A Comparative Study of Superoxide Dismutase Activity in Poiymorphonuclear Leukocytes, Monocytes, and Alveolar Macrophages of the Guinea Pig MANFRED RISTER2J AND ROBERT L. BAEHNER Division of Pediatric Hematology-Oncology, James Whitcomb Riley Hospital for Children and the Department of Pediatrics, Indiana University School of Medicine, Indianapolis, Indiana 46202
ABSTRACT Superoxide dismutase, an enzyme which catalyzes the dismutation of superoxide radical formed during the univalent reduction of oxygen, was quantitated by observing the inhibition of cytochrome C reduction in three cell fractions in guinea pig peritoneal P M N s and monocytes and compared to alveolar macrophages. No differences were found in the 16,000 x g pellets containing mitochondria, membranes, and granules and representing 96% of total SOD activity in PMNs and monocytes but only 48% total SOD activity in alveolar macrophages. The 100,000 x g microsomal pellet of alveolar macrophages contained 8% of total SOD activity and two-five times more activity than the respective fractions from monocytes and PMNs. However, there was 70 times more SOD in the 100,000 x g supernatant from alveolar macrophages containing 44% of total enzyme activity than in the same fraction of P M N s and monocytes containing less than 2 % total SOD activity. SOD activity is mainly located in the 16,000 x g particulate fraction of P M N and monocytes but more equally distributed between the particulate fractions and cytosol of alveolar macrophages. Superoxide dismutase, an enzyme found in all aerobic organisms but lacking in strict anaerobes (McCord et al., '71), catalyzes the following reaction: 0;
+ 0; + 2H+-
b HzOz
+ 02
It is assumed that the physiological function of superoxide dismutase (SOD) is to protect the cell agajnst highly reactive superoxide radical (Oi) which is generated during the univalent reduction of oxygen (Fridovich, '72) (Saltzmann and Fridovich, '73). Two kinds of SOD are found in most cells; one located in the cytosol is cyanide sensitive and contains copper and zinc whereas the other, associated with the mitochondria, is cyanide insensitive and contains manganese (McCord and Fridovich, '69; Weisiger and Fridovich, '73). Although the presence of SOD has been confirmed in a wide variety of species (Beckmann et al., '73; DeChatelet et al., '74; Patriarca et al., '74; Salin and McCord, '74; Stansell and Deutsch, '65; Winterborn et al., '75), a study comparing SOD activity in various tissues from the same species has not been performed. Since peritoneal cavity and lung tissues J. CELL.PWYSIOL., 87: 345-356.
are exposed to different tensions of oxygen, SOD activity of PMNs, monocytes, and alveolar macrophages from guinea pigs was compared to determine whether environmental differences in oxygen concentration may influence SOD activity. Furthermore, since alveolar macrophages, in contrast to PMNs and monocytes, contain prominent mitochondria and demonstrate a biological dependence on oxidative phosphorylation, we also compared the sensitivity of SOD activity to cyanide in the three types of cell sonicates. EXPERIMENTALPROCEDURE
Preparation of P M N s , peritoneal, and alveolar macrophages PMNs and peritoneal macrophages ("monocytes") were obtained by a method previously described (Oren et al., '63). Received May 20, '75. Accepted Sept. 18, '75. 1 This project was supported by National Institutes of Health Grant Number AI-10892-02 and a grant from the Riley Memorial Association. 2 Dr. Rister is the recipient of Deutsche Forschungsgemeinsch aft. 3 Address reprints to: Dr. Manfred Rister, Division of Pediatric Hematology-Oncology, James Whitcomb Riley Hospital for Children, 1100 West Michigan Street, Indianapolis, Indiana 46202.
345
346
MANFRED RISTER AND ROBERT L. BAEHNER
PMNs were harvested 18 hours after injection of 12% caseine into the peritoneal cavity of guinea pigs; “monocytes” were obtained three and one-half days after intraperitoneal injection of 1.2% caseine. Alveolar macrophages were recovered from the same guinea pigs by a modification of the technique described by Maxwell (’64). The upper portion of the trachea was exposed by dissection and a polyethylene catheter attached to a three-way stop cock was inserted in the transected trachea. Two 60 ml Luer lock syringes were then attached to the stop cock. According to the method of Brain and Frank (’73), one was filled with buffered saline pH 7.4 warmed to 37°C. Aliquots of 5 to 10 mls were injected into the lungs and the stop cock valve was then adjusted to aspirate the macrophage-rich fluid from the lung into the other syringe. Cell suspensions were centrifuged at 200 X g at 4 ° C and washed thrice in cold Krebs-Ringer-phosphate buffer (KRP) pH 7.4. Between the second and third washing, red cells were lysed by exposure to deionized water for 20 seconds. Isotonicity was then restored by the addition of an appropriate volume of 3.5% saline. The “monocyte” and alveolar macrophage suspension was freed of contaminating PMNs by a Ficoll-Hypaque gradient
(Boyum, ’67). Using this method, a purity and viability assessed by trypan blue of greater than 90% was achieved for each type of cell preparation. This was documented on a smear stained with Wright’s and monocyte-specific alpha naphthyl esterase stains (Rosenszajn et al., ’68) (figs. 1, 2). The final samples of PMNs and “monocytes” were resuspended in KRP buffer to a concentration of either 2 X 1 0 7 cellslml or 2 x 10*cellslml. Since 20 times more PMNs and “monocytes” than alveolar macrophages were required for a single SOD assay, the SOD activity from a contamination of 10% PMNs in the final alveolar macrophage suspension would not be detectable.
Preparation of cell sonicate fractions The cells were sonicated at 40°C for one minute with a Sonifier Cell Disruptor Model W 140 by Branson Sonic Power Co. with an output control setting of 3 . They were centrifuged a t 16,000 X g for 15 minutes at 4 “C. The resulting supernatant was centrifuged at 100,000 x g for one hour in a Beckman Ultracentrifuge, Model L3-50 with a fixed angle rotor, type 30. The 16,000 X g and 100,000 X g pellets were resuspended in ice-cold 0.2% (volurnelvolume) Triton X 100 to lyse mito-
Fig. 1 Final monocyte preparation stained with monocyte specific alpha naphthyl esterase stain; X 1,200.
34 7
SOD ACTIVITY IN GUINEA PIG LEUKOCYTES
chondria, lysosomes, and other cell organelles. Both the supernatant and the solubilized pellets were assayed for cyanide sensitive and cyanide insensitive SOD activity. The protein concentration of each cell fraction was determined by the Folin phenol reagent procedure of Lowry with human albumin as a standard (Lowry et al., '51). Preparation of cell homogenates P M N s and alveolar macrophages were resuspended in ice-cold buffered sucrose at pH 7.4 and homogenized for four minutes until a cell breakage greater than 99% was achieved. The homogenates were centrifuged and treated with Triton X 100 like the sonicates.
Electron microscopy studies Electron micrographs were prepared from the 16,000 X g and 100,000 X g pellets by fixing them in 4 % glutaraldehyde in phosphate buffer pH 7.2 for two hours and then placing them in 1% osmium overnight. After dehydrating them with ethanol and propylene oxide, they were embedded in Epon 812 according to the Luft method (Luft, '61). Sections were prepared on a LKB ultratome, stained with uranyl acetate and lead citrate stain, and examined on a Phillips 300 electron microscope.
