bluuebunny/crossref_metadata_2025 huggingface dataset:
Further studies on the structural requirements for polypeptide-mediated histamine release from rat mast cells
Authors: Jasani B , Kreil G , Mackler B F , Stanworth D R
Published: August 2015

Structure-activity studies have been performed on a series of naturally occurring and ‘tailor-made’ polypeptides, by measurement of ability to induce selective histamine release from normal rat peritoneal mast cells in vitro. Compounds investigated include corticotropin and melittin derivatives, mast-cell-degranulating peptide from bee venom, polymyxin B, bradykinin and various synthetic poly(amino acids) and short-chain peptides. It was confirmed that a cluster of four basic residues (lysine or arginine) was optimal for histamine release by corticotropin and melittin polypeptides, provided that the C-terminal carboxyl group was substituted (by, for instance, amidation). In contrast, the presence of a free C-terminal carboxyl group or nearby dicarboxylic acid residues led to a considerable diminution in histamine-releasing activity. Likewise, polypeptides comprised essentially of acidic amino acids were inactive. On the basis of these observations it has been possible to predict that synthetic peptides comprising a particular sequence within the Fc region of human immunoglobulin E, the immunoglobulin class particularly involved in mediation of allergic reactions of the immediate type, would possess potent histamine-releasing activity when similarly made to react with normal rat mast cells. The further study of such a structure should throw new light on the molecular basis of allergen-antibody triggering of mast cells.
Multiple forms of 2-deoxy-d-glucoside-2-sulphamate sulphohydrolase from human placenta
Authors: Paschke E , Kresse H
Published: August 2015

2-Deoxyglucoside-2-sulphamate sulphohydrolase was purified about 10 000-fold from the soluble extract of human placenta by using as substrate [N-sulpho-35S]heparin. Differently charged enzyme forms were observed on chromatography on DEAE-cellulose, all of which had an apparent mol.wt. of 110 000 as determined by gel filtration. By using immobilized heparan sulphate as affinity matrix the sulphamate sulphohydrolase could be separated into two forms, a minor one with low and a major one with high affinity for the adsorbent. When tested with [N-sulpho-35S]heparan sulphate the low-affinity form had a Km of 0.2 mM, and the high-affinity form a Km of 0.03 mM. Both forms exhibited the same Km of 10 microM towards [N-sulpho-35S]heparin and were equally well adsorbed to immobilized heparin. The two forms could be distinguished by their pH-optima and by the influence of KCl on heparan sulphate sulphohydrolase activity.
Metabolism and biological activity of 25-fluorocholecalciferol, 24-dehydrocholecalciferol and 25-dehydrocholecalciferol in the rat
Authors: Onisko B L , Schnoes H K , DeLuca H F , Glover R S
Published: August 2015

Three side-chain analogues of cholecalciferol (vitamin D3) modified at C-25, namely 25-fluorocholecalciferol, 24-dehydrocholecalciferol and 25-dehydrocholecalciferol, conceived as potential inhibitors of the cholecalciferol 25-hydroxylase have been prepared and tested in the rat. These compounds markedly diminish conversion in vivo of cholecalciferol into 25-hydroxycholecalciferol, but are not antagonists of vitamin D action, because they themselves possess significant biological activity in vivo. Each compound is capable of stimulating the intestinal transport of calcium and the mobilization of calcium from bone in vitamin D-deficient rats. Biological responses equivalent to those generated by a physiological dose of cholecalciferol (0.05 microgram) are produced, however, only when the analogues are administered at high doses (5-50 microgram). The biological activity of 24-dehydrocholecalciferol and 25-dehydrocholecalciferol is shown to result from conversion, in vivo, to the natural hormone, 1 alpha,25-dihydroxycholecalciferol, whereas 25-fluorocholecalciferol is metabolically activated in the rat by hydroxylation to 1 alpha-hydroxy-25-fluorocholecalciferol. This latter conversion is the first reported example of the 1 alpha-hydroxylation of a vitamin D compound lacking the 25-hydroxy group.
Lactose synthesis in the rat, and the effects of litter size and malnutrition
Authors: Wilde C J , Kuhn N J
Published: August 2015

1. The rate of lactose synthesis per g of mammary tissue, measured in vivo by a radioisotopic technique, rose 13-fold between parturition and day 16 of lactation in the rat, but was unaffected by wide variation in litter size. 2. The increase reflected a greater tissue content of galactosyltransferase (EC 2.4.1.22), and was augmented by a rise in the total weight of mammary tissue. Superimposed on this were unpredictable changes in the functional efficiency of the enzyme. 3. Lactose synthesis in 14-day-lactating rats, permitted only 76% of the food intake of paired control rats over the previous 3 weeks, showed a pronounced diurnal variation at an overall rate markedly below that in control rats. 4. Such nutritional deficiency did not affect the tissue content of galactosyltransferase, but impaired its functional efficiency in a manner reversed by renewed feeding or by the preparation and incubation of acini in vitro. 5. Plasma insulin concentrations decreased at parturition and with increasing litter size, and remained relatively unchanged during lactation and malnutrition.
Calcium ions and cell fusion. Effects of chemical fusogens on the permeability of erythrocytes to calcium and other ions
Authors: Blow A M J , Botham G M , Lucy J A
Published: August 2015

