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1.
In the previous paper [Xu, J., Oshima, T., & Yoshida, M. (1990) J. Mol. Biol. 215, 597-606], we reported that phosphofructokinase from Thermus thermophilus is allosterically inhibited by phosphoenolpyruvate, which induces dissociation of the active four-subunit enzyme into an inactive two-subunit form. When T. thermophilus was cultured in a glucose-containing medium, another phosphofructokinase (PFK2) appeared in addition to the reported one (PFK1). The molecular weights of the native PFK2 molecule (132,000) and its subunit (34,500), which are slightly smaller than those of PFK1, suggest that PFK2 is also composed of four identical subunits. However, the hyperbolic kinetics and molecular form of PFK2 are not affected at all by phosphoenolpyruvate. The NH2-terminal amino acid sequences of subunits of PFK1 and PFK2 revealed that they are composed of very similar but different polypeptides. 相似文献
2.
N P Balaban M R Sharipova V I Vershinina L V Znamenskaia E B Chernokal'skaia E R Romakhina T I Volkova S V Lopatnev 《Nauchnye doklady vysshe? shkoly. Biologicheskie nauki》1992,(4):68-74
Active and inactive highly purified extracellular alkaline ribonucleases (barnase) from recombinant Escherichia coli have been prepared according to the industrial technology of Bacillus intermedius ribonuclease purification. Electrophoretic parameters and ultraviolet spectra characterize these preparations as homogeneous proteins. Immunochemical test system of identification of inactive RNAse for its purification has been worked out. 相似文献
3.
Crude and purified xanthine dehydrogenase preparations from rat liver were examined for the existence of a naturally occurring inactive form. Reduction of the purified enzyme by xanthine under anaerobic conditions proceeded in two phases. The enzyme was inactivated by cyanide, which caused the release of a sulfur atom from the molybdenum center as thiocyanate. The amount of thiocyanate released was almost in parallel with the initial specific activity. The active and inactive enzymes could be resolved by affinity chromatography on Sepharose 4B/folate gel. These results provided evidence that the purified enzyme preparation from rat liver contained an inactive form. A method for the determination of the active and inactive enzymes in crude enzyme preparations from rat liver was devised based on the fact that only active enzyme could react with [14C]allopurinol and both active and inactive enzymes could be immunoprecipitated quantitatively by excess specific antibody to xanthine dehydrogenase. The amount of [14C]alloxanthine (derived from [14C]allopurinol) bound to the active sulfo enzyme in crude rat liver extracts was about 0.5 mol/mol of FAD. As this content is closely similar to that in the purified enzyme, these results suggest the existence of an inactive desulfo form in vivo. 相似文献
4.
Treatment of the Ascaris suum phosphofructokinase (PFK) with 2',3'-dialdehyde ATP (oATP) results in an enzyme form that is inactive. The conformational integrity of the active site, however, is preserved, suggesting that oATP modification locks the PFK into an inactive T state that cannot be activated. A rapid, irreversible first-order inactivation of the PFK is observed in the presence of oATP. The rate of inactivation is saturable and gives a KoATP of 1.07 +/- 0.27 mM. Complete protection against inactivation is afforded by high concentrations of ATP, and the dependence of the inactivation rate on the concentration of ATP gives a Ki of 326 +/- 26 microM for ATP which is 22-fold higher than the Km for ATP at the catalytic site but close to the binding constant for ATP to the inhibitory site. Fructose 6-phosphate, fructose 2,6-bisphosphate, and AMP provide only partial protection against modification. The pH dependence of the inactivation rate gives a pKa of 8.4 +/- 0.1. Approximately 2 mol of [3H]oATP is incorporated into a subunit of PFK concomitant with 90% loss of activity, and ATP prevents the derivatization of 1 mol/subunit. The oATP-modified enzyme is not activated by AMP or fructose 2,6-bisphosphate. oATP has no effect on the activity of a desensitized form of PFK in which the ATP inhibitory site is modified with diethyl pyrocarbonate but with the active site intact [Rao, G.S.J., Wariso, B.A., Cook, P.F., Hofer, H.W., & Harris, B.G. (1987) J. Biol. Chem. 262, 14068-14073].(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
5.
