bluuebunny/crossref_metadata_2025 huggingface dataset:
High-pressure liquid chromatographic analysis of anaerobic photoproducts of bilirubin-IX α in vitro and its comparison with photoproducts in vivo
Authors: Onishi S , Kawade N , Itoh S , Isobe K , Sugiyama S
Published: August 2015

To carry out photochemical experiments under conditions similar to those prevailing for neonatal bilirubin metabolism in jaundice phototherapy, we have studied photoproducts produced by the action of light on a bilirubin—albumin solution and further clarified the relationship between the photoproducts obtained from experiments in vitro and in vivo. (1) An accurate and sensitive separation method by high-pressure liquid chromatography for photoproducts of bilirubin under anaerobic irradiation of visible light is described. (2) There were two main photoproducts obtained from experiments both in vivo and in vitro. (3) Exact correspondence of retention time on high-pressure liquid chromatography, diazo-reactivity, thermal reversion and absorption-spectrum maxima was observed between unknown pigment and photobilirubin-IX alpha from biological fluids, and the comparable peaks 2 and 3 from experiments in vitro. (4) The behaviour of photoproducts in various solutions in the absence of light and O2 is described. (5) A lower affinity of photoproducts, especially unknown pigment, for human serum albumin than with bilirubin-IX alpha for the albumin was demonstrated by the gel-filtration method.
Demonstration of a geometric isomer of bilirubin-IX α in the serum of a hyperbilirubinaemic newborn infant and the mechanism of jaundice phototherapy
Authors: Onishi S , Isobe K , Itoh S , Kawade N , Sugiyama S
Published: August 2015

Photobilirubin-IX alpha in the serum of a hyperbilirubinaemic newborn infant was demonstrated for the first time by reversed-phase high-pressure liquid chromatography. The concentrations increased significantly during phototherapy. Corresponding to these changes in the serum, biliary unconjugated bilirubin concentrations increased markedly but photobilirubin-IX alpha was not detected in the bile. Moreover, large amounts of ‘unknown pigment’ that are diazo-negative appeared in the bile during phototherapy.
Some studies on the composition and surface properties of oil bodies from the seed cotyledons of safflower (Carthamus tinctorius) and linseed (Linum ustatissimum)
Authors: Slack C R , Bertaud W S , Shaw B D , Holland R , Browse J , Wright H
Published: August 2015

1. The average oil-body diameter in intact cells of developing linseed (Linum usitatissimum) and safflower (Carthamus tinctorius) cotyledons was similar (about 1.4 micrometer), and there was little change in size after oil bodies were isolated and repeatedly washed. 2. The glycerolipid composition of washed oil bodies from both developing and mature cotyledons of the two species was similar; oil bodies from ten different batches of cotyledons contained 4.3 +/- 0.16 mumol of 3-sn-phosphatidylcholine and 25.2 +/- 1.7 mumol of diacylglycerol per 1000 mumol of triacylglycerol. During four successive washings of a once-washed oil-body preparation, the proportion of diacylglycerol to triacylglycerol remained constant and that of 3-sn-phosphatidylcholine to triacylglycerol decreased by only 20%. 3. The protein content of thrice-washed oil bodies from the two species was similar, about 2.4% of the weight of glycerolipids, and appeared to be independent of the stage of cotyledon maturity. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated that the protein of purified oil bodies from the two species consisted mainly of only four polypeptides and that two of the polypeptides from each species had apparent mol.wts. of 17500 and 15500. Similar patterns of polypeptides were obtained after the hydrolysis of the 15500-mol.wt. polypeptides from linseed and safflower oil bodies by Staphylococcus aureus V8 proteinase, whereas the proteolysis of the 17500-mol.wt. polypeptides from the two species produced different patterns of polypeptides. 4. The 3-sn-phosphatidylcholine in oil-body preparations was hydrolysed about 85% by bee-venom phospholipase A2 without any apparent coalescence of the oil bodies. Incubation with lipase from Rhizopus arrhizus caused rapid coalescence of the oil bodies, and this lipase appeared to initially hydrolyse diacylglycerols in preference to triacylglycerol. 5. Oil bodies from both species were almost completely dispersed in suspensions of pH between 7.1 and 8.3, but formed large aggregates at pH values between 6.7 and 3.9; pH-induced aggregation caused no coalescence. Aggregates formed under acidic conditions were dispersed by re-adjusting the pH of suspensions to 8.3. 6. A freeze-etch electron-microscopic examination of isolated oil bodies indicated that these organelles were bounded by some form of membrane with a particle-free outer surface.
The effects of oestrogens and progesterone on oestrogen receptors in female rat liver
Authors: Marr W , Elder M G , Lim L
Published: August 2015

The administration of oestradiol-17 beta or ethynyloestradiol as well as the synthetic progestogen norethisterone acetate resulted in translocation of the oestrogen receptor. Progesterone and the synthetic progestogen (+)-norgestrel were ineffective. The increases in nuclear oestrogen receptor content 1 h after injection of each steroid were similar but different subsequently. The increase with oestradiol-17 beta extended for 3—6 h and for at least 9 h with ethynyloestradiol. With norethisterone acetate, nuclear content was still increased after 24 h. Oestrogen injection resulted in cytosol receptor depletion and a ‘deficit’ in receptor content extending for 6 h, whereas norethisterone acetate-induced translocation was quantitative. With injections of norethisterone acetate + ethynyloestradiol the increase at 1 h and retention of the nuclear receptors were similar to that with norethisterone acetate alone. In contrast, the depletion of cytosol receptor and its restoration were similar to that seen with ethynyloestradiol alone, suggesting that norethisterone acetate did not interfere with the oestrogen receptor replenishment. Specific binding in vitro of [3H]oestradiol-17 beta in liver cytosols was inhibited by (+)-norgestrel and norethisterone acetate, but not progesterone, at concentrations of 10—100 microM. Nuclear receptors present after norethisterone acetate injection bound oestrogen with high affinity (Kd = 1.52 nM), similar to receptors of oestrogen-injected animals. In the uterus, differential retention of nuclear receptors in response to oestrogens is associated with different cellular responses. The differences in the response of the receptor system in liver to the various steroids suggests that the corresponding tissue responses may also be dissimilar. These results are discussed in relation to the problems of liver dysfunction in oral-contraceptive users.
Insulin stimulates synthesis of soluble proteins in isolated rat hepatocytes
Authors: Clark R L , Hansen R J
Published: August 2015

