scholarly journals The molecular composition of the volutin granule of yeast

1982 ◽  
Vol 201 (3) ◽  
pp. 473-479 ◽  
Author(s):  
L Jacobson ◽  
M Halmann ◽  
J Yariv
Keyword(s):  
Molecular Weight ◽  
Organic Solvent ◽  
Density Gradient ◽  
Gradient Centrifugation ◽  
Linear Chain ◽  
Total Cell ◽  
Molecular Composition ◽  
Analytical Ultracentrifuge ◽  
Molecular Weights ◽  
Freeze Dried

The volutin granule was isolated from yeast by disruption of freeze-dried cells in an organic solvent and density-gradient-gradient centrifugation. The granule is composed of two types of macromolecule, a linear-chain polyphosphate and four basic proteins, of molecular weights ranging from 10 000 to 20 000. In the dissolved granule these macromolecules are in a complex that is uniform by hydrodynamic criteria (s20,w = 22.3 S). The polyphosphate separated from this complex gives a single 31P n.m.r. resonance and in the analytical ultracentrifuge behaves as a monodisperse solute of molecular weight 245 000 +/- 1000. In the 31P n.m.r. spectrum of yeast used for its isolation, this polyphosphate accounts for 14% of total cell polyphosphate.

Characterization of proteins associated with nuclear ribonucleoprotein particles by two-dimensional polyacrylamide gel electrophoresis

1979 ◽  
Vol 57 (1) ◽  
pp. 32-42 ◽  
Author(s):  
D. Suria ◽  
C. C. Liew
Keyword(s):  
Molecular Weight ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Two Dimensional ◽  
Molecular Weights ◽  
Electrophoretic Patterns ◽  
Phenol Extraction ◽  
Chromatin Proteins ◽  
Nuclear Ribonucleoprotein

Rat liver nuclear ribonucleoprotein particles were prepared by two different methods and defined as 40S ribonucleoprotein (40S RNP) and heterogeneous nuclear ribonucleoprotein (HnRNP) particles. The RNP particles were either solubilized in 8 M urea – 6 mM 2-mercaptoethanol – 20 mM glycine – 20 mM Tris–HCl (pH 8.4) or subjected to removal of RNA by phenol extraction prior to solubilizing the proteins in the urea buffer. The proteins associated with 40S RNP and HnRNP were heterogeneous and very similar in their electrophoretic patterns when analyzed by two-dimensional PAGE, except a protein with molecular weight of 62 000 and an isoelectric point (pI) of 6.2 was present only in HnRNP particles. At least 12 major and 22 minor components could be identified in both preparations. The major proteins were found at pI values varying from 6.0 to 8.5 and with molecular weights from 32 000 to 42 000, and a group of proteins with molecular weight approximately 65 000 were more prominent in HnRNP than in 40S RNP. The other components were found mainly at pI ranges from 5.0 to 6.5 with molecular weights from 43 000 to 65 000. The phenol method extracted essentially all proteins associated with either 40S RNP and HnRNP, but was less effective in extracting a group of proteins with pI values from 5.0 to 5.5 and more efficient for proteins with pI values from 7.5 to 8.5. When chromatin proteins isolated by phenol extraction were compared with HnRNP particle proteins isolated by the same method, the electrophoretic mobilities of the HnRNP particle proteins were found to be identical with a fraction of nonhistone chromatin proteins. The 40S RNP particles were further purified by metrizamide isopycnic density gradient centrifugation. The electrophoretic patterns of these proteins were very similar to those prepared by sucrose density gradient centrifugation. Therefore, we concluded that the proteins of RNP particles constituted part of the chromatin proteins.

Molecular Weight Determinations Of Low Molecular Weight (LMW) Heparins By Ultracentrifugation And High Pressure Liquid Chromatography

1981 ◽  
Author(s):  
Grant Barlow ◽  
N Sugisaka ◽  
F J Petracek
Keyword(s):  
Molecular Weight ◽  
High Pressure ◽  
Liquid Chromatography ◽  
High Pressure Liquid Chromatography ◽  
Low Molecular Weight ◽  
Analytical Ultracentrifuge ◽  
Molecular Weights ◽  
Molecular Weight Distributions ◽  
Weight Distributions ◽  
Heparin Fractions

