Effects of [CO2] and nitrogen fertilization on soils planted with ponderosa pine

The effects of six years treatment with elevated [CO₂] (350, 525, and 700 μl l^-1) and nitrogen (N) (0, 10, and 20 g N m^-2 yr^-1) on soils, soil solution, and CO₂ efflux in an open-top chamber study with ponderosa pine (Pinus ponderosa Laws.) are described. The clearest [CO₂] effect was in year 6, when a pattern of lower soil N concentration and higher C/N ratio with elevated [CO₂] emerged. Statistically significant effects of elevated [CO₂] on soil total C, extractable P, exchangeable Mg²⁺, exchangeable Ca²⁺, base saturation, and soil solution HCO₃ ^- and NO₃ ^- were also found in various treatment combinations and at various times; however, these effects were inconsistent among treatments and years, and in many cases (P, Mg²⁺, Ca²⁺, base saturation) reflected pre-treatment differences. The use of homogenized buried soil bags did not improve the power to detect changes in soil C and N or help resolve the inconsistencies in soil C patterns. Nitrogen fertilization had the expected negative effects on exchangeable Ca²⁺, K⁺, and Mg²⁺ in year 6, presumably because of increased NO₃ ^- leaching, but had no consistent effect on soil C, N, or extractable P. This revised version was published online in June 2006 with corrections to the Cover Date.     

Metal requirement of the isolated red cell Ca-pump ATPase after elimination of calmodulin dependence by trypsin attack

Mild proteolysis by trypsin activates the purified (Ca²⁺ + Mg²⁺) - ATPase protein from human red cells in a way which is similar to the effect obtained by addition of calmodulin. The trypsin concentration required to reach half maximal effect in 3 minutes at 37°C is 2.5 – 3.5 μg/ml. SDS-poly-acrylamide gel electrophoresis reveals a degradation of the main protein (150'000 Dalton) into a large fragment (95'000 – 100'000 Dalton) and a small fragment (35'000 – 40'000 Dalton). Increasing ATPase activity correlates with the degree of proteolysis.The _Ca of the digested (Ca²⁺ + Mg²⁺)-ATPase is 0.85 ± 0.1 μM Ca²⁺ as compared to 8.0 ± 0.75 μM Ca²⁺ before digestion and is statistically significantly different from _Ca = 1.66 ± 0.22 μM Ca²⁺ observed in activation by a saturating calmodulin concentration. Addition of calmodulin to the trypsinized enzyme has neither an effect on the Ca²⁺-affinity nor achieves any large increase of the maximal rate.High Ca²⁺ concentrations (above 0.05 – 0.1 mM) after trypsin treatment still inhibit the (Ca²⁺ + Mg²⁺)-ATPase activity. Mg²⁺ activates in the same concentration range ( _Mg = 25 μM) as in the undigested preparation ( _Mg = 27 μM) and retains its competitive behaviour towards Ca²⁺ after trypsin treatment.It is concluded that (1) trypsin treatment unmasks high affinity sites for Ca²⁺ ( _Ca 1 μM) and that, therefore, such sites are not added to the system by calmodulin, and (2) that inhibition by high Ca²⁺-concentrations is not due to Ca - Mg competition at sites located on the calmodulin molecule.     

31P-nmr studies of the effect of various metals on substrate binding to yeast enolase

