bluuebunny/crossref_metadata_2025 huggingface dataset:
H+ production by antimycin-inhibited mitochondria
Authors: Wrigglesworth J M , Nicholls P
Published: August 2015

1. Oxygen-pulse experiments on antimycin-treated liver mitochondria from young rats in the absence of added substrates indicate the presence of an endogenous hydrogenated reductant on the oxygen side of the antimycin inhibition site. This reductant has been estimated to be present in a 20-50-fold molar excess over cytochrome aa3. Oxidation of the reductant by small bursts of electron flow to oxygen gives a ratio H+ translocated: O atoms taken up of 2.1 +/- 0.2. 2. The endogenous hydrogenated reductant competes with added ferrocyanide and with ascorbate/NNN‘N’-tetramethyl-p-phenylenediamine as a source of reducing equivalents to oxygen. 3. When the contribution of endogenous substrate to H+ production is taken into account, there still remains some respiration-dependent H+ translocation consistent with a H+ pumping activity of cytochrome oxidase. The stoichiometry of this ‘extra’ H+ translocation is variable and depends on the rate of electron flow.
Structure and biological activity of finback-whale (Balaenoptera physalus L.) heparin octasaccharide. Chemical, carbon-13 nuclear-magnetic-resonance, enzymic and biological studies
Authors: Ototani N , Kikuchi M , Yosizawa Z
Published: August 2015

Finback-whale (Balaenoptera physalus L.) heparin was partially digested with a purified heparinase and an octasaccharide with high affinity for antithrombin III was isolated from the digest by gel filtration, followed by affinity chromatography on a column of antithrombin III immobilized on Sepharose 4B. This octasaccharide possessed high inhibitory activity for Factor Xa in the presence of antithrombin III, but was essentially inactive for thrombin-antithrombin III reaction. The anticoagulant activity determined by the activated-partial-thromboplastin-time method was very low (40-70 units/mg), although the initial whale heparin exhibited high activity (252 units/mg). On the basis of the results of chemical analyses, 13C n.m.r. spectrum and enzymic studies with purified heparinase, heparitinases 1 and 2, the predominant structure of the octasaccharide was proposed as follows: delta UA(2S) alpha 1 leads to 4GlcNS alpha 1 leads to 4IdUA alpha 1 leads to 4GlcNAc(6S) alpha 1 leads to 4GlcUA beta 1 leads to 4GlcNS(3S) alpha 1 leads to 4IdUA(2S) alpha 1 leads to 4GlcNS. Comparing this structure with those of the heparin octasaccharides so far reported, the presence of the critical structural elements for binding to antithrombin III was suggested in the pentasaccharide region situated at the reducing end of this octasaccharide. Binding to antithrombin III of the critical structural elements alone would appear to elicit the acceleration of the Factor Xa-antithrombin III reaction. Additional structural elements required for the acceleration of the thrombin-antithrombin III reaction and for the manifestation of high anticoagulant activity are discussed.
Evolution of α2-macroglobulin. The demonstration in a variety of vertebrate species of a protein resembling human α2-macroglobulin
Authors: Starkey P M , Barrett A J
Published: August 2015

Plasma or serum samples from a large number of vertebrate species were screened for the presence of a papain-binding protein resembling human alph a 2-macroglobulin (alpha 2M). The screening method depended on the unique property of alpha 2M of binding proteinases in such a way that the enzyme retains partial activity against low-molecular-weight substrates. A papain-binding protein was detected in serum from members of all the major vertebrate taxa. In mammals, birds, reptiles and amphibians the protein had an Mr similar to that of human alpha 2M (725 000), but in fish, including dipnoans, actinopterygians, elasmobranchs and cyclostomes, the papain-binding protein was of Mr about 360 000. Of the invertebrate species tested, all of which were arthropods, two were negative, but the horseshoe crab, an arachnid, did possess a papain-binding protein, although this was heterogeneous in electrophoresis and differed from alpha 2M in resisting inactivation by methylamine. From the results, and a detailed study of the properties of the fish papain-binding protein described in an accompanying paper [Starkey, Fletcher & Barrett (1982) Biochem. J. 205, 97-104], it seems that alpha 2M first appeared in an ancestor of all modern vertebrates as a protein of Mr 360 000 and that the larger macroglobulin (Mr 725 000) first appeared in an ancestor of the tetrapods.
Kinetics and thermodynamics of activation by pretreatment with guanosine 5′-[βγ-imido]triphosphate of smooth-muscle adenylate cyclase
Authors: Krall J. Frederick , Leshon Steven C. , Korenman Stanley G.
Published: August 2015

Catalytic subunits (C) of uterine smooth-muscle adenylate cyclase were activated (C*) by incubating the enzyme with the GTP analogue guanosine 5′-[βγ-imido]triphosphate (p[NH]ppG), followed by treatment with GTP and washing at 2°C. Activation (C→C*) proceeded in a time- and temperature-dependent manner as disclosed by subsequent assay of the pretreated particles at 37°C. The properties of the activated subunits were a function of the pretreatment temperature and not those of the enzyme assay performed at 37°C. Over the range 6–24°C, activation by pretreatment with p[NH]ppG followed simple Michaelis–Menten kinetics, and increase in temperature increased the concentration of catalytic subunits in the C* state and decreased Km for the guanosine nucleotide. Characterization of the temperature-dependent effects of pretreatment with p[NH]ppG suggested that activation of the catalytic subunit at the temperature in situ (37°C) was moderately endergonic (ΔH0 ∼8kJ·mol−1) and accompanied by an increase in entropy (ΔS0 ∼146J·mol−1·K−1). The β-adrenergic catecholamine receptor, reflected by isoproterenol's effect on activation by pretreatment with p[NH]ppG, increased the concentration of catalytic subunits in the C* state but had an insignificant (P>0.05) effect on the Km at every temperature. This result suggested that formation of the receptor–hormone complex produced an increase in the first-order rate constant without an appreciable effect on the actual catalytic-subunit activation step. The primary function of the β-adrenergic catecholamine receptor under these conditions appeared to be regulation of the concentration of activation sites available for binding of p[NH]ppG.
Isolation of the mature subunit of δ-aminolaevulinate synthase from embryonic chick liver
Authors: Ades I Z , Harpe K G
Published: August 2015

