Showing papers on "Protein quaternary structure published in 1977"
TL;DR: The need for proteins to maintain particular quaternary structures constrains variability in amino acid sequence, which results in monomeric enzymes being more variable than dimeric forms, which in turn are expected to be moreVariable than tetrameric forms.
Abstract: The need for proteins to maintain particular quaternary structures constrains variability in amino acid sequence. Monomeric enzymes are then expected to be more variable than dimeric forms, which in turn are expected to be more variable than tetrameric forms. These predictions are confirmed by analysis of available data on enzyme variation. Theories relating enzyme heterozygosity to metabolic function are discussed in the light of these findings.
104 citations
TL;DR: Although each apoprotein has the ability to form the dimer, β-crustacyanin, on combination with astaxanthin, association between pairs of apoproteins, one from each set, occurs more readily.
Abstract: 1. 1. The five apoprotein subunits of α-crustacyanin have been resolved and their amino acid compositions and molecular weights determined. Two similar sets of apoproteins are distinguished. Although each apoprotein has the ability to form the dimer, β-crustacyanin, on combination with astaxanthin, association between pairs of apoproteins, one from each set, occurs more readily. 2. 2. β-crustacyanin has been separated into a number of components of distinct apoprotein subunit composition. 3. 3.|A proposed mode of binding of astaxanthin involving anhydride bond formation between carboxyl groups of the protein and keto groups of the carotenoid (Zagalsky & Herring, 1972) has not been substantiated.
49 citations
TL;DR: Methods for the fractionation of the protein complement of bisimidoester-treated 30 S subunits, the isolation of cross-linked complexes, the cleavage of bissamidine cross-links and the identification of released proteins are described.
45 citations
TL;DR: 31P NMR spectroscopy is used to compare the environment of DNA phosphate groups in the 140 base-pair core particle with that of the phosphates in the extracted DNA, and infrared spectroscopes are used to comparison the secondary structure of core protein in situ with that in the complex isolated in the solvent.
36 citations
TL;DR: It is concluded that the active enzyme is a dodecamer whereas the subfragments correspond to dimeric and hexameric forms.
Abstract: The smallest active form of aminopeptidase I (EC 3.4.11.1) from yeast has a molecular weight of 6.4 X 10(5). At neutral pH the active enzyme is in equilibrium with two inactive subfragments (Mr = 3.2 X 10(5) and 1.1 X 10(5)) as well as with higher aggregates (Mr greater than or equal 1.2 X 10(6)). All of these species may be dissociated to give a single type of subunits with a molecular weight of 5.3 X 10(4). It is concluded that the active enzyme is a dodecamer whereas the subfragments correspond to dimeric and hexameric forms.
25 citations
TL;DR: In this paper, a single-crystal microspectrophotometry of d -glyceraldehyde-3-phosphate dehydrogenase was obtained from the Mediterranean lobster Palinurus vulgaris and the reaction of the crystalline holo-enzyme with the chromophoric acylating reagent β-(2-furyl)acryloyl phosphate was investigated.
24 citations
TL;DR: It is demonstrated that covalent cross-linkers can be used as sensitive indicators of conformational changes induced in pyruvate kinase by substrates and allosteric ligands.
Abstract: Pyruvate kinase (EC 2.7.1.40) of Neurospora, a tetramer composed of apparently identical subunits, has been shown to be a dimer of dimers by interprotomeric cross-linking experiments in which bifun...
23 citations
TL;DR: Kinetic studies of the intact hemoglobin and its isolated domains are consistent with the oxygen equilibrium studies, and the relatively high oxygen affinities of this pigment and its domains are associated with low oxygen dissociation velocity constants.
23 citations
TL;DR: Electrophoretically homogeneous ribulose-1,5-bisphosphate (RuBP) carboxylase was obtained from autotropically grown Hydrogenomonas eutropha by sedimentation of the 105,000 X g supernatant in a discontinuous sucrose gradient and by ammonium sulfate fractionation followed by another Sucrose gradient centrifugation.
