Effect of ions and other compatible solutes on enzyme activity, and its implication for biocatalysis using ionic liquids

The effect of ions on enzyme activity and stability usually follows the Hofmeister series (or the kosmotropicity order): kosmotropic anions and chaotropic cations stabilize enzymes while chaotropic anions and kosmotropic cations destabilize them. The effect of ionic liquids (ILs) on the enzyme activity/stability/enantioselectivity is complicated especially when there is no or little water presence in the IL media. However, when aqueous solutions of hydrophilic ILs are employed as reaction media, the enzyme seems to follow the Hofmeister series since ILs dissociate into individual ions in water.     

Study of the nature of lysosomes and of their acid phosphatase

Summary Treatment with M/2 CaCl₂ or fresh frozen sections of various organs of mice inhibits the subsequent histochemical demonstration of acid phosphatase by various procedures. This inhibition can be reversed by treatment with M/I NaCl. Fixation in cold formalin solution, or treatment with fat solvents weakens or abolishes this effect with the result that phosphatase staining is not markedly influenced by pretreatment with salts. The effect of surfactants in inhibiting acid phosphatase staining is diminished by fixation in formalin. Treatment with M/2 CaCl₂ of fresh frozen sections of mice organs partly inhibited the histochemical staining of phospholipids.The findings indicate that acid phosphatase molecules are bound to polar lipids and that under the influence of excess of Ca ions the lipoproteic molecules of acid phosphatase form droplets bounded by hydrophobic groups of the lipids. In such droplets the enzyme molecules are not available to act on substrates dissolved in the medium, and the droplets may be regarded as lysosomes. Under the influence of excess Na ions in the milieu the lipoprotein layer breaks into droplets bounded on the outside by the enzyme protein and the hydrophilic groups of the phospholipids. Such particles probably represent microsomes. The implications of these conclusions are discussed.A preliminary communication dealing with the present study has been published (in Hebrew) in the Harefuah (Wolman, 1964).     

Structure-Activity Relationship of a U-Type Antimicrobial Microemulsion System

The structure-activity relationship of a U-type antimicrobial microemulsion system containing glycerol monolaurate and ethanol at a 1∶1 mass ratio as oil phase and Tween 20 as surfactant were investigated along a water dilution line at a ratio of 80∶20 mass% surfactant/oil phase, based on a pseudo-ternary phase diagram. The differential scanning calorimetry results showed that in the region of up to 33% water, all water molecules are confined to the hydrophilic core of the reverse micelles, leading to the formation of w/o microemulsion. As the water content increases, the water gains mobility, and transforms into bicontinuous in the region of 33–39% water, and finally the microemulsion become o/w in the region of above 39% water. The microstructure characterization was confirmed by the dynamic light scattering measurements and freeze-fracture transmission electron microscope observation. The antimicrobial activity assay using kinetics of killing analysis demonstrated that the microemulsions in w/o regions exhibited relatively high antimicrobial activity against Escherichia coli and Staphylococcus aureus due to the antimicrobial oil phase as the continuous phase, while the antimicrobial activity started to decrease when the microemulsions entered the bicontinuous region, and decreased rapidly as the water content increased in the o/w region, as a result of the dilution of antimicrobial oil droplets in the aqueous continuous phase.     

In vivo release kinetics of octreotide acetate from experimental polymeric microsphere formulations using oil/water and oil/oil processes

The purpose of the present study was to characterize the in vivo release kinetics of octreotide acetate from microsphere formulations designed to minimize peptide acylation and improve drug stability. Microspheres were prepared by a conventional oil/wate (o/w) method or an experimental oil/oil (o/o) dispersion technique. The dosage forms were administered subcutaneously to a rat animal model, and serum samples were analyzed by radioimmunoassay over a 2-month period. An averaged kinetic profile from each treatment group, as a result, was treated with fractional differential equations. The results indicated that poly(l-lactide) microspheres prepared by the o/o dispersion technique provided lower area under the curve (AUC) values during the initial diffusion-controlled release phase, 7.79 ng×d/mL, versus 75.8 ng/sxd/mL for the o/w batch. During the subsequent erosion-controlled release phase, on the other hand, the o/o technique yielded higher AUC values, 123 ng×d/mL, versus 42.2 ng×d/mL for the o/w batch. The differences observed between the 2 techniques were attributed to the site of drug incorporation during the manufacturing process, given that microspheres contain both porous hydrophilic channels and dense hydrophobic matrix regions. An o/o dispersion technique was therefore expected to produce microspheres with lower incorporation in the aqueous channels, which are responsible for diffusion-mediated drug release.     

