Rat Liver Cell Sap Research Articles

Two methods to avoid the effect of endogenous protein inhibitors during the assay of protein kinase C activity in tissue extracts.

Using H1 as substrate the protein kinase C activity of rat liver cell sap was increased about fourfold by treatment with DEAE-cellulose at pH 7.5 at an intermediate ionic strength due to removal of protein inhibitors. The activity of cell sap from rat spleen, brain or muscle was about doubled by the same treatment. In contrast, when a specific synthetic peptide substrate was used the corresponding increase of enzyme activity was not obtained when the inhibitors were removed. This shows that this type of substrates should be preferred for reliable assays of protein kinase C in crude extracts. The possible role of the protein inhibitors for the substrate specificity of protein kinase C is briefly discussed.

Hepatic L-type pyruvate kinase: separation of unphosphorylated, phosphorylated and proteolytically modified in vivo forms.

After partial chromatographic purification of rat liver cell sap on DEAE-cellulose, including the removal of type M2 pyruvate kinase, different forms of type L pyruvate kinase were separated by chromatofocusing. Three fractions of pyruvate kinase activity were found, eluting at pH 5.0, 5.2, and 5.3, respectively. The first one was identified as phosphorylated and the second one as unphosphorylated pyruvate kinase. There were strong indications that the third fraction represented a proteolytically modified form of the enzyme, since it co-migrated with a form modified in vitro and had a similarly increased apparent Km for phosphoenolpyruvate. To rule out the possibility of this being a phosphorylated form of pyruvate kinase, the enzyme was incubated with a phosphoprotein phosphatase and then phosphorylated with cAMP-dependent protein kinase. The enzyme was not phosphorylated, like pyruvate kinase modified with subtilisin or calcium-activated protease. There is some evidence that a proteolytically modified pyruvate kinase exists in vivo. This enzyme form has not previously been demonstrated in cell sap, prior to exposure to proteolytic enzymes. The relative amounts of the three forms were determined in livers from starved rats and rats fed on a normal or a carbohydrate-rich diet.

Protein Synthesis by Gastric Mucosal Ribosomes During Development in Rats

SummaryOxyntic gland mucosal ribosomes from 7− to 30‐day‐old rats were asayed for their ability to synthesize endogenous messenger RNA (mRNA) and poly(U)‐directed protein in a cell‐free system. Normal rat liver cell sap (105,000 g supernatant) was used as a source for transfer RNA (tRNA) and activating enzymes. Total mucosal DNA and ribosome content [as assessed by ribosomal RNA (rRNA)] were also determined. Although both DNA and rRNA content increased throughout the 4 post‐natal weeks, the rRNA/DNA ratio rose sharply during the 2nd and 4th week after birth. The ability of mucosal polyribosomes to synthesize endogenous mRNA‐directed protein in a cell‐free system remained elevated up to the age of 18 days, then decreased markedly within the next 3 days, and thereafter, declined more slowly. In 21− and 30‐day‐old rats, the rate of endogenous mRNA‐directed protein synthesis by mucosal ribosomes was 50–60% below that of the 7‐day‐old suckling rats. The protein/ polyphenylalanine ratio, which represents a ratio of polysomes to monosomes, was found to be about 57% lower for ribosomes from 30‐day‐old rats as compared to 7‐day‐old suckling animals. The ability of run‐off ribosomes (devoid of endogenous mRNA) to translate poly(U) was also found to be 33% lower for 30‐day‐old rats as compared to the 7‐day‐old suckling animals.

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Cytoplasmic free and bound polysomes isolated from bovine adrenal cortex were used to program protein synthesis in rat liver cell sap (175,000 X g soluble material) and wheat germ lysate S-175 (175,000 X g soluble material) systems. Both free and bound polysomes were translated with high fidelity and equal efficiency in the cell-free systems. Synthesis of adrenodoxin reductase (AdR) and adrenodoxin (Ad) in the cell-free systems was determined by the immunoprecipitation of the translation products using monospecific antibodies, and we found that AdR was synthesized by free polysomes whereas Ad was synthesized by both free and bound polysomes as a large precursor (pAd) having a molecular weight of approximately 22,000 daltons, which is about 10,000 daltons larger than mature Ad. The existence of a large precursor of Ad was also confirmed by its synthesis in a cell-free system programmed by total RNA isolated from bovine adrenal cortex. Bovine adrenal cortex mitochondria imported in vitro-synthesized AdR and pAd to the inside of the organelle in the absence of protein synthesis in a cell-free system, and processed pAd to the mature form during the import. These results suggest that cytoplasmically synthesized AdR and Ad are translocated posttranslationally into the matrix of mitochondria in adrenal cortex cells.

