0020-71
Ix:79/0101-ooo7s02.w/0
PYRIMIDINE METABOLISM IN LYMPHOCYTES AND ERYTHROCYTES OF MAN, HORSE AND CATTLE W. J. M. TAX, 6. J. PETFZRS and J. H. VEERKAMP Department of Biochemistry, Geert Grooteplein N 21, Nijmegen, The Netherlands (Receioed 10 Jury 1978) Abstract-l. No conversion of uridine to uracil or uridine nucleotides was found in erythrocytes of man, horse and monkey. 2. Activity of uridine kinase was about similar in lymphocyte extracts of man, cattle and horse, but the activities of orotate phosphoribosyltransferase, orotidine phosphate decarboxylase and uridine phosphorylase were markedly lower in lymphocyte extracts of horse than in those of man and cattle. 3. Incorporation of uridine into nucleotides and RNA was of the same order of magnitude when human lymphocytes were compared with those of horse. 4. Rates of phosphorylation and phosphorolysis of uridine in intact lymphocytes were only a few percent of the corresponding enzyme activities measured in lymphocyte extracts.
5. The results are discussed in relation to adenosine-induced pyrimidine starvation and the phenomenon that horses have a normal immunoresponse despite a low activity of adenosine deaminase in their lymphocytes.
INTRODUCTION
ied in intact lymphocytes of man and horse. In addi-
tion, the activity of uridine phosphoryl~e (EC 2.4.2.3) was assayed in lymphocytes and erythrocytes, since it was reported that uridine is rapidly converted to uracil in blood of rat (Dahnke & Mosebach, 1975), dog (Tseng et al., 1971) and mouse (Yngner et ai., 1977). The activity of uridine kinase was also determined in erythrocytes of man and horse, since we found earlier (Tax et at, 1976) marked differences in the activities of OPRT and ODC between both species.
~r~idine nucleotides can be formed by de now synthesis or by reutilization (salvage) of nucleosides. The relative contribution of both pathways to total production is different in various types of cells and in various conditions (Roux, 1973; Levine et al., 1974: Cihak & Rada, 1976). Both pathways are present in human lymphocytes (Lucas, 1967; Lucas, 1969; Ito & Uchino, 1971), but there is some disagreement about the extent, to which they participate in pyrimidine nucleotide synthesis in resting (peripheral) lymphocytes (Lucas, 1967;’ Kay & Handmaker, 1970). Stimutation with phytohemagglutinin appeared to increase the reutilization pathway markedly more than the de nova synthesis (Lucas, 1969; Ito & Uchino, 19’76). In a previous investigation (Tax & Veerkamp, 1978) we found that the activity of adenosine deaminase (ADA, EC 3.5.4.4) in horse lymphocyte was comparable to that in lymphocytes of patients with severe combined immunodeficiency associated with ADA deficiency. (Polmar et al., 1976). The mechanism by which ADA deficiency impairs immunologic function is still unknown, but several investigations (Ishii & Green, 1973; Ullman et al, 1976; Gudas et al., 1978) suggat that this enzyme deficiency may interfere with pyrimidine nucleotide synthesis de nouo. Such an interference could be bypassed in horse lymphocytes, when pyrimidine nucleotides are preferentially formed by the salvage pathway in these cells. Carbamyl phosphate synthetase (EC 2.7.2.5) and orotate phosphoribosyitranfer~e (OPRT, EC 2.4.2.10), and uridine kinase (EC 2.7.1.48) represent key enzymes of the de novo and the salvage pathway, respectively. In this study we measured the activity of the latter two enzymes together with that of orotidine phosphate decarboxylase (ODC, EC 4.1.1.23) in extracts of peripheral lymphocytes from man, horse and cattle. The metabolism of uridine was also stud-
MATERIALSAND METHODS Materials Ficoll (MW 400,000) was obtained from Pharmacia and Isopaque from Nyegaard & Co, Oslo, Norway. A FicollIsopaque solution was prepared by dissolving 9.56 8 Ficoll in 130 ml distilled water and adding 20 ml Isopaque; distilled water was added until a fina specific gravity of 1.077 was obtained. Minimum essential medium for suspension cultures (MEMS), human serum and horse serum were obtained from Flow Lab. [Carboxyl-“Clorotic acid and rcarboxyl-*4C]orotidine-S-monophosphate 8nd Omnifluor were obtained from New England Nuclear Corporation, [2-‘4C]-uridine from the Radiochemical Centre, Amersh~ and hydroxide of Hyamine 10-X from Packard. Thin-layer plate; containing _polyethyleneiminecellu1ose (PEI-cellulose) were obtained from Merck. Phosphoribosyl-l-pyrophosphate (PRPP) was purchased from Boehringer, Mannheim.
