Tore Hultin

Activation of ribosomes in sea urchin eggs in response to fertilization

Abstract 1. 1. Soon after fertilization the incorporation of labeled amino acids into protein by Paracentrotus eggs showed a rapid increase. This effect was equally manifest in cell-free incorporation systems. 2. 2. The increased incorporation activity was mainly localized in the ribosome components of the systems. It was equally pronounced irrespectively of whether these particles were free or associated with lipoprotein membranes. 3. 3. Activation effects similar to those released by fertilization were obtained after parthenogenetic activation of the eggs with butyric acid, in spite of the fact that such treatment is insufficient for inducing normal cell divisions. 4. 4. Attempts to activate microsomes from unfertilized eggs by varied treatments in vitro were only partially successful.

Preparation and amino acid incorporating ability of ribonucleoprotein-particles from different tissues of the rat.

Abstract RNP-particles were prepared from different tissues of the rat by treatment of a mitochondria-free suspension with DOC in high magnesium concentrations. The fractions obtained were able to incorporate amino acids into proteins in the presence of cell sap and an energy generating system. The RNP-particles from different tissues showed approximately the same capacity for incorporating amino acids. The incorporation into these particles was strongly affected by the content of potassium and magnesium ions. Lecithinase A enhanced the incorporation of amino acids in the isolated particles. It was observed that dichlorophenolindophenol at 10−3 M inhibited up to 80–90 per cent of the incorporation.

Incorporation in vivo of 15N-labeled glycine into liver fractions of newly hatched chicks.

Abstract In the livers of newly hatched chicks, 15N from labeled glycine is incorporated in vivo into the microsome proteins earlier and at a more rapid rate than into the proteins of any other cell fraction isolated by differential centrifugation. The microsomes, moreover, probably contribute in a high degree to the surprisingly high isotope level of the considerable amounts of cell fluid proteins. Since the microsomes are especially rich in ribonucleic acid, and the production of phosphate-bound energy is centered in the mitochondria, the experiments indicate that the immediate availability of ribonucleic acid is more important for the formation of proteins than a close proximity to energy-producing enzyme systems.

Inductive effects of 3-methylcholanthrene on enzyme activities and amino acid incorporation capacity of rat liver microsomes☆

Abstract Liver microsomes from rats treated with 3-methylcholanthrcne (MC) 2 showed an increased activity in metabolizing aromatic amines. Under the same conditions a significant, although less pronounced increase was observed in the TPNH diaphorase and TPNH cytochrome b 5 reductase activities. Microsomes from regenerating liver showed a positive response also with respect to the cytochrome b 5 content. The activities of glucose-6-phosphatase, DPNH and TPNH cytochrome c reductase, DPNH diaphorase, and DPNH cytochrome b 5 reductase were not increased under the influence of MC. Liver microsomes from MC-treated rats showed an increased RNA to protein ratio and a higher capacity for incorporating l -leucine-C 14 into protein in vitro .

Crosslinking of initiation factor eIF-2 to proteins of the small subunit of rat liver ribosomes.

Eukaryotic mltiation factor eIF-2 binds Met-tRNA, and GTP to 40 S ribosomal subunits [l-7] . The bmdmg site for eIF-2 on the 40 S subunit was studied with 14C-labeled eIF-2 and ‘H-labeled ribosomal subunits, using DBI as a crosslinking reagent. High molecular weight protein conjugates were isolated which predominantly contained ribosomal protems S2, S3 and S18 (according to [ 1 l] ) covalently bound to the mitiation factor.

The activity of microsomes from regenerating rat liver in amino acid incorporating systems

Microsomal suspensions were prepared in parallel from normal and regenerating rat livers, and their activity for amino acid incorporation was determined in incorporation systems containing C14-l-leucine as the labeled component and standard amounts of normal cell fluid as the source of activating enzymes. The concentrations of the microsomal suspensions were determined by analyses of total nitrogen, protein, ribonucleic acid (RNA) and glucose-6-phosphatase. When the relative microsomal activities for amino acid incorporation were compared at different periods after hepatectomy a rapid rise was observed after a lag period of about 12–14 hours. This rise occurred simultaneously with a change in the composition of the microsomal material. On the basis of equal protein concentrations the amounts of RNA increased while the activity of glucose-6-phosphatase decreased. The change may indicate modified proportions between the granular and lamellar constituents of the endoplasmic reticulum, from which the microsomal material emanates. The increased microsomal activities during the regeneration period may be conditioned by a higher average concentration of nucleo-protein granules connected with the reticular membranes.

The enzymatic composition of rat liver microsomes during liver regeneration.

