Self-association of rabbit muscle phosphofructokinase: effects of ligands

The effects of ligands on the self-association of rabbit muscle phosphofructokinase (PFK) were investigated by velocity sedimentation at pH 7.0 and 23 degrees C. The concentration dependence of the weight-average sedimentation coefficient was monitored in the presence of these ligands. The mode of association and equilibrium constants characterizing each association step were determined by theoretical fitting of the sedimentation data. The simplest mode of association for the PFK system is M in equilibrium M2 equilibrium M4 in equilibrium M16. Ligands and temperature would perturb the various equilibrium constants without altering the mode of association. The apparent equilibrium constants for the formation of tetramer, K4app, are increased in the presence of 0.1 mM ATP and 1.0 mM fructose 6-phosphate. The value of the sedimentation coefficient for the tetramer, S4 degrees, that would best fit the data is 12.4 S instead of 13.5 S determined in the absence of substrates, thus implying a structural change in the tetramer induced by substrates. Only an insignificant amount of dimer is present under the experimental conditions. The presence of activators, ADP or phosphate, enhances the formation of tetramers, and S4 degrees assumes a value of 13.5 S. Similar results are obtained with decreasing concentrations of proton. The presence of the inhibitor, citrate, however, favors the formation of dimers. The equilibrium constants determined as a function of ADP concentration were further analyzed by the linked-function theory derived by Wyman [Wyman, J. (1964) Adv. Protein Chem. 19, 224--285], leading to the conclusion that the formation of a tetramer involves the binding of two additional molecules of ADP per monomer. Similar analysis results in a conclusion that the formation of a dimer involves the binding of one additional molecule of citrate per phosphofructokinase subunit.     

Structural properties of an active form of rabbit muscle phosphofructokinase

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.     

Large-scale purification, oligomerization equilibria, and specific interaction of the LexA repressor of Escherichia coli

A rapid large-scale procedure for the purification of the LexA repressor of Escherichia coli is described. This procedure allows one to get more than 100 mg of purified protein from 100 g of bacterial paste with a purity of at least 97%. This method is comparable to earlier, far more complicated purification procedures giving clearly smaller yields. It is shown that the LexA protein may be identified spectroscopically by a large A235/A280 ratio and very pronounced ripples in the absorption spectrum arising from a high amount of phenylalanine residues with respect to that of the other aromatic amino acids. Polyacrylamide gel electrophoresis has been used to study the specific interaction of LexA with a recA operator fragment. The quaternary structure of LexA has been studied by equilibrium ultracentrifugation and sedimentation velocity measurements. The sedimentation coefficient increases with increasing LexA concentration, indicating that LexA is involved in self-association. This finding has been confirmed by equilibrium ultracentrifugation. The results are best described by a monomer-dimer and a subsequent dimer-tetramer equilibrium, with an association constant of 2.1 X 10(4) M-1 for the dimer and 7.7 X 10(4) M-1 for the tetramer formation. These relatively small association constants determined under near-physiological pH and salt conditions suggest that in vivo LexA should be essentially in the monomeric state. The degree to which LexA decreases the electrophoretic mobility of a 175 base pair fragment harboring the recA operator suggests that the recA operator interacts nevertheless with a LexA dimer. However, our results may be also explained by the binding of a LexA monomer with a simultaneous bending of the DNA fragment.     

Purification and characterization of two high-affinity (adenosine 3',5'-monophosphate)-binding proteins from yeast. Identification as multiple forms of glyceraldehyde-3-phosphate dehydrogenase

Two high-affinity cAMP-binding proteins (I and II) have been purified to homogeneity from baker's yeast by a procedure avoiding proteolytic damage. These proteins have been identified as multiple forms of glyceraldehyde-3-phosphate dehydrogenase. The two cAMP-binding proteins are similar in affinities for cAMP, have identical elution volumes on gel filtration, and contain one type of subunit (Mr 37 500). The form II of glyceraldehyde-3-phosphate dehydrogenase is free of NAD+ and has a Kd of 1.3 X 10(-6) M with respect to cAMP. A marked concentration-dependent self-association of the subunits of the form-II protein was revealed by Yphantis sedimentation equilibrium studies. Significant monomer concentrations are present at total concentrations less than 0.02 mg/ml. Conventional sedimentation equilibrium analyses indicated a tetramer Mr of 170 000. The high-affinity binding of cAMP to glyceraldehyde-3-phosphate dehydrogenase may significantly reduce intracellular cAMP levels and is also discussed in relation to the nature of eukaryote cAMP-binding proteins with similar native or subunit Mr values which are at present functionally undefined.     

