bluuebunny/crossref_metadata_2025 huggingface dataset:
Absence of positive co-operativity in the binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin to its cytosolic receptor protein
Authors: Farrell K , Safe S
Published: August 2015

The role of positive co-operativity in stabilizing the binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to the rat hepatic cytosolic TCDD receptor protein (Ah receptor) was investigated. The binding mechanism of TCDD was determined by kinetic means through equilibrium and saturation binding studies, and Scatchard and Hill plot analysis. In all studies, the slope of the Hill plot was close to 1.0, indicating the absence of positive co-operativity. Interpretation of the Scatchard plot was however complicated by the fact that both linear and nonlinear plots were experimentally obtained. The nonlinearity was shown to be an experimental artifact and a consequence not of co-operativity, but of high levels of nonspecific binding. The high level of nonspecific binding could be attributed to: (1) lipophilicity of the TCDD ligand, and (2) inefficient competition of receptor-bound [3H]TCDD. When nonspecific binding was minimized, the Scatchard slope was linear and in agreement with the Hill coefficient, thus indicating the lack of positive co-operativity in the binding of TCDD to the Ah receptor.
A monoclonal antibody to the phosphorylated form of phenylalanine hydroxylase. Definition of the phosphopeptide epitope
Authors: Smith S C , McAdam W J , Kemp B E , Morgan F J , Cotton R G
Published: August 2015

Monoclonal antibody PH7 has specificity for the phosphorylated form of the human liver phenylalanine hydroxylase and negligible reactivity towards the dephosphorylated form of the native enzyme by enzyme-linked immunoassay. PH7 binds specifically to the phosphorylated form of the liver enzyme after SDS/polyacrylamide-gel electrophoresis and transfer to nitrocellulose. Competitive blocking assays have been applied in conjunction with reversed-phase h.p.l.c. of purified tryptic fragments of human liver phenylalanine hydroxylase to localize the epitope. The major immunoreactive tryptic peptide cross-reacting with PH7 had an amino acid analysis corresponding to the first 41 amino acids of the human liver phenylalanine hydroxylase sequence and included the serine residue that is thought to be the phosphorylation site. The monoclonal antibody recognized the phosphorylated form of the synthetic decapeptide corresponding to the local phosphorylation-site sequence Gly-Leu-Gly-Arg-Lys-Leu-Ser(P)-Asp-Phe-Gly, but not the dephosphodecapeptide. Thermolysin digestion of the peptide demonstrated the monoclonal antibody bound to the pentapeptide Leu-Ser(P)-Asp-Phe-Gly. Monoclonal antibody PH7 recognized the phosphodecapeptide at concentrations 10(3)-fold higher than with phenylalanine hydroxylase, compared with 10(4)-10(7)-fold higher for other phosphopeptides and phosphoproteins. The results demonstrate that monoclonal antibody PH7 has specificity for the phosphorylated form of phenylalanine hydroxylase at the phosphorylation site.
Effects of prolonged elevation of plasma adrenaline concentration in vivo on insulin-sensitivity in soleus muscle of the rat
Authors: Budohoski L , Challiss R A , Dubaniewicz A , Kaciuba-Usciłko H , Leighton B , Lozeman F J , Nazar K , Newsholme E A , Porta S
Published: August 2015

1. Prolonged elevation of the plasma adrenaline concentration was produced in rats by implantation of adrenaline-releasing retard-tablets. With this technique, a hyperadrenalinaemic state is maintained for at least 5 days. 2. At 6 h after implantation of the retard-tablet it was found that plasma glucose and fatty acid concentrations increased and insulin concentration decreased compared with values obtained from placebo-tablet-implanted rats. Administration of a subcutaneous glucose load demonstrated an impaired glucose tolerance in vivo, and incubation of soleus muscle strips from 6 h-hyperadrenalinaemic rats in vitro demonstrated a decreased sensitivity of the rates of glycolysis and glucose transport to insulin. 3. The sensitivities of the rates of glycolysis, glucose transport and glycogen synthesis to insulin were determined for the incubated soleus muscle preparation isolated from animals after 48 h, 72 h and 120 h duration of hyperadrenalinaemia. At 48 h after retard-tablet implantation, the sensitivity of the processes of glucose transport and glycolysis was decreased; at 72 h, the insulin-sensitivities of the rates of glycolysis and glucose transport in skeletal muscle were similar to those determined for control animals; at 120 h, however, the sensitivities of the processes of glucose transport and glycolysis were both statistically significantly increased. In contrast, no changes in the sensitivity of the process of glycogen synthesis were observed at any of the time intervals studied. 4. The possible biochemical basis for the observed changes in skeletal-muscle insulin-sensitivity with prolonged hyperadrenalinaemia is discussed.
The preparation of monoclonal antibodies to human bone and liver alkaline phosphatase and their use in immunoaffinity purification and in studying these enzymes when present in serum
Authors: Bailyes E M , Seabrook R N , Calvin J , Maguire G A , Price C P , Siddle K , Luzio J P
Published: August 2015