Abstract: Electrophoretically homogeneous ribulose-1,5-bisphosphate (RuBP) carboxylase was obtained from autotropically grown Hydrogenomonas eutropha by sedimentation of the 105,000 X g supernatant in a discontinuous sucrose gradient and by ammonium sulfate fractionation followed by another sucrose gradient centrifugation. The molecular weight of the enzyme determined by light scattering was 490,000 +/- 15,000. The enzyme could be dissociated by sodium dodecyl sulfate into three types of subunits, and the molecular weights (+/- 10%) could be measured. There were two species of large subunits, L and L' (molecular weight 56,000 and 52,000, respectively) and one species of small subunits (molecular weight, 15,000). The mole ratio of L to L' was 5:3, and the overall mole ratio of the small to large subunits was 1.08. The simplest quaternary structure of the enzyme is L5L'3S8. The enzyme contained RuBP oxygenase activity as evidenced by the O2-dependent production of phosphoglycolate and 3-phosphoglyceric acid in equimolar quantities from RuBP.
22 citations
TL;DR: The results obtained from the examination of tryptic maps, the number and amino acid composition of cysteine-containing peptides and the uniqueness of the N-terminal sequence, strongly suggest that the 2 subunits of Escherichia coli K12 aspartokinase II--homoserine dehydrogenase II are identical.
Abstract: The quaternary structure of Escherichia coli K 12 aspartokinase II–homoserine dehydrogenase II has been examined. This multifunctional protein has a molecular weight Mr= 176000. It is composed of two subunits having the same molecular weight and the same charge.
The results obtained from the examination of tryptic maps, the number and amino acid composition of cysteine-containing peptides and the uniqueness of the N-terminal sequence, strongly suggest that the 2 subunits are identical. The properties of aspartokinase II–homoserine dehydrogenase II can be compared to those of the much better known protein aspartokinase I–homoserine dehydrogenase I.
20 citations
28 Sep 1977
TL;DR: At low enzyme concentrations second order steps gain importance which indicates that the enzymic activity is significantly modified by the formation of the native quaternary structure.
Abstract: The denaturation of aldolase from rabbit muscle in various solvents leads to significant qualitative and quantitative differences with respect to the structural disintegration of the enzyme. The differences refer to the quaternary structure and to the conformation which is changed only slightly in MgCl2 while in guanidine-HCl or urea at pH approximately 2 the molecule is close to the state of the random coil. Using the enzymic activity as a quantitative measure for the refolding process, the reaction order and the rate constants of the processes of structure formation (vi leads to N*) are found to be identical. This observation suggests a common intermediate D in the process of renaturation after denaturation and dissociation in the different solvent media. D may be considered an intermediate state with a defined number of nucleation centers whose rapid formation is predetermined by the aminoacid sequence. As taken from the first order kinetics in the given range of enzyme concentration, transconformation reactions are rate limiting in the obligatory pathway of refolding. At low enzyme concentrations second order steps gain importance which indicates that the enzymic activity is significantly modified by the formation of the native quaternary structure.
TL;DR: The influence of the quaternary structure on the functional and conformational properties of tryptophanase and the nature of the conformational change involved in the activation of the enzyme by its cofactor and specific cations are discussed.
Abstract: It is first shown that the inactivation of Escherichia coli apotryptophanase by N-ethylmaleimide results from the labeling of a single particularly reactive cysteine per protomer.
The reactivity of this cysteine under various conditions is investigated and the results indicate that the protein can exist under two classes of conformation: one, corresponding to inactive protein, in which the cysteine is reactive, and a second, corresponding to active enzyme, where the cysteine is masked. The rate of the isomerization step involved in this change in conformation is measured by the stopped-flow technique (τ= 0.4 s).
Finally, the reactivity of the cysteine is used to characterize the conformation of dimeric holotryptophanase (i.e. a dissociated form of the enzyme obtained as a transient species between dimeric apoenzyme and the natural tetrameric holoenzyme). By this criterion, it is shown that dimeric holotryptophanase falls in the class of ‘inactive’ conformations. These results are used to discuss the influence of the quaternary structure on the functional and conformational properties of tryptophanase and the nature of the conformational change involved in the activation of the enzyme by its cofactor and specific cations.
TL;DR: It is indicated that reformation of stable tetramers occurs relatively slowly, qualitatively paralleling the regain of enzyme activity, and that tetramer formation may be necessary for enzymatic activity.
