scholarly journals Physicochemical properties of Planorbis corneus erythrocruorin

1975 ◽  
Vol 149 (2) ◽  
pp. 437-445 ◽  
Author(s):  
E J Wood ◽  
L J Mosby
Keyword(s):  
Molecular Weight ◽  
High Speed ◽  
Sedimentation Coefficient ◽  
Sedimentation Equilibrium ◽  
Alkaline Conditions ◽  
Planorbis Corneus ◽  
A Subunit ◽  
Partial Dissociation ◽  
Equilibrium Ultracentrifugation ◽  
Intermediate Value

The erythrocruorin from the snail Planorbis corneus had a sedimentation coefficient, s020,w, of 33.5 ± 0.31S, and a molecular weight of 1.65 × 10(6) ± 0.04 × 10(6) by high-speed sedimentation-equilibrium ultracentrifugation. The amino acid composition and absorption spectrum of the protein are reported. A very low number of half-cystine residues was found, corresponding to 0.4 residue per haem group. The haem content was 2.76 ± 0.22%, corresponding to a protein molecular weight of about 22300. Under both acid and alkaline conditions partial dissociation took place to yield mixtures of products that could not be identified. A subunit corresponding to that containing one haem group was not obtained under any of the dossociating conditions tried. Electron microscopy revealed a ring-shaped molecule about 12.2 ± 0.5 nm in diameter. The native erythrocruoerin bound O2 co-operatively, the intermediate value of h in Hill plots having values between 1.7 and 3.4 depending on the conditions.

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.

scholarly journals Thiol reduction of human α2-macroglobulin. The subunit structure

1972 ◽  
Vol 127 (1) ◽  
pp. 187-197 ◽  
Author(s):  
J. M. Jones ◽  
J. M. Creeth ◽  
R. A. Kekwick
Keyword(s):  
Molecular Weight ◽  
Large Scale ◽  
Sedimentation Equilibrium ◽  
Subunit Structure ◽  
Experimental Conditions ◽  
A Value ◽  
Α2 Macroglobulin ◽  
A Subunit ◽  
Normal Human ◽  
Ph Range

1. Human α2-macroglobulin was prepared from a fraction obtained during the large-scale separation of normal human plasma proteins for clinical use. 2. Sedimentation-equilibrium measurements indicated a molecular weight of 725000. A value of 18.1S was obtained for s020,w. 3. The dissociation that occurs in the pH range 4.5–2.5 and in the region of neutrality in urea-containing solutions is consistent with a dimeric structure of the molecule. 4. The effects of the thiol reagents mercaptoethanol, mercaptoethylamine and N-acetylcysteine were investigated over a range of experimental conditions. Distinct components having sedimentation coefficients of 15, 12 and 8.5S were identified. 5. Conditions were found under which limited reduction with thiol liberated a subunit with a molecular weight approximately one-quarter of that of the intact molecule. This subunit retains the serological specificity of the whole molecule.

scholarly journals Molecular weights of the Thy-1 glycoproteins from rat thymus and brain in the presence and absence of deoxycholate

1978 ◽  
Vol 169 (2) ◽  
pp. 411-417 ◽  
Author(s):  
P W Kuchel ◽  
D G Campbell ◽  
A N Barclay ◽  
A F Williams
Keyword(s):  
Molecular Weight ◽  
Equilibrium Dialysis ◽  
Sedimentation Coefficient ◽  
Sedimentation Equilibrium ◽  
Membrane Glycoproteins ◽  
Guanidinium Chloride ◽  
Molecular Weights ◽  
Methionine Residue ◽  
Disulphide Bonds ◽  
Binding Data

1. The Thy-1 membrane glycoproteins from rat thymus and brain bound deoxycholate to 24% of their own weight as measured by equilibrium dialysis. The binding occurred co-operatively at the critical micelle concentration of deoxycholate, suggesting that the glycoproteins bind to a micelle, and not to the detergent monomer. 2. From sedimentation-equilibrium and deoxycholate-binding data the molecular weights of the glycoprotein monomers were calculated to be 18700 and 17500 for thymus and brain Thy-1 glycoprotein monomers were calculated to be 18700 and 17500 for thymus and brain Thy-1 glycoproteins respectively. The molecular weight of the polypeptide part of the glycoprotein is thus 12500. 3. In the absence of deoxycholate, brain or thymus Thy-1 glycoprotein formed large homogeneous complexes of mol. wt. 270000 or 300000 respectively. The sedimentation coefficient of these was 12.8 S. The complex was only partially dissociated by 4M-guanidinium chloride. 4. After cleavage of brain or thymus Thy-1 glycoprotein with CNBr, two peptides were clearly identified. They were linked by disulphide bonds and both contained carbohydrate. This cleavage suggests there is only one methionine residue per molecule, which is consistent with the above molecular weights and the known amino acid composition.

