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 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 Association of nascent polypeptide with 30S ribosomal subunits

1973 ◽  
Vol 136 (4) ◽  
pp. 859-863 ◽  
Author(s):  
Michael Cannon ◽  
M. Amin A. Mirza ◽  
Margaret L. M. Anderson
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Protein Synthesis ◽  
Trichloroacetic Acid ◽  
Sucrose Gradient ◽  
Gradient Centrifugation ◽  
Ribosomal Subunits ◽  
Nascent Polypeptide ◽  
Sucrose Gradient Centrifugation ◽  
Crude Extracts

1. Crude extracts of Escherichia coli were used to synthesize nascent peptides under the direction of endogenous mRNA and in the presence of radioactive amino acids. Analysis of such extracts by sucrose-gradient centrifugation in low Mg2+concentration has shown that after 2min of incubation approximately 14% of the total labelled protein recovered on the gradient, in association with whole ribosomes, sediments with 30S ribosomal subunits; this value rises to approximately 24% after 30min of incubation. The labelled protein associated with 30S ribosomal subunits is insoluble in hot trichloroacetic acid. 2. Similar results were also obtained in extracts that synthesized polypeptides under the direction of either of the synthetic polyribonucleotides poly(A) or poly(A,G,C,U). In contrast, however, analysis of crude extracts programmed in protein synthesis by poly(U) has indicated that under these conditions 30S ribosomal subunits have no associated polyphenylalanine; similarly there is little associated peptide after programming of extracts by poly(U,C).

ANALYSIS OF RADIOIMMUNOASSAY BY DISC ELECTROPHORESIS AND SUCROSE GRADIENT CENTRIFUGATION

1971 ◽  
Vol 66 (2) ◽  
pp. 257-265 ◽  
Author(s):  
Lubomir Valenta ◽  
Michel L. Aubert
Keyword(s):  
Sucrose Gradient ◽  
Solid Phase ◽  
Gradient Centrifugation ◽  
Human Growth ◽  
Disc Electrophoresis ◽  
Separate Analysis ◽  
Adrenocorticotrophic Hormone ◽  
Sucrose Gradient Centrifugation ◽  
Before And After ◽  
Polypeptide Hormones

ABSTRACT Radioiodine-labelled synthetic adrenocorticotrophic hormone (ACTH), human growth hormone (HGH), human chorionic somato-mammotrophin (HCS), and human (HTSH) and bovine (BTSH) thyroid stimulating hormones were studied by disc-electrophoresis and sucrose gradient centrifugation before and after incubation with corresponding antisera. All antisera contained 7 S antibodies. After incubation, soluble antigenantibody complexes besides a small amount of precipitate were observed in the incubation mixture, characteristic of each hormone. The complexes migrated like gamma globulins or more slowly on disc-electrophoresis. and on sucrose gradient centrifugation showed patterns dependent on the time of incubation. Light 7 or 9 S, or < 12 S complexes occurred mostly after incubation for several minutes (up to 30 min) before analysis. When incubation was prolonged to 24 h and more, these relatively light complexes disappeared or diminished in favour of heavier soluble or precipitating complexes. Reproducibly obtainable sedimentation patterns of the soluble complexes suggested some definite recombination of antigen molecules with 7 S antibodies. The complexes did not occur on incubation with other sera than an antiserum to a given hormone. They were not influenced by EDTA. Displacement of the radioactivity of the complexes into the free hormone peak was obtained by addition of a non-labelled hormone identical with the labelled one. Sucrose gradient centrifugation and disc-electrophoresis are recommended for the study of immunoreaction of diluted materials and for a separate analysis of different steps of the radioimmunoassay. Radioimmunoassay was introduced for the measurement of protein hormones by Yalow & Berson (1960). The method, described originally for insulin, was later adapted to the detection of a number of protein and polypeptide hormones. On incubation of the hormone with its antiserum, a soluble antigenantibody complex is formed, which is separated from an excess of the free hormone by various methods, e. g. chromatoelectrophoresis, precipitation with a second antibody, adsorption on a solid phase etc. (Hunter 1967). Sucrose gradient centrifugation and disc-electrophoresis were occasionally used to follow some isolated aspect of radioimmunoassay (Fitschen 1965; Monjardino et al. 1968). We are demonstrating that these methods made it possible to analyze the radioimmunoassay step by step and thus may be useful for practical purposes as well as in a study of the immunoreaction of diluted materials.

scholarly journals LYSOSOMAL PHOSPHOLIPASES A1 AND A2 OF NORMAL AND BACILLUS CALMETTE GUERIN-INDUCED ALVEOLAR MACROPHAGES

1973 ◽  
Vol 56 (3) ◽  
pp. 621-627 ◽  
Author(s):  
Richard C. Franson ◽  
Moseley Waite
Keyword(s):  
Acid Phosphatase ◽  
Alveolar Macrophages ◽  
Sucrose Gradient ◽  
Gradient Centrifugation ◽  
Marker Enzymes ◽  
Sucrose Gradient Centrifugation ◽  
Control Distribution ◽  
Specific Activities ◽  
Selective Increase ◽  
Phospholipases A

