Chromatography of soluble proteins from rat heart on phosphocellulose columns separates two 5'-nucleotidases. The first to emerge from the column shows a preference for AMP over IMP as substrate, whereas the second shows a preference for IMP over AMP. The properties of the IMP-preferring enzyme, including the conditions under which it is eluted from phosphocellulose columns, show it to be the enzyme studied by Itoh, Oka & Ozasa [Biochem. J. (1986) 235, 847-851]. The kinetic properties of the AMP-preferring enzyme indicate that it is likely to be the enzyme responsible for the production of adenosine under conditions of hypoxia and increased work load, and with metabolic stresses such as a high load of acetate.

5'-Nucleotidase I (N-I) from rabbit heart was purified to homogeneity. After ammonium sulfate precipitation, the purification involved chromatography on phosphocellulose, DEAE-Sepharose, AMP-agarose, and ADP-agarose. The pure enzyme has a specific activity of 318 mumol (mg of protein)-1 min-1. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate yields a subunit molecular weight of 40,000. N-I is activated by ADP but not by ATP, in contrast to the 5'-nucleotidase (N-II) purified by Itoh et al. (1986), which is activated by ATP and, less well, by ADP. N-I displays sigmoidal saturation kinetics in the absence of ADP and hyperbolic kinetics in the presence of ADP. Partially purified N-I was previously shown to prefer AMP over IMP as substrate (Truong et al., 1988); this has been confirmed for pure N-I. Comparison of AMP and ADP concentrations reported to occur in heart with the kinetic behavior of N-I implicates N-I as the enzyme responsible for producing adenosine under conditions leading to a rise in ADP and AMP, such as hypoxia or increased workload. N-I is not activated by the ADP analogue adenosine 5'-methylenediphosphonate (AOPCP) and is only weakly inhibited by relatively high concentrations of AOPCP, in contrast to 5'-nucleotidase from plasma membrane, which is powerfully inhibited by this analogue. N-I shows an absolute dependence on Mg2+ ions. Mn2+ and Co2+ ions can replace Mg2+ ions as activator; Ni2+ and Fe2+ are much less effective, while Ca2+, Ba2+, Zn2+, and Cu2+ fail to activate the enzyme.

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, J a p a n T h e r e n a l handlings o f oxypurinol a n d oxypurines by normal subjects after intake o f allopurinol w e r e investigated b y studies o n the influences o f uricosuric agents (probenecid a n d benzbromarone) a n d pyrazinamide. Pyrazinamide decreased t h e fractional clearance o f oxypurinol, hypoxanthine a n d xanthine 0.46-, 0.52-a n d 0.40-fold, respectively. Probenecid increased t h e fractional clearance o f oxypurinol 1.8-fold, decreased t h a t o f xanthine 0.61-fold and d i d n o t a f f e c t that of hypoxanthine. Benzbromarone increased t h e fractional clearance o f oxypurinol 2.17-fold b u t d i d n o t affect those o f hypoxanthine and xanthine. T h e s e results suggest t h a t oxypurinol i s reabsorbed a t least a t a postsecretory s i t e o f t h e renal tubules, t h a t t h e m a i n renal transport o f xanthine i s secretory w h e n conversion o f oxypurines t o u r i c acid i s decreased a n d t h a t the m a i n renal transport o f hypoxanthine t h a t i s n o t affected by these three agents may o c c u r w h e n conversion o f oxypurines t o uric acid i s decreased. MODULATION OF DIPHTHAMIDE SYNTHESIS BY METHYLTHIOAOENOSINE. Hisashi Yamanaka* andInstitute of Scripps Clinic. USA Exogenous addition of methylthioadenosine (MeSAdo) inhibits proliferation of cells which are deficient in MeSAdo phosphorylase. A series of evidences has shown that the target of toxic mechanism of MeSAdo is not totally dependent on polyamine synthesis. Eukaryotic cells contain unique modified amino acid in elongation factor 2 (EF-2). designated as diphthamide. This residue is the specific target of mono(ADP-ribosy1)ation catalyzed by diphtheria toxin (DT) or Pseudomonous toxin.The structure of diphthamide is 2-[3-carboxyamido-3-( t r l m e t h y l a m o n i o ) p r o p y l ] h i s t i d i n e , and the first reaction to modify histidine involves the transfer of an aminocarboxypropyl group from S-adenosylmethionine. MeSAdo should be the nucleoside product of this reaction. By these reasons. we have analyzed the effect of MeSAdo on the biosynthesis of diphthamide. A mutant cell line H3 which is deficient in MeSAdo phosphorylase and resistant to MeSAdo, has been isolated from murine lymphoma cell line R1.l. for the study. As measured by susceptibility to DT induced ADP-ribosylation, MeSAdo inhibits the formation of diphthamide in a dose dependent manner. In addition. MeSAdo substantially protected H3 cells from the lethal effect of DT. These results suggest that the modulation of diphthamide synthesis can be. at least a part of. the mechanism of MeSAdo toxicity toward eukaryotic cells. Poly(ADP-ribose) polymerase (ADPRP) is highly activated by DNA with strand breaks and transfer poly (ADP-ribose)chains out0 nuclear proteins. Poly (ADP-ribosyl )action rapidly consumes cellular NAD and leads cells to die. This reaction has been implied to be involved in the deoxyadenosine toxicity toward resting human lymphocytes, and could be an important mechanism in tne toxicity of all DNA-damaging agents. To analyze ...