bluuebunny/crossref_metadata_2025 huggingface dataset:
Biosynthesis and processing of legumin-like storage proteins in Lupinus angustifolius (lupin)
Authors: Johnson E D , Knight J , Gayler K R
Published: August 2015

Synthesis, secretion and post-translational proteolysis of the storage proteins in cotyledons of Lupinus angustifolius L. (lupin) have been examined in vivo and in vitro by using a combination of pulse-chase experiments with [3H]- or [35S]-labelled amino acids, subcellular fractionation and cell-free translation from poly(A)+ (polyadenylylated) RNA or membrane-bound polyribosomes. Related polypeptides were identified by immunoprecipitation, separation on sodium dodecyl sulphate/polyacrylamide gels and fluorography. The synthesis and processing of two proteins were compared. Conglutin alpha, the 11 S protein, was found as a family of precursor polypeptides of Mr 68000-88000 when translated from poly(A)+ RNA under conditions where signal segments were not cleaved, and Mr 64000-85000 both when sequestered into the endoplasmic reticulum and when accumulated in the protein bodies. Pulse-chase labelling showed that cotyledons from early stages of development were completely incapable of further proteolysis of these precursors. Nevertheless, in the same juvenile cotyledons, the precursors of the minor storage protein conglutin gamma, two polypeptides with Mr 50000-51000, were proteolytically cleaved to mature subunits of Mr 32000 and 17000 within 2 h. Further cleavage of the precursors of conglutin alpha into families of mature subunits of Mr 21000-24000 and 42000-62000 was detected in more mature cotyledons. A model is proposed which suggests that the mature subunits are produced by a single proteolytic cleavage of each of the three major precursors of conglutin alpha and also suggests that a close similarity exists between these subunits and those of other legumin-like proteins. The enzyme responsible for this cleavage, which appears at a specific stage in the middle of cotyledonary development, seems to be an integral part of the programmed developmental sequence in these pods.
Degradative inactivation of the peroxisomal enzyme, alcohol oxidase, during adaptation of methanol-grown Candida boidinii to ethanol
Authors: Hill D J , Hann A C , Lloyd D
Published: August 2015

Adaptation of methanol-grown C. boidinii to ethanol-utilization in non-growing cells resulted in decreased activity of the peroxisomal enzyme alcohol oxidase. Re-appearance of alcohol oxidase activity was dependent on protein synthesis de novo. Degradation of alcohol oxidase protein was shown to parallel the decrease in activity. Adaptation of methanol-grown cells to ethanol-utilization resulted in increased absorbance due to cytochromes and decreased absorbance due to flavoprotein. Decrease in alcohol oxidase activity was associated with loss of the flavin coenzyme, FAD, from the organisms and the appearance of flavins (FAD, FMN, riboflavin) in the surrounding medium. Electron microscopic observations showed that general degradation of whole peroxisomes rather than specific loss of crystalline cores (alcohol oxidase protein) occurred during the adaptation.
General method for exact evaluation of parameters of the elementary steps of coupled reactions. Invariant analysis
Authors: Havsteen B H
Published: August 2015

A general method has been devised for the exact evaluation of the rate constants of the elementary steps of a system consisting of any number of coupled reactions. The method is independent of the structure of the network of coupled steps. The precision of the data evaluation is solely dependent on the quality of the detection unit. The application of the method is illustrated with data collected for the binding of the competitive inhibitor proflavine to chymotrypsin under such conditions that five states of the enzyme are required to interpret the results. In the absence of a substance possessing the binding specificity, the enzyme is present as an equilibrium between an active and an inactive conformer. The latter prevails. The binding of the specific inhibitor releases a slow proton transfer from the medium to the alpha-amino group of Ile-16. Subsequently, the enzyme-inhibitor (or enzyme-substrate) complex re-arranges to the catalytically active form, which is retained until the supply of specific substrate is exhausted. The control features described are general, but are particularly conspicuous under the special environmental conditions used here. A comparison between data for alpha- and delta-forms of chymotrypsin showed that the chain ends of the former impeded the substrate binding and that the activity controlling conformational change occurred in the interior of the enzyme molecule.
Purification and comparison of the structures of human liver acidic α-d-mannosidases A and B
Authors: Cheng S H , Malcolm S , Pemble S , Winchester B
Published: August 2015

Human liver alpha-D-mannosidases A and B were purified 11 500-fold and 2000-fold respectively. Both showed microheterogeneity when analysed by isoelectric focusing. Alpha-D-Mannosidases A and B are immunologically identical but differ in their range of pI values, molecular masses, uptake into fibroblasts and subunit compositions. Alpha-D-Mannosidase A consists of equimolar proportions of subunits of molecular masses 62 kDa and 26 kDa, which are linked by disulphide bridges in the intact enzyme. Alpha-D-Mannosidase B also contains a small subunit, of molecular mass 26 kDa, and a variable mixture of larger subunits, of molecular masses 58 kDa and 62 kDa. The 62 kDa and 58 kDa subunits, but not the 26 kDa one, contain concanavalin A-recognizing glycans. The 58 kDa subunit has a lower pI, contains less high-mannose glycans but probably contains more mannose 6-phosphate than the 62 kDa subunit. It is postulated that the differences in structure and properties of alpha-D-mannosidases A and B are due to differences in the state of processing of the large subunit. This suggestion is consistent with a single locus on chromosome 19 for lysosomal alpha-D-mannosidase.
Effects of dexamethasone and insulin on the synthesis of triacylglycerols and phosphatidylcholine and the secretion of very-low-density lipoproteins and lysophosphatidylcholine by monolayer cultures of rat hepatocytes
Authors: Mangiapane E H , Brindley D N
Published: August 2015

