ATPASE AND PHOSPHATIDYLINOSITOL KINASE ACTIVITIES OF ADRENAL CHROMAFFIN VESICLES

Abstract— The hypothesis that the ATPase and phosphatidyhnositol (PI) kinase activities of chromaffin vesicle membranes are catalysed by same enzyme was investigated. The two activities exhibited entirely different responses to variations in Mg²⁺ or Mn²⁺ concentrations. In the presence of 1 mM ATP, maximal ATPase activity occurred with 1 mM Mg²⁺ while maximal PI kinase activity required 100 mM Mg²⁺ Similar differences were observed with Mn²⁺ with the exception that maximal ATPase activity occurred with 0.5 mM Mn²⁺ and maximal PI kinase activity occurred with 5 mM Mn²⁺ Mn²⁺ was more effective than Mg²⁺ in stimulating PI kinase activity at low concentrations, but at optimal concentrations of each, the maximal activity obtained with Mg²⁺ was 5-fold greater than the maximal activity obtained with Mn²⁺ The heat stabilities of the two enzymes are vastly different. At 50°C the ATPase activity of the intact membranes was stable for up to 20 min while the t _l/2 of PI kinase was less than 2 min. After solubilization in Lubrol PX or at higher temperatures both enzymes were less heat stable, but PI kinase was still inactivated at a much greater rate than the ATPase. The evidence suggests that the ATPase and the PI kinase are different proteins.
The major phosphorylated product was diphosphatidylinositol and once formed, it was stable. Phosphorylation of membrane protein accounted for less than 10% of the total ³²P-incorporated into chromaffin vesicles. SDS gel electrophoresis of the solubilized membranes showed the presence of at least 2 major phosphorylated high molecular weight components.     

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.     

DNA POLYMERASE ACTIVITY FROM THE BRAIN OF OCTOPUS VULGARIS LAM

Abstract— The DNA polymerase of the optic lobes of Octopus vulgaris Lam has been solubilized and some of its properties have been investigated. The enzyme catalyses the incorporation of [³H]thymidine triphosphate into a DNase-sensitive, acid-insoluble product in the presence of DNA, the four deoxynucleoside triphosphates and Mg²⁺. The pH optimum is between pH 9.2 and 9.6, the optimal concentration of Mg²⁺ is between 10 and 30 mM and the optimal temperature is in the range 27°-30°C. The content of enzyme per mg protein is highest in the vertical and optic lobes and lowest in the suboesophageal lobe.     

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 L-asparaginase from Bacillus sp. isolated from an intertidal marine alga (Sargassum sp.)

B.R. MOHAPATRA, R.K. SANI AND U.C. BANERJEE. 1995. The bacterial flora associated with an intertidal marine alga ( Sargassum sp.) were screened for the presence of extracellular L-asparaginase; one out of five Bacillus strains was found positive. The maximum L-asparaginase activity was found at 37°C and pH 8.0. The optimum NaCl concentration for enzyme activity was found to be 2% (w/v). The enzyme activity was not affected by the addition of different metal ions (Ca²⁺, Co²⁺, Fe²⁺, Mg²⁺and Ni²⁺) at 10 mmol 1^-1, but was strongly inhibited by EDTA.     

Ontogenesis of Ca++ and Mg++ Contents of Embryonic Chick Heart

The Ca⁺⁺ and Mg⁺⁺ contents of embryonic chick heart were studied by atomic absorption spectrophotometry during a period from 48 h of foetal development until 2-3 days post-hatching. The hearts were isolated and incubated for 40 min at 22°C in three different media aerated with 95% 0₂-5% C0₂. The media included: normal Ringer's; Ca⁺-free Ringer's with 3 mM EGTA; and Ca⁺⁺-free Ringer's with 3 mM EDTA. At 48 h, the tubular myocardium contained 7-3 mM Ca⁺⁺ per wet weight which decreased rapidly to 1-2 mM by 10 days of development and remained between 0-9 and 1-1 mM until hatching. The Ca⁺⁺ content paralleled the changes in Na⁺ content reported earlier. Treatment with excess chelators, EGTA or EDTA, resulted in removal of 65-75% of the Ca⁺⁺ content throughout development until the time of hatching, when 50% of the Ca⁺⁺ became firmly bound. In contrast to the results with Ca⁺⁺, myocardial Mg⁺⁺ content rose rapidly from an initial value of 3.2 mM at 48 h to 6.7 mM by the 5th day of development, and then gradually declined throughout the remaining foetal development to 4.8 mM 2-3 days post-hatching. The Mg⁺⁺ contents closely paralleled changes in K⁺ content during development, which were reported earlier. Treatment with EGTA and EDTA removed 13-22% and 19-28% of the myocardial Mg⁺⁺, respectively, during development until just prior to hatching, when only 10-12% could be removed by chelation.     

