bluuebunny/crossref_metadata_2025 huggingface dataset:
Intracellular localization, isolation and characterization of two distinct varieties of superoxide dismutase from Neurospora crassa
Authors: Henry Landis E. A. , Cammack Richard , Schwitzguebel Jean-Paul , Palmer John M. , Hall David O.
Published: August 2015

1. Neurospora crassa was found to contain two distinct superoxide dismutases. 2. Most of the activity is associated with the cytosolic fraction and was shown to be the Cu/Zn-containing form of the protein. 3. Mitochondria isolated from Neurospora crassa showed two distinct superoxide dismutases: a cyanide-sensitive Cu/Zn-containing protein and a cyanide-insensitive form which probably contains manganese. 4. Localization experiments, using selective marker enzymes and digitonin fractionation, indicated that the cyanide-sensitive form is localized in the intermembrane space, whereas the cyanide-insensitive form is confined to the mitochondrial matrix space. 5. The cytosolic Cu/Zn-containing superoxide dismutase was isolated in high yields and extensively characterized by using e.p.r. spectroscopy, isoelectric focusing and analytical ultracentrifugation. 6. E.p.r. spectroscopy was used to monitor changes in the copper environment of the native protein after the addition of a number of potential inhibitors and after high-pH treatment. 7. Both of the cyanide-sensitive Cu/Zn-containing enzymes (cytosolic and mitochondrial) appeared to have identical properties which in turn were different from the cyanide-insensitive enzyme. 8. It is probable that the cyanide-insensitive enzyme was not previously detected, owing to its low amount (less than 10% of the total activity), greater lability than the cyanide-sensitive enzyme and the necessity of obtaining a mitochondrial-enriched fraction before its isolation.
Optical properties of japanese-lacquer-tree (Rhus vernicifera) laccase depleted of type 2 copper(II). Involvement of type-2 copper(II) in the 330nm chromophore
Authors: Morpurgo L , Graziani M T , Finazzi-Agrò A , Rotilio G , Mondovì B
Published: August 2015

1. Spectroscopic and functional properties of Japanese-lacquer-tree (Rhus vernicifera) laccase were re-investigated, with special emphasis on the relationships between the different types of copper centres (Types 1, 2, and 3). 2. On removal of the Type 2 Cu(II), a decrease of absorbance occurred in the wavelength region above 650 nm (delta epsilon 750 = 300 M-1 . cm-1) and around 330 nm (delta episom 330 up to 2200 M-1 . cm-1). 3. Reductive titrations with ascorbic acid or ferrocyanide showed that the electron-accepting capacity of the partial apoprotein is one electron-equivalent lower than that of the native protein, i.e. the protein two-electron acceptor is present in the oxidized state in spite of absorbance loss at 330 nm. 4. The 330 nm chromophore apparently depends on the presence of both the Type 2 and the Type 3 copper in the oxidized state. 5. This finding may have implications in the relative location of Type 2 and 3 copper centres and on the redox behaviour of laccase.
The stoichiometry and location of troponin I- and troponin C-like proteins in the myofibril of the bay scallop, Aequipecten irradians
Authors: Lehman W , Head J F , Grant P W
Published: August 2015

Localization and quantification studies were carried out on bay-scallop (Aequipecten irradians) striated-muscle troponin C- and troponin I-like proteins. Indirect immunofluorescence microscopy of scallop myofibrils stained with either rabbit anti-(scallop troponin I) or anti-(scallop troponin C) antibodies shows staining of all I-bands observed. The results of quantification studies using sodium dodecyl sulfate poly-acrylamide-gel electrophoresis of untreated scallop myofibrils, washed scallop myofibrils, and isolated scallop thin filaments indicate an actin/tropomyosin/troponin-C molar rationn of 7:1:1. The molar ratio for troponin I could not be determined in untreated myofibrils because of interfering bands; in washed myofibrils a value of 0.6 mol of troponin I/mol of tropomyosin was found. Purified scallop troponin C binds Ca2+ and interacts with scallop troponin I to relieve troponin I-induced inhibition of actomyosin ATPase. Although scallop troponin C is an acidic protein, it appears to be less acidic than troponin C from higher organisms. A calmodulin-like protein has been isolated from scallop striated muscle that activates bovine brain phosphodiesterase to the same extent as does brain calmodulin. Its amino acid composition and its electrophoretic mobility on alkaline 6 M-urea/polyacrylamide gels differs from that of scallop troponin C, and it appears not to be associated with thin filaments.
Subnuclear fractionation by mild micrococcal-nuclease treatment of nuclei of different transcriptional activities causes a partition of expressed and non-expressed genes
Authors: Dimitriadis G J , Tata J R
Published: August 2015

