Effects of chemical and physico-chemical factors on sugar-beet invertase activity

Sugar-beet invertase preparation was found to be highly temperature-dependent in its activity (T_opt = 40 °C), to be decreased by bivalent metal salts (especially CaCl₂ and MgCl₂ and relatively unaffected by heavy metal cations from the end of the periodic table. Inhibitors of SH groups, such as iodoacetamide andp-chloromercuribenzoate, were highly inhibitory while ouabain was a medium-strength inhibitor, with 2,4-dinitrophenol having no effect. The intracellular pH of 7.3, corresponding to the optimum for invertase (at 7.2) could be substantially decreased (to 5.8) by exposure of cells of whole sugar-beet roots to carbon dioxide in aqueous solution.     

Two classes of arginine vasopressin binding sites on rat brain membranes

Specific binding sites for arginine vasopressin (AVP) were demonstrated on rat brain membranes using [³H]AVP of high specific activity. At pH 7.4 in the presence of 5 mM MgCl₂, one class of sites was measured with aK _D of 0.56 nM and aB _max of 4.3 fmol/mg protein. At pH 8.0 in the presence of MgCl₂, two distinct sites were observed, havingK _D values of 0.42 and 13 nM andB _max values of 5.6 and 68 fmol/mg protein, respectively, and similar results were obtained at pH 7.4 after repeatedly freezing and thawing the membranes. Binding increased with pH, apparently representing increased occupancy of the high capacity, lower affinity site. Binding to the lower affinity site was also enhanced by freezing and thawing membranes, or by adding 5 mM NiCl₂ or 10 M ZnCl₂ to the incubation medium, whereas binding to the high affinity site was dependent on the addition of Mg. AVP was over 35 times more active in displacing 0.4 nM AVP than oxytocin or arginine-vasotocin, and 10,000 times more active than somatostatin. A number of other peptides had no effect on [³H]AVP binding at concentrations up to 10^–5 M. Autoradiography and regional dissection studies revealed a marked concentration of high affinity AVP-binding sites in the supraoptic and paraventricular nuclei of the hypothalamus, and Mg significantly enhanced the binding in these regions.     

Characterization of an Invertase with pH Tolerance and Truncation of Its N-Terminal to Shift Optimum Activity toward Neutral pH

Most invertases identified to date have optimal activity at acidic pH, and are intolerant to neutral or alkaline environments. Here, an acid invertase named uninv2 is described. Uninv2 contained 586 amino acids, with a 100 amino acids N-terminal domain, a catalytic domain and a C-terminal domain. With sucrose as the substrate, uninv2 activity was optimal at pH 4.5 and at 45°C. Removal of N-terminal domain of uninv2 has shifted the optimum pH to 6.0 while retaining its optimum temperaure at 45°C. Both uninv2 and the truncated enzyme retained highly stable at neutral pH at 37°C, and they were stable at their optimum pH at 4°C for as long as 30 days. These characteristics make them far superior to invertase from Saccharomyces cerevisiae, which is mostly used as industrial enzyme.     

Trehalase,invertase, and amylase activities in the black scale,Saissetia oleae,and their relation to host adaptability

In young females of the black scale, Saissetia oleae, the optimum conditions for invertase activity involve a reaction mixture of pH 5.5 and 2% sucrose at 37°C for 60 min; for amylase, pH 6.0 and 0.5% starch at 37°C for 45 min; and for trehalase, pH 5.5 and 1.5% trehalose at 37°C for 60 min. At optimal conditions and using standard enzyme activity units, both invertase and trehalase activities were much higher (about 8-fold) than that of amylase, indicating the importance of these enzymes in food digestion and energy supply.The enzyme activities were strongly affected by various host plants. Trehalase activity in scales reared on potato sprouts was about 3.5- and 4-fold that obtained in scales reared on oleander and citrus plants, respectively. An increase of about 40% for invertase and 60% amylase activity was obtained in scales reared on potato sprouts as compared with those reared on oleander or citrus plants.A good correlation was observed between enzyme activity-especially of trehalase-and scale development. The duration of one generation of the black scale reared on potato sprouts was 2.5 to 3 months, on oleander 4 to 5 months, and on citrus above 6 months. These results suggest that trehalase and to some extent invertase could be used as parameters to assess the adaptability of the black scale to its host plant.     