The activating enzyme of the inactive form of Fraction I of delta-aminolevulinate (ALA) synthetase [EC 2.3.1.37] in Rhodopseudomonas (R.) spheroides was purified about 1,000-fold from an extract of R. spheroides cells grown anaerobically in the light. The purification of the activating enzyme was achieved by fractionating the 100,000 X g supernatant fraction of the crude extract with ammonium sulfate and acetone, followed by Sephadex G-200 chromatography, pyridoxamine phosphate-Sepharose 4B chromatography, and preparative gel electrophoresis. The final preparation of the activating enzyme still contained a minor contaminant (less than 20%) as judged by disc gel electrophoresis. The activating enzyme exhibited cystathionase [EC 4.4.1.1] activity throughout the purification. These two enzyme activities were not separated at all during any step of the purification. An apparently homogeneous preparation of cystathionase [EC 4.4.1.8] purified from rat liver also exhibited activating activity in the presence of L-cystine. It was concluded that the activating enzyme is a cystathionase. 相似文献
6.
Human erythrocyte and muscle phosphofructokinase (PFK) were purified completely by improved procedures. SDS-acrylamide gel electrophoresis in a discontinuous buffer system revealed two subunits (R and M) of erythrocyte PFK, the slower one (M) corresponding to the single subunit of muscle PFK. The staining intensity ratio R:M of the two bands of erythrocyte PFK was 2:1 or less. This suggests that native erythrocyte PFK contains multiple isoenzymes with different proportions of R and M, some being lost during purification. Nevertheless, isoelectric focusing showed single peaks of erythrocyte PFK (pI 5.0) and muscle PFK (pI 6.6), perhaps because of aggregation of erythrocyte PFK isoenzymes. Erythrocyte PFK from a patient with muscle PFK deficiency had a pI of 4.6 and could not be precipitated by antiserum against muscle PFK, findings compatible with the putative structure R4. 相似文献
7.
Choline kinase in rat liver has been shown to be induced up to 2-fold by the administration of polycyclic aromatic hydrocarbon carcinogens such as 3-methylcholanthrene and 3,4-benzo[a]pyrene (Ishidate, K., Tsuruoka, M. and Nakazawa, Y., (1980) Biochem. Biophys. Res. Commun. 96, 946-952). In order to characterize the nature of choline kinase induction by these carcinogens, the 3-methylcholanthrene-induced form as well as the normal form of choline kinase were partially purified from rat liver cytosol through acid treatment, (NH4)2SO4 precipitation and DEAE-cellulose column chromatography with linear KCl-gradient elution, and the catalytic properties were compared between the two preparations. Both enzyme activities were purified about 17-fold with a yield of 50% through the purification steps and there appeared no detectable difference in the elution pattern from either DEAE-cellulose column or Sephadex G-200 gel filtration. On the other hand, some differences were observed in catalytic properties between the two enzyme preparations; (1) the induced form showed a higher apparent Km value for choline (0.19 mM) when compared to the normal form (0.11 mM) and (2) the addition of polyamines caused a considerable increase in the maximum reaction velocity for the normal form whereas no remarkable change for the induced form, when the activities were plotted as a function of choline concentration. The overall results suggest that the 3-methylcholanthrene-induced form of choline kinase in rat liver could be different from the normal form, or that there exist several isoenzymes of choline kinase in rat liver, and one or some of them are inducible by the administration of polycyclic aromatic hydrocarbons. 相似文献
8.
X. T. Wang M. J. Dumoulin O. Befani B. Mondovi M. A. Mateescu 《Preparative biochemistry & biotechnology》2013,43(3-4):237-250
Abstract A purification procedure leading to a joint separation of two serum copperenzymes: ceruloplasmin (EC 1.16.3.1) and amineoxidase (EC 1.4.3.6), is described. Both enzymes are obtained in electrophoretically homogeneous form and their specific activities are higher than those obtained by previously described purification techniques. Two common steps: precipitation of bovine plasma proteins with ammonium sulphate (at 35 % and 55 % saturation) followed by column chromatography on AE-Agarose (obtained by treatment of agarose beads with 1-chloro-2-ethylamine), lead to an electrophoretically homogeneous ceruloplasmin. At the same time, the ceruloplasmin-free protein preparation eluted in a first peak, following further Q-Sepharose and Con A-Sepharose chromatography, leads to purified bovine serum amine oxidase (BSAO) with an improved yield. The emphasis was given to a mutual improving effect as a consequence of the integration of the two enzymes purification procedures. 相似文献
9.
