bluuebunny/crossref_metadata_2025 huggingface dataset:
Altered lipolytic response to glucagon and adenosine deaminase in adipocytes from starved rats
Authors: Honnor R C , Saggerson E D
Published: August 2015

1. Adipocytes isolated from epididymal adipose tissue of fed or 24 h-starved rats were incubated with a range of glucagon concentrations in the presence and absence of adenosine deaminase (4 munits/ml). 2. With adenosine deaminase present, the lipolytic response to low concentrations of glucagon (1-6 ng/ml) was considerably enhanced in cells from starved rats. 3. The effect of adenosine deaminase on basal lipolysis was altered after starvation. 4. D-3-Hydroxybutyrate (5 mM) decreased the sensitivity of lipolysis to glucagon. 5. The possible involvement of glucagon-stimulated lipolysis in the regulation of ketogenesis is briefly discussed.
Evidence that the active centre of chymopapain A is different from the active centres of some other cysteine proteinases and that the Brønsted coefficient (βnuc.) for the reactions of thiolate anions with 2,2′-dipyridyl disulphide may be decreased by reagent protonation
Authors: Brocklehurst K , Baines B S , Mushiri M S
Published: August 2015

The active centres of chymopapains A and B (jointly designated EC 3.4.22.6) and papaya (Carica papaya L.) peptidase A were investigated by using 2,2′-dipyridyl disulphide and 5,5′-dithiobis-(2-nitrobenzoic acid) as thiol-specific reactivity probes. Whereas the first active-centre pKa values for chymopapain B and papaya peptidase A are less than 5, is as the case for papain (EC 3.4.22.2) and ficin (EC 3.4.22.3), that for chymopapain A is about 6.8. The reason why the reactions of thiols of pKa approx. 6.5 with 2.2′-dipyridyl disulphide are essentially pH-independent in the pH range around the thiol pKa is delineated. The value of the Brønsted coefficient (beta nuc.) for the reactions of thiolate ions with the 2,2′-dipyridyl disulphide monocation appears to be smaller than its value for the corresponding reactions with the neutral disulphide.
The kinetics of effector binding to phosphofructokinase. The influence of effectors on the allosteric conformational transition
Authors: Roberts D , Kellett G L
Published: August 2015

1. The extent of the allosteric transition from the R into the T conformation of rabbit skeletal muscle phosphofructokinase induced by Mg2+-1,N6-etheno-ATP was determined by stopped-flow fluorimetry from the amplitude of the slow phase of the Mg2+-1,N6-etheno-ATP fluorescence enhancement [Roberts & Kellet (1979) Biochem. J. 183, 349–360]. 2. The amplitude of the slow phase was decreased by low concentrations of the activators cyclic AMP and fructose 1,6-bisphosphate, but increased in a complex manner by the inhibitor citrate. 3. Mg2+-1,N6-etheno-ATP and Mg2+-ATP are unable to induce the T conformation to a detectable extent in the presence of saturating cyclic AMP, but can do so readily in the presence of saturating fructose 1,6-bisphosphate. 4. The conformational transitions induced in enzyme alone by different ligands were observed by changes in intrinsic protein fluorescence. In general, an R-type conformation has diminished protein fluorescence compared with a T-type conformation. 5. Mg2+-ATP exerts a complex effect on protein fluorescence; both the enhancement at low concentrations and the quenching at high concentrations of Mg2+-ATP result from the binding of Mg2+-ATP to the inhibitory site and the ensuing allosteric transition. Enhancement reflects the extent of the allosteric transition and involves both tyrosine and tryptophan, probably in the region of the active site; quenching reflects occupation of the inhibitory site and involves tyrosine at the inhibitory site. 6. The mechanism of the allosteric transition from the R into the T conformation induced by Mg2+-1,N6-etheno-ATP at low concentrations occurs predominantly by a ‘prior-isomerization’ pathway; at higher concentrations a limited contribution from a ‘substrate-guided’ pathway occurs. 7. The allosteric behaviour of phosphofructokinase with respect to Mg2+-ATP and Mg2+-1,N6-ethenol-ATP binding may be accounted for in terms of the simple, concerted model.
Inhibition of protohaem ferro-lyase by N-substituted porphyrins. Structural requirements for the inhibitory effect
Authors: De Matteis F , Gibbs A H , Smith A G
Published: August 2015

N-Methyl mesoporphyrin was a powerful inhibitor of protohaem ferro-lyase in vitro, whereas N-ethyl mesoporphyrin and N-methyl coproporphyrin were not and neither was the newly described green pigment produced by giving rats ethylene. This suggests that the size of the substituent at a pyrrole nitrogen and also the number of carboxylic acid side chains of the substituted porphyrin are important for the inhibitory effect. Evidence that N-methyl mesoporphyrin inhibited the enzyme, whereas the ethylene-derived pigment did not, was also obtained in vivo.
Role of cytoplasmic thioltransferase in cellular regulation by thiol-disulphide interchange
Authors: Mannervik B , Axelsson K
Published: August 2015