1. Liver and bone alkaline phosphatase isoenzymes were solubilized with the zwitterionic detergent sulphobetaine 14, and purified to homogeneity by using a monoclonal antibody previously raised against a partially-purified preparation of the liver isoenzyme. Both purified isoenzymes had a specific activity in the range 1100-1400 mumol/min per mg of protein with a subunit Mr of 80,000 determined by SDS/polyacrylamide gel electrophoresis. Butanol extraction instead of detergent solubilization, before immunoaffinity purification of the liver enzyme, resulted in the same specific activity and subunit Mr. The native Mr of the sulphobetaine 14-solubilized enzyme was consistent with the enzyme being a dimer of two identical subunits and was higher than that of the butanol-extracted enzyme, presumably due to the binding of the detergent micelle. 2. Pure bone and liver alkaline phosphatase were used to raise further antibodies to the two isoenzymes. Altogether, 27 antibody-producing cell lines were cloned from 12 mice. Several of these antibodies showed a greater than 2-fold preference for bone alkaline phosphatase in the binding assay used for screening. No antibodies showing a preference for liver alkaline phosphatase were successfully cloned. None of the antibodies showed significant cross-reaction with placental or intestinal alkaline phosphatase. Epitope analysis of the 27 antibodies using liver alkaline phosphatase as antigen gave rise to six groupings, with four antibodies unclassified. The six major epitope groups were also observed using bone alkaline phosphatase as antigen. 3. Serum from patients with cholestasis contains soluble and particulate forms of alkaline phosphatase. The soluble serum enzyme had the same size and charge as butanol-extracted liver enzyme on native polyacrylamide-gel electrophoresis. Cellulose acetate electrophoresis separated the soluble and particulate serum alkaline phosphatases as slow- and fast-moving forms respectively. In the presence of sulphobetaine 14 all the serum enzyme migrated as the slow-moving form on cellulose acetate electrophoresis. Monoclonal anti-(alkaline phosphatase) immunoadsorbents did not bind the particulate form of alkaline phosphatase in cholestatic serum but bound the soluble form. In the presence of sulphobetaine 14 all the cholestatic serum alkaline phosphatase bound to the immunoadsorbents. 4. The electrophoretic and immunological data are consistent with both particulate and soluble forms of alkaline phosphatase in cholestatic serum being derived from the hepatocyte membrane.
Characterization of monoclonal antibodies that recognize band 4.5 polypeptides associated with nucleoside transport in pig erythrocytes
Authors: Good A H , Craik J D , Jarvis S M , Kwong F Y P , Young J D , Paterson A R P , Cass C E
Published: August 2015

Three monoclonal antibodies have been raised against partially purified band 4.5 polypeptides [Steck (1974) J. Cell Biol. 62, 1-19] from pig erythrocyte membranes. The antibodies were capable of binding to both intact pig erythrocytes and protein-depleted membrane preparations and recognized detergent-solubilized polypeptides from adult and neonatal pig erythrocytes that were photolabelled with [G-3H]nitrobenzylthioinosine (NBMPR), a potent specific inhibitor of nucleoside transport. The antibodies did not recognize polypeptides from neonatal pig erythrocytes that were photolabelled with the glucose-transport inhibitor [3H]cytochalasin B. Reactivity with polypeptides of apparent Mr 64,000 [10% (w/v) acrylamide gels] was demonstrated by Western-blot analysis. The antibodies recognized pig band 4.5 polypeptides after prolonged treatment with endoglycosidase F, a finding consistent with reactivity against polypeptide, rather than carbohydrate, determinants. Trypsin digestion of NBMPR-labelled protein-depleted pig erythrocyte membranes generated two labelled polypeptide fragments (Mr 43,000 and 26,000). Two of the antibodies recognized both fragments on Western blots, whereas the third bound to the larger, but not to the smaller, fragment. The antibodies had no significant effect on reversible binding of NBMPR to protein-depleted pig erythrocyte membranes and did not bind to NBMPR-labelled polypeptides in human, rabbit or mouse erythrocytes.
Epidermal growth factor, but not insulin, stimulates tyrosine phosphorylation of an endogenous protein of Mr 95,000 in triton extracts of human placental syncytiotrophoblast membranes
Authors: Tavaré J M , Diggle T A , Denton R M
Published: August 2015

1. Triton extracts of syncytiotrophoblast membranes were incubated with [gamma-32P]ATP, MgCl2 and MnCl2. Addition of epidermal growth factor (EGF) resulted in increased phosphorylation not only of the EGF receptor and a Mr-35,000 protein as previously described, but also a protein of Mr 95,000 on both tyrosine and serine residues. In addition, a small increase in the phosphorylation of a protein of Mr 105,000 was observed. Spermine had a similar effect on the phosphorylation of the Mr-95,000 protein, without affecting the phosphorylation of the other proteins. In the absence of MnCl2, the effect of spermine on the phosphorylation of Mr-95,000 protein was still evident, whereas that of EGF was greatly diminished. 2. The Mr-95,000 protein bound poorly to wheat-germ-lectin-Sepharose and was not precipitated by antisera specific for insulin and EGF receptors. The protein continued to exhibit serine and tyrosine phosphorylation on addition of [gamma-32P]ATP, MgCl2 and MnCl2 to a glycoprotein-depleted fraction prepared by chromatography on wheat-germ-lectin-Sepharose. The extent of phosphorylation was no longer increased by spermine or EGF, but was inhibited by heparin. 3. It is suggested that the Mr-95,000 protein not only is a possible direct substrate for the EGF-receptor (but not the insulin receptor) tyrosine kinase but is a substrate for other endogenous kinases, including a protein tyrosine kinase which is probably not a glycoprotein, and a protein serine kinase with properties similar to those of casein kinase II.
The effects of structural modifications on the insulin-releasing activity of β-cell-tropin
Authors: Dunmore S J , Panico M , Etienne A T , Morris H R , Beloff-Chain A
Published: August 2015

The minimal effective concentration of the pituitary insulin secretagogue beta-cell-tropin (beta-CT) on the in vitro perfused pancreas was established and the effects of various modifications of the peptide on its potency were tested: iodination with 127I and acetylation reduced the insulin-releasing activity of beta-cell-tropin, and the C-terminal fragments beta-CT-(2-18), beta-CT-(3-18) and beta-CT-(6-18) were all less potent than the intact molecule; beta-CT-(1-6) was not active and did not inhibit beta-CT-induced insulin release.
Fractional synthesis rates in vivo of skeletal-muscle myosin isoenzymes
Authors: Gregory P , Low R B , Stirewalt W S
Published: August 2015