Abstract Microsomal suspensions were prepared in parallel from normal rat livers and from livers after partial hepatectomy. The activities of different enzymes were compared in the course of the regeneration cycle on the basis of equal protein content. The growth induction had only limited effects on the activities of glucose-6-phosphatase, DPNH- and TPNH cytochrome c reductases and DPNH- and TPNH diaphorases. The activity of oxidative demethylation of MAB on the other hand rapidly decreased after a lag period of about 12–14 hours. In the presence of liver microsomes carcinogenic amines become bound to proteins. The same conditions as for metabolic oxidation are required. The binding to proteins of isotope from labeled carcinogenic amines was studied in different systems: (a) mitochondria-free homogenates fortified with a TPNH-generating system, (b) isolated microsomes combined with a TPNH-generating system, (c) isolated microsomes in the presence of added TPNH. In all of these systems the activity of the microsomes to effect a binding of isotope to proteins showed a marked decrease during the liver regeneration. In some experiments the effusion from the microsomes of reactive metabolites of the labeled amines was measured separately by means of added, soluble acceptor proteins which were isolated again after the incubation. The binding of isotope to these proteins also decreased during regeneration, but not quite as much as the binding to the microsomal proteins. The reasons for this are discussed. The content of cytochrome b5 was determined in a number of the microsomal preparations. In the microsomes from regenerating livers a pronounced decrease was observed.

On the acid formation, breakdown of cytoplasmic inclusions, and increased viscosity in Paracentrotus egg homogenates after the addition of calcium

1. 1. In fresh Paracentrotus egg homogenates a rapid acid formation was found after the addition of small concentrations of CaCl2. At the same time certain cytoplasmic inclusions swelled and were more or less completely broken down. The viscosity of the homogenates increased 100–200 per cent. 2. 2. The acid formation was most likely due not only to an incorporation of Ca++ ions into complexes with acid groups in the homogenate. It was not supposed to be caused by carbohydrate metabolites. An acid formation very similar to the calcium-produced acid formation was caused by papain. A breakdown of certain cytoplasmic granules, and an increased viscosity was observed also under the influence of papain. The calcium effects mentioned above could not be superimposed on the papain effects. 3. 3. Isolated cytoplasmic granules, resuspended in isotonic, Ca-free Bialaszewicz solution of KCl, were no longer influenced by calcium. Calcium is therefore supposed to act through a factor in the cell fluid. As a working hypothesis, this factor is considered to be identical with the acid-producing factor and possibly of enzymatic nature. 4. 4. The acid formation under the influence of calcium is one constituent of the complex acid formation, which was found by Runnstrom (19) to occur in whole sea urchin eggs in hypertonic sea water.

The incorporation in vitro of labeled amino acids into the proteins of regenerating rat liver.

Abstract The incorporation into proteins of C 14 - l -leucine and C 14 -glycine was studied in mitochondria-free rat liver homogenates at different periods after partial hepatectomy. As a response to the hepatectomy the enzymatic systems concerned with the incorporation of these labeled amino acids into proteins became activated. The activity maximum was reached about 40 hours after the operations. At that period the activity of the homogenates were 2.5–3 times higher than in control homogenates from non-operated animals. The increased activity involved the amino acid activating enzymes in the cell fluid as well as the microsomal systems. However, the effects of the microsomal activation on the amino acid incorporation were more pronounced. An increased rate of incorporation of C 14 -glycine into glutathione was observed in the homogenates from regenerating liver.

Reactions of C14-labeled carcinogenic azo dyes with rat liver proteins

Abstract 1. 1. When the carcinogenic azo dyes dimethylaminoazobenzene (DAB) and monomethylaminoazobenzene (MAB), uniformely labeled with C 14 in the aniline ring, were administered to rats by intravenous injections, a particularly high isotope content was observed in the proteins isolated from the microsomal fractions of the liver. 2. 2. In the presence of TPNH or a TPNH-regenerating system MAB was demethylated in vitro by suspensions of liver microsomes. Soluble cytoplasmic proteins had no effect in this reaction. 3. 3. Under the same experimental conditions isotope from C 14 -MAB became bound to the proteins of the microsomes. The bound isotope was not removed by prolonged extraction with methanol in a continuous extractor, and only to a minor extent by treatment with 5 per cent trichloroacetic acid (90 °C). In the absence of reduced coenzyme no binding of isotope was observed. 4. 4. In the demethylation and isotope incorporation experiments TPNH could only to a limited extent be replaced by DPNH. 5. 5. When soluble proteins from the cell fluid fraction were present in microsomal systems metabolizing labeled MAB, an isotope incorporation into the soluble proteins took place after a short delay. When bovine albumin was added to the microsomal systems instead of the soluble liver proteins only minor amounts of bound isotope were observed in the proteins of the medium after the incubation. Without microsomes, however, the soluble liver proteins did not bind any isotope. 6. 6. A metabolic degradation of MAB, and a binding to protein of C 14 from labeled MAB was observed also in microsomal systems isolated from mouse liver. 7. 7. It is believed that reactive oxidation intermediates, possibly with the character of free radicals, are formed in microsomal units metabolizing MAB. These postulated intermediates may react with protein components in the same structures, but may soon pass into the cytoplasmic fluid, where they gradually become trapped by soluble protein components.