HYPOXANTHINE TRANSPORT THROUGH HUMAN ERY- 6 n 5'-DEOXY-5'-METHYLTH IOADENOS INE (MTA) PHOSPHORY- 25 THROCYTE MEMBRANES. E. Capuozzo, M. C. Gigante, Lu LASE DEFICIENCY IN LEUKEMIA: GENETICS AN0 C.Salerno, C.Crifb BIOCHEMICAL ASPECTS. Car losbrrer~wb WIIIis.RobertM~hllcote.mKubota.nadL Institutes of Biological Chemistry and Rheumatology, -. Scrl pps Cl Inlc and Research Foundat Ion, Dept. of University of Rome, and C.N.R. Centre for Molecular Basic and Cl l nlcal Research, La Jol la, CA. Biology, Rome, Italy. MTA Is produced In eukaryotlc cells durlng the synthesis of polyamlnes from decarboxylated S-adenosylmethlonlne. The The uptake of (8-14c)hypoxanthine by human erythro- nucleoslde is rapldly cleaved to adenlne and methylthlo- cytes suspended in a phosphate-free medium has been rlbose-1-P b MTA phosphory lase (MTAse). We have asslgned studied. The erythrocytes freshly drawn from human the gene & to chromosome 9pter->9q12 by enzymatlc and electrophoretic analysls of somatlc cell hybrids. healty donors were washed twice and resuspended in iso- Al l normal tlssues and non-malglnant cel l l lnes contain tonic NaCl medium containing 1-30 uM labelled hypoxan- MTAse. However, several human leukemlc cell l lnes are deflclent In the enz me, and 5 patlents wlth acute lympho- thine to give a 50% hematocrit. At regular intervals, blastic leukemla (AIL) have been shown thus far to lack the cells were separated from the bathing medium by MTAse In their ma1 lgnant cel Is. Kar otyplc abnormal ltles rapid centrifugation through a layer of dibutyl phtha- involving fragl le slte 9p21 occur In A!L wlth lymphomatous cllnlcal features. One of 5 such patients studled prospec- late in order to determine hypoxanthine uptake. Under tively lacked MTAse In her leukemic cells but not In normal these conditions, more than 90% of labelled hypoxan- blood cel Is at remlsslon. No Inactlve enzyme proteln has thine taken up by the cells was present in the cytosol been detected by fmmunoadsorptlon among 7 leukemlc I lnes tested. The MTAse def l c l ent cel l I 1nes excrete MTA up to as such. The time course of hypoxanthine uptake beha- 0.32 nmol/hr/mg protein. In mlce, the rowth of MTAse ved as a single exponential process, the relaxation def lclent mutant lymphoma cel Is (but not ~~fseposltlve wlId time being reversely related to the temperature of the type cells) causes plasma MTA to rise from undetectable levels to > 800 nM pre-terminally. Assay of plasma or urlne cell suspension (5= 130 sec at o'c). Hypoxanthine MTA ma thus prove useful to screen leukem lc patients for was not appreciably taken up by erythrocytes when the MTAse leflc lent ma l lgnant cel l clones. external site of the cell membranes was partially de- gested by proteinase K.