1. Fusogenic and non-fusogenic chemicals were tesetd for their ability to allow 45Ca2+ and 3H2O to enter hen and human erythrocytes. 2. The ratio of 45Ca2+/3H2O in treated cells to that in untreated cells is referred to as the entry ratio. 3. Within 1 min at 37 degrees C both water-soluble and lipid-soluble fusogens increased the value of the entry ratio, which reached maximum values in 5–10 min. 4. Values of the entry ratio in the range of 4–12 were found under conditions that led to cell fusion. 5. Closely related but non-fusogenic chemicals did not significantly alter the entry ratio. 6. The entry ratios for 86Rb+, 22Na+ and 35SO42- were also significantly increased by both lipid-soluble and water-soluble fusogens, though the increases were not as large as those for 45Ca2+. 7. It is suggested that fusogenic compounds increase the permeability of biological membranes to ions, and that an increase in the concentration of intracellular Ca2+ initiates or facilitates events that lead to the chemically induced fusion of erythrocytes.
Interactions in vivo between oxidation of non-esterified fatty acids and gluconeogenesis in the newborn rat
Authors: Ferré P , Pégorier J P , Williamson D H , Girard J
Published: August 2015

Metabolic interactions between fatty acid oxidation and gluconeogenesis were investigated in vivo in 16h-old newborn rats under various nutritional states. As the newborn rat has no white adipose tissue, starvation from birth induces a low rate of hepatic fatty acid oxidation. Hepatic gluconeogenesis in inhibited in the starved newborn rat when compared with the suckling rat, which receives fatty acids through the milk, at the steps catalysed by pyruvate carboxylase and glyceraldehyde 3-phosphate dehydrogenase. These inhibitions are rapidly reversed by triacylglycerol feeding. Inhibition of fatty acid oxidation by pent-4-enoate in the suckling animal mimics the effect of starvation on the pattern of hepatic gluconeogenic metabolites. It is concluded that, in the newborn rat in vivo, hepatic fatty acids oxidation can increase the gluconeogenic flux by providing the acetyl-CoA necessary for the reaction catalysed by pyruvate carboxylase and the reducing equivalents (NADH) to displace the reversible reaction catalysed by glyceraldehyde 3-phosphate dehydrogenase in the direction of gluconeogenesis.
Quantitative analysis of proton-linked transport systems. The lactose permease of Escherichia coli
Authors: Booth I R , Mitchell W J , Hamilton W A
Published: August 2015

Evidence is presented that lactose uptake into whole cells of Escherichia coli occurs by symport with a single proton over the range of external pH 6.5–7.7. The proton/lactose stoicheiometry has been measured directly over this pH range by comparison of the initial rates of proton and lactose uptake into anaerobic resting cell suspensions of E. coli ML308. Further, the relationship between the protonmotive force and lactose accumulation has been studied in E. coli ML308-225 over the range of external pH 5.9–8.7. At no point was the accumulation of the beta-galactoside in thermodynamic equilibrium with the protonmotive force. It is concluded that the concentration of lactose within the cell is governed by kinetic factors rather than pH-dependent changes in the proton/substrate stoicheiometry. The relevance of these findings to the model of pH-dependent proton/substrate stoicheiometries derived from studies with E. coli membrane vesicles is discussed.
A comparison of methods for extracting ribonucleic acid polymerases from rat liver nuclei
Authors: Beebee T J C
Published: August 2015

Nuclei were prepared from rat liver after homogenization of the tissue in hyperosmotic sucrose and RNA polymerases (EC 2.7.7.6) extracted by two methods applied sequentially. Optimal conditions for washing loosely bound enzymes out of nuclei were determined first, and involved short (10 min) incubations at 0 degrees C in the presence of 5 mM-Mg2+ and 60 mM-(NH4)2SO4. Subsequent sonication of the residual nuclear pellet after resuspension and lysis at high ionic strength resulted in further release of RNA polymerases. The primary wash yielded about 2 × 10(4) molecules of RNA polymerases I and III (altogether) and 1 × 10(4) molecules of form-II enzymes per original nucleus, whereas subsequent sonication released 2 × 10(4)-2.5 × 10(4) form-I and -III enzyme molecules (altogether) and a further 7 × 10(3)-8 × 10(3) form-II enzyme molecules, as measured by end-labelling of nascent RNA. RNA polymerase II was partially purified from both types of extracts and shown to initiate very poorly on high-molecular-weight homologous DNA irrespective of the source of the enzyme.
The nature of the hydroxyapatite-binding site in salivary acidic proline-rich proteins
Authors: Bennick A , Cannon M , Madapallimattam G
Published: August 2015

Protein A and C, which are major components of the acidic proline-rich proteins in human saliva, were digested, before or after adsorption to hydroxyapatite, with alkaline phosphatase, trypsin, thermolysin and a proteinase preparation from salivary sediment. The results demonstrate that the binding site is located in the proline-poor N-terminal part of the protein, possibly between residues 3 and 25. Phosphoserine is necessary for maximal adsorption of the proteins to hydroxyapatite. When proteins A and C are adsorbed to hydroxyapatite before proteolytic digestion there is a protection of some of the susceptible bonds in the N-terminal part of the proteins and a gradual removal of the proline-rich C-terminal part. Thermolysin can cleave susceptible bonds in the part of the protein that remains bound to hydroxyapatite, but at least some of the resulting peptides are retained on the mineral. Since the ability of the proteins to inhibit hydroxyapatite formation and to bind calcium is located in the N-terminal proline-poor part, it is possible that these activities are retained after proteolytic digestion of the adsorbed proteins.
The incorporation of tritiated retinyl moiety into the active-site lysine residue of bacteriorhodopsin
Authors: Mullen E , Gore M G , Akhtar M
Published: August 2015