Abstract: After denaturing either type M or L pyruvate kinase by guanidine hydrochloride, urea, or low pH, enzymatic activity and quaternary structure can be recovered by diluting the enzyme into buffer containing beta-mercaptoethanol. After denaturation of type M pyruvate kinase by guanidine hydrochloride, the yield and polarization of the intrinsic protein fluorescence, as well as most of the circular dichroism characteristic of the native enzyme, were regained very rapidly, while enzymatic activity was recovered much more slowly. Under the conditions used, about 50% of the original M and 30-50% of the original type L activity were typically recovered. Average half-times for recovery of enzymatic activity were 37 min for type M and 104 min for type L but depended somewhat on the renaturation buffer and on protein concentrations in the renaturation medium. If types M and L pyruvate kinases are renatured together, an approximately random recombination of the two subunits types results in a five-membered hybrid set. We have used this hybridizability to determine the kinetics of reformation of the native tetramer by denaturing each isozyme and beginning its renaturation separately at various times mixing the two isozymes and continuing their renaturation together. These studies indicate that reformation of stable tetramers occurs relatively slowly, qualitatively paralleling the regain of enzymatic activity, and that tetramer formation may be necessary for enzymatic activity. Using a similar technique to test for spontaneous dissociation of the native isozymes in buffer, we find that type L, but not type M, reversibly dissociates into dimers and monomers in buffer solutions. This dissociation is decreased by the presence of the substrate, phosphoenolpyruvate, by Mg2+ ions, or by the allosteric effector, fructose bisphosphate.
TL;DR: A model is proposed in which one of the major subunits, alpha, is first dissociated and unfolded reversibly by the denaturants, followed by the irreversible unfolding and dissociation of the other major subunit, beta, from subunit delta and/or the components of relative mobility.
Abstract: 1. The fluorescence and circular dichroism of four homogeneous preparations of ATPase (adenosine triphosphatase) from Micrococcus lysodeikticus differing in molecular structure and enzymic properties were examined at pH 7.5 and 25 degrees. Emission was maximum at 325 and 335 nm and the relative intensities at these wavelengths may be used to characterize the different ATPase preparations. The circular-dichroism spectra exhibited negative extrema at 208 and 220 nm, and the relative value of the molar ellipticity at these wavelengths was also different for each molecular form of the enzyme. 2. The four preparations undergo two consecutive major unfolding transitions in guanidinium chloride (midpoints at 0.94 and 1.5 M denaturant), with concomitant destruction of the quaternary structure of the protein. A comparatively minor alteration in the ATPase structure also occurred in 0.05-0.2M-guanidine and led to complete inactivation of the enzyme. The inactivation and the first unfolding transition were reversible by dilution of the denaturant; the transition with midpoint at 1.5M-guanidine was irreversible. 3. Similar results were obtained in urea, except that the successive transitions had midpoints at concentrations of denaturant of 0.4, 2.0 and 4.5M. Low concentrations of urea caused a noticeable activation of the enzyme activity and alterations of the electrophoretic mobility of the ATPase. 4. A model is proposed in which one of the major subunits, alpha, is first dissociated and unfolded reversibly by the denaturants, followed by the irreversible unfolding and dissociation of the other major subunit, beta, from subunit delta and/or the components of relative mobility 1.0 in dodecyl sulphate/polyacrylamide-gel electrophoresis (rho).
TL;DR: Electron micrographs of native aminopeptidase I and of isolated subfragments were taken and it is concluded that the active enzyme is a dodecamer possessing pseudo-D3 symmetry with the dimer as the smallest symmetric unit.
Abstract: Aminopeptidase, Yeast, Electron Microscopy, Quaternary Structure Electron micrographs of native aminopeptidase I and of isolated subfragments were taken after negative staining with uranyl formate. From these studies and from the chemical evidence sum marized in the preceding paper it is concluded that the active enzyme is a dodecamer possessing pseudo-D₃ symmetry with the dimer as the smallest symmetric unit.
TL;DR: A model for the planar arrangement of the trimeric species in the quaternary structure of glutamate dehydrogenase is discussed, which includes both isologous and heterologous contact areas between the polypeptide chains.