We presented evidence indicating that the established procedure for purifying delta-aminolaevulinate (ALA) synthase from embryonic-chick liver yielded an enzyme with a partially degraded subunit of molecular weight 51000 [Ades & Harpe (1981) J. Biol. Chem. 256, 9329-9333]. We now report the purification from livers of porphyric embryos of a preparation of ALA synthase which consisted primarily of a 63000-Da polypeptide and a component migrating as a smear of polypeptides with a minimum molecular weight of 52 000. Neither component could be recovered from liver mitochondria of normal embryos, where the amounts of ALA synthase were relatively low. The 52 000-Da component had been established to be the partially degraded subunit of the enzyme. Peptide-mapping analyses indicated that the 63 000- and the 52 000-Da components possessed significant structural homologies, and it was concluded that the 63 000-Da polypeptide represented the mature subunit of ALA synthase.
Characterization of human placental neuraminidases. Stability, substrate specificity and molecular weight
Authors: McNamara D , Beauregard G , Nguyen H V , Yan D L , Bélisle M , Potier M
Published: August 2015

1. At least two components of neuraminidase can be distinguished on the basis of thermolability and sedimentability by using the artificial fluorogenic substrate 4-methylumbelliferyl N-acetyl-alpha-D-neuraminate. 2. In crude homogenates, thermodenaturation at 25 degrees C showed a biphasic curve corresponding to component A (half-life, 21 min) and B (half-life, 85 min). The two components were partially resolved by centrifugation. A being soluble and B sedimentable. Both had similar pH-activity curves (pH optimum, 4.4), Km values (A, 0.10 mM; B, 0.06 mM) and molecular weight as determined by radiation inactivation (A, 67000; B, 63000). 3. The soluble A form was still aggregated or bound to membranous debris since almost all neuraminidase activity was eluted near or at the void volume of a Sephacryl S-300 column. 4. Both soluble and sedimentable fractions of placenta hydrolysed the GD1A ganglioside and N-acetyl-neuraminyl-D-lactose linearly for 12 h but no fetuin hydrolysis was detected. 5. The neuraminidase activity with the artificial fluorogenic substrate was inhibited by N-acetylneuraminyl-D-lactose but not by the GD1A ganglioside. These preliminary results suggest that there exist two closely related enzymes hydrolysing both the artificial substrate and N-acetylneuraminyl-D-lactose and a third one hydrolysing the GD1A ganglioside exclusively.
A computergraphical method of describing the shapes of subunit interfaces of oligomers. Analysis of the quaternary structure of concanavalin A and of prealbumin
Authors: Milner-White E J
Published: August 2015

A new method is described for mapping the shape of the intersubunit contacts of oligomers with identical subunits or pairs of subunits. Plots are generated for the dimer-dimer interactions of concanavalin A and prealbumin, which are both tetramers. Both contacts lie at the junction between the faces of two apposed beta-sheets, but in most other respects the interactions are seen to be different.
Purification and characterization of the multiple forms of aldehyde reductase in ox kidney
Authors: Daly A K , Mantle T J
Published: August 2015

Aldehyde reductase from ox kidney cytosol has been fractionated into four forms, two of which have been purified to apparent homogeneity. One of the minor forms is shown to be heterogenous on polyacrylamide-gel electrophoresis. The substrate specificities of the four forms using a variety of aldehydes and ketones are presented. The sensitivity of the various forms to inhibition by sodium valproate, sodium barbitone and various benzodiazepines has been determined. The relationship of these forms to the previously described hexonate dehydrogenase, aldose reductase and prostaglandin dehydrogenase is discussed.
Cross-linking and 1H n.m.r. spectroscopy of the pyruvate dehydrogenase complex of Escherichia coli
Authors: Packman Leonard C. , Perham Richard N. , Roberts Gordon C. K.
Published: August 2015

The pyruvate dehydrogenase complex of Escherichia coli was treated with o-phenylene bismaleimide in the presence of the substrate pyruvate, producing almost complete cross-linking of the lipoate acetyltransferase polypeptide chains as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This took place without effect on the catalytic activities of the other two component enzymes and with little evidence of cross-links being formed with other types of protein subunit. Limited proteolysis with trypsin indicated that the cross-links were largely confined to the lipoyl domains of the lipoate acetyltransferase component of the same enzyme particle. This intramolecular cross-linking had no effect on the very sharp resonances observed in the 1H n.m.r. spectrum of the enzyme complex, which derive from regions of highly mobile polypeptide chain in the lipoyl domains. Comparison of the spin–spin relaxation times, T2, with the measured linewidths supported the idea that the highly mobile region is best characterized as a random coil. Intensity measurements in spin-echo spectra showed that it comprises a significant proportion (probably not less than one-third) of a lipoyl domain and is thus much more than a small hinge region, but there was insufficient intensity in the resonances to account for the whole lipoyl domain. On the other hand, no evidence was found in the 1H n.m.r. spectrum for a substantial structured region around the lipoyl-lysine residues that was free to move on the end of this highly flexible connection. If such a structured region were bound to other parts of the enzyme complex for a major part of its time, its resonances might be broadened sufficiently to evade detection by 1H n.m.r. spectroscopy.
The interaction of anthraquinone dyes with the plasmid-mediated OXA-2 β-lactamase
Authors: Monaghan C , Holland S , Dale J W
Published: August 2015