Fig. 2
Superoxide dismutase assays Since we failed to obtain reproducible results with either the inhibition of the auto-oxidation of epinephrine (Misra and Fridovich, '72) or with the inhibition of' the nitroblue tetrazolium reduction (Nashikimi et al., '72), all data reported in this paper were obtained with an assay method of McCord and Fridovich ('69). This assay depends upon the capacity of SOD to inhibit cytochrome C reduction mediated by superoxide radical (02) generated during the oxidation of xanthine catalyzed by xanthine oxidase. Xanthine grade 3, xanthine oxidase grade 4, and horse heart cytochrome C type VI were obtained from Sigma Chemical Corp., St. Louis, Missouri. The assays were performed at room temperature in 0.05 M potassium phosphate buffer pH 7.8 contain-ing 0.1 mM EDTA. Superoxide radical (02) was generated by incubating 5 X 1 0 - 5 M xanthine with 0.01 units of xanthine oxidase. Since SOD and cytochrome C both compete for 02 generated in this solution, maximum activity of the SOD is obtained at low cytochrome C concentrations (Salin and McCord, '74). In preliminary experiments, we determined that 5 pM cytochrome C was most sensitive for our assay. Purified bovine SOD obtained from Truett Laboratories, Dallas, Texas was used a s the
Alveolar macrophages with esterase stain;
X
1,200.
348
MANFRED RISTER AND ROBERT L. BAEHNER
standard. The change of the optical density was read a t 550 nm with the Gilford Model 2400 recording spectrophotometer with ten-fold scale expansion. Each reaction mixture contained 1 mM sodium azide to inhibit cytochrome C oxidases which otherwise would decrease the reduction of cytochrome C which, in turn, would produce a f a h e inhibition rate of cytochrome C reduction resulting in a false elevation of SOD activity. Total SOD activity was measured without cyanide in PMNs and “monocytes” and the manganese containing enzyme was measured by observing the remaining SOD activity after addition of 1 mM cyanide to the reaction mixture. In contrast, a greater inhibition of cytochrome C reduction was obtained in cell fractions of alveolar macrophages treated with 1 mM cyanide. For this reason, in alveolar macrophage fractions, cyanide insensitive SOD was measured at the final cyanide concentration of 1 pM. A progressive increase of cyanide concentration from 1 pM to 1 mM produced a higher inhibition of cytochrome C reduction which was independent of the concentration of SOD. This could be explained by a cyanide-ferricytochrome C complex which occurs at equimolar concentrations (Horecker and Kornberg, ’46). In preliminary studies we determined that 0.01 pM SOD/ml, a n amount in excess of that found in our samples, inhibited the cytochrome C reduction to 65 & 2 % . Since 1 mole of SOD binds two moles of cyanide, the binding capacity of 1 pM cyanide is 0.5 p M SOD. This amount of SOD is approximately 50 times in excess of that amount usually found in our assayed samples. Therefore, the cyanide concentration is sufficient to inhibit the cyanide sensitive SOD found in our samples. In a xanthine-xanthine oxidase free reaction mixture, we observed no reaction between the cell sonicates and homogenates, cytochrome C and cyanide. The difference between total SOD activity and cyanide insensitive SOD activity was calculated as cyanide sensitive SOD activity. Whereas assays of the 100,000 X g supernatant of the alveolar macrophage required 2 x 1 0 6 cells/ml, 2 X 108 cells/ ml of PMNs and “monocytes” were needed for a single assay of SOD activity in the 100,000 X g supernatant.
Beta g lucuronidas e Beta glucuronidase was assayed with p-nitrophenyl-beta-D-glucuronide(Sigma Chemical Co., St. Louis, Mo.) as a substrate (Michell et al., ’70). The reaction mixture contained a final concentration 50 mM sodium acetate buffer, pH 5 . 0 , O . 1% (volume/volume) Triton X 100, 1 mM nitrophenyl-beta-glucuronide, and between 0.02 mg and 0.2 mg of enzyme protein. Incubations were in a total volume of 1 ml for two hours at 37°C. The reaction was stopped by the addition of 2 ml of 0.1 N sodium hydroxide and the optical density determined at 410 nm. Results were calculated by using a molar extinction coefficientof 1.70 X lo4. Succinate dehydrogenase This enzyme was assayed using p-iodonitrotetrazolium violet as electron acceptor (Michell et al., ’70). The assay medium contained at final concentration: 1% (weight/volume) iodonitrotetrazolium violet, 50 mM sodium succinate, pH 7.4, 50 mM potassium phosphate buffer, pH 7.4, 2 mM sodium EDTA, and 0.1-0.5 mgs enzyme protein. Incubations were in a total volume of 1 ml for 45 minutes at 37°C. The reaction was stopped with 1 ml 50% TCA. The formazan was extracted with 1.5 ml ethyl-acetate using a Vortex mixer. Optical densities were measured at 490 n m and results calculated using the extinction coefficient of 20.1 X 103. All assays were corrected for blanks in which succinate and enzyme were omitted. RESULTS
All SOD activity found in each homogenate was compared to the amount of protein which caused a 30% inhibition of cytochrome C reduction. In our system three units bovine erythrocyte SOD (0.1 mcg of bovine erythrocyte SOD/ml reaction mixture) was needed for 30% inhibition. Knowing the amount of protein per reaction mixture, we were able to calculate and express the results as units SOD/ milligram protein. The protein distribution in the different cell fractions was similar between PMNs, “monocytes,” and alveolar macrophages. Forty-eight percent & 3% of total recovered protein was found in the
SOD ACTIVITY I N G U I N E A P I G L E U K O C Y T E S
Fig. 3 16,000 x mitochondria.
Fig. 4 16,000 are present.
g pellet
X g
of alveolar macrophages; X 31,250; note several damaged
pellet of polymorphonuclear leukocytes;
X
25,000; less mitochondria
349
350
MANFRED RISTER AND ROBERT L. BAEHNER
16,000 X g pellet, 12 & 100,000 X g pellet, and 40 supernatant.
2 % in the 4% in the
?
PMNs The 16,000 X g pellet of the PMNs obtained from a cell preparation of 2 x l o x cellslml was found to be very turbid and this turbidity prevented a sensitive enzyme assay. For this reason, we determined SOD activity in this fraction prepared from a 2 X lo7 cell preparation. In that case, we were able to assay SOD activity and found 16.2 & 1.2 units SOD/ mg of protein of this fraction. After addition of cyanide an identical protein concentration produced the same inhibition rate, indicating that all of the SOD activity was insensitive to cyanide, a characteristic of the mitochondria1 manganese enzyme. Ninety-six percent of total recovered SOD was found in this cell fraction. Greater than 1 mg of protein was needed to assay SOD activity in the 100,000 x g pellet and required a cell preparation of 2 X 107/ml. This pellet con-
tained 1.2 & 0.12 units SOD/mg of protein and with cyanide 0.9 i 0.18 units SOD/ mg protein, which represents 2.7% of total recovered SOD activity. Greater than 10 mgs protein was needed for detection of SOD activity in the 100,000 X g supernatant and required a cell preparation of 2 X 10*cells/ml; only 0.177 & 0.003 units SOD/mg protein without cyanide and 0.147 i 0.006 units SOD/mg protein with cyanide was found in the supernatant. Although these differences are significant (p < 0.01, student’s t-test) the 17% inhibition of SOD activity by cyanide indicates that SOD activity present in this supernatant fraction was largely the manganese containing enzme. Only 1.3% of total recovered SOD activity was found in the cytosol representing cell fraction.
“Monocytes” Similar to P M N , assay of SOD activity in the 16,000 X g pellet required a monocyte concentration of 2 X lo7 cell/ml and contained 28.5 k 1.5 units SOD/mg pro-
Fig. 5 100,000 X g pellet of polymorphonuclear leukocytes; x 20,500; no mitochondria are present.
