Isolation and characterization of cytoplasmic NADP+-malic enzyme from Lupinus luteus leaves

NADP⁺-dependent malic enzyme (L-malate : NADP⁺ oxidoreductase, decarboxylating, EC 1.1.1.40) was extracted from the leaves of yellow lupine. The purification procedure included fractionation with (NH₄)₂SO₄ and Sephadex G-25 chromatography, followed by purification on DEAE-cellulose and Sephadex G-200 columns. The enzyme was purified 122-fold. The enzyme affinity towards L-malate was found to be significantly higher with Mn²⁺ than with Mg²⁺. The Hill coefficient for Mg²⁺ depended on concentration and was 1.6 for the lower and 3.9 for the higher concentrations. The dependence of the enzyme activity on NADP⁺ followed a hyperbolic curve. K_m values and Hill coefficients for NADP⁺ were similar with both Mn²⁺ and Mg²⁺. The enzyme activity was strictly dependent on divalent cations and followed a sigmoidal curve at least for Mg²⁺. The enzyme had 4-fold higher affinity towards Mn²⁺ than towards Mg²⁺, the K_m values being 0.3 and 1.15 m M respectively. Of several tested organic acids, oxalate was the most effective inhibitor followed by oxaloacetate while succinate was the strongest activator.     

Identification and Characterization of Two DNA Polymerase Activities Present in Trypanosoma brucei Mitochondria

We have identified and partially purified two DNA polymerase activities from purified Trypanosoma brucei mitochondrial extracts. The DNA polymerase activity eluted from the single-stranded DNA agarose column at 0.15 M KCI (polymerase MI) was significantly inhibited by salt concentrations greater than 100 mM, utilized Mg²⁺ in preference to Mn²⁺ as a cofactor on deoxyribonucleotide templates with deoxyribose primers, and in the presence of Mn²⁺ favored a ribonucleotide template with a deoxyribose primer. A 44 kDa peptide in this fraction crossreacted with antisera against the Crithidia fasciculata β-like mitochondrial polymerase. In activity gels the catalytic peptide migrated at an apparent molecular weight of 35 kDa. The DNA polymerase activity present in the 0.3 M KCI DNA agarose fraction (polymerase M2) exhibited optimum activity at 120-180 mM KCI, used both Mg²⁺ and Mn²⁺ as cofactors, and used deoxyribonucleotide templates primed with either deoxyribose or ribose oligomers. Activity gel assays indicate that the native catalytic peptide(s) is ˜ 80 kDa in size. The two polymerases showed different sensitivities to several inhibitors: polymerase MI shows similarities to the Crithidia fasciculata β-like mitochondrial polymerase while polymerase M2 is a novel, salt-activated enzyme of higher molecular weight.     

Magnesium, manganese and mutual depletion systems in halophilic bacteria

Abstract The multi-ionic equilibria between enzymes, substrates and monovalent and divalent cations are related in such a way that a change in concentration of one element modifies the repartition of all the concentrations of the other elements, leading to a mutual depletion system. The pyruvate kinase reaction is a good application of the mutual depletion model: this cytoplasmic enzyme utilizes magnesium (Mg) and potassium (K) as cofactors and reacts with free phosphoenolpyruvate and MgADP, substrates involved in the binding of protons, K⁺ and Mg²⁺. Pyruvate kinase from Vibrio costicola , a moderately halophilic eubacterium, obeys the mutual depletion system and is competitively inhibited by physiological concentrations of potassium ions. This effect is relieved by manganese which forms more stable complexes than magnesium. Pyruvate kinase from Halobacterium cutirubrum cannot be described unambiguously by the mutual depletion model.
Cytoplasmic concentrations of potassium ions are elevated in halophilic bacteria and may thus inhibit the formation of the divalent cation complexes necessary in the enzymatic machinery of halophilic bacteria. Accordingly, the contents of the most abundant divalent cation, Mg²⁺, and of the trace element manganese, Mn²⁺, are higher in the halophilic bacteria, V. costicola, Halobacterium volcanii , and H. cutirubrum , and their increase is proportional to the ionic strength of the extracellular media. The Mn²⁺ increase is more marked than the Mg²⁺ increase, although the Mn²⁺ content is about two orders of magnitude lower than the Mg²⁺ content.     