Structure and reactivity of multiple forms of cytochrome oxidase as evaluated by X-ray absorption spectroscopy and kinetics of cyanide binding 总被引:3,自引:0,他引:3
The extended X-ray absorption fine structure (EXAFS) data show differences between the active site structures of different cytochrome oxidase preparations. In the resting (as isolated) state of the Yonetani preparation, the bridging atom between Fe3+a3 and Cu2+a3 is present [Powers, L., Chance, B., Ching, Y., & Angiolillo, P. (1981) Biophys. J. 34, 465], whereas in another preparation (e.g., Hartzell-Beinert), this atom seems to be bound only to Fe3+a3 in a significant fraction of the molecules. Both preparations bind cyanide in a multiphasic fashion, suggesting that the resting cytochrome oxidase is not homogeneous but rather is a mixture of several forms. The proportion of these forms as detected by cyanide binding kinetics differs for different preparations. However, upon reduction and reoxidation (conversion to the "oxygenated" form) the cyanide binding kinetics become monophasic and all preparations of the oxygenated form bind cyanide at the same rate. Thus, a combination of structural and kinetic approaches seems necessary for evaluation of the nature of the active site of cytochrome oxidase in its various forms. 相似文献
10.
Affinity purification of cytosolic epoxide hydrolase using derivatized epoxy-activated Sepharose gels 总被引:2,自引:0,他引:2
Improved affinity chromatography procedures for the purification of cytosolic epoxide hydrolase are described. An earlier affinity purification method using immobilized 7-methoxycitronellyl thiol (MCT) sporadically produced final enzyme preparations containing major impurities. To eliminate these impurities, we tested alternate ligands, spacer arms, and ligand concentrations. A series of alkyl and aryl thiols coupled to epoxy-activated Sepharose were found to exhibit markedly different binding characteristics as compared with commercially available alkyl- and aryl-Sepharose gels. Using one of these new matrices, benzylthio-Sepharose, cytosolic epoxide hydrolase from mouse liver was purified over 100-fold, appeared homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was obtained with 60-90% recovery of enzyme activity. The impurities previously observed with the MCT-Sepharose procedure were reduced or eliminated by using an MCT ligand concentration of 5 microequivalents per gram or less. MCT-Sepharose and benzylthio-Sepharose provide rapid and convenient one-step procedures for obtaining purified cytosolic epoxide hydrolase from numerous species and tissues. 相似文献
11.
H Okamura S Raguet A Bell J Gagnon P A Kelly 《The Journal of biological chemistry》1989,264(10):5904-5911
Prolactin receptors were purified from rat liver membranes by single-step immunoaffinity chromatography using a specific monoclonal antibody to the rat liver prolactin receptor. Scatchard analysis of 125I-human growth hormone binding to the purified receptor revealed two classes of specific binding sites with Ka = 18.5 x 10(9) and 1.2 x 10(9) M-1. Considering that both classes of binding sites are responsible for high affinity prolactin binding, the partially purified receptor preparation had a binding activity of 1.69 nmol/mg protein, representing 1000-fold purification over microsomal receptors with a recovery of 52%. From three separate purifications, 6 mg of partially purified prolactin receptor were obtained with a purity of approximately 4 to 6.5%. Thus, the use of monoclonal antibody for affinity chromatography resulted in a large improvement of prolactin receptor purification compared to previous hormone affinity chromatography (300-fold purification, 15% recovery). The purified receptor was run on preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis, and a homogeneous preparation of prolactin receptor was obtained by electroelution from gel slices corresponding to Mr 38,000-43,000. Immunoblot analysis using a radiolabeled monoclonal antibody revealed two separate but closely located bands of Mr 42,000 and 40,000 in microsomal, partially purified, and electroeluted preparations. The homogeneous receptor protein was extensively digested with L-1-tosylamido-2-phenylethyl chloromethyl ketone trypsin, and 10 internal amino acid sequences of the rat liver prolactin receptor were determined by gas-phase sequence analysis. Oligonucleotide probes were prepared against two of these internal sequences, and a prolactin receptor cDNA was isolated from a rat liver library using one of these probes (Boutin, J. M., Jolicoeur, C., Okamura, H., Gagnon, J., Edery, M., Shirota, M., Banville, D., Dusanter-Fourt, I., Djiane, J., and Kelly, P. A. (1988) Cell 53, 69-77). The amino acid sequence deduced from the cDNA reveals three potential sites of N-linked glycosylation, two of which were confirmed during protein sequencing. The prolactin receptor was characterized by affinity labeling with 125I-human growth hormone. Cross-linking of microsomes revealed a single band for the hormone-receptor complex with Mr 62,000. On the other hand, cross-linking of Triton X-100-solubilized or partially purified receptor with labeled hormone resulted in the appearance of two bands with Mr 62,000 and 102,000, suggesting the existence of a subunit structure of the prolactin receptor, or alternatively, the existence of two types of prolactin receptor.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
12.
