ARCHIVES
OF
BIOCHEMISTRY
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BIOPHYSICS
NADH-Cytochrome
176, 119-126 (1976)
c Reductase Activity Human Lymphocytes
in Cultured
Similarity to the Liver Microsomal NADH-Cytochrome b5 Reductase and Cytochrome b5 Enzyme System RUSSELL A. PROUGH’, RICHARD L. IMBLUM2 Department
AND
RICHARD A. KOURP
ofBiochemistry and the UTHSC Cancer Center, The University of Texas Health Science Center at Hines Boulevard, Dallas, Texas 75235, and ZDepartment of Biochemical Oncology, Microbiological Associates, 4733 Bethesda Avenue, Bethesda, Maryland 20014
Dallas, 5323 Harry
Received January 22, 1976 A NADH-cytochrome c reductase activity was increased upon mitogen stimulation of human lymphocytes. The activity was not inhibited by antimycin A or rotenone but was specifically inhibited by antibodies elicited against rat liver NADH-cytochrome b, reductase or cytochrome b,. The activity was linear with cellular homogenates up to 5.2 x lo6 cells/ml and had a broad pH optimum of 7.7. The presence of 3-methylcholanthrene in mitogen stimulation media had no effect on the NADH-cytochrome c reductase activity but differentially induced the benzo(a)pyrene hydroxylase (AHH) activity. The reductase activity was present in nonstimulated cells and appears not to be significantly increased in activity per cell upon mitogen-stimulation of the peripheral lymphocyte.
Nowell has demonstrated that phytohemagglutinin (PHA)3 stimulates blast-like cell formation in cultures of human peripheral blood (1) and the response of lymphocytes to various activators (specific and nonspecific) has subsequently been reviewed by Waithe and Hirshhorn (2). Current interest in human genetics and disease states have prompted studies on the genetic regulation of aromatic hydrocarbon hydroxylase (AHH) activity in stimulated human lymphocyte populations (3) and on the correlation of AHH activity in human lymphocytes to the plasma half-life of drugs such as antipyrine and phenylbutazone (4). The biochemical components of lymphocytes or thymocytes are not well defined. 1 To whom reprint requests should be sent. 3 Abbreviations used: PHA, phytohemagglutinin; AHH, aromatic hydrocarbon hydroxylase; 3-MC, 3methylcholanthrene; Hanks’ BSS, Hanks’ balanced salt solution; FCS, fetal calf serum; HEPES, N-2hydroxyethylpiperazine-N’-2-ethanesulfonic acid; BP, benzo(a)pyrene; and L3H]TdR, [3Hlthymidine. 119
Copyright 0 1976 by Academic Press, All rights of reproduction in any form
Inc. reserved.
Several research groups have fractionated stimulated lymphocytes into subcellular components and have noted several enzymes to be present, such as 5’-nucleosidase, e&erase, NADH diaphorase, succinate dehydrogenase, or acid phosphatase activities (5, 6). To date, these enzyme activities have not been studied in detail due to the low activities and quantities of the enzymes in lymphocytes relative to other tissue. Recently, Kister and Gracy have reported that an isozyme of triosephosphate isomerase is preferentially observed in mitogen-stimulated human lymphocytes (7). This report will characterize a rotenoneinsensitive NADH-cytochrome c reductase activity in human lymphocytes which is mitogen-activated in culture by phytohemagglutinin and pokeweed mitogen. The enzymes involved are immunochemically similar to the NADH-cytochrome b, reductase and’ cytochrome b, which have been shown to exist in liver microsomes (8, 9) and erythrocytes (10). Lymphocyte cul-
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tures in the presence of 3-methylcholanthrene (3-MC) were shown to possesshigh levels of AHH activity, but the NADHcytochrome c reductase activity was unaffected by this treatment. MATERIALS
AND
METHODS
Materials. 3-methylcholanBenzo(a)pyrene, threne, NADH, and NADPH were purchased from Sigma Chemical Co., St. Louis, MO. RPM1 medium 1640, fetal calf serum, glutamine, penicillin-streptomycin mixture, and Ficoll-Hypaque solution (sp gr 1.080) were from Microbiological Associates, Inc., Bethesda, Md. Pokeweed mitogen was obtained from Grand Island Biological Co., Grand Island, N.Y. and phytohemagglutinin was purchased from Burroughs-Wellcome Co., Research Triangle Park, N.C. Plastic flasks and tubes were products of the Becton, Dickinson, and Co., Los Angeles, Calif. Fifteen-milliliter 125 x 15 mm glass screw-top culture tubes were from Kimble Products Division, OwenIllinois Glass Co., Toledo, Ohio. Acetone, hexane, and Handifluor were obtained from Mallinckrodt, Inc., St. Louis, MO. Liver microsomes and heart mitochondrial particles were prepared as described (11, 12). Preparation of specific antibodies to microsomal proteins. NADH-cytochrome b, reductase was prepared from rat liver microsomes by the method of Takesue and Omura (13) and had a specific activity equal to that reported for the homogeneous enzyme. NADPH-cytochrome c (P-450) reductase was purified from rat liver microsomes using pancreatic lipase as described by Prough and Masters (14) and had a specific activity equal to that of the homogeneous enzyme from pig liver (15). Both reductases had a single major protein band (>98%) on disc electrophoresis using 7.5% polyacrylamide gels. The major band of either reductase were excised from the gels, homogenized in 0.9% NaCl, and used as the challenging antigens. Cytochrome b, was prepared using the trypsin digestion method of Omura and Takesue and had a specific content equal to that reported by Omura and Takesue (16). Since a single and identical protein and heme band was noted on disc gel electrophoresis, the cytochrome b5 was used without performing disc gel electrophoresis purification The antibodies to all three proteins were elicited in separate young male white rabbits as described earlier (17, 18). An expression of titer was obtained by measuring the percentage inhibition of liver microsomal NADH-cytochrome c reductase activity by serum from rabbits injected with either NADH-cytochrome b, reductase or cytochrome b, and the percentage inhibition of liver microsomal NADPH-cytochrome c reductase activity by serum from rabbits injected with NADPH-cytochrome c (P-450) reduc-
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KOURI
tase. Sera, with high antibody titer for each antigen, were pooled and the immunoglobins were prepared by precipitation with 1.75 M ammonium sulfate (17, 18). The precipitates were dialyzed against buffer and stored frozen. The protein concentration was obtained by measuring the 280-nm absorption of the solutions assuming that a 1 mg/ml solution has an absorbance of 1.35. The specificity of the antibodies was tested by employing them as inhibitors against NADH- and NADPH-dependent microsome-catalyzed cytochrome c reduction. The anti-NADH-cytochrome b, reductase and anti-cytochrome b, globulins inhibited only the NADH-dependent reaction (92 and 90%, respectively) and the anti-NADPH-cytochrome c (P-450) reductase globulin inhibited only the NADPH-dependent reaction (93%). The effects of these antibodies and their specificity toward other microsomal reactions have been described (17, 18). Cytochrome c reductase activity of a reaction mixture containing 1 x lo-* M purified NADH-cytochrome b5 reductase, 1 x 10e7 M purified cytochrome b,, 4 x 1O-s M cytochrome c, and 1 x 1O-4 M NADH was inhibited only by either the anti-NADH-cytochrome b, reductase or anti-cytochrome b, globulin, while only anti-NADPH-cytochrome c (P-450) reductase globulin inhibited cytochrome c reductase activity of a reaction mixture containing 1 X 10m8M purified NADPH-cytochrome c (P-450) reductase, 4 x lo-” M cytochrome c, and 1 x 10e4 M NADPH. When examined by Ouchterlony double diffusion tests in agar gel (19), all three