Rat hepatocytes in monolayer culture were preincubated for 19 h with 1 microM-dexamethasone, and the incubation was continued for a further 23 h with [14C]oleate, [3H]glycerol and 1 microM-dexamethasone. Dexamethasone increased the secretion of triacylglycerol into the medium in particles that had the properties of very-low-density lipoproteins. The increased secretion was matched by a decrease in the triacylglycerol and phosphatidylcholine that remained in the hepatocytes. Preincubating the hepatocytes for the total 42 h period with 36 nM-insulin decreased the amount of triacylglycerol in the medium and in the cells after the final incubation for 23 h with radioactive substrates. However, insulin had no significant effect on the triacylglycerol content of the cell and medium when it was present only in the final 23 h incubation. Insulin antagonized the effects of dexamethasone in stimulating the secretion of triacylglycerol from the hepatocytes, especially when it was present throughout the total 42 h period. The labelling of lysophosphatidylcholine in the medium when hepatocytes were incubated with [14C]oleate and [3H]glycerol was greater than that of phosphatidylcholine. The appearance of this lipid in the medium, unlike that of triacylglycerol and phosphatidylcholine, was not stimulated by dexamethasone, or inhibited by colchicine. However, the presence of lysophosphatidylcholine in the medium was decreased when the hepatocytes were incubated with both dexamethasone and insulin. These findings are discussed in relation to the control of the synthesis of glycerolipids and the secretion of very-low-density lipoproteins and lysophosphatidylcholine by the liver, particularly in relation to the interactions of glucocorticoids and insulin.
The mechanism of regulatory light chain dissociation from scallop myosin
Authors: Bennett A J , Bagshaw C R
Published: August 2015

The dissociation of the regulatory light chains from scallop myosin subfragments, on addition of EDTA, was investigated by using the fluorophore 8-anilinonaphthalene-1-sulphonate as a probe. The rate of this process (0.014 s-1) was partially limited by the rate of Mg2+ dissociation (0.058 s-1) from the non-specific high-affinity site. The dissociation of the regulatory light chain subfragment 1 was less extensive than from heavy meromyosin. Reassociation of the scallop regulatory light chain was induced on addition of Mg2+, but it appeared to be limited by a first-order step. The nature of this step was revealed by the kinetics of Mercenaria regulatory light chain association. Scallop heavy meromyosin, denuded of its regulatory light chains, exists in a refractory state, whose reversal to the nascent state limits the rate of light chain association (0.006 s-1). The formation of the refractory state is the driving force for the net dissociation of regulatory light chains from scallop heavy meromyosin. This mechanism is discussed with reference to existing structural information on light-chain-denuded myosin.
Structural variations in actins. A study of the immunological reactivity of the N-terminal region
Authors: Roustan C , Benyamin Y , Boyer M , Cavadore J C
Published: August 2015

The antigenicity of the N-terminal region of skeletal-muscle actin was analysed. Two epitopes, corresponding to the 1-7 and 18-28 sequences, were determined. The antibodies specific for the first epitope discriminate skeletal-muscle actin from cardiac-muscle and smooth-muscle actins. The antibodies specific for the second epitope interact with all the actins tested, ranging from invertebrate to higher-vertebrate actins.
Comparative binding of bovine, human and rat insulin-like growth factors to membrane receptors and to antibodies against human insulin-like growth factor-1
Authors: Read L C , Ballard F J , Francis G L , Baxter R C , Bagley C J , Wallace J C
Published: August 2015

The immunological properties of human, bovine and rat insulin-like growth factors (IGF) and insulin were compared in competitive binding studies with Tr10 and NPA polyclonal antisera raised in rabbits against human IGF-1. Bovine IGF-1 was 11-19% as effective as human IGF-1 in competing for binding with 125I-labelled human IGF-1, whereas IGF-2 reacted poorly and insulin did not compete. Similar competitive binding curves were obtained with the mouse monoclonal anti-(human IGF-1) antibody 3D1, except that bovine IGF-1 showed a severalfold greater affinity for the monoclonal antibody than for either polyclonal antiserum. Membranes isolated from human placenta, sheep placenta and foetal-human liver were used as sources of cellular receptors. In human placental membranes, most of the binding of IGF-1 tracers could be attributed to a type-1 receptor, because insulin inhibited up to 65% of tracer binding. The other two tissues apparently contain only type-2 receptors, as evidenced by the very low potency of bovine or human IGF-1 in competing for binding with IGF-2 tracers and the absence of any competition by insulin. In competition for binding with labelled bovine or human IGF-1 to human placental membranes, bovine IGF-1 had a similar potency to human IGF-1, whereas bovine IGF-1 was more potent in binding studies with tissues rich in type-2 receptors. Rat IGF-2 was considerably less effective than human IGF-2 in competition for receptors on any of the membrane preparations.
Mechanistic origin of the sigmoidal rate behaviour of glucokinase
Authors: Pettersson G
Published: August 2015

Model studies are presented which demonstrate that reactions proceeding by a random ternary-complex mechanism may exhibit most pronounced deviations from Michaelis-Menten kinetics even if the reaction is effectively ordered with respect to net reaction flow. In particular, the kinetic properties and reaction flow characteristics of glucokinase can be accounted for in such terms. It is concluded that insufficient evidence has been presented to support the idea that glucokinase operates by a ‘mnemonical’ type of mechanism involving glucose binding to distinct conformational states of free enzyme. The sigmoidal rate behaviour of glucokinase can presently be more simply explained in terms of glucose binding to differently ligated states of the enzyme.
Determination of rates of protein synthesis, gain and degradation in intact hind-limb muscle of lambs
Authors: Oddy V H , Lindsay D B
Published: August 2015

