and/or stimulation by calcium of the low Mg2+-ATPase activity which remains in the DOC-treated microsomes. Using the standard ATP concentration of 0.5 mM, maximum (Ca2+ + Mg2+)-ATPase activity occurred at 1 mM magnesium. Higher magnesium concentrations were inhibitory; activity was reduced by 18% at 5 mM magnesium. These data yield an apparent K, for magnesium of 125 PM and a V of 47 nmol 32P/mg/min. Effect of potassium on (Ca’+ + Mg*+)ATPase activity. Omission of potassium
from the standard incubation buffer resulted in a 78% reduction of the (Ca2+ + Mg2+)-ATPase activity in DOC-treated microsomes, analogous to the inhibition of calcium stimulation in untreated microsomal preparations (Table I) and in contrast to the effect of potassium on Mg2+-ATPase activity. The (Ca 2+ + Mg2+)-ATPase activity was a function of potassium concentration, with maximum activity reached at 100 mM potassium (Fig. 7). Analysis of the data by double-reciprocal plots yields an apparent K, of 10 mM and a V of 43.5 nmol 32P/mg/min. Temperature dependency of + Mg2+)-ATPase activity. The
(Ca2+
(Ca2+
500
I I
2 MAGNESIUM
3 CONCENTRATON
4 (mid)
I 5
FIG. 6. Dependence of (Ca2+ + Mg2+)-ATPase activity on concentration of magnesium. @a*+ + Mg2+)ATPase activity was determined using the standard incubation buffer with 0.5-5.0 ITIM magnesium. Each point represents the mean value obtained from three DOC-treated microsomal preparations, each assayed in duplicate. The inset is a double-reciprocal plot of the data.
AND JARETT
I 50 POTASSIUM
I 103
4
150 CONCENTRATION
I MO [mM)
FIG. 7. Dependence of (CaZ+ + MgZ+)-ATPase activity on concentrations of potassium. (Ca*+ + MgZ+)ATPase activity was determined using the standard incubation buffer with no added potassium or 5-200 !IIM potassium. Each point represents the mean value obtained from three DOC-treated microsomal preparations, each assayed in duplicate. The inset is a doublereciprocal plot of the data.
+ Mg2f)-ATPase activity was highly dependent upon temperature between 20 and 40°C. Analysis of the data by Arrhenius plots (Fig. 3) yields a Q10 of 3.3 and an activation energy of 21.5 kcal/mol. These values contrast to the respective values for Mg2+-ATPase of 1.6 and 8.6 kcal/mol. DISCUSSION
Adipocyte microsomes contain two types of ATPase activity which require the presence of divalent cations. The predominant ATPase activity of the preparation (Mg2+ATPase) can be stimulated by either calcium or magnesium in the absence of the other cation. The Mg2+-ATPase has an absolute requirement for divalent cations, since EDTA completely inhibits ATP hydrolysis. This divalent cation requirement is relatively nonspecific since calcium at saturating concentrations gives 70% as much activity as optimal concentrations of magnesium (Fig. 2). The fact that 50 PM calcium elicits progressively less ATP hydrolysis as magnesium is increased to saturating levels suggests that calcium and magnesium act at the same site. Presumably, either cation plus ATP forms a complex which serves as the true substrate of the reaction as proposed for Ca2+-, Mg2+ATPases of other cell types (26).
ATPase
ACTIVITY
OF
ADIPOCYTE
The microsomal preparation contains a second ATPase activity (Ca2+ + Mg*+)ATPase which is distinguished from the Mg2+ATPase by an absolute dependence on calcium. This dependence is revealed as a 7% stimulation of ATP hydrolysis by 10 pM calcium under conditions where Mg’+ATPase is saturated with magnesium. The concept that this calcium-stimulated activity represents a second ATPase is supported by the opposite effect of addition of PCMBS or omission of potassium on Mg2+ATPase and calcium-stimulated Mg2+-ATPase activity (Table I). Since the endoplasmic reticulum of these microsomal preparations is contaminated with small amounts of mitochondria and plasma membrane, it was necessary to determine the extent to which the latter organelles contribute to microsomal ATPase activity. Mitochondria represent a minor contaminant (maximum of 6%) of the preparation and thus could not contribute significantly to microsomal (Ca2+ + Mg2+)ATPase activity (Table I). Consistent with this conclusion, a concentration of sodium azide (10 mM) which inhibited mitochondrial ATPase activity by 73% had no effect on (Ca2+ + Mg2+)-ATPase activity in DOCtreated microsomes (unpublished observation). Highly enriched plasma membranes contain no detectable (Ca2+ + Mg2+)-ATPase activity (Table I) indicating that plasma membrane contaminants do not contribute to the (Ca2+ + Mg2+)-ATPase activity of adipocyte microsomes. The high Mg2+ATPase activity of plasma membranes (approximately 1100 nmol 32P/mg/min.) could, however, account for part of the microsomal Mg2+-ATPase activity. If Mg2+ATPase activity is a combination of endoplasmic reticulum and plasma membrane components, the Mg2+-ATPase of the two organelles must have similar properties since kinetic analysis and Arrhenius plots indicate the presence of only one enzyme. Previous studies of adipocyte ATPase have also reported the presence of high Mg2+ATPase activity in both endoplasmic reticulum and plasma membrane fractions (24, 27). Therefore, microsomal Mg2+-ATPase activity may be derived from both endoplasmic reticulum and plasma membrane
ENDOPLASMIC
RETICULUM
99
elements, whereas the (Ca2+ + Mg*+)-ATPase activity is localized in the endoplasmic reticulum. The Mg*+-ATPase of adipocyte microsomes resembles a magnesium- or calciumstimulated ATPase activity common to many plasma membrane and endoplasmic reticulum preparations. Such a nonspecific, divalent cation ATPase has been described in plasma membrane and endoplasmic reticulum from a variety of tissues, including skeletal, cardiac, and smooth muscle (28-32), liver (33, 34), kidney (35), placenta (26), and brain (13). The Mg2+- or Ca2+ATPases of these tissues are similar in that they do not require sodium or potassium, but can be stimulated by either magnesium or calcium with maximum activity reached at millimolar concentrations. Reported K,,, values range from 0.3-1.5 mM for magnesium or calcium, and from 0.05-0.3 mM for ATP. The Mg2+-ATPase of adipocyte microsomes shares these properties with no apparent monovalent cation requirement, maximum stimulation at 1 IrIM magnesium or calcium, and K,s of 0.33 mM for magnesium, 0.20 mM for calcium, and 0.11 lllM for ATP. The cellular function of these nonspecific Mg2+- or Ca2+-ATPases is not known, but speculations have included a role in magnesium or calcium transport (26, 35). The Mg2+-ATPase of adipocyte endoplasmic reticulum does not appear to function as a calcium transport enzyme, since the calcium transport system of this organelle requires the presence of both magnesium and calcium and has a much lower K, for calcium than does Mg2+ATPase activity (14, 22). The (Ca2+ + Mg2+)-ATPase of adipocyte endoplasmic reticulum represents a small percentage of the total ATPase present, similar to the calcium-stimulated Mg2+ATPase activity of other noncontractile cell microsomes (13, 15, 36). Treatment of adipocyte microsomes with DOC increases the relative and specific activity of (Ca’+ + Mg*+)-ATPase which allows kinetic analysis of the enzyme. A similar partial purification with low concentrations of DOC has been described for (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum (37) and brain microsomes (13). Treatment with deter-
