bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition by sporidesmin of hepatocyte bile acid transport
Authors: Cordiner S J , Jordan T W
Published: August 2015

Exposure of isolated rat hepatocytes (approx. 2 × 10(7)–5 × 10(7) cells/10ml of incubation mixture) to 0.5 mg of the mycotoxin sporidesmin for 30—60 min at 37 degrees C produced loss of plasma-membrane microvilli with some disruption of organelle distribution in the sub-surface region. There was accompanying inhibition of [14C]cholate and [14C]taurocholate transport, but bile acid conjugation was not altered. Inhibition of cholate uptake was maximal after exposure of hepatocytes to sporidesmin for 1 min, and was not reversed by washing cells free of extracellular sporidesmin. N-Ethylmaleimide (0.1 mM) or dithiothreitol (1 mM) partially protected hepatocytes from sporidesmin inhibition of bile acid uptake. Significant protection was not given by other thiols or by zinc sulphate, cholesterol, ascorbate or α-tocopherol. The results are discussed in terms of sporidesmin action on cell membranes and the toxin's effect on bile secretion.
Clearance and binding of native and defucosylated lactoferrin
Authors: Imber M J , Pizzo S V
Published: August 2015

These studies explore the role of carbohydrate recognition systems and the direct involvement of terminal alpha 1-3-linked fucose in the clearance of lactoferrin from the murine circulation and in the specific binding of lactoferrin to receptors on murine peritoneal macrophages. As previously reported, radiolabelled lactoferrin cleared very rapidly (t1/2 less than 1 min) after intravenous injection into mice. However, competing levels of ligands specific for the hepatic galactose receptor (asialo-orosomucoid), the hepatic fucose receptor (fucosyl-bovine serum albumin), and the mononuclear-phagocyte system pathway recognizing mannose, N-acetylglucosamine and fucose (mannosyl-, N-acetylglucosaminyl- and fucosyl-bovine serum albumin) did not block radiolabelled lactoferrin clearance in vivo or binding to mouse peritoneal macrophage monolayers in vitro. Almond emulsin alpha 1-3-fucosidase was used to prepare defucosylated lactoferrin in which 88% of the alpha 1-3-linked fucose was hydrolysed. No difference in clearance or receptor binding was observed between radiolabelled native and defucosylated lactoferrin. Fucoidin, a fucose-rich algal polysaccharide, completely inhibits the clearance in vivo and macrophage binding in vitro of lactoferrin. This effect, however, is probably not the result of competition for binding to the fucose receptor, since gel-filtration studies demonstrated formation of a stable complex between lactoferrin and fucoidin. The present results indicate that the lactoferrin-clearance pathway is distinct from several pathways mediating glycoprotein clearance through recognition of terminal galactose, fucose, N-acetylglucosamine or mannose. Furthermore, alpha 1-3-linked fucose on lactoferrin is not essential for lactoferrin clearance in vivo or specific binding to macrophage receptors in vitro.
Measurement of specific radioactivities in labelled hormones by self-displacement analysis
Authors: Calvo J C , Radicella J P , Charreau E H
Published: August 2015

A graphical method is described that allows the determination of specific radioactivities of radioactively labelled hormones. This method combines the self-displacement technique, plotting bound/free ratios versus mass of unlabelled hormone or total radioactivity of labelled preparation added to the receptor preparation, and the maximal binding capacity of the labelled hormone. The procedure presented herein provides a more realistic specific radioactivity for use in all binding experiments. Application of the method is demonstrated for 125I-labelled ovine prolactin, and data are presented for 125I-labelled human choriogonadotropin and [3H]testosterone.
Hormonally produced changes in caeruloplasmin synthesis and secretion in primary cultured rat hepatocytes. Relationship to hepatic copper metabolism
Authors: Weiner A L , Cousins R J
Published: August 2015

Hormonally produced changes in the synthesis and secretion of the serum copper-containing protein caeruloplasmin were studied in primary cultures of rat liver parenchymal cells isolated by the collagenase-perfusion technique. A rabbit antibody directed against rat caeruloplasmin was used to immunoprecipitate labelled caeruloplasmin. Isolated liver cells synthesized and secreted caeruloplasmin over a period of 3 days. Synthesis and secretion of this protein was enhanced when cells were treated with dexamethasone. The accumulation of copper was also moderately enhanced with glucocorticoid treatment. Inclusion of adrenaline in the culture medium resulted in elevated incorporation of copper into newly synthesized caeruloplasmin as well as an increase in 64Cu-labelled caeruloplasmin in the culture medium. However, adrenaline did not seem to increase the secretion of 3H-labelled protein, despite the elevation in secreted 64Cu-caeruloplasmin. This may be due to a large increase in the intracellular pool of 64Cu caused by enhanced accumulation of this metal when adrenaline is included in the incubation medium. Enhanced copper accumulation was also seen when cells were treated with glucagon. Adrenaline-stimulated accumulation of 64Cu could be inhibited by including phenoxybenzamine, an alpha-adrenergic blocker, in the culture medium. Elevation of extracellular copper caused enhancement in the detection of labelled caeruloplasmin in the medium of cultured cells, probably owing to the ability of this metal to stabilize the protein.
Stimulation of microsomal lipid peroxidation by iron and cysteine. Characterization and the role of free radicals
Authors: Searle A J F , Willson R L
Published: August 2015