Distribution of ATPases in wheat root membranes separated by phase partition

The distribution of divalent cation stimulated ATPase activity in relation to the distribution of other enzyme activities was studied for membrane fractions from wheat roots ( Tritium aestivum L . cv. Svenno). A homogenate from dark grown plants was fractionated by differential centrifugation at 1000 g , 10,000 g , 30,000 g and 60,000 g (1, 10, 30 and 60 KP fractions), followed by partition in an aqueous polymer two-phase system, using polyethylene glycol 4000/dextran T500 concentrations of 5.7/5.7, 5.9/5.9, 6.1/6.1, 6.3/6.3 and 6.5/6.5% (w/w). The 30 KP fraction was also separated by counter-current distribution id a 6.3/6.3% two-phase system. Protein and activities of Ca²⁺, Mg²⁺, and Mn²⁺ stimulated ATPases. cytochrome oxidase, light induced absorbance change (LIAC) related to cyt b reductions, inosine diphosphatase and NADH dependent antimycin A insensitive cytochrome c reductase were measured.
The partition of ATPase activities stimulated by Ca²⁺, Mg²⁺ or Mn²⁺ was similar at all polymer concentrations tested, indicating: a low cation specificity of the dominating ATPases. The distribution of ATPases. agreed with different marker enzymes in different centrifuge fractions. Divalent cation stimulated ATPases were evidently related to several of the organelles. In the different fractions the distribution of ATPase activity should then follow that of the marker enzyme of the dominant organelle. From studies with different polymer concentrations the 6.3/6.3-system was selected for further separation of the membranes in the 30 KP fraction by counter-current distribution. By this method one fraction was obtained, which probably consisted of plasmalemma and was free from mitochondrial material. Indications for plasmalemma in this fraction were a) similar partition as protoplasts and b) high LIAC activity.     

Sidedness of (Calcium, Magnesium) Adenosine Triphosphatase of Purified Torpedo Synaptic Vesicles

Abstract: Purified Torpedo synaptic vesicles contain ouabain-insensitive Mg²⁺_τ and Ca²⁺-stimulated ATPase activity. The sidedness of the ATPase on the vesicular membranes was investigated. Addition of ATP and Mg²⁺ or Ca²⁺ to intact vesicles results in activation of the ATPase. Exposure of the vesicles to low concentrations of Lubrol-PX and Triton X-100, which do not solubilize the activity, results in the concurrent release of the vesicular contents and in an increase of the Mg²⁺-ATPase activity, whereas the Ca²⁺-dependent activity is drastically decreased. p -Chloromercuribenzene sulphonate (PCMBS) almost completely inhibits the activity of detergent-treated vesicles whereas that of the native material is only slightly affected. Tryptic digestion of intact vesicles and of vesicular ghosts results in partial reduction of the ATPase activity. These results suggest that the vesicles contain an outward oriented Ca²⁺/Mg²⁺ ATPase activity which can be modulated by detergents.     

Transport of Mg2+ and Ca2+, and Mg2+-stimulated adenosine triphosphatase activity in oat roots as affected by mineral nutrition

Mg²⁺- and Ca²⁺-uptake was measured in dark-grown oat seedlings ( Avena sativa L. cv. Brighton) cultivated at two levels of mineral nutrition. In addition the stimulation of the ATPase activity of the microsomal fraction of the roots by Mg²⁺ was measured. Ca²⁺-uptake by the roots was mainly passive. Mg²⁺-uptake mainly active; the passive component of Mg²⁺-uptake was accompanied by Ca²⁺-efflux up to 60% of the Ca²⁺ present in the roots.
In general Mg²⁺ -uptake of oat roots was biphasic. The affinity of the second phase correspond well with that of the Mg²⁺-stimulation of the ATPase activity, in low-salt roots as well as in high-salt roots and in roots of plants switched to the other nutritional condition. Linear relationships were observed when [phase 2] Mg²⁺-uptake was plotted against Mg²⁺-stimulation of the ATPase activity of the microsomal fraction of the roots. In 5 days old high-salt plants 1 ATP (hydrolysed in the presence of Mg²⁺ J corresponded with active uptake of a single Mg²⁺ ion, but in older high-salt roots and in low-salt roots more ATP was hydrolysed per net uptake of a Mg²⁺ ion. The results are discussed against the background of regulation of the Mg²⁺-level of the cytoplasm of root cells by transport of Mg²⁺ by a Mg²⁺-ATPase to the vacuole, to the xylem vessels, and possibly outwards.     