Abstract: Cross-linking of the unimer of glutamate dehydrogenase from beef liver (consisting of six polypeptide chains each having a molecular weight of 56000) with dimethyladipimidate and subsequent analysis by sodium dodecylsulfate electrophoresis shows predominantly the trimeric species (molecular weight 168000). Treatment with dimethylimidates of other chain length yields significantly less trimeric species indicating that the amino groups being cross-linked are within a distance of about 0.85 nm. Comparison of the molar amount of incorporated [14C]dimethyladipimidate with the number of modified amino groups (determined with trinitrobenzenesulfonic acid) shows that although 8-9 of the 34 amino groups have reacted, only 2-3 of them are involved in cross-links. Reaction with dimethylimidates inactivates the enzyme. The loss of the activity is partly concomitant to cross-linking to the trimeric species and not simply due to the modification of essential lysine residues. This is supported by the fact that, although more lysine residues react with mono-functional methylimidates, the loss of activity is reduced. Purified chymotryptic and tryptic peptides of the radioactive-labeled trimeric species were subjected to sequence analysis. Six peptides containing 75% of the total label were identified: one involves the amino-terminal residue alanine-1 and the others involve lysine-105, lysine-154, lysine-269, lysine-358 and lysine-399. Quantitative analysis of the specific radioactivity of each peptide/mol lysine leads to the conclusion that only lysine-105, lysine-154, lysine-269 and lysine-358 participate in cross-links, lysine-269 and lysine-358, respectively, being at isologous and lysine-105 cross-linked with lysine-154 at heterologous contact domains of the enzyme. A model for the planar arrangement of the trimeric species in the quaternary structure of glutamate dehydrogenase is discussed. It includes both isologous and heterologous contact areas between the polypeptide chains.
TL;DR: The dimeric quaternary structure was demonstrated by dissociation-recombination procedures of sunflower seed acid phosphatase and the formation of the hybrid isozyme occurred during 12 hr dialysis against a pH 7.0 buffer.
Abstract: Formal genetic studies of sunflower (Helianthus annuus) seed acid phosphatase (ACP, E.C. 3.1.3.2) had suggested that the functional enzyme consists of two polypeptide subunits. The dimeric quaternary structure was demonstrated by dissociation-recombination procedures. Dissociation of electrophoretically distinct homodimers was effected upon freezing of extracts in a pH 8–9 buffer containing 1 m NaCl and 0.1 m 2-mercaptoethanol. Reassociation, as indicated by the formation of the hybrid isozyme, occurred during 12 hr dialysis against a pH 7.0 buffer.
TL;DR: The new bifunctional reagent was used to investigate the quaternary structure of yeast alcohol dehydrogenase and the result suggests that the four protomers of the enzyme could be arranged as a dimer of dimers.
Abstract: The new bifunctional reagent, N-(4-chloromercuriphenyl)-4-chloro-3,5-dinitrobenzamide (I) was used to investigate the quaternary structure of yeast alcohol dehydrogenase.
The four essential – SH groups of the enzyme were substituted by the mercuriphenyl moiety of compound I in the course of the reaction of one mole of protein with four moles of the reagent (one molecule of compound I incorporated by yeast alcohol dehydrogenase monomer). In a second step only two of the four chlorodinitrophenyl fragments bound to the protein established intermonomeric cross-links with non-essential – NH2 groups. The resulting dimers could be re-dissociated with mercaptoethanol. This result suggests that the four protomers of the enzyme could be arranged as a dimer of dimers.
TL;DR: It is proposed that thebeta-93 probes sense the dramatic change in beta subunit structure resulting from the quaternary structure change (T leads to R) upon heme saturation as well as tertiary structure changes at the alpha1-beta2 contact region resulting from ligand binding to the beta-heme group.