The stimulation of microsomal lipid peroxidation by FeSO4 and cysteine has been investigated. Although both FeSO4 and cysteine alone promoted an increase in malonaldehyde production, when these agents were added together to microsomes the resultant level of malonaldehyde was greater than the sum of the amounts formed by these pro-oxidants when acting individually. A further indication of an interaction between FeSO4 and cysteine was shown by the inhibitory action of chelating agents. Stimulation of peroxidation was shown to be independent of microsomal protein, including cytochrome P-450. The system has been characterized for the effects of cysteine, Fe2+ and O2 concentrations, pH, temperature and antioxidants. The results indicate that the high level of peroxidation attained with this system, its non-enzymic character and the involvement of hydroxyl radicals make it particularly useful for the investigation of the action of antioxidants. Furthermore it may also be a model of way in which decompartmentalized, delocalized or ‘free’ iron initiates peroxidation in vivo.
Diet fat influences liver plasma-membrane lipid composition and glucagon-stimulated adenylate cyclase activity
Authors: Neelands P J , Clandinin M T
Published: August 2015

Rats were fed diets that differed in fatty acid composition or in the proportion of energy derived from fat to determine if alteration of dietary fat intake influences the structural lipid composition of liver plasma membrane and the expression of an associated hormone-receptor-mediated function. Weanling rats were fed 9% (w/w) or 20% (w/w) low-erucic acid rape-seed oil or 9% (w/w) soya-bean oil for 24 days. Plasma membranes were isolated and the effect of diet fat on the fatty acid composition of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and sphingomyelin was determined. Diet fat significantly altered total saturated and (omega-9) and (omega-6)-unsaturated fatty acid composition in addition to the (omega-6)- to (omega-3)-unsaturated fatty acid ratio in these polar lipids. Feeding the high-fat diet increased the (omega-6)- to (omega-3)-unsaturated fatty acid ratio and the (omega-9)-unsaturated fatty acid content in all lipids except sphingomyelin. Assay of glucagon-stimulated adenylate cyclase activity at both high and low glucagon concentrations indicated that high-fat intake also decreased cyclic AMP formation. In a second experiment the fat intake was held constant (40% of energy) and oleic acid was substituted for linoleic acid by blending high- and low-linoleic acid-type safflower oils. This experiment established that a dose-response relationship exists between dietary intake of fatty acid and the fatty acid composition of plasma-membrane phospholipids. Specific diet-induced transitions in membrane phospholipid fatty acid composition were paralleled by changes in glucagon-stimulated adenylate cyclase activity. This study suggests that transitions in dietary fat intake can alter a hormone-receptor-mediated enzyme function in vivo by changing the surrounding lipid environment.
Metabolic control of hepatic gluconeogenesis during exercise
Authors: Dohm G L , Newsholme E A
Published: August 2015

Prolonged exercise increased the concentrations of the hexose phosphates and phosphoenolpyruvate and depressed those of fructose 1,6-bisphosphate, triose phosphates and pyruvate in the liver of the rat. Since exercise increases gluconeogenic flux, these changes in metabolite concentrations suggest that metabolic control is exerted, at least, at the fructose 6-phosphate/fructose 1,6-bisphosphate and phosphoenolpyruvate/pyruvate substrate cycles. Exercise increased the maximal activities of glucose 6-phosphatase, fructose 1,6-bisphosphatase, pyruvate kinase and pyruvate carboxylase in the liver, but there were no changes in those of glucokinase, 6-phosphofructokinase and phosphoenolpyruvate carboxykinase. Exercise changed the concentrations of several allosteric effectors of the glycolytic or gluconeogenic enzymes in liver; the concentrations of acetyl-CoA, ADP and AMP were increased, whereas those of ATP, fructose 1,6-bisphosphate and fructose 2,6-bisphosphate were decreased. The effect of exercise on the phosphorylation-dephosphorylation state of pyruvate kinase was investigated by measuring the activities under conditions of saturating and subsaturating concentrations of substrate. The submaximal activity of pyruvate kinase (0.5 mM-phosphoenolpyruvate), expressed as percentage of Vmax., decreased in the exercised animals to less than half that found in the controls. These changes suggest that hepatic pyruvate kinase is less active during exercise, possibly owing to phosphorylation of the enzyme, and this may play a role in increasing the rate of gluconeogenesis.
The bivalent-cation dependence of phosphatidylinositol synthesis in a cell-free system from lymphocytes
Authors: Moore J P , Smith G A , Hesketh T R , Metcalfe J C
Published: August 2015

The bivalent-cation requirements of two enzymes involved in phosphatidylinositol synthesis were defined for pig lymphocyte membranes using a citric acid buffer. CTP:phosphatidic acid cytidylyltransferase (EC 2.7.7.41) is activated by free Mn2+ concentrations above 20nM and by free Mg2+ concentrations above 10 microM. When activated by Mg2+, the enzyme is weakly inhibited by Ca2+ (Ki greater than 250 microM), but Ca2+ has no effect when Mn2+ is used to stimulate CDP-diacylglycerol synthesis. The synthesis of phosphatidylinositol from phosphatidic acid is also stimulated by Mn2+ and Mg2+ concentrations similar to those above and is inhibited by free Ca2+ concentrations above 500nM, probably by its action on CDP-diacylglycerol:inositol 3-phosphatidyltransferase (EC 2.7.8.11). Taken together, these studies suggest that under physiological conditions phosphatidylinositol synthesis is activated by Mg2+ and it is possible that it is further regulated by the free concentrations of Ca2+ and/or Mn2+.
The purine nucleotide cycle and ammonia formation from glutamine by rat kidney slices
Authors: Strzelecki T , Rogulski J , Angielski S
Published: August 2015

