SEARCH · PubMed Health

Results for “Sodium Cholate”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 199 records · Page 11Linked to original sources

PubMed2004

Solubilization of negatively charged DPPC/DPPG liposomes by bile salts.

The interactions of the bile salts sodium cholate (NaC) and sodium deoxycholate (NaDC) in 0.1 M NaCl (pH 7.4) with membranes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylglycerol (DPPG) and mixtures of DPPC and DPPG at molar ratios of 3:1 and 1:1 were studied by means of high-sensitivity isothermal titration calorimetry (ITC), dynamic light scattering (DLS), and differential scanning calorimetry (DSC). The partition coefficients and the transfer enthalpies for the incorporation of bile salt molecules into the phospholipid membranes were determined by ITC. The vesicle-to-micelle transition was investigated by ITC, DLS, and DSC. The phase boundaries for the saturation of the vesicles and their complete solubilization established by ITC were in general agreement with DLS data, but systematic differences could be seen due to the difference in detected physical quantities. Electrostatic repulsion effects between the negatively charged bile salt molecules and the negatively charged membrane surfaces are not limiting factors for the vesicle-to-micelle transition. The membrane packing constraints of the phospholipid molecules and the associated spontaneous curvature of the vesicles play the dominant role. DPPG vesicles are transformed by the bile salts into mixed micelles more easily or similarly compared to DPPC vesicles. The saturation of mixed DPPC/DPPG vesicles requires less bile salt, but to induce the solubilization of the liposomes, significantly higher amounts of bile salt are needed compared to the concentrations required for the solubilization of the pure phospholipid systems. The different solubilization behavior of DPPC/DPPG liposomes compared to the pure liposomes could be due to a specific "extraction" of DPPG into the mixed micelles in the coexistence region.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2003

The effect of taurine on the cholesterol metabolism in rats fed diets supplemented with cholestyramine or high amounts of bile acid.

The effects of taurine on serum cholesterol levels and hepatic cholesterol 7alpha-hydroxylase activity (CYP7A1) were studied in rats fed cholestyramine or high amounts of sodium cholate in order to alter the intestinal pool of bile acids. Rats were fed a diet supplemented with 1% cholesterol and 0.25% sodium cholate (high cholesterol, control; C), and C supplemented with 4% cholestyramine (CH) or 0.75% sodium cholate (BA) for 14 d. Taurine groups were fed the diet supplemented with 3% taurine (CT, CHT and BAT). Compared to rats fed C and BA diets, serum cholesterol levels were significantly reduced in rats fed CT and BAT diets, but a significant reduction of serum cholesterol by taurine feeding was not observed in the CHT group as compared to the CH group. An increase in hepatic CYP7A1 activity due to taurine intake was observed in the CT and BAT groups. However, the simultaneous administration of cholestyramine and taurine (CHT group) did not increase hepatic CYP7A1 activity compared the intake of cholestyramine only (CH group). A significant increase in fecal bile acid excretion due to taurine intake was found only in rats fed the CT diet. In conclusion, it is suggested that taurine facilitates hepatic CYP7A1 activity regardless of the enlarged intestinal pool of bile acids due to increased intake of exogenous bile acid, and then reduces the serum cholesterol concentration.

Animals↗

PubMed1999

Hyperlipidemia and atherosclerotic lesion development in LDL receptor-deficient mice fed defined semipurified diets with and without cholate.

