bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies on acetyl-CoA carboxylase and fatty acid synthase from rat mammary gland and mammary tumours
Authors: Ahmad P M , Feltman D S , Ahmad F
Published: August 2015

The activities of two lipogenic enzymes, acetyl-CoA carboxylase and fatty acid synthase, were determined in two transplantable mammary adenocarcinomas (13762 and R3230AC) carried by non-pregnant, pregnant and lactating rats, and in mammary tissue of control animals (non-tumour-carrying) of comparable physiological states. During mammary-gland differentiation of control or tumour-carrying animals, the activities of acetyl-CoA carboxylase and fatty acid synthase in the lactating gland increased by about 40-50-fold over the values found in non-pregnant animals. On the other hand, in tumours carried by lactating dams there were only modest increases (1.5-2-fold) in acetyl-CoA carboxylase and fatty acid synthase compared with the neoplasms carried by non-pregnant animals. On the basis of the Km values for different substrates and immunodiffusion and immunotitration data, the fatty acid synthase of neoplastic tissues appeared to be indistinguishable from the control mammary-gland enzyme. However, a comparison of the immunotitration and immunodiffusion experiments indicated that the mammary-gland acetyl-CoA carboxylase might differ from the enzyme present in mammary neoplasms.
The effect of chloroquine on the distribution of newly synthesized and old β-hexosaminidase in fibroblasts
Authors: Vladutiu Georgirene D.
Published: August 2015

Most of the newly synthesized β-N-acetyl-d-glucosaminidase (EC 3.2.1.30; β-hexosaminidase) in normal fibroblast cultures is excreted during 24h incubation with serum-free medium. In this study, this new enzyme only comprises about one-half of the excreted pool as determined by a near total inhibition of [14C]leucine incorporation into the excreted enzyme in the presence of cycloheximide, with only a 46% reduction in enzyme activity. These data indicate that nearly equal fractions of new and old enzyme are normally excreted by fibroblasts. Incubation of normal fibroblast cultures with chloroquine (25 μm) for 24h doubled the amount of extracellular β-hexosaminidase activity from 15% to 37% of total culture activity while reducing the incorporation of [14C]leucine into intra- and extracellular enzyme by 66 and 29% of control, respectively. Therefore, it appears that chloroquine inhibited enzyme synthesis while enhancing the excretion of old as well as newly synthesized enzyme. Chloroquine and cycloheximide together reduced the [14C]leucine incorporation into intracellular enzyme by more than either agent alone, indicating a combined effect on enzyme synthesis and/or degradation. β-Hexosaminidase-deficient fibroblasts that contained endocytosed enzyme spontaneously excreted 10% of their enzyme during 24h incubation with serum-free medium and 18% in the presence of mannose 6-phosphate (2 mm). These results indicated that about one-half of the excreted enzyme still possessed its phosphomannosyl recognition residues and actually re-entered the cells. Chloroquine stimulated the excretion of an addition 15% of the total endocytosed enzyme at 48 and 72h after endocytosis. These data suggest that new, old and endocytosed β-hexosaminidase are all excreted by fibroblasts, that this excretion is enhanced by chloroquine, and that a fraction of the excreted enzyme retains its phosphomannosylated residues needed for re-uptake and transport inside the cells.
Regulation of lipogenic capacity in lactating rats
Authors: Grigor M R , Geursen A , Sneyd M J , Warren S M
Published: August 2015

1. The rate of mammary-gland lipogenesis measured in vivo from 3H2O was suppressed after decreasing the milk demand by decreasing the number of pups from ten to two or three, as well as by giving diets containing lipid [Grigor & Warren (1980) Biochem. J. 188, 61-65]. 2. The specific activities of the lipogenic enzymes fatty acid synthase, glucose 6-phosphate dehydrogenase and ‘malic’ enzyme increased between 6- and 10-fold in the mammary gland and between 2- and 3-fold in the livers during the first 10 days of lactation. The increases in specific activity coupled with the doubling of liver mass which occurred during pregnancy and lactation resulted in considerable differences in total liver activities when compared with virgin animals. 3. Although consumption of a diet containing 20% peanut oil suppressed the activities of the three lipogenic enzymes in the livers, only the ‘malic’ enzyme was affected in the mammary glands. 4. In contrast, decreased milk demand did not affect the specific activities of any of the liver enzymes, whereas it resulted in suppression of all three lipogenic enzymes of the mammary glands. There was no effect on either the cytoplasmic malate dehydrogenase or the lactate dehydrogenase of the mammary gland. 5. In all the experiments performed, the activity of the fatty acid synthase correlated with the amount of material precipitated by the rabbit antibody raised against rat fatty acid synthase.
Effect of cytosol on transport of RNA in vitro during cardiac hypertrophy
Authors: Meenakshi S , Thirunavukkarasu C , Rajamanickam C
Published: August 2015

An enhanced RNA-transport activity was observed in vitro from nuclei obtained from animals with cardiac hypertrophy as compared with that of sham-operated controls. The 100 000 g supernatants obtained from hypertrophic hearts stimulated the RNA transport from nuclei of sham-operated controls, and this stimulation was maximum with 40% supernatant. Ca2+- and nucleic acid-dependent ATPase and alkaline phosphatase activities, which may be involved in an energy-dependent transport, were high in nuclei from hypertrophic hearts, and the nuclei of sham-operated animals showed higher activities of these enzymes after incubation with supernatant from hypertrophic hearts, which stimulates the RNA transport in vitro from nuclei of sham-operated animals.
The regulatory properties of rabbit muscle pyruvate kinase. The influence of effector concentrations
Authors: Gregory R B , Ainsworth S , Kinderlerer J
Published: August 2015