A model of leucine metabolism in the hind-limb muscles of the milk-fed lamb was developed which permitted simultaneous estimation of the rates of protein synthesis (Ks, days-1), degradation (Kd) and therefore gain (Kg) of muscle in vivo. The conclusions drawn from the model were: the rate of protein synthesis in muscle was related to uptake of leucine; the rate of degradation of protein was related to leucine output, as leucine, or its corresponding oxo acid, 4-methyl-2-oxopentanoic acid, or CO2. These findings support findings drawn from a wide range of studies in vitro. There was no correlation between rate of protein synthesis and rate of protein degradation, which suggests that the method can allow independent estimates of each. Estimates of protein synthesis obtained from the model (of leucine metabolism in muscle) were compared with those obtained simultaneously by constant infusion of radioisotope and analysis of incorporation into tissue. There were no significant differences between the mean values obtained for synthesis (Ks), gain (Kg) and degradation (Kd) by either method (Ks 0.051 +/- 0.002, 0.046 +/- 0.007; Kg 0.016 +/- 0.002, 0.004 +/- 0.008; Kd 0.035 +/- 0.004, 0.041 +/- 0.008 day-1, respectively, for tissue analysis and the model). However, Ks obtained from the model was significantly and positively correlated with uptake of leucine from plasma, whereas Ks obtained from tissue analysis was not.
Phosphopeptide analysis of phenylalanine hydroxylase isolated from liver cells exposed to hormonal stimuli
Authors: Fisher M J , Dickson A J , Pogson C I
Published: August 2015

Hormonal control of the phosphorylation of phenylalanine hydroxylase was studied by using rat liver cells incubated with [32P]Pi. After immunoprecipitation from cell extracts, the hydroxylase was subjected to proteinase digestion and subsequent sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. V8-proteinase digestion yielded one major 32P-labelled fragment, of approx. 9 kDa. Chymotrypsin digestion gave five 32P-labelled fragments ranging from approx. 39 kDa to approx. 10 kDa. Noradrenaline (10 microM) and glucagon (0.1 microM) enhanced the 32P content of all peptide fragments uniformly. Phorbol ester, in contrast with ionophore A23187, did not stimulate enzyme phosphorylation or enhance phenylalanine metabolism in liver cells. These results are discussed in relation to the nature of the protein kinase(s) that mediate phosphorylation of phenylalanine hydroxylase in liver cells.
Interrelationships in rats of tissue pools of cholecalciferol and 25-hydroxycholecalciferol formed in u.v. light
Authors: Lawson D E M , Sedrani S H , Douglas J
Published: August 2015

Vitamin D-deficient rats were irradiated with u.v. light three times weekly for 30 min for several weeks. D3 (cholecalciferol) and 25(OH)D3 (25-hydroxycholecalciferol) concentrations in skin, plasma, muscle and adipose tissue were measured. In other experiments, isolated skin or the whole animal was irradiated once and the cholecalciferol response monitored. Only a small fraction of the 7-dehydrocholesterol in skin is converted into D3 (less than 2%), and the presence of fur decreases the proportion converted into 20% of that occurring in shaved rat skin. D3 formed in the skin disappears relatively slowly, so that about 90% has gone after 7 days. In normal rats 10 micrograms of D3 formed over 2 h irradiation only caused a small rise in plasma D3 concentration over the following week, indicative of a high rate of clearance from this tissue. Irradiation of vitamin D-deficient rats for a prolonged period raised plasma D3 and 25(OH)D3 concentrations to a constant value. D3, but not 25(OH)D3, could be found in adipose tissue and muscle. Prolonged irradiation of normal rats showed these tissues and plasma could hold very large amounts of D3. Pharmacokinetic analysis of the changes in D3 concentration in rats showed that the disposition kinetics of D3 was explained by a two-compartment model with half-lives of 13.8 and 7.7 days. The volume of distribution of the more-slowly-turning-over compartment was 500 ml, which presumably reflects the large amounts of D3 that can accumulate in adipose tissue. Rat skin can synthesize about 0.85 ng of D3/mJ of u.v. light energy, but it seems that not all this is available to the rat. Adipose-tissue D3 is available for use by the rat, the t1/2 being 12.0 days.
Production of platelet thromboxane A2 inactivates purified human platelet thromboxane synthase
Authors: Hall E R , Tuan W M , Venton D L
Published: August 2015

Human platelet thromboxane synthase was partially purified by DEAE-cellulose, Affi-Gel Blue, and Sephacryl S-300 chromatography to a specific activity of 259 nmol of thromboxane B2/min per mg. Thromboxane synthase retained 75-90% of its enzymic activity when bound to phenyl-Sepharose. The immobilized enzyme was inactivated at pH 3.0 and inhibited by 1-benzylimidazole and U-63,557A. The ability of the enzyme to produce thromboxane A2 from prostaglandin H2 was dramatically reduced by multiple additions of prostaglandin H2. Our data suggest that the production of thromboxane A2 by the enzyme is self-limiting and that the enzyme is inactivated during the reaction.
Inactivation of macromolecules by ionizing radiation. Deterministic single-hit or stochastic multievent process?
Authors: Swillens S
Published: August 2015

A stochastic theory concerning the radiation inactivation of macromolecules such as enzymes or receptors is elaborated. In contrast with the single-hit theory, which assumes a complete inactivation of the target as the result of one hit, the stochastic theory postulates that the degree of inactivation by one hit is a random variable. This distinguishing feature has been considered in order to give a possible interpretation to the observed effect of temperature on the radiation-sensitivity of enzymes. As a consequence of the progressive inactivation during irradiation, the binding affinity of a ligand for the macromolecule is impaired by irradiation. Although this property might discriminate the stochastic theory from the classical single-hit theory on the basis of a statistical analysis of experimentally obtained data, it is shown that the commonly obtained degree of inaccuracy may render the statistical test non-conclusive.
Interaction between calmodulin and five different spin-labelled chlorophenothiazines
Authors: Olivier J L , Rainteau D , Bereziat G , Wolf C
Published: August 2015

