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1.
This report describes a purification procedure for a cholesteryl ester hydrolase (CEH) from female rat liver microsomes, and some structural, immunological, kinetic, and regulatory properties of the enzyme that distinguish the microsomal CEH from other hepatic cholesteryl ester-splitting enzymes. CEH was purified 12.4-fold from reisolated microsomes using sequential solubilization by sonication, polyethylene glycol precipitation, fractionation with hydroxyapatite, anion exchange chromatography, and chromatography on hydroxyapatite, with an overall yield of 3.2%. CEH activity was purified 141-fold over nonspecific esterase activity and 56-fold over triacylglycerol lipase activity. In sharp contrast with most esterases and lipases, CEH did not bind to concanavalin A-Sepharose and heparin-Sepharose. After polyacrylamide gel electrophoresis, the purified enzyme exhibited two silver-stained bands, but only the protein electroeluted from the low mobility band had CEH activity. Affinity-purified polyclonal antibodies raised to electroeluted CEH inhibited 90% of the activity of liver microsomal CEH and reacted with a 106 kDa protein band on Western blot analysis. This 106 kDa CEH contains a unique N-terminal amino acid sequence. The purified enzyme had optimal activity at pH 6 and no taurocholate requirements, and was inhibited by the serine active site inhibitor phenylmethylsulfonyl fluoride and by free sulfhydryl specific reagents. It hydrolyzed cholesteryl oleate much more efficiently than trioleine, and hydrolytic activity with p-nitrophenyl acetate was higher than with p-nitrophenyl butyrate. These results indicate that rat liver microsomes contain a bile salt-independent catalytic protein that is relatively specific for cholesteryl ester hydrolysis. 相似文献
2.
The solubilization and morphological change of human platelets in various detergents 总被引:1,自引:0,他引:1
The solubilization of human gel-filtered platelets by octyl glucoside, Triton X-100, dodecylsulfate, and deoxycholate was compared from the analysis of (1) cell lysis, (2) marker leakiness, and (3) component solubility. These analyses all revealed that the effect of detergent concentration on the solubilization of platelets by these detergents was exerted in three stages, i.e., the prelytic, lytic, and complete platelet-lysis stages. These analyses also indicated several differences among platelets in these detergents. (i) The ratio of the platelet-saturation concentration (PSC) to critical micellar concentration (CMC) was about 1/2 for octyl glucoside. Triton X-100 and dodecylsulfate, while it was close to 1 for deoxycholate. (ii) Platelets in octyl glucoside. Triton X-100, and dodecylsulfate all showed parallel curves in cell lysis, protein solubilization and marker leakiness, while the platelet lysis in deoxycholate was identical to the phospholipid solubilization. (iii) The solubility curves of various components in Triton X-100 and deoxycholate were parallel. However, the solubility of cholesterol in octyl glucoside was lower than that of protein and phospholipid. In dodecylsulfate, the solubility of phospholipid and cholesterol was very low in comparison with that of protein. In addition, morphological studies using scanning electron microscopy (scanning EM) revealed that the solubilization by octyl glucoside or Triton X-100 might occur via membrane area expansion. On the other hand, the solubilization by dodecylsulfate or deoxycholate showed membrane vesiculation prior to cell lysis. Moreover, in the prelytic stage, the morphological change in platelets in octyl glucoside showed only concentration dependence by swelling to an ellipsoid and then to a sphere. However, the morphological change in platelets in the other three detergents was dependent not only on the detergent concentration but also on prolonged incubation. Specifically, in Triton X-100, the cells initially changed to spiculate discs and then reached their final shape as swollen discs with surface invagination. In dodecylsulfate and deoxycholate the morphological changes were almost the same. The cell initially deformed in shape to a spiculate disc and finally to a stretched-out flat form. The results are discussed according to the bilayer couple hypothesis. Also, in the prelytic stage, these detergents caused inhibition of the response of platelets to collagen and ADP-fibrinogen. 相似文献
3.
