INFrzTION AND IMMUNITY, Feb. 1977, p. 436-443 Copyright O 1977 American Society for Microbiology

Vol. 15, No. 2 Printed in U.S.A.

Magnesium-Dependent Adenosine Triphosphatase as a Marker Enzyme for the Plasma Membrane of Human Polymorphonuclear Leukocytes JOSEPH HARLAN, LAWRENCE R. DECHATELET,* DAVID B. IVERSON, AND CHARLES E. McCALL Departments ofBiochemistry* and Medicine, The Bowman Gray School of Medicine, Winston-Salem, North Carolina 27103 Received for publication 23 August 1976

The adenosine triphosphatase (ATPase) activities of human polymorphonuclear leukocytes (PMNL) were studied with an assay that monitored the release of 32P-labeled inorganic pyrophosphate (32P1) from y-[32P]adenosine 5'-triphosphate (ATP). In cell homogenates, (Nat + K+)-sensitive, ouabain-inhibitable ATPase comprised an insignificant fraction of the total ATPase activity. Additions ofp-nitrophenyl phosphate and (-glycerophosphate (substrates for nonspecific acid and alkaline phosphatases) and of tartrate (inhibitor of acid phosphatase) gave no indication of inhibition. This suggested that the assay was relatively specific for ATP hydrolysis. The activity was found to have a pH optimum of 8.7 and a K. for ATP of 0.6 mM. There was an absolute requirement for Mg2e, with other divalent cations substituting less efficiently. When the Mg2+-dependent ATPase activity of intact cells was compared with that in homogenized cells, no significant difference was observed. The activity in intact cells was linear with respect to incubation time up to at least 60 min. Trypan blue staining and lactate dehydrogenase assays revealed that greater than 92% of the PMNL remained intact and viable during the assay. No soluble ATPase was released from the cells under assay conditions. In following the distribution ofy-[32P]ATP and 32Pi during the assay, no 32p1 counts became cell associated. Since the experimental evidence supports the observations that PMNL remain intact and viable and that ATP does not penetrate the cell under assay conditions, it is proposed that greater than 90% of the Mg2+-dependent ATPase of the human PMNL is associated with a plasma membrane enzyme. This would qualify the enzyme for the role of a plasma membrane marker for future fractionation and isolation attempts.

Studies directed toward isolation of the plasma membrane of the human polymorphonuclear leukocytes (PMNL) have been limited, due to the lack of an acceptable marker en-

zyme.

In other tissues investigators have identified certain enzymes whose activities are localized in the plasma membrane. Those most frequently reported include 5'-nucleotidase, alkaline phosphatase, adenyl cyclase, and (Nat + K+)-sensitive, ouabain-inhibitable adenosine triphosphatase (ATPase). The characterization and localization of these activities in various tissues have recently been reviewed in substantial detail (7, 19, 21). Since the plasma membrane has one of its surfaces facing the external environment, any enzyme that has its active site on the external surface of the cell offers the possible advantage

of being accurately characterized before cell disruption and exposure of subcellular components. DePierre and Karnovsky have established rather rigid criteria for the use of such "ecto-enzymes" in plasma membrane isolation (6-10). One of the most ubiquitous and best-described plasma membrane markers is 5'-nucleotidase. Two independent studies have demonstrated that this enzyme is absent in human PMNL (17a; 20). In the rabbit alveolar macrophage, which also lacks 5'-nucleotidase, Wang et al. have identified alkaline phosphodiesterase 1 as a monitor for plasma membrane (22). This enzyme also proved to be absent in the human PMNL (L. R. DeChatelet, unpublished data). The purpose of this report is to characterize a Mg2e-dependent ATPase activity in the human