The initial velocity of the reaction catalysed by rabbit muscle pyruvate kinase was studied as a function of the concentrations of the modifiers phenylalanine and fructose 1,6-bisphosphate under conditions where the relationships between the initial velocities and the concentrations of substrates are non-hyperbolic. It is shown that these data can be represented by the exponential model for a regulatory enzyme.
Purification and properties of the membrane-bound by hydrogenase from Desulfovibrio desulfuricans
Authors: Lalla-Maharajh W V , Hall D O , Cammack R , Rao K K , Le Gall J
Published: August 2015

The membrane-bound hydrogenase from the anaerobic sulphate-reducing bacterium Desulfovibrio desulfuricans (Norway strain) has been purified to homogeneity, with an overall 80-fold purification and a specific activity of 70 mumol of H2 evolved/min per mg of protein. The hydrogenase had a relative molecular mass of 58 000 as determined by gel filtration and was estimated to contain six iron atoms and six acid-labile sulphur groups per molecule. The absorption spectrum of the enzyme was characteristic of an iron-sulphur protein. The E400 and E280 were 28 500 and 109 000 M-1.cm-1 respectively. The e.s.r. of the oxidized protein indicated the presence of [4Fe-4S]3+ or [3Fe-3S]3+, and another paramagnetic centre, probably Ni(III). The hydrogenase was inhibited by heavy-metal salts, carbon monoxide and high ionic strength. However, it was resistant to inhibition by thiol-blocking and metal-complexing reagents. N-Bromosuccinimide totally inhibited the enzyme activity at low concentrations. The enzyme was stable to O2 over long periods and to high temperatures. It catalyses both H2-evolution and H2-uptake with a variety of artificial electron carriers. D. desulfuricans cytochrome C3, its natural electron carrier, had a high affinity for the enzyme (Km = 2 microns). Rate enhancement was observed when cytochrome C3 was added to Methyl Viologen in the H2-evolution assay. The pH optimum for H2-evolution was 6.5.
Low-frequency vibrations of helical structures in protein molecules
Authors: Chou K C
Published: August 2015

A physically intuitive and mathematically easily handled formula is presented for calculating the low-frequency vibrations of helical structures in protein molecules. alpha-Chymotrypsin is taken as an example, and the calculated result shows precise agreement with observations of the low-frequency Raman spectra. As reflected in the formula, this kind of low frequency is very sensitive to the conformation of a biomacromolecule, and can therefore serve as a vehicle to investigate the mechanism of action of a biomacromolecule from the viewpoint of dynamics. On this basis a feasible experiment is suggested by which one can examine the relationship between a presumed mode of low-frequency vibration in a biomacromolecule and its activity.
Metabolism of 3-deoxy-3-fluoro-d-mannose and 4-deoxy-4-fluoro-d-mannose by Saccharomyces cerevisiae S288C
Authors: Grier T J , Rasmussen J R
Published: August 2015

Incubation of Saccharomyces cerevisiae S288C with 4-deoxy-4-fluoro-D-[1-14C]-mannose resulted in the formation of three metabolites that were characterized as 4-deoxy-4-fluoro-D-[1-14C]mannose 1,6-bisphosphate, 4-deoxy-4-fluoro-D-[1-14C]-mannose 6-phosphate and GDP-4-deoxy-4-fluoro-D-[1-14C]mannose. In addition, radioactive material was incorporated into a particulate fraction composed primarily of cell-wall polysaccharides. Compared with the 4-fluoro sugar, 3-deoxy-3-fluoro-D-[1-14C]mannose was not transported into yeast cells as well, and its conversion into sugar nucleotide was much less efficient. Metabolites that were isolated after incubation with the 3-fluoro analogue were identified as 3-deoxy-3-fluoro-D-[1-14C]mannose 1,6-bisphosphate, 3-deoxy-3-fluoro-D-[1-14C]mannose 6-phosphate and GDP-3-deoxy-3-fluoro-D-[1-14C]mannose. Little radioactivity was transferred into the cell-wall fraction.
Partial characterization of the phosphotransferase system of human central-nervous-system myelin
Authors: Wu N C , Yourist J E , Rector W D , Ahmad F
Published: August 2015

The phosphotransferase system of human central-nervous-system myelin was investigated. Evidence obtained indicated the presence of at least two different phosphotransferase systems (cyclic nucleotide-dependent and -independent) in myelin, which were found to be firmly associated with the membrane. The cyclic AMP-dependent kinase of myelin and white-matter cytosol preferentially phosphorylated certain histone fractions and displayed only modest activity with basic protein as substrate. On the other hand, the cyclic nucleotide-independent system showed specificity toward basic protein. Its activity was not only dependent on Mg2+ but it was greatly enhanced by this bivalent cation. Whereas the cyclic nucleotide-dependent kinase could be extracted with buffers containing Triton X-100, the bivalent cation-regulated kinase resisted solubilization from myelin under these conditions.
Exogenous phospholipase enzymes mimic effects of phenylephrine on Ca2+ transport in hepatocytes
Authors: Barritt G J , Whiting J A
Published: August 2015

Phospholipase C from Clostridium perfringens induced the release of 45Ca2+ from isolated rat hepatocytes incubated at 0.1 mM extracellular Ca2+ with a time course similar to that for the action of phenylephrine. Under the conditions of these experiments, no significant damage to the plasma membrane was detected in the presence of phospholipase C. Little 45Ca2+ release was induced by bee venom phospholipase A2. At 1.3 mM extracellular Ca2+, both phospholipase enzymes stimulated the initial rate of 45Ca2+ exchange. Concentrations of phospholipase C comparable with those that stimulated 45Ca2+ release increased the rates of glucose release and O2 utilization by 70 and 20% respectively. An increase in the rate of O2 utilization but not glucose release was observed after the addition of phospholipase A2 to hepatocytes. The possible role for a cellular phospholipase C in the mechanism by which phenylephrine stimulates glycogenolysis in the liver cell is briefly discussed.
Differences in polyamine availability and insertion into fibronectins released from normal and transformed cells
Authors: Roch A M , Quash G , Ripoll H , Mardon M
Published: August 2015