The synthesis rates of different myosin isoenzymes in a single muscle, and of the same isoenzymes in different muscles (soleus, masseter and plantaris), were measured. The rate of total protein synthesis was significantly higher in the soleus [greater than 95% slow myosin (SM)] than in the plantaris [greater than 95% fast myosin (FM)]. Two fast isoenzymes, FM2 and FM3, were synthesized at different rates in the masseter, and SM was synthesized at a faster rate than FM. Intermediate myosin had a synthesis rate similar to that of FM. There was a small but significant difference between the synthesis rates of the SM isoenzymes of the soleus and masseter muscles. FM3 was synthesized faster in the masseter than in the plantaris, whereas FM2 was synthesized faster in the plantaris than in the masseter.
Cytochrome b2, an electron carrier between flavocytochrome b2 and cytochrome c. Rapid kinetic characterization of the electron-transfer parameters with ionic-strength-dependence
Authors: Capeillère-Blandin C , Albani J
Published: August 2015

The oxidation-reduction properties of free cytochrome b2 isolated by controlled proteolysis from flavocytochrome b2, i.e. the flavodehydrogenase-bound cytochrome b2, were investigated by using stopped-flow spectrophotometry. The rapid kinetics of the reduction of cytochrome b2 by flavocytochrome b2 in the presence of L-lactate are reported. The self-exchange rate constant between reduced cytochrome b2 bound to the flavodehydrogenase and free cytochrome b2 was determined to be 10(5) M−1 X S−1 at 5 degrees C, I 0.2 and pH 7.0. The specific electron-transfer reaction between reduced cytochrome b2 and cytochrome c was also studied, giving an apparent second-order rate constant of 10(7) M−1 X S−1 at 5 degrees C, I 0.2 and pH 7.0. This electron-exchange rate is slightly modulated by ionic strength, following the Debye-Hückel relationship with a charge factor Z1Z2 = −1.9. Comparison of these data with those for the reduction of cytochrome c by flavodehydrogenase-bound cytochrome b2 [Capeillère-Blandin (1982) Eur. J. Biochem. 128, 533-542] leads to the conclusion that the intramolecular electron exchange between haem b2 and haem c within the reaction complex occurs at a rate very similar to that determined experimentally in presence of the flavodehydrogenase domain. The low reaction rate observed with free cytochrome b2 is ascribed to the low stability of the reaction complex formed between free cytochrome b2 and cytochrome c.
Differential sensitivity of the insulin-receptor kinase to thiol and oxidizing agents in the absence and presence of insulin
Authors: Wilden P A , Pessin J E
Published: August 2015

The purified human placental insulin-receptor beta-subunit autophosphorylating activity was found to be inhibited, in a time- and concentration-dependent manner, by the specific thiol-alkylating agents N-ethylmaleimide and 5,5′-dithiobis-(2-nitrobenzoic acid). The insulin-receptor kinase was observed to be more sensitive to inhibition by N-ethylmaleimide in the presence [IC50 (concn, giving 50% inhibition) = 25 +/- 3 microM] than in the absence (IC50 = 73 +/- 6 microM) of insulin. Similarly, inhibition by 5,5′-dithiobis-(2-nitrobenzoic acid) occurred with IC50 = 30 +/- 6 microM in the presence and 155 +/- 35 microM in the absence of insulin. Examination of the exogenous-substrate protein kinase activity demonstrated that the differential sensitivity to N-ethylmaleimide was due to direct inhibition of protein kinase activity, as opposed to blockade of the phospho-acceptor properties of the insulin receptor. In contrast, iodoacetamide had essentially no effect on the insulin-receptor beta-subunit autophosphorylating activity and was able to protect partially against the N-ethylmaleimide inhibition in both the presence and the absence of insulin. Consistent with these findings, none of the thiol-specific agents were able to alter significantly insulin binding at concentrations which maximally inhibited the beta-subunit autophosphorylation. Further, in the presence of insulin, the insulin-receptor kinase activity was also observed to be more sensitive to oxidation by H2O2 and FeCl3/ascorbate compared with insulin receptors in the absence of insulin. These results indicate that there is a critical thiol group(s) necessary for the beta-subunit autophosphorylating activity of the insulin-receptor kinase and that in the presence of insulin is more susceptible to exogenously added thiol and oxidizing agents.
Enzyme-substrate interactions in the hydrolysis of peptides by cathepsins B and H from rat liver
Authors: Brömme D , Bescherer K , Kirschke H , Fittkau S
Published: August 2015

The number of possible subsites of the rat liver cysteine proteinases cathepsin B and cathepsin H was determined in the N-terminal direction from the scissile bond. An elongation of the substrate peptide chain of up to four amino acid residues enhances the hydrolysis rate of both cathepsins. The greatest increase in activity was observed by elongation to the dipeptide substrate for cathepsin B and to the tetrapeptide substrate for cathepsin H. Both proteinases discriminate proline from their subsites S1 and S2, but accept it well in S3. A quantitative distinction between the endopeptidase and the peptidyl dipeptidase activity of cathepsin B was feasible by using two model peptides: (Formula: see text) (Z = benzyloxycarbonyl; X = NH2 or OH; the arrow shows the cleavage site). Whereas the peptide acid, representing the peptidyl dipeptidase substrate, was hydrolysed by cathepsin B twice as fast as the peptide amide as an endopeptidase substrate, cathepsin H clearly had a preference for the amide substrate.
Biliary lipid secretion and its control. Effect of taurodehydrocholate
Authors: Rahman K , Coleman R
Published: August 2015