Cytoplasmic thioltransferase purified from rat liver [Axelsson, Eriksson & Mannervik (1978) Biochemistry 17, 2978–2984] catalyses the formation and decomposition of mixed disulphides of proteins and glutathione. The enzyme was found to catalyse the reversible thiol-disulphide interchange between glutathione disulphide and a crude thiol-containing protein fraction from rat liver. This finding indicates a role of the thioltransferase in the regulation of the ‘glutathione status’ of the cell. Specifically, it was found that thioltransferase catalyses the reactivation of pyruvate kinase from rat liver that had previously been inactivated by glutathione disulphide. It is suggested that thioltransferase may have a general role in regulatory processes involving thiol-disulphide interchange.
Intermediates in the assembly of ribosomes by a mutant of Escherichia coli
Authors: Butler P D , Sims P F G , Wild D G
Published: August 2015

Escherichia coli strain 15–28 is a mutant that accumulates ribonucleoprotein (‘47 S’) particles during exponential growth. These particles contain mature 23 S rRNA, but lack three of the proteins of the larger ribosomal subunit, to which they are a precursor. In organisms growing at 20 degrees C, assembly of 47 S particles involves three intermediates that contain precursor 23 S rRNA, one of which has the same sedimentation properties as 47 S particles. Assembly of 50 S ribosomal subunits in the parent strain is ‘normal’. There are three intermediates; each contains precursor 23 S rRNA, and one cannot be distinguished from completed subunits by sedimentation. Synthesis of 30 S ribosomal subunits in parent and mutant strains is qualitatively similar, but quantitatively different. When growth is at 37 degrees C, assembly in the mutant alters. There are now two sequential precursors to 47 S particles. Both contain precursor 23 S rRNA; one has the same sedimentation coefficient as 47 S particles. In some respects, synthesis in the mutant proceeds as though 47 S particles, rather than 50 S ribosomal subunits, are the end-product of assembly.
Synthesis of glycosaminoglycans by human skin fibroblasts cultured on collagen gels
Authors: Gallagher J T , Gasiunas N , Schor S L
Published: August 2015

A comparison has been made of the synthesis of glycosaminoglycans by human skin fibroblasts cultured on plastic or collagen gel substrata. Confluent cultures were incubated with [3H]glucosamine and Na235SO4 for 48h. Radiolabelled glycosaminoglycans were then analysed in the spent media and trypsin extracts from cells on plastic and in the medium, trypsin and collagenase extracts from cells on collagen gels. All enzyme extracts and spent media contained hyaluronic acid, heparan sulphate and dermatan sulphate. Hyaluronic acid was the main 3H-labelled component in media and enzyme extracts from cells on both substrata, although it was distributed mainly to the media fractions. Heparan sulphate was the major [35S]sulphated glycosaminoglycan in trypsin extracts of cells on plastic, and dermatan sulphate was the minor component. In contrast, dermatan sulphate was the principal [35S]sulphated glycosaminoglycan in trypsin and collagenase extracts of cells on collagen gels. The culture substratum also influenced the amounts of [35S]sulphated glycosaminoglycans in media and enzyme extracts. With cells on plastic, the medium contained most of the heparan sulphate (75%) and dermatan sulphate (> 90%), whereas the collagenase extract was the main source of heparan sulphate (60%) and dermatan sulphate (80%) from cells on collagen gels; when cells were grown on collagen, the medium contained only 5-20% of the total [35S]sulphated glycosaminoglycans. Depletion of the medium pool was probably caused by binding of [35S]sulphated glycosaminoglycans to the network of native collagen fibres that formed the insoluble fraction of the collagen gel. Furthermore, cells on collagen showed a 3-fold increase in dermatan sulphate synthesis, which could be due to a positive-feedback mechanism activated by the accumulation of dermatan sulphate in the microenvironment of the cultured cells. For comparative structural analyses of glycosaminoglycans synthesized on different substrata labelling experiments were carried out by incubating cells on plastic with [3H]glucosamine, and cells on collagen gels with [14C]glucosamine. Co-chromatography on DEAE-cellulose of mixed media and enzyme extracts showed that heparan sulphate from cells on collagen gels eluted at a lower salt concentration than did heparan sulphate from cells on plastic, whereas with dermatan sulphate the opposite result was obtained, with dermatan sulphate from cells on collagen eluting at a higher salt concentration than dermatan sulphate from cells on plastic. These differences did not correspond to changes in the molecular size of the glycosaminoglycan chains, but they may be caused by alterations in polymer sulphation.
Regulation of S-adenosylmethionine decarboxylase by polyamines in Ehrlich ascites-carcinoma cells grown in culture
Authors: Alhonen-Hongisto Leena
Published: August 2015