of cellsuspensions and cell extracts Blood samples from human adults, healthy adult horses and cattle were collected in heparinized tubes. Erythrocytes were isolated by centrifugation, washed twice with 50mM Tris-HCl (pH 7.4) containing 1OOmM NaCl. and resuspended in a solution containing 4OmM potassium phosphate (pIi 7.4), 110 mM NaCI. 5.5 mM alucose and 1 mM MgCl,: Lymphocytes were isolated at ;oom temperature by Ficoll-Isopaque gradient centrifugation of diluted blood (Bayurn, 1968) and counted in a hemo~tometer. Preparation
7
W.
J. M. TAX_G. J.
PETERS
and
J. H. VEERKAMP
Table 1. Activities of OPRT and ODC and ODC/OPRT ratio in sonicated extracts of lymphocytes from man. cattle and horse
M&WI Cattle Horse
OPRT activity
ODC activity
ODC/OPRT
0.39 & 0.19(11) 0.47 f 0.16 (9) 0.061 + 0.012(5)
1.37 $- 0.44(Q) 0.68 + 0.19(9) 0.16 + 0.03 (5)
4.80 + 2.67(9) 1.51 * 0.34(9) 2.70 f 0.58 (5)
Enzyme activities are given in nmole/hr pet lo6 cells as the means + SD. The numbers in parentheses refer to the number of individuals. Far experiments with intact lymphocytes. ceIIs were suspended in a mixture of toy{;,homologous serum and !%I?$ MEMS containing 2mM glutamin and penicillin (100U/ml) and streptomycin (100 &ml). Hemolysates were prepared by freezing and thawing the erythrocytes twice. Extracts of lymphocytes weri obtained by sonication (branson sonifier. 8 bursts of 5 set at maximal output) in MmM Tris-HCl/l mM EDTA (pH 7.4). For uridine kinase and phosphorylase assay extracts were centrifuged at T.OO@gfor t5min at 4°C.
All enzyme activities in hemolysates and lymphocyte extracts were assayed at 37°C by radiochemical methods. Enzyme activities for lymphocytes are expressed in nmoles product formed in 1 hr/106 cells. Linearity of the reactions with respect to time and amount of cell extract was ascertained. The incubation mixture (7O#j for uridine kinase assay contained 4OmM Tris-HCI (pH 7.4j. t mM EDTA. IOmM MgC’I,, 5mM ATP and 70pM [2-**C}uridine (!?imCi/ mmole). Reaction was terminated after 60min by heating the incubation mixture at 95°C for 5min (Eppendorf heater). After centrifugatian and addition of carriers, uracil and uridine nucleotides were separated from uridine by thin-layer chromatography on PEI-cellulose (Reyew 1972). Assay conditions for uridine phosphoryiase were the same as for uridine kinase except that 40mM o~bophusphate (pW 7.4j was added, The incubation mixture (0.55mIj for the OPRT assay contained 0.12 mM [carboxyt-%)orotic acid (7.5 mCii mmole), 0.55 mM PRPP. 4SmM MgCl*, 1 mM EDTA and 50mM Tris-HCI buffer (pH 7.4). The amount of lymphocyte extract used was equivalent to 616.10b cells for man, to 20-45.106 cells for horse and to 6-10a106 cells for cattle. Production of %X& was measured ailer 6Omin incubation as described previously (Tax et al., 19761, The reaction mixture for the QD& assay (52.5$j contained 88 @i ~~rboxy~-14~]orot~~~-~-monoph~phate (l.Om~i/mmo~ej, I mM EDTA, XtmM Tris-Ha (pH 7.41 and simifar amounts of lymphocyte extract as used for the OPRT assay. Production of ‘*CO1 was measured after 60min as described previously (Tax et al.. 1976). Protein was determined according to Lowry et nl. (1951). Metabolism
at 9.500~ (5 min. 4°C). Extract together with the wash fluid was neutralized with 5N KOH and after anttifugation of KCIO. samples were analyzed on TLC sheets coated with PEl-cellulose. 0.2ml of I N hydroxide of Hyamine was added to the acid-insoluble cell material and left ovcrnight at room temperature. Radioactiviiy was then counted with lOm1 of toluene containing 4g of omnifluot per liter.
