Modifications and additions to the ISO-DALT system for two-dimensional gel electrophoresis.

Modifications of ISO-DALT devices that further enhance the efficiency and reproducibility of two-dimensional mapping of proteins are described. The principal changes in ISO system devices include the introduction of a gel casting trough with a removable panel to permit the removal of excess gel without introducing air into the electrofocusing gels and the introduction of an upper electrode compartment with a separate watertight septum for each electrofocusing tube to permit tube removal for cleaning and replacement. The principal changes in DALT system devices include the use of modified powder funnels to introduce acrylamide solutions into the slab gel gradient former without aeration; the introduction of a flexible outlet system for the gradient former to facilitate the removal of air bubbles; the introduction of an inexpensive two-part mixing chamber to permit disassembly for cleaning; the use of split gel holders to eliminate deformation and breakage of electrofocusing gels during loading onto slab gels; the introduction of an inexpensive integrated slab gel casting/rotating apparatus; and the introduction of a simple, water-cooled slab gel electrophoresis apparatus to reduce the volume of running buffer used in electrophoresis.

A general method for the isolation of haptoglobin 1-1, 2-1, and 2-2 from human plasma.

A general method for the isolation of haptoglobin 1-1, 2-1, and 2-2 human plasma is described. Plasma is fractionated by affinity chromatography on chicken hemoglobin-Sepharose using the full capacity of the column; then after washing the column thoroughly, haptoglobin is eluted with 8 M urea and the eluate is collected in fractions to separate active and denatured haptoglobin. The urea-free, active fractions of haptoglobin are fractionated by affinity chromatography on Affi-Gel Con A to remove nonglycoproteins, principally apolipoprotein A-I, and the haptoglobin is eluted with 0.5 M glucose. Then the haptoglobin-containing fractions are fractionated by negative immunoadsorption chromatography on anti-chicken hemoglobin-protein A-Sepharose to remove chicken hemoglobin-human haptoglobin complexes. Haptoglobin prepared by this three-step procedure is biologically active and nearly homogeneous. The recovery is approximately 70%, irrespective of phenotype. The procedure can be completed in 3 days. A partial purification of apolipoprotein A-I is obtained simultaneously by this method.

Isolation of undegraded free and membrane-bound polysomal mRNA from rat brain.

A new procedure is described for the isolation of undegraded free and membrane-bound polysomal mRNA from rat brain in which polysomes are simultaneously separated from soluble components of the cell and slowly sedimenting ribosome species and concentrated by rate-zonal centrifugation on short linear sucrose gradients. This avoids the problem encountered in conventional procedures of pelleting polysomes along with membranous materials that are not solubilized by detergents and that appear to contain traces of nucleases. It also permits a more thorough analysis of the mRNA populations actively engaged in protein synthesis, since both polyadenylated and nonpolyadenylated mRNAs are isolated together. Moreover, the likelihood of sedimenting nonpolysomal mRNP particles along with polysomes is reduced by using rate-zonal rather than pelleting centrifugation. The translational activity in vitro of free and membrane-bound polysomal RNA prepared in this way is high and is about 1.5 times that of RNA prepared by a conventional pelleting technique. The difference is attributable to better preservation of large mRNAs, as inferred from two-dimensional gel electrophoretic analysis of translation product abundance. The recovery of both classes of polysomal RNA is about 90%. The method is simple, efficient, and adapted for isolation of small amounts of polysomal RNA.

Blockade of morphine dependence-related enhancement of secretory protein synthesis in the pons-medulla and striatum-septum by naltrexone.

The effects of several schedules of multiple morphine pellet implantation on body weight and dependence development in rats were studied. One schedule designated 6/7 (total number of pellets implanted per total number of treatment days), consisting of 1 pellet on day 0, 2 pellets on day 2 and 3 pellets on day 4 and a total treatment period of 7 days, produced high levels of dependence without affecting either body weight or rates of protein synthesis in whole brain or liver. The components of this schedule, 1/2 (one pellet on day 0 and a total treatment period of 2 days), 3/4 (one pellet on day 0 and two pellets on day 2 and a total treatment period of 4 days) and 6/7 (see above), produced a linear increase in the degree of dependence as measured by loss of body weight following naloxone-precipitated withdrawal. Hence, they were used to investigate the relationship between the degree of dependence and the rate of translation in vivo on free and membrane-bound polysomes in seven brain regions (cerebrum, cerebellum, mesencephalon, pons-medulla, striatum-septum, hippocampus-amygdala and thalamus-hypothalamus) following a 6 min pulse with a pool expansion dose of [3H]leucine. The rate of translation on free and membrane-bound polysomes was unaffected except on the bound polysomes of the pons-medulla and striatum-septum, where increased rates of translation were found to be closely correlated with degrees of dependence, measured as loss of body weight. Concomitant administration of naltrexone prevented both the stimulation of protein synthesis and the development of dependence, whereas administration of naltrexone alone did not affect translation rates in either polysome compartment of any region. Thus, it is concluded that the mechanisms underlying opiate dependence involve the stimulation of secretory protein synthesis in the pons-medulla and striatum-septum, presumably by the interaction between morphine and opiate receptors.