351
SOD ACTIVITY I N GUINEA PIG L E U K O C Y T E S
*
represented 14% of total SOD activity and contained 4.65 0.75 units SOD/mg protein without cyanide and 4.8 1.2 units SOD/mg protein with cyanide, indicating that the predominating enzyme activity was the mitochondrial manganese SOD. The 100,000 X g supernatant of the macrophages contained very high activity of both cyanide sensitive and insensitive SOD and 38% of total recovered SOD activity. Without cyanide 13.44 0.69 units SOD/ mg protein and with cyanide 8.58 0.6 units SOD/mg protein was found in the supernatant. Thirty-six percent of total activity was cyanide sensitive. To determine the effect of sonication on the distribution and activity of SOD we assayed the enzyme activity in homogenized cells. In each cell fraction of P M N s and alveolar macrophages there was the same cyanide sensitive and insensitive SOD activity as found in the cell sonicates. Table 1 summarizes these results and indicates the units SOD/rng protein and the Alveolar macrophages percentage of total recovered SOD in the Alveolar macrophages showed the high- various cell fractions. In addition, the effect of sonication on est SOD activity. For a single assay, more than 50 pg protein from the 16,000 X g a known granule enzyme, p-glucuronidase, pellet and a cell concentration of 2 X 106/ and a known mitochondrial enzyme, suc1.2 units SOD/mg cinate dehydrogenase, was also determined. ml was needed; 21.9 protein without cyanide and 21.42 0.9 Beta glucuronidase units SOD/mg protein with cyanide was found in the 16,000 X g pellet which conThe 16,000 X g pellet of the macrotained 48% of total recovered SOD activity. phage contained 8 2 % , the 100,000 x g Again, there was no significant difference pellet 8 % , and the supernatant 10% of between the results performed with and total recovered beta glucuronidase activwithout cyanide. The 100,000 X g pellet ity. In the PMNs, only 6 % was found in
tein and 25.8 1.5 units SOD/mg protein after addition of cyanide indicating that all SOD activity was cyanide insensitive. More than 500 pg protein and a cell concentration of 2 X 107/ml was needed to detect SOD activity in the 100,000 X g “monocyte” pellet which had 2.25 & 0.15 units SOD/mg protein without cyanide and 0.3 units SOD/mg protein with 2.49 cyanide. There was no significant difference between the inhibition rates with and without cyanide. The 100,000 X g supernatant contained 0.06 i 0.01 units SOD/mg protein and required 2 X 108 monocytes/ml for 30% inhibition. All of the SOD activity found in the 100,000 X g supernatant of the monocytes was cyanide sensitive. The distribution of SOD activity was similar to the PMNs; 97% of total recovered SOD activity was found in the 16,000 X g pellet, 2 % in the 100,000 X g pellet, and only 1% in the supernatant .
*
*
*
*
*
*
*
TABLE 1
U n i t s SODImg protein in cell f r a c t i o n s of P M N , “monocytes” and alveolar macrophages 16,000 X g
pellet
PMN No cyanide With cyanide 70
’
“Monocytes” No cyanide With cyanide % ’
Alveolar macrophages No cyanide With cyanide % I 1
100,000
x
9
pellet
100,000 x g supernatant
16.2? 1.2 16.22 1.2 96
1.2k0.12 0.9f 0.18 2.7
0.177f 0.009 0.147f 0.006 1.3
28.5f 1.5 25.8f 1.5 97
2.25f0.15 2.49-+- 0.30 2
0.18f 0.003 no activity 1
21.90f 1.2 21.42k0.9 48
4.65f 0.75 4.80f 1.20 14
Percent of total recovered SOD-activity
13.44f 0.69 8.58f 0.60 38
352
MANFRED RISTER AND ROBERT L. BAEHNER
and three times more than “monocytes” (fig. 6). The difference is highly significant and is due to the larger amount of SOD activity found in the cytosol of alveolar macrophages, since the protein distribuSuccinate dehydrogenase tion in each fraction between each cell The 16,000 X g pellet of the alveolar type is similar. Although sonication caused macrophages contained only 36% of total a release of the Kreb’s cycle enzyme sucrecovered succinate dehydrogenase activ- cinate dehydrogenase from the mitochonity. The residual activity was found in the dria, it did not disturb the true distribution 100,000 X g supernatant. The 100,000 of SOD. Even with gentle homogenation X g pellet showed no succinate dehydrog- in 0.34 M buffered sucrose, the distribution enase activity. Similar results were ob- of SOD was the same as that obtained tained in the PMNs; the 16,000 X g pellet with sonication. This observation agrees contained 2 5 % , the supernatant 7 5 % of with previous studies showing that Kreb’s cycle enzymes are more easily extracted total recovered enzyme activity. in soluble form than are mitochondria1 DISCUSSION enzymes which remain in the membrane Guinea pig PMNs, “monocytes,” and (Ziegler and Linnane, ’58). Sonication did alveolar macrophages contain SOD activity not cause a release of the granule enthat is predominantly insensitive to cya- zyme, p-glucuronidase, since the distrinide, a characteristic of the manganese bution obtained by sonication was similar enzyme usually found in mitochondria. Al- to that observed on gentle homogenization veolar macrophages have at least five times (Michell et al., ’70). higher total SOD activity than the PMNs In each cell type the largest SOD ac-
the 16,000 X g pellet, 7 3 % in the 100,000 x g pellet, and 21% of total recovered beta glucuronidase activity in the supernatant.
O
p1
p2
100 0 SUPER
PMN
p1
p2
100 0
SUPER MONO
p1
p2
100
SUPER ALV.MAC.
% TOTAL PROTEIN
Fig. 6 Total SOD activity of PMNs, monocytes, and alveolar macrophages. The filled column represents the cyanide insensitive amount of total SOD activity. Since alveolar macrophages contain 2.5 times more proteinlcell than PMN and monocytes, the total area of the columns represented for alveolar macrophages must be multiplied by 2.5 to obtain total alveolar macrophage SOD activity. PI: 16,000 x g pellet; P,: 100,000 X g pellet; super: 100,000 X g supernatant.
353
SOD ACTIVITY I N GUINEA PIG LEUKOCYTES
tivity was found in the 16,000 X g pellet of the PMNs, “monocytes,” and alveolar macrophages. There was no significant difference between SOD activity with and without cyanide indicating that the activity was due to the manganese mitochondrial enzyme. The electron microscopy studies showing large numbers of mitochondria especially in alveolar macrophages support these biochemical studies. The 16,000 X g pellet from sonicates of alveolar macrophages contain 1.3 times and the monocytes 1.7 times as much SOD activity as do P M N s (fig. 7a). There was no significant difference detectable between SOD activity in the 100,000 X g pellet with or without cyanide indicating that only mitochondrial enzyme was present. Since electron microscopy studies show no mitochondria in this fraction, the SOD activity found in it could be explained either by attachment of the enzyme to the microsomal particles during the preparation of the sonicates, release of SOD from mitochondria in vivo, or by association of newly synthesized enzyme of the microsomes. This cell fraction of the monocyte contains 2.2 times more enzyme activity compared to the 100,000 X g pellet of the PMNs. The same cell fraction of the alveolar macrophages contained four times more activity than the P M N s (fig. 7b). The 100,000 X g supernatant of alveolar macrophages contained 75 times more SOD than P M N s and “monocytes,” In contrast to both pellets, there was cyanide sensitive and insensitive SOD activity in + 16,000 X g Pellet
PMN
MONO AL.MAC.
