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1.
A small hydrophobic domain that localizes human erythrocyte acetylcholinesterase in liposomal membranes is cleaved by papain digestion 总被引:3,自引:0,他引:3
A small hydrophobic domain in isolated human erythrocyte acetylcholinesterase is responsible for the interaction of this enzyme with detergent micelles and the aggregation of the enzyme on removal of detergent. Papain has been shown to cleave this hydrophobic domain and to generate a fully active hydrophilic enzyme that shows no tendency to interact with detergents or to aggregate [Dutta-Choudhury, T.A., & Rosenberry, T.L. (1984) J. Biol. Chem. 259, 5653-5660]. We report here that the intact enzyme could be reconstituted into phospholipid liposomes while the papain-disaggregated enzyme showed no capacity for reconstitution. More than 80% of the enzyme reconstituted into small liposomes could be released by papain digestion as the hydrophilic form. Papain was less effective in releasing the enzyme from large liposomes that were probably multilamellar. In a novel application of affinity chromatography on acridinium resin, enzyme reconstituted into small liposomes in the presence of excess phospholipid was purified to a level of 1 enzyme molecule per 4000 phospholipid molecules, a ratio expected if each enzyme molecule was associated with a small, unilamellar liposome. Subunits in the hydrophilic enzyme form released from reconstituted liposomes by papain digestion showed a mass decrease of about 2 kilodaltons relative to the intact subunits according to acrylamide gel electrophoresis in sodium dodecyl sulfate, a difference similar to that observed previously following papain digestion of the soluble enzyme aggregates. The data were consistent with the hypothesis that the same hydrophobic domain in the enzyme is responsible for the interaction of the enzyme with detergent micelles, the aggregation of the enzyme in the absence of detergent, and the incorporation of the enzyme into reconstituted phospholipid membranes. 相似文献
2.
Malic enzyme activity in differentiated 3T3-L1 cells was about 20-fold greater than activity in undifferentiated cells. A new steady-state level was achieved about 8 days after initiating differentiation of confluent cultures with a 2-day exposure to dexamethasone, isobutylmethylxanthine, and insulin. This increase in enzyme activity resulted from an increase in the mass of malic enzyme as detected by immunotitration of enzyme activity with goat antiserum directed against purified rat liver malic enzyme. Malic enzyme synthesis was undetectable in undifferentiated cells and increased to about 0.2% of soluble protein in differentiated cells, suggesting that the increase in enzyme mass was due primarily to an increase in enzyme synthesis. Thyroid hormone, a potent stimulator of malic enzyme activity in hepatocytes in culture and in liver and adipose tissue in intact animals, decreased or increased malic enzyme activity in differentiating 3T3-L1 cells by about 40% when it was removed or added to the medium, respectively. Insulin, another physiologically important regulator of malic enzyme activity in vivo, had no effect on the initial rate of accumulation of malic enzyme activity in the differentiating cells and caused a 30 to 40% decrease in the final level of enzyme activity in the fully differentiated cells. Cyclic AMP, a potent inhibitor of malic enzyme synthesis in hepatocytes in culture, inhibited this process in 3T3-L1 cells by 30%. Malic enzyme is like several other enzymes in that the large increase in its concentration which accompanies differentiation of 3T3-L1 cells is due to increased synthesis of enzyme protein. However, the hormonal modulation of malic enzyme characteristic of liver and adipose tissue in intact animals does not appear to occur in differentiated 3T3-L1 cells, suggesting that differentiated 3T3-L1 cells may not be an appropriate model system in which to study the hormonal modulation of malic enzyme that occurs in liver and adipose tissue of intact animals. 相似文献
3.