The ³¹P nuclear magnetic resonance (nmr) spectra of product (phosphoenolpyruvate) and substrate (2-phosphoglycerate) binding to 1:1 molar ratios ot yeast enolase were obtained as functions of the level of various metal ions. Levels sufficient to produce substrate and product binding but not catalysis ( 1 equivalent/subunit), produced shifts (with respect to 86% H₃PO₄) to lower shielding of ca. 30 ppm in the case of Co²⁺, 5–8 ppm in the case of Mg²⁺, and 2–3 ppm in the case ofCa²⁺, but virtual obliteration in the case of Mn²⁺. The effects of Mn²⁺ and Co²⁺ are consistent with a close approach of the metal ions to the phosphate groups. The effects of the physiological cofactor and optimum activator Mg²⁺ and the nonactivator Ca²⁺ are interpreted as indicating different degrees of distortion of the R-O-P bond angle in the two metal-enzyme-substrate complexes. Levels of Mg²⁺ sufficient for optimal or near optimal catalysis (2 equivalents/subunit) produce shifts to higher shielding in the ³¹P resonances of both substrate and product. These shifts are intermediate between those in the presence of 1 equivalent/subunit and those of the free ligands. Addition of a second equivalent of Ca²⁺ produces a slight shift to lower shielding of the phosphoenolpyruvate resonance and a small shift to higher shielding in the resonance for 2-phosphoglycerate. Similar levels of Co²⁺ eliminate the resonances for both substrate and product. These effects are interpreted as arising from direct coordination between substrate-dependent metal ion binding and the phosphate esters. Higher levels of Ca²⁺, Mg²⁺, or Co²⁺ or addition of KF, all of which inhibit enzyme activity, have only minor effects on the spectra. The spectrum of inorganic phosphate, a competitive inhibitor, was also examined. KF strongly enhances binding, as does excess Mg²⁺, and the binding is accompanied by a chemical shift to lower shielding of ca 2 ppm. This is not due to formation of a magnesium-fluorophosphate complex, consistent with the findings of other workers.     

On the Divalent Metal Ion Dependence of DNA Cleavage by Restriction Endonucleases of the EcoRI Family

Restriction endonucleases of the PD…D/EXK family need Mg²⁺ for DNA cleavage. Whereas Mg²⁺ (or Mn²⁺) promotes catalysis, Ca²⁺ (without Mg²⁺) only supports DNA binding. The role of Mg²⁺ in DNA cleavage by restriction endonucleases has elicited many hypotheses, differing mainly in the number of Mg²⁺ involved in catalysis. To address this problem, we measured the Mg²⁺ and Mn²⁺ concentration dependence of DNA cleavage by BamHI, BglII, Cfr10I, EcoRI, EcoRII (catalytic domain), MboI, NgoMIV, PspGI, and SsoII, which were reported in co-crystal structure analyses to bind one (BglII and EcoRI) or two (BamHI and NgoMIV) Me²⁺ per active site. DNA cleavage experiments were carried out at various Mg²⁺ and Mn²⁺ concentrations at constant ionic strength. All enzymes show a qualitatively similar Mg²⁺ and Mn²⁺ concentration dependence. In general, the Mg²⁺ concentration optimum (between ∼ 1 and 10 mM) is higher than the Mn²⁺ concentration optimum (between ∼ 0.1 and 1 mM). At still higher Mg²⁺ or Mn²⁺ concentrations, the activities of all enzymes tested are reduced but can be reactivated by Ca²⁺. Based on these results, we propose that one Mg²⁺ or Mn²⁺ is critical for restriction enzyme activation, and binding of a second Me²⁺ plays a role in modulating the activity. Steady-state kinetics carried out with EcoRI and BamHI suggest that binding of a second Mg²⁺ or Mn²⁺ mainly leads to an increase in K_m, such that the inhibitory effect of excess Mg²⁺ or Mn²⁺ can be overcome by increasing the substrate concentration. Our conclusions are supported by molecular dynamics simulations and are consistent with the structural observations of both one and two Me²⁺ binding to these enzymes.     

