scholarly journals Studies on the subunit structure of 4-deoxy-5-oxoglucarate hydro-lyase (decarboxylating) from Pseudomonas acidovorans

1975 ◽  
Vol 145 (2) ◽  
pp. 305-309 ◽  
Author(s):  
R Jeffcoat
Keyword(s):  
Molecular Weight ◽  
High Speed ◽  
Guanidine Hydrochloride ◽  
Sodium Dodecyl ◽  
Potassium Phosphate ◽  
Sedimentation Equilibrium ◽  
Subunit Structure ◽  
Before And After ◽  
Lysine Residues ◽  
Peptide Map

1. Homogeneous preparations of D-4-deoxy-5-oxoglutarate hydro lyase (decarboxylating)(EC4.2.1.41) were analysed in the ultracentrifuge by the high-speed sedimentation-equilibrium method of Yphantis (1964). The molecular weight in 0.1 M-potassium phosphate buffer, pH 7.2, in 6M-guanidine hydrochloride and in 0.1 M-beta-mercaptoethanol in 6M-guanidine hydrochloride was 113,000, 56,000 and 30,400 respectively. Polyacrylamidegel electrophoresis in the presence of sodium dodecyl sulphate indicated a minimum molecular weight of 30,500. 2. Measurement of the thiol content of the enzyme, before and after reduction with NaBH4 or dithiothreitol under denaturing conditions, indicated the presence of eight thiol residues and two interchain disulphide bridges/enzyme molecule. 3. Amino acid analysis showed that the intact enzyme contains a total of approximately 100 arginine and lysine residues, but digestion of the enzyme with trypsin yielded about 49 peptides staining with ninhydrin in a peptide “map”. 4. With the knowledge that the enzyme contains only two substrate-binding sites, it is suggested that the enzyme probably consists of four polypeptide chains arranged in an alpha2beta2 confirmation.

scholarly journals Physical properties and subunit structure of l-asparaginase isolated from Erwinia carotovora

1972 ◽  
Vol 126 (2) ◽  
pp. 361-379 ◽  
Author(s):  
K. A. Cammack ◽  
D. I. Marlborough ◽  
D. S. Miller
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Molecular Conformation ◽  
Sodium Dodecyl ◽  
Erwinia Carotovora ◽  
Optical Rotatory Dispersion ◽  
Spherical Particles ◽  
Sedimentation Equilibrium ◽  
Subunit Structure ◽  
Equilibrium Ultracentrifugation

1. l-Asparaginases from Erwinia carotovora and Escherichia coli (EC2 enzyme) are both capable of inhibiting and eliminating certain types of tumour cells. The Er. carotovora enzyme is a more basic protein, however, and in contrast with the EC2 enzyme it contains neither tryptophan nor cystine, and disulphide bonds are therefore absent. The molecule is very stable in solution from pH3.0 to about pH12.0, and is somewhat more stable at alkaline pH than is the Esch. coli enzyme. Calculations based on a s020,w 7.43S and a sedimentation-equilibrium molecular weight of 135000±10000 give a frictional ratio (f/f0) of 1.08. The molecular conformation is therefore very compact in solution, and the electron microscope shows the negatively stained molecules as almost spherical particles with a diameter of 7.2±0.7nm. 2. Sedimentation-velocity and equilibrium ultracentrifugation, in 5–8m solutions of urea and guanidinium chloride, and also electrophoresis in sodium dodecyl sulphate–polyacrylamide gel, reveal a dissociation of the native protein molecule into four subunits of similar molecular weight in the range 32500–38000. The enzymically inactive subunits can be physically reassembled into an active tetramer when urea is removed by dialysis. Although the subunit structures of the Er. carotovora enzyme and the Esch. coli enzyme molecules are similar, the secondary bonding forces holding the subunits together in the tetramer are somewhat stronger in the Er. carotovora enzyme. 3. The optical-rotatory-dispersion (o.r.d.) parameters that characterize the Cotton effects arising from ordered structure in the molecule are [m′]233=−3522±74° and [m′]200=9096±1700°. These show very marked changes as the secondary structure is disrupted and the molecule dissociates into subunits. A correlation pathway was traced on the basis of o.r.d. parameters and enzyme activity as the polypeptide chains were denatured and renatured (and reconstituted) into active molecules after the dilution of solutions in urea. Subunits resulting from treatment with sodium dodecyl sulphate do not show the typically disordered o.r.d. profile, but nevertheless they are inactive.