To test the significance of the purine nucleotide cycle in renal ammoniagenesis, studies were conducted with rat kidney cortical slices using glutamate or glutamine labelled in the alpha-amino group with 15N. Glucose production by normal kidney slices with 2 mM-glutamine was equal to that with 3 mM-glutamate. With L-[15N]glutamate as sole substrate, one-third of the total ammonia produced by kidney slices was labelled, indicating significant deamination of glutamate or other amino acids from the cellular pool. Ammonia produced from the amino group of L-[alpha-15N]glutamine was 4-fold higher than from glutamate at similar glucose production rates. Glucose and ammonia formation from glutamine by kidney slices obtained from rats with chronic metabolic acidosis was found to be 70% higher than by normal kidney slices. The contribution of the amino group of glutamine to total ammonia production was similar in both types of kidneys. No 15N was found in the amino group of adenine nucleotides after incubation of kidney slices from normal or chronically acidotic rats with labelled glutamine. Addition of Pi, a strong inhibitor of AMP deaminase, had no effect on ammonia formation from glutamine. Likewise, fructose, which may induce a decrease in endogenous Pi, had no effect on ammonia formation. The data obtained suggest that the contribution of the purine nucleotide cycle to ammonia formation from glutamine in rat renal tissue is insignificant.
Rapid accumulation of inositol trisphosphate reveals that agonists hydrolyse polyphosphoinositides instead of phosphatidylinositol
Authors: Berridge M J
Published: August 2015

The agonist-dependent hydrolysis of inositol phospholipids was investigated by studying the breakdown of prelabelled lipid or by measuring the accumulation of inositol phosphates. Stimulation of insect salivary glands with 5-hydroxytryptamine for 6 min provoked a rapid disappearance of [3H]phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and [3H]phosphatidylinositol 4-phosphate (PtdIns4P) but had no effect on the level of [3H]phosphatidylinositol (PtdIns). The breakdown of PtdIns(4,5)P2 was associated with a very rapid release of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], which reached a peak 5 1/2 times that of the resting level after 5 s of stimulation. This high level was not maintained but declined to a lower level, perhaps reflecting the disappearance of PtdIns(4,5)P2. 5-Hydroxytryptamine also induced a rapid and massive accumulation of inositol 1,4-bisphosphate [Ins(1,4)P2]. The fact that these increases in Ins(1,4,5)P3 and Ins(1,4)P2 precede in time any increase in the level of inositol 1-phosphate or inositol provides a clear indication that the primary action of 5-hydroxytryptamine is to stimulate the hydrolysis of PtdIns(4,5)P2 to yield diacylglycerol and Ins(1,4,5)P3. The latter is then hydrolysed by a series of phosphomonoesterases to produce Ins(1,4)P2, Ins1P and finally inositol. The very rapid agonist-dependent increases in Ins(1,4,5)P3 and Ins(1,4)P2 suggests that they could function as second messengers, perhaps to control the release of calcium from internal pools. The PtdIns(4,5)P2 that is used by the receptor mechanism represents a small hormone-sensitive pool that must be constantly replenished by phosphorylation of PtdIns. Small changes in the size of this small energy-dependent pool of polyphosphoinositide will alter the effectiveness of the receptor mechanism and could account for phenomena such as desensitization and super-sensitivity.
Biochemical differences in the mechanism of macrophage lysosomal exocytosis initiated by zymosan particles and weak bases
Authors: Riches D W H , Watkins J L , Stanworth D R
Published: August 2015

By utilizing compounds with different inhibitory properties, discrete biochemical differences were found in the mechanism of selective lysosomal enzyme secretion by macrophages in response to stimulation with zymosan particles and methylamine. Pretreatment of macrophages with trypsin markedly impaired the capacity of the cells to respond to stimulation with zymosan particles, but had no effect on methylamine-stimulated lysosomal enzyme secretion. Similarly, the addition of phenylmethanesulphonyl fluoride or EDTA to the incubation medium substantially inhibited zymosan-induced lysosomal enzyme secretion, whereas the methylamine-stimulated response was unaffected by these agents. The addition of 2-deoxyglucose to incubation media, however, strongly inhibited both zymosan- and methylamine-stimulated β-galactosidase secretion. These findings are consistent with a mechanism for lysosomal enzyme secretion by macrophages, based on a receptor-dependent uptake of zymosan particles and a receptor-independent uptake of methylamine.
Role of diamine oxidase during the treatment of tumour-bearing mice with combinations of polyamine anti-metabolites
Authors: Kallio A , Jänne J
Published: August 2015

Treatment of mice bearing L1210 leukaemia with 2-difluoromethylornithine, a specific inhibitor of ornithine decarboxylase (EC 4.1.1.17), produced a profound depletion of putrescine and spermidine in the tumour cells. Sequential combination of methylglyoxal bis(guanylhydrazone), an inhibitor of adenosylmethionine decarboxylase (EC 4.1.1.50), with difluoromethylornithine largely reversed the polyamine depletion and led to a marked accumulation of cadaverine in the tumour cells. Experiments carried out with the combination of difluoromethylornithine and aminoguanidine, a potent inhibitor of diamine oxidase (EC 1.4.3.6), indicated that the methylglyoxal bis(guanylhydrazone)-induced reversal of polyamine depletion was mediated by the known inhibition of diamine oxidase by the diguanidine. In spite of the normalization of the tumour cell polyamine pattern upon administration of methylglyoxal bis(guanylhydrazone) to difluoromethylornithine-treated animals, the combination of these two drugs produced a growth-inhibitory effect not achievable with either of the compounds alone.
Evidence that Mg2+- or Ca2+-activated adenosine triphosphatase in rat pancreas is a plasma-membrane ecto-enzyme
Authors: Hamlyn J M , Senior A E
Published: August 2015