Feinstrukturelle Untersuchungen zur Entwicklung der Ölzellen bei dem LebermoosRiella

Summary Liverworts are characterized by the possession of typical cell elements, the oil bodies. In the submerse, thalloid liverwortRiella helicophylla (Sphaerocarpales) oil bodies are existing in idioblastic cells only, called oil body cells. Each oil body cell contains only one oil body. The oil body originates from small vacuoles. Their membranes are extremely high in contrast and asymmetric. The lipophilic substances are probably produced inside the oil bodies. At the end of the development of an oil body cell lipophilic and hydrophilic material will be separated from each other inside the oil body. The result is an oil body, consisting of one large spherical oil globule surrounded by a thin layer of hydrophilic matrix.

     

Temperature-Sensitive Microemulsion Gel: An Effective Topical Delivery System for Simultaneous Delivery of Vitamins C and E

Microemulsions (ME)—nanostructured systems composed of water, oil, and surfactants—have frequently been used in attempts to increase cutaneous drug delivery. The primary objective addressed in this work has been the development of temperature-sensitive microemulsion gel (called gel-like ME), as an effective and safe delivery system suitable for simultaneous topical application of a hydrophilic vitamin C and a lipophilic vitamin E. By changing water content of liquid o/w ME (o/w ME), a gel-like ME with temperature-sensitive rheological properties was formed. The temperature-driven changes in its microstructure were confirmed by rotational rheometry, viscosity measurements, and droplet size determination. The release studies have shown that the vitamins’ release at skin temperature from gel-like ME were comparable to those from o/w ME and were much faster and more complete than from o/w ME conventionally thickened with polymer (o/w ME carbomer). According to effectiveness in skin delivery of both vitamins, o/w ME was found the most appropriate, followed by gel-like ME and by o/w ME carbomer, indicating that no simple correlation between vitamins release and skin absorption could be found. The cytotoxicity studies revealed good cell viability after exposure to ME and confirmed all tested microemulsions as nonirritant. This work was supported by a grant of Slovenian Research Agency.     

A model to explain ion-induced differentiation in zoospores ofPhytophthora palmivora

Zoospores ofPhytophthora palmivora undergo synchronous encystment followed by germination when exposed to sodium ions at 3–10 mM, calcium ions at 5–30 mM, and strontium ions at 0.3–10 mM. Lithium ions induce encystment at 3–10 mM but do not induce germination. Ferric, manganese, and barium ions.act as chaotropic agents, damaging the zoospore plasma membrane under both isoosmotic and hypoosmotic conditions, at concentrations as low as 30 μM, ferric ions and 1 mM, barium ions. Cesium and ammonium ions are cytotoxic and induce lysis under hypoosmotic conditions. The capacity of cations to induce zoospore differentiation can be explained in terms of the following model. It is suggested that differentiation inP. palmivora requires the entry of sodium at one or more specific sites. Access to these sites is regulated by a calcium gated monovalent ion translocator with a high specificity for sodium. Calcium not only regulates sodium ion access but also regulates the cell's osmoregulatory system. Divalent cations act by competing for the calcium binding site, monovalent cations by restricting fodium ion access.     

Stable ionotropic gel for cell immobilization using high molecular weight pectic acid

It is shown that Ca- and Al-pectate gel beads prepared by use of high molecular weight polygalacturonic acid (viz. polygalacturonic acid having a high STAUDINGER index) are well suited for cell immobilization. The pectate beads are much more insensitive to those ions and chemical agents which destructively act on alginate beads (such as phosphate, citrate, gluconate, lactate as well as a high excess of sodium, potassium, and/or ammonium ions), even without addition of gelling cations to the liquid phase.     