Multiple short pulses of taurocholate (TC) brought about, in isolated perfused rat livers, the secretion of phospholipid and cholesterol into bile; the lipids showed an appreciable lag period behind the bile-salt secretion, and there was considerable variability in response, both between low and high dose pulses of TC and, at the higher dose, even between individual livers. When a background continuous infusion of taurodehydrocholate (a hydrophilic non-micelle-forming bile-salt analogue) was superimposed upon the short TC pulses, the lipid secretion showed much better control, and the lipid peaks were of more uniform size, following more closely, or more coincident with, the bile-salt output peaks. Taurodehydrocholate may provide a signal for the control of the supply and delivery of lipid vesicles to the bile-canalicular membrane, from where the lipid vesicles are then removed by the action of the pulses of TC.
Studies on the interactions of human pancreatic elastase 2 with human α1-proteinase inhibitor and α1-antichymotrypsin
Authors: Davril M , Laine A , Hayem A
Published: August 2015

The interactions of human pancreatic elastase 2 with alpha 1-proteinase inhibitor and alpha 1-antichymotrypsin were compared by studies in vitro. The equimolar complexes obtained between the enzyme and either inhibitor were relatively stable at 25 degrees C since they could be visualized for up to 5 days by an electrophoretic method. However, in both cases, a slow dissociation occurred with release of active enzyme. As the kass. rate constants are of the same order of magnitude, with a slightly lower value for alpha 1-proteinase inhibitor when compared with alpha 1-antichymotrypsin [(5.6 +/- 1.2) X 10(5) and (8.9 +/- 1.3) X 10(5) M-1.s-1 respectively], partition of human pancreatic elastase 2 between both inhibitors in human plasma is mainly dependent on their respective concentrations. A comparative study by crossed immunoelectrophoresis of the interactions of this enzyme with the two inhibitors contained in normal human plasma and in a mimetic mixture of pure inhibitors was carried out. This allowed the visualization of complexes with either inhibitor. Formation of such a complex with alpha 1-antichymotrypsin had never been demonstrated previously. The patterns obtained are similar when working with normal plasma or with the synthetic mixture, suggesting that, in the conditions used, alpha 1-proteinase inhibitor and alpha 1-antichymotrypsin are the main inhibitors of human pancreatic elastase 2 in the plasma sample. However, it is also shown that part of the enzyme may be taken up by alpha 2-macroglobulin, which is responsible for the remaining enzyme activity on a synthetic substrate. The present work suggests that, according to the delay times of inhibition of human pancreatic elastase 2 calculated from the normal plasma concentrations of alpha 1-proteinase inhibitor and alpha 1-antichymotrypsin, a significant role can be assigned to both inhibitors. Moreover, the role of alpha 1-antichymotrypsin would be enhanced in alpha 1-proteinase-inhibitor deficiency.
A model for the uptake and release of Ca2+ by sarcoplasmic reticulum
Authors: Gould G W , McWhirter J M , East J M , Lee A G
Published: August 2015

On addition of ATP to vesicles derived from the sarcoplasmic reticulum (SR) of skeletal muscle, Ca2+ is accumulated from the external medium. Following uptake, spontaneous release of Ca2+ occurs in the presence or in the absence of ATP. These processes of Ca2+ uptake and release were simulated by using the models derived for ATPase activity [Gould, East, Froud, McWhirter, Stefanova & Lee (1986) Biochem. J. 237, 217-227; Stefanova, Napier, East & Lee (1987) Biochem. J. 245, 723-730] and for Ca2+ release from passively loaded vesicles [McWhirter, Gould, East & Lee (1987) Biochem. J. 245, 713-722]. The simulations are consistent with measurements of the effects of pH, K+, Ca2+ and Mg2+ on uptake and release of Ca2+. The increase in maximal Ca2+ accumulation observed in the presence of maleate is explained in terms of complexing of Ca2+ and maleate within the SR. The calculated concentration of ADP generated by hydrolysis of ATP has a large effect on the simulations. The effects of an ATP-regenerating system on the measured Ca2+ uptake is explained in terms of both removal of ADP and precipitation of Ca3(PO4)2 within the vesicles. It is concluded that both the process of Ca2+ uptake and the process of Ca2+ release seen with SR vesicles can be interpreted quantitatively in terms solely of the properties of the Ca2+ + Mg2+-activated ATPase.
Effect of cations and temperature on kinetics of desmin assembly
Authors: Stromer M H , Ritter M A , Pang Y Y , Robson R M
Published: August 2015

Smooth-muscle desmin, which was isolated from avian gizzard, was purified and used to form reconstituted intermediate filaments. Filament assembly was done in the presence of physiological cations, Ca2+, Mg2+, Na+, and Na+ plus Mg2+, and with non-physiological cations Cu2+ and Ni2+. Assembly was done at 2 degrees, 22 degrees and 37 degrees C, and was monitored by absorbance and by electron microscopy. Absorbance increased most rapidly during the first 2-5 min and then increased at a slower rate with the physiological cations, but decreased after that time with the non-physiological cations. For each physiological cation, absorbance increased with increasing temperature. This was particularly evident with Ca2+, which produced the lowest absorbance at 2 degrees C and the highest at 37 degrees C. When ionic strength was comparable, filament-forming buffers that contained bivalent cations were associated with higher absorbance values. Filament diameters were significantly smaller 60 min after assembly initiation than after 5 min. Average filament diameters, when formed in the presence of Cu2+ or Ni2+, were 10% greater than in the presence of the physiological cations and did not show a consistent tendency to decrease as time increased. These results demonstrate the importance, not only of the pH and ionic composition of the filament-forming buffer, but also of the temperature and duration of dialysis for reconstitution of desmin filaments.
Formation and metabolism of inositol 1,4,5-trisphosphate in human platelets
Authors: Daniel J L , Dangelmaier C A , Smith J B
Published: August 2015