Expression,purification and immobilization of the intracellular invertase INVA,from <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> on crystalline cellulose and Nylon-6

This paper presents two immobilization methods for the intracellular invertase (INVA), from Zymomonas mobilis. In the first method, a chimeric protein containing the invertase INVA, fused through its C-terminus to CBD _Cex from Cellulomonas fimi was expressed in Escherichia coli strain BL21 (DE3). INVA was purified and immobilized on crystalline cellulose (Avicel) by means of affinity, in a single step. No changes were detected in optimal pH and temperature when INVA-CBD was immobilized on Avicel, where values of 5.5 and 30 °C, respectively, were registered. The kinetic parameters of the INVA-CBD fusion protein were determined in both its free form and when immobilized on Avicel. K _m and V _max were affected with immobilization, since both showed an increase of up to threefold. Additionally, we found that subsequent to immobilization, the INVA-CBD fusion protein was 39% more susceptible to substrate inhibition than INVA-CBD in its free form. The second method of immobilization was achieved by the expression of a 6xHis-tagged invertase purified on Ni-NTA resin, which was then immobilized on Nylon-6 by covalent binding. An optimal pH of 5.5 and a temperature of 30 °C were maintained, subsequent to immobilization on Nylon-6 as well as with immobilization on crystalline cellulose. The kinetic parameters relating to V _max increased up to 5.7-fold, following immobilization, whereas K _m increased up to 1.7-fold. The two methods were compared showing that when invertase was immobilized on Nylon-6, its activity was 1.9 times that when immobilized on cellulose for substrate concentrations ranging from 30 to 390 mM of sucrose.     

Invertases in Oat Seedlings: SEPARATION, PROPERTIES, AND CHANGES IN ACTIVITIES IN SEEDLING SEGMENTS

The soluble invertase activity in etiolated Avena seedlings was highest at the apex of the coleoptile and much lower in the primary leaf, mesocotyl, and root. The activity in all parts of the seedling consisted of two invertases (I and II) which were separated by chromatography on diethylaminoethylcellulose. Both enzymes appeared to be acid invertases, but they differed in molecular size, pH optimum, and the kinetic parameters K_m and V_max of their action on sucrose, raffinose, and stachyose. Invertase II had low stability at pH 3.5 and below, and exhibited high sensitivity to Hg²⁺, with complete inhibition by 2 micromolar HgCl₂. Segments of coleoptiles incubated in water lost about two-thirds of the total invertase activity after 16 hours. The loss of activity was due primarily to a decrease in the level of invertase II. The loss of invertase was decreased by indoleacetic acid, 2,4-dichlorophenoxyacetic acid, and α-naphthaleneacetic acid but not by β-naphthaleneacetic acid and p-chlorophenoxyisobutyric acid. Conditions that inhibited auxin-induced growth of the segments (20 millimolar CaCl₂ and 200 millimolar mannitol) also blocked the auxin effect on invertase loss.     

Ouabain binding sites and the (Na+,K+)-ATPase of brain microsomal membranes

Beef brain microsomes bound approximately 180–220 pmoles of [³H]ouabain per mg of protein in the presence of either MgCl₂ and inorganic phosphate or ATP, MgCl₂ and NaCl. The ouabain-binding capacity and the ouabain-membrane complex were more stable than the (Na⁺,K⁺)-ATPase activity to treatment with agents known to affect the membrane integrity, such as, NaClO₄, sodium dodecyl sulfate, $\text{p-}\text{chloromercuribenzoate}$, urea. ultrasonication, heating, pH and phospholinase C.The presence of binding sites that were normally inaccessible to ouabain in brain microsomes was demonstrated. These sites appeared after disruption of microsomes with 2 M NaClO₄ as evidenced by increased binding of [³H]ouabain. These sites may be buried during the subcellular fractionation procedure and could be accessible in the intact cell.     

