Ti10 TYPES OF ,'lOUSE FM3A CELL IILTANTS DEFICIE>'T I:I RELATIONSHIP BETWEEN EXTRACELLULAR AND INTRACELLIILAR r A811NOIi41DAZOLECAR30XA:11DE RiBOTI3E TRANSFORdYLASE NUCLEOTIDE METABOLISM IN HUMAN LYMPHOCYTES. 3 XND TKEIR TRAiISFORYANTS ISOLATED 3Y CHROi4OSOME- 8 Barankiewicz, Hans-Michael Dosch, Rov Cheung and BPlQS MEDiATED GENE TRANSFER. 3ai Ayuzawd, Xiniko Shi:lizu, C&e& The Hospital for Sick Children, Division of i'l~salakeYamcuchi 1, Michio3-i 1snGfakeshi Im~~nology/Rheumatology,Research Institute, Toronto, ~enoi.S~iCama Cancer~Res~Instituie. Ina-nachl. Ontario, Canada. =aka-ken 362, . ,pan, l~heIns tituCe of lied ,licrobioioSy, In our previous studies it has been shown that lymphocytes of B I;niversi.Ly of Tokyo, Tokyo 113, Japan and 2~aiionalInstiCut; of lineage but not T lymphocytes are able to degrade extracellular Gcneeics, .lishina, Shi~uoia-ken 411, Jcpan. ATP, normally present in hman blood plasma (20-30,uM). Adenosine eie isola-Led chre zi:nin2 su::o'irophic mutants (Ade 1-3) ;f produced by B lymphocytes front extracellular ATP can easily enter mouse Fc13;I cells 6eficicn.t in iminoinidazo1ecarboxar.iide ribdbiti:. the different cells and therefore extracellular nucleotide de- (,1ICAR) transfornylas-. Ade 1 and Ad;. 2 3180 lacited inosinico;z gradation may significantly affect intracellular metabolism. .jctivity. Ad2 2 2nd Ad: 3 complemenCei in cell-cell hybrids, Adenosine produced extracellularly by cells is mainly deaminated al.chough Adc 1 did no; either of then. An intermediate AICAR intracellularly to inosine whereas a minor part is incorporated was found .Lo ~ccumulatein ill .<he mutants indicating ihat to intracellular nucleotides - mainly ATP. The cells which are earlitr steps than hat of formylation of AICAX in -;h~ purine unable to degrade extracellular ATP such as T lymphocytes or red biosynthetic pathway seem normal. Primary and secontiiry irans- blood cells also incorporate adenosine extracellularly produced formants wers isclated fron Ade ;and Ade 3 using hunjn by B cells to their nucleotides and this incorporation is chroc3son+mediatei Ccnc transf?r. All the ,transformants 'Lesiej especially efficient in red blood cells. Although extracellular were found Lo produce boih huma-type XICAR transformylase and ATP has no effect on intracellular nucleotide metabolism, its inosi nicasz. dnexpsc;edly, human DNA intergrated in the host degradation product adenosine significantly inhibits purine bio- Tenons of :he cerlain primary transformant was reduced to as synthesis de novo as well as the salvage of adenine, hypoxanthine, little is scveral hundred kilobase pairs in length. Uhen guanosine or thymidine in both B and T lymphocytes. On the other digest~dwith certain restriction endo~wcleases, identical human liand, adenosine fonnec' extracellularly may act as a physiological DNA bands ss probed with a human :Alu sequence were detected in mdulator through specific membrane adenosine receptors, and the transfornants of Ad i and Atin. Since only human regulate cyclic AMP levels. chromoson: 2 was cnough to rescue :he senetic defects in the threc mutani-s, these mueanls seem to have arisen by ;r,iu;aiion in the AICAR iransfornylase Sene or i Ls zdjacent gene. CELL SPECIES-DEPENDENT METABOLISM OF THE POTENT STUDIES OF MUTANT HUHAN ADENYLOSUCCINATE LYASE. ANTI-HIV (HUMAN IMMUNODEFICIENCY VIRUS) COMPOUNDS Bruce A. Barsho . Arthur S. Alberts, and Harr E. 6 3'-AZIDO-2' ,3'-DIDEOXYTH IDINE AND 2' ,3'-DIDEOXY- 9 Gruber, Univerzity of California San D&~, CTD E Jan Balzarr,'C Erik De clercq*, Samuel Department of Medicine, La Jolla. CA, USA. Brode) and D.G. Johns . *~ega Institute for Medical Rfsearch, Katholieke Universiteit Leuven, Leuven, Residual adenylosuccinate lyase activity was Belgium and National Cancer Institute, National Institutes of studied in cultured lymphoblasts from a pair of siblings who Health, Bethesda, Maryland, U.S.A. have mental retardation and autism, and who have been pre- The cytostatic and anti-retrovirus activity, as well as the viously shown to exhibit a deficiency of the enzyme. Utiliza- intracellular metabolism, of 3'-azido-2',3'-dideoxythymidine tion of formate by intact cells showed de novo synthesis in the (AzddThd) and 2',3'-dideoxycytidine (ddCyd) have been evaluated mutant lymphoblasts to be similar to normal, consistent with a using cell lines derived from different species. AzddThd and partial deficiency of this enzyme as previously reported. The ddCyd are considerably more cytostatic to human cell lines than steady-state kinetics and thermal stability of adenylosuccinate murine cells. In human lymphoid (ATHB, MoltI4F) cells AzddThd lyase were examined in the lymphoblast lysates. The pH-activity accumulates principally as its 5'-monophosphate metabolite curves were nearly identical for the mutant and normal cells, (AzddTMP), whereas in murine leukemia L1210 cells it is readily with an optimum in the region of pH 7.8. While the substrate metabolized to AzddTTP. The rapid conversion of AzddThd to affinity was not