After incubation of erythrocytea of man, horse or monkey with [2-14C]uridine, no significant radioactivity was found in either uracil or uridine hucleotides. Virtually all radioactivity was recovered in uridine. Hemolysates from these three species also showed no conversion of uridine. Human and bovine ~rnp~t~ contain comparabte activities of OPRT, but ODC was in the former about 2-fold higher (Table I). In horse lymphocytes markedly lower activities of OPRT and ODC appeared to be present. Uridine kinase activity is similar in lymphocyte extracts of man, cattle and horse (Table 2). Uridine phosphorylase activity is higher than uridine kinase activity in lymphocyte extracts from man and cattle, but lower in ~ympho~e extracts from horse. When intact lymphocytes from man and horse were incubated with uridine, product Formation waS linear with time for at least 2hr. No conversion of uridine could be detected after incubation with a mixture of 90% MEMS and 10% human serum or horse serum. A significant amount of uracil was found in the medium after incubation of human lymphocytes with uriditle but not after incubation of horse lymphocytes. Both with human and horse lymphocytes most of the intr~~~u~~ acid-sdubb radioactivity was associated with uridine nudeotides, Radioactivity was atso present in the acid-insohzble materiaf which is assumed to contain the cellular RNA, Table 3 shows that both the conversion of uridine to uridine nucleotides and its incorporation into RNA are of the same Rhesus
of uridine by intact cells
Suspensions of erythrocytes (25x, by vol) or lympbocvtes (30 x IO6ceIls/mlt were Breincubated for 30 min at 3j”C with gentle shltkiig. [2-**C&ridine was then added to a final ~~n~ntration of 70pM. After appropriate times (ud to 3 hrj 50111sam~ies were taken and mixed with 1Opl idmM ur~dind/lOm]ilI uracil. Cells were separated from the medium by centrifwgation at 3,5OO# (5min, 4”C, Burkard Koolspin centrifuge) and the cells were washed with 50~1 saline. Medium and wash fluid were combined, deproteinized, and analyzed by means of thin-layer chromatography on PEK-cellulose. Cells were extracted with 50.~1 0.5 N HCK& and acid-insoluble material was washed once with Mpl 0.5 N HCIO+ Centrifnga~~on was
Table 2. Activities of uridine kinase and uridine phosphoty&se in lymphocyte extracts of man, cattle and horse
Species
Uridine kinase
Man Cattle Horse
0.51 & O.lS(6) 0.48 f 0.35 (8) 0.57 f 0.15 (6)
2.13 P 0.32(4) 1.90 * 0.91(4) 0.33 f 0.19 (4)
Enzyme activities are given in nmole/hr per lo6 cells as the means f S.D. Numbers in parentheses refer to the number of individuals. Extracts from lymphocytes were prepared as described in Materials and Metbods.
9
Pyrimidine metabolism in lymphocytes Table 3. Uridihe metabolism in intact lymphocytes of man and horse
Species Man
Horse
Phosphorylation RNA Nucleotides 1860 f 199(4) 1970 f 462 (4)
974 f 172 (4) 1123 f 63(4)
Phosphordysis Uracil 7661 + 1539(3) < looo(4)
Intact lymphocytes were incubated with 70pM [2-“C]uridine (55 mCi/mmole) for periods up to 3 hr. Radioactivity in cells and medium was analyzed as described in Materials and Methods. The conversion of uridine to nucleotides or uracil and the incorporation into RNA are given in dpm/hr per 10” cells. Values are the means + SD. of 3-4 experiments in quadruplicate.
