scholarly journals Purification and subunit analysis of wheat-germ ribonucleic acid polymerase II

1974 ◽  
Vol 139 (3) ◽  
pp. 771-777 ◽  
Author(s):  
Jerome J. Jendrisak ◽  
Wayne M. Becker
Keyword(s):  
Enzyme Activity ◽  
Rna Polymerase ◽  
Wheat Germ ◽  
Sucrose Gradient ◽  
Gradient Centrifugation ◽  
Deae Cellulose ◽  
Protamine Sulphate ◽  
Molecular Weights ◽  
Molar Ratios ◽  
High Salt Buffer

A procedure is described for the purification of the α-amanitin-sensitive DNA-dependent RNA polymerase [EC 2.7.7.6] from wheat germ. Solubilization of the enzyme activity was achieved by sonication of a crude extract in a high-salt buffer. Purification involved precipitation with protamine sulphate and (NH4)2SO4, chromatography on DEAE-cellulose and phosphocellulose, and sucrose gradient centrifugation. Under denaturing conditions the enzyme dissociated into five polypeptides with molecular weights and molar ratios of 220000 (0.9), 170000 (0.1), 140000 (1.0), 45000 (0.2), and 40000 (0.4). Approx. 1mg of purified RNA polymerase was obtained as a routine from 100g of starting material.

Development of a monoclonal antibody to the rabbit 8.5S uterine progestin receptor

1985 ◽  
Vol 63 (1) ◽  
pp. 33-40 ◽  
Author(s):  
Kiyohide Nakao ◽  
John E. Myers ◽  
Lee E. Faber
Keyword(s):  
Sucrose Gradient ◽  
Solid Phase ◽  
Gradient Centrifugation ◽  
Protein A ◽  
Deae Cellulose ◽  
Enzyme Linked Immunosorbent Assay ◽  
Progestin Receptors ◽  
Sucrose Gradient Centrifugation ◽  
Progestin Receptor ◽  
Solid Phase Radioimmunoassay

Nonactivated (8.5S) rabbit uterine progestin receptor was enriched 10- to 30-fold by chromatography on columns of spheroidal hydroxylapatite and DEAE-cellulose. A total of`~25 μg of receptor (purity ~1%) was injected at multiple sites into a BALB/c mouse. After several injections, splenic lymphocytes were fused with P3x63Ag8.653 mouse myeloma cells. This fusion produced in excess of 240 hybridomas, which were screened by an enzyme-linked immunosorbent assay (ELISA), solid-phase radioimmunoassay, and sucrose gradient centrifugation. One colony (KN 382/EC1) produced a mouse immunoglobulin G1 which bound rabbit 8.5S uterine progestin receptor. The cell line has been repeatedly cloned under conditions of limiting dilution and expanded in mice as ascitic tumors. Antibody was purified by (NH4)2SO4 precipitation, DEAE-cellulose chromatography, and affinity chromatography with protein A – Sepharose CL-4B. Specificity of the antibody was determined by sucrose gradient centrifugation and solid-phase radioimmunoassay. The antibody bound to progestin receptors from rabbit uterus and MCF-7 breast cancer cells. It did not react with progestin receptors from rat uterus, guinea pig uterus, or chick oviduct, nor did it bind to estrogen receptors from any of the tissues we tested.

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.

scholarly journals Structure and biochemical composition of desmosomes and tonofilaments isolated from calf muzzle epidermis.

1978 ◽  
Vol 79 (2) ◽  
pp. 427-443 ◽  
Author(s):  
P Drochmans ◽  
C Freudenstein ◽  
J C Wanson ◽  
L Laurent ◽  
T W Keenan ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Lipid Composition ◽  
Gradient Centrifugation ◽  
Fatty Acid Pattern ◽  
High Ratio ◽  
Molecular Weights ◽  
Molar Ratios ◽  
Stratum Spinosum ◽  
High Concentrations ◽  
Low Ionic Strength

Complexes of plasma membrane segments with desmosomes and attached tonofilaments were separated from the stratum spinosum cells of calf muzzle by means of moderately alkaline buffers of low ionic strength and mechanical homogenization. These structures were further fractionated by the use of various treatments including sonication, sucrose gradient centrifugation, and extraction with buffers containing high concentrations of salt, urea, citric acid, or detergents. Subfractions enriched in desmosome-tonofilament-complexes and tonofilament fragments were studied in detail. The desmosome structures such as the midline, the trilaminar membrane profile, and the desmosomal plaque appeared well preserved and were notably resistant to the various treatments employed. Fractions containing desmosome-tonofilament complexes were invariably dominated by the nonmembranous proteins of the tonofilaments which appeared as five major polypeptide bands (apparent molecular weights: 48,000; 51,000; 58,000; 60,000; 68,000) present in molar ratios of approx. 2:1:1:2:2. Four of these polypeptide bands showed electrophoretic mobilities similar to those of prekeratin polypeptides from bovine hoof. However, the largest polypeptide (68,000 mol wt) migrated significantly less in polyacrylamide gels than the largest component of the hoof prekeratin (approximately 63,000 mol wt). In addition, a series of minor bands, including carbohydrate-containing proteins, were identified and concluded to represent constituents of the desmosomal membrane. The analysis of protein-bound carbohydrates (total 270 microgram/mg phospholipid in desmosome-enriched subfractions) showed the presence of relatively high amounts of glucosamine, mannose, galactose, and sialic acids. These data as well as the lipid composition (e.g., high ratio of cholesterol to phospholipids, relatively high contents of sphingomyelin and gangliosides, and fatty acid pattern) indicate that the desmosomal membrane is complex in protein and lipid composition and has a typical plasma membrane character. The similarity of the desmosome-associated tonofilaments to prekeratin filaments and other forms of intermediate-sized filaments is discussed.

