High-Resolution Density Gradient Sedimentation Analysis

Britten, Roy J.; Roberts, Richard B.

doi:10.1126/science.131.3392.32

Cited by 771 publications

(115 citation statements)

References 3 publications

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“…(h) Sucrose Density Gradient Analysis Linear sucrose density gradients (Britten and Roberts, 1960) (12 .5 ml) were prepared in tubes of the SB283 rotor of the International B .60 centrifuge (International Equipment Company) . Centrifugation, without braking, was carried out at 2°C and at 40,000 rpm for the times indicated in the figure legends (usually for 100-150 min) .…”

Section: (E) Isolation and Purification Of Tetramers From Crude Ribosmentioning

confidence: 99%

Ribosome Crystallization in Chicken Embryos

Morimoto

Blobel

Sabatini

1972

The Journal of Cell Biology

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Isolated tetrameric particles (1665) derived from the crystalline lattices known to appear in hypothermic chicken embryos consist of mature 80S ribosomes which contain all species of ribosomal RNA and a complete set of ribosomal proteins .Ribosome tetramers are not a special type of polysomes since in solutions of high ionic strengths (500 mm KC1 and 50 mm triethanolamine-HC1 buffer) containing 5 MM MgCl2 they dissociate into 40S and 60S ribosomal subunits, without the need of puromycin, and at a concentration of Mg++ higher than 3 mm they are not disassembled by mild RNase treatment . Tetramers spontaneously disassemble into 80S monomers when the Mg++ concentration is lowered to 1 mm at relatively low ionic strength . Tetramers failed to couple in vitro puromycin-1 H into an acid-insoluble product, indicating the lack of nascent polypeptide chains . Although tetramers have no endogenous messenger RNA activity, they can be programmed in vitro with polyuridylic acid (poly U) to synthesize polyphenylalanine . All ribosomes within a tetramer can accept poly U, without the need of disassembly of the tetramers into monomers or subunits .

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Section: (E) Isolation and Purification Of Tetramers From Crude Ribosmentioning

confidence: 99%

Ribosome Crystallization in Chicken Embryos

Morimoto

Blobel

Sabatini

1972

The Journal of Cell Biology

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“…4 ml of this suspension was layered over 22 ml of a continuous sucrose gradient ranging from 0 .29 M to 0.73 M . The linear sucrose gradients were prepared with a mixing chamber (15) . Subfractionation was performed in an SW 27 rotor (Christ Omega II ultracentrifuge) at 56,000 g for 75 min .…”

Section: Sub Fractionation Of Smooth Microsomesmentioning

confidence: 99%

Subfractionation of Smooth Microsomes From Rat Liver

Glaumann

Dallner

1970

The Journal of Cell Biology

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Total smooth microsomes from rat liver isolated on a Cs+-containing sucrose gradient were concentrated and subsequently fractionated by zone centrifugation on a stabilizing sucrose gradient. The prerequisite for fractionation is to prepare total smooth microsomes in a nonaggregated condition, as well as to utilize a procedure which counteracts enzyme inactivation . The median equilibrium density of the various smooth microsomal vesicles ranges from 1 .10 to 1 .18 . The phospholipid/protein ratio is identical in all subfractions, but cholesterol, on a PLP basis, is enriched in the subfractions with the highest sedimentation velocity . The enzyme distribution pattern reveals a pronounced heterogeneity. A number of NADH-and NADPH-oxidizing enzymes are concentrated in the upper part of the gradient and exhibit a certain degree of separation from G6Pase . Mg++-ATPase and AMPase are enriched in the lower part of the gradient . No specific enrichment of newly synthesized NADPH-cytochrome c reductase activity occurs in any of the subfractions after phenobarbital treatment . These data demonstrate that smooth microsomes, by adequate fractionation procedure, can be separated into subfractious of heterogeneous composition .

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“…The incubation procedures, method for protein isolation and counting and RNA estimation were as described by Bullock, White & Worthington (1968). Sucrose density gradients (20 ml) were prepared from 15%,' (w/v) to 50% (w/v) sucrose in medium containing 9 mM MgCl2, 75 mM KCl, 35 mM tris-KCl buffer pH 7-4 at 370 C, by the method of Britten & Roberts (1960). Ribosomal suspensions (10 ml) were layered on the gradients which were then centrifuged for 1-5 h at 128,000 g in a 3 x 23 ml MSE swing-out rotor.…”

Section: Injurymentioning

confidence: 99%

Intracellular enzymes and protein synthesis in rabbit skin after thermal injury

Lewis¹,

Peters²,

White³

1971

British J Pharmacology

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Summary The concentrations of several intracellular enzymes in rabbit skin have been measured 5 min, 2, 6 and 24 h after thermal injury. At 5 min and 2 h after a burn (60° C for 1 min) there was a significant fall in the enzyme activities whereas at 6 h their activities were higher than control. It appears that the increase in enzyme concentrations in the lymph during the first few hours after thermal injury is associated with a fall in the enzyme concentrations in the tissues and therefore might be the result of leakage of enzyme from storage sites in the injured cells. The second increase in enzyme concentrations in the lymph which has been observed 6–18 h after thermal injury occurs at a time when there is also an increase in the enzyme concentrations in the tissue. It seems unlikely that these increased activities are due to new synthesis since there was no apparent correlation between tissue enzyme concentrations and protein synthetic activity, and the changes still occurred after administration of cycloheximide. There was a change in the LDH isoenzyme pattern after injury towards a predominance of LDH‐1. This change did not occur immediately after the burn, but was present at 2 and 6 h, and returned to normal 24 h later.

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High-Resolution Density Gradient Sedimentation Analysis

Abstract: The principle of stability for a sample layered in a density-gradient liquid column is discussed, and a method for separating ribonucleoprotein particles by means of sedimentation in the ultracentrifuge is described.

Cited by 771 publications

References 3 publications

Ribosome Crystallization in Chicken Embryos

Ribosome Crystallization in Chicken Embryos

Subfractionation of Smooth Microsomes From Rat Liver

Intracellular enzymes and protein synthesis in rabbit skin after thermal injury

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