Cholesterol metabolism in the rat lactating mammary gland: The role of cholesteryl ester hydrolase

An acid cholesteryl ester hydrolase activity associated with a fraction containing mitochondria and lysosomes from rat lactating mammary glands was found to have a pH optimum of 5.0. Its sedimentation pattern was closely related to that of the lysosomal enzyme markers acid phosphatase and β-glucuronidase, suggesting that the activity is associated with the lysosomes. The enzyme was strongly inhibited by Cu²⁺, but was inhibited little by other divalent metal ions. Acid cholesteryl ester hydrolase activity was almost completely abolished byp-hydroxymercuribenzoate, but this effect was reversed in the presence of an equimolar concentration of reduced glutathione (GSH), indicating that the enzyme requires free sulfhydryl groups for activity. These properties are similar to those of acid, lysosomal cholesteryl ester hydrolases found in other tissues. Acid cholesteryl ester hydrolase activity was 8–14 fold higher in mammary tissue from lactating as compared to virgin rats. Neutral cholesteryl ester hydrolase activities associated with the microsomal and cytosolic subcellular fractions were also increased in lactating glands, but to a lesser extent. In addition, a 2-fold increase in the activities of both the acid and microsomal neutral enzymes was seen during the first few days of lactation, while the cytosolic neutral activity remained constant. These results suggest that mammary gland cholesteryl ester hydrolases have a role in the regulation of cholesterol metabolism in mammary cells, and in the provision of cholesterol for secretion into milk.     

The activity and properties of a hepatic acid cholesteryl ester hydrolase obtained from rats of different age groups

The activity of lysosomal acid cholesteryl ester hydrolase (acid CEH, EC 3.1.1.13) in rat liver was determined at 3, 5, 7, 10 and 20 wk following birth. The levels of acid CEH activity showed a marked decrease as rats grew older, whereas those of other lysosomal marker enzymes, such as acid phosphatase, β-glucuronidase and cathepsin B and D, showed only a slight decrease. On the other hand, acid CEH activity was detected in all subcellular fractions obtained from rat liver, but the enzyme activity in these fractions did not show the age-related decrease observed in the lysosomal fraction. The results presented here suggest that the marked alteration of lysosomal acid CEH activity that accompanies aging may be related to its possible involvement in the regulation of cholesterol concentration in rat liver.     

Regulation of rat liver microsomal cholesterol ester hydrolase by reversible phosphorylation

The regulation of neutral cholesterol ester hydrolase activity by changes in its phosphorylation state was studied in rat liver microsomes. Treatment with cAMP-dependent protein kinase resulted in increased enzyme activity, which was further enhanced by the addition of cAMP and MgATP. Consistent activations were also achieved with MgCl₂ and MgATP, the magnesium effect being abolished by ethylenediaminetetraacetic acid and adenosine triphosphate. Cholesterol ester hydrolase was activated twofold by free calcium and Ca²⁺/calmodulin; this latter effect was blocked by the chelator ethyleneglycol-bis(β-aminoethyl ether)N,N,N′,N′-tetraacetic acid and the calmodulin antagonist trifluoperazine. The phosphatase inhibitors pyrophosphate and glycerophosphate led to marked and dose-dependent increases in esterase activity, whereas okadaic acid elicited no effect. Furthermore, pyrophosphate and okadaic acid did not change the increases in enzyme activity promoted by Ca²⁺, Ca²⁺/calmodulin, Mg²⁺ and MgATP. Cholesterol ester hydrolase was inactivated in a concentration-dependent manner by nonspecific alkaline phosphatases. In cAMP-dependent protein kinase/cAMP- or Ca²⁺/calmodulin-activated microsomes, a time-dependent loss of activation in cholesteryl oleate hydrolysis was caused by alkaline phosphatase. These findings suggest that microsomal cholesterol ester hydrolase is activated through cAMP and Ca²⁺/calmodulin phosphorylation, whereas enzyme deactivation is dependent on phosphatase action.     

