bluuebunny/crossref_metadata_2025 huggingface dataset:
GDP binding to brown-adipose-tissue mitochondria of diabetic-obese (db/db) mice. Decreased binding in both the obese and pre-obese states
Authors: Goodbody A E , Trayhurn P
Published: August 2015

GDP binding to brown-adipose-tissue mitochondria from adult diabetic–obese (db/db) mice was significantly less than with lean siblings. Binding was also decreased in the mutant mice before obesity had begun to develop. Decreased GDP binding was found to result from a decrease in the number of binding sites.
Stimulation of glycolysis and accumulation of a stimulator of phosphofructokinase in hepatocytes incubated with vasopressin
Authors: Hue L , Van Schaftingen E , Blackmore P F
Published: August 2015

Vasopressin stimulates glycolysis in hepatocytes prepared from fed rats, or from starved rats when incubated with glucose. It causes the stimulation of phosphofructokinase activity and the accumulation of a stimulator of phosphofructokinase, which is probably fructose 2,6-bisphosphate, the recently discovered stimulatory of phosphofructokinase.
Glycoprotein biosynthesis in animal cells grown in suspension culture. Assembly of lipid-linked saccharides and formation of protein-bound ‘high-mannose’ oligosaccharides
Authors: Bailey D S , Burke J , Sinclair R , Mukherjee B B
Published: August 2015

Glycoprotein biosynthesis was studied with mouse L-cells grown in suspension culture. Glucose-deprived cells incorporated [3H]mannose into ‘high-mannose’ protein-bound oligosaccharides and a few relatively high-molecular-weight lipid-linked oligosaccharides. The latter were retained by DEAE-cellulose and turned over quite slowly during pulse--chase experiments. Increased heterogeneity in size of lipid-linked oligosaccharides developed during prolonged glucose deprivation. Sequential elongation of lipid-linked oligosaccharides was also observed, and conditions that prevented the assembly of the higher lipid-linked oligosaccharides also prevented the formation of the larger protein-bound ‘high-mannose’ oligosaccharides. In parallel experiments, [3H]mannose was incorporated into a total polyribosome fraction, suggesting that mannose residues were transferred co-translationally to nascent protein. Membrane preparations from these cells catalysed the assembly from UDP-N-acetyl-D-[6-3H]glucosamine and GDP-D-[U-14C]mannose of polyisoprenyl diphosphate derivatives whose oligosaccharide moieties were heterogeneous in size. Elongation of the N-acetyl-D-[6-3H]glucosamine-initiated glycolipids with mannose residues produced several higher lipid-linked oligosaccharides similar to those seen during glucose deprivation in vivo. Glucosylation of these mannose-containing oligosaccharides from UDP-D-[6-3H]glucose was restricted to those of a relatively high molecular weight. Protein-bound saccharides formed in vitro were mainly smaller in size than those assembled on the lipid acceptors. These results support the involvement of lipid-linked saccharides in the synthesis of asparagine-linked glycoproteins, but show both in vivo and in vitro that protein-bound ‘high-mannose’ oligosaccharide formation can occur independently of higher lipid-linked oligosaccharide synthesis.
Ionic control of immobilized enzymes. Kinetics of acid phosphatase bound to plant cell walls
Authors: Ricard J , Noat G , Crasnier M , Job D
Published: August 2015

When an enzyme is bound to an insoluble polyelectrolyte it may acquire novel kinetic properties generated by Donnan effects. It the enzyme is homogeneously distributed within the matrix, a variation of the electrostatic partition coefficient, when substrate concentration is varied, mimics either positive or negative co-operativity. This type of non-hyperbolic behaviour may be distinguished from true co-operativity by an analysis of the Hill plots. If the enzyme is heterogeneously distributed within the polyelectrolyte matrix, an apparent negative co-operativity occurs, even if the electrostatic partition coefficient does not vary when substrate concentration is varied in the bulk phase. If the partition coefficient varies, mixed positive and negative co-operativities may occur. All these effects must be suppressed by raising the ionic strength in the bulk phase. Attraction of cations by fixed negative charges of the polyanionic matrix may be associated with a significant decrease of the local pH. The magnitude of this effect is controlled by the pK of the fixed charges groups of the Donnan phase. The local pH cannot be much lower than the value of this pK. This effect may be considered as a regulatory device of the local pH. Acid phosphatase of sycamore (Acer pseudoplatanus) cell walls is a monomeric enzyme that displays classical Michaelis-Menten kinetics in free solution. However, when bound to small cell-wall fragments or to intact cells, it has an apparent negative co-operativity at low ionic strength. Moreover a slight increase of ionic strength apparently activates the bound enzymes and tends to suppress the apparent co-operativity. At I0.1, or higher, the bound enzyme has a kinetic behavior indistinguishable from that of the purified enzyme in free solution. These results are interpreted in the light of the Donnan theory. Owing to the repulsion of the substrate by the negative charges of cell-wall polygalacturonates, the local substrate concentration in the vicinity of the bound enzyme is smaller than the corresponding concentration in bulk solution. The kinetic results obtained are consistent with the view that there exist at least three populations of bound enzyme with different ionic environments: a first population with enzyme molecules not submitted to electrostatic effects, and two other populations with molecules differently submitted to these effects. The theory allows one to estimate the proportions of enzyme belonging to these populations, as well as the local pH values and the partition coefficients within the cell walls.
State of methaemoglobin in normal adults and in patients with hereditary methaemoglobinaemia
Authors: Perutz M F
Published: August 2015