1. Fibronectin released from transformed rat kidney cells compared with that released from normal rat kidney cells shows a 50% increase in amino group availability. 2. No such changes were observed in thiol and carboxy group availability or in sialic acid content. 3. The increased amino group availability is not due to a greater polyamine content, which was about 0.04 pmol/mg of protein. 4. Transglutaminase mediated the insertion of spermidine into normal cell fibronectin with linear kinetics. With fibronectin from transformed cells (temperature-sensitive mutant or wild-type), kinetics typical of substrate inhibition were observed. 5. Immunochemical analysis with an anti-polyamine antiserum and an anti-(human fibronectin) antiserum showed that fibronectins from normal and transformed cells react differently. The significance of these results is discussed in the light of changes in the secondary structure between the two fibronectins.
Absence of down-regulation of the insulin receptor by insulin. A possible mechanism of insulin resistance in the rat
Authors: Walker A P , Flint D J
Published: August 2015

Insulin resistance occurs in rat adipocytes during pregnancy and lactation despite increased or normal insulin binding respectively; this suggests that a post-receptor defect exists. The possibility has been examined that, although insulin binding occurs normally, internalization of insulin or its receptor may be impaired in these states. Insulin produced a dose-dependent reduction in the number of insulin receptors on adipocytes from virgin rats maintained in culture medium, probably due to internalization of the hormone-receptor complex. In contrast, adipocytes from pregnant and lactating rats did not exhibit this ‘down-regulation’ phenomenon. Down regulation was, however, apparent in all groups when the experiments were performed in Tris buffer (where receptor recycling is inhibited), suggesting that in pregnant and lactating rats insulin receptors are rapidly recycled back to the plasma membrane, whereas in virgin rats this recycling process is less effective. Internalization of insulin was also determined by using 125I-labelled insulin. Adipocytes from pregnant and lactating rats appeared to internalize similar amounts of insulin to virgin rats. In the presence of the lysosomal inhibitor chloroquine, adipocytes from pregnant rats internalized more insulin than virgin or lactating rats. These results suggest that adipocytes from pregnant and lactating rats internalize insulin and its receptor normally, whereas intracellular processing of the insulin receptor may differ from that in virgin rats. In addition the rate of lysosomal degradation of insulin may be altered in adipocytes from pregnant rats.
Oxidative phosphorylation in Escherichia coli. Characterization of mutant strains in which F1-ATPase contains abnormal β-subunits
Authors: Senior A E , Langman L , Cox G B , Gibson F
Published: August 2015

To facilitate study of the role of the beta-subunit in the membrane-bound proton-translocating ATPase of Escherichia coli, we identified mutant strains from which an F1-ATPase containing abnormal beta-subunits can be purified. Seventeen strains of E. coli, characterized by genetic complementation tests as carrying mutations in the uncD gene (which codes for the beta-subunit), were studied. The majority of these strains (11) were judged to be not useful, as their membranes lacked ATPase activity, and were either proton-permeable as prepared or remained proton-impermeable after washing with buffer of low ionic strength. A further two strains were of a type not hitherto reported, in that their membranes had ATPase activity, were proton-impermeable as prepared, and were not rendered proton-permeable by washing in buffer of low ionic strength. Presumably in these two strains F1-ATPase is not released in soluble form by this procedure. F1-ATPase of normal molecular size were purified from strains AN1340 (uncD478), AN937 (uncD430), AN938 (uncD431) and AN1543 (uncD484). F1-ATPase from strain AN1340 (uncD478) had 15% of normal specific Mg-dependent ATPase activity and 22% of normal ATP-synthesis activity. The F1-ATPase preparations from strains AN937, AN938 and AN1543 had respectively 1.7%, 1.8% and 0.2% of normal specific Mg-dependent ATPase activity, and each of these preparations had very low ATP-synthesis activity. The yield of F1-ATPase from the four strains described was almost twice that obtained from a normal haploid strain. The kinetics of Ca-dependent ATPase activity were unusual in each of the four F1-ATPase preparations. It is likely that these four mutant uncD F1-ATPase preparations will prove valuable for further experimental study of the F1-ATPase catalytic mechanism.
A requirement for Ca2+ in the extraction of O2-evolving Photosystem 2 preparations from the cyanobacterium Anacystis nidulans
Authors: England R R , Evans E H
Published: August 2015

Ca2+ has been shown to be essential for the retention of maximal O2-evolving activity in Photosystem 2 particles extracted by using dodecyldimethylamine oxide from Anacystis nidulans thylakoids. The effect cannot entirely be mimicked by using Mg2+. Ca2+ stimulates electron transport from diphenylcarbazide to 2,6-dichloroindophenol catalysed by lead-inhibited cation-free preparations, showing the presence of two cation-binding sites in these particles. Photosystem 2 preparations extracted in Ca2+-containing buffer show the presence of three polypeptides at mol. wt. 30000, 33000 and 36000, which are absent or much decreased in preparations extracted in Mg2+-containing buffer. The calmodulin antagonist chlorpromazine inhibits activity of the Photosystem 2 preparation, suggesting the presence of a Ca2+-binding protein.
The effect of phenobarbital on the transcriptional activity of liver
Authors: Hardwick J P , Schwalm F , Richardson A
Published: August 2015