Activation of rat liver cholesterol ester hydrolase by cAMP-dependent protein kinase and protein kinase C

Short term regulation of hepatic cholesterol ester hydrolase by reversible phosphorylation is described. Two different kinase systems seem to be involved in this regulation. The addition of ATP, cyclic AMP and Mg²⁺ to rat liver 104,000× g supernatant (S104) produced a 100–140% increase in cholesterol ester hydrolase activity. This stimulation was abolished when protein kinase inhibitor was added prior to the addition of ATP, cyclic AMP and Mg²⁺. Cholesterol ester hydrolase activity was also stimulated when calcium ions, phosphatidylserine, and diolein were added to S104 along with ATP and Mg²⁺. Diolein in this reaction could be substituted by phorbol 12-myristate 13-acetate. Preincubation of S104 with alkaline phosphatase resulted in a deactivation of cholesterol ester hydrolase. The addition of increasing concentrations of Mg²⁺ to S104 produced increasing inhibition of cholesterol ester hydrolase activity, and this effect was blocked by NaF. It is suggested that rat liver cholesterol ester hydrolase is activated by cyclic AMP dependent protein kinase and protein kinase C. Deactivation is accomplished by dephosphorylation catalyzed by a phosphoprotein phosphatase, dependent on Mg²⁺. This work was presented at the Twenty-Third Southeastern Regional Lipid Conference, held October 26–28, in Cashiers, North Carolina.     

Evidence that cholesteryl ester hydrolase and triglyceride lipase are different enzymes in rat liver

Studies on intracellular cholesteryl ester hydrolase (CEH) and triglyceride lipase (TGL) from rat adipose tissue and adrenal cortex have suggested that a single protein is responsible for both activities. To determine whether one hepatic protein catalyzes both reactions, we studied several properties of CEH and TGL in rat liver. During liver perfusion with heparin, perfusate peaks of TGL and CEH did not consistently coincide, and TGL activity was considerably higher and less heat-stable than that of CEH. Significant TGL, but not CEH, activity was released during incubation of isolated hepatocytes. Although microsomes isolated from hepatocytes contained both activities, the specific activities of CEH and TGL in cytosol from hepatocytes were 95% and 3%, respectively, of those found in cytosol from whole liver. Preincubation of liver cytosol with 5 mM Mg²⁺ decreased CEH, but not TGL, activity. Intracellular CEH and TGL activities were completely separated by prep-disc gel electrophoresis. Finally, both cytosolic and microsomal TGL, but not CEH, activities were inhibited by antiserum against rat hepatic TGL. We conclude that extracellular TGL does not have CEH activity and intracellular CEH differs from TGL.     

Properties of an acid cholesteryl ester hydrolase inhibitor from rat serum

The inhibitory effect of a protein isolated from rat serum on lysosomal acid cholesteryl ester hydrolase (acid CEH; EC.3.1.1.13) activity was studied. An inhibitor was purified from rat serum following ultracentrifugation and heat treatment using column chromatography on Sephacryl S-200 and ultrafiltration. The purified inhibitor appeared as a single protein band in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The molecular weight of the inhibitor was 28,000 Daltons as judged by gel filtration on Sephacryl S-200 and SDS-polyacrylamide gel electrophoresis. The purified inhibitor was shown to be apolipoprotein A-I (apo A-I), the major apolipoprotein of high-density lipoprotein (HDL), using immunoprecipitation with rat anti-apo A-I immunoglobulin (Ig)G. Inhibition of acid CEH activity by apo A-I was dependent on the concentration of apo A-I. The values of V_max obtained were similar with or without apo A-I. Apo A-I of various other mammalian species, including human, bovine and rabbit, also inhibited acid CEH activity. Other apolipoproteins, such as apo A-II and apo B, also showed inhibiting activity. On the other hand, apo A-I had no effect on the activity of other enzymes found in lysosomes, such as cathepsin D, β-glucuronidase and acid phosphatase. The results suggest that apolipoproteins may play a role in the regulation of hydrolysis of cholesteryl esters in lipoproteins, that have been transferred to the liver, and that the inhibition of acid CEH activity by apo A-I may be a characteristic of the lipid-binding protein or be due to changes of the lipid/water interface.     