1. The mechanism of stimulation of S-adenosylmethionine decarboxylase (EC 4.1.1.50) activity by inhibitors of ornithine decarboxylase (EC 4.1.1.17), namely dl-α-difluoromethylornithine, 1,3-diaminopropane and 1,3-diaminopropan-2-ol, was studied in Ehrlich ascites-tumour cells grown in suspension cultures. 2. Difluoromethylornithine and diaminopropane, although decreasing the content of putrescine and spermidine, markedly stimulated adenosylmethionine decarboxylase activity after exposure of the cells to the drugs for 8h, whereas the effect of diaminopropanol only became apparent many hours later. In tumour cells exposed to any of the inhibitors, a close negative correlation existed between the activity of adenosylmethionine decarboxylase and the intracellular concentration of spermidine and/or spermidine plus spermine, suggesting that a depletion of higher polyamines triggered enhancement of adenosylmethionine decarboxylase activity. 3. The mechanism of difluoromethylornithine- and diaminopropane-induced stimulation of adenosylmethionine decarboxylase involved (a) a marked increase in the apparent half-life of the enzyme and (b) an induction of enhanced enzyme synthesis. Diaminopropanol seemed to act solely via an induction mechanism. 4. The increased adenosylmethionine decarboxylase activity elicited by difluoromethylornithine could be restored to control values by micromolar concentrations of exogenous spermidine and spermine in 4h and by putrescine in 22h. In addition to the natural polyamines, elevated adenosylmethionine decarboxylase activity could be repressed by 3,3′-iminodipropylamine, a close analogue of spermidine, but not by non-physiological diamines. 5. Addition of spermidine and actinomycin D to cultures treated with difluoromethylornithine produced a comparable decay of enhanced adenosylmethionine decarboxylase activity (with an apparent half-life of about 2.5h), whereas the effect of cycloheximide was much more rapid. The present results suggest that polyamines may regulate adenosylmethionine decarboxylase at the transcriptional level of gene expression.
Unoccupied nuclear oestrogen receptors in the female rat hypothalamus. Increases on oestrogen administration
Authors: White J O , Lim L
Published: August 2015

A major proportion of the hypothalamic nuclear oestrogen receptors were available for complexing with radioactive oestradiol in vitro at 4 degrees C and were apparently unoccupied . At 6 h after oestradiol administration the content of unoccupied nuclear receptors had increased 2.5-fold and represented 71% of the total nuclear receptor content. These results suggest that unoccupied receptors may be active elements in the ‘long-term’ receptor population of the hypothalamus. Androgenized females had lower contents of these receptors.
The antibody response to myoglobin is independent of the immunized species. Analysis in terms of replacements in the antigenic sites and in environmental residues of the cross-reactions of fifteen myoglobins with sperm-whale myoglobin antisera raised in different species
Authors: Twining S S , Lehmann H , Atassi M Z
Published: August 2015

The recent determination of the entire antigenic structure of sperm-whale myoglobin with rabbit and goat antisera has permitted the examination of whether the antigenic structure recognized by antibodies depends on the species in which the antisera are raised. Also, by knowledge of the antigenic structure, the molecular factors that determine and influence antigenicity can be better understood in terms of the effects of amino acid substitutions occurring in the antigenic sites and in the environmental residues of the sites. In the present work, the myoglobins from finback whale, killer whale, horse, chimpanzee, sheep, goat, bovine, echidna, viscacha, rabbit, dog, cape fox, mouse and chicken were examined for their ability to cross-react with antisera to sperm-whale myoglobin. By immunoadsorbent titration studies with radioiodinated antibodies, each of these myoglobins was able to bind antibodies to sperm-whale myoglobin raised in goat, rabbit, chicken, cat, pig and outbred mouse. It was found that the extent of cross-reaction of a given myoglobin was not dependent on the species in which the antisera were raised. This indicated that the antibody response to sperm-whale myoglobin (i.e. its antigenic structure) is independent of the species in which the antisera are raised and is not directed to regions of sequence differences between the injected myoglobin and the myoglobin of the immunized host. Indeed, in each antiserum from a given species examined, that antiserum reacted with the myoglobin of that species. The extent of this auto-reactivity for a given myoglobin was comparable with the general extent of cross-reactivity shown by that myoglobin with antisera raised in other species. The cross-reactivities and auto-reactivities (both of which are of similar extents for a given myoglobin) can be reasonably rationalized in terms of the effects of amino acid substitutions within the antigenic sites and within the residues close to these sites. These findings confirm that the antigenicity of the sites is inherent in their three-dimensional locations.
Purification and properties of l-3-glycerophosphate dehydrogenase from pig brain mitochondria
Authors: Cottingham I R , Ragan C I
Published: August 2015

L-3-Glycerophosphate dehydrogenase (EC 1.1.99.5) was purified from pig brain mitochondria by extraction with deoxycholate, ion-exchange chromatography and (NH4)2SO4 fractionation in cholate, and preparative isoelectric focusing in Triton X-100. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis shows that the purified enzyme consists of a single subunit of mol.wt. 75 000. The enzyme contains non-covalently bound FAD and low concentrations of iron and acid labile sulphide. No substrate reducible e.p.r. signals were detected. The conditions of purification, particularly the isoelectric focusing step, lead to considerable loss of FAD and possibly iron-sulphur centres. It is therefore not possible to decide with certainty whether the enzyme is a flavoprotein or a ferroflavoprotein. The enzyme catalyses the oxidation of L-3-glycerophosphate by a variety of electron acceptors, including ubiquinone analogues. A number if compounds known to inhibit ubiquinone oxidoreduction by other enzymes of the respiratory chain failed to inhibit L-3-glycerophosphate dehydrogenase, except at very high concentrations.
Studies on the incorporation of [32P]phosphate into pyruvate dehydrogenase in intact rat fat-cells. Effects of insulin
Authors: Hughes W A , Brownsey R W , Denton R M
Published: August 2015