Conditions for the solubilization of membrane-bound neutral proteinase associated with monkey liver microsomes were investigated. Among the reagents tested, deoxycholate, cholate, and some nonionic detergents, including Triton X-100, with hydrophilic-lipophilic balance values of around 13, were effective. The solubilization profile indicated that the enzyme is bound to the microsomal membranes by strong hydrophobic interaction. The enzyme was partially purified from monkey liver microsomal fraction, previously washed with 1 M KCl and 0.05% sodium dodecyl sulfate, by Triton X-100 extraction, followed by chromatography on columns of hydroxylapatite and Sepharose CL-6B. The apparent molecular weight of the enzyme was estimated to be about 88,000 from the elution position on Sepharose CL-6B column chromatography in the presence of 0.5% sodium cholate. It was optimally active at pH 8.0 with heat-denatured casein as a substrate. It was strongly inhibited by diisopropyl phosphorofluoridate and phenylmethanesulfonyl fluoride, indicating that the enzyme is a serine proteinase. EDTA, EGTA, and chymostatin also inhibited the enzyme strongly. Among urea-denatured protein substrates tested, calf thymus histone was hydrolyzed most rapidly, followed by casein, hemoglobin, and bovine serum albumin, whereas practically no hydrolysis occurred with denatured ovalbumin, fibrinogen, and gamma-globulin as substrates. 相似文献
4.
A mathematical model of erythrocyte lysis by detergents is developed which takes into consideration the kinetics of detergent binding to plasma membrane. Experimentally obtained sigmoidal kinetic and concentration curves of hemolysis are well described by the model. A comparative study is carried out in terms of the model of hemolytic action for five detergents: Triton X-100, sodium dodecylsulfate, sodium deoxycholate, cetyltrimethylammonium bromide, and cetylpyridinium chloride. The amount of detergent which should be bound to an erythrocyte membrane to induce lysis was found to be roughly the same for all detergents studied. However, detergents vary in their affinity to the membrane. Cetylpyridinium displays the highest affinity (and consequently the highest hemolytic activity), whereas deoxycholate has the least one. 相似文献
5.
The enzyme guanylate cyclase is present in both particulate and soluble form in rat lung homogenates. As previously reported, the soluble enzyme can be activated by preincubation in the presence of O2. The inactive (nonactivated) soluble enzyme is also stimulated by nonionic detergents, in the order Tween 20 > Lubrol PX > Triton X-67 > Triton X-100. The activated enzyme, however, was inhibited by these detergents in the reverse order. Sodium deoxycholate and lysolecithin were potent inhibitors of both inactive and activated enzyme. The activity of the particulate enzyme was stimulated by Lubrol PX > Triton X-100 > Triton X-67 > Tween 20. At a low concentration of lysolecithin or deoxycholate the particulate activity was increased; however, when detergent/protein > 1, inhibition was seen. In the case of deoxycholate, the inhibition could be reversed if excess deoxycholate was removed either by chromatography or by forming mixed micelles with Lubrol PX; however, deoxycholate inhibition of the soluble enzyme was irreversible. The stimulation by detergents of the particulate enzyme was apparently the result of solubilization. The effects upon the activity of the soluble enzyme were interpreted in terms of a model which assumes two hydrophobic regions on the enzyme surface. The two regions differ in hydrophobicity with the more hydrophobic region only being exposed as a result of activation. Interaction of a nonionic detergent with the less hydrophobic region stimulates activity, while interaction with the more hydrophobic region results in inhibition. 相似文献
6.
The solubilization of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from the membrane fraction was studied in whole leaf extracts and chloroplasts from pea. The amount of membrane-bound Rubisco was dependent on the pH of the chloroplastic lysate buffer. Maximum binding was found at pH 8.0, with about 8% of total leaf Rubisco being bound. The binding of Rubisco to the membranes was strong, and it was not released by repeated washing with hypotonic buffer or by changing ionic strength. Detergents such as Triton X-100, Tween 20, deoxycholate and dodecylsulfate were effective in solubilizing the membrane-bound Rubisco. Triton X-100 was most effective in the range of 0.04% to 0.2% and it solubilized Rubisco from the membrane without any decrease in enzyme activity.Abbreviations BSA
bovine serum albumin
- CABP
carboxyarabinitol-1,5-bisphosphate
- DTT
dithiothreitol
- LDS
lithium dodecylsulfate
- LHC
light-harvesting chlorophyll protein complex
- RuBP
ribulose-1,5-bisphosphate
- Rubisco
RuBP carboxylase/oxygenase
- SDS
sodium dodecylsulfate
- SDS-PAGE
SDS-polyacrylamide gel electrophoresis 相似文献
7.