The incorporation of [3H]leucine into soluble cellular protein was measured in isolated hepatocytes at extracellular leucine concentrations ranging from 0.15 to 20.0 mM. Insulin caused a 12—15% stimulation of [3H]leucine incorporation in the presence of high extracellular leucine concentrations. It is concluded that insulin causes a small but significant increase in the rate of hepatic protein synthesis.
Huntington's-chorea fibroblasts. Cellular protein glycosylation
Authors: Hung W Y , Mold D E , Tourian A
Published: August 2015

Five cell cultures of Huntington's-chorea fibroblasts exhibit greater than normal protein and lipid glycosylation when labelled with [U-14C]glucosamine. Oligosaccharide—polypeptide chains from all molecular-weight ranges are labelled differentially on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This difference in protein glycosylation is not accompanied by any apparent difference in general cellular protein synthesis or by a differential rate of glucosamine uptake or decreased degradation of [14C]glycosylated macromolecules. Additionally [U-14C]glucosamine exclusively labels hexosamines and sialic acid of cellular macromolecules.
Isotope-dilution analysis of the effects of deoxyguanosine and deoxyadenosine on the incorpoŕation of thymidine and deoxycytidine by hydroxyurea-treated thymus cells
Authors: Scott Fraser W. , Forsdyke Donald R.
Published: August 2015

It is presumed that the dGTP and dATP needed for replicative DNA synthesis can be formed by way of either ‘salvage’ pathways or biosynthesis de novo. This was examined by adding hydroxyurea to cultures of rat thymus cells to inhibit ribonucleoside diphosphate reductase, a key enzyme of the ‘de novo’ pathway. Most of the inhibition of the incorporation of [Me-3H]thymidine and deoxy[5-3H]cytidine by low concentrations of hydroxyurea (100–500μm) was prevented by substrates of the salvage pathway (400μm-deoxyguanosine and, to a lesser extent, 200μm-deoxyadenosine). However, isotope-dilution studies indicated that the purine deoxyribonucleosides prevented inhibition by decreasing pyrimidine deoxyribonucleotide competitor pools. Evidence was obtained that a hydroxyurea-induced increase in the thymidine-competitor pool (probably dTTP) was prevented to an equal extent by deoxyguanosine and by the inhibitor of thymidylate synthase, deoxy-5-fluorouridine. These compounds had almost identical effects on hydroxyurea dose–response curves and on thymidine isotope-dilution plots. The evidence suggests that exogenous purine deoxyribonucleosides cannot prevent the inhibition by hydroxyurea of thymus-cell DNA synthesis. This could mean that, with respect to the metabolism of purine deoxyribonucleotides, ribonucleoside diphosphate reductase is tightly coupled to DNA polymerase in a multienzyme complex. The complex would not permit entry of exogenous metabolic intermediates into the ‘de novo’ pathway, but would still be subject to the regulatory effects of these intermediates. Thus dGTP and dATP formed from exogenous purine deoxyribonucleosides by salvage pathways might deplete pyrimidine deoxyribonucleotide competitor pools by inhibiting relatively hydroxyurea-insensitive activities of ribonucleoside diphosphate reductase.
Spontaneous lysosomal enzyme secretion by a murine macrophage-like cell line
Authors: Jessup W , Dean R T
Published: August 2015

Lysosomal enzyme secretion by the murine macrophage-like cell line, P388D1, was compared with that of normal peritoneal macrophages. Unlike macrophages, lysosomal hydrolase secretion by P388D1 cells occurred spontaneously in vitro and was not further stimulated by the presentation of inflammatory agents such as zymosan and asbestos.
Ecto-enzymes of mammary gland and its tumours. Ca2+- or Mg2+-stimulated adenosine triphosphatase and its perturbation by concanavalin A
Authors: Carraway C A C , Corrado F J , Fogle D D , Carraway K L
Published: August 2015

Intact viable 13762 mammary-adenocarcinoma ascites cells hydrolyse added ATP. The localization of hydrolysis product and inactivation by the slowly penetrating chemical reagent diazotized sulphanilic acid indicate that this ATPase is at the external surface of the cell. A number of features differentiate this enzyme from mitochondrial, myosin and cation-transport ATPases. It is stimulated by either Ca2+ or Mg2+ and has little or no activity in their absence. It is insensitive to ouabain, oligomycin and azide. It is the major ATPase activity found in homogenates of gently disrupted 13762 cels. The ATPase activity is inhibited at high substrate concentrations and shows an apparent stimulation by concanavalin A in isolated membranes, but not in intact cells. The stimulation by concanavalin A results predominantly from a release from substrate inhibition.
Immunochemical comparison of UDP-glucuronyltransferase from Gunn- and Wistar-rat livers
Authors: Weatherill P J , Kennedy S M , Burchell B
Published: August 2015