1. Intact rat epididymal fat-cells were incubated with 32Pi, and the intracellular proteins were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. One of the separated bands of phosphorylated proteins had an apparent subunit mol.wt. of 42 000, which is the same as that of the alpha-subunit of the pyruvate dehydrogenase complex. By using a combination of subcellular fractionation, immunoprecipitation with antiserum raised against pyruvate dehydrogenase complex and two-dimensional electrophoresis it was apparent that the incorporation into alpha-subunits accounted for 35–45% of the total incorporation into this band of phosphoproteins. 2. The increase in the initial activity of pyruvate dehydrogenase that follows brief exposure of fat-cells to insulin was shown to be associated with a decrease in the steady-state incorporation of 32P into the alpha-subunits of pyruvate dehydrogenase. 3. Tryptic peptide analysis of pyruvate dehydrogenase [32P]phosphate, labelled in intact fat-cells, indicated that three serine residues on the alpha-subunit were phosphorylated, corresponding to the three sites phosphorylated when purified pig heart pyruvate dehydrogenase was incubated with [gamma-32P]ATP. The relative phosphorylation of all three serine residues appeared to be similar in 32P-labelled alpha-subunits in both control and insulin-treated fat-cells.
Relative distribution of post-nuclear poly(A)-containing RNA abundance groups within the nuclear and post-nuclear polyadenylated and non-polyadenylated RNA populations of the lactating guinea-pig mammary gland
Authors: Bathurst Ian C. , Craig Roger K. , Herries David G. , Campbell Peter N.
Published: August 2015

1. RNA isolated from the post-nuclear supernatant of the lactating guinea-pig mammary gland was fractionated with oligo(dT)–cellulose into three populations; those that bound at ‘low salt’ [long poly(A) tracts, 78–32 nucleotides]; those that bound at ‘high salt’ [shorter poly(A) tracts, 48–21 nucleotides]; and those that did not bind [no poly(A) or short poly(A) tracts, <20 nucleotides]. Nuclear RNA was fractionated into two populations, those that bound in ‘low salt’ and those that did not bind. All the post-nuclear RNA fractions directed the synthesis of milk proteins in a Krebs II ascites cell-free system. 2. 3H-labelled DNA complementary to the post-nuclear poly-(A)-containing RNA population (low-salt fraction) was fractionated into abundant (milk-protein mRNA), moderately abundant and scarce sequences. This complementary DNA was then used to investigate the distribution of the mRNA sequences in the different RNA populations. This showed that all sequences were present in polyadenylated and non-polyadenylated fractions, but that major quantitative differences were apparent. The abundant milk-protein mRNA sequences predominated in the ‘low-salt’ post-nuclear poly(A)-containing RNA fraction, whereas the moderately abundant sequences predominated in the non-polyadenylated post-nuclear RNA fraction. In total cellular RNA, those sequences deemed initially to be moderately abundant within the ‘low-salt’ poly(A)-containing RNA population were present at a concentration very similar to those of the abundant milk-protein mRNA (approx. 6×105 copies of each sequence/cell). Similarly, analysis of the nuclear RNA populations showed that the ‘abundant’ and so-called ‘moderately abundant’ sequences were present in essentially identical concentrations (2×103 copies of each sequence/cell). The majority of these (90–95%) were non-polyadenylated. 3. The results are discussed in terms of the post-transcriptional mechanisms involved in the regulation of gene expression in the lactating guinea-pig mammary gland.
The preparation and purification of isolated rat corpus-luteum cells and their use in studying the relationship between cholesterol biosynthesis and the lutropin-stimulated formation of progesterone
Authors: McNamara B C , Cranna C E G , Booth R , Stansfield D A
Published: August 2015

Isolated luteal cells, prepared from superovulated rat ovaries by digestion with collagenase, were subjected to density-gradient centrifugation on Percoll to give a more highly purified preparation of luteal cells than has been reported previously. The cells formed progesterone when incubated in vitro; lutropin stimulated this steroidogenesis. Progesterone formation was linear for at least 2 h; a minimal lutropin concentration of 1.0 ng/ml was needed for stimulation and concentrations of 3.0 and 100 ng/ml gave half-maximal and maximal responses respectively. The cells were unresponsive towards hormones other than lutropin. Exposure to lutropin raised the cellular cyclic AMP concentration, and dibutyryl cyclic AMP, but not dibutyryl cyclic GMP, was as effective in stimulating steroidogenesis as was lutropin. Aminoglutethimide, an inhibitor of cholesterol side-chain cleavage, completely blocked progesterone formation by the cells, showing cholesterol side-chain cleavage to be an obligatory step in steroidogenesis by these cells. Neither the activity of 3-hydroxy-3-methylglutaryl-CoA reductase nor the incorporation of radioactively labelled acetate or mevalonate into cholesterol by cells incubated in vitro were detectable unless the rats had been treated previously with 4-aminopyrazolo[3,4-d]pyrimidine. In cells from rats so treated, compactin was found to block almost completely the incorporation of radioactively labelled acetate, but not of mevalonate, into cholesterol, indicating that this inhibitor acts in corpus luteum in the same way as it does in other tissues. In cells from rats not treated with 4-aminopyrazolo[3,4-d]pyrimidine compactin had no effect on progesterone formation in vitro, showing cholesterol biosynthesis to be unnecessary for the rapid steroidogenic response by luteal cells to lutropin.
The effect of complex-formation with polyanions on the redox properties of cytochrome c
Authors: Petersen L C , Cox R P
Published: August 2015