immunoglobulins elicited single precipitation lines against the respective purified microsomal proteins. No spurring of the precipitant band of a given antibody was seen when all three proteins were added separately to adjacent wells of the Ouchterlony plates. These results suggest that the three antibodies are monospecific globulins to their respective antigens. Cell culture. Lymphocytes were isolated from fresh (less than 2 h old) whole blood as described previously (19) with several modifications. All operations were performed at room temperature. Venous blood (about 100 ml) was collected in sterile 200-ml evacuated containers in which 5000 units of sodium heparin per 100 ml of blood had been added previously. The blood was diluted 40% with sterile Hanks’ BSS buffer supplemented with penicillin and streptomycin and layered in lo-ml portions onto 5 ml of a solution of Ficoll-Hypaque (sp gr 1.080) in 16 x 100 mm plastic tubes. The Ficoll-Hypaque tubes were centrifuged at 600g in an swinging bucket rotor for 45 min. The lymphocyte band at the interface between the Ficoll-Hypaque and plasma was collected with sterile Pasteur pipets and transferred to 50-ml centrifuge tubes. The cells were pelleted by centrifugation and suspended in RPM1 1640 medium supplemented with 17% fetal calf serum (FCS), 2 mM glutamine, 25 mM HEPES (pH 7.4), 50 units of penicil-
LYMPHOCYTE
CYTOCHROME
lin/ml, 50 wg of streptomycin/ml, 1% phytohemagglutinin, and 1% pokeweed mitogen. Cells were counted in an Autocytometer 11 (Fisher Scientific, Pittsburgh, Pa.) and adjusted to a concentration of 0.5 x 106 cells/ml. Eight-milliliter aliquots of cells were added to Falcon 3013 plastic flasks and incubated at 37”C, 5% CO,-95% air. For induction of AHH activity, 3-methylcholanthrene (~-MC) was added to cells at a final concentration of 1.5 @M at 0 time, the time at which cultures were initiated. In certain experiments cell viability was tested using the trypan blue dye exclusion test and was greater than 80% in all cases. Enzyme assays. AHH activity was assayed on whole cells by modification of published procedures (20). Prior to assay the cells were collected by centrifugation in Kimble disposable 125 x 15 mm glass screw-top culture tubes (one flask per tube) and washed once with Hanks’ BSS. The cell pellet was stored at -70°C after addition of 0.2 ml of 100 mM sodium phosphate, 0.1% bovine serum albumin (pH 8.5). The stored cells were assayed by first thawing the frozen cell pellet, mixing until a uniform cell suspension was obtained, then adding 0.8 ml of 50 mM Tris-HCl (pH 8.5),3 mM MgCl,, 200 mM sucrose, 1.7 mM NADPH, and 1.3 mM NADH. The reaction was started by the addition of 80 nmol of benzo(a)pyrene (BP) in 10 ~1 of acetone and terminated after 45 min of incubation at 37°C by the addition of 4.25 ml of cold hexane-acetone (3.25:1). After shaking for 10 min at 27”C, the phases were separated by centrifugation and 3 ml of the upper hexane phase was transferred to a clean tube. The aqueous phase (and other material remaining in the assay tube) was saved for DNA assays. The hexane phase was extracted with 1 ml of 1 N NaOH by vigorous mixing for 30 s and centrifuged to separate the phases. The alkaline phase was removed and its fluorescence was measured at an excitation wavelength of 396 nm and an emission wavelength of 512 nm. The fluorimeter was calibrated with a quinine sulfate standard. Relative fluorescence units were converted to picomoles of 3-hydroxybenzo(a)pyrene by use of a standard curve prepared by measuring the fluorescence of known concentrations of the phenol. One enzyme unit of AHH activity is that catalyzing the formation of the fluorescent equivalent of 1.0 pmol of 3-hydroxybenzo(a)pyrene/min under the above conditions. Assays of control cultures had fluorescence readings 210 times that of 0 time assays, those of 3-MC-induced cultures had lo-100 times. Under the above conditions the reaction was linear from 1 x lo6 to 8 x 10” cells (19) and with incubation time to at least 50 min (Kouri, Imblum, McKinney, and Sosnowski, unpublished results). NADH-cytochrome c reductase activities were measured by monitoring the rate of reduction of exogenous cytochrome c at 550 nm (E = 21,000 M-’
c REDUCTASE
ACTIVITY
121
cm-‘) in an dual-beam spectrophotometer. The reaction mixtures consisted of 4.5 x 10e5 M cytochrome c, 1.0 X 1oe4 M NASH, 1.0 X 1oe4 M EDT.4 in 0.05 M potassium phosphate buffer, pH 7.7 in a l-ml cuvette (l-cm path). The temperature was 25°C in all reductase assays. The influence of cytochrome oxidase on the reoxidation of cytochrome c was tested by addition of 5.0 x 10m4 M potassium cyanide and potassium cyanide was included in the reaction mixtures when appreciable reoxidation of cytochrome c was observed. Succinate-cytochrome c reductase activity was measured using the previous condition except 1 x 10e3 M succinate replaced NADH. The lymphocytes diluted to a l-ml volume with buffer were homogenized with a Tekman SDT-100N homogenizer prior to analysis and 0.1-0.4 ml of homogenate added to the cuvette. DNA assay and [3Hlthymidine incorporation. DNA content and incorporation of 13H]thymidine ([3H1TdR) into cellular DNA was measured on the same samples used for the enzyme assays. Cultures were exposed to 0.5 &i of [3H]TdR/ml for 24 h prior to assay. Portions of the cell homogenates from the NADH-cytochrome c reductase assay were diluted with 0.5 vol of 20% trichloroacetic acid and the aqueous phase of the AHH assays was diluted to 8 ml with 2% potassium acetate in 95% ethanol prior to mixing. All samples were stored overnight at 5°C and centrifuged at 800g for 20 min. The precipitates were hydrolyzed with 1.0 ml of 1 N perchloric acid for 20 min at 80°C. Aliquots of the hydrolysate were counted by liquid scintillation spectrometry in 5 ml of Handifluor. The remainder of the hydrolysate was used to determine DNA content by the method of Burton (21). RESULTS
Figure 1 shows the effect of cell homogenate concentration on the reduction of exogenous cytochrome c. The rate of reduction was linear from 1.25 x 1O+5to 5.2 x lO+‘j cells/ml of reaction mixture. The absorbance change per minute at 1.25 x lO+j cells/ml was approximately 0.01 A/min with NADH present; no change in absorbance was noted when NADH was omitted. Concentrations of NADH or cytochrome c larger than 1 x lop4 or 4.5 x lop5 M, respectively, did not increase the rate of cytochrome c reduction. The reaction had a broad pH optima at pH 7.7 and the rate at pH 7.2 or 8.2 was approximately 80% the rate at pH 7.7. There was a very weak NADPH-cytochrome c reductase activity which was 3-6% as active as the NADHdependent reaction. Specific inhibitors of mitochondrial
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also seen in the human erythrocyte using antibodies elicited against the NADH-cytochrome b, reductase and cytochrome b, (22). These results indicate that the principal NADH-cytochrome c reductase activity of stimulated human lymphocytes is immunochemically similar to the rotenone-insensitive activity of liver microsomes, outer mitochondrial membrane, and erythrocytes which consist of a flavoprotein, NADH-cytochrome b, reductase, and cytochrome b,. The small NADPH-
a
IO’ cells/ml FIG. 1. The dependence of NADH-cytochrome c reductase activity on cell concentration. The activity was measured as described in the Methods section on 120-h cultured cells. The concentration of homogenized lymphocytes per milliliter was determined by counting the number of cells from one large culture with an Autocytomter 11 prior to homogenization; suitable aliquots of cell homogenate were added to a final cuvette volume of 1 ml prior to initiation of the assay with NADH.
