- Email: [email protected]
Enzymes of nucleic acid metabolism as biochemical markers of T cell development in mouse
Cell Biology International
401
Reports, Vol. 8, No. 5, May 1984
ENZYMES OF NUCLEIC ACID METABOLISM AS BIOCHEMICAL OF T CELL DEVELOPMENTIN MOUSE Subbanaidu Department School
MARKERS
Ramagopal*
of Pharmacology, New York University of Medicine, New York, NY 10016 SUMMARY
The levels of three purine salvage enzymes, adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP) and 5'-nucleotidase (5*-N), which are known to be associated with certain immunodeficiThese were determined in mouse T lymphocytes. ency disorders enzymes showed characteristic changes depending on the stage of T The activity of ADA was 5-fold higher in cell development. On the other hand, the thymocytes compared to splenic T cells. splenic T cells displayed a 2-fold and a 4-8 fold greater activity The apparof PNP and 5'-N, respectively than those of thymocytes. for all the 3 enzymes ent Km and Vmax values have been determined in the immature and mature T cells. The data demonstrate that the absolute and relative activity of these enzymes may be used as biochemical markers to characterize the T lymphocytes during different stages of differentiation. INTRODUCTION The discovery of an apparent relationship between the activities of some purine salvage enzymes and certain inborn errors of metabolism has elicited the attention of several immunologists in recent years. Deficiencies of adenosine deaminase and purine nucleoside phosphorylase have been found in children with severe combined immunodeficiency disease (SCID) and severe T cell dysfunction, respectively [1,2]. Another enzyme, 5'-nucleotidase, was deficient in various primary immunodeficiency states such as congenital X-linked agammaglobulinemia (CAG), adult onset common variable hypogammaglobulinemia (CVHG) and chronic lymphocytic leukemia (CLL) [3,4]. ADA (E.C. 3.5.4.4) catalyzes the deamination of adenosine and deoxyadenosine, PNP (E.C.2.4.2.1) functions in the reversible phosphorolysis of purine nucleosides except adenosine and 5'-N (E.C.3.1.3.5) dephosphorylates riboand deoxyribonucleoside monophosphates. How the deficiency of these enzymes relates to immunodeficiency disorders is still unsolved. *Present USA
address:
USDA-ARS, 800 Buchanan
0309-16511841050401-05/$03.00/O
Street,
@ 1984 Academic
Berkeley
CA 94710
Press Inc. (London)
Ltd.
402
Cell Biolog y International
Reports, Vol. 8, No. 5, May 7984
One way of approaching this problem is to examine the expression of the above enzyme activities during the various stages of lymphocyte differentiation. Recently, we showed that these enzymes thymocyte differentiation displayed characteristic changes during in human beings [5]. The lymphoid cells of mouse offer an attractive choice in this regard as the genetics of the immune system in this organism have been characterized extensively. Except for limited information on 5'-N [6], the status of other enzymes in mouse is presently unknown. In the present study, I report the changes in ADA, PNP and 5*-N in the immature (thymocytes) and mature (splenic T cells) mouse lymphocytes. Sensitive, radiometric assays were employed to estimate the level of each of of T cell development. the three enzymes at the two stages MATERIALS AND METHODS Animals and preparation of cells: Mouse strains C57BL/6, A/J The thymus and spleen and AKR of 6-16 weeks of age were used. were removed asceptically soon after cervical dislocation of the animals and transferred to RPM1 1640 with 1% bovine serum albumin. from each organ by gentle A single cell suspension was prepared teasing and passed through a nylon sieve to remove the tissue debris. The red blood cells were lysed with 0.16 M NH4C1, 0.05 M Tris-HCl (pH 7.5) and 0.1 mM Na2EDTA (pH 7.4) and the lymphocytes Splenic were washed again with the above medium by centrifugation. T cells were enriched by a nylon wool column [7]. The nylon wool was preactivated at 37°C overnight and splenic lymphocytes were After incubating for 45 min to 1 hr at applied to the column. 