1. The stable complex formed between mammalian cytochrome c and phosvitin at low ionic strength was studied by partition in an aqueous two-phase system. Oxidized cytochrome c binds to phosvitin with a higher affinity than reduced cytochrome c. The difference was equivalent to a decrease of the redox potential by 22 mV on binding. 2. Complex-formation with phosvitin strongly inhibited the reaction of cytochrome c with reagents that react as negatively charged species, such as ascorbate, dithionite, ferricyanide and tetrachlorobenzoquinol. Reaction with uncharged reagents such as NNN‘N’-tetramethylphenylenediamine and the reduced form of the N-methylphenazonium ion (present as the methylsulphate) was little affected by complex-formation, whereas oxidation of the reduced cytochrome by the positively charged tris-(phenanthroline)cobalt(III) ion was greatly stimulated. 3. A similar pattern of inhibition and stimulation of reaction rates was observed when phosvitin was replaced by other macromolecular polyanions such as dextran sulphate and heparin, indicating that the results were a general property of complex-formation with polyanions. A weaker but qualitatively similar effect was observed on addition of inositol hexaphosphate and ATP. 4. It is suggested that the effects of complex-formation with polyanions on the reactivity of cytochrome c with redox reagents are mainly the result of replacing the positive charge on the free cytochrome by a net negative charge. Any steric effects on polyanion binding are small in comparison with such electrostatic effects.
Ornithine decarboxylase activity in insulin-deficient states
Authors: Conover Cheryl A. , Rozovski S. Jaime , Belur Eva R. , Aoki Thomas T. , Ruderman Neil B.
Published: August 2015

The activity of ornithine decarboxylase, the rate-controlling enzyme in polyamine biosynthesis, was determined in tissues of normal control rats and rats made diabetic with streptozotocin. In untreated diabetic rats fed ad libitum, ornithine decarboxylase activity was markedly diminished in liver, skeletal muscle, heart and thymus. Ornithine decarboxylase was not diminished in a comparable group of diabetic rats maintained on insulin. Starvation for 48h decreased ornithine decarboxylase activity to very low values in tissues of both normal and diabetic rats. In the normal group, refeeding caused a biphasic increase in liver ornithine decarboxylase; there was a 20-fold increase in activity at 3h followed by a decrease in activity, and a second peak between 9 and 24h. Increases in ornithine decarboxylase in skeletal muscle, heart and thymus were not evident until after 24–48h of refeeding, and only a single increase occurred. The increase in liver ornithine decarboxylase in diabetic rats was greater than in normal rats after 3h of refeeding, but there was no second peak. In peripheral tissues, the increase in ornithine decarboxylase with refeeding was diminished. Skeletal-muscle ornithine decarboxylase is induced more rapidly when meal-fed rats are refed after a period without food. Refeeding these rats after a 48h period without food caused a 5-fold increase in ornithine decarboxylase in skeletal muscle at 3h in control rats but failed to increase activity in diabetic rats. When insulin was administered alone or together with food to the diabetic rats, muscle ornithine decarboxylase increased to activities even higher than in the refed controls. In conclusion, these findings indicate that the regulation of ornithine decarboxylase in many tissues is grossly impaired in diabetes and starvation. They also suggest that polyamine formation in vivo is an integral component of the growth-promoting effect of insulin or some factor dependent on insulin.
Differential inhibition of ketogenesis by malonyl-CoA in mitochondria from fed and starved rats
Authors: Cook G A , Otto D A , Cornell N W
Published: August 2015

Rates of ketogenesis in mitochondria from fed or starved rats were identical at optimal substrate concentrations, but responded differently to inhibition by malonyl-CoA. Kinetic data suggest that the K1 for malonyl-CoA is greater in the starved animal. These results indicate that, for the regulation of ketogenesis in the starved state, the lower sensitivity of carnitine palmitoyltransferase to inhibition by malonyl-CoA may be more important than the concentration of malonyl-CoA.
Distribution of receptor sites on large glycoprotein molecules by electron microscopy. Application to epiglycanin
Authors: Slayter H S , Codington J F
Published: August 2015

Electron microscopy was used to map the loci of immunochemically active sites on individual glycoprotein molecules. The positions of specific galactose residues and asparagine-linked carbohydrate chains containing specific mannose residues in epiglycanin, a glycoprotein of extended conformation from the surface of TA3 mouse mammary tumour cells, were observed in complexes with Ricinus communis toxin and concanavalin A respectively. The maximum number of Ricinus communis toxin molecules attached to a single epiglycanin molecule was 23, and the average number was 16. Only one concanavalin A molecule was observed attached to any epiglycanin molecule, and this at one end of the molecule, suggesting the presence of only one receptor for this lectin. By means of this new approach for mapping specific residues, evidence has been obtained that suggests microheterogeneity in epiglycanin with respect to the locations of carbohydrate chains containing receptors for Ricinus communis toxin.
Kinetic and physical properties of the l-malate-NAD+ oxidoreductase from Methanospirillum hungatii and comparison with the enzyme from other sources
Authors: Storer A C , Sprott G D , Martin W G
Published: August 2015