,
the 100,000 X g supernatant indicating that both mitochondrial mangano as well as cytosol copper-zinc enzymes were present in these preparations. In the P M N 100,000 X g supernatant 17% of SOD was cyanide sensitive whereas 36% of SOD in alveolar macrophages was cyanide sensitive. All SOD activity was cyanide sensitive in the 100,000 X g supernatant fraction from “monocytes” (fig. 7c). The difference in cyanide sensitivity for SOD activity in 100,000 X g supernatant suggests that there is a shift of mitochondrial SOD into the cytosol of P M N s and alveolar macrophages during maturation in vivo. The disrupted cristae and damaged mitochondria noted by electron microscopy studies of the 16,000 X g pellet indicates the effect of sonication which released succinate dehydrogenase but not SOD into the cytosol. As noted in figure 8, the 16,000 X g pellets represent 96% of total recovered SOD activity in P M N s and “monocytes” respectively but only 48% of total recovered SOD activity in alveolar macrophages. The 100,000 X g supernatant of P M N and “monocytes” contained only 1% of total recovered enzyme activity compared to 44% of total recovered SOD activity in alveolar macrophages. Therefore, SOD activity of P M N and “monocytes” is mainly located in the 16,000 X g particulate fraction but is equally distributed between the particulate and cytosol fractions of alveolar macrophages. The high SOD activity in the cytosol of
100,000X g Pellet
PMN
MONO AL.MAC.
+ 100,000X g Supernatant
PMN
MONO AL.MAC.
Fig. 7 The relative SOD activity of each cell fraction compared to the same cell fraction of the PMNs. The filled column represents the cyanide insensitive SOD activity and the open column represents the cyanide sensitive SOD activity. (a) 16,000 X g pellet; (b) 100,000 X g pellet; (c) 100,000 X g supernatant.
354
MANFRED RISTER AND ROBERT L. BAEHNER
501
Crussi for performing the electron microscopy studies and Mrs. Mary Deck, Mrs. Karen Watkins, and Mr. Al Whitlow for their technical assistance. We also thank Mrs. Elaine Carroll and Mrs. Joyce Partlow for typing the manuscript.
n
LITERATURE CITED
i 1 6 P S
1 6 P S
PMN
MONO
16
P
S
ALV. MAC
Fig. 8 The distribution of total recovered SOD activity in PMNs, “monocytes,” and alveolar macrophages. 16, 16,000 X g pellet; P, 100,000 x g pellet; S, 100,000 X g supernatant.
alveolar macrophages could be explained by exposure of these cells to higher oxygen concentrations. Since many enzymes are induced by their substrates, it is possible that the oxygen concentration of the alvecli stimulates a n increase generation of 0,which, in turn, induces superoxide dismutase to enable the cell to survive in the presence of oxygen. This phenomena could be shown in studies performed on yeast and bacteria (Gregory and Fridovich, ’73). SOD activity was induced in these microorganisms by exposing them to high oxygen concentrations. Saccharomyces cerevisiae and E. coli grown under 100% or hyperbaric oxygen contained significant more SOD activity than similar cells grown in anaerobic environment. Since the bacteria grown under 100% oxygen had an elevated level of SOD they were most resistant to the damaging effects of hyperbarric oxygen (Gregory et al., ’74). Recent studies on rats, guinea pigs, and hamsters exposed to 80% oxygen have indicated that they are protected against oxygen toxicity as their lung tissue contained high SOD activity (Crapo and Tierney, ’74). These results and our data support the suggestion that SOD eliminates the toxic superoxide radical and protects biological membranes against oxidative damage. Further in vitro and in vivo studies are in progress in this laboratory to study the phenomena in more detail. ACKNOWLEDGMENTS
We gratefully acknowledge Dr. Frank
Beckmann, G., E. Lundgren and A. Tiirnvik 1973 Superoxide dismutase isozymes in different human tissues, their genetic control and intracellular localisation. Human Hered., 23: 338-345. Boyum, A. 1967 Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Lab. Invest., 97: 7 7 4 9 . Brain, J. D., and D. Frank 1973 Alveolar makrophage adhesion: wash elecirolyte composition and free cell yield. J. Appl. Physiol., 34: 75-80. Crapo, J. D., and D. F. Tierney 1974 Superoxide dismutase and pulmonary oxygen toxicity. Am. J. Physiol., 226 (6): 1401-1407. DeChatelet, L. R., C. E. McCall, L. C. McPhail and R. B. Johnston 1974 Superoxide dismutase activity in leukocytes. J . Clin. Invest., 53: 11971201. Fee, J. A., and P. Gaber 1972 Anion binding to bovine erythrocyte-superoxide dismutase. J. Biol. Chem., 247: 60-65. Fridovich, J. 1972 Superoxide radical and superoxide dismutase. Accounts Chem. Res., 5: 321326. Gregory, E. M., and J. Fridovich 1973 Induction of superoxide dismutase by molecular oxygen. J. Bacteriol., 114: 543548. Gregory, E. M., S. A. Goscin and J. Fridovich 1974 Superoxide dismutase and oxygen toxicity in a eukaryote. J. Bacteriol., 1 1 7: 456460. Horecker, B. L., and A. Kornberg 1946 The cytochrome C-Cyanide complex. J. Biol. Chem., 165: 11-20. Lowry, 0. H., N. J. Rosenbrough, A. L. Farr and R. J. Randall 1951 Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265-275. Luft, J. H. 1961 Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol., 9: 409. Maxwell, U. S. 1964 In situ method for harvesting guinea pig alveolar macrophages. Amer. Rev. Resp. Dis., 89: 5 7 9 5 8 0 . McCord, J. M., and J. Fridovich 1969 Superoxide dismutase: an enzymatic function for erythrocuprein. J. Biol. Chem., 25: 6049-6055. McCord, J. M., B. B. Keele and J. Fridovich 1971 An enzyme-based theory of obligate anaerobiosis: the physiological function of superoxide dismutase. Proc. Nat. Acad. Sci., 68: 10241027. Michell, R. H., M. J. Karnovsky and M. L. Karnovsky 1970 The distribution of some granuleassociated enzymes in guinea pig polymorphonuclear leucocytes. Biochem. J., 116: 207-216. Misra, H. P., and I. Fridovich 1972 The role of superoxide anion in the autooxidation of epinephrine and a simple assay for superoxide dismutase. J. Biol. Chem., 247: 31703175, Nashikimi, M., N. A. Rao and K. Yagi 1972 The occurrence of suueroxide anion in the reaction of reduced methosulfate and molecular
SOD ACTIVITY IN GUINEA PIG LEUKOCYTES
oxygen. Biochem. Biophys. Res. Commun., 46: 849454. Oren, R., A. E. Farnham, K. Saito, E. Milofsky and M. L. Karnovsky 1963 Metabolic patterns i n three types of phagocytizing cells. J. Cell. Biol., 17: 487501. Patriarca, P., P . Dri and F. Rossi 1974 Superoxide dismutase in leukocytes. Fed. Europ. Biochem. SOC.,43: 247-251. Rosenszajn, L., M. Leibovich, D. Shoham and J. Epstein 1968 The esterase activity in megaloblasts, leukemic and normal hematopoietic cells. J. Haemat., 14: 605410. Salin, M. L., and J. M. McCord 1974 Superoxide dismutase in polymorphonuclear leukocytes. J. Clin. Invest., 54: 1005-1009. Saltzmann, H. A , , and J. Fridovich 1973 Oxy-
355
gen toxicity, introduction to a protective enzyme: superoxide dismutase. Circulation XLVIII: 921923. Stansell, M. J., and H. F. Deutsch 1965 Preparation of crystalline erythrocuprein and catalase from human erythrocytes. J. Biol. Chem., 240: 42994305, Weisiger, R. A,, and J. Fridovich 1973 Superoxide dismutase organelle specificity. J. Biol. Chem., 248: 35823592, Winterborn, C. C., R. E. Hawkins, M. Brian and R. W. Carrel1 1975 The estimation of red cell SOD activity. J. Lab. Clin. Med., 85: 337341, Ziegler, D. M., and A. W. Linnane 1958 Mitochondrial structure and dehydrogenase activity in isolated mitochondria. Biochim. Biophys. Acta, 30: 53-61.