distinguishable from normal, there was a sig- AzddTTP in murine cells may explain why AzddThd has a more pro- nificant difference (p < 0.05) in the Vmax of the mutant cells. nounced activity against Moloney murine sarcoma virus (MSV)-in- The lyase from normal cells exhibited an apparent Km of 3.3 + duced transformation of murine C3H cells than human immunodefi- 0.8 pM and a Vmax of 13.8 + 0.9 nm01.m~-"min-'. In the mutant ciency virus-induced cytopathogenicity in ATHB cells. In con- cells, the lyase had an apparent Km of 2.6 2 0.5 pM and Vmax of trast, ddCyd is less active in murine than human cells, most li- 6.7 2 1.1 nmol mg-'.min-'. There was a significant difference kely due to a differential conversion of ddCyd to its 5'-tri- between the normal and mutant cell lyases with regard to ther- phosphate in murine, as compared to human cells. When [%I- mal stability. At 37'C, the half-time for inactivation was 22.2 AzddThd and [3~~dd~ydwere compared for their metabolism in hu- hours for the normal cells and 15.7 hours for the mutant cell man (ATH8, MoltlhF) cells, substantial levels of [~H]~~cTPbuilt lysates, and at 60°C the half-times were 5.0 and 0.49 minutes up gradually, whereas lictle (3~l~zdd~~~was formed even after a for normal and mutant cell lysates, respectively. There was no 48 hr-incubation period. Thus, much higher ddCTP than AzddTTP evidence of inhibitory activity in the lysates of the mutant levels were achieved in human lymphoid cells, an observation cells. The results are consistent with a structural mutation that may be particularly relevant from a therapeutic viewpoint. in the adenylosuccinate lyase gene of the affected individuals. EXTRACELLULAR NUCLEOTIDE HETABOLISM AS THE MAJOR S0UI:CS OF AOENOSINE IN WMAN LVMPIIOCYTES. Jerzv CHARACTERIZATION OF A FOLATE-METHOTREXATE 7 -, Hans-Michael and Amos Cohen, The 10 TRANSPORT-DEF I C i ENT CLONE OF LE l SHMAN l A DONOVAN l Hospital for Sick Children, Division of Immunology/ --Joanne Beck and Buddy Ul lman, Rheumatology. Research Institute, Toronto, Ontario, The Oregon Health Sciences University, Canada. Biochemistry Department, Portland, Oregon, U.S.A. Cells of B lymphocyte lineage (non-E-rosetting B lymphocytes from Leishmania donovani is the causative agent of visceral peripheral blood or tonsils and different B lymphoblastoid cell leishmaniasis or kala azar. A clonal mutant strain of lines) showed the presence of very active ectonucleotidase sites Leishmania donovani was derived in a single step from a wild type population by virtue of its resistance to 1 millimoiar on their external surface. Thus, B cells are able to metabolize methotrexate. This cell line, MTXA5, was cross-resistant to extracellular ATP in extracellular pathway via AMP* ADP- AMP + aminopterin but equally sensitive to two other inhibitors of adenosine. Since adenosine is not produced during intracellular certain dihydrofolate reductases, pyrimethamine and trimetho- ATP degradation, the degradation of extracellular ATP may be the prim. In contrast to the wild type parental cells, MTXA5 ceils only source of adenosine production in lymphocytes. The expres- were incapable of taking up or transporting radiolabelled sion of ectoATPase, ectoADPase and ectoAMPase seems to be closely methotrexate and folate from the cell culture mediun. associated with B cell development. EctoATPase and ectoAPPase activities increase contin~~ouslyduringB cell maturation(early Surprisingly, however, both wild type and mutant ceils grew equally woll in increasing concentrations of folate, although preB, late preB, mature B cells), whereas ectoAMPase reaches only the wild type parental cells were capable of growing in maximal activity in late preB cells. Extracellular nucleotide folate-deficient growth mediwn supplemented with either catabolism seems to have low specificity with comparahle degrada- tion rate of dATP, CTP, dCTP and CTP. Extracellular ATP degrada- biopterin or neopterin. In order to attempt to-analyze the transport system of Leishmania biochemically, an affinity tion is stimulated by M~?+but not by ~a2+. These ectoenzymes are labelling technique was developed using radiolabelled metho- not released from cell surface to extracellular space. In con- trexate and folate that had been 'activated' with 1-ethyl-3(3- trast to B cells, T lymphocytes (E rosetting lymphocytes from dimethylaminopropyl)carbodiimide. Using this protocol, a peripheral blood or tonsils were unable to degrade extracellular protein with MW = 46 kd was labelled in wild type cells. This nucleotides. These results showed significant difference between 46 kd protein was associated with plasma membrane fractions. extracellular and intracellular ATP catabolism. Intracellular ATP No band was observed in wild type cytosolic fractions. These degradation proceeds exclusively via AMP deamination, whereas data have biochemically and genetically identified a common extracellular via AMP dephosphc.rylation resulting in adenosine foiate/methotrexate carrier in this genus of parasites. format ion. .