Sedimentation equilibrium measurements of recombinant DNA derived human interferon gamma

Recombinant DNA derived human interferon gamma (IFN-gamma) from Escherichia coli was examined by equilibrium ultracentrifugation. Short-column equilibrium experiments at pH 6.9 in 0.1 M ammonium acetate buffer gave a z-average molecular weight of 33,500 +/- 1400 at infinite dilution, corresponding to 1.98 +/- 0.08 times the formula weight. Long- (2.6 mm) column experiments at pH 7.5 in 0.04 M imidazole buffer gave a molecular weight of 33,400 +/- 500. Under the latter conditions IFN-gamma behaves somewhat nonideally, with the departure from ideality accounted for by an effective (Donnan) charge of about 6+. No association of this dimer to form tetramer or higher polymers was observed, with the association constant for formation of tetramer from dimer K24 found to be less than 34 L mol-1. Similarly, no dissociation to monomers was observable, with the dissociation constant to monomer K21 being less than 5 X 10(-8) mol L-1. At pH 3.55 in 0.02 M buffer (acetate plus acetic acid), there was virtually complete dissociation of the dimer to monomer. Extreme nonideality was seen in this low ionic strength system, and the effective charge on the protein was estimated to be about 11+. The reduced molecular weight M(1 -upsilon rho) of the monomer was found to be about 4.09 +/- 0.20 kg mol-1; this corresponds to a molecular weight of 16,410 +/- 820, with the Scatchard definition of components. A small amount of a polymer with a molecular weight of about 0.5 X 10(6) was detected under these conditions.     

An ultracentrifugal study of the self-association of canine apolipoprotein A-I in solution.

The sedimentation behavior of canine apolipoprotein (apo) A-I in 0.02 M EDTA, pH 8.6, was studied as a function of protein concentration by the techniques of sedimentation velocity and sedimentation equilibrium in the analytical ultracentrifuge. At concentrations of less than 1 g/liter, apo-A-I exhibited a monomodal sedimentation pattern, with apparent sedimentation coefficients which varied from 2.3 to 3.5 S with increasing protein concentrations. Above 1.5 g/liter, apo-A-I had two well resolved peaks with s20,w values of 4.15 S and 5.75 S. The proportion of the 5.75 S component increased with increasing apo-A-I concentrations, with a concomitant decrease of the 4.15 S component. By sedimentation equilibrium ultracentrifugation with both the conventional and meniscus-depletion methods, the apparent weight-average molecular weight of apo-A-I was found to be concentration-dependent. At a protein concentration of 5.25 g/liter, an apparent weight average molecular weight of 138,000 was determined, indicating that molecular species larger than a tetramer (monomer molecular weight = 28,000) were present in solution. When analyzed in terms of a reversible self-associating system, the experimental data could best be described according to a monomer-dimer-tetramer-octamer model, as previously reported from human apo-A-I (Vitello, L. B., and Scanu, A. M. (1975) J. Biol. Chem. 251, 1131-1136). The equilibrium constants were: K2 = 4.5 liters/g, K4 = 470 liters3/g3, and K8 = 41,600 liters7/g7, respectively.     