The binding to purified calmodulin of five spin-labelled derivatives of chlorophenothiazine was investigated by e.s.r. spectrometry and by the antagonizing potency on the calmodulin-dependent activation of myosin light chain kinase. The results of a comparative study and the influence of pH and ionic strength on the binding support the occurrence of an electrostatic binding involving the terminal amino group of the side-chain of the chlorophenothiazine. These results are discussed in relation to the specificity of the interaction that holds the antipsychotic drug-calmodulin complex together.
Equations describing the effects of the transient stability of subpopulations of nascent hnRNAs on the kinetics of turnover of hnRNA
Authors: Kleene K C
Published: August 2015

The equations that have been used previously to analyse the rate of decay of hnRNA implicitly assume that nascent hnRNAs are degraded stochastically. This assumption is inconsistent with electron-microscopic studies of transcription cited here, which show that nascent hnRNAs are not degraded during transcription, implying that hnRNA degradation occurs only after termination of transcription and release of the hnRNA from chromatin. Equations are derived describing the accumulation of radioactivity hnRNA during continuous labelling assuming that nascent hnRNAs are stable and that hnRNAs decay with first-order kinetics only after completion of transcription. The effects of the transient stability of nascent hnRNAs on the kinetics of hnRNA turnover can become important when the half-life of the hnRNA is shorter than the time to transcribe an hnRNA from the point of initiation to the point of termination. These equations should prove useful in studies of hnRNA turnover that require a precise description of the labelling kinetics of nascent and completed subpopulations of hnRNA.
Utilization of [15N]glutamate by cultured astrocytes
Authors: Yudkoff M , Nissim I , Hummeler K , Medow M , Pleasure D
Published: August 2015

The metabolism of 0.25 mM-[15N]glutamic acid in cultured astrocytes was studied with gas chromatography-mass spectrometry. Almost all 15N was found as [2-15N]glutamine, [2-15N]glutamine, [5-15N]glutamine and [15N]alanine after 210 min of incubation. Some incorporation of 15N into aspartate and the 6-amino position of the adenine nucleotides also was observed, the latter reflecting activity of the purine nucleotide cycle. After the addition of [15N]glutamate the ammonia concentration in the medium declined, but the intracellular ATP concentration was unchanged despite concomitant ATP consumption in the glutamine synthetase reaction. Some potential sources of glutamate nitrogen were identified by incubating the astrocytes for 24 h with [5-15N]glutamine, [2-15N]glutamine or [15N]alanine. Significant labelling of glutamate was noted with addition of glutamine labelled on either the amino or the amide moiety, reflecting both glutaminase activity and reductive amination of 2-oxoglutarate in the glutamate dehydrogenase reaction. Alanine nitrogen also is an important source of glutamate nitrogen in this system.
Structural and kinetic studies on β-lactamase K1 from Klebsiella aerogenes
Authors: Emanuel E L , Gagnon J , Waley S G
Published: August 2015

beta-Lactamase K1 from Klebsiella aerogenes 1082E hydrolyses both penicillins and cephalosporins comparably and is inhibited by mercurials but not by cloxacillin. These properties distinguish it from those other beta-lactamases that have been allotted to classes on the basis of their amino sequences. beta-Lactamase K1 has been isolated by affinity chromatography; its composition shows resemblances to class A beta-lactamases. Moreover, the N-terminal sequence is similar to those of class A beta-lactamases: there is about 30% identity over the first 32 residues. Furthermore, a putative active-site octapeptide has been isolated and its sequence is similar to the region around the active-site serine residue in class A beta-lactamases. There is one thiol group in beta-lactamase K1; it is not essential for activity. The pH-dependence of kcat. and kcat./Km for the hydrolysis of benzylpenicillin by beta-lactamase K1 were closely similar, suggesting that the rate-determining step is cleavage of the beta-lactam ring.
Thermodynamic and kinetic studies on saccharide binding to soya-bean agglutinin
Authors: Swamy M J , Krishna Sastry M V , Khan M I , Surolia A
Published: August 2015

The fluorescence of N-dansylgalactosamine [N-(5-dimethylaminonaphthalene-1-sulphonyl)galactosamine] was enhanced 11-fold with a 25 nm blue-shift in the emission maximum upon binding to soya-bean agglutinin (SBA). This change was used to determine the association constants and thermodynamic parameters for this interaction. The association constant of 1.51 × 10(6) M-1 at 20 degrees C indicated a very strong binding, which is mainly due to a relatively small entropy value, as revealed by the thermodynamic parameters: delta G = −34.7 kJ × mol-1, delta H = −37.9 kJ × mol-1 and delta S = −10.9 J × mol-1 × K-1. The specific binding of this sugar to SBA shows that the lectin can accommodate a large hydrophobic substituent on the C-2 of galactose. Binding of non-fluorescent ligands, studied by monitoring the fluorescence changes when they are added to a mixture of SBA and N-dansylgalactosamine, indicates that a hydrophobic substituent at the anomeric position increases the affinity of the interaction. The C-6 hydroxy group also stabilizes the binding considerably. Kinetics of binding of N-dansylgalactosamine to SBA studied by stopped-flow spectrofluorimetry are consistent with a single-step mechanism and yielded k+1 = 2.4 × 10(5) M-1 × s-1 and k-1 = 0.2 s-1 at 20 degrees C. The activation parameters indicate an enthalpicly controlled association process.
Formation of hydroxyl radicals in the presence of ferritin and haemosiderin. Is haemosiderin formation a biological protective mechanism?
Authors: O'Connell M , Halliwell B , Moorhouse C P , Aruoma O I , Baum H , Peters T J
Published: August 2015