The demonstration in rat liver cell sap of protein kinase and phosphoprotein phosphatase active on fructose-bisphosphatase

A protein kinase active on fructose-bisphosphatase ( d-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) was demonstrated in rat liver cell sap. The protein kinase activity was stimulated by cyclic AMP and coincided with the activity of cyclic AMP-dependent protein kinase type I. In addition, three different peaks of phosphoprotein phosphatase active on [ 32P]phosphofructose-bisphosphatase were found on chromatography of rat liver cell sap on a DEAE-cellulose column. These phosphatases needed divalent cations for full activity. 5′-AMP, a negative modulator of fructose-bisphosphatase, had no effect on the phosphorylation-dephosphorylation reactions of the enzyme. ATP and Ca 2+ did not influence the dephosphorylation reaction of fructose-bisphosphatase.

In vitro synthesis of mitochondrial cytochromes P-450(scc) and P-450(11-β) and microsomal cytochrome P-450(C-21) by both free and bound polysomes isolated from bovine adrenal cortex

In vitro synthesis of mitochondrial cytochromes P-450(scc) and P-450(11-β), and microsomal cytochrome P-450(C-21) programmed by bovine adrenal cortex polysomes was carried out using rat liver cell sap and wheat germ lysate systems. Synthesis of P-450 proteins in the cell-free systems was determined by immunoprecipitation and immunoadsorption using mono-specific antibodies to each species of P-450, and the sizes of the in vitro products were analyzed by SDS-polyacrylamide gel electrophoresis. Both free and bound polysomes synthesized these three species of P-450 in the cell-free systems. P-450(scc) and P-450(C-21) were synthesized apparently as the mature size products, whereas P-450(11-β) was synthesized as a putative precursor approximately 5,000 daltons larger than the mature form. Mitochondrial and microsomal P-450 proteins seem to share common sites of synthesis in the cytoplasm of adrenal cortex cells.

In vitro synthesis of adrenodoxin and adrenodoxin reductase: Existence of a putative large precursor form of adrenodoxin

Cytoplasmic free and bound polysomes were isolated from bovine adrenal cortex, and used to program in vitro protein synthesis in rat liver cell sap and wheat germ lysate systems. Synthesis of adrenodoxin(Ad) and adrenodoxin reductase(AdR) in the cell-free systems was determined by immunoprecipitation using monospecific antibodies, and the sizes of the in vitro products were analyzed by SDS-polyacrylamide gel electrophoresis. Ad was synthesized by both free and bound polysomes as a putative large precursor having molecular weight of approximately 20,000 daltons, which was processed to mature size Ad (MW 12,000 daltons) by in vitro incubation with adrenal cortex mitochondria. On the other hand, AdR was synthesized only by free polysomes apparently as the mature size product.

The in vitro modification of phosphorylated pyruvate kinase by a Ca2+-activated protease from rat liver.

A Ca/+-activated protease from rat liver cell sap was prepared. It was shown to act on rat liver pyruvate kinase that had been phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase, the activity being optimum at neutral pH. The modified pyruvate kinase had the same Vmax as the phosphoenzyme but showed a lower affinity for the substrate phosphoenolpyruvate. The possibility that this proteolytic attack is the step that initiates further degradation in the cell is discussed.

Identification of L-type pyruvate kinase as a major phosphorylation site of endogenous cyclic AMP-dependent protein kinase in rat liver soluble fraction