NADH-dependent cytochrome c reductase activity were used to determine the nature of this activity (Table I). Although rotenone (1 x low6 M) and antimycin A (9.1 x lop6 M) strongly inhibited heart mitochonridal NADH-cytochrome c reductase activity, these compounds had negligible effect on the human lymphocyte or liver microsomal NADH-cytochrome c reductase activity. The human lymphocyte homogenate did contain appreciable activity of an antimycin-sensitive succinate-cytochrome c reductase activity similar to the heart mitochondrial particles. Figure 2 shows the effect of specific antibodies to several liver microsomal enzymes on the human lymphocyte NADHcytochrome c reductase activity. Specific immune globulins elicited against purified rat liver microsomal NADH-cytochrome b, reductase (EC 1.6.2.2) and cytochrome b, inhibited the human lymphocyte activity 60% while nonimmune globulins or the immune globulin elicited to liver microsomal NADPH-cytochrome c (P-450) reductase had no effect. A similar lack of complete inhibition of NADH-cytochrome c reductase activity in human tissue was
TABLE
I
THE EFFECT OF INHIBITORS OF MITOCHONDRIAL ELECTRON TRANSPORT ON LYMPHOCYTE NADHCYTOCHROME c REDUCTASE ACTIVITY” Substrate
Inhibitor
Cytochrome c Reductase Activity omloll min/ml)
NADH
Beef heart mitochondrial Rotenone Antimycin A
Succinate Rotenone Antimycin
A
particles 10.7 2.8 2.8 8.0 7.8 0.5
(% inhibition) 0 75 75 0 3 93
Rat liver microsomes 6.2 6.5 6.2
0 0 0
lymphocyte homogenate 2.9 Rotenone 2.7 Antimycin A 2.8 1.0 Rotenone 1.0 Antimycin A 0.3
0 7 4 0 0 70
NADH Rotenone Antimycin Human NADH
Succinate
A
a The reaction mixtures contained 5.0 x 10e4 M KCN, 1 x 10e4 M NADH, or 1 x 1O-3 M succinate, 1 x 10e4 M EDTA, and 4.5 x lo-” M cytochrome c in 0.05 M potassium phosphate buffer, pH 7.7, in a final volume of 1 ml. The rotenone and antimycin A concentrations were 1 x lo-” M and 9.1 x lo-” M, respectively. The beef heart mitochondrial particles were present at 0.075 mg protein/ml, the liver microsomes were present at 0.02 mg protein/ml, and the lymphocyte homogenate (120-h stimulated) was present at approximately 7.5 X lo-” cells/ml. The rate of cytochrome c reduction was monitored at 550 nm. No succinate-dependent cytochrome c reductase activity was noted in rat liver microsomes.
LYMPHOCYTE
CYTOCHROME
c REDUCTASE
123
ACTIVITY
out mitogens and AHH activity was observed to be much higher in 3-MC-treated cultures. The inducibilities (the ratio of AHH activity in 3-MC-treated cultures to the activity in control cultures) for four different individuals assayed as described in Figs. 3 and 4 was observed to be between 6-12. A more complete description
4 2 mg globulin/ml
6
FIG. 2. The effect of anti-rat liver NADH-cytochrome b5 reductase or cytochrome b, globulin on the NADH-cytochrome c reductase activity of stimulated lymphocytes. The activity of uninhibited lymphocyte activity was 2.8 nmol of cytochrome c reduced/min/l x lo6 cells. The immune and nonimmune globulins were added to aliquots of homogenized stimulated lymphocytes (1.4 x lo6 cells or 63 pg protein) and incubated for 5 min prior to addition of cytochrome c and NADH. Nonimmune globulin, x; anti-NADPH-cytochrome c reductase globulin, Cl; anti-NADH-cytochrome b, reductase globulin, 0; and anti-cytochrome b, globulin, A.
dependent cytochrome c reductase activity was inhibited (65%) only by the antiNADPH-cytochrome c reductase globulin (results not shown). The levels of NADH-cytochrome c reductase and AHH activity during the time period from 24 to 120 h of mitogen activation are shown in Fig. 3. Total activity of NADH-cytochrome c reductase increases between 48 and 120 h of culture and the addition of 3-MC to the culture media has no effect on reductase activity. The mean specific activity of this individual’s lymphocyte reductase (units/pg DNA) for the period from 24 to 120 h is 0.32 (coefficient of variation = 0.06). However, there is some variability of the specific activity between individuals and a large number of samples will be required for a statistical analysis. Experiments are in progress to establish whether the reductase activity per cell remains constant throughout the 120-h activation period. AHH activity increased two- to tenfold as the result of mitogen activation compared to lymphocytes cultured 96 h with-
HOURS
FIG. 3. The time course of mitogen induction of lymphocyte NADH-cytochrome c reductase (A) and aryl hydrocarbon hydroxylase (B) activity in the presence and absence of 3-methylcholanthrene. Frozen samples were analyzed for the two activities in duplicate samples of stimulated lymphocytes of one individual. Cells cultured in absence of 3-MC, 0, and cells cultured in the presence of 3-MC, A, starting at zero time.
1 A
50
B p-o
i
13
1 Y 24
48
72
96
120
24
48
72
96
120
HOURS
FIG. 4. The time course of mitogen induction of lymphocyte DNA content (A) and DNA synthesis (B) in the presence and absence of J-methylcholanthrene. The samples from Fig. 3 were analyzed in duplicate for total DNA and for the rate of [3Hlthymidine incorporation into DNA. Cells cultured in absence of 3-MC, 0, and cells cultured in the presence of 3-MC, A, starting at zero time.
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of the factors yielding these relatively high inducibility ratios will be described elsewhere (Kouri, Imblum, McKinney, and Sosnowski, unpublished results). The time-dependent increase in total DNA and rates of DNA synthesis for these same cultures is shown in Fig. 4. Under these culture conditions, the increase in total DNA and rates of DNA synthesis is four- to fivefold during 48 to 96 h of culture. Although there is some variability involved in determining DNA in the nonstimulated cells, analysis of reductase activities of five different individuals at 0 time (no mitogen activation) and 96 h of mitogen activation resulted in mean reductase activities per microgram of DNA, which were not significantly different. These results suggest that the levels of NADH-cytochrome c reductase activity are the same in nonmitogen activated, as well as, in mitogen-activated lymphocytes. Stimulated lymphocytes from eight individuals cultured at three different times gave similar reductase activities per lo6 cells as seen in Table II. Although some variation exists, all individuals had similar levels of activity and there appear to be no significant differences between cells cultured in the presence or absence of 3MC. DISCUSSION
The current interest in human genetics and disease has prompted a number of studies related to the genetic regulation of carcinogen or drug metabolism and to the correlation with enzyme activities in stimulated human lymphocyte populations (3, 4). These human studies have been based on observations in model animal systems where the inducibility of the aryl hydrocarbon hydroxylase activity and the associated monooxygenases segregate as a single autosomal dominant gene (20, 23) and a single autosomal co-dominant gene (24, 25), or where the phenotype, noninducibility of AHH activity, can be the dominant trait (25), depending on the strain of inbred mice employed. Further, the susceptibility of these mice to 3-methylcholanthrene- (26, 28) or benzo(a)pyrene-
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KOURI TABLE
II
COMPARISON OF STIMULATED LYMPHOCYTE HOMOGENATE NADH-CYTOCHROME c REDLJCTASE ACTIVITY FROM HUMAN PERIPHERAL BLOOD” Sample
3-MC in culture
C
+ + + + + + + +
T H I MI RK MH AV
Nanomoles per minute per IO6 cells 3.7 3.9 5.0 4.6 4.8 5.1 3.4 5.3 4.6 4.1 5.2 4.4 3.9 3.3 4.5 4.9
k 0.1 k 0.1 2 0.2 k 0.2 k 0.2 k 0.1 ‘- 0.2 k 0.3 k 0.1 t 0.1 + 0.1 5 0.1 -r- 0.1 k 0.1 2 0.1 + 0.1
u The reaction mixture contained 1 x 10m4 M NADH, 4.5 x 10e5 M cytochrome c, 1 x 1O-4 M EDTA, and 1.4 x lo6 homogenized cells in 0.05 M potassium phosphate buffer, pH 7.7, in 1 ml. The rate of cytochrome c reduction was monitored at 550 nm. The average of the assays and the average deviation was calculated from three determinations of the NADH-cytochrome c reductase activity of each sample. The following samples were cultured separate from each other for 120 h: C, T, H, and I; MI; RK; MH; or AV.