ABSTRACT Superoxide dismutase, an enzyme which catalyzes the dismutation of superoxide radical formed during the univalent reduction of oxygen, was quantitated by observing the inhibition of cytochrome C reduction in three cell fractions in guinea pig peritoneal P M N s and monocytes and compared to alveolar macrophages. No differences were found in the 16,000 x g pellets containing mitochondria, membranes, and granules and representing 96% of total SOD activity in PMNs and monocytes but only 48% total SOD activity in alveolar macrophages. The 100,000 x g microsomal pellet of alveolar macrophages contained 8% of total SOD activity and two-five times more activity than the respective fractions from monocytes and PMNs. However, there was 70 times more SOD in the 100,000 x g supernatant from alveolar macrophages containing 44% of total enzyme activity than in the same fraction of P M N s and monocytes containing less than 2 % total SOD activity. SOD activity is mainly located in the 16,000 x g particulate fraction of P M N and monocytes but more equally distributed between the particulate fractions and cytosol of alveolar macrophages. Superoxide dismutase, an enzyme found in all aerobic organisms but lacking in strict anaerobes (McCord et al., '71), catalyzes the following reaction: 0;
+ 0; + 2H+-
b HzOz
+ 02
It is assumed that the physiological function of superoxide dismutase (SOD) is to protect the cell agajnst highly reactive superoxide radical (Oi) which is generated during the univalent reduction of oxygen (Fridovich, '72) (Saltzmann and Fridovich, '73). Two kinds of SOD are found in most cells; one located in the cytosol is cyanide sensitive and contains copper and zinc whereas the other, associated with the mitochondria, is cyanide insensitive and contains manganese (McCord and Fridovich, '69; Weisiger and Fridovich, '73). Although the presence of SOD has been confirmed in a wide variety of species (Beckmann et al., '73; DeChatelet et al., '74; Patriarca et al., '74; Salin and McCord, '74; Stansell and Deutsch, '65; Winterborn et al., '75), a study comparing SOD activity in various tissues from the same species has not been performed. Since peritoneal cavity and lung tissues J. CELL.PWYSIOL., 87: 345-356.
are exposed to different tensions of oxygen, SOD activity of PMNs, monocytes, and alveolar macrophages from guinea pigs was compared to determine whether environmental differences in oxygen concentration may influence SOD activity. Furthermore, since alveolar macrophages, in contrast to PMNs and monocytes, contain prominent mitochondria and demonstrate a biological dependence on oxidative phosphorylation, we also compared the sensitivity of SOD activity to cyanide in the three types of cell sonicates. EXPERIMENTALPROCEDURE
Preparation of P M N s , peritoneal, and alveolar macrophages PMNs and peritoneal macrophages ("monocytes") were obtained by a method previously described (Oren et al., '63). Received May 20, '75. Accepted Sept. 18, '75. 1 This project was supported by National Institutes of Health Grant Number AI-10892-02 and a grant from the Riley Memorial Association. 2 Dr. Rister is the recipient of Deutsche Forschungsgemeinsch aft. 3 Address reprints to: Dr. Manfred Rister, Division of Pediatric Hematology-Oncology, James Whitcomb Riley Hospital for Children, 1100 West Michigan Street, Indianapolis, Indiana 46202.
345
346
MANFRED RISTER AND ROBERT L. BAEHNER
PMNs were harvested 18 hours after injection of 12% caseine into the peritoneal cavity of guinea pigs; “monocytes” were obtained three and one-half days after intraperitoneal injection of 1.2% caseine. Alveolar macrophages were recovered from the same guinea pigs by a modification of the technique described by Maxwell (’64). The upper portion of the trachea was exposed by dissection and a polyethylene catheter attached to a three-way stop cock was inserted in the transected trachea. Two 60 ml Luer lock syringes were then attached to the stop cock. According to the method of Brain and Frank (’73), one was filled with buffered saline pH 7.4 warmed to 37°C. Aliquots of 5 to 10 mls were injected into the lungs and the stop cock valve was then adjusted to aspirate the macrophage-rich fluid from the lung into the other syringe. Cell suspensions were centrifuged at 200 X g at 4 ° C and washed thrice in cold Krebs-Ringer-phosphate buffer (KRP) pH 7.4. Between the second and third washing, red cells were lysed by exposure to deionized water for 20 seconds. Isotonicity was then restored by the addition of an appropriate volume of 3.5% saline. The “monocyte” and alveolar macrophage suspension was freed of contaminating PMNs by a Ficoll-Hypaque gradient
(Boyum, ’67). Using this method, a purity and viability assessed by trypan blue of greater than 90% was achieved for each type of cell preparation. This was documented on a smear stained with Wright’s and monocyte-specific alpha naphthyl esterase stains (Rosenszajn et al., ’68) (figs. 1, 2). The final samples of PMNs and “monocytes” were resuspended in KRP buffer to a concentration of either 2 X 1 0 7 cellslml or 2 x 10*cellslml. Since 20 times more PMNs and “monocytes” than alveolar macrophages were required for a single SOD assay, the SOD activity from a contamination of 10% PMNs in the final alveolar macrophage suspension would not be detectable.
Preparation of cell sonicate fractions The cells were sonicated at 40°C for one minute with a Sonifier Cell Disruptor Model W 140 by Branson Sonic Power Co. with an output control setting of 3 . They were centrifuged a t 16,000 X g for 15 minutes at 4 “C. The resulting supernatant was centrifuged at 100,000 x g for one hour in a Beckman Ultracentrifuge, Model L3-50 with a fixed angle rotor, type 30. The 16,000 X g and 100,000 X g pellets were resuspended in ice-cold 0.2% (volurnelvolume) Triton X 100 to lyse mito-
Fig. 1 Final monocyte preparation stained with monocyte specific alpha naphthyl esterase stain; X 1,200.
34 7
SOD ACTIVITY IN GUINEA PIG LEUKOCYTES
chondria, lysosomes, and other cell organelles. Both the supernatant and the solubilized pellets were assayed for cyanide sensitive and cyanide insensitive SOD activity. The protein concentration of each cell fraction was determined by the Folin phenol reagent procedure of Lowry with human albumin as a standard (Lowry et al., '51). Preparation of cell homogenates P M N s and alveolar macrophages were resuspended in ice-cold buffered sucrose at pH 7.4 and homogenized for four minutes until a cell breakage greater than 99% was achieved. The homogenates were centrifuged and treated with Triton X 100 like the sonicates.
Electron microscopy studies Electron micrographs were prepared from the 16,000 X g and 100,000 X g pellets by fixing them in 4 % glutaraldehyde in phosphate buffer pH 7.2 for two hours and then placing them in 1% osmium overnight. After dehydrating them with ethanol and propylene oxide, they were embedded in Epon 812 according to the Luft method (Luft, '61). Sections were prepared on a LKB ultratome, stained with uranyl acetate and lead citrate stain, and examined on a Phillips 300 electron microscope.
