Evidence from nitrogen-15 and solvent deuterium isotope effects on the chemical mechanism of adenosine deaminase

We have determined 15N isotope effects and solvent deuterium isotope effects for adenosine deaminase using both adenosine and the slow alternate substrate 7,8-dihydro-8-oxoadenosine. With adenosine, 15N isotope effects were 1.0040 in H2O and 1.0023 in D2O, and the solvent deuterium isotope effect was 0.77. With 7,8-dihydro-8-oxoadenosine, 15N isotope effects were 1.015 in H2O and 1.0131 in D2O, and the solvent deuterium isotope effect was 0.45. The inverse solvent deuterium isotope effect shows that the fractionation factor of a proton, which is originally less than 0.6, increases to near unity during formation of the tetrahedral intermediate from which ammonia is released. Proton inventories for 1/V and 1/(V/K) vs percent D2O are linear, indicating that a single proton has its fractionation factor altered during the reaction. We conclude that a sulfhydryl group on the enzyme donates its proton to oxygen or nitrogen during this step. pH profiles with 7,8-dihydro-8-oxoadenosine suggest that the pK of this sulfhydryl group is 8.45. The inhibition of adenosine deaminase by cadmium also shows a pK of approximately 9 from the pKi profile. Quantitative analysis of the isotope effects suggests an intrinsic 15N isotope effect for the release of ammonia from the tetrahedral intermediate of approximately 1.03 for both substrates; however, the partition ratio of this intermediate for release of ammonia as opposed to back-reaction is 14 times greater for adenosine (1.4) than for 7,8-dihydro-8-oxoadenosine (0.1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Cardiovascular effects of training for a marathon run in unfit middle aged men.

The effects of a 30 week exercise programme on serum lipid values, blood pressure, and cardiac function were assessed in a group of sedentary men aged 35-50 training for their first marathon. Mean serum cholesterol concentration (n = 33) fell by 12% from 6.54 (SE 0.18) to 5.76 (0.15) mmol/l (mean fall 0.78 mmol/l; 95% confidence interval 0.52 to 1.04 mmol/l), serum triglyceride concentration (n = 33) by 22% from 1.56 (0.17) to 1.21 (0.09) mmol/l (mean fall 0.34 mmol/l; 95% confidence interval 0.12 to 0.56 mmol/l), and mean blood pressure (n = 27) by 10% from 102 (2) to 92 (2) mm Hg (mean fall 10 mm Hg; 95% confidence interval 7 to 13 mm Hg). These changes were not explained by changes in body composition. Peak exercise left ventricular end diastolic volume (n = 16) increased with training; as a result of this and an increased exercise left ventricular ejection fraction peak exercise cardiac output increased from 19.9 (1.2) to 23.1 (3.0) l/min (mean rise 3.2 l/min; 95% confidence interval 1.5 to 5.0 l/min). Maximum oxygen consumption increased from 33.9 (1.6) to 39.0 (1.3) ml/kg/min (mean rise 5.0 ml/kg/min; 95% confidence interval 1.8 to 8.2 ml/kg/min). This study showed favourable effects on coronary risk factors and cardiac function and supports the place of regular exercise in coronary prevention programmes.     

Auxin Physiology of the Tomato Mutant diageotropica

The tomato (Lycopersicon esculentum, Mill.) mutant diageotropica (dgt) exhibits biochemical, physiological, and morphological abnormalities that suggest the mutation may have affected a primary site of auxin perception or action. We have compared two aspects of the auxin physiology of dgt and wild-type (VFN8) seedlings: auxin transport and cellular growth parameters. The rates of basipetal indole-3-acetic acid (IAA) polar transport are identical in hypocotyl sections of the two genotypes, but dgt sections have a slightly greater capacity for IAA transport. 2,3,5-Triiodobenzoic acid and ethylene reduce transport in both mutant and wild-type sections. The kinetics of auxin uptake into VFN8 and dgt sections are nearly identical. These results make it unlikely that an altered IAA efflux carrier or IAA uptake symport are responsible for the pleiotropic effects resulting from the dgt mutation. The lack of auxin-induced cell elongation in dgt plants is not due to insufficient turgor, as the osmotic potential of dgt cell sap is less (more negative) than that of VFN8. An auxin-induced increase in wall extensibility, as measured by the Instron technique, only occurs in the VFN8 plants. These data suggest dgt hypocotyls suffer a defect in the sequence of events culminating in auxin-induced cell wall loosening.     