Cyclic Nucleotides and the Release of Vasopressin from the Rat Posterior Pituitary Gland

Abstract: The effect of ATP, Mg²⁺, or MgATP on the release of luteinizing hormone-releasing hormone (LH-RH) from hypothalamic granules was examined under in vitro conditions. Granules, isolated from adult male hypothalami, were incubated at 37°C in a buffered (pH 7.8) medium containing 0.15 m -KCl. The addition of ATP to the incubation mixture did not stimulate the release of LH-RH. In contrast, the addition of MgATP stimulated the release of LH-RH, the release being 62% greater than control. The addition of Mg²⁺ to the incubated granules also stimulated the release of LH-RH. However, the magnitude of this Mg²⁺-stimulated release of LH–RH was significantly ( P < 0.01) lower than that of the MgATP-stimulated release, indicating that ATP stimulates LH-RH release in a Mg²⁺-dependent manner. As both MgATP and Mg²⁺ alone stimulated LH-RH release, we characterized further these two release processes by incubating the granules under one of the following conditions: incubation at 4°C in a buffered medium containing 0.15 m -KCl or incubation at 37°C in a medium that does not contain KCl. Under these two incubation conditions, the MgATP-stimulated release of LH-RH was not manifested, whereas the Mg²⁺-stimulated release of LH-RH was manifested. On the basis of these differences, we propose that two different processes can lead to the release of LH-RH from isolated hypothalamic granules: one process involves ATP and Mg²⁺ (MgATP) and another process involves Mg²⁺ alone.     

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.     

β-Amylase from Clostridium thermocellum SS8 - a thermophilic, anaerobic, cellulolytic bacterium

The extracellular amylase produced by Clostridium thermocellum strain SS8 on starch was characterized as a β-amylase based on blue value reduction test and the production of maltose from starch. The enzyme had a temperature and pH optima of 60°C and 6.0, respectively. Of the metal ions tested, Ca^{2 +} and Mg^{2 +} had little effect on enzyme activity, but their presence increased its thermal stability. Ca^{2 +} displayed a higher stabilizing effect and at 10 mmol 1^-1 Ca^{2 +}, the enzyme retained 86% activity even after exposure at 70°C for 30 min. The amylase was induced on starch or maltose but was repressed strongly by glucose.     

Parallel stimulation of mitochondrial H+ (Mg2+-ATPase and rate of germ tube outgrowth from conidia of Neurospora crassa

Abstract Mitochondrial H⁺ (Mg²⁺)-ATPase (EC 3.6.1.3) activity of Neurospora crassa has been studied during conidial germination in the presence and absence of 2,4-dinitrophenol and dicoumarol. The activity was optimal at pH 8.5–9.0 and was inhibited (80–90%) by oligomycin, N , N '-dicyclo-hexylcarbodiimide and azide.
The enzyme activity already present in mitochondria isolated from dry- and wet-harvested conidia did not change significantly during the 2.5 h pregermination at 25°C (20% outgrowth), but increased sharply up to 7.5 h corresponding to the end of germination period and remained steady until the end of log phase (15 h).
In uncoupler-treated conidia, the stimulation of germ tube outgrowth at 2.5 h was found to parallel the activation (40%) of mitochondrial ATPase.     

EFFECTS OF SULFHYDRYL REAGENTS ON BRAIN MICROTUBULE-ASSOCIATED ATPase ACTIVITY IN VITRO

Abstract— The effects of two sulfhydryl reagents, PCMBS ( p -chloromercuribenzene sulfonic acid) and NEM ( N -ethylmaleimide) on microtubule-associated Mg²⁺ -and Ca²⁺ -ATPase activity were studied in a MTP (microtubule proteins) preparation and in a MAP (microtubule-associated proteins) fraction. In the MTP preparation at pH 6.8, PCMBS stimulated the Mg²⁺ -ATPase activity at low concentrations and inhibited at higher, whereas the Ca²⁺ -ATPdse activity was only inhibited. NEM affected the activity in a similar way. At pH 8.0 PCMBS was only inhibitory. NEM showed stimulatory effects over a broader concentration range.
Preincubation in the presence of ATP counteracted the stimulatory effects of both PCMBS and NEM on Mg²⁺ -ATPase at pH 6.8.
In the MAP fraction at pH 6.8 PCMBS and NEM caused similar but less pronounced effects on the Mg²⁺ -and Ca²⁺ -ATPase.
The results show that brain microtubule-associated ATPase activity is similar to dynein and myosin ATPases with respect to biphasic alteration by sulfhydryl reagents.     