Extremely mild treatment with micrococcal nuclease of isolated nuclei yields subnuclear fractions in which the majority of RNA polymerase II transcriptional complexes formed in vivo are segregated [Tata & Baker (1978) J. Mol. Biol. 118, 249-272]. We now describe different approaches followed to established whether or not the nuclei are thus resolved into transcribed and non-transcribed DNA. First, we have compared the sensitivity to deoxyribonuclease I, which is known to digest preferably expressed genes as present in nuclei or chromatin, of three micrococcal-nuclease-derived fractions from nuclei of different transcriptional activities. In transcriptionally active nuclei (rat liver, hen liver and oviduct, and Xenopus liver), the DNA in a polynucleosomal fraction comprising 6-15% of DNA and the majority of template-engaged RNA polymerase II (fraction P2) was 10-50 times as sensitive to deoxyribonuclease I as the DNA in the other two fractions (fractions P1 and S, comprising 78-88% of total nuclear DNA as large polynucleosomal aggregates and 2-6% of DNA mostly as mononucleosomes, respectively). In transcriptionally inactive nuclei obtained from hen erythrocytes, micrococcal nuclease did not separate DNA into fractions exhibiting such differential sensitivities. Second, we have monitored the partition of an expressed gene. Hybridization of complementary DNA to Xenopus albumin mRNA revealed a 5-10-fold enrichment of the albumin (but not the globin) gene in the P2 fraction of nuclei from Xenopus liver in which this gene is fully expressed. Third, a large part of the nascent rapidly labelled RNA synthesized in vivo in rat liver nuclei was recovered in the micrococcal-nuclease-derived fraction that is more susceptible to digestion with deoxyribonuclease I. It is concluded that mild micrococcal-nuclease treatment of nuclei causes their separation into transcribed and non-transcribed DNA as determined by a number of very different criteria.
A new human erythrocyte variant (Ph) containing an abnormal membrane sialoglycoprotein
Authors: Tanner Michael J. A. , Anstee David J. , Mawby William J.
Published: August 2015

1. A new human erythrocyte variant (Ph) is described. The variant contains an unusual sialic acid-rich glycoprotein in addition to the blood-group-MN([unk])- and blood-group-Ss(δ)-active sialoglycoproteins found in normal erythrocytes. 2. The unusual component Ph has an apparent mol.wt. of 32000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The Ph component is not degraded during trypsin treatment of intact erythrocytes. 3. The Ph component was labelled by lacto-peroxidase-mediated radioiodination of intact erythrocytes and was found to be present in amounts approximately equimolar to α-sialoglycoprotein in the variant erythrocytes. 4. The Ph component had receptors for the lectins from Maclura aurantiaca (osage orange) and Triticum vulgaris (wheat-germ), but lacked a receptor for the Phaseolus vulgaris (red kidney bean) lectin, suggesting that it carries only O-linked oligosaccharides. 5. The presence of the Ph component in these erythrocytes does not correspond to any of the known blood-group-MNSs-related antigens examined. 6. We suggest that this component may be a hybrid polypeptide containing the N-terminal portion derived from normal δ-sialoglycoprotein, and the C-terminal portion from normal α-sialoglycoprotein, in a manner similar to the anti-Lepore haemoglobin.
Peptide fragmentation suitable for solid-phase microsequencing. Use of N-bromosuccinimide and BNPS-skatole {3-bromo-3-methyl-2-[(2-nitrophenyl)thio]-3H-indole}
Authors: Hunziker P E , Hughes G J , Wilson K J
Published: August 2015

