Only the Complete NADH:Nitrate Reductase Activity is Inhibited by Magnesium, not the Partial Activities

In response to in situ dark modulation, or in vitro ATP preincubationof higher plant nitrate reductase, Mg²⁺ inhibits NADH:nitratereductase activity but not MV:nitrate reductase activity incrude extracts. Also for the purified enzyme the complete NADH:nitratereductase activity is inhibited by Mg²⁺, but not the partialMV:nitrate reductase or Cyt c reductase activities. (Received October 13, 1993; Accepted January 24, 1994)     

Effects of ATP, Mg2+, and redox agents on the Ca2+ dependence of RyR channels from rat brain cortex

Despite their relevance for neuronal Ca²⁺-induced Ca²⁺ release (CICR), activation by Ca²⁺ of ryanodine receptor (RyR) channels of brain endoplasmic reticulum at the [ATP], [Mg²⁺], and redox conditions present in neurons has not been reported. Here, we studied the effects of varying cis-(cytoplasmic) free ATP concentration ([ATP]), [Mg²⁺], and RyR redox state on the Ca²⁺ dependence of endoplasmic reticulum RyR channels from rat brain cortex. At pCa 4.9 and 0.5 mM adenylylimidodiphosphate (AMP-PNP), increasing free [Mg²⁺] up to 1 mM inhibited vesicular [³H]ryanodine binding; incubation with thimerosal or dithiothreitol decreased or enhanced Mg²⁺ inhibition, respectively. Single RyR channels incorporated into lipid bilayers displayed three different Ca²⁺ dependencies, defined by low, moderate, or high maximal fractional open time (P_o), that depend on RyR redox state, as we have previously reported. In all cases, cis-ATP addition (3 mM) decreased threshold [Ca²⁺] for activation, increased maximal P_o, and shifted channel inhibition to higher [Ca²⁺]. Conversely, at pCa 4.5 and 3 mM ATP, increasing cis-[Mg²⁺] up to 1 mM inhibited low activity channels more than moderate activity channels but barely modified high activity channels. Addition of 0.5 mM free [ATP] plus 0.8 mM free [Mg²⁺] induced a right shift in Ca²⁺ dependence for all channels so that [Ca²⁺] <30 µM activated only high activity channels. These results strongly suggest that channel redox state determines RyR activation by Ca²⁺ at physiological [ATP] and [Mg²⁺]. If RyR behave similarly in living neurons, cellular redox state should affect RyR-mediated CICR. Ca²⁺-induced Ca²⁺ release; Ca²⁺ release channels; endoplasmic reticulum; thimerosal; 2,4-dithiothreitol; ryanodine receptor     

Passive Interactions of Ca2+ and other Multivalent Cations with the Membranes of Isolated Corn Mitochondria

The presence of Ca²⁺ in a hypo-osmotic reaction medium reducessuccinate: cytochrome c reductase activity and the release ofouter membrane-specific antimycin A-insensitive NADH: cytochromec reductase. The action of Ca²⁺ is non-competitive and approximately30 mmol m^–3 Ca²⁺ affords half-maximal (I₅₀) protection.The effect of a range of inorganic and organic multivalent cationson succinate: cytochrome c reductase activity suggests thatthe action of Ca²⁺ is non-specific and probably involves Ca²⁺binding to outer membrane component(s) which may be proteins. Valinomycin- or gramicidin-induced passive swelling of isolatedcorn mitochondria in isotonic K.C1 is also non-competitivelyinhibited by up to 50% with Ca²⁺. Half-maximal inhibition (I₅₀)occurs at 0-35 mol m^–3 Ca²⁺ for valinomycin and 1-0 molm^–3 Ca²⁺ for gramicidin. Other divalent cations, Mg²⁺,Sr²⁺ and Ba²⁺, seem to inhibit similarly while the trivalentcations La³⁺ and Ho³⁺ show a maximum inhibition of up to 85%,with an I₅₀ of 0.1 mol m^–3 for valinomycin. It is suggestedthat non-specific cation binding may reduce membrane fluiditythereby slowing down the rate of ionophore penetration throughthe inner membrane. Key words: Calcium, Mitochondria, Membranes     

Rapid Modulation of Spinach Leaf Nitrate Reductase Activity by Photosynthesis : I. Modulation in Vivo by CO(2) Availability