Urea-induced inactivation, dissociation, and unfolding of the allosteric phosphofructokinase from Escherichia coli

The influence of urea on the allosteric phosphofructokinase from Escherichia coli has been studied by measuring the changes in enzymatic activity, protein fluorescence, circular dichroism, and retention in size-exclusion chromatography. Tetrameric, dimeric, and monomeric forms of the protein can be discriminated by their elution from a high-performance liquid chromatography gel filtration column. Three successive steps can be detected during the urea-induced denaturation of phosphofructokinase: (i) the dissociation of the native tetramer into dimers which abolishes the activity; (ii) the dissociation of dimers into monomers which exposes the unique tryptophan, Trp-311, to the aqueous solvent; (iii) the unfolding of the monomers which disrupts most of the secondary structure. This pathway involves the ordered dissociation of the interfaces between subunits and supports a previous hypothesis (Deville-Bonne et al., 1989). Phosphofructokinase can be quantitatively renatured from urea solutions, provided that precautions are taken to avoid the aggregation of one insoluble monomeric state. The renaturation of phosphofructokinase from urea implies three steps: an initial folding reaction within the monomeric state is followed by two successive association steps. The faster association step restores the native fluorescence, and the slower regenerates the active enzyme. The renaturation and denaturation of phosphofructokinase correspond to the complex pathway: tetramer in equilibrium dimer in equilibrium folded monomer in equilibrium unfolded monomer. It is found that the subunit interface which forms the regulatory site is more stable and associates 40 times more rapidly than the subunit interface which forms the active site.     

Functional and subunit assembly properties of hemoglobin Alberta (alpha 2 beta 2(101) Glu----Gly)

Hemoglobin Alberta has an amino acid substitution at position 101 (Glu----Gly), a residue involved in the alpha 1 beta 2 contact region of both the deoxy and oxy conformers of normal adult hemoglobin. Oxygen equilibrium measurements of stripped hemoglobin Alberta at 20 degrees C in the absence of phosphate revealed a high affinity (P50 = 0.75 mm Hg at pH 7), co-operative hemoglobin variant (n = 2.3 at pH 7) with a normal Bohr effect (- delta log P50/delta pH(7-8) = 0.65). The addition of inositol hexaphosphate resulted in a decrease in oxygen affinity (P50 = 8.2 mm Hg at pH 7), a slight increase in the value of n and an enhanced Bohr effect. Rapid mixing experiments reflected the equilibrium results. A rapid rate of carbon monoxide binding (l' = 7.0 X 10(5) M-1 S-1) and a slow rate of overall oxygen dissociation (k = 15 s-1) was seen at pH7 and 20 degrees C in the absence of phosphate. Under these experimental conditions the tetramer stability of liganded and unliganded hemoglobin Alberta was investigated by spectrophotometric kinetic techniques. The 4K4 value (the liganded tetramer-dimer equilibrium dissociation constant) for hemoglobin Alberta was found to be 0.83 X 10(-6) M compared to a 4K4 value for hemoglobin A of 2.3 X 10(-6) M, indicating that the Alberta tetramer was less dissociated into dimers than the tetramer of hemoglobin A. The values of 0K4 (the unliganded tetramer-dimer equilibrium dissociation constant) for hemoglobin Alberta and hemoglobin A were also measured and found to be 2.5 X 10(-8) M and 1.5 X 10(-10) M, respectively, demonstrating a greatly destabilized deoxyhemoglobin tetramer for hemoglobin Alberta compared to deoxyhemoglobin A. The functional and subunit dissociation properties of hemoglobin Alberta appear to be directly related to the dual role of the beta 101 residue in stabilizing the tetrameric form of the liganded structure, while concurrently destabilizing the unliganded tetramer molecule.     

Molecular size of nerve growth factor in dilute solution.

Nerve growth factor (NGF) is a protein composed of two identical chains of mass 13,259. An analysis of the sedimentation equilibrium, sedimentation velocity, and gel filtration behavior of dilute solutions of NGF indicates the existence of a rapidly reversible monomer in equilibrium dimer equilibrium and that the association constant K for the reaction at neutral pH is 9.4 X 10(6)M-1. Reaction mixtures consist of equal concentrations of monomer and dimer at a total protein concentration as high as 1.4 mug/ml, and at 1 ng/ml, monomer accounts for greater than 99% of the total. The latter concentration is 20 to 30 times that required for the biological activity of NGF. Several lines of evidence suggest that the dimerization reaction is highly stereospecific, although its biological significance is not known.     