Calcium and the mechanical properties of soybean hypocotyl cell walls: Possible role of calcium and protons in cell-wall loosening

The role of calcium in the mechanical strength of isolated cell walls of soybean (Glycine max (L.) Merr. cv. Wayne) hypocotyls has been investigated, using the Instron technique to measure the plastic extensibility (PEx) of methanol-boiled, bisected hypocotyl sections and epidermal strips, and atomic absorption spectroscopy to measure wall calcium. Plastic extensibility was closely correlated with the growth rate of intact soybean hypocotyls. Removal of calcium from isolated cell walls by ethylene glycol-bis(2-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA) or low pH increased PEx, while addition of calcium decreased PEx; both effects were reversible. The amount of calcium removed and the increase in PEx at pH 4.5 were strongly dependent upon the chelating ability of the buffer anion. There was a direct correlation between the amount of calcium removed from the wall by EGTA or acid and the increase in PEx. Removal of up to 60% of the calcium increased PEx of half-section up to two fold, but further loss of calcium caused a much greater increase in PEx. With epidermal strips, PEx increased only when calcium was reduced below a threshold. At pH 3.5, there was an additional increase in PEx after a lag of about 2 h; this additional increase may be the result of acid-induced cleavage of a different set of load-bearing bonds. We conclude that calcium bridges are part of the load-bearing bonds in soybean hypocotyl cell walls, and that breakage of these crosslinks by apoplastic acid participates in wall loosening. Acid-induced solubilization of wall calcium may be one mechanism involved in wall loosening of dicotyledonous stems.Abbreviations EGTA ethylene glycol-bis(2-aminoethyl ether)-N,N,N,N-tetraacetic acid - PEx Instron plastic extensibility     

The pH profile for acid-induced elongation of coleoptile and epicotyl sections is consistent with the acid-growth theory

The acid-growth theory predicts that a solution with a pH identical to that of the apoplast of auxintreated tissues (4.5–5.0) should induce elongation at a rate comparable to that of auxin. Different pH profiles for elongation have been obtained, however, depending on the type of pretreatment between harvest of the sections and the start of the pH-incubations. To determine the acid sensitivity under in vivo conditions, oat (Avena sativa L.) coleoptile, maize (Zea mays L.) coleoptile and pea (Pisum sativum L.) epicotyl sections were abraded so that exogenous buffers could penetrate the free space, and placed in buffered solutions of pH 3.5–6.5 without any preincubation. The extension, without auxin, was measured over the first 3 h. Experiments conducted in three laboratories produced similar results. For all three species, sections placed in buffer without pretreatment elongated at least threefold faster at pH 5.0 than at 6.0 or 6.5, and the rate elongation at pH 5.0 was comparable to that induced by auxin. Pretreatment of abraded sections with pH-6.5 buffer or distilled water adjusted to pH 6.5 or above gave similar results. We conclude that the pH present in the apoplast of auxin-treated coleoptile and stems is sufficiently low to account for the initial growth response to auxin.Abbreviations FS free space - IAA indole-3-acetic acid This research was supported by a grant from the National Adonautics and space Administration (NASA), NAGW 1394 to R.E.C., NASA grant NAGW-297 to M.L.E., and NASA grant NAG 1849 to D.L.R.     