The predominance of valency hybrids over fully oxidized methaemoglobin in the haemolysate from a methaemoglobinaemia patient can be explained by random oxidation of the haems, followed by re-assortment and co-migration of equally charged alpha beta-dimers in the electric field of the isoelectric-focusing gel.
Demonstration of five major glycoproteins in myelin and myelin subfractions
Authors: Mena E E , Moore B W , Hagen S , Agrawal H C
Published: August 2015

Myelin was found to contain five major glycoproteins with molecular weights of 120000, 95000, 88000, 43000 and 38000. Light myelin contained only 5-7% of the amount of these glycoproteins in whole myelin, whereas heavy myelin and the membrane fraction contained amounts nearly identical with whole myelin. Since all the major and minor glycoproteins, with the exception of 120000-mol-wt. glycoprotein, were detected only after treating the myelin membrane with neuraminidase, N-acetylneuraminic acid is a terminal sugar residue in these glycoproteins.
Charge screening by cations affects the conformation of the mitochondrial inner membrane. A study of exogenous NAD(P)H oxidation in plant mitochondria
Authors: Møller I M , Palmer J M
Published: August 2015

Cations caused a decrease in the apparent Km and an increase in the Vmax. for the oxidation of exogenous NADH by both Jerusalem-artichoke (Helianthus tuberosus) and Arum maculatum (cuckoo-pint) mitochondria prepared and suspended in a low-cation medium (approximately or equal to 1 mM-K+). In Arum mitochondria the addition of cations caused a much greater stimulation of the oxidation of NAD(P)H via the cytochrome oxidase pathway than via the alternative, antimycin-insensitive, pathway. This shows that cations affected a rate-limiting step in the electron-transport chain at or beyond ubiquinone, the branch-point of electron transport in plant mitochondria. The effects were only dependent on the valency of the cation (efficiency C3+ greater than C2+ greater than C+) and not on its chemical nature, which is consistent with the theory of the diffuse layer. The results are interpreted to show that the screening of fixed negative membrane changes on lipids and protein complexes causes a conformational change in the mitochondrial inner membrane, leading to a change in a rate-limiting step of NAD(P)H oxidation. More specifically, it is proposed that screening removes electrostatic restrictions on lateral diffusion and thus accelerates diffusion-limited steps in electron transport.
Interactions of tervalent lanthanide ions with bacterial collagenase (clostridiopeptidase A)
Authors: Evans Christopher H.
Published: August 2015

Tervalent cations of the lanthanide (rare-earth) elements reversibly inhibit bacterial collagenase (clostridiopeptidase A; EC 3.4.24.3). Sm3+, whose ionic radius is closest to that of Ca2+, is the most effective inhibitor, completely suppressing clostridiopeptidase activity at a concentration of 100μm in the presence of 5mm-Ca2+. Er3+ and Lu3+, which both have ionic radii smaller than either Ca2+ or Sm3+, inhibit less efficiently, and La3+, which is slightly larger than Ca2+ or Sm3+, inhibits only weakly. These findings indicate a closely fitting, stereospecific, Ca2+-binding pocket in clostridiopeptidase, which excludes ions that are only slightly larger than Ca2+ [ionic radius 0.099nm (0.99 Ȧ)]. By contrast, trypsin, an enzyme whose activity does not depend on Ca2+, requires lanthanide concentrations 50–100-fold greater for inhibition. Furthermore, the relative efficiency of inhibition of trypsin by lanthanides increases as the lanthanide ions become smaller and the charge/volume ratio increases. At a concentration of 50μm, Sm3+ lowers the apparent Km for the hydrolysis of Pz-peptide by clostridiopeptidase from 5.4mm to 0.37mm and the apparent Vmax. from 0.29 Wünsch–Heidrich unit to 0.018 unit. Thus Sm3+ enhances the affinity of this enzyme for its substrate; inhibition of hydrolysis of Pz-peptide may result from the excessive stability of the enzyme–Sm3+–substrate complex. Inhibition by Sm3+ is competitive with regard to Ca2+. The apparent dissociation constant, Kd, of Ca2+ is 0.27mm, where the Ki for Sm3+ is 12μm. Clostridiopeptidase is more thermolabile in the absence of Ca2+. With Sm3+, thermoinactivation of the enzyme at 53°C or 60°C is initially accelerated, but then becomes retarded as heating continues. Lanthanide ions bind to gelatin and collagen. In so doing, they appear to protect these substrates from lysis by clostridiopeptidase through mechanisms additional to supplanting Ca2+ at its binding site on the enzyme. Collagen and gelatin sequester sufficient lanthanide ions to gain partial protection from clostridiopeptidase in the absence of an extraneous source of these inhibitors.
Effect of prevention of procollagen triple-helix formation on proline 3-hydroxylation in freshly isolated chick-embryo tendon cells
Authors: Majamaa K
Published: August 2015

Inhibition of procollagen triple-helix formation by the addition of cis-hydroxyproline or azetidine-2-carboxylic acid increased the synthesis of 3-hydroxy[14C]proline 1.7-1.8-fold in pulse-chase experiments with freshly isolated chick-embryo tendon cells. The amount of 3-hydroxy[14C]proline, expressed as a percentage of the total 14C radioactivity in hydroxyproline, reached 8.4%. Control experiments indicated that the two analogues had no effect on the prolyl 3-hydroxylase activity of these cells. The data suggest that the time available before triple-helix formation in part regulates the extent of the 3-hydroxylation of proline in the biosynthesis of collagen in intact cells.
Influence of steroid-hormone-receptor-protein complexes on initiation of ribonucleic acid synthesis in liver nuclei isolated from rats of various ages
Authors: Miller J K , Bolla R
Published: August 2015