Past studies of atherosclerosis in mice have used chow-based diets supplemented with cholesterol, lipid, and sodium cholate to overcome species resistance to lesion formation. Similar diets have been routinely used in studies with LDL receptor-deficient (LDLR(-/-)) mice. The nonphysiological nature and potential toxicity of cholate-containing diets have led to speculation that atherogenesis in these mice may not accurately reflect the human disease process. We have designed a semipurified AIN-76A-based diet that can be fed in powdered, pelleted, or liquid form and manipulated for the precise evaluation of diet-genetic interactions in murine atherosclerosis. LDLR(-/-) mice were randomly assigned among 4 diets (n=6/diet) as follows: 1, control, 10% kcal lipid; 2, high fat (40% kcal), moderate cholesterol (0.5% by weight); 3, high fat, high cholesterol (1.25% by weight); and 4, high fat, high cholesterol, and 0.5% (wt/wt) sodium cholate. Fasting serum cholesterol was increased in all cholesterol-supplemented mice compared with controls after 6 or 12 weeks of feeding (P<0.01). The total area of oil red O-stained atherosclerotic lesions was determined from digitally scanned photographs. In contrast to the control group, all mice in cholesterol-supplemented dietary groups 2 to 4 had lesions involving 7.01% to 12.79% area of the thoracic and abdominal aorta at 12 weeks (P<0.002, for each group versus control). The distribution pattern of atherosclerotic lesions was highly reproducible and comparable. The histological features of lesions in mice fed cholate-free or cholate-containing diets were similar. This study shows that sodium cholate is not necessary for the formation of atherosclerosis in LDLR(-/-) mice and that precisely defined semipurified diets are a valuable tool for the examination of diet-gene interactions.

Animal Feed↗

PubMed1993

Role of the rac1 p21-GDP-dissociation inhibitor for rho heterodimer in the activation of the superoxide-forming NADPH oxidase of macrophages.

Activation of the superoxide (O2-)-generating NADPH oxidase of phagocytes requires the interaction of membrane-associated cytochrome b559 with three cytosolic components; p47-phox, p67-phox and sigma 1. We proposed that sigma 1 was a heterodimer composed of proteins of 22 kDa and 24 kDa that were tentatively identified as the small GTP-binding protein (G protein) rac1 p21 and GDP-dissociation inhibitor for rho (rho GDI). We now describe a modified procedure for the rapid purification of sigma 1 and demonstrate that the NADPH-oxidase-activating capacity is associated, throughout the purification sequence, with a protein binding 35S-labelled guanosine 5'-[3-O-thio]triphosphate. SDS/PAGE analysis confirmed the absolute association of sigma 1 activity with the presence of both the 22 kDa and 24 kDa proteins. Immunoblotting with a battery of antibodies against the small G proteins demonstrated that the 22-kDa protein was only recognized by antibodies reacting with rac1 p21; no reaction was found with anti-(rac2 p21), anti-[v-ras(H) p21] and anti anti-(rap1 p21). Free rac1 p21 (not in complex with rho GDI) was not detected at any stage of cytosol fractionation. The proteins comprising the sigma 1 heterodimer could be separated by reverse-phase chromatography and amino acid sequencing was performed on peptides derived by trypsin digestion of each of the isolated proteins. This demonstrated the identity of the 22-kDa protein with rac1 p21 and that of the 24-kDa protein with rho GDI. Purified heterodimeric sigma 1 did not require exogenous GTP for activity under conditions that assured the absence of free nucleotides. Treatment of the sigma 1 heterodimer with 1% sodium cholate, followed by gel filtration or anion-exchange chromatography in the presence of 1% sodium cholate, effectively separated rac1 p21 from rho GDI. Monomeric rac1 p21, obtained by these procedures, was able to stimulate cell-free O2- generation. Artificial heterodimeric sigma 1, capable of NADPH oxidase activation, could be reconstituted in vitro by recombining purified monomeric rac1 p21 and rho GDI and removing the sodium cholate used to dissociate the native sigma 1 dimer. Monomeric rac1 p21 exhibited an almost absolute dependence on exogenous GTP following removal of the endogenous nucleotide in low Mg2+ solution. Under similar conditions, heterodimeric sigma 1 was resistant to nucleotide exchange.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1976

[The non-surgical treatment of postoperatively retained hepato-choledochal calculi].

Postoperatively retained stones in the extrahepatic bile ducts can be chemically dissolved or removed mechanically. One method of chemically dissolving stones is sodium cholate perfusion through the T-tube. In the light of one personal case of successful treatment with sodium cholate, this relatively little-used method is described and recommended as a useful alternative to reoperation. Before reoperation for residual cholesterol gallstones in the extrahepatic bile ducts, dissolving of stones with sodium cholate should at least be attempted.