The mechanism by which hormones such as glucagon and catecholamines stimulate gluconeogenesis in liver has as yet not been elucidated. Injection of glucagon or epinephrine has been reported to lead to a lowered activity of the glycolytic enzymes phosphofructokinase and pyruvate kinase, whereas the gluconeogenic enzyme fructose-l ,6-diphosphatase shows an increased activity; all enzymes being measured under one particular condition [ 1,2]. The mechanism of these changes in enzyme activity has not been investigated. The most well-known action of glucagon on liver cells is the stimulation of cyclic AMP-dependent protein kinases. It is obvious to explain the action of glucagon on gluconeogensis by phosphorylation of the above-mentioned enzymes which results in a change of enzyme activity. This seems indeed to be the case for pyruvate kinase, as judged from in vitro studies (reviewed in [3]) and from in vivo experiments [4]. Phosphorylation of purified phosphofructokinase [S] and fructose-l ,6diphosphatase [6] with concurrent change in enzyme activity has been reported. Experimental evidence for in vivo regulation of these enzymes by phosphorylation-dephosphorylation reaction is up till now absent. Phosphorylation of rat liver cell sap [779] or rat hepatocyte soluble proteins [lo] has been studied in the presence of added exogenous protein kinase. The effect of glucagon and catecholamines on the phosphorylation of supernatant proteins has been studied [lo] in intact hepatocytes where endogenous protein kinase was used. Although it was not proven, pyruvate kinase was indicated as one of the proteins of which the phosphorylation is greatly stimulated by these hormones. No evidence for a stimulated phosphorylation of fructose-l ,6-diphosphatase was found. We investigated the cyclic AMP-dependent phosphorylation of rat liver soluble proteins by endogenous protein kinase(s). Phosphorylated proteins were analysed by SDS-polyacrylamide gel electrophoresis and subsequent autoradiography. With this system we found that L-type pyruvate kinase is the major protein which incorporates 32P from [y-32P]ATP in a cyclic AMP-stimulated reaction. The phosphorylation of pyruvate kinase is greatly diminished in the presence of the substrate phosphoenolpyruvate and not influenced by the allosteric activator fructose-l ,6-diphosphate. Phosphorylation of fructose-l ,6-diphosphatase was not detectable in this system.

The importance of arginyl residues for phosphorylation of rat liver cell sap proteins.

Arginyl residues in phosvitin, histone and cell sap protein were blocked by 1,2-cyclohexanedione, resulting in markedly impaired phosphorylation of histone and cell sap. Interestingly, the phosphate incorporation into phosvitin was not changed by this treatment. Intact arginyl residues in the protein kinase substrates seemed to be essential for more than half of the cell sap phosphorylation at 5 mM ATP. Furthermore both phosvitin kinase and histone kinase activities in cell sap were inhibited by arginyl residue blockade, indicating that these enzymes had functional arginyl residues.

Endogenous substrates of protein kinase in rat liver cell sap under different dietary conditions

Liver cell sap from normally fed rats, rats fed with a highly-carbohydrate diet and fasted rats was chromatographed on DEAE-cellulose (pH 7.0). The chromatogram from each diet group was analyzed for pyruvate kinase activity and endogenous substrates of cyclic AMP-stimulated protein kinase. The materials were pooled into five phosphorylatable fractions, in each of which phosphate incorporation at 0.1 mM and 1.0 mM [ 32P]ATP in the presence of cyclic AMP and protein kinase was determined. For characterization of the phosphorylatable components, thin-layer gel chromatography on Sephadex G-200 and polyacrylamide gel electrophoresis in detergent were used for determination of native and minimal molecular weights, respectively. Except for pyruvate kinase, eight components which incorporated at least 0.05 nmol of [ 32P]phosphate/g of liver were detected. The phosphorylation of four of them was stimulated by cyclic AMP. Their minimal molecular weights were 42 000, 21 000, 52 000 and 49 000. The component with a minimal molecular weight of 42 000 seemed to have a native molecular weight of 160 000. Both the 21 000 and the 52 000 component had a native molecular weight of about 110 000–120 000. The protein with a minimal molecular weight of 49 000 could not be correlated with certainty to a native molecular weight. The proteins whose phosphorylation was not stimulated by cyclic AMP had minimal molecular weights of 54 000, 39 000, 34 000 and 22 000.

Rapid proteolytic removal of phosphopeptides and phosphorylatable sites from proteins in rat liver cell SAP

Rapid proteolytic removal of phosphopeptides and phosphorylatable sites from proteins in rat liver cell sap

Relative in vivo and in vitro incorporation of labelled leucine into pig liver and muscle proteins

