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.     

The involvement of a G-protein in phytochrome-regulated, Ca2+-dependent swelling of etiolated wheat protoplasts

The red light (R)-induced swelling of mesophyll protoplasts, isolated from dark-grown wheat ( Triticum aestivum L. cv. Arminda) leaves, was inhibited by guanosine-5'-0-(2-thiodiphosphate) (GDP-β-S). In darkness or after control irradiation with far-red light (FR), guanosine-5'-O-(3-thiotriphosphate) (GTP-γ-S) induced swelling to the same extent as after R. Both GDP-β-S and GTP-γ-S were introduced into the cytoplasm by means of electroporation. The possibility of R-induced activation of the phosphatidylinositol pathway of transmembrane signalling was investigated. Neomycin, Li⁺ and l-(5-isoquinolinesulfonyl)-2-methylpiperazine (H₇) inhibited the R-induced swelling. Phorbol 12-myristate 13-acetate (PMA) induced swelling after control irradiation with FR. Neomycin and Li⁺ also inhibited GTP-γ-S-induced swelling. These results suggest that a GTP-binding protein is involved in the phytochrome-regulated swelling response. Addition of N⁶, 2'-0-dibutyryladenosine 3':5'-cyclic monophosphate (DB-cAMP) induced swelling to the same extent as R-irradiation. The calmodulin antagonist N-(6-aminohexyl)5-chloro-l-naphthalenesulfonamide (W₇) induced swelling after FR, while R-induced swelling was not affected. The less active analogue N-(6-aminohexyl)-l-naphthalenesulfonamide (W₅) induced no swelling after FR. It is speculated that the protoplast volume is correlated with the cytoplasmic concentration of free Ca²⁺.     

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.     

Characterisation of maltase from enteropathogenic Escherichia coli

Abstract Enteropathogenic strains of faecal Escherichia coli produced significantly ( P < 0.01) more maltase than the non-pathogenic strains of the organism. The enzyme was induced by maltose but repressed by glucose and fructose. The maltase was partially purified by ammonium sulphate precipitation, followed by dialysis and gel permeation chromatography. The partially purified maltase had an M _r of 144500 and an apparent K _m of approx. 7.6 mM for maltose. The enzyme was stimulated by Ca²⁺, inhibited by Cu²⁺, Hg²⁺, Uo²⁺, IAA and EDTA, and exhibited optimum activity at pH 6.5 at 30°C.     

Anti-Candida activity of four antifungal benzothiazoles

Abstract Anti- Candida activity of 6-amino-2- n -pentylthiobenzothiazole (I), benzylester of (6-amino-2-benzothiazolylthio)acetic acid (II) and of 3-butylthio-(1,2,4-triazolo)-2,3-benzothiazole (III) was followed and compared to that of 2-mercaptobenzothiazole (IV). I and II exhibited good activity against the C. albicans yeast form, similar to IV. They were inhibitorily active against other Candida strains, IC₅₀ values being of the order of 10⁻⁵ M, which means better activity than IV. Compound I also exhibited inhibitory activity on germ-tube formation and mycelial growth in the C. albicans strains, while II, III and IV were not active in these tests. III was the least active form of the compounds tested, IC₅₀ values being of the order of 10⁻⁴ M. All the compounds tested were highly active on a nystatin-resistant C. albicans mutant, with IC₅₀s of the order of 10⁻⁶ M−10⁻⁵ M.     