100
BLACK,
MCDONALD,
gents has been found to alter the properties of some membrane-bound ATPases (13, 38, 39). Although detailed characterization of untreated adipocyte microsomes is precluded by the low (Ca2+ + Mg*+)-ATPase to total ATPase ratio, the effect of omitting potassium (Table I, Fig. 7) or adding PCMBS (Table I, unpublished observation) is similar in both untreated and DOCtreated preparations, suggesting that no major alteration of enzyme properties has occurred. Analysis of the kinetics and ionic requirements of (Ca2+ + Mg2+)-ATPase in DOCtreated microsomes reveals a striking similarity to the (Ca2+ + Mg*+)-ATPase of sarcoplasmic reticulum. Both enzymes require calcium, magnesium, and potassium for full activity. The K, for calcium of 0.61.2 PM for microsomal (Ca’+ + Mg*+)ATPase (Fig. 4) is comparable to that of 0.1-1.0 pM reported for sarcoplasmic reticulum (3). In the presence of 0.5 mM ATP, maximal activity with magnesium is reached between 0.5 and 1.0 mM magnesium (Fig. 6), suggesting that Mg2+-ATP is the true substrate of the enzyme as is the case for sarcoplasmic reticulum (Ca*+ + Mg*+)ATPase (3, 40). The inhibition observed with excess magnesium is also characteristic of the sarcoplasmic reticulum enzyme (40, 41). Both the apparent K, for potassium (10 mM) and the concentration of potassium required for maximal activity (100 mM) are similar to the corresponding values of lo-20 and 50-100 mM reported for (Ca*+ + Mg*+)-ATPase of sarcoplasmic reticulum (42, 43). Moreover, microsomal and sarcoplasmic reticulum (Ca*+ + Mg2+)ATPase shows a similar dependence upon temperature, with activation energies of 21.5 and 17.9-26.2 kcalimol, respectively (28, 44, 45). Analysis of the substrate dependency of (Ca*+ + Mg*+)-ATPase in DOC-treated microsomes indicates two apparent K,,s for ATP (Fig. 4), again similar to the (Ca2+ + Mg*+)-ATPase of sarcoplasmic reticulum (28,40,46,47). The biphasic double-reciproco1 plot of velocity vs ATP concentration cannot be explained by a nonlinear variation of the complexed Mg2+-ATP since 95- 100% of the total ATP is in the form of Mg2+-ATP between ATP concentrations of 0.01-0.5
AND
JARETT
as calculated by the method of Katz (25). Use of the Hill plot to analyze substrate kinetics gives a Hill coefficient of 0.8 for ATP. This suggests that the nonlinear double-reciprocal plot could be due to negative cooperativity as recently suggested for Mg*+-ATP interactions with the (Ca*+ + Mg2+)-ATPase of sarcoplasmic reticulum (47). The (Ca2+ + Mg2+)-ATPase of adipocyte endoplasmic reticulum probably functions as a calcium transport protein. This concept is supported by studies of passive calcium binding to adipocyte endoplasmic reticulum which suggests that high affinity binding sites represent binding of calcium to the transport sites of the membrane (48). In sarcoplasmic reticulum, calcium binding to sites with corresponding high affinity has been identified as calcium binding to the (Ca”+ + Mg*+)-ATPase, and represents the first step in energy-dependent calcium transport (3). The kinetics and ionic requirements of microsomal (Ca2+ + Mg*+)-ATPase are comparable to those of the calcium transport system in adipocyte endoplasmic reticulum (14, 22). Furthermore, a comparison of calcium transport rates (22) to (Ca” + Mg2+)-ATPase activity in untreated microsomes (Table I) indicates that one or two calcium molecules are transported per ATP hydrolyzed, similar to the 2:l ratio characteristic of the sarcoplasmic reticulum calcium transport system (3). The (Ca2+ + Mg2+)-ATPase of adipocyte endoplasmic reticulum is qualitatively similar to (Ca2+ + MgZ+)-ATPase of sarcoplasmic reticulum, but the specific activity of the enzymes differs by one to two orders of magnitude (3, 44). The lower specific activity of endoplasmic reticulum (Ca2+ + Mg2+)ATPase might reflect a lower concentration of (Ca*+ + Mg*+)-ATPase proteins associated with the membrane. The (Ca2+ + Mg2+)-ATPase in sarcoplasmic reticulum membranes can be visualized as intramembranous particles by freeze-fracture electron microscopic technique (3). Freezefracture studies of adipocyte microsomal vesicles have revealed an intramembranous particle density l- 10% of the value reported for sarcoplasmic reticulum (49), consistent with the concept that endoplasmic reticulum contains a lower density of mM,
ATPase ACTIVITY
OF ADIPOCYTE
calcium transport proteins than sarcoplasmic reticulum. Such a quantitative difference might be anticipated since the sarcoplasmic reticulum membrane is highly specialized for calcium transport, whereas the endoplasmic reticulum of noncontractile cells has a wide range of functions. The demonstation of (Ca2+ + Mg*+)ATPase activity in adipocyte endoplasmic reticulum strengthens the concept that this organelle plays a role in controlling the distribution of intracellular calcium. The cellular mechanism(s) which control (Ca*+ + Mg*+)-ATPase activity are obscure, but might include phosphorylationldephosphorylation of the endoplasmic reticulum membrane, analogous to regulation of the calcium transport system in sarcoplasmic reticulum (50-52). An investigation of such potential regulatory systems could provide insight into the mechanism by which cytoplasmic calcium concentrations are altered and calcium homeostasis is maintained in noncontractile cells. REFERENCES 1. RASMUSSEN, H. (1970) Science 170, 404-412. 2. WEBER, A. M., HERZIG, R., AND REISS, I. (1966) Biochem. 2. 345, 329-369. 3. MACLENNAN, D. H., AND HOLLAND, D. C. (1975) Annu. Rev. Biophys. Bioenerg. 4, 377-402. 4. TADA, M., KIRCHBERGER, M. A., REPKE, D. I., AND KATZ, A. M. (1975) J. Biol. Chem. 249, 6174-6180. 5. FITZPATRICK, D. F., LANDON, E. J., DEBBAS, G., AND HURWITZ, L. (1972) Science 21, 305-306. 6. MOORE, I,., HURWITZ, L., DAVENPORT, G. R., AND LANDON, E. J. (1975) Biochim. Biophys. Acta 413, 432-443. 7. SCHATZMANN, H. J. (1973) J. Physiol. (London) 235, 551-569. 8. MOORE, I,., CHEN, T., KNAPP, H. R., AND LANDOIL’, E. J. (1975) b. Biol. Chem. 250, 4562-4568. 9. FARBER, J. L., EL-Mo~Y, S. K., SCHANNE, F. A. X., ALEO, J. J., AND SERRONI, A. (1977) Arch. Biochem. Biophys. 178, 617-624. 10. MOORE, L., AND PASTAN, I. (1976) J. Cell. Physiol. 91, 289-296. 11. MOORE, L., AND PASTAN, I. (19’77) J. Biol. Chem. 252, 6304-6309. 12. MOORE, L., FITZPATRICK, D. F., CHEN, T. S., AND LANDON, E. J. (1974) Biochim. Biophys. Acta 345, 405-418. 13. ROBINSON, .J. D. (1976) Arch. Biochem. Biophys. 176, 366-374.