Dissociation of the lactose repressor protein tetramer using high hydrostatic pressure

Dissociation of lac repressor tetramer by high hydrostatic pressures was monitored with intrinsic tryptophan fluorescence. With the assumption of complete dissociation to monomer, tryptophan polarization data gave delta V a approximately 170 mL/mol and the concentration for 50% tetramer dissociation, C1/2, was 3.8 X 10(-8) M. Upon addition of inducer, the calculated delta V a increased to approximately 220 mL/mol and the C1/2 decreased to approximately 1 X 10(-8) M, a free energy difference of approximately 0.7 kcal. These results indicate a modest stabilization of the tetramer by the presence of inducer. Monitoring the average energy of tryptophan emission demonstrated that tetramer dissociation takes place over the same range of pressures as evidenced by the polarization data and IPTG dissociation can be more or less superimposed upon tetramer dissociation depending upon the ligand concentration used. Although the two transitions cannot be separated entirely, the delta V a for the region of the pressure dependence dominated by ligand dissociation was 69 mL/mol, an unexpectedly large value. For tetramer modified with methyl methanethiosulfonate, subunit dissociation was shifted to much higher pressures and IPTG dissociation did not occur. The delta V a for subunit association was calculated as approximately 160 mL/mol, and the C1/2 was 3.5 X 10(-9) M. Interactions at the subunit interface of the modified protein are apparently stronger than in the unmodified protein. The absence of inducer dissociation from the MMTS-modified tetramer by the application of high hydrostatic pressure suggests that the volume change for inducer binding to the modified protein is much smaller than that observed for the unmodified repressor.     

Influence of inositol hexaphosphate binding on subunit dissociation in methemoglobin.

The tetramer-dimer equilibria of various forms of methemoglobin have been measured by sedimentation equilibrium to test the hypothesis of Perutz that high spin derivatives can be switched by inositol hexaphosphate (Inos-P6) from the R state to the T state more readily than low spin derivatives. Since transitions from the R state to the T state are accompanied by a decrease in the tetramer-dimer dissociation constant (K4,2), this parameter is a quantitative indicator of the conformational state. Measurements of K4,2 were performed using an analytical ultracentrifuge with absorption optics and a scanner-computer system. Statistical analysis of the sedimentation data indicated that the stoichiometry if Inos-P6 binding is 1 molecule/hemoglobin tetramer and 2 molecules/hemoglobin dimer. The apparent affinity of the dimer sites for Inos-P6 is much lower than the corresponding value for the tetramer site. As a result of the stoichiometries, at low concentrations Inos-P6 shifts the tetramer-dimer equilibrium in favor of the tetramer, but at high concentrations Inos-P6 shifts the equilibrium in favor of the dimer. Te tetramer binding site for Inos-P6 of various liganded forms of hemoglobin appears to be the same as has been established for deoxyhemoglobin, since the effect of Inos-P6 on subunit dissociation is reduced in pyridoxylated derivatives. Values of K4,2 for aquo-, azido- and cyanomethemoglobin in 0.01 M 2,2-bis(hydroxymethyl)-2,2',2'-nitroethanol buffer, pH 6.0/0.1 M NaCl, are all near 2 X 10(-5) M. Upon addition of 50 muM Inos-P6 the values of K4,2 for all three forms are shifted to near 10(-9) M. Since the aquo derivative is high spin, while the azido and cyano derivatives are low spin, the similarity of values for the derivatives in the presence and absence of Inos-P6 indicate that the changes in K4,2 are not spin-spin state dependent. For another high spin derivative, fluoromethemoglobin, such high concentrations of NaF are required that ionic strength effects are encountered. When data at several NaF concentrations are extrapolated to 0.1 M NaF to correct for the ionic strength effects, values of K4,2 of 7 X 10(-6) M and 10(-8) M are obtained for solutions in the absence and in the presence of 50 muM Inos-P6, respectively. Therefore the results with the fluoro derivative, in conjunction with the other forms of methemoglobin, support the view that high spin derivatives do not exhibit a greater response to Inos-P6 than low spin derivatives.     

Monomer concentrations and dimerization constants in crystallizing lysozyme solutions by dialysis kinetics.