Fig. 2
Superoxide dismutase assays Since we failed to obtain reproducible results with either the inhibition of the auto-oxidation of epinephrine (Misra and Fridovich, '72) or with the inhibition of' the nitroblue tetrazolium reduction (Nashikimi et al., '72), all data reported in this paper were obtained with an assay method of McCord and Fridovich ('69). This assay depends upon the capacity of SOD to inhibit cytochrome C reduction mediated by superoxide radical (02) generated during the oxidation of xanthine catalyzed by xanthine oxidase. Xanthine grade 3, xanthine oxidase grade 4, and horse heart cytochrome C type VI were obtained from Sigma Chemical Corp., St. Louis, Missouri. The assays were performed at room temperature in 0.05 M potassium phosphate buffer pH 7.8 contain-ing 0.1 mM EDTA. Superoxide radical (02) was generated by incubating 5 X 1 0 - 5 M xanthine with 0.01 units of xanthine oxidase. Since SOD and cytochrome C both compete for 02 generated in this solution, maximum activity of the SOD is obtained at low cytochrome C concentrations (Salin and McCord, '74). In preliminary experiments, we determined that 5 pM cytochrome C was most sensitive for our assay. Purified bovine SOD obtained from Truett Laboratories, Dallas, Texas was used a s the
Alveolar macrophages with esterase stain;
X
1,200.
348
MANFRED RISTER AND ROBERT L. BAEHNER
standard. The change of the optical density was read a t 550 nm with the Gilford Model 2400 recording spectrophotometer with ten-fold scale expansion. Each reaction mixture contained 1 mM sodium azide to inhibit cytochrome C oxidases which otherwise would decrease the reduction of cytochrome C which, in turn, would produce a f a h e inhibition rate of cytochrome C reduction resulting in a false elevation of SOD activity. Total SOD activity was measured without cyanide in PMNs and “monocytes” and the manganese containing enzyme was measured by observing the remaining SOD activity after addition of 1 mM cyanide to the reaction mixture. In contrast, a greater inhibition of cytochrome C reduction was obtained in cell fractions of alveolar macrophages treated with 1 mM cyanide. For this reason, in alveolar macrophage fractions, cyanide insensitive SOD was measured at the final cyanide concentration of 1 pM. A progressive increase of cyanide concentration from 1 pM to 1 mM produced a higher inhibition of cytochrome C reduction which was independent of the concentration of SOD. This could be explained by a cyanide-ferricytochrome C complex which occurs at equimolar concentrations (Horecker and Kornberg, ’46). In preliminary studies we determined that 0.01 pM SOD/ml, a n amount in excess of that found in our samples, inhibited the cytochrome C reduction to 65 & 2 % . Since 1 mole of SOD binds two moles of cyanide, the binding capacity of 1 pM cyanide is 0.5 p M SOD. This amount of SOD is approximately 50 times in excess of that amount usually found in our assayed samples. Therefore, the cyanide concentration is sufficient to inhibit the cyanide sensitive SOD found in our samples. In a xanthine-xanthine oxidase free reaction mixture, we observed no reaction between the cell sonicates and homogenates, cytochrome C and cyanide. The difference between total SOD activity and cyanide insensitive SOD activity was calculated as cyanide sensitive SOD activity. Whereas assays of the 100,000 X g supernatant of the alveolar macrophage required 2 x 1 0 6 cells/ml, 2 X 108 cells/ ml of PMNs and “monocytes” were needed for a single assay of SOD activity in the 100,000 X g supernatant.
Beta g lucuronidas e Beta glucuronidase was assayed with p-nitrophenyl-beta-D-glucuronide(Sigma Chemical Co., St. Louis, Mo.) as a substrate (Michell et al., ’70). The reaction mixture contained a final concentration 50 mM sodium acetate buffer, pH 5 . 0 , O . 1% (volume/volume) Triton X 100, 1 mM nitrophenyl-beta-glucuronide, and between 0.02 mg and 0.2 mg of enzyme protein. Incubations were in a total volume of 1 ml for two hours at 37°C. The reaction was stopped by the addition of 2 ml of 0.1 N sodium hydroxide and the optical density determined at 410 nm. Results were calculated by using a molar extinction coefficientof 1.70 X lo4. Succinate dehydrogenase This enzyme was assayed using p-iodonitrotetrazolium violet as electron acceptor (Michell et al., ’70). The assay medium contained at final concentration: 1% (weight/volume) iodonitrotetrazolium violet, 50 mM sodium succinate, pH 7.4, 50 mM potassium phosphate buffer, pH 7.4, 2 mM sodium EDTA, and 0.1-0.5 mgs enzyme protein. Incubations were in a total volume of 1 ml for 45 minutes at 37°C. The reaction was stopped with 1 ml 50% TCA. The formazan was extracted with 1.5 ml ethyl-acetate using a Vortex mixer. Optical densities were measured at 490 n m and results calculated using the extinction coefficient of 20.1 X 103. All assays were corrected for blanks in which succinate and enzyme were omitted. RESULTS
All SOD activity found in each homogenate was compared to the amount of protein which caused a 30% inhibition of cytochrome C reduction. In our system three units bovine erythrocyte SOD (0.1 mcg of bovine erythrocyte SOD/ml reaction mixture) was needed for 30% inhibition. Knowing the amount of protein per reaction mixture, we were able to calculate and express the results as units SOD/ milligram protein. The protein distribution in the different cell fractions was similar between PMNs, “monocytes,” and alveolar macrophages. Forty-eight percent & 3% of total recovered protein was found in the
SOD ACTIVITY I N G U I N E A P I G L E U K O C Y T E S
Fig. 3 16,000 x mitochondria.
Fig. 4 16,000 are present.
g pellet
X g
of alveolar macrophages; X 31,250; note several damaged
pellet of polymorphonuclear leukocytes;
X
25,000; less mitochondria
349
350
MANFRED RISTER AND ROBERT L. BAEHNER
16,000 X g pellet, 12 & 100,000 X g pellet, and 40 supernatant.
2 % in the 4% in the
?
PMNs The 16,000 X g pellet of the PMNs obtained from a cell preparation of 2 x l o x cellslml was found to be very turbid and this turbidity prevented a sensitive enzyme assay. For this reason, we determined SOD activity in this fraction prepared from a 2 X lo7 cell preparation. In that case, we were able to assay SOD activity and found 16.2 & 1.2 units SOD/ mg of protein of this fraction. After addition of cyanide an identical protein concentration produced the same inhibition rate, indicating that all of the SOD activity was insensitive to cyanide, a characteristic of the mitochondria1 manganese enzyme. Ninety-six percent of total recovered SOD was found in this cell fraction. Greater than 1 mg of protein was needed to assay SOD activity in the 100,000 x g pellet and required a cell preparation of 2 X 107/ml. This pellet con-
tained 1.2 & 0.12 units SOD/mg of protein and with cyanide 0.9 i 0.18 units SOD/ mg protein, which represents 2.7% of total recovered SOD activity. Greater than 10 mgs protein was needed for detection of SOD activity in the 100,000 X g supernatant and required a cell preparation of 2 X 10*cells/ml; only 0.177 & 0.003 units SOD/mg protein without cyanide and 0.147 i 0.006 units SOD/mg protein with cyanide was found in the supernatant. Although these differences are significant (p < 0.01, student’s t-test) the 17% inhibition of SOD activity by cyanide indicates that SOD activity present in this supernatant fraction was largely the manganese containing enzme. Only 1.3% of total recovered SOD activity was found in the cytosol representing cell fraction.
“Monocytes” Similar to P M N , assay of SOD activity in the 16,000 X g pellet required a monocyte concentration of 2 X lo7 cell/ml and contained 28.5 k 1.5 units SOD/mg pro-
Fig. 5 100,000 X g pellet of polymorphonuclear leukocytes; x 20,500; no mitochondria are present.