Characterization of a cytosolic protein inhibiting lysosomal acid cholesteryl ester hydrolase

An inhibitor of lysosomal acid cholesteryl ester hydrolase (Acid CEH), (EC 3.1.1.13) was found in the cytosolic fraction of rat liver and various other tissues. The extent of the inhibitory effect was dependent on the concentration of the cytosolic protein. The Acid CEH inhibitor was heat-labile, nondialyzable, and its inhibitory activity significantly decreased by trypsin or chymotrypsin digestion, but not by lipase digestion. The inhibitor had no effect on the activity of cathepsin D, β-glucuronidase and acid phosphatase, which are other enzymes found in lysosomes. The present findings suggest that the inhibitor may be involved in the regulation of the hydrolysis of cholesteryl esters in lipoproteins that have been transferred into the liver.     

A diurnal variation of hepatic acid cholesteryl ester hydrolase activity in the rat

The diurnal variation in lysosomal acid cholesteryl ester hydrolase (Acid CEH), (EC 3.1.1.13) has been examined in fed, fasted and adrenalectomized rats. The Acid CEH activity of normal rat liver exhibits a diurnal rhythm with maxima at 06.00 hours and minima at 18.00 hours, but such a rhythm was not observed in spleen and brain. This rhythm was abolished after fasting for two days, and the resulting Acid CEH activity remained constant at the minimum level. However, adrenalectomy did not abolish the diurnal rhythm. These results indicate that the Acid CEH activity varies according to a diurnal rhythm with maxima and minima separated by approximately 12 hr. Further, it is evident that the appearance of this rhythm is dependent upon dietary, but not adrenal hormone influence.     

Preparation of novel bio-matrix by the complexation of DNA and metal ions

Large amounts of DNA-enriched biomaterials, such as salmon milts and shellfish gonads, are discarded as industrial waste around the world. We could convert the discarded DNA into a novel bio-matrix by the hybridization of DNA and metal ions, such as Al³⁺, Cr³⁺, Fe²⁺, Fe³⁺, Cu⁺, Cu²⁺, Zn²⁺, and Cd²⁺. These water-insoluble DNA-metal ion matrices could be created in various desirable forms, such as a gel, capsule, film, or fiber. DNA-Al³⁺ matrices were found to maintain a B-form DNA structure, which was the native double-stranded DNA structure in water. The DNA-Al³⁺ fiber showed flexibility with the molecular orientation in the direction of drawing. When a DNA-Cu²⁺ matrix was incubated in an aqueous hydroquinone or ascorbic acid solution, benzoquinone or dehydroascorbic acid was produced, respectively, by the oxidative effect of Cu²⁺ in the DNA-metal ion matrix. These results suggest that metal ions in the DNA-metal matrix maintained the oxidative function. The water-insoluble DNA-metal ion matrices may have a potential utility as a functional bio-material, such as an antibacterial, oxidative, bio-sensor, and ion conductive materials.     

Selective Recovery of Copper,Cobalt, and Nickel from Aqueous Chloride Media using Solvent Impregnated Resins

Selective recovery of copper, cobalt, and nickel from acidic chloride media was investigated with solvent impregnated resins (SIRs) containing acidic organophosphorus extractants. The adsorption of each metal with the SIR proceeds via a cation exchange mechanism. An SIR containing 2-ethylhexylphosphonic acid mono-2-ethylhexyl ester (PC-88A) has high selectivity for Cu²⁺, and the order of selectivity is Cu²⁺ > Co²⁺ > Ni²⁺. Effective adsorption-elution can be achieved by chromatographic operation for the selective recovery of Cu²⁺ from a ternary metal solution. High selectivity for Co²⁺ was observed using a SIR comprising bis-2,4,4-trimethylpentylphosphinic acid (Cyanex 272) and can be effectively separated from a Co and Ni binary metal solution by column adsorption. A simple and effective process flow sheet is proposed for selective recovery of Cu²⁺, Co²⁺, and Ni²⁺ from aqueous chloride media using the two SIRs.     