induced (28) cancers appears to be linked to the increased ability of the genetically responsive mice to metabolize these carcinogens. The use of stimulated lymphocytes as a tool in these experiments has presented some technical difficulties since a number of factors seem to effect the stimulation of lymphocytes or the induction of AHH activity. Several factors, such as source of fetal calf serum, pokeweek mitogen, and phytohemagglutinin have been noted to effect the stimulation of lymphocyte growth or AHH activity (Kouri, Imblum, McKinney and Sosnowski, unpublished results). Total lymphocyte DNA content or rates of DNA synthesis determined from the rate of 13HlTdR incorporation generally have been determined to normalize the activity measured in lymphocytes;
LYMPHOCYTE
CYTOCHROME
however, certain criticisms of these measurements have been presented (2). This report presents evidence for the existence of a NADH-cytechrome c reductase activity in lymphocytes which is immunochemically similar to the NADH-cytochrome b, reductase and cytochrome b, found in liver microsomes, spleen microsomes, erythrocytes, and the outer mitochondrial membrane. The activity of this enzyme system can be easily measured and can be observed in nonmitogen-stimulated cultures. The increase in total activity appears to be dependent upon the number of mitogen-activated cells and is independent of the 3-MC-dependent induction of AHH activity. The role of this enzyme activity in metabolically quiescent peripheral blood lymphocytes is a matter for conjecture at this time. It has recently been noted that the ratio of polyenoic acids (18:2 and 20:4) to saturated fatty acids was doubled on stimulation of rabbit lymphocytes and thymocytes with concanavalin A in vitro or mycobacterium Calmette Guerin in viuo (29). The turnover of phospholipids in lymphocyte cell membranes has been shown to increase after PHA treatment and net increases in membrane phospholipids can be measured within several hours (30). Since the increased phospholipid turnover has been shown to be independent of either RNA or protein synthesis, it is suspected that the enzyme systems involved in lipid turnover are initially present in the resting cells. In liver and spleen, one biochemical function for the NADH-cytochrome c reductase system has been shown to be the desaturation of fatty acid derivatives like stearoyl CoA (31, 32) and the synthesis of ethanolamine plasmalogen, an unsaturated phospholipid (33-35). The lymphocyte NADH-cytochrome b, reductase and cytochrome b, may be involved in the early changes in lipid turnover. Experiments are in progress to determine if the reductase activity in mitogen-activated cells is identical to that in nonstimulated cells. The measurement of lymphocyte NADH-cytochrome c reductase may be a useful biochemical tool in the study of lymphocyte function. The activity appears to
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be directly related to the amount (and possibly viability) of cells present, may measure the microsomal content of the cells, and may be related to the degree of mitogen responsiveness. Since it is easily measured, the level of NADH-cytochrome c reductase activity could be a good parameter on which to base the measurement of any other lymphocyte activity. Normalization of the AHH activity in 3-MC-treated lymphocyte cultures using the NADH-cytochrome c reductase activity as a standard for stimulated lymphocytes should allow the effective comparison of the hydrocarbon metabolizing capacity of different individuals; such studies are in progress. ACKNOWLEDGMENTS This report was supported in part by National Cancer Institute Contracts NO1 CP 33362 (RAP), NOl-CP-T-43309-57 (RK), and NOl-CP-55605 (RK) and Contracts from the Council for Tobacco Research. REFERENCES 1. NOWELL, P. C. (1960) CancerRes. 20,462-466. 2. WAITHE, W. I., AND HIRSCHHORN, K. (1973) Handbook of Experimental Immunology, Vol. 2, pp. 25.1-25.10, Blackwell Scientific, Oxford. 3. KELLERMAN, G., LUYTEN-KELLERMAN, M., AND SHAW, C. R. (1973) Amer. J. Hum.
Genet. 25,
327-331. 4. KELLERMAN, G., LUYTEN-KELLERMAN, M., HORNING, M. G., AND STAFFORD, M. (1975) Drug Metab. Disp. 3, 47-50. 5. LANCE, E. M., FORD, P. J., AND RUSZKIEWICZ, M. (1968) Zmmunology 15, 571-580. 6. HALBFASO, H. J., PARAVINCINI, D., SCHAFER, H., MICHAELIS, W., AND STAIB, I. (1971) EuFOP. surg. Res. 3, 122-129. 7. KESTER, M. V., AND GRACY, R. W. (1975) Biothem. Biophys. Res. Commun. 65, 1270-1277. 8. STRITTMATTER, P., AND VELICK, S. F. (1956) J. Biol. Chem. 221, 253-264. 9. STRITTMATTER, P., AND VELICK, S. F. (1957) J. Biol. Chem. 228, 785-799. 10. KUMA, F., KIRAYAMA, K., ISHIZAWA, S., AND NAKAJIMA, H. (1972) J. Biol. Chem. 247, 550555. 11. REMMER, H., GREIM, H., SCHENKMAN, J. B., AND ESTABROOK, R. W. (1967) in Methods in
Enzymology (Colowick, S.P., and Kaplan, N.O., eds.), Vol. 10, pp. 703-708, Academic Press, New York.
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12. KING, T. E. (1967) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 10, pp. 202-208, Academic Press, New York. 13. TAKESUE, S., AND OMURA, T. (1970) J. Biothem. 67, 267-276. 14. PROUGH, R. A., AND MASTERS, B. S. S. (1973) Ann. N. Y. Acad. Sci. 212, 89-93. 15. MASTERS, B. S. S., AND ZIEGLER, D. M. (1971) Arch. Biochem. Biophys. 145, 358-364. 16. OMURA, T., AND TAKESUE, S. (1970) J. Biochem. 67, 249-257. 17. HRYCAY, E. G., AND PROUGH, R. A. (1974)Arch. Biochem. Biophys. 165, 331-339. 18. PROUGH, R. A., AND BURK, M. D. (1975) Arch. Biochem. Biophys. 170, 160-168. 19. KOURI, R. E., RATRIE, H., ATLAS, S. A., NIWA, A., AND NEBERT, D. W. (1974) Life Sci. 15, 1585-1595. 20. GIELEN, J. E., GOUJON, R. M., AND NEBERT, D. W., (1972) J. Biol. Chem. 247, 1125-1137. 21. BURTON, K. (1968) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 12B, pp. 163-166, Academic Press, New York. 22. KUMA, F., PROUGH, R. A., AND MASTERS, B. S. S. (1976) Arch. Biochem. Biophys. 172, 600607. 23. THOMAS, P. E., KOURI, R. E., AND HUTTON, J. J. (1972) B&hem. Genet. 6, 157-168.
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24. THOMAS, P. E., AND HUTTON, J. J. (1973) Biothem. Genet. 8, 249-257. 25. ROBINSON, J. R., CONSIDINE, N., AND NEBERT, D. W. (1974) J. Biol. Chem. 249, 5851-5859. 26. KOURI, R. E., RATRIE, H., AND WHITMIRE, C. E. (1973) J. Nat. Cancer Inst. 51, 197-200. 27. KOURI, R. E., RATRIE, H., AND WHITMIRE, C. E. (1975) Znt. J. Cancer 13, 714-720. 28. KOURI, R. E. (1976) in Symposium on Polynuclear Aromatic Hydrocarbons, (Freudenthal and Jones, eds.), in press. 29. FERBER, E., DEPASQUALE, G. G., AND RESCH, K. (1975) Biochim. Biophys. Acta 398, 364-376. 30. HARDY, D. A., AND LING, N. R. (1973) in The Cell Cycle in Development and Differentiation (Balls, M., and Billett, F. S., eds.), pp. 397-436, Cambridge Univ. Press, New York. 31. OSHINO, N., IMAI, Y., AND SATO, R. (1966) Biochim. Biophys. Acta 128, 13-28. 32. STRITTMATTER, P., SPATZ, L., CORCORAN, D., ROGERS, M. J., SETLOW, B., AND REDLINE, R., (1974) Proc. Nat. Acad. Sci. USA 71, 45654569. 33. WYKLE, R. L., BLANK, M. L., MALONE, B., AND SNYDER, F. (1972) J. Biol. Chem. 247, 54425447. 34. PALTAUF, F., AND HOLASEK, A. (1973) J. Biol. Chem. 246, 1609-1615. 35. PALTAUF, F., PROUGH, R. A., MASTERS, B. S., AND JOHNSTON, J. M. (1974) J. Biol. Chem. 249, 2661-2662.