Subunit Structure and some Properties of Pyruvate Kinase of Neurospora

1975 ◽  
Vol 53 (2) ◽  
pp. 109-119 ◽  
Author(s):  
M. Kapoor
Keyword(s):  
Molecular Weight ◽  
Pyruvate Kinase ◽  
Guanidine Hydrochloride ◽  
Gel Filtration ◽  
Sedimentation Equilibrium ◽  
Phosphoryl Group ◽  
Subunit Structure ◽  
Equilibrium Studies ◽  
Variable Extent ◽  
High Concentrations

Pyruvate kinase isolated from Neurospora and purified to homogeneity has been shown to be a tetramer of molecular weight around 242 000 by gel filtration studies and 239 000 daltons by sedimentation equilibrium measurements. The monomer produced by treatment with guanidine hydrochloride is found to be 51 000–52 000 daltons by sedimentation equilibrium studies; a molecular weight of 62 000 was determined for the monomer generated by SDS treatment by electrophoresis in SDS–polyacrylamide gels. The enzyme has an isoelectric point of 6.35–6.41. Substrate saturation kinetics of PEP show a variable extent of cooperativity depending upon the buffer ions employed in the assay. ADP is the most effective phosphoryl group acceptor, GDP and IDP being poor substitutes. A divalent cation, Mg2+, is required for activity. At low concentrations, Ca2+ acts as an activator of pyruvate kinase but it is inhibitory at high concentrations. Fructose 1,6-diphosphate is the most potent allosteric activator, fructose 6-phosphate being next in order of effectiveness. Valine is a powerful inhibitor. Phenylalanine, tyrosine, and tryptophan are without any effect individually, but their simultaneous presence results in a considerable activation. Alanine does not affect this enzyme appreciably.

scholarly journals Erythrocruorin from the water-flea Daphnia magna. Quaternary structure and arrangement of subunits

1982 ◽  
Vol 207 (2) ◽  
pp. 297-303 ◽  
Author(s):  
E Ilan ◽  
E Weisselberg ◽  
E Daniel
Keyword(s):  
Molecular Weight ◽  
Daphnia Magna ◽  
Quaternary Structure ◽  
Slow Component ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Sedimentation Equilibrium ◽  
Subunit Structure ◽  
Single Chain

The subunit structure of erythrocruorin from the cladoceran Daphnia magna was studied. The native protein was found to have a sedimentation coefficient (S2(20), w) of 17.9 +/- 0.2 S and a molecular weight, as determined by sedimentation equilibrium, of 494 000 +/- 33 000. Iron and haem determinations gave 0.312 +/- 0.011% and 3.84 +/- 0.04%, corresponding to minimal molecular weights of 17900 +/- 600 and 16 100 +/- 200 respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis gave one band with mobility corresponding to a molecular weight of 31 000 +/- 1 500. The molecular weight of the polypeptide chain determined by sedimentation equilibrium in 6 M-guanidinium chloride and 0.1 M-2-mercaptoethanol is 31 100 +/- 1300. On a molecular-weight basis, Daphnia erythrocruorin is composed of 16 identical polypeptide chains carrying two haem groups each. The native structure is stable between pH5 and 8.5. At alkaline and acidic pH, a gradual decrease in the sedimentation coefficient down to 9.8S occurs. Above pH 10 and below pH4, a slow component with S20, w between 2.7S and 4.0S is observed. The 2.7S, 4.0S and 9.8S species are identified as single-chain subunits, subunit dimers and half-molecules respectively. We propose a model for the molecule composed of 16 2.7S subunits grouped in two layers stacked in an eclipsed orientation, the eight subunits of each layer occupying the vertices of a regular eight-sided polygon. Support for this arrangement is provided from electron microscopy and from analysis of the pH-dissociation pattern.

