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1.
Ultraviolet difference spectroscopy has been used to study the binding of the transition state analog saccharo-1,4-lactone to purified rat preputial gland beta-glucuronidase. At pH 4.5 (the pH optimum), the inhibitor induces a difference spectrum indicative of a change in the environment of tryptophyl residues. Based on the magnitude of the induced difference spectrum as a quantitative measure of inhibitor binding, a titration curve for saccharo-1,4-lactone was obtained. A Scatchard plot of the titration data indicates that 4 molecules of inhibitor bind to the enzyme tetramer at a K-I of 4 times 10-7 M. The inhibitor also induces a similar difference spectrum at pH 7.5, although the binding is considerably weaker at this pH than at pH 4.5. When the native enzyme at pH 4.5 is compared with the native enzyme at pH 7.5, a difference spectrum, distinct from that of the binding of saccharo-1,4-lactone, is observed, indicating that the enzyme exists in different conformations at these pH values. The indication that tryptophyl residues are perturbed upon binding of saccharo-1,4-lactone was supported by studies carried out with N-bromosuccinimide. At pH 4.3, this reagent was found to oxidize 6 tryptophyl residues in the native enzyme but only three in the saccharo-1,4-lactone-inhibited enzyme. A spectrophotometric titration of the enzyme indicated that of the 33 tyrosyl residues per subunit, only 5 to 6 ionize at the pK expected for free phenolic groups. 相似文献
2.
1. An NAD-specific L(+)-lactate dehydrogenase (EC 1.1.1.27) from the mycelium of Phycomyces blakesleeanus N.R.R.L. 1555 (-) was purified approximately 700-fold. The enzyme has a molecular weight of 135,000-140,000. The purified enzyme gave a single, catalytically active, protein band after polyacrylamide-gel electrophoresis. It shows optimum activity between pH 6.7 and 7.5. 2. The Phycomyces blakesleeanus lactate dehydrogenase exhibits homotropic interactions with its substrate, pyruvate, and its coenzyme, NADH, at pH 7.5, indicating the existence of multiple binding sites in the enzyme for these ligands. 3. At pH 6.0, the enzyme shows high substrate inhibition by pyruvate. 3-hydroxypyruvate and 2-oxovalerate exhibit an analogous effect, whereas glyoxylate does not, when tested as substrates at the same pH. 4. At pH 7.5, ATP, which inhibits the enzyme, acts competitively with NADH and pyruvate, whereas at pH 6.0 and low concentrations of ATP it behaves in a allosteric manner as inhibitor with respect to NADH, GTP, however, has no effect under the same experimental conditions. 5. Partially purified enzyme from sporangiophores behaves in entirely similar kinetic manner as the one exhibited by the enzyme from mycelium. 相似文献
3.
B M Turner 《Biochimica et biophysica acta》1979,578(2):325-336
alpha-L-Fucosidase has been purified 12 000 fold from human placenta. The enzyme is a glycoprotein containing, by weight: 0.9% galactose; 1.9% mannose, 1.9% N-acetylglucosamine and 1.9% N-acetylneuraminic acid. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate separated proteins with molecular weights ot 55 000, 51 400 and 25 000. Resolution of the two larger protein bands varied with the gel system and these proteins may differ only in carbohydrate content. Gel filtration of te purified enzyme failed to separate the three proteins. Treatments with the cross-linking reagent dimethyl suberimidate prior to electrophoresis, resulted in a diminution of the original protein bands and the formation of oligomers with molecular weights of 80 000, 100 000, 130 000, and 144 000. These results suggest that the heavy (55 000 and 51 400) and light (25 000) proteins are structurally associated. The molecular weight of the native enzyme, measured by gel filtration, was dependent on the pH of the eluting buffer. At pH 5.0 or 6.0 a catalytically active peak was observed, with a molecular weight of 305 000. At pH 7.5 this peak was completely absent and the enzyme eluted as an asymmetrical peak with an apparent molecular weight of about 60 000. The reduction in apparent molecular weight at pH 7.5 was reversible by dialysis of isolated fractions at pH 6.0. In agreement with these findings the sedimentation coefficient was 8.5 S at pH 5.0 but only 3.6 S at pH 7.5. The results can be accounted for by the existence of a pH-dependent equilibrium between aggregated and dissociated forms of the enzyme or by pH-depedent conformational changes. 相似文献
4.