PROTEIN PHOSPHORYLATION IN VITRO IN BRAIN TUBULIN PREPARATIONS: EFFECTS OF Zn2+ AND CYCLIC NUCLEOTIDES

Abstract— Endogenous protein phosphorylation has been studied during in vitro polymerization of microtubules by incubating a purified tubulin preparation at 37°C in the presence of radioactive ATP. At optimal conditions the rate of phosphorylation was found to follow the course of polymerization by a shift to a lower rate at the polymerization plateau.
Zn²⁺ at 0.5 m m was shown to stimulate phosphorylation, mainly of tubulin-associated proteins (mol wt 110,000 and 175,000,) and to a lesser extent of tubulin. The effect occurred at Zn²⁺-concentrations which induce formation of tubulin sheet polymers, which suggests that the state of aggregation of tubulin is of importance for the phosphorylation. In contrast to Zn²⁺, Mg²⁺ only increased phosphorylation of the high molecular weight proteins, and to a lesser degree. The stimulation by Zn²⁺ or Mg²⁺ was potentiated by cyclic AMP or cyclic GMP.
A low concentration of Zn²⁺ (5 μ m ) or cyclic GMP at 10 μ m inhibited phosphorylation, possibly by interaction with a co-existing protein phosphatase.     

Characterization of urethanase from Micrococcus species associated with the marine sponge (Spirasfrella species)

B.R. Mohapatra and M. Bapuji. 1997. Eighty per cent of the urethanase activity of Micrococcus sp. isolated from the marine sponge Spirastrella sp. was cell associated. The urethanase had optimal activity at pH 5.0 and 45 °C. The activation and deactivation energies of the partially purified enzyme, calculated from an Arrhenius plot, were 9.31 and 34.01 kcal mol⁻¹, respectively. The enzyme was not affected by EDTA and the major cations of sea water, such as Ca²⁺, Mg²⁺ and Na⁺. The enzyme was resistant to 20% (v/v) ethanol and may be practically applicable in the removal of urethane from alcoholic beverages.     

The influences of calcium and magnesium ions on the exchange of monovalent cations in Neurospora crassa

Low-K⁺, high-Na⁺ cells of strain RL21a of Neurospora crassa , in steady state with 25 m M Na⁺, were used to study K⁺/Na⁺ exchanges in the presence or absence of Ca²⁺ and Mg²⁺. In the presence of Ca²⁺ and Mg²⁺, a low concentration of K⁺ (0.3 m M ) triggered a rapid exchange, but in the absence of the divalents, a high K⁺ concentration (30 m M ) was required to initiate the exchange at a rapid rate. In the absence of Ca²⁺ and Mg²⁺, K⁺ uptake did not occur at low K⁺ concentration, internal K⁺ did not regulate Na⁺ influx in the presence of external K⁺, and the efflux of Na⁺ proceeded at maximum activity at very low-K⁺ contents.     

Effects of sub-minimal inhibitory concentrations of EDTA on growth of Escherichia coli and the release of lipopolysaccharide

Abstract Release of lipopolysaccharide from E. coli was studied in the presence of sub-minimal inhibitory concentrations of ethylenediaminetetraacetic acid (EDTA). In untreated cells no release was detected with 50 mM Mg²⁺ in the medium, but a steady release of over 50% of the synthesized lipopolysaccharide was observed with 0.1 mM Mg²⁺. EDTA at MIC/8 led to a 2- to 3-fold higher release, presumably by an adjustment of the concentration of unchelated Mg²⁺ to a value still sustaining normal growth but giving rise to a highly unstable outer membrane. No structural difference was observed between cell-bound and released lipopolysaccharide.     