Aged↗

PubMed2003

Characterisation of a Bifidobacterium strain with acquired resistance to cholate--a preliminary study.

The susceptibility levels against bile salts (ox gall) and sodium cholate of 19 Bifidobacterium strains from different origins ranged between 0.125% and 2.0% and 0.05% and 0.4%, respectively. Seven of these strains were subsequently selected for the isolation of sodium cholate resistant derivatives by exposure to gradually increasing concentrations of this compound. Derivatives resistant to at least 1.2% of cholate were obtained from Bifidobacterium bifidum CECT 4549 and B. bifidum M6. Further analysis of the CECT 4549 derivative indicated that the phenotype induced was stable; the enzymatic activities analysed remained unchanged and no major rearrangements of chromosomal DNA were produced. However, the cells of the resistant derivative were smaller and more regular in average size than those of the original strain. In addition, the derivative had lost the ability to use lactose, fructose and galactose, and showed reduced expression levels of two membrane proteins of 78 and 114 kDa. Finally, the resistant strain displayed higher survival and lower growth inhibition in the presence of cholate than the original strain.

Bifidobacterium↗

PubMed2000

Lecithin vesicular carriers for transdermal delivery of cyclosporin A.

Two kinds of vesicles with and without the presence of sodium cholate (flexible vesicles and conventional vesicles) were prepared, using cyclosporin A as model drug. When applied onto the excised abdominal skin of mice non-occlusively, the enhancing effects of vesicles on the penetration of cyclosporin A were assessed by an in vitro permeation technique. The effect of sodium cholate micelles was also studied. In vivo study was carried out by topical application of vesicles onto the mice skin and drug serum concentration was detected. Results showed that after 8 h of administration, flexible vesicles transported 1.16 microg of cyclosporin A through per cm(2) mice skin and amounted to 1.88 microg 24 h later. The residual amount in the skin was 1.78+/-0.51 microg/cm(2). However, flexible vesicles failed to transport measurable amount of drug through pre-hydrated skin while deposited 2.39+/-0.26 microg/cm(2) into the skin. Conventional vesicles failed to transfer cyclosporin A into the receiver while accumulated 0. 72+/-0.19 microg/cm(2) of drug in the skin. Furthermore, 1 and 40% sodium cholate micelles precluded the transport of cyclosporin A. In vivo studies indicated that with the application of flexible vesicles, serum drug concentration of 53.43+/-9.24 ng/ml was detected 2 h later. After the stratum corneum of mouse skin has been destroyed by shaving, flexible vesicles transferred large amount of drug into blood, up to 187.32+/-53.21 ng/ml after 1 h of application. Conventional vesicles failed to deliver measurable amount of drug into the blood under normal skin condition. In conclusion, flexible vesicle is better than conventional vesicle as the carrier for transdermal delivery of cyclosporin A. Penetration and fusion have been suggested to be two major functional mechanisms. Hydration is detrimental to the enhancement effect. Stratum corneum constitutes main barrier to the transport of lipophilic cyclosporin A.

Administration, Cutaneous↗

PubMed1999

Isolation and characterization of two 20S proteasomes from the endoplasmic reticulum of rat liver microsomes.