Mitochondrial ATP-Mg/phosphate carriers transport divalent inorganic cations in complex with ATP

The ATP-Mg/phosphate carriers (APCs) modulate the intramitochondrial adenine nucleotide pool size. In this study the concentration-dependent effects of Mg²⁺ and other divalent cations (Me²⁺) on the transport of [³H]ATP in liposomes reconstituted with purified human and Arabidopsis APCs (hAPCs and AtAPCs, respectively, including some lacking their N-terminal domains) have been investigated. The transport of Me²⁺ mediated by these proteins was also measured. In the presence of a low external concentration of [³H]ATP (12 μM) and increasing concentrations of Me²⁺, Mg²⁺ stimulated the activity (measured as initial transport rate of [³H]ATP) of hAPCs and decreased that of AtAPCs; Fe²⁺ and Zn²⁺ stimulated markedly hAPCs and moderately AtAPCs; Ca²⁺ and Mn²⁺ markedly AtAPCs and moderately hAPCs; and Cu²⁺ decreased the activity of both hAPCs and AtAPCs. All the Me²⁺-dependent effects correlated well with the amount of ATP-Me complex present. The transport of [¹⁴C]AMP, which has a much lower ability of complexation than ATP, was not affected by the presence of the Me²⁺ tested, except Cu²⁺. Furthermore, the transport of [³H]ATP catalyzed by the ATP/ADP carrier, which is known to transport only free ATP and ADP, was inhibited by all the Me²⁺ tested in an inverse relationship with the formation of the ATP-Me complex. Finally, direct measurements of Mg²⁺, Mn²⁺, Fe²⁺, Zn²⁺ and Cu²⁺ showed that they are cotransported with ATP by both hAPCs and AtAPCs. It is likely that in vivo APCs transport free ATP and ATP-Mg complex to different degrees, and probably trace amounts of other Me²⁺ in complex with ATP.     

Nitrate absorption and assimilation in ryegrass as influenced by calcium and magnesium

The absorption and assimilation patterns of ¹⁵NO₃⁻ supplied as the Ca²⁺ and Mg²⁺ salts to intact ryegrass (Lolium perenne) seedlings were compared. No statistically significant effect of ambient cation on the amounts of ¹⁵NO₃⁻ absorbed was observed in the initial six hours, but during the subsequent six hours, absorption from Ca(¹⁵NO₃)₂ exceeded that from Mg (¹⁵NO₃)₂.     

Interactions of polyamines with the DNA octamers d(m5CG)4 and d(GGAATTCC): A 1H‐NMR investigation

The interaction of two DNA octamers, d(m⁵CG)₄ and d(GGAATTCC), with the polyamines spermine⁴⁺ and spermidine³⁺, has been studied by means of ¹H‐nmr nuclear Overhauser effect (NOE) difference measurements. The experiments were performed at 10°C and for a polyamine charge to DNA charge (i.e., phosphate) ratio of 0.4, where the solution of d(m⁵CG)₄ contains about 50% Z‐form of the DNA. The results show that the polyamine intramolecular NOEs for the protons on the propyl chains are similarly negative with the two oligonucleotides, while those on the butyl chain show slightly more negative NOE with d(m⁵CG)₄ than with d(GGAATTCC). The fully N‐methylated analogues of spermine (Me₁₀Spn⁴⁺) and spermidine (Me₈Spd³⁺) as well as the diamines 1,3‐diaminopropane (DAP²⁺) and 1,4‐diaminobutane (putrescine²⁺) have been studied for the ability to transform d(m⁵CG)₄ from the B‐ to the Z‐form. ¹H‐nmr spectra showed the order spermine⁴⁺ > spermidine³⁺ > Me₁₀Spn⁴⁺ > Me₈Spd³⁺ > 1,3‐diaminopropane²⁺ > putrescine²⁺, with spermine showing the largest relative amount of Z‐DNA. ¹H‐nmr pulsed‐gradient self‐diffusion measurements of the triamines showed a large difference in the interaction of Spd and Me₈Spd with the two different duplexes. With the same duplex (either of the two), however, no difference between Spd and Me₈Spd can be seen. Within a two‐state model this is interpreted as a larger fraction of bound polyamines with d(m⁵CG)₄ than with d(GGAATTCC). © 1999 John Wiley & Sons, Inc. Biopoly 49: 41–53, 1999     