BNPS-skatole (3-bromo-3-methyl-2-(2-nitrophenyl)thiol-3H-indole) and N-bromosuccinimide have been used to specifically cleave at peptide bonds after amino acids with available C-gamma=C-delta double bonds, i.e. tryptophan, tyrosine and histidine. The resulting C-terminal lactones conveniently attach to amino-glass supports for sequencing with DABITC (4-NN-dimethylaminoazobenzene 4′-isothiocyanate). Also, peptides having such C-termini, i.e. from a chymotryptic digest, can be readily made to react with these reagents and thus be easily attached and sequenced by solid-phase methods.
The carbohydrate moiety of band-3 glycoprotein of human erythrocyte membranes
Authors: Tsuji T , Irimura T , Osawa T
Published: August 2015

Band-3 glycoprotein was purified from human blood-group-A erythrocyte membranes by selective solubilization and gel chromatography on Sepharose 6B in the presence of sodium dodecyl sulphate. The purified glycoprotein was subjected to hydrazinolysis in order to release the carbohydrate moiety. The released oligosaccharides were N-acetylated and applied to a column of DEAE-cellulose. Most of the band-3 oligosaccharides obtained were found to be free of sialic acids. When this neutral fraction was subjected to gel chromatography on a column of Sephadex G-50, two broad peaks were observed indicating that the band-3 glycoprotein was heterogeneous in the size of the oligosaccharide moieties. All fractions from gel chromatography were found to contain galactose, mannose, N-acetylglucosamine and fucose. The higher-molecular-weight (mol.wt. 3000-8000) peak consisted of fucose, mannose, galactose, N-acetylglucosamine and N-acetylgalactosamine in a molar proportion of 1.6:3.0:8.4:10.5:0.2. Most of these oligosaccharides were digested with a mixture of beta-galactosidase and beta-N-acetylhexosaminidase after alpha-L-fucosidase treatment to give a small oligosaccharide with the structure alpha Man2-beta Man-beta GlcNAc-GlcNAc. Methylation studies and limited degradation by nitrous acid deamination showed that the oligosaccharides contained the repeating disaccharide Gal beta 1→4GlcNAc beta 1→3, with branching points at C-6 of some of the galactose residues. These results indicate that a major portion of the band-3 oligosaccharide has a common core structure, with heterogeneity in the numbers of the repeating disaccharides, and contains fucose residues both in the peripheral portion and in the core portion. Haemagglutination tests were also carried out to determine the blood-group specificities of the glycoprotein and the results demonstrated the presence of both blood-group-H and I antigenic activities.
The isolation of human-erythrocyte band-3 polypeptide labelled with a photosensitive hydrophobic probe
Authors: Wells E , Findlay J B
Published: August 2015

To investigate the intramembranous domains of the major band-3 polypeptide, human erythrocyte membranes were labelled with 1-azido-4-[125I]iodobenzene. The anion-exchange protein has been isolated by a new procedure that decreases possible contamination by other integral membrane proteins of similar molecular weight. After chemical modification, the protein was subjected to proteolytic cleavage by several proteinases to yield a distinct pattern of peptides, a few of which contained radioactivity.
Dietary regulation of mammary lipogenesis in lactating rats
Authors: Grigor M R , Warren S M
Published: August 2015

The proportion of medium-chain fatty acids (C8:0, C10:0 and C12:0) in rat milk increased significantly between day 4 and day 8 of lactation and for the remainder of lactation these acids comprised 40-50mol% of the total fatty acids. The milk fatty acid composition from day 8 was markedly dependent on the presence of dietary fat and altered to include the major fatty acids of the fats (peanut oil, coconut oil and linseed oil). The distribution of fatty acids made within the gland, however, was independent of dietary lipid and C8:0, C10:0 and C12:0 acids accounted for over 70% of the fatty acids made. The rates of lipogenesis in both the mammary gland and liver determined in vivo after the administration of 3H2O were affected by the presence of dietary lipid. In the mammary gland the rate for rats fed a diet containing peanut oil for 7 days was only one fifth that for rats fed a fat-free diet. Coconut oil also suppressed lipogenesis. Both dietary fats also suppressed lipogenesis in the liver.
Influence of nucleotides, cations and nucleoside triphosphatase inhibitors on the release of ribonucleic acid from isolated rat liver nuclei
Authors: Agutter P S
Published: August 2015

