bluuebunny/crossref_metadata_2025 huggingface dataset:
The selective release of phospholipase A2 by resident mouse peritoneal macrophages
Authors: Wightman P D , Dahlgren M E , Davies P , Bonney R J
Published: August 2015

Resident mouse peritoneal macrophages have three phospholipase activities: a phospholipase A2 active at pH 4.5, a Ca2+-dependent phospholipase A2 active at pH 8.5 and a phosphatidylinositol-specific phospholipase C activity. When macrophages are exposed to zymosan in culture, the cellular activity of pH-4.5 phospholipase A2 is diminished in a manner dependent on zymosan concentration and time of exposure, whereas the cellular activities of pH-8.5 phospholipase A2 and phospholipase C remain unchanged. The depletion of pH-4.5 phospholipase A2 activity from the cell is paralleled by a quantitative recovery of this activity in the culture medium in a manner similar to the cellular depletion and extracellular recovery of two lysosomal enzymes. This release is specifically elicited by an inflammatory substance such as zymosan, since macrophages incubated with 6 micrometer latex spheres retain pH-4.5 phospholipase A2 activity and lysosomal enzyme activities intracellularly.
Rat liver microsomes catalyse mannosyl transfer from GDP-d-mannose to retinyl phosphate with high efficiency in the absence of detergents
Authors: Shidoji Yoshihiro , De Luca Luigi M.
Published: August 2015

In the absence of detergent, the transfer of mannose from GDP-mannose to rat liver microsomal vesicles was highly stimulated by exogenous retinyl phosphate in incubations containing bovine serum albumin, as measured in a filter binding assay. Under these conditions 65% of mannose 6-phosphatase activity was latent. The transfer process was linear with time up to 5min and with protein concentration up to 1.5mg/0.2ml. It was also temperature-dependent. The microsomal uptake of mannose was highly dependent on retinyl phosphate and was saturable against increasing amounts of retinyl phosphate, a concentration of 15μm giving half-maximal transfer. The uptake system was also saturated by increasing concentrations of GDP-mannose, with an apparent Km of 18μm. Neither exogenous dolichyl phosphate nor non-phosphorylated retinoids were active in this process in the absence of detergent. Phosphatidylethanolamine and synthetic dipalmitoylglycerophosphocholine were also without activity. Several water-soluble organic phosphates (1.5mm), such as phenyl phosphate, 4-nitrophenyl phosphate, phosphoserine and phosphocholine, did not inhibit the retinyl phosphate-stimulated mannosyl transfer to microsomes. This mannosyl-transfer activity was highest in microsomes and marginal in mitochondria, plasma and nuclear membranes. It was specific for mannose residues from GDP-mannose and did not occur with UDP-[3H]galactose, UDP- or GDP-[14C]glucose, UDP-N-acetyl[14C]-glucosamine and UDP-N-acetyl[14C]galactosamine, all at 24μm. The mannosyl transfer was inhibited 85% by 3mm-EDTA and 93% by 0.8mm-amphomycin. At 2min, 90% of the radioactivity retained on the filter could be extracted with chloroform/methanol (2:1, v/v) and mainly co-migrated with retinyl phosphate mannose by t.l.c. This mannolipid was shown to bind to immunoglobulin G fraction of anti-(vitamin A) serum and was displaced by a large excess of retinoic acid, thus confirming the presence of the β-ionone ring in the mannolipid. The amount of retinyl phosphate mannose formed in the bovine serum albumin/retinyl phosphate incubation is about 100-fold greater than in incubations containing 0.5% Triton X-100. In contrast with the lack of activity as a mannosyl acceptor for exogenous dolichyl phosphate in the present assay system, endogenous dolichyl phosphate clearly functions as an acceptor. Moreover in the same incubations a mannolipid with chromatographic properties of retinyl phosphate mannose was also synthesized from endogenous lipid acceptor. The biosynthesis of this mannolipid (retinyl phosphate mannose) was optimal at MnCl2 concentrations between 5 and 10mm and could not be detected below 0.6mm-MnCl2, when synthesis of dolichyl phosphate mannose from endogenous dolichyl phosphate was about 80% of optimal synthesis. Under optimal conditions (5mm-MnCl2) endogenous retinyl phosphate mannose represented about 20% of dolichyl phosphate mannose at 15min of incubation at 37°C.
The effects of partial and selective reduction in the components of the proton-motive force on lactose uptake in Escherichia coli
Authors: Ahmed S , Booth I R
Published: August 2015

The relationship between the steady state lactose accumulation (delta plac) and the magnitude of the membrane potential (delta psi) and pH gradient (delta pH) has been studied at pHo5.5 and pHo7.5. An attempt has been made to differentiate between two possible means by which lactose accumulation may be reduced below the proton-motive force (delta p). Firstly, that delta psi and delta pH are not equivalent in driving lactose transport and secondly, that ‘slip’ reactions (beta-galactoside exit via the carrier without a proton) may reduce accumulation. The data support the latter; however, our conclusions are tempered by the observation that the apparent stoichiometry (delta plac/delta p) increases to a value of at least 2 at values of delta p below 130 mV.
Caspase 3 attenuates XIAP (X-linked inhibitor of apoptosis protein)–mediated inhibition of caspase 9
Authors: Denault Jean-Bernard , Eckelman Brendan P. , Shin Hwain , Pop Cristina , Salvesen Guy S.
Published: August 2015