Long-term and seasonal changes in the chemical composition of precipitation and surface waters of lakes and tarns in the English Lake District

SUMMARY. The composition of bulk (wet and dry) precipitation in 1975 and 1976 was similar to that found 22 years previously. In 1975, mean values for nine precipitation samplers at one site (Wraymires) ranged from pH 4.3 to 4.5; similar values were obtained in 1976. Samplers covered with fine-mesh plastic gauze caught substantially more Ca²⁺ and K⁺ than open samplers, but pH and SO^2-₄+ NO₃^- concentrations were similar in open and covered samplers. In precipitation, c. 50% of H⁺ was balanced by NO₃^- and c. 50% by SOi; 80% of the SO^2-₄ was balanced by Ca²⁺ and Mg²⁺. Conccntrations of major cations (H⁺, Na⁺, K⁺,Ca²⁺, Mg²⁺) and anions (CI^-, NO₃^- SO^2-₄ and alkalinity [Alk—largely HCO₃^-]) in upland water- bodies were similar to those found in precipitation, but pH levels were generally higher and above 6.0 m some tarns. At lower altitudes, on base-rich roeks and soils, Ca²⁺ and Alk become dominant. Results of a survey of lakes and tarns in 1974–78 are compared with a survey in 1953–56 and published data (chiefly for pH and Alk) for 1947–50, 1932 and 1928. Comparisons are also made with other measurements of Alk in three productive lakes (Blelham Tarn, Esthwaite Water and Windermere) for 1936–39 and 1945–80. Winter levels of NO₃-N, PO₄-P and Si are given for these lakes; although the first two have increased during the late 1960s and the 1970s there has been no significant change in the last. NO₃^- and probably some SO^2-₄. In productive lakes a substantial (c. 50%) rise in mean Alk occurred during the late 1960s and the 1970s, possibly related to increased winter levels of NO₃-N and PO₄-P derived from sewage and fertilizers. In this period the maximum pH levels reached in summer were notably high, sometimes exceeding pH 10. The rise in Alk, conductivity and pH of surface waters is influenced by climatic factors (a decade of drier years), sewage input and biological productivity within the lakes. Considerable seasonal fluctuations in the concentrations of major ions, a characteristic feature of surface waters in the English Lake District, are illustrated and some implications for cation-anion balance briefly discussed. Mid-winter concentrations are usually high forNa⁺, K⁺, Cl^-. NO₃^-and low for Ca²⁺, Mg²⁺, Alk. SO^2-₄. Alkalinity. pH and conductivity of Lake District tarns and lakes show no signs of acidification during the period 1928–80. On the contrary, productive lakes have become more alkaline and some unproductive low-alkalinity (< 100 μ-equiv. 1^-1) lakes also show signs of alkalization, with increased mean concentrations of Na⁺. Ca²⁺ and Mg²⁺, balanced by Alk.     

模拟酸沉降对鼎湖山季风常绿阔叶林地表径流水化学特征的影响

通过模拟酸沉降实验,研究了旱季期间(10-3月份)鼎湖山季风常绿阔叶林在4种不同pH模拟酸雨处理(对照、pH 4.0、pH 3.5、pH 3.0)下地表径流水化学输出特征.结果显示:(1)地表径流pH随酸处理强度增强呈“U”型变化模式,酸沉降对地表径流pH的影响不显著(P＞0.05),表明模拟酸沉降尚未引起地表水的酸化.(2)地表径流中NO3-、SO24-浓度随酸处理强度增强略有增加;HCO3-浓度的变化模式与地表径流pH类似.酸根离子浓度与地表径流pH相关性分析表明,SO24-、HCO3-有助于提高地表水抗酸化能力而NO3-则有助于促进地表水酸化.(3)地表径流中盐基离子对酸沉降的响应不尽相同.pH 3.0处理显著提高地表径流中Ca2+、Na+浓度;Mg2+浓度具有随酸处理梯度增强而增加的趋势;K+受模拟酸度的影响小.表明强酸(pH3.0)处理将导致土壤Na+、Ca2+、Mg2+盐基离子流失.(4)酸沉降具有诱发土壤可溶性有机碳(DOC)流失的倾向,增加地表水受有机污染的风险.     