The reasons underlying reported discrepancies in the effects of ATP, ADP, adenosine 5′-[beta gamma-methylene]triphosphate, AMP + PPi, P-chloromercuribenzoate and F- on RNA efflux from isolated rat liver nuclei and on nuclear envelope nucleoside triphosphatase activity were investigated. The stimulatory effect of ADP was attributed to myokinase activity associated with the nuclei; this activity was eluted on repeated washing with nuclear incubation medium. In the absence of Ca2+ and Mn2+, ATP, adenosine 5′[beta gamma-methylene]triphosphate and AMP +PPi were found to promote release of both DNA and RNA. In the presence of 0.5 mM-Ca2+ and 9.3 mM-Mn2+, only ATP promoted RNA efflux to a significant extent. In the absence of spermidine, Ca2+ and Mn2+, nuclei released large quantities of DNA and RNA into the medium; this effect was promoted by p-chloromereuribenzoate. In the presence of the three cations, however, p-chloromercuribenzoate inhibited RNA efflux. F- caused a slight leakage of DNA from nuclei. The results are discussed in terms of models for the effects of ATP and analogues on RNA efflux and nuclear stability.
Stimulation of the alternative oxidase of Neurospora crassa by Nucleoside phosphates
Authors: Vanderleyden J , Peeters C , Verachtert H , Bertrand H
Published: August 2015

The alternative-oxidase-mediated succinate oxidase activity of Neurospora crassa decreases drastically when mitochondria are fractionated into submitochondrial particles or treated with deoxycholate. The activity, however, can be completely restored in the presence of nucleoside 5′-monophosphates. The purine nucleoside 5′-monophosphates are more effective than the pyrimidine homologues. 5′-GMP gives a 10-fold stimulation of the alternative-oxidase-mediated succinate oxidase activity in submitochondrial particles. A comparison is made with the results obtained earlier with Moniliella tomentosa [Hanssens & Verachtert (1976) J. Bacteriol. 125, 825–835; Vanderleyden, Van Den Eynde & Verachtert (1980) Biochem. J. 186, 309–316].
Consequences of hormone-induced desensitization of adenylate cyclase in intact cells
Authors: Swillens S , Lefort E , Barber R , Butcher R W , Dumont J E
Published: August 2015

A hypothesis on the role of the hormone-induced desensitization of adenylate cyclase is proposed. It is suggested that the desensitization process could provide the cell with a highly efficient cyclic AMP system for transmitting hormone stimulus without requiring a large energy consumption. Theoretical considerations show that in fact the desensitization phenomenon allows the cyclic AMP system to present a good compromise between the efficiency and economy requirements of the cells.
Alloxan reversibly impairs glucagon release and glucose oxidation by pancreatic A2-cells
Authors: Östenson Claes-Göran
Published: August 2015

Alloxan is known as a selective B-cell cytotoxic substance, and there is so far little evidence for a direct toxic effect on the other islet cell types. To elucidate further whether such effects occur, the actions of alloxan on glucagon release and glucose oxidation were studied in isolated normal or A2-cell-rich pancreatic islets of the guinea pig. The A2-cell-rich islets were obtained from animals injected with streptozotocin 1–2 weeks before islet isolation. After exposure to alloxan (2 or 5mm) in vitro for 30min at 4°C, the islets were incubated in media containing either 1.7mm-glucose or 16.7mm-glucose plus 30m-i.u. of bovine insulin/ml. In both types of islet, alloxan abolished the ability of glucose and insulin both to decrease glucagon release and to increase the rate of glucose oxidation. A high concentration of glucose (28mm) during exposure to alloxan protected against these injurious effects. Tissue culture of alloxan-treated islets for 24h in 5.5mm-glucose restored neither the suppressive effect of glucose on glucagon release nor the inhibition of glucose oxidation of the A2-cells. However, culture for 1 week completely normalized both the glucagon-secretory response and glucose oxidation by both kinds of islets. It is therefore concluded that alloxan affects the secretory mechanism of not only the B-cell but also of the islet A2-cell, although this latter cell type is not primarily destroyed by the drug. The data furthermore support the concept of a relationship between glucose metabolism and the glucose-mediated glucagon release of the A2-cell.
Effect of carnitine on mitochondrial oxidation of palmitoylcarnitine
Authors: Brass Eric P. , Hoppel Charles L.
Published: August 2015