The effect of age on the induction of the initiation of RNA synthesis was investigated in liver nuclei isolated from adrenalectomized rats of various ages after binding of a dexamethasone-receptor-protein complex. Binding of this complex to nuclear chromatin resulted in increased initiation of nuclear RNA synthesis at all ages; however, an age-associated decline in the extent of this induction was observed. This suggests an age-related decrease of total rat liver nuclear RNA synthesis with a decreased response to glucocorticoid hormones.
MgATP-induced inhibition of the adenosine triphosphatase activity of the chloroform-released mitochondrial adenosine triphosphatase
Authors: Lowe P N , Beechey R B
Published: August 2015

1. Preincubation of the ox heart chloroform-released mitochondrial ATPase with MgATP results in a time-dependent inhibition of ATPase activity. No re-activation occurs when MgATP remains in the preincubation medium. The enzyme activity returns when all the MgATP in the preincubation system has been hydrolysed. 2. The mechanism of the MgATP-induced inhibition was examined. Inhibition occurs on incubation with MgATP or other hydrolysable nucleotides. Incubation with MgADP or Pi does not cause any inhibition. Neither freshly bound adenine nucleotide nor Pi is associated with inhibited enzyme. The rate of MgATP-induced inhibition correlates with the rate of ATP hydrolysis in the preincubation medium. Changing the rate of ATP hydrolysis at a fixed concentration of ATP also changes the rate of MgATP-induced inhibition by the same proportion. The inhibition is thus related to the ATP-hydrolysis process itself. 3. We propose that intermediate enzyme species of the ATP-hydrolytic sequence can undergo a conformational change to form inhibited species. The kinetics of the inhibition suggest that a substrate-activation step is involved in ATP hydrolysis and MgATP-induced inhibition. 4. The effects of the nature of the preincubation medium on the process of MgATP-induced inhibition and its reversal were examined.
MgATP-induced inhibition of the adenosine triphosphatase activity of submitochondrial particles
Authors: Lowe P N , Beechey R B
Published: August 2015

1. The ATP-hydrolytic activity of ox heart submitochondrial particles can be increased from 2-3 mumol/min per mg of protein to 10-12 mumol/min per mg of protein by incubation in media containing 50 mM-Na2B4O7. This process appears to be due to the partial release of inhibitor protein from the particles. 2. The ATPase activity of submitochondrial particles can be inhibited by incubation with the substrate, MgATP. This inhibition is not due to the accumulation of the hydrolysis products, MgADP and Pi, but could involve the process of ATP hydrolysis. 3. The mechanism of MgATP-induced inhibition of ATPase activity is proposed to involve a conformational change in one of the intermediate enzyme species of the ATP-hydrolytic sequence. 4. MgATP inhibits the ATPase activity of control submitochondrial particles at a higher rate and to a greater extent than it does that of inhibitor-protein-depleted submitochondrial particles, suggesting that the conformational change involves the endogenous inhibitor protein.
Cholesterol in sarcoplasmic reticulum and the physiological significance of membrane fluidity
Authors: Johannsson A , Keightley C A , Smith G A , Metcalfe J C
Published: August 2015

Vesicles of sarcoplasmic reticulum from rabbit muscle can be loaded with cholesterol to at least 20 mol% with respect to endogenous sarcoplasmic-reticulum phospholipid without effect on the ATPase activity at 32 degrees C. This applies both to sarcoplasmic-reticulum vesicles in which the ATPase activity is stably coupled to Ca2+ accumulation, and to sarcoplasmic-reticulum vesicles in which the sarcoplasmic-reticulum ATPase is activated severalfold by fully uncoupling the enzyme from net Ca2+ accumulation. Since the incorporation of cholesterol causes a large decrease in fluidity of sarcoplasmic-reticulum phospholipid bilayer, these results for sarcoplasmic reticulum raise the more general question of whether bilayer fluidity is important in modulating the function of membrane proteins under physiological conditions as is widely assumed, or whether the function of membrane proteins may be effectively buffered under normal operating conditions against changes in bilayer fluidity due to extraneous agents.
Heterogeneity of guinea-pig caseins synthesized and sequestered by cell-free protein-synthesizing systems
Authors: Pascall J C , Boulton A P , Parker D , Hall L , Craig R K
Published: August 2015

1. Individual mRNA species encoding guinea-pigs caseins A, B and C, and alpha-lactalbumin, were purified by hydridization to recombinant milk-protein plasmid DNA immobilized on diazobenzyloxymethyl-paper or diazobenzyloxymethyl-cellulose. Addition of the purified mRNA species to a reticulocyte-lysate cell-free system, in the presence or absence of a dog pancreas microsomal membrane fraction, established a precursor-product relationship between the primary translation products and those sequestered within microsomal vesicles, as determined by polyacrylamide-gel analysis in one and two dimensions. 2. Three sequestered variants of sequestered casein A were identified, but only single forms of sequestered casein B and alpha-lactalbumin. Sequestered variants of casein C proved to be unexpectedly basic, and did not focus on the pH gradient utilized. 3. Comparative analysis of milk proteins synthesized in the reticulocyte-lysate and wheat-germ cell-free systems by two-dimensional gel electrophoresis demonstrated both quantitative and qualitative differences. In particular, marked but variable heterogeneity was apparent within the primary translation products of casein A and casein B. Pre-casein C did not focus. Limited N-terminal processing of the primary translation products was also evident. These observations are discussed in relation to (i) unscheduled post-translational modifications by cell-free protein-synthesizing systems and (ii) multiplicity of signal sequences. 4. Overall we demonstrate that complex precursor-product relationships between primary translation products and their sequestered variants, programmed in vitro by a mixed mRNA population, may be readily analysed by using individual mRNA sequences purified by hybridization to immobilized cloned complementary-DNA sequences.
Effect of lutropin and cycloheximide on lutropin receptors and cyclic AMP production in Leydig tumour cells in vitro
Authors: Dix C J , Cooke B A
Published: August 2015

