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... The densities of wet spores were determined in a linear density gradient made from Metrizamide/water solutions . The density of a small quantity of the Metrizamide solution beside a floating particle was determined according to the known relationship between density and refractive index. ...
The water contents and effective water activity of the core, cortex, and coat ofBacillus stearothermophilus spores in water, as well as the masses of the core, cortex, and coat in the dry state, were calculated from volumes, dry densities, and water absorption isotherms of the sporal components. The calculation depended upon the solution of simple simultaneous equations for the dry mass, dry volume, wet mass, and wet volume of the spore and its components. The effective water activity of the core and cortex was found to be 0.83.
In the 1970s great progress was made in the study of the mechanisms of gene exPression, although the bulk of the existing information is from tissue other than the brain.1–4 However, since reliable data on brain nuclei also became available after the publication of Rappoport’s work, this chapter primarily describes the data from brain. Reports on brain nuclei published before 1970 have been reviewed by Rappoport et al.
5 and McEwen and Zigmond.6
This chapter focuses on the fractionation of chromatin by buoyant density–gradient sedimentation in metrizamide. Most of the methods that have been used in attempts to fractionate chromatin have been designed to take advantage of the differences observed in vivo between heterochromatin and euchromatin—namely, the former is granulated and highly condensed, while the latter appears to exist mainly as uncondensed, extended fibrils. It has been realized that certain X-ray contrast media and their derivatives possess many of the attributes required of a density–gradient solute suitable for the isopycnic sedimentation of unfixed nucleoproteins. Metrizamide is totally nonionic in aqueous solutions and has been most widely exploited for experiments involving the isopycnic banding of macromolecules, macromolecular complexes, cell organelles, whole cells, and viruses. The chapter discusses the use of metrizamide for the isopycnic sedimentation of unfixed chromatin and describes the important properties of metrizamide. Formation of gradients of metrizamide and fractionation and analysis of metrizamide gradients are described in the chapter.
A theory of isopycnic sedimentation of interacting systems provides a theoretical explanation for the isopycnic behavior of catalase and other proteins in metrizamide gradients of pH 7.0–7.5, particularly the bimodal banding patterns reported by some investigators. As for whether or not the model is realistic, a spectroscopic investigation provides an unambiguous demonstration of a reversible interaction between metrizamide and the catalase dimers formed by dissociation of catalase tetramer at pH 3. The metrizamide-catalase dimer complex(es) is of high bouyant density. In addition, metrizamide at pH 7.4 interacts with heat-denatured ovalbumin. These observations suggest that the denser band in the experimental bimodal isopycnic patterns might be a complex(es) of metrizamide with a comformationally altered state of the protein.
A large-scale purification procedure for messenger ribonucleoprotein (mRNP) particles from rabbit reticulocyte polysomes is described. The mRNP particles were dissociated from polysomes by treatment with urea and separated by differential centrifugation under conditions of high ionic strength. Zonal centrifugation in a metrizamide buoyant density gradient was the final purification step. One major class of mRNA particle was observed. The RNA was defined as mRNA by polyacrylamide gel electrophoresis and by globin production in a cell-free protein-synthesizing system. The twenty-three different proteins associated with the particle were a discrete set of proteins, which ranged in molecular weight from 175,000 to 23,500. The relative amount of each peptide in the particle was determined from a gel scan of the stained protein by computer simulation. None of the polypeptides comigrated with proteins from the 40-S and 60-S ribosomal subunits when analyzed by two-dimensional polyacrylamide gel electrophoresis.
A simple and rapid nonenzymatic method has been developed to isolate myocardial cell nuclei from whole heart tissue. This method consists of a controlled disruption of cells followed by isopycnic gradient centrifugation. We have reviewed and compared our method to others more lengthy and laborious. By using a number of criteria, such as morphometric measurements, chemical composition, functional studies, specific nuclear protein markers, and mathematical analysis, we show that the nonenzymatic digestion method provides a most useful technique for the study of the biochemistry of the myocardial cell nucleus.