Effective solvent systems for separation and an improved detection method for amino acids by paper chromatoghraphy

Binding of poly(A)-containing RNP to oligo(dT)-cellulose has been investigated as a function of mono- and divalent ion concentration. 80–90% binding was obtained either in high (500 mM) or in moderate NaCl concentrations in the presence of 5 mM MgCl₂. At 40 mM NaCl and 5 mM MgCl₂ poly(A)⁺-RNP exhibit approximately t he same stability as poly(A)⁺-RNA in binding to oligo(dT)-cellulose with a melting temperature of 41 and 45°C, respectively, indicating that the protein moeity has no effect on the ribonucleoprotein binding in these conditions. Differences were observed int he elution of poly(A)⁺-RNA and poly(A)⁺-RNP from oligo(dT)-cellulose in buffer without salts. Poly(A)⁺-RNA was completely removed at 4°C whereas the melting temperature of poly(A)⁺-RNP was only decreased to 34°C. The isolation of poly(A)⁺-RNP by thermal elution from oligo(dT)-cellulose is described.     

Binding to the high-affinity substrate site of the (Na+ + K+)-dependent ATPase

The (Na⁺ + K⁺)-dependent ATPase exhibits substrate sites with both high affinity (K _m near 1 µM) and low affinity (K _m near 0.1 mM) for ATP. To permit the study of nucleotide binding to the high-affinity substrate sites of a canine kidney enzyme preparation in the presence as well as absence of MgCl₂, the nonhydrolyzable - imido analog of ATP, AMP-PNP, was used in experiments performed at 0–4°C by a centrifugation technique. By this method theK _D for AMP-PNP was 4.2 µM in the absence of MgCl₂. Adding 50 µM MgCl₂, however, decreased theK _D to 2.2 µM; by contrast, higher concentrations of MgCl₂ increased theK _D until, with 2 mM MgCl₂, theK _D was 6 µM. The half-maximal effect of MgCl₂ on increasing theK _D occurred at approximately 1 mM. This biphasic effect of MgCl₂ is interpreted as Mg²⁺ in low concentrations favoring AMP-PNP binding through formation at the high-affinity substrate sites of a ternary enzyme-AMP-PNP-Mg complex; inhibition of nucleotide binding at higher MgCl₂ concentrations would represent Mg²⁺ acting through the low-affinity substrate sites. NaCl in the absence of MgCl₂ increased AMP-PNP binding, with a half-maximal effect near 0.3 mM; in the presence of MgCl₂, however, NaCl increased theK _D for AMP-PNP. KCl decreased AMP-PNP binding in the presence or absence of MgCl₂, but the simultaneous presence of a molar excess of NaCl abolished (or masked) the effect of KCl. ADP and ATP acted as competitors to the binding of AMP-PNP, although a substrate for the K⁺-dependent phosphatase reaction also catalyzed by this enzyme,p-nitrophenyl phosphate, did not. This lack of competition is consistent with formulations in which the phosphatase reaction is catalyzed at the low-affinity substrate sites.     

The Regulation of Invertase Activity in the Latex of Hevea brasiliensis Muell. Arg: THE EFFECTS OF GROWTH REGULATORS, BARK WOUNDING, AND LATEX TAPPING

Treatments which increase latex yield, e. g. bark scraping,latex tapping, and bark application of 2, 4-D or 2-chloroethylphosphonicacid (CEPA) were found to enhance the activity of latex invertase.In previously untapped trees, both the introduction of tappingand the application of 2, 4-D brought about an increase in thelevel of invertase. In regularly tapped trees, the amount oflatex invertase is several times higher than in untapped treesand evidence was obtained that its activity is regulated bythe variation of latex pH. The pH of latex of the clone investigated(PR 107) was shown to vary between 6.3 and 7.1 whereas the activityof invertase, as assayed directly in the latex, has a sharpoptimum at pH 7.5 and falls rapidly with the shift of pH tothe acid side. There was no increase in the content of latexinvertase when trees adapted to regular tapping were treatedwith 2, 4-D. The effect of auxin on actual invertase activitywas essentially mediated through related increase of latex pH.The CEPA and bark scraping were also shown to increase latexpH in tapped trees. The treatment of the bark of tapped trees with CEPA increasedthe level of latex sucrose, as did auxins. Bark scraping alsohad a slight stimulatory effect. The K_m of latex invertase asa function of pH was found to change in the same way as V_max,being highest at pH optimum.     