Poly(Ester Amide) with Pendent 8-Quinolinol Moiety as a Novel Oligomeric Ligand

The poly(ester amide) (PEA) was prepared by polycondensation of 1,4-phenylenebisphthalamic acid and the epoxy resin diglycidyl ether of bisphenol-A. The PEA was then treated with 5-chloromethyl-8-quinolinol hydrochloride. The resultant oligomer, designated as PEA-8-quinolinol (PEAQ), was characterized by elemental analysis, IR spectral studies and thermogravimetry. The metal chelates of PEAQ with Cu²⁺, Zn²⁺, Mn²⁺, Ni²⁺, and Co²⁺ metal ions were prepared and characterized by metal:ligand ratio, IR and reflectance studies, magnetic properties, thermogravimetry and microbicidal activity.     

Hepatic zonation of the formation and hydrolysis of cholestery esters in periportal and perivenous parenchymal cells

The periportal (PP) and perivenous (PV) zones of the liver acinus differ in enzyme complements and capacities for cholesterol and bile acid synthesis and other metabolic processes. The aim of this investigation was to determine the acinar distribution of the catalytic activity of the enzymes governing the formation and hydrolysis of cholesteryl esters using PP and PV hepatocytes from normal or cholestyramine-fed rats. The hepatocyte subpopulations were isolated by centrifugal elutriation, characterized according to the distribution pattern of a number of cell parameters and marker enzymes, and assayed for acyl-CoA: cholesterol acyltransferase (ACAT) and lysosomal, cytosolic and microsomal cholesteryl ester hydrolase (CEH). In normally fed rats, no zonation was found in the activity of lysosomal CEH and ACAT, and the activity of both cytosolic and microsomal CEH zonated toward the PV zone of the acinus. Concentrations of free and esterified cholesterol in homogenates, cytosol, and microsomes of PP and PV cells were, however, similar. Cholestyramine raised significantly the PV/PP ratio of ACAT because of an exclusive PP reduction of activity and abolished the heterogeneity in microsomal CEH because of a greater inhibitory PV response, whereas the PV dominance of cytosolic CEH and the homogeneous distribution of lysosomal CEH were unaffected. These results demonstrated homogeneity within the liver acinus for the enzymatic degradation of endocyted lipoprotein-derived cholesteryl esters, a structural zonation of the cytosolic CEH and a dynamic zonation of ACAT and the microsomal CEH, with a PV dominance of the enzymatic capacity for the degradation of stored cholesteryl esters in normal livers.     

Introduction of amidoxime groups into cellulose and its ability to adsorb metal ions

Introduction of amidoxime groups into cellulose substrate was investigated by reaction of cyano-group-containing celluloses such as cyanoethylcellulose (CE-Cell) and acrylonitrilegrafted cellulose (G-Cell) with hydroxylamine at 70°C in water medium (pH = 7.0). Dissolving pulp from softwood was used as the cellulose sample, and photografting was applied to the preparation of G-Cell, where hydrogen peroxide was used as a photoinitiator. Degree of substitution (DS) of CE-Cell and percent grafting of G-Cell employed were less than 1.5 and 40, respectively. The amidoximation of CE-Cell proceeded easily, and the amidoxime content increased with increasing the reaction time and increasing the concentration of hydroxylamine, but the amidoxime content of G-Cell decreased significantly at longer reaction time. The amidoximated samples prepared by CE-Cell exhibited an ability to adsorb metal ions such as Cu²⁺, Ni²⁺, and Co²⁺, and the adsorbed amount of Cu²⁺ was highest among the three metal ions. Moreover, the adsorption of Cu²⁺ varied depending on the DS of CE-Cell. That is, the absorption ability was reduced when the sample was prepared using CE-Cell with higher DS. The amidoximated samples prepared from G-Cell showed adsorption of Cu²⁺ similar to samples prepared by CE-Cell with lower DS, irrespective of percent grafting. © 1995 John Wiley & Sons, Inc.     

Purification and properties of lipase from the anaerobepropionibacterium acidi-propionici

A lipase secreted by the anaerobePropionibacterium acidipropionici was purified 52-fold with 27% recovery by employing a three-step purification protocol. The enzyme has a small molecular mass (Mr = 6000–8000) as determined by gel filtration and ultracentrifugation. It hydrolyzed palm oil, coconut oil, castor oil, olive oil, groundnut oil and tributyrin. Enzyme activity was inhibited by Ni²⁺, Ba²⁺, Mg²⁺, Cu²⁺, ethylenediaminetetraacetic acid, iodoacetamide, N-acetylimidazole and nonidet P-40 but stimulated by Ca²⁺, Co²⁺, K⁺, Fe²⁺, sodium dodecyl sulfate and N-bromosuccinamide. The enzyme showed substrate inhibition for both tributyrin andp-nitrophenyl acetate.     