Preparations of enzymically dispersed rat pancreatic cells hydrolyse externally added nucleoside triphosphates and diphosphates at high rates in the presence of Mg2+ or Ca2+. The lack of response to specific inhibitors and activators differentiates this hydrolytic activity from that of other well-characterized ion-transporting ATPases. Studies based on inactivation of this hydrolytic activity by the covalently reacting, slowly permeating probe diazotized sulphanilic acid indicated that this nucleoside tri- and di-phosphatase is primarily a plasma-membrane ecto-enzyme. It is the major ATPase activity associated with intact cells, homogenates and isolated plasma-membrane fractions. Concanavalin A stimulates this ATPase activity of intact cells and isolated plasma-membrane fractions. The insensitivity of this ATPase activity to univalent ions and inhibitors of pancreatic electrolyte secretion, taken together with the evidence that the active site is externally located, suggests that this enzyme is not directly involved in HCO3- secretion in the pancreas. Its actual function remains unknown.
Challenge of hepatocytes by glucagon triggers a rapid modulation of adenylate cyclase activity in isolated membranes
Authors: Heyworth C M , Houslay M D
Published: August 2015

Membrane fractions obtained from hepatocytes treated with glucagon exhibited a decreased glucagon (with or without GTP)-stimulated adenylate cyclase activity. A maximum effect was seen in around 5 min. No change in the rate of cyclic AMP production was observed for the basal, NaF-, p[NH]ppG (guanosine 5′-[beta, gamma-imido]-triphosphate)- and GTP-stimulated states of the enzyme. The lag observed in the p[NH]ppG-stimulated adenylate cyclase activity of native membranes was abolished when membranes from glucagon-pretreated cells were used. When Mn2+ replaced Mg2+ in the assays, the magnitude of the apparent desensitization was decreased. Mn2+ abolished the lag of onset of p[NH]ppG-stimulated activity in native membranes. The desensitization process was dose-dependent on glucagon, which exhibited a Ka of 4 X 10(-10) M. Depletion of intracellular ATP did not affect this process. It is suggested that this desensitization occurs at the level of the guanine nucleotide-regulatory protein.
The influence of GDP on Ca2+ uptake by mitochondria of brown adipose tissue from lean and genetically obese (ob/ob) mice
Authors: Trayhurn P , Fraser D R
Published: August 2015

The specific binding capacity for purine nucleotides in brown-adipose-tissue mitochondria is thought to indicate the capacity of the proton-conductance pathway which leads to uncoupled respiration. This functional relationship was investigated in studies measuring initial Ca2+-uptake rates and membrane potential in the presence or absence of GDP in brown-adipose-tissue mitochondria with different GDP-binding capacities. The mitochondria from pre-obese and obese ob/ob mice were less able than those from lean control mice to dissipate membrane potential in the absence of GDP. Mitochondria from the obese animals also maintained a higher Ca2+-uptake rate without GDP in comparison with the rate found with mitochondria from the lean mice. The GDP-dependence of Ca2+ uptake was greater in brown-adipose-tissue mitochondria from cold-adapted animals than in those from animals kept at 22 degrees C or at thermoneutrality (33 degrees C). It is concluded that Ca2+-uptake rate and membrane-potential values are depressed in the absence of GDP and indicate indirectly the influence of purine nucleotides on maintaining the proton electrochemical gradient in brown-adipose-tissue mitochondria. It is also apparent that the lower GDP-binding capacity in mitochondria from ob/ob mice is related to a decreased ability to dissipate the proton electrochemical gradient.
Biosynthesis and secretion of procollagenase by rabbit synovial fibroblasts. Inhibition of procollagenase secretion by monensin and evidence for glycosylation of procollagenase
Authors: Nagase H , Brinckerhoff C E , Vater C A , Harris E D
Published: August 2015

Monolayer cultures of rabbit synovial fibroblasts stimulated with phorbol myristate acetate to produce large amounts of collagenase (EC 3.4.24.7) were used to study the biosynthesis and secretion of this enzyme. [3H]Leucine was added to cell cultures for pulse-chase and continuous-labelling experiments. The labelled procollagenase synthesized was identified by immunoprecipitation followed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and fluorography. The amounts of intracellular and extracellular proenzyme were quantified by measuring radioactivity incorporated into the proteins. procollagenase was synthesized as doublet proteins of Mr 57 000 and Mr 61 000. Immunoprecipitable proenzyme proteins were first detected in culture medium 35 min after [3H]leucine was added to the cells. Monensin treatment of the cells inhibited procollagenase secretion and led to intracellular accumulation of the proenzyme. Cells treated with tunicamycin produced only the 57 000-Mr form, indicating that in rabbit synovial cells the 61 000-Mr form was post-translationally modified by addition of oligosaccharides to asparagine residues. The ratios of glycosylated to unglycosylated forms in cell lysates and in culture medium were 0.22:1 and 0.07:1 respectively.
Spontaneous Nε-methylation and Nε-formylation reactions between l-lysine and formaldehyde inhibited by l-ascorbic acid
Authors: Trézl L , Rusznák I , Tyihák E , Szarvas T , Szende B
Published: August 2015

For the inhibition of spontaneous N epsilon-methylation and N epsilon-formylation reactions between L-lysine and formaldehyde, L-ascorbic acid proved to be most suitable. The inhibition was not complete unless the molar concentration of ascorbic acid exceeded that of formaldehyde. T.l.c., potentiometric titration, n.m.r. spectroscopy and radiometric analysis were applied in the study of the inhibition process. Formaldehyde was reduced by L-ascorbic acid to ethylene glycol.
Binding and endocytosis of glycoproteins and neoglycoproteins by isolated rabbit hepatocytes
Authors: Connolly D T , Townsend R R , Kawaguchi K , Hobish M K , Bell W R , Lee Y C
Published: August 2015