The effect of phenobarbital on the transcriptional activity of liver was studied by measuring the synthesis of RNA by suspensions of hepatocytes isolated from rats treated with phenobarbital for various time periods. The absolute rates of RNA synthesis by isolated hepatocytes were determined by measuring the incorporation of [3H]orotic acid into RNA as UMP and the specific radioactivity of the UTP pool. The specific radioactivity of the UTP extracted from hepatocytes isolated from phenobarbital-treated rats was consistently lower than that of the UTP pool of hepatocytes from untreated rats. Phenobarbital treatment increased the rate of RNA synthesis 10-fold over that observed for hepatocytes from untreated rats. The maximum rate of RNA synthesis was observed 16-18 h after phenobarbital administration. Phenobarbital treatment also affected the nuclear-cytoplasmic transport of RNA by isolated hepatocytes. Immediately after phenobarbital treatment, the transport of RNA decreased; however, 24 h after phenobarbital administration, the transport of RNA was increased 4-fold. An increase in the synthesis of RNA in vivo by liver was found 18 h after phenobarbital treatment, and the incubation of suspensions of hepatocytes with various concentrations of phenobarbital increased RNA synthesis significantly.
Identification and origin of oestradiol-binding protein in immature rat skin. Demonstration of oestrophilic α-foetoprotein synthesis and secretion by developing rat skin explants in culture
Authors: Ali M , Mujoo K , Sahib M K
Published: August 2015

Oestrophilic alpha-foetoprotein (alpha FP) is found in high concentrations in developing rat skin cytosol. Elevated levels of alpha FP observed in foetal-rat skin decreased during development, and the protein became undetectable after 3 weeks of postnatal life. The developmental profile of alpha FP in skin is different from that in foetal blood. alpha FP in skin arises as a result of its synthesis in situ in the epidermal cells. Synthesis of alpha FP in skin is demonstrated by linear incorporation of [14C]leucine into immunoprecipitable, intracellular alpha FP by skin explants during 6 h in culture. Secretion is demonstrated by incorporation into alpha FP in culture medium. The rate of alpha FP synthesis in skin also declined with age and its synthesis is completely switched off 2 weeks after birth. The skin alpha FP level during development is regulated by controlling the rate of its synthesis in skin. alpha FP synthesized and secreted by skin is immunologically, electrophoretically and, with respect to molecular weight and oestradiol-binding properties, similar to that found in foetal serum. alpha FP was also identified as the major oestradiol-binding protein present in newborn-rat skin or secreted by newborn-rat skin explants in culture.
Transport of myo-inositol into endoneurial preparations of sciatic nerve from normal and streptozotocin-diabetic rats
Authors: Gillon K R W , Hawthorne J N
Published: August 2015

myo-Inositol transport by a viable rat sciatic-nerve preparation is described. Such ‘endoneurial’ nerve preparations accumulated myo-inositol by an energy-dependent saturable system. Streptozotocin-diabetes reduced myo-inositol transport into sciatic nerve by approx. 40%. Elevated medium glucose concentration reduced myo-inositol transport into control nerves to a similar extent. Fructose and sorbitol did not inhibit myo-inositol transport. Inclusion of an aldose reductase inhibitor in the medium counteracted the reduced myo-inositol transport caused by elevated glucose concentration. The importance of these results to the problem of diabetic neuropathy is discussed.
Specificity and properties of the nucleotide carrier in chromaffin granules from bovine adrenal medulla
Authors: Weber A , Westhead E W , Winkler H
Published: August 2015

1. The influence of various substances on the uptake of [3H]ATP and [14C]-noradrenaline into isolated bovine chromaffin granules was investigated. The carrier-mediated [3H]ATP uptake is specifically inhibited by SO42-, PO43- and phosphoenolpyruvate. Compounds with carboxylic acid or sulphonic acid groups had no significant inhibitory effects on either uptake. 2. 35SO42-, 32PO43- and phosphoenol[14C]pyruvate are taken up into chromaffin granules by a temperature-dependent process that is inhibited by atractyloside, uncouplers of oxidative phosphorylation and lipid-permeant anions. The apparent Km of 35SO42- uptake is 0.4 mM. 3. These results indicate that the nucleotide carrier in chromaffin granules has a broad specificity, transporting compounds with two strong negative charges. 4. Amino acid probes influence the uptake of ATP and catecholamines differently. Pyridoxal phosphate inhibits both uptake processes, 4,4′-di-isothiocyanostilbene-2,2′-disulphonic acid preferentially blocks ATP uptake, whereas phenylglyoxal blocks only ATP transport. It is suggested that the nucleotide carrier possesses arginine residues in a functionally important position. 5. The significance of these results obtained on isolated granules for the function of chromaffin granules within the cell is discussed.
The contribution of phenylalanine to tyrosine metabolism in vivo. Studies in the post-absorptive and phenylalanine-loaded rat
Authors: Moldawer L L , Kawamura I , Bistrian B R , Blackburn G L
Published: August 2015