Immobilized Sclerotinia sclerotiorum invertase to produce invert sugar syrup from industrial beet molasses by product

The fungus Sclerotinia sclerotiorum produces invertase activity during cultivation on many agroindustrial residues. The molasses induced invertase was purified by DEAE-cellulose chromatography. The molecular mass of the purified enzyme was estimated at 48 kDa. Optimal temperature was determined at 60 °C and thermal stability up to 65 °C. The enzyme was stable between pH 2.0 and 8.0; optimum pH was about 5.5. Apparent K_m and V_max for sucrose were estimated to be respectively 5.8 mM and 0.11 μmol/min. The invertase was activated by β-mercaptoethanol. Free enzyme exhibited 80 % of its original activity after two month’s storage at 4 °C and 50 % after 1 week at 25 °C. In order to investigate an industrial application, the enzyme was immobilized on alginate and examined for invert sugar production by molasses hydrolysis in a continuous bioreactor. The yield of immobilized invertase was about 78 % and the activity yield was 59 %. Interestingly the immobilized enzyme hydrolyzed beet molasses consuming nearly all sucrose. It retained all of its initial activity after being used for 4 cycles and about 65 % at the sixth cycle. Regarding productivity; 20 g/l of molasses by-product gave the best invert sugar production 46.21 g/day/100 g substrate related to optimal sucrose conversion of 41.6 %.     

A thermally induced alteration in lysosome membranes: Salt permeability at 0 and 37 °C

Preparations of radioactive lysosomes were obtained from mouse kidney after injection of radioactive iodine-labeled bovine ribonuclease. Stability of these lysosomes in various media was estimated from measurements of proteolytic activity towards the ribonuclease, and of ribonuclease retention in particles. The lysosomes were stable at 37 °C in isotonic, sucrose-free solutions of KCl, NaCl and potassium acetate, and in mixtures of these with MgCl₂, showing that these salts are relatively impermeant through the lysosomal membranes. The membranes were less permeable to Na⁺ than to K⁺. Both KCl and NaCl exerted their optimal protective effects over a broad concentration range above 0.125 M in 0.025 M acetate buffer. Mg²⁺ enhanced the protective effect of both K⁺ and Na⁺; the osmotic effect of 0.075 M NaCl-0.05 M MgCl₂ was indistinguishable during the entire course of ribonuclease digestion from that of isotonic sucrose. Osmotic protection by KCl-MgCl₂ was demonstrated over the pH range 5.5–7.0. A marked alteration in membrane properties occurs at lower temperatures in 0.11 M KCl-0.01 M MgCl₂ such that, at 0 °C, K⁺ permeability is much higher than at 37 °C, as shown by a several-fold decrease in stability at the lower temperature.     

Broad nucleotide cofactor specificity of DNA ligase from the hyperthermophilic crenarchaeon Hyperthermus butylicus and its evolutionary significance

The nucleotide cofactor specificity of the DNA ligase from the hyperthermophilic crenarchaeon Hyperthermus butylicus (Hbu) was studied to investigate the evolutionary relationship of DNA ligases. The Hbu DNA ligase gene was expressed under control of the T7lac promoter of pTARG in Escherichia coli BL21-CodonPlus(DE3)-RIL. The expressed enzyme was purified using the IMPACT?-CN system (intein-mediated purification with an affinity chitin-binding tag) and cation-ion (Arg-tag) chromatography. The optimal temperature for Hbu DNA ligase activity was 75 °C, and the optimal pH was 8.0 in Tris–HCl. The activity was highly dependent on MgCl₂ or MnCl₂ with maximal activity above 5 mM MgCl₂ and 2 mM MnCl₂. Notably, Hbu DNA ligase can use ADP and GTP in addition to ATP. The broad nucleotide cofactor specificity of Hbu DNA ligase might exemplify an undifferentiated ancestral stage in the evolution of DNA ligases. This study provides new evidence for possible evolutionary relationships among DNA ligases.     