Differential effect of chronic ethanol administration on rates of protein synthesis on free and membrane-bound polysomes in vivo in rat liver during dependence development.

Administration of ethanol thrice daily to rats in amounts sufficient to induce a high degree of physical dependence resulted in a 20% decrease in the rate of protein synthesis on liver membrane-bound polysomes in vivo after 3 days of treatment without affecting the rate on free polysomes. The inhibition was attributable to a decrease in the rate of polypeptide elongation as evidence by comparable decreases in nascent chain synthesis and completed protein release without any change in leucine uptake by liver. Chronic ethanol treatment did not affect the quantity or distribution of free and membrane-bound polysomes, the DNA concentration, or the weight of liver. The inhibition of protein synthesis on membrane-bound polysomes cannot, therefore, be readily ascribed to ethanol-induced nutritional deficiencies or to some nonspecific toxic effect of ethanol.

Changes in in vivo rates of protein synthesis on free and membrane-bound polysomes in rat brain during the development of physical dependence on ethanol and after the withdrawal of ethanol.

Administration of ethanol and nutrients to rats thrice daily by gavage for 3 days produced a linear increase in physical dependence during the first 3 days and a 2% increase in body weight. Rates of protein synthesis on free and membrane-bound polysomes in whole brain and in 7 brain regions, comprising the entire brain, were measured in vivo under pool expansion conditions with [3H]leucine at intervals during the development and decay of ethanol dependence. During dependence development there was a progressive decrease in the rate of protein synthesis on free polysomes, but this change was not significant (P less than 0.05) until the third day, and a decrease in the rate on membrane-bound polysomes after 3 days. The inhibition of protein synthesis is attributable to a decreased rate of polypeptide elongation. There was no change in brain weight, DNA content, ribosome content, ribosome distribution of mRNA pool size. There was, however, a decrease in leucine uptake after 3 days. In an attempt to distinguish between the acute effects of ethanol on regional rates of protein synthesis and those changes associated with dependence development, rates were measured 1.5 h after administering a 5 g/kg dose of ethanol to both control and dependent rats. Rates on free polysomes in the hippocampus-amygdala and thalamus-hypothalamus and on membrane-bound polysomes in the cerebellum and hippocampus-amygdala of dependent rats were reduced; however, there was a general reduction in the rates in control rats that may have obscured reductions in other regions from dependent rats. During early withdrawal, 12 h after the last dose of ethanol, there was an increase in the rate of free polysomes in the pons-medulla and striatum-septum and on membrane-bound polysomes in the hippocampus-amygdala, and a decrease in the rate on free polysomes in the cortex and thalamus-hypothalamus and on membrane-bound polysomes in the cortex. After 24 h, there was an increase in the rate on free polysomes in all regions (cerebellum, cortex, mesencephalon, striatum-septum and thalamus-hypothalamus) except the hippocampus-amygdala and pons-medulla and an increase in the rate on membrane-bound polysomes in all regions (cortex, hippocampus-amygdala, mesencephalon, pons-medulla and striatum-septum) except the cerebellum and thalamus-hypothalamus. The possible relationship of these changes to the homeostat hypothesis of ethanol dependence is discussed.

Differences in size, structure and function of free and membrane-bound polyribosomes of rat liver. Evidence for a single class of membrane-bound polyribosomes.