Horse spleen and human spleen ferritins increase the formation of hydroxyl radicals (OH) at both pH 4.5 and pH 7.4 in reaction mixtures containing ascorbic acid and H2O2. The generation of OH is inhibited by the chelator desferrioxamine. Human spleen haemosiderin also accelerates OH generation in identical reaction mixtures, but is far less effective (on a unit iron basis) than ferritin under all reaction conditions. It is proposed that conversion of ferritin into haemosiderin in iron overload is biologically advantageous in that it decreases the ability of iron to promote oxygen-radical reactions.
Studies of the influence of chloro-substituent sites and conformational energy in polychlorinated biphenyls on uroporphyrin formation in chick-embryo liver cell cultures
Authors: Sassa S , Sugita O , Ohnuma N , Imajo S , Okumura T , Noguchi T , Kappas A
Published: August 2015

Treatment of cultured chick-embryo liver cells with polychlorinated biphenyls (PCB) results in decreased uroporphyrinogen decarboxylase activity and increased uroporphyrin accumulation. In the present study we examined the effect of the chloro- or bromo-substituent sites in biphenyls (BP) on uroporphyrin accumulation in cultured hepatocytes and the three-dimensional structure of these congeners determined by molecular orbital calculations using a MNDO (‘modified neglect of diatomic overlap’) method. Among 20 congeners examined, those which were effective in stimulating porphyrin accumulation contained at least two Cl or Br atoms at the lateral adjacent positions in each phenyl ring, e.g. 3,4,3′,4′-tetrachloro-, 2,4,3′,4′-tetrachloro-, 3,4,5,3′,4′,5′-hexachloro- and 3,4,5,3′,4′,5′-hexabromobiphenyl, whereas those which contained less than two halogen atoms or more than three halogen atoms in each phenyl ring or those which contained halogen atoms at 2,2′-positions were not effective. On the basis of the conformational energy (delta E, difference from the most stable conformational energy), which is calculated as a function of the dihedral angle (theta) between the two phenyl rings, biphenyl congeners can be classified into four groups with different conformations. The conformation of active PCB was relatively flexible, whereas inactive species had a rigidly angulated conformation. Furthermore, the calculated probability of the conformation distribution for each congener indicated that the probability of co-planarity was higher for active biphenyls than for inactive congeners. These structural characteristics suggest the significance of both the chloro-substituent sites and the conformational energy reflecting the phenyl-ring twist angles in determining the inhibitory effect of PCB on uroporphyrinogen decarboxylase activity.
Regulation of concentrations of mRNA for amylase, trypsinogen I and chymotrypsinogen B in rat pancreas by secretagogues
Authors: Renaud W , Giorgi D , Iovanna J , Dagorn J C
Published: August 2015

Regulation of pancreatic gene expression by the secretory stimulants cholecystokinin-pancreozymin, caerulein and pilocarpine was studied in the rat, by using cloned cDNA probes to quantify concentrations of specific mRNAs (amylase, trypsinogen I and chymotrypsinogen B). It is concluded that long-term pancreatic stimulation results in pre-translational regulation of secretory-protein gene expression, with a preferential accumulation of RNA transcripts encoding serine proteinases, compared with that for amylase.
Quantification of tissue-type plasminogen activator (t-PA) mRNA in human endothelial-cell cultures by hybridization with a t-PA cDNA probe
Authors: van Zonneveld A J , Chang G T G , van den Berg J , Kooistra T , Verheijen J H , Pannekoek H , Kluft C
Published: August 2015

We describe the construction of a recombinant DNA plasmid, consisting of the vector pBR322 and full-length tissue-type plasminogen-activator (t-PA) cDNA, by using polyadenylated RNA from cultured Bowes melanoma cells as substrate. A 1280-base-pair PstI restriction fragment, covering the 3′ untranslated region and part of the coding region for the t-PA L-chain, was used as a radiolabelled probe to determine the size and the number of t-PA mRNA molecules in cultured endothelial cells of different origin from the same individual. Northern blotting showed that in all these cells a t-PA mRNA is synthesized of about 2500 nucleotides, indicating that transcriptional initiation, splicing and polyadenylation is similar. The number of t-PA mRNA molecules per cell measured, by using a dot-blotting technique and t-PA mRNA made in vitro, with a plasmid DNA preparation harbouring a specific promotor of the Salmonella typhimurium bacteriophage SP6, t-PA cDNA and SP6 RNA polymerase as standard, is approx. 10,000 in all cultured endothelial cells from adult vessels. However, the amount of t-PA antigen synthesized and/or secreted differs by a factor of 6-20. Relatively large amounts of t-PA antigen secreted were detected in conditioned medium from vena-cava-derived cells, whereas low amounts were found in conditioned medium from arteria-iliaca-derived cells.
Chondroitin sulphate proteoglycan in the substratum adhesion sites of Balb/c 3T3 cells. Fractionation on various ion-exchange and affinity columns
Authors: Wightman B C , Weltman E A , Culp L A
Published: August 2015