Use of primary deuterium and 15N isotope effects to deduce the relative rates of steps in the mechanisms of alanine and glutamate dehydrogenases

We have used deuterium and 15N isotope effects to study the relative rates of the steps in the mechanisms of alanine and glutamate dehydrogenases. The proposed chemical mechanisms for these enzymes involve carbinolamine formation, imine formation, and reduction of the imine to the amino acid [Grimshaw, C.E., Cook, P.F., & Cleland, W.W. (1981) Biochemistry 20, 5655; Rife, J.E., & Cleland, W.W. (1980) Biochemistry 19, 2328]. These steps are almost equally rate limiting for V/Kammonia with alanine dehydrogenase, while with glutamate dehydrogenase carbinolamine formation, imine formation, and release of glutamate after hydride transfer provide most of the rate limitation of V/Kammonia. Release of oxidized nucleotide is largely rate limiting for Vmax for both enzymes. When beta-hydroxypyruvate replaces pyruvate, or 3-acetylpyridine NADH (Acpyr-NADH) or thio-NADH replaces NADH with alanine dehydrogenase, nucleotide release no longer limits Vmax, and hydride transfer becomes more rate limiting. With glutamate dehydrogenase, replacement of alpha-ketoglutarate by alpha-ketovalerate makes hydride transfer more rate limiting. Use of Acpyr-NADPH has a minimal effect with alpha-ketoglutarate but causes an 8-fold decrease in Vmax with alpha-ketovalerate, with hydride transfer the major rate-limiting step. In contrast, thio-NADPH with either alpha-keto acid causes carbinolamide formation to become almost completely rate limiting. These studies show the power of multiple isotope effects in deducing details of the chemistry and changes in rate-limiting step(s) in complicated reaction mechanisms such as those of alanine and glutamate dehydrogenases.     

Flowering and Endogenous Levels of Plant Hormones in Lemna Species

The occurrence and endogenous level of various plant hormoneswere measured for the short-day plants Lemna paucicostata 151and 381 and the long-day plant Lemna gibba G3 to determine whetherany of them are involved in the photoperiodic control of flowering.ABA, IAA, GA₁, GA₂₉, GA₃₄, GA₅₃, trans- and cis-zeatin, trans-and cis-ribosyl zeatin, N⁶-(²-isopentenyl) adenine and N⁶-(²-isopentenyl)adenosine were definitely detected in each species, while GA₄was only detected in L. gibba G3 and GA₂₀ was only detectedin L. paucicostata 151. The endogenous levels of ABA and IAAwere in the range of 1–7 ng/g fr wt and were not significantlydifferent in vegetative and flowering plants. The endogenousgibberellin levels were generally higher in Lemna grown underlong-day rather than short-day conditions. The endogenous cytokininlevels were almost the same in both flowering and vegetativeplants of L. paucicostata 151 and 381. In L. gibba G3, however,the level of cis-ribosyl zeatin, N⁶-(²-isopentenyl) adenineand N⁶-(²-sopentenyl) adenosine were higher in vegetative thanin flowering plants. These results indicate that there is not necessarily a directrelation between endogenous plant hormone levels and flowering,and that the chemical basis for the photoperiodic control offlowering cannot be explained solely by changes in hormone levels.The possibility remains, however, that one or more of the planthormones has some influence of secondary importance on the floweringprocess in Lemna. (Received January 29, 1986; Accepted July 12, 1986)     

Inhibition of Flowering in the Long-Day Plant Lemna gibba G3 by Hutner's Medium and its Reversal by Medium Modification