Stimulated secretion of lysosomal enzymes by cells in culture

F9 mouse teratocarcinoma and PyS-2 cells in culture incubated with monovalent cations in buffered sucrose solution (0.25 M) can secrete as much as 40% of their total lysosomal enzymes into the medium within 30 min. Longer incubation does not lead to further loss of enzyme, suggesting that only a certain fraction of lysosomes is capable of discharge. The simultaneous presence of sucrose and cation, each at the respective optimal concentrations of 0.25 and 0.15 M, is required for lysosomal discharge (i.e. twice isoosmolarity). The cells remain fully viable. Sodium ions are more effective than lithium and potassium ions, whereas amines and divalent cations are less effective. Other sugars including glucose can replace sucrose to varying extents. Secretion is accompanied by a rapid short-lived rise in the level of cAMP. Forskolin as well as agents that activate G protein such as cholera toxin, AlF4-, and vanadate ions also increase the rate of secretion. Sucrose-Na+ stimulation takes place independently of changes in influx or efflux of calcium ions or changes in the levels of extracellular or free intracellular calcium ions. Neomycin, an inhibitor of phospholipase C, has little effect on secretion. Our results suggest that the secretion observed is mediated by a cAMP-dependent mechanism involving G proteins. Calcium ions and phospholipase C appear to play little or no part in the activation process.     

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.     

The removal by phospholipase C of a layer of lanthanum-staining material external to the cell membrane in embryonic chick cells

Fixation of embryonic chick cells (heart, neural retina, and limb bud) in the presence of lanthanum ions shows the presence of an electron-opaque layer, about 50 A thick, external to the cell membrane. This layer, designated LSM (for lanthanum-staining material), is not removable by trypsin, pronase, EDTA, DNase, α-amylase, neuraminidase, or N-acetyl-L-cysteine. However, phospholipase C, in concentrations as low as 0.001 mg/ml, succeeds in stripping the LSM from the cell surface. Heating the enzyme preparation does not inhibit this activity, but removal of divalent cations does; both of these results are consistent with the known properties of phospholipase C. The LSM is present at the cell surface in the control tissues and on cells dissociated from the tissues by proteolytic enzymes and EDTA. These results are interpreted to mean that the LSM is probably an integral part of the cell and not an extraneous coat. How this phenomenon bears on the problem of cellular adhesion is discussed, as is the possible chemical composition of the LSM.     

Activation of the capacity for sodium-induced pinocytosis in Amoeba proteus

Amoebae treated with cycloheximide or starved for 8-10 days lose their pinocytotic response to Na+. Their capacity for Na+-induced pinocytosis was activated after application of various physical or chemical stimuli (electrical stimulation, mechanical shearing forces, osmotic pressure, UV-light, alkali metal ions, capsaicin, and indole). The degree of activation was related to the intensity and duration of the stimulus and lasted several hours after the stimulus had been withdrawn. The dose-response curves of activating stimuli were always biphasic. Strong activating agents reduced the sensitivity of the amoeba to the inducer. At concentrations lower than those which induced pinocytosis, but in the same order of efficacy, inorganic cations were potent activating agents. Like induction of pinocytosis, activation by cations required minute amounts of Ca2+ and was inhibited by high concentrations of this ion. Activation may therefore be an early event during the induction of pinocytosis. Capsaicin and indole were potent activators, indicating that specific chemical stimuli may increase the capacity for pinocytosis. The activation may be the result of a secretory process adding area and structures to the old membrane which are necessary for the induction of pinocytosis.     

Differences in the interaction of inorganic and organic (hydrophobic) cations with phosphatidylserine membranes

The interaction of phosphatidylserine dispersions with “hydrophobic”, organic cations (acetylcholine, tetraethylammonium ion) is compared with that of simple inorganic cations (Na⁺, Ca²⁺); differences in the hydration properties of the two classes of ions exist in the bulk phase as evident from spin-lattice relaxation time T₁, measurements. It is shown that the reaction products (cation-phospholipid) differ markedly in their physicochemical behaviour. With increasing concentration both classes of ions reduce the ζ-potential of phosphatidylserine surfaces, the monovalent inorganic cations being only slightly more effective than the hydrophobic cations. Inorganic cations cause precipitation of the lipid once the surface charge of the bilayer is reduced to a certain threshold value. This is not the case with the organic cations. The difference is probably associated with the different hydration properties of the resulting complexes. Thus binding of Ca²⁺ causes displacement of water of hydration and formation of an anhydrous, hydrophobic calcium-phosphatidylserine complex which is insoluble in water, whereas the product of binding of the organic cations is hydrated, hydrophilic and water soluble. The above findings are consistent with NMR results which show that the phosphodiester group is involved in the binding of both classes of cations as well as being the site of the primary hydration shell. Besides affecting interbilayer membrane interactions such as those involved in cell adhesion and membrane fusion, the binding of both classes of cation can affect the molecular packing within a bilayer.     