The effects of carnitine on the metabolism of palmitoylcarnitine were studied by using isolated rat liver mitochondria. Particular attention was given to carnitine acyltransferase-mediated interactions between carnitine and the mitochondrial CoA pool. Carnitine concentrations less than 1.25mm resulted in an increased production of acetylcarnitine during palmitoylcarnitine oxidation. Despite this shunting of C2 units to acetylcarnitine formation, no change was observed in the rate of oxygen consumption or major product formation (citrate or acetoacetate). Further, no changes were observed in the mitochondrial content of acetyl-CoA, total acid-soluble CoA or acid-insoluble acyl-CoA. These observations support the concept, based on studies in vivo, that the carnitine/acylcarnitine pool is metabolically sluggish and the acyl-group flux low as compared with the CoA/acyl-CoA pool. Acid-insoluble acyl-CoA content was decreased and CoA content increased at carnitine concentrations greater than 1.25mm. When [14C]carnitine was used in the incubations, it was demonstrated that this resulted from acid-insoluble acylcarnitine formation from intramitochondrial acid-insoluble acyl-CoA mediated by carnitine palmitoyltransferase B. Again, the higher carnitine concentrations resulted in no changes in the rates of oxygen consumption or major product formation. The above effects of carnitine were observed whether citrate or acetoacetate was the major product of oxidation. In contrast, an increase in acetyl-CoA concentration was observed at high carnitine concentrations only when acetoacetate was the product. Since the rate of acetoacetate production was not changed, these higher acetyl-CoA concentrations suggest that a new steady state had been established to maintain acetoacetate-production rates. Since there was no change in acetyl-CoA concentration when citrate was the major product, a change in the activity of the pathway utilizing acetyl-CoA for ketone-body synthesis and the potential regulation of this pathway must be considered.
Quantitative analysis of proton-linked transport system. β-Galactoside exit in Escherichia coli
Authors: Booth I R , Hamilton W A
Published: August 2015

The exit of lactose and thiomethyl-beta-D-galactoside from Escherichia coli ML308-225 has been studied to determine the role of carrier-dependent (zero-trans efflux) and carrier-independent (leak) processes. On the basis of its sensitivity to p-chloromercuribenzene sulphonate the exit of lactose was found to be almost wholly mediated by the carrier. Consistent with this conclusion was the finding that the rate of exit of this sugar was dependent on the external pH, being considerably slower at acid pH. On the other hand exit of thiomethyl-beta-D-galactoside was found to be composed of both carrier-dependent and carrier-independent processes. Both processes exhibited first-order kinetics with the rate constants for zero-trans efflux and leak being 0.137 min-1 and 0.079 min-1, respectively. The relevance of these findings for out earlier proposal for the methods of attenuation of solute accumulation is discussed [Booth, Mitchell & Hamilton (1979) Biochem. J. 182, 687–696].
A developmental change in dolichyl phosphate mannose synthase activity in pig brain
Authors: Harford Joe B. , Waechter Charles J.
Published: August 2015

The initial rate of dolichyl phosphate mannose biosynthesis was measured in white-matter membranes from pig brain at various ages from before birth throughout the period of most rapid brain development. Dolichyl phosphate mannose synthase activity increased from prenatal values to a maximum in 3 week-old animals, and gradually decreased to adult values after 8 weeks of age. The nature of the developmental change was investigated by enzymic and biochemical comparisons of the membrane preparations from the most active age (3 weeks) and adult controls. The specific activity of dolichyl phosphate mannose synthase in preparations from actively myelinating animals was approx. 3-fold higher than adults when mannolipid formation was assayed with saturating concentrations of GDP-[14C]mannose and utilizing only endogenous acceptor lipid. No major variations were found in the apparent Km values for GDP-mannose or exogenous dolichyl monophosphate. However, the ratio of dolichyl phosphate mannose synthase activity for myelinating animals/adult animals decreased significantly when large amounts of exogenous dolichyl monophosphate were added to the incubation mixtures. Dolichyl phosphate mannose synthase activity was also compared in white-matter membranes depleted of endogenous dolichyl monophosphate by enzymic mannosylation or treatment with butanol. When these preparations were assayed with identical amounts of exogenous dolichyl monophosphate, the dolichyl monophosphate-depleted membranes from actively myelinating animals contained only 20–30% more dolichyl phosphate mannose synthase activity. Overall, these studies strongly suggest that the developmental change in dolichyl phosphate mannose synthase activity is due primarily to the presence of a relatively lower amount of endogenous dolichyl monophosphate being accessible to the mannosyltransferase in the white-matter membranes from adult animals.
Changes in the synthesis of ribosomal ribonucleic acid and of poly(A)-containing ribonucleic acid during the differentiation of intestinal epithelial cells in the rat and in the chick
Authors: Morrison A , Porteous J W
Published: August 2015