OF
BIOCHEMISTRY
AND
BIOPHYSICS
NADH-Cytochrome
176, 119-126 (1976)
c Reductase Activity Human Lymphocytes
in Cultured
Similarity to the Liver Microsomal NADH-Cytochrome b5 Reductase and Cytochrome b5 Enzyme System RUSSELL A. PROUGH’, RICHARD L. IMBLUM2 Department
AND
RICHARD A. KOURP
ofBiochemistry and the UTHSC Cancer Center, The University of Texas Health Science Center at Hines Boulevard, Dallas, Texas 75235, and ZDepartment of Biochemical Oncology, Microbiological Associates, 4733 Bethesda Avenue, Bethesda, Maryland 20014
Dallas, 5323 Harry
Received January 22, 1976 A NADH-cytochrome c reductase activity was increased upon mitogen stimulation of human lymphocytes. The activity was not inhibited by antimycin A or rotenone but was specifically inhibited by antibodies elicited against rat liver NADH-cytochrome b, reductase or cytochrome b,. The activity was linear with cellular homogenates up to 5.2 x lo6 cells/ml and had a broad pH optimum of 7.7. The presence of 3-methylcholanthrene in mitogen stimulation media had no effect on the NADH-cytochrome c reductase activity but differentially induced the benzo(a)pyrene hydroxylase (AHH) activity. The reductase activity was present in nonstimulated cells and appears not to be significantly increased in activity per cell upon mitogen-stimulation of the peripheral lymphocyte.
Nowell has demonstrated that phytohemagglutinin (PHA)3 stimulates blast-like cell formation in cultures of human peripheral blood (1) and the response of lymphocytes to various activators (specific and nonspecific) has subsequently been reviewed by Waithe and Hirshhorn (2). Current interest in human genetics and disease states have prompted studies on the genetic regulation of aromatic hydrocarbon hydroxylase (AHH) activity in stimulated human lymphocyte populations (3) and on the correlation of AHH activity in human lymphocytes to the plasma half-life of drugs such as antipyrine and phenylbutazone (4). The biochemical components of lymphocytes or thymocytes are not well defined. 1 To whom reprint requests should be sent. 3 Abbreviations used: PHA, phytohemagglutinin; AHH, aromatic hydrocarbon hydroxylase; 3-MC, 3methylcholanthrene; Hanks’ BSS, Hanks’ balanced salt solution; FCS, fetal calf serum; HEPES, N-2hydroxyethylpiperazine-N’-2-ethanesulfonic acid; BP, benzo(a)pyrene; and L3H]TdR, [3Hlthymidine. 119
Copyright 0 1976 by Academic Press, All rights of reproduction in any form
Inc. reserved.
Several research groups have fractionated stimulated lymphocytes into subcellular components and have noted several enzymes to be present, such as 5’-nucleosidase, e&erase, NADH diaphorase, succinate dehydrogenase, or acid phosphatase activities (5, 6). To date, these enzyme activities have not been studied in detail due to the low activities and quantities of the enzymes in lymphocytes relative to other tissue. Recently, Kister and Gracy have reported that an isozyme of triosephosphate isomerase is preferentially observed in mitogen-stimulated human lymphocytes (7). This report will characterize a rotenoneinsensitive NADH-cytochrome c reductase activity in human lymphocytes which is mitogen-activated in culture by phytohemagglutinin and pokeweed mitogen. The enzymes involved are immunochemically similar to the NADH-cytochrome b, reductase and’ cytochrome b, which have been shown to exist in liver microsomes (8, 9) and erythrocytes (10). Lymphocyte cul-
120
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IMBLUM
tures in the presence of 3-methylcholanthrene (3-MC) were shown to possesshigh levels of AHH activity, but the NADHcytochrome c reductase activity was unaffected by this treatment. MATERIALS
AND
METHODS
Materials. 3-methylcholanBenzo(a)pyrene, threne, NADH, and NADPH were purchased from Sigma Chemical Co., St. Louis, MO. RPM1 medium 1640, fetal calf serum, glutamine, penicillin-streptomycin mixture, and Ficoll-Hypaque solution (sp gr 1.080) were from Microbiological Associates, Inc., Bethesda, Md. Pokeweed mitogen was obtained from Grand Island Biological Co., Grand Island, N.Y. and phytohemagglutinin was purchased from Burroughs-Wellcome Co., Research Triangle Park, N.C. Plastic flasks and tubes were products of the Becton, Dickinson, and Co., Los Angeles, Calif. Fifteen-milliliter 125 x 15 mm glass screw-top culture tubes were from Kimble Products Division, OwenIllinois Glass Co., Toledo, Ohio. Acetone, hexane, and Handifluor were obtained from Mallinckrodt, Inc., St. Louis, MO. Liver microsomes and heart mitochondrial particles were prepared as described (11, 12). Preparation of specific antibodies to microsomal proteins. NADH-cytochrome b, reductase was prepared from rat liver microsomes by the method of Takesue and Omura (13) and had a specific activity equal to that reported for the homogeneous enzyme. NADPH-cytochrome c (P-450) reductase was purified from rat liver microsomes using pancreatic lipase as described by Prough and Masters (14) and had a specific activity equal to that of the homogeneous enzyme from pig liver (15). Both reductases had a single major protein band (>98%) on disc electrophoresis using 7.5% polyacrylamide gels. The major band of either reductase were excised from the gels, homogenized in 0.9% NaCl, and used as the challenging antigens. Cytochrome b, was prepared using the trypsin digestion method of Omura and Takesue and had a specific content equal to that reported by Omura and Takesue (16). Since a single and identical protein and heme band was noted on disc gel electrophoresis, the cytochrome b5 was used without performing disc gel electrophoresis purification The antibodies to all three proteins were elicited in separate young male white rabbits as described earlier (17, 18). An expression of titer was obtained by measuring the percentage inhibition of liver microsomal NADH-cytochrome c reductase activity by serum from rabbits injected with either NADH-cytochrome b, reductase or cytochrome b, and the percentage inhibition of liver microsomal NADPH-cytochrome c reductase activity by serum from rabbits injected with NADPH-cytochrome c (P-450) reduc-
AND
KOURI
tase. Sera, with high antibody titer for each antigen, were pooled and the immunoglobins were prepared by precipitation with 1.75 M ammonium sulfate (17, 18). The precipitates were dialyzed against buffer and stored frozen. The protein concentration was obtained by measuring the 280-nm absorption of the solutions assuming that a 1 mg/ml solution has an absorbance of 1.35. The specificity of the antibodies was tested by employing them as inhibitors against NADH- and NADPH-dependent microsome-catalyzed cytochrome c reduction. The anti-NADH-cytochrome b, reductase and anti-cytochrome b, globulins inhibited only the NADH-dependent reaction (92 and 90%, respectively) and the anti-NADPH-cytochrome c (P-450) reductase globulin inhibited only the NADPH-dependent reaction (93%). The effects of these antibodies and their specificity toward other microsomal reactions have been described (17, 18). Cytochrome c reductase activity of a reaction mixture containing 1 x lo-* M purified NADH-cytochrome b5 reductase, 1 x 10e7 M purified cytochrome b,, 4 x 1O-s M cytochrome c, and 1 x 1O-4 M NADH was inhibited only by either the anti-NADH-cytochrome b, reductase or anti-cytochrome b, globulin, while only anti-NADPH-cytochrome c (P-450) reductase globulin inhibited cytochrome c reductase activity of a reaction mixture containing 1 X 10m8M purified NADPH-cytochrome c (P-450) reductase, 4 x lo-” M cytochrome c, and 1 x 10e4 M NADPH. When examined by Ouchterlony double diffusion tests in agar gel (19), all three immunoglobulins elicited single precipitation lines against the