Lecithin-cholesterol acyltransferase was purified from rat plasma and the properties of this enzyme during the purification procedures and those of the purified enzyme were investigated in comparison with the human enzyme. The rat enzyme was not adsorbed on hydroxyapatite, which was employed for the purification of the human enzyme. When purified human enzyme was incubated at 37 degrees C in 0.1 mM phosphate buffer (pH 7.4; ionic strength, 0.00025), no alteration of enzyme activity was observed for up to 6 h. In the case of the rat enzyme, however, approximately 40% of the enzyme activity was lost under the same conditions. The human enzyme and rat enzyme were both retained on a Sepharose 4B column to which HDL3 was covalently linked, in 39 mM phosphate buffer, pH 7.4. Although the human enzyme was eluted from the column in 1 mM phosphate buffer, the rat enzyme was dissociated from the column at a lower buffer concentration (0.1 mM phosphate buffer). These findings indicate that the rat enzyme effectively associated with HDL3 in 39 mM phosphate buffer, pH 7.4, but the association was more sensitive to increase of ionic strength compared with that of the human enzyme. 相似文献
4.
Membrane-bound L-lactate dehydrogenase was freed from the detergent used during purification. The detergent-free enzyme had about one-half the specific activity of the enzyme in 1.0% Tween 80, and was only partially sensitive to the specific antibody. This enzyme was activated about 3-fold with phosphatidylglycerol, cardiolipin, or a mixture of phospholipids. The phospholipid-activated enzyme had a similar Km value for L-lactate to that of the membrane enzyme and was completely inhibited by the specific antibody. On heat treatment, the phospholipid-activated enzyme was more stable than detergent-free enzyme and was as stable as membrane-bound enzyme. The alpha helical content of the enzyme increased 1.7-fold during preincubation with these lipids and the alpha helix became more stable during heat treatment than that of the detergent-free enzyme. These results suggest that the enzyme showed monomolecular dispersion in the lipid bilayer and that its conformation, including its active site and secondary structure, was different from that of the detergent-free enzyme. Phosphatidylethanolamine, dilauroyl lecithin and lecithin from egg yolk had none of the above effects on the activity or the secondary structure of the enzyme. On the other hand, mixtures of each of these lipids and cholate had essentially similar effects to phosphatidylglycerol. 相似文献
5.
A homogeneous preparation of ;malic' enzyme (EC 1.1.1.40) from livers of thyroxine-treated rats was used to prepare in rabbits an antiserum to the enzyme that reacts monospecifically with the ;malic' enzyme in livers of rats in several physiological states. Changes in enzyme activity resulting from modification of the state of the animal are hence due to an altered amount of enzyme protein. The antiserum has been used to precipitate out ;malic' enzyme from heat-treated supernatant preparations of livers from both adult and neonatal rats, in a number of physiological conditions, that had been injected 30min earlier with l-[4,5-(3)H]leucine. The low incorporations of radioactivity into the immunoprecipitable enzyme have permitted the qualitative conclusion that changed enzyme activity in adult rats arises mainly from alterations in the rate of enzyme synthesis. The marked increase in ;malic' enzyme activity that occurs naturally or as a result of thyroxine treatment of the weanling rat is likewise due to a marked increase in the rate of enzyme synthesis possibly associated with a concurrent diminished rate of enzyme degradation. 相似文献
6.