A sensitive assay for 5 alpha-reductase was introduced which is capable of detecting at least 0.2 U of activity per sample. The assay was used in developing a method for the solubilization of human prostatic 5 alpha-reductase. Homogenisation conditions were devised under which 95% of the total prostatic 5 alpha-reductase was released into the microsomal fraction. A combination of 0.1 M sodium citrate, 0.1 M KCl, 20% (v/v) glycerol, 0.5 mM NADPH and 1 microM testosterone was found to stabilise 5 alpha-reductase in the presence of detergents. The effect of the presence of low concentrations of detergents in the assay on the activity of 5 alpha-reductase was studied. Triton X-100, Lubrol PX and Nonidet P-40, caused a concentration-dependent inhibition of activity. The ability of several detergents (Triton X-100 MEGA-9, Tween 20, Tween 80, digitonin, Lubrol PX and Nonidet P-40) to solubilise 5 alpha-reductase was studied. All detergents caused a concentration-dependent solubilization of 5 alpha-reductase. Significant amounts of active solubilized enzyme were recovered only with Lubrol PX at concentrations less than 1.1 mg/ml. Seventy percent of the 5 alpha-reductase was solubilized in an active form by extracting the membranes 3 times with 0.8 mg/ml Lubrol PX. 相似文献
8.
Triton X-100 is increasingly effective in solubilizing human liver glycoprotein (asialofetuin) sialytransferase (CMP-N-acetylneuraminate:D-galactosyl-glycoprotien N-acetylneuraminyltransferase, EC 2.4.99.1) activity as its concentration is increased in the homogenizing buffer. At the optimal concentration of 1.5% (v/v), essentially all of the homogenate sialyltransferase activity was solubilized into the supernatant fluid. Higher concentrations of Triton X-100 inhibited sialyltransferase activity. Several kinetic properties of the solubilized asialofetuin-sialyltransferase activity were compared to those of the membrane-bound enzyme(s) (in homogenates made without Triton X-100 or in resuspended pellets). No major difference was apparent, suggesting that solubilization has not significantly altered the properties of sialyltransferase. The solubilized sialyltransferase activity is quite unstable, losing approximately 50% of its activity after one week of storage at 4 degrees C. Various detergents (Zwittergent, sodium taurocholate and sodium deoxycholate) are differentially effective in stabilizing the solubilized activity. Sodium taurocholate (1.5%, w/v) was most effective with no loss in activity after 40 days and minimal loss (14%) after 60 days storage at 4 degrees C. The solubilized sialyltransferase preparation retains full activity after storage in the frozen state (-20 degrees C) for at least 159 days. 相似文献
9.
The ability of eight stripping agents to solubilize five marker enzymes from rat renal brush border membranes isolated by three different preparative methods was examined. Protein and enzyme activities - alkaline phosphatase (APase), L-leucine aminopeptidase (LAPase), gamma-glutamyl transpeptidase (GGTase), gamma-glutamyl hydrolase (GGHase) and maltase - solubilized by the treatments were expressed as percent of total activity recovered in excess of control values. The relative enzyme activity and the solubilization factor were determined for each marker enzyme in every treated sample and the treatments with the eight agents compared. Trypsin treatment released > 80% of LAPase and < 10% of total membrane protein. Papain treatment released only 16--23% of total membrane protein but most of the enzyme activities except APase. Neuraminidase had no solubilizing effect. 4--10% of total membrane protein was solubilized by LiCl treatment but no marker enzyme activities were released. Less total membrane protein was released by treatment with proteolytic enzymes or LiCl than with the detergents Triton X-100, hexadecyltrimethylammonium bromide, sodium deoxycholate, and sodium dodecylsulfate. APase activity was the least readily solubilized. Correlating the degree of solubilization for five marker enzymes with the types of stripping agents used and with the appearance of the membrane surface when examined by electron microscopy led to the suggestion that LAPase, GGTase, GGHase and maltase molecules are part of an interwoven surface layer of membrane proteins which can be disrupted by transamidation and transesterification reactions. APase appears to be more strongly associated with the intact lipid matrix than the bulk of the membrane protein. 相似文献
10.