order of magnitude in human and horse lymphocytes. The amount of uridine which is metabolized by intact lymphocytes (Table 3) can be calculated in nmoles/hr per lo6 cells from the specific activity of uridine (122,000 dpm/nmole), when the original intracellular uridine pool is neglected. Both the rate of phosphorylation (about 24 pmoles/hr per lo6 cells) and of phosphorolysis (about 62 pmoles/hr per lo6 cells in human lymphocytes and
Ix:79/0101-ooo7s02.w/0
PYRIMIDINE METABOLISM IN LYMPHOCYTES AND ERYTHROCYTES OF MAN, HORSE AND CATTLE W. J. M. TAX, 6. J. PETFZRS and J. H. VEERKAMP Department of Biochemistry, Geert Grooteplein N 21, Nijmegen, The Netherlands (Receioed 10 Jury 1978) Abstract-l. No conversion of uridine to uracil or uridine nucleotides was found in erythrocytes of man, horse and monkey. 2. Activity of uridine kinase was about similar in lymphocyte extracts of man, cattle and horse, but the activities of orotate phosphoribosyltransferase, orotidine phosphate decarboxylase and uridine phosphorylase were markedly lower in lymphocyte extracts of horse than in those of man and cattle. 3. Incorporation of uridine into nucleotides and RNA was of the same order of magnitude when human lymphocytes were compared with those of horse. 4. Rates of phosphorylation and phosphorolysis of uridine in intact lymphocytes were only a few percent of the corresponding enzyme activities measured in lymphocyte extracts.
5. The results are discussed in relation to adenosine-induced pyrimidine starvation and the phenomenon that horses have a normal immunoresponse despite a low activity of adenosine deaminase in their lymphocytes.
INTRODUCTION
ied in intact lymphocytes of man and horse. In addi-
tion, the activity of uridine phosphoryl~e (EC 2.4.2.3) was assayed in lymphocytes and erythrocytes, since it was reported that uridine is rapidly converted to uracil in blood of rat (Dahnke & Mosebach, 1975), dog (Tseng et al., 1971) and mouse (Yngner et ai., 1977). The activity of uridine kinase was also determined in erythrocytes of man and horse, since we found earlier (Tax et at, 1976) marked differences in the activities of OPRT and ODC between both species.
~r~idine nucleotides can be formed by de now synthesis or by reutilization (salvage) of nucleosides. The relative contribution of both pathways to total production is different in various types of cells and in various conditions (Roux, 1973; Levine et al., 1974: Cihak & Rada, 1976). Both pathways are present in human lymphocytes (Lucas, 1967; Lucas, 1969; Ito & Uchino, 1971), but there is some disagreement about the extent, to which they participate in pyrimidine nucleotide synthesis in resting (peripheral) lymphocytes (Lucas, 1967;’ Kay & Handmaker, 1970). Stimutation with phytohemagglutinin appeared to increase the reutilization pathway markedly more than the de nova synthesis (Lucas, 1969; Ito & Uchino, 19’76). In a previous investigation (Tax & Veerkamp, 1978) we found that the activity of adenosine deaminase (ADA, EC 3.5.4.4) in horse lymphocyte was comparable to that in lymphocytes of patients with severe combined immunodeficiency associated with ADA deficiency. (Polmar et al., 1976). The mechanism by which ADA deficiency impairs immunologic function is still unknown, but several investigations (Ishii & Green, 1973; Ullman et al, 1976; Gudas et al., 1978) suggat that this enzyme deficiency may interfere with pyrimidine nucleotide synthesis de nouo. Such an interference could be bypassed in horse lymphocytes, when pyrimidine nucleotides are preferentially formed by the salvage pathway in these cells. Carbamyl phosphate synthetase (EC 2.7.2.5) and orotate phosphoribosyitranfer~e (OPRT, EC 2.4.2.10), and uridine kinase (EC 2.7.1.48) represent key enzymes of the de novo and the salvage pathway, respectively. In this study we measured the activity of the latter two enzymes together with that of orotidine phosphate decarboxylase (ODC, EC 4.1.1.23) in extracts of peripheral lymphocytes from man, horse and cattle. The metabolism of uridine was also stud-
MATERIALSAND METHODS Materials Ficoll (MW 400,000) was obtained from Pharmacia and Isopaque from Nyegaard & Co, Oslo, Norway. A FicollIsopaque solution was prepared by dissolving 9.56 8 Ficoll in 130 ml distilled water and adding 20 ml Isopaque; distilled water was added until a fina specific gravity of 1.077 was obtained. Minimum essential medium for suspension cultures (MEMS), human serum and horse serum were obtained from Flow Lab. [Carboxyl-“Clorotic acid and rcarboxyl-*4C]orotidine-S-monophosphate 8nd Omnifluor were obtained from New England Nuclear Corporation, [2-‘4C]-uridine from the Radiochemical Centre, Amersh~ and hydroxide of Hyamine 10-X from Packard. Thin-layer plate; containing _polyethyleneiminecellu1ose (PEI-cellulose) were obtained from Merck. Phosphoribosyl-l-pyrophosphate (PRPP) was purchased from Boehringer, Mannheim.
