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)     

Solvent isotope effects on the reaction catalyzed by yeast hexokinase

The pH dependence of the maximum velocity (V) for the phosphorylation of glucose, the V/Kglucose and the V/KMgATP have been obtained in H2O and 2H2O. In H2O, V decreases below a pK of 5.8, V/Kglucose decreases below a pK of 6.1 and V/KMgATP decreases below a pK of 6.7. In 2H2O, complex behavior is observed for these parameters as a function of pD. The ratios of the parameters in H2O and 2H2O above their respective pK values give solvent deuterium isotope effects of about 1.5-1.7 for all three parameters. When 1,5-anhydromannitol is used as an alternative substrate, an isotope effect different than unity is obtained only for V/K1,5-anhydromannitol which gives a value of about 0.7. Both the complex pH profiles and the relative magnitude of the isotope effects are interpreted in terms of a pH-dependent change in the E X glucose complex.     

Effect of Ferricyanide, Ferrocyanide and KCN on Growth and Flowering in the Short-Day Plant Lemna paucicostata 6746

Flowering in the short-day plant Lemna paucicostata 6746 canbe induced under continuous light by the addition of ferricyanie,ferrocyanide or KCN to M-sucrose medium. Each substance is nearly10 times more effective when the flasks are covered by glassbeakers than when cotton plugs are used. By contrast, when floweringis induced under continuous light by copper or by short-daytreatment, neither flowering nor growth are affected by whetherglass beakers or cotton plugs are used. Ferricyanide, ferrocyanideand KCN are also able to induce long-day flowering when theplants are grown on Msucrose medium in small beakers that areplaced in a covered storage dish that also contains a solutionof one of these compounds. Addition of a KOH trap to the storagedish completely blocks the flowering induced by these compounds.If [¹⁴C]ferrocyanide is added to the storage dish both the M-sucrosemedium and the plants contain significant amounts of radioactivity,the amount of radioactivity being proportional to the floweringresponse. These results indicate that ferricyanide, ferrocyanideand KCN break down to release HCN and that it is the HCN whichis responsible for inducing flowering in L. paucicostata 6746under continuous light. ¹Present address: Department of Biology, Osaka Kyoiku University,Ikeda, Osaka 563, Japan. ²Present address: Institute of Horticulture, The Volcani Center,P. O. B. 6, Bet-Dagan, Israel. (Received January 17, 1983; Accepted March 24, 1983)     

Instability of the growth-limiting proteins of the Avena coleoptile and their pool size in relation to auxin

Summary The stability and pool size of the growth-limiting proteins (GLP) of the Avena coleoptile have been studied by measuring the time required for cycloheximide to inhibit the growth of auxin-treated segments. Inhibition of growth follows inhibition of protein synthesis by 20–25 min regardless of the growth rate. This indicates that the growth inhibition is due to inherent instability of the GLP rather than to exhaustion of the pool through growth. A study of the amount and rate of auxin-induced growth which occurs when cycloheximide is added just before or after the auxin indicates that the rate of elongation is determined by the size of the GLP pool, and that the pool of GLP is low in the absence of auxin, but rapidly expands and reaches a maximum 20–25 min after addition of auxin. Three ways in which auxin might expand the pool of GLP are discussed.     

Exercise-induced translocation of protein kinase C and production of diacylglycerol and phosphatidic acid in rat skeletal muscle in vivo. Relationship to changes in glucose transport

Contraction-induced translocation of protein kinase C (Richter E.A., Cleland, P.J.F., Rattigan, S., and Clark, M.G. (1987) FEBS Lett. 217, 232-236) implies a role for this enzyme in muscle contraction or the associated metabolic adjustments. In the present study, this role is further examined particularly in relation to changes in glucose transport. Electrical stimulation of the sciatic nerve of the anesthetized rat in vivo led to a time-dependent translocation of protein kinase C and a 2-fold increase in the concentrations of both diacylglycerol and phosphatidic acid. Maximum values for the latter were reached at 2 min and preceded the maximum translocation of protein kinase C (10 min). Stimulation of muscles in vitro increased the rate of glucose transport, but this required 20 min to reach maximum. There was no reversal of translocation or decrease in the concentrations of diacylglycerol and phosphatidic acid even after 30 min of rest following a 5-min period of stimulation in vivo. Translocation was not influenced by variations in applied load at maximal fiber recruitment but was dependent on the frequency of nontetanic stimuli, reaching a maximum at 4 Hz. The relationship between protein kinase C and glucose transport was also explored by varying the number of tetanic stimuli. Whereas only one train of stimuli (200 ms, 100 Hz) was required for maximal effects on protein kinase C, diacylglycerol, and phosphatidic acid, more than 35 trains of stimuli were required to activate glucose transport. It is concluded that the production of diacylglycerol and the translocation of protein kinase C may be causally related. However, if the translocated protein kinase C is involved in the activation of glucose transport during muscle contractions, an accumulated exposure to Ca2+, resulting from multiple contractions, would appear to be necessary.     