Although beta-lactamases do not require any nucleotide co-substrates, the OXA-2 type is inhibited competitively by Cibacron Blue 3GA, and by other anthraquinone dyes, including some simpler compounds with no side chain. The enzyme causes a red shift in the spectrum of Cibacron Blue. The beta-lactamase can be adsorbed in Blue Sepharose and specifically eluted by benzylpenicillin. These results indicate that the binding of anthraquinone dyes is a specific effect similar to that seen with many nucleotide-binding enzymes.
Isolation and re-association of the subunits from the pro-(carboxypeptidase A)–pro-(proteinase E) binary complex from pig pancreas
Authors: Vendrell Josep , Aviles Francesc X. , San Segundo Blanca , Cuchillo Claudi M.
Published: August 2015

The component subunits of the pro-(carboxypeptidase A)–pro-(proteinase E) binary complex from pig pancreas were separated with a high recovery (80–95%) of their original potential activity. The isolated subunits and the reconstituted complex have properties similar to those of the corresponding natural species. The tryptic activation course of the pro-(carboxypeptidase A) subunit is substantially modified when bound to pro-(proteinase E), whereas the activation of pro-(proteinase E) is not dependent on this association.
Change in subunit composition of the iron protein of nitrogenase from Rhodospirillum rubrum during activation and inactivation of iron protein
Authors: Preston G G , Ludden P W
Published: August 2015

The subunit composition of the Fe protein of nitrogenase from Rhodospirillum rubrum during activation and inactivation was investigated. It was found that the upper subunit (on gel electrophoresis) of the two-subunit Fe protein was converted into the lower subunit during activation in vitro. When the Fe protein was inactivated in vivo by the addition of NH4Cl and alpha-oxoglutarate to the cells, a phosphate-labelled upper band appeared. During activation in vitro by the activating enzyme, some of the phosphate of the upper band remained with the protein and appeared in the lower band. Activations in vitro were performed on inactive Fe protein obtained from cells grown with glutamate as the nitrogen source. Both native and oxygen-denatured Fe protein exhibited the loss of upper band during treatment with activating enzyme.
Studies on the temperature-dependent autoinhibition of human plasma kallikrein I
Authors: Levison P R , Tomalin G
Published: August 2015

At 37 degrees C, human plasma kallikrein I follows Michaelis-Menten behaviour and exhibits a normal linear relationship between the initial velocity of hydrolysis of Ac-Pro-Phe-Arg-OMe, HCl and enzyme concentration in the range 0-150 pM. At temperatures of 30 degrees C and below substantial deviations from linearity are observed over the same enzyme concentration range. The temperature-dependent autoinhibition of kallikrein I activity is reversible and is not due to low-molecular-weight endogenous inhibitors or cofactors. The kinetic effect is apparently due to aggregation and can be abolished by the addition of sodium deoxycholate.
Selective elution of immunoadsorbed anti-(human prolactin) immunoglobulins with enhanced immunochemical properties
Authors: Hodgkinson S C , Lowry P J
Published: August 2015

Specific anti-(human prolactin) immunoglobulin, isolated by immunoadsorption from a resolubilized Na2SO4 precipitate of a sheep antiserum, has been fractionated into antibody populations with a 10-fold range of affinity for antigen by using a new elution procedure. The procedure utilized acetonitrile (20%, v/v) in a gradient of pH, decreasing from pH 7 to pH2. The recovered immunoglobulin (74.3%) was eluted as a single peak after chromatography on Sephacryl S-300 and on radioiodination retained high immunoreactivity (91%). One antibody population, of low abundance, had an affinity constant (Ka = 8.6×10(10) l/mol) 5.7 times that of the unfractionated antiserum and, when used in radioimmunoassay standard curves, resulted in a 3.8-fold increase in the sensitivity of the assay. The bulk of adsorbed immunoglobulin was eluted at pH 4.3, thereby avoiding exposure to strong acid conditions and maintaining the integrity of the immunoglobulin molecule. In contrast, elution of immunoglobulin in the absence of acetonitrile could only be achieved at low pH (pH2) with minimal fractionation, and resulted in a poor recovery (37.1%) and loss of immunoreactivity (53%).
Isolation and partial characterization of a lectin from a false brome grass (Brachypodium sylvaticum)
Authors: Peumans W J , Spaepen C , Stinissen H M , Carlier A R
Published: August 2015

A lectin has been isolated from embryos of a false brome grass species (Brachypodium sylvaticum) by affinity chromatography on immobilized N-acetylglucosamine. It is a dimeric protein of two identical subunits of mol.wt. 18 000. Although it resembles cereal lectins with respect to its biochemical and physicochemical properties, it differs structurally in several aspects from wheat-germ-agglutinin-like lectins.
Kinetic properties of the Ca2+-accumulation system of a rat liver microsomal fraction
Authors: Dawson A P
Published: August 2015