1. 1. Relative incorporation of 3H-leucine in vivo into pig liver proteins expressed as counts/min/ per mg protein, was 4.0 times greater than that incorporated into skeletal muscle proteins after the administration of the labelled amino acid. RNA concentration was 4.4 times greater in the liver than in the muscle. 2. 2. Relative incorporation of C 14-leucine into proteins by rat liver post-mitochondrial fraction in cell-free amino acid incorporating system was 2.8 and 4.1 times greater than that incorporated into liver and muscle proteins by pig liver and muscle post-mitochondrial fractions. 3. 3. Amino acid incorporating activity of pig liver post-mitochondrial fraction could be destroyed by incubating the preparations with 0.2 fig/red pancreatic ribonuclease. Homogenization of pig liver in rat liver cell-sap (which exhibits relatively high ribonuclease inhibitor activity) in the presence and absence of yeast RNA increased the relative incorporation by 30% and 20%, respectively. 4. 4. The relationship between RNA concentration and intensity of protein synthesis and the difficulties encountered in comparing amino acid incorporating activity of the liver and muscle post-mitochondrial preparations in cell-free systems have been discussed.

Status of tRNA charging, trinucleotide acceptor sequence and tRNA nucleotidyltransferase activity in the human placenta

Samples of tRNA isolated from the cell sap of full-term human placenta were found to have a low capacity for accepting amino acids in the presence of partially purified synthetase preparations made from placental or rat liver cell sap. Gel electrophoresis of placental tRNA showed that part of this could be accounted for by gross degradation. The proportion of chargeable tRNA carrying amino acids was estimated by periodate oxidation followed by stripping and then charging with labeled amino acids. Only 50% of chargeable placental tRNA was in the charged state when isolated, whereas 87% of freshly isolated rat liver tRNA was found to be charged with amino acids. A fraction from placental cell sap was shown to have tRNA nucleotidyltransferase activity. When placental tRNA was incubated with this fraction and [3H]ATP or [3H]CTP, ATP was incorporated into about 12% of the tRNA molecules and CTP into 5-7%. When rat liver tRNA was used in place of placental tRNA, [3H]ATP was incorporated into less than 5% of the tRNA molecules. By using snake-venom diesterase over short periods of incubation, it was confirmed that the ATP had been incorporated terminally as AMP into the placental tRNA. These observations show that, in contrast to rat liver tRNA, tRNA prepared from human placenta is poorly charged with amino acids, many of the molecules lack the acceptor trinucleotide and there is extensive degradation beyond this stage.

On the mode of action of clofibrate on lipid metabolism. Inhibition of rat liver microsomal fatty acid synthesis

Evidence is presented indicating that clofibrate in rat liver cell sap, at a concentration below 10 m m, inhibits de novo fatty acid synthesis from malonyl-CoA by about 30%. Microsomal chain elongation, the main system responsible for hepatic synthesis of saturated and unsaturated fatty acids with a chain length higher than C 16, at the same concentration, is reduced by about 70%. On the other hand, the mitochondrial chain elongation system is not significantly affected at all the concentrations used. These results point out that the hypolipidemic effect of clofibrate, besides inhibition of de novo formation of palmitate, may be due to a reduced synthesis of higher and unsaturated fatty acids, which represent the main components of plasma lipids. Moreover they furnish further support that fatty acid chain elongation in rat liver microsomes and mitochondria is carried out by two different enzymatic systems.

The response of the small intestine to vitamin D. Isolation and properties of chick intestinal polyribosomes.

Undegraded polyribosome preparations may be obtained from chick intestinal mucosa if ribonuclease activity is strictly controlled. This is best achieved by homogenization of the mucosa directly in rat liver cell-sap. 2. The extent of amino acid incorporation by chick intestinal polyribosomes is greatly influenced by the source of the cell-sap. Sephadex-treated intestinal cell-sap caused impaired incorporation and release of completed polypeptide chains, whereas Sephadex-treated rat liver cell-sap promoted the polymerization of up to 90 amino acids per ribosome. Under optimum conditions 30-35% of the nascent polypeptide chains are completed and released. 3. The preparation of an antiserum against the calcium-binding protein formed in response to vitamin D is described. It is shown that the antiserum is highly specific for calcium-binding protein. 4. This antiserum was used to investigate the ability of chick intestinal polyribosomes to synthesize calciumbinding protein. Only polyribosomes from chicks receiving vitamin D have the ability to synthesize calcium-binding protein. Moreover, the product formed in vitro has the same electrophoretic mobility as calcium-binding protein synthesized in vivo. 5. It is concluded that one of the main functions of vitamin D in the small intestine is to induce the synthesis de novo of calcium-binding protein.