Characterization of Senescence in Lemna gibba G3: A Determinate Growth System

Frond senescence in Lemna gibba G3 was characterized, and itscontrol by light, ABA and kinetin investigated. The plant exhibitsa determinate growth pattern with a frond producing a set numberof daughter fronds before undergoing senescence and death regardlessof whether or not it flowers. When a frond was cut in half,the distal half (half frond) which lacks any meristem underwentrapid senescence as compared with intact fronds. In both intactand half fronds, the onset of senescence was accelerated byABA and retarded by kinetin. Continuous white light acceleratedsenescence in both intact and half fronds over the dark controls.Under different photoperiodic light regime, the pace of daughterfrond production is accelerated in proportion to the lengthof light period. In half fronds, however, very short photoperiodiclight treatments (e.g. 1L: 23D or 3L: 21D) rather delayed senescenceover the dark controls. Two separate light control systems operatingin opposite directions in Lemana senescence appear to exist. ¹Present address: Department of Biology, Yonsei University,Seoul 120-749, Korea ²Present address: U.S. Department of Agriculture, Aero SpaceBuilding, Rm. 323, 901 D Street, S.W., Washington, D.C. 20251-2200, U.S.A. (Received July 13, 1989; Accepted May 8, 1990)     

The contribution of threonine 55 to catalysis in aspartate transcarbamoylase.

Heavy-atom isotope effects and steady-state kinetic parameters were measured for the catalytic trimer of an active site mutant of aspartate transcarbamoylase, T55A, to assess the role of Thr 55 in catalysis. The binding of carbamoyl phosphate to the T55A mutant was decreased by 2 orders of magnitude relative to the wild-type enzyme whereas the affinities for aspartate and succinate were not markedly altered. This indicates that Thr 55 plays a significant role in the binding of CbmP. If, as had been suggested previously, Thr 55 assists in the polarization of the carbonyl group of CbmP, the carbon isotope effect for the T55A mutant should increase relative to that observed for the wild-type enzyme. However, the opposite is seen, indicating that Thr 55 is not involved in stabilizing the oxyanion in the transition state. Quantitative analysis of a series of 13C and 15N isotope effects suggested that the rate-determining step in the reaction catalyzed by T55A trimer may be a conformational change in the protein subsequent to formation of the Michaelis complex. Thus, Thr 55 may facilitate a conformational change in the enzyme that is a prerequisite for catalysis. An altered active site environment in the binary and Michaelis complexes with T55A trimer is reflected in the pH profiles for log V, log (V/K)asp, and pK(i) succinate, show a displacement in the pK values of ionizing residues involved in aspartate binding and catalysis relative to the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Long-term treatment of isolated rat soleus muscle with phorbol ester leads to loss of contraction-induced glucose transport.

Muscle contraction involves mobilization of intracellular Ca2+ and is associated with several metabolic adjustments, including increased glucose transport. In the present study isolated rat soleus muscles were exposed to 12-O-tetradecanoylphorbol 13-acetate, and responses to both insulin and contraction in terms of glucose transport were assessed. Muscles treated with this phorbol ester for 12 h showed an increased basal rate of 3-O-methylglucose uptake, and responded partially to insulin but did not respond to contraction. Phorbol-ester-treated and non-treated (vehicle-only) muscles were indistinguishable in terms of pre-contraction content of adenine nucleotide, phosphocreatine, lactate and glycogen, as well as contractile performance and contraction-induced glycogenolysis. Phorbol ester treatment of isolated solei for 12 h resulted in the loss of 90% of protein kinase C activity as determined with histone IIIs as substrate, and 70% as determined by using phorbol ester binding. It is concluded that treatment of solei with phorbol ester gives rise to a marked loss of contraction-induced glucose transport.     

Plasmodesmal-mediated cell-to-cell transport in wheat roots is modulated by anaerobic stress

Summary Cell-to-cell transport of small molecules and ions occurs in plants through plasmodesmata. Plant roots are frequently subjected to localized anaerobic stress, with a resultant decrease in ATP. In order to determine the effect of this stress on plasmodesmal transport, fluorescent dyes of increasing molecular weight (0.46 to 10 kDa) were injected into epidermal and cortical cells of 3-day-old wheat roots, and their movement into neighboring cells was determined by fluorescence microscopy. Anaerobiosis was generated by N₂ gas or simulated by the presence of sodium azide, both of which reduced the ATP levels in the tissue by over 80%. In the absence of such stress, the upper limit for movement, or size exclusion limit (SEL), of cortical plasmodesmata was <1 kDa. The ATP analogue TNP-ADP (mw 681) moved across the plasmodesmata of unstressed roots, indicating that plasmodesmata may be conduits for nucleotide (ATP and ADP) exchange between cells. Upon imposition of stress, the SEL rose to between 5 and 10 kDa. This response of plasmodesmata to a decrease in the level of ATP suggests that they are constricted by an ATP-dependent process so as to maintain a restricted SEL. When roots are subjected to anaerobic stress, an increase in SEL may permit enhanced delivery of sugars to the affected cells of the root where anaerobic respiration could regenerate the needed ATP.Abbreviations F-dextran fluorescein-coupled dextran - LYCH Lucifer Yellow CH - SEL size exclusion limit - TNP-ADP 2-O-(trinitrophenyl)adenosine-5-diphosphate