Structure and properties of rat thymus deoxyribonucleoprotein. Solubility properties and effects of precipitation

1. Deoxyribonucleoprotein was prepared from rat thymus and was studied chiefly by the method of electric birefringence. The birefringence depends on the electrical and optical properties of the molecules and on their volume; the decay time of birefringence (after the removal of the electric field) depends on molecular length. 2. A comparison was made of the properties of deoxyribonucleoprotein redissolved after precipitation in 0.15m-sodium chloride with those of the original material, the main object being to find if structural changes have occurred in the former. As a preliminary, the dependence of the solubility of the deoxyribonucleoprotein on the concentration of added salt was investigated, and the birefringence properties were also studied after the addition of sodium chloride at low concentrations, after the alteration of pH and after dialysis. 3. It was found that precipitation of deoxyribonucleoprotein occurs in two fractions, beginning at about 0.4mm-sodium chloride. The solubility is minimal at about 0.15m-sodium chloride. 4. The electric birefringence and decay time of the deoxyribonucleoprotein decrease with increasing concentration of added sodium chloride, and the birefringence also decreases after dialysis. Prolonged dialysis leads to precipitation. 5. The properties of deoxyribonucleoprotein redissolved after precipitation with 0.15m-sodium chloride differ considerably from those of the original deoxyribonucleoprotein. This is attributed to some type of disorganization process occurring during precipitation. It is concluded that the organization of the original structure is very specific.     

Electric birefringence of native DNA in an alternating field

The relaxation of the birefringence of native DNA in solution was investigated in a pulsed sine-wave electric field. Relaxation times were calculated from the degree of damping of the birefringence signal and were studied as a function of the strength and frequency of the applied field, the molecular weight of the DNA, and the viscosity and ionic strength of the solvent. Relaxation times decrease with increasing field strength. For high-molecular weight DNA (>10⁶ daltons), the relaxation times decreased with frequency and increased less than linearly with viscosity. For low-molecular-weight DNA (<6 × 10⁵ daltons), the relaxation times were independent of frequency, increased linearly with viscosity, and varied with the 1.65 ± 0.1 power of the molecular weight. The average birefringence of high-molecular-weight DNA decreased with frequency in 0.001M Na₂ EDTA plus NaOH, pH 7.0, but is much less frequency-dependent if the EDTA concentration is reduced tenfold, while the average birefringence of sonicated DNA increases in both solvents with increasing frequency.     

Electric birefringence of eukaryotic ribosomes

Eukaryotic 60S ribosomal subunits were studied by transient electric birefringence. Conformations of subunits in active and inactive states upon changes in magnesium concentration were compared by electric birefringence and orientational relaxation time measurements. Active subunits exhibit a positive birefringence and a relaxation time of the order of 8 microseconds. In the presence of EDTA, inactive subunits show no birefringence. When Mg2+ is reverted in the cold to its initial level, the electro-optical properties of the subunits are partially restored although the particles remain biologically inactive.     

Higher order structure of chromatin: influence of ionic strength and proteolytic digestion on the birefringence properties of polynucleosomal fibers

Effects of ionic strength and proteolytic digestion on the conformation of chromatin fibers were studied by electric birefringence and relaxation measurements. The results confirm that at low ionic strength chromatin presents structural features reflecting those observed in the presence of cations. Soluble chromatin prepared from rat liver nuclei by brief nuclease digestion exhibits a positive birefringence. As the salt concentration is increased, the transition to a compact solenoidal structure is deduced from changes in electro-optical properties: the positive birefringence gradually decreases and the observed reduction in 40 mM NaCl is nearly 95%; the relaxation time decreases dramatically and the character of the kinetic changes since the decay of birefringence described initially by a spectrum of relaxation times becomes monoexponential. On digestion with proteases at low ionic strength we observe at first a rapid increase of the positive birefringence concomitant with an increase of the relaxation time. Then the birefringence decreases and becomes negative. Chromatin undergoes two successive transitions: the first transition is explained by a lengthening of nucleosomal chains without modification of the orientation of nucleosomes within the superstructure and the second one by the unwinding of the DNA tails and internucleosomal segments. When chromatin is digested at 30 mM NaCl we find a single unfolding transition characterized by the decrease of birefringence and a slight increase in the relaxation time. The results imply that the positive birefringence of chromatin does not depend on the presence of whole histone H1 and that a salt concentration of 30 mM NaCl is sufficient to modify the initial site or/and the effects of proteolytic attack.     