Proteoglycans on the cell surface play critical roles in the adhesion of fibroblasts to a fibronectin-containing extracellular matrix, including the model mouse cell line Balb/c 3T3. In order to evaluate the biochemistry of these processes, long-term [35S]sulphate-labelled proteoglycans were extracted quantitatively from the adhesion sites of 3T3 cells, after their EGTA-mediated detachment from the substratum, by using an extractant containing 1% octyl glucoside, 1 M-NaCl and 0.5 M-guanidinium chloride (GdnHCl) in buffer with many proteinase inhibitors. Greater than 90% of the material was identified as a large chondroitin sulphate proteoglycan (Kav. = 0.4 on a Sepharose CL2B column), and the remainder was identified as a smaller heparan sulphate proteoglycan; only small amounts of free chains of glycosaminoglycan were observed in these sites. These extracts were fractionated on DEAE-Sepharose columns under two different sets of elution conditions: with acetate buffer (termed DEAE-I) or with acetate buffer supplemented with 8 M-urea (termed DEAE-II). Under DEAE-I conditions about one-half of the material was eluted as a single peak and the remainder required 4 M-GdnHCl in order to recover it from the column; in contrast, greater than 90% of the material was eluted as a single peak from DEAE-II columns. Comparison of the elution of [35S]sulphate-labelled proteoglycan with that of 3H-labelled proteins from these two columns, as well as mixing experiments, indicated that the GdnHCl-sensitive proteoglycans were trapped at the top of columns, partially as a consequence of their association with proteins in these adhesion-site extracts. Affinity chromatography of these proteoglycans on columns of either immobilized platelet factor 4 or immobilized plasma fibronectin revealed that most of the chondroitin sulphate proteoglycan and the heparan sulphate proteoglycan bound to platelet factor 4 but that only the heparan sulphate proteoglycan bound to fibronectin, providing a ready means of separating the two proteoglycan classes. Affinity chromatography on octyl-Sepharose columns to test for hydrophobic domains in their core proteins demonstrated that a high proportion of the heparan sulphate proteoglycan but none of the chondroitin sulphate proteoglycan bound to the hydrophobic matrix. These results are discussed in light of the possible functional importance of the chondroitin sulphate proteoglycan in the detachment of cells from extracellular matrix and in light of previous affinity fractionations of proteoglycans from the substratum-adhesion sites of simian-virus-40-transformed 3T3 cells.
A re-evaluation of evidence attributing the difference in cleavage rates of restriction endonuclease at different sites in the substrate to differences in Km values
Authors: Karlovský P
Published: August 2015

The only result supporting the hypothesis that the differences in restriction endonuclease cleavage rates at various target sites are caused by differences in Km values was reported by Forsblom, Rigler, Ehrenberg, Petterson & Philipson [(1976) Nucleic Acids Res. 3, 3255-3269]. The present work shows that the kinetic analysis in that paper is based on incorrect derivation and in fact provides no support for the hypothesis mentioned.
Isolation and characterization of latherin, a surface-active protein from horse sweat
Authors: Beeley J G , Eason R , Snow D H
Published: August 2015

A protein, latherin, with unusual surface activity was isolated from horse sweat by gel filtration and ion-exchange chromatography. The protein has a Stokes radius, determined by gel filtration, of 2.47 nm, and in the ultracentrifuge sediments as a single species with S20,W 2.05 S, indicating an Mr of 24,400. On SDS/polyacrylamide-gel electrophoresis the molecule behaves as a single peptide chain of apparent Mr 20,000. Latherin contains a high proportion of hydrophobic amino acids (37.2%), and the leucine content (24.5%) is exceptionally high. The unusual composition of the protein may account for apparent anomalies in the Mr of latherin determined by empirical methods. Evidence indicating that latherin is responsible for much of the surface activity of horse sweat was obtained by a simple assay for surface tension and by contact-angle measurements. Latherin adsorbs very readily at hydrophobic surfaces, rendering them wettable. A possible role for latherin in thermoregulation is proposed.
Interactions of the neurotoxic amine 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine with monoamine oxidases
Authors: Singer T P , Salach J I , Castagnoli N , Trevor A
Published: August 2015

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a thermal breakdown product of a meperidine-like narcotic used by drug abusers as a heroin substitute, produces Parkinsonian symptoms in humans and primates. The nigrostriatal toxicity is not due to MPTP itself but to one or more oxidation products resulting from the action of monoamine oxidase (MAO) on this tertiary allylamine. Both MAO A and B catalyse the oxidation of MPTP to the 1-methyl-4-phenyl-2,3-dihydropyridinium species (MPDP+), which undergoes further oxidation to the fully aromatic 1-methyl-4-phenylpyridinium species (MPP+). These bio-oxidations are blocked by selective inhibitors of MAO A and B. Additionally, MPTP, MPDP+ and MPP+ are competitive inhibitors of MAO A and B. The A form of the enzyme is particularly sensitive to this type of reversible inhibition. Both MAO A and B also are irreversibly inactivated by MPTP and MPDP+, but not by MPP+. This inactivation obeys the characteristics of a mechanism-based or ‘suicide’ process. The inactivation, which is accompanied by the incorporation of radioactivity from methyl-labelled MPTP, is likely to result from covalent modification of the enzyme.
The role of Ca2+ in steroidogenesis in Leydig cells. Stimulation of intracellular free Ca2+ by lutropin (LH), luliberin (LHRH) agonist and cyclic AMP
Authors: Sullivan M H F , Cooke B A
Published: August 2015