It has been shown recently that in spinach leaves (Spinacia oleracea) net photosynthesis and nitrate reduction are closely linked: when net photosynthesis was low because of stomatal closure, rates of nitrate reduction decreased (WM Kaiser, J Förster [1989] Plant Physiol 91: 970-974). Here we present evidence that photosynthesis regulates nitrate reduction by modulating nitrate reductase activity (NRA, EC 1.6.6.1). When spinach leaves were exposed to low CO₂ in the light, extractable NRA declined rapidly with a half-time of 15 minutes. The inhibition was rapidly reversed when leaves were brought back to air. NRA was also inhibited when leaves were wilted in air; this inhibition was due to decreased CO₂ supply as a consequence of stomatal closure. The modulation of NRA was stable in vitro. It was not reversed by gel filtration. In contrast, the in vitro inhibition of nitrate reductase (NR) by classical inhibitors such as cyanide, hydroxylamin, or NADH disappeared after removal of free inhibitors by gel filtration. The negative modulation of NRA in —CO₂-treated leaves became manifest as a decrease in total enzyme activity only in the presence of free Mg²⁺ or Ca²⁺. Mg²⁺ concentrations required for observing half-maximal inhibition were about 1 millimolar. In the presence of EDTA, the enzyme activity was always high and rather independent of the activation status of the enzyme. NRA was also independent of the pH in the range from pH 7 to pH 8, at saturating substrate and Mg²⁺ concentrations. The apparent substrate affinities of NR were hardly affected by the in vivo modulation of NR. Only V_max changed.     

Properties of glutamate dehydrogenase purified from green tobacco callus tissue

Glutamate dehydrogenase [L-glutamate : NAD(P) oxidoreductase(deaminating) EC 1.4.1.3 [EC] .] has been purified from the mitochondrialfraction of green tobacco callus tissue. The enzyme was stableat –20?C for several months. The pH optimum for the aminationreaction was 7.8. But the optimum for the deamination reactionwas indistinct because it was in an extremely alkaline domain.Relative activities of the enzyme for amination were 50 withNADH and 10 with NADPH, and those for deamination were 5 withNAD and 1 with NADP at pH 7.9. The enzyme was inactivated by EDTA, but its activity partiallyrestored by the addition of divalent cations such as Ca²⁺, Mn²⁺,Zn²⁺, Cu²⁺ and Mg²⁺. Ca²⁺, Mn²⁺ and Zn²⁺ activated the reductiveamination 141, 122 and 39% respectively, but these divalentcations scarcely affected the oxidative deamination. Citrate and fumarate acted as inhibitors for reductive amination,and oxaloacetate for oxidative deamination of the enzyme reaction.These inhibitions were counteracted by the addition of Ca²⁺.ATP and ADP exerted an inhibitory effect on both directionsof the enzyme reaction. The inhibitory effect was hardly preventedby the addition of AMP. Ca²⁺ caused considerable recovery fromthe inhibition of ATP and ADP. Amino acids scarcely affectedthe enzyme activity. Michaelis constants were 0.28 mM for NAD, 0.065 mM for NADH,2.19 mM for a-ketoglutarate, 43.6 mM for ammonium chloride and4.24 mM for L-glutamate. ¹To whom requests for reprints should be addressed. (Received June 25, 1980; )     

Effects of Calcium on NAD(H)-Glutamate Dehydrogenase from Turnip (Brassica rapa L.) Mitochondria

The effect of Ca²⁺ and ammonia on mitochondrial NADH-glutamatedehydrogenase (GDH: EC 1.4.1.2 [EC] ) isolated from turnip root (Brassicarapa L.) activity was examined. Increasing the ammonia [(NH₄)₂SO₄]concentration led to significant substrate inhibition whichcould be reversed by micromolar levels of Ca²⁺. The sensitivityof the enzyme to ammonia inhibition and its reversal by Ca²⁺was affected by proteolysis. After treatment with various proteases,lower concentrations of Ca²⁺ were capable of fully activatingthe enzyme or overcoming the inhibitory effects of high ammonium,compared to non-treated enzyme. However, the protease-treatedenzyme was still sensitive to ethylene glycol-bis(ß-aminoethylether) N,N,N',N'-tetraacetate (EGTA). In contrast, NADH-GDHactivity was inhibited approx. 30% by organic mercurials (200µm), but the residual activity was not affected by thesubsequent additions of EGTA. NADH-GDH activity could also bestimulated by additions of high concentrations of NaCl (300mM) in the absence of added Ca²⁺. These results suggest thathydrophobic and -SH groups may be involved in the regulationof mitochondrial NADH-GDH activity by Ca²⁺. ² Present address: CSIRO Division of Horticulture, Urrbrae,S.A. 5064, Australia (Received April 18, 1990; Accepted July 23, 1990)     

