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1.
Yeast phosphofructokinase was subjected to limited proteolysis by trypsin in the presence of different effectors. It could be demonstrated that the substrates MgATP and fructose-6-phosphate are able to protect the enzyme from inactivation by trypsin. Other effectors like AMP, ADP, phosphoenolpyruvate, citrate and ammonium ions exhibit only negligible effects. During the first step of degradation consisting in the conversion of the subunits from Mr 120,000 to 90,000 no significant effects of the substrates and effectors on the proteolytic inactivation of yeast phosphofructokinase can be observed. In the presence of ATP as well as of ADP the sensitivity of the enzyme against ATP inhibition is either not or only slightly influenced by proteolytic modification. The modified enzyme retains its sensitivity against activation by AMP, independently of whether effectors are present or absent during proteolysis. The kinetic parameters of the enzyme modified by subtilisin in the presence of ATP or of fructose-6-phosphate have been determined. 相似文献
2.
UV irradiation of the sarcoplasmic reticulum (SR) ATPase in the presence of vanadate cleaves the enzyme at either of two different sites. Under conditions favoring the presence of monovanadate, and in the presence of Ca(2+), ADP, and Mg(2+), cleavage results in two fragments of 71- and 38-kDa electrophoretic mobility. On the other hand, under conditions permitting formation of decavanadate, and in the absence of Ca(2+) and ADP, cleavage results in two fragments of 88- and 21-kDa electrophoretic mobility. The amino terminus resulting from cleavage is blocked and resistant to Edman degradation. However, the initial photo-oxidation product can be reduced with NaB(3)H(4,) resulting in incorporation of radioactive (3)H label. Extensive digestion of the labeled protein with trypsin then yields labeled peptides that are specific for the each of the photo-oxidation conditions, and can be sequenced after purification. Collection of the Edman reaction fractional products reveals the radioactive label and demonstrates that Thr(353) is the residue oxidized by monovanadate at the phosphorylation site (i.e. Asp(351)). Correct positioning of monovanadate at the phosphorylation site requires binding of Mg(2+) and ADP to the Ca(2+)-dependent conformation of the enzyme. Subsequent hydrolytic cleavage is likely assisted by the neighboring Asp(601), and yields the 71- and 38-kDa fragments. On the other hand, Ser(186) (and possibly the following three residues: Val(187), Ile(188), and Lys(189)) is the residue that is photo-oxidized by decavanadate in the absence of ADP. Hydrolytic cleavage of the oxidized product at this site is likely assisted by neighboring acidic residues, and yields the 88- and 21-kDa fragments. The bound decavanadate, which we find to produce steric interference with TNP-AMP binding, must therefore extend to the A domain (i.e. small cytosolic loop) in order to oxidize Ser(186). This protein conformation is only obtained in the absence of Ca(2+). 相似文献
3.
Glycogen phosphorylase is progressively degraded during irradiation with near UV light in the presence of vanadate. The pattern of protein cleavage by monovanadate is characterised by fewer peptides than that by decavanadate, which leads to fragmentation in a ligand dependent way. In both instances, the initial cleavage releases a peptide of 82,000 daltons which accounts for the N-terminal portion of the subunit, including the regulatory phosphorylation site. 相似文献
4.
M N Tijane A F Chaffotte F J Seydoux C Roucous M Laurent 《The Journal of biological chemistry》1980,255(21):10188-10193
Yeast phosphofructokinase contains 83 +/- 2 cysteinyl residues/enzyme oligomer. On the basis of their reactivity toward 5,5-dithiobis(2-nitrobenzoic acid), the accessible cysteinyl residues of the native enzyme may be classified into three groups. For titrations performed with N-ethylmaleimide, subdivisional classes of reactivity are evidenced. In each case, the 6 to 8 most reactive cysteines are not protected by fructose 6-phosphate from chemical labeling and do not seem involved in subsequent enzyme inactivation. Differential labeling studies as well as direct protection experiments in the presence of fructose 6-phosphate, indicate that 12 -SH groups/enzyme oligomer (i.e. three -SH groups per binding site) are protected by the allosteric substrate from the chemical modification. Specific labeling by the differential method of the cysteinyl residues protected by fructose 6-phosphate and further separation of the two types of subunits constituting yeast phosphofructokinase, show that the substrate binding sites are localized exclusively on subunits of beta type. Thus, alpha subunits are not implicated directly in the catalytic mechanism of yeast phosphofructokinase reaction. 相似文献
5.