Two new forms of proteasomes, designated as the endoplasmic reticulum (ER) membrane-associated proteasome (ERa proteasome) and ER membrane-bound proteasome (ERb proteasome), were purified to homogeneity from 0.0125 and 2.5% sodium cholate extracts, respectively, of a rat liver microsomal fraction. SDS-PAGE analysis revealed that the purified ERa and ERb proteasomes were composed of multiple subunits similar to the cytosolic 20S proteasome. However, electrophoretic, structural and immunochemical differences between the ERa, ERb and cytosolic 20S proteasomes were observed on native PAGE, two-dimensional (2D) PAGE, and immunoblot analyses. Purification of ERb from a 2.5% sodium cholate extract of the trypsin-treated microsomal fraction yielded a trypsin-modified form of ERb (tERb), which lacked the C2 subunit at least. On the other hand, no ERa proteasome was obtained from the 0.0125% sodium cholate extract of the trypsin-treated microsomes, suggesting that ERa and ERb are ER membrane-associated and -bound proteasomes, respectively. The ERa, ERb, and cytosolic 20S proteasomes exhibited similar specificities as to peptide hydrolyzing activity, although differences in their activities were noted in the presence of SDS and phospholipid. With respect to the proteolysis of protein substrates, only the ERb proteasome cleaved beta-casein, and it also degraded reduced and carboxymethylated lysozyme considerably faster than the cytosolic 20S and ERa proteasomes. Collectively our results suggest that the ERa and ERb proteasomes may play roles in intracellular proteolysis distinct from that of the cytosolic 20S proteasome.

Amino Acid Sequence↗

PubMed1982

[Lipase localization in Serratia marcescens cells].

The localization of lipase was studied in the cells of Serratia marcescens 345. When the cells were treated with sodium cholate, the enzyme bound to the cellular structures was released in the solution. The supernatant obtained after the cells were treated with sodium cholate contained no glucose-6-phosphate dehydrogenase, a well known endocellular enzyme; therefore, the cytoplasmic membrane was not damaged by the treatment. Ultrathin sections of the intact cells and the cells treated with sodium cholate were examined using electron microscopy, and no disorders were detected in the cytoplasmic and outer membranes. It appears therefore that the lipase of S. marcescens 345 is located mainly on the exterior surface of the outer membrane.

Cell Fractionation↗

PubMed1980

Aldrin epoxidation catalyzed by purified rat-liver cytochromes P-450 and P-448. High selectivity for cytochrome P-450.

Aldrin epoxidation was studied in monooxygenase systems reconstituted from purified rat liver microsomal cytochrome P-450 or P-448, NADPH-cytochrome c reductase, dilauroylphosphatidylcholine and sodium cholate. Cytochrome P-450, purified from hepatic microsomes of phenobarbital-treated rats, exhibited a high rate of dieldrin formation. The low enzyme activity observed in the absence of the lipid and sodium cholate was increased threefold by addition of dilauroylphosphatidylcholine and was further stimulated twofold by addition of sodium cholate. The apparent Km for aldrin in the complete system was 7 +/- 2 microM. SKF 525-A, at a concentration of 250 microM, inhibited aldrin epoxidation by 65%, whereas 7,8-benzoflavone had no inhibitory effect at concentrations up to 250 microM. Addition of ethanol markedly increased epoxidase activity. The increase was threefold in the presence of 5% ethanol. When cytochrome P-448 purified from hepatic microsomes of 3-methylcholanthrene-treated rats was used, a very low rate of epoxidation was observed which was less than 3% of the activity mediated by cytochrome P-450 under similar assay conditions. Enzyme activity was independent of the lipid factor dilauroylphosphatidylcholine. The apparent Km for aldrin was 27 +/- 7 microM. The modifiers of monooxygenase reactions, 7,8-benzoflavone, SKF 525-A and ethanol, inhibited the activity mediated by cytochrome P-448. The I50 was 0.05, 0.2 and 800 mM, respectively. These results indicate that aldrin is a highly selective substrate for cytochrome P-450 species present in microsomes of phenobarbital-treated animals and is a poor substrate for cytochrome P-448. The two forms of aldrin epoxidase can be characterised by their turnover number, their apparent Km and their sensitivity to modifiers, like 7,8-benzoflavone and ethanol.

Aldrin↗

PubMed1979

Conversion of deoxycorticosterone to 3-keto-4-etienic acid, catalyzed by a partially purified preparation from bovine adrenocortical mitochondria.