SALT-DEPENDENT PROPERTIES OF HATCHING ENZYME FROM EMBRYOS OF THE SEA URCHIN, HEMICENTROTUS PULCHERRIMUS

The effect of salts on hatching enzyme and protease from the embryo of the sea urchin, Hemicentrotus pulcherrimus , was investigated. The culture medium containing hatching enzyme secreted from the hatched blastula was dialyzed against Tris-HCl (pH 8.0) with or without salts. Both hatching enzyme and protease were activated and stabilized by CaCL₂, NaCI and KCI, while inhibited by MgCI₂. Protease activity was maximal at about 0.25 M NaCI. KCI, NH₄, CI and LiCI. Maximal activity of hatching enzyme was obtained at 0.5 M NaCl, KCI and NH₄ CI, while activity was inhibited by any concentration of LiC1. Among monovalcnt cations, the order of activation was NaCI, KCI > NH₄Cl. The activity of hatching enzyme was stabilized by dialysis against 1 M NaCI or KCI in the presence of CaCl.₂, but was rapidly lost by dialysis against lower concentrations of salts. Reactivation of hatching enzyme was not achieved by redialysis against I M NaCI. On the other hand, protease was reactivated by I M NaCI or KCI. From these results, hatching enzyme of the sea urchin may be called a moderate halophilic enzyme. It was assumed that at least two enzymes exist in the crude enzyme preparation and that they may have different functions.     

Purification and characterization of an endoamylase from tulip (Tulipa gesneriana) bulbs

Amylase activity extracted from tulip ( Tulipa gesneriana L. cv. Apeldoorn) bulbs that had been stored for 6 weeks at 4°C was resolved to 3 peaks by anion-exchange chromatography on diethylaminoethyl-Sephacel. These 3 amylases exhibited different relative mobilities during non-denaturing polyacrylamide gel electrophoresis (PAGE). The most abundant amylase form (amylase I) was purified to apparent homogeneity using hydrophobic interaction chromatography, gel filtration and chromatofocusing. The apparent molecular mass of the purified amylase was estimated to be 51 kDa by sodium dodecyl sulfate-PAGE and 45 kDa by gel filtration chromatography. The purified amylase was determined to be an endoamylase (EC 3.2.1.1) based on substrate specificity and end-product analysis. The enzyme had a pH optimum of 6.0 and a temperature optimum of 55°C. The apparent K_m value with soluble starch (potato) was 1.28 mg ml⁻¹. The presence of Ca²⁺ increased the activity and thermal stability of the enzyme. The presence of dithiothreitol enhanced the activity, while β -mercaptoethanol and reduced glutathione had no significant effect. When pre-incubated in the absence of the substrate, N-ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoic acid) partially inhibited the enzyme. α -cyclodextrins or β -cyclodextrins had no effect on enzyme activity up to 10 m M . In addition to CaCl₂, CoCl₂ slightly enhanced activity, while MgCl₂ and MnCl₂ had no significant effect at a concentration of 2 m M . ZnCl₂, CuSO₄, AgNO₃ and EDTA partially inhibited enzyme activity, while AgNO₃ and HgCl₂ completely inhibited it at 2.0 m M .     

Purification and characterization of 3-isopropylmalate dehydrogenase from a thermoacidophilic archaebacterium Sulfolobus sp. strain 7

Abstract 3-Isopropylmalate dehydrogenase was purified (about 2000-fold) to homogeneity for the first time from an archaebacterium, Sulfolobus sp. strain 7. The enzyme showed an apparent molecular mass of about 110 kDa by gel filtration and a single 36-kDa polypeptide band on SDS-PAGE, suggesting tri- or tetrameric structure. The p I value was 6.9. The N-terminal amino acid sequence was similar to enzymes from other sources. The enzyme activity was greatly stimulated by the presence of Mn²⁺, Cd²⁺, Mg²⁺, or Co²⁺. In contrast to 3-isopropylmalate dehydrogenase from other sources, monovalent cations such as K²⁺ and Na²⁺ were neither essential for activity nor stability of the protein. The enzyme was extraordinarily thermostable.     