Recently reported decameric vanadate (V(10)) high affinity binding site in myosin S1, suggests that it can be used as a tool in the muscle contraction regulation. In the present article, it is shown that V(10) species induces myosin S1 cleavage, upon irradiation, at the 23 and 74 kDa sites, the latter being prevented by actin and the former blocked by the presence of ATP. Identical cleavage patterns were found for meta- and decavanadate solutions, indicating that V(10) and tetrameric vanadate (V(4)) have the same binding sites in myosin S1. Concentrations as low as 50 muM decavanadate (5 muM V(10) species) induces 30% of protein cleavage, whereas 500 muM metavanadate is needed to attain the same extent of cleavage. After irradiation, V(10) species is rapidly decomposed, upon protein addition, forming vanadyl (V(4+)) species during the process. It was also observed by NMR line broadening experiments that, V(10) competes with V(4) for the myosin S1 binding sites, having a higher affinity. In addition, V(4) interaction with myosin S1 is highly affected by the products release during ATP hydrolysis in the presence or absence of actin, whereas V(10) appears to be affected at a much lower extent. From these results it is proposed that the binding of vanadate oligomers to myosin S1 at the phosphate loop (23 kDa site) is probably the cause of the actin stimulated myosin ATPase inhibition by the prevention of ATP/ADP exchange, and that this interaction is favoured for higher vanadate anions, such as V(10). 相似文献
6.
L (liver) type phosphofructokinase subunits purified from human leukocytes are slightly lighter than L subunits from liver and red blood cells. A mild treatment of red blood cell L4 enzyme with subtilisin converts its subunits into forms of similar molecular weight to leukocyte enzyme. From a kinetical point of view, subtilisin-treated L4 phosphofructokinase and leukocyte enzymes are characterized by a decrease of the allosteric properties as compared to non-treated red cell L4 phosphofructokinase. 相似文献
7.
Phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) from human muscle, brain, heart and granulocytes has been purified using a two or three step purification procedure. The main step is Blue Dextran-Sepharose 4B chromatography with selective elution of phosphofructokinase by formation of the ternary complex ADP or ATP-fructose-6-P-enzyme. Muscle and heart contain only enzyme subunits with a molecular weight of 85,000. This type of subunit is predominnant in brain, where it co-exists with subunits of about 80,000 daltons. A single type of subunits is found in the granulocytes, with a molecular weight of 80,000. Anti-muscle phosphofructokinase antiserum reacts only with M-type enzyme. Anti-granulocyte enzyme antiserum, absorbed by pure brain phosphofructokinase, exhibits a narrow specificity against the so-called L-type enzyme. Anti-brain antiserum, absorbed by pure muscle phosphofructokinase and partly purified red cell enzyme, exhibits a narrow specificity against a phosphofructokinase form predominant in fibroblasts and present in brain (F-type). 相似文献
8.
Polyvanadate solutions obtained by extracting vanadium pentoxide with dilute alkali over a period of several hours contained increasing amounts of decavanadate as characterized by NMR and ir spectra. Those solutions having a metavanadate:decavanadate ratio in the range of 1-5 showed maximum stimulation of NADH oxidation by rat liver plasma membranes. Reduction of decavanadate, but not metavanadate, was obtained only in the presence of the plasma membrane enzyme system. High simulation of activity of NADH oxidation was obtained with a mixture of the two forms of vanadate and this further increased on lowering the pH. Addition of increasing concentrations of decavanadate to metavanadate and vice versa increased the stimulatory activity, reaching a maximum when the metavanadate:decavanadate ratio was in the range of 1-5. Increased stimulatory activity can also be obtained by reaching these ratios by conversion of decavanadate to metavanadate by alkaline phosphate degradation, and of metavanadate to decavanadate by acidification. These studies show for the first time that both deca and meta forms of vanadate present in polyvanadate solutions are needed for maximum activity of NADH oxidation. 相似文献
9.
Phosphofructokinase is known to associate with the human erythrocyte membrane both in vitro and in vivo. Such association activates the enzyme in vitro by relieving the allosteric inhibition imposed by ATP (Karadsheh, N.S., and Uyeda, K. (1977) J. Biol. Chem. 252, 7418-7420). We now demonstrate that ADP, ATP, and NADH, all of which are known to bind to the enzyme's adenine nucleotide activation site, are particularly potent in eluting the enzyme from the membrane. In addition, both inside-out red cell membrane vesicles and a 23-kDa fragment containing the amino terminus of the membrane protein, band 3, cause a slow, partial, and reversible inactivation of phosphofructokinase. The dependence of the residual phosphofructokinase activity on phosphofructokinase concentration demonstrates that inactivation occurs through the dissociation of active tetramers to inactive dimers. Dimers of phosphofructokinase associate with the membrane more avidly than tetramers. The kinetics of phosphofructokinase inactivation are consistent with the dissociation of tetramers in solution followed by the binding of dimers to the membrane. There is no indication of an association equilibrium between tetramers and dimers of phosphofructokinase bound to the membrane. Taken together, these results suggest that the amino-terminal segment of band 3 binds to the adenine nucleotide activation site, which is thought to be located in a cleft between the dimeric subunits of phosphofructokinase. As a result, band 3 not only rapidly activates the phosphofructokinase tetramer but also slowly inactivates the enzyme by preferentially binding its dissociated subunits. 相似文献
10.