K Prakash S D Patil S N Hegde 《Archives internationales de physiologie et de biochimie》1982,90(4):255-264
In the pigeon, 70-80% of the activities of maltase (alpha-D-glucoside glucohydrolase EC 3.2.1.20), sucrase (alpha-glucohydrolase, EC 3.2.1.48), isomaltase (dextran 6-alpha-D-glucan hydrolase, EC 3.2.1.10) and glucoamylase (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) were found to be localized in the brush-border membrane of intestinal epithelial cells. Of the total glycosidase activities in the mucosal homogenate, nearly 60 to 70% were recovered in the microsomal (105 000 X g) fraction, about 30% in the mitochondrial (22 000 X g) fraction and less than 5% from the cytosol (105 000 X g supernatant) fraction. The hydrolases were solubilized by digestion with papain but not with trypsin, and the phosphate ion had a protective effect in the solubilization. Amongst detergents, Triton X-100 but not sodium deoxycholate, was found to truly solubilize these enzymes. 相似文献
11.
1. The lipid requirement for maximum desaturase activity was investigated using acetone/water mixtures. It was shown that for maximum stearoyl-CoA desaturase activity of hen liver microsomes neither the total neutral lipid fraction nor 44% of the phospholipid fraction were required. 2. The effect of sodium deoxycholate, Triton X-100, Nonidet P-40 and Bio-solv on the enzyme activity indicated that the neutral detergents had a milder effect than the ionic detergent but both classes could cause considerable irreversible loss of activity. 3. The treatment of the microsomes with 2.5% (v/v) water in acetone greatly improved the effective solubilising power of Triton X-100. The yield of desaturase in the 100 000 X g supernatant obtained by treating the microsomal fraction in this way was strongly dependent upon protein concentration. Maximum solubilisation was achieved with25 mg protein per ml 1% (w/v) Triton X-100 in 0.1 M potassium phosphate buffer pH 7.4. 4. A comparison of the properties of the solubilised and membrane-bound enzyme was made by an investigation of: (i) the temperature and pH optimum, (ii) activation energy and (iii) the effect of inhibitors on the enzyme activity. 相似文献
12.
1. A number of local anaesthetics was shown to inhibit rat liver cholesterol esterase activity towards radioactively labelled cholesterol oleate. The anaesthetics inhibited in the order dibucaine greater than chlorpromazine greater than tetracaine greater than benzocaine greater than procaine greater than lidocaine greater than cocaine. 2. The mode of inhibition was seen to be non-competitive with respect to the substrate and is probably independent of any involvement of Ca2+. 3. The inhibition by tetracaine is partially reversed by sodium deoxycholate. However, all ionic and non-ionic detergents studied, sodium deoxycholate, sodium taurocholate, Triton X-100, and cetyltrimethylammonium bromide are capable of inhibiting the rat liver cholesterol esterase in a concentration dependent manner. Only sodium taurocholate stimulates enzymic activity. 相似文献
13.
The kinetics of palmitoyl-CoA hydrolase were influenced by both the availability of the substrate and formation of micelles. At palmitoyl-CoA concentrations below the critical micelle concentration, addition of non-ionic detergent increased the activity until the critical micelle concentration of the mixed micelles was reached. At palmitoyl-CoA concentrations above the critical micelle concentration, inhibitor of the activity was observed, but addition of detergents of the Triton X series reversed the inhibition. Maximum palmitoyl-CoA hydrolase activity was found when the ratios (w/v) of palmitoyl-CoA: Triton X-100 and palmitoyl-CoA: Triton X-405 were approximately 0.35 and 0.05, respectively. At these above the mixed critical micelle concentration. The results indicate that monomer palmitoyl-CoA is the substrate and that monomer forms of the non-ionic detergents of the Triton X series activate the enzyme. Isolated microsomal lipids activated the microsomal palmitoyl-CoA hydrolase, suggesting that a hydrophobic environment is advantageous for interaction between enzyme and substrate in vivo. The maximum activity in the presence of mixed micelles is discussed in relation to a model where mixed micelles are regarded as artificial membranes to which the enzyme may adhere in an equilibrium with the monomer substrate and detergent in the monomer form. It is suggested that intracellular membranes may resemble mixed micelles in equilibrium with detergent-active substrates such as palmitoyl-CoA. 相似文献
14.