37"C, splenic T cells were eluted with RPM1 plus 1% BSA. The cells were concentrated by centrifugation and washed with the above medium. This procedure (7) greatly enriches splenic T cells and We have not estimated the actual proportion of was used as such. any contaminating B or null cells. The frozen cells were resuspended in Extraction of enzymes: 10 mM potassium phosphate (pH 7.2), 0.1 mM &mercaptoethanol, 1 mM Na2EDTA (pH 7.2) and 0.25% Kyro-EOB and lysed by incubating The superat 37°C for 10 min and brief mixing in a Vortex mixer. natant obtained after centrifugation at 10,000 rpm (Sorvall SS34 The enzyme rotor) for 30 min was used as the enzyme source. extracts were divided into small aliquots and stored frozen at -76OC. The protein concentration was estimated by the Lowry procedure [8] with bovine serum albumin as the standard. Published procedures [g-11] were modified. Enzyme assays: ADA was assayed in 50 mM potassium phosphate, pH 7.2, 0.1 mM reaction (0.02 uCi) and the extract. The [8-14C] adenosine PNP,i.n the extract was mixture was incubated for 10 min at 37'C. determined in 50 mM Tris-HCl (pH 7.8), 5 mM MgC12, 2 mM dithioand 0.08 mM [8-14C] 1.15 mM ribose-l-phosphate threitol, hypoxanthine (0.25 $i). The incubation was for 15 min at 37OC. for 5'-N consisted of 50 mM Tris-HCl The incubation mixture
(pN
7‘5),
10
mM
M&$12,
0.5%
(V/V)
Triton
x-100,
20
mM p-
glpcerophoaphate, 5 mM sodium potassium tartarate, 1 mM [14CJ adeaosine S'monophosphate (0.1 @IX.) and the extract; it was volume was 50 elf. and incubated far f hr at 3?*C. The reactfon the enzyme reactions were terminated by adding l/10 volume of 1Opl [8-24C] Adenosine (54.7 mCi/mmole), [8-l4C] hypoformic acid. xanthine (52.8 mCi/mmole) and 1:"4CJ adenoeine S'monophosphate (479 Ci/m.mole) were purchased from New England Nuclear Corp., Boston. The enzyme actfvftles were linear with incubation tfme and The times noted above in amount of protein (data not shown; 5). Routinely, 2-4 the standard assays were in the linear range. different concentrations of protein were tested for each point, Thin Layer Ch~~~tograp~y: Usually, 5 &I of the reaction mfxture wae mixed with unlabeled chemicals for UV identification, applied to polyethyleneimine cellulose plate (FBI-cellulose, Brinkman) and developed in bu~ano~:methanol:B2O:N~4~H (~~:20:2~: 1) for 6% hr [12J. The spots were cut out and counted in a liquid scfntillatfon couriter.
Table X. shows the activity data for AD& PNP and 5'-N in three different mouse streinsl The enzymes were prepared from the thymocytes and splenic T cells from each &rain. In general, the enzyme levels rln the two cell types were similar in all the mice. ADA activity fn thymocytes was 5-fold greater than that in spleen. On the other hand, PI@ level was a-fold higher fn the mature T cells (3-6 fold) in the af spleen. Similarly, the S'-N was greater splenic T cells. Since non-specific phosphetases also interfere in the eet%mation of 5*-N activity, we have included 8-glycerophosphate and sod&urn potassium tartarate during; fneubation to prevent In Table 2, we have further sach aetivitfes. examined the 5'-N activity J;n the presence of AOPCP, a specific inhibitor of this enzyme [13]. The thymocyte enzyme was inhibited by about 70% and the splenic T cell enzyme was inhfbfted by 90%. A consideration of the AOP&Pm~nhibitab~e 5'-N activity indicates an 8-fold difference between the thymua and spleen. The 5'-N activity in whole cells was also examined (5) and the results (ectivitfes in nmol/hr/mg protefn were 89&i for thymes and 816-Z for spleen) were s&milar to those crf cell lysates as in human lymphocytes. All the enzymo.s from the two stages of development displayed MLchaefis-Menten kinetics. The kinetic properties are listed in Table 3.. The apparent Km data for ADA and PNP were similar between the thymus and spleen (Table 3); however, the Rm for 5'-N was Z-fold greater in the spleen. The differences in V,, for the 3 enzymes confirm the data in Table 1.
