Urea Dissociation Research Articles

An approach to the elucidation of the quaternary structure role in the activity of pyruvate kinase studies on the immobilized enzyme

1. 1. Studies on pyruvate kinase immobilized via -S-S- linkages showed that single subunits of this tetrameric enzyme are inactive. 2. 2. Pyruvate kinase was coupled with different preparations of CNBr-activated Sepharose via one and more than one bond, and after removal of non-covalently bound subunits by denaturant treatment the subunit derivatives were obtained composed of different pyruvate kinase species. 3. 3. Heat inactivation studies of immobilized enzyme derivatives suggested that not only tetramers of pyruvate kinase are active. 4. 4. This idea is further supported by experiments with 2 M urea dissociation of immobilized tetrameric enzyme carried out in the presence of Mg 2+ ions.

Molecular studies of liver aldolase B in hereditary fructose intolerance using blotting and immunological techniques.

Hereditary fructose intolerance is due to a deficiency of liver aldolase (aldolase B). Little is known about its molecular mechanisms. We have tried to demonstrate the presence of the molecule and have explored the possibility of genetic heterogeneity. Liver samples from fifteen cases of hereditary fructose intolerance due to aldolase B deficiency were studied by various electrophoretic techniques. After electrophoresis on polyacrylamide gels, proteins were electrophoretically transferred on to nitrocellulose filters. They were treated with specific antialdolase B antibodies, and then with radioiodinated protein A, followed by autoradiography. Investigations included: (a) sodium dodecyl sulphate electrophoresis, in order to detect the presence of immunologically reactive molecules and to estimate the subunit size; (b) attempts to discover charge anomalies of the native molecule and of its subunits, by the use of: Isoelectric focusing of the native enzyme. Isoelectric focusing and non-equilibrium pH gradient electrophoresis (NEPHGE) after dissociation in urea. The major results were the following: (1) In all cases a cross-reacting material was found, with a molecular subunit size of 38000, indistinguishable from that of controls. (2) Evidence for molecular heterogeneity of the disease was provided by two types of data: amount of apparent immunologically reactive protein, which varied from less than 3% to 100% of that of controls; and charge data, aldolase B from seven patients showing an increased negative charge and from one patient a normal charge.

Preparation of frog myosin. Isolation and characterization of the light chains

Frog myosin can be prepared with a good yield by precipitation of a high ionic strength extract between I 0.20 and 0.05 or by ammonium sulphate fractionation of actomyosin in the presence of Mg-ATP. Two alkali light chains, LC1 and LC3, along with one DTNB light chain LC2 have been isolated by chromatography on ion exchange cellulose after urea dissociation. A supplementary light chain LC1d present in variable amounts from one preparation to the other corresponds to a proteolysis product of LC1. Their stoichiometry, molecular weight, amino acid composition, isoelectric point and peptide map have been determined. Their general proportions and structural properties show many similarities with rabbit skeletal muscle light chains. Amino acid compositions and peptide maps confirm that the additional band LC1d comes from a proteolytic degradation affecting the N-terminal part of LC1.

Isolation, characterization, and in vitro assembly of the tetragonally arrayed layer of Bacillus sphaericus

The tetragonally arranged cell wall layer (T-layer) of Bacillus sphaericus NTCC 9602 was isolated and characterized. Parallel studies were made on a spontaneous variant of the wild-type strain which had a T-layer subunit of altered molecular weight. A purification method for the T-layers was devised which involved separation of the cell walls from the cytoplasmic contents, urea dissociation of the T-layer from the cell walls, removal of soluble contaminants by differential centrifugation, and finally selective adsorption of uncleaved subunits to sacculi. The purified subunits retained the capacity to form an assembly in vitro with the same lattice parameters as that observed on whole cells or cell walls and could readsorb to the cell walls from which they had been extracted. Both the wild-type and the variant subunits behaved as single, homogeneous polypeptide chains. Carbohydrate assay and isoelectric point determinations revealed that both subunit types were acidic glycoproteins. Values obtained for thebuoyant density, isoelectric point, and extinction coefficient differed minimally; major differences were observed in the molecular weight and the characterisitc width of cylinders formed by in vitro-assembled T-layer of the wild-type and variant. Assembled T-layer was subject to alkaline or acid dissociation and in acid titration dissociated at its isoelectric point.

