scholarly journals Effects of lipid removal on the molecular size and kinetic properties of bovine plasma arylesterase

1973 ◽  
Vol 135 (1) ◽  
pp. 93-99 ◽  
Author(s):  
Barry J. Kitchen ◽  
Colin J. Masters ◽  
Donald J. Winzor
Keyword(s):  
Molecular Weight ◽  
Guanidine Hydrochloride ◽  
Average Molecular Weight ◽  
Sedimentation Equilibrium ◽  
Partial Specific Volume ◽  
Detectable Effect ◽  
Kinetic Properties ◽  
Maximal Velocity ◽  
Yellow Colour ◽  
Bovine Plasma

A purified arylesterase preparation from bovine plasma was characterized to the extent that it has a partial specific volume of 0.91ml/g and an apparent z-average molecular weight of 440000. The relatively large magnitude of the former reflects the presence of phospholipids, cholesterol, triglycerides and β-carotene, the last-named being responsible for the pronounced yellow colour of the preparation. Removal of the lipid material is accompanied by a decrease in the apparent z-average molecular weight to 120000, the size of the smallest species detected by high-speed sedimentation equilibrium being in the vicinity of 70000 daltons: denaturation of the lipid-free preparation with 6m-guanidine hydrochloride caused essentially complete breakdown into subunits of this size. In kinetic studies on the enzyme the maximal velocity for the hydrolysis of phenyl acetate was found to increase by 60% on addition of 1 mm-Ca2+, with the Km showing a concomitant decrease from 6.6 to 2.1 mm. Removal of lipid had no detectable effect on Vmax. or Km in either the presence or the absence of Ca2+. It is concluded that the bovine plasma arylesterase preparation is either a lipoprotein or an enzyme–lipoprotein complex with properties very similar to those of the α1-lipoprotein or high-density lipoprotein (HDL2) fraction of serum.

scholarly journals THE MITOTIC APPARATUS

1967 ◽  
Vol 32 (2) ◽  
pp. 255-275 ◽  
Author(s):  
R. E. Stephens
Keyword(s):  
Molecular Weight ◽  
Disulfide Bonds ◽  
Tertiary Structure ◽  
Sea Urchins ◽  
Guanidine Hydrochloride ◽  
Average Molecular Weight ◽  
Strongylocentrotus Droebachiensis ◽  
Optical Rotatory Dispersion ◽  
Partial Specific Volume ◽  
Mitotic Apparatus

The major 22S protein of the hexylene glycol-isolated mitotic apparatus has been characterized from spindle isolates and extracts of whole eggs and acetone powders of eggs from the sea urchins Strongylocentrotus purpuratus, Strongylocentrotus droebachiensis, and Arbacia punctulata. The protein is free of nucleotide, lipid, and ATPase activity. Essentially identical in amino acid composition, proteins from these species show a relatively high content of glutamic and aspartic acids and are fairly rich in hydrophobic amino acids. Optical rotatory dispersion studies indicate a helical content of about 20%, a value consistent with the proline content of the protein. The purified proteins have sedimentation rates in the range of 22–24S, diffusion constants of 2.4–2.5F, intrinsic viscosities of 3.7–4.3 ml/g, a partial specific volume of 0.74, and an average molecular weight of 880,000. Electron microscopy indicates a globular molecule with dimensions of approximately 150 by 200 A; such size and symmetry are consistent with hydrodynamic measurements. The 22S protein yields 6–7S, 9–10S, and 13–14S subunits below pH 4 or above pH 11. The 13–14S component has an estimated molecular weight of 600,000–700,000. A 5–6S particle is formed in 8 M urea or 5 M guanidine hydrochloride, while at pH 12 the 6–7S subunit is seen; each particle has a molecular weight of 230,000–240,000. In 8 M urea plus 2% mercaptoethanol or at pH 13, the molecular weight becomes 105,000–120,000; under these conditions the particle sediments at 2.5–3S and 4S, respectively. On the basis of these molecular weights, the 6–7S, 9–10S, 13–14S, and the parent 22S particle should be dimer, tetramer, hexamer, and octamer, respectively, of the 105,000–120,000 molecular weight subunit. The various subunits will reform the 22S particle when returned to neutral buffer, with the exception of the mercaptoethanol-treated urea subunit where breakage of disulfide bonds results in a polydisperse aggregate. The 22S particle itself is not susceptible to sulfhydryl reagents, implying either that the disulfide bonds are inaccessible or that they are unnecessary for maintenance of tertiary structure once the 22S particle has formed from subunits.

scholarly journals Molecular weight of Escherichia coli β-galactosidase in concentrated solutions of guanidine hydrochloride

1970 ◽  
Vol 120 (2) ◽  
pp. 255-261 ◽  
Author(s):  
Robert P. Erickson
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Guanidine Hydrochloride ◽  
Average Molecular Weight ◽  
Sedimentation Velocity ◽  
Sedimentation Equilibrium ◽  
Weight Average Molecular Weight ◽  
Random Coils ◽  
Equilibrium Sedimentation ◽  
Apparent Weight

The molecular weight of Escherichia coli β-galactosidase was determined in 6m- and 8m-guanidine hydrochloride by meniscus-depletion sedimentation equilibrium, sedimentation velocity and viscosity. Sedimentation equilibrium revealed heterogeneity with the smallest component having a molecular weight of about 50000. At lower speeds, the apparent weight-average molecular weight is about 80000. By use of a calculation based on an empirical correlation for proteins that are random coils in 6m-guanidine hydrochloride, sedimentation velocity gave a molecular weight of 91000, and the intrinsic viscosity indicated a viscosity-average molecular weight of 84000. Heating in 6m-guanidine hydrochloride lowered the viscosity of β-galactosidase in a variable manner.