Bovine adrenocortical mitochondria were sonicated and subjected to extraction with sodium cholate. The extract contained not only cytochrome P-450 activities, but also an activity which catalyzed the conversion of deoxycorticosterone to an unknown steroid (designated X). The latter activity was concentrated by (NH4)2SO4 fractionation in the presence of sodium cholate, and separated from P-450 by taking advantage of their different solubilities in phosphate buffer without sodium cholate. The specific activity of the partially purified enzyme fraction was 70 times higher than that of sonicated mitochondria. The conversion of deoxycorticosterone to steroid X required NAD or NADP. The conversion rate was dependent on the concentration of deoxycorticosterone. The major product, steroid X, was isolated from the reaction mixture by means of silicic acid and Iatrobeads column chromatography. The steroid was characterized as 3-keto-4-etienic acid (3-oxoandrost-4-ene-17beta-carboxylic acid). This result suggests that an enzyme system for the conversion of deoxycorticosterone to 3-keto-4-etienic acid exists in adrenocortical mitochondria.

Adrenal Cortex↗

PubMed1993

sn-1,2-diacylglycerol cholinephosphotransferase from pig liver: mixed micellar assay and kinetic analysis of the partially pure enzyme.

sn-1,2-Diacylglycerol cholinephosphotransferase from pig liver microsomes was partially purified through a procedure involving solubilization with sodium cholate and chromatography on Sepharose 6B. The resulting preparation was 19-fold enriched with respect to microsomes and was shown to be very sensitive to different detergents. Sodium cholate gave the best yields in activity. In a mixed micellar assay with Triton X-100 a strong dependence of the enzyme activity on the concentration of mixed micelles was observed, due to Triton X-100 acting as an inactivator. Soja phosphatidylcholine added exogenously protected the enzyme against detergent inactivation and stimulated the enzyme activity. Dioleoyl-phosphatidylcholine had a similar stimulatory effect, whereas didecanoyl- or dioctanoyl-phosphatidylcholine did not; thus long-chain phosphatidylcholines seem to be essential in the activation of cholinephosphotransferase. In a mixed micellar assay with sodium cholate no inactivation of the enzyme could be detected and it was found that soja phosphatidylcholine stimulates the activity in a greater extent than in Triton X-100 mixed micelles. The phospholipid activates the enzyme in a noncompetitive way with an activation constant of 176 mol%. Km was estimated as 1.54 mol% with a Vmax = 30 nmol/min/mg protein. Those results support an activation mechanism by phosphatidylcholine interacting at sites different from the active center. The high activation constant led to the conclusion that cholinephosphotransferase requires a lipidic boundary for full activation. No activation by substrate was observed. Short-chain diacylglycerides such as dihexanoyl-, dioctanoyl-, or didecanoylglycerol can be used as substrates although the enzyme in this case has only 5 to 10% of the activity it has for dioleoylglycerol or egg diglycerides.

Animals↗

PubMed1995

The role of palmitoylation of the guanine nucleotide binding protein G11 alpha in defining interaction with the plasma membrane.

Mutations of Cys-9 to serine, Cys-10 to serine and a combination of both alterations were produced in a cDNA encoding murine G11 alpha to potentially interfere with the ability of the expressed polypeptides to act as substrates for post-translational palmitoylation. Each of these mutants and the wild-type protein were expressed in simian COS-1 cells. Mutation of either cysteine-9 or cysteine-10 decreased the degree of palmitoylation of the protein by some 80% compared with the wild-type, while the double mutant totally failed to incorporate [3H]palmitate. By contrast, in all transfections the endogenously expressed simian G11 alpha incorporated [3H]palmitate to similar levels. Particulate and cytoplasmic fractions from these cells were subjected to SDS/PAGE under conditions which allow resolution of primate and rodent forms of G11 alpha. Immunoblotting of these fractions demonstrated that in all cases the endogenously expressed simian G11 alpha was exclusively associated with the particulate fraction, as was the transfected and expressed wild-type murine G11 alpha. By contrast, each of the mutated forms of murine G11 alpha displayed a distribution in which approx. 70% of the expressed protein was present in the particulate fraction and 30% in the supernatant. To examine the conformation of the particulate expressed forms of murine G11 alpha, these fractions were treated with various concentrations of sodium cholate and immunoblots were subsequently performed on the solubilized and remaining particulate proteins. Whereas essentially all of the endogenous simian G11 alpha was solubilized by treatment with 1% (w/v) sodium cholate and some 50% with 0.32% cholate, expressed wild-type murine G11 alpha was more recalcitrant to solubilization. However, that fraction of wild-type murine G11 alpha which was solubilized behaved identically to the endogenous simian G11 alpha on Superose-12 gel-exclusion chromatography. The particulate fraction of the C9S/C10S double mutant of murine G11 alpha was highly resistant to solubilization by sodium cholate, whereas the particulate fractions of the two single cysteine to serine mutants were intermediate to the wild-type and double mutant in their ability to be solubilized by this detergent. These data demonstrate that the palmitoylation status of the cysteine residues at positions 9 and 10 in murine G11 alpha plays a central role in defining membrane association of this G-protein and indicate that much of the particulate fraction of the expressed palmitoylation-resistant mutants is likely to represent non-functional rather than correctly folded protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1999