Abstract: The binding of oxygen and 1-oxyl-2,2,6,6-tetramethylpiperidine 4-triphosphate (spin-labeled triphosphate) to normal adult human hemoglobin (HbA) covalently labeled at the beta-93 sulfhydryl groups with N-(2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide (I) was studied. HbA-I was used as a model for HbA labeled at the beta-93 SH groups with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide (II) since the binding of SLTP to HbA-II could not be measured conveniently, in the presence of the paramagnetic resonance signal of II. Both HbA-I and HbA-II can be treated as variant hemoglobins with abnormal beta chains. The oxygen and SLTP binding data from HbA-I and oxygen binding data from HbA-II are consistent with a concerted transition model for cooperativity which assumes nonequivalence between alpha and beta subunits (GCT model). The distribution of environments "seen" by conformation sensitive probes such as II and trifluoracetone (19F NMR probe) attached to the beta-93 sulfhydryl groups of HbA can also be accounted for by the GCT model. It is proposed that the beta-93 probes sense the dramatic change in beta subunit structure resulting from the quaternary structure change (T leads to R) upon heme saturation as well as tertiary structure changes at the alpha1-beta2 contact region resulting from ligand binding to the beta-heme group. Structural changes caused by ligation of the alpha-hemes are not discussed.
Journal Article•
TL;DR: In this paper, the quaternary structure and some kinetic properties were studied for NAD-kinase from the rabbit skeletal muscles, and the molecular weight of the 150-300-fold purified enzymic preparation was determined by separation in the Sephadex G-200 thin layer, by means of separating the column gel-filtration and by the method of electrophoresis in the gradient of polyacrylamide gel concentration.
Abstract: The quaternary structure and some kinetic properties were studied for NAD-kinase from the rabbit skeletal muscles. The molecular weight of the 150-300-fold purified enzymic preparation was determined by separation in the Sephadex G-200 thin layer, by means of Sephadex G-200 column gel-filtration and by the method of electrophoresis in the gradient of polyacrylamide gel concentration. Some molecular forms of NAD-kinase with a molecular weight of 31000-305000 are found in the enzymic preparation and possibility to change from one form to another is shown. On the basis of the established quaternary structure and complex kinetic characteristics of the enzyme a conclusion is drawn on the existence of the rabbit skeletal muscle NAD-kinase as an equilibrium system of the oligomeric forms possessing different catalytic activity and consisting of different combinations of subunits with a molecular weight of 31000.
Journal Article•
TL;DR: Study of kinetic manifestation of "native", dissociated and reassociated enzyme forms has revealed that the enzyme in the "normal" state has probably at least two active sites, "negative" homotropic cooperative interaction being establish-d between them at the definite degree of substrate saturation.
Abstract: Previous data on oligomeric nature and values of molecular masses of "native" and dissociated forms of cytoplasmic protein with ribonulcease activity from wheat seedling leaves (Bezenchukskaya 98) are confirmed by means of polyacrylamide gel disc electrophoresis. Possible relation of the "native" enzyme form with 3':5'-cAMP is demonstrated by paper chromatography and the enzyme reassociation by the nucleotide. Study of kinetic manifestation of "native", dissociated and reassociated enzyme forms has revealed that the enzyme in the "normal" state has probably at least two active sites, "negative" homotropic cooperative interaction being establish-d between them at the definite degree of substrate saturation. The cooperativity disappears under dissociation, and it is discovered again under reassociation.
Journal Article•
TL;DR: The mechanisms of activation and inhibition of enzymes by antibodies and the use of immunochemical methods for the study of the active sites structure, conformational changes during interaction with substrates and other ligands and the quaternary structure of enzymes are discussed.
Abstract: The binding of antibodies to enzymes-antigens markedly affects the activity and structure of the latters. The mechanisms of activation and inhibition of enzymes by antibodies and the use of immunochemical methods for the study of the active sites structure, conformational changes during interaction with substrates and other ligands and the quaternary structure of enzymes are discussed.
TL;DR: Use of the method in determining the subunit geometry of oligomeric proteins is discussed and a small number of molecules in a population which has been intramolecularly cross-linked with polylysine retain important features of their tertiary and quaternary structure when negatively stained and examined in the electron microscope.
Abstract: Oligomeric proteins can be intramolecularly cross-linked with polylysine in a reaction in which a water soluble carbodiimide mediates an amide linkage between the protein carboxyl groups and the e-amino groups of polylysine. Studies carried out with a cytochrome P-450 indicate that a small number of molecules in a population which has been cross-linked in this way retain important features of their tertiary and quaternary structure when negatively stained and examined in the electron microscope. Use of the method in determining the subunit geometry of oligomeric proteins is discussed.