D W Marseno K Hori K Miyazawa 《Comparative biochemistry and physiology. B, Comparative biochemistry》1992,102(2):247-253
A preliminary examination for the purification and characterization of 5'-nucleotidase of fish muscle was carried out and the following results were obtained. 1. The activities of 5'-nucleotidase in the muscles of marine vertebrates and invertebrates (total 11 species) were determined. The highest activity of 5'-nucleotidase was found in Blackrock fish Sebastes inermis, which was then used as a material for estimation of subcellular distribution and solubilization of the enzyme. 2. The 5'-nucleotidase of ordinary muscle of the fish Sebastes inermis was found in nuclear, microsomal and cytosolic fractions. About half of the total activity was found in the nuclear fraction, whereas the highest specific activity was observed in the microsomal fraction. 3. Complete solubilization of the enzyme was attained by using a high concentration of detergent such as Triton X-100, CHAPS, octylglucoside, octylthioglucoside and sodium deoxycholate, suggesting that the enzyme was tightly bound to the membrane. 4. Based on the results of solubility and stability tests, Triton X-100 seemed suitable for solubilizing 5'-nucleotidase from the membrane. 5. Microsomal 5'-nucleotidase was an Mg(2+)-activated enzyme, and no inactivation was observed up to 50 mM of Mg2+. 相似文献
15.
The possibilities to solubilize the rat brain cortex muscarinic acetylcholine receptor and its complex with [3H]-L-quinuclidinyl benzilate (QNB) were studied, using 14 detergents. It was shown that the native muscarinic cholinoreceptor was solubilized in addition to digitonin, also by CHAPS, with a 6% yield. Besides, the receptor-QNB complex was solubilized with the detergents Triton X-100, -102, -114, -165 (with 30% and 50% yields) and within a narrow concentration range with sodium dodecyl sulfate (50% yield). Some detergents of the Tween series, e.g., Triton X-45 and -305, as well as sodium deoxycholate and sodium oxycholate, did not solubilize the native receptor and its complex with QNB. It was found that yield of receptor solubilization did not exceed half of the total number of the receptor sites in the membranes, despite the fact that different concentrations of detergents were applied. The solubilization yield did not increase, when different mixtures of detergents were used. It was assumed that incomplete solubilization of the receptor protein reflects its heterogeneity in the membrane structure. 相似文献
16.
A purification procedure for rat brain phosphatidylinositol synthetase (PI synthetase; CDP-1,2-diacyl-sn-glycerol:myo-inositol 3-phosphatidyltransferase; EC 2.7.8.11) is described. The enzyme was purified 200-250-fold from the homogenate by solubilization with Triton X-100 from microsomal membranes and affinity chromatography on CDP-diacylglycerol-Sepharose. Elution of enzyme activity required the presence of Triton X-100, CDP-diacylglycerol, and either phosphatidylcholine or asolectin. The product that was obtained in 5-10% yield from whole brain and in 70% yield from the microsomal fraction contained three protein bands as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The final preparation contained levels of CDP-diacylglycerol hydrolase and CDP-diacylglycerol: sn-glycero-3-phosphate 3-phosphatidyltransferase activities that were less than 1% of PI synthetase activity. The purified enzyme displayed a pH optimum of 8.5-9.0, required either Mg2+ or Mn2+ and exhibited a Km of 4.6 mM for myo-inositol. 相似文献
17.