Optico-hydrodynamic properties of high molecular weight DNA. II. Effect of aqueous glycerol solvents

The optical and hydrodynamic properties of T2 bacteriophage DNA have been determined by steady-state flow birefringence and viscosity in glycerol–aqueous buffer solvents at 25°C. Flow birefringence and extinction angle data were obtained over a velocity gradient range of 0.1 to 5 sec^?1 and at concentrations from 3 to 55 μg/ml in solvents containing approximately 30, 42, and 48 vol-% glycerol. Large optical backgrounds were observed in the mixed solvent flow birefringence studies which presented special experimental difficulties; these are described and their effect upon the flow birefringence data are discussed. The data on extinction angle provide no evidence for an internal viscosity effect on the stationary-state hydrodynamic properties of high molecular weight DNA over a range of solvent viscosity from 0.9 to 4.6 cP. Both the optical and hydrodynamic properties under present conditions of measurement appear to be self-consistent in terms of the values for these quantities in neutral aqueous buffer solution. Interpretation of the birefringence is complicated by uncertainties inherent in calculating the form anisotropy of DNA in non-aqueous solvents, but the data imply no large changes in helical structure with increasing glycerol concentration. Both intact and slightly degraded DNA samples were investigated, and no significant polydispersity effects were observed under the experimental conditions described.     

Interaction of the alkylating agent mechlorethamine with DNA in presence and in absence of the radioprotector WR-1065: A transient electric birefringence study

A transient electric birefringence computerized spectrometer inducing Kerr effect in anisotropically polarizable solutions as DNA has been used to investigate, at ambient temperature, the mechlorethamine-DNA interaction in presence and in absence of the WR-1065 radioprotector. The birefringence, and the time relaxation variations versus drug concentration are reported. An interpretation including DNA electrooptic properties is proposed.     

An electro-optical study of the mechanisms of DNA condensation induced by spermine

Condensation of DNA by spermine has been studied by electric dichroism, electric birefringence and rotational relaxation times at 1 mM ionic strength. Using Manning's theory, we found that condensation occurs for a fraction of neutralized phosphate charges (r) equal to 0.90, in good agreement with previous studies using spermidine, synthetic polyamines and trivalent cations (e.g. Co(NH3)36 +, Tb3 +). Our results are compatible with the presence in solution of torus-shaped condensed structures in a narrow range of spermine concentration; further addition of the polyamine produced precipitation due to the self-aggregation of several toroids. For spermine concentrations lower than that required for collapse, important changes of the orientation mechanism in the electric field and of DNA stiffness were observed. Whereas free DNA was mainly oriented by a fast-induced polarizability mechanism, DNA-spermine complexes displayed an important permanent dipole component, in the spermine concentration range where extension of the DNA molecules was present. The birefringence relaxation times suggested that, in the first step, the stiffness of the DNA molecules increased, and then, at higher spermine concentration, bending of the DNA molecules occurred so that condensation into toroidal particles became possible.     

Electric and flow birefringence properties of myosin in aqueous urea and sodium pyrophosphate.

The electric dipole moment of rabbit skeletal myosin was estimated from the electric and flow birefringence properties. Myosin formed small polydisperse aggregates (0.2-1.1 microM in length) with an apparent electric dipole moment of 5,000-20,000 Debye in aqueous urea or sodium pyrophosphate at low ionic strength. Permanent dipole moment contributed substantially to the apparent dipole moment. An anti-parallel association of myosin was suggested from the dependence of the apparent dipole moment on myosin concentration. Some interactions between myosin and C-protein were detected in 1 M urea by flow birefringence and analytical ultracentrifugation studies. The apparent dipole moment of myosin aggregates was less dependent on myosin concentration in the presence of C-protein.     