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.

scholarly journals Haemoglobin from the tadpole shrimp, Lepidurus apus lubbocki Characterization of the molecule and determination of the number of polypeptide chains

1979 ◽  
Vol 183 (2) ◽  
pp. 325-330 ◽  
Author(s):  
E Ilan ◽  
E Daniel
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Sedimentation Equilibrium ◽  
Guanidinium Chloride ◽  
Tadpole Shrimp ◽  
Polypeptide Chains

Haemoglobin from the tadpole shrimp, Lepidurus apus lubbocki, was found to have a sedimentation coefficient (s020,w) of 19.3 +/- 0.2 S and a molecular weight, as determined by sedimentation equilibrium, of 798000 +/- 20000. The amino acid composition showed the lack of cysteine and cystine residues. A haem content of 3.55 +/- 0.03% was determined, corresponding to a minimal mol.wt. of 17400 +/- 200. The pH-independence in the range pH 5-11 of the sedimentation coefficient indicates a relatively high stability of the native molecule. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis gave one band with mobility corresponding to a mol.wt. of 34000 +/- 1500. The molecular weight of the polypeptide chain was determined to be 32800 +/- 800 by sedimentation equilibrium in 6 M-guanidinium chloride and 0.1 M-2-mercaptoethanol. The findings indicate that Lepidurus haemoglobin is composed of 24 identical polypeptide chains, carrying two haem groups each.

scholarly journals Isolation of a sixth dynein subunit adenosine triphosphatase of Chlamydomonas axonemes.

1988 ◽  
Vol 106 (1) ◽  
pp. 133-140 ◽  
Author(s):  
G Piperno
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Adenosine Triphosphatase ◽  
Sucrose Gradient ◽  
Soluble Fraction ◽  
Sedimentation Coefficient ◽  
Apparent Molecular Weight ◽  
A Subunit ◽  
Dynein Arms ◽  
Different Levels

This study of the axoneme led to the identification of a previously unknown adenosine triphosphatase (ATPase), which is likely a major component of inner dynein arms. The ATPase was isolated from a soluble fraction of axonemes obtained from pf 28, a Chlamydomonas mutant lacking the outer dynein arms. The activity hydrolyzed up to 2.3 mumol of ATP.min-1.mg-1 of protein (at pH 7.2, in the presence of both Ca++ and Mg++), had a sedimentation coefficient of 11S in sucrose gradient, and cosedimented with four polypeptides of apparent molecular weight 325,000, 315,000 140,000, and 42,000. Several arguments indicate that the new ATPase is a component of the inner dynein arms. Three or four polypeptides cosedimenting with the activity belong to a group of axonemal components that are deficient in the axonemes of pf 23 and pf 30, two mutants that display different levels of inner dynein arm deficiency. The 42,000 component is axonemal actin, a subunit of two other inner dynein ATPases. The two polypeptides of molecular weight greater than 300,000 have electrophoretic mobility similar to that of high molecular weight components of outer and inner dynein arms. In spite of some similarities each ATPase isolated from inner or outer arms is composed of a different set of polypeptides. Different ATPases may be required for the modulation of localized sliding of adjacent outer double microtubules in the axoneme.

Physiochemical Properties of Thermomycolase, the Thermostable, Extracellular, Serine Protease of the Fungus Malbranchea pulchella

1974 ◽  
Vol 52 (11) ◽  
pp. 981-990 ◽  
Author(s):  
Gerrit Voordouw ◽  
G. Maurice Gaucher ◽  
Rodney S. Roche
Keyword(s):  
Molecular Weight ◽  
Calcium Ion ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Ion Concentration ◽  
Sedimentation Equilibrium ◽  
Globular Structure ◽  
Ion Concentrations ◽  
Malbranchea Pulchella ◽  
Calcium Ion Concentration

