bluuebunny/crossref_metadata_2025 huggingface dataset:
Modification of an arginine residue in pig kidney general acyl-coenzyme A dehydrogenase by cyclohexane-1,2-dione
Authors: Jiang Z Y , Thorpe C
Published: August 2015

The flavoenzyme pig kidney general acyl-CoA dehydrogenase (EC 1.3.99.3) is inactivated by cyclohexane-1,2-dione in borate buffer in a reaction that exhibits pseudo-first-order kinetics. Strong protection is afforded by the substrate octanoyl-CoA, as well as by heptadecyl-CoA, a potent competitive inhibitor of the dehydrogenase that does not reduce enzyme flavin. Enzyme exhibiting 10% residual activity in borate buffer contains about 1.3 modified arginine residues per flavin molecule. Very little reduction of the modified enzyme in borate buffer occurs at high concentrations of octanoyl-CoA, in marked contrast with the stoicheiometric reduction of the native enzyme. However, in phosphate buffer alone, the modified enzyme exhibits 55% residual activity and, although binding of substrate is still seriously impaired (apparent Kd=14 microM), excess substrate effects the formation of the characteristic reduced flavin X enoyl-CoA charge-transfer complex. These results suggest that the susceptible arginine residue, though not catalytically essential, is probably within the acyl-CoA-binding site of general acyl-CoA dehydrogenase.
Association of human erythrocyte membrane glycoproteins with blood-group Cad specificity
Authors: Cartron J P , Blanchard D
Published: August 2015

Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of erythrocyte membranes from a blood-group-B individual with the rare Cad phenotype indicates a lower-than-normal mobility of the main sialoglycoproteins, suggesting an increase in apparent molecular mass of 3kDa and 2kDa respectively for glycoprotein alpha (synonym glycophorin A) and glycoprotein delta (synonym glycophorin B). Since the chief structural determinant of Cad specificity is N-acetylgalactosamine, the membrane receptors have been isolated by affinity binding on immobilized Dolichos biflorus (horse gram) lectin. The predominant species eluted from the gel was the abnormal glycoprotein alpha, whereas in control experiments no material could be recovered from the adsorbent incubated with group-B Cad-negative erythrocyte membranes. After partition of the membranes with organic solvents, the blood-group-Cad activity was found in aqueous phases containing the sialoglycoproteins, but not in the organic phases containing simple or complex glycolipids, which, however, retained the blood-group-B activity. The carbohydrate composition of highly purified lipid-free glycoprotein alpha molecules prepared from Cad and control erythrocytes was determined. Interestingly the molar ratio of N-acetylneuraminic acid to N-acetylgalactosamine was equal to 2:1 in the case of controls and equal to 1:1 in the case of Cad erythrocytes. Taken together these results suggest that Cad specificity is defined by N-acetylgalactosamine residues carried by the alkali-labile oligosaccharide chains attached to the erythrocyte membrane sialo-glycoproteins.
Differential energy requirements for platelet responses. A simultaneous study of aggregation, three secretory processes, arachidonate liberation, phosphatidylinositol breakdown and phosphatidate production
Authors: Holmsen Holm , Kaplan Karen L. , Dangelmaier Carol A.
Published: August 2015

Previous studies have indicated different energy requirements for some platelet responses; these differences could, however, be due to inadequate methodology and differences in platelet preparation. The present study describes the effect of decreasing ATP availability on seven platelet responses measured in gel-filtered human platelets. The cells, prelabelled with 5-hydroxy[3H]tryptamine, [3H]- or [14C]adenine, [32P]Pi or [3H]arachidonate, were incubated with antimycin A and 2-deoxy-d-glucose. Platelet responses induced by thrombin and collagen (secretion only), level of metabolic ATP and the adenylate energy charge (AEC) were determined at various times during incubation. Platelet aggregation was rapidly inhibited after a lag of 5–15 min and with 50% inhibition at AEC = 0.55–0.60. Secretion of 5-hydroxy[14C]tryptamine and ATP + ADP from dense granules and of fibrinogen and β-thromboglobin from α-granules were inhibited in parallel, without a lag and with 50% inhibition at AEC = 0.65–0.70. The inhibition of secretion of platelet factor 4 from the α-granules followed another pattern with 50% inhibition at AEC = 0.70–0.80. Breakdown of [3H]-phosphatidylinositol, formation of [3H]- and [32P]-phosphatidate, liberation of [3H]arachidonate and secretion of acid hydrolases were inhibited in parallel and inhibition was present at the start of incubation with 50% inhibition at AEC = 0.80–0.87. These results suggest that the responses have different energy requirements, increasing in the order: aggregation < dense granule and α-granule secretion < acid hydrolase secretion, phosphatidylinositol breakdown, phosphatidate formation and arachidonate liberation. The powerful inhibition of phosphatidylinositol breakdown by metabolic inhibitors suggests that energy-requiring steps are involved in the activation of phospholipase C.
Characteristics of the desensitization and resensitization of the cyclic AMP-independent glycogenolytic response in rat liver cells
Authors: Keppens S , De Wulf H
Published: August 2015

Vasopressin and alpha-adrenergic agonists were shown previously [Bréant, Keppens & De Wulf (1981) Biochem. J. 200, 509-514] to induce a heterologous, dose-dependent and receptor-mediated desensitization of the cyclic AMP-independent glycogenolytic response in isolated hepatocytes. The desensitized state of the hepatocytes can be preserved as long as the agonist is bound to its receptor. Conversely, washing the cells with a hormone-free buffer or displacement of the agonist from its receptor by a specific antagonist restores the responsiveness. The desensitization and its reversibility (i.e. resensitization) are obtained within minutes. The desensitization can be clearly elicited at temperatures as low as 5 degrees C, whereas the glycogenolytic response and the enhancement of the 45Ca flux are only obtained above 15 degrees C; the resensitization requires even higher temperatures. A tentative model is proposed to account for the observed effects.
The use of acetyl-CoA carboxylase activity and changes in wall composition as measures of embryogenesis in tissue cultures of oil palm (Elaeis guineensis)
Authors: Turnham E , Northcote D H
Published: August 2015