A system to study lutropin-induced desensitization of tumour Leydig cells in vitro has been investigated. Tumour Leydig cells were purified on a Percoll gradient and then incubated for 30 min with lutropin (0-1000ng/ml). The cells were then washed and incubated in suspension media at 32 degrees C. 125I-labelled human choriogonadotropin binding and basal and lutropin-stimulated cyclic AMP production were determined at various times. Initially the cells showed a dose-dependent decrease in human choriogonadotropin binding (1.18 and 0.13fmol/10(6) cells respectively) followed by an increase at 1 h (2.32 and 0.87fmol/10(6) cells respectively). Human choriogonadotropin binding remained elevated in the cells pre-incubated without lutropin, whereas the cells pre-incubated with lutropin showed a dose-dependent decrease over the next 10 h (2.20-0.18fmol/10(6) cells respectively). Basal production of cyclic AMP initially reflected the pre-incubation conditions (1.17-21.19ng/10(6) cells per h for 0-1000ng of lutropin/ml respectively). However, by 1 h there was a marked rise in basal cyclic AMP production which returned to the initial lower values by 4 h. At all time intervals studied, lutropin-induced cyclic AMP production showed a decrease that was proportional to lutropin concentration in the pre-incubated media. The decreases in human choriogonadotropin binding produced by pre-incubations with lutropin (100ng/ml) was partially inhibited by the presence of cycloheximide in the pre-incubation media and totally prevented by the continuous presence of cycloheximide. These results demonstrate that desensitization of tumour Leydig cells occurs after exposure to lutropin in vitro. This desensitization involves both a loss of plasma membrane receptors for lutropin and lutropin-stimulated adenylate cyclase. These events can be prevented by cycloheximide and are therefore probably dependent on protein synthesis.
Role of calcium in the modulation of ornithine decarboxylase activity in isolated pig granulosa cells in vitro
Authors: Veldhuis Johannes D. , Hammond James M.
Published: August 2015

We examined the role of Ca2+ in the control of basal and hormone-stimulated ornithine decarboxylase activity in isolated pig granulosa cells maintained under chemically defined conditions in vitro. Omission of Ca2+ from the incubation medium (measured Ca2+ concentration 5μm) decreased basal enzymic activity, and significantly (P<0.01) impaired the response to maximally stimulating doses of either lutropin or follitropin. No significant alteration occurred in the concentration of either gonadotropin required to elicit half-maximal effects. The addition of EGTA (1.27–2.0mm) to chelate residual extracellular Ca2+ further decreased hormone-induced rises in ornithine decarboxylase activity. Despite the presence of 1.27mm concentrations of extracellular Ca2+, the administration of presumptive Ca2+ antagonists, believed to impair trans-membrane Ca2+ influx [verapamil (10–100μm), nifedipine (1–100μm) or CoCl2 (1mm)] suppressed hormone-stimulated ornithine decarboxylase activity. The inhibitory effects of verapamil or of Ca2+ omission from the medium were not overcome by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.25mm), or by cholera toxin, or by an exogenously supplied cyclic AMP analogue, 8-bromo cyclic AMP. Conversely, micromolar concentrations of a putative bivalent-cation ionophore, A23187, increased significantly the stimulation of ornithine decarboxylase activity by saturating concentrations of lutropin or 8-bromo cyclic AMP. Thus the present observations implicate Ca2+ ions in the modulation of hormone action and cellular function in normal ovarian cells.
Induction by phenobarbital of the mRNA for a specific variant of rat liver microsomal cytochrome P-450
Authors: Phillips I R , Shephard E A , Mitani F , Rabin B R
Published: August 2015

The treatment of rats for 4 days with phenobarbital causes an apparent 3-fold increase in the amount of total liver cytochrome P-450. By sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, metyrapone binding and immunoprecipitation, this increase was found to be due to a much larger increase in a restricted number of specific cytochrome P-450 variants. A radioimmunoassay technique demonstrated that the major phenobarbital-inducible variant, of molecular weight 52 000, is induced 24-fold by phenobarbital. Immunoprecipitation analysis of products of translation in vitro with an antibody specific to the 52 000-mol.wt. cytochrome P-450 showed that phenobarbital induces the mRNA in polyribosomes for this variant 20-fold. Evidence is presented for the action of phenobarbital at the transcriptional and translational levels.
Participation of a transmembrane proton gradient in 5-hydroxytryptamine transport by platelet dense granules and dense-granule ghosts
Authors: Wilkins J A , Salganicoff L
Published: August 2015

Dense granules, the storage organelles for 5-hydroxytryptamine in blood platelets, have been isolated from porcine platelets and are shown to transport 5-hydroxytryptamine in response to a transmembrane proton gradient (delta pH). Transport in the absence of delta pH is minimal, and it is shown that a rapid increase in transport takes place as delta pH increases. Direct measurements with [14C]methylamine show a delta pH of 1.1 units (acid inside) for intact granules. Osmotically active ghosts of dense granules from which 95% of the endogenous 5-hydroxytryptamine content has been released have also been prepared. Ghosts swell in the presence of ATP and Mg2+, and this swelling is shown to be due to the entry of protons via a process linked to ATP hydrolysis. Proton entry is also apparently linked to anion penetration in ghosts. Steady-state 5-hydroxytryptamine transport in ghosts is stimulated approx. 3-fold on the addition of ATP to the incubation medium, and the stimulation of 5-hydroxytryptamine transport in ghosts correlates with the formation of a transmembrane delta pH. Ghosts generate a delta pH of 1.1-1.3 pH units (acid inside) in the presence of 5 mM-ATP/2.5 mM-MgSO4. delta pH is generated within 3 min at 37 degrees C and is dissipated by the ionophore nigericin and by NH4Cl. It is shown that an Mg2+-stimulated ATPase activity is present on the ghost membrane, and inhibition of the ATPase leads to a corresponding decrease in 5-hydroxytryptamine transport. The results presented support the idea that 5-hydroxytryptamine transport into platelet dense granules is dependent on the presence of a transmembrane delta pH and, together with previous findings by others, suggest a generalized mechanism for biogenic amine transport into subcellular storage organelles.
The protonmotive potential difference across the vacuo-lysosomal membrane of Hevea brasiliensis (rubber tree) and its modification by a membrane-bound adenosine triphosphatase
Authors: Marin B , Marin-Lanza M , Komor E
Published: August 2015