1. myo-[3H]Inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], when added to lysed platelets, was rapidly converted into [3H]inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4], which was in turn converted into [3H]inositol 1,3,4-trisphosphate [Ins(1,3,4)P3]. This result demonstrates that platelets have the same metabolic pathways for interconversion of inositol polyphosphates that are found in other cells. 2. Labelling of platelets with [32P]Pi, followed by h.p.l.c., was used to measure thrombin-induced changes in the three inositol polyphosphates. Interfering compounds were removed by a combination of enzymic and non-enzymic techniques. 3. Ins(1,4,5)P3 was formed rapidly, and reached a maximum at about 4 s. It was also rapidly degraded, and was no longer detectable after 30-60 s. 4. Formation of Ins(1,3,4,5)P4 was almost as rapid as that of Ins(1,4,5)P3, and it remained detectable for a longer time. 5. Ins(1,3,4)P3 was formed after an initial lag, and this isomer reached its maximum, which was 10-fold higher than that of Ins(1,4,5)P3, at 30 s. 6. Comparison of the intracellular Ca2+ concentration as measured with fura-2 indicates that agents other than Ins(1,4,5)P3 are responsible for the sustained maintenance of a high concentration of intracellular Ca2+. It is proposed that either Ins(1,3,4)P3 or Ins(1,3,4,5)P4 may also be Ca2+-mobilizing agents.
Participation of ATP in the binding of a yeast replicative complex to DNA.
Authors: Jazwinski S M
Published: August 2015

The activity that replicates yeast DNA in vitro can be isolated from cells of the budding yeast Saccharomyces in a high-Mr (approximately 2 × 10(6] form. Several lines of evidence indicate that this fraction contains a multiprotein replicative complex. A functional assay has been developed for the analysis of the interaction of the replicating activity with DNA. Binding of the activity required Mg2+, but did not require the addition of ATP or the other ribo- or deoxynucleoside triphosphates. However, the ATP analogues adenosine 5′-[gamma-thio]triphosphate and adenosine 5′-[beta gamma-imido]triphosphate blocked the binding, suggesting that ATP participates in the interaction at some stage. The binding was template (origin)-specific in either the presence or the absence of ATP and the other nucleoside triphosphates; however, ATP stabilized the replicating activity. The preferential inhibition of binding that was observed in the presence of the DNA topoisomerase II inhibitor coumermycin suggests that the requirement for ATP may be at least partially accounted for by the involvement of this enzyme in the initial interaction of the replicating activity with DNA. Finally, the binding was rapid. In contrast, DNA synthesis displayed a lag when assayed directly without first allowing a period for the replicating activity to bind to the DNA. In addition, binding was ‘tight’, as judged by the resistance of the protein–DNA complexes to salt in comparison with the relative sensitivity of binding. The replicating activity was not readily displaced from the complexes by exogenous DNAs, either possessing or lacking yeast origins of replication. The results suggest that the interaction of the replicating activity with the DNA occurs in more than one stage.
High-affinity binding of glycogen-synthase phosphatase to glycogen particles in the liver. Role of glycogen in the inhibition of synthase phosphatase by phosphorylase a
Authors: Mvumbi L , Stalmans W
Published: August 2015

1. Post-mitochondrial supernatants were prepared from the livers of 24 h-fasted rats. Upon centrifugation at high speed, the major part of the glycogen-synthase phosphatase activity sedimented with the microsomal fraction. However, two approaches showed that the enzyme was associated with residual glycogen rather than with vesicles of the endoplasmic reticulum. Indeed, the activity was entirely solubilized when the remaining glycogen was degraded either by glucagon treatment in vivo or by alpha-amylolysis in vitro. No evidence could be found for an association of glycogen-synthase phosphatase with the smooth endoplasmic reticulum, as isolated with the use of discontinuous sucrose gradients. 2. After solubilization by glucagon treatment in vivo, synthase phosphatase could be transferred to glycogen particles with very high affinity. Half-maximal binding occurred at a glycogen concentration of about 0.25 mg/ml, whereas glycogen synthase and phosphorylase required 1.5-2 mg/ml. 3. In gel-filtered extracts prepared from glycogen-depleted livers, the activation of glycogen synthase was not inhibited at all by phosphorylase alpha. The inhibition was restored when the liver homogenates were prepared in a glycogen-containing buffer. The effect was half-maximal at a glycogen concentration of about 0.25 mg/ml, and virtually complete at 1 mg/ml. These findings explain long-standing observations that in fasted animals the liver contains appreciable amounts of both synthase and phosphorylase in the active form.
Collagen activates superoxide anion production by human polymorphonuclear neutrophils
Authors: Monboisse J C , Bellon G , Dufer J , Randoux A , Borel J P
Published: August 2015

Human polymorphonuclear neutrophils (PMNs), purified on Ficoll-Hypaque cushions, were incubated for 5 min with calf skin acid-soluble collagen and the released superoxide anions (O2-) measured spectrophotometrically by reduction of ferricytochrome c or by chemiluminescence analysis. This collagen stimulated the release of O2- unless it had been treated with pepsin. The stimulatory activity remained in denatured collagen, was contained only in the alpha 1(I) chain and was present in the alpha 1(I)-CB 6 (CNBr-cleaved) peptide, which is C-terminal. The activity was linearly dependent on the collagen concentration up to about 200 micrograms/ml. In addition, this collagen induced a release of beta-glucuronidase and N-acetyl-beta-glucosaminidase from PMNs.
Desferrioxamine (Desferal) and superoxide free radicals. Formation of an enzyme-damaging nitroxide
Authors: Davies M J , Donkor R , Dunster C A , Gee C A , Jonas S , Willson R L
Published: August 2015