Soluble chromatin can be banded isopycnically in metrizamide gradients without prior fixation. The chromatin—DNA of minimally sheared chromatin (750–1000 base-pairs) bands as a single, sharp peak, almost completely separated from the chromatin ribonucleoprotein particles. More extensive shearing (to about 360 base-pairs) leads to a bimodal distribution of the chromatin—DNA in the gradients; all of the DNA is complexed with proteins.Examination of some of the properties of the two fractions of extensively sheared chromatin showed that the major difference between them is in the proportion of protein complexed with the DNA. Escherichia coli RNA polymerase binds to both fractions, but there is no more than a 2-fold difference in their template activities. Also, comparison of the concentrations of globin genes in both fractions of chromatin from cells synthesising haemoglobin and a tissue which does not synthesise haemoglobin showed no significant difference. These findings are incompatible with the two fractions being heterochromatin and euchromatin, as these are usually defined. The data presented, in agreement with those from other studies, show that soluble chromatin contains short protein-rich regions, the function of which is unknown at present.
The behaviour of isolated cell nuclei of different tissues in the mediterranean meal moth has been studied in metrizamide density gradients to find whether differences exist in the bouyant density between nuclei derived from different specialized tissues. The different nuclei studied have buoyant densities ranging form 1.228-1.295 g/cm3 in metrizamide density gradients and clear differences exist between different populations of nuclei. The different buoyant densities make it possible to separate isolated nuclei from a heterogeneous population of nuclei according to their characteristic densities. This has been tested by the separation of silk gland nuclei from follicle cell nuclei by the density centrifugation method. The degree of polyploidy of the nuclei is not correlated to a decrease or increase of the buoyant density, which suggests that different amounts of nuclear proteins, ribonucleoproteins or RNA are responsible for the differences determined. These tissue-specific differences of the nuclear densities might be correlated to different transcriptional activities. In addition to this, the isopycnic sedimentation technique in metrizamide density gradient provides a method to purify nuclei very carefully without any visible alteration of their morphology and integrity.
Metrizamide, a tri-iodinated benzamido-derivative of glucose, is a density-gradient material which gives dense aqueous solutions of low viscosity and osmolality (1). These advantages, coupled with its nonionic nature, chemical inertness, and low degree of hydration in solution, promise that it will be increasingly used for the separation of biological macromolecules, subcellular components, viruses, and cells by equilibrium density gradient centrifugation (2–4). In the course of our attempts to use Metrizamide to facilitate the fractionation of membranous organelles of pituitary homogenates by equilibrium density centrifugation (5), we have found that Metrizamide, even at concentrations as low as 1%, significantly interferes with protein determination by the Lowry method. We report here that the interference of Metrizamide can be circumvented by the use of appropriate blanks and calibration curves.
Metrizamide(2-(3-acetamido-5-N-methylacetamido-2,4,6-triiodobenzamido)-2-deoxy-d-glucose) dissolved in D2O was found to be a very suitable medium for the separation of labeled and unlabeled proteins by equilibrium gradient sedimentation. It is nontoxic, and has little influence on the activity of enzymes. Solutions in the density range of 1.3–1.45 g cm−3 have low viscosities. Since the spontaneous equilibrium gradient, which is dependent on the angular velocity, occurs only after a long time of centrifugation in metrizamide solutions, the equilibrium density gradient sedimentation of proteins can be performed at the highest available speed with any preformed shallow gradient. Examples for the separation of proteins of different densities are given.
The properties of nuclei, which had been isolated from freeze-dried rat liver in anhydrous glycerol, and purified by a variety of steps, were investigated. The nuclei retained high levels of a number of nuclear enzymes including NAD pyrophosphorylase, and DNA and RNA polymerases. The nuclei isolated by these methods contained considerably higher amounts of soluble and non-histone proteins than did those isolated in aqueous solutions of sucrose. Some specific differences in the profiles of the proteins in these two categories were noted between nuclei prepared by these two methods. Rat liver nuclei isolated in non-aqueous solvents may be fractionated either in gradients prepared by dissolving metrizamide in glycerol-dimethyl-sulfoxide or in gradients composed of propane-1,3-diol and 3 chloro-1,2-propanediol. The fractionation provides a partial resolution of diploid stromal from diploid parenchymal nuclei and of diploid nuclei from the tetraploid nuclei.