1. Antiserum was raised against purified Wistar-rat liver UDP-glucuronyltransferase. 2. UDP-glucuronyltransferase activities towards 4-nitrophenol, bilirubin, 1-naphthol and morphine were co-immunoprecipitated from solubilized Wistar-rat liver preparations. 3. UDP-glucuronyltransferase activities towards 1-naphthol, 2-aminophenol and 4-nitrophenol were precipitated from solubilized Gunn-rat liver preparations by this antiserum. 4. UDP-glucuronyltransferase activities towards 1-naphthol, 4-nitrophenol and bilirubin, from Wistar-rat liver, were slightly inhibited by antiserum, whereas 1-naphthol UDP-glucuronyltransferase activity from Gunn-rat livers was greatly inhibited. 5. Measurable Wistar-rat liver glucuronyltransferase activities in washed immunoprecipitates indicate that the enzyme(s) were not merely inhibited by antiserum. 6. Immunoglobulin G purified from this antiserum immunoprecipitated transferase activities towards 4-nitrophenol, bilirubin and 1-naphthol. 7. The washed immunoprecipitates from both rat strains, containing UDP-glucuronyltransferase activity, appear to be similar when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 8. Radial-immunodiffusion studies suggest that a smaller amount of UDP-glucuronyltransferase protein is present in Gunn-rat liver than in Wistar-rat liver. 9. The significance of these results in relation to the genetic deficiency in the Gunn rat is discussed.
Purification and characterization of chromatin-bound DNA-dependent RNA polymerase I from parsley (Petroselinum crispum). Influence of nucleoside triphosphates
Authors: Grossmann K , Friedrich H , Seitz U
Published: August 2015

The isolation and purification of DNA-dependent RNA polymerase I (EC 2.7.7.6) from parsley (Petroselinum crispum) callus cells grown in suspension culture is described. The enzyme was solubilized from isolated chromatin. Purification was achieved by using DEAE- and phospho-cellulose in batches, followed by column chromatography on DEAE- and phospho-cellulose (two columns) and density-gradient centrifugation. The highly purified enzyme was stable over several months. The properties of purified parsley RNA polymerase I were investigated. Optimum concentration for Mn2+ was 1 mM, and for Mg2+ 4-6 mM, Mn2+ was slightly more stimulatory than Mg2+. The enzyme was most active at low ionic strengths [10-20 mM-(NH4)SO4]. The influence of various phosphates was tested: pyrophosphate inhibited RNA polymerase at low concentrations, whereas orthophosphate had no effect on the enzyme activity. ADP was slightly inhibitory, and AMP had no effect on the enzyme reaction. Nucleoside triphosphates and bivalent cations in equimolar concentrations in the range 4-11 mM did not influence the RNA synthesis in vitro. Free nucleoside triphosphates in excess of this 1:1 ratio inhibited the enzyme activity, unlike free bivalent cations, which stimulated RNA polymerase I.
Interaction of pseudomonic acid A with Escherichia coli B isoleucyl-tRNA synthetase
Authors: Hughes J , Mellows G
Published: August 2015

Sodium pseudomonate was shown to be a powerful competitive inhibitor of Escherichia coli B isoleucyl-tRNA synthetase (Ile-tRNA synthetase). The antibiotic competitively inhibits (Ki 6 nM; cf. Km 6.3 microM), with respect top isoleucine, the formation of the enzyme . Ile approximately AMP complex as measured by the pyrophosphate-exchange reaction, and has no effect on the transfer of [14C]isoleucine from the enzyme . [14C]Ile approximately AMP complex to tRNAIle. The inhibitory constant for the pyrophosphate-exchange reaction was of the same order as that determined for the inhibition of the overall aminoacylation reaction (Ki 2.5 nM; cf. Km 11.1 microM). Sodium [9′-3H]pseudomonate forms a stable complex with Ile-tRNA synthetase. Gel-filtration and gel-electrophoresis studies showed that the antibiotic is only fully released from the complex by 5 M-urea treatment or boiling in 0.1% sodium dodecyl sulphate. The molar binding ratio of sodium [9′-3H]pseudomonate to Ile-tRNA synthetase was found to be 0.85:1 by equilibrium dialysis. Aminoacylation of yeast tRNAIle by rat liver Ile-tRNA synthetase was also competitively inhibited with respect to isoleucine, Ki 20 microM (cf. Km 5.4 microM). The Km values for the rat liver and E. coli B enzymes were of the same order, but the Ki for the rat liver enzyme was 8000 times the Ki for the E. coli B enzyme. This presumably explains the low toxicity of the antibiotic in mammals.
Affinity-chromatographic purification of human α2-antiplasmin
Authors: Wiman B
Published: August 2015

A new simple and efficient purification method for alpha 2-antiplasmin is described that is based on the interaction between alpha 2-antiplasmin and a fragment from elastase-digested plasminogen constituting the three N-terminal triple-loop structures in the plasmin A-chain (LBSI). After a single-step adsorption of the alpha 2-antiplasmin from plasminogen-depleted plasma to LBSI-Sepharose and elution with 6-aminohexanoic acid, an 80-90% pure preparation with a yield of 50-60% is obtained. The major impurity is fibrinogen, which can easily be removed by gel filtration, and, as a result, a homogeneous fully active alpha 2-antiplasmin preparation is obtained that has the same properties as previously described for alpha 2-antiplasmin. Evidence is put forward that a form of alpha 2-antiplasmin with less affinity for the lysine-binding sites in plasminogen may exist, even in unfractionated plasma.
Resolution of protein disulphide-isomerase and glutathione-insulin transhydrogenase activities by covalent chromatography
Authors: Hillson D A , Freedman R B
Published: August 2015