During apoptosis, the initiator caspase 9 is activated at the apoptosome after which it activates the executioner caspases 3 and 7 by proteolysis. During this process, caspase 9 is cleaved by caspase 3 at Asp330, and it is often inferred that this proteolytic event represents a feedback amplification loop to accelerate apoptosis. However, there is substantial evidence that proteolysis per se does not activate caspase 9, so an alternative mechanism for amplification must be considered. Cleavage at Asp330 removes a short peptide motif that allows caspase 9 to interact with IAPs (inhibitors of apoptotic proteases), and this event may control the amplification process. We show that, under physiologically relevant conditions, caspase 3, but not caspase 7, can cleave caspase 9, and this does not result in the activation of caspase 9. An IAP antagonist disrupts the inhibitory interaction between XIAP (X-linked IAP) and caspase 9, thereby enhancing activity. We demonstrate that the N-terminal peptide of caspase 9 exposed upon cleavage at Asp330 cannot bind XIAP, whereas the peptide generated by autolytic cleavage of caspase 9 at Asp315 binds XIAP with substantial affinity. Consistent with this, we found that XIAP antagonists were only capable of promoting the activity of caspase 9 when it was cleaved at Asp315, suggesting that only this form is regulated by XIAP. Our results demonstrate that cleavage by caspase 3 does not activate caspase 9, but enhances apoptosis by alleviating XIAP inhibition of the apical caspase.
Finding the needles in the haystack: mapping constitutive proteolytic events in vivo
Authors: Bogyo Matthew
Published: August 2015

Our quest to understand the complex inner workings of the cell depends on the development of new technologies that allow the study of global regulatory events as they happen within their native cellular environment. Post-translational processing of proteins by proteases is one such regulatory process that can control many aspects of basic cell biology. In this issue of the Biochemical Journal, Timmer et al. describe a new proteomic approach that can be used to globally monitor constitutive proteolytic events in vivo. Using bacterial, human, yeast and mouse cells, the authors show that this methodology provides a comprehensive map of constitutive trimming events mediated by regulatory proteases such as methionine aminopeptidase. This study also identifies previously uncharacterized processing events that highlight potential novel regulatory mechanisms mediated by proteolysis.
Decreased protein and puromycinyl-peptide degradation in livers of senescent mice
Authors: Lavie L , Reznick A Z , Gershon D
Published: August 2015

Liver protein-degradation rates were determined in young and old C57B1 mice by the method of Swick & Ip [(1974) J. Biol. Chem. 249, 6836-6841]. The results indicated a marked age-related increase in the half-lives of short-lived proteins in the nuclear, mitochondrial, lysosomal and 100000 g-supernatant cellular fractions and in total trichloroacetic acid-precipitable proteins. The efficiency of the degradation system in removing aberrant proteins from livers of young and old mice was tested. The time required for 50% disappearance of puromycinyl-peptides changed from about 20 min in 6-month-old mice to approx. 150 min in 24-month-old animals. These findings suggest that in old animals the proteolytic activity involved in degradation of aberrant proteins, and presumably of "native proteins, is markedly defective.
Evidence that insulin activates fat-cell acetyl-CoA carboxylase by increased phosphorylation at a specific site
Authors: Brownsey R W , Denton R M
Published: August 2015

1. A new rapid method for the purification of fat-cell acetyl-CoA carboxylase is described; the key step is sedimentation after specific polymerization by citrate. 2. Incubation of epididymal fat-pads or isolated fat-cells with insulin or adrenaline leads to a rapid increase or decrease respectively in the activity of acetyl-CoA carboxylase measured in fresh tissue extracts. The persistence of the effect of insulin through high dilution of tissue extracts and through purification involving precipitation with (NH4)2SO4 suggests that the enzyme undergoes a covalent modification after exposure of intact tissue to the hormone. The opposed effects of insulin and adrenaline are not adequately explained through modification of a common site on acetyl-CoA carboxylase, since these hormones bring about qualitatively different alterations in the kinetic properties of the enzyme measured in tissue extracts. 3. The state of phosphorylation of acetyl-CoA carboxylase within intact fat-cells exposed to insulin was determined, and results indicate a small but consistent rise in overall phosphorylation of the Mr-230000 subunit after insulin treatment. 4. Acetyl-CoA carboxylase from fat-cells previously incubated in medium containing [32P]phosphate was purified by immunoprecipitation and then digested with performic acid and trypsin before separation of the released phosphopeptides by two-dimensional analysis. Results obtained show that the exposure of fat-cells to insulin leads to a 5-fold increase in incorporation of 32P into a peptide which is different from those most markedly affected after exposure of fat-cells to adrenaline. 5. These studies indicate that the activation of acetyl-CoA carboxylase in cells incubated with insulin is brought about by the increased phosphorylation of a specific site on the enzyme, possibly catalysed by the membrane-associated cyclic AMP-independent protein kinase described by Brownsey, Belsham & Denton [(1981) FEBS Lett. 124, 145-150].
Alteration of inner-membrane components and damage to electron-transfer activities of bovine heart submitochondrial particles induced by NADPH-dependent lipid peroxidation
Authors: Narabayashi H , Takeshige K , Minakami S
Published: August 2015