The physicochemical properties of the extracellular protease of the fungus Malbranchea pulchella, for which we have adopted the name thermomycolase, were investigated. The molecular weight of diisopropylphosphorylthermomycolase was found to be 32 000–33 000 by sedimentation equilibrium and sodium dodecyl sulfate (SDS) gel electrophoresis. Its sedimentation coefficient (s020, w = 2.97 S), intrinsic viscosity ([η] = 3.0 cc/g), and frictional ratio (f/f0 = 1.09) characterize the enzyme as a typical globular protein. The circular dichroism spectrum of the protein is also consistent with its globular structure. Active thermomycolase autolyzes extensively, especially at low calcium ion concentrations, producing low molecular weight peptide material. In the presence of SDS, a different autolytic degradation is observed, resulting in much higher molecular weight polypeptide products (16 500, 12 500,11 000, 8 500). The results indicate that, in the presence of SDS, thermomycolase has three sites that are highly susceptible to autolysis. At calcium ion concentrations of 10−3 M and 10−2 M the enzyme undergoes a sharp thermal denaturation with transition temperatures at 69 °C and 75 °C, respectively, and with complete loss of enzyme activity. At 70 °C the enzyme appeared to be maximally thermostable at a calcium ion concentration of 10−2 M.

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.

Rabbit Cardiac Myosin. I. Physical and Chemical Characterization of the Native Molecule

1975 ◽  
Vol 53 (2) ◽  
pp. 164-174 ◽  
Author(s):  
William T. Wolodko ◽  
Cyril M. Kay
Keyword(s):  
Molecular Weight ◽  
Atpase Activity ◽  
High Speed ◽  
Deae Cellulose ◽  
Sedimentation Equilibrium ◽  
Experimental Program ◽  
Activity Measurement ◽  
Cardiac Myosin ◽  
Skeletal Myosin ◽  
Physical And Chemical

Rabbit cardiac myosin, isolated from frozen tissue, was effectively purified by batchwise treatment with DEAE-cellulose in addition to using dilution–precipitation techniques. An extensive experimental program was subsequently carried out with respect to the enzymic, amino acid, optical, and physicochemical properties of native cardiac myosin. This program has included the following: examination of the effects of pH and varying concentrations of ATP, CaCl2, MgCl2, and PCMB on its ATPase activity; measurement of its circular dichroic spectrum in solvent buffers, at different pH or containing ATP in the absence or presence of Ca2+ or Mg2+ ions; study of the concentration dependence of its viscosity and sedimentation velocity at low temperatures; and investigation of its molecular weight by the Archibald method and low- and high-speed sedimentation equilibrium. The results of these studies were consistent with the interpretation that cardiac myosin is comprised of highly asymmetric, semi-rigid molecules with a molecular weight in the order of 4.7 × 105, which display non-ideality even in solvent buffers of high ionic strength at neutral pH. In addition, computer analysis of the high-speed sedimentation equilibrium data has provided evidence for the presence of a self-association reaction at low protein concentration. Even though the specific ATPase activity of cardiac myosin was found to be approximately one-third that reported for skeletal myosin in all cases, it was concluded, on the basis of the essentially analogous physical and chemical properties of rabbit cardiac and skeletal myosin, that the two proteins are very similar in terms of molecular size, shape, and secondary structure.

scholarly journals Characterization of the extracellular haemoglobin of Haemopsis sanguisuga (L.)

1976 ◽  
Vol 153 (3) ◽  
pp. 589-596 ◽  
Author(s):  
E J Wood ◽  
L J Mosby ◽  
M S Robinson
Keyword(s):  
Molecular Weight ◽  
Electron Microscope ◽  
Gel Electrophoresis ◽  
Root Nodules ◽  
Sedimentation Coefficient ◽  
Soya Bean ◽  
Sedimentation Equilibrium ◽  
Helical Content ◽  
Polypeptide Chains

The haemoglobin from the blood of the horseleech, Haemopsis sanguisuga (L.), had a sedimentation coefficient, SO20, w, of 59.11 +/- 0.55 S, and a molecular weight as determined by sedimentation equilibrium of 3.71 × 10(6)+/-9904 × 10(6). In the electron microscope the molecule appeared to be made up of two hexagonal plates, as is found with other worm haemoglobins, with dimensions 24.4+/-2.0 nm (across the hexagon) and 15.2+/-1.4 nm (height). The amino acid composition and spectrum were closely similar to those of the haemoglobins of other annelids (e.g. Lumbricus). The α-helical content, calculated from circular-dichroism measurements in the far-u.v. region, was 56-63%. The haem content was 2.49%, corresponding to a minimum molecular weight per haem group of 24 800, but detergent-gel electrophoresis indicated the presence of polypeptide chains of mol.wts. 12 600, 14 800, 15 500 and 25 100. The pH-induced dissociation of the native molecule yielded compotosol of Soya-bean root nodules.

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