Insulin action in isolated fat cells: II. Effects of divalent cations on stimulation by insulin of protein synthesis,on inhibition of lipolysis by insulin,and on the binding of 125I-labelled insulin to isolated fat cells

The effects of omission of Ca²⁺ and Mg²⁺ from the incubation medium on three aspects of insulin action in isolated fat cells have been investigated. In the (Ca²⁺ + Mg²⁺)-free incubation medium incorporation of L-[¹⁴C]leucine into fat cell protein was reduced in the absence of insulin. Insulin stimulated L-[¹⁴C]-leucine incorporation only in the presence of added CaCl₂ or MgCl₂. Incubation of the cells in the (Ca²⁺ + Mg²⁺)-free medium reduced but did not abolish the ability of adrenaline to stimulate lipolysis or the ability of insulin to inhibit the adrenaline-stimulated lipolysis. Specific binding of ¹²⁵I-labelled insulin to the fat cells was reduced in the absence of Ca²⁺ and Mg²⁺ but was not abolished, even in the presence of EDTA. Ca²⁺ was routinely the most effective divalent cation in supporting these aspects of insulin action, but similar responses were obtained with Mg²⁺, Sr²⁺ and Ba²⁺.Since insulin still binds to the cells under conditions in which some of the cellular effects of the hormone are abolished, it is suggested that divalent cations may have a role, either direct or indirect, in the processes linking the insulin-insulin receptor complex to certain effector systems in the cells. It is tentatively suggested that this action occurs at the level of the fat cell plasma membrane.     

Ionophorous properties of the 20,000-dalton fragment of (Ca2++Mg2+)-ATPase in phosphatidylcholine: Cholesterol membranes

Summary The purified 20,000-dalton fragment of sarcoplasmic reticulum (Ca²⁺+Mg²⁺)-ATPase has been shown by us (A.E. Shamoo, T.E. Ryan, P.S. Stewart, D.H. MacLennan, 1976. J. Biol. Chem.251:4147) to have Ca²⁺-selective ionophoric activity. The Ca²⁺-ionophoric fragment has been purified by either SDS-column chromatography or SDS-preparative gel electrophoresis. The Ca²⁺-ionophoric fragment has been subjected to prolonged dialysis to insure the removal of bound SDS from the fragment. The selectivity sequence of this fragment in black lipid membranes (BLM) formed from either oxidized cholesterol or phosphatidylcholine/cholesterol is the same,P _Ba>P _Ca>P _Sr>P _Mg>P _Mn. This selectivity sequence is the same as that for the intact (Ca²⁺ +Mg²⁺)-ATPase. Treatment of the fragment with cholate to absolutely insure the removal of bound SDS resulted in the fragment having a selectivity sequence as above except thatP _Mn>P _Mg. This and other data indicate that the 20,000-dalton fragment is the site containing the Ca²⁺-ionophoric activity of the (Ca²⁺+Mg²⁺)-ATPase.     

Calmodulin stimulation of calcium uptake and (Ca2+-Mg2+)-ATPase activities in microsomes from canine tracheal smooth muscle

The role of calmodulin in the regulation of microsomal ⁴⁵Ca²⁺ transport in canine tracheal smooth muscle was studied. Calmodulin stimulated ATP-dependent ⁴⁵Ca²⁺ uptake and (Ca²⁺Mg²⁺)-ATPase activities in microsomes treated with 0.5 mM EDTA and 0.5 mM EGTA. Oxalate also stimulated ATP-dependent ⁴⁵Ca²⁺ uptake and (Ca²⁺Mg²⁺)-ATPase activities and the stimulation was additive to the effects of calmodulin. The (Ca²⁺Mg²⁺)-ATPase and ATP-dependent ⁴⁵Ca²⁺ uptake activities are probably related as they exhibited similar [Ca²⁺]_free- and [calmodulin]-dependencies. These results indicate that calmodulin may play a role in the control of the cytosolic [Ca²⁺]_free in canine tracheal smooth muscle.     