B. Adu-Amankwa A. Constantinides W. R. Vieth 《Biotechnology and bioengineering》1981,23(11):2609-2627
Penicillium duponti enzyme was immobilized on reconstituted collagen by macromolecular complication, impregnation, and covalent crosslinking techniques. The immobilization of the enzyme on collagen has a twofold purpose: (1) providing a protein microenvironment for the proteolytic enzyme; and (2) extending the useful life the enzyme once immobilized on the collagen matrix. Two types of collagen were used, one produced by the United States Department of Agriculture and the other produced by FMC. The USDA collagen contained unhydrolyzed telepeptide linkages and required pretreatment to reduce collagenaselike activity of the enzyme. Activity analysis of the immobilized enzyme complex showed that membranes with enzyme loading less than 10 mg enzyme/g of wet membrane in the reactor were dimensionally stable. The degree of crosslinking was an important parameter. Membranes with structural opening up to three times the initial dry thickness were found to be the maximum limit for controlled release of enzyme from the collagen membrane during enzymatic reaction. Higher activities and better stability of the enzyme in collagen membrane were found for covalent crosslinking of the enzyme to treated collagen films. The hydrolysis of soybean vegetable protein with the immobilized enzyme in a recycle reactor at enzyme loading of mg/g of wet membrane at 40°C, pH 3.4, produced 56.5% of soluble protein in 10h. The production is equivalent to 1.84 h total contact time between the substrate and the immobilized enzyme. The average productivity based on a stable enzyme activity and 20g of dry membrane was 329 mg of protein/g/mg of active enzyme immobilized. The productivity of the free enzyme in a batch reactor was 62.5 mg protein/h/mg enzyme. 相似文献
7.
DISTRIBUTION AND PROPERTIES OF ANGIOTENSIN CONVERTING ENZYME OF RAT BRAIN 总被引:29,自引:19,他引:10
Abstract— Angiotensin converting enzyme of rat brain was studied using Hip-His-Leu as substrate. The highest specific activity of the enzyme was associated with the microsomal fraction. The specific activity of the microsomal enzyme in several regions of the rat brain varied significantly. For example, the specific activities of the striatal and pituitary enzymes were about 10-fold greater than that of the cerebral cortical enzyme. The enzyme required chloride ion; moreover, activity was inhibited in the presence of disodium EDTA or O-phenanthroline, effects suggesting that the converting enzyme of brain is a metalloprotein. SQ-20881, a nonapeptide that inhibits converting enzyme in peripheral tissue, was a potent inhibitor of the enzyme of brain. In addition to Hip-His-Leu, the microsomal fraction was capable of liberating C terminal dipeptides from angiotensin I, Hip-Gly-Gly and Z-Gly- Gly-Val. The broad substrate specificity of the enzyme suggests that, in addition to the possible contribution of the enzyme to the brain renin-angiotensin system, other naturally occurring peptides might also be substrates for the enzyme. 相似文献
8.
Two DNA polymerases were detected in Tetrahymena pyriformis, strain GL. One (enzyme I) was sensitive to N-ethylmaleimide, while the other (enzyme II) was insensitive. The molecular weight of the enzymes, as determined by glycerol gradient centrifugation analysis, were approximately 130,000 and 70,000, respectively. Optimal concentration of MgCl2 was 10mM for enzyme I and 18mM for enzyme II. KCl inhibited enzyme I but stimulated enzyme II. Poly (dA-dT) served effectively as a template for enzyme I, while poly(dA).(dT)12-18 was an effective template for enzyme II. Enzyme I activity increased with cell growth and sharply declined after the cells reached the stationary phase. On the other hand, enzyme II activity appeared only at the end of log phase. In cells synchronized by starvation-refeeding technique enzyme I was markedly stimulated in correspondence to the rate of DNA synthesis, whereas the level of enzyme II activity changed to lesser extent. By ethidium bromide treatment, only enzyme I activity was induced. 相似文献
9.
Dihydrofolate reductase (E.C. 1.5.1.3) from Plasmodium falciparum and from its host, the owl monkey (Aotus trivirgatus), were partially purified and characterized. The molecular weight of the parasite enzyme was estimated to be over 10 times as high as that of the host enzyme. The host enzyme had 2 pH optima whereas the parasite enzyme only one. The activity of the host enzyme was greatly stimulated by KCl and urea, while that of the parasite enzyme was inhibited at high concentrations of such chaotropic agents. Km of the parasite enzyme was significantly higher than that of the host enzyme. The parasite enzyme had much lower Ki for pyrimethamine than the host enzyme. Dihydrofolate reductases isolated from pyrimethamine-resistant and pyrimethamine sensitive strains of P. falciparum were found to be similar. 相似文献
10.