With some lines of oil-palm tissue cultures embryogenesis occurs spontaneously within the callus grown on a medium containing 2.5 mg of 3-naphthylacetic acid/litre. One of the initial biochemical events that occurs just before the embryoid can be seen is the accumulation of fat droplets within the cells. This accumulation of lipid is correlated with an increase in acetyl-CoA carboxylase activity. The carboxylase is thus probably a rate-limiting step in fatty acid synthesis in these cells and can be used as a quantitative marker of somatic embryogenesis within the tissue. During the development of the embryoid tissue there is an increase in cell division and the differentiation of vascular cells with secondary thickened walls. These stages of the differentiation may be monitored by measuring the ratio of pectin synthesis (polygalacturonic acid formation) to hemicellulose synthesis (xylan formation).
Activation of the chick oviduct progesterone receptor by heparin in the presence or absence of hormone
Authors: Yang C R , Mester J , Wolfson A , Renoir J M , Baulieu E E
Published: August 2015

Activation (transformation) of the chick oviduct progesterone receptor was found to be induced at 0 degrees C by heparin free in solution as well as by chromatography on a column of heparin linked to acrylamide/agarose. The transformed molecule displayed properties of the activated form of [3H]progesterone-receptor complex obtained by heat treatment or by high ionic strength: smaller size (s20,w = 3.9 S, Stokes radius = 5.2 nm), lower rate of dissociation (t 1/2 approx. 50 h at 0 degrees C compared with approx. 20 h for the ‘native’ form) and increased binding to phosphocellulose. In all cases, molybdate was an effective inhibitor of transformation and stabilized a large ‘native’ form (s20,w = 7.9 S, Stokes radius = 7.6 nm). Transformation by neither KCl nor heparin depended on the presence of ligand bound to the receptor, and the properties of the receptor molecule produced by treatment of ligand-free receptor with high ionic strength or with heparin were identical with those of the activated progesterone-receptor complex, demonstrating that receptor activation can be obtained experimentally in the absence of hormone. Our data are compatible with a model in which activation implies separation of the 4 S units, which compose the approx. 8 S ‘native’ form.
Fatty acid oxidation by liver and muscle preparations of exhaustively exercised rats
Authors: Barakat H A , Kasperek G J , Dohm G L , Tapscott E B , Snider R D
Published: August 2015

The influence of exhaustive exercise on the capacity of liver and muscle of rats to oxidize fatty acids was investigated in vitro. The rate of oxidation of fatty acids by liver preparations was significantly elevated as a result of exhaustion. Concurrently, the concentrations of beta-hydroxybutyrate were elevated in the plasma of the exhausted rats, suggesting that oxidation of fatty acids was also elevated in vivo. These findings are analogous to the findings of increased oxidation of fatty acids that results from training. In muscle, oxidation of palmitate, palmitoylcarnitine and beta-hydroxybutyrate by homogenates and isolated mitochondria was depressed with exercise. Despite the decrease in the oxidative capacity of the muscle preparations, the activities of several enzymes of beta-oxidation were either increased or unchanged as a result of exercise, suggesting that the depression in fatty acid oxidation may not be related to alterations in the process of beta-oxidation. Further studies showed that oxidation of [2-(14)C]pyruvate by muscle was depressed, whereas oxidation of [1-(14)C]pyruvate was not changed as a result of exercise. These results suggest that the decrease in fatty acid oxidation may be related to aberrations in the oxidation of acetyl-CoA. The changes in fatty acid oxidation that were observed, which are at variance with what is reported to occur with training, may have resulted from increased fragility of muscle mitochondria as a result of exercise. This increased fragility may render the mitochondria more susceptible to experimental manipulations in vitro and a subsequent loss of normal function.
Directional Ca2+ effect on stimulation of mucin secretion from chicken trachea in vitro
Authors: Mian Nasi , Anderson Caroline E. , Pope Andrew J. , Smith Anthony R. , Richardson Paul S. , Balfre Kim , Kent Paul W.
Published: August 2015

Chicken tracheal mucosa in vitro transported and incorporated radioactive precursors into mucins, which were secreted at a steady rate into the tracheal lumen. Secretion of mucins labelled with 35S and 3H after pulse-labelling of the mucosal layer with Na235SO4 and d-[1-3H]glucosamine as precursors was an energy-dependent process, as it was strongly inhibited by the action of respiratory-chain inhibitors, an uncoupler of oxidative phosphorylation, a metabolic blocker and a temperature shift from 41°C to 5°C. On the other hand, both cholinergic and parasympathomimetic agents considerably increased the secretion of dual-radiolabelled mucins when applied on the submucosal side of the trachea. The effect of Ca2+ was directional, since only high submucosal (3.6 or 18mm) or low luminal (zero or 0.18mm) Ca2+ massively enhanced the secretion of radiolabelled mucin compared with the mucin output measured under physiological Ca2+ conditions (1.8mm). Whereas application of ionophore A23187 on either side of the trachea significantly increased mucin output, its presence in the appropriate tracheal compartment and under appropriate Ca2+ conditions further accentuated the output of radiolabelled mucins. Addition of acetylcholine under appropriate conditions also had an additive effect on the Ca2+-stimulated secretion of mucins. Ca2+ stimulation of mucin secretion appears to be dependent on the metabolic integrity of the mucosal cells. Mucins secreted in response to high submucosal and low luminal [Ca2+] appear to consist of a number of different types of glycoproteins, as judged from their ion-exchange-chromatographic behaviour.
The effect of N-methylprotoporphyrin IX on the synthesis of photosynthetic pigments in Cyanidium caldarium. Further evidence for the role of haem in the biosynthesis of plant bilins
Authors: Brown Stanley B. , Holroyd J. Andrew , Vernon David I. , Troxler Robert F. , Smith Kevin M.
Published: August 2015