Rabbit hepatocytes were isolated by a collagenase perfusion technique, and used to study the binding and endocytosis of the glycoprotein, asialo-orosomucoid, and the neoglycoprotein, Gal39-bovine serum albumin. Both of these proteins contain exposed galactosyl residues, and were avidly bound by the lectin on the hepatic parenchymal cell surface. Steady state and kinetic experiments performed at 2 degrees C and at 37 degrees C revealed the presence of two apparent classes of binding sites totalling 4.7 X 10(5) sites/cell at 2 degrees C, and 6.3 X 10(5) sites/cell at 37 degrees C. At 37 degrees C, both classes of sites participated in internalization of bound ligand. The cells were capable of internalizing about 60 000 molecules/min per cell. The process appeared to be first-order, with a rate constant k = 0.098 min-1 and t1/2 = 7.1 +/- 0.6 min. Binding could be inhibited by galactose-containing compounds, EGTA, and by anti-(hepatic lectin) immunoglobulin G. The inhibition by antibody appeared to be reversible upon removal of antibody-containing medium.
Protein synthesis in skeletal muscle of the perfused rat hemicorpus compared with rates in the intact animal
Authors: Preedy V R , Garlick P J
Published: August 2015

The rate of protein synthesis was measured in muscles of the perfused rat hemicorpus, and values were compared with rates obtained in whole animals. In gastrocnemius muscle of fed rats the rate of synthesis measured in the hemicorpus was the same as that in the whole animal. However, in plantaris, quadriceps and soleus muscles rates were higher in the hemicorpus than those in vivo. In the hemicorpus, starvation for 1 day decreased the rate of protein synthesis in gastrocnemius and plantaris muscles, in parallel with decreases in the RNA content, but the soleus remained unaffected. Similar effects of starvation were observed in vivo, so that the relationships between rates in vivo and in the hemicorpus were the same as those in fed rats. Proteins of quadriceps and plantaris muscles were separated into sarcoplasmic and myofibrillar fractions. The rate of synthesis in the sarcoplasmic fraction of the hemicorpus from fed rats was similar to that in vivo, but synthesis in the myofibrillar fraction was greater. In the plantaris of starved rats the rates of synthesis in both fractions were lower, but the relationships between rates measured in vivo and in the perfused hemicorpus were similar to those seen in fed rats. The addition of insulin to the perfusate of the hemicorpus prepared from 1-day-starved animals increased the rates of protein synthesis per unit of RNA in gastrocnemius and plantaris muscles to values above those seen in fed animals when measured in vivo or in the hemicorpus. Insulin had no effect on the soleus. Overall, the rates of protein synthesis in the hemicorpus differed from those in vivo. However, the effect of starvation when measured in the whole animal was very similar to that measured in the isolated rat hemicorpus when insulin was omitted from the perfusate.
Regulation of mitochondrial pyruvate carboxylation in isolated hepatocytes by acute insulin treatment
Authors: Chisholm A B , Allan E H , Titheradge M A
Published: August 2015

The effect of acute insulin treatment of hepatocytes on pyruvate carboxylation in both isolated mitochondria and cells rendered permeable by filipin was examined. Challenging the cells with insulin alone had no effect on either the basal rate of pyruvate carboxylation or gluconeogenesis, although it did suppress the responses to both glucagon and catecholamines. Insulin treatment was unable to antagonize the enhanced rate of pyruvate carboxylation caused by stimulation of the cells with either angiotensin or vasopressin. Neither insulin nor the gluconeogenic hormones altered the total extractable pyruvate carboxylase activity in the isolated mitochondria, suggesting that the effect of hormones at the level of the isolated intact organelle was mediated via alterations in the intramitochondrial concentrations of effector molecules, notably ATP and the [ATP]/[ADP] ratio and substrate availability. The alterations in pyruvate carboxylation correlate well with glucose synthesis in terms of sensitivity to effector molecules, putative second messengers and time of onset of the response, indicating that alterations in the flux through this enzyme are compatible with it being an important site in the control of gluconeogenesis from C3 precursors.
Haematoporphyrin and OO'-diacetylhaematoporphyrin binding by serum and cellular proteins. Implications for the clearance of these photochemotherapeutic agents by cells
Authors: Smith A , Neuschatz T
Published: August 2015

Haematoporphyrin derivative (HpD), a mixture of porphyrins, is currently used as a photochemotherapeutic agent in the treatment of neoplasias. The interaction of purified components of HpD with serum and cellular proteins was investigated using absorption and fluorescence spectroscopy. The interactions of haematoporphyrin and OO'-diacetylhaematoporphyrin with human albumin and with haemopexin, the two major serum porphyrin-binding proteins, show stoichiometries of 1 mol of porphyrin bound per mol of protein. The apparent dissociation constants, Kd, are in the range of 1-2 microM for albumin and 3-4 microM for haemopexin. These two major components of HpD would, after intravenous injection, bind to albumin and circulate in serum as albumin complexes. Free porphyrin rather than porphyrin bound to albumin interacts with Morris hepatoma tissue culture cells. A rapid high-affinity saturable transport system operates at free porphyrin concentrations of less than 2 microM. In addition, fluorescence spectra show that components in rat liver cytosol can bind haematoporphyrin and OO'-diacetylhaematoporphyrin and distinguish these binders from those present in rat serum.
Activation of protein kinase in the bovine corpus luteum by phospholipid and Ca2+
Authors: Davis J S , Clark M R
Published: August 2015