Changes of membrane-associated Mg2+-activated ATPase of cucumber roots during calcium starvation

The properties of membrane-associated ATPase of cucumber (Cucumis sativus cv. Seiriki No. 2) roots cultured in a complete medium (complete enzyme) and in a medium lacking Ca²⁺ (Ca²⁺-deficient enzyme) were investigated. The basal activity of membrane-associated ATPase increased during Ca²⁺ starvation, while Mg²⁺-activation of the enzyme decreased and even resulted in inhibition by high Mg²⁺ concentration at the late stage of the Ca²⁺ starvation. The complete enzyme had low basal activity and showed a Mg²⁺-activated hyperbolic reaction curve in relation to ATP concentration. Ca²⁺-deficient enzyme with high basal activity showed a biphasic reaction curve and Mg²⁺-activation was seen only at high ATP concentrations. Activation of membrane-associated ATPase by various cations was decreased or lost during Ca²⁺ starvation. The basal ATPase activity of Ca²⁺-deficient enzyme increased for various substrates including pyrophosphate, p-nitrophenyl phosphate, glucose-6 phosphate, β-glycerophosphate, AMP, ADP and ATP. Mg²⁺-activation was found only for ADP and ATP in both the complete and Ca²⁺-deficient enzymes, but the activation for ATP was greatly reduced by Ca²⁺ starvation. The heat inactivation curves for basal and Mg²⁺-activated ATPase did not differ much between the complete and Ca²⁺-deficient enzyme. The delipidation of membrane-associated enzyme by acetone affected the protein content and the basal activity slightly, but inhibited the Mg²⁺-activated ATPase activity clearly with somewhat different behaviour between the complete and Ca²⁺-deficient enzyme.     

Repression of the K+ Uptake and Cation-Stimulated ATPase Activity Associated with the Plasma Membrane-Enriched Fraction of Cucumber Roots Due to Ca2+ Starvation

The uptake of K⁺ by cucumber plants decreased markedly duringCa²⁺ starvation. A plasma membrane-enriched fraction, judgedfrom the distribution of marker enzymes, was prepared from controland Ca²⁺-starved roots. The Mg²⁺- and K⁺-Mg²⁺-ATPase activitiesassociated with the plasma membrane-enriched fraction of controlroots were maxima at pH 6.5. Various monovalent cations andpotassium salts of monovalent anions stimulated Mg²⁺-ATPaseactivity. Vanadate, DES and DCCD inhibited K⁺- Mg²⁺-ATPase activity.Of the divalent cations and phosphate esters tested, Mg²⁺ andATP were most effective for the stimulation of ATPase by K⁺,whereas Ca²⁺ was ineffective in replacing Mg²⁺. Mg²⁺- and K⁺-Mg²⁺-ATPase activities associated with the plasmamembrane enriched fraction of Ca²⁺-starved roots were much lowerthan those of control roots. K_m values of K⁺-Mg²⁺-ATPase forATP were comparable for control and Ca²⁺-starved roots. The K⁺-stimulated activity of Mg²⁺-ATPase in Ca²⁺-starved rootswas approximately one fourth that of the control, whereas therate of stimulation was only slightly lower in Ca²⁺-starvedroots. (Received May 9, 1984; Accepted September 17, 1984)     

Changes of Some Membrane-Associated Enzyme Activities Degradation of Membrane Phospholipids in Cucumber Roots Due to Ca2+ Starvation