404
Cell Biolog y International
Reports, Vol. 8, No. 5, May 1984
The above results demonstrate that there is a characteristic shift in the levels of ADA, PNP and 5'-N during the development of T cells in mice. Thus, during the early stages of development the activity of ADA is high and at later stages, the activities of PNP and 5'-N are high. Similar changes of these enzymes have been Taken demonstrated in rat [14] and recently, in humans [5,15]. together these data suggest that the expression of the above three enzymes is developmentally regulated in the T lymphocytes of varithey must serve as useful biochemical Therefore, ous organisms. markers in the future studies on T cell ontogeny. The results also indicate an alteration in the metabolism of nucleic acids during T cell differentiation which in turn may affect the development of various immunocompetent cells. ACKNOWLEDGMENTS I am grateful to R. Basch of the Pathology Department for the mice and help in the cell preparation and I would like to thank Marc Kaisman for technical assistance. REFERENCES 1.
Giblett, E.R., Anderson, J.E., Cohen, Meuwissen, H.J. (1972) Lancet 2:1067.
2.
Giblett, E.R., Ammann, D., L.K. (1975) Lancet E:lOlO.
Wara, W., Sandman, R. and Diamond,
Edwards, N.L., Magilaury, (1978) Science 201:628.
D.B.,
3.
F.,
Cassidy,
Pollara,
J.T.
4.
Silber, R., Conkyln, M., Grusky, (1975) J. Clin. Invest. -56:1234.
5.
Ramagopal, S. and G. Reem (1982) Thymus 4:163.
6.
Uusitalo, Cytochem.
7.
Trixio,
8.
Lowry, O.H., Rosebrough, N.J., (1951) J. Biol. Chem. 193:265.
9.
Carson, -57:274.
R.J. and 25:87. -
Karnovsky,
D. and Cudkowicz,
D.A.
10.
Milman, G., Anton, -15:4967.
D.L.
11.
Chatterjee, S.K., Battacharya, Anal. Biochem. -95 : 497
and Fox,
I.H.
G., and Zucker-Franklin,
M.J.
G. (1974)
and Seegmiller,
B. and
Parr, J.E.
(1977) J. A.L. (1976)
and Weber, J.L.
D.
J.
Histochem.
Immunol.
113:1093.
and Randall, J. (1976)
M. and Barlow,
Clin.
R.J. Invest.
Biochemistry J.J.
(1979)
Cell Biolog y International
and
E.
Randerath,
(1967)
12.
Randerath, -18:323.
13.
Edwards, N.L., Gelfand, E.W., (1979) Proc. Natl. Acad, Sci.
14.
Barton, R., Martiniuk, F., Hirschhorn, I. (1980) Cell. Immunol. -49:208.
15.
K.
Reports, Vol. 8, No. 5, May 1984
Burke, L., Dosch, USA -76:3474.
Ma, D. D. F., Sylwestrowicz, Franks, R., Janossy, G. and Immunol. 129:1430.
Received:
Methods
Table Activities of various
of ADA, PNP and 5'-N mouse strains.
Mouse Strains
T cell source
17th April
A/J AKR
Thymus Spleen Thymus Spleen Thymus Spleen
311.6 58 288.5 82.9 267.0 56.0
f 26.7 f 4.1 + 103.9 +_ 21.5
I.H.
M., J.
1984.
1 and splenic
PNP
T cells
5'-N
(nmoles/min/mg) C57BL/6
Hand Fox,
T., Granger, S., Massia, Hoffbrand, A. V. (1982)
in thymocytes
ADA
Enzymol.