Dialysis kinetics measurements have been made to study the effect of ionic strength on the dimerization of lysozyme in acidic solutions that lead to the growth of tetragonal lysozyme crystals. Using glutaraldehyde cross-linked dimers of lysozyme, we have determined that both monomers and dimers can escape from 25,000 molecular weight cutoff dialysis membranes with velocity constants of 5.1 x 10(-7) and 1.0 x 10(-7) s(-1) for the monomer and dimer species, respectively. The flux from 25K MWCO membranes has been measured for lysozyme in pH 4.0 buffered solutions of 1, 3, 4, 5, and 7% NaCl over a wide range of protein concentrations. Assuming that dimerization is the first step in crystallization, a simple monomer to dimer equilibrium was used to model the flux rates. Dimerization constants calculated at low protein concentrations were 265, 750, 1212, and 7879 M(-1) for 3, 4, 5, and 7% NaCl, respectively. These values indicate that dimerization increases with the ionic strength of the solution suggesting that aggregation is moderated by electrostatic interactions. At high protein concentrations and high supersaturation, the dimerization model does not describe the data well. However, the Li model that uses a pathway of monomer <-> dimer <-> tetramer <-> octamer <-> 16-mer fits the measured flux data remarkably well suggesting the presence of higher order aggregates in crystallizing solutions.     

Solution physicochemical properties of bovine beta 2-microglobulin. Aggregation states

Bovine beta 2-microglobulin (beta 2-m), the light chain of the histocompatibility antigen, was isolated in crystalline form from colostrum. Previous studies from this laboratory on the solution properties of this protein suggest the existence of a time-dependent multiple aggregation phenomenon. To clarify the molecular states of beta 2-m, its solution properties were studied by ultracentrifugation and spectropolarimetry. Sedimentation equilibrium experiments at pH 5.0 (0.08 M NaCl, 0.02 M sodium phosphate) at concentrations less than 0.3 mg/ml give Mr = 11,800. From sedimentation velocity results, we conclude that bovine beta 2-m is a much more symmetrical and compact molecule than either guinea pig or human beta 2-m. At concentrations above 0.4 mg/ml under the same conditions, sedimentation equilibrium experiments show that a monomer to tetramer reversible self-association occurs. Also, the tetramerization increases with decreasing temperature. beta 2-Microglobulin undergoes an irreversible temperature-dependent association to a much larger aggregate over a period of 7 days, as evidenced by sedimentation equilibrium and velocity results. The rate of this aggregation decreases as the pH approaches the isoelectric point (pH 7) from either side. Furthermore, circular dichroism measured at pH 5.0 under time-dependent aggregating conditions showed a marked increase in the percentage of disordered structure, leading to the conclusion that this effect is a denaturation phenomenon.     

Phosphorylation of phosphofructokinase from skeletal muscle: correlations between phosphorylation and muscle function.

The specific radioactivity of [³²P]-phosphate incorporated into muscle phosphofructokinase was in equilibrium with the specific radioactivity of the γ-phosphate group of ATP. The incorporation was independent of the presence of cycloheximide. The total content of covalently bound phosphate in phosphofructokinase was correlated with the functional state of the muscle from which the enzyme was purified. Muscle dissected post mortem led to phosphofructokinase containing less than 2 phosphate groups per tetramer. Muscle dissected in vivo gave phosphofructokinase with 4 phosphates per tetramer when kept at rest and 8 phosphates per tetramer when stimulated to contract.     

Affinity of hemoglobin for the cytoplasmic fragment of human erythrocyte membrane band 3. Equilibrium measurements at physiological pH using matrix-bound proteins: the effects of ionic strength, deoxygenation and of 2,3-diphosphoglycerate