The vacuo-lysosomes of Hevea brasiliensis (rubber tree) constitute a suitable model system for the study of active transport and energization at the level of the membrane of plant vacuoles. The pH gradient (delta pH) and the membrane potential (delta psi) of vacuo-lysosomes were determined by means of the weak base methylamine and the lipophilic cation tetraphenylphosphonium. The values obtained depended strongly on the experimental conditions such as medium pH or K+ concentration. Under experimental conditions, i.e., pH 7.5 outside and low K+, the delta pH amounts to about 0.9 unit, interior acid, and the delta psi to -120 mV, interior negative. The delta psi is presumably caused by the imposed K+ gradient, and the internal acidification might be a consequence of the passive proton inflow along the electric field. This explanation is sustained by the ineffectiveness of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in destroying the delta pH and delta psi, whereas higher K+ concentration decreased both. Under conditions existing in vivo, the membrane potential might be significantly lower. The presence of ATP increased the acidification of the intravesicular space by 0.5pH unit to a delta pH of up to 1.4 and shifts the membrane potential at least 60mV to a more positive value. The change of the protonmotive potential did not occur with ADP; the pH-dependence of the change was identical with the pH-dependence of a vacuo-lysosomal membrane-bound ATPase, and the effect of ATPase was prevented by the presence of the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The change of protonmotive potential difference, brought about by the ATPase, was at least 90 mV. This is evidence that a vacuo-lysosomal ATPase in plants can function as an electrogenic proton pump that transfers protons into the vacuo-lysosomal space.
Foetal glutamate as a possible precursor of placental glutamine in the guinea pig
Authors: Bloxam D L , Tyler C F , Young M
Published: August 2015

The role of foetal glutamate as a source of placental glutamine was investigated in the near-term pregnant guinea-pig placenta perfused in situ through the umbilical vessels. With normal foetal amino acid concentrations there was a significant two-way exchange of glutamate between the placenta and foetal perfusate, but a net release of the amino acid from the placenta. Radioactively labelled glutamate carbon entering the placenta by this exchange was freely incorporated into intracellular glutamine, but only 1.5% of it was found in glutamine transported out into the foetal circulation. In the guinea pig, therefore, foetal glutamate does not appear to be a precursor of glutamine released from the placenta on the foetal side.
Early dolichyl sugar synthesis during differentiation of non-growing brine-shrimp (Artemia salina) embryos
Authors: Quesada-Allue L A
Published: August 2015

Incubation of cell-free extracts of Artemia salina with sugar nucleotides resulted in the formation of dolichyl derivatives. These enzymic activities were detected early in the development of the encysted Artemia embryos. Dolichyl sugars seemed to act as intermediates in the glycosylation of proteins, which may be involved in the initiation of cellular differentiation.
Binding of fluid-phase complement components C3 and C3b to human lymphocytes
Authors: Sim E , Sim R B
Published: August 2015

It is known that a population of B-lymphocytes has receptors for the third component of complement, C3, and that these lymphocytes may be identified by their ability to form rosettes with sheep erythrocytes coated with covalently bound fragments of complement component C3. Human tonsil lymphocytes, enriched for B-cells, form rosettes with sheep erythrocytes coated with antibody and complement components C1, C4b and C3b (EAC143b cells). Fluid-phase C3 will inhibit rosette formation between EAC143b and human tonsil lymphocytes over the same concentration range as fluid-phase C3b. C3 is not cleaved to C3b during incubation with lymphocytes or with lymphocytes and EAC143b cells. Fluid-phase 125I-labelled C3 and 125I-labelled C3b bind to lymphocytes in a specific manner. The characteristics of binding of both radioiodinated C3 and radioiodinated C3b are very similar, but the binding oc C3 is again not a result of cleavage to C3b. Salicylhydroxamic acid does not inhibit binding of 125I-labelled C3 to tonsil lymphocytes at concentrations that completely inhibit binding of 125I-labelled C3 to EAC142 cells via the nascent binding site of C3b. It is concluded that C3 and C3b share a common feature involved in binding to lymphocytes bearing receptors for the third component of complement.
Rates of pinocytic capture of simple proteins by rat yolk sacs incubated in vitro
Authors: Livesey G , Williams K E
Published: August 2015

The rates of pinocytic uptake of a number of small 125I-labelled simple proteins (insulin, ribonuclease A and lysozyme) by rat yolk sacs incubated in vitro were determined both before and after treating these proteins with reagents that are known to increase the rate of capture of 125I-labelled bovine serum albumin. Uptake of the untreated forms of all three proteins was extremely rapid, indicating that adsorptive pinocytosis is the principal mechanism by which yolk-sac cells capture these simple proteins, but these rates show no simple correlation with molecular charge. In contrast with albumin, the rates of uptake of treated proteins were either unchanged or lower than that of the corresponding untreated protein preparations; polymeric forms of 125I-labelled lysozyme larger than dimers were ingested at rates significantly lower than that of the monomer.
Modification of human prekeratin during epidermal differentiation
Authors: Bowden P E , Cunliffe W J
Published: August 2015