N-Methylprotoporphyrin IX strongly inhibits synthesis of phycocyanobilin, but not chlorophyll a, in the dark. In the light, both phycocyanin and chlorophyll a synthesis are inhibited in parallel. These results are consistent with the intermediacy of haem in algal bilin synthesis and suggest a control mechanism for chlorophyll a synthesis, previously unknown.
The metabolism of the fluorescent probe cholesta-5,7,9(11)-trien-3β-ol by rat liver
Authors: Wilton David C.
Published: August 2015

The chemical synthesis and metabolism of [3α-3H]cholesta-5,7,9(11)-trien-3β-ol is described. This radioactive fluorescent sterol probe cannot be rearranged or converted to cholesterol by rat liver but is readily esterified.
A new procedure for haptenizing adenosine leading to a more specific radioimmunoassay method
Authors: Newby A C , Sala G B
Published: August 2015

Adenosine was coupled to human serum albumin by two different procedures that preserved the purine and ribose rings. The conjugates were evaluated for antigenicity in rabbits and guinea pigs. A conjugate containing 2′(3′)-O-succinoyladenosine failed to elicit antibodies, whereas one containing laevulinic acid (O2′, 3′-adenosine-acetal) elicited antibodies in all animals injected. The affinity and specificity of binding of adenosine to three selected antisera were evaluated. Dissociation constants of 31-187 nM were observed. Displacement of adenosine binding to all antisera by adenine 5′-nucleotides, adenine, inosine and hypoxanthine required more than 1000-fold higher concentrations than of adenosine itself. Similar affinities for adenosine and 2′-deoxyadenosine were observed. By exploiting the high specificity of the antisera, a radioimmunoassay method was established that was capable of detecting down to 1 pmol of adenosine (20 nM) in unfractionated heart perfusates and cell extracts. The acetal-mediated coupling procedure is applicable to other biologically important cis-diols.
A new unextracted-sample radioimmunoassay method for hepatic endogenous nuclear l-tri-iodothyronine content. Validity of its use in determining nuclear receptor binding characteristics
Authors: Yagura Toshihiro , Walfish Paul G.
Published: August 2015

Endogenous l-tri-iodothyronine content in an hepatic nuclear extract was measured by a new unextracted-sample radioimmunoassay method using 8-anilinonaphthalene-1-sulphonic acid to inhibit the l-[125I]tri-iodothyronine binding to the nuclear l-tri-iodothyronine receptor within the extract. For this method, the lower sensitivity limit was 3.125 pg/tube, the recovery of added l-tri-iodothyronine was 90–120%, and the between-assay coefficient of variation was 10%. The amount of endogenous l-tri-iodothyronine was 10–40 pg/0.2 ml of hepatic nuclear extract from euthyroid rats, compared with less than 3.125 pg/0.2 ml from thyroidectomized rats. The results obtained by this new method were compared with a Sephadex G-25 column extracted-sample radioimmunoassay method and showed a good agreement. The values for the endogenous l-tri-iodothyronine content were utilized to correct for the l-tri-iodothyronine concentration within the binding assay mixture in order to accurately determine by Scatchard analysis the binding characteristics of the nuclear l-tri-iodothyronine receptor. The validity of the correction for endogeneous l-tri-iodothyronine was demonstrated by using a nuclear extract from a thyroidectomized rat which was preincubated with a small known amount of l-tri-iodothyronine before determining the nuclear l-tri-iodothyronine receptor binding characteristics. When the Scatchard plots were corrected for the preincubated dose, the results obtained were similar to true values, but they were falsely lower when not corrected. It is concluded that the necessity and validity of using endogenous l-tri-iodothyronine corrections in the Scatchard analytical computations of the nuclear l-tri-iodothyronine receptor binding characteristics has been demonstrated, being particularly more important for affinity constant than maximum binding capacity.
The rate of protein degradation in developing brain. Methodological considerations
Authors: Dunlop D S , McHale D M , Lajtha A
Published: August 2015

Recently we reported that the rate of protein breakdown decreases during development. Breakdown rates were calculated from the rates of protein synthesis and the changes in brain protein content with age. A different study, measuring breakdown by monitoring the loss of label from brain protein after an H14CO3- pulse, came to the opposite conclusion: that the rate of breakdown is low in immature brain and increases during development. We have now investigated some of the factors (the distribution of label in protein and the potential for recycling) that might introduce errors into these measurements. The specific radioactivities of both protein-bound and free amino acids were determined in the brains of young rats several days after an intraperitoneal pulse of H14CO3-. For a number of amino acids the specific radioactivity of the free amino acid is high compared with that of the protein-bound amino acid, and therefore recycling could result in an underestimate of the degradation rate. Because glutamic acid had a relatively low specific-radioactivity ratio, [1-14C]glutamic acid was used in a pulse-labelling experiment to measure degradation. The rate so obtained, 0.6% . h-1, is twice the rate found with H14CO3- labelling (based on total protein-bound radioactivity). Insofar as recycling is a possible complication, 0.6% . h-1 may be a minimum value. Although somewhat higher degradation rates are found after labelling with an intracranial pulse, which was considered as a possible route to limit recycling, there are difficulties in interpreting these data.
Effects of thyroidectomy and starvation on the activity and properties of hepatic carnitine palmitoyltransferase
Authors: Saggerson E D , Carpenter C A , Tselentis B S
Published: August 2015