Ribonuclease T1 [EC 3.1.4.8] was coupled to a water-insoluble cross-linked polyacrylamide (Enzacryl AH) by the acid azide method. The immobilized enzyme exhibited about 45% and 77% of the original activity toward yeast RNA and 2', 3-cyclic GMP, respectively, as substrates. Although the specific activity was lowered by the coupling, the immobilized enzyme was found to be far more stable to heat and extremes of PH than the native enzyme. The immobilized enzyme was active toward RNA even above pH 9 (at 37 degree C) or above 60 degree C (at pH 7.5), where the native enzyme was inactive. The immobilized enzyme retained much of its activity as assayed at 37 degree C after incubation in the range of pH 1 to 10 at 37 degree C, or after heating at 100 degree C (at pH 7.5) under conditions where the native enzyme was inactivated to a considerable extent. The enzyme derivative could be repeatedly recovered and reused without much loss of activity. The active site glutamic acid-58 in the immobilized enzyme appeared to be nearly as reactive with iodoacetate as that in the native enzyme. 相似文献
5.
Steady-state kinetic analysis of the quinoprotein methylamine dehydrogenase from Paracoccus denitrificans. 总被引:1,自引:0,他引:1
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V L Davidson 《The Biochemical journal》1989,261(1):107-111
A steady-state kinetic analysis was performed of the reaction of methylamine and phenazine ethosulphate (PES) with the quinoprotein methylamine dehydrogenase from Paracoccus denitrificans. Experiments with methylamine and PES as varied-concentration substrates produced a series of parallel reciprocal plots, and when the concentrations of these substrates were varied in a constant ratio a linear reciprocal plot of initial velocity against PES concentration was obtained. Nearly identical values of V/Km of PES were obtained with four different n-alkylamines. These data suggest that this reaction proceeds by a ping-pong type of mechanism. The enzyme reacted with a variety of n-alkylamines but not with secondary, tertiary or aromatic amines or amino acids. The substrate specificity was dictated primarily by the Km value exhibited by the particular amine. A deuterium kinetic isotope effect was observed with deuterated methylamine as a substrate. The enzyme exhibited a pH optimum for V at pH 7.5. The absorbance spectrum of the pyrroloquinoline quinone prosthetic group of this enzyme was also effected by pH at values greater than 7.5. The enzyme was relatively insensitive to changes in ionic strength, and exhibited a linear Arrhenius plot over a range of temperatures from 10 degrees C to 50 degrees C with an energy of activation 46 kJ/mol (11 kcal/mol). 相似文献
6.
The regulatory properties of purified maize leaf NADP-malic enzyme (EC 1.1.1.40) were studied at three different pHs and the following results were obtained. (a) At pH 7.5 enzyme activity reaches a maximum at 0.4–0.8 mm malate depending on the Mg2+ concentration, and higher levels of malate result in marked substrate inhibition; with increasing pH the degree of substrate inhibition is reduced to where at pH 8.4 little or no inhibition is observed. (b) The inhibitory effect of malate is more pronounced at 1 mm Mg2+ than at 5–10 mm Mg2+ in the pH range of 7.5 to 8.4; a plot of enzyme activity vs Mg2+ concentration at 3 mm malate follows Michaelis-Menten kinetics at both pH 7.5 and 8.4; the apparent affinity of the enzyme for Mg2+ at pH 8.4 was threefold greater than that at pH 7.5. (c) The activity of NADP-malic enzyme decreases as the ratio of increases, and this effect is enhanced at lower pH. (d) Various α-keto acids including glyoxylate, oxaloacetate, and α-ketoglutarate inhibit NADP-malic enzyme activity, whereas HCO3?, pyruvate, and other organic acids, sugar phosphates, and amino acids have little or no effect on the activity of the enzyme. Based on these experimental findings, the regulatory properties of maize leaf NADP-malic enzyme are discussed with respect to its key role in net CO2 fixation in maize bundle sheath chloroplasts during C4 photosynthesis. 相似文献
7.