In neutral solutions, desferrioxamine (Desferal) can react with the superoxide free radical, O2.- (possibly through its protonated form HO2.), to form a relatively stable nitroxide free radical, which can have a half-life of approx. 10 min at room temperature. The formation of the radical can be largely prevented by the presence of superoxide dismutase. The radical reacts rapidly with cysteine, methionine, glutathione, vitamin C and a water-soluble derivative of vitamin E. It also reacts rapidly with alcohol dehydrogenase, causing a loss of enzyme activity. The implications of these findings for mechanistic free-radical biochemistry and iron-chelation therapy could be considerable.
myo-Inositol phosphorothioates, phosphatase-resistant analogues of myo-inositol phosphates. Synthesis of dl-myo-inositol 1,4-bisphosphate and dl-myo-inositol 1,4-bisphosphorothioate
Authors: Hamblin M R , Flora J S , Potter B V L
Published: August 2015

Syntheses of a metabolite of the second messenger myo-inositol 1,4,5-trisphosphate, myo-inositol 1,4-bisphosphate, and an analogue, the 1,4-bisphosphorothioate, are reported, by using phosphite chemistry on (+/-)-1,2:4,5-di-isopropylidene-myo-inositol. The synthesis of (+/-)-1,2:4,5-di-isopropylidene 3,6-bis[di-(2-cyanoethyl)]phosphite provides an intermediate that can be oxidized to either the corresponding bisphosphate or bisphosphorothioate. myo-Inositol phosphorothioates are proposed as novel analogues of myo-inositol phosphates; their resistance to phosphatase-catalysed breakdown is reported.
Control of respiration rate in non-growing cells of Paracoccus denitrificans
Authors: Kucera I , Lampardová L , Dadák V
Published: August 2015

By means of fluorimetric measurement and by direct determination of intracellular NAD+ and NADH contents, it was proved that the respiration rate of Paracoccus denitrificans cells utilizing glucose is limited by processes preceding NADH oxidation in the respiratory chain, so that the membrane NADH dehydrogenase is not saturated by its substrate. In the separated membrane fraction on saturation with exogenous NADH the main limiting factor is represented by NADH: ubiquinone oxidoreductase.
The role of vitamin A in the glycosylation reactions of glycoprotein synthesis in an ‘in vitro’ system
Authors: Chan V T , Wolf G
Published: August 2015

Microsomal membrane preparations from rat livers, when incubated with labelled sugar-nucleotides, were shown to synthesize labelled oligosaccharide-lipids in the presence of excess exogenous dolichyl phosphate. Under the incubation conditions defined in the present study, dolichyl pyrophosphoryl(DolPP)GlcNAc2-Man5, DolPPGlcNAc2Man9 and DolPPGlcNAc2Man9Glc3 were the principal oligosaccharide-lipids formed by both control and vitamin A-deficient membranes. However, deficient membranes synthesized 3.2 +/- 0.8 times as much oligosaccharide-lipids and 2.6 +/- 0.7 times as much dolichyl phosphate mannose (DolPMan) and dolichyl phosphate glucose (DolPGlc) as the controls. The transfer of the oligosaccharide chain from the dolichol carrier to the endogenous protein acceptors in vitamin A-deficient microsomes (microsomal fractions) was only 57.5 +/- 9.5% of that of controls. After endo-beta-N-acetylglucosaminidase treatment, only one oligosaccharide species was isolated from both control and vitamin A-deficient microsomal glycoproteins, and was characterized as GlcNAcMan9Glc3. We conclude that the decreased incorporation of labelled mannose and glucose from sugar-nucleotides into the glycoproteins must be due to decreased transfer of GlcNAc2Man9Glc3 from the dolichol carrier to the protein acceptors. This conclusion was further substantiated by the finding that control membranes transferred 4-6 times as much labelled oligosaccharides from exogenously added dolichol-linked substrate (DolPPGlcNAc2Man9Glc3) to endogenous microsomal protein acceptors as compared with the vitamin A-deficient membranes. Attempts to reverse this defect by addition of retinol or retinyl phosphate (a source of retinyl phosphate mannose) to the incubations were unsuccessful.
Evidence that the flux control coefficient of the respiratory chain is high during gluconeogenesis from lactate in hepatocytes from starved rats. Implications for the hormonal control of gluconeogenesis and action of hypoglycaemic agents
Authors: Pryor H J , Smyth J E , Quinlan P T , Halestrap A P
Published: August 2015

1. Increasing concentrations of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a mild respiratory-chain inhibitor [Halestrap (1987) Biochim. Biophys. Acta 927, 280-290], caused progressive inhibition of glucose production from lactate + pyruvate by hepatocytes from starved rats incubated in the presence or absence of oleate and gluconeogenic hormones. 2. No significant changes in tissue ATP content were observed, but there were concomitant decreases in ketone-body output and cytochrome c reduction and increases in NADH fluorescence and the ratios of [lactate]/[pyruvate] and [beta-hydroxybutyrate]/[acetoacetate]. 3. The inhibition by DCMU of palmitoylcarnitine oxidation by isolated liver mitochondria was used to calculate a flux control coefficient of the respiratory chain towards gluconeogenesis. In the presence of 1 mM-oleate, the calculated values were 0.61, 0.39 and 0.25 in the absence of hormone and in the presence of glucagon or phenylephrine respectively, consistent with activation of the respiratory chain in situ as previously suggested [Quinlan & Halestrap (1986) Biochem. J. 236, 789-800]. 4. Cytoplasmic oxaloacetate concentrations were shown to decrease under these conditions, implying inhibition of pyruvate carboxylase. 5. Inhibition of gluconeogenesis from fructose and dihydroxyacetone was also observed with DCMU and was accompanied by an increased output of lactate + pyruvate, suggesting that activation of pyruvate kinase was occurring. With the latter substrate, measurements of tissue ADP and ATP contents showed that DCMU caused a small fall in [ATP]/[ADP] ratio. 6. Two inhibitors of fatty acid oxidation, pent-4-enoate and 2-tetradecylglycidate, were shown to abolish and to decrease respectively the effects of hormones, but not valinomycin, on gluconeogenesis from lactate + pyruvate, without changing tissue ATP content. 7. It is concluded that the hormonal increase in mitochondrial matrix volume stimulates fatty acid oxidation and respiratory-chain activity, allowing stimulation of pyruvate carboxylation and thus gluconeogenesis to occur without major changes in [ATP]/[ADP] or [NADH]/[NAD+] ratios. 8. The high flux control coefficient of the respiratory chain towards gluconeogenesis may account for the hypoglycaemic effect of mild respiratory-chain inhibitors.
The cross-linking of tyrosine residues in apo-ovotransferrin by treatment with periodate anions
Authors: Hsuan J J
Published: August 2015