respective purified microsomal proteins. No spurring of the precipitant band of a given antibody was seen when all three proteins were added separately to adjacent wells of the Ouchterlony plates. These results suggest that the three antibodies are monospecific globulins to their respective antigens. Cell culture. Lymphocytes were isolated from fresh (less than 2 h old) whole blood as described previously (19) with several modifications. All operations were performed at room temperature. Venous blood (about 100 ml) was collected in sterile 200-ml evacuated containers in which 5000 units of sodium heparin per 100 ml of blood had been added previously. The blood was diluted 40% with sterile Hanks’ BSS buffer supplemented with penicillin and streptomycin and layered in lo-ml portions onto 5 ml of a solution of Ficoll-Hypaque (sp gr 1.080) in 16 x 100 mm plastic tubes. The Ficoll-Hypaque tubes were centrifuged at 600g in an swinging bucket rotor for 45 min. The lymphocyte band at the interface between the Ficoll-Hypaque and plasma was collected with sterile Pasteur pipets and transferred to 50-ml centrifuge tubes. The cells were pelleted by centrifugation and suspended in RPM1 1640 medium supplemented with 17% fetal calf serum (FCS), 2 mM glutamine, 25 mM HEPES (pH 7.4), 50 units of penicil-
LYMPHOCYTE
CYTOCHROME
lin/ml, 50 wg of streptomycin/ml, 1% phytohemagglutinin, and 1% pokeweed mitogen. Cells were counted in an Autocytometer 11 (Fisher Scientific, Pittsburgh, Pa.) and adjusted to a concentration of 0.5 x 106 cells/ml. Eight-milliliter aliquots of cells were added to Falcon 3013 plastic flasks and incubated at 37”C, 5% CO,-95% air. For induction of AHH activity, 3-methylcholanthrene (~-MC) was added to cells at a final concentration of 1.5 @M at 0 time, the time at which cultures were initiated. In certain experiments cell viability was tested using the trypan blue dye exclusion test and was greater than 80% in all cases. Enzyme assays. AHH activity was assayed on whole cells by modification of published procedures (20). Prior to assay the cells were collected by centrifugation in Kimble disposable 125 x 15 mm glass screw-top culture tubes (one flask per tube) and washed once with Hanks’ BSS. The cell pellet was stored at -70°C after addition of 0.2 ml of 100 mM sodium phosphate, 0.1% bovine serum albumin (pH 8.5). The stored cells were assayed by first thawing the frozen cell pellet, mixing until a uniform cell suspension was obtained, then adding 0.8 ml of 50 mM Tris-HCl (pH 8.5),3 mM MgCl,, 200 mM sucrose, 1.7 mM NADPH, and 1.3 mM NADH. The reaction was started by the addition of 80 nmol of benzo(a)pyrene (BP) in 10 ~1 of acetone and terminated after 45 min of incubation at 37°C by the addition of 4.25 ml of cold hexane-acetone (3.25:1). After shaking for 10 min at 27”C, the phases were separated by centrifugation and 3 ml of the upper hexane phase was transferred to a clean tube. The aqueous phase (and other material remaining in the assay tube) was saved for DNA assays. The hexane phase was extracted with 1 ml of 1 N NaOH by vigorous mixing for 30 s and centrifuged to separate the phases. The alkaline phase was removed and its fluorescence was measured at an excitation wavelength of 396 nm and an emission wavelength of 512 nm. The fluorimeter was calibrated with a quinine sulfate standard. Relative fluorescence units were converted to picomoles of 3-hydroxybenzo(a)pyrene by use of a standard curve prepared by measuring the fluorescence of known concentrations of the phenol. One enzyme unit of AHH activity is that catalyzing the formation of the fluorescent equivalent of 1.0 pmol of 3-hydroxybenzo(a)pyrene/min under the above conditions. Assays of control cultures had fluorescence readings 210 times that of 0 time assays, those of 3-MC-induced cultures had lo-100 times. Under the above conditions the reaction was linear from 1 x lo6 to 8 x 10” cells (19) and with incubation time to at least 50 min (Kouri, Imblum, McKinney, and Sosnowski, unpublished results). NADH-cytochrome c reductase activities were measured by monitoring the rate of reduction of exogenous cytochrome c at 550 nm (E = 21,000 M-’
c REDUCTASE
ACTIVITY
121
cm-‘) in an dual-beam spectrophotometer. The reaction mixtures consisted of 4.5 x 10e5 M cytochrome c, 1.0 X 1oe4 M NASH, 1.0 X 1oe4 M EDT.4 in 0.05 M potassium phosphate buffer, pH 7.7 in a l-ml cuvette (l-cm path). The temperature was 25°C in all reductase assays. The influence of cytochrome oxidase on the reoxidation of cytochrome c was tested by addition of 5.0 x 10m4 M potassium cyanide and potassium cyanide was included in the reaction mixtures when appreciable reoxidation of cytochrome c was observed. Succinate-cytochrome c reductase activity was measured using the previous condition except 1 x 10e3 M succinate replaced NADH. The lymphocytes diluted to a l-ml volume with buffer were homogenized with a Tekman SDT-100N homogenizer prior to analysis and 0.1-0.4 ml of homogenate added to the cuvette. DNA assay and [3Hlthymidine incorporation. DNA content and incorporation of 13H]thymidine ([3H1TdR) into cellular DNA was measured on the same samples used for the enzyme assays. Cultures were exposed to 0.5 &i of [3H]TdR/ml for 24 h prior to assay. Portions of the cell homogenates from the NADH-cytochrome c reductase assay were diluted with 0.5 vol of 20% trichloroacetic acid and the aqueous phase of the AHH assays was diluted to 8 ml with 2% potassium acetate in 95% ethanol prior to mixing. All samples were stored overnight at 5°C and centrifuged at 800g for 20 min. The precipitates were hydrolyzed with 1.0 ml of 1 N perchloric acid for 20 min at 80°C. Aliquots of the hydrolysate were counted by liquid scintillation spectrometry in 5 ml of Handifluor. The remainder of the hydrolysate was used to determine DNA content by the method of Burton (21). RESULTS
Figure 1 shows the effect of cell homogenate concentration on the reduction of exogenous cytochrome c. The rate of reduction was linear from 1.25 x 1O+5to 5.2 x lO+‘j cells/ml of reaction mixture. The absorbance change per minute at 1.25 x lO+j cells/ml was approximately 0.01 A/min with NADH present; no change in absorbance was noted when NADH was omitted. Concentrations of NADH or cytochrome c larger than 1 x lop4 or 4.5 x lop5 M, respectively, did not increase the rate of cytochrome c reduction. The reaction had a broad pH optima at pH 7.7 and the rate at pH 7.2 or 8.2 was approximately 80% the rate at pH 7.7. There was a very weak NADPH-cytochrome c reductase activity which was 3-6% as active as the NADHdependent reaction. Specific inhibitors of mitochondrial
122
PROLJGH,
IMBLUM
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KOURI
also seen in the human erythrocyte using antibodies elicited against the NADH-cytochrome b, reductase and cytochrome b, (22). These results indicate that the principal NADH-cytochrome c reductase activity of stimulated human lymphocytes is immunochemically similar to the rotenone-insensitive activity of liver microsomes, outer mitochondrial membrane, and erythrocytes which consist of a flavoprotein, NADH-cytochrome b, reductase, and cytochrome b,. The small NADPH-
a
IO’ cells/ml FIG. 1. The dependence of NADH-cytochrome c reductase activity on cell concentration. The activity was measured as described in the Methods section on 120-h cultured cells. The concentration of homogenized lymphocytes per milliliter was determined by counting the number of cells from one large culture with an Autocytomter 11 prior to homogenization; suitable aliquots of cell homogenate were added to a final cuvette volume of 1 ml prior to initiation of the assay with NADH.