1. Protein disulphide-isomerase (EC 5.3.4.1) and glutathione-insulin transhydrogenase (EC 1.8.4.2) were resolved by covalent chromatography. Both activities, in a partially purified preparation from bovine liver, bind covalently as mixed disulphides to activated thiopropyl-Sepharose 6B, in a new stepwise elution procedure protein disulphide-isomerase is displaced in mildly reducing conditions whereas glutathione-insulin transhydrogenase is only displaced by more extreme reducing conditions. 2. This together with evidence for partial resolution of the two activities by ion-exchange chromatography, conclusively establishes that the two activities are not alternative activities of a single bovine liver enzyme. 3. Protein disulphide-isomerase, partially purified by a published procedure, has now been further purified by covalent chromatography and ion-exchange chromatography. The final material is 560-fold purified relative to a bovine liver homogenate; it has barely detectable glutathione-insulin transhydrogenase activity. 4. The purified protein disulphide-isomerase shows a single major band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis corresponding to a mol.wt. of 57000. 5. The purified protein disulphide-isomerase has Km values for ‘scrambled’ ribonuclease and dithiothreitol of 23 microgram/ml and 5.4 microM respectively and has a sharp pH optimum at 7.5. The enzyme has a broad thiol-specificity, and several monothiols, at 1mM, can replace dithiothreitol. 6. The purified protein disulphide-isomerase is completely inactivated after incubation with a 2-3 fold molar excess of iodoacetate. The enzyme is also significantly inhibited by low concentrations of Cd2+ ions. These findings strongly suggest the existence of a vicinal dithiol group essential for enzyme activity. 7. When a range of thiols were used as co-substrates for protein disulphide-isomerase activity, the activities were found to co-purify quantitatively, implying the presence of a single protein disulphide-isomerase of broad thiol-specificity. Glutathione-disulphide transhydrogenase activities, assayed with a range of disulphide compounds, did not co-purify quantitatively.
Bovine liver thiol-protein disulphide oxidoreductases. An alternative method for differential purification and resolution of protein disulphide-isomerase and glutathione-insulin transhydrogenase
Authors: Hillson D A , Freedman R B
Published: August 2015

1. Protein disulphide-isomerase (EC 5.3.4.1) and glutathione-insulin transhydrogenase (EC 1.8.4.2) activities in bovine liver were studied in parallel during purification of ‘thiol-protein disulphide oxidoreductase’ by the procedure of Carmichael, Morin & Dixon [(1977) J Biol. Chem. 252, 7163-7167]. The two activities showed no quantitative co-purification and were partially resolved by (NH4)SO4 precipitation, indicating that distinct enzymes are present. 2. Protein disulphide-isomerase was purified by a relatively rapid method involving a combination of the early stages of the Carmichael procedure and covalent chromatography, with a new stepwise elution procedure. Ion-exchange chromatography yields a homogeneous preparation of mol.wt. 57 000. 3. The relationship between protein disulphide-isomerase, glutathione-insulin transhydrogenase and ‘thiol-protein disulphide oxidoreductase’ is discussed.
Purification of glutamate dehydrogenase from ox brain and liver. Evidence that commercially available preparations of the enzyme from ox liver have suffered proteolytic cleavage
Authors: McCarthy A D , Walker J M , Tipton K F
Published: August 2015

1. A rapid procedure, involving ion-exchange chromatography on DEAE-cellulose and affinity chromatography on GTP-Sepharose, was used to purify glutamate dehydrogenase from ox brain and liver. 2. Preparations purified in this way differed from those of the ox liver enzyme that were obtained from commercial suppliers in their mobilities on polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. This difference appears to result from the occurrence of limited proteolysis during the preparation of the latter enzyme samples. 3. N-Terminal sequence analysis showed the presence of four amino acid residues in the enzyme prepared in this study that were not present in those obtained from the commercial sources and which have not been reported in previous studies on the sequence of the ox liver enzyme. 4. A preliminary examination of the enzyme prepared in this way indicated that the Michaelis constants for the substrates are similar to those obtained from the commercial preparation, but that the response to allosteric effectors was modified.
Characterization of glycopeptides labelled from d-[2-3H]mannose and l-[6-3H]fucose in intestinal epithelial cell membranes during differentiation
Authors: Herscovics A , Bugge B , Quaroni A , Kirsch K
Published: August 2015

The labelled glycopeptides obtained by Pronase digestion of rat intestinal epithelial cell membranes were examined by gel filtration after injection of D-[2-3H]mannose and L-[6-3H]fucose. Three labelled fraction were eluted in the following order from Bio-Gel P-6, Fraction I, which was excluded from the gel, was labelled mostly with [3H]fucose and slightly with [3H]mannose. Fraction II contained “complex” asparagine-linked oligosaccharides since it was labelled with [3H]mannose and [3H]fucose, was stable to mild alkali treatment, and resistant to endo-beta-N-acetyl-glucosaminidase H. Fraction III contained “high-mannose” asparagine-linked oligosaccharides, which were labelled with [3H]mannose, but not with [3H]fucose; these were sensitive to endo-beta-N-acetylglucosaminidase H, and were adsorbed on concanavalin A-Sepharose and subsequently eluted with methyl alpha-D-mannopyranoside. The time course of incorporation of [3H]mannose into these glycopeptides in microsomal fractions showed that high-mannose oligosaccharides were precursors of complex oligosaccharides. The rate of this processing was faster in rapidly dividing crypt cells than in differentiated villus cells. The ratio of radioactively labelled complex oligosaccharides to high-mannose oligosaccharides, 3h after [3H]mannose injection, was greater in crypt than in villus-cell lateral membranes. Luminal membranes of both crypt and villus cells were greatly enriched in labelled complex oligosaccharides compared with the labelling in lateral-basal membranes. These studies show that intestinal epithelial cells are polarized with respect to the structure of the asparagine-linked oligosaccharides on their membrane glycoproteins. During differentiation of these cells quantitative differences in labelled membrane glycopeptides, But no major qualitative change, were observed.
The H+/e− stoicheiometry of respiration-linked proton translocation in the cytochrome system of mitochondria
Authors: Papa Sergio , Guerrieri Ferruccio , Lorusso Michele , Izzo Gianfranco , Boffoli Domenico , Capuano Ferdinando , Capitanio Nazzareno , Altamura Nicola
Published: August 2015