Deprivation of Ca²⁺ from a complete culture medium affectedthe enzyme activities associated with five membrane fractionsof cucmber roots obtained by discontinuous sucrose density gradientcentrifugation. The total activity of K⁺-ATPase, Cyt. c oxidaseand NADPH-Cyt. c reductase of Ca²⁺-deficient roots, starvedfor only 4 days, had decreased to 14, 38 and 60% of the activityof the control roots. In general, loss of enzyme activitieswas accompanied by a shift of activity distribution from theheavier density fractions to lighter ones. The amounts of Ca²⁺ associated with membranes from Ca²⁺-starvedroots decreased to 50–60% of those of the control roots.Both phospholipid and neutral lipid contents in the membranesdecreased markedly while the protein content was not changedby Ca²⁺ deficiency. Phospholipid analysis indicated a drasticdrop in the percent composition of phosphatidylinositol butan increase of phosphatidic acid. Also, phospholipase D activityincreased remarkably during Ca²⁺ starvation, paralleling theappearance of Ca²⁺-deficiency symptoms. Thus, the major effects of Ca²⁺ deficiency appear to be to stimulatephospholipase D activity and a reduction in membrane bound Ca²⁺.These effect may be involved in disorganization of the membranestructure and the changes of enzyme activities associated withthe altered membranes. ¹Rubber Research Institute of Sri Lanka, Dartonfield, Agalwatta,Sri Lanka. (Received July 15, 1985; Accepted November 21, 1985)     

Taste responses to divalent cations in the frog glossopharyngeal nerve: competitive inhibition of the Mg2+ response by Ca2+

In the frog glossopharyngeal nerve, single water fibers respondto low CaCl₂ (1–2 mM) and relatively high MgCl₂ (100 mM).In the present study, it was found that stimulation by a mixtureof low CaCl₂ and relatively high MgCl₂ led to a small response.This suggests that the Ca⁺ response is inhibited by the presenceof Mg²⁺ and the Mg²⁺ response is inhibited by the presence ofCa²⁺. Hence, it is suggested that there are different receptorsites for divalent cations in single water fibers of the frogglossopharyngeal nerve, a calcium receptor site (X_Ca) responsiblefor the Ca²⁺ response and a magnesium receptor site (X_Mg) responsiblefor the Mg²⁺ response. It has been reported that Mg²⁺ inhibitsthe Ca²⁺ response by competing with Ca²⁺ for X_Ca (Kitada andShimada, 1980). In the present study, the inhibition of theMg²⁺ response by Ca²⁺ was examined quantitatively under theassumption that the magnitude of the neural response is proportionalto the amount of MgX_Mg complex minus a constant (the thresholdconcentration of the MgX_Mg complex). The results obtained indicatethat Ca²⁺ competes with Mg²⁺ for X^Mg. The apparent dissociationconstants for MgX_Mg complex and CaX_Mg complex, which were obtainedfrom the present study, were 8.0 x 10^–2 M and 7.2 x 10^–4M, respectively. Thus, competition between Ca⁺ and Mg²⁺ forthe distinct receptor sites involved in taste reception wasdemonstrated by the results described in this paper. Since thedivalent cations do not always bring about activation of tastereceptors, the responses to salts in the frog glossopharyngealnerve cannot be explained in terms of changes in the surfacepotential outside the taste cells. The present results suggestthat there exist multiple specific receptor sites for cationsinvolved in salt taste responses, and only the binding of eachseparate cation to its appropriate receptor sites leads to activationof the receptor and the initiation of impulses in sensory nerveendings.     

Matrix free Mg2+ and the regulation of mitochondrial volume

Mitochondria must maintain volume homeostasis inorder to carry out oxidative phosphorylation. It has been postulatedthat the concentration of freeMg²⁺([Mg²⁺]) serves as thesensor of matrix volume and regulates aK⁺-extrudingK⁺/H⁺antiport (K. D. Garlid. J. Biol. Chem.255: 11273-11279, 1980). To test this hypothesis, the fluorescentprobe furaptra was used to monitor[Mg²⁺] and freeCa²⁺ concentration ([Ca²⁺]) in the matrix ofisolated beef heart mitochondria, andK⁺/H⁺antiport activity was measured by passive swelling in potassium acetate. Concentrations that result in 50% inhibition of maximum activity of 92 µM matrix [Mg²⁺] and 2.2 µM[Ca²⁺] were determined for theK⁺/H⁺ antiport. Untreated mitochondria average670 µM matrix [Mg²⁺], a value that would permit <1%of maximumK⁺/H⁺antiport activity. Hypotonic swelling results in large decreases inmatrix [Mg²⁺], butswelling due to accumulation of acetate salts does not alter[Mg²⁺]. Swelling inphosphate salts decreases matrix[Mg²⁺], but not tolevels that permit appreciable antiport activity. We conclude that1) it is unlikely that matrix[Mg²⁺] serves as themitochondrial volume sensor, 2) ifK⁺/H⁺antiport functions as a volume control transporter, it is probably regulated by factors other than[Mg²⁺], and3) alternative mechanisms formitochondrial volume control should be considered.