1. Hepatic carnitine palmitoyltransferase activity was measured over a range of concentrations of palmitoyl-CoA and in the presence of several concentrations of the inhibitor malonyl-CoA. These measurements were made in mitochondria obtained from the livers of fed and starved (24 h) normal rats and of fed and starved thyroidectomized rats. 2. In the fed state thyroidectomy substantially decreased overt carnitine palmitoyltransferase activity and also decreased both the Hill coefficient and the s0.5 when palmitoyl-CoA concentration was varied as substrate. Thyroidectomy did not appreciably alter the inhibitory effect of malonyl-CoA on the enzyme. 3. Starvation increased overt carnitine palmitoyltransferase activity in both the fed and the thyroidectomized state. In percentage terms this response to starvation was substantially greater after thyroidectomy. In both the hypothyroid and normal states starvation decreased sensitivity to inhibition by malonyl-CoA.
Regulation of malate oxidation in plant mitochondria. Response to rotenone and exogenous NAD+
Authors: Palmer J M , Schwitzguébel J P , Møller I M
Published: August 2015

Exogenous NAD+ stimulated the rotenone-resistant oxidation of all the NAD+-linked tricarboxylic acid-cycle substrates in mitochondria from Jerusalem artichoke (Helianthus tuberosus L.) tubers. The stimulation was not removed by the addition of EGTA, which is known to inhibit the oxidation of exogenous NADH. It is therefore concluded that added NAD+ gains access to the matrix space and stimulates oxidation by the rotenone-resistant NADH dehydrogenase located on the matrix surface of the inner membrane. Added NAD+ stimulated the activity of malic enzyme and displaced the equilibrium of malate dehydrogenase; both observations are consistent with entry of NAD+ into the matrix space. Analysis of products of malate oxidation showed that rotenone-resistant oxygen uptake only occurred when the concentration of oxaloacetate was low and that of NADH was high. Thus it is proposed that the concentration of NADH regulates the activity of the two internal NADH dehydrogenases. Evidence is presented to suggest that the rotenone-resistant NADH dehydrogenase is engaged under conditions of high phosphorylation potential, which restricts electron flux through the rotenone-sensitive dehydrogenase (coupled to ATP synthesis).
The association of FAD with the cytochrome b–245 of human neutrophils
Authors: Cross Andrew R. , Jones Owen T. G. , Garcia Rudolfo , Segal Anthony W.
Published: August 2015

A plasma membrane fraction prepared from human neutrophils had a fluorescence resembling that of a fluorescent flavoprotein, with emission maximum near 520nm and excitation maxima near 380 and 460nm. The fluorescence emission and excitation properties of Triton N-101-solubilized membrane fraction resembled those of FAD. FAD was present in the membranes at a concentration of 417pmol/mg of protein and cytochrome b–245 at a concentration of 407pmol/mg of protein. In a 110-fold purified preparation of cytochrome b–245 the ratio of FAD:cytochrome b was 1:1. Analytical gradient centrifugation of neutrophil homogenates shows a coincidence of two cytochrome b peaks and two peaks of fluorescence, corresponding with plasma membrane and specific granule fractions; most of the FAD was non-fluorescent and located in fractions lighter than the plasma membrane. Plasma membrane fractions prepared from neutrophils of patients suffering from the X-linked form of chronic granulomatous disease lacked cytochrome b and contained 194pmol of FAD/mg of protein; plasma membrane fractions prepared from neutrophils of patients with the autosomal recessive form of chronic granulomatous disease contained both cytochrome b–245 and FAD in the normal range of concentrations in a ratio of 1:1. Phagocytic vesicles were prepared from normal neutrophils and found to contain FAD and cytochrome b in a ratio 2.22:1, suggesting that activation of neutrophils many involve the incorporation of an additional flavin into the membrane. Under anaerobic conditions in the presence of EDTA to act as an electron donor to a flavin, the cytochrome b–245 of neutrophil membranes was partly (12%) photoreducible, an effect increased to 100% by the addition of FMN. The extent of reduction of cytochrome b in an anaerobic neutrophil homogenate containing NADH increased from 30% to 70% on illumination. We suggest that these results indicate a close association between FAD and cytochrome b–245 and support a scheme for electron transport thus: [Formula: see text]
Characterization of calcium binding to brush-border membranes from rat duodenum
Authors: Miller A , Li S T , Bronner F
Published: August 2015