1. Rates of appearance and oxidation of plasma L-leucine, L-phenylalanine and L-tyrosine, as well as conversion of plasma phenylalanine into plasma tyrosine, were determined in 90-120 g rats after overnight starvation and while receiving 115-120 mumol of L-phenylalanine/h. 2. In the post-absorptive state, plasma tyrosine and phenylalanine appearances were similar, despite the fact that 22% of plasma tyrosine appearance could be attributed to the hydroxylation of phenylalanine. 3. A constant infusion of 115-120 mumol of L-phenylalanine/h did not significantly alter plasma leucine kinetics, but increased appearance of plasma phenylalanine and tyrosine. The percentage of phenylalanine and tyrosine appearance that was oxidized increased from 12.1% and 24.4% to 37.3% and 48.0% respectively. In phenylalanine-loaded rats, 72% of plasma tyrosine appearance could be attributed to the conversion of phenylalanine. 4. Whole-body tyrosine oxidation measured from a continuous infusion of either L-[14C]tyrosine or L-[14C]phenylalanine differed by 165%. 5. It can be concluded that, in the post-absorptive state, phenylalanine hydroxylation makes a substantial contribution to the plasma appearance of tyrosine and is significantly increased when phenylalanine is administered. The disposal of excess infused phenylalanine is a result of a greater percentage of plasma phenylalanine being converted into tyrosine and a greater proportion of tyrosine being further oxidized. However, apparent tyrosine oxidation rates estimated from plasma tyrosine specific radioactivities and appearance of expired 14CO2 during administration of [14C]tyrosine are underestimates of true rates, in part because tyrosine generated from phenylalanine hydroxylation is catabolized without freely equilibrating with the plasma compartment.
The effect of cysteine oxidation on isolated hepatocytes
Authors: Viña J , Saez G T , Wiggins D , Roberts A F C , Hems R , Krebs H A
Published: August 2015

Isolated hepatocytes incubated with 4mM-cysteine lose reduced glutathione, adenine nucleotides and intracellular enzymes, thus showing extensive membrane damage. The toxic effects of cysteine are enhanced by NH4Cl. Lactate, ethanol and unsaturated fatty acids afford significant protection against cysteine-induced cytoxicity. Addition of catalase to the incubation medium also protected against cysteine toxicity, indicating that H2O2 formed during the oxidation of cysteine is involved in the toxic effects observed. Under anaerobic conditions cysteine did not cause leakage of lactate dehydrogenase from cells, confirming that rapid autoxidation is an essential condition for development of the toxic effects of cysteine.
Chloramphenicol stimulates acid phosphatase activity in germinating cotton (Gossypium hirsutum) embryos
Authors: Bhargava R , Sachar R C
Published: August 2015

Low dosages of chloramphenicol (25-50 micrograms/ml) brought about a 2-4-fold stimulation of acid phosphatase activity in 48 h-germinated cotton (Gossypium hirsutum) embryos. However, at high concentrations of chloramphenicol (100-1000 micrograms/ml), there was a progressive decline in enzyme activity. The stimulatory effect of the drug on acid phosphatase activity was relatively specific, since no significant stimulation of activities of proteinase, deoxyribonuclease, ribonuclease, o-diphenolase and peroxidase was observed in germinating cotton embryos. Chloramphenicol, however, did promote the activities of isocitric lyase and alkaline phosphatase. Sephadex G-200 chromatography of the enzyme fraction revealed high (230 000)- and low (106 000)-molecular-weight multiple forms of acid phosphatase in the chloramphenicol-treated embryos, in contrast with a single molecular form (mol.wt. 106 000) in the untreated embryos. Thus the treatment of cotton embryos with chloramphenicol induced both a qualitative and a quantitative change in the acid phosphatase activity. Chloramphenicol-stimulated acid phosphatase activity was strongly inhibited when Pi was included in the germination medium. However, the control embryos showed less pronounced inhibition of enzyme activity in presence of Pi ions.
Biosynthesis of intestinal microvillar proteins. Processing of aminopeptidase N by microsomal membranes
Authors: Danielsen E M , Norén O , Sjöström H
Published: August 2015

The biosynthesis of small-intestinal aminopeptidase N (EC 3.4.11.2) was studied in a cell-free translation system derived from rabbit reticulocytes. When dog pancreatic microsomal fractions were present during translation, most of the aminopeptidase N synthesized was found in a membrane-bound rather than a soluble form, indicating that synthesis of the enzyme takes place on ribosomes attached to the rough endoplasmic reticulum. The microsomal fractions process the Mr-115 000 polypeptide, which is the primary translation product of aminopeptidase N, to a polypeptide of Mr 140 000. This was found to be sensitive to the action of endo-beta-N-acetylglucosaminidase H (EC 3.2.1.96), showing that aminopeptidase N undergoes transmembrane glycosylation during synthesis. The position of the signal sequence in aminopeptidase N was determined by a synchronized translation experiment. It was found that microsomal fractions should be added before about 25% of the polypeptide was synthesized to ensure processing to the high-mannose glycosylated form. This suggests that the signal sequence is situated in the N-terminal part of the aminopeptidase N. The size of the cell-free translation product in the absence of microsomal fractions was found to be similar to that on one of the forms of the enzyme obtained from tunicamycin-treated organ-cultured intestinal explants.
Effect of pH on malonyl-CoA inhibition of carnitine palmitoyltransferase I
Authors: Stephens T W , Cook G A , Harris R A
Published: August 2015

Malonyl-CoA inhibition of carnitine palmitoyltransferase I was found to be very pH-dependent. Malonyl-CoA concentrations causing 50% inhibition (I50) at pH 6.0, 6.5, 7.0, 7.5 and 8.0 were 0.04, 1, 9, 40 and 200 microM respectively. It is suggested that a lowering of intracellular pH, such as might occur in ketoacidosis, may attenuate hepatic fatty acid oxidation by increasing malonyl-CoA sensitivity of carnitine palmitoyltransferase I.
Metabolism of cartilage proteins in cultured tissue sections
Authors: Paulsson M , Sommarin Y , Heinegård D
Published: August 2015