A new species of protein kinase has been identified in cytosol preparations from bovine corpora lutea. Enzyme activity required the simultaneous presence of Ca2+ and phospholipid, and was also enhanced by glyceryl dioleate. Phosphatidylserine was the most effective phospholipid for stimulating histone phosphorylation. Other phospholipids capable of supporting enzymic activity were, in order of decreasing activity, phosphatidylinositol, phosphatidic acid, cardiolipin and phosphatidylglycerol. Several other phospholipids tested were ineffective. A cyclic AMP-dependent protein kinase was also present in the luteal cytosol. This enzyme activity was eliminated by protein kinase inhibitor without affecting the Ca2+- and phospholipid-stimulated activity. Lysine-rich histone (IIIS) was a much better substrate than type-IIA histone for Ca2+- and phospholipid-dependent phosphorylation. Ca2+ and phospholipid also enhanced phosphorylation of endogenous luteal cytosol protein. Calmodulin, alone or in the presence of Ca2+, was unable to increase phosphorylation. Trifluoperazine inhibited protein kinase activity stimulated by Ca2+ and phospholipid. These data suggest that a phospholipid-sensitive, Ca2+-dependent protein kinase may provide an important link between hormonally-induced changes in phospholipid metabolism and corpus-luteum function.
Myofibrillar protein turnover. Synthesis rates of myofibrillar and sarcoplasmic protein fractions in different muscles and the changes observed during postnatal development and in response to feeding and starvation
Authors: Bates P C , Millward D J
Published: August 2015

Measurement of rates of synthesis of skeletal-muscle proteins in adult rats shows that the faster overall rate of turnover in diaphragm and soleus muscles compared with several other, more glycolytic, muscles is also exhibited by the myofibrillar proteins, since the ratio of sarcoplasmic to myofibrillar protein synthesis is similar for all muscles. Further, throughout postnatal development, when the overall turnover rate falls with age, parallel changes occur for the myofibrillar proteins, as indicated by a constant ratio of sarcoplasmic to myofibrillar protein synthesis (2.06) in the steady state after overnight starvation. Only in the youngest (4 weeks old) rats is a slightly lower ratio observed (1.72). These results indicate that, when changes in the overall turnover rate of muscle proteins occur, the relative turnover of the two major protein fractions stays constant. However, measurements in the non-steady state during growth and after starvation for 4 days show that the relative synthesis rates of the two fractions change as a result of a disproportionate increase in myofibrillar protein synthesis during growth and decrease during starvation. Thus the synthesis rate of the slower-turning-over myofibrillar protein fraction is more sensitive to nutritional state than is that of the sarcoplasmic protein. It is suggested that such responses may help to maintain constant tissue composition during non-steady-state conditions of growth and atrophy.
Biosynthesis and transport of lysosomal enzymes in human monocytes and macrophages. Effects of ammonium chloride, zymosan and tunicamycin
Authors: Imort M , Zühlsdorf M , Feige U , Hasilik A , von Figura K
Published: August 2015

Human monocytes and macrophages synthesize lysosomal enzymes as larger precursors. The polypeptide patterns of several lysosomal-enzyme precursors and their mature forms are similar to those observed in human fibroblasts. Like fibroblasts, the monocytes and macrophages release small amounts of lysosomal-enzyme precursors. The lysosomotropic NH4+ cation enhances this release. In contrast, zymosan, a degranulating agent, causes release of both the mature and the precursor forms of the lysosomal enzymes. Both NH4Cl and zymosan inhibit maturation of the precursors. The fractional amounts of mature cathepsin D and beta-hexosaminidase released in the presence of zymosan are strikingly different. Probably, in the macrophages several lysosomal organelles are packaged with different relative contents of lysosomal enzymes. The transport of the precursors of cathepsin D into lysosomes is inhibited by tunicamycin. Therefore oligosaccharide side chains are likely to function as signals in packaging of lysosomal enzymes in macrophages also.
Evidence for the vitamin K-dependent γ-carboxylation of the first glutamic acid residue in peptide substrates containing a diglutamyl sequence
Authors: Burgess A I , Esnouf M P , Rose K , Offord R E
Published: August 2015

The peptide substrate commonly used in vitamin K-dependent carboxylation, Phe-Leu-Glu-Glu-Val, has been shown, by the use of high-voltage paper electrophoresis, to be degraded from the N-terminus by a microsomal leucine amino-peptidase. The replacement of phenylalanine with a N-t-butoxycarbonyl group resulted in a tetrapeptide substrate with a blocked N-terminus resistant to enzymic degradation. Vitamin K-dependent carboxylation of this non-degradable substrate gave a unique carboxylated product, which was separated from microsomal protein and unchanged substrate by using DEAE-Sephadex A25 as a final step. The carboxylated product was subsequently decarboxylated in 2HCl and analysed by using g.l.c. coupled to a mass spectrometer. This showed that only the first glutamic acid residue in the peptide substrate was carboxylated.
Haem disorder in modified myoglobins. Effect of reconstitution procedures
Authors: Ahmad M B , Kincaid J R
Published: August 2015

Apomyoglobin was reconstituted with deuterohaem derivatives under various conditions. The fraction of disordered component, which is characterized by a 180 degree rotation of the haem group, for the various preparations was determined by n.m.r. spectroscopy. By using the procedures described, it was shown that the fraction of disordered component is minimized if the reconstitution is carried out with high-spin ferric haem derivatives within an experimentally determined optimum pH range of 8-9.5. Use of low-spin derivatives in either the ferrous or ferric forms leads to substantial increases in the fraction of disordered form. Attempted removal of the disordered form by selective oxidation and chromatographic purification was not effective.
Benzyl p-guanidinothiobenzoate hydrochloride, a new active-site titrant for trypsin and trypsin-like enzymes
Authors: Cook R R , Powers J C
Published: August 2015