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VOL. 15, 1977

PMNL and to provide supporting evidence that greater than 90% of this activity is associated with a membrane enzyme. MATERIALS AND METHODS Isolation of PMNL. This procedure was a modification of that described by Spitznagel et al. (18). Briefly, heparinized venous blood (10 USP units per ml of whole blood) was obtained from healthy adult volunteers. The blood was mixed with Plasmagel (Roger Bellon Laboratories, Neuilly-sur, France) in a 3:1 (vol/vol) ratio and sedimented for 30 min at room temperature. The supernatant was centrifuged at 170 x g for 10 min, and the resulting pellet was suspended in Dulbecco phosphate-buffered saline (Grand Island Biological Co., Grand Island, N.Y.), pH 7.4, to a volume 15% of the original blood volume. This suspension was layered over a portion of Lymphoprep (Nyegaard and Co., Oslo, Norway) in a 4:3 (vol/vol) ratio and centrifuged at 380 x g for 35 min. The pellet (granulocytes plus erythrocytes) was washed with 0.34 M sucrose to remove any remaining platelets. The erythrocytes were disrupted by a 30-s hypotonic lysis. The PMNL were finally washed and then suspended in isotonic sucrose (0.25 M) and stored at 4°C. After the Lymphoprep procedure, the cells were maintained at 4°C. All pelleting of cells involved centrifugation at 170 x g for 10 min. Routine differential cell counts on a hemocytometer yielded less than 2% mononuclear cells in the final PMNL suspension. Wright staining of slides prepared fronr a cytocentrifuge gave ranges of 93 to 96% PMNL, 3 to 7% eosinophils, and 0 to 2% mononuclear cells. All whole-cell studies were completed within 4 1 after isolation. The conclusion that these isolated PMNL were intact and viable was supported by the observation of an appropriate stimulation of the hexose monophosphate shunt (by the procedure of DeChatelet et al. [5]) when the cells were exposed to a phagocytic stimulus and by the fact that greater than 90% of the cells excluded trypan blue dye (11). Cell disruption. Isolated PMNL were suspended in 0.34 M sucrose at 108 cells per ml and homogenized on ice with a motor-driven Teflon pestle in a Potter-Elvejhem homogenizer. The motor, a Tri-R stirrer model S63-C, was driven at 12,000 rpm (full speed). Total homogenization time equaled 5 min. Phase-microscopic monitoring consistently revealed greater than 90% cell rupture. Alternatively, cells were disrupted by sonication using a Sonifier cell disrupter, model W-140, with a microtip attachment with the output setting control on 6. A total sonication time of 1 min yielded greater than 95% cell rupture by phase microscopy. Biochemicals. All chemicals used were of reagent grade and purchased from commercial sources. y-[3P]adenosine 5'-triphosphate (ATP) (New England Nuclear Corp., Boston, Mass.) was further purified by passage over a Dowex 1-Cl resin column. The resin was activated overnight at pH 8.0. The column was then packed and rinsed with water until the eluant was free of Cl-, as monitored by lack of AgCl precipitation upon the addition of AgNO3. 'y-

Mg2+ ATPase IN HUMAN PMNL

437

[32P]ATP, in 50 mM tris(hydroxymethyl)aminomethane (Tris)-hydrochloride, pH 7.4, and 6 mM MgCl2, was then applied to the column. Rinsing with 0.02 M NH4Cl-0.02 N HCl removed all adenosine 5'-monophosphate (AMP), adenosine 5'-diphosphate (ADP), and inorganic pyrophosphate (P.) from the column. Extensive washing with water diluted out any remaining NH4+. Subsequent elution of the column with a small volume of 0.25 N HCl yielded a purified y-[32P]ATP solution that was suitable for the ATPase assay. ATPase assay. The standard assay mixture (2.0 ml) contained 100 mM Tris-hydrochloride, pH 7.4, 2 mM MgCl2, and 2 mM ATP, plus sufficient y[32P]ATP to give 100,000 cpm per assay. Various metal cations, substrate analogues, and other modifiers were added to this standard mixture as described for particular experiments. Conditions were established to ensure approximately 10% hydrolysis of substrate; this required 0.5 mg of protein and an incubation time of 60 min. The assay was stopped by the addition of 1.0 ml of cold 10% (wt/vol) trichloroacetic acid. Samples were then centrifuged at 2,000 x g for 10 min to remove the protein. Separation of released Pi from remaining ATP, ADP, and AMP was accomplished by complexing Pi with molybdate in an acid environment and extracting the resultant phosphomolybdic acid into an organic solvent (17). A 2.0-ml portion of supernatant obtained after precipitation of the protein with trichloroacetic acid was combined with 0.5 ml of 2.5% (wt/vol) ammonium molybdate in 3 N H2SO4. This was extracted twice with 2.5 ml of water-saturated isobutanol. Control studies demonstrated that extraction with two portions of isobutanol was specific for Pi and that 98% of the Pi present could be recovered in the organic phase. The two organic phases were mixed and 32 Pi determined on a Beckman model LS-100C liquid scintillation system using Aquasol (New England Nuclear Corp.) as the fluor. A control incubation lacking enzyme was performed with each assay, and all assays were performed at least in duplicate. Other assays. Protein was determined by the method of Lowry et al. (14), using the average results of bovine serum albumin and lysozyme as a standard. Lactate dehydrogenase (LDH) was quantitated by a spectrophotometric assay which monitored the oxidation of nicotinamide adenine dinucleotide, reduced form (decreasing optical density at 340 nm) as pyruvate substrate was converted to lactate (12). Cell viability was determined by the exclusion of trypan blue. Four parts of 0.2% (wt/vol) trypan blue in water was mixed with 1 part of 4.25% (wt/vol) NaCl in water immediately before viability studies. PMNL portions were mixed with trypan blue solution in leukocyte-diluting pipettes, and viability counts were determined with a hemocytometer.