Free loosely bound and tightly bound polyribosomes were separated from rat liver homogenate by salt extraction followed by differential centrifugation, and several of their structural and functional properties were compared to resolve the existence of loosely bound polyribosomes and verify the specificity of the separation. The free and loosely bound polyribosomes have similar sedimentation profiles and polyribosome contents, their subunit proteins have similar electrophoretic patterns and their products of protein synthesis in vitro show a close correspondence in size and amounts synthesized. In contrast, the tightly bound polyribosomes have different properties from those of the free and loosely bound polyribosomes; their average size is significantly smaller; their polyribosome content is higher; their 60 S-subunit proteins lack two components and contain four or more components not found elsewhere; their products of protein synthesis in vitro differ in size and amounts synthesized. These observations show that rat liver membranes entrap a large fraction of the free polyribosomes at low salt concentrations and that these polyribosomes are similar to those of the free-polyribosome fraction and are different from those of the tightly bound polyribosome fraction in size, structure and function.

Effect of starvation of the distribution of free and membrane-bound ribosomes in rat liver and on the content of phospholipid and glycogen in purified ribosomes.

The distribution of free and membrane-bound ribosomes in liver in response to starvation has not been clearly defined. An investigation has been made of the effects of starvation on the content of DNA, RNA, protein, phospholipid and glycogen in rat liver, on the distribution of free and membrane-bound ribosomes, and on the content of phospholipid and glycogen in free and bound ribosome fractions. The results indicate that starvation can produce up to a 50% reduction in hepatic ribosomes without altering either the fraction of rRNA relative to the total RNA or the distribution of free and membrane-bound ribosomes. In addition, the degree of contamination of isolated ribosomes with membranous material does not fluctuate with changes in the nutritional status of the animal. The results suggest that the relative capacities for protein synthesis among the two ribosome compartments are maintained during the early stages of starvation. Further, co-sedimentation with glycogen is not responsible for the presence of membranous materials in purified ribosomes.

A procedure for the quantitative recovery of homogeneous populations of undegraded free and bound polysomes from rat liver.

A procedure is described for the preparation of free and bound polysomes from whole homogenates of rat liver tissue. Liver is homogenized in a conventional medium containing glutathione; then after a 12-min centrifugation at 131000g, the free polysomes in the supernatant are saved, while the membrane-bound polysomes in the pellet are suspended in a mixture of ribonuclease inhibitors (cell sap, 250 mM KCl, and glutathione), homogenized in the presence of detergent (Triton X-100), centirfuged for 5 min at 1470g, decanted, and treated with deoxycholate; the polysomes in the two supernatants are harvested by centrifugation through sucrose gradients containing 250 mM KCl and cell sap. Free and bound polysomes prepared in this manner are undegraded, equally active in cell-free protein synthesis, and virtually free of ribonuclease, membranous material, glycogen, deoxycholate, completed protein, and cross-contamination. The recovery of polysomes is approximately 95% and the distribution between the free and membrane-bound state is 25 and 75%, respectively. The molecular weight profiles after sodium dodecyl sulfate-acrylamide gel electrophoresis of the polypeptides completed and released by free and bound polysomes in vitro are different, indicating that there are quantitative differences in the synthesis of various size polypeptides between the two polysome classes. The differential centrifugation procedure is rapid and reproducible, requires much less ultracentrifugation than the isopycnic technique, and provides a nearly quantitative means of separating free and bound polysomes.

Effects of phenobarbital on protein synthesis and polysome levels in rat liver.

Administration of phenobarbital to rats increases the rate of synthesis of certain microsomal drug-metabolizing enzymes in a selective manner and promotes proliferation of smooth endoplasmic reticulum in the liver. Phenobarbital increased a number of factors by which protein synthesis could be enhanced in the liver. It produced a 30% increase in the amount of ribosomes and mRNA per cell. The proportion of ribosomes associated with polysomes was increased by 5-10% over normal liver. There was a 10-30% increase in the rate of ploypeptide elongation and a small increase or no change in polysome size, indicating that the rate of polypeptide initiation was increased proportionately. The product of these effects accounts for the 1.5-fold increase in the rate of total protein synthesis previously reported. The average polysome size, and the size of free polysomes in particular, was maintained when actinomycin D was administered to phenobarbital-pretreated rats, suggesting that the rate of mRNA degradation was decreased selectively. Phenobarbital did not, however, affect the distribution of ribosomes between the free and membrane-bound states or the activity of ribonucleases associated with isolated free and bound polysomes. Thus, we conclude that phenobarbital stimulates protein synthesis by expanding the mRNA pool, at least partially through effects on mRNA degradation, and by augmenting the rate of mRNA translation.