The L-malate-NAD+ oxidoreductase of Methanospirillum hungatii was purified to homogeneity by using Blue Sepharose and ADP-Sepharose affinity chromatography. The molecular weight was estimated as 61 700 +/- 1900 by gel filtration and 64 200 +/- 1200 by ultracentrifugation. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated that the protein is composed of two polypeptide chains, each corresponding to 31 350 +/- 2150 daltons. Inhibition patterns obtained for malate, alpha-oxoglutarate and ADP established that the sequential reaction mechanism was ordered, with NADH serving as the first substrate. Intracellular concentrations of oxaloacetate approximated the Km value of 27 microM, but NADH was present at less than Km values. Comparison of the amino-acid composition of the L-malate-NAD+ oxidoreductase of M. hungatii and 22 others from prokaryotic and eukaryotic cells revealed a significant direct relationship between average hydrophobicity and the frequency of non-polar side chains, as well as a significant indirect relationship between average hydrophobicity and the polarity ratio. Calculations based on amino-acid-composition data indicated significant composition similarity between pairs of mammalian-cytoplasmic or pairs of mitochondrial L-malate-NAD+ oxidoreductases from various sources, but no significant composition similarity between any of the pairs of bacterial species examined.
Selective binding of zinc ions to heparin rather than to other glycosaminoglycans
Authors: Parrish R F , Fair W R
Published: August 2015

The relative binding affinity of Zn2+ to several glycosaminoglycans was determined by gel-filtration chromatography. Binding was observed only between Zn2+ and heparin. No binding was observed between Zn2+ and chondroitin 4-sulphate, chondroitin 6-sulphate, dermatan sulphate of hyaluronic acid. All of the glycosaminoglycans contained carboxy groups, but only heparin bound Zn2+. This observation suggests that, contrary to a previously proposed hypothesis, simple electrostatic interactions between the negatively charged carboxy groups of the glycosaminoglycans and the positively charged Zn2+ cannot explain the observed binding.
The preparation and properties of folate-binding protein from cow's milk
Authors: Salter D N , Scott K J , Slade H , Andrews P
Published: August 2015

An improved affinity-chromatographic method for the preparation of folate-binding protein from cow's milk is described. Under dissociating conditions the protein appeared homogeneous in the ultracentrifuge, with a molecular weight of 35 000 +/- 1500, but it was heterogeneous on electrophoresis and ion-exchange chromatography and evidently consisted of several glycoproteins with similar molecular weights that all bound folic acid. Overall, the protein contained a high proportion of half-cystine (18 residues/molecule) and 10.3% of carbohydrate. At saturation it bound approx. 1 mol of folate/mol of protein at pH 7.2. Equilibrium-dialysis measurements of the binding of folic acid and 5-methyltetrahydrofolate to the purified protein gave non-linear Scatchard plots, the shapes of which depended on pH. The results were interpreted in terms of ligand binding to a polymerizing system in which the affinity of ligand for monomer was greater than its affinity for polymer. When the protein concentration was similar to that in cow's milk, dissociation constants (Kd) for folate and 5-methyltetrahydrofolate were 3 nM and 5 nM respectively at pH 7.2 and 37 degrees C, whereas Kd for the binding of folate to monomer was about 50 pM. The properties of the binding protein are discussed in relation to its possible role in folate absorption in the gut.
Bile pigment synthesis in plants. Incorporation of haem into phycocyanobilin and phycobiliproteins in Cyanidium caldarium
Authors: Brown S B , Holroyd J A , Troxler R F , Offner G D
Published: August 2015

A procedure was developed whereby haem was taken up by dark-grown cells of the unicellular rhodophyte Cyanidium caldarium. These cells were subsequently incubated either in the dark with 5-aminolaevulinate, which results in excretion of phycocyanobilin into the suspending medium or incubated in the light, which results in synthesis and accumulation of phycocyanin and chlorophyll a within the cells. Phycocyanobilin was isolated from phycocyanin by cleavage from apoprotein in methanol. Phycocyanobilin prepared from phycocyanin or excreted from cells given 5-aminolaevulinate was methylated and purified by t.l.c. By using 14C labelling either in the haem or in 5-aminolaevulinate administered, haem incorporation into phycocyanobilin was demonstrated in both dark and light systems. Since chlorophyll a synthesized in the light in the presence of labelled haem contained no radioactivity, it was clear that haem was directly incorporated into phycocyanobilin and not first converted into protoporphyrin IX. These results clearly demonstrate phycocyanobilin synthesis via haem and not via magnesium protoporphyrin IX as has also been postulated.
The accumulation of amino acids by mouse ascites-tumour cells. Dependence on but lack of equilibrium with the sodium-ion electrochemical gradient
Authors: Hacking C , Eddy A A
Published: August 2015

1. The fluorescent dye 3,3′-dipropyloxadicarbocyanine was used to show that the tumour cells absorbed 2-aminoisobutyrate, glycine, L-leucine and L-isoleucine and certain other amino acids electrogenically. The Km values with respect to amino acid concentration ([A]o), obtained from the fluorescence assays, varied through the above series from 0.8 to 26 mM, with Vmax. fairly constant. 2. Similar Km values described the uptake of the 14C-labelled amino acids in five instances where this was measured. 3. Each amino acid lowered the membrane potential (E) by 10-20 mV when its cellular concentration ([A]i) had reached a steady value and [A]o was 10mM. In these experiments energy metabolism was maintained by glycolysis, 2,4-dinitrophenol was present and cellular respiration was inhibited. The corresponding net flow of amino acid through the Na+ symport was deduced by making use of the fact that the depolarization an amino acid initially caused was roughly proportional to the net influx of amino acid itself. 4. The steady-state depolarization was attributed to the presence of a leak pathway for the amino acid with a rate coefficient PA. As assayed in the absence of Na+, PA was about 5-fold larger for isoleucine than for glycine. 5. Direct estimates of Vmax./PA were similar to those inferred from the extent of depolarization in the steady state and [A]i. 6. A mathematical model was used to predict [A]i/[A]o in term of the measured values of [Na]o, [Na]i, E, Km and Vmax./PA. The predicted and observed values agreed fairly well when [A]o was 1 mM or 10 mM. 7. [A]i/[A]o varied from about 2.5 for 10 mM-isoleucine to 30 for 1 mM-2-aminoisobutyrate when delta microNa, expressed as a ratio, was ostensibly in the range 19-43. 8. The concentration of 2-aminoisobutyrate from a 0.1 mM solution in the presence or absence of ouabain was consistent with the model, whereas the concentration of isoleucine from a 0.1 mM solution exceeded the predicted values 2-5-fold. 9. The tumour cells concentrated 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid by a non-electrogenic mechanism, with which isoleucine may also interact.
Early effects of dimethylnitrosamine on the initiation of protein synthesis in mouse liver
Authors: Nygård O , Hultin T
Published: August 2015