Effect of Ca(2+) and Mg(2+) on the Mn-superoxide dismutase from rat liver and heart mitochondria

Summary.  The manganese superoxide dismutase (Mn-SOD) converts superoxide anions to hydrogen peroxide plus oxygen, providing the first line of defense against oxidative stress in mitochondria. Heart mitochondria exhibited higher Mn-SOD activity than liver mitochondria. In mitochondria from both tissues Mn-SOD activity decreased after incubation at low oxygen concentration (hypoxic mitochondria). The effects of free Ca²⁺ ([Ca²⁺]_f) and free Mg²⁺ ([Mg²⁺]_f) on normoxic and hypoxic mitochondria from either organ were tested. In normoxic mitochondria from either tissue, both [Ca²⁺]_f and [Mg²⁺]_f activated the enzyme, although [Mg²⁺]_f was less efficient as an activator and the effect was lower in heart than in liver mitochondria. When added simultaneously, high [Ca²⁺]_f and [Mg²⁺]_f exhibited additive effects which were more pronounced in heart mitochondria and were observed regardless of whether mitochondria had been incubated under normal or low oxygen. The data suggest that [Ca²⁺]_f plays a role in regulating Mn-SOD in concert with the activation of aerobic metabolism. Received April 2, 2001 Accepted August 16, 2001     

Dietary and physiological studies to investigate the relationship between calcium and magnesium signalling in the mammalian myocardium

This study employs both dietary and physiological studies to investigate the relationship between calcium (Ca²⁺) and magnesium (Mg²⁺) signalling in the mammalian myocardium. Rats maintained on a low Mg²⁺ diet (LMD; 39 mg Kg^-1 Mg²⁺ in food) consumed less food and grew more slowly than control rats fed on a control Mg²⁺ diet (CMD; 500 mg Kg^-1 Mg²⁺ in food). The Mg²⁺ contents of the heart and plasma were 85 ± 3% and 34 ± 6.5%, respectively relative to the control group. In contrast, Ca²⁺ contents in the heart and plasma were 177 ± 5% and 95 ± 3%. The levels of potassium (K⁺) was raised in the plasma (129 ± 16%) and slightly decreased in the heart (88 ± 6%) compared to CMD. Similarly, sodium (Na⁺) contents were slightly higher in the heart and lowered in the plasma of low Mg²⁺ diet rats compared to control Mg²⁺ diet rat. Perfusion of the isolated Langendorff's rat heart with a physiological salt solution containing low concentrations (0-0.6 mM) of extracellular magnesium [Mg²⁺]₀ resulted in a small transient increase in the amplitude of contraction compared to control [Mg²⁺]₀ (1.2 mM). In contrast, elevated [Mg²⁺]₀ (2-7.2 mM) caused a marked and progressive decrease in contractile force compared to control. In isolated ventricular myocytes the L-type Ca²⁺ current (I_Ca,L was significantly (p < 0.001) attenuated in cells dialysed with 7.1 mM Mg²⁺ compared to cells dialysed with 2.9 µM Mg²⁺. The results indicate that hypomagnesemia is associated with decrease levels of Mg²⁺ and elevated levels of Ca²⁺ in the heart and moreover, internal Mg²⁺ is able to modulate the Ca²⁺ current through the L-type Ca²⁺ channel which in turn may be involved with the regulation of contractile force in the heart.     