The long-day plant Lemna gibba G3 flowers normally in E medium(Hoagland-type medium plus 30 µM EDTA) but in 0.5 H mediumthere is no flowering. Ammonium is present in 0.5 H medium andis known to inhibit flowering in L. gibba G3, but even in NH₄⁺-free0.5 H medium there is virtually no flowering under continuouslight. Increasing the phosphate concentration of the NH₄⁺-free0.5 H medium from 1.15 ITIM to 12 or 16 mM results in substantialflowering. Decreasing the EDTA concentration from 850 µIMto 250 µM, or raising the nitrate concentration from 4mM to 12 mM, results in only a small increase in flowering.If the decrease in EDTA and increase in nitrate are combinedwith the increase in phosphate, however, the flowering responseis nearly as good as that obtained using E medium. Thus, withthese three changes the inhibitory effect of NH₄⁺free 0.5 Hmedium for flowering in L. gibba G3 is almost completely reversed In the above studies flowering was not limited by daylength.When plants were grown on E medium under an 11 hour daylengthwhere flowering is limited by daylength, decreasing the phosphateconcentration in the medium reduced flowering, but increasingthe phosphate concentration in the medium did not stimulateflowering. Thus, when flowering is limited by daylength, highphosphate will not cause flowering, but a certain level of phosphateappears to be necessary for the expression of photoinductionunder long days. (Received January 14, 1986; Accepted June 24, 1986)     

Symmetry and asymmetry in mandelate racemase catalysis

Kinetic properties of mandelate racemase catalysis (Vmax, Km, deuterium isotope effects, and pH profiles) were all measured in both directions by the circular dichroic assay of Sharp et al. [Sharp, T. R., Hegeman, G. D., & Kenyon, G. L. (1979) Anal. Biochem. 94, 329]. These results, along with those of studying interactions of mandelate racemase with resolved, enantiomeric competitive inhibitors [(R)- and (S)-alpha-phenylglycerates], indicate a high degree of symmetry in both binding and catalysis. Racemization of either enantiomer of mandelate in D2O did not show an overshoot region of molecular ellipticity in circular dichroic measurements upon approach to equilibrium. Both the absence of such an overshoot region and the high degree of kinetic symmetry are consistent with a one-base acceptor mechanism for mandelate racemase. On the other hand, results of irreversible inhibition with partially resolved, enantiomeric affinity labels [(R)- and (S)-alpha-phenylglycidates] reveal a "functional asymmetry" at the active site. Mechanistic proposals, consistent with these results, are presented.     

A comparative description of mitochondrial DNA differentiation in selected avian and other vertebrate genera

Levels of mitochondrial DNA (mtDNA) sequence divergence between species within each of several avian (Anas, Aythya, Dendroica, Melospiza, and Zonotrichia) and nonavian (Lepomis and Hyla) vertebrate genera were compared. An analysis of digestion profiles generated by 13-18 restriction endonucleases indicates little overlap in magnitude of mtDNA divergence for the avian versus nonavian taxa examined. In 55 interspecific comparisons among the avian congeners, the fraction of identical fragment lengths (F) ranged from 0.26 to 0.96 (F = 0.46), and, given certain assumptions, these translate into estimates of nucleotide sequence divergence (p) ranging from 0.007 to 0.088; in 46 comparisons among the fish and amphibian congeners, F values ranged from 0.00 to 0.36 (F = 0.09), yielding estimates of P greater than 0.070. The small mtDNA distances among avian congeners are associated with protein-electrophoretic distances (D values) less than approximately 0.2, while the mtDNA distances among assayed fish and amphibian congeners are associated with D values usually greater than 0.4. Since the conservative pattern of protein differentiation previously reported for many avian versus nonavian taxa now appears to be paralleled by a conservative pattern of mtDNA divergence, it seems increasingly likely that many avian species have shared more recent common ancestors than have their nonavian taxonomic counterparts. However, estimates of avian divergence times derived from mtDNA- and protein-calibrated clocks cannot readily be reconciled with some published dates based on limited fossil remains. If the earlier paleontological interpretations are valid, then protein and mtDNA evolution must be somewhat decelerated in birds. The empirical and conceptual issues raised by these findings are highly analogous to those in the long-standing debate about rates of molecular evolution and times of separation of ancestral hominids from African apes.