Hypaque (sodium diatrizoate), when added to cells growing in culture (two HeLa S3 strains, rat glioblastoma C6 and mouse lymphoma L5178Y), increased the intracellular level of adenosine 3',5'-cyclic monophosphate. A transient elevation of adenosine 3',5'-cyclic monophosphate was also observed when L5178Y cells were subjected to a procedure recommended for separation of lymphocytes from peripheral blood. The effect of Hypaque did not appear to be related to the increase in osmolality of the medium.
Use of Urografin and Conray for the equilibrium centrifugation of viruses is described. These pharmaceuticals, which consist of iodinated arylic compounds, reach densities of 1.6 g/cm3 and have low intrinsic viscosities. Poliovirus, Newcastle disease virus, and lymphocytic choriomeningitis virus were centrifuged to equilibrium in gradients made of these substances. Viral infectivities were not measurably affected, which is especially noteworthy in the case of the very labile lymphocytic choriomeningitis virus. Buoyant densities were found to be significantly lower than densities obtained with gradients made of CsCl and sucrose.
Hyaluronic acid (HA) isolated from synovial fluid by CsCl density-gradient ultracentrifugation show a decrease in the viscoelasticity. Metrizamide, a substance used recently for non-ionic density-gradient ultracentrifugation, do not change the rheology of synovial fluid and HA. Thus it may be possible to isolate HA without altering rheological properties.
Hypaque (sodium diatrizoate), when added to cells growing in culture (two HeLa S3 strains, rat glioblastoma C6 and mouse lymphoma L5178Y), increased the intracellular level of adenosine 3′,5′-cyclic monophosphate. A transient elevation
of adenosine 3′,5′-cyclic monophosphate was also observed when L5178Y cells were subjected to a procedure recommended for
separation of lymphocytes from peripheral blood. The effect of Hypaque did not appear to be related to the increase in osmolality
of the medium.
This chapter discusses the density labeling of proteins. A simple way of demonstrating enzyme synthesis is the method of density labeling of the enzyme protein with heavy isotopes followed by equilibrium density gradient sedimentation. This method is based on the same rationale as the classic method of density labeling and isopycnic centrifugation of nucleic acids and is the same method by which the semiconservative reduplication of DNA was demonstrated. If a protein is centrifuged for sufficient time in a density gradient, it will form a band at the point in the gradient where the density of the gradient corresponds to its own buoyant density. After centrifugation, the content of the centrifuge tube is divided into fractions of 3–5 drops. The density slope of the gradient can be conveniently determined by measuring the refractive index in some fractions. The other fractions are assayed for enzymatic activity. Density labeling can be combined with radioactive tracer methods to determine the turnover of proteins in general or in individual enzymes.
Nuclear proteins from barley have been separated by two-dimensional gel electrophoresis. Changes in the patterns of phosphorylated and unphosphorylated nuclear proteins which accompany germination have been examined. Only about half the nuclear proteins present in ungerminated embryos are still detectable after 24 hr germination.
This chapter describes isopycnic centrifugation in nonionic media. The nonionic density-gradient media represent the most commonly used compounds for isopycnic centrifugation under nonionic conditions. The centrifugation conditions necessary for isopycnic banding depend on a number of factors, including density and size of the particle and the viscosity of the gradient. This technique has been widely used for the fractionation of nucleic acids. However, its use has been severely limited by the types of density-gradient solute available. For example, most cellular and sub-cellular structures are sensitive to both the ionic content and osmotic strength of the surrounding medium. In addition, the solutions of carbohydrate gradient media are extremely viscous at high concentrations and, unless modified by the introduction of heavy atoms, have only a very limited density range. However, the recent upsurge in interest in this field has led to the introduction of other types of nonionic density-gradient media with markedly different properties.
Since the development of the first quantitative technique for fractionating liver tissue by Albert Claude (1946a,b), successive improvements have been introduced by several workers that have led to reproducible methods for disaggregating the tissue into a suspension of subcellular components,* called tissue homogenate, and for resolving this homogenate by differential centrifugation into four fractions containing mainly but not exclusively (1) nuclei and large cell debris; (2) mitochondria and other large granules; (3) small granules, designated microsomes (Claude, 1943), which were found later to derive largely from the endoplasmic reticulum of the intact cell (Palade and Siekevitz, 1956); and (4) soluble constituents of the tissue. The door was thus open wide to a new domain in cytology: the intracellular topography of biochemical functions, i.e., the study of the chemical composition, metabolic functions, and other properties of subcellular organelles.