Manganese induces sustained Ser40 phosphorylation and activation of tyrosine hydroxylase in PC12 cells

Manganese (Mn²⁺) is an essential metal involved in normal functioning of a range of physiological processes. However, occupational overexposure to Mn²⁺ causes neurotoxicity. The dopaminergic system is a particular target for Mn²⁺ neurotoxicity. Tyrosine hydroxylase (TH) is the rate limiting enzyme for dopamine synthesis and is regulated acutely by phosphorylation at Ser40 and chronically by protein synthesis. In this study we used pheochromocytoma 12 cells to investigate the effects of Mn²⁺ exposure on the phosphorylation and activity of TH. Mn²⁺ treatment for 24 h caused a sustained increase in Ser40 phosphorylation and TH activity at a concentration of 100 μM, without altering the level of TH protein or PC12 cell viability. Inhibition of protein kinase A and protein kinase C and protein kinases known to be involved in sustained phosphorylation of TH in response to other stimuli did not block the effects of Mn²⁺ on Ser40 phosphorylation. A substantial increase in H₂O₂ production occurred in response to 100 μM Mn²⁺. The antioxidant Trolox^TM completely inhibited H₂O₂ production but did not block TH phosphorylation at Ser40, indicating that oxidative stress was not involved. Sustained TH phosphorylation at Ser40 and the consequent activation of TH both occurred at low concentrations of Mn²⁺ and this provides a potential new mechanism for Mn²⁺-induced neuronal action that does not involve H₂O₂-mediated cell death.     

Plasmalemma from the roots of cucumber: Isolation by two-phase partitioning and characterization

Plasmalemma was isolated from the roots of 2-week-old cucumber plants ( Cucumis sativus L. cv. Rhensk druv) by utilizing an aqueous polymer two-phase system with 6.5%:6.5% (w/w) Dextran T500 and polyethylene glycol (PEG) 3350 at pH 7.8. The plasmalemma fraction comprised ca 6% of the membrane proteins contained in the microsomal fraction. The specific activity of the plasma membrane marker enzyme (K⁺, Mg²⁺-ATPase) was 14- to 17-times higher in the upper (PEG-rich) than in the lower (Dextran-rich) phase, and the reverse was true for marker enzymes (cytochrome c oxidase, EC 1.9.3.1, and antimycin A-resistant NADPH cytochrome c reductase) of intracellular membranes. The ATPase was highly stimulated by the addition of detergent (Triton X-100), so that the isolated plasmalemma vesicles appear tightly sealed and in a right-side-out orientation. Further characterization of the ATPase activities showed a pH optimum at 6.0 in the presence of Mg²⁺. This optimum was shifted to pH 5.8 after addition of K⁺. K⁺ stimulated the ATPase activity below pH 6 and inhibited above pH 6. The ATPase activity was specific for ATP and sensitive to N,N-dicyclohexylcarbodiimide and sodium vanadate, with K⁺ enhancing the vanadate inhibition. The enzyme was insensitive to sodium molybdate, NO⁻₃, azide and oligomycin. No Ca²⁺-ATPase was detected, and even as little as 0.05 m M Ca²⁺ inhibited the Mg²⁺-ATPase activity.     