Malate thiokinase has been purified to apparent homogeneity by employing conventional purification techniques along with affinity chromatography. The enzyme is composed of two nonidentical subunits (alpha subunit Mr=34,000, beta subunit Mr=42,500) to yield an alpha 4 beta 4 structure for the native enzyme. Phosphorylation of the enzyme by ATP occurs exclusively on the alpha subunit. The phosphorylated enzyme is acid labile and base stable consistent with phosphorylation of a histidine residue. Dephosphorylation of the enzyme is promoted by ADP, succinate, malate, and coenzyme A plus inorganic phosphate. Phosphorylation of the enzyme leads to a reversible change in the sedimentation properties of the enzyme; the native enzyme exhibits an S20,w of approximately 10, whereas the phosphoenzyme exhibits an S20,w of approximately 7. Formation of the 7 S form of the enzyme is also observed when coenzyme A and succinyl-CoA interact with the enzyme. The ratio of alpha to beta subunits in both the 10 S and 7 S forms of the enzyme is approximately 1.0, suggesting that the 7 S form of the enzyme has an alpha 2 beta 2 structure. 相似文献
11.
Immunological characterization of maize starch branching enzymes 总被引:1,自引:1,他引:0
Highly purified fractions of three starch branching enzymes from developing maize (Zea mays L.) endosperm were used to prepare antisera in rabbits. In double diffusion experiments, no immunoprecipitate was observed when branching enzyme IIa or IIb was tested against branching enzyme I antiserum. No immunoprecipitate was formed when branching enzyme I was tested against branching enzyme IIa or IIb antiserum. Increasing amounts of antisera in the above combinations also failed to inhibit enzyme activity. Branching enzyme IIa antiserum cross-reacted and formed spurs with branching enzyme IIb when compared with branching enzyme IIa antigen. Comparison of branching enzyme IIb antiserum with branching enzyme IIa also resulted in an immunoprecipitate. Increasing levels of branching enzyme IIa antiserum inhibited branching enzyme IIb as did the reciprocal combination. The data indicated that branching enzymes IIa and IIb are immunologically similar while branching enzyme I is distinct. The data supports the classification of starch branching enzymes based on genetic, kinetic, and chromatographic properties. 相似文献
12.
Lysine-60 in the regulatory chain of Escherichia coli aspartate transcarbamoylase is important for the discrimination between CTP and ATP 总被引:2,自引:0,他引:2
Lysine-60 in the regulatory chain of aspartate transcarbamoylase has been changed to an alanine by site-specific mutagenesis. The resulting enzyme exhibits activity and homotropic cooperativity identical with those of the wild-type enzyme. The substrate concentration at half the maximal observed specific activity decreases from 13.3 mM for the wild-type enzyme to 9.6 mM for the mutant enzyme. ATP activates the mutant enzyme to the same extent that it does the wild-type enzyme, but the concentration of ATP required to reach half of the maximal activation is reduced approximately 5-fold for the mutant enzyme. CTP at a concentration of 10 mM does not inhibit the mutant enzyme, while under the same conditions CTP at concentrations less than 1 mM will inhibit the wild-type enzyme to the maximal extent. Higher concentrations of CTP result in some inhibition of the mutant enzyme that may be due either to hetertropic effects at the regulatory site or to competitive binding at the active site. UTP alone or in the presence of CTP has no effect on the mutant enzyme. Kinetic competition experiments indicate that CTP is still able to displace ATP from the regulatory sites of the mutant enzyme. Binding measurements by equilibrium dialysis were used to estimate a lower limit on the dissociation constant for CTP binding to the mutant enzyme (greater than 1 x 10(-3) M). Equilibrium competition binding experiments between ATP and CTP verified that CTP still can bind to the regulatory site of the enzyme. For the mutant enzyme, CTP affinity is reduced approximately 100-fold, while ATP affinity is increased by 5-fold.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
13.