Enzymic studies performed with chemically modified yeast hexokinase (ATP : D-hexose-6-phosphotransferase) confirm previous results indicating that the sulfhydryl, imidazol and most of the reactive amino groups do not seem to be directly implicated in the enzyme active site. On the other hand the modification of these functional groups of the enzyme does not affect the transition between the acidic inactive form to an active enzyme form after deprotonation. The chemically modified forms of hexokinase and the native enzyme are affected in the same way by activators (citrate, D-malate, 3-phosphoglycerate and Pi) when the activity was measured at pH 6.6. Moreover the loss of enzyme activity observed in the course of the chemical modifications is accompanied by an increase of the activation effect. This increase must be related to some reorganization of the enzyme active site in presence of the effectors, since the same effect was observed when hexokinase was denatured with 3M urea at pH 7.5. However no increase in the activation effect was observed when the denaturation was carried out at pH 6.5 At this pH the loss in activity and the change of optical absorption at 286 nm were much slower than at pH 7.5, which indicates a great difference in the protein structure between these pHs. 相似文献
8.
1. Phospholipase D [EC 3.1.4.4] from Streptomyces hachijoensis was purified about 570-fold by column chromatography on DEAE-cellulose and Sephadex G-50 followed by isoelectric focusing. 2. The purified preparation was found to be homogeneous both by immunodiffusion and polyacrylamide disc gel electrophoresis. 3. The isoelectric point was found to be around pH 8.6 and the molecular weight was about 16,000. 4. The enzyme has maximal activity at pH 7.5 at 37 degrees. The optimal temperature is around 50 degrees at pH 7.5, using 20 min incubation. 5. The enzyme was stable at 50 degrees for 90 min. At neutral pH, between 6 and 8, the enzyme retained more than 95% of its activity on 24 hr incubation at 25 degrees. However, the enzyme lost 80% of its activity under the same conditions at pH 4.0. 6. The enzyme was stimulated slightly by Ca2+, Mn2+, and Co2+, and significantly by Triton X-100 and ethyl ether. It was inhibited by Sn2+, Fe2+, Fe3+, Al3+, EDTA, sodium dodecyl sulfate, sodium cholate, and cetylpyridinium chloride. 7. This phospholipase D hydrolyzes phosphatidylethanolamine, phosphatidylcholine, cardiolipin, sphingomyelin, phosphatidylserine, and lysophosphatidylcholine, liberating the corresponding bases. 8. The Km value was 4mM, determined with phosphatidylethanolamine as a substrate. 相似文献
9.
The oxidation-reduction potential of chloroperoxidase, an enzyme which catalyzes peroxidative chlorination, bromination, and iodination reactions, has been investigated. In addition to catalyzing biological halogenation reactions, chloroperoxidase is unusual in that the carbon monoxide complex of ferrous chloroperoxidase shows the typical long wavelength Soret absorption associated with P-450 hemoproteins. The pH dependence of the chloroperoxidase oxidation-reduction potential shows a discontinuity around pH 4.7. Similarly, measurements of the affinity of ferrous chloroperoxidase for carbon monoxide monitored both by spectroscopic and potentiometric titration exhibit a discontinuity in the pH 4.7 region. Oxidation-reduction potential measurements on chloroperoxidase in a CO atmosphere also show a discontinuous pH profile. These results suggest that ferrous chloroperoxidase undergoes reversible modification at low pH and that these changes are reflected in the oxidation-reduction potential. The oxidation-reduction potential of chloroperoxidase at pH 6.9 is - 140 mV, close to that measured for cytochrome P-450cam in the presence of substrate. The oxidation-reduction potential of chloroperoxidase at pH 2.7, the pH optimum for enzymatic chlorination, is +150 mV. The oxidation-reduction potentials of the halide complexes of chloroperoxidase (chloride, bromide, and iodide) are essentially identical with the potential measurements on the native enzyme. These observations suggest that, although halide anions bind to the enzyme, they probably do not bind as an axial ligand to the heme ferric iron. 相似文献
10.