Characteristics of ATPase from sugarcane protoplast and vacuole membranes

Characteristics of membrane-associated ATPase from commercial Hawaiian varieties of sugarcane ( Saccharum spp. hybrids) were investigated in preparations from sugarcane cell suspension culture and from stalk tissues of the intact plant. In order to examine comparable preparations, protoplasts and vacuoles, in turn, were obtained from both sources. ATPase from preparations of crude protoplast membranes and tonoplast had a pH optimum of 6 to 6.5. The relative effectiveness of divalent cations in stimulating ATPase was Mg²⁺ > Mn²⁺≥ Co²⁺ > Ca²⁺≥ Zn²⁺. Enzyme activity was not stimulated by K⁺, nor by other monovalent cations. Protoplasts and vacuoles from both sources showed significant acid phosphatase activity. Acid phosphatase activity was inhibited by molybdate, but ATPase activity was unaffected. Membrane preparations from protoplasts contained inorganic pyrophosphatase, but enzyme activity was low or not present in tonoplast preparations. Cell suspension and stalk tissue preparations hydrolyzed a large number of nucleoside di- and triphosphates. The hydrolysis is most likely due to a series of enzymes rather than a single enzyme. ATPase from protoplast and tonoplast preparations was inhibited 30–50% by diethylstilbestrol and sodium ortho-vanadate and was unaffected by ionophores. This study illustrates the complexity of phosphohydrolase activities in membrane preparations from sugarcane. The study, however, also illustrates substantial similarity in the behavior of these enzymes, whether they are derived from the plant itself or from cell cultures originating from comparable tissues of the plant.     

Uptake of Magnesium by Chromaffin Granules In Vitro: Role of the Proton Electrochemical Gradient

Abstract: The chromaffin granule membrane in vitro is impermeable to protons as well as to Mg²⁺; however, when granules are incubated in the presence of the proton ionophore carbonyl cyanide p -trifluoromethoxy-phenylhydrazone or an inhibitor of the granule membrane Mg²⁺-dependent ATPase, the metal ion is accumulated inside the granules. This accumulation is dependent upon the granule transmembrane potential. The simultaneous presence of the ATPase inhibitor and the proton ionophore markedly increases metal ion incorporation. Mg²⁺ incorporation is also promoted by nigericin in the presence of potassium or sodium ions, indicating that Mg²⁺ accumulation is also dependent upon the transmembrane pH gradient. Concomitant with the Mg²⁺ accumulation, there is a significant loss of endogenous catecholamines. It is concluded that Mg²⁺ accumulation is determined by the electrochemical gradient maintained across the membrane. Once the metal ion has accumulated into the granules it displaces catecholamines from their storage sites.     

Calcium-Dependent Binding of Cytosolic Proteins by Chromaffin Granules from Adrenal Medulla

Abstract: Purified chromaffin granules from bovine adrenal medulla bound a small group of medullary cell cytosol proteins at micromolar levels of Ca²⁺ and physiological levels of K⁺, Mg²⁺, and Mg-ATP. The bound proteins had molecular weights of 33,000-37,000 and 70,000-71,000 on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and did not correspond with any previously reported cytosolic components of chromaffin cells. The new proteins were eluted from intact granules or resealed granule membranes at 0.1 μ M Ca²⁺; binding was half-maximal at 2.6 μ M . Adsorption and elution in this manner resulted in a high degree of purification of the new proteins that were minor components of the original cytosol. Partially purified fractions enriched in the 33,000-37,000 and 70,000-71,000 proteins bound ⁴⁵Ca²⁺ at submicromolar levels in the presence of millimolar Mg²⁺. Calmodulin was also bound by the granule membranes and was present in trace amounts in cytosol eluates from granules, but it did not bind to the new proteins in the presence of calcium ions. The possible significance of the new proteins to calcium-mediated secretion from chromaffin cells is discussed.     