Benzyl p-guanidinothiobenzoate hydrochloride was synthesized and demonstrated to be useful for active-site titration of bovine trypsin, bovine thrombin, human lung tryptase, bovine activated protein C, human Factor XIIa fragment and bovine Factor Xa beta. The titration is based on rapid formation of a stable acyl-enzyme with a stoichiometric release of benzyl thiol. Thiol production is measured quantitatively by including 4,4′-dithiodipyridine in the reaction mixture and measuring the increase in absorbance at 324 nm. Ellman's reagent has also been successfully employed, allowing measurement at 410 nm. Unlike p-nitrophenyl p'-guanidinobenzoate, the thioester titrant reacts slowly with chymotrypsin A alpha thus eliminating interference by this enzyme in most titrations. Advantages of this reagent as a titrant include: flexibility in detection of the released thiol, selectivity between trypsin and chymotrypsin-like enzymes, minimal pH-dependence of the epsilon of the absorbing species, relative stability of the reagent under titration conditions, and high epsilon at pH 7.2 with either 4,4′-dithiodipyridine or Ellman's reagent. The reagent should prove useful as an alternative to p-nitrophenyl p'-guanidinobenzoate hydrochloride for the determination of active-site concentrations of the enzymes employed, as well as of other related enzymes.
The core protein of fibroblast proteoheparan sulphate consists of disulphide-bonded subunits
Authors: Cöster L , Malström A , Carlstedt I , Fransson L Å
Published: August 2015

Fibroblast proteoheparan sulphate has a disulphide-bonded subunit structure. The core protein appears to consist of two polypeptides each of Mr 80 000-100 000. As shown elsewhere [Carlstedt, Cöster, Malmström & Fransson (1983) J. Biol. Chem. in the press], both polypeptide molecules carry four to six heparan sulphate side chains (approx. Mr 20 000) and an unknown number of oligosaccharide units, giving the whole macromolecule an Mr in the range 300 000-400 000.
Chemical modification of Escherichia coli succinyl-CoA synthetase with the adenine nucleotide analogue 5′-p-fluorosulphonylbenzoyladenosine
Authors: Prasad A R S , Ybarra J , Nishimura J S
Published: August 2015

Escherichia coli succinyl-CoA synthetase (EC 6.2.1.5) was irreversibly inactivated on incubation with the adenine nucleotide analogue 5′-p-fluorosulphonylbenzoyladenosine (5′-FSBA). Optimal inactivation by 5′-FSBA took place in 40% (v/v) dimethylformamide. ATP and ADP protected the enzyme against inactivation by 5′-FSBA, whereas desulpho-CoA, an analogue of CoA, did not. Inactivation of succinyl-CoA synthetase by 5′-FSBA resulted in total loss of almost four thiol groups per alpha beta-dimer, of which two groups appeared to be essential for catalytic activity. 5′-FSBA at the first instance appeared to interact non-specifically with non-essential thiol groups, followed by a more specific reaction with essential thiol groups in the ATP(ADP)-binding region. Plots of the data according to the method of Tsou [(1962) Sci. Sin. 11, 1535-1558] revealed that, of the two slower-reacting thiol groups, only one was essential for catalytic activity. When succinyl-CoA synthetase that had been totally inactivated by 5′-FSBA was unfolded in acidic urea and then refolded in the presence of 100 mM-dithiothreitol, 85% of the activity, in comparison with the appropriate control, was restored. These data are interpreted to indicate that inactivation of succinyl-CoA synthetase by 5′-FSBA involves the formation of a disulphide bond between two cysteine residues. Disulphide bond formation likely proceeds via a thiosulphonate intermediate between 5′-p-sulphonylbenzoyladenosine and one of the reactive thiol groups of the enzyme.
Fractionation of the mycobacillin-synthesizing enzyme system
Authors: Ghosh S K , Mukhopadhyay N K , Majumder S , Bose S K
Published: August 2015

The mycobacillin-synthesizing enzyme system was highly purified by fractionation at 30-55% (NH4)2SO4 saturation. The enzyme concentrate on Sephadex G-200 gel chromatography was resolved into three distinct fragments. Each of the fragments on further purification by DEAE-cellulose ion-exchange chromatography behaved as a single-component system, as clearly indicated by the sharpness of the peaks in the elution diagram. None of the fragments alone nor any two of them in all possible combinations possessed mycobacillin-synthesizing activity, which was restored only when the three fragments were used together in the test system.
The serine proteinase chain of human complement component C̅1s. Cyanogen bromide cleavage and N-terminal sequences of the fragments
Authors: Carter P E , Dunbar B , Fothergill J E
Published: August 2015

Human complement component C1s was purified from fresh blood by conventional methods of precipitation and chromatography. The single-chain zymogen form was activated by treatment with C1r. Reduction and carboxymethylation then allowed the light chain and heavy chain to be separated on DEAE-Sepharose CL-6B in 8 M-urea. Liquid-phase sequencing of the light chain determined 50 residues from the N-terminus. CNBr-cleavage fragments of the light chain were separated by high-pressure liquid chromatography on gel-permeation and reverse-phase columns. N-Terminal sequencing of these fragments determined the order of a further 138 residues, giving a total of 188 residues or about 75% of the light chain. Seven of these eight sequences could be readily aligned with the amino acid sequences of other serine proteinases. The typical serine proteinase active-site residues are clearly conserved in C1s, and the specificity-related side chain of the substrate-binding pocket is aspartic acid, as in trypsin, consistent with the proteolytic action of C1s on C4 at an arginine residue. Somewhat surprisingly, when the C1s sequence is compared with that of complement subcomponent C1r, the percentage difference (59%) is approximately the same as that found between the other mammalian serine proteinases (56-71%).
2,3-diamino-2,3-dideoxy-d-glucofuranurono-6,3-lactam from the hydrolysate of Pseudomonas aeruginosa P14 lipopolysaccharide
Authors: Okuda S , Suzuki N
Published: August 2015