Purple membranes were isolated from Halobacterium halobium bleached and regenerated with all-trans-[15-3H]retinal. The incorporation of label was 1.2 mol of retinal/mol of bacterio-opsin. The [3H]retinyl-bacterio-opsin obtained from regeneration was hydrolysed to give tritiated retinyl-lysine, which, on hydrogenation to N-epsilon-perhydro[3H]retinyl-lysine and reaction with 1-fluoro-2,4-dinitrobenzene, gave bis-(2,4-dinitrophenyl)-N-epsilon-perhydro[3H]retinyl-lysine. This result confirmed that the retinyl moiety of the chromophore is attached to an epsilon-amino group of lysine.
Reactivities of neutral and cationic forms of 2,2′-dipyridyl disulphide towards thiolate anions. Detection of differences between the active centres of actinidin, papain and ficin by a three-protonic-state reactivity probe
Authors: Brocklehurst K , Stuchbury T , Malthouse J P G
Published: August 2015

The second-order rate constants (k) for the reactions of 2,2′-dipyridyl disulphide (pKa2,45) with 2-mercaptoethanol (pKa9.6) and with benzimidazol-2-ylmethanethiol (pKa values 5.6 and 8.3) were determined at 25 degrees C at I 0.1 by stopped-flow spectral analysis over a wide range of pH. These were used to calculate the pH-independent second-order rate constants (k) for the reactions of neutral 2,2′-dipyridyl disulphide and of its monocation with the 2-mercaptoethanol thiolate anion (associated pKa9.6) and with the benzimidazol-2-ylmethanethiol zwitterion (associated pKa5.6). For both thiolate ions, the rate-enhancement factor (kmonocation/kneutral disulphide) is about 1.5×10(3). The dependence on pH in acidic media of k for the reaction of 2,2′-dipyridyl disulphide with actinidin, the thiol proteinase from Actinidia chinensis, was shown to differ from the forms of pH-dependence observed for the analogous reactions with papain (EC 3.4.22.2) and ficin (3.4.22.3). The reactivity of the 2,2′-dipyridyl disulphide dication and its apparent sensitivity to the presence and location of a positive charge in the attacking thiol are discussed.
Wheat-germ aspartate transcarbamoylase. Steady-state kinetics and stereochemistry of the binding site for l-aspartate
Authors: Grayson J E , Yon R J , Butterworth P J
Published: August 2015

1. The steady-state kinetics of the bisubstrate reaction catalysed by aspartate transcarbamoylase purified from wheat (Triticum vulgare)-germ have been studied at 25 degrees C, pH 8.5 AND I 0.10-0.12. Initial-velocity and product-inhibition results are consistent with an ordered sequential mechanism in which carbamoyl phosphate is the first substrate to bind, followed by L-aspartate, and carbamoyl aspartate is the first product to leave, followed by Pi. The order of substrate addition is supported by dead-end inhibition studies using pyrophosphate and maleate as inhibitory analogues of the substrates. Product inhibition permitted a minimum value for the dissociation constant of L-aspartate from the ternary complex to be estimated. This minimum is of the same order as the dissociation constant (Ki) of succinate. 2. A range of dicarboxy analogues of L-aspartate were tested as possible inhibitors of the enzyme. These studies suggested that L-aspartate is bound with its carboxy groups in the eclipsed configuration, and that the stereochemical constraints around the binding site are very similar to those reported for the catalytic subunit of the enzyme from Escherichia coli [Davies, Vanaman & Stark (1970) J. Biol. Chem. 245, 1175-1179].
Partial purification and characterization of chick-embryo prolyl 3-hydroxylase
Authors: Tryggvason K , Majamaa K , Risteli J , Kivirikko K I
Published: August 2015

Prolyl 3-hydroxylase was purified up to about 5000-fold from an (NH4)2SO4 fraction of chick-embryo extract by a procedure consisting of affinity chromatography on denatured collagen linked to agarose, elution with ethylene glycol and gel filtration. The molecular weight of the purified enzyme is about 160000 by gel filtration The enzyme is probably a glycoprotein, since (a) its activity is inhibited by concanavalin A, and (b) the enzyme is bound to columns of this lectin coupled to agarose and can be eluted with a buffer containing methyl alpha-D-mannoside. The Km values for Fe2+, 2-oxoglutarate, O2 and ascorbate in the prolyl 3-hydroxylase reaction were found to be very similar to those previously reported for these co-substrates in the prolyl 4-hydroxylase and lysyl hydroxylase reactions.
Specific interaction of human Tamm-Horsfall gylcoprotein with leucoagglutinin, a lectin from Phaseolus vulgaris (red kidney bean)
Authors: Serafini-Cessi F , Franceschi C , Sperti S
Published: August 2015

Human Tamm-Horsfall glycoprotein inhibits lymphocyte transformation induced by leucoagglutinin and haemagglutinin from Phaseolus vulgaris (red kidney bean). The glycoprotein interacts with the two lectins, giving insoluble precipitates. The interaction with leucoagglutinin is highly specific, and the shape of the precipitin curve is that of an antigen-antibody reaction; precipitation is specifically inhibited by N-acetyl-D-galactosamine. Results are discussed, and it is suggested that inhibition of lymphocyte transformation is due to competition between human Tamm-Horsfall glycoprotein and carbohydrate receptors on lymphocytes for the two lectins. The interaction between human Tamm-Horsfall glycoprotein and Phaseolus vulgaris lectins has been used to develop a one-step procedure for the separation of the two lectins by affinity chromatography on (human Tamm-Horsfall-glycoprotein)-Sepharose.
Nucleotide sequences of chloroplast 5S ribosomal ribonucleic acid in flowering plants
Authors: Dyer T A , Bowman C M
Published: August 2015

Evidence for the sequence of duckweed (Lemna minor) chloroplast 5S rRNA was derived from the analysis of partial and complete enzymic digests of the 32P-labelled molecule. The possible sequence of the chloroplast 5S rRNA from three other flowering plants was deduced by complete digestion with T1 ribonuclease and comparison of the sequences of the oligonucleotide products with homologous sequences in the duckweed 5S rRNA. This analysis indicates that the chloroplast 5S rNA species differ appreciably from their cytosol counterparts but bear a strong resemblance to one another and to the 5S rRNA species of prokaryotes. Structural features apparently common to all 5S rRNA molecules are also discussed.
Interference with glycosylation of glycoproteins. Inhibition of formation of lipid-linked oligosaccharides in vivo
Authors: Datema R , Schwarz R T
Published: August 2015