of cellsuspensions and cell extracts Blood samples from human adults, healthy adult horses and cattle were collected in heparinized tubes. Erythrocytes were isolated by centrifugation, washed twice with 50mM Tris-HCl (pH 7.4) containing 1OOmM NaCl. and resuspended in a solution containing 4OmM potassium phosphate (pIi 7.4), 110 mM NaCI. 5.5 mM alucose and 1 mM MgCl,: Lymphocytes were isolated at ;oom temperature by Ficoll-Isopaque gradient centrifugation of diluted blood (Bayurn, 1968) and counted in a hemo~tometer. Preparation
7
W.
J. M. TAX_G. J.
PETERS
and
J. H. VEERKAMP
Table 1. Activities of OPRT and ODC and ODC/OPRT ratio in sonicated extracts of lymphocytes from man. cattle and horse
M&WI Cattle Horse
OPRT activity
ODC activity
ODC/OPRT
0.39 & 0.19(11) 0.47 f 0.16 (9) 0.061 + 0.012(5)
1.37 $- 0.44(Q) 0.68 + 0.19(9) 0.16 + 0.03 (5)
4.80 + 2.67(9) 1.51 * 0.34(9) 2.70 f 0.58 (5)
Enzyme activities are given in nmole/hr pet lo6 cells as the means + SD. The numbers in parentheses refer to the number of individuals. Far experiments with intact lymphocytes. ceIIs were suspended in a mixture of toy{;,homologous serum and !%I?$ MEMS containing 2mM glutamin and penicillin (100U/ml) and streptomycin (100 &ml). Hemolysates were prepared by freezing and thawing the erythrocytes twice. Extracts of lymphocytes weri obtained by sonication (branson sonifier. 8 bursts of 5 set at maximal output) in MmM Tris-HCl/l mM EDTA (pH 7.4). For uridine kinase and phosphorylase assay extracts were centrifuged at T.OO@gfor t5min at 4°C.
All enzyme activities in hemolysates and lymphocyte extracts were assayed at 37°C by radiochemical methods. Enzyme activities for lymphocytes are expressed in nmoles product formed in 1 hr/106 cells. Linearity of the reactions with respect to time and amount of cell extract was ascertained. The incubation mixture (7O#j for uridine kinase assay contained 4OmM Tris-HCI (pH 7.4j. t mM EDTA. IOmM MgC’I,, 5mM ATP and 70pM [2-**C}uridine (!?imCi/ mmole). Reaction was terminated after 60min by heating the incubation mixture at 95°C for 5min (Eppendorf heater). After centrifugatian and addition of carriers, uracil and uridine nucleotides were separated from uridine by thin-layer chromatography on PEI-cellulose (Reyew 1972). Assay conditions for uridine phosphoryiase were the same as for uridine kinase except that 40mM o~bophusphate (pW 7.4j was added, The incubation mixture (0.55mIj for the OPRT assay contained 0.12 mM [carboxyt-%)orotic acid (7.5 mCii mmole), 0.55 mM PRPP. 4SmM MgCl*, 1 mM EDTA and 50mM Tris-HCI buffer (pH 7.4). The amount of lymphocyte extract used was equivalent to 616.10b cells for man, to 20-45.106 cells for horse and to 6-10a106 cells for cattle. Production of %X& was measured ailer 6Omin incubation as described previously (Tax et al., 19761, The reaction mixture for the QD& assay (52.5$j contained 88 @i ~~rboxy~-14~]orot~~~-~-monoph~phate (l.Om~i/mmo~ej, I mM EDTA, XtmM Tris-Ha (pH 7.41 and simifar amounts of lymphocyte extract as used for the OPRT assay. Production of ‘*CO1 was measured after 60min as described previously (Tax et al.. 1976). Protein was determined according to Lowry et nl. (1951). Metabolism
at 9.500~ (5 min. 4°C). Extract together with the wash fluid was neutralized with 5N KOH and after anttifugation of KCIO. samples were analyzed on TLC sheets coated with PEl-cellulose. 0.2ml of I N hydroxide of Hyamine was added to the acid-insoluble cell material and left ovcrnight at room temperature. Radioactiviiy was then counted with lOm1 of toluene containing 4g of omnifluot per liter.