ENDOPLASMIC
RETICULUM
101
14. BRUNS, D. E., MCDONALD, J. M., ANDJARETT, L. (1976) J. Biol. Chem. 251, 7191-7197. 15. ROUFOGALIS, B. D. (1973) Biochim. Biophys. Acta 318, 360-370. 16. OHTSUKI, I. (1969) J. Biochem. 66, 645-650. 17. BEUTLER, E., AND GUINTO, E. (1976) J. Lab. Clin. Med. 88, 520-524. 18. JARE~T, L. (1974) in Methods in Enzymology (Fleischer, S., ed.), Vol. 31, pp. 60-71, Academic Press, New York. 19. JARE~, L., AND SMITH, R. M. (1975) Proc. Nat. Acad. Sci. USA 72, 3526-3530. 20. MCKEEL, D. W., AND JARETT, L. (1970) J. Cell Biol. 44, 417-432. 21. SEALS, J. R., MCDONALD, J. M., BRUNS, D., AND JARE?T, L. (1978)AnaI. Biochem. 90,785-795. 22. BLACK, B. L., MCDONALD, J. M., AND JARETT, L. (1978) Fed. Proc. 37, 1300. 23. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. L., AND MCKEEL, D. (1970) Arch. 24. JARE~, Biochem. Biophys. 140, 362-370. 25. KATZ, A. M., REPKE, D. I., UPSHAW, J. E., AND POLASCIK, M. A. (1970) Biochim. Biophys. Acta 205, 473-490. 26. SHAMI, Y.. AND RADDE, I. C. (1971) Biochim. Biophys. Acta 249, 345-352. 27. AVRUCH, J., AND WALLACH, D. F. H. (1971) Biochim. Biophys. Acta 233, 334-337. 28. YAMAMOTO, T., AND TONOMURA, Y. (1967) J. Btichem. (Tokyo) 62, 558-575. 29 SULAKE, R. V., DRUMMOND, G. I., ANDNG, D. C. (1973) J. Biol. Chem. 248, 4150-4157. 30. OLIVEIRA, M. M., AND HOLZHACKER, S. (1974) Biuchim. Biophys. Acta 332, 221-232. 31. SEAVER. M., AND DUGGAN, P. F. (1974) Biochem. Sot. Trans. 2, 1378-1380. 32. ANAND, M. B., CHAUHAN, M. S., AND DHALLA, N. S. (1977) J. Btichem. 82, 1731-1739. 33. ERNSTER, L., AND JONES, L. C. (1962) J. Cell Biol. 15, 563-576. 34. EMMELOT, P., AND Bos, C. J. (1965) Biochim. Biophys. Acta 121, 375-385. 35. PARKINSON, D. K., AND RADDE, I. C. (1971) B&him. Biophys. Acta 242, 238-246. 36. DEMEIS, L., RUBIN-ALTSCHUL, B. M., AND MACHADO, R. D. (1970) J. Biol. Chem. 245, 1883 - 1889. 37. MEISSNER, G., CONNER, G. E., AND FLEISCHER, S. (1973) Biochim. Biophys. Acta 298,246-269. 38. DAHMS, A. S., AND BOYER, R. D. (1973) J. Biol. Chem. 248, 3155-3162. 39. INESI, G., COHEN, J. A., AND COAN, C. R. (1976) Biochemistry 15, 5293-5298. 40. VIANNA, -4. L. (1975) Biochim. Biophys. Acta 410, 389-406. 41. FROEHLICH, J. P., AND TAYLOR, E. W. (1975) J. Biol. Chem. 250, 2013-2021.
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42. DUGGAN, P. F. (1977) J. Bid. 1627.
Chem. 252, 1620-
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43. JONES, L. R., BESCH, H. R., AND WATANABE, A. M. (19’77) J. Biol. Chem. 252. 3315-3323. 44. INESI, G. (1972)Annu. Rev. Biophys. Bioenerg. 1, 191-210. 45. SUKO, J. (1973) Experientia 29, 396398. 46. INESI, G. (1967) J. Bid. Chem. 242, 4637. 47. NEET, K. E., AND GREEN, N. M. (1977) Arch. B&hem. Btiphys. 178, 588-597. 48. BRUNS, D. E., MCDONALD, J. M., ANDJARETT, L. (1977) J. Biol. Chem. 252, 927-932. 49. BRUNS, D. E., BLACK, B. L., MCDONALD, J. M., AND JAREIT, L. (1977) in Calcium Binding
AND JARETT Proteins and Calcium Function (Wasserman, R. H., Carradino, R. A., Carafoli, E., Kretsinger, R. H., MacLennan, D. H., and Siegel, F. L., eds.), pp. 181-184, Elsevieri North-Holland, New York. 50. TADA, M., KIRCHBERGER, M. A., LI, H., AND KATZ, A. M. (1975) in Calcium Transport in Contraction and Secretion (Carafoli, G., Clementi, F., Drabikowski, W., and Margreth, A., eds.), p. 373, North-Holland, Amsterdam. 51. NISHIKARI, K., TAKANAKA, T., AND MAENO, H. (1977) iViol. k’harmacol. 13, 671-678. 52. HORL, W. H., JENNISSEN, H. P., AND HEILMEYER, L. M. G. (1978) Biochemistry 17, 759-766.
Characterization
BETTY Division
AND BIOPHYSICS pp. 92-102, 1980
of Mg2+- and (Ca2+ + Mg2+)-ATPase Endoplasmic Reticulum1 L.
BLACK,z
of Laboratory School
JAY
Medicine,
of Medicine,
M.
MCDONALD,3
Departments and Barnes Received
AND
qf Pathology Hospital, July
LEONARD
Activity
in Adipocyte
JARETT
and Medicine, Washington St. Louis, Missouri 63110
IJniversity
16, 1979
The presence of an energy-dependent calcium uptake system in adipocyte endoplasmic reticulum (D. E. Bruns, J. M. McDonald, and L. Jarett, 1976, J. Biol. Chem. 251, ‘71917197) suggested that this organelle might possess a calcium-stimulated transport ATPase. This report describes two types of ATPase activity in isolated microsomal vesicles: a nonspecific, divalent cation-stimulated ATPase (Mg*+-ATPase) of high specific activity, and a specific, calcium-dependent ATPase (Ca ‘+ + Mg*+-ATPase) of relatively low activity. Mg*+-ATPase activity was present in preparations of mitochondria and plasma membranes as well as microsomes, whereas the (Ca*+ + Mg’+)-ATPase activity appeared to be localized in the endoplasmic reticulum component of the microsomal fraction. Characterization of microsomal Mg2+-ATPase activity revealed apparent K, values of 115 PM for ATP, 333 yM for magnesium, and 200 PM for calcium. Maximum Mg*+-ATPase activity was obtained with no added calcium and 1 mM magnesium. Potassium was found to inhibit MgZ+-ATPase activity at concentrations greater than 100 mM. The energy of activation was calculated from Arrhenius plots to be 8.6 kcal/mol. Maximum activity of microsomal (Ca2+ + Mg2+)-ATPase was 13.7 nmol “*P/mgimin, which represented only 7% of the total ATPase activity. The enzyme was partially purified by treatment of the microsomes with 0.09% deoxycholic acid in 0.15 M KC1 which increased the specific activity to 37.7 nmol “‘Pimglmin. Characterization of (Ca2+ + Mg2+)-ATPase activity in this preparation revealed a biphasic dependence on ATP with a Hill coefficient of 0.80. The apparent K,s for magnesium and calcium were 125 and 0.6-1.2 FM, respectively. (Ca ?+ + Mg*+)-ATPase activity was stimulated by potassium with an apparent K, of 10 mM and maximum activity reached at 100 mM potassium. The energy of activation was 21.5 kcal/mol. The kinetics and ionic requirements of (Ca*+ + Mg2+)-ATPase are similar to those of the (Ca 2+ + Mg*+)-ATPase in sarcoplasmic reticulum. These results suggest that the (Ca*+ + Mg2+)-ATPase of adipocyte endoplasmic reticulum functions as a calcium transport enzyme.