Purification and characterization of glutamate decarboxylase from Neurospora crassa conidia. 总被引:7,自引:0,他引:7
L-Glutamate decarboxylase, an enzyme under the control of the asexual developmental cycle of Neurospora crassa, was purified to homogeneity from conidia. The purification procedure included ammonium sulfate fractionation and DEAE-Sephadex and cellulose phosphate column chromatography. The final preparation gave a single band on sodium dodecyl sulfate-polyacrylamide gels with a molecular weight of 33,200 +/- 200. A single band coincident with enzyme activity was found on native 7.5% polyacrylamide gels. The molecular weight of glutamate decarboxylase was 30,500 as determined by gel permeation column chromatography at pH 6.0. The enzyme had an acidic pH optimum and showed hyperbolic kinetics at pH 5.5 with a Km for glutamic acid of 2.2 mM and a Km for pyridoxal-5'-phosphate of 0.04 microM. 相似文献
11.
Vincenzo Zappia Maria Cartenì-Farina Gennaro Della Pietra 《The Biochemical journal》1972,129(3):703-709
1. The presence of S-adenosylmethionine decarboxylase in human prostate gland is reported. A satisfactory radiochemical enzymic assay was developed and the enzyme was partially characterized. 2. Putrescine stimulates the reaction rate by up to 6-fold at pH7.5: the apparent activation constant was estimated to be 0.13mm. The stimulation is pH-dependent and a maximal effect is observed at acid pH values. 3. Putrescine activation is rather specific: other polyamines, such as spermidine and spermine, did not show any appreciable effect. 4. The apparent K(m) for the substrate is 4x10(-5)m. The calculated S-adenosylmethionine content of human prostate (0.18mumol/g wet wt. of tissue) demonstrates that the cellular amounts of sulphonium compound are saturating with respect to the enzyme. 5. The enzyme is moderately stable at 0 degrees C and is rapidly inactivated at 40 degrees C. The optimum pH is about 7.5, with one-half of the maximal activity occurring at pH6.6. 6. Several carboxy-(14)C-labelled analogues and derivatives of S-adenosylmethionine were tested as substrates. The enzyme appears to be highly specific: the replacement of the 6'-amino group of the sulphonium compound alone results in a complete loss of activity. 7. Inhibition of the enzyme activity by several carbonyl reagents suggests an involvement of either pyridoxal phosphate or pyruvate in the catalytic process. 8. The inhibitory effect of thiol reagents indicates the presence of ;essential' thiol groups. 相似文献
12.
1. Glutamate dehydrogenase was subject to rapid inactivation when irradiated in the presence of Rose Bengal or incubated in the presence of ethoxyformic anhydride. 2. Inactivation in the presence of Rose Bengal led to the photo-oxidation of four histidine residues. Oxidation of three histidine residues had little effect on enzyme activity, but oxidation of the fourth residue led to the almost total loss of activity. 3. Acylation of glutamate dehydrogenase with ethoxyformic anhydride at pH6.1 led to the modification of three histidine residues with a corresponding loss of half the original activity. Acylation at pH7.5 led to the modification of two histidine residues and a total loss of enzyme activity. 4. One of the histidine residues undergoing reaction at pH6.1 also undergoes reaction at pH7.5. 5. The presence of either glutamate or NAD(+) in the reaction mixtures at pH6.1 had no appreciable effect. At pH7.5 glutamate caused a marked decrease in both the degree of alkylation and degree of inactivation. NAD(+) had no effect on the degree of inactivation at pH7.5 but did modify the extent of acylation. 6. The normal response of the enzyme towards ADP was unaffected by acylation at pH6.1 or 7.5. 7. The normal response of the enzyme towards GTP was altered by treatment at both pH6.1 and 7.5. 相似文献
13.
Efficient purification and molecular properties of spinach chloroplast fructose 1,6-bisphosphatase. 总被引:12,自引:0,他引:12
A relatively straightforward procedure has been developed for the purification of chloroplast fructose bisphosphatase from spinach leaves to apparent homogeneity and with 80% yield. The molecular weight of the enzyme was about 160 000. Chloroplast fructosebisphosphatase consists of four possibly identical subunits and, at pH 8.8, EASILY DISSOCIATES INTO EQUAL HALVES WITH LOWered activity. Sigmoid saturation curves with Hill coefficients between 3.0 and 3.7 were obtained for fructose 1,6-bisphosphate and Mg2+. Incubation of the enzyme with 20 mM dithiothreitol slowly altered the response to pH from no activity measured at pH 7.5 and full activity at pH 8.8 to equal activity at each of these pH values; at the same time the number of freely available sulphydryl groups increased from four to twelve per molecule. These properties are considered in the context of the observed activation of this enzyme following illumination of chloroplasts. 相似文献
14.