An unknown amino sugar, U-7, which had been detected in the hydrolysate of the polysaccharide fraction (F-A) of Pseudomonas aeruginosa P14 lipopolysaccharide, was isolated from the hydrolysate of whole cells of this micro-organism and converted into the N-acetyl derivative (U-7NAc). On the basis of i.r.-absorption spectrometry, 13C-n.m.r. and 1H-n.m.r. spectroscopy and mass spectrometry, the structure of compound U-7NAc was identified as 2-acetamido-3-amino-2,3-dideoxyhexofuranurono-6,3-lactam. The configuration of compound U-7NAc was then unequivocally identified as 2-acetamido-3-amino-2,3-dideoxy-D-glucofuranurono-6,3-lactam by comparing the synthetic and natural compounds. Compound U-7 and synthetic 2,3-diamino-2,3-dideoxy-D-glucofuranurono-6,3-lactam showed the same behaviour on chromatography. G.l.c.-mass-spectral analyses of fraction F-A and synthetic 2,3-diacetamido-2,3-dideoxy-D-glucuronic acid after methanolyses and trimethylsilylations showed the presence of the same derivative. It was concluded that the amino sugar U-7 was produced from the 2,3-diacetamido-2,3-dideoxy-D-glucuronic acid residue present in fraction F-A.
Polymeric structure of a high-molecular-weight glycoprotein from bovine cervical mucus
Authors: Meyer F A
Published: August 2015

A 16 X 10(6)-Mr glycoprotein isolated from bovine oestrus cervical mucus when reduced under conditions where disulphide-bond cleavage is essentially quantitative produces chains whose Mr from light-scattering and from sedimentation and diffusion data is some 4 X 10(6)-5 X 10(6). Pronase digestion of the chains indicates that glycosylated sequences of Mr 0.3 X 10(6)-0.5 X 10(6) are interspersed with enzyme-susceptible non-glycosylated peptide sequences.
Biosynthesis of intestinal microvillar proteins. Role of the Golgi complex and microtubules
Authors: Danielsen E M , Cowell G M , Poulsen S S
Published: August 2015

The effect of monensin and colchicine on the biogenesis of aminopeptidase N (EC 3.4.11.2), aminopeptidase A (EC 3.4.11.7), dipeptidyl peptidase IV (EC 3.4.14.5), sucrase (EC 3.2.1.48)-isomaltase (EC 3.2.1.10) and maltase-glucoamylase (EC 3.2.1.20) was studied in organ-cultured pig small-intestinal explants. On the ultrastructural level, monensin (1 microM) caused an increasingly extensive dilation and vacuolization of the Golgi complex during 4h exposure of the explants. On the molecular level, the effect of monensin was twofold. (1) The processing from the initial high-mannose-glycosylated form to the mature complex-glycosylated form was arrested. For some of the enzymes studied, intermediate stages between the high-mannose and complex forms could be seen, probably corresponding to ‘trimmed’ or partially complex-glycosylated polypeptides. (2) Labelled microvillar enzymes failed to reach their final destination. These findings suggest the involvement of the Golgi complex in the post-translational processing and transport of microvillar enzymes. The presence in the growth medium of colchicine (50 micrograms/ml) caused a significant inhibition of the appearance of newly synthesized enzymes in the microvillar membrane during a 3 h labelling period. Since synthesis and post-translational modification of the microvillar enzymes were largely unaffected by colchicine, the results obtained suggest that microtubules play a role in the final transport of the enzymes from the Golgi complex to the microvillar membrane.
Effect of taurochenodeoxycholate or tauroursodeoxycholate upon biliary output of phospholipids and plasma-membrane enzymes, and the extent of cell damage, in isolated perfused rat livers
Authors: Barnwell S G , Lowe P J , Coleman R
Published: August 2015

Isolated perfused rat livers were used to study the effects of taurochenodeoxycholate (TCDC) and tauroursodeoxycholate (TUDC) upon some aspects of biliary composition. After depletion of the endogenous bile salt pool of the liver, introduction of either bile salt brought about increases in bile flow, bile salt output and biliary phospholipid output. Taurochenodeoxycholate needed a lower biliary concentration to produce phospholipid output than did tauroursodeoxycholate. TCDC perfusion caused a substantial output of plasma-membrane enzymes (5′-nucleotidase and alkaline phosphodiesterase) into the bile, whereas TUDC caused little output of either enzyme; this may represent a characteristic difference between the effects of the two bile salts on the hepatobiliary system. The results from TUDC perfusion indicate also that much of the output of biliary phospholipid promoted by bile salts, may be independent of the output of plasma-membrane enzymes promoted by bile salts.
Transfer of exogenous cholesterol to microsomes of hepatocytes investigated with [3H]desmosterol tracer
Authors: Goh E H
Published: August 2015

The feasibility of using exogenous [3H]desmosterol as a mass metabolic tracer for exogenous non-esterified cholesterol in hepatocytes is investigated with albumin-bound non-esterified cholesterol containing [3H]desmosterol and [14C] cholesterol tracers. The amounts of uptake and metabolism of exogenous cholesterol monitored by either tracer are the same. In addition, the conversion of [3H]desmosterol into [3H]cholesterol by the delta 24-sterol reductase in the microsomes can be used as an estimate for the mass transfer of exogenous cholesterol to the microsomes. The results obtained indicate that only a small fraction of exogenous cholesterol that was transferred to the microsomes was metabolized into bile acids and steryl esters. The technique of estimating the mass transfer of exogenous cholesterol to the microsomes with [3H]desmosterol may be of importance in investigations dealing with the effect of exogenous plasma cholesterol on changes in the physiological functions of the endoplasmic reticulum in the cells.