P Reddy N Fredd-Kuldell E Liberman A Peterkofsky 《Protein expression and purification》1991,2(2-3):179-187
We present methods for the rapid, simple purification of Enzyme I, HPr, and Protein IIIGlc of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system (PTS) using plasmids overproducing gene products. The gene for HPr (ptsH) was cloned into the expression vector pKC30. A simple procedure was devised for the purification to homogeneity of this protein from extracts of heat-induced cells containing pKC30/ptsH recombinant clone. The genes for Enzyme I (ptsI) and Protein IIIGlc (crr) were cloned separately into the expression vector pRE1. Rapid purification procedures were developed for the isolation of homogeneous preparations of these two proteins from extracts of heat-induced cells containing pRE1/ptsI and pRE1/crr recombinants. From about 6 g of cells, these procedures yielded 100, 86, and 50 mg of Enzyme I, HPr, and Protein IIIGlc, respectively. The activity of the proteins purified by these methods was comparable to that of the proteins isolated by previously published less efficient procedures. 相似文献
13.
Keiko Kimura Ihei Iwata Hiroyuki Nishimura 《Bioscience, biotechnology, and biochemistry》2013,77(5):1387-1389
Phenoloxidase was purified from pupae of the housefly, Musca domestica L. The purification procedures included ammonium sulfate precipitation, affinity chromatography and Sephadex G-200 gel filtration. The final preparations appear to be homogeneous based on results obtained from polyac-rylamide gel electrophoresis. The molecular weight of phenoloxidase was estimated to be 330,000, as determined by gel filtration. The kinetic properties of phenoloxidase were studied using six catecholamines as substrates. The preferred order of substrates for phenoloxidase was found to be N-β-alanyldopamine > dopamine > TV-acetyldopamine > norepinephrine > epinephrine > DOPA. 相似文献
14.
Purification of cytochrome P-450, NADPH-cytochrome P-450 reductase,and epoxide hydratase from a single preparation of rat liver microsomes 总被引:1,自引:0,他引:1
A simplified procedure is presented for the simultaneous purification of the enzymes cytochrome P-450, epoxide hydratase (EC 3.3.2.3), and NADPH-cytochrome P-450 reductase (EC 1.6.2.4) from a single preparation of rat liver microsomes. All three enzymes can be recovered after chromatography of detergent-solubilized microsomes on a column of n-octylamino-Sepharose 4B. The major form of cytochrome P-450 (of phenobarbitaltreated rats) is purified by subsequent DEAE-cellulose chromatography, epoxide hydratase is purified by DEAE- and O-(carboxymethyl)-cellulose chromatography, and NADPH-cyto-chrome P-450 reductase is purified using 2′,5′-ADP agarose chromatography. The nonionic detergent Lubrol PX and the ionic detergents sodium cholate and deoxycholate are used in these procedures to permit utilization of uv-absorbance measurements in monitoring protein during purification. Overall yields of the three enzymes are approximately 20, 25, and 60%, respectively. All three enzymes are apparently homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and are functionally active. The same procedure can be used to obtain the major cytochrome P-450 present in liver microsomes isolated from β-naphthoflavone (5,6-benzoflavone)- or 3-methylcholanthrene-treated rats. Thus, the described procedures permit the rapid and reproducible purification of three major rat liver microsomal enzymes which can be coupled to study bioactivation and detoxification of a variety of xenobiotics in reconstituted systems. 相似文献
15.