A total of 59 bacteria samples from Antarctic sea water were collected and screened for their ability to produce alpha-amylase. The highest activity was recorded from an isolate identified as an Alteromonas species. The purified alpha-amylase shows a molecular mass of about 50,000 Da and a pI of 5.2. The enzyme is stable from pH 7.5 to 9 and has a maximal activity at pH 7.5. Compared with other alpha-amylases from mesophiles and thermophiles, the "cold enzyme" displays a higher activity at low temperature and a lower stability at high temperature. The psychrophilic alpha-amylase requires both Cl- and Ca2+ for its amylolytic activity. Br- is also quite efficient as an allosteric effector. The comparison of the amino acid composition with those of other alpha-amylases from various organisms shows that the cold alpha-amylase has the lowest content in Arg and Pro residues. This could be involved in the principle used by the psychrophilic enzyme to adapt its molecular structure to the low temperature of the environment. 相似文献
15.
A galactosyltransferase, which converts blood group O red bloodcells to B-cells, was purfied to homogeneity from plasma of blood group B subjects. The stepwise purification procedures include: (a) column chromatography with CM-Sephadex, followed by ammonium sulfate fractionation; (b) Sephadex G-200 gel filtration; (c) column chromatogr,phy with DEAE-Sephadex; and (d) column chromatography with hydroxylapatite. The procedures provided about a 400,000-fold increase of specific activity with a 40 to 50% yield. Further purification of the enzyme was performed by small scale preparative acrylamide gel electrophoresis at pH 4.3. The final enzyme preparation showed a single protein band which coincided with enzyme activity, in acrylamide gel electrophoresis, and revealed a single protein band in sodium dodecyl sulfate-gel electrophoresis. Judging from the molecular weight, which was estimated by Sephadex gel filtration, and subunit size estimated by sodium dodecyl sulfate-gel electrophoresis, the enzyme is presumably in a dimeric form. The enzyme required Mn2+ for its activity and had a pH optimum at 7.0 to 7.5. 相似文献
16.
Rabbit testis arylsulphatase A was purified 140-fold with a recovery of 20% from detergent extracts of an acetone-dried powder by using DE-52 cellulose column chromatography, gel filtration on Sephadex G-200 and preparative isoelectric focusing. The purified enzyme showed one major band with one minor contaminant on electrophoresis in a 7.5% (w/v) polyacrylamide gel at pH8.3. On sodiumdodecyl sulphate/polyacrylamidegel electrophoresis, a single major band was observed with minor contaminants. The final preparation of enzyme was free from general proteolytic, esterase, hyaluronidase, beta-glucuronidase and beta-galactosidase activities. Rabbit testicular arylsulphatase A exists as a dimer of mol.wt. 110000 at pH7.1. At pH5.0 the enzyme is a tetramer of mol.wt. 220000. Arylsulphatase A appears to consist of two identical subunits of mol.wt. 55000 each. The highly purified enzyme has pI4.6. The enzyme hydrolyses p-nitrocatechol sulphate with Km and Vmax, of 4.1 mM and 80nmol/min respectively, but has no activity toward p-nitrophenyl sulphate. The pH optimum of the enzyme varies with the incubation time. By applying Sephacex G-200 chromatography and preparative isoelectric focusing, one form of enzyme was obtained. The enzyme has properites common to arylsulphatase A of other sources with respect to the anomalous time-activity relationship, pI, inhibition by PO42-, SO32- and Ag+ ions and substrate affinity to p-nitrocatechol sulphate. However, the enzyme shows the temperature optimum of arylsulphatase B of other species. 相似文献
17.