The iron-binding ability of apotransferrins is rapidly abolished in the reaction with periodate anions, which destroys 4 mol of tyrosine per mol of protein. Treatment of ovotransferrin with cyanogen bromide and tryptic digestion of the glycopeptide fragment demonstrated the existence of an intramolecular cross-link in the C-terminal domain of the oxidized protein. The cross-linked residues were identified as Tyr-421 and Tyr-524 and the product is similar in structure to 3,3′-dityrosine.
Studies on fatty acid-binding proteins. The binding properties of rat liver fatty acid-binding protein
Authors: Wilkinson T C , Wilton D C
Published: August 2015

1. The fluorescent fatty acid probe 11-(dansylamino)undecanoic acid binds to rat liver fatty acid-binding protein with a 1:1 stoichiometry. 2. The binding of the fluorescent probe is competitive with long-chain fatty acids. 3. Binding displacement studies were performed with a wide range of fatty acids and other ligands and identified C16 and C18 fatty acids as the preferred fatty acids for rat liver fatty acid-binding protein. No preference was observed for unsaturated fatty acids within this group. 4. Fatty acyl-CoA binds less well than the corresponding fatty acid.
Stimulation of Ca2+ efflux from sarcoplasmic reticulum by preincubation with ATP and inorganic phosphate
Authors: Shoshan-Barmatz V
Published: August 2015

Preincubation of sarcoplasmic reticulum with 1 mM-ATP completely inhibits Ca2+ accumulation and stimulates ATPase activity by over 2-fold. This effect of ATP is obtained only when the preincubation is carried out in the presence of Pi, but not with arsenate, chloride or sulphate. The inhibition by ATP of Ca2+ accumulation is pH-dependent, increasing as the pH is increased above 7.5. Inhibition of Ca2+ accumulation is observed on preincubation with ATP, but not with CTP, UTP, GTP, ADP, adenosine 5′-[beta gamma-methylene]triphosphate or adenosine 5′-[beta gamma-imido]triphosphate. The presence of Ca2+, but not Mg2+, during the preincubation, prevents the effect of ATP + Pi on Ca2+ accumulation. The ATP + Pi inhibition of Ca2+ accumulation is not due to modification of the ATPase catalytic cycle, but rather to stimulation of a rapid Ca2+ efflux from actively or passively loaded vesicles. This Ca2+ efflux is inhibited by dicyclohexylcarbodi-imide. Photoaffinity labelling of sarcoplasmic-reticulum membranes with 8-azido-[alpha-32P]ATP resulted in specific labelling of two proteins, of approx. 160 and 44 kDa. These proteins were labelled in the presence of Pi, but not other anions.
Binding of aromatic isonitriles to haemoglobin and myoglobin
Authors: Wood M A , Dickinson K , Willey G R , Dodd G H
Published: August 2015

A series of aromatic isonitriles were synthesized and their binding to sheep haemoglobin and horse heart myoglobin was investigated. The disubstituted ligands 2,6-dimethylphenylisonitrile and 2,6-diethylphenylisonitrile were found to bind to horse-heart myoglobin with affinities ranging from 500 to 5000 times greater than that of ethylisonitrile (4.6 × 10(-6) M) which has been the tightest binding isonitrile ligand for myoglobin thus far reported. The tight binding was not found to vary significantly with pH or temperature. An explanation for the unexpectedly high affinity is offered in terms of the electronic structure of aromatic isonitriles.
Cellular free cholesterol in Hep G2 cells is only partially available for down-regulation of low-density-lipoprotein receptor activity
Authors: Havekes L M , de Wit E C M , Princen H M G
Published: August 2015

We have previously shown that in Hep G2 cells and human hepatocytes, as compared with fibroblasts, the low-density lipoprotein (LDL) receptor activity is only weakly down-regulated after incubation of the cells with LDL, whereas incubation with high-density lipoproteins (HDL) of density 1.16-1.20 g/ml (heavy HDL) strongly increased the LDL-receptor activity. To elucidate this difference between hepatocytes and fibroblasts, we studied the cellular cholesterol homoeostasis in relation to the LDL-receptor activity in Hep G2 cells. (1) Interrupting the cholesteryl ester cycle by inhibiting acyl-CoA: cholesterol acyltransferase (ACAT) activity with compound 58-035 (Sandoz) resulted in an enhanced LDL-mediated down-regulation of the receptor activity. (2) The stimulation of the receptor activity by incubation of the cells with cholesterol acceptors such as heavy HDL was not affected by ACAT inhibition. (3) Incubation of the Hep G2 cells with LDL, heavy HDL or a combination of both grossly affected LDL-receptor activity, but did not significantly change the intracellular content of free cholesterol, suggesting that in Hep G2 cells the regulatory free cholesterol pool is small as compared with the total free cholesterol mass. (4) We used changes in ACAT activity as a sensitive (indirect) measure for changes in the regulatory free cholesterol pool. (5) Incubation of the cells with compactin (2 microM) without lipoproteins resulted in a 4-fold decrease in ACAT activity, indicating that endogenously synthesized cholesterol is directed to the ACAT-substrate pool. (6) Incubation of the cells with LDL or a combination of LDL and heavy HDL stimulated ACAT activity 3-5 fold, whereas incubation with heavy HDL alone decreased ACAT activity more than 20-fold. Our results suggest that in Hep G2 cells exogenously delivered (LDL)-cholesterol and endogenously synthesized cholesterol are primarily directed to the cholesteryl ester (ACAT-substrate) pool or, if present, to extracellular cholesterol acceptors (heavy HDL) rather than to the free cholesterol pool involved in LDL-receptor regulation.
Effect of perchlorate on calcium uptake and insulin secretion in mouse pancreatic islets
Authors: Sehlin J
Published: August 2015

