Postnuclear Supernatant Fraction Research Articles

Cellular polyamine depletion reduces DNA synthesis in isolated lymphocyte nuclei

The accumulation of putrescine, spermidine and spermine in activated bovine lymphocytes was blocked by the combined action of two inhibitors of polyamine biosynthesis, methylglyoxal bis(guanylhydrazone) (MGBG) and α-methylornithine. Lymphocytes were cultured under three conditions: (1) α-methylornithine alone, (2) MGBG alone, or (3) α-methylornithine plus MGBG. DNA synthesis in nuclei isolated from these cells was reduced from control rates by approx. 10, 55 and 75%, respectively. In each case, the degree of inhibition was similar to that observed with the intact cells. Stimulation of nuclear DNA synthesis with the postnuclear supernatant fraction was not affected by polyamine depletion of the cells. Several experiments indicate that the reduced rate of in vitro DNA synthesis was caused by the lack of polyamines and not by alternate effects of the drugs. No inhibition was observed (1) when spermidine was added to inhibited cultures 12 h before harvest and nuclear isolation, (2) when the drugs were added after polyamines had accumulated, and (3) when the drugs were added directly to the in vitro assay. In addition, the degree of inhibition of in vitro DNA synthesis correlated with the degree of polyamine deficiency. These in vitro studies confirm the results obtained with whole cells and support the hypothesis that DNA synthesis is one cellular site of action of the naturally occurring polyamines.

Studies on the enzymatic hydrolysis of polyglutamyl folates by chicken liver folyl poly-γ-glutamyl carboxypeptidase I. Intracellular localization, purification and partial characterization of the enzyme

I. Intracellular distribution, purification and properties of a folyl poly-γ-glutamyl carboxypeptidase (peptidyl- l-glutamate hydrolase, EC 3.4.12.10) from chicken liver have been investigated. The post-nuclear particulate and cell supernatant fractions showed activity. The particulate enzyme exhibited characteristics suggestive of its lysosomal origin; on solubilization, however, it cannot be distinguished from the cell cytosol activity. The bulk enzyme was purified about 80-fold to apparent homogeneity by 50–90% ammonium sulfate fractional precipitation, dialysis and chromatography on a ‘mixed column’ of Sephadex G-100 superimposed on CM-Sephadex C-50. The purified enzyme behaved homogeneously on Sephadex G-100 chromatography, sucrose density gradient centrifugation analyses and polyacrylamide gel electrophoresis. However, polyacrylamide gel electrophoresis in the presence of mercaptoethanol dissociated the native enzyme into two separable isoenzymic components. The enzyme exhibits two pH optima (4.1 and 5.2) and a temperature optimum of 35–40°C. The reaction is linear for 20 min. The enzyme sequentially cleaves the terminal γ-glutamyl residues of polyglutamylfolates, finally releasing a monoglutamyl end-product. An apparent K m value of 0.83 · 10 −6 M and a V of 1.50 mmol/min were determined for N 5 -methyltetrahydropteroyltetraglutamate with 0.20 mg enzyme protein/ml reaction. The enzyme is substantially stimulated in the presence of mercaptoethanol, Na +, Mn 2+ and low concentrations of denaturing agents (urea). Citrate potentyl inhibits and phosphate inactivates the enzyme.

Purification and Polypeptide Composition of Semliki Forest Virus RNA Polymerase

A purification method for Semliki Forest virus-specified RNA-dependent RNA polymerase from BHK cells is described. The procedure entails (i) the preparation of a crude cell lysate by Dounce homogenization of cells 3-5 h post-infection, (ii) differential centrifugation to give a 15 000 g 'mitochondrial' pellet, (iii) equilibrium centrifugation on discontinuous sucrose gradients (Friedman et al. 1972) to give a membranous band of density 1-16 g/ml, (iv) solubilization with Triton N-101 and velocity centrifugation to give a 25S solubilized polymerase complex and (v) affinity chromatography through an oligo (dT)-cellulose matrix bearing immobilized 42S virus particle RNA. The overall purification was approx. 360-fold with a 5% recovery of activity. Of the various intermediate fractions in the purfication procedure, only the relatively crude post-nuclear supernatant fraction was competent to synthesize the major single-stranded RNAs found in infected cells. Other fractions incorporated precursor only into replicative intermediate (RI) or replicative from (RF). Analysis of the product RF showed that it was of the same size and could bind to the same extent to oligo (dT)-cellulose as the RF isolated directly from lysates of infected cells. Displacement hybridization and ribonuclease digestion suggested that the purified polymerase could only complete previously initiated progeny positive strands using negative strands as template and, even in its most highly purified form, was still tightly bound to its template. Analysis on polyacrylamide slab gels revealed the presence of three 35S-labelled polypeptides in the purified polymerase preparation, but a polypeptide which had identical electrophoretic mobility to the lowest mol. wt. polypeptide of the purified polymerase was also present in material from mock-fected cells which had been taken through the purification procedure. From these results we conclude that only two virus-specified polypeptides are present in the polymerase. A scheme for the synthesis of these polypeptides is presented in the accompanying paper.