NADH-dependent cytochrome c reductase activity were used to determine the nature of this activity (Table I). Although rotenone (1 x low6 M) and antimycin A (9.1 x lop6 M) strongly inhibited heart mitochonridal NADH-cytochrome c reductase activity, these compounds had negligible effect on the human lymphocyte or liver microsomal NADH-cytochrome c reductase activity. The human lymphocyte homogenate did contain appreciable activity of an antimycin-sensitive succinate-cytochrome c reductase activity similar to the heart mitochondrial particles. Figure 2 shows the effect of specific antibodies to several liver microsomal enzymes on the human lymphocyte NADHcytochrome c reductase activity. Specific immune globulins elicited against purified rat liver microsomal NADH-cytochrome b, reductase (EC 1.6.2.2) and cytochrome b, inhibited the human lymphocyte activity 60% while nonimmune globulins or the immune globulin elicited to liver microsomal NADPH-cytochrome c (P-450) reductase had no effect. A similar lack of complete inhibition of NADH-cytochrome c reductase activity in human tissue was
TABLE
I
THE EFFECT OF INHIBITORS OF MITOCHONDRIAL ELECTRON TRANSPORT ON LYMPHOCYTE NADHCYTOCHROME c REDUCTASE ACTIVITY” Substrate
Inhibitor
Cytochrome c Reductase Activity omloll min/ml)
NADH
Beef heart mitochondrial Rotenone Antimycin A
Succinate Rotenone Antimycin
A
particles 10.7 2.8 2.8 8.0 7.8 0.5
(% inhibition) 0 75 75 0 3 93
Rat liver microsomes 6.2 6.5 6.2
0 0 0
lymphocyte homogenate 2.9 Rotenone 2.7 Antimycin A 2.8 1.0 Rotenone 1.0 Antimycin A 0.3
0 7 4 0 0 70
NADH Rotenone Antimycin Human NADH
Succinate
A
a The reaction mixtures contained 5.0 x 10e4 M KCN, 1 x 10e4 M NADH, or 1 x 1O-3 M succinate, 1 x 10e4 M EDTA, and 4.5 x lo-” M cytochrome c in 0.05 M potassium phosphate buffer, pH 7.7, in a final volume of 1 ml. The rotenone and antimycin A concentrations were 1 x lo-” M and 9.1 x lo-” M, respectively. The beef heart mitochondrial particles were present at 0.075 mg protein/ml, the liver microsomes were present at 0.02 mg protein/ml, and the lymphocyte homogenate (120-h stimulated) was present at approximately 7.5 X lo-” cells/ml. The rate of cytochrome c reduction was monitored at 550 nm. No succinate-dependent cytochrome c reductase activity was noted in rat liver microsomes.
LYMPHOCYTE
CYTOCHROME
c REDUCTASE
123
ACTIVITY
out mitogens and AHH activity was observed to be much higher in 3-MC-treated cultures. The inducibilities (the ratio of AHH activity in 3-MC-treated cultures to the activity in control cultures) for four different individuals assayed as described in Figs. 3 and 4 was observed to be between 6-12. A more complete description
4 2 mg globulin/ml
6
FIG. 2. The effect of anti-rat liver NADH-cytochrome b5 reductase or cytochrome b, globulin on the NADH-cytochrome c reductase activity of stimulated lymphocytes. The activity of uninhibited lymphocyte activity was 2.8 nmol of cytochrome c reduced/min/l x lo6 cells. The immune and nonimmune globulins were added to aliquots of homogenized stimulated lymphocytes (1.4 x lo6 cells or 63 pg protein) and incubated for 5 min prior to addition of cytochrome c and NADH. Nonimmune globulin, x; anti-NADPH-cytochrome c reductase globulin, Cl; anti-NADH-cytochrome b, reductase globulin, 0; and anti-cytochrome b, globulin, A.
dependent cytochrome c reductase activity was inhibited (65%) only by the antiNADPH-cytochrome c reductase globulin (results not shown). The levels of NADH-cytochrome c reductase and AHH activity during the time period from 24 to 120 h of mitogen activation are shown in Fig. 3. Total activity of NADH-cytochrome c reductase increases between 48 and 120 h of culture and the addition of 3-MC to the culture media has no effect on reductase activity. The mean specific activity of this individual’s lymphocyte reductase (units/pg DNA) for the period from 24 to 120 h is 0.32 (coefficient of variation = 0.06). However, there is some variability of the specific activity between individuals and a large number of samples will be required for a statistical analysis. Experiments are in progress to establish whether the reductase activity per cell remains constant throughout the 120-h activation period. AHH activity increased two- to tenfold as the result of mitogen activation compared to lymphocytes cultured 96 h with-
HOURS
FIG. 3. The time course of mitogen induction of lymphocyte NADH-cytochrome c reductase (A) and aryl hydrocarbon hydroxylase (B) activity in the presence and absence of 3-methylcholanthrene. Frozen samples were analyzed for the two activities in duplicate samples of stimulated lymphocytes of one individual. Cells cultured in absence of 3-MC, 0, and cells cultured in the presence of 3-MC, A, starting at zero time.
1 A
50
B p-o
i
13
1 Y 24
48
72
96
120
24
48
72
96
120
HOURS
FIG. 4. The time course of mitogen induction of lymphocyte DNA content (A) and DNA synthesis (B) in the presence and absence of J-methylcholanthrene. The samples from Fig. 3 were analyzed in duplicate for total DNA and for the rate of [3Hlthymidine incorporation into DNA. Cells cultured in absence of 3-MC, 0, and cells cultured in the presence of 3-MC, A, starting at zero time.
124
PROUGH,
IMBLUM
of the factors yielding these relatively high inducibility ratios will be described elsewhere (Kouri, Imblum, McKinney, and Sosnowski, unpublished results). The time-dependent increase in total DNA and rates of DNA synthesis for these same cultures is shown in Fig. 4. Under these culture conditions, the increase in total DNA and rates of DNA synthesis is four- to fivefold during 48 to 96 h of culture. Although there is some variability involved in determining DNA in the nonstimulated cells, analysis of reductase activities of five different individuals at 0 time (no mitogen activation) and 96 h of mitogen activation resulted in mean reductase activities per microgram of DNA, which were not significantly different. These results suggest that the levels of NADH-cytochrome c reductase activity are the same in nonmitogen activated, as well as, in mitogen-activated lymphocytes. Stimulated lymphocytes from eight individuals cultured at three different times gave similar reductase activities per lo6 cells as seen in Table II. Although some variation exists, all individuals had similar levels of activity and there appear to be no significant differences between cells cultured in the presence or absence of 3MC. DISCUSSION
The current interest in human genetics and disease has prompted a number of studies related to the genetic regulation of carcinogen or drug metabolism and to the correlation with enzyme activities in stimulated human lymphocyte populations (3, 4). These human studies have been based on observations in model animal systems where the inducibility of the aryl hydrocarbon hydroxylase activity and the associated monooxygenases segregate as a single autosomal dominant gene (20, 23) and a single autosomal co-dominant gene (24, 25), or where the phenotype, noninducibility of AHH activity, can be the dominant trait (25), depending on the strain of inbred mice employed. Further, the susceptibility of these mice to 3-methylcholanthrene- (26, 28) or benzo(a)pyrene-
AND
KOURI TABLE
II
COMPARISON OF STIMULATED LYMPHOCYTE HOMOGENATE NADH-CYTOCHROME c REDLJCTASE ACTIVITY FROM HUMAN PERIPHERAL BLOOD” Sample
3-MC in culture
C
+ + + + + + + +
T H I MI RK MH AV
Nanomoles per minute per IO6 cells 3.7 3.9 5.0 4.6 4.8 5.1 3.4 5.3 4.6 4.1 5.2 4.4 3.9 3.3 4.5 4.9
k 0.1 k 0.1 2 0.2 k 0.2 k 0.2 k 0.1 ‘- 0.2 k 0.3 k 0.1 t 0.1 + 0.1 5 0.1 -r- 0.1 k 0.1 2 0.1 + 0.1
u The reaction mixture contained 1 x 10m4 M NADH, 4.5 x 10e5 M cytochrome c, 1 x 1O-4 M EDTA, and 1.4 x lo6 homogenized cells in 0.05 M potassium phosphate buffer, pH 7.7, in 1 ml. The rate of cytochrome c reduction was monitored at 550 nm. The average of the assays and the average deviation was calculated from three determinations of the NADH-cytochrome c reductase activity of each sample. The following samples were cultured separate from each other for 120 h: C, T, H, and I; MI; RK; MH; or AV.