1. The →H+/e− quotients for proton release from mitochondria associated with electron flow from succinate and duroquinol to O2, ferricyanide or ferricytochrome c, and from NNN′N′-tetramethyl-p-phenylenediamine+ascorbate to O2, were determined from rate measurements of electron flow and proton translocation. 2. Care was taken to avoid, or to take into account, unrelated electron flow and proton translocation, which might take place in addition to the oxido-reductions that were the subject of our analysis. Spectrophotometric techniques were chosen to provide accurate measurement of the rate of consumption of oxidants and reductants. The rate of proton translocation was measured with fast pH meters with a precision of 10−3 pH unit. 3. The →H+/O quotient for succinate or duroquinol oxidation was, at neutral pH, 4, when computed on the basis of spectrophotometric determinations of the rate of O2 consumption or duroquinol oxidation. Higher →H+/O quotients for succinate oxidation, obtained from polarographic measurements of O2 consumption, resulted from underestimation of the respiratory rate. 4. The →H+/2e− quotient for electron flow from succinate and duroquinol to ferricyanide or ferricytochrome c ranged from 3.9 to 3.6. 5. Respiration elicited by NNN′N′-tetramethyl-p-phenylenediamine+ascorbate by antimycin-inhibited mitochondria resulted in extra proton release in addition to that produced for oxidation of ascorbate to dehydroascorbate. Accurate spectrophotometric measurement of respiration showed that the →H+/e− ratio was only 0.25 and not 0.7–1.0 as obtained with the inadequate polarographic assay of respiration. Proton release was practically suppressed when mitochondria were preincubated aerobically in the absence of antimycin. Furthermore, the rate of scalar proton consumption for water production was lower than that expected from the stoicheiometry. Thus the extra proton release observed during respiration elicited by NNN′N′-tetramethyl-p-phenylenediamine+ascorbate is caused by oxidation of endogenous hydrogenated reductants. 6. It is concluded that (i) the →H+/O quotient for the cytochrome system is, at neutral pH, 4 and not 6 or 8 as reported by others; (ii) all the four protons are released during electron flow from quinol to cytochrome c; (iii) the oxidase transfers electrons from cytochrome c to protons from the matrix aqueous phase and does not pump protons from the matrix to the outer aqueous phase.
Protein degradation in skin fibroblasts from patients with Duchenne muscular dystrophy
Authors: Statham H E , Witkowski J A , Dubowitz V
Published: August 2015

The rates of degradation of [3H]leucine-labelled proteins have been measured in cultures of skin fibroblasts obtained from normal controls (five subjects) and patients with Duchenne muscular dystrophy (six subjects). Cultures were incubated with [3H]leucine (10 microCi/ml) for 60 min to label “short-lived” proteins, and with [3H]leucine (5 microCi/ml) for 60 h to label “long-lived” proteins. Optimal wash procedures were devised for removal of [3H]leucine from the extracellular space and from cell pools before beginning degradation measurements. Re-utilization of [3H]leucine released from degraded labelled proteins was prevented by supplementing the medium with 4mM-leucine. Rates of degradation did not depend on the growth state of the cells or on cell age over the range used (passages eight-20). Degradation of long-lived proteins was approximately linear over a 24h period, at a rate of 1.0% per h. 30% of short-lived protein was degraded within 6h. No differences were observed between protein degradation in normal fibroblasts and in those from patients with Duchenne muscular dystrophy.
Localization and some properties of phosphate-dependent glutaminase in disrupted liver mitochondria
Authors: McGivan J D , Lacey J H , Joseph S K
Published: August 2015

1. Glutaminase activity in frozen and thawed liver mitochondria was activated by NH4+, phosphate and HCO3-ions and also by ATP . 2. NH4+ and HCO3-ions decreased the requirement of the enzyme for phosphate. The activation by ATP was observed only in the presence of NH4+ or HCO3-ions. 3. In frozen-and-thawed mitochondria, the enzyme was loosely bound to the inner membrane, the Arrhenius plot showing a break at 23 degrees C. On sonication, glutaminase was detached from the membrane and the Arrhenius plot became linear. 4. The apparent Km for glutamine of the membrane-bound form was 6 mM, and that of the soluble form was 21 mM. 5. It is likely that the properties of glutaminase in the intact cell are dependent on the association of this enzyme with the mitochondrial membrane.
Reduced lysyl oxidase activity in skin fibroblasts from patients with Menkes' syndrome
Authors: Royce P M , Camakaris J , Danks D M
Published: August 2015