R. and Goldschneider,
Accepted:
7th March 1984.
in
(nmoles/hr/mg) 22.9 k 40.4 2 5.8 + 12.7 f n.d.
1.8 5.4 2.2 4.2
51.6 343.5 82.2 430 98.4 322.4
4 6.4 f 32.1 t 41.4 ? 217.6
The enzyme activities were determined as described in Materials and Methods. The mean of 3 (C57BL/6 of 6-8 weeks of age) or 5 (A/J of 6-16 weeks of age) experiments f standard deviation is shown. Only one experiment was conducted with AKR strain; the mean of 3 incubations is shown. n.d. not determined.
Cell Biolog y lnterna tional Reports, Vol. 8, No. 5, May 1984
406
Table 2 Effect of AOPCP on 5'-N activity of T cells from the thymus and spleen
Treatment
Thymus
Spleen
(nmoles/hr/mg) (% Inhibition) (nmoles/hr/mg) (X Inhibition) None AOPCP (3mM) AOPCP (6mM)
60.5 18.7 23.8
384.0 43.0 39.1
69 61
89 90
The 5'-N activity was estimated as in Table 1 except that in some reactions AOPCP (a,&methylene adenosine 5'-diphosphate, P.L. Biochemicals) was also present at the indicated concentration.
Table 3 Kinetic properties of ABA, PNP and 5'-N from thymocytes and splenic T cells.
ABA
PNP
5'-N
Source T cells
Km
"max
Km
"max
Thymus Spleen
67 59
285.7 64.5
500 500
122.0 227.3
Km
"max
75 154
64.5 526.3
The data were obtained from Lineweaver-Burk plots for the activities of ABA, PNP and 5'-N in thymocytes and splenic T cells. Adenosine (0.01 - lmM), hypoxanthine (0.05-0.25 mM) and adenosine 5'-monophosphate (O.l-2mM) were used as substrates for ABA, PNP The Km values are in w and V,, values and 5'-N, respectively. are in nmoles/min/mg protein (ABA and PNP) or nmoles/hr/mg protein (5'-N).
401
Reports, Vol. 8, No. 5, May 1984
ENZYMES OF NUCLEIC ACID METABOLISM AS BIOCHEMICAL OF T CELL DEVELOPMENTIN MOUSE Subbanaidu Department School
MARKERS
Ramagopal*
of Pharmacology, New York University of Medicine, New York, NY 10016 SUMMARY
The levels of three purine salvage enzymes, adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP) and 5'-nucleotidase (5*-N), which are known to be associated with certain immunodeficiThese were determined in mouse T lymphocytes. ency disorders enzymes showed characteristic changes depending on the stage of T The activity of ADA was 5-fold higher in cell development. On the other hand, the thymocytes compared to splenic T cells. splenic T cells displayed a 2-fold and a 4-8 fold greater activity The apparof PNP and 5'-N, respectively than those of thymocytes. for all the 3 enzymes ent Km and Vmax values have been determined in the immature and mature T cells. The data demonstrate that the absolute and relative activity of these enzymes may be used as biochemical markers to characterize the T lymphocytes during different stages of differentiation. INTRODUCTION The discovery of an apparent relationship between the activities of some purine salvage enzymes and certain inborn errors of metabolism has elicited the attention of several immunologists in recent years. Deficiencies of adenosine deaminase and purine nucleoside phosphorylase have been found in children with severe combined immunodeficiency disease (SCID) and severe T cell dysfunction, respectively [1,2]. Another enzyme, 5'-nucleotidase, was deficient in various primary immunodeficiency states such as congenital X-linked agammaglobulinemia (CAG), adult onset common variable hypogammaglobulinemia (CVHG) and chronic lymphocytic leukemia (CLL) [3,4]. ADA (E.C. 3.5.4.4) catalyzes the deamination of adenosine and deoxyadenosine, PNP (E.C.2.4.2.1) functions in the reversible phosphorolysis of purine nucleosides except adenosine and 5'-N (E.C.3.1.3.5) dephosphorylates riboand deoxyribonucleoside monophosphates. How the deficiency of these enzymes relates to immunodeficiency disorders is still unsolved. *Present USA
address:
USDA-ARS, 800 Buchanan
0309-16511841050401-05/$03.00/O
Street,
@ 1984 Academic
Berkeley
CA 94710
Press Inc. (London)
Ltd.
