Distribution of histones in alkali-denatured chromatin studied by isopycnic centrifugation in alkaline metrizamide density gradients.

Three types of density gradients - neutral metrizamide, alkaline NaOH-metrizamide and alkaline triethanolamine-metrizamide - were used for studying the distribution of histones between the two DNA strands in alkali-denatured chromatin. It was found possible to avoid both protein redistribution and dissociation by using triethanolamine-metrizamide density gradients at pH 10.5. Under these conditions an alkali-denatured mixture of DNA and chromatin was well separated into the original DNA and DNP. When native or sonicated chromatin was denatured at pH 12.2 and centrifuged in a triethanolamine-metrizamide density gradient at pH 10.5 no peak of free DNA appeared. These results show that both DNA strands remain associated with histone molecules upon alkaline denaturation of chromatin.     

A study of the interaction of histones with DNA using isopycnic centrifugation in metrizamide gradients.

Isopycnic sedimentation in metrizamide gradients has shown that mouse-liver histones bind co-operatively to both homologous and bacterial DNA's. However, at low input ratios of histone to DNA, two types of stable complex are formed, depending on the histone concentration. One complex contains half as much histone as DNA while the other contains approximately equal amounts of histone and DNA. At high input ratios of histone to DNA extra histone is bound giving complexes containing up to twice as much histone as DNA. Poly-L-lysine and protamine were also found to bind co-operatively to DNA.     

Isolation of rat liver lysosomes by isopycnic centrifugation in a metrizamide gradient

A preparation, similar to the light mitochondrial fraction of rat liver (L fraction of de Duve et al, (1955, Biochem. J. 60: 604-617), was subfractionated by isopycnic centrifugation in a metrizamide gradient and the distribution of several marker enzymes was established. The granules were layered at the top or bottom of the gradient. In both cases, as ascertained by the enzyme distributions, the lysosomes are well separated from the peroxisomes. A good separation from mitochondria is obtained only when the L fraction if set down underneath the gradient. Taking into account the analytical centrifugation results, a procedure was devised to purify lysosomes from several grams of liver by centrifugation of an L fraction in a discontinuous metrizamide gradient. By this method, a fraction containing 10--12% of the whole liver lysosomes can be prepared. As inferred from the relative specific activity of marker enzymes, it can be estimated that lysosomes are purified between 66 and 80 times in this fraction. As ascertained by plasma membrane marker enzyme activity, the main contaminant could be the plasma membrane components. However, cytochemical tests for 5'AMPase and for acid phosphatase suggest that a large part of the plasma membrane marker enzyme activity present in the purified lysosome preparation could be associated with the lysosomal membrane. The procedure for the isolation of rat liver lysosomes described in this paper is compared with the already existing methods.     

Isopycnic centrifugation of sheared L-cell chromatin in metrizamide gradients

Isopycnic gradient centrifugation of L-929 cell chromatin in a 38% (W/V) metrizamide solution yields two distinct fractions. The fraction banding at a density of 1.24 gm/cm² (H chromatin) contains about 10% of the DNA present in the fraction banding at a density of 1.18 gm/cm² (L chromatin). Both fractions contain the same proportions of satellite to main band DNA''s. Some differences can be seen in the DNA: protein ratios and types of proteins present in the H and L chromatin fractions.     

Quantitative aspects of the binding of nuclear non-histone proteins to DNA as determined by centrifugation in metrizamide gradients

Density-gradient centrifugation in metrizamide gradients has been used to study the binding of nuclear proteins to DNA. The unique advantage of this method is that the nucleoprotein complexes can be isolated free of non-complexed DNA and proteins. The chromatin non-histone proteins bound to native DNA in a non-random manner. The extent of binding was dependent on the ionic strength of the medium and was decreased in the presence of RNA.     

Purification of eukaryotic ribosomes by isopycnic centrifugation in sucrose

A simple and inexpensive procedure for the isolation and purification of ribosomes from eukaryotes is described. The method avoids pelleting of ribosomes at high centrifugal forces and involves isopyenic centrifugation of the post-mitochrondrial supernatant in sucrose, precipitation of ribosomes with 10% polyethylene glycol, and zonal sucrose gradient centrifugation. The ribosomes obtained in this way are very pure and thus especially suited for the measurements of physical properties. The isopycnic centrifugation can also be used for the purification of other macromolecules and is only limited by a maximum density of sucrose of 1.40 g/cm³ obtained at the bottom of the centrifugation tubes.     

Analysis by isopycnic centrifugation of isolated nucleoids of Escherichia coli.