The Ca2+-binding properties of isolated brush-border membranes at physiological ionic strength and pH were examined by rapid Millipore filtration. A comprehensive analysis of the binding data suggested the presence of two types of Ca2+-binding sites. The high-affinity sites, Ka = (6.3 +/- 3.3) X 10(5) M-1 (mean +/- S.E.M.), bound 0.8 +/- 0.1 nmol of Ca2+/mg of protein and the low-affinity sites, Ka = (2.8 +/- 0.3) X 10(2) M-1, bound 33 +/- 3.5 nmol of Ca2+/mg of protein. The high-affinity site exhibited a selectivity for Ca2+, since high concentrations of competing bivalent cations were required to inhibit Ca2+ binding. The relative effectiveness of the competing cations (1 and 10 mM) for the high-affinity site was Mn2+ approximately equal to Sr2+ greater than Ba2+ greater than Mg2+. Data from the pH studies, treatment of the membranes with carbodi-imide and extraction of phospholipids with aqueous acetone and NH3 provided evidence that the low-affinity sites were primarily phospholipids and the high-affinity sites were either phosphoprotein or protein with associated phospholipid. Two possible roles for the high-affinity binding sites are suggested. Either high-affinity Ca2+ binding is involved with specific enzyme activities or Ca2+ transport across the luminal membrane occurs via a Ca2+ channel which contains a high-affinity Ca2+-specific binding site that may regulate the intracellular Ca2+ concentration and gating of the channel.
The breakdown of Tetrahymena ribosomes in vivo. The effects of inhibitors
Authors: Klemperer H G , Pilley D J
Published: August 2015

1. When Tetrahymena were deprived of nutrients 50% of the polysomes disaggregated within 20 min and 20% of the total RNA broke down in 2 h. Ribosomal RNA accounted for 75% of the RNA breakdown. 2. RNA labelled by a long incubation with [14C]uridine was stable in growing cells and in the presence of actinomycin D, but broke down at the same rate as bulk RNA in starved cells. 3. The following substances inhibited the loss of RNA during starvation: cycloheximide (which inhibited both polysome disaggregation and protein synthesis), inhibitors of energy metabolism and puromycin (all of which caused polysome disaggregation and inhibited protein synthesis), and chloroquine and 7-amino-1-chloro-3-L-tosylamidoheptan-2-one (‘TLCK’) (neither of which affected polysomes or protein synthesis). 4. Starvation appears to activate a ribosome degradation mechanism that may involve lysosomal and non-lysosomal enzymes.
Pulse-radiolysis studies on the interaction of one-electron-reduced species with ascorbate oxidase in aqueous solution
Authors: O'Neill P , Fielden E M , Finazzi-Agrò A , Avigliano L
Published: August 2015

The interaction of e-aq., CO2-. and one-electron reduced nitroaromatics (RNO2-.) with ascorbate oxidase (AAO) was studied in aqueous solution at pH 6.0 and 7.5 by using the technique of pulse radiolysis. From observations at 330, 410 and 610 nm, interaction of e-aq. and CO2-. with AAO results in non-specific reduction of the protein followed by reduction of Type 1 Cu in a rate-determining intramolecular step. Only a few per cent of the reducing equivalents ultimately results in reduction of Type 1 Cu. With large excesses of reducing equivalents (e-aq. and CO2-.) with respect to the copper concentration, the amount of Type 1 copper reduced never exceeds 50% of the total amount of Type 1 copper after a single radiation pulse. With less-powerful reducing agents, e.g. RNO2-. reduction of Type 1 Cu occurs via a bimolecular step, and there is no evidence for formation of radicals on protein residues. From observations at 330 nm it is evident that Type 2 and/or Type 3 Cu may also be reduced along with Type 1 Cu. Almost stoichiometric reduction of AAO by RNO2-. was observed, e.g. the protein accepts 6-7 reducing equivalents. It is inferred that the various types of redox couples Cu2+/Cu+ are in equilibrium and that intramolecular electron transfer between the different types of Cu is not rate-determining when using RNO2-. as reducing agent.
Medium-chain fatty acid synthesis by goat mammary-gland fatty acid synthetase. The effect of limited proteolysis
Authors: Grunnet I , Knudsen J
Published: August 2015

Fatty acid synthetase from goat mammary gland was subjected to limited proteolysis by trypsin and elastase. Both proteolytic enzymes selectively cleaved the chain-terminating thioester hydrolase component from the enzyme complex, leaving all other partial activities intact in the core peptides. Trypsin, but not elastase, caused extensive degradation of the released thioester hydrolase. The released thioester hydrolase could be purified to homogeneity by gel filtration. The molecular weight was estimated as 29 000 and the enzyme showed only significant hydrolytic activity toward long-chain acyl-CoA esters. The core peptides retained the ability to synthesize medium-chain acyl-CoA esters in the presence of 2,6-di-O-methyl-alpha-cyclodextrin. The results conclusively show that the terminating thioester hydrolase of goat mammary-gland fatty acid synthetase is not involved in termination of medium-chain-length fatty acid synthesis by this enzyme.
Activation of liver alcohol dehydrogenase by glycosylation
Authors: Tsai C S , White J H
Published: August 2015

D-Fructose and D-glucose activate alcohol dehydrogenase from horse liver to oxidize ethanol. One mol of D-[U-14C]fructose or D-[U-14C]glucose is covalently incorporated per mol of the maximally activated enzyme. Amino acid and N-terminal analyses of the 14C-labelled glycopeptide isolated from a proteolytic digest of the [14C]glycosylated enzyme implicate lysine-315 as the site of the glycosylation. 13C-n.m.r.-spectroscopic studies indicate that D-[13C]glucose is covalently linked in N-glucosidic and Amadori-rearranged structures in the [13C]glucosylated alcohol dehydrogenase. Experimental results are consistent with the formation of the N-glycosylic linkage between glycose and lysine-315 of liver alcohol dehydrogenase in the initial step that results in an enhanced catalytic efficiency to oxidize ethanol.
The Nα-acetylenkephalin carboxypeptidase activity of N-acetyltyrosine deacetylase from monkey kidney. Purification, characterization and substrate specificity
Authors: George S T , Balasubramanian A S
Published: August 2015