Partial amino acid sequence of the wheat germ Ec protein. Comparison with another protein very rich in half-cystine and glycine: wheat germ agglutinin

1984 ◽  
Vol 62 (9) ◽  
pp. 908-913 ◽  
Author(s):  
Theo Hofmann ◽  
David I. C. Kells ◽  
Byron G. Lane
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Wheat Germ Agglutinin ◽  
High Speed ◽  
Wheat Germ ◽  
Sodium Dodecyl ◽  
Sedimentation Equilibrium ◽  
Partial Sequence ◽  
Edman Degradation

A wheat germ protein (Ec), the dominant site of cysteine incorporation during early (E) germination of isolated wheat embryos, has been partially sequenced by automated Edman degradation. The sequence of residues 1–59 is not significantly similar to the amino acid sequence known for any other protein, including wheat germ agglutinin which, like Ec, is very rich in half-cystine and glycine. The partial sequence for Ec contains an almost identical pattern of half-cystine residues in segments 6–20 and 35–48, a duplication which includes 10 of the 12 half-cystine residues in the sequence. The partial sequence of Ec may include a large part of the complete sequence, but this remains uncertain because it has not been possible to arrive at a definitve estimate of molecular weight using different physical techniques. Protein Ec can be prepared from a reticulocyte lysate in which cell-free synthesis is programmed by bulk wheat germ mRNA. Determination of the distribution of half-cystine moieties between residues 1 and 20 by Edman degradation of the [35S]cysteine-labeled product of cell-free synthesis shows that it is devoid of an N-terminal extension. Unlike wheat germ agglutinin, Ec does not seem to arise by processing of a conspicuously larger precursor protein. Unlike Ec, another wheat germ protein, Em, the most conspicuous methionine-labeled protein when cell-free protein synthesis is directed by wheat germ mRNA, is refractory to direct sequence analysis by Edman degradation. However, again unlike Ec, uncertainty about the molecular weight of Em, based on its mobility in different sodium dodecyl sulphate – polyacrylamide gel systems, has been resolved by virtue of Em being ideally suited to study by the Yphantis high-speed sedimentation equilibrium method.

scholarly journals The molecular weight and properties of a neutral metallo-endopeptidase from rabbit kidney brush border

1974 ◽  
Vol 137 (3) ◽  
pp. 489-495 ◽  
Author(s):  
M. A. Kerr ◽  
A. J. Kenny
Keyword(s):  
Molecular Weight ◽  
Brush Border ◽  
Sodium Dodecyl Sulphate ◽  
Guanidine Hydrochloride ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Atomic Absorption Spectrophotometry ◽  
Sedimentation Equilibrium ◽  
Rabbit Kidney

1. Some properties of a brush-border neutral endopeptidase purified from rabbit kidney were investigated. The peptidase was assayed by its ability to hydrolyse [125I]iodoinsulin B chain. 2. The enzyme was found to be homogeneous when studied in the analytical ultracentrifuge and stained as a single glycoprotein band after electrophoresis in polyacrylamide gels. 3. The molecular weight was estimated by gel filtration in columns of Sephadex G-200, by polyacrylamide-gel electrophoresis in the presence of 2-mercapto-ethanol and sodium dodecyl sulphate and by sedimentation equilibrium in the ultra-centrifuge. The estimates fell within the range 87000–96000. The mean from two sedimentation equilibrium experiments was 93000, though this estimate may be slightly inflated because of the carbohydrate component of the enzyme. No evidence of dissociation into smaller subunits was obtained in the presence of thiol, sodium dodecyl sulphate or guanidine hydrochloride. 4. The endopeptidase was maximally active at pH6.0, although in phosphate buffer, which was strongly inhibitory, an optimum above pH8 was observed. 5. The enzyme was not affected by di-isopropyl phosphofluoridate nor by several thiol reagents. It was, however, strongly inhibited by many thiols and by EDTA and other chelating agents. 6. Although activity of the EDTA-treated enzyme could be partially restored by various bivalent metal ions, the optimum concentration for its reactivation by Zn2+ was lower than that for other ions. This metal was detected in the enzyme preparation by atomic absorption spectrophotometry in an amount equivalent to approximately one atom/mol. 7. The enzyme is the only endopeptidase shown to be located in the kidney brush border and is the first mammalian example of a neutral Zn2+- activated endopeptidase to be characterized.

