A. P. Mathias

THE USES OF METRIZAMIDE IN THE FRACTIONATION OF NUCLEI FROM BRAIN AND LIVER TISSUE BY ZONAL CENTRIFUGATION

1. Introduction Sucrose is the most commonly used solute for density gradient centrifugation. It has two disadvantages, firstly its permeability to cellular membranes and secondly its high viscosity. Although the first may be overcome by the use of polymers such as Ficoll, such materials have the drawback of a high viscosity com- bined with a low upper limit of the density of their solutions. The disadvantage of solutions of sucrose are most apparent in the separations involving isopycnic centrifugation, for example, the nuclei of brain tissue which requires the use of 2.8 M sucrose [ 11. We have investigated the use of a new material which offers the advantages of a considerably higher maximum solution density without the severe problems of viscosity, and which is also biochemically inert. The compound, which is given the trivial name metrizamide, is N-(2,4,6-triiodo, 3-N methyl acetyl amino, 5-acetylamino benzoyl) glucosamine (mol. wt. 788.1). 2. Methods 2.1.

The chemical reactivity of the thiol group in the active centre of ficin

1. 1. The irreversible inhibition of ficin (EC 3.4.4.12) by chloroacetamide or iodoacetamide is due to the alkylation of the thiol group of one particular cysteine residue in the enzyme. 2. 2. The variation of the rate of alkylation of ficin with pH shows that the reactive thiol group of ficin behaves essenitally as a freely ionising roup, pKa′ = 8.55 (25 °; I, 0.1). Hence it is concluded that this group is unlikely to exist in a hydrogen-bonded form in the free enzyme. 3. 3. The rates of reaction of ficin with chloroacetamide or iodacetamide were accelerated in the presence of substrates or products of catalytic activity without the stoichiometry of the overall reaction being affected. The rate of addition of the essential thiol of ficin to N-ethylmaleimide was unaffected by substrate or substrate analogues. 4. 4. The nucleophilicity of the essential thiol group of ficin in SN2 reactions was found to be greater than that of simple thiol compounds whereas in the addition reaction to N-ethylmaleimide the converse was found. 5. 5. A possible reason for the high nucleophilicyt of ficin in SN2 reactions and the acceleration caused by the presence of substrate or substrate analogues is given.

The role of rat liver nuclear DNA polymerase and its distribution in various classes of liver nuclei

Rate zonal centrifugation has proved a useful technique for the separation of rat liver nuclei into various classes [l] . Studies of labelling in vivo of nuclei following injection of 3H-thymidine and 14C-orotic acid enable an identification of nuclei active in synthesis of DNA and RNA [2] . The distribution of a number of nuclear enzymes including RNA polymerase, NMN-adenylyl-transferase and polyADPR ligase have been investigated [2,3] and certain significant correlations have come to light. One of the most important functions of the nucleus is the replication of its DNA, and DNA polymerase may be involved in this process. If nuclei are isolated from aqueous media only about 20% of the total DNA polymerase is recovered in the nuclear fraction, the greater part being in the cytosol [4] , whereas a substantially greater proportion of the enzyme is found in nuclei prepared in non-aqueous media [ 5,6] . In view of the continuing interest in mammalian DNA polymerase [7,8,9] we have investigated its distribution in fractionated rat liver nuclei and especially its relationship to the nuclei active in vivo in DNA synthesis. These experiments show that the specific activity of DNA polymerase is substantially reduced in nuclei with the maximal labelling.

The interaction of ribonuclease with cytidine nucleotides.

Abstract We wish to report some spectrophotometric studies on the interaction of bovine pancreatic ribonuclease A (RNAse) and an enzymically inactive derivative of RNAse with cytidine nucleotides and Zn(II) ions.

Characterization of rat liver nuclei isolated in anhydrous media and their fractionation using non-aqueous gradients.

Abstract 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.