Paramecium bursaria chlorella virus, a large DNA virus that replicates in unicellular Chlorella-like algae, encodes an RNA triphosphatase which is involved in the synthesis of the RNA cap structure found at the 5' end of the viral mRNAs. The Chlorella virus RNA triphosphatase is the smallest member of the metal-dependent RNA triphosphatases that include enzymes from fungi, DNA viruses, protozoans and microsporidian parasites. In the present study, we investigated the ability of various vanadate oxoanions to inhibit the phosphohydrolase activity of the enzyme. Fluorescence spectroscopy and CD studies were used to directly monitor the binding of decavanadate to the enzyme. Moreover, competition assays show that decavanadate is a potent non-competitive inhibitor of the phosphohydrolase activity, and mutagenesis studies indicate that the binding of decavanadate does not involve amino acids located in the active site of the enzyme. In order to provide additional insight into the relationship between the enzyme structure and decavanadate binding, we correlated the effect of decavanadate binding on protein structure using both CD and guanidinium chloride-induced denaturation as structural indicators. Our data indicated that no significant modification of the overall protein architecture was occurring upon decavanadate binding. However, both fluorescence spectroscopy and CD experiments clearly revealed that the binding of decavanadate to the enzyme significantly decreased the structural stability of the enzyme. Taken together, these studies provide crucial insights into the inhibition of metal-dependent RNA triphosphatases by decavanadate. 相似文献
11.
A procedure for the purification of Escherichia coli phosphofructokinase by affinity chromatography is described. The results of amino acid analyses of the purified protein and tryptic peptide mapping suggest that the tetrameric phosphofructokinase is composed of chemically identical subunits. In addition, the reaction product, ADP, was observed to bind to 4.1 +/- 0.1 equal and independent sites on the enzyme. 相似文献
12.
The hybrid isozyme of phosphofructokinase, A2B2, was formed by incubation of rabbit muscle enzyme. A4, and rabbit liver enzyme, B4, in the presence of sodium citrate at neutral pH. The enzyme composition of the resulting mixture of A2B2 and the homoprotomeric forms was identical to that found in rabbit adipose tissue extracts. Hybrid formation, which apparently proceeds by way of dimers, can be blocked by fructose-1,6-P2, fructose-6-P, and high concentrations of MgATP. The A2B2 isozyme was separated from A4 and B4 by ion exchange chromatography. The kinetic regulatory properties of A2B2 were compared with those of A4, B4, and a 1:1 mixture of A4 and B4. ATP inhibition of A2B2 was intermediate between that observed with A4 and B4 and was clearly not identical to a simple summing of the effects of A and B subunits. Similar comparisons were made using other phosphofructokinase inhibitors, citrate, 2,3-P2-glycerate, and P-creatine. In each case the observed inhibition was intermediate between the observed with A4 and B4. The existence in a number of tissues of phosphofructokinase A2B2 provides added diversity to the regulatory mechanisms of glycolysis. 相似文献
13.
Influence of fructose 2,6-bisphosphate on the aggregation properties of rat liver phosphofructokinase 总被引:2,自引:0,他引:2
G D Reinhart 《The Journal of biological chemistry》1983,258(18):10827-10830
The influence that fructose 2,6-bisphosphate (Fru-2,6-BP) has on the aggregation properties of rat liver phosphofructokinase has been studied by observing the fluorescence polarization of the enzyme covalently bound to the fluorescent probe pyrenebutyric acid. Fru-2,6-BP dramatically slows the dissociation of the high molecular weight aggregate forms of the enzyme when the enzyme is diluted to 3.2 micrograms/ml (4 X 10(-8) M subunits). Furthermore, Fru-2,6-BP is a strong promoter of reassociation to tetramer and larger forms if the enzyme has been previously allowed to dissociate to the dimer in its absence. Unlike many other positive effectors of liver phosphofructokinase, Fru-2,6-BP is also able to overcome the tendency of MgATP to promote tetramer formation and instead stabilize a very high degree of high molecular weight aggregate formation even in the presence of MgATP. The apparent affinity of liver phosphofructokinase for Fru-2,6-BP was measured by its ability to promote reassociation and compared to that for Fru-1,6-BP. The apparent dissociation constant for Fru-2,6-BP under these conditions is 36 microM, about 40-fold lower than the value of 1.4 mM measured for Fru-1,6-BP. Both ligands demonstrate synergism with the substrate Fru-6-P, which can lower the dissociation constant for Fru-2,6-BP 9-fold to 4 microM and that for Fru-1,6-BP 5-fold to 0.28 mM. These data are interpreted to suggest that influencing the aggregation state of rat liver phosphofructokinase may be one way in which Fru-2,6-BP achieves its effects on the enzyme in vivo. 相似文献
14.