RESULTS Initial studies were conducted to examine Mg2e-dependent ATPase activity in human

438

HARLAN ET AL.

INEECT. IMMUN.

PMNL homogenates as a function of both protein concentration and incubation time. As shown in Fig. 1 and 2, the ATPase assay was linear over a protein range from 0.2 to 2.0 mg and up to 60 min of incubation time. Differentiation of Mg2+-dependent ATPase from (Nat + K+)-sensitive, ouabain-inhibitable ATPase. (Na+ + K+)-sensitive, ouabaininhibitable ATPase is a transport enzyme which is generally believed to be a component of the plasma membrane of most mammalian cells. Since this enzyme also has an absolute requirement of Mg2+ ions a differential assay is inevitable. Bonting (1) reviewed the nature of this enzyme in various mammalian tissues and recommended certain assay conditions. He defined the (Na+ + K+)-sensitive, ouabain-inhibitable ATPase activity as the difference in ATP hydrolysis between a completed ion sys-

tem (60 mM NaCl + 5 mM KC1 + 2 mM MgCl2 + 0.1 mM ethylenediaminetetraacetate

[EDTA]) and the

average

of three control

sys-

tems (one that lacked KC1, one that lacked KC1 but contained 0.5 mM ouabain, and one that contained both KC1 and 0.5 mM ouabain). The

of these three controls was necessitated by some tissues the Mg2+-dependent ATPase activity was stimulated by Nat ions, and second the Km for K+ in the (Na+ + K+)-sensitive, ouabain-inhibitable ATPase was often so small that substantial activity was contributed by K+ endogenous to the tissue preparation. The controls allow for adequate monitoring of these factors. For both whole-cell suspensions and disrupted cells, there was no significant difference between the completed ion system and any of the controls (Table 1). Under the conditions defined in this report, (Na+ + K+)-sensitive, use

two observations. First, in

I 0.60-

1.00

0.40

.k 0.75 ~

~

~

R 0.50

0.20

z

15

30 Time (minutes)

45

60

Reaction no.

Assay mixture

I-

,

0.5

FIG. 1. Effect of incubation time on the activity of the Mg2+-dependent ATPase of human PMNL. Homogenates from two different PMNL preparations (open versus closed circles) were assayed for ATPase activity (as described under Materials and Methods) over a range of incubation times. Each point represents the mean ofat least duplicate determinations.

TABLE 1. Detection of (Na+

0.25

+

1.0 protein (mg)

1.5

2.0

FIG. 2. Effect of protein concentration on the Mg2+-dependent ATPase activity. ATPase activity was measured at several protein levels on each of two separate PMNL homogenates (open and closed circles). Each point is the average of duplicate determinations.