The polypeptide-chain components of human epidermal prekeratin and keratin were analysed by high-resolution SDS (sodium dodecyl sulphate)/polyacrylamide-gradient-gel electrophoresis. Size heterogeneity existed amongst prekeratin components and at least ten polypeptides, in the molecular-weight range 46,000-70,000, were observed in 0.1 M-citric acid/sodium citrate buffer (pH 2.65) extracts of scale epidermis. Prekeratin from scalp pilosebaceous ducts was identical with that from the contiguous epidermis, and no prekeratin was found in extracts of scale dermis. Prekeratin from plantar epidermis contained additional polypeptide chains, but only slight anatomical variation existed between the non-callus sites examined. Keratin differed from prekeratin in at least two major respects: (a) many major components did not co-electrophorese on high-resolution SDS/polyacrylamide slab gels, and (b) keratin, but not prekeratin, required denaturing and reducing conditions for extraction. Keratin extracted from scale epidermis after complete removal of prekeratin was identical with forearm stratum-corneum keratin. Palmar and plantar keratin contained additional polypeptide chains and had a different size distribution compared with forearm and scalp keratin components. Modification of prekeratin components to produce the keratin polypeptide profile occurred during epidermal differentiation, and these changes appeared to take place in the granular-layer region of the epidermis.
Microbial oxidation of amines. Distribution, purification and properties of two primary-amine oxidases from the yeast Candida boidinii grown on amines as sole nitrogen source
Authors: Haywood Geoffrey W. , Large Peter J.
Published: August 2015

1. The yeast Candida boidinii was grown on glucose as carbon source with a range of amines and amino acids as nitrogen sources. Cells grown on amines contained elevated activities of catalase. If the amines contained N-methyl groups, formaldehyde dehydrogenase, formate dehydrogenase and S-formylglutathione hydrolase were also elevated in activity compared with cells grown on (NH4)2SO4. 2. Cells grown on all the amines tested, but not those grown on urea or amino acids, contained an oxidase attacking primary amines, which is referred to as methylamine oxidase. In addition, cells grown on some amines contained a second amine oxidase, which is referred to as benzylamine oxidase. 3. Both amine oxidases were purified to near homogeneity. 4. Benzylamine oxidase was considerably more stable at 45 and 50°C than was methylamine oxidase. 5. Both enzymes had a pH optimum in the region of 7.0, and had a considerable number of substrates in common. There were, however, significant differences in the substrate specificity of the two enzymes. The ratio V/Kapp.m increased with increasing n-alkyl carbon chain length for benzylamine oxidase, but decreased for methylamine oxidase. 6. Both enzymes showed similar sensitivity to carbonyl-group reagents, copper-chelating agents and other typical ‘diamine oxidase inhibitors’. 7. The stoicheiometry for the reaction catalysed by each enzyme was established. 8. The kinetics of methylamine oxidase were examined by varying the methylamine and oxygen concentrations in turn. A non-Ping Pong kinetic pattern with intersecting double-reciprocal plots was obtained, giving Km values of 10mum for O2 and 198mum for methylamine. The significance of this unusual kinetic behaviour is discussed. Similar experiments were not possible with the benzylamine oxidase, because it seemed to have an even lower Km for O2. 9. Both enzymes had similar subunit Mr values of about 80000, but the benzylamine oxidase behaved as if it were usually a dimer, Mr 136000, which under certain conditions aggregated to a tetramer, Mr 288000. Methylamine oxidase was mainly in the form of an octamer, Mr 510000, which gave rise quite readily to dimers of Mr 150000, and on gel filtration behaved as if the Mr was 286000.
The β-glucosidase from Botryodiplodia theobromae. Mechanism of enzyme action
Authors: Umezurike G M
Published: August 2015

1. In the presence of a high concentration of p-nitrophenyl beta-D-glucopyranoside (donor) the rates of production of p-nitrophenol and a transglucosylation product (1-glyceryl beta-D-glucopyranoside) increased, whereas the rate of production of glucose decreased with increasing concentration of glycerol in reactions catalysed by the high-molecular-weight beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) obtained from culture filtrates of Botryodiplodia theobromae Pat. 2. When [donor] greater than Km the rate of production of p-nitrophenol was higher in the presence of glycerol than in its absence, whereas when [donor] less than Km the rate of production of p-nitrophenol was lower in the presence of glycerol than in its absence. 3. Glycerol increased both the Michaelis constant (Km) and maximum velocity (Vmax.), whereas dioxan increased Km but decreased Vmax. 4. Up to 1 mM-AgNO3 had no effect on enzyme activity. 5. A 2H-solvent-isotope-effect [Vmax. (H2O)/V max. (2H2O)] value of 1.40 +/- 0.05 was found at pH (or p2H) 5.8 6. alpha-2H-kinetic isotope-effect (kappa H/kappa 2H) values of 1.03 +/- 0.01 and 1.05 +/- 0.01 were found in the absence and presence of glycerol respectively. 7. Although maltose was a non-competitive inhibitor of beta-glucosidase activity, the ratio of velocity in the presence of glycerol to that in its absence increased, after an initial decline, with increasing concentration of maltose. 8. These results are discussed in terms of a mechanism involving a solvent-separated glucosyl cation-carboxylate ion-pair, which has greater affinity for alcoholic glucosyl acceptors, and an intimate ion-pair, which has greater affinity for water as a glucosyl acceptor and which could collapse reversibly and rapidly into a preponderance of an unreactive covalent glucosyl-enzyme.
The stereochemical course of yeast hexokinase-catalysed phosphoryl transfer by using adenosine 5′[γ(S)-16O,17O,18O]triphosphate as substrate
Authors: Lowe G , Potter B V L
Published: August 2015