Control of intracellular calcium levels appears to be crucial for the maintenance of normal cell function (1). Regulation of cytoplasmic calcium has been most extensively studied in muscle where the calcium concentration plays the major role in the contraction-relaxation cycle. In skeletal (2, 3),
cardiac (4), and smooth muscle (5, 6), cytoplasmic calcium concentrations are controlled primarily by the calcium transport system of sarcoplasmic reticulum. A similar calcium transport system has been described in plasma membranes of erythrocytes where calcium is extruded from the cells against a concentration gradient (7). Calcium transport in these systems is mediated by a calcium-stimulated Mg2+-ATPase with one to two calcium ions transported per ATP hydrolyzed, and the K, for calcium is in the micromolar range, consistent with estimated cytoplasmic concentrations of 10m7-
1 Presented in part at the 69th Annual Meeting of the American Society of Biological Chemists and supported by United States Public Health Service Research Grants AM 11892, AM 20097, AM 20579, and postdoctoral fellowship IF32 AM05666. 2 Present address: Department of Zoology, North Carolina State University, Raleigh, N. C. 27650. 3 To whom reprint requests should be sent. Telephone No. 314-454-2169. 0003.9861/80/010092-11$02.00/O Copyright 0 1980 by Academic Press, Inc. A11 rights of reproduction in any form reserved.
lo-6
M (1).
Energy-dependent 92
calcium transport
has
ATPase
ACTIVITY
OF ADIPOCYTE
recently been described in microsomal preparations from hepatocytes (8, 9>, fibroblasts (10, ll), kidney cells (12), brain tissue (13), and adipocytes (14). Although calcium-stimulated ATPase activity has been detected in some of these preparations, such studies have been complicated by the lability and low relative activity of the enzyme, by the presence of multiple ATPases, and by the heterogeneity of the microsomal preparations (13, 15, 16). Furthermore a relationship between enzyme activity and calcium transport has not been established. If these calcium transport systems are analogous to the well-characterized red cell and sarcoplasmic reticulum calcium pumps, then there must exist a specific calcium-stimulated ATPase which mediates the transport of calcium. The present study is part of a series of investigations designed to elucidate the mechanism and hormonal regulation of calcium transport in adipocyte endoplasmic reticulum. This paper characterizes the ATPase activity associated with this organelle and reveals the presence of two calciumstimulated enzymes: a nonspecific, divalent cation-stimulated ATPase (Mg2+-ATPase) of high specific activity and a specific, calcium-dependent ATPase ((Ca2+ + Mg’+)ATPase) of relatively low activity. Analysis of the kinetics and ionic requirements of both enzymes indicates that the Mg’+ATPase lacks the characteristics of a calcium transport ATPase, whereas all properties of the (Ca + Mg2+)-ATPase are strikingly similar to those of the transport (Ca + Mg2+)-ATPase of sarcoplasmic reticulum. EXPERIMENTAL
PROCEDURES
Materials. Male Wistar rats of 120 g were purchased from National Laboratory Animal Company (O’Fallon, MO.). Collagenase (Type I) from Clostridium histolyticum, bovine serum albumin (fraction V), and ATP (disodium salt) were purchased from Sigma Chemical Company (St. Louis, MO.). [Y-~~P]ATP was obtained from AmershamiSearle (Arlington Heights, Ill.), or was synthesized according to the method of Beutler and Guinto (17) from s2P, purchased from New England Nuclear (Boston, Mass.). All other reagents were of reagent grade quality and were purchased from standard sources. All reagents were prepared with water deionized by a double-chambered mixed bed ion
ENDOPLASMIC
RETICULUM
93
exchange resin system (Culligan, Inc., Northbrook, Ill.) which was filtered prior to use with a 0.25~pm pore size filter (Ultipor, PTM Corp., Cortland, N. Y.). Adipocyte fractionation. Adipocytes were isolated from rat epididymal fat pads as previously described (18). A modified method of obtaining microsomes was utilized since microsomes prepared by standard procedures did not transport calcium (14). Isolated adipocytes were homogenized in 0.25 M sucrose with 10 mM Tris-HCl, pH 7.4 at 4°C (Tris-sucrose). The homogenate was centrifuged at 20,OOOg for 15 min. ATP (0.5 mM) and MgCl, (0.25 mM) were added to the resulting supernatant (S,) which was centrifuged at 160,OOOg for 10 min to obtain a microsomal pellet. The pellet was resuspended in Tris-sucrose by brief homogenization. Previous characterization of microsomes isolated by a 60.min centrifugation of S, indicated that the preparation was highly enriched in endoplasmic reticulum (19. 20). The rapidly obtained microsomal preparation was found to be equally enriched in endoplasmic reticulum by assays of cytochrome c reductase activity which indicated an B.&fold enhancement of activity in the microsomes as compared to values in the homogenate. Based on the activity of this marker enzyme, 30% of the endoplasmic reticulum present in the homogenate was recovered in the rapidly prepared microsomes, similar to a 35% recovery in microsomes prepared by the standard 60-min centrifugation (N = 5). Microsomes were either immediately treated with DOCj or rapidly frozen in small aliquots with dry ice/ethanol and stored at -70°C until used. Mitochondrial and plasma membrane fractions were obtained as previously described (18. 20), except that EDTA was omitted throughout the procedure (14). DOG’-treated microsornes. Freshly isolated microsomes at a concentration of l-2 mg protein/ml were added to an equal volume of DOC in KC1 giving a final concentration of 0.09% DOC-0.15 M KCl. The DOCmicrosomes solution was stirred for 5 min at 24°C. diluted 5-10x with Tris-sucrose plus 0.5 mM ATP and 0.25 mM MgCl,, and centrifuged at 160,000~ for 10 min. The resulting pellet was resuspended m Trissucrose, rapidly frozen, and stored at ~ 70°C until used, ATPnae assays. ATP hydrolysis by microsomal, mitochondrial, and plasma membrane fractions was measured by an isotopic assay method (21). Standard incubations were performed in duplicate in conical glass tubes with constant shaking at 37°C. The Assam was initiated by addition of lo-20 wg of protein to a standard incubation medium containing 50 mM TrisHCl, pH 6.8 at 37”C, 100 mM KCl, 1 mM MgCl,, 5 rnM K+-oxalate or Tris-oxalate, 0.5 mM Na,ATP with tracer [y-“*P]ATP, 0.2 mM EGTA, and 0.18 mifl CaCl, 4 Abbreviations used: DOC. deoxycholic acid; EGTA, ethylene glycol bis (P-aminoethyl ether) N,N ‘-tetraacetic acid; PCMBS, p-chloromercuribenzene sulfonate; S, supernatant.