K+)-sensitive, ouabain-inhibitable ATPasea Whole cells no. 1

Homo-

Sonic

genate no. 1

extract no. 1

Whole. cells no. 2

Homogenate no. 2

+5 mM KCl (total system) 0.466 0.487 0.336 0.516 0.586 -5 mM KCl 0.504 0.528 0.333 NDb 0.579 +5 mM KCl + 0.5 mM ouabain 0.452 0.516 0.318 ND 0.583 -5 mM KCl + 0.5 mM ouabain 0.421 0.516 0.324 0.492 0.498 Avg of 2, 3, 4 0.459 0.520 0.325 0.492 0.553 a The ATPase assay was carried out as described in the text except that 60 mM NaCl and 0.5 mM EDTA were added to each mixture. Further assay modifications are indicated for particular reactions. Since (Na+ + K+)-sensitive ATPase also requires Mg2+, a differential assay of ATPase activities is required. The activity of the (Na+ + K+)-sensitive, ouabain-inhibitable ATPase is defined as the ATPase activity of the total ion system (Mg2+ + Na+ + K+) less the average activity of three appropriate controls. Each number is the mean of triplicate determinations and is expressed as specific activity (micromoles of ATP cleaved per milligram per hour). b ND, not determined. 1 2 3 4 5

VOL. 15, 1977

Mg2+ ATPase IN HUMAN PMNL

ouabain-inhibitable ATPase did not comprise a significant percentage of the total ATPase activity. Substrate specificity. Due to the existence of both acid and alkaline phosphatases in the human PMNL (3), it appeared necessary to demonstrate that this assay was specific for ATP hydrolysis and was not due to hydrolysis by nonspecific phosphatases. The addition of 5 mM p-nitrophenyl phosphate (a preferred substrate for alkaline and acid phosphatases) had no effect on the ATPase activities of whole cells, homogenates, or sonic extracts (Table 2). Table 2 also illustrates that 5 mM ,B-glycerophosphate (an alternate substrate for nonspecific phosphatases) or 5 mM tartrate (an inhibitor of acid phosphatase) had no significant effect on the measurable ATPase. Cation requirements. The ATPase activity as a function of the concentration of MgC12 is illustrated in Fig. 3. It can be seen that a molar ratio of 1:1 for ATP/Mg2+ was optimal for this reaction. Moreover, Mg2e appears to be an ab-

100

_

8,0

1.0

3.0

50 mg

IQO C2 (mM)

15.0

2Q0

FIG. 3. Effect of Mg2+ concentration on ATPase activity. Standard assay conditions were used (see Materials and Methods) except for altered levels of MgCl2 in the incubation mixture. Each point is the average of triplicate determinations.

439

solute requirement for enzymatic activity, since the addition of 4 mM EDTA to the standard assay mixture inhibited the hydrolysis of ATP by 95% (Table 3). Other divalent cations could apparently substitute for Mg2+ but were less efficient. Table 3 reveals their effectiveness (Mg2+ > Mn 2+ > Ca 2+ > Zn2+). As increasing concentrations of Ca 2+ were added to the assay in the presence of 2 mM Mg2e, the resulting specific activities progressively decreased from that seen with Mg2+ alone and approached the activity observed with Ca2+ alone (data not shown). pH optimum. A typical pH curve for the Mg2+-dependent ATPase is presented in Fig. 4. In three separate experiments the pH optimum was observed between 8.5 and 9.0. Specific activities decreased as pH conditions approached those defined for acid and alkaline phosphatases. In one experiment, the pH curve for ATPase activity was run in the presence and absence of 5 mM p-nitrophenyl phosphate. The presence of this compound did not affect the measured enzyme activity at any pH value tested, in agreement with the results presented in Table 2 (data not shown). Enzyme kinetics. The Km of the Mg2+-dependent ATPase activity was 5.9 x 10-4 M, with an average deviation of 0.7 x 10-4 M, as determined from three separate experiments each run in triplicate. Figure 5 presents a TABLE 3. Effect of divalent cations on ATPase activity Divalent cationa % Activity 2 mM MgCl2 100 2 mM MnCl2 74 2 mM CaC12 50 2 mM ZnCl2 32 4 mM EDTA 4 2 mM MgCl2 + 4 mM EDTA 6 a Various divalent cations and EDTA were substituted for 2 mM MgCl2 in the standard ATPase assay. Values presented are the average of triplicate determinations.