The effect of sodium butyrate on acetylation in vitro of chromosomal proteins in three classes of liver nuclei from different ages of rats

The optimum conditions of in vitro incorporation of sodium [3H]acetate into sliced rat liver were studied. The incubations with sliced liver from three different ages of rats were performed in the presence of sodium n-butyrate. It was found that butyrate decreases the incorporation of sodium [3H]acetate into the homogenate, isolated nuclei, non-histone chromosomal proteins and histones for all age groups. The acetylations of non-histone chromosomal proteins and histones increase with age upto 2-months and decrease in 4-month-old rats both in the absence and presence of butyrate. Liver nuclei were fractionated by the simple method of zonal centrifugation into three classes, namely diploid stromal, diploid parenchymal and tetraploid parenchymal nuclei. The acetylations of non-histone chromosomal proteins and histones in three classes of nuclei of three ages of rats were studied in the presence and absence of butyrate. Butyrate can decrease the overall acetylations of non-histone chromosomal proteins and histones but increase the amount of polyacetylated histone H4 in all classes of nuclei of the three ages.

Effects of gene modulators on the acetylation of chromosomal proteins of rat liver slices

Abstract Rat liver slices were used to study the incorporation of sodium [ 3 H]-acetate into isolated nuclei, histones and non-histone chromosomal proteins. The modulations of the incorporation by cycloheximide, actinomycin D, cordycepin and spermine were compared. Actinomycin D and cordycepin, which affect transcription, change the level of acetylation much more dramatically than the modulator of translation, cycloheximide, does. The results suggest that acetylation of histones is involved in the control of gene expression at the level of transcription rather than translation. The decrease of the acetylation of histones when transcription is blocked is probably due to a decrease in histone acetyltransferase activity rather than an increase in histone deacetylase activity.

The labelling in vivo of nuclear and cytoplasmic RNA of pigeon reticulocytes

Abstract 1. 1. The time course of incorporation of 32 P 1 in vivo into the RNA of pigeon reticulocytes has been investigated. Nuclear RNA extracted after treatment with hot phenol and cytoplasmic RNA obtained by treatment of polysomes with sodium dodecyl sulphate were analysed on sucrose density gradients. The labelling of the nuclear RNA was complex. At early times (3 h), RNA sedimenting faster than 28 S was rapidly labelled. As the period of incorporation was extended, the relative amount of this material decreased and ribosomal RNA (rRNA) became labelled. Successive aqueous extractions of nuclei treated with phenol yielded samples of RNA of increasing specific activity containing rapidly labelled components sedimenting at 7–13 S, 23 S and faster than 28 S. 2. 2. The labelling of the cytoplasmic RNA lagged 1–2 h behind the nuclear RNA. The only cytoplasmic fraction labelled more rapidly than transport RNA or rRNA sedimented at about 8 S.

The fractionation of suspensions of isolated hepatocytes by rate zonal centrifugation

Parenchymal cells, isolated from rat liver by a simple non-enzymic technique, were fractioned according to ploidy class by rate zonal centrifugation on sucrose density gradients. This method of fractionation applied to liver cells prelabelled in vivo with tritiated thymidine separated different size classes of cells synthesising DNA.

The synthesis of rapidly labelled RNA in a β-galactosidase constitutive mutant of Escherichia coli

Abstract 1. Extrusion from a French Press at low pressure was used to prepare extracts of Escherichia coli containing about half their ribosomes as polysomes. A fraction enriched in polysomes was obtained from these extracts by centrifugation through two sucrose layers. 2. Double-label experiments, using [ 3 H]- and [ 14 C]uracil were carried out to compare the distribution of rapidly labelled RNA in the polysomes of a β-galactosidase constitutive mutant with that in the wild-type strain. Sucrose gradient analysis of both lysates and resuspended polysome fractions showed an excess of material from the constitutive strain sedimenting in the 30–50 S region. 3. RNA was extracted from the polysome pellet using the sodium lauryl sulphate-phenol procedure. Two regions of excess RNA levels from the constitutive mutant were apparent both on sucrose gradient centrifugation and agarose gel electrophoresis. These regions corresponded to RNA molecules with s values of about 18 and 25 S. 4. These results are consistent with the view that gratuitous synthesis of β-galactosidase is associated with enhanced levels of ribosomal precursors.