The biosynthesis and turnover of cartilage proteins was studied in organ cultures of bovine tracheal-cartilage sections. In cultures labelled with [3H]leucine, more than 99% of the labelled macromolecules were retained in the sections. About half of the [3H]leucine-labelled protein was extracted with 4M-guanidinium chloride. The incorporation of [3H]leucine into protein extractable with guanidinium chloride was linear with time, after an initial delay of 20-25 min. The 148kDa and 36kDa cartilage proteins were major labelled components in this extract. The elimination of the proteins was studied by using a pulse-chase protocol. The 148kDa protein was found to have a very slow turnover, similar to that of proteoglycans, whereas the 36kDa protein was eliminated more rapidly.
The effects of starvation and re-feeding on glycogen metabolism in mouse tail skin
Authors: Harmon C S , Phizackerley P J R
Published: August 2015

Although the glycogen content of mouse tail skin was decreased during starvation and was restored on re feeding, the proportion of glycogen synthase in the I form remained constant throughout at about 10% of the total. During the phase of net glycogen synthesis 1.5h after access to food was restored, the concentration of UDP glucose was markedly increased and the proportion of phosphorylase in the a form was significantly decreased.
Effects of dibutyryl cyclic AMP on the syntheses of dolichol-linked saccharides and glycoproteins in cultured hepatoma cells. Correlation with the effect on the adhesiveness of the cells
Authors: Okamoto Y , Sakai H , Sato J , Akamatsu N
Published: August 2015

When the hepatoma cells (AH 70Btc, Clone 10-5) were cultured in the presence of 1 mM-dibutyryl cyclic AMP for 2 days, the incorporation of [14C]glucosamine into protein was increased over 2-fold. At the same time, dibutyryl cyclic AMP increased the incorporation of [14C]glucosamine into dolichol-linked N-acetylglucosamine and NN′-diacetylchitobiose about 1.5-fold and into dolichol-linked oligosaccharides about 3-fold. Analysis of cellular glycoproteins by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis after reduction showed that dibutyryl cyclic AMP specifically enhanced the glycosylation of a fibronectin-like glycoprotein with an apparent mol.wt. of 220 000 and two other high-molecular-weight glycoproteins (apparent mol.wts. 270 000 and 185 000). Increased glycosylation of the glycoproteins with mol.wts. of 220 000 and 185 000 was shown to be linked to increased synthesis of the polypeptide portion. In addition to the above effects, dibutyryl cyclic AMP enhanced the adhesiveness of AH 70Btc cells to glass surfaces. Both the effects on the glycosylation pathway and on adhesiveness of cells were reversed by further treatment of the cells with 1 microgram of tunicamycin/ml. The results indicated that dibutyryl cyclic AMP increased the synthesis of dolichol-linked oligosaccharides and N-glycosylation of proteins in AH 70Btc cells. The enhancement of adhesiveness may be mediated by the increased synthesis of dolichol-linked oligosaccharides and also may be related to the increased synthesis of fibronectin.
Insulin and growth factors stimulate the phosphorylation of a Mr-22000 protein in 3T3-L1 adipocytes
Authors: Blackshear P J , Nemenoff R A , Avruch J
Published: August 2015

Insulin, epidermal growth factor (EGF), platelet-derived growth factor, multiplication-stimulating activity and 10% foetal-calf serum each stimulated the phosphorylation of a cytosolic Mr-22000 acidic heat-stable protein in Swiss mouse 3T3-L1 adipocytes. Phosphorylation of this protein was not stimulated by isoprenaline or dibutyryl cyclic AMP. The effect of insulin was maximal (3-fold increase) by 10 min; half-maximal stimulation was observed at 70 pM-insulin. Both [32P]phosphoserine and [32P]phosphothreonine residues were present in the Mr-22000 protein after insulin- and growth-factor-stimulated phosphorylation, but no [32P]phosphotyrosine. The major site of insulin- and EGF-stimulated phosphorylation appeared to be a threonine residue, in contrast with previously studied insulin-stimulated phosphorylation of serine residues. Insulin treatment appeared to result in a shift of the protein toward the anode on isoelectric focusing. Insulin and EGF present simultaneously did not lead to phosphorylation beyond that seen with each hormone singly. We surmise that insulin, EGF and perhaps other growth factors may activate a common protein kinase or inhibit a common protein phosphatase in 3T3-L1 adipocytes which acts on the Mr-22000 protein.
Induction of glycolytic enzyme synthesis in proliferating fibroblasts. Study of phosphofructokinase, glucose phosphate isomerase and pyruvate kinase
Authors: Meienhofer M C , Dreyfus J C , Kahn A
Published: August 2015

Specific activity of phosphofructokinase is 7-8-fold higher in exponentially growing human fibroblasts than in quiescent cells, but the difference is considerably less pronounced for two other glycolytic enzymes, glucose phosphate isomerase and pyruvate kinase. The ratio of the F-type to L-type phosphofructokinase subunits is essentially the same in growing and resting cells, 4:1. F-type-phosphofructokinase-related antigen concentration is decreased in resting cells as compared with proliferating fibroblasts, but relatively less than the enzyme activity; the ratio of the enzyme activity to the antigen concentration (immunological specific activity) is therefore lower in resting than in growing fibroblasts. Synthesis of phosphofructokinase, as a percentage of the total protein synthesis, is about 30-fold greater during the proliferative phase than in quiescent cells, but this difference is only 3-4-fold for glucose phosphate isomerase and pyruvate kinase. Modulation of the synthesis of phosphofructokinase therefore seems to be responsible for the changes of its specific activity in function of cell proliferation. The appearance of some inactive cross-reacting material in quiescent cells is probably due to post-translational alteration of the pre-synthesized molecules. Compared with other glycolytic enzymes, such as glucose phosphate isomerase and pyruvate kinase, phosphofructokinase seems to be the (or one of the) preferential target of glycolytic induction in proliferating cells.
The effects of 24R,25-dihydroxycholecalciferol and of 1 α,25-dihydroxycholecalciferol on ornithine decarboxylase activity and on DNA synthesis in the epiphysis and diaphysis of rat bone and in the duodenum
Authors: Sömjen D , Binderman I , Weisman Y
Published: August 2015