We investigated the changes of the inner-membrane components and the electron-transfer activities of bovine heart submitochondrial particles induced by the lipid peroxidation supported by NADPH in the presence of ADP-Fe3+. Most of the polyunsaturated fatty acids were lost as a result of the peroxidation, and phospholipids were changed to polar species. Ubiquinone was also modified to polar substances as the peroxidation proceeded. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis showed the disappearance of 27000-Mr and 30000-Mr proteins and the appearance of highly polymerized substances. Flavins and cytochromes were not diminished, but the respiratory activity was lost. The reactions of NADH oxidase and NADH-cytochrome c reductase were most sensitive to the peroxidation, followed by those of succinate oxidase and succinate-cytochrome c reductase. Succinate dehydrogenase and duroquinol-cytochrome c reductase were inactivated by more extensive peroxidation, but cytochrome c oxidase was only partially inactivated. NADH-ferricyanide reductase was not inactivated. The pattern of the inactivation indicated that the lipid peroxidation affected the electron transport intensively between NADH dehydrogenase and ubiquinone, and moderately at the succinate dehydrogenase step and between ubiquinone and cytochrome c.
Simulations of the roles of multiple cyclic nucleotide phosphodiesterases
Authors: Reynolds C H
Published: August 2015

1. Simulations were performed using a model for cellular cyclic AMP metabolism involving a hormone-activated adenylate cyclase and two cyclic nucleotide phosphodiesterases with different Michaelis constants. 2. The response curves of cyclic AMP concentration as a function of hormone concentration were affected by regulating the phosphodiesterases. The maximum velocity of the high-affinity phosphodiesterase (V1) was important in determining the position of the response curve; when v1 was less than the maximal activity of adenylate cyclase (Vc), sigmoid response curves were readily produced. The maximum attainable concentration of cyclic AMP was determined primarily by V1 when Vc less than V1, and primarily by the activity of the low-affinity enzyme when Vc greater than V1 (V2 much greater than Vc in all cases). 3. The glucagon-stimulated adenylate cyclase and insulin-stimulated phosphodiesterase of the rat liver plasma membrane were simulated using experimentally determined values for the enzyme-kinetic parameters, and a considerable potential for regulation of the system by insulin was demonstrated. 4. Other possible functions for the regulation of phosphodiesterases are considered, in particular the value of increasing the speed of response to decreases in hormone concentration.
The stimulus-secretion coupling of glucose-induced insulin release. Environmental influences on l-glutamine oxidation in pancreatic islets
Authors: Sener A , Malaisse-Lagae F , Malaisse W J
Published: August 2015

L-Glutamine at a near-physiological concentration (1.0mM) was rapidly taken up and metabolized in rat pancreatic islets. The rate of glutamine deamidation much exceeded that of glutamate conversion into 2-oxoglutarate, the latter conversion being mediated mainly by transamination reactions. The production of 14CO2 from L-[U-14C]glutamine, which reflected the generation of ATP through the metabolism of exogenous glutamine, appeared to be regulated by the redox state of nicotinamide nucleotides and the ATP content of the islet cells. The influence of environmental factors on glutamine oxidation was examined in order to identify ATP-requiring processes. Glutamine oxidation was decreased in the absence of extracellular Ca2+, under conditions aiming at inhibition of the (Na+ + NA+)-dependent ATPase and, provided that glucose was present in the incubation medium, by cycloheximide. These findings were interpreted to suggest that the handling of Ca2+ by the islet cells, the active transport of univalent cations and the biosynthesis of proinsulin represent three major ATP-consuming processes in this fuel-sensor organ.
The polyadenylated RNA directing the synthesis of the rat myelin basic proteins is present in both free and membrane-bound forebrain polyribosomes
Authors: Hall C , Mahadevan L C , Whatley S A , Ling T S , Lim L
Published: August 2015

Free and membrane-bound polyribosomes were isolated from the forebrain of actively myelinating 24-day-old rats. The poly(A)+ RNA (polyadenylated RNA) extracted from both fractions was translated in vitro in reticulocyte lysates [Hall & Lim (1981) Biochem. J. 196. 327-336] in the presence or absence of a heterologous microsomal membrane fraction from dog pancreas. The rat myelin basic proteins synthesized in vitro were isolated by CM-cellulose chromatography and by immunoprecipitation with purified anti-(myelin basic protein) antibody. The large (mol.wt. 18 500) and small (mol.wt. 16 000) myelin basic proteins were translational products of poly(A)+ RNA from both free and membrane-bound polyribosomes. The identity of the myelin basic proteins was verified by analysis of peptides generated by the cathepsin D digestion of the immunoprecipitated proteins synthesized in vitro, in comparison with authentic rat myelin basic proteins. Although several other translational products of membrane-bound polyribosomal poly(A)+ RNA were modified when microsomal membranes were present during translation, molecular weights of the myelin basic proteins themselves were unchanged. The myelin basic proteins synthesized in vitro also did not differ significantly in size from the authentic myelin basic proteins, indicating that these membrane proteins are unlikely to be synthesized as substantially larger precursor molecules. The presence of the specific mRNA species on both free and membrane-bound polyribosomes is compatible with the extrinsic location of the myelin basic proteins on the cytoplasmic surface of the myelin membrane.
Differential sensitization to deoxyribonuclease I of Xenopus vitellogenin and albumin genes during primary and secondary induction of vitellogenesis by oestradiol
Authors: Dimitriadis G J , Tata J R
Published: August 2015