The Effect of Divalent Cations on Aggregation of Dictyostelium discoideum

Following the initiation of development, amoebae of Dictyostelium discoideum aggregate chemotactically toward cyclic AMP (cAMP). Adenyl cyclase, cAMP phosphodiesterase, and cAMP binding sites all increase 20–40 fold during the first few hours of development. It has been shown that addition of 1 mM EDTA and 5 mM MgCl₂ accelerates the aggregation process. Likewise, the calcium ionophore, A23187, leads to precocious aggregation while 4 × 10⁻⁵ M progesterone considerably delays it These treatments have now been shown to result in increased accumulation of adenyl cyclase in the case of EDTA and Mg²⁺ or the ionophore and greatly decreased accumulation in the case of the steroid.
Treatment with EDTA and Mg²⁺ or the ionophore has been shown not only to accelerate aggregation in wild-type amoebae but to overcome complete blocks to aggregation in certain mutant strains. We have found that addition of Mn²⁺ will also permit aggregation of mutant cells otherwise unable to aggregate. This divalent ion, unlike EDTA and Mg²⁺ or the ionophore, was shown to directly stimulate adenyl cyclase. Calcium ions were also found to affect the enzyme such that at Ca²⁺ concentrations found within the cells the great majority of the activity is inhibited. Manganese ions can overcome the inhibition by Ca²⁺.
These findings show that conditions which stimulate aggregation result in increased activity of adenyl cyclase either by increased accumulation of the enzyme or by increased activity of the available enzyme, and support the proposed central role of adenyl cyclase in aggregation.     

STUDIES ON MEVALONATE KINASE, PHOSPHOMEVALONATE KINASE AND PYROPHOSPHOMEVALONATE DECARBOXYLASE IN DEVELOPING RAT BRAIN

Abstract— We have in the present study investigated the properties of mevalonate kinase, phosphomevalonate kinase and pyrophosphomevalonate decarboxylase in the 105,000 g supernatant fractions from rat brain, and determined whether the activities of these enzymes change during brain development. All three enzymes in brain showed a specific requirement for ATP for optimal activity. The presence of Mg²⁺ as divalent cation was also required for optimal activity of mevalonate kinase and phosphomevalonate kinase. Both Mg²⁺ and Mn²⁺ were equally effective divalent metal ions for pyrophosphomevalonate decarboxylase in brain. Mevalonate kinase as well as phosphomevalonate kinase were active in a broad pH range of 6.5–8 while the pH curve for pyrophosphomevalonate decarboxylase showed a peak activity at approx 6. No age-dependent change occurred in the activities of mevalonate kinase and phosphomevalonate kinase in developing brain, whereas pyrophosphomevalonate decarboxylase activity in brain increased during the 1st week after birth, reached a peak value at about the 8th day of age and declined slowly thereafter. The K_m for brain mevalonate kinase in 2, 13 and 52 day old rats were 312, 400 and 434 μM, respectively. The V _max for the kinase in 2, 13 and 52 day old rats were in the range of 45–52 nmol/h/mg protein, respectively. This suggests that, like in liver (R amachandran & S hah , 1976), pyrophosphomevalonate decarboxylase in brain may also be one regulatory step for cholesterol synthesis.     

Properties of NADP+-malic enzyme from pod walls of chickpea (Cicer arietinum)

NADP⁺-malic enzyme ( l -malate: NADP⁺ oxidoreductase, decarboxylating EC 1.1.1.40) from pod walls of chickpea was purified 51-fold by ammonium sulphate fractionation, DEAE- cellulose chromatography and gel filtration through Sepharose 4B. The purified enzyme required a divalent cation, either Mn²⁺ or Mg²⁺, for its activity. K_m values at pH 7.8 for malate, NADP⁺ and Mn²⁺ were 4.0, 0.031 and 0.71 m M , respectively. Mn²⁺-dependent activity was inhibited by heavy metal ions such as Cd²⁺, Zn²⁺, Hg²⁺, and to a lesser extent by Pb²⁺ and Al³⁺. Among the organic acids examined, sodium salts of oxalate and oxaloacetate were inhibitory. Kinetics of the reaction mechanism showed sequential binding of malate and NADP⁺ to the enzyme. Products of reaction, viz. pyruvate, bicarbonate and NADPH, inhibited the enzyme activity. At limiting concentrations of NADP⁺, pyruvate and bicarbonate induced a positive cooperative effect by malate. It is proposed that the activity of NADP⁺-malic enzyme is controlled by intracellular concentrations of substrates and products.     