351
SOD ACTIVITY I N GUINEA PIG L E U K O C Y T E S
*
represented 14% of total SOD activity and contained 4.65 0.75 units SOD/mg protein without cyanide and 4.8 1.2 units SOD/mg protein with cyanide, indicating that the predominating enzyme activity was the mitochondrial manganese SOD. The 100,000 X g supernatant of the macrophages contained very high activity of both cyanide sensitive and insensitive SOD and 38% of total recovered SOD activity. Without cyanide 13.44 0.69 units SOD/ mg protein and with cyanide 8.58 0.6 units SOD/mg protein was found in the supernatant. Thirty-six percent of total activity was cyanide sensitive. To determine the effect of sonication on the distribution and activity of SOD we assayed the enzyme activity in homogenized cells. In each cell fraction of P M N s and alveolar macrophages there was the same cyanide sensitive and insensitive SOD activity as found in the cell sonicates. Table 1 summarizes these results and indicates the units SOD/rng protein and the Alveolar macrophages percentage of total recovered SOD in the Alveolar macrophages showed the high- various cell fractions. In addition, the effect of sonication on est SOD activity. For a single assay, more than 50 pg protein from the 16,000 X g a known granule enzyme, p-glucuronidase, pellet and a cell concentration of 2 X 106/ and a known mitochondrial enzyme, suc1.2 units SOD/mg cinate dehydrogenase, was also determined. ml was needed; 21.9 protein without cyanide and 21.42 0.9 Beta glucuronidase units SOD/mg protein with cyanide was found in the 16,000 X g pellet which conThe 16,000 X g pellet of the macrotained 48% of total recovered SOD activity. phage contained 8 2 % , the 100,000 x g Again, there was no significant difference pellet 8 % , and the supernatant 10% of between the results performed with and total recovered beta glucuronidase activwithout cyanide. The 100,000 X g pellet ity. In the PMNs, only 6 % was found in
tein and 25.8 1.5 units SOD/mg protein after addition of cyanide indicating that all SOD activity was cyanide insensitive. More than 500 pg protein and a cell concentration of 2 X 107/ml was needed to detect SOD activity in the 100,000 X g “monocyte” pellet which had 2.25 & 0.15 units SOD/mg protein without cyanide and 0.3 units SOD/mg protein with 2.49 cyanide. There was no significant difference between the inhibition rates with and without cyanide. The 100,000 X g supernatant contained 0.06 i 0.01 units SOD/mg protein and required 2 X 108 monocytes/ml for 30% inhibition. All of the SOD activity found in the 100,000 X g supernatant of the monocytes was cyanide sensitive. The distribution of SOD activity was similar to the PMNs; 97% of total recovered SOD activity was found in the 16,000 X g pellet, 2 % in the 100,000 X g pellet, and only 1% in the supernatant .
*
*
*
*
*
*
*
TABLE 1
U n i t s SODImg protein in cell f r a c t i o n s of P M N , “monocytes” and alveolar macrophages 16,000 X g
pellet
PMN No cyanide With cyanide 70
’
“Monocytes” No cyanide With cyanide % ’
Alveolar macrophages No cyanide With cyanide % I 1
100,000
x
9
pellet
100,000 x g supernatant
16.2? 1.2 16.22 1.2 96
1.2k0.12 0.9f 0.18 2.7
0.177f 0.009 0.147f 0.006 1.3
28.5f 1.5 25.8f 1.5 97
2.25f0.15 2.49-+- 0.30 2
0.18f 0.003 no activity 1
21.90f 1.2 21.42k0.9 48
4.65f 0.75 4.80f 1.20 14
Percent of total recovered SOD-activity
13.44f 0.69 8.58f 0.60 38
352
MANFRED RISTER AND ROBERT L. BAEHNER
and three times more than “monocytes” (fig. 6). The difference is highly significant and is due to the larger amount of SOD activity found in the cytosol of alveolar macrophages, since the protein distribuSuccinate dehydrogenase tion in each fraction between each cell The 16,000 X g pellet of the alveolar type is similar. Although sonication caused macrophages contained only 36% of total a release of the Kreb’s cycle enzyme sucrecovered succinate dehydrogenase activ- cinate dehydrogenase from the mitochonity. The residual activity was found in the dria, it did not disturb the true distribution 100,000 X g supernatant. The 100,000 of SOD. Even with gentle homogenation X g pellet showed no succinate dehydrog- in 0.34 M buffered sucrose, the distribution enase activity. Similar results were ob- of SOD was the same as that obtained tained in the PMNs; the 16,000 X g pellet with sonication. This observation agrees contained 2 5 % , the supernatant 7 5 % of with previous studies showing that Kreb’s cycle enzymes are more easily extracted total recovered enzyme activity. in soluble form than are mitochondria1 DISCUSSION enzymes which remain in the membrane Guinea pig PMNs, “monocytes,” and (Ziegler and Linnane, ’58). Sonication did alveolar macrophages contain SOD activity not cause a release of the granule enthat is predominantly insensitive to cya- zyme, p-glucuronidase, since the distrinide, a characteristic of the manganese bution obtained by sonication was similar enzyme usually found in mitochondria. Al- to that observed on gentle homogenization veolar macrophages have at least five times (Michell et al., ’70). higher total SOD activity than the PMNs In each cell type the largest SOD ac-
the 16,000 X g pellet, 7 3 % in the 100,000 x g pellet, and 21% of total recovered beta glucuronidase activity in the supernatant.
O
p1
p2
100 0 SUPER
PMN
p1
p2
100 0
SUPER MONO
p1
p2
100
SUPER ALV.MAC.
% TOTAL PROTEIN
Fig. 6 Total SOD activity of PMNs, monocytes, and alveolar macrophages. The filled column represents the cyanide insensitive amount of total SOD activity. Since alveolar macrophages contain 2.5 times more proteinlcell than PMN and monocytes, the total area of the columns represented for alveolar macrophages must be multiplied by 2.5 to obtain total alveolar macrophage SOD activity. PI: 16,000 x g pellet; P,: 100,000 X g pellet; super: 100,000 X g supernatant.
353
SOD ACTIVITY I N GUINEA PIG LEUKOCYTES
tivity was found in the 16,000 X g pellet of the PMNs, “monocytes,” and alveolar macrophages. There was no significant difference between SOD activity with and without cyanide indicating that the activity was due to the manganese mitochondrial enzyme. The electron microscopy studies showing large numbers of mitochondria especially in alveolar macrophages support these biochemical studies. The 16,000 X g pellet from sonicates of alveolar macrophages contain 1.3 times and the monocytes 1.7 times as much SOD activity as do P M N s (fig. 7a). There was no significant difference detectable between SOD activity in the 100,000 X g pellet with or without cyanide indicating that only mitochondrial enzyme was present. Since electron microscopy studies show no mitochondria in this fraction, the SOD activity found in it could be explained either by attachment of the enzyme to the microsomal particles during the preparation of the sonicates, release of SOD from mitochondria in vivo, or by association of newly synthesized enzyme of the microsomes. This cell fraction of the monocyte contains 2.2 times more enzyme activity compared to the 100,000 X g pellet of the PMNs. The same cell fraction of the alveolar macrophages contained four times more activity than the P M N s (fig. 7b). The 100,000 X g supernatant of alveolar macrophages contained 75 times more SOD than P M N s and “monocytes,” In contrast to both pellets, there was cyanide sensitive and insensitive SOD activity in + 16,000 X g Pellet
PMN
MONO AL.MAC.