Hermie J. M. Harmsen Eva M. Kubicek-Pranz Max Röhr Jaap Visser Christian P. Kubicek 《Applied microbiology and biotechnology》1992,37(6):784-788
Summary Phosphofructokinase 2 (PFK 2) was isolated from mycelia of the citric-acid-accumulating fungus Aspergillus niger, and partially purified by Trisacryl-Blue chromatography and Mono Q fast protein liquid chromatography. The appearance of a 96/94-kDa double band correlated with PFK 2 activity during purification. Purified PFK 2 had a half-life of 240 min at 4° C. The enzyme exhibited Michaelis-Menten type kinetics with respect to its substrates fructose-6-phosphate and ATP, required inorgaic phosphate for activity, and was only weakly inhibited by phospho(enol)pyruvate, AMP and citrate. The enzyme activity was not influenced by incubating partially purified PFK 2 preparations with ATP, MG2+ and the catalytic subunit of bovine heart protein kinase, although such treatment phosphorylated the 96/94-kDa protein. Consistently, treatment with alkaline phosphatase had no effect on PFK 2 activity. Also, no influence on PFK 2 activity was observed when cell-free extracts (containing A. niger protein kinases) from either glucose or citrate-grown mycelia were incubated with ATP and Mg2+ alone. It is concluded that, in A. niger, regulation of PFK 2 by phosphorylation/dephosphorylation does not occur, and this is related to the development of high glycolytic flow and citrate accumulation under conditions of supplying high sugar concentrations.
Correspondence to: C. P. Kubicek 相似文献
16.
Two glycosulfatases [EC 3.1.6.3], I and II, were purified 31.3- and 33.9-fold respectively, from a crude extract of the liver of Charonia lampas. The purification was carried out by the following chromatographic procedures; phosphocellulose, Sephadex G-150, Concanavalin A-Sepharose and isoelectric focussing. The enzyme preparations obtained were practically free from arylsulfatase [EC 3.1.6.1] contamination. Both glycosulfatases are probably glycoproteins differing in their carbohydrate moieties. The molecular weights of glycosulfatase I and II were estimated to be about 112,000 and 79,000 respectively. They had the same optimum pH of 5.5, and the same Km value of 25.0 mM for glucose 6-sulfate. 相似文献
17.
S Pontremoli E Melloni F Salamino M Michetti L H Botelho H A El-Dorry D K Chu C E Isaacs B L Horecker 《Archives of biochemistry and biophysics》1978,191(2):825-827
Fructose 1,6-bisphosphatase (Fru-P2ase,EC 3.1.3.11) purified from livers of fed rabbits has been reported to contain tryptophan, which is not present in the enzyme purified from livers of fasted animals. We now show that the tryptophan arises from small amounts of active or inactive rabbit liver aldolase in the Fru-P2ase preparations. Fru-P2ase free of tryptophan may conveniently be prepared by raising the temperature of the heat step in the purification procedure to 67 °C. 相似文献
18.