TABLE 2. Effect of various compounds on Mg2+-dependent ATPasea Reaction mixture

Whole cells

Homogenate

Sonic extract

No additions 0.540 + 0.078 (4) 0.560 ± 0.060 (5) 0.336 (1) +5 mMp-nitrophenyl phosphate 0.627 (1) 0.586 ± 0.039 (4) 0.337 (1) +5 mM B-glycerophosphate NDb ND 0.343 (1) +5 mM Na-K tartrate ND ND 0.346 (1) a The usual assay conditions for ATPase were used with added substrate analogues as described for particular reactions. Values represent specific activities in micromoles of ATP cleaved per milligram per hour. Data are presented as mean + standard deviation (number of experiments). The value from each experiment is the result of triplicate determinations. b ND, not determined.

440

HARLAN ET AL.

INFECT. IMMUN.

[

I

0.80 _ 0.70-

~0.60

i0.50 k0.400.30

8o

7.0

6.0

0.0

50

11.0

pH

FIG. 4. Effect ofpH on the Mg2+-dependent ATPase. This represents a typical pH curve for ATPase activity under described assay conditions. Tris-maleate was used as the buffer from pH 6.0 to 7.0, whereas Tris-hydrochloride was used from pH 7.0 to 9.0, and 2-amino-2-methyl-1-propanol-HC1 from pH 9.0 to 10.5. All buffer concentrations were 100 mM. The buffers themselves did nkt effect the ATPase activity as indicated by the fact that the points ofpH overlap between two buffer systems consistently yielded the same specific activities. This figure is one of three separate experiments with each point the result of duplicate trials. The pH optimum was always between 8.5 and 9.0.

ize the site of ATP hydrolysis in the human PMNL. Table 4 compares the ATPase activity of whole-cell suspensions to that observed in homogenates and sonic extracts. A Student paired t test revealed that there was no significant difference (P > 0.05) between parallel determinations on homogenates and whole cells from the same donor. The decreased activity in sonic extracts may represent enzymatic denaturation or the trapping of membrane fragments within small vesicles formed during such a rigorous disruption procedure. The addition of increasing concentrations of Mg2+ to the sonic extract (up to 10 mM) did not increase the activity (data not shown). The possibility that the ATPase activity observed in intact PMNL could be due to cell lysis during the course of the incubation was examined. Two methods were used to determine the status of the whole-cell suspensions under the assay conditions. Utilizing trypan blue exclusion as a monitor for viability (11) and quantitating the cell counts with a hemocytometer, it was possible to assess both cell viability and cell lysis by making determinations before and after the assay incubation. Table 5 demonstrates that 93% of the cells remained intact TABLE 4. Mg2+-dependent ATPase of intact cells compared with disrupted cellsa Sample

Whole cells

Homogenate

Sonic extract

Mg2+-depen- 0.530 + 0.099 0.594 ± 0.074 0.308 ± 0.029 dent ATPase (9) (11) (3) a Values are obtained the by described ATPase procedure and represent specific activity in micromoles of ATP cleaved per milligram per hour. Data are presented as mean ± standard deviation (number of experiments). Each point is the average of at least duplicate determinations in each experiment.

T a4

as

I/ [SI

12

1.6

2.0

FIG. 5. Lineweaver-Burk plot demonstrating enzyme kinetics for the Mg2+-dependent ATPase. The substrate concentration for Mg2t-ATP (S) is given as millimolar and the reaction velocity (V) is expressed as micromoles of ATP hydrolyzed per milligram of protein per hour. The described assay conditions were used with the amount of[y-32P]ATP varied proportionately with the Mg2t-ATP concentration to avoid isotope dilution effects. This is one of three experiments with each point determined in triplicate.

typical Lineweaver-Burk plot from which these values were derived. Higher concentrations of substrate (>3 mM) appeared to inhibit the enzyme.