N alpha-Acetylenkephalin carboxypeptidase was co-purified with N-acetyltyrosine deacetylase from monkey kidney. Almost 90% of the activity from the homogenate was recovered in a high-speed supernatant without the use of detergents. The crucial steps in the purification were Cibacron Blue F3GA-Sepharose chromatography (involving negative and positive binding sequentially) and metal chelate affinity chromatography. The purified enzyme showed three bands on gel electrophoresis under non-denaturing conditions. All the three bands exhibited both N-acetyltyrosine deacetylase and N-acetylenkephalin carboxypeptidase activity, indicating their co-migration, Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in the presence and absence of 2-mercaptoethanol gave a single protein band of mol.wt. 34 000. The native enzyme was a dimer of mol.wt. 66 000 as observed on Bio-Gel P-300 gel filtration. The carboxypeptidase removed two amino acids from the C-terminal end of either N-acetyl[Met5]- or N-acetyl[Leu5]-enkephalin. Non-acetylated enkephalins were less active as substrates. Peptides with their carboxy end blocked were inactive as substrates. Models suggested for carboxypeptidase A [Hartsuck & Lipscomb (1971) Enzymes 3, 1-56] support the idea that the kidney N-acetylated aromatic amino acid deacetylase or acylase III [Endo (1978) Biochim. Biophys. Acta 523, 207-217] can act as a carboxypeptidase on peptides having hydrophobic amino acids at the C-terminal end.
The purification of a novel amylase from Bacillus subtilis and its inhibition by wheat proteins
Authors: Orlando A R , Ade P , Di Maggio D , Fanelli C , Vittozzi L
Published: August 2015

A new alpha-amylase (EC 3.2.1.1) from Bacillus subtilis was purified by affinity chromatography. The molecular weight of the purified enzyme, estimated from sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, was 93000, which is very different from the molecular weights of two well-characterized amylases from B. subtilis. Electrofocusing showed an isoelectric point of 5. Amylase shows a broad maximum of activity between pH 6 and 7; maximal inhibition of enzyme by wheat-protein alpha-amylase inhibitors is displayed at pH 7.
Localization of β-glucan synthases on the membranes of cultured Lolium multiflorum (ryegrass) endosperm cells
Authors: Henry R J , Schibeci A , Stone B A
Published: August 2015

The distribution of beta-glucan synthases between plasma membranes and intracellular membranes of suspension-cultured Italian-ryegrass (Lolium multiflorum Lam.) endosperm cells was examined. Highly purified plasma membranes prepared from protoplasts were only slightly enriched in beta-glucan synthases assayed at 10 microM- and 1 mM-UDP-glucose. Most beta-glucan synthase was associated with intracellular membranes. These membranes were fractionated on a linear sucrose density gradient and were resolved into different membrane fractions containing beta-glucan synthases. Beta-Glucan synthases assayed at 10 microM-UDP-glucose were found in a fraction banding at a density of 1.11 g . cm-3, but most of the beta-glucan synthase assayed at 1 mM-DDP-glucose was at a density of 1.04 g . cm-3.
Stringent requirement for Ca2+ in the removal of Z-lines and α-actinin from isolated myofibrils by Ca2+-activated neutral proteinase
Authors: Reddy M K , Rabinowitz M , Zak R
Published: August 2015

Treatment of isolated myofibrils with Ca2+-activated neutral proteinase (CANP) results in specific removal of Z-line and of alpha-actinin. To investigate the ionic requirement for these processes, we measured Z-line removal by phase-contrast and interference microscopy and alpha-actinin removal by sodium dodecyl sulphate/polyacrylamide-gel electrophoretic analysis of myofibrillar proteins. The proteolytic digestion of native purified proteins was measured directly on polyacrylamide gels and by the fluorescamine technique. We found that the removal of Z-line and alpha-actinin as well as the release of proteolytic degradation products from isolated myofibrils by CANP occur only in the presence of Ca2+; Sr2+, Ba2+, Mn2+, Mg2+, Co2+ and Zn2+ are all ineffective. In contrast with this stringent requirement for Ca2+, the proteolytic activity of CANP measured with denatured casein, native and denatured haemoglobin, native actin and tropomyosin also occurs in the presence of other bivalent cations, in the following order: Ca2+ greater than Sr2+ greater than Ba2+. These data suggest that only Ca2+ can produce the conformational change in myofibrils that renders them susceptible to the action of CANP, whereas its proteolytic activity is stimulated by several bivalent ions.
Isolation and properties of a lectin from the seeds of the Indian bean or lablab (Dolichos lablab L.)
Authors: Güran A , Tichá M , Filka K , Kocourek J
Published: August 2015

The lectin of the Indian bean or lablab (Dolichos lablab L.) was purified by affinity chromatography on two types of affinity carriers: O-alpha-D-mannopyranosyl-Separon and Separon-bound ovomucoid. The lectin is homogeneous in the ultracentrifuge: S20, w = 6.14 S, Mr = 110 000; the molecule appears to comprise two pairs of two types of subunits (Mr 16 000 and 40 000), and contains 2% neutral sugar and 0.2 Mn and 0.5 Zn atom respectively. The lectin agglutinates human erythrocytes non-specifically with regard to ABO grouping at a limit concentration of 8 micrograms/ml, and this activity is inhibited most effectively by N-acetyl-D-glucosamine, methyl alpha-D-mannopyranoside and ovomucoid, but not by free D-mannose.
A marked gradation in active-centre properties in the cysteine proteinases revealed by neutral and anionic reactivity probes. Reactivity characteristics of the thiol groups of actinidin, ficin, papain and papaya peptidase A towards 4,4′-dipyridyl disulphide and 5,5′-dithiobis-(2-nitrobenzoate) dianion
Authors: Brocklehurst K , Mushiri S M , Patel G , Willenbrock F
Published: August 2015