Properties of a new form of DNA from whole calf thymus nuclei: evidence for reactive, special sites in DNA

A new method for DNA preparation from whole calf thymus nuclei, which was designed to minimize enzymatic activity throughout to the greatest possible extent, is presented. The isolated DNA shows a number of interesting properties differing from those of DNA prepared by standard literature methods. The most important property is that the DNA prepared from our whole, purified nuclei, having originally a high molecular weight of 20.5 million, cleaved on EDTA treatment into a number of relatively homogeneous DNA fractions with a mean molecular weight of 2.3 million. In contrast, the standard DNA did not cleave on EDTA treatment. On the basis of this finding it was concluded that in the DNA backbone special sites exist at which cleavage can occur. These sites were further shown to occur in one of two possible forms: (1) fully covalent and (2) noncovalent. The proportion of the sites in form 1 increases with increasing ATP level. In standard DNA all special sites are most likely in the covalent form. It was also shown that for the conversion from one form to the other enzymes of the nucleus are necessary. The ATP level very probably controls this enzymatic activity in vivo.     

Higher Order Structure of Chromatin: Influence of Ionic Strength and Proteolytic Digestion on the Birefringence Properties of Polynucleosomal Fibers

Abstract

Effects of ionic strength and proteolytic digestion on the conformation of chromatin fibers were studied by electric birefringence and relaxation measurements. The results confirm that at low ionic strength chromatin presents structural features reflecting those observed in the presence of cations. Soluble chromatin prepared from rat liver nuclei by brief nuclease digestion exhibits a positive birefringence. As the salt concentration is increased, the transition to a compact solenoidal structure is deduced from changes in electro-optical properties: the positive birefringence gradually decreases and the observed reduction in 40 mM NaCl is nearly 95%; the relaxation time decreases dramatically and the character of the kinetic changes since the decay of birefringence described initially by a spectrum of relaxation times becomes monoexponential.

On digestion with proteases at low ionic strength we observe at first a rapid increase of the positive birefringence concomitant with an increase of the relaxation time. Then the birefringence decreases and becomes negative. Chromatin undergoes two successive transitions: the first transition is explained by a lengthening of nucleosomal chains without modification of the orientation of nucleosomes within the superstructure and the second one by the unwinding of the DNA tails and internucleosomal segments.

When chromatin is digested at 30 mM NaCl we find a single unfolding transition characterized by the decrease of birefringence and a slight increase in the relaxation time. The results imply that the positive birefringence of chromatin does not depend on the presence of whole histone HI and that a salt concentration of 30 mM NaCl is sufficient to modify the initial site or/and the effects of proteolytic attack.     

Microsomal chemiluminescence of benzo[a]pyrene-7,8-dihydrodiol and its synthetic analogues trans- and cis-1-methoxyvinylpyrene

Low-density lipoproteins (LDL) have been shown to have a number of effects on the function of various cell types. To appreciate whether the in vitro effects of LDL have in vivo relevance, it is necessary to demonstrate that the biologic action described can be accounted for by native LDL and not by an alteration in the molecule or an addition to the preparation occurring during isolation. EDTA is frequently added to LDL during preparation to prevent oxidation. The effect of EDTA dialysis on LDL-mediated inhibition of lymphocyte responses was therefore examined. LDL alone did not inhibit mitogen-induced initial lymphocyte activation but rather suppressed lymphocyte DNA synthesis and subsequent proliferation in a transferrin-reversible manner. LDL dialysed with EDTA also inhibited lymphocyte responsiveness but the inhibition was not reversed by transferrin. Further experiments demonstrated that after dialysis EDTA in the LDL accounted for the change in its inhibitory effects. EDTA did not alter the lipoprotein but itself inhibited lymphocyte responses by chelating zinc necessary for DNA synthesis. These data indicate that LDL preparations may exhibit at least two separate effects on lymphocyte function. LDL is directly suppressive, while small amounts of contaminating EDTA can additionally be suppressive by chelating zinc. Thus, EDTA present in LDL preparations can alter their apparent biologic effects.     