1. By using Ca-EGTA buffers, the Km for Ca2+ uptake into rat liver heavy microsomes (microsomal fraction) was found to be 0.2 microM free Ca2+. 2. In the absence of oxalate, these vesicles accumulate about 20 nmol of Ca2+/mg of protein. Efflux of Ca2+ from the vesicles is much faster at pH 7.6 than at pH 6.8, but does not apparently show saturation kinetics or any stringent requirement for external ions. 3. The steady-state distribution of Ca2+ between the microsomes and the medium in the presence of ATP and the absence of oxalate is dependent on Ca2+ load. When the vesicles are loaded to 50% capacity, the external free Ca2+ concentration is 70 nM. 4. The affinity of heavy microsomes for Ca2+ is such that is seems likely that they has a dominant role in the determination of cytoplasmic free Ca2+ concentrations.
Pyruvate dehydrogenase kinase/activator in rat heart mitochondria, Assay, effect of starvation, and effect of protein-synthesis inhibitors of starvation
Authors: Kerbey A L , Randle P J
Published: August 2015

Purified pig heart pyruvate dehydrogenase complex is denuded of its intrinsic pyruvate dehydrogenase kinase activity by sedimentation from dilute solution (60 munits/ml). Kinase activity is restored by a supernatant fraction prepared by high-speed centrifugation of rat heart mitochondrial extracts; the factor responsible is referred to as kinase/activator. Kinase/activator was also assayed by its ability to accelerate NgATP-induced inactivation in dilute solutions of unprocessed complex (50 munits/ml). With this assay it has been shown that the activity of kinase/activator in heart mitochondria is increased 3-6 fold by starvation of rats for 48 h. This increase was prevented completely by cycloheximide treatment and prevented partially by puromycin treatment of rats during starvation. The concentration of kinase/activator in heart mitochondria fell during 20 h of re-feeding of 48 h-starved rats; this fall was correlated with an increase in the proportion of complex in the active form. Kinase/activator was also extracted from ox kidney mitochondria, and on gel filtration (Sephadex G-100, superfine grade) was eluted close to the void volume. Kinase/activator (ox kidney or rat heart) was thermolabile, non-diffusable on dialysis, and inactivated by trypsin. The results of this study appear to show increased cytoplasmic synthesis in starvation of pyruvate dehydrogenase kinase and/or of an activator of the kinase.
Isolation of cell-surface glycopeptides from bovine cerebral cortex that inhibit cell growth and protein synthesis in normal but not in transformed cells
Authors: Kinders R J , Johnson T C
Published: August 2015

Glycopeptides were isolated from the cell surfaces of bovine cerebral cortex that could inhibit increase in cell numbers in tissue culture and cellular protein synthesis. This cell growth inhibition apparently affected all cells exposed, could completely block cell division in a reversible manner and synchronized BHK-21 cell cultures. Polyoma-virus-transformed BHK-21 cells were completely insensitive to the inhibitor. Fractionation of the inhibitor on a Bio-Gel P-100 column revealed two peaks of biologically active material eluting at apparent molecular weights of 45 000 and 10 000 with A 1cm,280 1% 11.0. Affinity purification of the inhibitory fractions on a Ulex europaeus agglutination I lectin column resulted in retention of the inhibitory activity, suggesting the inhibitor material was a glycopeptide. Subsequent elution with 0.10 M-fucose resulted in a 244-fold increase in the specific biological activity over the starting material. Although purified from bovine brain, the material could inhibit baby-hamster kidney cell protein synthesis by 50% at a concentration of 5×10(6) molecules per target cell. Analysis by competitive radioimmunoassay or immunoadsorption indicated that the bovine inhibitor was structurally related to, although not necessarily identical with, a similar inhibitory glycopeptide preparation that we had previously isolated from mouse brain.
Regulation of glycolysis and fatty acid synthesis from glucose in sheep adipose tissue
Authors: Robertson James P. , Faulkner Anne , Vernon Richard G.
Published: August 2015

1. The following were measured in adipose-tissue pieces, obtained from 7–9 month-old sheep, before or after the tissue pieces had been maintained in tissue culture for 24 h: the rates of synthesis from glucose of fatty acids, acylglycerol glycerol, pyruvate and lactate; the rate of glucose oxidation to CO2; the rate of glucose oxidation via the pentose phosphate pathway; the activities of hexokinase, glucose 6-phosphate dehydrogenase, phosphofructokinase, pyruvate kinase, pyruvate dehydrogenase and ATP citrate lyase; the intra- and extra-cellular water content; the concentration of various metabolites and ATP, ADP and AMP. 2. The proportion of glucose carbon converted into the various products in sheep adipose tissue differs markedly from that observed in rat adipose tissue. 3. There was a general increase in the rate of glucose utilization by the adipose-tissue pieces after maintenance in tissue culture; largest changes were seen in the rates of glycolysis and fatty acid synthesis from glucose. These increases are paralleled by an increase in pyruvate kinase activity. There was no change in the activities of the other enzymes as measured, although the net flux through all the enzymes increased. 4. Incubation of fresh adipose-tissue pieces for 2–6h led to an increase in the affinity of pyruvate kinase for phosphoenolpyruvate. 5. The rate of pyruvate production by glycolysis was greater than the activity of pyruvate dehydrogenase of the tissue. 6. The results suggest that both pyruvate kinase and pyruvate dehydrogenase have important roles in restricting the utilization of glucose carbon for fatty acid synthesis in sheep adipose tissue.
Fatty acid desaturation and microsomal lipid fatty acid composition in experimental hypothyroidism
Authors: Faas F H , Carter W J
Published: August 2015