94
BLACK,
MCDONALD, TABLE
ATPase Subcellular fraction Microsomes
Mitochondria Plasma membranes
ACTIVITYINADIPOCYTE
Assay Standard Standard Standard Standard Standard
medium medium medium medium medium
I
RESULTS
Subcellular
The Mg2+-ATPase activity (207 2 18.9 nmol 32P/mg/min) of the rapidly obtained microsomal fraction (Table I) was comparable to that found in the original adipocyte microsomal preparation of Jarett and McKee1 (24). Addition of 10 FM free calcium to the standard incubation medium increased total ATPase activity by approximately ‘7%, yielding an additional 13.7 nmol
FRACTIONLP
Mg*+-ATPase (nmol 32P/mg/min)
(12)* plus 50 PM PCMBS (5) minus potassium (5) (8) (10)
(approximately 10 PM free calcium) in a total of 500 ~1. The oxalate was included to make the buffer identical to that used in calcium transport studies (22). The reaction was terminated after 2-20 min by addition of 1% sodium lauryl sulfate. The hydrolyzed 32P was extracted in xylene:isobutanol and quantitated by liquid scintillation counting in toluene:omnifluor: ethanol. In all assays, appropriate control tubes without protein were included to correct for nonenzymatic ATP hydrolysis. To measure (Ca2+ + Mg2+)ATPase activity, assays were performed with and without calcium. Values obtained in the absence of calcium (Mg*+-ATPase activity) were subtracted from the total ATPase activity in the presence of calcium to give a value for (Ca*+ + Mg2+)-ATPase activity. Other assays. Protein was measured by the method of Lowry et al. (23) using bovine serum albumin as a standard. NADH-cytochrome c reductase and succinic dehydrogenase activity were measured as previously described (20). Calculations. Free calcium concentrations in the incubation buffer were calculated from the EGTAI calcium ratios as described by Schatzmann (7). Data are expressed as means with standard errors given in cases where the number of preparations is greater than 4.
in Adipocyte
JARETT
SUBCELLULAR
conditions
n Mg*+-ATPase and (CaZ+ + MgZ+)-ATPase activities described under Experimental Procedures. b Values represent the mean (-tSEM) of the number
ATPase Activity Fractions
AND
were
207 183 260 613 1094 assayed
of preparations
+ k +? *
(Ca*+ + Mg2+)-ATPase (nmol 3*P/mg/min)
18.9 20.0 20.0 92.0 149
13.7 -0.4 0.6 13.9 -1.6
in the standard listed
incubation
5 t t + 2
1.8 0.2 3.6 12.5 14.5 medium
as
in parentheses.
32P/mg/min. This additional ATPase activity was specific for calcium ((Ca2+ + Mg2+)ATPase) since increasing the magnesium concentration from 1 mM to 2 mM in the absence of calcium did not increase the total ATPase activity (data not shown). Addition of 50 j..&M PCMBS to the standard incubation buffer or omission of potassium slightly altered Mg2+-ATPase activity to 88 and 126% of control values, respectively (Table I). In contrast, (Ca2+ + Mg2+)-ATPase activity was virtually eliminated under these conditions. ATPase activity was assayed in highly enriched mitochondrial and plasma membrane preparations to ascertain whether the Mg2+-ATPase or (Ca2+ + Mg2+)-ATPase activity in the microsomal fraction could be accounted for by small amounts of mitochondrial or plasma membrane contaminants of the microsomal preparations. Mitochondrial Mg2+-ATPase activity was three-fold higher than that of the microsomes (Table I). The addition of 10 PM free calcium stimulated mitochondrial ATPase by 13.9 nmol 32P/mg/min. The maximum mitochondrial contamination of the microsomal fraction is 6%, based on assays of succinic dehydrogenase activity which gave values of 116.8 + 22 and 7.2 k 1.4 nmol succinate oxidizedlmglmin (N = 8) for mitochondrial and microsomal fractions, respectively. Thus mitochondrial (Ca”+ + Mg2+)-ATPase could account for a maximum of 5.8% (approximately 0.8 nmol/mg/ min) of the microsomal (Ca2+ + Mg2+)ATPase activity. The Mg2+-ATPase specific
ATPase
ACTIVITY
OF ADIPOCYTE
FIG. 1. Dependence of Mg2+-ATPase activity on concentration of ATP. Mg2+-ATPase activity was determined using an assay buffer containing 50 mM Tris-HCl, pH 6.8 at 37”C, 1 mM MgCl,, and 0.05-1.0 mM ATP. Each point represents the mean value obtained from four microsomal preparations, each assayed in duplicate. The inset is a double-reciprocal plot of the data.
activity of plasma membranes was five-fold higher than that of the microsomal fraction, but calcium-stimulated ATPase activity was not detectable under the standard incubation conditions. Therefore, plasma membrane contaminants could not contribute to the (Ca2+ + Mg2+)-ATPase activity in the microsomal fraction. On the other hand, because of the extremely high specific activity of Mg2+-ATPase in the mitochondrial and plasma membrane fractions, even a small amount of contamination of the microsomal preparation by these organelles could account for a substantial portion of the microsomal Mg2+-ATPase activity.
ENDOPLASMIC
RETICULUM
95
concentration is shown in Fig. 1. Analysis by double-reciprocal plots yields an apparent K, of 115 pM. Mg2+ATPase activity was dependent upon the presence of divalent cations. In the absence of any added divalent cations, ATPase activity was approximately 2% of that found under optimal conditions (data not shown). This residual activity could be eliminated by the addition of 150 pM EDTA and thus was presumed to reflect the presence of divalent cations in the microsomal membranes or incubation medium. ATP hydrolysis as a function of either calcium or magnesium concentration was dose dependent and saturable (Fig. 2). Analysis of doublereciprocal plots yielded an apparent K, for magnesium of 333 pM and a V of 556 nmol “ZPlmglmin. The addition of 200 pM EGTA to the incubation buffer did not alter these parameters, indicating that magnesium is effective in the complete absence of calcium. Calcium proved to be a fair substitute for magnesium. In the absence of added magnesium, the apparent K, for calcium was 200 PM and V was 370 nmol 32Plmglmin. As nonsaturating concentrations of magnesium were increased from 10 pM to ZOO pM, stimulation of activity by 50 pM calcium was reduced from 400 to 21% (data not shown), indicating that calcium stimulation was inhibited by magnesium. No calcium stimulation occurred at magnesium concentrations above 500 PM. Thus, high rates of ATP hydrolysis could be supported by
Characterization of Mg2+-ATPase Activity in Microsomes
The Mg2+-ATPase activity of the microsomal fraction was further characterized in an incubation buffer containing 50 mM TrisHCl, pH 6.8 at 37”C, 0.5 mM ATP, and 1 mM MgCl,. Potassium was omitted from the buffer to eliminate (Ca2+ + Mg2+)-ATPase activity (Table I). Omission of EGTA under these assay conditions had no significant effect on Mg2+-ATPase activity. The Mg2+ATPase activity was linear for 2-3 min at protein concentrations between 10 and 40 Fg/ml (data not shown). ATP, magnesium, and calcium dependency of Mg2+-ATPase activity. Mg2+-
ATPase
activity
as a function
of ATP
FIG. 2. Dependence of Mg2+-ATPase activity on concentrations of magnesium and calcium. M$+ATPase activity was determined using an assay buffer containing 50 mM Tris-HCl, pH 6.8 at 37”C, 0.5 mM ATP, and 0.05-2.0 mM magnesium (e) or calcium (0). Each point represents the mean value obtained from four microsomal preparations, each assayed in duplicate.