scholarly journals Structural studies on individual components of bovine transferrin

1973 ◽  
Vol 135 (1) ◽  
pp. 87-92 ◽  
Author(s):  
N. E. Richardson ◽  
N. Buttress ◽  
A. Feinstein ◽  
A. Stratil ◽  
R. L. Spooner
Keyword(s):  
Molecular Weight ◽  
Sialic Acid ◽  
Guanidine Hydrochloride ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Equilibrium ◽  
Sialic Acid Content ◽  
Equilibrium Ultracentrifugation ◽  
Peptide Maps ◽  
Bovine Transferrin

The single-banding components of bovine transferrin from animals homozygous for the four transferrin variants found in the U.K. were isolated. Sedimentation equilibrium ultracentrifugation and sodium dodecyl sulphate–polyacrylamide-gel electrophoresis showed that the bands of a single variant have molecular weights of 77500 and 73300 respectively. The different bands of a single variant and single bands of different variants show no evidence of size heterogeneity or of low-molecular-weight peptides being split off after reduction in 6m-guanidine hydrochloride. The two slower bands of a single variant, which both contain 2 molecules of sialic acid/molecule of protein, have the same molecular weight and amino acid composition, and give identical peptide ‘maps’, although differences in composition and peptide ‘maps’ occur between the different variants. The results support the concept that bovine transferrin is essentially a single polypeptide chain, but they do not explain differences in electrophoretic mobility between bands of the same variant which are not produced by differing sialic acid content.

Soluble wool Proteins. I. Light scattering and viscosity in Aqueous solution

1958 ◽  
Vol 11 (4) ◽  
pp. 581 ◽  
Author(s):  
BS Harrap ◽  
EF Woods
Keyword(s):  
Molecular Weight ◽  
Light Scattering ◽  
Intrinsic Viscosity ◽  
High Speed ◽  
Guanidine Hydrochloride ◽  
Sodium Dodecyl ◽  
Relative Viscosity ◽  
Molecular Weights ◽  
Solvent Component ◽  
Wool Proteins

S-Carboxymethylkerateine 2 (SCMK2) and cc-keratose, two proteins derived from wool, have been characterized by nitrogen content, ultraviolet absorption, refractive index increment, light scattering, and intrinsic viscosity. Variations in the physical properties of different batches are attributed to different degrees of aggregation during the preparation. The relative viscosity decreased with time and was generally accompanied by an increase in turbidity, indicating aggregation. The effect of heating was to accelerate the fall in viscosity and increase in turbidity. Light scattering investigations showed that dissociation occurred on dilution and in some cases this could be detected by viscosity measurements. The molecular weight of several millions for SCMK2 at pH 6.7 was reduced to less than 1 million by removal of large aggregates by high-speed centrifuging, with an increase in both dissymmetry and intrinsic viscosity. In alkaline buffers at pH 10.5 the proteins were further dissociated and gave molecular weights of the order of 450,000. The behaviour of α-keratose was similar to that of SCMK2. Measurements on SCNK2 carried out in the presence of sodium dodecyl sulphate gave a molecular weight of 142,000 for the detergent-protein complex, corresponding to 95,000 for the protein, the dissymmetry was near unity and the intrinsic viscosity 0.115 dl/g. In 10M acetic acid, 8M urea, and 641 guanidine hydrochloride the apparent molecular weights were 95,000, 140,000, and 210,000 respectively, but these values are only upper limits because of possible selective solvation of the solvent component in such three-component systems.