The requirements of purified rat Leydig cells for intra- and extra-cellular Ca2+ during steroidogenesis stimulated by LH (lutropin), cyclic AMP analogues and LHRH (luliberin) agonist were investigated. The intracellular Ca2+ concentrations ([Ca2+]i) were measured by using the fluorescent Ca2+ chelator quin-2. The basal [Ca2+]i was found to be 89.4 +/- 16.6 nM (mean +/- S.D., n = 25). LH, 8-bromo cyclic AMP and dibutyryl cyclic AMP increased [Ca2+]i, by 300-500 nM at the highest concentrations of each stimulator, whereas LHRH agonist only increased [Ca2+]i by a maximum of approx. 60 nM. Low concentrations of LH (less than 1 pg/ml) and all concentrations of LHRH agonist increased testosterone without detectable changes in cyclic AMP. With amounts of LH greater than 1 pg/ml, parallel increases in cyclic AMP and [Ca2+]i occurred. The steroidogenic effect of the LHRH agonist was highly dependent on extracellular Ca2+ concentration ([Ca2+]e), whereas LH effects were only decreased by 35% when [Ca2+]e was lowered from 2.5 nM to 1.1 microM. No increase in [Ca2+]i occurred with the LHRH agonist in the low-[Ca2+]e medium, whereas LH (100 ng/ml) gave an increase of 52 nM. It is concluded that [Ca2+]i can be modulated in rat Leydig cells by LH via mechanisms that are both independent of and dependent on cyclic AMP, whereas LHRH-agonist action on [Ca2+]i is independent of cyclic AMP. The evidence obtained suggests that, at sub-maximal rates of testosterone production, Ca2+, rather than cyclic AMP, is the second messenger, whereas for maximum steroidogenesis both Ca2+- and cyclic-AMP-dependent pathways may be involved.
Direct evidence for the involvement of domain III in the N-F transition of bovine serum albumin
Authors: Khan M Y
Published: August 2015

The domain III of bovine serum albumin containing residues 377-582 of the protein sequence was isolated and its behaviour in acid solution was studied. The fragment was found to undergo structural transformations over the pH range 3.5-4.5 known to cause N-F transition in serum albumin. On the other hand, an albumin fragment that was devoid of domain III was unable to exhibit such a transition. These results were consistent with a mechanism where N-F transition involves the separation of domain III from the rest of the albumin starts at about pH 4.3 and is completed at pH 3.5.
Polyamine-mediated turnover of ornithine decarboxylase in Chinese-hamster ovary cells
Authors: Glass J R , Gerner E W
Published: August 2015

We have used Chinese-hamster ovary (CHO) cells maintained in a chemically defined medium to study the regulation of ornithine decarboxylase (ODC) by polyamines. Cells maintained in the defined medium had no detectable putrescine, and approx. 1-3 units of ODC activity/10(6) cells, where 1 unit corresponds to 1 nmol of substrate decarboxylated in 30 min. The defined medium is ornithine-deficient, thus limiting the exogenous substrate for ODC, and subsequently decreasing intracellular polyamine accumulation. Restoration of intracellular putrescine and increased formation of spermidine by addition of exogenous ornithine or putrescine led to a marked decrease in ODC activity, which was paralleled by a decrease in a alpha-DL-difluoromethyl[3,4-3H]ornithine (DFMO)-binding protein of Mr approx. 53,000, which is precipitable with anti-ODC antibody. Calculation of DFMO binding per unit of activity showed no change in the specific activity of the enzyme. We identified [35S]methionine-labelled peptides corresponding to ODC by immunoprecipitation of radiolabeled whole cell proteins. Only one protein was precipitated, of Mr approx. 53 000, which co-migrated with the DFMO-binding protein. Immunoprecipitation of radiolabelled proteins from cells incubated in the presence of exogenous ornithine indicated that the observed activity decrease was not due to an inhibition of ODC protein synthesis. Analysis of immunoprecipitable ODC protein from cells that had been pulse-labelled with [35S]methionine, and then treated for 5 h with 100 microM-ornithine, -putrescine or -spermidine, revealed a distinct disappearance of labelled ODC protein after restoration of intracellular polyamine pools. No detectable turnover of ODC was observed in the absence of exogenous polyamine treatment. These data support the hypothesis that ODC protein, and subsequent activity, is regulated by intracellular polyamine content through mechanisms that influence turnover of the enzyme.
Hydrogenosomes in the rumen fungus Neocallimastix patriciarum
Authors: Yarlett N , Orpin C G , Munn E A , Yarlett N C , Greenwood C A
Published: August 2015

Sedimentable hydrogenase activity was demonstrated in cell-free extracts from both zoospores and vegetative growth of the anaerobic rumen fungus Neocallimastix patriciarum. Electron micrographs of the fraction enriched in hydrogenase activity contained finely granular microbody-like organelles, about 0.5 micron in diameter and having an equilibrium density of about 1.2 g X ml-1 in sucrose, 1.12 g X ml-1 in Percoll and 1.27-1.28 g X ml-1 in Metrizamide. These organelles, which are sedimentable at 10(5) g-min, bear no similarity to mitochondria, but are morphologically similar to hydrogen-evolving organelles possessed by certain anaerobic protozoa and termed ‘hydrogenosomes’. Other typical hydrogenosomal enzymes, namely ‘malic’ enzyme, pyruvate:ferredoxin oxidoreductase and NADPH:ferredoxin oxidoreductase, were enriched in the same particle fraction as hydrogenase. The synthesis of pyruvate:ferredoxin oxidoreductase was found to be suppressed when the organism was cultured under an atmosphere of CO2, and an alternative pathway is proposed for growth under these conditions.
Stimulation of phosphoinositide metabolism in hamster brown adipocytes exposed to α1-adrenergic agents and its inhibition with phorbol esters
Authors: Schimmel R J , Dzierzanowski D , Elliott M E , Honeyman T W
Published: August 2015