This chapter highlights the practical aspects of rate-zonal centrifugation. Rate-zonal centrifugation is used to separate two or more populations of particles from each other by virtue of a difference in their sedimentation rates. The rate at which the particles sediment depends on their size, shape, and density and also depends on the density and viscosity at each point in the gradient. Sedimentation is opposed by viscous drag; the magnitude of which is related to the surface area of the particle so that large or near-spherical particles, those with a small ratio of surface area to mass, will sediment faster than small or extended particles. Sedimentation is slower in density gradients because local increases in density where particles have been concentrated against the wall are stabilized against convection. The effect will depend on the angle between the direction of sedimentation and the wall and on the particle concentration. With low concentrations, there may be no increase in the sedimentation rates of large or small particles due to wall effects.
This chapter presents the protocols for the separation of a number of particles representing a wide range of particles and techniques. Isopycnic sedimentation in swinging buckets presents few technical problems. The chapter discusses the separation of neoplastic mast cells according to their stage of development. It presents a procedure for the analytical separation of polysomes from rat liver. This procedure was used with particular success in the dissection of the mechanism of protein synthesis. The chapter also presents a study of E. coli ribosomal proteins, in which a limiting step was the resolution of the ribosomes into the 30S and 50S subunits. The chapter discusses the theoretical and practical limits of gradient capacity in the B-XV zonal rotor, which finds that up to 2 gm of crude ribosomes could be resolved into subunits in a single run.
This chapter discusses the properties of the common gradient materials. Density gradients are usually prepared from solutions or sols of materials, which are considerably denser than water. The density of most gradient solutions is greater than 1.0 gm/cm3. Excluding nonaqueous gradients for the present, the properties of the ideal solute for gradients are—freely soluble in water, very dense, nonviscous, negligible osmotic pressure, physiologically and chemically inactive, transparent in visible and ultraviolet light, and cheap. Common sugar is easily the most widely used gradient material. Although sucrose gradients have been used more than any other for the separation of the membrane-bound organelles, they are almost certainly inferior to silica and Ficoll. Mannitol was greatly favored over sucrose by early physiologists as an osmoticum, presumably because it penetrated cell membranes less rapidly than sucrose. More recently, mannitol has been recommended over sucrose in the isolation of mitochondria. Ficoll has become the most generally useful among polymeric gradient materials.
Myocardial cells were isolated after treatment with collagenase (0.05%) and hyaluronidase (0.1%) by discontinuous-gradient centrifugation on 3% Ficoll. Nuclei derived from these myocardial cells were then fractionated on a discontinuous sucrose density gradient with the following steps: (I) 2.0M/2.3M, (II) 2.3M/2.4M, (III) 2.4M/2.5M, (IV) 2.5M/2.6M, and (V) 2.6M/2.85M. The myocardial nuclei were sedimented in the interfaces of gradient fractions (II) and (III). Nuclei from whole ventricles that had been treated with the enzymes before isolation sedimented into five major subsets of nuclei. These findings suggest that nuclei sedimented in the isopycnic gradient at fractions (II) and (III) are most probably derived from myocardial cells. However, this procedure is laborious and lengthy, and the recovery of myocardial-cell nuclei is low. An alternative method was developed to isolate an enriched fraction of myocardial-cell nuclei from whole ventricular tissue without exposing the tissues to enzyme digestion. These ventricular nuclei could be fractionated into five nuclear subsets by using the same discontinuous sucrose density gradient as that described above. The content of DNA, RNA and protein per nucleus for each band was determined. Although the DNA content per nucleus was constant (10pg), that of RNA varied from 1.5 to 4.5pg and that of protein from 16 to 24pg. Nuclei from each band were examined by light-microscopy: large nuclei occurred in the ligher regions whereas smaller nuclei were found in the denser regions of the gradient. From the size distribution pattern of myocardial-cell nuclei compared with that of total ventricular nuclei, it was found that nuclear subsets (II), (III), and (IV) were similar to myocardial nuclei. Electrophoretic analyses of the proteins solubilized in sodium dodecyl sulphate/phenol or Tris/EDTA/2-mercaptoethanol/phenol obtained from each nuclear subset indicate that these fractions are similar, with limited qualitative differences. These findings indicate that isolation of an enriched fraction of myocardial-cell nuclei could be achieved by discontinuous-sucrose-density-gradient centrifugation.