Influenza-virus-infected cells were labelled with radioactive sugars and extracted to give fractions containing lipid-linked oligosaccharides and glycoproteins. The oligosaccharides linked to lipid were of the ‘high-mannose’ type and contained glucose. In the glycoprotein fraction, radioactivity was associated with virus proteins and found to occur predominantly in the ‘high-mannose’ type of glycopeptides. In the presence of the inhibitors 2-deoxy-D-glucose, 2-deoxy-2-amino-D-glucose (glucosamine), 2-deoxy-2-fluoro-D-glucose and 2-deoxy-2-fluoro-D-mannose incorporation of radiolabelled sugars into lipid- and protein-linked oligosaccharides was decreased. Kinetic analysis showed that the inhibitors affected first the assembly of lipid-linked oligosaccharides and then protein glycosylation after a lag period. During inhibition by deoxyglucose and the fluoro sugars lipid-linked oligosaccharides were formed that contained oligosaccharides of decreased molecular weight. No such aberrant forms were found during inhibition by glucosamine. In the case of inhibition by deoxyglucose it was shown that the aberrant oligosaccharides were not transferred to protein. Inhibition of formation of lipid-linked oligosaccharides by deoxyglucose and fluoro sugars was antagonized by mannose, in which case oligosaccharides of normal molecular weight were formed. The inhibition by glucosamine was reversed by its removal from the medium. The reversible effects of these inhibitors exemplify their usefulness as tools in the study of glycosylation processes.
Effect of ionophores on carrier-mediated electron translocation in ferricyanide-containing liposomes
Authors: Miller Mette , Petersen Lars Chr. , Hansen Finn B. , Nicholls Peter
Published: August 2015

Ferricyanide-containing liposomes were used as a system to compare the electron- and proton-translocating properties of six redox reagents commonly used as electron donors for biochemical systems. The effects of different ionophore combinations on the ferricyanide-reduction rate were generally consistent with the expected proton- and electron-translocating properties of the mediators. The transmembrane pH gradient produced by hydrogen carriers was demonstrated. Nigericin or valinomycin plus carbonyl cyanide p-trifluoromethoxyphenylhydrazone are capable of collapsing this gradient and of stimulating ferricyanide reduction mediated by this type of carrier. No pH gradient is produced with the electron carrier 1,1′-dibutylferrocene. In the presence of tetraphenylboron anion, which is needed for this carrier to act as an efficient mediator, addition of valinomycin alone is sufficient to obtain full stimulation of ferricyanide reduction. NNN′N′-Tetramethyl-p-phenylenediamine does not behave as a simple electron carrier. During NNN′N′-tetramethyl-p-phenylenediamine-mediated ferricyanide reduction protons are translocated across the membrane and accumulated in the vesicles. This is not due to the presence of demethylated impurities in the NNN′N′-tetramethyl-p-phenylenediamine sample, but may be the result of an accumulation of oxidation products other than the Wurster's Blue radical. These results suggest a reconsideration of studies on protonmotive forces across membranes where NNN′N′-tetramethyl-p-phenylenediamine is used as a mediator.
Structure–activity relationship of inhibitors of hydroxyapatite formation
Authors: Williams G , Sallis J D
Published: August 2015

The relative abilities of some compounds to inhibit the transformation of amorphous calcium phosphate into hydroxyapatite were determined. Organic diphosphates, fructose 1,6-bisphosphate, pyrophosphate, imidodiphosphate and 3-phospholycerate all inhibited to various degrees. Strong inhibition was observed with phosphonoformate, and phosphocitrate proved to be the most powerful of the inhibitors examined. On the basis of these new findings an improved concept of the structure–activity relationship of inhibitors is proposed and mechanisms for inhibitor action are discussed.
Tissue and subcellular distribution of the lectin from Datura stramonium (thorn apple)
Authors: Kilpatrick D C , Yeoman M M , Gould A R
Published: August 2015

Plants of Datura stramonium (thorn-apple) were dissected into their component tissues and examined for the presence of the Datura lectin. This lectin was easily detected in seeds and in various parts of the flowers of adult plants. Traces were also found in green (emerged) cotyledons and roots of seedlings. The specific lectin activity in seeds contained within the fruits increased as the seeds matured. Mature seeds were homogenized in sucrose and separated by differential centrifugation into four fractions, three of which were clearly of distinct composition. Most of the lectin activity sedimented with the low-speed (cell-wall/protein-body) pellet, but a similar specific activity was recovered from the other fractions. However, if EDTA was included in the homogenization medium, three or four times more lectin activity was recovered in the soluble fraction. Immunofluorescent staining of formaldehyde-fixed sections showed that the lectin was localized in the cytoplasm, with little associated with cell walls. The possible relevance of these results to the function of the lectin in plant cells is discussed.
Phospholipase A activity in the skin. Modulators of arachidonic acid release from phosphatidylcholine
Authors: Ziboh V A , Lord J T
Published: August 2015