Lysyl oxidase activity against both collagen and elastin substrates has been examined in the culture medium of skin fibroblasts derived from unrelated patients with Menkes' syndrome and from control subjects. The medium of three Menkes' fibroblast lines showed 3–30% of the activity present in the medium of control fibroblasts, against a purified collagen substrate. Lysyl oxidase activity in the culture medium of two of the Menkes' fibroblast lines was also examined by using a crude aortic-elastin substrate and was similarly decreased in comparison with that in the medium of control fibroblasts. Lysyl oxidase activity in the medium of a fourth fibroblast line, derived from a foetus with Menkes' syndrome, was 42% of that in the medium of control fibroblasts derived from a 1-day-old baby against a collagen substrate, and 26% of that in control fibroblast medium against an elastin substrate. The copper content of the cell layers of the Menkes' fibroblast cultures was elevated in comparison with normal fibroblast cultures, as has previously been reported to be characteristic of such cells. It is suggested that the decrease in lysyl oxidase activity would help to explain the connective tissue defects observed in Menkes' syndrome, and that this reduction, in conjunction with the elevated concentrations of cellular copper, would support the hypothesis that a functional intracellular copper deficiency exists in Menkes' syndrome.
Efficiency of ferredoxins and flavodoxins as mediators in systems for hydrogen evolution
Authors: Fitzgerald M P , Rogers L J , Rao K K , Hall D O
Published: August 2015

1. The efficiencies of ferredoxins and flavodoxins from a range of sources as mediators in systems for hydrogen evolution were assessed. 2. In supporting electron transfer from dithionite to hydrogenase of the bacterium Clostridium pasteurianum, highest activity was shown by the ferredoxin from the cyanobacterium Chlorogloeopsis fritschii and flavodoxin from the bacterium Megasphaera elsdenii. The latter was some twenty times as active as comparable concentrations of Methyl Viologen. Ferredoxins from the cyanobacterium Anacystis nidulans and the red alga Porphyra umbilicalis also showed high activity. 3. In mediating electron transfer from chloroplast membranes to Clostridium pasteurianum hydrogenase the flavodoxin from Anacystis nidulans proved the most active with Nostoc strain MAC flavodoxin and Porphyra umbilicalis ferredoxin also being appreciably more active than other cyanobacterial and higher plant ferredoxins. 4. In both hydrogenase systems the ferredoxin and flavodoxin from the red alga Chondrus crispus and the ferredoxin from another red alga Gigartina stellata showed very low activity. 5. There appeared to be no apparent correlation of efficiency in supporting hydrogenase activity with midpoint redox potential (Em) of the mediators, though some correlation of Em with the efficiency of the mediators in supporting NADP+ photoreduction by chloroplasts, or pyruvate oxidation by a Clostridium pasteurianum system, was evident. 6. Activity of the mediators in the hydrogenase systems therefore primarily reflects differences in tertiary structure conferring differing affinities for the other components of the systems.
A rapid and convenient technique for measuring the rate of protein synthesis in tissues by injection of [3H]phenylalanine
Authors: Garlick P J , McNurlan M A , Preedy V R
Published: August 2015

A rapid procedure for measuring the specific radioactivity of phenylalanine in tissues was developed. This facilitates the accurate determination of rates of protein synthesis in a wide range of tissues by injection of 150 mumol of L-[4-(3)H]phenylalanine/100 g body wt. The large dose of amino acid results in a rapid rise in specific radioactivity of free phenylalanine in tissues to values close to that in plasma, followed by a slow but linear fall. This enables the rate of protein synthesis to be calculated from measurements of the specific radioactivity of free and protein-bound phenylalanine in tissues during a 10 min period after injection of radioisotope.
Effect of bile salts on the hydrolysis of gangliosides, glycoproteins and neuraminyl-lactose by the neuraminidase of Clostridium perfringens
Authors: Gatt S , Gazit B , Barenholz Y
Published: August 2015

Studies were done on the effect of bile salts on the rates of hydrolysis of the N-acetylneuraminyl linkages of several sialic acid-containing compounds by the neuraminidase of Clostridium perfringens. When GM3-ganglioside, two glycolipids (glycophorin and orosomucoid) and neuraminyl-lactose were used as substrates, hydrolysis was obtained even in the absence of bile salts, but addition of this detergent, below its critical micellar concentration, increased the reaction rates; above the critical micellar concentration of the detergent rates decreased again. When a second ganglioside, GM1, was used as substrate, the requirement for bile salts was absolute; hydrolysis was not observed at all without this detergent. With increasing concentrations of bile salt and in the presence of high concentrations of enzyme, rates of hydrolysis increased, reaching maximal values at fixed ratios of bile salt to GM1-ganglioside. Physical measurements showed that mixtures of bile salt and GM1-ganglioside form mixed micelles that have a higher critical micellar concentration, a lower molecular weight and greater axial ratio than the corresponding micelles of pure GM1-ganglioside.
Chemical properties of the functional groups of insulin
Authors: Chan Y K , Oda G , Kaplan H
Published: August 2015