     

Enhancing effects of transition metals on the salt taste responses of single fibers of the frog glossopharyngeal nerve: specificity of and similarities among Ca2+, Mg2+ and Na+ taste responses

Fibers of the frog glossopharyngeal nerve (water fibers) thatare sensitive to water also respond to CaCl₂, MgCl₂ and NaCl.In the present study, interaction among cations (Ca²⁺, Mg²⁺and Na⁺) on taste cell membrane in frogs was studied using transitionmetals (NiCl₂, CoCl₂ and MnCl₂), which themselves are barelyeffective in producing neural response at concentrations below5 mM. Unitary discharges from single water fibers were recordedfrom fungiform papillae with suction electrode. Transition metalions (0.05–5.0 mM) had exclusively enhancing effects onthe responses to 50 mM Ca²⁺, 100 mM Mg²⁺ and 500 mM Na⁺. Theeffects of transition metal ions were always reversible. Therank order of effectiveness of transition metals at 1 mM inthe enhancement of the responses to 50 mM CaCl₂, 100 mM MgCl₂and 500 mM NaCl was NiCl₂ > CoCl² > MnCl₂. The concentrationof transition metal ions effective to enhance salt responsewas almost the same among Ca²⁺, Mg²⁺ and Na⁺ responses. Theresults suggest that a common mechanism is involved in the enhancementof Ca²⁺, Mg²⁺ and Na⁺ taste responses. The enhanced Mg²⁺ responseand the enhanced Na⁺ response were greatly inhibited by theaddition of Ca²⁺ ions, and the enhanced Ca²⁺ response was inhibitedby the addition of Mg²⁺ or Na⁺ ions, suggesting that competitiveantagonism occurs between Ca²⁺ and Mg²⁺ ions and between Ca²⁺and Na⁺ ions in the presence of Ni²⁺ ions. Ni²⁺ ions had a dualeffect on the Ca²⁺ response induced by low concentration (0.1mM) of CaCl₂: enhancement at lower concentrations (0.02–0.1mM) of NiCl₂ and inhibition at higher concentrations (0.5–5mM)of NiCl₂. The present results suggest that transition metalions do not affect the receptor-antagonist complex, but affectonly the receptor-agonist complex.     

Mg2+ activates the ryanodine receptor type 2 (RyR2) at intermediate Ca2+ concentrations

To clarify whether activity of the ryanodine receptor type 2 (RyR2) is reduced in the sarcoplasmic reticulum (SR) of cardiac muscle, as is the case with the ryanodine receptor type 1 (RyR1), Ca²⁺-dependent [³H]ryanodine binding, a biochemical measure of Ca²⁺-induced Ca²⁺ release (CICR), was determined using SR vesicle fractions isolated from rabbit and rat cardiac muscles. In the absence of an adenine nucleotide or caffeine, the rat SR showed a complicated Ca²⁺ dependence, instead of the well-documented biphasic dependence of the rabbit SR. In the rat SR, [³H]ryanodine binding initially increased as [Ca²⁺] increased, with a plateau in the range of 10–100 µM Ca²⁺, and thereafter further increased to an apparent peak around 1 mM Ca²⁺, followed by a decrease. In the presence of these modulators, this complicated dependence prevailed, irrespective of the source. Addition of 0.3–1 mM Mg²⁺ unexpectedly increased the binding two- to threefold and enhanced the affinity for [³H]ryanodine at 10–100 µM Ca²⁺, resulting in the well-known biphasic dependence. In other words, the partial suppression of RyR2 is relieved by Mg²⁺. Ca²⁺ could be a substitute for Mg²⁺. Mg²⁺ also amplifies the responses of RyR2 to inhibitory and stimulatory modulators. This stimulating effect of Mg²⁺ on RyR2 is entirely new, and is referred to as the third effect, in addition to the well-known dual inhibitory effects. This effect is critical to describe the role of RyR2 in excitation-contraction coupling of cardiac muscle, in view of the intracellular Mg²⁺ concentration. [³H]ryanodine binding; CICR; stimulation by physiological Mg²⁺, excitation-contraction coupling in the heart     