Electric birefringence of restriction enzyme fragments of DNA: optical factor and electric polarizability as a function of molecular weight

The electric birefringence of restriction enzyme fragments of DNA has been investigated as a function of DNA concentration, buffer concentration, and molecular weight, covering a molecular weight range from 80 to 4364 base pairs (bp) (6 × 10⁴–3 × 10⁶ daltons). The specific birefringence of the DNA fragments is independent of DNA concentration below 20 μg DNA/ml, but decreases with increasing buffer concentration, or conductivity, of the solvent. At sufficiently low field strengths, the Kerr law is obeyed for all fragments. The electric field at which the Kerr law ends is inversely proportional to molecular weight. In the Kerr law region the rise of the birefringence is accurately symmetrical with the decay for fragments ≤ 389 bp, indicating an induced dipole orientation mechanism. The optical factor calculated from a 1/E extrapolation of the high field birefringence data is ?0.028, independent of molecular weight; if a 1/E² extrapolation is used, the optical factor is ?0.023. The induced polarizability, calculated from the Kerr constant and the optical factor, is proportional to the square of the length of the DNA fragments, and inversely proportional to temperature. Saturation curves for DNA fragments ≤ 161 bp can be described by theoretical saturation curves for induced dipole orientation. The saturation curves of larger fragments are broadened, because of a polarization term which is approximately linear in E, possibly related to the saturation of the induced dipole in high electric fields. This “saturated induced dipole” is found to be 6400 D, independent of molecular weight. The melting temperature of a 216-bp sample is decreased 6°C in an electric field of 8 kV/cm, because the lower charge density of the coil form of DNA makes it more stable in an electric field than the helix form.     

Factors affecting the zinc content of E. coli alkaline phosphatase

Through experiments with radioactively labeled EDTA, it has been shown that alkaline phosphatasc from E. coli has a high affinity for binding EDTA, requiring extensive dialysis for removal. This paper reviews the results of zinc analyses of E. coli alkaline phosphatase prepared in the presence and absence of EDTA. The presence of EDTA in most preparations of alkaline phosphatase accounts for previous overestimates of the zine content of the enzyme.With radioactively labeled EDTA, direct evidence for the binding of EDTA to the metal-free alkaline phosphatase is presented. It has been shown that the apoprotein binds two EDTA molecules rather strongly. Addition of four metal ions are necessary for reactivation of this EDTA-contaminated apoenzyme. However, when the EDTA-contaminated apoenzyme is subject for extensive dialysis and EDTA is removed, the addition of two zinc ions restores the enzyme activity completely.     

Interphase chromosomal deoxyribonucleoprotein isolated as a discrete structure from cultured cells

A new procedure is described for the preparation of interphase chromatin from cultured mouse cells (line P815). The primary objective of this procedure was to eliminate exchanges of histones between deoxynucleoprotein molecules; this objective is shown experimentally to have been attained. The chromatin is released from cells by the non-ionic detergent Nonidet P40 in medium of low ionic strength (0.1 mM-KNa₂PO₄), and may then be sedimented as a structure which conserves the general form and ultrastructural characteristics of chromatin within the cell. The nuclear envelope cannot be detected in these structures by electron microscopy, and their content of choline-containing phospholipids is less than 10% of that of nuclei. The maintenance of form in this structure must thus depend on properties of the chromatin itself, and possibly on the more compact peripheral chromatin.Soluble DNP² prepared by shearing these structures has the same relative contents of DNA, histones, non-histone proteins and RNA as DNP prepared by standard methods. Analyses by electrophoresis on polyacrylamide gels of the non-histone proteins reveals certain differences from the pattern of these proteins in DNP prepared by a salt precipitation method. The template activity for RNA synthesis, in the presence of Escherichia coli RNA polymerase of sheared, soluble DNP prepared by this procedure, is comparable to that of DNP prepared by other methods. However, in the absence of exogenous RNA polymerase the rate of RNA synthesis by structured (unsheared) chromatin is about ten times higher than the rate using sheared DNP.The rapid removal of the nuclear envelope in this lysis procedure allowed experimental examination of the origin of the histones and non-histone proteins of DNP. When DNP was prepared from a mixture of two populations of cells, one containing DNA distinguishable by a density label and the other containing radioactively labelled proteins, radioactive proteins were found exclusively in DNP of normal density, and not in dense DNP and vice versa. It is concluded that the proteins of DNP prepared in this way are not acquired during the preparation procedure but were already associated with DNA in vivo, and that other proteins are not bound non-specifically to DNA during the preparation of DNP. When a mixture of DNP molecules prepared, in this way is precipitated in 150 mm-NaCl and redissolved, some radioactively labelled histones migrate onto dense DNA molecules.This procedure is suitable for routine, quantitative isolation of chromosomal DNP from small numbers of cells; it is also applicable to cells of other cultured lines.     