Micellar electrokinetic capillary chromatography of macrolide antibiotics. Separation of tylosin, erythromycin and their related substances.

The separation of tylosin by micellar electrokinetic capillary chromatography with a mixed micelle system is described. Good selectivity was obtained with sodium phosphate buffer (80 mM, pH 7.5) containing 20 mM sodium cholate and 7 mM cetyltrimethylammonium bromide (CTAB). This method permits tylosin to be separated from its closely related substances within 15 min. The influences of type of buffer, buffer pH, the concentrations of sodium cholate and CTAB were investigated. The robustness of the method was examined for tylosin by means of a full-fraction factorial design. Quantitative results are presented. Using a similar buffer system (80 mM sodium phosphate, pH 6.0, 20 mM sodium cholate and 5 mM CTAB), separation of erythromycin and its main related substances was also obtained. However, detection sensitivity and resolution are not sufficient for analysis of related substances in erythromycin commercial samples.

Anti-Bacterial Agents↗

PubMed2004

Cytotoxicity studies of Dynasan 114 solid lipid nanoparticles (SLN) on RAW 264.7 macrophages-impact of phagocytosis on viability and cytokine production.

Solid lipid nanoparticles (SLN) based on Dynasan 114 (D114) were tested using RAW 264.7 cells. The influence of different surfactants on the cytotoxicity of this type of SLN was examined, expressed as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) viability and the production of cytokines such as interleukin 6 (IL-6), IL-12 and tumour necrosis factor-alpha (TNF-alpha). Results were compared with previously obtained data when peritoneal mouse macrophages were used. SLN produced with stabilizers/surfactants such as poloxamer 188, sodium cholate, Lipoid S75, Tween 80, Poloxamine 908 and sodium dodecylsulfate were shown to be nontoxic towards RAW 264.7 cells. Cytokine production was reduced and stimulation, expressed in elevated cytokine levels, could not be found. Using cetylpyridinium chloride (CPC) as stabilizing surfactant, SLN became cytotoxic in a concentration-dependent manner. Not only were the viabilities reduced but also cytokine production. Cytotoxic effects of CPC stabilized SLN could be antagonized using cytochalasin B to block phagocytosis. D114-SLN produced with pharmaceutically accepted surfactants for intravenous injection (poloxamer 188, Lipoid S75, sodium cholate, Tween 80) were very well tolerated by the cells. Even sodium dodecylsulfate-stabilized D114-SLN did not exert toxic effects. Comparison of the RAW 264.7 data with previously obtained data from toxicity studies of D114-SLN towards peritoneal mouse macrophages showed similar results. This offers the possibility of using the RAW 264.7 cell line for cytotoxicity studies of colloidal drug carrier systems, rather than using laboratory animals as source of macrophages for these kinds of studies.

Animals↗

PubMed1976

Effects of detergents and choline-containing phospholipids on human spleen glucocerebrosidase.