Physicochemical and biological characterizations of pregnant mare serum gonadotropin and its subunits

Pregnant mare serum gonadotropin and its subunits have been further characterized. Ultracentrifugation of the gonadotropin at pH 1.3 and 11.5 showed little evidence of dissociation compared to pH 8.2. Highly purified subunits are obtained by urea dissociation and ion-exchange chromatography followed by gel-filtration. Circular dichroism spectra of the gonadotropin and its subunits are much like those of ovine lutropin and its subunits in that there is little evidence for secondary structure and one or more tyrosine residues are inaccessible in the intact gonadotropin compared to the subunits. The α-subunit possesses almost 3 times as much total carbohydrate as the β-subunit; the individual sugar composition of each was determined as well as the amino acid composition. The α-subunit begins with the sequence NH 2-Phe-Pro (Gly or Pro) … and terminates with isoleucine. The β-subunit has the sequence NH 2-Ser-Pro-Gly …; no C-terminal residue is detectable by either carboxypeptidase or hydrazinolysis. Biological studies show the gonadotropin to be active in assays specific for both lutropin and follitropin. Precipitin test in agar with rabbit antiserum against the gonadotropin show that the beta subunit cross-reacts whereas the alpha subunit does not.

The inhibition of rat liver chromatin protease by congeners of the phenylboronic acids

A group of arylalkylboronic acids were synthesized in order to investigate the inhibitory potential of these compounds for rat liver chromatin protease (EC 3.4.-.-). The effect of side chain length, side chain substitution and aromatic substitution on proteolytic activity in chromatin dissociation in salt and urea was assayed. It was determined that a side chain length two carbons long provided the greatest inhibitory effect with complete inhibition attainable at 20 mM concentration of phenylethylboronic acid. Aryl substitution in the ortho position proved to be the most potent structural change with complete inhibition attained by 1 mM concentration of o-methylphenylethylboronic acid. The binding of these two inhibitors proved to be reversible.

Chromosomal proteins: Tightly bound nucleic acid and its bearing on the measurement of nonhistone protein phosphorylation

Nonhistone proteins isolated by salt—urea dissociation of purified HeLa cell chromatin contain a small amount of tightly bound nucleic acid. The association between these proteins and copurifying nucleic acid is exceptionally strong, surviving exposure to guanidine · HCl, urea, sodium dodecyl sulfate, and concentrated cesium sulfate (for equilibrium density gradient centrifugation). Tightly bound nucleic acid is a feature of approximately 60% of the nonhistone protein (mass), while the remainder behaves as pure protein. The bound nucleic acid coelectrophoreses with the nonhistone proteins in polyacrylamide gels containing sodium dodecyl sulfate. These observations demonstrate that gel electrophoresis per se is not a valid analytical end point for measuring nonhistone protein phosphorylation. A simple technique is described for eliminating this problem.

Dissociation and Reassociation of Poliovirus I. Effect of Urea on the Virion

Poliovirus is dissociated by urea in the presence of mercaptoethanol or dithiothreitol into RNA and protein(s). Differences exist in the sensitivity of poliovirus type I, II or III toward these agents. The decrease in infectivity titer of the virion is in the range of 10(9). After appropriate dissociation no infectious virions are found in urea-treated poliovirus concentrates. The released viral RNA is infectious. Its infectivity (1 times 10(6) PFU/mug RNA) is comparable to that of phenol extracted RNA. Therefore, urea dissociation is a fast and reliable method for the preparation of infectious poliovirus RNA even at the microliter scale. The dissociation of poliovirus is dependent upon the concentration, temperature and pH. The decrease in virion infectivity is time dependent. It is preceded by a lag phase of different lengths for poliovirus type I, II or III. During the lag phase the virion undergoes configurational changes without losing infectivity. From the observed hyperchromic effect is it obvious that the secondary structure of the RNA inside the virion breaks down, giving rise to expanded forms of poliovirus particles. After the lag phase the RNA is released and the empty capsid dissociated into the appropriate polypeptides.