scholarly journals STUDIES ON THE POSTERIOR SILK GLAND OF THE SILKWORM BOMBIX MORI

1970 ◽  
Vol 46 (1) ◽  
pp. 1-16 ◽  
Author(s):  
Yutaka Tashiro ◽  
Eiichi Otsuki
Keyword(s):  
Guanidine Hydrochloride ◽  
Lithium Bromide ◽  
Posterior Part ◽  
Silk Gland ◽  
Sedimentation Equilibrium ◽  
Partial Specific Volume ◽  
Anterior Portion ◽  
Equilibrium Analyses ◽  
Fibroin Solution ◽  
Posterior Silk Gland

Ultracentrifugal analyses of the native silk proteins extracted from the various parts of the middle silk gland of the mature silkworm have revealed that there exist four components with S°20,w values of 10S, 9–10S, 9S, and 4S in the extract. It is suggested that the fastest 10S component is the native fibroin synthesized in the posterior silk gland and transferred to the middle silk gland to be stored there, while the slower three components probably correspond to inner, middle, and outer sericins which were synthesized in the posterior, middle, and anterior portion of the middle silk gland, respectively. Native fibroin solution was prepared from the most posterior part of the middle silk gland. Ultracentrifugal analyses have shown that the solution contains considerable amounts of aggregates in addition to the main 10S component. Treatment with lithium bromide (LiBr), urea, or guanidine hydrochloride solution up to 6 M all have failed to dissociate the 10S component. From the sedimentation equilibrium analyses and partial specific volume of 0.716, the molecular weight of the 10S component of the native fibroin solution was found to be between 3.2 – 4.2 x 105, with a tendency to lie fairly close to 3.7 x 105.

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.

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 THE NUMBER OF POLYPEPTIDE CHAINS IN BOVINE PLASMA ALBUMIN

1956 ◽  
Vol 34 (2) ◽  
pp. 160-169 ◽  
Author(s):  
M. E. Reichmann ◽  
J. Ross Colvin
Keyword(s):  
Molecular Weight ◽  
Light Scattering ◽  
Sodium Chloride ◽  
Sedimentation Velocity ◽  
Performic Acid ◽  
Sedimentation Equilibrium ◽  
Plasma Albumin ◽  
Disulphide Bonds ◽  
Bovine Plasma ◽  
Polypeptide Chains

The molecular weight of performic acid oxidized bovine plasma albumin, dispersed in 0.08 M borate +0.2 M sodium chloride buffer, pH 7.4, was estimated as 30,000 by light-scattering and sedimentation equilibrium methods, 19,000 by osmotic pressure. Sedimentation velocity analyses and electrophoresis showed that the component polypeptide chains of the material are similar in mass and charge density so the polydispersity must be attributed to labile aggregates. The results indicate that here are at least three and probably four similar polypeptide chains in the molecule of native bovine plasma albumin, held together by disulphide bonds.

scholarly journals Osmotic Pressure Measurements on Insulin: Anomalous Results Indicate that the Monomer is Preferentially Adsorbed

1986 ◽  
Vol 39 (4) ◽  
pp. 319 ◽  
Author(s):  
Peter D Jeffrey
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
Osmotic Pressure ◽  
Hydrophobic Interactions ◽  
Average Molecular Weight ◽  
Adsorption Properties ◽  
Sedimentation Equilibrium ◽  
Pressure Measurements ◽  
Number Average Molecular Weight ◽  
Adsorption Analysis

The concentration dependence of the number average molecular weight of insulin at pH 2, ionic strength 0'05, and 20�C as determined by osmotic pressure measurements indicates that the .hormone is a homogeneous protein of molecular weight close to that of the dimer. Since sedimentation equilibrium experiments confirm what is well known, namely that insulin is a self-associating protein dissociating to monomer under these conditions, an explanation for the anomaly was sought in the possible loss of protein from solution by adsorption. Analysis of the results strongly supports this conclusion and consideration of the adsorption properties of insulin in terms of hydrophobic interactions shows them to be consistent with the behaviour of insulin as a self-associating protein. The monomer appears to be the primary molecular species responsible for insulin adsorption.

scholarly journals Isolation and characterization of dermatan sulphate proteoglycan from human uterine cervix

1983 ◽  
Vol 209 (2) ◽  
pp. 497-503 ◽  
Author(s):  
N Uldbjerg ◽  
A Malmström ◽  
G Ekman ◽  
J Sheehan ◽  
U Ulmsten ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Uterine Cervix ◽  
Proteinase Inhibitors ◽  
Gradient Centrifugation ◽  
Average Molecular Weight ◽  
Deae Cellulose ◽  
Sedimentation Equilibrium ◽  
Dermatan Sulphate ◽  
Guanidinium Chloride ◽  
Isolation And Characterization

Proteoglycans were extracted from human uterine cervix with 4 M-guanidinium chloride in the presence of proteinase inhibitors. They were purified by density-gradient centrifugation in 4 M-guanidinium chloride/CsCl (starting density 1.32 g/ml) followed by DEAE-cellulose and Sepharose chromatography. Only one polydisperse proteoglycan was found. s020,w was 2.1S and the weight-average molecular weight was 73 000 (sedimentation-equilibrium centrifugation) to 110 500 (light-scattering). The core protein was monodisperse, with an apparent molecular weight of 47 000. The proteoglycan contained about 30% protein and probably two or three glycosaminoglycan side chains per molecule. High contents of aspartate, glutamate and leucine were found. The glycan moiety of the proteoglycan was exclusively dermatan sulphate, with a co-polymeric structure with approximately equal quantities of iduronic acid- and glucuronic acid-containing disaccharides.

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.

Sign in / Sign up

Export Citation Format

Share Document