Rotary dialysis: its application to the preparation of large liposomes and large proteoliposomes (protein-lipid vesicles) with high encapsulation efficiency and efficient reconstitution of membrane proteins

An apparatus for rotary dialysis is introduced and described in detail. The component parts are inexpensive, widely available, and relatively easy to modify and assemble. The apparatus achieves increased mixing of the contents of dialysis bags by constant end-over-end rotation. This technique is particularly useful in systems where maximum contact is desired between substances which would tend to partition under standard dialysis conditions. We have applied rotary dialysis to two liposome production methods. These are (i) the calcium-EDTA-chelation method of Papahadjopoulos et al. (1), which produces large unilamellar liposomes from negatively charged phospholipids, and (ii) a procedure for the reconstitution of membrane proteins into liposomes with a large internal aqueous space, which we have developed using the calcium-EDTA-chelation technique as a point of departure. In both techniques, vesicle formation occurs when a calcium-phospholipid precipitate is dissolved by the addition of EDTA. Instead of adding a 150 mM EDTA solution directly, as described in the original method, we have used overnight rotary dialysis against buffer containing 10 mM EDTA at the vesicle formation stage. Materials are encapsulated within the aqueous interior of the vesicles at much higher efficiencies when rotary dialysis is used in either method, compared to efficiencies obtained with direct addition of EDTA (up to 37% of added material vs a maximum published efficiency of 10% for direct addition). Rotary dialysis also promotes the reconstitution of a higher proportion of the membrane proteins present in the dialysis mixture into the bilayer of large liposomes (79 vs 41.6%). It also affects the content of liposomes qualitatively, allowing better reconstitution of the Sendai virus F glycoprotein than does direct addition of EDTA. These effects may be due to the slow time course, the extensive mixing of components, and the low volume-to-phospholipid ratios maintained during vesicle formation.     

Tissue distribution of EDTA encapsulated within liposomes of varying surface properties

Liposomes containing ethylenediaminetetraacetic acid (EDTA) were prepared with different surface properties by varying the liposomal lipid constituents. Positively charged liposomes were prepared with a mixture of phosphatidylcholine, cholesterol, and stearylamine. Negatively charged liposomes were prepared with a mixture of phosphatidylcholine, cholesterol, and phosphatidylserine. Neutral liposomes were prepared with phosphatidylcholine alone, dipalmitoyl phosphatidylcholine alone, or with a mixture of phosphatidylcholine and cholesterol. Distributions of ¹⁴C-labeled EDTA were determined in mouse tissues from 5 min to 24 h after a single intravenous injection of liposome preparation. Differences in tissue distribution were produced by the different liposomal lipid compositions. Uptake of EDTA by spleen and marrow was highest from negatively charged liposomes. Uptake of EDTA by lungs was highest from positively charged liposomes; lungs and brain retained relatively high levels of EDTA from these liposomes between 1 and 6 h after injection. Liver uptake of EDTA from positively or negatively charged liposomes was similar; the highest EDTA uptake by liver was from the neutral liposomes composed of a mixture of phosphatidylcholine and cholesterol. Liposomes composed of dipalmitoyl phosphatidylcholine produced the lowest liposomal EDTA uptake observed in liver and marrow but modrate uptake by lungs. Tissue uptake and retention of EDTA from all of the liposome preparations were greater than those of non-encapsulated EDTA. The results presented demonstrate that the tissue distribution of a molecule can be modified by encapsulation of that substance into liposomes of different surface properties. Selective delivery of liposome-encapsulated drugs to specific tissues could be effectively used in chemotherapy and membrane biochemistry.     