Divalent Ions and the Surface Potential of Charged Phospholipid Membranes

Phospholipid bilayer membranes were bathed in a decimolar solution of monovalent ions, and the conductance produced by neutral carriers of these monovalent cations and anions was used to assess the electric potential at the surface of the membrane. When the bilayers were formed from a neutral lipid, phosphatidylethanolamine, the addition of alkaline earth cations produced no detectable surface potential, indicating that little or no binding occurs to the polar head group with these ions. When the bilayers were formed from a negatively charged lipid, phosphatidylserine, the addition of Sr and Ba decreased the magnitude of the surface potential as predicted by the theory of the diffuse double layer. In particular, the potential decreased 27 mv for a 10-fold increase in concentration in the millimolar-decimolar range. A 10-fold increase in the Ca or Mg concentration also produced a 27 mv decrease in potential in this region, which was again due to screening, but it was necessary to invoke some specific binding to account for the observation that these cations were effective at a lower concentration than Ba or Sr. It is suggested that the ability of the alkaline earth cations to shift the conductance-voltage curves of a nerve along the voltage axis by 20–26 mv for a 10-fold increase in concentration may be due to essentially a screening rather than a binding phenomenon.     

Rapid stimulation of liver palmitoyl-CoA synthetase, carnitine palmitoyltransferase and glycerophosphate acyltransferase compared to peroxisomal beta-oxidation and palmitoyl-CoA hydrolase in rats fed high-fat diets

Key enzymes involved in oxidation and esterification of long-chain fatty acids were investigated in male rats fed different types and amounts of oil in their diet. A diet with 20% (w/w) fish oil, partially hydrogenated fish oil (PHFO) and partially hydrogenated soybean oil (PHSO) was shown to stimulate the mitochondrial and microsomal palmitoyl-CoA synthetase activity (EC 6.2.1.3) compared to soybean oil-fed animals after 1 week of feeding. Rapeseed oil had no effect. Partially hydrogenated oils in the diet resulted in significantly higher levels of mitochondrial glycerophosphate acyltransferase compared to unhydrogenated oils in the diet. Rats fed 20% (w/w) rapeseed oil had a decreased activity of this mitochondrial enzyme, whereas the microsomal glycerophosphate acyltransferase activity was stimulated to a comparable extent with 20% (w/w) rapeseed oil, fish oil or PHFO in the diet. Increasing the amount of PHFO (from 5 to 25% (w/w)) in the diet for 3 days led to increased mitochondrial and microsomal palmitoyl-CoA synthetase and microsomal glycerophosphate acyltransferase activities with 5% of this oil in the diet. The mitochondrial glycerophosphate acyltransferase was only marginally affected by increasing the oil dose. Administration of 20% (w/w) PHFO increased rapidly the mitochondrial and microsomal palmitoyl-CoA synthetase, carnitine palmitoyltransferase and microsomal glycerophosphate acyltransferase activities almost to their maximum value within 36 h. In contrast, the glycerophosphate acyltransferase and palmitoyl-CoA hydrolase (EC 3.1.2.2) activities of the mitochondrial fraction and the peroxisomal beta-oxidation reached their maximum activities after administration of the dietary oil for 6.5 days. This sequence of enzyme changes (a) is in accordance with the proposal that an increased cellular level of long-chain acyl-CoA species act as metabolic messages for induction of peroxisomal beta-oxidation and palmitoyl-CoA hydrolase, i.e., these enzymes are regulated by a substrate-induced mechanism, and (b) indicates that, with PHFO, a greater part of the activated fatty acids are directed from triacylglycerol esterification and hydrolysis towards oxidation in the mitochondria. It is also conceivable that the mitochondrial beta-oxidation is proceeding before the enhancement of peroxisomal beta-oxidation.     

Structural Changes in Halophilic and Non-halophilic Proteases in Response to Chaotropic Reagents

Halophilic enzymes have been established for their stability and catalytic abilities under harsh operational conditions. These have been documented to withstand denaturation at high temperature, pH, organic solvents, and chaotropic agents. However, this stability is modulated by salt. The present study targets an important aspect in understanding protein–urea/GdmCl interactions using proteases from halophilic Bacillus sp. EMB9 and non-halophilic subtilisin (Carlsberg) from Bacillus licheniformis as model systems. While, halophilic protease containing 1 % (w/v) NaCl (0.17 M) retained full activity towards urea (8 M), non-halophilic protease lost about 90 % activity under similar conditions. The secondary and tertiary structure were lost in non-halophilic but preserved for halophilic protein. This effect could be due to the possible charge screening and shielding of the protein surface by Ca²⁺ and Na⁺ ions rendering it stable against denaturation. The dialyzed halophilic protease almost behaved like the non-halophilic counterpart. Incorporation of NaCl (up to 5 %, w/v or 0.85 M) in dialyzed EMB9 protease containing urea/GdmCl, not only helped regain of proteolytic activity but also evaded denaturing action. Deciphering the basis of this salt modulated stability amidst a denaturing milieu will provide guidelines and templates for engineering stable proteins/enzymes for biotechnological applications.     