1. Glucocerebrosidase, extracted from human spleen lysosomal membrane by sodium cholate and recovered in a high speed centrifugation supernatant, aggregated following removal of the detergent. 2. Re-solubilization of the enzymatic activity from the aggregate was achieved by treatment with the non-ionic detergents Triton X-100 and Tween 20. The anionic detergents sodium cholate and sodium taurocholate and the cationic detergents cetyltrimethylammonium bromide and cetylpyridinium chloride were also effective. The solubilizing capacity of the anionic detergents was smaller than that of the nonionic detergents. Quantitative evaluation of the solubilizing capacity of the cationic detergents was not feasible because of their being potent inhibitors of glucocerebrosidase activity. 3. Treatment of the enzyme aggregate with acetone rendered it buffer-soluble. 4. In addition to the above cationic detergents some choline-containing and highly hydrophobic phospholipids were found to inhibit the glucocerebrosidase activity.

Cholic Acids↗

PubMed1996

Optimization studies of components in enzymatic cholesterol reagents containing cholesterol oxidase from Nocardia erythropolis, Streptomyces sp, or Pseudomonas fluorescens.

Although enzymatic methods for serum cholesterol determination are widely used in clinical laboratories, little is known about the optimization of each component in enzymatic reagents. We investigated the optimal components in the reagents containing cholesterol oxidase isolated from Nocardia erythropolis, Streptomyces sp, or Pseudomonas fluorescens. The optimal components in the reagents are: cholesterol oxidase 250 (Nocardia erythropolis), 250 (Streptomyces sp), or 300 (Pseudomonas fluorescens) U/L, cholesterol esterase 200 U/L, peroxidase 10,000 U/L, sodium cholate 3 mmol/L, 4-aminoantipyrine 0.5 mmol/L, phenol 20 mmol/L, Triton X-100 2 mL/L, and phosphate buffer, pH 7.0. Lower reaction sensitivity and lower cholesterol linearity, < 18.1 mmol/L (700 mg/dL), could be obtained by using lower components than those suggested above. Pseudomonas fluorescens were an improper source for cholesterol oxidase; either Nocardia erythropolis or Streptomyces was suitable cholesterol oxidase. We prefer using Streptomyces sp cholesterol oxidase because of its economical cost and longest reagent stability. Sodium cholate must be included in the enzymatic reagent to prevent turbidity. However, sodium cholate of > 5 mmol/ L will suppress the reaction resulting in low cholesterol linearity.

Ampyrone↗

PubMed1987

Neurotoxic esterase: characterization of the solubilized enzyme and the conditions for its solubilization from chicken brain microsomal membranes with ionic, zwitterionic, or nonionic detergents.

Neurotoxic esterase (NTE) is a membrane-bound protein found in highest concentration in brain and lymphocytes. The enzyme has no known physiological function, but its organophosphorylation and aging in neural tissue are thought to trigger the pathogenesis of organophosphorus-induced delayed neuropathy (OPIDN). Solubilization of NTE from microsomal membranes from hen or chick brain was studied with ten detergents encompassing ionic, zwitterionic, or nonionic types. Corrected yields of NTE solubilized over a range of [detergent]/[protein] ratios were determined by dividing the activity not sedimenting in detergent at 100,000 g for 60 min at 4 degrees by the activity in the original microsomal fraction with no detergent present. Highest corrected yields were obtained with sodium cholate (44%), Triton X-100 (48%), and nonyl-GPS (57%). Partial loss of NTE activity occurred in the presence of detergent which could be prevented by the inclusion of asolectin in the solubilization preparation. NTE could not be solubilized by omitting detergent or by substituting 2 M NaCl for detergent. Mipafox pI50 values obtained from complete titration curves carried out on NTE solubilized in Triton X-100, sodium cholate, or sodium cholate/asolectin were indistinguishable from the value for native enzyme from brain homogenate. These results indicate that NTE exhibits the properties of an integral membrane protein with lipid dependence. The enzyme can be solubilized in good yield with a variety of detergents with retention of its characteristic differential inhibition by paraoxon and mipafox, a necessary prelude to bulk purification of the enzymatically active protein.

Animals↗