1. The nuclei of the cells of the whole rat brain have been fractionated in a B-XIV zonal rotor with a discontinuous gradient of sucrose. Five fractions were obtained. Zone (I) contained neuronal nuclei (70%) and astrocytic nuclei (23%). Zone (II) contained astrocytic nuclei (81%) and neuronal nuclei (15%). Zone (III) contained astrocytic nuclei (84%) and oligodendrocytic nuclei (15%). Zone (IV) contained oligodendrocytic nuclei (92%) and zone (V) contained only oligodendrocytic nuclei. 2. The content of DNA, RNA and protein per nucleus was determined for each zone. Although the amount of DNA per nucleus is constant (7pg) the RNA varies from 4.5 to 2.5pg/nucleus and the protein from 38 to 17.6pg/nucleus. The neuronal nuclei have the greatest amounts of protein. The oligodendrocytic nuclei have the least content of RNA and protein. 3. The effects of pH, ionic strength, and Mg(2+) and Mn(2+) concentration on the activity of the nuclear system for synthesis in vitro of RNA have been investigated for unfractionated nuclei. From these studies a standard set of conditions for the assay of nuclear RNA polymerase has been established. 4. The activity of the RNA polymerase in each of the zonal fractions has been determined in the presence and in the absence of alpha-amanitin. Zone (II) is the most active, followed by zone (I). The nuclei of zones (IV) and (V) have comparable activity, which is 40% of that of zone (II). 5. The extent of incorporation of each of the four labelled nucleoside triphosphates by the nuclei from each zone has been measured. These values have been used to calculate the base composition of the RNA synthesized in vitro in each class of nucleus. 6. The effect of changes in the condition of assay of RNA polymerase in the different classes of nuclei has been investigated. Significant differences in the response to concentrations of metal ions and ammonium sulphate have been observed. 7. Homopolymer formation in each zone of brain nuclei has been determined. The extent of formation of the four homopolymers roughly parallels the RNA polymerase activity.
1. Purified liver nuclei from adult rats separate into two main zones when centrifuged in the slow-speed zonal rotor. One zone contains diploid nuclei, the other tetraploid. 2. The effect of age on the pattern of rat liver ploidy was examined. Tetraploid nuclei are virtually absent from young animals. They increase in proportion steadily with age. Partial hepatectomy disturbs the pattern of ploidy. 3. The zonal centrifuge permits the separation of diploid, tetraploid, octaploid and hexadecaploid nuclei from mouse liver. 4. Rat liver nuclei are isopycnic with sucrose solutions of density 1.35 at 5 degrees .
Since 1922 when Wu proposed the use of the Folin phenol reagent for
the measurement of proteins (l), a number of modified analytical pro-
cedures ut.ilizing this reagent have been reported for the determination
of proteins in serum (2-G), in antigen-antibody precipitates (7-9), and
in insulin (10).
Although the reagent would seem to be recommended by its great sen-
sitivity and the simplicity of procedure possible with its use, it has not
found great favor for general biochemical purposes.
In the belief that this reagent, nevertheless, has considerable merit for
certain application, but that its peculiarities and limitations need to be
understood for its fullest exploitation, it has been studied with regard t.o
effects of variations in pH, time of reaction, and concentration of react-
ants, permissible levels of reagents commonly used in handling proteins,
and interfering subst.ances. Procedures are described for measuring pro-
tein in solution or after precipitation wit,h acids or other agents, and for
the determination of as little as 0.2 y of protein.
SE. Kerr and K. Seraidarian, J. Biol. Chem. 159 (1945)
J BIOL CHEM
SE. Kerr and K. Seraidarian, J. Biol. Chem. 159 (1945)
Birnie Subcellular components: preparation fractionation 1972 Butterworths and Co London 53