The cytoplasmic fragment of band 3 protein isolated from the human erythrocyte membrane was linked to a CNBr-activated Sepharose matrix in an attempt to measure, in batch experiments, its equilibrium binding constant with oxy- and deoxyhemoglobin at physiological pH and ionic strength values and in the presence or the absence of 2,3-diphosphoglycerate. All the experiments were done at pH 7.2, and equilibrium constants were computed on the basis of one hemoglobin tetramer bound per monomer of fragment. In 10 mM-phosphate buffer, a dissociation constant KD = 2 X 10(-4)M was measured for oxyhemoglobin and was shown to increase to 8 X 10(-4)M in the presence of 50 mM-NaCl. Association could not be demonstrated at higher salt concentrations. Diphosphoglycerate-stripped deoxyhemoglobin was shown to associate more strongly with the cytoplasmic fragment of band 3. In 10 mM-bis-Tris (pH 7.2) and in the presence of 120 mM-NaCl, a dissociation constant KD = 4 X 10(-4)M was measured. Upon addition of increasing amounts of 2,3-diphosphoglycerate, the complex formed between deoxyhemoglobin and the cytoplasmic fragment of band 3 was dissociated. On the reasonable assumption that the hemoglobin binding site present on band 3 fragment was not modified upon linking the protein to the Sepharose matrix, the results indicated that diphosphoglycerate-stripped deoxyhemoglobin or partially liganded hemoglobin tetramers in the T state could bind band 3 inside the intact human red blood cell.     

Conformational studies of aqueous melittin: thermodynamic parameters of the monomer-tetramer self-association reaction

The self-association reaction in which four melittin molecules associate to form an aqueously soluble tetramer was studied by fluorescent spectroscopy. At 23 degrees C, pH 7.15, gamma/2 0.50, the dissociation constant, Kd, is 3.20 x 10(-16) M3. At 23 degrees C, gamma/2 0.60, melittin has an amino acyl group with a proton ionization constant at ca. 10(-6) M, which must be un-ionized for tetramer formation to occur. The change in Kd with temperature indicates the forward reaction (tetramer formation) proceeds primarily by entropic changes, with delta H degrees = -20.3 kJ/mol of monomer and delta S degrees = 211 J/(K . mol of monomer). The observed enthalpic and entropic values for the tetramerization reaction are consistent with the expected contributions of both nascent hydrogen bonds and hydrophobic stabilization to the reaction. The ionic strength dependence of the tetramerization reaction was found to be consistent with an Edsall-Wyman treatment of activity coefficients. Specifically, the calculated charge of melittin varied from 2.5 (pH 10.53, gamma/2 less than 0.08) to ca. 6 (pH 7.15, gamma/2 greater than 0.3) and showed a strong dependence on gamma/2.     

The association−dissociation behavior of the ApoE proteins: kinetic and equilibrium studies

The apolipoprotein E family consists of three major protein isoforms: apolipoprotein E4 (ApoE4), ApoE3, and ApoE2. The isoforms, which contain 299 residues, differ only by single-amino acid changes, but of the three, only ApoE4 is a risk factor for Alzheimer’s disease. At micromolar concentrations, lipid-free ApoE exists predominantly as tetramers. In more dilute solutions, lower-molecular mass species predominate. Using fluorescence correlation spectroscopy (FCS), intermolecular fluorescence resonance energy transfer (FRET), and sedimentation methods, we found that the association?dissociation reaction of ApoE can be modeled with a monomer?dimer?tetramer process. Equilibrium constants have been determined from the sedimentation data, while the individual rate constants for association and dissociation were determined by measurement of the kinetics of dissociation of ApoE and are in agreement with the equilibrium constants. Dissociation kinetics as measured by intermolecular FRET show two phases reflecting the dissociation of tetramer to dimer and of dimer to monomer, with dissociation from tetramer to dimer being more rapid than the dissociation from dimer to monomer. The rate constants differ for the different ApoE isoforms, showing that the association?dissociation process is isoform specific. Strikingly, the association rate constants are almost 2 orders of magnitude slower than expected for a diffusion-controlled process. Dissociation kinetics were also monitored by tryptophan fluorescence in the presence of acrylamide and the data found to be consistent with the monomer?dimer?tetramer model. The approach combining multiple methods establishes the reaction scheme of ApoE self-association.     

Effect of the ionic environment on the molecular structure of bacteriophage SPP1 portal protein.