ΔGθ, ΔHθ, ΔSθ de transfert de H2O et du couple H+, OH− de l'eau à ses mélanges contenant jusqu'à 40% de EtOH, tert-BuOH, DMSO, acétone et urée à 25 °C

We have measured the heat of neutralization of dilute solutions of HCl in aqueous–organic mixtures containing up to 40% by weight of t-BuOH, DMSO, acetone, and urea; autoprotolysis constants of the mixtures H2O–DMSO and H2O–urea also were determined. Thus the usual thermodynamic quantities ΔGθ, ΔHθ, and ΔSθ could be determined for the dissociation of water in these mixtures. The results for the H2O-urea mixtures must be accepted with caution because the dissociation of urea itself is not negligible compared to that of water. Using various literature data we have calculated values of the Gibbs free energy and the enthalpy for the transfer of a molecule of water from the pure liquid to the mixtures noted above. (We also have calculated for these mixtures the coefficients of isobaric expansion and those for the variation with temperature of the dielectric constant.) In addition, the values for the transfer of the ion pair H+, OH− have been determined as well as those for the transfer from water to H2O–EtOH mixtures. These are com pared to those already known for HCl and the alkali halides. It is not possible to establish a simple correlation between the differing variations of these quantities with the structural prop erties usually considered to exist in the media studied. [Journal translation]

Urea dissociation. Measure of steric hindrance in secondary amines

Urea dissociation. Measure of steric hindrance in secondary amines

Complementation in vitro between mutationally altered beta2 subunits of Escherichia coli tryptophan synthetase.

Cross-reacting beta(2) subunits (CRMs) were purified from eight trpB missense mutants to test for complementation in vitro after urea dissociation and reaggregation. One CRM (B290, demonstrating "repairability," i.e., the appearance of enzymatic activity on combination with alpha subunits) was clearly positive with four others, all "non-repairable" CRMs resulting from mutations at three different but neighboring sites. One complementing pair, B290-B248, was studied in more detail and found, upon mixing purified proteins, to give complementation in the absence of denaturants. Complementation activity was low in each case. To study the mechanism of the modest increases in activity, we used a reduced beta(2) subunit as an artificial CRM to form hybrids where both the amount of activity due to complementation and the amount of hybrid could be measured. (In a reduced beta(2) subunit, the two pyridoxal phosphate cofactors have been chemically reduced by sodium borohydride and are covalently attached to lysine residues. This abolishes activity in the tryptophan synthetic reaction and causes the protein to migrate much faster than normal in acrylamide gel electrophoresis.) Reduced beta(2) subunit formed hybrid dimers with the non-repairable CRMs B244 and B248 at pH 6.0, but no enzymatic activity appeared. On the other hand, when reduced beta(2) subunit was mixed with B290 CRM at pH 6.0 to 6.6, an activity increase was seen that was proportional to the amount of hybrid. We conclude that hybrid formation is essential for complementation and that the mechanism of complementation in this system is the correction of a repairable active site on the B290 beta chain by a conformational change occuring when hybrid dimer is formed. This type of complementation must be restricted to a small class of CRMs having a conformationally deformed active site. From the amount of hybrid present and the increase in activity, a specific activity of 50 U/mg was calculated for the hybrid containing reduced and B290 beta chains. This value is slightly less than but close to the activity of the hybrid formed between reduced and normal beta chains, shown earlier to have half the specific activity of the normal dimer.

The molecular basis of the heterogeneity of the MM isozyme of rabbit muscle creatine phosphokinase

Purified rabbit muscle creatine phosphokinase MM isozyme was resolved into a pattern of six subbands by starch gel electrophoresis at pH 6.3 in a Tris-citrate buffer system. Following urea dissociation and denaturation, urea-starch gel electrophoresis resolved six distinct, enzymatically inactive subunits of molecular weight 43 000 daltons as determined by sodium dodecyl sulfate-acrylamide gel electrophoresis. This indicates that six different subunit types are involved in rabbit MM isozyme subbanding. The six subbanded pattern obtained following renaturation and reassociation from urea was identical to that exhibited by the purified, untreated MM isozyme. The exact nature of the subbanding is not clear; but it does not appear to be due to sulfhydryl group interactions or to alternative conformations of a single M-type subunit. Rather, it appears to be due to conformational differences resulting from alterations in primary structure, deamidation or ligand heterogeneity.