,
the 100,000 X g supernatant indicating that both mitochondrial mangano as well as cytosol copper-zinc enzymes were present in these preparations. In the P M N 100,000 X g supernatant 17% of SOD was cyanide sensitive whereas 36% of SOD in alveolar macrophages was cyanide sensitive. All SOD activity was cyanide sensitive in the 100,000 X g supernatant fraction from “monocytes” (fig. 7c). The difference in cyanide sensitivity for SOD activity in 100,000 X g supernatant suggests that there is a shift of mitochondrial SOD into the cytosol of P M N s and alveolar macrophages during maturation in vivo. The disrupted cristae and damaged mitochondria noted by electron microscopy studies of the 16,000 X g pellet indicates the effect of sonication which released succinate dehydrogenase but not SOD into the cytosol. As noted in figure 8, the 16,000 X g pellets represent 96% of total recovered SOD activity in P M N s and “monocytes” respectively but only 48% of total recovered SOD activity in alveolar macrophages. The 100,000 X g supernatant of P M N and “monocytes” contained only 1% of total recovered enzyme activity compared to 44% of total recovered SOD activity in alveolar macrophages. Therefore, SOD activity of P M N and “monocytes” is mainly located in the 16,000 X g particulate fraction but is equally distributed between the particulate and cytosol fractions of alveolar macrophages. The high SOD activity in the cytosol of
100,000X g Pellet
PMN
MONO AL.MAC.
+ 100,000X g Supernatant
PMN
MONO AL.MAC.
Fig. 7 The relative SOD activity of each cell fraction compared to the same cell fraction of the PMNs. The filled column represents the cyanide insensitive SOD activity and the open column represents the cyanide sensitive SOD activity. (a) 16,000 X g pellet; (b) 100,000 X g pellet; (c) 100,000 X g supernatant.
354
MANFRED RISTER AND ROBERT L. BAEHNER
501
Crussi for performing the electron microscopy studies and Mrs. Mary Deck, Mrs. Karen Watkins, and Mr. Al Whitlow for their technical assistance. We also thank Mrs. Elaine Carroll and Mrs. Joyce Partlow for typing the manuscript.
n
LITERATURE CITED
i 1 6 P S
1 6 P S
PMN
MONO
16
P
S
ALV. MAC
Fig. 8 The distribution of total recovered SOD activity in PMNs, “monocytes,” and alveolar macrophages. 16, 16,000 X g pellet; P, 100,000 x g pellet; S, 100,000 X g supernatant.
alveolar macrophages could be explained by exposure of these cells to higher oxygen concentrations. Since many enzymes are induced by their substrates, it is possible that the oxygen concentration of the alvecli stimulates a n increase generation of 0,which, in turn, induces superoxide dismutase to enable the cell to survive in the presence of oxygen. This phenomena could be shown in studies performed on yeast and bacteria (Gregory and Fridovich, ’73). SOD activity was induced in these microorganisms by exposing them to high oxygen concentrations. Saccharomyces cerevisiae and E. coli grown under 100% or hyperbaric oxygen contained significant more SOD activity than similar cells grown in anaerobic environment. Since the bacteria grown under 100% oxygen had an elevated level of SOD they were most resistant to the damaging effects of hyperbarric oxygen (Gregory et al., ’74). Recent studies on rats, guinea pigs, and hamsters exposed to 80% oxygen have indicated that they are protected against oxygen toxicity as their lung tissue contained high SOD activity (Crapo and Tierney, ’74). These results and our data support the suggestion that SOD eliminates the toxic superoxide radical and protects biological membranes against oxidative damage. Further in vitro and in vivo studies are in progress in this laboratory to study the phenomena in more detail. ACKNOWLEDGMENTS
We gratefully acknowledge Dr. Frank
Beckmann, G., E. Lundgren and A. Tiirnvik 1973 Superoxide dismutase isozymes in different human tissues, their genetic control and intracellular localisation. Human Hered., 23: 338-345. Boyum, A. 1967 Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Lab. Invest., 97: 7 7 4 9 . Brain, J. D., and D. Frank 1973 Alveolar makrophage adhesion: wash elecirolyte composition and free cell yield. J. Appl. Physiol., 34: 75-80. Crapo, J. D., and D. F. Tierney 1974 Superoxide dismutase and pulmonary oxygen toxicity. Am. J. Physiol., 226 (6): 1401-1407. DeChatelet, L. R., C. E. McCall, L. C. McPhail and R. B. Johnston 1974 Superoxide dismutase activity in leukocytes. J . Clin. Invest., 53: 11971201. Fee, J. A., and P. Gaber 1972 Anion binding to bovine erythrocyte-superoxide dismutase. J. Biol. Chem., 247: 60-65. Fridovich, J. 1972 Superoxide radical and superoxide dismutase. Accounts Chem. Res., 5: 321326. Gregory, E. M., and J. Fridovich 1973 Induction of superoxide dismutase by molecular oxygen. J. Bacteriol., 114: 543548. Gregory, E. M., S. A. Goscin and J. Fridovich 1974 Superoxide dismutase and oxygen toxicity in a eukaryote. J. Bacteriol., 1 1 7: 456460. Horecker, B. L., and A. Kornberg 1946 The cytochrome C-Cyanide complex. J. Biol. Chem., 165: 11-20. Lowry, 0. H., N. J. Rosenbrough, A. L. Farr and R. J. Randall 1951 Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265-275. Luft, J. H. 1961 Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol., 9: 409. Maxwell, U. S. 1964 In situ method for harvesting guinea pig alveolar macrophages. Amer. Rev. Resp. Dis., 89: 5 7 9 5 8 0 . McCord, J. M., and J. Fridovich 1969 Superoxide dismutase: an enzymatic function for erythrocuprein. J. Biol. Chem., 25: 6049-6055. McCord, J. M., B. B. Keele and J. Fridovich 1971 An enzyme-based theory of obligate anaerobiosis: the physiological function of superoxide dismutase. Proc. Nat. Acad. Sci., 68: 10241027. Michell, R. H., M. J. Karnovsky and M. L. Karnovsky 1970 The distribution of some granuleassociated enzymes in guinea pig polymorphonuclear leucocytes. Biochem. J., 116: 207-216. Misra, H. P., and I. Fridovich 1972 The role of superoxide anion in the autooxidation of epinephrine and a simple assay for superoxide dismutase. J. Biol. Chem., 247: 31703175, Nashikimi, M., N. A. Rao and K. Yagi 1972 The occurrence of suueroxide anion in the reaction of reduced methosulfate and molecular
SOD ACTIVITY IN GUINEA PIG LEUKOCYTES
oxygen. Biochem. Biophys. Res. Commun., 46: 849454. Oren, R., A. E. Farnham, K. Saito, E. Milofsky and M. L. Karnovsky 1963 Metabolic patterns i n three types of phagocytizing cells. J. Cell. Biol., 17: 487501. Patriarca, P., P . Dri and F. Rossi 1974 Superoxide dismutase in leukocytes. Fed. Europ. Biochem. SOC.,43: 247-251. Rosenszajn, L., M. Leibovich, D. Shoham and J. Epstein 1968 The esterase activity in megaloblasts, leukemic and normal hematopoietic cells. J. Haemat., 14: 605410. Salin, M. L., and J. M. McCord 1974 Superoxide dismutase in polymorphonuclear leukocytes. J. Clin. Invest., 54: 1005-1009. Saltzmann, H. A , , and J. Fridovich 1973 Oxy-
355
gen toxicity, introduction to a protective enzyme: superoxide dismutase. Circulation XLVIII: 921923. Stansell, M. J., and H. F. Deutsch 1965 Preparation of crystalline erythrocuprein and catalase from human erythrocytes. J. Biol. Chem., 240: 42994305, Weisiger, R. A,, and J. Fridovich 1973 Superoxide dismutase organelle specificity. J. Biol. Chem., 248: 35823592, Winterborn, C. C., R. E. Hawkins, M. Brian and R. W. Carrel1 1975 The estimation of red cell SOD activity. J. Lab. Clin. Med., 85: 337341, Ziegler, D. M., and A. W. Linnane 1958 Mitochondrial structure and dehydrogenase activity in isolated mitochondria. Biochim. Biophys. Acta, 30: 53-61.