Interaction of α-Phenyl-N-tert-Butyl Nitrone and Alternative Electron Acceptors with Complex I Indicates a Substrate Reduction Site Upstream from the Rotenone Binding Site

Abstract: Mitochondrial complexes I, II, and III were studied in isolated brain mitochondrial preparations with the goal of determining their relative abilities to reduce O₂ to hydrogen peroxide (H₂O₂) or to reduce the alternative electron acceptors nitroblue tetrazolium (NBT) and diphenyliodonium (DPI). Complex I and II stimulation caused H₂O₂ formation and reduced NBT and DPI as indicated by dichlorodihydrofluorescein oxidation, nitroformazan precipitation, and DPI-mediated enzyme inactivation. The O₂ consumption rate was more rapid under complex II (succinate) stimulation than under complex I (NADH) stimulation. In contrast, H₂O₂ generation and NBT and DPI reduction kinetics were favored by NADH addition but were virtually unobservable during succinate-linked respiration. NADH oxidation was strongly suppressed by rotenone, but NADH-coupled H₂O₂ flux was accelerated by rotenone. α-Phenyl- N-tert -butyl nitrone (PBN), a compound documented to inhibit oxidative stress in models of stroke, sepsis, and parkinsonism, partially inhibited complex I-stimulated H₂O₂ flux and NBT reduction and also protected complex I from DPI-mediated inactivation while trapping the phenyl radical product of DPI reduction. The results suggest that complex I may be the principal source of brain mitochondrial H₂O₂ synthesis, possessing an "electron leak" site upstream from the rotenone binding site (i.e., on the NADH side of the enzyme). The inhibition of H₂O₂ production by PBN suggests a novel explanation for the broad-spectrum antioxidant and antiinflammatory activity of this nitrone spin trap.     

Characterization of alpha-galactosidase from Lactobacillus fermentum

α-Galactosidase activity was studied in Lactobacillus fermentum strains. The optimum temperature was found to be 45°C. The enzyme was inactivated at temperatures higher than 55°C, but remained active during storage at low temperatures (0, -30 and -70°C) for 5 months. Enzyme activity was observed within a 5.0–6.5 pH range, while optimum pH was dependent on the particular strain assayed. The addition of Zn²⁺ to the reaction buffer exerted a slight negative effect upon the activity, while Hg²⁺ and p -chloromercuribenzoate produced a strong inhibition. These results would indicate the presence of -SH groups in the catalytic site of the enzyme.     

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.     

Characterization of a Novel Phospholipase A2 Activity in Human Brain

Abstract: Phospholipases A₂ (PLA₂) are a family of enzymes that catalyze the removal of fatty acid residues from phosphoglycerides. The enzyme is postulated to be involved in several human brain disorders, although little is known regarding the status of PLA₂ activity in human CNS. We therefore have characterized some aspects of the PLA₂ activity present in the temporal cortex of human brain. More PLA₂ activity was found in the membrane (particulate) fraction than in the cytosolic fraction. The enzyme could be solubilized from particulate material using 1 M potassium chloride, and was capable of hydrolyzing choline phosphoglyceride (CPG) and ethanolamine phosphoglyceride (EPG), with a preference (approximately eightfold) for EPG over CPG. When the solubilized particulate enzyme was subjected to gel filtration chromatography, PLA₂ activity eluted in a high molecular mass fraction (∼180 kDa). PLA₂ activity was weakly stimulated by dithiothreitol, strongly stimulated by millimolar concentrations of calcium ions, and inhibited by brief heat treatment at 57°C, bromophenacyl bromide, the arachidonic acid derivative AACOCF₃, γ-linolenoyl amide, and N -methyl γ-linolenoyl amide. Thus, whereas the human brain enzyme(s) characterized in our study displays some of the characteristics of previously characterized PLA₂s, it differs in several key features.     