induced (28) cancers appears to be linked to the increased ability of the genetically responsive mice to metabolize these carcinogens. The use of stimulated lymphocytes as a tool in these experiments has presented some technical difficulties since a number of factors seem to effect the stimulation of lymphocytes or the induction of AHH activity. Several factors, such as source of fetal calf serum, pokeweek mitogen, and phytohemagglutinin have been noted to effect the stimulation of lymphocyte growth or AHH activity (Kouri, Imblum, McKinney and Sosnowski, unpublished results). Total lymphocyte DNA content or rates of DNA synthesis determined from the rate of 13HlTdR incorporation generally have been determined to normalize the activity measured in lymphocytes;
LYMPHOCYTE
CYTOCHROME
however, certain criticisms of these measurements have been presented (2). This report presents evidence for the existence of a NADH-cytechrome c reductase activity in lymphocytes which is immunochemically similar to the NADH-cytochrome b, reductase and cytochrome b, found in liver microsomes, spleen microsomes, erythrocytes, and the outer mitochondrial membrane. The activity of this enzyme system can be easily measured and can be observed in nonmitogen-stimulated cultures. The increase in total activity appears to be dependent upon the number of mitogen-activated cells and is independent of the 3-MC-dependent induction of AHH activity. The role of this enzyme activity in metabolically quiescent peripheral blood lymphocytes is a matter for conjecture at this time. It has recently been noted that the ratio of polyenoic acids (18:2 and 20:4) to saturated fatty acids was doubled on stimulation of rabbit lymphocytes and thymocytes with concanavalin A in vitro or mycobacterium Calmette Guerin in viuo (29). The turnover of phospholipids in lymphocyte cell membranes has been shown to increase after PHA treatment and net increases in membrane phospholipids can be measured within several hours (30). Since the increased phospholipid turnover has been shown to be independent of either RNA or protein synthesis, it is suspected that the enzyme systems involved in lipid turnover are initially present in the resting cells. In liver and spleen, one biochemical function for the NADH-cytochrome c reductase system has been shown to be the desaturation of fatty acid derivatives like stearoyl CoA (31, 32) and the synthesis of ethanolamine plasmalogen, an unsaturated phospholipid (33-35). The lymphocyte NADH-cytochrome b, reductase and cytochrome b, may be involved in the early changes in lipid turnover. Experiments are in progress to determine if the reductase activity in mitogen-activated cells is identical to that in nonstimulated cells. The measurement of lymphocyte NADH-cytochrome c reductase may be a useful biochemical tool in the study of lymphocyte function. The activity appears to
c REDUCTASE
ACTIVITY
125
be directly related to the amount (and possibly viability) of cells present, may measure the microsomal content of the cells, and may be related to the degree of mitogen responsiveness. Since it is easily measured, the level of NADH-cytochrome c reductase activity could be a good parameter on which to base the measurement of any other lymphocyte activity. Normalization of the AHH activity in 3-MC-treated lymphocyte cultures using the NADH-cytochrome c reductase activity as a standard for stimulated lymphocytes should allow the effective comparison of the hydrocarbon metabolizing capacity of different individuals; such studies are in progress. ACKNOWLEDGMENTS This report was supported in part by National Cancer Institute Contracts NO1 CP 33362 (RAP), NOl-CP-T-43309-57 (RK), and NOl-CP-55605 (RK) and Contracts from the Council for Tobacco Research. REFERENCES 1. NOWELL, P. C. (1960) CancerRes. 20,462-466. 2. WAITHE, W. I., AND HIRSCHHORN, K. (1973) Handbook of Experimental Immunology, Vol. 2, pp. 25.1-25.10, Blackwell Scientific, Oxford. 3. KELLERMAN, G., LUYTEN-KELLERMAN, M., AND SHAW, C. R. (1973) Amer. J. Hum.
Genet. 25,
327-331. 4. KELLERMAN, G., LUYTEN-KELLERMAN, M., HORNING, M. G., AND STAFFORD, M. (1975) Drug Metab. Disp. 3, 47-50. 5. LANCE, E. M., FORD, P. J., AND RUSZKIEWICZ, M. (1968) Zmmunology 15, 571-580. 6. HALBFASO, H. J., PARAVINCINI, D., SCHAFER, H., MICHAELIS, W., AND STAIB, I. (1971) EuFOP. surg. Res. 3, 122-129. 7. KESTER, M. V., AND GRACY, R. W. (1975) Biothem. Biophys. Res. Commun. 65, 1270-1277. 8. STRITTMATTER, P., AND VELICK, S. F. (1956) J. Biol. Chem. 221, 253-264. 9. STRITTMATTER, P., AND VELICK, S. F. (1957) J. Biol. Chem. 228, 785-799. 10. KUMA, F., KIRAYAMA, K., ISHIZAWA, S., AND NAKAJIMA, H. (1972) J. Biol. Chem. 247, 550555. 11. REMMER, H., GREIM, H., SCHENKMAN, J. B., AND ESTABROOK, R. W. (1967) in Methods in
Enzymology (Colowick, S.P., and Kaplan, N.O., eds.), Vol. 10, pp. 703-708, Academic Press, New York.
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PROUGH,
IMBLUM
12. KING, T. E. (1967) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 10, pp. 202-208, Academic Press, New York. 13. TAKESUE, S., AND OMURA, T. (1970) J. Biothem. 67, 267-276. 14. PROUGH, R. A., AND MASTERS, B. S. S. (1973) Ann. N. Y. Acad. Sci. 212, 89-93. 15. MASTERS, B. S. S., AND ZIEGLER, D. M. (1971) Arch. Biochem. Biophys. 145, 358-364. 16. OMURA, T., AND TAKESUE, S. (1970) J. Biochem. 67, 249-257. 17. HRYCAY, E. G., AND PROUGH, R. A. (1974)Arch. Biochem. Biophys. 165, 331-339. 18. PROUGH, R. A., AND BURK, M. D. (1975) Arch. Biochem. Biophys. 170, 160-168. 19. KOURI, R. E., RATRIE, H., ATLAS, S. A., NIWA, A., AND NEBERT, D. W. (1974) Life Sci. 15, 1585-1595. 20. GIELEN, J. E., GOUJON, R. M., AND NEBERT, D. W., (1972) J. Biol. Chem. 247, 1125-1137. 21. BURTON, K. (1968) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 12B, pp. 163-166, Academic Press, New York. 22. KUMA, F., PROUGH, R. A., AND MASTERS, B. S. S. (1976) Arch. Biochem. Biophys. 172, 600607. 23. THOMAS, P. E., KOURI, R. E., AND HUTTON, J. J. (1972) B&hem. Genet. 6, 157-168.
AND
KOURI
24. THOMAS, P. E., AND HUTTON, J. J. (1973) Biothem. Genet. 8, 249-257. 25. ROBINSON, J. R., CONSIDINE, N., AND NEBERT, D. W. (1974) J. Biol. Chem. 249, 5851-5859. 26. KOURI, R. E., RATRIE, H., AND WHITMIRE, C. E. (1973) J. Nat. Cancer Inst. 51, 197-200. 27. KOURI, R. E., RATRIE, H., AND WHITMIRE, C. E. (1975) Znt. J. Cancer 13, 714-720. 28. KOURI, R. E. (1976) in Symposium on Polynuclear Aromatic Hydrocarbons, (Freudenthal and Jones, eds.), in press. 29. FERBER, E., DEPASQUALE, G. G., AND RESCH, K. (1975) Biochim. Biophys. Acta 398, 364-376. 30. HARDY, D. A., AND LING, N. R. (1973) in The Cell Cycle in Development and Differentiation (Balls, M., and Billett, F. S., eds.), pp. 397-436, Cambridge Univ. Press, New York. 31. OSHINO, N., IMAI, Y., AND SATO, R. (1966) Biochim. Biophys. Acta 128, 13-28. 32. STRITTMATTER, P., SPATZ, L., CORCORAN, D., ROGERS, M. J., SETLOW, B., AND REDLINE, R., (1974) Proc. Nat. Acad. Sci. USA 71, 45654569. 33. WYKLE, R. L., BLANK, M. L., MALONE, B., AND SNYDER, F. (1972) J. Biol. Chem. 247, 54425447. 34. PALTAUF, F., AND HOLASEK, A. (1973) J. Biol. Chem. 246, 1609-1615. 35. PALTAUF, F., PROUGH, R. A., MASTERS, B. S., AND JOHNSTON, J. M. (1974) J. Biol. Chem. 249, 2661-2662.