Enhancement of the metal-binding properties of chitosan through synthetic addition of sulfur- and nitrogen-containing compounds

Several new chitosan derivatives were synthesized with the intent of forming polymers that could be used in hazardous waste remediation as toxic metal-binding agents in aqueous environments. The ability of these derivatives to bind Cu²⁺, Pb²⁺, Cd²⁺, and Fe²⁺ was tested and compared to chitosan. Four of the new compounds, the products of the reaction of chitosan with mercaptosuccinic acid, thiirane, pyridoxal hydrochloride, and succinamide, show promising results as binding agents for the above metal ions. The compound with mercaptosuccinic acid bound twice as much Cd²⁺, five times as much Pb²⁺, and virtually no Fe²⁺ when compared to chitosan. The compound with thiirane bound three times as much Pb²⁺, whereas the pyridoxal hydrochloride derivative bound 30% more Cu²⁺ and twice the Pb²⁺. The succinamide derivative gave results comparable to chitosan, but with decreased solubility at low pH. © 1993 John Wiley & Sons, Inc.     

The Potential Use of Low‐Grade Phosphate Rocks as Adsorbent

This study evaluates the suitability of using low‐grade phosphate for Cu²⁺ removal. The study also investigates the effects of the presence of ethylene‐diamine‐tetra‐acetic acid (EDTA), citric acid, tartaric acid and sodium chloride in a solution containing Cu²⁺ on the adsorption capacity of Cu²⁺ onto low‐grade phosphate. For aqueous solutions with 100 ppm Cu²⁺, the percentage removal of Cu²⁺ at pH 4 onto 0.2 g of 0.063 mm low‐grade phosphate was found to be 96.6 % after one hour. Using one mmol of either Cu²⁺, Cu‐NaCl, Cu‐tartaric acid, Cu‐EDTA, or Cu‐citric acid aqueous solutions, the present work shows trend of the percentage removal of Cu²⁺ at equilibrium time: Cu²⁺ > Cu‐NaCl > Cu‐tartaric acid > Cu‐EDTA > Cu‐citric acid. It is also found that increasing the concentration of ligand to copper ratio decreases the percentage removal of Cu²⁺ significantly. This means that the adsorption capacity is ligand type and concentration dependent.     

Synthesis and characterization of poly(hydroxamic acid)–poly(amidoxime) chelating ligands from polymer‐grafted acacia cellulose

Graft copolymerization of methyl acrylate (MA) and acrylonitrile (AN) onto acacia cellulose was carried out using free radical initiating process in which ceric ammonium nitrate (CAN) was used as an initiator. The optimum grafting yield was determined by the certain amount of acacia cellulose (AGU), mineral acid (H₂SO₄), CAN, MA, and AN at 0.062, 0.120, 0.016, 0.397, and 0.550 mol L^?1, respectively. The poly(methyl acrylate‐co‐acrylonitrile)‐grafted acacia cellulose was obtained at 55°C after 2‐h stirring, and purified acrylic polymer‐grafted cellulose was characterized by FTIR and TG analysis. Therein, the ester and nitrile functional groups of the grafted copolymers were reacted with hydroxylamine solution for conversion into the hydroxamic acid and amidoxime ligands. The chelating behavior of the prepared ligands toward some metal ions was investigated using batch technique. The metal ions sorption capacities of the ligands were pH dependent, and the sorption capacity toward the metal ions was in the following order: Zn²⁺ > Fe³⁺ > Cr³⁺ > Cu²⁺ > Ni²⁺. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Selective Extraction of Cu2+ and Ag+ Ions from Sulfuric Acid by Synergistic Combinations of Tetradentate Thia Macrocycles with Dioodecylnaphthalene Sulfonic Acid