The distribution of the hydrolysis of 1-acyl-2-[1-14C]arachidonoyl-sn-glycero-3-phosphocholine and the simultaneous biosynthesis of prostaglandins by subcellular fractions from human and rat skin membrane preparations were determined. The phospholipase A2 activity was distributed among the subcellular particulate preparations with the highest specific activity in the 105000g particulate fraction. The activity was optimal at pH 7.5 in the presence of 1.0 mM-CaCl2 and was inhibited by EDTA. The hydrolysis of phosphatidylcholine by the skin 105000g particulate fraction was inhibited by cortisol and triamcinolone acetonide and it was stimulated by histamine, bradykinin, retinoic acid and cholera enterotoxin (freeze-dried Vibrio cholerae). Furthermore hydrolysis of phosphatidylcholine by the skin phospholipase A was also enhanced by low concentrations of prostaglandin E2 and prostaglandin F2 alpha. These last results suggest that the amplication of the hydrolysis of phosphatidylcholine by prostaglandin E2 and prostaglandin F2 alpha, with the consequent release of arachidonic acid (the substrate of prostaglandin synthesis) is likely a positive-feedback regulation of the arachidonic acid-prostaglandin cascade.
Androgen-sensitive spermine-binding protein of rat ventral prostate. Purification of the protein and characterization of the hormonal effect
Authors: Mezzetti G , Loor R , Liao S
Published: August 2015

The rat ventral prostate contains a cytosol protein that can non-covalently bind spermine much more tightly than spermidine or other natural diamines. The protein has been purified to homogeneity, as judged by electrophoresis in urea- and sodium dodecyl sulphate-containing polyacrylamide gels. The protein, with or without spermine bound to it, sediments at 3 S in a sucrose gradient with or without 0.4 M-KCl. The molecular weight of the protein is about 30 000. Each molecule of the binding protein can bind one molecule of spermine. In the prostate of rats injected with cycloheximide, the protein appears to have a half-life of about 3.5 h. The spermine-binding activity of an acidic fraction obtained by DEAE-cellulose chromatography of the prostate cytosol proteins is reduced by about 40–60% within 20–40 h after castration. This effect is reversed very rapidly within 15–30 min by intraperitoneal injection of 5 alpha-dihydrotestosterone. The hormonal effect is androgen-specific and is not mimicked by dexamethasone or oestradiol-17 beta. The androgen effect was reduced significantly when rats were injected with cycloheximide or actinomycin D, suggesting that the acidic protein may be one of the earliest proteins induced by androgen in the rat ventral prostate.
The tricarboxylic acid cycle in Dictyostelium discoideum. Metabolite concentrations, oxygen uptake and 14c-labelled amino acid labelling patterns
Authors: Kelly P J , Kelleher J K , Wright B E
Published: August 2015

Some aspects of tricarboxylic acid-cycle activity during differentiation and aging in Dictyostelium discoideum were examined. The concentrations of glutamate, aspartate, alanine, citrate, 2-oxoglutarate, succinate, fumarate, malate, oxaloacetate, pyruvate and acetyl-CoA were determined at four stages over the course of differentiation. The rate of O2 utilization was also determined over differentiation. In addition, experiments are described in which the specific radioactivities of citrate, 2-oxoglutarate, succinate, fumarate and malate were determined during a 30 min labelling of cells from the preculmination stage of development with [14C]glutamate, [14C]aspartate or [14C]alanine. A similar experiment was also performed with cells from the aggregation stage of development using [14C]glutamate.
Prostaglandins and their precursors in tissues from rats fed on trans, trans-linoleate
Authors: Kinsella J E , Hwang D H , Yu P , Mai J , Shimp J
Published: August 2015

Feeding trans, trans-9,12-linoleate to rats as 50 and 100% of the dietary fat decreased the concentrations of n-6 fatty acids, i.e. 18:2, 20:3 and 20:4, in heart, kidney, lung, adipose tissue and platelets of rats. The concentrations of prostaglandin products prostaglandins E1, E2 and F2 alpha in serum were significantly decreased in rats receiving high concentrations of dietary trans, trans-linoleate.
Factors influencing proteoglycan size in rachitic-chick growth cartilage
Authors: Roughley P , Dickson I
Published: August 2015

1. Proteoglycan isolated from rachitic-chick growth cartilage was of smaller size than that isolated from tissue of normal chicks. 2. The two proteoglycan populations were of similar average chemical composition and similar in the size of their chondroitin sulphate chains. 3. The size of the proteoglycans was not affected by reduction and alkylation. 4. Labelling studies in vivo with Na235SO4 and [3H]leucine suggest that the difference in size in the rachitic state results from an alteration in synthesis rather than extracellular proteolytic degradation of the normal proteoglycan, but the direct cause of this alteration remains unestablished.
Purification and properties of different forms of modeccin, the toxin of Adenia digitata. Separation of subunits with inhibitory and lectin activity
Authors: Barbieri L , Zamboni M , Montanaro L , Sperti S , Stirpe F
Published: August 2015

1. The subunits were isolated of modeccin (subsequently referred to as modeccin 4B), the toxin purified from the roots of Adenia digitata by affinity chromatography on Sepharose 4B [Gasperi-Campani, Barbieri, Lorenzoni, Montanaro, Sperti, Bonetti & Stirpe (1978) Biochem J. 174, 491-496]. They are an A subunit (mol.wt. 26 000), which inhibits protein synthesis, and a B subunit (mol.wt. 31 000), which binds to cells. Both sununits, as well as intact modeccin, gave single bands on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, but showed some heterogeneity on isoelectric focusing and on polyacrylamide-gel electrophoresis at pH 9.5. 2. A second form of modeccin, not retained by Sepharose 4B, was purified by affinity chromatography on acid-treated Sepharose 6B: this form is subsequently termed modeccin 6B 3. Modeccin 6B has a molecular weight indistinguishable from that of modeccin 4B, and consists of two subunits of mol.wts. 27 000 and 31 000, joined by a disulphide bond. The subunits were not isolated because of their high insolubility in the absence of sodium dodecyl sulphate. 4. As compared with modeccin 4B, modeccin 6B is slightly less toxic to animals, does not agglutinate erythrocytes, and is a more potent inhibitor of protein synthesis in a lysate of rabbit reticulocytes, giving 50% inhibition at the concentration of 0.31 microgram/ml.
Properties of the non-specific calcium-binding sites of rabbit skeletal-muscle myosin
Authors: Wikman-Coffelt J
Published: August 2015