We have studied the influence of experimental hypothyroidism in the rat on the synthesis of unsaturated fatty acids and on liver microsomal lipid fatty acid composition. Hypothyroid rats demonstrated an 80% decrease in delta 9 (stearate) desaturation and a 43% decrease in delta 6 (linoleate) desaturation. Liver microsomal fatty acid composition was altered in the hypothyroid animals with a significantly decreased proportion of arachidonate and increased proportions of linoleate, eicosa-8,11,14-trienoate, eicosapentaenoate and docosahexaenoate. The bulk of these changes occurred in both of the two major phospholipid components, phosphatidylcholine and phosphatidylethanolamine. All of the changes were corrected by treatment of the hypothyroid rat with 25 micrograms of tri-iodothyronine/100 g body wt. twice daily. The diminished delta 9 desaturation did not lead to any changes in fatty acid composition. The increased linoleate and decreased arachidonate levels may be due to the diminished delta 6 desaturase activity, the rate-controlling step in the conversion of linoleate into arachidonate. The increases in the proportions of the other polyunsaturated fatty acid components cannot be explained by changes in the synthesis of unsaturated fatty acids, but are probably due to diminished utilization of these fatty acids.
1H n.m.r. of the DNA-dependent RNA polymerase from Escherichia coli
Authors: Cellai Lucian , Segre Annalaura , Heumann Hermann
Published: August 2015

The1H n.m.r. study of the DNA-dependent RNA polymerase from Escherichia coli has revealed that the holoenzyme (ββ′α2σ) displays two mobile regions: one, observable also in the core enzyme (ββ′α2), is characterized by basic amino acids and its appearance and form depend on ionic strength; the other, specific to the holoenzyme, is characterized by threonine residues and its appearance does not depend on ionic strength.
Evidence for similar conformational changes in α2-macroglobulin on reaction with primary amines or proteolytic enzymes
Authors: Björk Ingemar , Fish Wayne W.
Published: August 2015

Reactions of α2-macroglobulin (α2M) with primary amines (ammonium chloride, methylammonium chloride and ethylammonium chloride) or proteolytic enzymes (trypsin, chymotrypsin and thrombin) resulted in changes of the absorption, fluorescence and circular-dichroism spectra and of the sedimentation coefficient of the inhibitor. All physico-chemical changes caused by the inactivation of α2M by the amines were identical with, or highly similar to, those induced by the formation of the enzyme–inhibitor complexes. This suggests that similar conformational changes of the inhibitor occur in the two types of reactions. The frictional ratio, calculated from the increase in sedimentation coefficient, decreased from 1.67 for untreated α2M to 1.57 for the amine- or proteinase-treated inhibitor. This change is due to a decrease in either asymmetry or hydration of the protein, resulting in a slightly smaller hydrodynamic volume. The circular-dichroism analyses indicated that the reaction of α2M with either amines or proteinases is accompanied by a loss of the small amount (about 5%) of α-helix of the untreated protein. The changes of u.v. absorption and fluorescence suggested that about one out of the eight to ten tryptophan residues of each α2M subunit is buried as a result of the conformational change. All spectroscopic and hydrodynamic changes that were observed are compatible with a spatial rearrangement of the subunits of α2M, as implicated by the ‘trap’ hypothesis for the mechanism of inhibition of proteinases. However, a conformational change involving a decrease in the hydrodynamic volume of each subunit cannot be excluded.
Amino acid sequences of three phospholipases A I, III and IV from the venom of the sea snake Laticauda semifasciata
Authors: Nishida S , Kim H S , Tamiya N
Published: August 2015

Amino acid sequences of three phospholipases A, I, III and IV, from the venom of the sea snake Laticauda semifasciata were elucidated. Each protein consisted of a single chain of 118 amino acid residues, including 14 half-cystine residues. They showed high homology among themselves, and with the other snake-venom phospholipases A and with the enzymes from mammalian pancreas. Phospholipases A III and IV were especially similar to each other, with only four differences out of their 118 amino acid residues. Phospholipase A I contained one tryptophan residue at position 64, which was important for enzymic activity, whereas III and IV did not contain tryptophan residues and their corresponding positions were occupied by leucine residues. The substitution by leucine resulted in a decreased, but definite, phospholipase A activity. The substituted enzymes have a more potent neuromuscular blocking activity. Full experimental details and evidence for the amino acid sequences of the proteins have been deposited as Supplementary Publication SUP 50118 (39 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J.(1981)193,5.
Evidence that the uptake of tri-iodo-l-thyronine by human erythrocytes is carrier-mediated but not energy-dependent
Authors: Docter Roelof , Krenning Eric P. , Bos Greetje , Fekkes Durk F. , Hennemann George
Published: August 2015