Production, purification, and characterization of alpha-amylase from Thermomonospora curvata. 总被引:3,自引:1,他引:2
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Thermomonospora curvata produces an extracellular alpha-amylase. Maximal amylase production by cultures in a starch-mineral salts medium occurred at pH 7.5 and 53 degrees C. The crude enzyme was unstable to heating (65 degrees C) at pH 4 to 6, and was activated when heated at pH 8. The enzyme was purified 66-fold with a 9% yield and appeared homogeneous on discontinuous gel electrophoresis. The pH and temperature optima for activity of the purified enzyme were 5.5 to 6.0 and 65 degrees C. The molecular weight was calculated to be 62,000. The Km for starch was 0.39 mg/ml. The amylolytic pattern consisted of a mixture of maltotetraose and maltopentaose. 相似文献
18.
A NADP-dependent group III alcohol dehydrogenase (ADH) was purified from the hyperthermophilic strictly anaerobic archaeon Thermococcus hydrothermalis, which grows at an optimum temperature of 85 degrees C and an optimum pH of 6. The gene encoding this enzyme was cloned, sequenced, and over-expressed in Escherichia coli. The recombinant enzyme was purified, characterized and compared with the native form of the enzyme. The enzyme structure is pH-dependent, being a 197-kDa tetramer (subunit of 45 kDa) at pH 10.5, the pH optimum for alcohol oxidation, and a 80.5-kDa dimer at pH 7.5, the pH optimum for aldehyde reduction. The kinetic parameters of the enzyme show that the affinity of the enzyme is greater for the aldehyde substrate and NADPH cofactor, suggesting that the dimeric form of the enzyme is probably the active form in vivo. The ADH of T. hydrothermalis oxidizes a series of primary aliphatic and aromatic alcohols preferentially from C2 to C8 but is also active towards methanol and glycerol and stereospecific for monoterpenes. T. hydrothermalis ADH is the first Thermococcale ADH to be cloned and overproduced in a mesophilic heterologous expression system, and the recombinant and the native forms have identical main characteristics. 相似文献
19.
An improved procedure is described for the purification of fructose 1,6-bisphosphatase (FbPase) from chicken liver. The purified enzyme shows a single band in gel electrophoresis either in the presence or absence of sodium dodecyl sulfate. From 200 g of frozen liver, we have obtained about 29 mg of homogeneous enzyme, with the pH profile indistinguishable from that of the enzyme in crude extracts. The overall recovery of enzyme activity is about 71%. The FbPase protein was estimated to represent approximately 0.36% of the total soluble protein of crude liver extract. Treatment of purified enzyme with papain or subtilisin results in a rapid increase in activity at pH 9.2 and a gradual decrease at pH 7.5, while digestion with trypsin or chymotrypsin results in a concomitant decrease in activities at both pH 9.2 and 7.5. The rates of hydrolysis by these four proteases are all markedly decreased in the presence of AMP. Both AMP and fructose 1,6-bisphosphate increase the thermal stability of the enzyme, and their effects are additive. Attempts were made to investigate the structural requirements for histidine activation. The results suggest that activation by this amino acid involves not only the imidazole ring but also the α-amino and α-carboxyl groups. 相似文献
20.
Tadpole collagenase hydrolyzed native and denatured collagen and synthetic peptides with sequences of 2,4-dinitrophenyl-L-prolyl-L-leucylglycyl-L-isoleucyl-L-alanylglycyl-L-arginie amide and 2,4-dinitrophenyl-L-prolyl-L-glutaminyl-glycyl-L-isoleucyl-L-alanylglycyl-L-glutaminyl-D-arginine. The specific enzyme activity against the latter substrate and collagen fibrils is found to be 933 nmol/min per mg protein and 8440 units (microgram collagen degraded/min), respectively. Optimum pH for the enzyme is 7.5-8.5. A collagenase complex with alpha2-macroglobulin did not hydrolyze collagen fibrils, but digested the synthetic substrates at the Gly-Ile bond. The amino acid composition of the enzyme was determined. Immunoelectrophoresis of the enzyme at pH 8.6 against anti-tadpole collagenase rabbit immunoglobulin G shows a single precipitin line at a position migrating faster than human serum albumin and corresponding to enzyme activity against collagen fibril and synthetic substrates. 相似文献