Once the characteristics of the enzyme were established, we initiated experiments to local-

TABLE 5. Status of intact PMNL after ATPase assay' Quantitative trypan blue No. of expt

Cells intact (%)

Cells viable (%)

Cellassociated LDH

(%) 92 + 4 92 + 3 93 + 3 a Portions of intact PMNL were evaluated for their integrity before and after a 60-min incubation at 370C in an ATPase assay mixture. Trypan blue exclusion was used to monitor viability while cell lysis was determined by quantitative cell counts of these aliquots on a hemocytometer. LDH was also used as a measure of cell viability. The cells were centrifuged at 170 x g for 10 min and LDH activity was assayed in the cell pellet and supernatant. Each point is the average of triplicate determinations. Data are presented as the mean + average deviation.

3

VOL. 15, 1977

Mg2+ ATPase IN HUMAN PMNL

and 92% of the intact cells were viable. These results correlated well with observations that utilized LDH (a known cytoplasmic enzyme) as a monitor of cell integrity. When whole-cell suspensions were incubated and then centrifuged at 185 x g for 10 min to pellet the cells, 92% of the LDH remained cell associated. These data suggest that the PMNL remained intact and viable under the conditions of the Mg2+-dependent ATPase assay. The Mg2e-dependent ATPase activity of intact PMNL was then measured as a function of the time of incubation. The linear relationship observed (Fig. 6) would not be anticipated if cell lysis or transport of ATP were occurring during the course of the assay. To more closely examine the possibility that the ATPase activity of whole cells was due to the release of an intracellular enzyme during the assay without consequent cell lysis or cell death, the following experiment was carried out. Whole-cell suspensions were incubated in buffer under isotonic conditions at 370C for up to 2 h and then centrifuged to pellet the intact cells. Only 7% of the total ATPase activity was found to be released into the supernatant. This activity correlated well with the fact that these suspensions released 6% of the total LDH activity. To assess the distribution of y-[nP]ATP and 32Pi during the ATPase assay, the following experiment was devised. A whole-cell suspension that yielded an intracellular volume which was greater than 5% of the total assay volume was incubated under assay conditions hydrolyz0.40

441

ing greater than 20% of the added substrate. [14C]sucrose, a reliable extracellular marker for human PMNL suspensions, was also added to the assay mixture. At the end of the incubation, the cells were centrifuged at 185 x g for 6 min. The supernatant was removed and the cell pellet was digested overnight in 0.10 N HNO3. Differential counts for 14C and 32p were then performed in portions of the supernatant and digested pellet. Essentially no 32P (80% y[32P]ATP + 20% 32P1 of 100,000 added cpm) was cell associated (Table 6). Two experiments are presented in Table 6, each of which was performed in triplicate, with an appropriate control incubated at-00C for 60 min. We ran one experiment using a higher cell concentration such that the cell volume was 20% of the total incubation volume. Under these conditions, the total hydrolysis of [32P]ATP was determined to be greater than 90%. The inorganic phosphate was extracted from both the cell pellet and supernatant as described in Materials and Methods. Only 7% of the inorganic 32P was cell associated; this corresponded exactly with the extracellular marker in that 7% of the [14C]sucrose was likewise found in the cell pellet (data not shown). DISCUSSION The first part of the research was designed to characterize the nature of the Mg2+-dependent ATPase and the specificity of the assay. (Nat + K+)-sensitive, ouabain-inhibitable ATPase did not contribute significantly to the total ATPase activity of intact or disrupted human PMNL. One might not expect to see (Nat

-

TABLz 6. Localization of[y-32P]ATP and 32pJ during the ATPase assays

0.30

% cpm in super-

natant (32P/14C)

% cpm in cell pellet (32P/'4C)

Sample 0.20

0.10

time (minutes)

FIG. 6. Effect of incubation time on Mg2+-dependent ATPase in intact PMNL. Standard ATPase assays were conducted on whole-cell suspensions at different incubation times using an amount of cells equivalent to 0.8 mg ofprotein. The experiment illustrated is representative of three separate experiments; each point was determined in triplicate in each experiment.