96
BLACK,
MCDONALD,
AND
JARETT
(Table I). At concentrations of 150 and 200 mM, Mg2+-ATPase activity was inhibited by 22 and 33%, respectively (data not shown). Temperature dependency of Mg2+-ATPase activity. The Mg2+-ATPase activity in-
creased with temperature between 15 and 35°C. Analysis of the data by Arrhenius plots revealed a Q1,, of 1.64 and an activation energy of 8.65 kcal/mol (Fig. 3). Characterization of the (Ca2+ + Mg2+)ATPase Activity FIG. 3. Arrhenius plots of ATPase activity. Mg*+ATPase activity (0) in microsomal preparations was determined at temperatures of 15-35°C using an incubation buffer containing 50 mM Tris-HCI, adjusted to pH 6.8 at each temperature, 1 mM MgCl,, and 0.5 mM ATP. (Ca2+ + My+)-ATPase activity (0) in DOC-treated microsomal preparations was determined at temperatures of ZO-40°C using the standard incubation buffer with the pH adjusted to 6.8 at each temperature. Each point represents the mean value obtained from three preparations, each assayed in duplicate.
either magnesium or calcium, but magnesium in the absence of calcium elicited maximum ATPase activity. Effect activity.
of potassium
on Mg2+-ATPase
The Mg2+-ATPase activity was slightly inhibited (20%) by 100 mM potassium, in contrast to (Ca’+ + Mg2+)-ATPase which required this cation for full activity TABLE COMPARISON
OF ATPase
ACTIVITY
ATPase after
microsomes” microsomes’
activity recovered DOC treatmentd
Optimal
conditions
for DOC treatment.
The effect of DOC treatment (see Experimental Procedures) on the ATPase activity of subsequently resuspended vesicles was tested for DOC concentrations between 0.06 and 0.12%. The Mg2+-ATPase activity was lost much more rapidly than (Ca2+ + Mg2+)-ATPase activity at DOC concentrations between 0.06 and 0.09%. The (Ca2+ + Mg2+)-ATPase to total ATPase ratio was highest with 0.09% DOC. Lowering the temperature during DOG treatment II
IN UNTREATED
Mg*+-ATPase (nmol 3ZP/mg/min) Untreated DOC-treated
It was necessary to reduce Mg2+-ATPase activity relative to (Ca2+ + Mg2+)-ATPase activity to further characterize the microsomal (Ca2+ + Mg*+)-ATPase, since the (Ca2+ + Mg2+)-ATPase activity represented only 7% of the total ATPase activity present. This was accomplished by a brief treatment of the microsomal vesicles with DOC.
vs DOGTREATED
(Ca’+ + Mg’+)ATPase (nmol “*P/mg/min)
207 t 18.9 60.9 ? 6.5
13.7 -t 1.8 37.7 -+ 2.1
7.8%
72.6%
MICROSOMES” (Ca2’
+ Mg*+)-ATPasei total ATPase (%I 6.7 t 1.0 40.9 2 1.0
a Mg*+-ATPase and (Ca2+ + Mg2+)-ATPase activities were assayed in the standard incubation medium as described under Experimental Procedures. * Values represent the mean (t SEM) of 12 preparations. c Values represent the mean (2 SEM) of 27 preparations. d The protein recovered in the DOC-treated microsomes was 26.4 + 1.3% (n = 12) of the protein in the original, untreated microsomal preparation.
ATPase
ACTIVITY
OF ADIPOCYTE
ENDOPLASMIC
RETICULUM
97
ATP dependency of (Ca2+ + Mg2+)ATPase activity. The (Ca’+ + Me+)-ATPase
FIG. 4. Dependence of (Ca” + Mg2+)-ATPase activity on concentration of ATP. (Ca*’ + Mg*-)ATPase activity was determined using the standard incubation buffer with 0.01-1.0 mM ATP. Each point represents the mean value of three DOC-treated microsomal preparations, each assayed in duplicate. The inset is a double-reciprocal plot of the data.
from 24°C to 4°C elicited a 10% increase in (Ca2+ + Mg2+)-ATPase activity, but Mg2+ATPase specific activity increased 6000/c, giving a low (Ca2+ + Mg’+)-ATPase to total ATPase ratio. The (Ca2+ + Mg2+)ATPase activity in preparations treated with 0.09% DOC at 24°C remained constant during 3-6 min of treatment, but dropped after longer exposure to the detergent. Therefore, subsequent studies of (Ca2+ + Me+)-ATPase utilized microsomes treated with 0.09% DOC at 24°C for 5 min. The specific activity of (Ca’+ + Mg2+)ATPase of microsomes prepared in this manner was almost threefold higher than that in untreated microsomes (Table II). The (Ca’+ + Mg’+)-ATPase as a percentage of total ATPase activity was increased from 7% to 41%. Recovery studies indicated that 75% of the original (Ca2+ + Mg2+)-ATPase activity present in the untreated microsomes was recovered, as compared to only 7-8% of the Mg*+-ATPase activity. Stability, linearity, and protein ency of (Ca2+ + Mg2i)-ATPase
activity was dose dependent and saturable as a function of ATP concentration (Fig. 4). Analysis of the data by double-reciprocal plots yielded a broken line with two apparent components. An apparent K, of 36 PM was found in the ATP concentration range of lo-50 PM, whereas the apparent K,, at higher concentrations was 118 FM. A Hill plot of the data yielded a straight line with a Hill coefficient of 0.80. Calcium and magnesium dependency of (Ca2+ + Mg2+)-ATPase activity. Calcium-
stimulated ATP hydrolysis in DOG-treated microsomes required the presence of both calcium and magnesium, in contrast to the requirements of the Mg’+-ATPase activity. In the presence of 1 mM magnesium, the addition of as little as 0.2 PM free calcium stimulated ATPase activity by lo-15%. Maximum stimulation by calcium occurred at 3-5 FM (Fig. 5). Double reciprocal plots revealed a K, for free calcium of 0.6 PM and V of 30.3 nmol ~“P/mg/min. The free calcium concentrations used in these calculations were obtained from the calcium/EGTA ratios in the incubation buffer as described by Schatzman (7). A similar K, for free calcium of 1.2 PM is calculated by estimating free calcium concentrations by the method of Katz (25) which includes a correction for the calcium complexed by ATP. The magnesium dependency of (Ca” I
.’
rl
dependactivity.
The (Ca2+ + Mg2+)-ATPase activity in DOC-treated microsomes was stable at 4°C for 25-30 min, but decreased to 82% by 60 min. Activity remained constant for at least 6 weeks when the preparation was rapidly frozen and stored at -70°C. In the standard incubation buffer at 37”C, (Ca2+ + Mg2+)-ATPase activity was linear with time for 20-40 min using protein concentrations from lo-40 pgiml (data not shown).
FIG. 5. Dependence tivity on concentration ATPase activity was incubation buffer with point represents the from 13 DOC-treated assayed in duplicate. plot of the data.
of (Ca2+ + Mg*+)-ATPase acof calcium. (Ca*+ + Mg2’)determined using the standard 0.5-10.0 pM free calcium. Each mean value (?SE) obtained microsomal preparations, each The inset is a double-reciprocal
98
BLACK,
MCDONALD.