PRIMARY GOUT IN AGING PATIENTS GENETIC ANALYSIS OF DEOXYADENOSINE TOXICITY IN 26 A. Carcassi, S. Boschi, F. D8Ubaldo and S. Campagna. 29 DIVIDING HUMAN LYMPEOBLASTS. Dennis 4 Carson, The onset of primary gout in aging patients has been rarer in --Masaru Kubota, D. Bruce Wasson. Erik Willis, and Taizo Iizasa. Scripps Clinic and Research Foundation, the past than it is today. Department of Basic and Clinical Research, La Jolla, We observed late onset-over 65 years of age- of gout in 15 California. patients or in 14% out of 104 patients. The accumulation of deoxyadenosine (dAdo) and its Comparative studies of clinic and physiopatological mani- metabolites may produce immunodeficiency in children who festations of these 15 patients revealed the following: lack adenosine deaminase. The metabolism of dAdo in prevalence of females was higher (26.5%), familial incidenceo'i dividing human lymphoblasts, and the mechanism of dAdo - toxicity, have aroused considerable controversy. To the disease was present only in 6%,overweight was uncommon (13%). investigate this problem, we have selected stable mutant In most cases hyperuricemia was present (mean value 8.3 mg%), human lymphoblastoid cell lines resistant to the anti- with significant reduction of uric acid clearance (mean value proliferative effects of dAdo and have compared their 6.7 ml/min) while uricosuria was normal (mean value 672 mg/24h.) biochemical phenotypes. The dAdo-resistant mutants differed The site of initial attack was the first metatarsal falangeal from wild type cells in one of three ways: (1) an increase joint (podagra) in 60%. Tophi were never found. Renal calculi in the activity of ribonucleotide reductase, (2) an increase in cytoplasmic nucleotidase activity, (3) a decrease in were observed only in 2 cases (13%). deoxycytidine kinase activity. All three genetic changes Cholesterol and tryglicerides mean levels were normal meanwhile caused a secondary rise in de novo deoxycytidine formation significantly lower levels of HDL-Cholesterol and of Apo-A Lipo- and excretion, and a reciprocal inability to phosphorylate proteins were observed in patients with coronary heart disease &Ado and to form dATP. Thus, human lymphocytes can avert (20%). dAdo toxicity by increasing deoxycytidylate synthesis and Hypertension was present in 33% of the patients. degradation, as well as by decreasing deoxycytidine kinase levels. The net result in each case is an impaired In these patients levels of ionized calcium were significantly functional capacity to phosphorylate deoxyadenosine. lower (mean value 2.04 mEq/l) than in patients with normal blood pressure (mean value 2.18 mEq/l) .

HYPOXANTHINE UPTAKE BY ISOLATED BRAIN MICRO- MECHANISM OF ADENOSINE TOXICITY TO ADENOSINE 27 VESSELS. P.Cardelli-Cangiano, A.Flori, A. 30 KINASE DEFICIENT MAMMALIAN CELLS. & Giacomello, R.Strom, C.Salerno Carson.LMKalander.Masaru& . Scrlpps Cllnlc and Research Foundatlon. Department of Human Biopathology, Institutes of Biolo- Dept. of Basic and Cl l nlcal Research, La Jol la, CA. gical Chemistry and Rheumatology, University of Rome, Somatlc cell genetlcs has been used to probe the and C.N.R. Centre for Molecular Biology, Rome, Italy. mechanism of adenos i ne (Ado) toxic1ty to mamma I Ian cel Is def lclent In Ado deam lnase and Ado k lnase. Ado reslstant Isolated bovine brain microvessels were used as in clones of an Ado klnase deflclent murlne lymphoblastold cell - line (R1.1) were isolated and characterlzed. In deox cofor- vitro model of blood/brain barrier. In a phosphate- mycin supplemented medlum, the mutant clones were 10-50 fold free Krebs Ringer medium at 37'~, (8-14~)hypoxanthine more resistant than parental cells to the antl-proliferative is taken up by the microvessels and converted for ab- actlons of Ado, 3-deaza-Ado, carbocycllc-Ado, adenlne. 5'- methyl thloadenoslne, and several other Ado analo s. The out 45% in the corresponding nucleotide. The initial mutants were normally sensltlve to the toxlc effects of rate of the uptake is a hyperbolic function of hypo- deoxyadenoslne and adenlne arablnoslde. Levels of S- xanthine concentration in the suspending medium. Inor- adenosy lmethlonlne (SAM) were 50 pmols/106 cel Is in the parental I fne, compared to 250-350 pmol s/lo6 cel Is In the ganic phosphate (up to 10 mM) decreases the apparent mutant. Methionlne adenosy Itransferase actlvity was 1.5-3.2 Km without affecting the apparent Vmax of hypoxanthine fold hlgher In the Ado-resistant cells than In parental uptake. The presence of inorganic phosphate, which (in lymphoblasts, and varled inversely wlth the medlum methlonlne concentration. Ado Induced the accumul atIon of the absence of hypoxanthine) causes a raise in the ATP equivalent amounts of S-adenosy l homocystelne (SAH) In both concentration in the endothelial cells, induces also cel l types. However, the SAH to SAM ratlo in the Ado- resistant mutants never exceeded 0.1. These results show an increase in the equilibrium levels of IMP and hypo- that (1) methionine adenosyltransferase Is an Inducible xanthine within the cells, the value of the nucleotide enzyme In mammallan cells, (11) the toxlc effects of Ado, /base ratio remaining approximately constant. adenlne, and man Ado analo s, can be averted by lncreaslng the veloclty of AM synthesgs.