The effect of cholecalciferol metabolites on ornithine decarboxylase activity and on DNA synthesis in developing long bones was investigated in vitamin D-depleted rats. In the epiphysis there was a 6.4-fold increase in ornithine decarboxylase activity 5 h after a single injection of 24R,25-dihydroxycholecalciferol but not of 24S,25-dihydroxycholecalciferol or other vitamin D metabolites. In comparison, in the diaphysis and duodenum, 1 alpha,25-dihydroxycholecalciferol, but not other vitamin D metabolites, caused a 3-3.5-fold increase in the enzyme activity. The enzyme activity in the tissues examined attained a maximal value at 5 h after the injection of the metabolites. The activity of ornithine decarboxylase in the epiphysial region increased dose-dependently as the result of a single injection of 24R,25-dihydroxycholecalciferol and attained a maximal value at a dose between 30 and 3000 ng. In addition, administration of 24R,25-dihydroxycholecalciferol, but not 24S,25-dihydroxycholecalciferol or other metabolites, caused within 24 h a 1.7-2.0-fold increase in [3H]thymidine incorporation into DNA of the epiphyses of tibial bones. In comparison, 1 alpha,25-dihydroxycholecalciferol caused a 1.5-fold increase in [3H]thymidine incorporation into DNA of the diaphyses and of the duodenum. The present data indicate that 24R,25-dihydroxycholecalciferol is involved in the regulation of epiphyseal growth, whereas 1 alpha,25,dihydroxycholecalciferol stimulates the proliferation of cells in the diaphysis of long bones and in the intestinal mucosa.
Biosynthesis and turnover of the phosphomannosyl receptor in human fibroblasts
Authors: Creek K E , Sly W S
Published: August 2015

The phosphomannosyl receptor mediates intracellular targeting of newly synthesized acid hydrolases to lysosomes, and is also expressed as a pinocytosis receptor on the cell surface of fibroblasts. We have purified the phosphomannosyl receptor from bovine liver and produced rabbit antibodies to the bovine receptor. The antibodies partially blocked pinocytosis of human spleen beta-glucuronidase by fibroblasts, a process mediated by the phosphomannosyl receptor. Affinity-purified antibodies to the phosphomannosyl receptor were used to study the biosynthesis and turnover of the receptor in human fibroblasts. Phosphomannosyl receptor immunoprecipitated after a 15 min pulse-labelling of fibroblasts with [35S]methionine exhibited an identical mobility on sodium dodecyl sulphate/polyacrylamide gels as purified bovine liver phosphomannosyl receptor. Pulse-chase experiments for up to 3 days provided no evidence for changes in molecular weight attributable to post-translational processing of the phosphomannosyl receptor. Turnover studies determined that the half-life of the phosphomannosyl receptor in normal human fibroblasts was 24-29 h. The half-life of the receptor was slightly longer (32 h) in I-cell disease fibroblasts and normal fibroblasts exposed to leupeptin (32 h), slightly shorter in fibroblasts exposed to NH4Cl (23 h) and saturating amounts of ligand (21 h) and unaffected in cells exposed to mannose 6-phosphate (24 h). These studies show that the turnover of the phosphomannosyl receptor in fibroblasts is very slow, in contrast with its rate of internalization in endocytosis, and that its rate of degradation is not greatly altered by a variety of agents that affect lysosomal protein turnover and/or receptor-mediated endocytosis. These results suggest that the degradative activities of the lysosomes do not play an important role in phosphomannosyl receptor turnover in cultured fibroblasts.
Postnatal hypoglycaemia and gluconeogenesis in the newborn rat. Delayed onset of gluconeogenesis in prematurely delivered newborns
Authors: Fernández E , Valcarce C , Cuezva J M , Medina J M
Published: August 2015

The concentrations of glucose and lactate in the blood and of liver glycogen, and the phosphoenolpyruvate carboxykinase activity in liver and kidney of term and preterm newborn rats, were studied during the first 6 h post partum. Rates of lactate turnover and gluconeogenesis in vivo from [U-14C]lactate at 3 h and 6 h post partum were also quantified. The development of the prolonged postnatal hypoglycaemia observed after birth in the premature newborn rat is associated with lower rates of glucose production through glycogenolysis and gluconeogenesis; liver glycogenolysis was the main contributing factor to the glucose available during the neonatal period studied in both groups. Delayed induction of liver phosphoenolpyruvate carboxykinase activity was observed in premature newborn rats. Renal phosphoenolpyruvate carboxykinase activity increased 72% from birth in preterm newborns, but only a 25% increase was found in term newborns during the same experimental period. The gluconeogenesis in vivo from [U-14C]lactate paralleled the appearance of cytosolic phosphoenolpyruvate carboxykinase activity in the liver of both groups of newborns. Blood lactate concentrations remained higher in preterm than in term newborns. The postnatal utilization of lactate via the gluconeogenic pathway in either group of newborns was always less than 20% of the total lactate used. The results presented are discussed in relation to the development of postnatal hypoglycaemia and gluconeogenesis in the premature newborn rat.
Measles-virus-persistent infection in BGM cells. Modification of the incorporation of [3H]arachidonic acid and [14C]stearic acid into lipids
Authors: Anderton P , Wild T F , Zwingelstein G
Published: August 2015