Bacteriophage SPP1 portal protein is a large cyclical homo-oligomer composed of 13 subunits. The solution structure and assembly behavior of this protein with high-point rotational symmetry was characterized. The purified protein was present as a monodisperse population of 13-mers, named gp6H, at univalent salt concentrations in the hundred millimolar range (>/= 250 mM NaCl) or in the presence of bivalent cations in the millimolar range (>/= 5 mM MgCl2). Gp6H had a slightly higher sedimentation coefficient, a smaller shape-dependent frictional ratio, and a higher rate of intersubunit cross-linking in the presence of magnesium than in its absence. In the absence of bivalent cations and at univalent salt concentrations below 250 mM, the 13-mer molecules dissociated partially into stable monomers, named gp6L. The monomer had a somewhat different shape from the subunit present in the 13-mer, but maintained a defined tertiary structure. The association-dissociation equilibrium was mainly between the monomer and the 13-mer with a minor population of intermediate oligomers. Their interconversion was strongly influenced by the ionic environment. Under physiological conditions, the concentration of Mg2+ found in the Bacillus subtilis cytoplasm (10-50 mM) probably promotes complete association of gp6 into 13-mer rings with a compact conformation.     

The isolation and characterization of phosphofructokinase from the epithelial cells of rat small intestine.

1. Only a single phosphofructokinase isoenzyme is present in the mucosa of rat small intestine. 2. Mucosal phosphofructokinase was purified to yield a homogeneous preparation of specific activity 175 units/mg of protein. 3. The native enzyme is a tetramer, with monomer Mr 84 500 +/- 5000. 4. The native enzyme may be degraded by the action of endogenous proteinases to give two products with the same specific activity as the native enzyme: degradation occurs in the order native enzyme leads to proteolytic product 1 leads to proteolytic product 2. 5. Proteolytic product 1 has a greater mobility in cellulose acetate electrophoresis at pH8 and binds more strongly to DEAE-cellulose than does native enzyme; the converse is true for proteolytic product 2. 6. Proteolytic product 1 is a tetramer with a monomer Mr about 74 300; proteolytic product 2 is also a tetramer. 7. Native enzyme can only be prepared in the presence of proteinase inhibitors; partial purifications based on simple fractionation of crude mucosal extracts in the absence of proteinases inhibitors contain proteolytic product 2 as the main component and proteolytic product 1 together with little native enzyme. 8. Purified native mucosal phosphofructokinase displayed little co-operativity with respect to fructose 6-phosphate at pH 7.0 and was only weakly inhibited by ATP.     

A kinetic study of the subunit dissociation and reassembly of rabbit muscle phosphofructokinase.

The kinetics of dissociation and reassembly of rabbit skeletal muscle phosphofructokinase has been studied using fluorescence, stopped-flow fluorescence and enzyme activity measurements. The dissociation of the fully active tetramer in 0.8 M guanidine hydrochloride (0.1 M potassium phosphate, pH 8.0) occurs in three kinetic phases as measured by changes in the protein fluorescence emission intensity: dissociation of tetramer to dimer with a relaxation time of a few milliseconds; dissociation of dimer to monomer with a relaxation time of a few seconds; and a conformational change of the monomer with a relaxation time of a few minutes. All three phases exhibit first-order kinetics; ATP (0.05 mM) retards the second step but does not influence the rate of the other two processes. The rate of the second process increases with decreasing temperature; this may be due to the involvement of hydrophobic interactions in the stabilization of the dimeric enzyme. A further unfolding of the monomer polypeptide chain occurs at higher guanidine concentrations, and the relaxation time associated with this process was found to be 83 ms in 2.5 M guanidine, 0.1 M potassium phosphate (pH 8.0) at 23 degrees C. The phosphofructokinase monomers were reassembled from 0.8 M guanidine chloride by 1:10 dilution of the guanidine hydrochloride concentration and yielded a protein with 70-94% of the original activity, depending on the protein concentration. The reactivation process follows second-order kinetics; ATP (5 mM) increases the rate of reactivation without altering the reaction order, while fructose 6-phosphate does not influence the rate of reaction. The rate-determining step is probably the association of monomers to form the dimer.