A 30-kDa Protein in the Surface Complex and Flagella of Euglena has Protein Kinase Activity

ABSTRACT A protein kinase (PK) was partially purified from NaCl extracts of the cell surface complex of Euglena using DEAE-cellulose chromatography. Tubulins extracted either from flagella or from the cell surface complexes of Euglena were readily phosphorylated when incubated with [γ-³²P]-ATP and the PK. Protein kinase activity was augmented with 5 mM Mn²⁺ or Mg² and was inhibited or had greatly reduced activity with 5 mM Ca²⁺, Co^2-, Cu²⁺ or Zn²⁺. Incorporation was much lower when [γ-³²P]-GTP was the phosphate donor. Serine and threonine were the major radiolabeled phosphoamino acids in tubulins; label was also found in phosphotyrosine. Alpha-tubulin solubilized from flagella was a relatively poor substrate for the PK, but a Euglena α-tubulin cDNA overexpressed as a Trx-fusion protein incorporated [γ-³²P]-ATP into serine and threonine when incubated with cell surface extracts. Alpha- and β-tubulins from cell surface complexes were equally good substrates for the PK. No incorporation was observed in intact microtubules either from the cell surface complex or from isolated flagella. In-gel assays identified a polypeptide of about 30 kDa that phosphorylated tubulins in extracts of both flagella and the cell surface complexes, and dephosphorylated casein was a competitive substrate for the partially purified kinase. In vivo incubation with [³²P]-orthophosphate produced numerous radiolabeled bands in acrylamide gels of NaCl extracts of the cell surface complex, but none of these bands could be positively related to tubulins extracted from surface complex microtubules.     

Interaction of divalent metal ions with the NADP+-malic enzyme from maize leaves

The effect of several metal ions on NADP⁺-malic enzyme (EC 1.1.1.40) purified from Zea mays L. leaves was studied Mg²⁺, Mn²⁺, Co²⁺ and Cd²⁺ were all active metal cofactors. The malic enzyme from maize has a moderately high intrinsic preference for Mn²⁺ relative to Mg²⁺ at pH 7.0 and 8.0 Negative cooperativity detected in the binding of Mg²⁺ at pH 7.0 and 8.0 and in the binding of Mn²⁺ at pH 7.0 suggests the existence of at least two binding sites with different affinity. All of the activating metal ions have preference for octahedral coordination geometry and have ionic radii of 0.86–1.09 Å. The ions that act as inhibitors are outside this range and/or are incapable of octahedral coordination. Ba²⁺, Sr²⁺, Cd²⁺, Ca²⁺, Be²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Co²⁺, Hg²⁺ showed mixed-type inhibition. The reciprocal of their K₁ values follow the order of their apparence in the Irving-Williams series of stability that derives in part from size effects. It is suggested that the size of the ions may play a partial role in determining the strength of the metal interaction.     

The Inositol Lipids of Paramecium tetraurelia and Preliminary Characterizations of Phosphoinositide Kinase Activity in the Ciliary Membrane

Inositol glycerolipids make up less than 10% of total phospholipids of Paramecium tetraurelia cells. Unlike inositol lipids found in mammalian and other cell types, these lipids from Paramecium lack arachidonic acid. It was demonstrated that kinase and possibly phosphatase enzymes that interconvert phosphatidylinositol (PI), phosphatidylinositol phosphate (PI-P) and phosphati-dylinositol-bis-phosphate (PI-P₂) exist in ciliary membranes of this ciliate. When exogenous soybean PI and [γ-³²P]ATP were provided as substrates, isolated cilia preparations exhibited PI and PI-P kinase activities as demonstrated by the incorporation of radiolabel into PI-P and PI-P₂. Kinase activity was activated by millimolar [Mg²⁺] and inhibited by millimolar [Ca²⁺]. Significant inhibition of kinase activity in the presence of unlabeled excess ATP suggested that ATP is the preferred phosphate donor for this reaction. Of 4 suborganellar fractions of isolated cilia, the membrane fraction had the greatest kinase activity indicating that the enzyme(s) is membrane-associated     