The sensitivity to DNAase (deoxyribonuclease) I (which preferentially digests transcribed sequences) of vitellogenin and albumin genes in liver and erythrocytes of male Xenopus after primary and secondary induction of vitellogenesis by oestrogen was measured by hybridization to cDNA (complementary DNA) of the residual DNA after enzymic digestion of isolated nuclei. Vitellogenin sequences were rendered selectively more sensitive to limited DNAase-I digestion (15-20% of DNA rendered acid-soluble) during primary hormonal activation (5 days) of vitellogenin genes in liver, but not erythrocyte, nuclei. Hormone withdrawal (25 days after first injection) did not result in reversion to a pre-activation gene configuration, nor did secondary hormonal stimulation (5 days after second and 25 days after first injection) augment the sensitivity of the genes to digestion by the nuclease. Similar hormone treatment did not affect the sensitivity of the constitutively expressed albumin genes in liver nuclei, nor their insensitivity in erythrocyte nuclei. Under the same conditions, globin genes remained indigestible in liver nuclei. It is concluded that primary induction of vitellogenesis in male Xenopus liver is accompanied by relatively long-lasting (3-4 weeks) change in the configuration of vitellogenin genes in hepatic nuclei which is not reversed or further modified during short-term oestrogen withdrawal or upon secondary stimulation.
Effect of excessive vitamin A intake on muscle protein turnover in the rat
Authors: Hillgartner F B , Morin D , Hansen R J
Published: August 2015

3-Methylhistidine excretion in vivo and in vitro was monitored in hypervitaminotic and pair-fed control rats. Feeding with excess of retinyl palmitate (40 000 i.u./day per 100 g body wt.) significantly increased urinary 3-methylhistidine and creatinine output during a 4-day treatment interval. 3-Methylhistidine release from perfused rat hindquarters was also elevated after 5 days of vitamin treatment. To determine whether the adrenals were involved in mediating the above response, a study was conducted on adrenalectomized and sham-operated rats. Excessive vitamin A intake stimulated 3-methylhistidine excretion in vivo and in vitro in both adrenalectomized and sham-operated animals, thus suggesting that the vitamin A-induced acceleration in myofibrillar protein breakdown was not mediated by the adrenals. In both groups of rats, vitamin A treatment had no effect on the rate of protein synthesis, on the basis of incorporation in vitro of [3H]phenylalanine into muscle protein. Additional studies revealed that the addition of excess retinol to the perfusion medium (10 i.u./ml) had no significant effect on the rates of 3-methylhistidine release or [3H]phenylalanine incorporation in vitro. Finally, high doses of cortisol (7 mg/day per 100g body wt.) administered to intact rats for 5 days significantly increased rates of 3-methylhistidine excretion, both in vivo and in vitro.
Immuno-electron-microscopic studies on the subcellular distribution of rat liver epoxide hydrolase and the effect of phenobarbitone and 2-acetamidofluorene treatment
Authors: Waechter F , Bentley P , Germann M , Oesch F , Stäubli W
Published: August 2015

The distribution of rat liver epoxide hydrolase in various subcellular fractions was investigated by immuno-electron-microscopy. Ferritin-linked monospecific anti-(epoxide hydrolase) immunoglobulins bound specifically to the cytoplasmic surfaces of total microsomal preparations and smooth and rough microsomal fractions as well as the nuclear envelope. Specific binding was not observed when the ferritin conjugates were incubated with peroxisomes, lysosomes and mitochondria. The average specific ferritin load of the individual subcellular fractions correlated well with the measured epoxide hydrolase activities. This correlation was observed with fractions prepared from control, phenobarbitone-treated and 2-acetamidofluorene-treated rats.
Evidence for the origin of the unoccupied oestrogen receptor in nuclei of a human breast-cancer cell line (MCF-7)
Authors: Geier Avraham , Haimsohn Michal , Lunenfeld Bruno
Published: August 2015

The origin of the unoccupied nuclear oestrogen receptor (Rn) was studied. Three working hypotheses were investigated. (a) Rn is a dissociation product of the oestrogen occupied nuclear receptor (ERn). (b) ERn is only partially occupied, so that additional binding may occur at 0°C (the temperature at which oestradiol saturates unoccupied sites). (c) Rn is derived from the penetration of unoccupied cytoplasmic receptor (Rc) into the nucleus. The MCF-7 cell line was used as a model in the present investigation. The amount of unoccupied receptors was measured by saturation with 7.5nm-[3H]oestradiol at 0°C, whereas the occupied receptors were measured by exchange at 30°C. The cells at preconfluency were exposed to a medium fortified with 10nm-[3H]oestradiol for 1h, washed and cultured up to 5 days in fresh growth medium. The distribution of oestradiol receptors was determined before exposure and during the following 5 days. After 1h exposure only ERn was found in the nuclear fraction. Thereafter ERn declined continuously so that on day 5 it approached 15% of its value measured 1h after exposure. Although after 3 days about 80% of ERn disappeared no Rn appeared, which contradicts hypotheses (a) and (b). On day 4 Rn and Rc appeared simultaneously. The appearance of Rn and Rc was not prevented by culturing the cells in an oestrogen-free medium, supporting hypothesis (c). Exposure of cells to increasing concentration of [3H]oestradiol (0.1–10nm) for 1h resulted in a parallel increase in ERn without increasing the amount of unoccupied binding sites, which contradicts hypothesis (b). The present study supports the hypothesis (c), i.e., Rc may also penetrate the nucleus without binding to oestradiol.
Is Percoll innocuous to cells?
Authors: Wakefield J S , Gale J S J , Berridge M V , Jordan T W , Ford H C
Published: August 2015