Molecular weights were independently determined on nitrous acid depolymerized LMW heparin fractions ranging from 2-15 daltons using the analytical ultracentrifuge and high pressure liquid chromatography (HPLC).Sedimentation-diffusion equilibria were obtained in the analytical ultracentrifuge using speeds ranging from 20,000 to 56,000 rpm. Near theta conditions were obtained using 0.5M NaCl as the solvent. Calculations of molecular weight distributions and, from those figures, weight average molecular weights were made using the method described by Scholte (N.Y. Acad Sci. 164, 156, 1969). The results show that weight average values as low as 2,000 daltons can be determined.Sedimentation-diffusion equilibria were obtained in the analytical ultracentrifuge using speeds ranging from 20,000 to 56,000 rpm. Near theta conditions were obtained using 0.5M NaCl as the solvent. Calculations of molecular weight distributions and, from those figures, weight average molecular weights were made using the method described by Scholte (N.Y. Acad Sci. 164, 156, 1969). The results show that weight average values as low as 2,000 daltons can be determined.The HPLC results were obtained using previously described methods (Fed Proc. 36, 89, 1977) and a new highly efficient gel column (TSK gels). Fractionated dextrans were used as reference standards.

scholarly journals Molecular weight and subunit size of rabbit mammary-gland fatty acid synthetase

1978 ◽  
Vol 173 (3) ◽  
pp. 929-933 ◽  
Author(s):  
I Grunnet ◽  
J Knudsen
Keyword(s):  
Molecular Weight ◽  
Mammary Gland ◽  
Fatty Acid ◽  
Sucrose Gradient ◽  
Gradient Centrifugation ◽  
Fatty Acid Synthetase ◽  
Mammary Glands ◽  
Sucrose Gradient Centrifugation ◽  
Molecular Weights ◽  
Subunit Size

1. The molecular weights of fatty acid synthetases isolated from lactating rabbit, rat, cow and goat mammary glands were estimated by sucrose gradient centrifugation and compared by chromatography on Sepharose 6B. 2. The values obtained for all four enzymes were in the same range (0.40 × 10(6)-0.55 × 10(6)) as that found for other mammalian and avian fatty acid synthetases. The molecular weight found for the rabbit mammary enzyme therefore differs from published values of approx. 0.9 × 10(6). 3. The molecular weights of the subunits of these four synthetases were 225000-242000. Again, the value for the rabbit mammary enzyme differs from published values.

Determination of Copolymer Molecular Weight

1968 ◽  
Vol 41 (1) ◽  
pp. 245-253 ◽  
Author(s):  
Paul Rempp ◽  
Henri Benoit
Keyword(s):  
Molecular Weight ◽  
Light Scattering ◽  
Density Gradient ◽  
Compositional Heterogeneity ◽  
Precise Information ◽  
Molecular Weights ◽  
Molecular Weight Distributions ◽  
Weight Distributions ◽  
Binary Copolymers

Abstract From this brief review it appears that determinations of molecular weight averages, of molecular weight distributions, and of compositional inhomogeneity of binary copolymers, require care in the choice of techniques and methods. Some of the most commonly used techniques for molecular weight determinations on homopolymers of various kinds are inadequate for the same determinations on copolymers. Others are more sensitive to fluctuations in composition than in molecular weights. Osmotic methods are the only one which are really insensitive to inhomogeneity, and which yield molecular weights. Ultracentrifugation in a density gradient yields precise information only on fluctuations in composition. Viscosity determinations require calibration, but even so, they may lead to erroneous values of the molecular weight in the case of copolymers. GPC is less sensitive to compositional heterogeneity, but cannot be applied for nonlinear copolymers. Finally, light scattering is a very powerful tool for studies on copolymers, since it leads to molecular weight averages and its helps characterize polydispersity and fluctuations in composition.

Aggregation States of a Regulatory Enzyme Complex Catalyzing the Early Steps of Pyrimidine Biosynthesis in Bakers' Yeast

1971 ◽  
Vol 49 (4) ◽  
pp. 403-411 ◽  
Author(s):  
P. F. Lue ◽  
J. G. Kaplan
Keyword(s):  
Molecular Weight ◽  
Density Gradient ◽  
Crude Extract ◽  
Analytical Ultracentrifugation ◽  
Specific Activity ◽  
Feedback Inhibition ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Regulatory Site ◽  
Aspartate Transcarbamylase