F Koller O Hoffmann-Ostenhof 《Hoppe-Seyler's Zeitschrift für physiologische Chemie》1979,360(4):507-513
Using the technique of affinity chromatography on a myo-inositol-substituted Sepharose, the myo-inositol oxygenase from rat kidneys was purified to homogeneity. The active enzyme contains iron, most probably in its divalent form. Electrophoresis on polyacrylamide gel containing sodium dodecylsulphate causes the cleavage of the enzyme protein into apparently identical subunits with a molecular weight of approximately 17,000. The smallest active unit consists of 4 subunits, and is in a pH-dependent equilibium with species consisting of 8, 12, and 16 subunits, respectively, which all show the same specific enzyme activity. In the presence of oxygen the enzyme is highly unstable; at the early stages of inactivation it can be reactivated by reducing agents like NaBH4. Under anaerobic conditions or under the influence of Fe2-chelating agents, the enzyme is also inactivated; this inactivation is caused by the loss of iron and concomitant cleavage into the subunits. It can be reversed by incubation with FeSO4 in the presence of air. If myo-inositol and FeSO4 are present, the reactivation involves an oligomerization to the species with 16 subunits with the uptake of 8 gram-atoms of iron per mole of this species. The enzyme reaction follows Michaelis-Menten kinetics; the Michaelis constants are 4.5 x 10(-2)M for myo-inositol and 9.5 x 10(-6)M for oxygen. 相似文献
15.
Purification of F4 phosphofructokinase from human platelets and comparison with the other phosphofructokinase forms 总被引:2,自引:0,他引:2
The phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) tetramers F4, F3L and F2L2 have been separated from human platelets, and purified to homogeneity by affinity chromatography on Dextran Blue-Sepharose 4B. The F subunits have a molecular weight of 85 000, identical to that of the M subunits. By contrast with L-type phosphofructokinase, the F-type enzyme seems to exist predominantly in a tetrameric form and not to aggregate to high molecular weight polymers. Specific activity of pure F4 phosphofructokinase is about 140 IU/mg of protein. Immunologically, it is easy to distinguish all the basic phosphofructokinase forms (i.e. M, L and F types); nevertheless a slight immunological cross-reactivity seems to exist between all these forms. 相似文献
16.
The quaternary structure of an active form of rabbit muscle phosphofructokinase was studied by sedimentation and electron microscopy. Active enzyme centrifugation studies at pH 7.0 and 23 +/- 1 degrees C showed that phosphofructokinase sediments as a single component with a sedimentation coefficient of 12.2 +/- 0.5 S. Identical results were obtained in two assay and three solvent systems. Boundary sedimentation studies of phosphofructokinase in the presence of 1.0 mM fructose 6-phosphate, 0.1 mM adenylyl imidodiphosphate at pH 7.0 and 23 +/- 1 degrees C were performed. The results showed that the sedimentation coefficient of phosphofructokinase remains constant within the range of protein concentration studied and assumes a value of 12.4 S. The molecular weights of the subunit and the 12.4 S component were measured by sedimentation equilibrium yielding values of 83,000 and 330,000 for the monomeric and polymeric species, respectively. It is, therefore, concluded that the active form of phosphofructokinase is indeed the tetrameric species. The structure of the phosphofructokinase tetramer was also studied by electron microscopy of negatively stained specimens. Particles identified as tetramers measured approximately 9 nm in diameter by 14 nm in length. The observed size and shape are consistent with the hydrodynamic measurements. Structural features within the tetramer were interpreted as due to the four individual subunits, each one approximately 4 X 6 X 6 nm in size, arranged with D2 symmetry. 相似文献
17.