scholarly journals The nucleoside triphosphate–ribonucleic acid nucleotidyltransferase (EC 2.7.7.6) of Agrobacterium tumefaciens (Smith and Townsend) Conn. Purification and properties of the enzyme from the tumorigenic strain B6806

1974 ◽  
Vol 143 (3) ◽  
pp. 511-520 ◽  
Author(s):  
U. C. Knopf
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Gradient Centrifugation ◽  
Deae Cellulose ◽  
Nucleoside Triphosphate ◽  
Subunit Structure ◽  
Bacterial Cells ◽  
Protein Subunit ◽  
Ph Optimum ◽  
Protamine Sulphate

The RNA nucleotidyltransferase (RNA polymerase) of the plant-tumorigenic bacterium Agrobacterium tumefaciens was purified. The method involves the disruption of the bacterial cells with glass beads in a Waring Blendor, treatment with DEAE-cellulose, fractionation with (NH4)2SO4, protamine sulphate precipitation, DEAE-cellulose column chromatography and either glycerol-gradient centrifugation or phosphocellulose chromatography. The subunit structure of the highly purified enzyme is similar to, although not identical with, the RNA nucleotidyltransferase of Escherichia coli. It can be described as β′, β, χ1 and α (mol.wts. 160000, 150000, 98000, and 41000±10% respectively). χ1 is the temporary designation for a protein subunit, which might have the same functions as the σ subunit in E. coli. The enzyme of A. tumefaciens is rifampicin-sensitive, has a temperature optimum in vitro of 41±1°C and a pH optimum of 8.2±0.1. Mg2+ and Mn2+ are activators. The enzyme transcribes with different efficiencies artificial, viral, bacterial, plant and animal templates.

scholarly journals Cyclic nucleotide phosphodiesterase activities in Neurospora crassa

1982 ◽  
Vol 203 (3) ◽  
pp. 611-616 ◽  
Author(s):  
M T Téllez-I ñón ◽  
G C Glikin ◽  
H N Torres
Keyword(s):  
Cyclic Amp ◽  
Neurospora Crassa ◽  
Cyclic Gmp ◽  
Cyclic Nucleotide ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Cyclic Nucleotide Phosphodiesterase ◽  
Sucrose Density Gradient Centrifugation ◽  
Molecular Weights

Cyclic nucleotide phosphodiesterase activities in soluble Neurospora crassa mycelial extracts were resolved into two peaks, phosphodiesterase I and II, by chromatography on DEAE-cellulose columns. Phosphodiesterase I hydrolysed cyclic AMP and cyclic GMP equally well. Phosphodiesterase II was active on cyclic GMP but scarcely active on cyclic AMP. Phosphodiesterase I was resolved by gel filtration and sucrose-density-gradient centrifugation into three peaks having molecular weights of about 57 000, 125 000 and 225 000. This suggests that this enzyme activity has at least three aggregation forms, tentatively defined as monomeric, dimeric and tetrameric. Similarly, phosphodiesterase II was resolved into two forms, having molecular weights of about 170 000 and 320 000. Evidence on the interconversion between phosphodiesterase I forms was obtained.

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.

Template Specificity and Subunits of RNA Polymerase from Asporogenous Mutants of Bacillus subtilis

1974 ◽  
Vol 52 (11) ◽  
pp. 966-973 ◽  
Author(s):  
H. Nishimoto ◽  
I. Takahashi
Keyword(s):  
Rna Polymerase ◽  
Morphological Changes ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Sedimentation Coefficient ◽  
Enzyme Preparations ◽  
Template Specificity ◽  
Α Subunits

In order to investigate relations between template specificity of RNA polymerase and sporulation, RNA polymerase activities in partially purified preparations from various asporogenous mutants were measured with poly[d(A-T)] or DNA from phage PBS 15 as template. Results obtained suggest that morphological changes occurring during sporulation may not be tightly linked temporally to transcriptional events.Subunits of RNA polymerase from these mutants were analyzed by sodium dodecyl sulfate – polyacrylamide gel electrophoresis after purification by (NH4)2SO4 precipitation, DEAE-cellulose chromatography, phosphocellulose chromatography, and glycerol gradient centrifugation. Phenylmethylsulfonyl fluoride was present throughout the purification procedure to prevent proteolytic degradation. It was found that β and β′ subunits were present in 1:1 ratio in all preparations. In addition to β, β′, and α subunits, a protein having a molecular weight of 95 000 was found in enzyme preparations from a wild-type strain and stage II mutants harvested at t5–t9. This protein was absent in stage 0 mutants and in all strains harvested in log phase. The enzyme containing this protein was eluted from phosphocellulose column with 0.6 M KCl rather than 0.35 M KCl, which eluted the enzyme without the 95 000 dalton protein. Furthermore the enzyme with this protein showed a sedimentation coefficient higher than that of the enzyme without the 95 000 dalton protein.