Effects of mitochondrial complex III disruption in the respiratory chain of Neurospora crassa

In mitochondria from most organisms, including Neurospora crassa , dimeric complex III was found associated with complex I. Additional association of complex IV with this core structure leads to the formation of a respirasome. It was recently described for bacteria and mammals that complex III is needed for the assembly/stability of complex I. To elucidate the role of complex III in the organization of the respiratory chain of N. crassa , we analysed strains devoid of either the Rieske iron-sulphur or the COREII polypeptide subunits. The mutants display reduced growth, are female sterile and lack active complex III. The supramolecular organization of the oxidative phosphorylation system was characterized by electrophoretic analyses and the efficiency of the respiratory chain analysed by oxygen consumption measurements. The results obtained indicate that absence of complex III activity is not associated with the absence of complex I or complex IV, and leads to the induction of alternative oxidase. Complex III mutant mitochondria are devoid of respirasomes but contain significant amounts of dimeric complex I (I₂) and of the supercomplex I₁IV₁. Moreso, for the first time the alternative oxidase was found associated with dimeric complex IV and with supercomplex I₁IV₁.     

Genetic differences in physiology, growth hormone levels and migratory behaviour of Atlantic salmon smolts

Out of five strains of Atlantic salmon Salmo salar of 1+ years released upstream of a fyke net in the River Gudenaa in 1996, three, Lagan, Ätran and Corrib, migrated immediately, 50% of the recaptured fish reaching the net in 3–6 days. Burrishoole and Conon fish migrated with a 15–19 day delay. Smolt development in 1997 at the hatchery showed a spring surge in gill Na⁺, K⁺-ATPase activity in all strains which was correlated with increased seawater tolerance. Differences in the timing of gill enzyme development matched the observed migration pattern well. Lagan, Ätran and Corrib strains reached high enzyme activity earlier than the Burrishoole and Conon strains, and strains with delayed enzyme development and migration showed a delayed regression of seawater tolerance compared with the early strains. Inter-strain differences in plasma growth hormone profiles could not be related to the observed patterns of Na⁺, K⁺-ATPase and seawater tolerance development. The study gives evidence of genetic influence on the timing and intensity of smolting and subsequent migration in Atlantic salmon.     

Reconstitution and rapid partial purification of the red beet plasma membrane ATPase

Red beet ( Beta vulgaris L., cv. Detroit Dark Red) plasma membrane ATPase solubilized from a deoxycholate-extracted plasma membrane fraction with Zwittergent 3–14 was reconstituted into liposomes. Detergent removal and reconstitution was carried out by column chromatography on Sephadex G-200 followed by centrifugation at 100 000 g for I h. Prior to reconstitution, optimal activity in the solubilized preparation was observed when dormant red beet tissue was used in the extraction/solubilization procedure. Following reconstitution into liposomes, ATP-dependent proton transport could be demonstrated by measuring the quenching of acridine orange fluorescence. Proton transport and ATPase activity in the reconstituted enzyme preparation were inhibited by orthovandate but stimulated by KNO₃. This stimulation most likely results from a reduction in the membrane potential generated during electrogenic proton transport by the reconstituted ATPase. The ATPase activity of the reconstituted ATPase was further characterized and found to have a pH optimum of 6.5 in the presence of both Mg²⁺ and K⁺. The activity was specific for ATP, insensitive to ouabain and azide but inhibited by N;N-dicyclohexylcarbodiimide and diethylstilbestrol. Stimulation of ATP hydrolytic activity occurred in the sequence: K⁺ Rb⁺ Na⁺ Cs⁺ Li⁺ and the kinetics of K⁺ stimulation of ATPase activity followed non-Michaelis-Menten kinetics as observed for both the membrane-bound and solubilized forms of the enzyme. Reconstitution of the plasma membrane ATPase from red beet allowed a substantial purification of the enzyme and resulted in the enrichment of a 100 kDa polypeptide representing the ATPase catalytic subunit.     