We investigated 3,3′,5-tri-iodo-l-thyronine transport by human erythrocytes and by ‘ghosts’ prepared from these cells. Uptake of tri-iodothyronine by erythrocytes at 37°C was time-dependent with a maximum reached after 60min. Tracer analysis after incubation for 1min revealed only one saturable binding site, with Km 128±19nm (mean±s.e.m.; n=7) and Vmax. 4.6±0.7pmol of tri-iodothyronine/min per 6×107 cells. After 10min incubation Km 100±16nm (n=10) was found with Vmax. 7.7±1.2pmol of tri-iodothyronine/10min per 6×107 cells. At 0°C the uptake system is still active, with Km 132±26nm and Vmax. 1.8±0.3pmol of tri-iodothyronine/10min per 6×107 cells. The Vmax. with intact cells is 5-fold greater than the Vmax. with membranes derived from the same amount of cells when uptake studies are performed in media with similar free tri-iodothyronine concentrations. This indicates that at least 80% of tri-iodothyronine taken up by the intact erythrocytes enters the cell. This saturable uptake system can be inhibited by X-ray-contrast agents in a dose-dependent fashion. (±)-Propranolol, but not atenolol, has the same effect, indicating that the membrane-stabilizing properties of (±)-propranolol are involved. Furthermore, there is no inhibition by ouabain or vanadate, which indicates that tri-iodothyronine uptake is not dependent on the activity of Na++K+-dependent adenosine triphosphatase. We have prepared erythrocyte ‘ghosts‘, resealed after 2.5min with 0mm-, 2mm- or 4mm-ATP inside. Inclusion of ATP and integrity of the membrane of the erythrocyte ‘ghosts’ were verified on the basis of an ATP-concentration-dependent functioning of the Ca2+ pump. No difference was found in the uptake of tri-iodothyronine by erythrocyte ‘ghosts’ with or without ATP included, indicating that uptake of tri-iodothyronine is not ATP-dependent. The following conclusions are drawn. (1) Tri-iodothyronine enters human erythrocytes. (2) There is only one saturable uptake system present for tri-iodothyronine, which is neither energy (i.e. ATP)-dependent nor influenced by the absence of an Na+ gradient across the plasma membrane. This mode of uptake of tri-iodothyronine by human erythrocytes is in sharp contrast with that of rat hepatocytes, which uptake system is energy-dependent and ouabain-sensitive [Krenning, Docter, Bernard, Visser & Hennemann (1978) FEBS Lett.91, 113–116; Krenning, Docter, Bernard, Visser & Hennemann (1980) FEBS Lett.119, 279–282]. (3) X-ray-contrast agents inhibit tri-iodothyronine uptake by erythrocytes in a similar fashion to that by which they inhibit the uptake of tri-iodothyronine by rat hepatocytes [Krenning, Docter, Bernard, Visser & Hennemann (1982) FEBS Lett.140, 229–233].
Effect of the fatty acid oxidation inhibitor 2-tetradecylglycidic acid on pyruvate dehydrogenase complex activity in starved and alloxan-diabetic rats
Authors: Caterson Ian D. , Fuller Stephen J. , Randle Philip J.
Published: August 2015

Intravenous administration of the fatty acid oxidation inhibitor 2-tetradecylglycidic acid had no effect on the proportion of pyruvate dehydrogenase complex in the active form in heart, diaphragm or gastrocnemius muscles or in liver, kidney or adipose tissue of fed normal rats. The compound reversed the effect of 48h starvation (which decreased the proportion of active complex) in heart muscle, partially reversed the effect of starvation in kidney, but had no effect in the other tissues listed. The compound failed to reverse the effect of alloxan-diabetes (which decreased the proportion of active complex) in any of these tissues. In perfused hearts of fed normal rats, 2-tetradecylglycidate reversed effects of palmitate (which decreased the proportion of active complex), but it had no effect in the absence of palmitate. In perfused hearts of 48h-starved rats the compound increased the proportion of active complex to that found in fed normal rats in the presence or absence of insulin. In perfused hearts of diabetic rats the compound normalized the proportion of active complex in the presence of insulin, but not in its absence. Palmitate reversed the effects of 2-tetradecylglycidate in perfused hearts of starved or diabetic rats. Evidence is given that 2-tetradecylglycidate only reverses effects of starvation and alloxan-diabetes on the proportion of active complex in heart muscle under conditions in which it inhibits fatty acid oxidation. It is concluded that effects of starvation and alloxan-diabetes on the proportion of active complex in heart muscle are dependent on fatty acid oxidation. Insulin had no effect on the proportion of active complex in hearts or diaphragms of fed or starved rats in vitro. In perfused hearts of alloxan-diabetic rats, insulin induced a modest increase in the proportion of active complex in the presence of albumin, but not in its absence.
Effects of colchicine and vinblastine on output of proteins into bile
Authors: Godfrey P P , Lembra L , Coleman R
Published: August 2015

The microtubule poisons colchicine and vinblastine caused a reduction in the biliary output of total protein, compared with controls, in bile-fistula rats. The various protein components of bile showed patterns of change in output differing from one another. Alkaline phosphodiesterase I, a ‘plasma-membrane’ enzyme, showed a decline in output during the first hour after drug administration. Immunoglobulin A output did not decline until after the first hour. In contrast with these reductions, the biliary output of (rat) plasma albumin into bile was increased. At no time was there any evidence (as measured by lactate dehydrogenase release into bile) for any cytolytic damage. These results are discussed in relation to current theories on the output of proteins into bile and the repair of the plasma membrane in the bile canaliculus.
Prekeratin biosynthesis in human scalp epidermis
Authors: Bladon P T , Bowden P E , Cunliffe W J , Wood E J
Published: August 2015

Analysis of human scalp epidermal prekeratin polypeptides by two-dimensional gel electrophoresis revealed that each of the bands observed in one-dimensional electrophoresis consisted of three to five polypeptides of the same molecular weight but differing in isoelectric points. It was possible to divide the polypeptides into two families, with isoelectric points in the ranges pH 6.0-8.0 and pH 5.0-5.5 respectively. Incorporation of radiolabelled amino acids into freshly excised pieces of scalp epidermis showed that some of the polypeptides had relatively greater contents of glycine and serine than others. Radiolabelled methionine and leucine were, in contrast, incorporated more or less uniformly into all the polypeptides. After incubation with 32P-labelled orthophosphate, relatively more intense labelling by 32P was observed in the higher molecular weight bands of each family. The most basic of the isoelectric variants in each case did not take up phosphate, implying that at least some of the variation in charge was due to different degrees of phosphorylation. Polyadenylated RNA isolated from scalp epidermis was translated in an RNA-dependent reticulocyte haemolysate system followed by immunoprecipitation and electrophoresis. The polypeptides isolated by using anti-(human scalp prekeratin) immunoglobulin G had similar electrophoretic mobilities in sodium dodecyl sulphate/polyacrylamide gels to authentic prekeratin polypeptides, but had different isoelectric properties. This suggested that the products of keratin gene expression undergo post-translational modification.
Absence of a direct role of phospholipid methylation in stimulus-secretion coupling and control of adenylate cyclase in guinea-pig and rat parotid gland
Authors: Padel U , Unger C , Söling H D
Published: August 2015