The method of competitive binding [Kaplan, Stevenson & Hartley (1971) Biochem. J. 124, 289-299] with 1-fluoro-2,4-dinitrobenzene as the labelling reagent [Duggleby & Kaplan (1975) Biochemistry 14, 5168-5175] was used to determine the chemical properties, namely pK and reactivity, of the amino groups, the histidine residues and the tyrosine residues of the dimeric form of pig zinc-free insulin at 20.0 degrees C. The N-terminal glycine residue of the A-chain has a pK of 7.7 and a slightly higher than normal reactivity. The N-terminal phenylalanine residue of the B-chain has a pK of 6.9 and is approximately an order of magnitude more reactive than a corresponding amino group with the same pK value. The lysine epsilon-amino group has an unusually low pK of 7.0 but has approximately the expected reactivity of such a group. In the case of the two histidine and four tyrosine residues only the average properties of each class were determined. The histidine residues have a pK value of approx. 6.6, but, however, their reactivity is at least an order of magnitude greater than that of a free imidazole group. The tyrosine residues have a pK value of approx. 10, but their average reactivities are substantially less than for a free phenolic group. At alkaline pH values above 8 the reactivity of all the functional groups show sharp discontinuities, indicating that insulin is undergoing a structural change that alters the properties of these groups.
Identification of ‘buried’ lysine residues in two variants of chloramphenicol acetyltransferase specified by R-factors
Authors: Packman L C , Shaw W V
Published: August 2015

Two variants of chloramphenicol acetyltransferase which are specified by genes on plasmids found in Gram-negative bacteria were subjected to amidination with methyl acetimidate to determine the relative reactivity of surface lysine residues and to search for unreactive or “buried” amino groups which might contribute to stabilization of the native tetramers. Representative examples of the type-I and type-III variants of chloramphenicol acetyltransferase were found to have one lysine residue each in the native state which appears to be inaccessible to methyl acetimidate. The uniquely unreactive residue of the type-I protein is lysine-136, whereas the lysine that is “buried” in the type-III enzyme is provisonally assigned to residue 38 of the prototype sequence. It is suggested that the lysine residue in each case participates in the formation of an ion pair at the intersubunit interface and that the two amino groups in question occupy functionally equivalent positions in the quaternary structures of their respective enzyme variants. Lysine-136 of type-I enzyme is also uniquely unavailable for modification by citraconic anhydride, a reagent used to disrupt the quaternary structure of the native enzyme. Contrary to expectation, exhaustive citraconylation fails to dissociate the tetramer, but does destroy catalytic activity. Removal of citraconyl groups from modified chloramphenicol acetyltransferase is accompanied by a full region of catalytic activity. Analysis of the rate of hydrolysis of citraconyl groups from the modified tetramer by amidination of unblocked amino groups with methyl [14C]acetamidate reveals difference in lability for several of the ten modified lysine residues. Although the unique stability of the quaternary structure of chloramphenicol acetyltransferase may be due to strong hydrophobic interactions, it is argued that lysine-136 may contribute to stability via the formation of an ion pair at the subunit interface.
Variant specific antigens of Trypanosoma brucei exist in solution as glycoprotein dimers
Authors: Auffret C A , Turner M J
Published: August 2015

Purified variant specific antigens of Trypanosoma brucei were shown to exist in solution as dimers, and occasionally as higher oligomers, as judged by gel filtration and by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis after treatment with bifunctional cross-linking reagents.
Identification of the apparently essential lysine residues in phospholipase C (Bacillus cereus)
Authors: Myrnes B J , Little C
Published: August 2015

Phospholipase C (Bacillus cereus) contains two apparently essential and very reactive lysine residues that may be labelled selectively by pyridoxal 5′-phosphate [Aurebekk & Little (1977) Biochem, J. 161, 159–165]. One of these lysine residues was found in the 25-amino acid N-terminal fragment liberated by CNBr digestion of the pyridoxal-labelled enzyme and identified as lysine-6. Two of the labelled peptides isolated from the chymotryptic digest of pyridoxal-labelled enzyme contained proline, suggesting that the other labelled lysine residue is situated in the same region of the primary structure as the single proline residue of the enzyme.
Amino acid sequence and oligosaccharide distribution of the haemagglutinin from an early Hong Kong influenza virus variant A/Aichi/2/68 (X-31)
Authors: Ward C W , Dopheide T A
Published: August 2015

The amino acid sequence and oligosaccharide distribution for the haemagglutinin from the early Hong Kong influenza virus A/Aichi/2/68 (X-31) was investigated. The two polypeptide chains, HA1 and HA2, were fragmented by CNBr and enzymic digestion, and the amino acid sequence of each small peptide was deduced by comparing its chromatographic behaviour, electrophoretic mobility, amino acid composition and N-terminus with that of the corresponding peptide of the haemagglutinin of known structure from the influenza-virus variant A/Memphis/102/72. Those peptides in which changes were detected were sequenced fully. The complete amino acid sequence of the haemagglutinin HA1 chain (328 residues) and 188 of the 221 residues of the HA2 chain were established by this approach, and revealed only twelve differences between the amino acid sequences of variant-A/Aichi/68 and -A/Memphis/72 haemagglutinins. These occurred at positions 2, 3, 122, 144, 155, 158, 188, 207, 242 and 275 in the HA1 chain and 150 and 216 in the HA2 chain. The highly aggregated hydrophobic region (residues 180-121) near the C-terminal end of the HA2 chain was not resolved by peptide sequencing. The oligosaccharide distribution in variant-A/Aichi/68 haemagglutinin was identical with that found in that of A/Memphis/72, with sugar units attached at asparagine residues 8, 22 38, 81, 165 and 285 in the HA1 chain and 154 on the HA2 chain. The monosaccharide compositions of the individual carbohydrate units on variant-A/Aichi/68 haemagglutinin differed from those of the corresponding units in variant-A/Memphis/72 haemagglutinin, and evidence was found for heterogeneity in the oligosaccharide units attached at single glycosylation sites.
N.m.r. and e.p.r. characterization of [4-carboxy-2,6-dinitrophenyl-lysine]cytochromes c
Authors: Falk K E , Jovall P A , Angström J
Published: August 2015

