The previously developed chelator O-aminophenol-N,N,O-triacetic acid (APTRA) (L. A. Levy, E. Murphy, B. Raju, and R. E. London. Biochemistry 27: 4041-4048, 1988) has been modified to yield a fluorescent analogue which can be utilized as an intracellular probe for ionized Mg2+. The fluorescent analogue, FURAPTRA, with a magnesium dissociation constant of 1.5 mM, is structurally analogous to the calcium chelator fura-2 and exhibits a similar excitation shift on magnesium complexation. Hence, data on the intracellular Mg2+ concentration can be obtained using an analogous ratio method. The acetoxymethyl form of the chelator is readily loaded into cells and has been used to determine a cytosolic free Mg2+ concentration of 0.59 mM for isolated rat hepatocytes. As a consequence of the relatively high levels of cytosolic Mg2+, the problem of ion buffering is much less severe than for the analogous calcium indicators.

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Topoisomerase 2 (TOP2) DNA transactions are essential for life, and proceed via formation of the TOP2 cleavage complex (TOP2cc), a covalent enzyme-DNA reaction intermediate that is vulnerable to trapping by potent anticancer TOP2 drugs. How genotoxic TOP2 DNA-protein crosslinks are resolved is unclear. Here, we show that the SUMO ligase ZATT (ZNF451) is a multifunctional DNA repair factor that controls cellular responses to TOP2 damage. ZATT binding to TOP2cc facilitates a proteasome-independent Tyrosyl-DNA phosphodiesterase 2 (TDP2) hydrolase activity on stalled TOP2cc. The ZATT SUMO ligase activity further promotes TDP2 interactions with SUMOylated TOP2, regulating efficient TDP2 recruitment through a "split-SIM" SUMO2 engagement platform. These findings uncover a ZATT–TDP2 catalyzed and SUMO2-modulated pathway for direct resolution of TOP2cc.

Changes in cytosolic free calcium concentration during myocardial ischemia were measured by 19F NMR in 5FBAPTA-loaded perfused rat hearts. The hearts were perfused with Krebs-Henseleit buffer containing 5 microM of the acetoxymethyl ester of 5FBAPTA, which was hydrolyzed by cytosolic esterases to achieve cytosolic concentrations of 5FBAPTA of 0.12 to 0.65 mM. Cytosolic free calcium concentrations were calculated as the product of the ratio of peak areas for bound and free 5FBAPTA in the NMR spectra and the dissociation constant (708 nM). The basal cytosolic calcium concentration, measured in potassium or magnesium arrested hearts, was 252 nM, and the time-average calcium concentration in beating hearts was 630 nM. Following the onset of total ischemia, there was no immediate substantial change in cytosolic calcium despite a rapid decline in creatine phosphate and ATP and a marked increase in inorganic phosphate as monitored by 31P NMR, but by 10 minutes, there was a substantial increase in free calcium concentration. The ratio of peak areas of bound and free 5FBAPTA returned to the preischemic value during reperfusion, and there was no detectable loss of 5FBAPTA from the heart. Creatine phosphate was also restored to its preischemic level during reperfusion. These results indicate that cytosolic free calcium increases during ischemia and is not immediately associated with lethal injury. This increase in cytosolic calcium may activate degradative enzymes that eventually could compromise myocyte viability.

The mechanism by which preconditioning (brief intermittent periods of ischemia and reflow) improves recovery of function and reduces enzyme release after a subsequent 30-minute period of ischemia was investigated in perfused rat hearts. Specifically, it was hypothesized that ischemia after preconditioning would result in a decreased production of H+ and therefore a smaller rise in [Na+]i and [Ca2+]i via Na(+)-H+ and Na(+)-Ca2+ exchange. To test this hypothesis we measured pHi, [Na+]i, [Ca2+]i, and cell high-energy phosphates during ischemia and reflow, and we correlated this with recovery of contractile function and release of creatine kinase during reflow. 31P nuclear magnetic resonance (NMR) was used to measure pHi and cell phosphates. [Na+]i was measured by 23Na NMR using the shift reagent thulium 1,4,7,10-tetraazacyclododecane-N,N,'N",N"'-tetramethylenephosph onate to distinguish intracellular from extracellular sodium. [Ca2+]i was measured by 19F NMR using hearts loaded with 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid, termed 5F-BAPTA. Basal time-averaged levels of pHi, [Na+]i, and [Ca2+]i were 7.07 +/- 0.08, 9.4 +/- 0.8 mM, and 715 +/- 31 nM, respectively. After 30 minutes of ischemia, in preconditioned hearts, pHi was 6.5 +/- 0.06, [Na+]i was 2.09 +/- 4.4 mM, [Ca2+]i was 2.1 +/- 0.4 microM, and ATP was negligible. In untreated hearts, after 30 minutes of ischemia, pHi was 6.3 +/- 0.08, [Na+]i was 26.7 +/- 3.8 mM, [Ca2+]i was 3.2 +/- 0.6 microM, and ATP was undetectable. During reperfusion after 30 minutes of ischemia, preconditioned hearts had significantly better recovery of contractile function than untreated hearts (71 +/- 9% versus 36 +/- 8% initial left ventricular developed pressure), and after 60 minutes of ischemia, preconditioned hearts had significantly less release of the intracellular enzyme creatine kinase (102 +/- 12 versus 164 +/- 17 IU/g dry wt). We also found that unpreconditioned hearts arrested with 16 mM MgCl2 (to inhibit calcium entry via calcium channels and Na(+)-Ca2+ exchange) before 30 minutes of ischemia recover function on reflow to the same extent as preconditioned hearts with or without magnesium arrest. Thus, preconditioning has no additional benefit in addition to magnesium arrest. In addition, in hearts that received 16 mM MgCl2 just before the 30-minute period of ischemia, preconditioning had no effect on the rise in [Ca2+]i during the 30-minute period of ischemia. These data support the hypothesis that preconditioning attenuates the increase in [Ca2+]i, [Na+]i, and [H+]i during ischemia, most likely because of reduced stimulation of Na(+)-H+ and Na(+)-Ca2+ exchange.(ABSTRACT TRUNCATED AT 400 WORDS)