Epithelial cells were isolated from rat and chick small intestine by techniques which separated subpopulations of differentiating villus and upper crypt cells from each other and from populations of mitotically dividing lower crypt cells. Incorporation of precursors into epithelial-cell DNA, cytoplasmic rRNA and cytoplasmic poly(A)-containing RNA occurred in the lower crypt cells in vivo when precursor was supplied from the vascular system of the intestine. Incorporation of precursor into 28S and 18S rRNA continued in the upper crypt cells, but occurred to only a very slight extent (if at all) in villus cells, whereas incorporation into poly(A)-containing RNA continued (at a diminishing rate) as the differentiating cells migrated along the villi. When precursor was supplied from the intestinal lumen, its incorporation into DNA and into rRNA of crypt cells was not very different from that observed with the other mode of precursor administration, but incorporation into villus-cell poly(A)-containing RNA then occurred at essentially the same rate in all intestinal epithelial cells in vivo. Cytoplasmic poly(A)-containing RNA appeared to turn over in rat crypt cells with a half-life not exceeding 24 h; crypt-cell rRNA showed no turnover and no evidence could be found for the presence of ‘metabolic DNA’.
Glutamate, glutamine, aspartate, asparagine, glucose and ketone-body metabolism in chick intestinal brush-border cells
Authors: Porteous John W.
Published: August 2015

1. Suspensions of isolated chick jejunal columnar absorptive (brush-border) cells respired on endogenous substrates at a rate 40% higher than that shown by rat brush-border cells. 2. Added d-glucose (5 or 10mm), l-glutamine (2.5mm) and l-glutamate (2.5mm) were the only individual substrates which stimulated respiration by chick cells; l-aspartate (2.5 or 6.7mm), glutamate (6.7mm), glutamine (6.7mm), l-alanine (1 or 10mm), pyruvate (1 or 2mm), l-lactate (5 or 10mm), butyrate (10mm) and oleate (1mm) did not stimulate chick cell respiration; l-asparagine (6.7mm) inhibited slightly; glucose (5mm) stimulated more than did 10mm-glucose. 3. Acetoacetate (10mm) and d-3-hydroxybutyrate (10mm) were rapidly consumed but, in contrast to rat brush-border cells, did not stimulate respiration. 4. Glucose (10mm) was consumed more slowly than 5mm-glucose; the dominant product of glucose metabolism during vigorous respiration was lactate; the proportion of glucose converted to lactate was greater with 10mm- than with 5mm-glucose. 5. Glutamate and aspartate consumption rates decreased, and alanine and glutamine consumption rates increased when their initial concentrations were raised from 2.5 to 6.7 or 10mm. 6. The metabolic fate of glucose was little affected by concomitant metabolism of any one of aspartate, glutamate or glutamine except for an increased production of alanine; the glucose-stimulated respiration rate was unaffected by concomitant metabolism of these individual amino acids. 7. Chick cells produced very little alanine from aspartate and, in contrast to rat cells, likewise produced very little alanine from glutamate or glutamine; in chick cells alanine appeared to be predominantly a product of transmination of pyruvate derived from glucose metabolism. 8. In chick cells, glutamate and glutamine were formed from aspartate (2.5 or 6.7mm); aspartate and glutamine were formed from glutamate (2.5mm) but only aspartate from 6.7mm-glutamate; glutamate was the dominant product formed from glutamine (6.7mm) but aspartate only was formed from 2.5mm-glutamine. 9. Chick brush-border cells can thus both catabolize and synthesize glutamine; glutamine synthesis is always diminished by concomitant metabolism of glucose, presumably by allosteric inhibition of glutamine synthetase by alanine. 10. Proline was formed from glutamine (2.5mm) but not from glutamine (2.5mm)+glucose (5mm) and not from 2.5mm-glutamate; ornithine was formed from glutamine (2.5mm)+glucose (5.0mm) but not from glutamine alone; serine was formed from glutamine (2.5mm)+glucose (5mm) and from these two substrates plus aspartate (2.5mm). 11. Total intracellular adenine nucleotides (22μmol/g dry wt.) remained unchanged during incubation of chick cells with glucose. 12. Intracellular glutathione (0.7–0.8mm) was depleted by 40% during incubation of respiring chick cells without added substrates for 75min at 37°C; partial restoration of the lost glutathione was achieved by incubating cells with l-glutamate+l-cysteine+glycine.
Different handling of parathyrin by basal-lateral and brush-border membranes of the bovine kidney cortex
Authors: Mohr H , Hesch R D
Published: August 2015