1. Dimethylnitrosamine (37.5 mg/kg body wt.) was administered to mice by a single intraperitoneal injection, and the early effects on protein synthesis and related functions were studied in a liver S-30 system. 2. The incorporation of [14C]leucine into protein decreased rapidly after dimethylnitrosamine administration. The effect was associated with a decreased ability of the system to utilize methionyl-tRNAfMet and formyl-methionyl-tRNAfMet for 80 S ribosomal initiation-complex formation (primary initiation), and a loss of poly(A)-containing RNA from the postmicrosomal fraction. All the three effects developed simultaneously, and were clearly demonstrable within 15 min. 3. Initiation-complex formation in the polyribosomal fraction (re-initiation) was decreased to the same extent as the primary initiation, indicating that the initiation defect was not a result of the decrease in free mRNA. 4. The inhibition of initiation was only manifest at the joining of the 40 S pre-initiation complex to 60 S ribosomal subunits. It was not a result of methionyl-tRNAfMet deacylation. The functions between the formation of the methionyl-tRNAfMet-containing 80 S ribosomal complex and the first translocation on the ribosome were not involved, since the incorporation of formylmethionine into N-terminal polypeptides decreased to the same extent as the 80 S initiation-complex formation. 5. Inhibitors of protein synthesis (cycloheximide and pactamycin) decreased poly(A)-containing RNA in the postmicrosomal fraction in a similar way to dimethylnitrosamine.
Cell-free conversion of isopenicillin N into deacetoxycephalosporin C by Cephalosporium acremonium mutant M-0198
Authors: Baldwin J E , Keeping J W , Singh P D , Vallejo C A
Published: August 2015

In a cell-free system prepared by lysis of protoplasts of Cephalosporium acremonium mutant M-0198, isopenicillin N was converted into a penicillinase-resistant material that behaved like deacetoxycephalosporin C on high-pressure liquid chromatography analysis. This activity was found to be unstable to storage at -80 degrees C; 70-80% of the activity was lost after 1 day.
Computer simulation of protein self-association during small-zone gel filtration. Estimation of equilibrium constants
Authors: Stevens F J , Schiffer M
Published: August 2015

A simulation is developed that qualitatively describes the small-zone-gel-filtration behaviour of a reversibly associating protein. The results reflect the dependence of the apparent molecular weight of a reversibly associating protein on the equilibrium constant (KD) and initial concentration of the protein as well as the column length. The behaviour of a protein on an individual column is characterized and thus a means is provided for estimation of KD. The procedure is extended to describe the behaviour of a mixture of two proteins capable of heterologous as well as homologous association. This computer simulation has been applied in association studies of immunoglobulin light chains [Stevens, Westholm, Solomon & Schiffer (1980) Proc. Natl. Acad. Sci. 77, 1144--1148]. The KD value determined for the Bence--Jones protein Au (10(5) M-1) is close to the value (6.6 X 10(4) M-1) determined by other methods [Maeda, Steffen & Engel (1978) Biophys. Chem. 9, 57-64].
Purification of sphingomyelinase to apparent homogeneity by using hydrophobic chromatography
Authors: Jones C S , Shankaran P , Callahan J W
Published: August 2015

Placental sphingomyelinase has been purified to apparent homogeneity by a procedure that makes extensive use of hydrophobic interaction chromatography on sphingosylphosphocholine-CH-, octyl-, hexyl- and Blue-Sepharoses. Enzyme purification is about 10000- 14000-fold over starting extract with excellent yield (usually greater than 28%). Purification of bis-4-methylumbelliferyl phosphate phosphodiesterase activity generally paralleled that of sphingomyelinase during the final stages of the procedure. The enzyme also hydrolysed bis-p-nitrophenyl phosphate, but at a lower rate compared with bis-4-methylumbelliferyl phosphate. A single major protein was observed under non-denaturing conditions. Sphingomyelinase, denatured by reduction and alkylation, is composed of a major polypeptide chain with an apparent molecular weight of 89 100 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Two minor lower-molecular-weight components were consistently obtained at 47 500 and 30 700. These results were also obtained after maleoylation of the reduced and alkylated sample. The enzyme contains a blocked-N-terminal amino acid. An extensive search for contaminating enzymes revealed the presence of minor amounts of acid phosphatase, which were removed from the final enzyme sample. The highly purified enzyme is stable for several weeks when stored with Triton X-100 at 4 degrees C. The pure enzyme aggregates under denaturing and electrophoretic conditions and special care must be taken to ensure that hydrophobic bonding of the protein is decreased as much as possible. The reproducibility and large scale of this procedure should facilitate further study on the structure and kinetic properties of the enzyme.
Effects of aliphatic fatty acids on the binding of Phenol Red to human serum albumin
Authors: Kragh-Hansen U
Published: August 2015