+ Mg2+)-ATPase activity is illustrated in Fig. 6. The low activity present in the absence of added magnesium presumably reflects the presence of contaminating magnesium ions and/or stimulation by calcium of the low Mg2+-ATPase activity which remains in the DOC-treated microsomes. Using the standard ATP concentration of 0.5 mM, maximum (Ca2+ + Mg2+)-ATPase activity occurred at 1 mM magnesium. Higher magnesium concentrations were inhibitory; activity was reduced by 18% at 5 mM magnesium. These data yield an apparent K, for magnesium of 125 PM and a V of 47 nmol 32P/mg/min. Effect of potassium on (Ca’+ + Mg*+)ATPase activity. Omission of potassium
from the standard incubation buffer resulted in a 78% reduction of the (Ca2+ + Mg2+)-ATPase activity in DOC-treated microsomes, analogous to the inhibition of calcium stimulation in untreated microsomal preparations (Table I) and in contrast to the effect of potassium on Mg2+-ATPase activity. The (Ca 2+ + Mg2+)-ATPase activity was a function of potassium concentration, with maximum activity reached at 100 mM potassium (Fig. 7). Analysis of the data by double-reciprocal plots yields an apparent K, of 10 mM and a V of 43.5 nmol 32P/mg/min. Temperature dependency of + Mg2+)-ATPase activity. The
(Ca2+
(Ca2+
500
I I
2 MAGNESIUM
3 CONCENTRATON
4 (mid)
I 5
FIG. 6. Dependence of (Ca2+ + Mg2+)-ATPase activity on concentration of magnesium. @a*+ + Mg2+)ATPase activity was determined using the standard incubation buffer with 0.5-5.0 ITIM magnesium. Each point represents the mean value obtained from three DOC-treated microsomal preparations, each assayed in duplicate. The inset is a double-reciprocal plot of the data.
AND JARETT
I 50 POTASSIUM
I 103
4
150 CONCENTRATION
I MO [mM)
FIG. 7. Dependence of (CaZ+ + MgZ+)-ATPase activity on concentrations of potassium. (Ca*+ + MgZ+)ATPase activity was determined using the standard incubation buffer with no added potassium or 5-200 !IIM potassium. Each point represents the mean value obtained from three DOC-treated microsomal preparations, each assayed in duplicate. The inset is a doublereciprocal plot of the data.
+ Mg2f)-ATPase activity was highly dependent upon temperature between 20 and 40°C. Analysis of the data by Arrhenius plots (Fig. 3) yields a Q10 of 3.3 and an activation energy of 21.5 kcal/mol. These values contrast to the respective values for Mg2+-ATPase of 1.6 and 8.6 kcal/mol. DISCUSSION
Adipocyte microsomes contain two types of ATPase activity which require the presence of divalent cations. The predominant ATPase activity of the preparation (Mg2+ATPase) can be stimulated by either calcium or magnesium in the absence of the other cation. The Mg2+-ATPase has an absolute requirement for divalent cations, since EDTA completely inhibits ATP hydrolysis. This divalent cation requirement is relatively nonspecific since calcium at saturating concentrations gives 70% as much activity as optimal concentrations of magnesium (Fig. 2). The fact that 50 PM calcium elicits progressively less ATP hydrolysis as magnesium is increased to saturating levels suggests that calcium and magnesium act at the same site. Presumably, either cation plus ATP forms a complex which serves as the true substrate of the reaction as proposed for Ca2+-, Mg2+ATPases of other cell types (26).
ATPase
ACTIVITY
OF
ADIPOCYTE
The microsomal preparation contains a second ATPase activity (Ca2+ + Mg*+)ATPase which is distinguished from the Mg2+ATPase by an absolute dependence on calcium. This dependence is revealed as a 7% stimulation of ATP hydrolysis by 10 pM calcium under conditions where Mg’+ATPase is saturated with magnesium. The concept that this calcium-stimulated activity represents a second ATPase is supported by the opposite effect of addition of PCMBS or omission of potassium on Mg2+ATPase and calcium-stimulated Mg2+-ATPase activity (Table I). Since the endoplasmic reticulum of these microsomal preparations is contaminated with small amounts of mitochondria and plasma membrane, it was necessary to determine the extent to which the latter organelles contribute to microsomal ATPase activity. Mitochondria represent a minor contaminant (maximum of 6%) of the preparation and thus could not contribute significantly to microsomal (Ca2+ + Mg2+)ATPase activity (Table I). Consistent with this conclusion, a concentration of sodium azide (10 mM) which inhibited mitochondrial ATPase activity by 73% had no effect on (Ca2+ + Mg2+)-ATPase activity in DOCtreated microsomes (unpublished observation). Highly enriched plasma membranes contain no detectable (Ca2+ + Mg2+)-ATPase activity (Table I) indicating that plasma membrane contaminants do not contribute to the (Ca2+ + Mg2+)-ATPase activity of adipocyte microsomes. The high Mg2+ATPase activity of plasma membranes (approximately 1100 nmol 32P/mg/min.) could, however, account for part of the microsomal Mg2+-ATPase activity. If Mg2+ATPase activity is a combination of endoplasmic reticulum and plasma membrane components, the Mg2+-ATPase of the two organelles must have similar properties since kinetic analysis and Arrhenius plots indicate the presence of only one enzyme. Previous studies of adipocyte ATPase have also reported the presence of high Mg2+ATPase activity in both endoplasmic reticulum and plasma membrane fractions (24, 27). Therefore, microsomal Mg2+-ATPase activity may be derived from both endoplasmic reticulum and plasma membrane
ENDOPLASMIC
RETICULUM
99
elements, whereas the (Ca2+ + Mg*+)-ATPase activity is localized in the endoplasmic reticulum. The Mg*+-ATPase of adipocyte microsomes resembles a magnesium- or calciumstimulated ATPase activity common to many plasma membrane and endoplasmic reticulum preparations. Such a nonspecific, divalent cation ATPase has been described in plasma membrane and endoplasmic reticulum from a variety of tissues, including skeletal, cardiac, and smooth muscle (28-32), liver (33, 34), kidney (35), placenta (26), and brain (13). The Mg2+- or Ca2+ATPases of these tissues are similar in that they do not require sodium or potassium, but can be stimulated by either magnesium or calcium with maximum activity reached at millimolar concentrations. Reported K,,, values range from 0.3-1.5 mM for magnesium or calcium, and from 0.05-0.3 mM for ATP. The Mg2+-ATPase of adipocyte microsomes shares these properties with no apparent monovalent cation requirement, maximum stimulation at 1 IrIM magnesium or calcium, and K,s of 0.33 mM for magnesium, 0.20 mM for calcium, and 0.11 lllM for ATP. The cellular function of these nonspecific Mg2+- or Ca2+-ATPases is not known, but speculations have included a role in magnesium or calcium transport (26, 35). The Mg2+-ATPase of adipocyte endoplasmic reticulum does not appear to function as a calcium transport enzyme, since the calcium transport system of this organelle requires the presence of both magnesium and calcium and has a much lower K, for calcium than does Mg2+ATPase activity (14, 22). The (Ca2+ + Mg2+)-ATPase of adipocyte endoplasmic reticulum represents a small percentage of the total ATPase present, similar to the calcium-stimulated Mg2+ATPase activity of other noncontractile cell microsomes (13, 15, 36). Treatment of adipocyte microsomes with DOC increases the relative and specific activity of (Ca’+ + Mg*+)-ATPase which allows kinetic analysis of the enzyme. A similar partial purification with low concentrations of DOC has been described for (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum (37) and brain microsomes (13). Treatment with deter-