Picomolar concentrations of tentoxin affect Mg2+- and Ca2+-ATPase activities of plasma membrane vesicles from roots of winter wheat seedlings

Purified plasmalemma vesicles were isolated in the presence of 250 m M sucrose from roots of 14-day-old seedlings of winter wheat ( Triticum aestivum L. Martonvásári-8) by phase partitioning of salt-washed microsomal fractions in a Dextran-polyethylene glycol two-phase system, and both Mg²⁺- and Ca²⁺-ATPase activities were detected. Orthovanadate-sensitive Mg²⁺-ATPase activity associated with the inside of right side-out plasmalemma (PM) vesicles (latency 98%) was inhibited 76% by 0.3 m M Ca²⁺, Ca²⁺-dependent ATPase activity located partly on the inside and partly on the outside of plasmalemma vesicles (latency 47%) was not affected by Mg²⁺.
Mg²⁺-ATPase activity was inhibited by 68% and inhibition of Mg²⁺ activation by 0.3 m M Ca²⁺ partly disappeared in the presence of 10 p M tentoxin, a fungal phytotoxin. Mg²⁺-ATPase activity remained inhibited up to 10 n M tentoxin while at 1 μ M tentoxin Mg²⁺ activation was as high as without tentoxin. K⁺-stimulation and vanadate inhibition was increased and decreased, respectively, by 100 p M -10 n M tentoxin. Ca²⁺-dependent ATPase activity was continuously increased by 1 p M -10 n M tentoxin, but at 1 μ M tentoxin the stimulation disappeared. The effects of p M tentoxin on plasma-lemma Mg²⁺-ATPase are discussed in relation to its influence on K⁺ transport in wheat seedlings.     

Characterization of the plasmalemma ATPase activity from roots of Plantago major ssp. pleiosperma, purified by the two-phase partitioning method

A purified plasmalemma preparation from roots of Plantago major L. ssp. pleiosperma (Pilger) was obtained by the two-phase partitioning method, using 6.5% (w/w) of Dextran T-500 and polyethylene glycol 3350, respectively. The distribution of murker enzymes proved the purity of the plasmalemma fraction. The ATPase activity was characterized by determining its sensitivity to anions, cations and inhibitors. The Mg²⁺-dependent ATPase activity peaked at pH 7.25, K⁺-stimulation at pH 6.75, and the Cl⁻ -stimulation both at pH 6.75 and 7.5 (all in the presence of 3 m M MgSO₄). The plasmalemma preparations hydrolyzed preferentially ATP (in the presence of Mg²⁺), although they were less specific for ATP at pH 7.5 than at pH 6.75. The Cl⁻ - stimulated ATPase is probably associated with and located on the plasmalemma. The question if the Cl⁻ -stimulated activity is due to an ATPase distinct from the classical K⁺-stimulated ATPase is considered.     

MAGNESIUM REQUIREMENT IN FERTILIZATION PROCESS OF SEA URCHINS

The Mg²⁺ requirement in fertilization was investigated in sea urchins. It was found that when sea urchin eggs were inseminated in sea water free of Mg²⁺, little fertilization took place. Even when spermatozoa pre-treated with dissolved egg-jelly to induce the acrosome reaction, which needs Ca²⁺, were used, the fertilization rate remained quite low in the absence of Mg²⁺. In Strongylo-centrotus intermedius , the lowest concentration of Mg²⁺ required for 50% fertilization was 0.05 mM in the presence of 10 mM Ca²⁺, whereas that of calcium was 3 mM in the presence of 49 mM Mg²⁺. These critical concentrations increased when the concentration of the other ion decreased. Removal of Mg²⁺ or Ca²⁺ or both from the suspending medium had little adverse effect on sperm motility. The elevation of the fertilization membrane was also induced by butyric acid independent of the presence or absence of Mg²⁺ and/or Ca²⁺. These results indicate that Mg²⁺ are required at least in some process(es) between acrosome reaction and fertilization membrane elevation, such as sperm penetration or membrane fusion.