Binding of Phenol Red to human serum albumin at pH 7.0 was studied by ultrafiltration (n1 = 1, K1 = 3.9 X 1-(4) M-1, n2 = 5, K2 = 9.6 X 10(2) M-1). The presence of 1 mol of octanoate or decanoate per mol of albumin caused a decrease in dye binding (dye/protein molar ratio 1:1), which, in contrast with additional fatty acid, was very pronounced: 1-8 mol of palmitate or stearate resulted in a small, and apparently linear, displacement of Phenol Red. The displacement effect of 1-5 mol of oleate, linoleate or linolenate per mol of albumin was comparable with that of the equimolar concentrations of palmitate or stearate. A higher molar ratios the unsaturated acids caused a drastic decrease in dye binding. The different Phenol Red-displacement effects of low molar ratios of medium-chain and long-chain fatty acids indicate that these acids have different high-affinity binding sites. In accordance with this proposal, low concentrations of stearate had only a small effect on the Phenol Red-displacement effect of octanoate. Phenol Red-binding curves in the presence of 1 mol of octanoate, 8 mol of stearate and 6 or 7 mol of linolenate per mol of albumin respectively indicated that the dye and the fatty acids do not complete for a common primary binding site. In contrast, a secondary Phenol Red-binding site could be identical with the primary octanoate-binding site. Furthermore, the primary Phenol Red-binding site could be the same as a secondary linolenate-binding site. Assignment of the different primary binding sites for Phenol Red and for medium-chain and long-chain fatty acids to a model of the secondary structure of albumin is attempted.
The organization of formate dehydrogenase in the cytoplasmic membrane of Escherichia coli
Authors: Graham A , Boxer D H
Published: August 2015

The arrangement of the proton-translocating formate dehydrogenase of the anaerobic respiratory chain of Escherichia coli within the cytoplasmic membrane was examined by direct covalent modification with non-membrane-permeant reagents. Three methods were employed, lactoperoxidase-catalysed radioiodination, labelling with diazotized [125I] di-iodosulphanilic acid and labelling with diazobenzene [35S] sulphonate. All three procedures yield consistent with the view that the two larger subunits of the enzyme, Mr 110000 and 32000, both occupy transmembranous locations within the membrane. In each case the modification of the Ca2+ or Mg2+-activated F1-ATPase was monitored, and all reagents employed correctly located this enzyme at the cytoplasmic face of the membrane. A procedure involving agglutination with specific antibodies is described which appears to fractionate membrane vesicles of mixed orientation into two populations, one with the same membrane orientation as that of spheroplasts and the other opposite orientation.
The prenatal and postnatal development of UDP-glucuronyltransferase activity towards bilirubin and the effect of premature birth on this activity in the human liver
Authors: Kawade N , Onishi S
Published: August 2015

Liver UDP-glucuronyltransferase activity towards bilirubin was studied in a total of 88 human subjects, including foetuses, premature and full-term newborn babies, infants, children, and adults. Determination of very low enzyme activity was performed by high-pressure liquid chromatography. Prenatal and postnatal changes of the activity can be divided into four developmental phases, i.e. middle foetal, late foetal, neonatal and early infantile, and mature. The activities of the first three phases corresponded to about 0.1, 0.1-1 and 1-100%, respectively, of the mature-phase values (mean +/- S.D.: 1320 +/- 514 microgram/h per g of liver, n = 27).
Choline metabolism and phosphatidylcholine biosynthesis in cultured rat hepatocytes
Authors: Pritchard P H , Vance D E
Published: August 2015

1. Adult rat hepatocytes were isolated by collagenase perfusion and were maintained in monolayer culture for 24h. 2. Choline metabolism and phosphatidylcholine biosynthesis were studied in these cells by performing pulse-chase studies at physiological concentrations (1-40 microM) of (Me-3H)-labelled or unlabelled choline in the culture medium. 3. During the 15 min pulse incubation, choline entering the cells was rapidly phosphorylated to phosphocholine or oxidized to betaine. Low concentrations of choline in the medium decreased the relative amount of choline oxidized. 4. During the 3 h chase period, the radioactivity in the phosphocholine pool was transferred to phosphatidylcholine. Very little radioactivity was associated with CDP-choline. These results provide good evidence that the rate-limiting step for phosphatidylcholine biosynthesis in these cultured hepatocytes is the conversion of phosphocholine into CDP-choline. Similar results were obtained for all concentrations of choline in the culture medium. 5. Cellular concentrations of phosphocholine were unaffected by the concentration of choline (1-40 microM) in the medium. 6. The majority of the label associated with betaine was secreted into the culture medium during the chase incubation. 7. From the pulse-chase studies, and the cellular phosphocholine concentrations, it was possible to estimate the rate of phosphatidylcholine biosynthesis (2.2, 2.8, 3.1 and 3.7 nmol/min per g wet weight of cells cultured in 1, 5, 10 and 40 microM-choline respectively for up to 4.25 h).