Adenosine 5′[gamma(S)-16O, 17O, 18O]triphosphate has been synthesized and used to determine the stereochemical course of phosphoryl transfer catalysed by yeast hexokinase. The chirality at phosphorus of the D-glucose 6-[16O,17O,18O]phosphate formed was analysed, after cyclization and methylation, by 31P n.m.r. spectroscopy. The phosphoryl transfer was found to occur with inversion of configuration, with a stereoselectivity in excess of 94%. The simplest interpretation of this result is that the phosphoryl group is transferred between substrates in the enzyme-substrate ternary complex by an ‘in line’ mechanism.
Purification and characterization of an endoglucanase (1,4-β-d-glucan glucanohydrolase) from Clostridium thermocellum
Authors: Ng T K , Zeikus J G
Published: August 2015

An endoglucanase (1,4-beta-D-glucan glucanohydrolase, EC 3.2.1.4) was purified from Clostridium thermocellum by procedures that included centrifugation, ultrafiltration, selective precipitation, ion-exchange Sephadex chromatography and preparative gel electrophoresis. The 22-fold-purified enzyme behaved as a homogeneous protein under non-denaturing conditions. The enzyme represented a significant component (greater than 25%) of total extracellular endoglucanase activity, but was purified in low yield by the procedures employed. The native molecular weight of the endoglucanase was determined by ultracentrifugational analysis, amino acid composition and polyacrylamide-gel electrophoresis, and varied between 83000 and 94000. The enzyme contained 11.2% carbohydrate and was isoelectric at pH 6.72. The pH and temperature optima of the endoglucanase were 5.2 and 62 degrees C respectively. The enzyme lacked cysteine and was low in sulphur-containing amino acids. The purified endoglucanase displayed: high activity towards carboxymethylcellulose, celloheptaose, cellohexaose and cellopentaose; low activity towards Avicel microcrystalline cellulose and cellotetraose; no detectable activity towards cellotriose or cellobiose; increased activity towards cello-oligosaccharides with increasing degree of polymerization. The internal glycosidic bonds of cello-oligosaccharides were cleaved by the enzyme in preference to external linkages. The apparent Michaelis constant ([S]0.5V) and Vmax. for cellopentaose and cellohexaose hydrolysis were 2.30 mM and 39.3 mumol/min per mg of protein, and 0.56 mM and 58.7 mumol/min per mg of protein, respectively.
Human complement component C4. Structural studies on the fragments derived from C4b by cleavage with C3b inactivator
Authors: Press E M , Gagnon J
Published: August 2015

1. One of the activation products of C4, C4b, was prepared, and the reactive thiol group on the alpha′-chain was radioactively labelled with iodo[2-14C]acetic acid. The alpha′-chain was isolated and the N-terminal amino acid sequence of the first 13 residues was determined. 2. C4b was cleaved by C3bINA in the presence of C4b-binding protein and C4d and C4c isolated. The radioactive label and therefore the reactive thiol group were located to C4d. 3. C4c was reduced and alkylated and the two alpha′-chain fragments of C4c were separated. 3. The molecular weights, amino acid analyses and carbohydrate content of the three alpha′-chain fragments were determined. C4d has a mol.wt. of 44500 and a carbohydrate content of 6%. The two alpha′-chain fragments of C4c have mol.wts. of 25000 (alpha 3) and 12000 (alpha 4) and carbohydrate contents of 10 and 22% respectively. 4. The N-terminal amino acid sequences of C4d, the alpha 3 and the alpha 4 fragments were determined for 18, 24 and 11 residues respectively and, by comparison with the N-terminal sequence of the C4b alpha′-chain, the 25000-mol.wt. fragment (alpha 3) was shown to be derived from the N-terminal part of the alpha′-chain. 5. C-Terminal analyses were done on the alpha′-chain and its three fragments. Arginine was found to be the C-terminal residue of C4d and of the alpha 3 fragment. The C-terminal residue of the alpha′-chain and of the alpha 4 fragment could not be identified. The order of the three fragments of the alpha′-chain is therefore: alpha 3(25000)--C4d(44500)--alpha 4(12000). The specificity of C3bINA is for an Arg--Xaa peptide bond.
Kinetic studies of the fatty acid synthetase multienzyme complex from Euglena gracilis variety bacillaris
Authors: Walker T A , Jonak Z L , Worsham L M S , Ernst-Fonberg M L
Published: August 2015

A fatty acid synthetase multienzyme complex was purified from Euglena gracilis variety bacillaris. The fatty acid synthetase activity is specifically inhibited by antibodies against Escherichia coli acyl-carrier protein. The Euglena enzyme system requires both NADPH and NADH for maximal activity. An analysis was done of the steady-state kinetics of the reaction catalysed by the fatty acid synthetase multienzyme complex. Initial-velocity studies were done in which the concentrations of the following pairs of substrates were varied: malonyl-CoA and acetyl-CoA, NADPH and acetyl-CoA, malonyl-CoA and NADPH. In all three cases patterns of the Ping Pong type were obtained. Product-inhibition studies were done with NADP+ and CoA. NADP+ is a competitive inhibitor with respect to NADPH, and uncompetitive with respect to malonyl-CoA and acetyl-CoA. CoA is uncompetitive with respect to NADPH and competitive with respect to malonyl-CoA and acetyl-CoA. When the concentrations of acetyl-CoA and malonyl-CoA were varied over a wide range, mutual competitive substrate inhibition was observed. When the fatty acid synthetase was incubated with radiolabelled acetyl-CoA or malonyl-CoA, labelled acyl-enzyme was isolated. The results are consistent with the idea that fatty acid synthesis proceeds by a multisite substituted-enzyme mechanism involving Ping Pong reactions at the following enzyme sites: acetyl transacylase, malonyl transacylase, beta-oxo acyl-enzyme synthetase and fatty acyl transacylase.
The action on cellulose and its derivatives of a purified 1,4-β-glucanase from Trichoderma koningii
Authors: Halliwell G , Vincent R
Published: August 2015