Purification and properties of a neutral invertase from the roots of Cichorium intybus

Multiple activity peaks of neutral invertase (EC 3.2.1.26) were found in chicory roots ( Cichorium intybus L. var. foliosum cv. Flash). The main activity peak was purified by a combination of anion-exchange chromatography, hydrophobic interaction chromatography, chromatofocusing and gel filtration. This protocol produced a 77-fold purification and a specific activity of 1.6 μmol (mg protein)⁻¹ min⁻¹. The mass of the enzyme was 260 kDa as estimated by gel filtration and 65 kDa on SDS-PAGE. Optimal activity was found between pH 7 and 7.5. The purified enzyme exhibited hyperbolic saturation kinetics with a K_m between 10 and 20 mM for sucrose. No other products than glucose and fructose could be detected. Raffinose was hydrolyzed at a rate of 2.4% relative to sucrose whereas the enzyme did not hydrolyze maltose, cellobiose, trehalose, 1-kestose, 1.1-nystose or inulin. Neutral invertase activity was completely inhibited by HgCl₂ and AgNO₃ and partially inhibited by CoCl₂, and ZnSO₄ (1 mM). Pyridoxal phosphate (K_i≅ 500 μ M ), Tris (K_i≅ 1.2 m M ), glucose and fructose (K_i≅ 16 m M ) were strong inhibitors of the enzyme. Fructose and Tris behaved as competitive inhibitors. A possible role for the enzyme's activity in vivo is discussed.     

Glycolate metabolism in cyanobacteria

A comparative analysis of glycolate excretion in 11 cyanobacteria showed that 8 strains, although grown and assayed in air, excreted glycolate. The largest quantities were excreted by the filamentous strains Plectonema boryanum 73110 and Anabaena cylindrica (Lemm). The carbon lost by excretion was at most 9% of the net fixed carbon in air for heterocystous cyanobacteria but increased (up to 60%) in some strains under a high pO₂ (0.03 kPa CO₂ in pure O₂). A. cylindrica excreted glycolate at a maximum level of 2 and 10 μmol (mg chl a )⁻¹ h⁻¹ in air and at high pO₂, respectively. The excretion continued for several hours. Increases in light intensity and pO₂ and a shift in pH from 7 to 9 increased the amount of glycolate excreted. A. cylindrica also showed the most O₂-sensitive fixation of CO₂. In vitro activity of phosphoglycolate phosphatase (EC 3.1.3.18) was found in all strains tested, with the highest activities noted for Gloeobacter violaceus 7.82 and Gloeothece 6909 and for young cultures of A. cylindrica . The lowest activities were found in Anabaena 7120 and Anacystis nidulans 625, strains excreting no or only minor quantities of glycolate.     

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.     

Phosphoryl Group Transfer by a Fraction of the Soluble Proteins of Catecholamine Storage Vesicles

Abstract: The terminal phosphate group of ATP was transferred to ADP by an enzyme present in the soluble core proteins of adrenal medulla catecholamine storage vesicles. It was purified 10–30-fold by DEAE Sephadex chromatography (Fraction I). The enzyme required divalent metal ions for activation; Mn²⁺ was almost as effective as Mg²⁺, but Ca²⁺ was only a weak activator. Activation by Mg²⁺ took place over a very narrow concentration range (0.5–3 m m ). The specificity of the enzyme activity to nucleoside triphosphates was broad, to the nucleoside diphosphates narrow, favouring adenosine diphosphate. In dependence on the pH the activity increased from pH 4 to pH 7 and remained constantly high between pH 7 and 9. The Arrhenius plot was linear between 5 and 70°C, with an activation energy of 11.1 kcal/mol. The phosphoryl group transfer reaction depended on the function of thiol groups; p -hydroxymercuribenzoate inhibited 50% of the enzyme activity; dithioerythritol reactivated it completely. Gel electrophoresis revealed that in Fraction I, a protein of molecular weight about 45,000, was enriched compared with the total soluble proteins. The enzyme-enriched Fraction I differed significantly in its relative amino acid composition from that of the total soluble proteins; in general, the acidic amino acids were reduced and the more basic acids enhanced.