The non-specific Ca2+-binding sites of skeletal-muscle myosin are located on the light chains; with the dissociation of light chains there is a corresponding decrease in the number of Ca2+-binding sites on light-chain-deficient myosin. The released light chains have a decreased binding affinity. Myosin heavy chains indirectly influence the Ca2+-binding properties of light chains by increasing the affinity of light chains for bivalent cations; this influence varies with pH. Because of light-chain dissociation at low Ca2+ and/or Mg2+ concentrations, anomalies may exist when analyses of non-specific Ca2+-binding properties of myosin are assessed by dialysis equilibrium.
Purification and some properties of a lectin from the fruit juice of the tomato (Lycopersicon esculentum)
Authors: Kilpatrick D C
Published: August 2015

In the tomato (Lycopersicon esculentum) plant, the fruit juice was found to be the richest source of agglutinating activity. The lectin responsible could be inhibited by oligomers of N-acetylglucosamine, and this property was exploited to purify the lectin by affinity adsorption on trypsin-treated erythrocytes. The lectin is a glycoprotein that cross-reacts immunologically with the lectin from Datura stramonium (thorn-apple).
Isolation and characterization of sulphated mucopolysaccharides from rat leukaemic (RBL-1) basophils
Authors: Metcalfe D D , Wasserman S I , Austen K F
Published: August 2015

Proteoglycans of 300 000 mol.wt. were isolated from dispersed rat basophil tumour cells after labelling of the sulphated mucopolysaccharides with 35S in vitro:90% of the 35S-labelled mucopolysaccharides were extracted at high salt concentration. Alkali degradation of the 35S-labelled proteoglycans yielded glycosaminoglycan chains of 40 000 mol.wt. The composition of the salt-extracted 35S-labelled mucopolysaccharides, as defined by parallel or sequential degradation with chondroitinase AC, chondroitinase ABC and heparinase and resolution of the disaccharide-digestion products obtained with chondroitinase AC, was 48-61% chondroitin 4-sulphate, 20-30% dermatan sulphate, 10-15% heparin and 7-9% chondroitin 6-sulphate. Most of the salt-extracted 35S-labelled mucopolysaccharides were highly charged, with heparin and chondroitin 6-sulphate being relatively uniform in this regard, whereas chondroitin 4-sulphate and dematan sulphate exhibited a range of charge characteristics. The diversity of sulphated mucopolysaccharides present in the rat leukaemic basophil is in contrast with the predominance of heparin in the rat mast cell.
Oxygen kinetics of frozen cytochrome oxidase. The capacity of the oxygen pocket
Authors: De Fonseka K , Chance B
Published: August 2015

Low-temperature kinetics of the reaction between O2 and cytochrome oxidase suggest the existence of an O2 pocket of limited capacity in membrane-bound cytochrome oxidase, and one of larger capacity in purified cytochrome oxidase. A model is proposed to explain the difference in capacity of the pockets.
Purification of Torpedo californica post-synaptic membranes and fractionation of their constituent proteins
Authors: Elliott J , Blanchard S G , Wu W , Miller J , Strader C D , Hartig P , Moore H P , Racs J , Raftery M A
Published: August 2015

A rapid methof for preparation of membrane fractions highly enriched in nicotinic acetylcholine receptor from Torpedo californica electroplax is described. The major step in this purification involves sucrose-density-gradient centrifugation in a reorienting rotor. Further purification of these membranes can be achieved by selective extraction of proteins by use of alkaline pH or by treatment with solutions of lithium di-idosalicylate. The alkali-treated membranes retain functional characteristics of the untreated membranes and in addition contain essentially only the four polypeptides (mol.wts. 40000, 50000, 60000 and 65000) characteristic of the receptor purified by affinity chromatography. Dissolution of the purified membranes or of the alkali-treated purified membranes in sodium cholate solution followed by sucrose-density-gradient centrifugation in the same detergent solution yields solubilized receptor preparations comparable with the most highly purified protein obtained by affinity-chromatographic procedures.
Ubiquinones have surface-active properties suited to transport electrons and protons across membranes
Authors: Quinn Peter J. , Esfahani Manouchehre A.
Published: August 2015

Surface-active properties of ubiquinones and ubiquinols have been investigated by monomolecular-film techniques. Stable monolayers are formed at an air/water interface by the fully oxidized and reduced forms of the coenzyme; collapse pressures and hence stability of the films tend to increase with decreasing length of the isoprenoid side chain and films of the reduced coenzymes are more stable than those of their oxidized counterparts. Ubiquinone with a side chain of two isoprenoid units does not form stable monolayers at the air/water interface. Mixed monolayers of ubiquinol-10 or ubiquinone-10 with 1,2-dimyristoyl phosphatidylcholine, soya phosphatidylcholine and diphosphatidylglycerol do not exhibit ideal mixing characteristics. At surface pressures less than the collapse pressure of pure ubiquinone-10 monolayers (approx. 12mN·m−1) the isoprenoid chain is located substantially within the region occupied by the fatty acyl residues of the phospholipids. With increasing surface pressure the ubiquinones and their fully reduced equivalents are progressively squeezed out from between the phospholipid molecules until, at a pressure of about 35mN·m−1, the film has surface properties consistent with that of the pure phospholipid monolayer. This suggests that the ubiquinone(ol) forms a separate phase overlying the phospholipid monolayer. The implications of this energetically poised situation, where the quinone(ol) is just able to penetrate the phospholipid film, are considered in terms of the function of ubiquinone(ol) as electron and proton carriers of energy-transducing membranes.
The effect of ascorbic acid on the nature and production of collagen and elastin by rat smooth-muscle cells
Authors: de Clerck Y A , Jones P A
Published: August 2015