In BGM cells chronically infected with measles virus, although the composition of the phospholipids is unaltered, the fatty acid composition is modified. Uninfected, lytic and persistently infected cells were labelled with [3H]arachidonic acid and [14C]stearic acid and their metabolic fate analysed. No difference in the total incorporation was observed in the different systems. However, the [14C]stearic acid and [3H]arachidonic acid were incorporated up to 2-fold and 13-fold respectively greater into the neutral lipid of persistently infected compared with that of uninfected cells. Both radioactive fatty acids were specifically accumulated in the triacylglycerol and non-esterified fatty acids fractions. Lytically infected cells were similar to uninfected cells. Although there was no significant difference in the incorporation of radioactivity into the total phospholipid in either system, there was a large decrease in [3H]arachidonic acid incorporated into phosphatidylethanolamine and to a lesser extent phosphatidylcholine and phosphatidylinositol in persistently infected cells. [14C]Stearic acid incorporation was also reduced in phosphatidylcholine and phosphatidylethanolamine fractions of persistently infected cells.
Metabolic homoeostasis of l-threonine in the normally-fed rat. Importance of liver threonine dehydrogenase activity
Authors: Bird M I , Nunn P B
Published: August 2015

Threonine dehydratase, threonine aldolase and threonine dehydrogenase activities were assayed in livers of rats that had been normally-fed, starved for 72 h, fed a high-protein diet or normally-fed and injected with glucagon or cortisone. A modified continuous spectrophotometric assay for threonine aldolase overcame interference resulting from threonine dehydratase activity and revealed that threonine aldolase activity was very low in rat liver, irrespective of the metabolic state of the animal. The concentration of free threonine was determined in livers of animals subjected to the same treatments as described above. Using Michaelis-Menten kinetics to estimate enzyme activities in vivo at intracellular threonine concentrations it was calculated that in the normally-fed state, 87% of the threonine degraded was catabolized by threonine dehydrogenase. In other metabolic states (except in glucagon-treated animals) threonine dehydratase was the major enzyme catalysing threonine catabolism. It was concluded that threonine dehydrogenase activity plays a hitherto unrecognized role in the metabolic homoeostasis of threonine in the normally-fed rat and that this enzyme activity, in association with 2-amino-3-oxobutyrate CoA-ligase, accounts for the known rate of glycine formation from threonine in the rat.
Microsomal electron-transport reductase activities and fatty acid elongation in rat brain. Developmental changes, regional distribution and comparison with liver activity
Authors: Takeshita M , Tamura M , Yubisui T
Published: August 2015

Gestational and postnatal changes of microsomal NADH:cytochrome b5 reductase and NADPH:cytochrome c reductase activities were examined in rat brain. The specific activity of NADH:cytochrome b5 reductase was high at 18-19 days of gestational age, decreased to a minimum at 4 to 6 days after birth and increased thereafter. An essentially similar developmental pattern was observed for the specific activity of NADPH:cytochrome c reductase. In contrast, the specific activities of these reductases in liver microsomes were low, did not display a peak during gestation and increased steadily to a maximum at 40-50 days after birth. The rate of incorporation of [2-14C]malonyl-CoA into palmitoyl-CoA in brain microsomes was found to be high in the foetus, sharply decreased to a minimum at the time of birth and increased thereafter. The activity of fatty acid elongation in liver microsomes was much less than that in brain during gestation and increased rapidly after birth to values at 50-60 days 20-fold greater than the foetal activity. NADH and NADPH were equally effective for brain microsomal fatty acid elongation. Regional distribution of cytochrome reductase activities and the activity of fatty acid elongation showed the lowest specific activity in cerebellum. These results suggest that brain microsomal electron transport may be correlated with the developmental alteration in fatty acid elongation.
Hormonal control of fructose 2,6-bisphosphate concentration in isolated rat hepatocytes
Authors: Bartrons R , Hue L , Van Schaftingen E , Hers H G
Published: August 2015

The ability of glucagon and of adrenaline to affect the concentration of fructose 2,6-bisphosphate in isolated hepatocytes was re-investigated because of important discrepancies existing in the literature. We were unable to detect a significant difference in the sensitivity of the hepatocytes with regard to the effect of glucagon to initiate the interconversion of phosphorylase, pyruvate kinase, 6-phosphofructo-2-kinase and fructose 2,6-bisphosphatase, and also to cause the disappearance of fructose 2,6-bisphosphate. In contrast, we have observed differences in the time-course of these various changes, since the interconversions of phosphorylase and of pyruvate kinase were at least twice as fast as those of 6-phosphofructo-2-kinase and of fructose 2,6-bisphosphatase. When measured in a cell-free system in the presence of MgATP, the cyclic AMP-dependent interconversion of pyruvate kinase was 5-10-fold more rapid than those of 6-phosphofructo-2-kinase and of fructose 2,6-bisphosphatase. These data indicate that 6-phosphofructo-2-kinase and fructose 2,6-bisphosphatase are relatively poor substrates for cyclic AMP-dependent protein kinase; they also support the hypothesis that the two catalytic activities belong to a single protein. Adrenaline had only a slight effect on the several parameters under investigation, except for the activation of phosphorylase. In the absence of Ca2+ ions from the incubation medium, however, adrenaline had an effect similar to that of glucagon.
Fructose 2,6-bisphosphate in livers of genetically obese rats
Authors: Hue L , van de Werve G , Jeanrenaud B
Published: August 2015

Livers of genetically obese (fa/fa) rats, starved for 24 h, contained more fructose 2,6-bisphosphate, glucose 6-phosphate, fructose 6-phosphate and glycogen, and more pyruvate kinase and phosphofructokinase 2 activities, than livers of control lean rats. These changes may be explained in terms of cyclic AMP concentration, which was decreased in livers of obese starved rats.