The present study was undertaken to investigate a possible involvement of phospholipid methyltransferases in the coupling of receptor-mediated stimulation to secretion. Phospholipid methyltransferases were assayed in isolated parotid acini in the presence of carbamoylcholine or isoprenaline. Carbamoylcholine reduced the incorporation of methyl groups into phospholipids, whereas isoprenaline showed no effect. Amylase secretion stimulated either by carbamoylcholine or by isoprenaline could not be affected by inhibitors of methyltransferases (3-deaza-adenosine alone or plus homocysteine thiolactone) under conditions where phospholipid methylation was strongly inhibited. The activity of adenylate cyclase in isolated parotid microsomal membranes was not inhibited or stimulated by S-adenosyl-homocysteine or -methionine respectively. These results indicate that phospholipid methylation does not play an essential role in stimulus-secretion coupling in the parotid gland.
Cytochrome c oxidase is preferentially synthesized in the rough endoplasmic reticulum-mitochondrion complex in rat liver
Authors: Parimoo S , Rao N , Padmanaban G
Published: August 2015

The specific activity and content of cytochrome oxidase in the rough endoplasmic reticulum-mitochondrion complex are higher than in the mitochondrial fraction. Radiolabelling studies with the use of hepatocytes and isolated microsomal and rough endoplasmic reticulum-mitochondrion fractions, followed by immunoprecipitation with anti-(cytochrome oxidase) antibody, reveal that the nuclear-coded cytoplasmic subunits of cytochrome oxidase are preferentially synthesized in the latter fraction. The results have a bearing on the mechanism of transport of these subunits into mitochondria.
The control of glucose 1,6-bisphosphatase by Ca2+ and calmodulin
Authors: Wakelam M J O , Emmerich M , Pette D
Published: August 2015

1. The control of glucose 1,6-bisphosphatase activity, the enzyme that degrades glucose 1,6-bisphosphate, a metabolite that regulates hexose phosphate metabolism, has been examined in a rat muscle extract. 2. The enzyme has been found to be activated by physiological Ca2+ concentrations. Ca2+ can be replaced by Sr2+ and Mn2+, but the effects are not so pronounced. 3. The Ca2+ effect is inhibited by EGTA. Trifluoperazine also inhibits enzyme activity. This effect can be reversed by adding exogenous calmodulin.
Influence of dietary fat on the lipid composition of rat brain synaptosomal and microsomal membranes
Authors: Foot M , Cruz T F , Clandinin M T
Published: August 2015

The modulation of rat brain microsomal and synaptosomal membrane lipid by diet fat was examined. Brain synaptosomal and microsomal membrane composition was compared for rats fed on diets containing either soya-bean oil (SBO), SBO plus choline, SBO lecithin, sunflower oil (SFO), chow or low-erucic acid rape-seed oil (LER) for 24 days. Cholesterol and phosphatidylcholine levels in both membranes were altered by diet. Diet fat also affected the microsomal content of sphingomyelin. Change in membrane phosphatidylcholine level was related to the relative balance of omega-6, omega-3 and monounsaturated fatty acids within the diets fed. The highest phosphatidylcholine levels appeared in membranes of animals fed on SBO lecithin and the lowest in those fed on LER. Microsomal membrane cholesterol and sphingomyelin content increased by feeding on SBO lecithin. In both synaptosomal and microsomal membranes a highly significant correlation was observed between membrane phosphatidylcholine and cholesterol content. The fatty acyl composition of phospholipids from both membranes also altered with diet and age. Alteration in fatty acid composition was observed in response to dietary levels of omega-6, omega-3 and monounsaturated fatty acids, but the unsaturation index of each phospholipid remained constant for all diet treatments. These changes in lipid composition suggest that dietary fat may be a significant modulator in vivo of the physicobiochemical properties of brain synaptosomal and microsomal membranes.
Purification and characterization of the inactive MoFe protein (NifB-Kp1) of the nitrogenase from nifB mutants of Klebsiella pneumoniae
Authors: Hawkes T R , Smith B E
Published: August 2015

The inactive MoFe protein of nitrogenase, NifB-Kp1, from two distinct nifB mutants of Klebsiella pneumoniae, Kp5058 (a nifB point mutant) and UNF1718 (a nifB, nifJ double mutant) has been purified and characterized. NifB-Kp1 can be activated by reaction with the iron-molybdenum cofactor, FeMoco, extracted from active MoFe protein. NifB-Kp1 purified from either source had similar properties and was contaminated with an approximately equimolar amount of protein of mol.wt. 21 000. Like active wild-type Kp1, it was an alpha 2 beta 2 tetramer, but it was far less stable than Kp1, deteriorating rapidly at temperatures above 8 degrees C or on mild oxidation. NifB-Kp1 preparations contained 0.4-0.9 Mo and 9.0 +/- 0.9 Fe atoms . mol-1 and, when activated by FeMoco, had a specific activity of approx. 500 units . mg-1. The Mo in our preparations was not associated with the e.p.r. signal normally observed from FeMoco. All preparations exhibited a weak gav. = 1.95 e.p.r. signal which was probably not associated with activatable protein.
Inhibition of reverse transcriptases by seminalplasmin
Authors: Reddy E S P , Das M R , Reddy E P , Bhargava P M
Published: August 2015