402
Cell Biolog y International
Reports, Vol. 8, No. 5, May 7984
One way of approaching this problem is to examine the expression of the above enzyme activities during the various stages of lymphocyte differentiation. Recently, we showed that these enzymes thymocyte differentiation displayed characteristic changes during in human beings [5]. The lymphoid cells of mouse offer an attractive choice in this regard as the genetics of the immune system in this organism have been characterized extensively. Except for limited information on 5'-N [6], the status of other enzymes in mouse is presently unknown. In the present study, I report the changes in ADA, PNP and 5*-N in the immature (thymocytes) and mature (splenic T cells) mouse lymphocytes. Sensitive, radiometric assays were employed to estimate the level of each of of T cell development. the three enzymes at the two stages MATERIALS AND METHODS Animals and preparation of cells: Mouse strains C57BL/6, A/J The thymus and spleen and AKR of 6-16 weeks of age were used. were removed asceptically soon after cervical dislocation of the animals and transferred to RPM1 1640 with 1% bovine serum albumin. from each organ by gentle A single cell suspension was prepared teasing and passed through a nylon sieve to remove the tissue debris. The red blood cells were lysed with 0.16 M NH4C1, 0.05 M Tris-HCl (pH 7.5) and 0.1 mM Na2EDTA (pH 7.4) and the lymphocytes Splenic were washed again with the above medium by centrifugation. T cells were enriched by a nylon wool column [7]. The nylon wool was preactivated at 37°C overnight and splenic lymphocytes were After incubating for 45 min to 1 hr at applied to the column. 37"C, splenic T cells were eluted with RPM1 plus 1% BSA. The cells were concentrated by centrifugation and washed with the above medium. This procedure (7) greatly enriches splenic T cells and We have not estimated the actual proportion of was used as such. any contaminating B or null cells. The frozen cells were resuspended in Extraction of enzymes: 10 mM potassium phosphate (pH 7.2), 0.1 mM &mercaptoethanol, 1 mM Na2EDTA (pH 7.2) and 0.25% Kyro-EOB and lysed by incubating The superat 37°C for 10 min and brief mixing in a Vortex mixer. natant obtained after centrifugation at 10,000 rpm (Sorvall SS34 The enzyme rotor) for 30 min was used as the enzyme source. extracts were divided into small aliquots and stored frozen at -76OC. The protein concentration was estimated by the Lowry procedure [8] with bovine serum albumin as the standard. Published procedures [g-11] were modified. Enzyme assays: ADA was assayed in 50 mM potassium phosphate, pH 7.2, 0.1 mM reaction (0.02 uCi) and the extract. The [8-14C] adenosine PNP,i.n the extract was mixture was incubated for 10 min at 37'C. determined in 50 mM Tris-HCl (pH 7.8), 5 mM MgC12, 2 mM dithioand 0.08 mM [8-14C] 1.15 mM ribose-l-phosphate threitol, hypoxanthine (0.25 $i). The incubation was for 15 min at 37OC. for 5'-N consisted of 50 mM Tris-HCl The incubation mixture
(pN
7‘5),
10
mM
M&$12,
0.5%
(V/V)
Triton
x-100,
20
mM p-
glpcerophoaphate, 5 mM sodium potassium tartarate, 1 mM [14CJ adeaosine S'monophosphate (0.1 @IX.) and the extract; it was volume was 50 elf. and incubated far f hr at 3?*C. The reactfon the enzyme reactions were terminated by adding l/10 volume of 1Opl [8-24C] Adenosine (54.7 mCi/mmole), [8-l4C] hypoformic acid. xanthine (52.8 mCi/mmole) and 1:"4CJ adenoeine S'monophosphate (479 Ci/m.mole) were purchased from New England Nuclear Corp., Boston. The enzyme actfvftles were linear with incubation tfme and The times noted above in amount of protein (data not shown; 5). Routinely, 2-4 the standard assays were in the linear range. different concentrations of protein were tested for each point, Thin Layer Ch~~~tograp~y: Usually, 5 &I of the reaction mfxture wae mixed with unlabeled chemicals for UV identification, applied to polyethyleneimine cellulose plate (FBI-cellulose, Brinkman) and developed in bu~ano~:methanol:B2O:N~4~H (~~:20:2~: 1) for 6% hr [12J. The spots were cut out and counted in a liquid scfntillatfon couriter.