Expt

Expt

Expt

Expt

no. 1 97.8/97.8

no. 2

no. 1

no. 2

0C x 60 min 98.4/98.4 2.2/2.2 1.6/1.6 37TC x 60 min 97.6/97.6 98.0/98.0 2.4/2.4 2.0/2.0 a Sufficient PMNL (2 x 108 to 3 x 106 cells) were added to ATPase assay mixtures to give intracellular volumes which were >5% of the total assay volumes. The experiment was set up with one mixture incubated at 37C for 60 min (20% hydrolysis of ATP) while a control was incubated at 4C for 60 min (0% hydrolysis of ATP). [t4C]sucrose (100,000 cpm) was added as a marker for extracellular volume. At the end of the incubation, the cells were centrifuged at 170 x g for 6 min, and the supernatant was separated from the pellet. The cell pellet was digested in 0.1 N HNO3 overnight. Differential counts for 14C and 3P were then made on the supernatant and digested cell pellet. Two experiments were run with identical results. Each point is the average of triplicate trials.

442 HARLAN ET AL. + K+)-sensitive, ouabain-inhibitable ATPase in whole-cell suspensions, since this enzyme typically has its active site on the cytoplasmic surface of the plasma membrane and ATP does not readily penetrate normal mammalian membranes. The observation of similar activities in whole and broken PMNL preparations minimized the likelihood that such an enzyme contributed substantially to the total ATPase of the PMNL. This is consistent with the observation that this enzyme is characteristically a small percentage of total ATPase in nonsecretory, non-neural tissues (1). The existence of a (Na+ + K+) ATPase has been previously reported in the human PMNL, but it consitituted only a small fraction of the total ATP-hydrolyzing ability when assayed spectrophotometrically for release of Pi (13). The additions ofp-nitrophenyl phosphate and ,3-glycerophosphate to incubation mixtures gave no indication of a competitive inhibition, which would be anticipated if the observed hydrolysis of ATP were due to nonspecific phosphatases (Table 2). The phosphatase inhibitor tartrate also had no effect on ATPase activity. These observations led to the conclusions that the enzyme being measured was not alkaline or acid phosphatase and that its affinity for ATP was not affected by the presence of general phosphatase substrates. Certain facts about the Mg2+-dependent ATPase of human PMNL are consistent with the nature of other Mg2e-dependent ATPases which have been reported. A divalent cation was essential for activity; Mg2e best fulfilled this cation requirement, with a Mg2e ATP molar ratio of 1:1 giving optimal activity. The pH curve was broad with an optimum between 8.5 and 9.0. This pH profile may be contrasted to the (Nat + K+)-sensitive, ouabain-inhibitable ATPases that give narrower pH curves with distinct optima near 7.5 (1). In other reports on PMNL from various sources, investigators have studied ATPase activities in the presence of Mg2+ compared with Ca2+ (4). The current data suggest that any measurable Ca2+-dependent ATPase in intact or homogenized human PMNL may be due to substitution for Mg2+, since Mg2+- and Ca2+ATPase activities were not additive. When progressive additions of Ca2+ were made to the standard ATPase assay mixture (2 mM Mg2+ + 2 mM ATP), the specific activity shifted from that seen with Mg2+ alone toward that observed with Ca2+ alone. The possibility that the Mg2+-dependent ATPase of the human PMNL might be located in the plasma membrane ofthe cell was investigated when it was observed that no significant

INFECT. IMMUN.