Human hexosaminidase A was covalently bound to soluble poly(N-vinylpyrrolidone), and the effect of this binding on the enzyme inactivation by various procedures was investigated. Whereas the polymer-bound hexosaminidase underwent inactivation to the same extent as the free enzyme, when exposed to heat or acidic pH, the conjugation to polymer appeared to protect the enzyme towards proteolysis. Thus, the polymer-bound enzyme exhibited considerably higher resistance to treatment of both pronase and macrophage cathepsins. The clearance rate from rabbit blood, of the polymer-bound enzyme (expressed as enzyme activity), was shown to be significantly slower than that of the free enzyme. 相似文献
14.
Phenylalanine hydroxylase, important in phenylalanine metabolism in mammals, is regulated through short-term (activation) and long-term (induction) mechanisms. To help elucidate the structure-function relationships involved in the activation of this enzyme, we have isolated and characterized full-length cDNA clones to rat phenylalanine hydroxylase. Recombinant rat phenylalanine hydroxylase was placed into an expression vector in Escherichia coli. The enzyme has been purified to homogeneity and its physical and catalytic properties have been characterized. The molecular weight and the fluorescence emission spectrum of the recombinant enzyme were identical to those of the native enzyme. The recombinant enzyme could be activated by incubation with phenylalanine or lysolecithin or by phosphorylation, as is the rat liver enzyme. The extent of activation is the same as that for the native enzyme in each case except for phenylalanine, which activates the recombinant enzyme only 5- to 10-fold rather than the 15- to 30-fold activation observed with the native enzyme. The kinetic constants determined for the recombinant enzyme are also essentially the same as those reported for the native enzyme. We conclude that this enzyme is essentially identical to the native enzyme and should be very useful in the future study of this important hydroxylase. 相似文献
15.
Synthesis and degradation of phenylalanine ammonia-lyase of Rhodosporidium toruloides. 总被引:1,自引:3,他引:1
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The regulation of the enzyme phenylalanine ammonia-lyase (PAL), which is of potential use in oral treatment of phenylketonuria, was investigated. Antiserum against PAL was prepared and was shown to be monospecific for the enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native enzyme and two inactive mutant forms of the enzyme were purified to homogeneity by immunoaffinity chromatography, using anti-PAL immunoglobulin G-Sepharose 4B. Both mutant enzymes contained intact prosthetic groups. The formation of PAL catalytic activity after phenylalanine was added to yeast cultures was paralleled by the appearance of enzyme antigen. During induction, uptake of [3H]leucine into the enzyme was higher than uptake into total protein. Our results are consistent with de novo synthesis of an enzyme induced by phenylalanine, rather than activation of a proenzyme. The half-lives of PAL and total protein were similar in both exponential and stationary phase cultures. No metabolite tested affected the rate of enzyme degradation. Glucose repressed enzyme synthesis, whereas ammonia reduced phenylalanine uptake and pool size and so may repress enzyme synthesis through inducer exclusion. The synthesis of enzyme antigen by a mutant unable to metabolize phenylalanine indicated that this amino acid is the physiological inducer of the enzyme. 相似文献
16.
RecBCD enzyme acts in the major pathway of homologous recombination of linear DNA in Escherichia coli. The enzyme unwinds DNA and is an ATP-dependent double-strand and single-strand exonuclease and a single-strand endonuclease; it acts at Chi recombination hotspots (5'-GCTGGTGG-3') to produce a recombinogenic single-stranded DNA 3'-end. We found that a small RNA with a unique sequence of approximately 24 nt was tightly bound to RecBCD enzyme and co-purified with it. When added to native enzyme this RNA, but not four others, increased DNA unwinding and Chi nicking activities of the enzyme. In seven similarly active enzyme preparations the molar ratio of RNA molecules to RecBCD enzyme molecules ranged from 0.2 to <0.008. These results suggest that, although this unique RNA is not an essential enzyme subunit, it has a biological role in stimulating RecBCD enzyme activity. 相似文献
17.