Table X. shows the activity data for AD& PNP and 5'-N in three different mouse streinsl The enzymes were prepared from the thymocytes and splenic T cells from each &rain. In general, the enzyme levels rln the two cell types were similar in all the mice. ADA activity fn thymocytes was 5-fold greater than that in spleen. On the other hand, PI@ level was a-fold higher fn the mature T cells (3-6 fold) in the af spleen. Similarly, the S'-N was greater splenic T cells. Since non-specific phosphetases also interfere in the eet%mation of 5*-N activity, we have included 8-glycerophosphate and sod&urn potassium tartarate during; fneubation to prevent In Table 2, we have further sach aetivitfes. examined the 5'-N activity J;n the presence of AOPCP, a specific inhibitor of this enzyme [13]. The thymocyte enzyme was inhibited by about 70% and the splenic T cell enzyme was inhfbfted by 90%. A consideration of the AOP&Pm~nhibitab~e 5'-N activity indicates an 8-fold difference between the thymua and spleen. The 5'-N activity in whole cells was also examined (5) and the results (ectivitfes in nmol/hr/mg protefn were 89&i for thymes and 816-Z for spleen) were s&milar to those crf cell lysates as in human lymphocytes. All the enzymo.s from the two stages of development displayed MLchaefis-Menten kinetics. The kinetic properties are listed in Table 3.. The apparent Km data for ADA and PNP were similar between the thymus and spleen (Table 3); however, the Rm for 5'-N was Z-fold greater in the spleen. The differences in V,, for the 3 enzymes confirm the data in Table 1.
404
Cell Biolog y International
Reports, Vol. 8, No. 5, May 1984
The above results demonstrate that there is a characteristic shift in the levels of ADA, PNP and 5'-N during the development of T cells in mice. Thus, during the early stages of development the activity of ADA is high and at later stages, the activities of PNP and 5'-N are high. Similar changes of these enzymes have been Taken demonstrated in rat [14] and recently, in humans [5,15]. together these data suggest that the expression of the above three enzymes is developmentally regulated in the T lymphocytes of varithey must serve as useful biochemical Therefore, ous organisms. markers in the future studies on T cell ontogeny. The results also indicate an alteration in the metabolism of nucleic acids during T cell differentiation which in turn may affect the development of various immunocompetent cells. ACKNOWLEDGMENTS I am grateful to R. Basch of the Pathology Department for the mice and help in the cell preparation and I would like to thank Marc Kaisman for technical assistance. REFERENCES 1.
Giblett, E.R., Anderson, J.E., Cohen, Meuwissen, H.J. (1972) Lancet 2:1067.
2.
Giblett, E.R., Ammann, D., L.K. (1975) Lancet E:lOlO.
Wara, W., Sandman, R. and Diamond,
Edwards, N.L., Magilaury, (1978) Science 201:628.
D.B.,
3.
F.,
Cassidy,
Pollara,
J.T.
4.
Silber, R., Conkyln, M., Grusky, (1975) J. Clin. Invest. -56:1234.
5.
Ramagopal, S. and G. Reem (1982) Thymus 4:163.
6.
Uusitalo, Cytochem.
7.
Trixio,
8.