Inhibition of sarcoplasmic reticulum Ca2+-ATPase by miconazole

The inhibition of sarcoplasmic reticulumCa²⁺-ATPase activity by miconazole was dependent on theconcentration of ATP and membrane protein. Half-maximal inhibition wasobserved at 12 µM miconazole when the ATP concentration was 50 µMand the membrane protein was 0.05 mg/ml. When ATP was 1 mM, a lowmicromolar concentration of miconazole activated the enzyme, whereashigher concentrations inhibited it. A qualitatively similar responsewas observed when Ca²⁺ transport was measured. Likewise,the half-maximal inhibition value was higher when the membraneconcentration was raised. Phosphorylation studies carried out aftersample preequilibration in different experimental settings shed lighton key partial reactions such as Ca²⁺ binding and ATPphosphorylation. The miconazole effect on Ca²⁺-ATPaseactivity can be attributed to stabilization of theCa²⁺-free enzyme conformation giving rise to a decrease inthe rate of the Ca²⁺ binding transition. The phosphoryltransfer reaction was not affected by miconazole.

     

Regulation of dynamic behavior of cardiac ryanodine receptor by Mg2+ under simulated physiological conditions

Mg²⁺, an important constituent of the intracellular milieu in cardiac myocytes, is known to inhibit ryanodine receptor (RyR) Ca²⁺ release channels by competing with Ca²⁺ at the cytosolic activation sites of the channel. However, the significance of this competition for local, dynamic Ca²⁺-signaling processes thought to govern cardiac excitation-contraction (EC) coupling remains largely unknown. In the present study, Ca²⁺ stimuli of different waveforms (i.e., sustained and brief) were generated by photolysis of the caged Ca²⁺ compound nitrophenyl (NP)-EGTA. The evoked RyR activity was measured in planar lipid bilayers in the presence of 0.6-1.3 mM free Mg²⁺ at the background of 3 mM total ATP in the presence or absence of 1 mM luminal Ca²⁺. Mg²⁺ dramatically slowed the rate of activation of RyRs in response to sustained (=" BORDER="0">10-ms) elevations in Ca²⁺ concentration. Paradoxically, Mg²⁺ had no measurable impact on the kinetics of the RyR response induced by physiologically relevant, brief (<1-ms) Ca²⁺ stimuli. Instead, the changes in activation rate observed with sustained stimuli were translated into a drastic reduction in the probability of responses. Luminal Ca²⁺ did not affect the peak open probability or the probability of responses to brief Ca²⁺ signals; however, it slowed the transition to steady state and increased the steady-state open probability of the channel. Our results indicate that Mg²⁺ is a critical physiological determinant of the dynamic behavior of the RyR channel, which is expected to profoundly influence the fidelity of coupling between L-type Ca²⁺ channels and RyRs in heart cells. excitation-contraction coupling; cardiac myocytes; magnesium; calcium signaling     

The Role of Calcium and Calmodulin in Carrot Somatic Embryogenesis

The role of Ca²⁺ and calmodulin in carrot somatic embryo formationwas examined. Embryogenic cell clumps were induced to form embryosin medium containing 0–3 mM Ca²⁺. Embryo formation wasnot affected until the concentration of Ca²⁺ was lower than200 µM and after this threshold was reached the percentof embryo formation decreased with lower Ca²⁺ concentrations.Treatment of developing embryos with either verapamil or nifedipine,Ca²⁺ -channel blockers, or the Ca²⁺ ionophore A23187 [GenBank] , inhibitedembryo formation. These results suggest that exogenous Ca²⁺or the maintenance of Ca²⁺ gradients are required for properembryo development. Analysis of membrane-associated Ca²⁺ andtotal membrane distribution using the fluorescent dyes chlorotetracyclineand N-phenyl-1-napthylamine, respectively, indicated changesin the distribution of membranes during embryogenesis withoutany significant alterations in the concentration of Ca²⁺ associatedwith the membranes. In heart- and torpedo-stage embryos, calmodulin-Ca²⁺complexes were localized in regions containing the developingmeristems of both the cotyledon tips and rhizoid regions whiletotal calmodulin protein appeared to be more uniformly distributed.Calmodulin mRNA levels increased slightly when cell clumps wereinduced to form embryos. (Received July 7, 1993; Accepted September 27, 1993)     