Action of Rice α-Glucosidase on Maltose and Starch

Rice seeds possess α-glucosidase I and II, and the action of the α-glucosidases on maltose and starch was studied. The activity on starch was increased 2.3~2.6 times in both enzymes at the concentration of 50 mM of potassium chloride. Such activation was also caused by mono and di-valent cations. The activity on maltose was not influenced by the cations. In mixed substrate experiments, liberation of ¹⁴C-glucose from ¹⁴C-maltose was not inhibited in the presence of starch, and this was also the case with that from ¹⁴C-starch in the existence of maltose. From these results, it was suggested that the α-glucosidases possess maltose-hydrolyzing site and starch-hydrolyzing site separately, and also probably regulatory. The α-glucosidases liberated only glucose from starch, and were presumed to complete hydrolysis of starch after longer incubation.     

Role of calcium ions in the control of embryogenesis of Xenopus. Changes in the subcellular distribution of calcium in early cleavage embryos after treatment with the ionophore A23187.

Treatment of stage 5 Xenopus embryos with the ionophore A23187 for only 10 min, in the absence of extracellular Mg2+ and Ca2+, causes cortical contractions and a high incidence of abnormal embryos during subsequent development. Cation analysis shows that divalent ions are not lost from the embryos, but that Ca2+ is redistributed within the subcellular fractions. Ca2+ is probably released from yolk platelets and/or pigment granules by the action of A23187, [Ca2+] rises in the cytosol, and the mitochondria attempt to take up this free Ca2+. The mitochondria concomitantly undergo characteristic ultrastructural transformations, changing towards energized-twisted and energized-zigzag conformations. A23187 allows these changes to be demonstrated in situ. Extracellular divalent cations (10(-4) M) interfere with this intracellular action of A23187. Intracellular accumulation of Na+ (by treatment with ouabain) or Li+ also causes abnormal development, probably by promoting a release of Ca2+ from the mitochondria. It is suggested (a) that all these treatments cause a rise in [Ca2+]i which interferes with normal, integrated cell division, so causing, in turn, abnormal embryogenesis, (b) that levels of [Ca2+]i are of importance in regulating cleavage, (c) that the mitochondria could well have a function in regulating [Ca2+]i during embryogenesis in Xenopus, and (d) that vegetalizing agents may well act by promoting a rise in [Ca2+]i in specific cells in the amphibian embryo.     

Enzymatic Synthesis of Lipophilic Caffeoyl Lipids Using Soybean Oil as the Novel Acceptor

Soybean oil-based caffeoyl lipids are the novel lipophilic derivatives of caffeic acid, which can be used as UV absorbers and antioxidants in the food and cosmetic industries. In the work, the novel lipophilic structured lipids were prepared using soybean oil as the novel caffeoyl acceptor by enzymatic transesterification. The effects of the reaction variables on the transesterification were investigated, and response surface methodology was used to optimize the reaction variables. Reactions were monitored by HPLC-UV. Different enzymes (Novozym 435, Lipozyme RMIM, and Lipozyme TLIM) were used as biocatalysts, and Novozym 435 showed the best performance for the reaction. The results showed that a high lipophilic soybean oil-based caffeoyl lipids yield (73.5 ± 1.2%) was achieved under the optimal conditions (reaction temperature 85°C, substrate molar ratio 1:6 (ethyl caffeate (EC)/soybean oil), enzyme load 25% (w/w), and 60 h at atmosphere pressure). The activation energies of EC conversion, hydrophilic glyceryl caffeates (GC) and lipophilic caffeoylated acylglycerol (CAG) formations were 32.92 kJ/mol, 17.21 kJ/mol and 57.36 kJ/mol, respectively. Km and Vm were 0.022 mol/L and 0.033 × 10^-3 mol/(Lmin), respectively.