Aspartate transcarbamylase (ATCase) of bakers' yeast has been purified 78-fold from a crude extract of a derepressed diploid strain; its specific activity was more than 300-fold that of a wild-type crude extract. During the last steps of the purification there was a parallel co-purification of carbamylphosphate synthetase (CPSase), and both activities retained full sensitivity to feedback inhibition by UTP; indeed the sensitivity of the ATCase to UTP increased during the purification doubtless due to discard of a feedback-insensitive ATCase subunit. The two enzyme activities co-eluted from gel filtration on Sepharose 6B together with the feedback site. Analytical ultracentrifugation revealed that the material was not homogeneous, showing two major peaks. Sucrose density gradient centrifugation in the presence of UTP, glutamine, and Mg2+ resulted in co-sedimentation of the two activities and the regulatory site, corresponding to a molecular weight of approximately 800 000 daltons. Omission of UTP from the gradient resulted in disappearance of the heavy peak and appearance of a new one, corresponding to a molecular weight of 380 000 and possessing both activities; the CPSase was still highly sensitive to UTP unlike the ATCase which was only slightly sensitive to retroinhibition. Omission of glutamine and Mg2+ from the sucrose density gradient caused a distinct CPSase peak to trail behind the ATCase; again, the CPSase (molecular weight 250 000) retained full sensitivity to feedback inhibition. This, together with genetic data, supports the view that the ura-2 gene which controls ATCase, CPSase, and the regulatory site is a polycistronic operon, coding for the production of two or three polypeptide chains; the CPSase subunit is inactive unless a regulatory site is present, whereas the ATCase subunit (molecular weight 140 000) is highly active but completely insensitive to feedback inhibition.

scholarly journals Nucleic acid enzymology of extremely halophilic bacteria. Gel-filtration and density-gradient-centrifugation studies of the molecular weights of Halobacterium cutirubrum polynucleotide phosphorylase and deoxyribonucleic acid- and ribonucleic acid-dependent ribonucleic acid polymerases

1971 ◽  
Vol 121 (4) ◽  
pp. 635-641 ◽  
Author(s):  
B. Gregory Louis ◽  
Pearl I. Peterkin ◽  
P. S. Fitt
Keyword(s):  
Rna Polymerase ◽  
Density Gradient ◽  
Ribonucleic Acid ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Halophilic Bacteria ◽  
Gel Filtration ◽  
Polynucleotide Phosphorylase ◽  
Sucrose Density Gradient Centrifugation ◽  
Molecular Weights

1. Conditions have been established for the estimation of molecular weights of proteins by analytical gel filtration and sucrose-density-gradient centrifugation in 2.5m-potassium chloride–1m-sodium chloride; Halobacterium cutirubrum polynucleotide phosphorylase, DNA-dependent RNA polymerase and RNA-dependent RNA polymerase have been studied by these methods. 2. The RNA-dependent polymerase has also been studied by density-gradient centrifugation in the absence of salt. 3. All three proteins are of unusually low molecular weight compared with similar enzymes from non-halophilic bacteria.

scholarly journals The structure and subunit composition of the particulate NADH-ubiquinone reductase of bovine heart mitochondria

1976 ◽  
Vol 154 (2) ◽  
pp. 295-305 ◽  
Author(s):  
C. I Ragan
Keyword(s):  
Molecular Weight ◽  
Complex I ◽  
Gradient Centrifugation ◽  
Sodium Dodecyl ◽  
Water Soluble ◽  
Heart Mitochondria ◽  
Molar Ratio ◽  
Polypeptide Composition ◽  
Molecular Weights ◽  
Bovine Heart