Peritoneal macrophages from mice, isolated rat liver Kupffer cells and rat testis Leydig cells ingested large numbers of Percoll particles, a gradient medium widely used for separation of cells and subcellular organelles by density-gradient centrifugation. A decrease in the percentage of macrophages adhering to plastic also occurred after exposure of the cells to Percoll, even at 4 degrees C, a temperature at which Percoll was not ingested. The effect of Percoll on macrophage adherence may involve a loose association between the density medium and the cell surface. Other cell-surface-related phenomena may also be affected by prior exposure of cells to Percoll.
Initial rates. A new plot
Authors: Boeker E A
Published: August 2015

Excellent estimations of initial rates can be obtained from plots of delta P/t versus product formed (where P is the instantaneous concentration of the product). delta P/t is the chord from P0,t0 to P,t on an ordinary P-versus-t plot. When the chord is plotted as a function of product, the intercept at P0 of the resulting curve is necessarily dP/dt0. This curve approximates to a straight line extremely closely in all cases tested thus far. If delta P/t versus product is calculated from the integrated rate equation for a first-order reaction, and if a straight line is fitted through points representing the first 50% of the reaction, the discrepancy between the true initial rate and dP/dt0 estimated from the plot is 0.68%. For the most common form of the integrated rate equation for catalysed reactions the discrepancy varies between 0 and 0.90%. Because of the complexities of the integrated rate equations, catalysed second-order reactions have not been evaluated directly; uncatalysed reactions have been done instead. For a reaction with one reactant and two products, the discrepancy varies from 0.68 to 2.02%. For two reactants and one product, it varies from 0 to 0.68%; for two and two, 0 to 2.02%. The larger discrepancies occur only when unfavourable equilibrium constants are being overcome by the initial conditions.
The kinetics of hydrolysis of some extended N-aminoacyl-l-arginine methyl esters by porcine pancreatic kallikrein. A comparison with human plasma Kallikrein
Authors: Levison P R , Tomalin G
Published: August 2015

The effects of subsite interactions in the S2-S4 region [Schechter & Berger (1967) Biochem. Biophys. Res. Commun. 27, 157-162] of porcine pancreatic kallikrein (EC 3.4.21.8) on its catalytic efficiency have been investigated. Kinetic constants (Kcat, Km) have been determined for a series of seven extended N-aminoacyl-L-arginine methyl esters whose sequence is based on either the C-terminal sequence of kallidin (-Pro-Phe-Arg) or (-Gly-)nArg. With these substrates it has been found that neither acylation nor deacylation of the enzyme is rate-limiting. Values of Kcat. range from 21.5 to 2320s-1, indicating that there are interactions with different residues in the N-aminoacyl chain and enzyme subsites in the S2-S4 region. It is shown that possible hydrogen-bonded interactions with the enzyme in the S3-S4 region have a significant effect on catalysis. The presence of L-phenylalanine at P2 has a very large effect on both Kcat, and Km, giving a greatly enhanced catalytic efficiency. Substrates with L-proline at P3 also have a marked effect, but in this case the overall effect is one of lowered catalytic efficiency. By comparison with the results of a similar study with human plasma kallikrein I (EC 3.4.21.8), it has been possible to demonstrate that there are considerable differences in kinetic behaviour between the two enzymes. These are related to relative differences in the rates of acylation and deacylation with ester substrates and also the roles of subsites S2 and S3 of the two enzymes.
Difference in thermal stability of type-I and type-III collagen from rat skin
Authors: Danielsen Carl Christian
Published: August 2015

Type-I and type-III collagens were obtained by differential salt fractionation of neutral-salt-soluble collagen from rat skin. Their thermal stabilities were determined by u.v. difference spectroscopy. The ‘melting’ temperature (Tm) in 5mm-acetic acid of type-III collagen was almost 2°C above that of type-I collagen. Intramolecular covalent cross-linking had no effect on the thermal stability.
The inhibitory effect of Zn2+ on poly(ADP-ribose) polymerase activity and its reversal
Authors: Larsen A G , Ostvold A C , Holtlund J , Kristensen T , Laland S G
Published: August 2015

Zn2+ inhibits purified poly(ADP-ribose) polymerase (50% inhibition at 10 microM). Furthermore poly (ADP-ribose) polymerase present in nuclei and metaphase chromosome clusters is also inhibited by Zn2+. The inactivated enzyme could be re-activated by dithiothreitol. The concentration of Zn2+ needed to affect the enzyme activity in the organelles is sufficiently low for it to have a possible role in controlling the activity of this chromatin-bound enzyme.
The characterization of lipoprotein lipase isolated from the post-heparin plasma of the rainbow trout, Salmo gairdneri Richardson
Authors: Skinner E R , Youssef A M
Published: August 2015

1. Intravenous injection of heparin into the trout resulted in the appearance in the plasma of a lipase with the properties of lipoprotein lipase. 2. The enzyme was purified to apparent electrophoretic homogeneity by means of heparin-Sepharose affinity chromatography. The enzyme was eluted with 1.5 M-NaCl and had a specific activity approx. 450-fold that of the post-heparin plasma. 3. The activity of the purified enzyme was inhibited by 1.0 M-NaCl and protamine sulphate and was stimulated between 3- and 8.8-fold by the addition of trout plasma. 4. The activity was strongly stimulated by trout very low density lipoproteins and to a lesser extent by high density lipoproteins. 5. The isolated enzyme fraction gave a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and had an apparent subunit M4 of 63 000. 6. These results suggest that the uptake of lipid by the tissues in the trout can occur by a process similar to that in mammals.
Evidence that peroxisomal acyl-CoA synthetase is located at the cytoplasmic side of the peroxisomal membrane
Authors: Mannaerts G P , Van Veldhoven P , Van Broekhoven A , Vandebroek G , Debeer L J
Published: August 2015