The present experiments were undertaken to investigate the role of the phosphoinositides phosphatidylinositol 4-phosphate (PtdIns-4-P) and phosphatidylinositol 4,5-biphosphate (PtdIns-4,5-P2) in the alpha 1-adrenergic stimulation of respiration in isolated hamster brown adipocytes. Exposure of isolated brown adipocytes to the alpha-adrenergic-receptor agonist phenylephrine provoked a breakdown of 30-50% of the PtdIns-4-P and PtdIns-4,5-P2 after prelabelling of the cells with [32P]Pi. Coincident with the breakdown of phosphoinositides was an accumulation of labelled phosphatidic acid, which continued for the duration of the cell incubation. The time course of phosphoinositide breakdown was defined more precisely by pulse-chase experiments. Under these conditions, phenylephrine caused radioactivity in phosphatidylinositol, PtdIns-4-P and PtdIns-4,5-P2 to fall by more than 50% within 30 s and to remain at the depressed value for the duration of the incubation (10 min). This phospholipid response to alpha-adrenergic stimulation was blocked by exposure of the cells to phorbol 12-myristate 13-acetate (PMA); likewise phenylephrine stimulation of respiration was prevented by PMA. beta-Adrenergic stimulation of respiration and inhibition of respiration by 2-chloroadenosine and insulin were, however, unaffected by treatment with PMA. On the assumption that PMA is acting in these cells as an activator of protein kinase C, these results suggest the selective interruption of alpha-adrenergic actions in brown adipocytes by activated protein kinase C. These findings suggest that breakdown of phosphoinositides is an early event in alpha-adrenergic stimulation of brown adipocytes which may be important for the subsequent stimulation of respiration. The results from the pulse-chase studies also suggest, however, that phenylephrine-stimulated breakdown of inositol phospholipids is a short-lived event which does not appear to persist for the entire period of exposure to the alpha 1-adrenergic ligand.
Competition of glycerol with other oxidizable substrates in rat brain
Authors: McKenna M C , Bezold L I , Kimatian S J , Tildon J T
Published: August 2015

The rate of conversion of [1,3-14C]glycerol into 14CO2 was measured in the presence and absence of unlabelled alternative substrates in whole homogenates from the brains of young (4-6 and 18-20 days old) and adult rats. Unlabelled glucose decreased 14CO2 production from [1,3-14C]glycerol by about 40% at all ages studied. Unlabelled 3-hydroxybutyrate significantly decreased the 14CO2 production from both low (0.2 mM) and high (2.0 mM) concentrations of glycerol in 4-6- and 18-20-day-old rat pups. However, the addition of 3-hydroxybutyrate had no effect on the rate of 14CO2 production from 2.0 mM-glycerol in adult rats, suggesting that the interaction of 3-hydroxybutyrate with glycerol in adult rat brain is complex and may be related to the biphasic kinetics previously reported for glycerol oxidation. Unlabelled glutamine decreased the production of 14CO2 by brain homogenates from 18-20-day-old and adult rats, but not in 4-6-day-old rat pups. In the converse situation, the addition of unlabelled glycerol to whole brain homogenates had little effect on the rate of 14CO2 production from [6-14C]glucose, 3-hydroxy[3-14C]butyrate and [U-14C]glutamine, although some significant differences were noted. Collectively these results suggest that glycerol and these other substrates may be metabolized in separate subcellular compartments in brain such that the products of glucose, 3-hydroxybutyrate and glutamine metabolism can dilute the oxidation of glycerol, but the converse cannot occur. The data also demonstrate that there are complex age-related changes in the interaction of glycerol with 3-hydroxybutyrate and glutamine. The fact that glycerol oxidation was only partially suppressed by the addition of 1-5 mM-glucose, -3-hydroxybutyrate or -glutamine could also suggest that glycerol may be selectively utilized as an energy substrate in some discrete brain region.
The role of acetyl-CoA carboxylase phosphorylation in the control of mammary gland fatty acid synthesis during the starvation and re-feeding of lactating rats
Authors: Munday M R , Hardie D G
Published: August 2015

Activation of acetyl-CoA carboxylase during incubation of crude extracts of lactating rat mammary gland with Mg2+ and citrate can be blocked by NaF, suggesting that it represents a dephosphorylation of the enzyme. The greater extent of activation in extracts from 24 h-starved rats (200%) compared with fed controls (70%) implies that the decrease in acetyl-CoA carboxylase activity in response to 24 h starvation may involve increased phosphorylation of the enzyme. Acetyl-CoA carboxylase was purified from the mammary glands of lactating rats in the presence of protein phosphatase inhibitors by avidin-Sepharose chromatography. Starvation of the rats for 24 h increased the concentration of citrate giving half-maximal activation by 75%, and decreased the Vmax. of the purified enzyme by 73%. This was associated with an increase in the alkali-labile phosphate content from 3.3 +/- 0.2 to 4.5 +/- 0.4 mol/mol of enzyme subunit. Starvation of lactating rats for 6 h, or short-term insulin deficiency induced by streptozotocin injection, did not effect the kinetic parameters or the phosphate content of acetyl-CoA carboxylase purified from mammary glands. The effects of 24 h starvation on the kinetic parameters and phosphate content of the purified enzyme were completely reversed by re-feeding for only 2.5 h. This effect was blocked if the animals were injected with streptozotocin before re-feeding, suggesting that the increase in plasma insulin that occurs on re-feeding was responsible for the activation of the enzyme. The effects of re-feeding 24 h-starved rats on the kinetic parameters and phosphate content of acetyl-CoA carboxylase could be mimicked by treating enzyme purified from 24 h-starved rats with protein phosphatase-2A in vitro. Our results suggest that, in mammary glands of 24 h-starved lactating rats, insulin brings about a dephosphorylation of acetyl-CoA carboxylase in vivo, which may be at least partly responsible for the reactivation of mammary lipogenesis in response to re-feeding.