The two parts of the bovine kidney cortex plasma membrane, the basal-lateral and the brush-border membrane, were simultaneously prepared from the same organ. Both types of membrane bound parathyrin, but only from the basal-lateral fraction was the hormone displaceable by its bioactive N-terminal fragment. In parallel, parathyrin-stimulated adenylate cyclase was predominantly found in basal-lateral membranes. The hormone was fragmented by both membrane types. Basal-lateral membranes generated fragments with a rather uniform size distribution (somewhat smaller than the intact peptide) and apparently preferred the hormone itself as a substrate. In contrast, the fragments produced by brush-border membranes were numberous small peptides.
Complex phosphorylation activity in neurosecretosomal membranes isolated from ox neurohypophyses
Authors: Treiman M , Worm-Petersen S , Thorn N A
Published: August 2015

Homogenates of neural lobes of bovine pituitary glands were fractionated on Ficoll gradients to yield neurosecretosomes (nerve endings). The neurosecretosomes were lysed in a hypo-osmotic buffer and the membranes were separated from the soluble components by centrifugation. On incubation with [gamma-32P]ATP this membrane preparation showed an endogenous phosphorylation activity, which was studied by means of gel electrophoresis in the presence of sodium dodecyl sulphate, and subsequent autoradiography. The major part of the [32P]Pi detected on the gel was shown to be incorporated into three protein bands, termed A, B and C, with minimal mol.wts. of 83 000, 59 000 and 47 000 respectively. The phosphorylation of these three proteins was studied under a variety of experimental conditions. The patterns obtained were partly similar. However, important individual differences were noted, particularly with respect to the effects of cyclic AMP, Mg2+ and Ca2+. On the basis of these differences, it is suggested that in this system the phosphorylation activity is heterogenous, bands A, B and C each reflecting the presence of a different site of phosphate turnover. The relationship of bands A, B and C to several of the previously described phosphoproteins in the brain is discussed.
An enzyme activity in normal and ataxia telangiectasia cell lines which is involved in the repair of γ-irradiation-induced DNA damage
Authors: Edwards M J , Taylor A M , Duckworth G
Published: August 2015

An enzyme that enhances the activity of DNA polymerase I (EC 2.7.7.7) for gamma-irradiated calf thymus DNA was demonstrated in cellular extracts of normal human fibroblasts and lymphoid-cell lines. This enzyme was found to be deficient in all cellular extracts of fibroblasts and lymphoid-cell lines examined from patients with the autosomal recessive disease ataxia telangiectasia. The activity in cellular extracts from normal fibroblasts was removed when heated to 100 degrees C for 2 min or when the assay was performed at 4 degrees C. No significant deficiency in primer-activating enzyme activity was observed in cell-free extracts of lymphoid lines from patients with xeroderma pigmentosum, Huntington's chorea or neurofibromatosis, or from an ataxia telangiectasia heterozygote.
Deterioration of rat liver mitochondria under conditions of metabolite deprivation
Authors: Parce J W , Spach P I , Cunningham C C
Published: August 2015