Phosphorylation of rat liver cell sap protein on incubation with [ 32P]ATP

1. 1. The aim of the present work was to obtain an overall view of the [ 32P]ATP-dependent phosphate turnover of the soluble rat liver phosphoproteins with phosphoryl groups bound to serine and threonine residues. Rat liver cell sap was therefore incubated with [ 32P]ATP under different conditions. The protein took up 32 P to a considerable extent. The highest 32P labelling of the protein was obtained on incubation with 5 mM [ 32P]ATP for 1 h at 30 °C, and corresponded to an incorporation of 55 nmoles of phosphate into the cell sap protein from 1 g of wet tissue. The total amount of protein-bound phosphate of this cell sap was 148 nmoles, as determined by chemical analysis. The ratio of phosphorylserine ([ 32P]Ser(P)) to phosphorylthreonine ([ 32P]Thr(P)) isolated from acid hydrolysates of [ 32P]ATP-incubated cell sap was 6–10. 2. 2. From the initial rates of the 32P labelling it was concluded that the phosphoryl could not depend appreciably on the formation of intermediate phosphoryl enzymes. The rate was compatible with that of protein kinase reactions. 3. 3. Cyclic 3′, 5′-AMP was shown to stimulate the 32P incorporation. 4. 4. The presence of high phosphoprotein phosphatase activity was apparent when all [ 32P]ATP had been consumed or removed by gel chromatography. 5. 5. It is assumed that most phosphoproteins of rat liver cell sap are enzymes and other proteins which are regulatively phosphorylated by protein kinases.

Cyclic 3',5'-AMP-stimulated and non-stimulated phosphorylation of protein fractions from rat-liver cell sap on incubation with (gamma-32P)ATP.

Rat-liver cell sap protein was separated into four fractions by pH 5.5 precipitation and ammonium sulfate fractionation. On incubation with (32P)ATP, protein of two of the fractions was phosphorylated, showing the presence of protein kinase activity and endogenous protein substrates. The phosphorylation of one of the fractions was markedly stimulated by cyclic 3′,5′-AMP. Strong evidence was obtained that this phosphate-incorporating material was neither active phosphorylase, phosphorylase kinase nor glycogen synthetase. The two phosphate-incorporating fractions were chromatographed on DEAE-cellulose with stepwise elution. Several subtractions contained material which was phosphorylated on incubation with (32P)ATP. The phosphorylation of two subfractions was greatly stimulated by cyclic 3′,5′-AMP. By polyacrylamide gel electrophoresis in detergent of (32P)ATP-incubated material from these fractions, three major 32P-labelled components with estimated molecular weights of 62000, 55000 and 47 000 were demonstrated. It is supposed that these components are derived from enzymes or other proteins with functions regulated by phosphorylation-dephosphorylation reactions.

Gel electrophoresis of RNA under denaturing conditions

1. We have studied the electrophoretic mobility of RNAs in acrylamide gels under denaturing conditions, using 8 M urea and low salt buffer at 60 °C. In these “urea gels” the mobilities of a number of RNAs in the molecular weight range of 0.5 · 10 6–1.5 · 10 6 are inversely related to their log molecular weights. 2. We have determined molecular weights in “urea gels” of a number of RNAs for which controversial estimates were available, including phage Q β RNA (1.4 · 10 6), cell-sap rRNAs of Tetrahymena pyriformis ( 0.59+0.59 · 10 6 and 0.52 · 10 6) and Crithidia luciliae ( 0.83+0.56 · 10 6 and 0.74 · 10 6) and some mitochondrial rRNAs. 3. We have redetermined the molecular weights of the large subunit rRNAs from Escherichia coli (1.13 · 10 6) and rat-liver cell sap (1.57 · 10 6), phage MS2 RNA (1.23 · 10 6) and the small subunit rRNA from yeast cell sap (0.72 · 10 6) by sedimentation equilibrium. Only the latter value is significantly different from that reported by others (0.62 · 10 6). 4. For a number of viral and rRNAs relative gel electrophoretic mobilities were determined at a range of temperatures, in a standard neutral salt buffer. No simple relationship was found between the variations in mobility and the gross characteristics of the RNAs. Differences in relative gel electrophoretic mobilities of RNAs in the Tris-phosphate-EDTA buffer introduced by Loening, and the Tris-borate-EDTA buffer introduced by Peacock and Dingman, can be attributed to differences in ionic concentrations.

Presence of an inhibitor of globin mRNA translation in rat liver cell sap

Presence of an inhibitor of globin mRNA translation in rat liver cell sap