1. The kinetics of the reactions of the catalytic-site thiol groups of actinidin (the cysteine proteinase from Actinidia chinensis), ficin (EC 3.4.22.3), papain (EC 3.4.22.2) and papaya peptidase A (the other monothiol cysteine proteinase component of Carica papaya) with 4,4′-dipyridyl disulphide (4-Py-S-S-4-Py) and with 5,5′-dithiobis-(2-nitrobenzoate) dianion (Nbs22-) were studied in the pH range approx. 6-10. These studies provided the pH-independent second-order rate constants (k) for the reactions of the two probe reagents with the catalytic-site thiolate anions each in the environment of a neutral histidine side chain where an active-centre carboxy group would be ionized. 2. The ratio R equal to kNbs22-/k4-Py-S-S-4-Py provides an index of the catalytic-site solvation properties of the four cysteine proteinases and varies markedly from one enzyme to another, being 0.80 for papaya peptidase A (0.86 for the model thiol, 2-mercaptoethanol), 29 for actinidin, 0.18 for ficin and 0.015 for papain. These differences appear to derive mainly from the response of the enzyme to the negative charge on Nbs22-. 3. Possible implications of these results for (a) mechanisms of cysteine proteinase catalysis and (b) the possibility of using series of functionally related enzymes in the study of mechanism are discussed.
Incorporation of adenine into the modifying group of inactive iron protein of nitrogenase from Rhodospirillum rubrum
Authors: Nordlund S , Ludden P W
Published: August 2015

Adenine was fed to cells of Rhodospirillum rubrum grown on glutamate. The adenine was found to be incorporated into the modifying group of the inactive form of iron protein. Adenine labelled in the 8-position ([8-3H]adenine) and the 2-position ([2-3H]adenine) was specifically incorporated into the electrophoretic ‘upper-band’ subunit of iron protein. Incorporation of label from the 2-position into many proteins was observed if histidine was not present in the medium. Label was removed by the activating enzyme for iron protein.
Changes in non-histone nuclear proteins during postnatal myocardial development
Authors: Jackowski G , Liew C C
Published: August 2015

Myocardial-cell nuclei isolated from ventricles of rats between 4 days and 15 months of age showed a progressive loss of DNA-synthetic activity in vitro. Correlated with this loss is the differential appearance of three major groups of non-histone nuclear proteins. Group a, with pI5.7-6.5 and Mr 67000, appeared in detectable amounts in rats more than 10 days of age, whereas group b, consisting of proteins with pI6.8-7.2 and Mr 45000, appeared in rats older than 33 days. The third group (group c), with pI6.5-8.5 and Mr 32000-42000, was present throughout postnatal development, but the amount of these proteins appeared to be greater in the 10-day-old rat than in the 15-month-old rat. All groups of these specific proteins were localized within the nucleus and did not reflect cytoplasmic constituents. Furthermore, the amino acid compositions of these polypeptides were also analysed and compared with those of actin and tubulin.
Inhibition of protein breakdown by epidermal growth factor in IMR90 human fibroblasts and other mammalian cell lines
Authors: Gunn J M , Bodner J B , Knowles S E , Ballard F J
Published: August 2015

1. Epidermal growth factor (EGF) inhibits intracellular protein breakdown in IMR90 human fibroblasts and other cell lines having EGF receptors. 2. Inhibition is achieved within 1 h of exposure to the growth factor and is reversed equally rapidly upon removal of EGF. 3. EGF inhibits protein breakdown and stimulates protein and DNA labelling with similar dependency on concentration. Half-maximal effects for all processes with IMR90 and AG2804 cell lines occur at 0.2 nM- and 0.05 nM-EGF respectively. 4. EGF and insulin effects on protein breakdown are additive only when the factors are included at suboptimal concentrations. 5. The apparent Kd for EGF binding in several cell lines is approximately 10-fold higher than the concentration needed for half-maximal inhibition of protein breakdown. 6. Down-regulation of EGF receptors in IMR90 cells produced a 60% decrease in the binding of 125I-labelled EGF. This was accompanied by a displacement of the concentration curve for EGF inhibition of protein breakdown by approximately two orders of magnitude, suggesting that protein breakdown can no longer respond to the down-regulated receptor-growth-factor complex. 7. Phorbol esters decrease the inhibitory effect of EGF, but not of insulin, on protein breakdown in IMR90 cells.
The involvement of protein phosphorylation in stimulus-secretion coupling in the mouse exocrine pancreas
Authors: Roberts M L , Butcher F R
Published: August 2015

Secretagogue-induced protein phosphorylation was studied in the mouse pancreas in vitro, by using polyacrylamide-gel electrophoresis to separate the labelled proteins. Muscarinic cholinergic agonists increased the phosphorylation of a single band, which corresponded to Mr 32000, when the tissue was incubated with Ca2+ present in the extracellular medium, but not in Ca2+-free Krebs solution. In the presence of Ca2+, ionophore A23187 stimulated phosphorylation of the same band. The dose-response curve for carbachol-induced phosphorylation was biphasic, with maximum response at 1.0 microM-carbachol, and lesser responses when greater concentrations were used. This resembles the dose-response curve for carbachol-induced amylase secretion. The data suggest that the muscarinic-agonist-induced protein phosphorylation is stimulated secondarily to elevation of cytosol [Ca2+] and do not support the idea that diacylglycerol formed from hydrolysis of phosphatidylinositol is the activator of the protein kinase. Derivatives of cyclic AMP stimulated phosphorylation of bands corresponding to Mr 95500, 32000 and 20000. The effects of dibutyryl cyclic AMP and bethanechol on the protein of Mr 32000 were not additive, suggesting that the two agents produced phosphorylation of the same site(s) on this protein. Since derivatives of cyclic AMP, which are not very effective secretagogues in the exocrine pancreas, stimulate phosphorylation of the protein of Mr 32000, it is difficult to argue that phosphorylation of this particular protein leads to protein secretion.
Biliary protein output by isolated perfused rat livers. Effects of bile salts
Authors: Barnwell S G , Godfrey P P , Lowe P J , Coleman R
Published: August 2015