Monosubstituted [4-carboxy-2,6-dinitrophenyl-lysine]cytochromes c were investigated by n.m.r. and e.p.r. Modification of Lys-13 or Lys-72 in ferricytochrome c by 4-chloro-3,5-dinitrobenzoate yields either of two different conformers that are rapidly exchanging in the native form. The equilibrium involves small local changes in the conformation of Met-80 (the sixth ligand) and Phe-82, as a result of whether Lys-13 is the ‘on’ or ‘off’ position in the Lys-13–Glu-90 salt bridge.
The stimulation of glutamine hydrolysis in isolated rat liver mitochondria by Mg2+ depletion and hypo-osmotic incubation conditions
Authors: Joseph S K , McGivan J D , Meijer A J
Published: August 2015

1. In respiring rat liver mitochondria EDTA stimulates glutaminase activity measured in the presence of phosphate and HCO3- ions. The stimulation can be reversed by the addition of low concentrations of MgCl2. EGTA does not stimulate glutamine hydrolysis. 2. Glutaminase activity assayed in disrupted mitochondria is not significantly affected by EDTA or MgCl2. 3. The addition of EDTA results in a decrease in the concentration of phosphate required for half-maximal glutaminase activity. 4. Depletion of mitochondrial Mg2+ by the addition of the ionophore A23187 also stimulates glutamine hydrolysis in both the presence and the absence of EDTA. The effect of the ionophore can be abolished by the addition of MgCl2. 5. Hypo-osmotic incubation conditions increase the rate of mitochondrial glutamine hydrolysis. The effect of hypo-osmoticity on glutaminase is much less when EDTA is present. 6. It is suggested that glutaminase is partially and indirectly inhibited by endogenous mitochondrial Mg2+ and that the inner membrane may play a role in the regulation of glutaminase activity.
Formation of the A/B cis ring junction of ecdysteroids in the locust, Schistocerca gregaria
Authors: Davies T G , Dinan L N , Lockley W J , Rees H H , Goodwin T W
Published: August 2015

1. The mechanism of formation of the A/B cis ring junction of ecdysteroids in the locust Schistocerca gregaria, was investigated by incorporation of [4-14C,3 alpha-3H], [4-14C,4 alpha-3H] and [4-14C,4 beta-3H]cholesterol into 20-hydroxyecdysone in fifth-instar larvae and into ecdysteroid conjugates in ovaries of maturing adult females. 2. In both systems there was retention of the 4 alpha-3H atom in the ecdysteroid and elimination of the 3 alpha- and 4 beta-3H atoms. 3. The 3H retained in the ecdysone formed from [4 alpha-3H]cholesterol in the ovarian system was probably located at C-4. The results are interpreted by postulating the involvement of a 3-oxo-delta 4 intermediate in ecdysteroid biosynthesis in insects.
Characterization of human somatotropin binding to detergent-solubilized lactogenic receptors from rat liver
Authors: Bonifacino J S , Sánchez S H , Paladini A C
Published: August 2015

Lactogenic receptors from rat liver microsomal fraction (‘microsomes’) were extracted by treatment with 1% (w/v) Triton X-100. Triton X-100 exerts an inhibitory effect on both the binding reaction and the separation of the free hormone from the complex. The association and dissociation of 125I-labelled human somatotropin are time- and temperature-dependent processes. The association rate constant, k1, is 6.7 × 10(6) mol . litre-1 . min-1 at 25 decrees C, and the dissociation rate constant, k-1, is 1.1 × 10(-3) min-1 at 25 degrees C. Scatchard analysis of saturation data reveals the existence of a single class of receptors and that solubilization leads to a slight decrease in affinity and a sharp increase in binding capacity. The dissociation constant, Kd, of the solubilized preparation is 0.22 nM and the binding capacity 2900 fmol/mg of protein. Similar results were obtained from competition experiments. Binding of 125I-labelled human somatotropin to the solubilized receptors is specifically inhibited by hormones with lactogenic activity. Incubation of the solubilized preparation with trypsin resulted in an 80% decrease in binding activity. The solubilized form of the receptor has a slightly increased sensitivity to the inactivation by trypsin, heat and extremes of pH, with respect to the membrane-bound form.
The effect of calmodulin on the phosphoprotein intermediate of Mg2+-dependent Ca2+-stimulated adenosine triphosphatase in human erythrocyte membranes
Authors: Jeffery D A , Roufogalis B D , Katz S
Published: August 2015

The effect of calmodulin on the formation and decomposition of the Ca2+-dependent phosphoprotein intermediate of the (Mg2+ + Ca2+)-dependent ATPase in erythrocyte membranes was investigated. In the presence of 60 microM-Ca2+ and 25 microM-MgCl2, calmodulin (0.5-1.5 microgram) did not alter the steady-state concentration of the phosphoprotein, but increased its rate of decomposition. Higher calmodulin concentrations significantly decreased the steady-state concentration of phosphoprotein. Calmodulin (0.5-1.7 microgram) increased Ca2+-transport ATPase activity by increasing the turnover rate of its phosphoprotein intermediate. Increasing the MgCl2 concentration from 25 microM to 250 microM increased the (Mg2+ + Ca2+)-dependent ATPase activity, but decreased the concentration of the phosphoprotein intermediate. Similarly to calmodulin, MgCl2 increased the turnover rate of the Ca2+-transport ATPase complex (about 3-fold). At the higher MgCl2 concentration calmodulin did not further affect the decomposition of the phosphoprotein intermediate. It was concluded that both calmodulin and MgCl2 increase the turnover of the Ca2+-pump by enhancing the decomposition of the Ca2+-dependent phosphoprotein intermediate.