The isolated, formaldehyde-fixed nucleoid of E. coli has been analyzed by isopycnic centrifugation in CsCl density gradients. The membrane-free nucleoid bands at a density of 1.69 +/- 0.02 g/cm3. The membrane-associated nucleoid bands at a density of 1.46 +/- 0.02 g/cm3. Both species sediment to equilibrium as nearly monodisperse bands in CsCl, suggesting that the nucleoid components of DNA, RNA and protein are present in relatively constant ratios. These ratios are constant regardless of the position of the nucleoids in the heterogeneous sedimentation profile of a preparative sucrose gradient. The fixed nucleoids remain condensed during isopycnic centrifugation and there is no detectable loss of RNA from the nucleoid.     

Isolation of periportal and centrolobular hepatocytes by isopycnic centrifugation

Suspensions of rat hepatocytes were separated by Ficoll discontinuous density gradient into four fractions. About 85% of the total quantity of cells sedimented within the range from 1.044 to 1.126 g/ml. The activity of key enzymes of glycolysis and gluconeogenesis were measured to determine the acinar origin of hepatocyte subpopulations. The activity of the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase, in hepatocytes with the density of 1.044 g/ml was twice as high as in hepatocytes with the density of 1.073 g/ml. In contrast, glycolytic enzyme, hexokinase, was 3 times more active in heavy than in light cells. The results indicate that light and heavy cells correspond to periportal and centrilobular hepatocytes, respectively.     

Separation of ribonucleoprotein particles (Hn-RNP) from nuclei of L cells, in two classes, by isopycnic centrifugation in metrizamide gradients]

Separation of nuclear Hn-RNP particles into two density classes, following isopy cnic centrifugation in metrizamide gradients, is reported. One class (density 1.31 g/ml) is rapidly labelled, and contains polydisperse heterogeneous high molecular weight particles, easily diffusible from intact nuclei under certain conditions. The other class (density 1.18 g/ml) is of lower molecular weight, not diffusible, and needs apparently a longer time to be labelled and/or to accumulate inside the nuclei.     

Repair replication in human cells studied by sodium iodide isopycnic centrifugation of DNA in a fixed-angle rotor

A method for purification of ethynyl steroids from biological fluids has been developed using silver-sulfoethyl cellulose column chromatography. Ethynyl steroids were applied in methanol, and the firm binding allowed the columns to be washed with methanol, ethyl acetate, or diethyl ether to remove endogenous materials. Nonethynylated steroids did not bind to silver-sulfoethyl cellulose. Release of ethynylated steroids was achieved with a saturated NaCl/methanol solution. Dehydration or de-ethynylation of ethynyl estradiol, ethynodiol diacetate, and ethynodiol was not observed. The utility of this technique for purification of ethynyl steroid metabolites from the urine of a beagle metabolizing norethynodrel was demonstrated.     

Purification of plant mitochondria by isopycnic centrifugation in density gradients of Percoll

Mitochondria from potato tubers have been separated from contaminating organelles and membrane vesicles on self-generated Percoll gradients and in a relatively short time. The Percoll-purified mitochondria devoid of carotenoids and galactolipids showed no contamination with intact plastids, microbodies, or vacuolar enzymes. Percoll-purified mitochondria exhibited intact membranes and a dense matrix. The intactness of purified mitochondrial preparations was ascertained by the measurement of KCN-sensitive ascorbate cyt c-dependent O₂ uptake. When compared with washed mitochondria, Percoll-purified mitochondria showed improved rates of substrate oxidation, respiratory control, and ADP:O ratios. The recovery of the cyt oxidase was 70–90% and on a cyt oxidase basis the rate of succinate oxidation by unpurified mitochondria was equal to that recorded for Percoll-purified mitochondria. The great flexibility of purification procedure involving silica sols was extended from mitochondria to the isolation of intact peroxisomes.     

Deterioration of rat-liver mitochondria during isopycnic centrifugation in an isoosmotic medium.

We have investigated the effect of the centrifugation speed on the behavior of rat-liver mitochondria during isopycnic centrifugation in an isoosmotic medium. The gradient was made with a macromolecular compound, glycogen dissolved in 0.25 M aqueous sucrose. The distribution curves of several mitochondrial enzymes change when the centrifugation reaches a certain speed: they are shifted toward regions of lower density. The results are plausibly explained by supposing that the inner mitochondrial membrane becomes permeable to sucrose at high centrifugation speeds, and that the granules swell. The main causal agent of the phenomenon is the hydrostatic pressure the mitochondria are subjected to during centrifugation. Morphological observations show that mitochondria are markedly deteriorated when centrifuged at high speed in the glycogen gradient; they are swollen and the outer membrane is broken; also frequently, a large electron-dense granule is seen in the matrix near the inner mambrane.