Electrophoretic patterns of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) from rat erythrocyte were studied. The enzyme was solubilized by the following treatments: a) Triton X-100, b) sodium deoxycholate, or c) ultrasonic irradiation. When the erythrocyte membrane was solubilized by Triton X-100 at concentrations higher than 0.3%, by 10 mM sodium deoxycholate, or by ultrasonic irradiation for more than 5 min, a single band of acetylcholinesterase activity appeared in the gel. Two bands of activity were stained in the gel when the membrane was solubilized by Triton X-100 at concentrations between 0.1--0.2%, or by ultrasound for 5 min. Electrophoretic patterns of acetylcholinesterase from rats fed a fat-sufficient diet were similar to those for the enzyme from animals fed a fat-free diet. The recombination of lipids with the enzyme eluted from the gels confirmed the "phenotypic allosteric desensitization phenomenon". 相似文献
18.
《Biochimica et Biophysica Acta (BBA) - Enzymology》1976,422(1):127-137
Electrophoretic patterns of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) from rat erythrocyte were studied. The enzyme was solubilized by the following treatments: a) Triton X-100, b) sodium deoxycholate, or c) ultrasonic irradiation. When the erythrocyte membrane was solubilized by Triton X-100 at concentrations higher than 0.3%, by 10 mM sodium deoxycholate, or by ultrasonic irradiation for more than 5 min, a single band of acetylcholinesterase activity appeared in the gel. Two bands of activity were stained in the gel when the membrane was solubilized by Triton X-100 at concentrations between 0.1–0.2%, or by ultrasound for 5 min.Electrophoretic patterns of acetylcholinesterase from rats fed a fat-sufficient diet were similar to those for the enzyme from animals fed a fat-free diet. The recombination of lipids with the enzyme eluted from the gels confirmed the “phenotypic allosteric desensitization phenomenon”. 相似文献
19.
Phosphatidylinositol biosynthesis in Saccharomyces cerevisiae: purification and properties of microsome-associated phosphatidylinositol synthase 总被引:17,自引:11,他引:6
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The membrane-associated phospholipid biosynthetic enzyme phosphatidylinositol synthase (cytidine 5'-diphospho-1,2-diacyl-sn-glycerol:myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11) was purified 1,000-fold from the microsomal fraction of Saccharomyces cerevisiae. The purification procedure included Triton X-100 solubilization of the microsomal membranes, CDPdiacylglycerol-Sepharose (Larson et al., Biochemistry 15:974-979, 1976) affinity chromatography, and chromatofocusing. The procedure resulted in the isolation of a nearly homogeneous protein preparation with an apparent minimum subunit molecular weight of 34,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Phosphatidylinositol synthase was dependent on manganese and Triton X-100 for maximum activity. The pH optimum was 8.0. Thioreactive agents inhibited enzyme activity. The energy of activation was found to be 35 kcal/mol (146,540 J/mol). The enzyme was reasonably stable at temperatures of up to 60 degrees C. 相似文献
20.
A rapid and simple purification of milligram amounts of 2,3-oxidosqualene cyclase, an integral membrane enzyme that catalyzes the cyclization of squalene epoxide to lanosterol, is reported. Several nonionic detergents (Triton X-100, Tween 80, Emulphogene, and lauryl maltoside) were evaluated for solubilization of oxidosqualene cyclase from rat liver microsomes. At a detergent concentration of 5 mg/ml, lauryl maltoside was approximately 10 times more effective than Emulphogene in the solubilization of oxidosqualene cyclase; Triton X-100 and Tween 80 were less effective than Emulphogene as judged by the relative specific activities of the solubilized enzyme. Treatment of microsomes with lauryl maltoside resulted in a selective solubilization of the cyclase with concomitant activation of the enzyme. The solubilized enzyme was purified to homogeneity by fast protein liquid chromatography. The purified enzyme consists of a single subunit that has an apparent molecular weight of 65,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme obeys saturation kinetics and the apparent Km of (2,3)-oxidosqualene is 15 microM; the apparent kcat/Km is 200 M-1.min-1. An improved assay of the enzyme that utilizes high performance liquid chromatography methods is also described. 相似文献