Cooperative regulation of Cav1.2 channels by intracellular Mg2+, the proximal C-terminal EF-hand,and the distal C-terminal domain

L-type Ca²⁺ currents conducted by Ca_v1.2 channels initiate excitation–contraction coupling in cardiac myocytes. Intracellular Mg²⁺ (Mg_i) inhibits the ionic current of Ca_v1.2 channels. Because Mg_i is altered in ischemia and heart failure, its regulation of Ca_v1.2 channels is important in understanding cardiac pathophysiology. Here, we studied the effects of Mg_i on voltage-dependent inactivation (VDI) of Ca_v1.2 channels using Na⁺ as permeant ion to eliminate the effects of permeant divalent cations that engage the Ca²⁺-dependent inactivation process. We confirmed that increased Mg_i reduces peak ionic currents and increases VDI of Ca_v1.2 channels in ventricular myocytes and in transfected cells when measured with Na⁺ as permeant ion. The increased rate and extent of VDI caused by increased Mg_i were substantially reduced by mutations of a cation-binding residue in the proximal C-terminal EF-hand, consistent with the conclusion that both reduction of peak currents and enhancement of VDI result from the binding of Mg_i to the EF-hand (K_D ≈ 0.9 mM) near the resting level of Mg_i in ventricular myocytes. VDI was more rapid for L-type Ca²⁺ currents in ventricular myocytes than for Ca_v1.2 channels in transfected cells. Coexpression of Ca_vβ_2b subunits and formation of an autoinhibitory complex of truncated Ca_v1.2 channels with noncovalently bound distal C-terminal domain (DCT) both increased VDI in transfected cells, indicating that the subunit structure of the Ca_v1.2 channel greatly influences its VDI. The effects of noncovalently bound DCT on peak current amplitude and VDI required Mg_i binding to the proximal C-terminal EF-hand and were prevented by mutations of a key divalent cation-binding amino acid residue. Our results demonstrate cooperative regulation of peak current amplitude and VDI of Ca_v1.2 channels by Mg_i, the proximal C-terminal EF-hand, and the DCT, and suggest that conformational changes that regulate VDI are propagated from the DCT through the proximal C-terminal EF-hand to the channel-gating mechanism.     

Neutral organic solute effects on the activity of the plasma membrane Ca2+-ATPase

We have compared effects of dimethylsulfoxide (Me₂SO) and two polyols on the Ca²⁺-ATPase purified from human erythrocytes. As studied under steady-state conditions over a broad solute concentration range and temperature, Me₂SO, glycerol, and xylitol do not inhibit the Ca²⁺-ATPase activity; this is in contrast to numerous other organic solutes that we have investigated. Under specific experimental conditions, Me₂SO (but not glycerol) substantially increases Ca²⁺-ATPase activity, suggesting a possible facilitation of enzyme oligomerization. The activation is more pronounced at low Ca²⁺ concentrations. In contrast to glycerol, Me₂SO shows no protective effect on enzyme structure as assessed by determining residual Ca²⁺-ATPase activity after exposing the enzyme to thermal denaturation at 45°C. Under these conditions several other organic solutes strongly enhance the denaturating effect of temperature. Because of the temperature dependence of its effect on the Ca²⁺-ATPase activity we believe that Me₂SO activates the Ca²⁺-ATPase by indirect water-mediated interactions.     