β-Galactosidase: IN VIVO α-COMPLEMENTATION

Partial diploids were prepared from fourteen β-galactosidase nonsense mutant strains of Escherichia coli by adding an episomal element containing a β-galactosidase structural gene with a small deletion in the α region. Active enzyme was obtained from all strains with the exception of one in which the site of mutation is very close to the operator locus. The complemented enzyme was stable during growth. Dissociation in urea of a complemented enzyme followed by centrifugation showed heterogeneity of the prematurely terminated fragment, suggesting that an exposed overlapping segment had been subjected to proteolysis. The amount of complemented enzyme formed varied from strain to strain and was dependent in part on the tertiary structure of the nonsense fragment itself. The results are discussed in relation to the mechanism of folding of polypeptide chains.

Denaturation of horse-liver alcohol dehydrogenase in urea studies by gel filtration and electrophoresis.

Partially denatured main isoenzyme com- Key Words ponent of alcohol dehydrogenase from horse liver separates Denaturation of horse into three peaks on chromatography on Sephadex G-150, liver alcohol dehydrogenase with relative elution volume (Ve/Vo) values of 1.7,1.3 and in urea 1.0, respectively. The peak with Ve/Vo value of 1.7 consists of the native enzyme. The components of the peak with Ve/Vo value of 1.3 are able to form the native enzyme under suitable techniques and, therefore, may represent enzyme subunits, or possibly protein aggregates. The peak with Ve/Vo value of 1.0 probably represents subunit aggregates. The fact that reassociable forms of the enzyme are excluded by Sephadex G-150 in front of the native enzyme suggests that dissociation in urea is accompanied by considerable unfolding of the native structure. The denaturation rate is faster at 4 than at 20°C and aggregate formation is also faster at 4 then at 20°C. The rate of dénaturation of the enzyme, as determined by electrophoresis and scanning, is highly dependent on the purity of urea.

Multiple forms of fumarase of pig heart

Crystalline pig heart fumarase was found to be highly heterogeneous by gel electrophoresis and ion-exchange chromatography. The enzyme was resolved into five active components by chromatography. These components in turn appear heterogeneous; one yielded four electrophoretic bands. Each chromatographic component has the same molecular weight as the original sample; contains four subunits of molwt 48,500; retains its particular chromatographic behavior on rechromatography; can be reconverted to active enzyme after urea dissociation of subunits. Each chromatographic component yielded three or four chromatographic peaks after dissociation and reassociation, but no two yielded the same pattern, showing that there are differences in primary structure between the subunits. The data are consistent with the presence of at least three different subunit types.

Dissociation of the Equimolar Human Plasmin-Streptokinase Complex: PARTIAL CHARACTERIZATION OF THE ISOLATED PLASMIN AND STREPTOKINASE MOIETIES

Abstract An isolated equimolar human plasmin-streptokinase complex (native complex) was dissociated either in 8 m urea or at pH 3.0, both before and after treatment with diisopropyl phosphorofluoridate (DIP complex). Dissociation of the native complex either in 8 m urea or at pH 3.0, resulted in an immediate loss of both the bovine plasminogen and human plasminogen activator activities with complete restoration of the original plasmin proteolytic activity. Urea or acid dissociation of the inactive DIP complex did not regenerate either activator or proteolytic activities. The weight average molecular weight of native complex was redetermined with the use of photoelectric scanning optics in the analytical untracentrifuge and found to be 123,900 ± 1,800. The sedimentation coefficient, s020,w, of native complex was redetermined to be 5.0 S. On immunoelectrophoretic analysis, native complex gave a single antigen-antibody band with specific rabbit antibodies to both plasminogen and streptokinase. The DIP complex dissociated with either urea or acid still reacted with both antibodies. However, urea dissociation of native complex caused the mixture to lose only its ability to react with the streptokinase antibody. Gel filtration on Sephadex G-150 in 0.001 m HCl-0.15 m NaCl, pH 3.0, and on Sephadex G-200 in 6 m urea-0.02 m Tris buffer, pH 8.0, was used to separate the dissociated plasmin and streptokinase moieties from the urea- and acid-dissociated complexes. The plasmin moiety isolated from the DIP complex dissociated at pH 3.0 was found to be homogeneous by electrophoretic and ultracentrifugal analysis with a weight average molecular weight of 84,000 ± 4,100 and a sedimentation coefficient, s20,w, of 4.2 S. Carboxyl-terminal amino acid analysis showed that this moiety contained 0.99 residue of asparagine, 0.47 residue of arginine, and 0.53 residue of lysine per mole of protein. It reacted with specific antibodies to both plasminogen and streptokinase. The weight average molecular weight of this moiety in 6 m guanidine-0.05 m phosphate buffer, pH 7.0, was determined to be 67,250. It was calculated that two species of molecules could be present in solution, in equimolar amounts, with molecular weights of 75,000 (plasmin) and 9,000 (streptokinase fragment). The plasmin moiety isolated from native complex dissociated in 8 m urea was found to be homogeneous by electrophoretic and ultracentrifugal analysis with a weight average molecular weight of 65,000 ± 4,000 and a sedimentation coefficient, s20,w, of 3.6 S. Carboxyl-terminal amino acid analysis indicated that considerable cleavage of the moiety had occurred at arginine- and lysine-peptide bonds. The streptokinase moieties isolated from both native and DIP complexes dissociated either in 8 m urea or at pH 3.0 were recovered primarily as fragments. In the ultracentrifuge, they appeared as mixtures of components with sedimentation coefficients between 2 and 0.8 S. This fragmentation occurs very shortly after formation of the complex without loss of any of the fragments from the complex.