1. Subfractionation by isopycnic density-gradient centrifugation in self-generating Percoll gradients of peroxisome-rich fractions prepared by differential centrifugation confirmed the presence of acyl-CoA synthetase in peroxisomes. Peroxisomes did not contain nicotinamide or adenine nucleotides other than CoA. 2. The gradient fractions most enriched in peroxisomes were pooled and the peroxisomes sedimented by centrifugation, resulting in a 50-fold-purified peroxisomal preparation as revealed by marker enzyme analysis. 3. Palmitate oxidation by intact purified peroxisomes was CoA-dependent, whereas palmitoyl-CoA oxidation was not, demonstrating that the peroxisomal CoA was available for the thiolase reaction, located in the peroxisomal matrix, but not for acyl-CoA synthetase. This suggests that the latter enzyme is located at the cytoplasmic side of the peroxisomal membrane. 4. Additional evidence for this location of peroxisomal acyl-CoA synthetase was as follows. Mechanical disruption of purified peroxisomes resulted in the release of catalase from the broken organelles, but not of acyl-CoA synthetase, indicating that the enzyme was membrane-bound. Acyl-CoA synthetase was not latent, despite the fact that at least one of its substrates appears to have a limited membrane permeability, as evidenced by the presence of CoA in purified peroxisomes. Finally, Pronase, a proteinase that does not penetrate the peroxisomal membrane, almost completely inactivated the acyl-CoA synthetase of intact peroxisomes.
Toxic effects of human eosinophil products on isolated rat heart cells in vitro
Authors: Tai P C , Hayes D J , Clark J B , Spry C J F
Published: August 2015

Rat heart cells and mitochondria were incubated with supernatants from eosinophils or neutrophils that had been stimulated with zymosan-C3b. Supernatants from eosinophils, but not neutrophils, were toxic to rat heart cells in a dose-dependent manner. This was associated with an increased O2 uptake, which was blocked by either 1 mM-cyanide or 100 microM-ouabain. Supernatants from eosinophils, but not neutrophils, caused a decrease in O2 uptake by rat heart mitochondria utilizing pyruvate (+ malate) but not other substrates. The activity of pyruvate dehydrogenase (EC 1.2.4.1) from rat heart was inhibited by Ca2+-free eosinophil supernatants. The activity of oxoglutarate dehydrogenase (EC 1.2.4.2) was also inhibited but not that of lipoamide dehydrogenase (EC 1.6.4.3). Prior incubation with heparin prevented these effects of eosinophil supernatants on heart cells, suggesting that they were caused by eosinophil cationic proteins. Other cationic proteins, including poly-L-lysine and poly-L-arginine were also toxic to rat heart cells, but these reduced O2 uptake. It was concluded that granulocyte secretion products containing eosinophil cationic proteins are toxic to isolated rat heart cells in vitro. This may be due to an initial increase in membrane permeability, which may lead to activation of (Na+ + K+)-dependent ATPase and increased O2 uptake. A second step may involve inhibition of pyruvate dehydrogenase by the same products, leading to a decreased O2 uptake. It is suggested that these mechanisms could contribute to the development of cardiac injury and myocardial disease in clinical situations where many degranulated eosinophils are present.
Quantitative alterations in the nucleolar and nucleoplasmic ribosomal ribonucleic acids in regenerating rat liver
Authors: Dabeva Mariana D. , Dudov Kalin P.
Published: August 2015

A quantitative analysis of the nuclear pre-rRNA (precursor to rRNA) and rRNA in normal and 12h-regenerating rat liver was carried out, and the absolute amounts of the identified pre-rRNA and rRNA species in the nucleolus and nucleoplasm were determined. Characteristic changes in the pre-rRNA and rRNA pool sizes in regenerating liver are found which reveal alternations in both pre-rRNA processing and nucleocytoplasmic transition of ribosomes.
The role of leucine in ketogenesis in starved rats
Authors: Thomas L K , Ittmann M , Cooper C
Published: August 2015

The quantitative significance of the conversion in vivo of L-[U-14C]leucine to ketone bodies was determined in rats starved for 3 or 48 h. In animals starved for 3 h, 4.4% of ketone-body carbon is derived from the metabolism of leucine, and in rats starved for 48 h the corresponding value is 2.3%. This conversion occurs rapidly, and the specific radioactivity of ketone bodies in blood is maximal at 2 min after the intravenous injection of labelled leucine for both periods of starvation. The flux of leucine in the blood is 1.01 and 1.04 mumol/min per 100 g body wt. respectively for animals starved for 3 and 48 h. The specific radioactivity of blood ketone bodies was compared at 2 min after the injection of labelled leucine, lysine and phenylalanine. The specific radioactivity was 4-5 fold higher with leucine than with lysine or phenylalanine.
Lipase-induced alterations of fatty acid synthesis by subcellular fractions from germinating pea (Pisum sativum L.)
Authors: Sanchez J , Jordan B R , Kay J , Harwood J L
Published: August 2015