Coordination of magnesium with adenosine 5′-diphosphate and triphosphate

³¹P NMR chemical shifts of salts of adenosine 5′-triphosphate and diphosphate: ATPH^2?₂2(Me₄N⁺) · H₂O, ATPH^2?₂2 Na⁺ · 3.5 H₂O, ATPH^2?₂Mg²⁺ · 4 H₂O, ATPH^2?₂Ca²⁺ · 2 H₂O, ADPH^2?2(Me₄N⁺) · H₂O and ADPH^2?Mg²⁺ · 4 H₂O have been measured in 0.02 M ²H₂O solutions at 145.7 MHz (22° C) at constant p²H values (8.20 and 6.20). The results are compared with those obtained from salts of adenosine 5′-monophosphate and other simpler phosphomonoesters, e.g. AMP^2?2(Me₄N⁺), AMP^2?Mg²⁺, AMPH^?Me₄N⁺ and (AMPH^?)₂Mg²⁺. It is concluded that the effects exerted by Mg²⁺ and Ca²⁺ on the ³¹P NMR shifts of dipoly- and tripolyphosphates relative to monovalent cations are due mainly to changes in conformation of the polyphosphate chain rather than to purely electronic factors associated with the binding of divalent cations to the phospho-oxyanions. The data are consistent with the existence of the following complexes at p²H 8.20: (MgP_αP_β)ADP^? and (MgP_αP_γ)ATP^2?af (MgP_αP_β)ATP^2?af (MgP_βP_γ)ATP^2? with the latter equilibrium relatively fast in the NMR time scale. Monoprotonation of the terminal phosphate appears to weaken the Mg²⁺-polyphosphate binding, particularly at P_β of MgADPH and at P_β and P_γ of MgATPH^?. The Mg²⁺-polyphosphate binding weakens further at p²H 3.70, i.e. in MgATPH₂. Possible implications of the results in the mechanism of actomyosin Mg²⁺-ATPase in muscle contraction are discussed.     

Enhancement of Nitrate Uptake and Growth of Barley Seedlings by Calcium under Saline Conditions

The effect of Ca²⁺ on NO₃⁻ assimilation in young barley (Hordeum vulgare L. var CM 72) seedlings in the presence and absence of NaCl was studied. Calcium increased the activity of the NO₃⁻ transporter under saline conditions, but had little effect under nonsaline conditions. Calcium decreased the induction period for the NO₃⁻ transporter under both saline and nonsaline conditions but had little effect on its apparent K_m for NO₃⁻ both in the presence and absence of NaCl. The enhancement of NO₃⁻ transport by Ca²⁺ under saline conditions was dependent on the presence of Ca²⁺ in the uptake solution along with the salt, since Ca²⁺ had no effect when supplied before or after salinity stress. Although Mn²⁺ and Mg²⁺ enhanced NO₃⁻ uptake under saline conditions, neither was as effective as Ca²⁺. In longer studies, increasing the Ca²⁺ concentration in saline nutrient solutions resulted in increases in NO₃⁻ assimilation and seedling growth.     

Renaissance of cytochemical localization of membrane ATPases in the myocardium

ATPases of cardiac cells are known to be among the most important enzymes to maintain the fluxes of vital cations by hydrolysis of the terminal high-energy phosphate of ATP. Biochemically the activities of Ca²⁺-pump ATPase, Ca²⁺/Mg²⁺-ecto ATPase, Na⁺,K⁺-ATPase and Mg²⁺-ATPase are determined in homogenates and isolated membranes as well as in myofibrillar and mitochondrial fractions of various purities. Such techniques permit estimation of enzyme activitiesin vitro under optimal conditions without precise enzyme topography. On the other hand, cytochemical methods demonstrate enzyme activityin situ, but not under optimal conditions. Until recently several cytochemical methods have been employed for each enzyme in order to protect its specific activity and precise localization but the results are difficult to interpret. To obtain more consistent data from biochemical and cytochemical point of view, we modified cytochemical methods in which unified conditions for each ATPase were used. The fixative solution (1% paraformaldehyde –0.2% glutaraldehyde in 0.1 M Tris Base buffer, pH 7.4), the same cationic concentrations of basic components in the incubation medium (0.1 M Tris Base, 2mM Pb(NO₂)₃, 5 mM MgSO₄, 5 mM ATP) and selective stimulators or inhibitors were employed. The results reveal improved localization of Ca²⁺-pump ATPase, Na⁺–K⁺ ATPase and Ca²⁺/Mg²⁺-ecto ATPase in the cardiac membrane.