The specific properties have been examined of the 1,4-beta-glucanase component of Trichoderma koningii that participates in an early and effective stage of random breakdown of native cellulose to short fibres. The enzyme was purified and freed from associated components of the cellulase complex (particularly beta-glucosidase) that interfere with, and complicate interpretation of, the action of such enzymes. Purification increased the specific activity 25-fold over culture filtrates; the enzyme hydrolysed CM-cellulose faster than the purified beta-glucosidase from the same organism hydrolysed any of its substrates (cellobiose or cellodextrins). The specificity of the glucanase was directed towards soluble derivatives of cellulose, CM-cellulose and cellodextrins, and not to insoluble cellulose or alpha-linked polymers. The approximate Km was 2.5 mg of CM-cellulose . ml-1 at 37 degrees C at the optimum pH, 5.5, where enzymic activity was maximal with 6--7 mg of CM-cellulose . ml-1 and inhibited by higher concentrations. The temperature optimum was 60 degrees C. The glucanase attacked larger cellodextrins (cellohexaose to cellotetraose, in that order) much more readily than smaller dextrins (cellobiose and cellotriose) and released a mixture of products, glucose up to cellopentaose, which was quantitatively determined after chromatography on charcoal. Similar examination of hydrolysates of the reduced cellodextrins showed clearly the high specificity of the enzyme for the central bond of its natural substrates (the cellodextrins), whatever their chain length, and indicated the nature of the enzyme as an endoglucanase. Outer bonds shared a weaker, but similar, susceptibility to enzymic cleavage. Transferase activity was absent and no larger dextrins than the initial substrate were formed.
The stereochemical course of phosphoryl transfer catalysed by Bacillus stearothermophilus and rabbit skeletal-muscle phosphofructokinase with a chiral [16O,17O,18O]phosphate ester
Authors: Jarvest R L , Lowe G , Potter B V L
Published: August 2015

Bacillus stearothermophilus and rabbit skeletal-muscle phosphofructokinases catalyse the transfer of the chiral [16O,17O,18O]phosphoryl group from D-fructose 1[(S)-16O,17O,18O],6-bisphosphate to ADP with inversion of configuration at the phosphorus atom. D-Fructose 1[(S)-16O,17O,18O],-bisphosphate was synthesized in situ from sn-glycerol 3[(S)-16O,17O,18O]phosphate. The simplest interpretation of these results is that the phosphoryl group is transferred between substrates in the enzyme substrate ternary complexes by an ‘in-line’ mechanism.
Characterization of a rat liver cyclic GMP-activated phosphodiesterase by chromatography on hexyl-agarose. Inhibition of phosphodiesterase activity by hexyl-agarose
Authors: Couchie D , Erneux C , Dumont J E
Published: August 2015

Chromatography on hexyl-agarose resolved a partially purified cyclic GMP-activated phosphodiesterase from rat liver into two peaks of activity: the first was eluted with 0.5 M-KCl and was cyclic AMP-specific. The second was tightly bound to hexyl-agarose and was not eluted with KCl (0--2.0 M), which enhanced the hydrophobic interactions of this form with the matrix. It was eluted with 0.5 M-Tris, hydrolysed cyclic AMP and cyclic GMP and was specifically activated by cyclic GMP. The cyclic GMP-activated phosphodiesterase was immobilized on hexyl-agarose. Enzyme activity, quantitatively bound to hexyl-agarose, was not released from the hydrophobic matrix in the presence of cyclic AMP or cyclic GMP, under our assay conditions. The immobilized form of the enzyme retained catalytic activity, was inhibited by 0.1 mM-cyclic AMP and was activated by micromolar concentrations of cyclic GMP to a lesser extent (7-fold) than the control, i.e. the enzyme mixed with unsubstituted agarose (15-fold). When the enzyme was immobilized, inhibition of cyclic AMP phosphodiesterase activity was only observed in the presence of cyclic GMP (at 3 microM); in its absence, activity remained unchanged. The kinetic behaviour of the immobilized enzyme is consistent with the hypothesis of a binding site distinct from the hydrolytic and activating sites.
The stereochemical course of hydrolysis catalysed by snake venom 5′-nucleotide phosphodiesterase
Authors: Jarvest R L , Lowe G
Published: August 2015

Adenosine 5′-(S)-[16O,17O,18O]phosphate was pyrophosphorylated by the combined action of adenylate kinase and pyruvate kinase. The isotopomers of adenosine 5′-[alpha-16O,17O,18O]triphosphate were hydrolysed by venom 5′-nucleotide phosphodiesterase (Crotalus adamanteus) in H2(17)O. Analysis by 31P nuclear magnetic resonance spectroscopy of the resulting adenosine 5′-[16O,17O,18O]phosphate, after cyclization and esterification, showed that the hydrolysis occurs with retention of configuration at phosphorus. The most likely explanation of this observation is that the enzymic hydrolysis involves a double displacement at phosphorus with a covalent nucleotidyl--enzyme intermediate on the reaction pathway.