Preparations of NADH-ubiquinone reductase from bovine heart mitochondria (Complex I) were shown to contain at least 16 polypeptides by gel electrophoresis in the presence of sodium dodecyl sulphate. 2. High-molecular-weight soluble NADH dehydrogenase prepared from Triton X-100 extracts of submitochondrial particles [Baugh & King (1972) Biochem. Biophys. Res. Commun. 49, 1165-1173] was similar to Complex I in its polypeptide composition. 3. Solubilization of Complex I by phospholipase A treatment and subsequent sucrose-density-gradient centrifugation did not alter the polypeptide composition. 4. Lysophosphatidylcholine treatment of Complex I caused some selective solubilization of a polypeptide of mol.wt. 33000 previosuly postulated to be the transmembrane component of Complex I in the mitochondrial membrane [Ragan (1975) in Energy Transducing Membranes: Structure, Function and Reconstitution (Bennun, Bacila & Najjar, eds.), Junk, The Hague, in the press]. 5. Chaotropic resolution of Complex I caused solubilization of polypeptides of molecular weights 75000, 53000, 29000, 26000 and 15500 and traces of others in the 10000-20000-mol.wt.range. 6. The major components of the iron-protein fraction from chaotropic resolution had molecular weights of 75000, 53000 and 29000, whereas the flavoprotein contained polypeptides of molecular weights 53000 and 26000 in a 1:1 molar ratio. 7. Iodination of Complex I by lactoperoxidase indicated that the water-soluble polypeptides released by chaotropic resolution, in particular those of the flavoprotein fraction, were largely buried in the intact Complex. 8. The polypeptides of molecular weights 75000, 53000, 42000, 39000, 33000, 29000 and 26000 were present in 1:2:1:1:1:1:1 molar proportions. The two subunits of molecular weight 53000 are probably non-identical.

scholarly journals An Efficient Procedure for Purification of an Isolate of Peanut Mottle Virus from Wild Peanut and Determination of Molecular Weights of the Viral Components1

1984 ◽  
Vol 11 (1) ◽  
pp. 40-42 ◽  
Author(s):  
John L. Sherwood
Keyword(s):  
Molecular Weight ◽  
Phosphate Buffer ◽  
Gradient Centrifugation ◽  
Potassium Phosphate ◽  
Sedimentation Coefficient ◽  
Infected Tissue ◽  
Mottle Virus ◽  
Efficient Procedure ◽  
Molecular Weights ◽  
Wild Peanut

Abstract An efficient procedure was developed for purification of peanut mottle virus (PMV) from pea (Pisum sativum cv. Little Marvel) that yielded 10–19 mg virus/ kg infected tissue. Virus was extracted from frozen infected tissue in 0.01 M potassium phosphate buffer, pH 8.0, with 0.001 M dithioerythritol, followed by clarification with chloroform (15%, v/v) and precipitation by KCl and polyethylene glycol. Virus was resuspended in 0.01 M borate-phosphate buffer, pH 8.3, with 0.2 M urea prior to density gradient centrifugation. Purified virus sedimented as a single component with a sedimentation coefficient of 149 S. The molecular weight of the single coat protein was estimated as 36,100 daltons in 12% polyacrylamide gels. The single nucleic acid isolated from PMV on sucrose gradients was degraded by RNase, but not DNase. The molecular weight of the RNA was estimated as 3.1 × 106 daltons on nondenaturing and denaturing sucrose gradients.

scholarly journals Isolation and characterization of human cervical-mucus glycoproteins

1983 ◽  
Vol 211 (1) ◽  
pp. 13-22 ◽  
Author(s):  
I Carlstedt ◽  
H Lindgren ◽  
J K Sheehan ◽  
U Ulmsten ◽  
L Wingerup
Keyword(s):  
Density Gradient ◽  
Proteinase Inhibitors ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Disc Electrophoresis ◽  
Guanidinium Chloride ◽  
Molecular Weights ◽  
Isolation And Characterization ◽  
Mucus Glycoproteins

Mucus glycoproteins (mucins) were extracted from human cervical pregnancy mucus by 6 M-guanidinium chloride in the presence of proteinase inhibitors. Purification was subsequently achieved by isopycnic density-gradient centrifugation in CsCl/ guanidinium chloride gradients. The purified macromolecules represented approx. 85% of the total and were devoid of nucleic acids and proteins, as judged by analytical density-gradient centrifugation, disc electrophoresis and u.v. spectroscopy. Sedimentation-velocity centrifugation revealed a single unimodal peak with S20,W 50.1S in 0.2M-NaCl and 37.0S in 6 M-guanidinium chloride. Molecular weights obtained by light-scattering were 9.7 × 10(6) and 5.9 × 10(6) in 0.2M-NaCl and 6 M-guanidinium chloride respectively. The chemical analyses were typical of those of epithelial mucins. The macromolecules contained approx. 20% (w/w) of protein, and 65% (w/w) was accounted for as carbohydrate. Serine and threonine constituted 32 mol/100 mol and proline 10 mol/100 mol of the amino acids. The major sugars found were N-acetylglucosamine (12.8%), N-acetylgalactosamine (9.7%), galactose (18.7%), sialic acid (15.0%) and fucose (7.5%).

Sign in / Sign up

Export Citation Format

Share Document