Regulation of NADH-Glutamate Dehydrogenase Activity by Phytochrome, Calcium and Calmodulin in Zea mays

Regulation by the active form of phytochrome (P_FR) and the effectof Ca²⁺ and calmodulin was examined with glutamate dehydrogenase(GDH) of Zea mays. A brief irradiation (5 min) to 5 day oldplants with red light resulted in 5-6 fold increase in GDH activity.This effect was nullified when red light was followed immediatelyby far-red light. The photoreversibility showed that P_FR regulatesGDH activity in vivo. To the enzyme extract obtained after EGTAtreatment, when Ca²⁺ was added in vitro, GDH was activated by6 fold. The maximum response by Ca²⁺ was obtained at 80 µM.Both P_AR and Ca²⁺ effects were found to be age dependent. Theenzyme activity was inhibited by compound 48/80 in partiallypurified extracts and the effect was reversed by calmodulin.The purified GDH, however, was not activated directly by calmodulin;it required the presence of another protein factor which wasseparated by gel permeation column by HPLC. Neither anticalmodulindrugs nor addition of calmodulin had any effect on nitrate re-ductaseactivity. (Received July 13, 1988; Accepted October 31, 1988)     

重组PICK1蛋白的多聚形式及其与Ca2+和Mg2+的结合

蛋白激酶Cα相互作用蛋白1(PICK1) 是从线虫到人的所有生物中非常保守的一类存在于细胞质中的膜结合蛋白,在蛋白质转运,以及细胞内信号转导过程中发挥重要作用.通过基因重组技术获得PICK1及其截短的 N-PDZ(1～110 残基)和 BAR-C(128～416残基)重组蛋白,结合变性与非变性聚丙烯酰胺凝胶电泳,以及分子排阻层析,表明溶液中的PICK1主要以二聚体形式存在.利用荧光光谱分析PICK1与金属离子Ca²⁺和Mg²⁺的结合情况.结果表明,在0.02 mol/L Hepes, pH 7.2,随着2种金属离子的不断滴加,PICK1在338 nm 处的最大荧光强度逐渐降低,PICK1与Ca²⁺结合常数为Ka1=（2.34±0.20）×10.6 L/mol^-1,Ka2=(7.75±0.62)×10.5 L/mol^-1,而Mg²⁺结合常数为Ka=（5.00±0.40）×10.6 L/mol^-1.另外,对PICK1的N端区域N-PDZ和C端区域BAR-C的重组片段与金属离子Ca²⁺和Mg²⁺结合情况进一步分析表明,Ca²⁺既能与PICK1的N 端N-PDZ结合,又可与C端BARC结合,而Mg²⁺只结合在PICK1的N-PDZ区域.比较Ca²⁺或Mg²⁺对PICK1结合脂质的影响,显示Ca²⁺能明显增强蛋白和脂质的结合.     

The phosphatase activity of the plasma membrane Ca pump. Activation by acidic lipids in the absence of Ca increases the apparent affinity for Mg

The purified PMCA supplemented with phosphatidylcholine was able to hydrolyze pNPP in a reaction media containing only Mg²⁺ and K⁺. Micromolar concentrations of Ca²⁺ inhibited about 75% of the pNPPase activity while the inhibition of the remainder 25% required higher Ca²⁺ concentrations. Acidic lipids increased 5-10 fold the pNPPase activity either in the presence or in the absence of Ca²⁺. The activation by acidic lipids took place without a significant change in the apparent affinities for pNPP or K⁺ but the apparent affinity of the enzyme for Mg²⁺ increased about 10 fold. Thus, the stimulation of the pNPPase activity of the PMCA by acidic lipids was maximal at low concentrations of Mg²⁺. Although with differing apparent affinities vanadate, phosphate, ATP and ADP were all inhibitors of the pNPPase activity and their effects were not significantly affected by acidic lipids. These results indicate that (a) the phosphatase function of the PMCA is optimal when the enzyme is in its activated Ca²⁺ free conformation (E2) and (b) the PMCA can be activated by acidic lipids in the absence of Ca²⁺ and the activation improves the interaction of the enzyme with Mg²⁺.