Immobilized phospholipase A2 from cobra venom. Prevention of substrate interfacial and activator effects 总被引:2,自引:0,他引:2
The activation of cobra venom phospholipase A2 by activators (containing phosphorylcholine moieties) appears to depend upon the aggregation state of the enzyme, and the presence of a lipid-water interface. The characteristics of this activation were studied by comparing the behavior of the enzyme immobilized on an agarose gel to that of the soluble enzyme. The immobilized enzyme displays only a few per cent of the soluble enzyme activity toward micellar dipalmitoyl-phosphatidylcholine (PC). However, the relative loss of activity is much less with micellar dipalmitoylphosphatidylethanolamine or soluble diheptanoyl-PC. The affinity for Ca2+ is increased about 10-fold by immobilization while the apparent pKa of the enzyme is decreased by 0.5-0.8 pH units. Activation energies are similar for the two enzyme forms and are independent of the physical state of the substrate used. Catalytic constants of the enzyme toward monomeric PC are not changed by immobilization. Yet, activators of the soluble enzyme have negligible effect on the immobilized enzyme, either in the presence or absence of an interface. Monomeric activators promote the binding of the soluble enzyme to the immobilized form. Apparently, immobilization mainly produces monomerically constrained enzyme which cannot be activated under any condition, whereas normally, activators in the presence of lipid-water interfaces induce the formation of enzyme dimers or possibly higher order aggregates. 相似文献
18.
Bolivar JM Cava F Mateo C Rocha-Martín J Guisán JM Berenguer J Fernandez-Lafuente R 《Applied microbiology and biotechnology》2008,80(1):49-58
The genome of Thermus thermophilus contains two genes encoding putative glutamate dehydrogenases. One of these genes (TTC1211) was cloned and overexpressed in Escherichia coli. The purified enzyme was a trimer that catalyzed the oxidation of glutamate to alpha-ketoglutarate and ammonia with either NAD+ or NADP+ as cofactors. The enzyme was also able to catalyze the inverse reductive reaction. The thermostability of the enzyme at neutral pH was very high even at 70 degrees C, but at acidic pH values, the dissociation of enzyme subunits produced the rapid enzyme inactivation even at 25 degrees C. The immobilization of the enzyme on glyoxyl agarose permitted to greatly increase the enzyme stability under all conditions studied. It was found that the multimeric structure of the enzyme was stabilized by the immobilization (enzyme subunits could be not desorbed from the support by boiling it in the presence of sodium dodecyl sulfate). This makes the enzyme very stable at pH 4 (e.g., the enzyme activity did not decrease after 12 h at 45 degrees C) and even improved the enzyme stability at neutral pH values. This immobilized enzyme can be of great interest as a biosensor or as a biocatalyst to regenerate both reduced and oxidized cofactors. 相似文献
19.
alpha-Amylase of the thermophilic actinomycete Thermomonospora vulgaris was partially purified. Maximal enzyme activity was obtained at 60degreeC and pH 6.0. KM value was l.4%. The effect of some metal salts on enzyme activity was studied. Enzyme activity was inhibited by by KCN, EDTA, and iodoacetate. Inhibition by EDTA was completely nullified by CaCl2, but the inhibition by iodoacetate was not overcome by 2-mercaptoethanol. Exposure of the enzyme to pH 7.0 and 9.0 for 2 hr. did not affect the enzyme, but exposure to pH 3.0 for few minutes completely inactivated the enzyme. Exposure of the enzyme to 60degreeC resulted in an appreciable inactivation and exposure to 80degreeC completely inactivated the enzyme. Addition of CaCl2, 2-mercaptoethanol, or enzyme substrate the 60degreeC exposed enzyme. However, bovine serym albumin had a protective effect when the enzyme was exposed to 60degreeC but not to 80degreeC. The enzyme was stable in the presence of 8 M urea. 相似文献