Co-ordinate synthesis of ribosomes and elongation factors in the liver of immature chicks following a metabolic shift up

Treatment of immature chicks with estradiol results in the induction of egg yolk protein synthesis in the liver ( Heald & McLachlan, 1963, 1965 ). There is a twofold increase in newly synthesized messenger RNA associated with polysomes within the first six hours and an increase in total liver weight, RNA, and protein within 48 hours ( Jost et al. , 1973 ). Conditions have been established for reliable and quantitative determination of elongation factor 1, elongation factor 2 and ribosomal RNA in the livers of immature chicks, in order to study the quantitative relationship between, these components of the protein synthesizing machinery during the “shift-up” situation following estrogen administration. Factor activity of high salt extracts was determined from initial rates of polyphenylalanine synthesis in the presence of excess complementary factor. Direct quantitation of EF-2 § was achieved by incorporation of [ 14 C]ADP-ribose from [ 14 C]NAD in the presence of diphtheria toxin. The reaction conditions were shown to give specific transfer of [ 14 C]ADP-ribose to EF-2 by analysis on gels containing sodium dodecyl sulfate. The fractionated [ 14 C]ADPrEF-2 was used to quantitate EF-2. The amounts of 18 S and 28 S rRNA were used as a measure of the ribosome content of the treated liver. Post-nuclear supernatant fractions were prepared in the presence of diethyl pyrocarbonate and Triton X100 and fractionated on sodium dodecyl sulfate/ sucrose gradients. These conditions were shown to given quantitative determination of 18 S and 28 S rRNA. Analyzed in the above manner, the levels of EF-1, EF-2 and ribosomes were found to increase co-ordinately following estrogen administration, although EF-1 activity was shown to be rate-limiting for poly-phenylalanine synthesis. The stoichiometry of EF-2: ribosome was maintained at unity. However, the ratio of 18 S rRNA: 28 S rRNA was found not to be unity, but 1·5:1.

DNA replication by a possible continuous-discontinuous mechanism in homogenates of Physarum polycephalum containing dextran

Nuclear DNA synthesis in homogenates of Physarum is greatly stimulated by the presence of dextran in the homogenizing medium. In this cell-free system, the DNA precursor is incorporated approximately equally into two classes of DNA intermediates. One of these is similar in size to that observed previously in the intact organism, i.e. its sedimentation rate in alkaline sucrose density gradients increases, presumably by chain elongation, as the organism progresses through the S phase. The other class (approx. 10 S) is similar to ‘Okazaki’ fragments. Thus, nuclear DNA synthesis in homogenates of Physarum may occur by a continuous-discontinuous mechanism. Substantial DNA-synthetic activity is obtained by the addition of dextran to dextran-free homogenates. Maximal activity in this system requires the presence of both the nuclear and post-nuclear supernatant fractions. It is possible that a partial separation and recombination of a DNA polymerase and the endogenous template is effected by this procedure.

Protein and glycoprotein electrophoretic patterns of enriched fractions of primary and secondary granules from guinea pig polymorphonuclear leukocytes.

The postnuclear supernatant fraction of sucrose homogenates of guinea pig polymorphonuclear leukocytes (PMNL) was subjected to differential centrifugation to obtain a total particulate fraction, a particle-free supernatant fraction, highly enriched fractions of primary and secondary granules, and a membrane-rich fraction. The various fractions were solubilized in buffer containing sodium dodecyl sulfate (SDS) and analyzed for protein and glycoproteincomponents by SDS -polyacrylamide gel electrophoresis. The major glycoprotein components of the postnuclear supernatant fraction were found mainly associated with the enriched fraction of secondary granules and, to a lesser extent, with the membrane-rich fraction. No major glycoprotein components were visible in the polypeptide electrophoretic patterns of the primary granule fraction or of the particle-free supernate. Attempts at separation of guinea pig granules by zonal sucrose density gradient centrifugation were only partially successful. Data supporting a species difference in this regard between rabbit and guinea pig PMNL granules are presented.