Seminalplasmin, an antibacterial protein present in bovine seminal plasma, is shown to be a potent inhibitor of reverse transcriptases (RNA-dependent DNA nucleotidyltransferases). Seminalplasmin inhibits RNA-directed, hybrid-directed, and DNA-directed DNA-polymerizing activities of purified reverse transcriptase from avian myeloblastosis virus and from crude viral lysates of several retroviruses by binding to the enzyme, at least in the case of avian myeloblastosis virus. Seminalplasmin does not inhibit significantly DNA synthesis either by Escherichia coli DNA polymerase I, or a mammalian alpha-DNA polymerase. The presence of seminalplasmin in the seminal fluid could provide protection to the male and/or the female reproductive tract against retroviruses.
Evidence on intramolecular electron transfer in the MoFe protein of nitrogenase from Klebsiella pneumoniae from rapid-freeze electron-paramagnetic-resonance studies of its oxidation by ferricyanide
Authors: Smith B E , Lowe D J , Chen G X , O'Donnell M J , Hawkes T R
Published: August 2015

A transient e.p.r. signal with g-values of 2.05, 1.95 and 1.81 was observed in rapid-freezing experiments when Kpl, the MoFe protein of nitrogenase from Klebsiella pneumoniae, was oxidized by ferricyanide or by some dyes. This e.p.r. signal was assigned to the ‘P’-centres and, since such signals are characteristics of [4Fe-4S]1+ clusters, provides further evidence for the ‘P’-centres being in the [4Fe-4S]0 oxidation level in the dithionite-reduced protein. When 4-10-fold excesses of ferricyanide were used as oxidants, the rate of disappearance of the transient e.p.r. signal was independent of the concentrations of ferricyanide, Kpl or ferrocyanide, i.e. its disappearance was by an intramolecular process. Under some circumstances the g = 3.7 e.p.r. signal from the FeMo-cofactors disappeared at a similar rate. It was concluded that, in these circumstances, the g = 3.7 e.p.r. signal disappears, owing to intramolecular electron transfer to the ‘P’-centres in the [4Fe-4S]2+ (P2+) oxidation level, whereas the gav. = 1.933 transient e.p.r. signal from the P1+ centres disappears, owing to a change in its spin state from S = 1/2 to S = 5/2 the rate of this process being maximal when there are two P1+ centres in the half-molecule. The rate of the intramolecular decay of the e.p.r. signals, 4.1 +/- 0.8 s-1, is the same as the rate of enzyme turnover. It is suggested that both processes may be linked to the same conformational change, which triggers, or is triggered by, intramolecular electron transfer.
Mutant strains of Acinetobacter calcoaceticus possessing additional mandelate dehydrogenases. Identification and preliminary characterization of the enzymes
Authors: Hills C A , Fewson C A
Published: August 2015

Acinetobacter calcoaceticus wild-type strain N.C.I.B. 8250 can grow on only the L(+)-isomer of mandelate but mutant strains have been isolated that can grow on D(-)-mandelate. These mutants contain a novel D(-)-mandelate dehydrogenase in addition to the original L(+)-mandelate dehydrogenase. A second wild-type strain, EBF 65/65, shows the opposite pattern and can grow on D(-)-mandelate but not on L(+)-mandelate; mutants have been isolated that possess an L(+)-mandelate dehydrogenase in addition to the original D(-)-mandelate dehydrogenase and can thus grow on L(+)-mandelate. Both L(+)- and D(-)-mandelate dehydrogenases, whether originally present or evolved, are very similar in many respects: they are membrane-bound and NAD(P)+-independent; their activities have similar dependence on temperature and pH; they are inhibited by oxalate but not by several metal-chelating agents; they are stereospecific in their action and are inhibited by the opposite stereoisomers. D(-)-Mandelate dehydrogenase is much more susceptible than L(+)-mandelate dehydrogenase to inhibition by HgCl2 and p-chloromercuribenzoate and is much more heat-labile.
Excretion of laccase by sycamore (Acer pseudoplatanus L.) cells. Purification and properties of the enzyme
Authors: Bligny R , Douce R
Published: August 2015

A laccase-type polyphenol oxidase is excreted by sycamore cells (Acer pseudoplatanus L.) cells. The enzyme has been purified by classical purification techniques. It is a blue copper protein of Mr 97 000, containing 45% carbohydrate and 0.24% copper. This protein consists of one single unit and the copper content corresponds to four copper atoms per protein molecule. The specific activity of the purified extracellular sycamore-cell laccase measured at pH 6.6 (optimum pH) and in the presence of 20mM-4-methhylcatechol (optimum substrate conditions) corresponded to an oxygen uptake of 32 000 nmol of O2/min per mg of protein. Under these conditions, the catalytic-centre activity of the enzyme reached 100 s-1. The excretion of laccase by sycamore cells is significant, being about 2% of the total protein synthesized by the cells during the exponential phase of growth, and is independent of cell growth. The physiological significance and the problems raised by the passage of this protein across the cytoplasmic membrane are discussed.
High-performance liquid chromatography of coproporphyrin isomers
Authors: Wright D J , Rideout J M , Lim C K
Published: August 2015

A reversed-phase system is described for the simultaneous isocratic separation of coproporphyrin I, II, III and IV isomers. The retention behaviour of coproporphyrin I and III is studied in detail. The method is suitable for both analytical and semi-preparative separation.