An increase in cytosolic free calcium (Ca;) has been shown to occur early during ischemia in perfused rat, ferret, and rabbit hearts. It has been proposed that this increase in Cal may occur as a result of exchange of Nai for Ca., which occurs as a result of an increase in Na; arising from exchange of Na0 for H+;. The latter exchange is stimulated by the intracellular acidification that occurs during ischemia. To test this hypothesis, we examined Ca;, Na;, ATP, and pH; during ischemia in rats in the presence and absence of 1 mM amiloride, a Na-H exchange inhibitor. Ca; was measured using '9F nuclear magnetic resonance (NMR) of 1,2-bis(2-amino-5-fluorophenoxy)ethane-N, N, N', N'-tetra-acetic acid (5F-BAPTA) -loaded rat hearts. Na; was measured using 23Na NMR, and the shift reagent 1, 4, 7, 10-tetraazacyclododecane-N, N', N", N"'-tetramethylenephosphonate (Tm[DOTPI 5) was used to separate Nai and Na0. ATP and pH were determined from 31P NMR measurements. During 20 minutes of ischemia, amiloride did not significantly alter the ATP decline but did significantly attenuate the rise in Na; and Cai. After 20 minutes of ischemia, time-averaged Caj was 1.0±0.2 ,uM (mean+SEM) in amiloride-treated hearts compared with 23±0.9 p,M in nontreated hearts. After 20 minutes of ischemia, Nai in the untreated heart was threefold greater than control, whereas in the amiloride-treated heart, Nai was not significantly different from control. These data are consistent with the involvement of Na-Ca exchange in the rise in Caj during ischemia. In addition, recovery of contractile function during reperfusion after 20 minutes of ischemia was significantly better in amiloride-treated hearts (71±10%) than in nontreated hearts (24±13%). These data are consistent with the hypothesis suggesting that the elevation in Ca; during ischemia may contribute to postischemic contractile dysfunction. (Circulation Research 1991;68:1250-1258 An increase in cytosolic free calcium (Cai)

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Type II dihydrofolate reductase (DHFR) is a plasmid-encoded enzyme that confers resistance to bacterial DHFR-targeted antifolate drugs. It forms a symmetric homotetramer with a central pore which functions as the active site. Its unusual structure, which results in a promiscuous binding surface that accommodates either the Dihydrofolate (DHF) substrate or the NADPH cofactor, has constituted a significant limitation to efforts to understand its substrate specificity and reaction mechanism. We describe here the first structure of a ternary R67 DHFR•DHF•NADP + catalytic complex, resolved to1.26 Å. This structure provides the first clear picture of how this enzyme, which lacks the active site carboxyl residue that is ubiquitous in Type I DHFRs, is able to function. In the catalytic complex, the polar backbone atoms of two symmetry-related I68 residues provide recognition motifs that interact with the carboxamide on the nicotinamide ring, and the N3-O4 amide function on the pteridine. This set of interactions orients the aromatic rings of substrate and cofactor in a relative endo geometry in which the reactive centers are held in close proximity. Additionally, a central, hydrogen-bonded network consisting of two pairs of Y69-Q67-Q67′-Y69′ residues provides an unusually tight interface, which appears to serve as a "molecular clamp" holding the substrates in place in an orientation conducive to hydride transfer. In addition to providing the first clear insight regarding how this extremely unusual enzyme is able to function, the structure of the ternary complex provides general insights into how a mutationallychallenged enzyme, i.e., an enzyme whose evolution is restricted to four-residues-at-a-time active site mutations, overcomes this fundamental limitation. KeywordsR67 DHFR; Dihydrofolate Reductase; X-ray crystallography; ternary complex; Type II DHFR Antifolate drug therapy plays a critical role in the treatment of pathogenic and neoplastic diseases. The evolution of a plasmid-encoded, Type II dihydrofolate reductase (DHFR) provides one mechanism for bacterial evasion of drugs such as trimethoprim that target the bacterial dihydrofolate reductase enzyme (1-4). Type II DHFR is an extremely unusual enzyme that exhibits no apparent structural or evolutionary relationship with the type I (chromosomal) enzyme. It is one of the smallest enzymes known to self-assemble into an active quaternary structure, forming a homotetramer consisting of four 78-residue peptides * To whom correspondence should be addressed. This type of active site structure also creates substantial evolutionary and mutational challenges to the enzyme. Since each mutation will alter four active site residues at a time, most of the evolutionary pressure that would normally optimize enzyme function is compromised by the need to balance the effects of substitutions at all four symmetry-related sites. For example, a residue substitution on one monomer, which might promote folate N5 protonation, may also interfere with NADPH binding when it is prese...