Properties of a Membrane-bound Phosphatase from the Thylakoids of Spinach Chloroplasts

A 3-phosphoglycerate phosphatase activity of about 2 micromoles per minute per milligram chlorophyll is associated with the thylakoid membranes of spinach chloroplasts. The K_m for 3-phosphoglycerate is 3 millimolar. The enzyme can be solubilized from thylakoid membranes by treatment with 0.33 molar MgCl₂ or sodium deoxycholate. The activity is not stimulated by sulfhydryl reagents or the addition of 10 millimolar MgCl₂. The enzymic activity is insensitive to ethylenediaminetetraacetate. The pH optimum is broad, between 5.5 to 7.5. Although the substrate specificity is broad, 3-phosphoglycerate is the best substrate of those tested at neutral pH. However, p-nitrophenyl phosphate was a more effective substrate at pH 5.5. The enzyme exhibits the general characteristics of an acid phosphatase.     

Determination of Fatty Acid in Surfactin and Elucidation of the Total Structure of Surfactin

Several properties of ATPase bound to the inner membrane of a psychrophilic marine bacterium Vibrio sp. strain ABE-1 were examined. The membrane-bound ATPase had two optimal peaks of the activity at pH 5.8 and 7.3. The ATPase activity was strongly inhibited by N,N’- dicyclohexylcarbodiimide (DCCD) and NaN₃ at pH 5.8 and 8.0, and stimulated by MgCl₂ and CaCl₂ at pH 8.0. At pH 8.0, the enzyme hydrolyzed GTP and ITP as well as ATP but not AMP or p-nitrophenylphosphate. CTP, UTP, and ADP were poor substrates. These characteristics indicate that there is a F₀F₁-type ATPase in the inner membrane of this bacterium. In addition, the ATPase activity was also significantly inhibited by Na₃ Vo₄, suggesting the coexistence of a P-type ATPase as a minor constituent. The membrane-bound ATPase activity was maximum at 50°C, but the strong DCCD-sensitivity observed at 20°C was greatly reduced at this temperature.     

Release of [14C]tyrosine from tubulinyl-[14C]tyrosine by brain extract. Separation of a carboxypeptidase from tubulin-tyrosine ligase

Summary The carboxypeptidase previously described³ that releases tyrosine from tubulinyl-tyrosine was obtained from rat brain preparation free of tubulin-tyrosine ligase. The enzyme was purified 24-fold. Its activity was increased by 2 mm MgCl₂ or 30 mm KCl. Mercaptoethanol (50 mm), colchicine (0.2 mm) and tyrosine (0.2 mm) showed practically no effect on the release of tyrosine whereas iodoacetate (2 mm), deoxycholate (0.5%), CuCl₂ (0.1 mm), ZnC1₂ (0.1 mm) and NaCl or KCI (240 mm) had a strong inhibitory effect. The optimal pH of this enzyme. was 6.3–7.A preparation containing tubulin-tyrosine ligase free of carboxypeptidase was also obtained. This preparation catalyzed the release of tyrosine from tyrosinated tubulin in the presence of ADP, Mg²⁺, K^– and Pi and the incorporation of tyrosine into tubulin. For the releasing activity the optimal concentration of MgCl₂ was 3–20 mm and of KCl was 10–30 mm. For ADP the maximal activity was at 0.3 mm or higher.An important difference between the activities of the carboxypeptidase and the ligase was that the former was active on denatured tubulin whereas the latter was not.     

Phosphoprotein phosphatase activity of Novikoff hepatoma nucleoli.

Nucleoli from Novikoff hepatoma ascites cells contain phosphatase activity that acts upon ³²P-labeled nucleolar protein substrates. The activity is optimal near pH 7.0 and is inhibited by increasing concentrations of NaCl. The divalent cations CaCl₂, MnCl₂ and CoCl₂ at 6 mM inhibited phosphatase activity from 30–60%. ZnCl₂ completely inhibited the activity above 2 mM while EDTA and MgCl₂ had little effect. The activity was stimulated by dithiothreitol and inhibited by N-ethylmaleimide indicating a requirement for free sulfhydryl groups.     