Purification and characterization of the reconstitutively active phosphate transporter from rat liver mitochondria 总被引:5,自引:0,他引:5
Procedures have been developed for the purification of a nearly homogeneous, highly active phosphate transport system from rat liver mitochondria in either a two-subunit (alpha, beta) or a single subunit (beta) form. Significantly, both forms display a similar high magnitude N-ethylmaleimide (NEM)-sensitive Pi/Pi exchange activity upon incorporation into phospholipid vesicles. The transport system is extracted from hypotonically shocked mitoplasts with Triton X-114 and purified in the presence of cardiolipin by sequential chromatography on hydroxylapatite, DEAE-Sepharose CL-6B, and Affi-Gel 501. Depending on the conditions used to elute the transporter from Affi-Gel 501, preparations are obtained which, when analyzed by high resolution sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis, consist of either a single 33-kDa protein (beta) or a 33-kDa (beta) plus a 35-kDa (alpha) component. In preparations yielding the latter result, both bands display a nearly equivalent Coomassie staining intensity. Furthermore, after alkylation with NEM, the two protein bands co-migrate. Fluorography indicates that the coalesced band contains [3H]NEM. Upon reconstitution of the purified Pi carrier into liposomes, direct measurement of both the initial transport rate and the amount of protein that actually incorporates into the phospholipid vesicles yields a specific transport activity of 22.6 mumol/min/mg of protein. The exchange is characterized by a first order rate constant of 0.85 min-1, a t1/2 of 49 s, and is inhibited by sulfhydryl reagents (i.e., NEM, p-chloromercuribenzoate, and mersalyl). It is also substantially inhibited by diethyl pyrocarbonate, N-acetylimidazole, phenylglyoxal, and 5-dimethylaminoaphthalene-1-sulfonyl chloride. In addition to providing a simple, rapid method for preparing the NEM-sensitive phosphate carrier in nearly homogeneous form, these studies provide new information about the catalytically active species of the carrier, its kinetic properties, and its inhibitor sensitivities. 相似文献
19.
Morris hepatoma 7777 previously has been shown to have no detectable pyridoxine- (pyridoxamine-) 5'-phosphate oxidase activity [Thanassi, J. W., Nutter, L. M., Meisler, N. T., Commers, P., & Chiu, J.-F. (1981) J. Biol. Chem. 256, 3370-3375]. In order to determine if this enzyme was missing in the hepatoma, we purified rat liver oxidase and raised antibodies to it in rabbits. Final purification of rat liver oxidase for use as an antigen was accomplished by affinity chromatography and gel electrophoresis. The rat liver enzyme is similar to rabbit liver oxidase [Kazarinoff, M. N., & McCormick, D. B. (1975) J. Biol. Chem. 250, 3436-3442] having two noncovalently linked subunits with molecular weights in the range of 25 000-28 000. Evidence indicating that inactive enzyme was simultaneously purified with native enzyme was obtained. The IgG fraction was purified from the serum of a rabbit that had been immunized with rat liver oxidase. This was used in the development of ELISA and immunoblot analyses for the presence of antigenically active pyridoxine- (pyridoxamine-) 5'-phosphate oxidase in cytosolic preparations from normal rat liver and Morris hepatoma 7777. The results indicated that there was no immunologically detectable oxidase protein in the tumor. An alternate pathway of pyridoxal 5'-phosphate synthesis, involving oxidation of pyridoxine to pyridoxal followed by phosphorylation, was ruled out. The implications of these findings with respect to acquisition of nutrients by tumors are discussed. 相似文献
20.
Rabbit muscle phosphofructokinase (PFK) is rapidly inactivated by a 2′,3′-dialdehyde derivative of adenosine triphosphate (dialdehyde-ATP). When allowed to react with 0.6 mm dialdehyde-ATP in 0.1 m borate buffer (pH 8.6) containing 0.2 mm EDTA and 0.5 mm dithiothreitol, PFK loses essentially all activity (99%) in 30 min. The modified PFK remains inactive following dialysis of the reaction mixture against sodium borate (pH 8.0) containing fructose diphosphate, EDTA, and dithiothreitol. Experiments with [14C]dialdehyde-ATP show that 99% inactivation of PFK corresponds to incorporation of 3 to 4 mol of the ATP analog per PFK protomer. The inactivation of PFK with dialdehyde reagent is not caused by dissociation of the 340,000 Mr, tetramer to the 170,000 Mr dimer, as determined by analytical ultracentrifugation. Adenosine diphosphate or ATP protect PFK from inactivation by dialdehyde-ATP at pH 8.6, but fructose 6-phosphate, cyclic 3′,5tadenosine monophosphate, or fructose diphosphate, which protect PFK from modification by pyridoxal phosphate, provide little protection from inactivation. Amino acid analyses of dialdehyde-inactivated PFK and of a control sample of the enzyme were compared following reaction of each with 2,4-dinitrofluorobenzene. The results show that three or four lysine residues per PFK protomer are modified by dialdehyde-ATP. Additional data indicate that these lysine residues react with dialdehyde-ATP to form dihydroxymorpholine-like adducts rather than Schiff bases. 相似文献