Microdissected beta-cell-rich pancreatic islets of non-inbred ob/ob mice were used in studies of how perchlorate (CIO4-) affects stimulus-secretion coupling in beta-cells. CIO4- at 16 mM potentiated D-glucose-induced insulin release, without inducing secretion at non-stimulatory glucose concentrations. The potentiation mainly applied to the first phase of stimulated insulin release. In the presence of 20 mM-glucose, the half-maximum effect of CIO4- was reached at 5.5 mM and maximum effect at 12 mM of the anion. The potentiation was reversible and inhibitable by D-mannoheptulose (20 mM) or Ca2+ deficiency. CIO4- at 1-8 mM did not affect glucose oxidation. The effects on secretion were paralleled by a potentiation of glucose-induced 45Ca2+ influx during 3 min. K+-induced insulin secretion and 45Ca2+ uptake were potentiated by 8-16 mM-CIO4-. The spontaneous inactivation of K+-induced (20.9 mM-K+) insulin release was delayed by 8 mM-CIO4-. The anion potentiated the 45Ca2+ uptake induced by glibenclamide, which is known to depolarize the beta-cell. Insulin release was not affected by 1-10 mM-trichloroacetate. It is suggested that CIO4- stimulates the beta-cell by affecting the gating of voltage-controlled Ca2+ channels.
Purification and characterization of a second form of acid lipase in human liver
Authors: Sjoberg E R , Hatton J D , O'Brien J S
Published: August 2015

We describe here the purification and characterization of a form of acid lipase from human liver (designated ALII), which differed from the more abundant Mr-29000 form (ALI). ALII was solubilized from frozen human liver with Triton X-100 and purified 8500-fold by chromatography over concanavalin A-sepharose, CM-cellulose and finally h.p.l.c. over a Mono S column. ALII migrated as a single band on polyacrylamide-gel electrophoresis in both the presence and the absence of SDS. The Mr of ALII was estimated to be 58,500 by SDS/polyacrylamide-gel electrophoresis. Gel filtration on Sephacryl S-200 gave an apparent Mr of 69,000. 4-Methylumbelliferyl (4MU) palmitate, cholesterol oleate and triolein were substrates for ALII, with apparent Vmax values of 5000, 1100 and 2500 nmol/min per mg respectively and Km values of 1.0, 1.5 and 1.8 mM respectively. Cholesterol oleate and triolein were hydrolysed optimally by ALII at pH 4.5, whereas 4MU palmitate was hydrolysed optimally at pH 5.3. Antisera were raised against ALI and ALII and, on immunoblot analysis, no antigenic similarity was observed between ALI and ALII. Cellulose acetate electrophoresis followed by reaction with 4MU palmitate revealed two forms of lipase, corresponding to ALI and ALII. The two enzymes were also separated by hydrophobic chromatography. The activity of ALII was stimulated by several proteins and was partially inhibited by millimolar concentrations of NaCl, CaCl2 and MgSO4.
Purification of Sarcophaga (fleshfly) lectin and detection of sarcotoxins in the culture medium of NIH-Sape-4, an embryonic cell line of Sarcophaga peregrina
Authors: Komano H , Kasama E , Nagasawa Y , Nakanishi Y , Matsuyama K , Ando K , Natori S
Published: August 2015

An established cell line originating from a Sarcophaga peregrina (fleshfly) embryo, NIH-Sape-4, was found to synthesize mRNAs for Sarcophaga lectin and sarcotoxin IA, but not those for storage protein or 25 kDa protein. These four proteins are known to be synthesized in the fat-body of third-instar larvae, and the two former in particular are known to participate in the defence mechanism of this insect and to be induced in response to injury of the body wall. Thus the embryonic cell line NIH-Sape-4 synthesizes certain defence proteins constitutively. This cell line will be useful for large-scale purification of Sarcophaga lectin, since 50 micrograms of purified Sarcophaga lectin could be obtained from about 400 ml of culture medium.
Protein metabolism in the mouse during pregnancy and lactation
Authors: Millican P E , Vernon R G , Pain V M
Published: August 2015

Protein synthesis was measured in vivo in the whole body and in a number of individual tissues in mice at various stages of pregnancy and lactation. The absolute rate of protein synthesis in the whole body increased from 640 mg/day in virgin mice to 1590 mg/day by day 18 of pregnancy, and to 2100 mg/day by day 15 of lactation. Large proportions of these increments were contributed by the rapidly growing foetuses and placentae in the pregnant animals and by protein synthesis in the mammary glands during lactation. In addition, a substantial stimulation of growth and protein synthesis was also observed in the liver and the gastrointestinal tract. Gastrocnemius muscle showed no changes in protein metabolism, indicating that in the well-fed mouse this tissue is not required to play a role as a protein reserve during pregnancy and lactation.