1. The effects of various concentrations of ascorbic acid on the quality and quantity of the insoluble extracellular matrices produced by two strains of cultured rat smooth-muscle cells were studied. 2. Ascorbic acid was necessary for the appearance of insoluble collagen in the extracellular matrix. 3. Secretion of soluble collagen continued in the absence of ascorbic acid, but this soluble collagen was markedly underhydroxylated. 4. The amount of insoluble collagen present in the matrix was directly related to the ascorbic acid concentration. 5. The insoluble collagen that appeared in the matrix under conditions where ascorbic acid was limiting was no more than 7% underhydroxylated. 6. In contrast, the amount of insoluble elastin produced was inversely proportional to the ascorbic acid concentration. 7. The elastin produced in the absence of ascorbic acid had the expected amino acid composition, but hydroxyproline was absent. 8. The hydroxyproline content of elastin was also directly dependent on the ascorbic acid concentration. 9. Ascorbic acid had variable effects on the quantity of glycoprotein(s) present in the matrix. 10. The appearance of insoluble collagen in the extracellular matrices produced by cultured human fibroblasts and calf endothelial cells was also completely dependent on the presence of ascorbic acid.
Seed extracts inhibiting protein synthesis in vitro
Authors: Gasperi-Campani A , Barbieri L , Morelli P , Stirpe F
Published: August 2015

Of 33 seed extracts examined, 12 inhibited protein synthesis in a rabbit reticulocyte lysate. This activity seems to be due to a protein, since (i) it was recovered with the (NH4)2SO4 precipitate, (ii) it was retained by dialysis membranes, and (iii) in all cases but one was destroyed by boiling. Only the extracts from the seeds of Adenia digitata and, to a lower extent, of Euonymus europaeus inhibited protein synthesis in intact cells.
Changes in bilirubins in human prenatal development
Authors: Blumenthal S G , Stucker T , Rasmussen R D , Ikeda R M , Ruebner B H , Bergstrom D E , Hanson F W
Published: August 2015

1. A densitometric method has been developed for the quantification of azodipyrroles derived from dog bile pigments treated with diazotized ethyl anthranilate. 2. This method was used to estimate the bilirubins in bile and meconium from foetuses of 14-36 weeks gestation. 3. The proportion of the bilirubins in foetal bile changed during gestation. (a) No bile pigments were found until 14 weeks. (b) Between 14 and 15 weeks bilirubin-IX beta was the only bile pigment detected. (c) At 16-17 weeks some unconjugated bilirubin-IX alpha was found in the bile, but up to 20 weeks bilirubin-IX beta was the predominant bilirubin in the bile. (d) At about 20 weeks glucose, xylose, and an unidentified bilirubin-IX alpha monoconjugate were found in the bile. (e) Between 20 and 23 weeks bilirubin-IX alpha glucuronide appeared in the bile. (f) At 30 weeks monoconjugates of bilirubin-IX alpha were the predominant bilirubins in the bile. (g) Only in full-term foetuses was bilirubin-IX alpha monoglucuronide the major bilirubin derivative.
Rates of glucose production and utilization by the foetus in chronically catheterized sheep
Authors: Hodgson J C , Mellor D J , Field A C
Published: August 2015

1. Six well-nourished Scottish Blackface ewes were each prepared at 110-112 days of gestation with indwelling catheters in an umbilical artery and vein and at 2 days before experiment with catheters in each maternal jugular vein. Two-day experiments were conducted between days 124 and 134 of gestation when [2-3H,U-14C]glucose was infused over 9 h into either the maternal or the foetal circulations, separate days being used for each infusion. 2. Plasma glucose specific radioactivities at plateau were used to determine, for a two-pool model representing the glucose system of the mother and foetus, the rates of glucose production, utilization and recycling by the mother and by the foetus and the rates of transfer of glucose between the mother and foetus. 3. The net rate of glucose utilization by the foetus, which we have called the ‘foetal glucose requirement’, was 7.6 +/- 0.6 mg/min per kg of foetus (n = 8). Compared with the corresponding rate in the mother of 1.1 +/- 0.1 mg/min per kg of mother (n = 8), this indicated a high rate of foetal metabolism. 4. The net rate of foetal uptake of glucose from the mother accounted for only 69 +/- 5% of the foetal glucose requirement, and it has been argued that the remainder was supplied by foetal gluconeogenesis. 5. The rate of glucose recycling within the foetus was high, equal to 108 +/- 16% of the net foetal glucose-utilization rate. The significance of this in terms of either Cori or futile cycling is discussed.
Syntheses of penicillin N, [6 α-3H]penicillin N and [10-14C,6 α-3H]penicillin N
Authors: Baldwin J E , Herchen S R , Singh P D
Published: August 2015