A single intravenous injection of 0.1 mg of heat-killed Bacillus Calmette Guérin (BCG) in 0.1 ml of Bayol F produced an accumulation of activated alveolar macrophages (BCG induced). Cells were collected 3.5–4.0 wk after injection. Phospholipases A and three lysosomal marker enzymes (acid phosphatase, ß-glucuronidase, and lysozyme) were measured in homogenates, and the distribution of the phospholipases A and lysosomal, mitochondrial, and microsomal marker enzymes were examined after sucrose gradient centrifugation of a postnuclear (1,000 g) supernatant. Homogenates of normal and BCG-induced macrophages contained phospholipases A1 and A2 which had optimal activity at pH 4.0 in the presence of 2.0 mM ethylenediaminetetraacetate (EDTA). These activities were inhibited 50–70% by 2.0 mM CaCl2. Homogenates of BCG-induced macrophages had specific activities of ß-glucuronidase, acid phosphatase, and lysozyme, which were increased 1.5- to 3.0-fold over the controls, whether expressed as activity per mg protein or activity per 107 cells. The specific activities of the phospholipases A, on the other hand, were consistently lower than those of the control. Distribution of the phospholipases A and the lysosomal marker enzymes after sucrose gradient centrifugation suggested that the phospholipases A active at pH 4.0 in the presence of EDTA are of lysosomal origin since: (a) BCG treatment caused a selective increase in the density of particles which contained both the phospholipases A and three lysosomal marker enzymes; and (b) since the density of mitochondria and microsomes were not affected by BCG treatment. The increase in the density of lysosomes seen here may be related to previously described morphologic changes of BCG-induced alveolar macrophages.

scholarly journals Degradation of surfactant-associated protein B (SP-B) during in vitro conversion of large to small surfactant aggregates

1993 ◽  
Vol 295 (1) ◽  
pp. 141-147 ◽  
Author(s):  
R A W Veldhuizen ◽  
K Inchley ◽  
S A Hearn ◽  
J F Lewis ◽  
F Possmayer
Keyword(s):  
Surface Area ◽  
Sucrose Gradient ◽  
Gradient Centrifugation ◽  
Sucrose Gradient Centrifugation ◽  
Aggregate Fraction ◽  
Surfactant Aggregates ◽  
Morphological Studies ◽  
Surface Active ◽  
Protein B

Pulmonary surfactant obtained from lung lavages can be separated by differential centrifugation into two distinct subfractions known as large surfactant aggregates and small surfactant aggregates. The large-aggregate fraction is the precursor of the small-aggregate fraction. The ratio of the small non-surface-active to large surface-active surfactant aggregates increases after birth and in several types of lung injury. We have utilized an in vitro system, surface area cycling, to study the conversion of large into small aggregates. Small aggregates generated by surface area cycling were separated from large aggregates by centrifugation at 40,000 g for 15 min rather than by the normal sucrose gradient centrifugation. This new separation method was validated by morphological studies. Surface-tension-reducing activity of total surfactant extracts, as measured with a pulsating-bubble surfactometer, was impaired after surface area cycling. This impairment was related to the generation of small aggregates. Immunoblot analysis of large and small aggregates separated by sucrose gradient centrifugation revealed the presence of detectable amounts of surfactant-associated protein B (SP-B) in large aggregates but not in small aggregates. SP-A was detectable in both large and small aggregates. PAGE of cycled and non-cycled surfactant showed a reduction in SP-B after surface area cycling. We conclude that SP-B is degraded during the formation of small aggregates in vitro and that a change in surface area appears to be necessary for exposing SP-B to protease activity.

Proteins in the Enamel Fluid of Immature Porcine Teeth

1987 ◽  
Vol 66 (12) ◽  
pp. 1721-1726 ◽  
Author(s):  
T. Aoba ◽  
T. Tanabe ◽  
E.C. Moreno
Keyword(s):  
Amino Acid ◽  
Amino Acid Composition ◽  
Protein Content ◽  
Acid Composition ◽  
Permanent Teeth ◽  
Major Protein ◽  
Protein Species ◽  
Terminal Sequence ◽  
Molecular Weights ◽  
Sds Electrophoresis

The fluid was separated from the immature soft enamel of porcine permanent teeth in the secretory stage according to procedures reported previously (Aoba and Moreno, 1987). The protein content of the fluid was about 2.8% w/v; its amino-acid composition was characterized by high contents of Pro, Glx, Leu, and His, showing composition similar to that of the 20 kilo-dalton (kd) amelogenin or its C-terminal segments. The two major protein species in the fluid had apparent molecular weights of 13 kd and 11 kd, as determined by SDS electrophoresis; the N-terminal residue of the former was Leu, while that of the latter was Ala. The C-terminal sequence of both of them was -Met-Phe-Ser. By comparison with the published sequence of 20-kd porcine amelogenin, it is concluded that the main fluid constituents were derived by cleavages of N-terminal segments from the 20-kd amelogenin.

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