The output of proteins into bile was studied by using isolated perfused rat livers. Replacement of rat blood with defined perfusion media deprived the liver of rat serum proteins (albumin, immunoglobulin A) and resulted in a rapid decline in the amounts of these proteins in bile. When bovine serum albumin was incorporated into the perfusion medium it appeared in bile within 20 min and the amount in the bile was determined by the concentration of the protein in the perfusion medium. The use of a defined perfusion medium also deprived the livers of bile salts and the amounts of these, and of plasma-membrane enzymes [5′-nucleotidase (EC 3.1.3.5) and phosphodiesterase I], in bile declined rapidly. Introduction of micelle-forming bile salts (taurocholate or glycodeoxycholate) to the perfusion medium 80 min after liver isolation markedly increased the output of plasma-membrane enzymes but had no effect on the other proteins. The magnitude of this response was dependent on the bile salt used and its concentration in bile; there was little effect on plasma-membrane enzyme output until the critical micellar concentration of the bile salt had been exceeded in the bile. A bile salt analogue, taurodehydrocholate, which does not form micelles, did not produce the enhanced output of plasma-membrane enzymes. This work supports the view that the output of plasma-membrane enzymes in bile is a consequence of bile salt output and also provides evidence for mechanisms by which serum proteins enter the bile.
Increased sensitivity of carnitine palmitoyltransferase I activity to malonyl-CoA inhibition after preincubation of intact rat liver mitochondria with micromolar concentrations of malonyl-CoA in vitro
Authors: Zammit V A
Published: August 2015

Carnitine palmitoyltransferase I in rat liver mitochondria preincubated with malonyl-CoA was more sensitive to inhibition by malonyl-CoA than was the enzyme in mitochondria preincubated in the absence of malonyl-CoA. For carnitine palmitoyltransferase I in mitochondria from starved animals this increase also resulted in the enzyme becoming significantly more sensitive than that in mitochondria assayed immediately after their isolation. Concentrations of malonyl-CoA that induced half the maximal degree of sensitization observed were 1-3 microM.
Disturbance of lysosomal glycogen metabolism by liposomal anti-alpha-glucosidase and some anti-inflammatory drugs
Authors: Geddes R , Otter D E , Scott G K , Taylor J A
Published: August 2015

The size-distribution of liver glycogen was shown to be distinctly affected by the anti-inflammatory drugs salicylate and indomethacin. By measurement of the incorporation of radioactive glucose into glycogen, salicylate was shown to have a depressing effect on overall liver glycogen metabolism. These effects appear to arise from the stabilizing of the lysosome by the drugs. The incorporation, via liposomes, of purified anti-1,4-alpha-glucosidase activity and in the content of high-molecular-weight glycogen. These changes are increased by prolonged liposomal antibody treatment and suggest that a possible feedback control mechanism operates in the incorporation of glycogen into lysosomes. These experiments may be useful as a model of glycogen turnover and its failure in glycogenosis type II (Pompe's disease).
Effect of starvation and refeeding on polyamine concentrations and ornithine decarboxylase antizyme in mammary gland of lactating rats
Authors: Brosnan M E , Farrell R , Wilansky H , Williamson D H
Published: August 2015

Starvation caused a marked increase in putrescine content in mammary gland of lactating rats, together with a marked decrease in activity of ornithine decarboxylase and appearance of measurable ornithine decarboxylase antizyme. 2. Refeeding for 5 h caused disappearance of free antizyme and ornithine decarboxylase activity returned to the value in fed animals. Putrescine concentration remained elevated. 3. There was no significant change in nucleic acid content of mammary gland from starved rats, but spermidine and spermine contents increased significantly. 4. Refeeding for 5 h returned the spermidine content of mammary glands to ‘fed’ values, and significantly decreased the content of spermine, although it did not reach control values. Thus changes in polyamine content of mammary gland in starved rats are clearly dissociated from changes in either RNA content or activities of polyamine-synthetic decarboxylases. 5. Starvation caused a fall in the content of spermidine in liver, with no change in spermine content. Refeeding for 5 h returned the spermidine content to ‘fed’ values.
The interaction between lipid peroxidation and prostaglandin synthesis in rabbit kidney-medulla slices
Authors: Fujimoto Y , Tanioka H , Keshi I , Fujita T
Published: August 2015

Lipid peroxidation induced by ascorbic acid and Fe2+ was inhibited by mepacrine (phospholipase A2 inhibitor) and aspirin (prostaglandin cyclo-oxygenase inhibitor) in rabbit kidney-medulla slices. Moreover, ascorbic acid and Fe2+ potentiated the inhibitory effect on prostaglandin E2 formation by mepacrine, but they had no influence on prostaglandin E2 production decreased by aspirin. Lipid peroxidation induced by ascorbic acid and Fe2+ appears to be affecting the activity of prostaglandin endoperoxide synthase. These results suggest that lipid peroxidation is connected closely with the prostaglandin-generating system, and it has the potential to modulate the turnover of arachidonic acid and prostaglandin synthesis.