difference existed between the ATP-hydrolyzing activity of whole cells and homogenates (Table 4). The finding that hydrolysis by intact cells was linear for at least 60 min was likewise consistent with this hypothesis. To validate such a proposal, it was necessary to demonstrate that the PMNL remained intact and viable during the course of the assay, that the ATPase remained cell associated, and that ATP did not penetrate the cell before or during hydrolysis. Two independent methods were used to monitor the integrity ofthe cells. Trypan blue staining utilized a histochemical basis and the cell association of the cytoplasmic enzyme LDH provided biochemical criteria. Both methods confirmed that greater than 92% of the added PMNL were intact and viable at the conclusion of the assay. It was considered important to rule out the possibility that an intracellular ATPase was being released from the intact cells during the assay, without actual loss of viability. The small amount of ATPase activity that was no longer cell associated after an assay incubation was found to equal extracellular LDH, suggesting that cell lysis was the only mechanism for release of the enzyme activity. Since the PMNL were found to remain intact and maintain enzymatic integrity, the only mechanism for an intracellular ATP hydrolysis would involve penetration of the cell by the substrate. To examine this possibility, the distribution of y-[32P]ATP and 32P, was monitored under assay conditions in which the intracellular volume comprised a significant fraction of the total assay volume. If intracellular hydrolysis or transport of ATP were occurring, one would expect to find cell-associated radioactivity when the assay was interrupted under linear conditions of hydrolysis. Such intracellular activity was not observed (Table 6). ATP hydrolysis by whole-cell suspensions has been observed in various tissues (7). Such "ecto-ATPase" activities have been reported in PMNL from guinea pig, rat, and rabbit sources. DePierre and Karnovsky characterized the ecto-enzymes of PMNL from guinea pig peritoneal exudates (9, 10). In contrast to the situation in human PMNL, they described an ectoATPase that comprised 50% of the assayable Mg2+-dependent ATPase of disrupted cells. The nature of this enzyme was quite similar to the ATPase we have described for the human PMNL, except that the Km for ATP in the guinea pig cells (0.03 mM) was substantially lower than that observed in human PMNL (0.6 mM). A Mg2e-dependent ATPase (Km for ATP =

VOL. 15, 1977

0.3 mM) has been reported in preparations of intact rat leukocytes (15). The specific activity of the enzyme was observed to decrease with cell disruption by homogenization, freeze-thaw, or sonication. No other attempts were made to localize the site of this enzyme. Coffey et al. studied ATPase activities in asthmatic children and reported a Mg2+-dependent ATPase in intact PMNL (4). They reported data on three adult controls, two of which had specific activities that fell within the 1-standard-deviation range of the activities reported in this paper. The results of the third control deviated 2 standard deviations from our values. Assay conditions were comparable. DePierre and Karnovsky provide an excellent summary of the possible functions for an ecto-enzyme (10). The data in the present paper are consistent with the hypothesis that the Mg2e ATPase of human PMNL is an ecto-enzyme, although absolute proof ofthis is lacking. No definitive statement can be made at this time concerning the role of a possible ecto-ATPase in the human PMNL. The ecto-ATPase activity of human platelets (2) and the tuftsinactivating ecto-aminopeptidase of the human PMNL (16) suggest potential roles for ecto-enzymes in the regulation of inflammation and

coagulation. The major thesis of this report is that the Mg2+-dependent ATPase activity of human PMNL appears to be a reliable marker enzyme for the plasma membrane. Because of the numerous surface phenomena of the PMNL (locomotion, chemotaxis, phagocytosis), particular interest has been directed toward evaluation of its plasma membrane. It is hoped that by coupling this marker enzyme with morphological monitoring and physical membrane probes it will be possible to isolate an enriched plasma membrane fraction from the human PMNL. ACKNOWLEDGMENTS This research was supported by a grant from the Forsyth Cancer Service and by Public Health Service grants AI10732 and AI-09169 from the National Institute of Allergy and Infectious Diseases, HL-16769 from the National Heart and Lung Institute, and CA-12197 from the National Cancer Institute. McCall is the recipient of Research Career Development award AI-70767 also from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Bonting, S. L. 1970. Sodium-potassium activated adenosinetriphosphatase and cation transport, p. 257-363. In E. E. Bittar (ed.), Membranes and ion transport, vol. 1. Wiley-Interscience, London. 2. Chambers, D. A., E. W. Salzman, and L. L. Neri. 1967. Characterization of ecto-ATPase of human blood platelets. Arch. Biochem. Biophys. 119:173-178. 3. Cline, M. J. 1965. Metabolism of the circulating leukocyte. Physiol. Rev. 45:674-720. 4. Coffey, R. G., J. W. Hadden, and E. Middleton, Jr. 1974. Increased adenosine triphosphatase in leuko-

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