1. The effect of exogenous lipases on fatty acid synthesis from [14C]malonyl-CoA by the microsomal and soluble fractions from germinating peas was studied. 2. Addition of phospholipase A2 or the lipase from Rhizopus arrhizus had no effect on total fatty acid synthesis by the soluble fraction but caused severe inhibition of that by the microsomal fraction. 3. The addition of enzymes with phospholipase activity particularly inhibited the microsomal stearate elongase. 4. Control studies indicated that the phospholipase-induced inhibition of fatty acid synthesis was due to the location of fatty acid synthetase, palmitate elongase and stearate elongase on the outside of the microsomal vesicles. 5. Experiments with a trypsin-like proteinase showed that approximately half the microsomal fatty acid synthesis was resistant to proteolysis. 6. Although addition of exogenous phospholipases had no effect on total fatty acid synthesis by the soluble fraction, it did increase alpha-hydroxylation of newly-formed palmitate and stearate. 7. The results provide further evidence for differences between the soluble and particulate fatty acid synthetase and palmitate elongase activities of germinating pea.
Role of thiols in degradation of proteins by cathepsins
Authors: Kooistra T , Millard P C , Lloyd J B
Published: August 2015

The effects of thiols on the breakdown of 125I-labelled insulin, albumin and formaldehyde-treated albumin by highly purified rat liver cathepsins B, D, H and L at pH 4.0 and 5.5 were studied. At both pH values degradation was strongly activated by the thiols cysteamine, cysteine, dithiothreitol, glutathione and 2-mercaptoethanol, and its rate increased with increasing thiol concentration. Preincubation of the protein substrates with 5 mM-glutathione did not affect concentration. Preincubation of the protein substrates with 5 mM-glutathione did not affect the rate of degradation by cathepsin D or L, and determination of free thiol groups after incubation of the proteins in the presence of glutathione but without cathepsin showed that their disulphide bonds were stable under the incubation conditions. Sephadex G-75 chromatography of the acid-soluble products of insulin digestion by cathepsin D or L suggested that thiols can reduce disulphide bonds in proteins after limited proteolysis. The resultant opening-up of the protein structure would lead to further proteolysis, so that the two processes (proteolysis and reduction) may act synergistically. By using the osmotic protection method it was shown that, at a physiological pH, cysteamine, and its oxidized form cystamine, can cross the lysosome membrane and thus may well be the physiological hydrogen donor for the reduction of disulphides in lysosomes. The results are discussed in relation to the lysosomal storage disease cystinosis.
A study of highly purified mucolipidosis III urinary N-acetyl-β-D-hexosaminidase B
Authors: Hirani S , Little L , Miller A L
Published: August 2015

Highly purified N-acetyl-beta-D-hexosaminidase B from normal urine and urine of a patient with mucolipidosis III was used to determine whether it has undergone any of the alterations associated with this genetic defect. Examination by sodium dodecyl sulphate/polyacrylamide gel electrophoresis showed that both the enzyme preparations contained protein components with apparent Mr values of 55 000 and 28 000. No differences in the binding and apparent KI (50%) to concanavalin A of the normal and mucolipidosis III enzymes were detected. However, the patient's N-acetyl-beta-D-hexosaminidase B had a slightly greater affinity for the lectin from Ricinus communis than did the normal enzyme. Two-dimensional tryptic peptide maps of the corresponding normal and the patient's N-acetyl-beta-D-hexosaminidase B subunits showed considerable homology. These results indicate that N-acetyl-beta-D-hexosaminidase b does not undergo the significant carbohydrate alterations characteristic of other acid hydrolases in mucolipidosis III.
Resensitization of lutropin-desensitized tumour Leydig-cell adenylate cyclase with human erythrocyte membranes
Authors: Dix C J , Cooke B A
Published: August 2015

Purified rat tumour Leydig cells were pretreated with or without lutropin (1 h at 32 degrees C). The plasma membranes were then isolated and the adenylate cyclase activity measured in the presence of freshly prepared or heat-inactivated (1 h at 60 degrees C) human erythrocyte membranes. In plasma membranes from control cells in the presence of heat-inactivated human erythrocyte membranes both guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG) plus lutropin and NaF caused a 45-50-fold increase in cyclic AMP production over 30 min compared with 12-13 fold p[NH[ppG and 2-3-fold with lutropin alone. In plasma membranes isolated from lutropin-pretreated cells the NaF- and the p[NH]ppG-stimulated cyclic AMP production rates were unchanged, but no effect of lutropin could be demonstrated with or without added p[NH]ppG. However, after mixing lutropin-desensitized Leydig tumour-cell plasma membranes with freshly prepared human erythrocyte plasma membranes, the adenylate cyclase activity in the presence of lutropin, p[NH]ppG, lutropin plus p[NH]ppG and NaF were similar to those of control cell plasma membranes treated in the same manner. The possible mechanisms of this reversal of lutropin-induced desensitization by human erythrocytes are discussed.
An Fc-receptor activity of plasma membranes from guinea-pig peritoneal polymorphonuclear leucocytes
Authors: Tsunawaki S , Mizuno D , Kakinuma K , Kasahara M
Published: August 2015

Plasma membranes prepared from guinea-pig peritoneal polymorphonuclear leucocytes showed an immune-complex-binding activity that corresponded well with the activity in intact cells. The characteristics of this activity were reversible binding, dependence on the Fc portion of antigen-complexed IgG (immunoglobulin G), competition with aggregated IgG and independence from energy metabolism. These results support the conclusion that the binding activity found in the isolated plasma membranes is an Fc-receptor activity of guinea-pig peritoneal polymorphonuclear leucocytes. The activity showed Kd = 6.7×10(-8) M-IgG and maximum binding of 17 pmol of IgG/mg of membrane protein when measured with an immune complex of alpha-amylase and homologous guinea-pig anti-(alpha-amylase) IgG. Inhibition of the Fc-receptor activity by a series of various salts indicated the contribution of the hydrophobic interaction to the binding. Inhibitory effects of salts or metal-chelating reagents on the Fc-receptor activity were also observed on superoxide generation by these cells induced by the immune complex, suggesting a role of the Fc receptor as the immune-complex-binding site responsible for the initiation of superoxide generation.
Effects of Mg2+, Ca2+ and Mn2+ on sheep liver cytoplasmic aldehyde dehydrogenase
Authors: Dickinson F M , Hart G J
Published: August 2015