Optical Properties of DNA in Aqueous Solution

In the study of DNA electric birefringence, it is usual to use theories that consider that molecules in solution are small in relation to the light wavelength. In this work, we study the DNA electric birefringence using a broken-rod macroion (BRM) model composed of two cylindrical arms which does not restrict the size of the molecules. To achieve this, we include the inhomogeneity effect of the light electric field through the molecule and the interaction between its different parts. To analyze the interaction between a molecule and the incident beam of light, we apply the discrete dipole approximation (DDA), according to which each molecule is described as a finite array of electronic coupled oscillators. The electric birefringence is calculated from the oscillator polarizability. This is obtained from experimental data of electric birefringence saturation and from the increment of the solution refraction index in relation to that of the solvent. Furthermore, the oscillator polarizability is also estimated from DNA absorption spectrum using the Kronig–Kramers relations. This allows us to analyze the contributions of the different absorption bands of DNA to the electric birefringence. We analyze the influence of the inhomogeneity of the light electric field and of the intramolecular interactions in the characterization of DNA optical properties using electric birefringence measurements.     

Effect of pulsed and reversing electric fields on the orientation of linear and supercoiled DNA molecules in agarose gels

When linear or supercoiled DNA molecules are imbedded in agarose gels and subjected to electric fields, they become oriented in the gel matrix and give rise to an electric birefringence signal. The sign of the birefringence is negative, indicating that the DNA molecules are oriented parallel to the electric field lines. If the DNA molecules are larger than about 1.5 kilobase pairs, a delay is observed before the birefringence signal appears. This time lag, which is roughly independent of DNA molecular weight, decreases with increasing electric field strength. The field-free decay of the birefringence is much slower for the DNA molecules imbedded in agarose gels than observed in free solution, indicating that orientation in the gel is accompanied by stretching. Both linear and supercoiled molecules become stretched, although the apparent change in conformation is much less pronounced for supercoiled molecules. When the electric field is rapidly reversed in polarity, very little change in the birefringence signal is observed for linear or supercoiled DNAs if the equilibrium orientation (i.e., birefringence) had been reached before field reversal. Apparently, completely stretched, oriented DNA molecules are able to reverse their direction of migration with little or no loss of orientation. If the steady-state birefringence had not been reached before the field reversal, complicated orientation patterns are observed after field reversal. Very large, partially stretched DNA molecules exhibit a rapid decrease in orientation at field reversal. The rate of decrease of the birefringence signal in the reversing field is faster than the field-free decay of the birefringence and is approximately equal to the rate of orientation in the field (after the lag period).(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and properties of brain acetylcholinesterase (EC 3.1.1.7)

Abstract— Approximately 50 per cent of the total AChE of brain tissue was solubilized by a simple procedure of repeated homogenization and centrifugation in 0.32 M-sucrose containing EDTA. Ion-exchange and molecular-sieve chromatography demonstrated two peaks of AChE activity which apparently differed with respect to charge properties and molecular size. The most active preparation of AChE, which hydrolysed 13,500 μMmoles of acetylthiocholine/mg of protein/h, had an estimated molecular weight of 291,000 and was contaminated by a single protein component, as judged by disc gel electrophoresis.     

Rotational diffusion of short DNA fragments in polyacrylamide gels: an electric birefringence study

Using electric birefringence we have examined the rotational diffusion of five short DNA fragments (55 to 256 base pairs) both in polyacrylamide gels as a function of gel concentration and in solution. The length dependence of the measured rotational relaxation times in the gels is in good agreement with the prediction from the Odijk theory for the dynamics of slightly flexible rods in a network. The rotational relaxation times were found to depend on the gel concentration, contrary to the prediction from the Odijk theory. Possible reasons for this observation are discussed. The birefringence decay curves for DNA fragments in the gel were single exponential only at small electric field strength.