100
BLACK,
MCDONALD,
gents has been found to alter the properties of some membrane-bound ATPases (13, 38, 39). Although detailed characterization of untreated adipocyte microsomes is precluded by the low (Ca2+ + Mg*+)-ATPase to total ATPase ratio, the effect of omitting potassium (Table I, Fig. 7) or adding PCMBS (Table I, unpublished observation) is similar in both untreated and DOCtreated preparations, suggesting that no major alteration of enzyme properties has occurred. Analysis of the kinetics and ionic requirements of (Ca2+ + Mg2+)-ATPase in DOCtreated microsomes reveals a striking similarity to the (Ca2+ + Mg*+)-ATPase of sarcoplasmic reticulum. Both enzymes require calcium, magnesium, and potassium for full activity. The K, for calcium of 0.61.2 PM for microsomal (Ca’+ + Mg*+)ATPase (Fig. 4) is comparable to that of 0.1-1.0 pM reported for sarcoplasmic reticulum (3). In the presence of 0.5 mM ATP, maximal activity with magnesium is reached between 0.5 and 1.0 mM magnesium (Fig. 6), suggesting that Mg2+-ATP is the true substrate of the enzyme as is the case for sarcoplasmic reticulum (Ca*+ + Mg*+)ATPase (3, 40). The inhibition observed with excess magnesium is also characteristic of the sarcoplasmic reticulum enzyme (40, 41). Both the apparent K, for potassium (10 mM) and the concentration of potassium required for maximal activity (100 mM) are similar to the corresponding values of lo-20 and 50-100 mM reported for (Ca*+ + Mg*+)-ATPase of sarcoplasmic reticulum (42, 43). Moreover, microsomal and sarcoplasmic reticulum (Ca*+ + Mg2+)ATPase shows a similar dependence upon temperature, with activation energies of 21.5 and 17.9-26.2 kcalimol, respectively (28, 44, 45). Analysis of the substrate dependency of (Ca*+ + Mg*+)-ATPase in DOC-treated microsomes indicates two apparent K,,s for ATP (Fig. 4), again similar to the (Ca2+ + Mg*+)-ATPase of sarcoplasmic reticulum (28,40,46,47). The biphasic double-reciproco1 plot of velocity vs ATP concentration cannot be explained by a nonlinear variation of the complexed Mg2+-ATP since 95- 100% of the total ATP is in the form of Mg2+-ATP between ATP concentrations of 0.01-0.5
AND
JARETT
as calculated by the method of Katz (25). Use of the Hill plot to analyze substrate kinetics gives a Hill coefficient of 0.8 for ATP. This suggests that the nonlinear double-reciprocal plot could be due to negative cooperativity as recently suggested for Mg*+-ATP interactions with the (Ca*+ + Mg2+)-ATPase of sarcoplasmic reticulum (47). The (Ca2+ + Mg2+)-ATPase of adipocyte endoplasmic reticulum probably functions as a calcium transport protein. This concept is supported by studies of passive calcium binding to adipocyte endoplasmic reticulum which suggests that high affinity binding sites represent binding of calcium to the transport sites of the membrane (48). In sarcoplasmic reticulum, calcium binding to sites with corresponding high affinity has been identified as calcium binding to the (Ca”+ + Mg*+)-ATPase, and represents the first step in energy-dependent calcium transport (3). The kinetics and ionic requirements of microsomal (Ca2+ + Mg*+)-ATPase are comparable to those of the calcium transport system in adipocyte endoplasmic reticulum (14, 22). Furthermore, a comparison of calcium transport rates (22) to (Ca” + Mg2+)-ATPase activity in untreated microsomes (Table I) indicates that one or two calcium molecules are transported per ATP hydrolyzed, similar to the 2:l ratio characteristic of the sarcoplasmic reticulum calcium transport system (3). The (Ca2+ + Mg2+)-ATPase of adipocyte endoplasmic reticulum is qualitatively similar to (Ca2+ + MgZ+)-ATPase of sarcoplasmic reticulum, but the specific activity of the enzymes differs by one to two orders of magnitude (3, 44). The lower specific activity of endoplasmic reticulum (Ca2+ + Mg2+)ATPase might reflect a lower concentration of (Ca*+ + Mg*+)-ATPase proteins associated with the membrane. The (Ca2+ + Mg2+)-ATPase in sarcoplasmic reticulum membranes can be visualized as intramembranous particles by freeze-fracture electron microscopic technique (3). Freezefracture studies of adipocyte microsomal vesicles have revealed an intramembranous particle density l- 10% of the value reported for sarcoplasmic reticulum (49), consistent with the concept that endoplasmic reticulum contains a lower density of mM,
ATPase ACTIVITY
OF ADIPOCYTE
calcium transport proteins than sarcoplasmic reticulum. Such a quantitative difference might be anticipated since the sarcoplasmic reticulum membrane is highly specialized for calcium transport, whereas the endoplasmic reticulum of noncontractile cells has a wide range of functions. The demonstation of (Ca2+ + Mg*+)ATPase activity in adipocyte endoplasmic reticulum strengthens the concept that this organelle plays a role in controlling the distribution of intracellular calcium. The cellular mechanism(s) which control (Ca*+ + Mg*+)-ATPase activity are obscure, but might include phosphorylationldephosphorylation of the endoplasmic reticulum membrane, analogous to regulation of the calcium transport system in sarcoplasmic reticulum (50-52). An investigation of such potential regulatory systems could provide insight into the mechanism by which cytoplasmic calcium concentrations are altered and calcium homeostasis is maintained in noncontractile cells. REFERENCES 1. RASMUSSEN, H. (1970) Science 170, 404-412. 2. WEBER, A. M., HERZIG, R., AND REISS, I. (1966) Biochem. 2. 345, 329-369. 3. MACLENNAN, D. H., AND HOLLAND, D. C. (1975) Annu. Rev. Biophys. Bioenerg. 4, 377-402. 4. TADA, M., KIRCHBERGER, M. A., REPKE, D. I., AND KATZ, A. M. (1975) J. Biol. Chem. 249, 6174-6180. 5. FITZPATRICK, D. F., LANDON, E. J., DEBBAS, G., AND HURWITZ, L. (1972) Science 21, 305-306. 6. MOORE, I,., HURWITZ, L., DAVENPORT, G. R., AND LANDON, E. J. (1975) Biochim. Biophys. Acta 413, 432-443. 7. SCHATZMANN, H. J. (1973) J. Physiol. (London) 235, 551-569. 8. MOORE, I,., CHEN, T., KNAPP, H. R., AND LANDOIL’, E. J. (1975) b. Biol. Chem. 250, 4562-4568. 9. FARBER, J. L., EL-Mo~Y, S. K., SCHANNE, F. A. X., ALEO, J. J., AND SERRONI, A. (1977) Arch. Biochem. Biophys. 178, 617-624. 10. MOORE, L., AND PASTAN, I. (1976) J. Cell. Physiol. 91, 289-296. 11. MOORE, L., AND PASTAN, I. (19’77) J. Biol. Chem. 252, 6304-6309. 12. MOORE, L., FITZPATRICK, D. F., CHEN, T. S., AND LANDON, E. J. (1974) Biochim. Biophys. Acta 345, 405-418. 13. ROBINSON, .J. D. (1976) Arch. Biochem. Biophys. 176, 366-374.
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