Abstract

The aqueous-insoluble thia macrocycles tetrathia-14-crown-4 (TT14C4) and tetrathia-16-crown-4 (TT16C4) strongly and selectively synergize the extraction of Cu²⁺ and Ag⁺ ions from aqueous sulfuric acid solutions by the organophilic cation exchanger didodecylnaphthalene sulfonlc acid (HDDNS) in toluene diluent. Over a range of sulfuric acid concentrations, the selectivity is given by the order Ag² > Cu²⁺ > Mn²⁺, Fe²⁺, Fe³⁺, Co²⁺, Ni²⁺, and Zn²⁺, where synergism occurs only for Ag⁺ and Cu²⁺. Selectivity factors greater than 300 have been achieved for Cu²⁺ over ubiquitous Fe³⁺. The synergistic extraction of Cu²⁺ and Ag⁺ was explored as a function of sulfuric acid concentration, relative concentration of macrocycle vs. HDDNS, and loading.     

Preparation of Chelating Resins Containing a Pair of Neighboring Carboxylic Acid Groups and the Adsorption Characteristics for Heavy Metal Ions

Abstract

For the functional enhancement of chelating resins containing carboxylic acids, copolymer beads were prepared by suspension polymerization of styrene (St), methyl methacrylate (MMA), and divinylbenzene (DVB) in the presence of toluene as diluent. The phenyl rings of the beads were directly chloromethylated, and the carboxylic ester groups of the beads were converted into hydroxymethyl groups by reduction followed by chlorination to give chloromethyl groups, respectively. The chelating resins containing a pair of neighboring carboxylic acid groups (NCAGs) were obtained by the alkylation of chloromethyl groups in copolymer beads with diethyl malonate in the presence of sodium hydride followed by hydrolysis using aqueous alkali solution. Accordingly, the structural effects of the resins on the adsorption of heavy metal ions were investigated. Poly(St‐co‐DVB)‐based chelating resin containing NCAGs showed adsorption abilities toward heavy metal ions like Pb²⁺, Cd²⁺, and Cu²⁺, whereas poly(MMA‐co‐DVB)‐based chelating resin containing NCAGs showed adsorption abilities toward heavy metal ions like Cu²⁺, Cd²⁺, and Co²⁺. On the other hand, poly(St‐co‐MMA‐co‐DVB)‐based chelating resin containing NCAGs showed adsorption abilities toward heavy metal ions like Pb²⁺, Cd²⁺, Hg²⁺, Co²⁺, and Cu²⁺: a synergistic effect on the adsorption of heavy metal ions like Pb²⁺, Cd²⁺, Hg²⁺, and Co²⁺ was observed. The adsorption ability of poly(St‐co‐MMA‐co‐DVB)‐based chelating resin among three kinds of chelating resins was relatively good.     

Regulation of the Peroxidase-Like Activity of nGO,MoS2 and WS2 Nanozymes by Using Metal Cations

Two dimensional nanoparticles (2D-NPs) along with other nanoscale materials have been deemed to be the next generation of artificial enzymes (nanozymes). The low-cost bulk-scale production, ease of storage and modification of such nanomaterials have given nanozymes an advantage over traditional enzymes. Many studies have been aimed at developing methods to increase the performance of these nanozymes, and also identify interfering agents. To investigate the interference of a number of metal cations, we studied the effect of Ti²⁺, Fe²⁺, Ag⁺, Hg²⁺, Co²⁺, Cu²⁺, Ni²⁺, Pb²⁺, Ca²⁺, Zn²⁺ and Mn²⁺ in a nanozyme assays of 2D-NPs using ABTS radical formation. Ti²⁺, Co²⁺, Cu²⁺, Ni²⁺, Ca²⁺, Zn²⁺ and Mn²⁺ ions did not display any notable effect on the peroxidase-like activity of nGO, MoS₂ and WS₂ 2D-NPs. However, Fe²⁺, Ag⁺, Hg²⁺ and Pb²⁺ ions’ effects on the overall ABTS reaction were significant enough to be visualised by partial least square discriminant analysis (PLSDA). We report that, similar to that of many natural enzymes, the nanozyme activity of 2D-NPs is regulated by a number of metal cations allowing their identification and discrimination by using a statistical analysis tool.