Hemoglobin Hiroshima (beta-143 histidine--aspartic acid): a newly identified fast moving beta chain variant associated with increased oxygen affinity and compensatory erythremia.

During a survey for hemoglobinopathies in over 9000 residents of Hiroshima Prefecture, Japan, a fast moving hemoglobin was identified in eight members of three generations in a Japanese family. The abnormal hemoglobin, named Hb Hiroshima, constitutes about 50% of the total hemoglobin in hemolysates from the carriers who have a mild erythremia but are otherwise apparently clinically unaffected. All preparations of Hb Hiroshima have increased affinity for oxygen, by either tonometric or oxygen electrode determinations. At pH 7.0, the oxygen pressure, P(50) required to half saturate an unfractionated hemolysate from a carrier was one-half that of Hb A, and the P(50) of a purified sample containing no Hb A was one-fourth that of Hb A. The pH dependence of the oxygen equilibrium (Bohr effect) is below normal, as shown by the absolute value of the Bohr effect factor which is about half that of Hb A, in the pH range between 7.0 and 7.4. The Hill constant, n, for Hb Hiroshima between pH 7.0 and 7.4 is 2-2.4, compared to 2.8-3 for Hb A under the same conditions, indicating reduction of, but not complete abolition of heme-heme interaction. Urea dissociation and canine hybridization tests located the biochemical lesion in the beta chain. Fingerprints (Ingram), carboxypeptidase digestion, and amino acid analysis demonstrated that the substitution was at residue 143 in the beta chain, where histidine was replaced by aspartic acid.In contrast to other recently described high oxygen affinity mutants that show intact Bohr effects, all three of the major characteristics of the reversible combination of hemoglobin with oxygen (oxygen equilibrium, heme-heme interaction, and pH dependence) are affected in Hb Hiroshima. A tentative interpretation of these effects, relating structure to function, is offered in terms of recently developed models of normal hemoglobin.

Protein constituents of plant ribosomes

The basic proteins obtained from plant ribosomes by dissociation in urea have been studied by disc-gel electrophoresis. Different plant species, unless closely related, gave recognisably different protein patterns. Cytoplasmic ribosomes from different organs of the pea plant gave similar patterns, but those from chloroplast ribosomes were quite different.

A study of the urea-produced subunits of β-galactosidase

The ability of urea reversibly to dissociate β-galactosidase into subunits is demonstrated. Urea dissociation is used to make hybrids between light radioactive and a large excess of heavy cold enzyme. By comparing the density of the hybrid to that of heavy and light molecules in a density gradient, it is shown that all urea subunits are one-quarter the size of the whole molecule.

On the Thermal Dissociation of Organic Compounds. V.5-8 The Effect of the Solvent (Fatty Acids) on the Thermal Dissociation of Urea

On the Thermal Dissociation of Organic Compounds. V.^5-8 The Effect of the Solvent (Fatty Acids) on the Thermal Dissociation of Urea