The isolation of surface array proteins from bacteria

1984 ◽  
Vol 62 (11) ◽  
pp. 1181-1189 ◽  
Author(s):  
S. F. Koval ◽  
R. G. E. Murray
Keyword(s):  
Molecular Weight ◽  
Protein Conformation ◽  
Gel Electrophoresis ◽  
Guanidine Hydrochloride ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Structural Features ◽  
Proteolytic Degradation ◽  
Low Concentrations ◽  
Single Polypeptide

The methods used for the isolation of regularly structured (RS) surface array proteins of a range of prokaryotes are described. Most RS proteins can be selectively solubilized from envelope preparations with low concentrations of urea or guanidine hydrochloride. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis analysis of the protein extracts shows that most RS arrays are composed of a single polypeptide that may contain carbohydrate. The molecular weight of the proteins varies from 41 000 to 200 000. Possible reasons for the presence of more than one polypeptide in RS protein preparations are discussed, as well as the evidence for proteolytic degradation of some RS proteins during isolation. Structural features of the RS proteins are described and the importance of protein conformation to assembly of the arrays is indicated.

Physicochemical Studies of Conglutin γ, a Storage Globulin From Seeds of Lupinus angustifolius

1980 ◽  
Vol 7 (1) ◽  
pp. 1 ◽  
Author(s):  
RJ Blagrove ◽  
JM Gillespie ◽  
GG Lilley ◽  
EF Woods
Keyword(s):  
Molecular Weight ◽  
Guanidine Hydrochloride ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Optical Rotatory Dispersion ◽  
Sedimentation Equilibrium ◽  
Native Protein ◽  
Equilibrium Studies ◽  
Physicochemical Studies ◽  
Α Helix

Physicochemical studies are reported for conglutin �, the minor globulin isolated from seeds of L. angustifolius cv. Uniwhite. Isoelectric focusing of the native protein in polyacrylamide gel slabs resolved major and minor broad bands near pH 8.0 and 7.8 respectively. Following reduction of disulfide bonds with β-mercaptoethanol in 8 M urea, the smaller polypeptide chain of known sequence focused near pH 6.9 while the larger chain focused near pH 8.0. Sedimentation equilibrium studies showed that the major component in aqueous buffers at neutral pH is a hexamer of molecular weight 280 000 which dissociates to the monomer of molecular weight 47 000 at pH 4.8. The sequence molecular weight of the small subunit polypeptide is 16 517 [Elleman, T.C. (1977). Aust. J. Biol. Sci. 30, 33-45]. The molecular weights determined for the larger chain by sedimentation equilibrium or column chromatography in 6 M guanidine hydrochloride, and by dodecyl sulfate-polyacrylamide gel electrophoresis, were in the range 28 000-30 000. Optical rotatory dispersion and circular dichroism measurements have been used to establish the approximate proportions of α-helix (15%), β-structure (35%), β-turns (18%) and unordered regions (32%) in the native protein. The denaturation curve for guanidine hydrochloride and the proportions of α-helix (50%), β-turns (18%) and unordered regions (32%) in 80 % trifluoroethanol have been determined.

Purification and studies of some physicochemical properties of glutamine synthetase of Neurospora crassa

1978 ◽  
Vol 56 (10) ◽  
pp. 927-933 ◽  
Author(s):  
W. S. Lin ◽  
M. Kapoor
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Glutamine Synthetase ◽  
Neurospora Crassa ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Affinity Column ◽  
Subunit Structure ◽  
Α Helix

Glutamine synthetase (EC 6.3.1.2) of Neurospora crassa was purified to near homogeneity by chromatography on a glutamate–Sepharose affinity column. Its properties, including molecular weight, subunit structure, amino acid composition, and approximate α-helix content, have been examined. In the native state, this enzyme has been demonstrated by gel filtration to be an octamer of molecular weight 360 000 and as having a sedimentation coefficient of 13.2 S by sedimentation velocity measurements. Circular dichroism spectra in the far ultraviolet range suggest an approximate α-helix content of 23–24%. The subunit generated by treatment with urea was found to be 45 000 daltons by gel filtration methods and a molecular weight of 46 000 was calculated for the monomer obtained by sodium dodecyl sulphate (SDS) treatment and electrophoresis in SDS-polyacrylamide gels. Interprotomeric cross-linking experiments, using diimidoesters, suggest the presence of two noncovalently linked tetramers comprising the native octameric structure. Amino acid analyses revealed the presence of six tryptophans, four half cystines, and nine methionine residues per monomer of 45 000 daltons.

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