After incubation of erythrocytea of man, horse or monkey with [2-14C]uridine, no significant radioactivity was found in either uracil or uridine hucleotides. Virtually all radioactivity was recovered in uridine. Hemolysates from these three species also showed no conversion of uridine. Human and bovine ~rnp~t~ contain comparabte activities of OPRT, but ODC was in the former about 2-fold higher (Table I). In horse lymphocytes markedly lower activities of OPRT and ODC appeared to be present. Uridine kinase activity is similar in lymphocyte extracts of man, cattle and horse (Table 2). Uridine phosphorylase activity is higher than uridine kinase activity in lymphocyte extracts from man and cattle, but lower in ~ympho~e extracts from horse. When intact lymphocytes from man and horse were incubated with uridine, product Formation waS linear with time for at least 2hr. No conversion of uridine could be detected after incubation with a mixture of 90% MEMS and 10% human serum or horse serum. A significant amount of uracil was found in the medium after incubation of human lymphocytes with uriditle but not after incubation of horse lymphocytes. Both with human and horse lymphocytes most of the intr~~~u~~ acid-sdubb radioactivity was associated with uridine nudeotides, Radioactivity was atso present in the acid-insohzble materiaf which is assumed to contain the cellular RNA, Table 3 shows that both the conversion of uridine to uridine nucleotides and its incorporation into RNA are of the same Rhesus
of uridine by intact cells
Suspensions of erythrocytes (25x, by vol) or lympbocvtes (30 x IO6ceIls/mlt were Breincubated for 30 min at 3j”C with gentle shltkiig. [2-**C&ridine was then added to a final ~~n~ntration of 70pM. After appropriate times (ud to 3 hrj 50111sam~ies were taken and mixed with 1Opl idmM ur~dind/lOm]ilI uracil. Cells were separated from the medium by centrifwgation at 3,5OO# (5min, 4”C, Burkard Koolspin centrifuge) and the cells were washed with 50~1 saline. Medium and wash fluid were combined, deproteinized, and analyzed by means of thin-layer chromatography on PEK-cellulose. Cells were extracted with 50.~1 0.5 N HCK& and acid-insoluble material was washed once with Mpl 0.5 N HCIO+ Centrifnga~~on was
Table 2. Activities of uridine kinase and uridine phosphoty&se in lymphocyte extracts of man, cattle and horse
Species
Uridine kinase
Man Cattle Horse
0.51 & O.lS(6) 0.48 f 0.35 (8) 0.57 f 0.15 (6)
2.13 P 0.32(4) 1.90 * 0.91(4) 0.33 f 0.19 (4)
Enzyme activities are given in nmole/hr per lo6 cells as the means f S.D. Numbers in parentheses refer to the number of individuals. Extracts from lymphocytes were prepared as described in Materials and Metbods.
9
Pyrimidine metabolism in lymphocytes Table 3. Uridihe metabolism in intact lymphocytes of man and horse
Species Man
Horse
Phosphorylation RNA Nucleotides 1860 f 199(4) 1970 f 462 (4)
974 f 172 (4) 1123 f 63(4)
Phosphordysis Uracil 7661 + 1539(3) < looo(4)
Intact lymphocytes were incubated with 70pM [2-“C]uridine (55 mCi/mmole) for periods up to 3 hr. Radioactivity in cells and medium was analyzed as described in Materials and Methods. The conversion of uridine to nucleotides or uracil and the incorporation into RNA are given in dpm/hr per 10” cells. Values are the means + SD. of 3-4 experiments in quadruplicate.
order of magnitude in human and horse lymphocytes. The amount of uridine which is metabolized by intact lymphocytes (Table 3) can be calculated in nmoles/hr per lo6 cells from the specific activity of uridine (122,000 dpm/nmole), when the original intracellular uridine pool is neglected. Both the rate of phosphorylation (about 24 pmoles/hr per lo6 cells) and of phosphorolysis (about 62 pmoles/hr per lo6 cells in human lymphocytes and