Lowry, O.H., Rosebrough, N.J., (1951) J. Biol. Chem. 193:265.
9.
Carson, -57:274.
R.J. and 25:87. -
Karnovsky,
D. and Cudkowicz,
D.A.
10.
Milman, G., Anton, -15:4967.
D.L.
11.
Chatterjee, S.K., Battacharya, Anal. Biochem. -95 : 497
and Fox,
I.H.
G., and Zucker-Franklin,
M.J.
G. (1974)
and Seegmiller,
B. and
Parr, J.E.
(1977) J. A.L. (1976)
and Weber, J.L.
D.
J.
Histochem.
Immunol.
113:1093.
and Randall, J. (1976)
M. and Barlow,
Clin.
R.J. Invest.
Biochemistry J.J.
(1979)
Cell Biolog y International
and
E.
Randerath,
(1967)
12.
Randerath, -18:323.
13.
Edwards, N.L., Gelfand, E.W., (1979) Proc. Natl. Acad, Sci.
14.
Barton, R., Martiniuk, F., Hirschhorn, I. (1980) Cell. Immunol. -49:208.
15.
K.
Reports, Vol. 8, No. 5, May 1984
Burke, L., Dosch, USA -76:3474.
Ma, D. D. F., Sylwestrowicz, Franks, R., Janossy, G. and Immunol. 129:1430.
Received:
Methods
Table Activities of various
of ADA, PNP and 5'-N mouse strains.
Mouse Strains
T cell source
17th April
A/J AKR
Thymus Spleen Thymus Spleen Thymus Spleen
311.6 58 288.5 82.9 267.0 56.0
f 26.7 f 4.1 + 103.9 +_ 21.5
I.H.
M., J.
1984.
1 and splenic
PNP
T cells
5'-N
(nmoles/min/mg) C57BL/6
Hand Fox,
T., Granger, S., Massia, Hoffbrand, A. V. (1982)
in thymocytes
ADA
Enzymol.
R. and Goldschneider,
Accepted:
7th March 1984.
in
(nmoles/hr/mg) 22.9 k 40.4 2 5.8 + 12.7 f n.d.
1.8 5.4 2.2 4.2
51.6 343.5 82.2 430 98.4 322.4
4 6.4 f 32.1 t 41.4 ? 217.6
The enzyme activities were determined as described in Materials and Methods. The mean of 3 (C57BL/6 of 6-8 weeks of age) or 5 (A/J of 6-16 weeks of age) experiments f standard deviation is shown. Only one experiment was conducted with AKR strain; the mean of 3 incubations is shown. n.d. not determined.
Cell Biolog y lnterna tional Reports, Vol. 8, No. 5, May 1984
406
Table 2 Effect of AOPCP on 5'-N activity of T cells from the thymus and spleen
Treatment
Thymus
Spleen
(nmoles/hr/mg) (% Inhibition) (nmoles/hr/mg) (X Inhibition) None AOPCP (3mM) AOPCP (6mM)
60.5 18.7 23.8
384.0 43.0 39.1
69 61
89 90
The 5'-N activity was estimated as in Table 1 except that in some reactions AOPCP (a,&methylene adenosine 5'-diphosphate, P.L. Biochemicals) was also present at the indicated concentration.
Table 3 Kinetic properties of ABA, PNP and 5'-N from thymocytes and splenic T cells.
ABA
PNP
5'-N
Source T cells
Km
"max
Km
"max
Thymus Spleen
67 59
285.7 64.5
500 500
122.0 227.3
Km
"max
75 154
64.5 526.3
The data were obtained from Lineweaver-Burk plots for the activities of ABA, PNP and 5'-N in thymocytes and splenic T cells. Adenosine (0.01 - lmM), hypoxanthine (0.05-0.25 mM) and adenosine 5'-monophosphate (O.l-2mM) were used as substrates for ABA, PNP The Km values are in w and V,, values and 5'-N, respectively. are in nmoles/min/mg protein (ABA and PNP) or nmoles/hr/mg protein (5'-N).