Induction of Fertilization Membrane Formation and Cyanide-insensitive Respiration in Sea Urchin Eggs by the Treatment with Dimethylsulfoxide Followed by an Incubation in an Ice Bath

In unfertilized eggs of the sea urchin Hemicentrotus pulcherrimus , fertilization membrane formation was induced by an incubation with dimethylsulfoxide (DMSO) for several min at 20°c followed by another incubation in an ice bath. The number of eggs with fertilization membrane, thus obtained, increased in relation to the concentration of DMSO between 1 and 3% (v/v) and was higher than 75% at concentrations above 3%. Fertilization membrane formation by this treatment occurred in Ca²⁺ free- or Ca²⁺, Mg²⁺ free- artificial sea water containing EGTA (50 mM) and was inhibited by verapamil. In the presence of DMSO, the membrane formation was also induced by 2, 4-dinitrophenol or cyanide in considerable number of eggs at 20°c. Eggs remained fertilizable, even when they were kept with DMSO for 1 hr at 20°c. DMSO slightly enhanced respiratory rate in unfertilized eggs and substantially reduced it in fertilized eggs. DMSO-treated eggs exhibited cyanide-insensitive respiratory burst following chilling in an ice bath or by adding DNP or cyanide, in a similar manner to the burst induced by sperm.     

Light modulation and in vitro effects of adenine nucleotides on leaf nitrate reductase activity in cucumber (Cucumis sativus)

Nitrate reductase (NR, EC 1.6.6.1) activity in attached cucumber ( Cucumis sativus L. cv. Ashley) leaves changed rapidly and reversibly during light/dark transitions, especially when assayed in the presence of free Mg²⁺. Light decreased and darkness increased the sensitivity of the enzyme to inhibition by Mg²⁺. The NR activation state, i.e. activity in the presence of Mg²⁺ relative to activity in the absence of Mg²⁺, increased with light intensity up to 400 μmol m⁻² s⁻¹ PAR (photosynthetically active radiation). When a desalted crude extract from illuminated leaves was preincubated with ATP, NR was gradually inactivated. Inactivation was only observed when activity was assayed in the presence of Mg²⁺. The ATP-inactivated NR remained inactive after removing the excess of ATP by gel filtration and it did not occur in partially purified NR preparations. NR extracted from darkened attached leaves was markedly activated when preincubated with 5'-AMP. These results support the view that inactivation/activation of cucumber-leaf NR in response to light/dark signals most likely involves phosphorylation/dephosphorylation of the enzyme catalysed by endogenous proteins. A substantial activation of NR by preincubation with 5'-AMP was also observed when activity was assayed in the absence of Mg²⁺, thus indicating that 5'-AMP can directly activate NR. Irradiation of an extract from darkened leaves containing FAD promoted a partial activation of NR. This effect was observed both in the +Mg²⁺ and in the −Mg²⁺ assay, indicating that activation was caused by photoexcited flavin and did not involve dephosphorylation of the enzyme.     

Molecular cloning of aromatic degradative genes from Pseudomonas stutzeri

Abstract Using dialysed cell-free extracts of the purple non-sulphur bacterium Rhodomicrobium vannielii protein kinase activities capable of transferring the gamma phosphate group from gamma [³²P]ATP to a variety of polypeptides were detected. The optimum concentration of Mg²⁺ for protein kinase activity was about 20 mM and the phosphorylation of one polypeptide ( M _r 47 kDa) was inhibited by chlorpromazine, a calmodulin antagonist, and also by Ca²⁺. The activity of at least one of the protein kinases (or a phosphatase) was regulated by ribulose 1,5-bisphosphate.     

Exoprotease activity of Leuconostoc oenos in stress conditions

Exoprotease activity during 48 h of total energy and nutrient starvation was examined in Leuconostoc oenos X₂L isolated from wine. Starved cells after 2 h of incubation at 30 °C in citrate buffer, 0.05 mmol 1⁻¹ pH 5, showed greater extracellular proteolytic activity than at the onset of starvation. In the presence of 60 mg l⁻¹ SO₂ and 8% or 12% ethanol, the proteolytic activity was higher ; 10 mmol l⁻¹ Ca²⁺ and Mg²⁺ produced an increase in protease activity during starvation. Glucose and 2-deoxyglucose (2-DOG) were found to repress synthesis by 80% and 100%, respectively. Cyclic adenosine 3'-5'-phosphate increased the exoprotease activity and reverted the repression by glucose and 2-DOG. De novo synthesis of proteins was required for the exoprotease activity by cells submitted to stress conditions. The absence of protease activity in the supernatant fluids from chloramphenicol-treated cells indicated that the activity is a result of deliberate release and not of passive cell lysis.     

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.