1. Penicillin N was synthesized by coupling alpha-amino-alpha-p-nitrobenzyl-N-p-nitro-benzyloxycarbonyl-D-adipate with 6-aminopenicillanic acid benzyl ester, followed by removal of the protecting groups through hydrogenolysis. 2. alpha-Amino-alpha-p-nitrobenzyl-N-p-nitrobenzyloxycarbonyl-D-[5-14C]adipate was prepared by treating alpha-p-nitrobenzyl-N-p-nitrobenzyloxycarbonyl-D-glutamic acid with [14C]diazomethane followed by rearrangement with silver trifluoromethanesulphonate. 3. Coupling of alpha-amino-alpha-p-nitrobenzyl-N-p-nitrobenzyloxycarbonyl-D-[5-14C]adipate with 6-aminopenicillanic acid benzyl ester gave triprotected [10-14C]penicillin N. 4. 3H was introduced at C-6 of the Schiff's base derivative (10) by oxidation followed by reduction with NaB3H4. 5. The so-derived (6 alpha-3H)-labelled Schiff's base was hydrolysed to give 6-amino [6 alpha-3H]penicillanic acid benzyl ester p-toluenesulphonic acid salt, which after coupling as the free amine with alpha-amino-alpha-p-nitrobenzyl-N-pnitrobenzyloxycarbonyl-D-adipate and then hydrogenolysis, yielded [6alpha-3H]penicillin N. 6. Triprotected [10-14C]penicillin N and triprotected [6alpha-3H]penicillin N in admixture were hydrogenolysed to give [10-14C,6alpha-3H]penicillin N.
Incorporation of 3H and 14C from (6 α-3H)penicillin N and (10-14C,6 α-3H) penicillin N into deacetoxycephalosporin C
Authors: Baldwin J E , Singh P D , Yoshida M , Sawada Y , Demain A L
Published: August 2015

1. In a cell-free system prepared by lysis of protoplasts of Cephalosporium acremonium mutant M-0198, 3H and 14C were incorporated from singly- and doubly-labelled penicillin N into deacetoxycephalosporin C. 2. The deacetoxcephalosporin C obtained from the above feeding experiments was converted into two different crystalline derivatives, namely N-phthalimidodeacetoxycephalosporin C bisbenzhydryl ester and N-phthalimidodeacetoxycephalosporin C bisdicyclohexylamine salt and recrystallized to constant specific activity or constant ratio of specific activity. 3. That 3H is incorporated at C-7 in the biosynthesized deacetoxycephalosporin C was shown by the loss of radioactivity (95.2%) after methoxylating the derived N-phthalimidodeacetoxycephalosporin C bisbenzyhydryl ester. 4. Deacetoxycephalosporin C was also the product of the cell-free reaction conducted in the presence of ferrous ions and ascorbic acid, as shown by two-dimensional paper electrophoresis-chromatography; these additives appreciably improved the efficiency of conversion.
UDP-galactose-ceramide galactosyltransferase in rat brain myelin subfractions during development
Authors: Koul O , Chou K H , Jungalwala F B
Published: August 2015

The localization and activity of the enzyme UDP-galactose-hydroxy fatty acid-containing ceramide galactosyltransferase is described in rat brain myelin subfractions during development. Other lipid-synthesizing enzymes, such as cerebroside sulphotransferase, UDP-glucose-ceramide glucosyltransferase and CDP-choline-1,2-diacylglycerol cholinephosphotransferase, were also studied for comparison in myelin subfractions and microsomal membranes. The purified myelin was subfractionated by isopycnic sucrose-density-gradient centrifugation. Four myelin subfractions, three floating respectively on 0.55 M- (light-myelin fraction), 0.75 M- (heavy-myelin fraction) and 0.85 M-sucrose (membrane fraction), and a pellet, were isolated and purified. At all ages, 70-75% of the total myelin proteins was found in the heavy-myelin fraction, whereas 2-5% of the protein was recovered in the light-myelin fraction, and about 7-12% in the membrane fraction. Most of the galactosyltransferase was associated with the heavy-myelin and membrane fractions. Other lipid-synthesizing enzymes studied appeared not to associate with purified myelin or myelin subfractions, but were enriched in the microsomal-membrane fraction. During development, the specific activity of the microsomal galactosyltransferase reached a maximum when the animals were about 20 days old and then declined. By contrast the specific activity of the galactosyltransferase in the heavy-myelin and membrane fractions was 3-4 times higher than that of the microsomal membranes in 16-day-old animals. The specific activity of the enzyme in the heavy-myelin fraction sharply declined with age. Chemical and enzymic analyses of the heavy-myelin and membrane myelin subfractions at various ages showed that the membrane fraction contained more proteins in relation to lipids than the heavy-myelin fraction. The membrane fraction was also enriched in phospholipids compared with cholesterol and contrined equivalent amounts of 2':3'-cyclic nucleotide 3'-phosphohydrolase compared with heavy- and light-myelin fractions. The membrane fraction was deficient in myelin basic protein and proteolipid protein and enriched in high-molecular-weight proteins. The specific localization of galactosyltransferase in heavy-myelin and membrane fractions at an early age when myelination is just beginning suggests that it may have some role in the myelination process.
Polyol-pathway enzymes of human brain. Partial purification and properties of aldose reductase and hexonate dehydrogenase
Authors: O'Brien M M , Schofield P J
Published: August 2015

Aldose reductase and hexonate dehydrogenase were isolated from human brain and partially purified. The two enzymes exhibited distinctive substrate-specificity profiles with a variety of aldoses, and aliphatic and aromatic aldehydes. Aldose reductase exhibited a high affinity for DL-glyceraldehyde (Km of 62 microM) and a low affinity (Km of 90 mM) for glucose, the physiological substrate of the polyol pathway. Hexonate dehydrogenase exhibited a relatively low affinity for D-glucuronate (Km of 4.6 mM) and a very low affinity for glucose (Km of 390 mM). Both enzymes exhibited a high specificity for NADPH, and both were inhibited competitively by NADP+. Hexonate dehydrogenase was inhibited by iodoacetate, iodoacetamide, N-ethylmaleimide and p-chloromercuribenzoate. Preincubation with 2-mercaptoethanol resulted in activation. Both enzymes were inhibited by a number of barbiturates (barbital, phenobarbital and pentobarbital) and by the central-nervous-system drugs diphenylhydantoin and ethosuccinimide. The substrate specificity and pattern of inhibition suggest that the two enzymes isolated correspond to two of four previously reported aldehyde reductases isolated from human brain.