J A Starling B L Allen M R Kaeini D M Payne H J Blytt H W Hofer B G Harris 《The Journal of biological chemistry》1982,257(7):3795-3800
A rapid and efficient procedure has been developed to purify phosphofructokinase from the muscle of the parasitic roundworm, Ascaris suum. The procedure can be accomplished in 1 day with a 420-fold purification and a 60% yield. The enzyme was shown to be homogeneous by two-dimensional electrophoresis, Sepharose 6B column chromatography, and high performance liquid chromatography utilizing a size exclusion column. The subunit molecular weight of the enzyme was found to be 95,000 by electrophoresis in the presence of sodium dodecyl sulfate. In solutions of low ionic strength, the native enzyme aggregated to species of higher molecular weight than did the rabbit muscle phosphofructokinase. In the presence of 0.2 M (NH4)2SO4, the minimum native molecular weight was determined to be 398,000 by high performance liquid chromatography and Sepharose 6B column chromatography. Therefore, the enzyme appears to be a tetramer with identical or near-identical subunits. The apparent isoelectric point of the enzyme was shown to be 7.3 to 7.4 by both column and gel isoelectric focusing. Amino acid analysis revealed a lower number of the aromatic residues Phe, Tyr, and Trp than in the rabbit muscle enzyme and this is in agreement with the lower extinction coefficient of E1%280 nm = 6.5. Analysis of the purified enzyme revealed 7.4 +/- 0.6 mol of phosphate/mol of enzyme. 相似文献
18.
The effect of glucagon on the phosphorylation and the enzymic activity of phosphofructokinase in rat liver in vivo was investigated. Glucagon stimulated the phosphorylation of liver phosphofructokinase approximately 3- to 5-fold and increased cAMP levels 5-fold and blood glucose levels 2-fold over the values obtained for control animals. The specific radioactivity of ATP isolated from liver was the same in both control and hormone-treated animals. During the purification of the 32P-labeled enzyme from both animals, no difference was observed in the total or specific enzyme activities of the enzymes from the various fractions. Thus, phosphofructokinase appears to be phosphorylated in vivo by a cyclic AMP-dependent protein kinase. Although phosphorylation does not affect the maximum catalytic activity of the enzyme, it does render the enzyme significantly more sensitive to ATP inhibition. Thus, at a given concentration of ATP, the phosphorylated phosphofructokinase exhibits considerably lower activity than the unphosphorylated enzyme. The possible relationship between our observations and glucagon-mediated control of glycolysis is discussed. 相似文献
19.
Phosphofructokinase (Pfk1; EC 2.7.1.11) is the third enzyme of the glycolytic pathway catalyzing the formation of fructose-1,6-bisphosphate from fructose-6-phosphate (F6P) and ATP. Schizosaccharomyces pombe Pfk1 is a homo-octameric enzyme of 800 kDa molecular weight, distinct from its yeast counterparts which are mostly hetero-octameric enzymes composed of two different subunits. Having an "open" conformation and a tendency to aggregate into higher oligomeric structures, the S. pombe enzyme shows similarities to the mammalian muscle Pfk1. It has been proposed that due to the distinct N-terminal region of the S. pombe subunit, the oligomeric organization of subunits in this enzyme is different from other yeast phosphofructokinases. Electron microscopy studies were carried out to reveal the quaternary structure of the homo-octameric Pfk1 from S. pombe in the F6P-bound and in the ATP-bound state. Random conical tilt data sets have been collected from deep stain preparations of the enzyme in both states. The 0 degrees tilt images have been separated into different classes and a 3D reconstruction has been calculated for each class from the high tilt images. Our results confirm the presence of a variety of views of the particle, most of which can be interpreted as views of the molecule rotating around its long axis. Despite the biochemical differences, the structure of phosphofructokinase from S. pombe in the presence of either F6P or ATP is similar to the hetero-octameric structure of phosphofructokinase from Saccharomyces cerevisiae. The molecule can be described as composed of two subdomains, connected by two well-defined densities. We have been able to establish a correlation between the kinetic behavior and the structural conformation of Pfk1. 相似文献
20.
In this paper, we describe an efficient procedure for the purification of yeast phosphofructokinase. This procedure eliminates any time delay and enables to obtain an enzyme with minimum proteolytic alterations. The molecular weights of the oligomeric enzyme and of its constitutive subunits were both evaluated by means of several independent methods. However, the accuracy of each measurement was not sufficient to discriminate between an hexameric and an octameric structure of the enzyme oligomer. On the other hand, crosslinking experiments demonstrated the octameric structure of yeast phosphofructokinase. Obviously, some methods of molecular weight determination have led to erroneous results. In particular, our experiments show that the reliability of molecular weight determinations performed by gel filtration of native proteins must be considered with caution. 相似文献