Structural proteins of a lipid-containing bacteriophage which replicates in Escherichia coli: phage PR4

1977 ◽  
Vol 23 (8) ◽  
pp. 1084-1087 ◽  
Author(s):  
Stephen P. Cadden ◽  
Jeffrey A. Sands
Keyword(s):  
Escherichia Coli ◽  
Viral Protein ◽  
Sucrose Gradient ◽  
Gradient Centrifugation ◽  
Structural Proteins ◽  
Major Protein ◽  
Protein Species ◽  
Sucrose Gradient Centrifugation ◽  
Molecular Weights ◽  
Escherichia Coli Phage

PR4 is a lipid-containing bacteriophage which is able to replicate in Escherichia coli. The virus was labeled with either [14C]leucine and [14C]threonine or H235SO4 and then purified by several rounds of sucrose gradient centrifugation. Autoradiographs showed the virus to be composed of six major protein species with molecular weights (1) 68 000, (2) 47 500, (3) 38 500, (4) 35 000, (5) 20 700, (6) 16 500 daltons. Electropherograms showed protein No. 2 to be the major protein, comprising about 43% of the total weight of viral protein.

scholarly journals Purification and characterization of chromatin-bound DNA-dependent RNA polymerase I from parsley (Petroselinum crispum). Influence of nucleoside triphosphates

1980 ◽  
Vol 191 (1) ◽  
pp. 165-171 ◽  
Author(s):  
K Grossmann ◽  
H Friedrich ◽  
U Seitz
Keyword(s):  
Enzyme Activity ◽  
Rna Polymerase ◽  
Gradient Centrifugation ◽  
Rna Polymerase I ◽  
Petroselinum Crispum ◽  
Isolation And Purification ◽  
Bivalent Cations ◽  
Nucleoside Triphosphates ◽  
Polymerase I

The isolation and purification of DNA-dependent RNA polymerase I (EC 2.7.7.6) from parsley (Petroselinum crispum) callus cells grown in suspension culture is described. The enzyme was solubilized from isolated chromatin. Purification was achieved by using DEAE- and phospho-cellulose in batches, followed by column chromatography on DEAE- and phospho-cellulose (two columns) and density-gradient centrifugation. The highly purified enzyme was stable over several months. The properties of purified parsley RNA polymerase I were investigated. Optimum concentration for Mn2+ was 1 mM, and for Mg2+ 4-6 mM, Mn2+ was slightly more stimulatory than Mg2+. The enzyme was most active at low ionic strengths [10-20 mM-(NH4)SO4]. The influence of various phosphates was tested: pyrophosphate inhibited RNA polymerase at low concentrations, whereas orthophosphate had no effect on the enzyme activity. ADP was slightly inhibitory, and AMP had no effect on the enzyme reaction. Nucleoside triphosphates and bivalent cations in equimolar concentrations in the range 4-11 mM did not influence the RNA synthesis in vitro. Free nucleoside triphosphates in excess of this 1:1 ratio inhibited the enzyme activity, unlike free bivalent cations, which stimulated RNA polymerase I.

The Carbohydrate Moiety of Mung Bean Vicilin

1976 ◽  
Vol 3 (6) ◽  
pp. 763 ◽  
Author(s):  
MC Ericson ◽  
MJ Chrispeels
Keyword(s):  
Mung Bean ◽  
Deae Cellulose ◽  
Indirect Evidence ◽  
Carbohydrate Moiety ◽  
Side Chain ◽  
Phaseolus Aureus ◽  
Molecular Weights ◽  
Molar Ratios ◽  
Peptide Moiety ◽  
Carbohydrate Unit

Mung bean (Phaseolus aureus Roxb.) vicilin, purified by DEAE-cellulose chromatography, is a glycoprotein (0.2% glucosamine and 1% mannose) which contains four different polypeptides with molecular weights of 63 500, 50000, 29 500 and 24 000 in the molar ratios 1 : 5 : 1 : 1. Two of these polypeptides (50 000 and 29 500) stain positively for carbohydrate. When vicilin is digested with pronase, the carbohydrate can be recovered as a glycopeptide containing mannose and glucosamine in the carbohydrate moiety and alanine, threonine and aspartic acid (or asparagine) in the peptide moiety. The total amount of carbohydrate (1.2 %) and the size of the carbohydrate unit (12 residues) suggest that each vicilin molecule contains only one carbohydrate side chain. Data indicate that this chain is attached via a glucosamine-asparagine link. The results raise the possibility that the presence of the two small polypeptides in vicilin preparations is the result of the breakdown of the major larger one. Indirect evidence suggests that vicilin may be a tetramer of four subunits, each with a molecular weight of 50 000, and that only one subunit has a carbohydrate side chain.

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