Xylulose 1,5-Bisphosphate Synthesized by Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase during Catalysis Binds to Decarbamylated Enzyme

Xylulose 1,5-bisphosphate (XuBP) is synthesized from ribulose 1,5-bisphosphate (RuBP) at carbamylated catalytic sites on ribulose 1,5-bisphosphate carboxylase (Rubisco) with significant amounts of XuBP being formed at pH less than 8.0. XuBP has been separated by high performance liquid chromatography and identified by pulsed amperometry from compounds bound to Rubisco during catalysis with the purified enzyme and from celery (Apium graveolens var Utah) leaf extracts. XuBP does not bind tightly to carbamylated sites, but does bind tightly to decarbamylated sites. Upon incubation of fully activated Rubisco with 5 micromolar XuBP, loss of activator CO₂ occurred before XuBP bound to the enzyme catalytic sites, even in the presence of excess CO₂ and Mg²⁺. Binding of XuBP to decarbamylated Rubisco sites was highly pH dependent. At pH 7.0 and 7.5 with 10 millimolar MgCl₂ and 10 millimolar KHCO₃, the apparent dissociation constant for XuBP, K_d, was 0.03 micromolar, whereas at pH 8.0 and 8.5, the apparent K_d was 0.35 and 2.0 micromolar, respectively. This increase in K_d with pH was a result of a decrease in the association rate constant and an increase in the dissociation rate constant of XuBP bound to decarbamylated sites on Rubisco. The K_d of 2-carboxyarabinitol 1-phosphate binding to carbamylated sites was only slightly pH dependent.     

Biochemical characterization and evaluation of invertases produced from Saccharomyces cerevisiae CAT-1 and Rhodotorula mucilaginosa for the production of fructooligosaccharides

Abstract

Invertases are used for several purposes; one among these is the production of fructooligosaccharides. The aim of this study was to biochemically characterize invertase from industrial Saccharomyces cerevisiae CAT-1 and Rhodotorula mucilaginosa isolated from Cerrado soil. The optimum pH and temperature were 4.0 and 70?°C for Rhodotorula mucilaginosa invertase and 4.5 and 50?°C for Saccharomyces cerevisiae invertase. The pH and thermal stability from 3.0 to 10.5 and 75?°C for R. mucilaginosa invertase, respectively. The pH and thermal stability for S. cerevisiae CAT-1 invertase from 3.0 to 7.0, and 50?°C, respectively. Both enzymes showed good catalytic activity with 10% of ethanol in reaction mixture. The hydrolysis by invertases occurs predominantly when sucrose concentrations are ≤5%. On the other hand, the increase in the concentration of sucrose to levels above 10% results in the highest transferase activity, reaching about 13.3?g/L of nystose by S. cerevisiae invertase and 12.6?g/L by R. mucilaginosa invertase. The results demonstrate the high structural stability of the enzyme produced by R. mucilaginosa, which is an extremely interesting feature that would enable the application of this enzyme in industrial processes.     

Binding of peroxiredoxin 6 to substrate determines differential phospholipid hydroperoxide peroxidase and phospholipase A2 activities

Peroxiredoxin 6 (Prdx6) differs from other mammalian peroxiredoxins both in its ability to reduce phospholipid hydroperoxides at neutral pH and in having phospholipase A₂ (PLA₂) activity that is maximal at acidic pH. We previously showed an active site C47 for peroxidase activity and a catalytic triad S32-H26-D140 necessary for binding of phospholipid and PLA₂ activity. This study evaluated binding of reduced and oxidized phospholipid hydroperoxide to Prdx6 at cytosolic pH. Incubation of recombinant Prdx6 with 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine hydroperoxide (PLPCOOH) resulted in peroxidase activity, cys47 oxidation as detected with Prdx6-SO₂₍₃₎ antibody, and a marked shift in the Prdx6 melting temperature by circular dichroism analysis indicating that PLPCOOH is a specific substrate for Prdx6. Preferential Prdx6 binding to oxidized liposomes was detected by changes in DNS-PE or bis-Pyr fluorescence and by ultrafiltration. Site-specific mutation of S32 or H26 in Prdx6 abolished binding while D140 mutation had no effect. Treatment of A549 cells with peroxides led to lipid peroxidation and translocation of Prdx6 from the cytosol to the cell membrane. Thus, the pH specificity for the two enzymatic activities of Prdx6 can be explained by the differential binding kinetics of the protein; Prdx6 binds to reduced phospholipid at acidic pH but at cytosolic pH binds only phospholipid that is oxidized compatible with a role for Prdx6 in the repair of peroxidized cell membranes.