Sheep liver cytoplasmic aldehyde dehydrogenase is strongly inhibited by Mg2+, Ca2+ and Mn2+. The inhibition is only partial, however, with 8-15% of activity remaining at high concentrations of these agents. In 50 mM-Tris/Hcl, pH 7.5, the concentrations giving half-maximal effect were: Mg2+, 6.5 micrometers; Ca2+, 15.2 micrometers; Mn2+, 1.5 micrometer. The esterase activity of the enzyme is not affected by such low metal ion concentrations, but appears to be activated by high concentrations. Fluorescence-titration and stopped-flow experiments provide evidence for interaction of Mg2+ with NADH complexes of the enzyme. As no evidence for the presence of increased concentrations of functioning active centres was obtained in the presence of Mg2+, it is concluded that effects of Mg2+ (and presumably Ca2+ and Mn2+ also) are brought about by trapping increased concentrations of NADH in a Mg2+-containing complex. This complex must liberate products more slowly than any of the complexes involved in the non-inhibited mechanism.
Isolation of plasma membrane from protoplasts of Lolium multiflorum (ryegrass) endosperm cells
Authors: Schibeci A , Fincher G B , Stone B A , Wardrop A B
Published: August 2015

Plasma membranes have been isolated from protoplasts of suspension-cultured ryegrass (Lolium multiflorum) endosperm cells. The protoplast membrane is coated before cell disruption with murine myeloma protein J539, a galactose-binding immunoglobulin A. The plasma membrane is labelled with 125I by using chemically or enzymically catalysed iodination techniques, or, more conveniently, by using 125I-labelled myeloma protein J539, which enables the membrane to be simultaneously coated and labelled. Protoplast lysis is effected by gentle mechanical means after swelling in hypo-osmotic medium. The plasma-membrane fraction is recovered at low centrifugal forces by fractionation of cell lysates on a discontinuous sucrose/sorbitol gradient. The plasma-membrane fraction is enriched 96-fold on a protein basis with respect to the specific radioactivity of 125I-labeled myeloma protein J539 in the homogenate. Electron microscopy showed long membrane profiles often associated with one another.
Sensitivity of carnitine acyltransferase I to malonyl-CoA inhibition in isolated rat liver mitochondria is quantitatively related to hepatic malonyl-CoA concentration in vivo
Authors: Robinson Ian N. , Zammit Victor A.
Published: August 2015

The sensitivity of carnitine acyltransferase I (EC 2.3.1.21) activity to malonyl-CoA inhibition in rat liver mitochondria isolated from animals in various physiological states was quantitatively proportional to the hepatic malonyl-CoA concentration in vivo. It is suggested that this relationship between the two parameters could result in a potent amplification mechanism for the reciprocal regulation of fatty acid synthesis and oxidation.
Characterization of the stimulatory effect of high-fat diets on peroxisomal β-oxidation in rat liver
Authors: Thomassen M S , Christiansen E N , Norum K R
Published: August 2015

1. The effect on rat liver peroxisomal beta-oxidation of feeding diets containing various amounts of dietary oils was investigated. With increasing amounts (5-25%, w/w) of soya-bean oil an apparent, but not statistically significant, increase of 1.5-fold was found both in specific activity, and in total liver activity. Increasing amounts of partially hydrogenated marine oil revealed a sigmoidal dose-response-curve, giving a 4-6-fold increase in the peroxisomal beta-oxidation activity at 20% or more of this oil in the diet. 2. Addition of small amounts of soya-bean oil to the marine-oil diet had no effect on the peroxisomal beta-oxidation activity, but decreased the C20:3(5,8,11) fatty acid/C20:4(5,8,11,14) fatty acid ratio in liver phospholipids from 0.74 to 0.01. 3. Starvation for 2 days led to a 1.5-1.8-fold increase in the peroxisomal beta-oxidation activity in rats previously fed on a standard pelleted diet, but had no effect in rats given high-fat diets. 4. Feeding partially hydrogenated marine oil or partially hydrogenated rape-seed oil resulted in higher activities than the corresponding unhydrogenated oils. 5. No significant differences in the effect on peroxisomal beta-oxidation could be detected between diets containing rape-seed oils with 15 or 45% erucic acid respectively. 6. These findings are discussed in relation to the possible effects of C22:1 and trans fatty acids in the process leading to increased peroxisomal beta-oxidation activity in the liver.
Changes in the blood level and affinity to concanavalin A of rat plasma glycoproteins during acute inflammation and hepatoma growth
Authors: Koj A , Dubin A , Kasperczyk H , Bereta J , Gordon A H
Published: August 2015

Plasma concentrations of ten individual proteins were measured by electroimmunoassay in young male Buffalo rats following injection of turpentine oil or implantation of Morris hepatoma 7777. The highest relative responses to inflammation and tumour growth were found for alpha 2-macroglobulin, alpha 1-acute-phase globulin and alpha 1-acid glycoprotein. As shown by crossed immuno-affinoelectrophoresis the concanavalin A-reactive fractions of the latter two glycoproteins were predominantly increased in plasma from injured and tumour-bearing rats.