In a previous study [Parce, Cunningham & Waite (1978) Biochemistry 17, 1634-1639] changes in mitochondrial phospholipid metabolism and energy-linked functions were monitored as coupled mitochondria were aged in iso-osmotic sucrose solution at 18 degrees C. The sequence of events that occur in mitochondrial deterioration under the above conditions have been established more completely. Total adenine nucleotides are depleted early in the aging process, and their loss parallels the decline in respiratory control. Related to the loss of total adenine nucleotides is a dramatic decrease in ADP and ATP translocation (uptake). The decline of respiratory control is due primarily to a decrease in State-3 respiration; loss of this respiratory activity can be related to the decline in ADP translocation. Mitochondrial ATPase activity does not increase significantly until State-4 respiration has increased appreciably. At the time of loss of respiratory control the ATPase activity increases to equal the uncoupler-stimulated activity. The H+/O ratio and P/O ratios do not decrease appreciably until respiratory control is lost. Similarly, permeability of the membrane to the passive diffusion of protons increases only after respiratory control is lost. There observations reinforce our earlier conclusion that there are two main phases in mitochondrial aging. The first phase is characterized by loss of the ability to translocate adenine nucleotides. The second phase is characterized by a decline in the ability of the mitochondrion to conserve energy (i.e. maintain a respiration-driven proton gradient) and to synthesize ATP.
The effect of vitamin A deficiency on hepatic, renal and pulmonary glutathione S-transferase activities in the rat
Authors: Siddik Z H , Mimnaugh E G , Trush M A , Gram T E
Published: August 2015

Feeding male weanling rats on a vitamin A-deficient diet for 6 weeks resulted in significant increases (44-57%) in glutathione S-aryl-, S-aralkyl- S-alkyl- and S-epoxidetransferase activities in the liver cytosol. Only the S-aralkyl- (27%) and S-alkyltransferase (14%) activities were significantly increased in the kidney as a result of deficiency. There was no effect on any of the pulmonary glutathione S-transferase activities. The increases in hepatic transferase activities were due primarily to increases (25-96%) in the apparent Vmax. There were no changes in the apparant Km of any of the four drug substrates employed. With 3,4-dichloronitrobenzene as the second substrate, the apparent Km for glutathione was increased by over 2-fold in vitamin A-deficient livers as compared with controls. The relationship between these results and enhanced susceptibility to chemical carcinogens in vitamin A deficiency is briefly discussed, and comparison is made between the effects of this nutritional state and pretreatment with drug inducers on the glutathione S-transferases.
Differential effects of nitrated ricin and nitrated and dithionite-reduced ricin on protein-synthesis inhibition and transmembrane tramsport in eukaryotic cells
Authors: Dalrymple P N , Houston L L
Published: August 2015

Ricin, was nitrated with tetranitromethane and reduced with sodium dithionite. Of the 8.0 nitro groups incorporated, 3.2 were on the A chain and 5.1 were on the B chain. Nitrated ricin1 was somewhat less active than nitrated and reduced ricin1 in inhibiting protein synthesis in vitro, but both were highly inhibitory. However, the modified toxins were less than 1% as active as ricin in inhibiting protein synthesis in cultured cells. Indirect immunofluorescence assays demonstrated tha both modified toxins were specifically bound to the cell surface and could be displaced by galactose.
Binding of anhydrotrypsin to α2-macroglobulin
Authors: Sayers C A , Barrett A J
Published: August 2015

Radiolabelled anhydrotrypsin was bound by alpha 2M (alpha 2-macroglobulin) sufficiently tightly to resist separation during gel electrophoresis; 2 mol of anhydrotrypsin were bound/mol of alpha 2M, but the interaction differed in important respects from that between active proteinases and alpha 2M. Anhydrotrypsin was bound by the electrophoretically ‘fast’ form of alpha 2M, although much less effectively than by the ‘slow’ form. The inactive enzyme was displaced from alpha 2M by trypsin inhibitor, the order of effectiveness being aprotinin > soya-bean trypsin inhibitor > benzamidine. Saturation of alpha 2M with anhydrotrypsin did not prevent subsequent binding and inhibition of active trypsin by the alpha 2M, and the anhydrotrypsin was not displaced during this reaction. Anhydrotrypsin bound by alpha 2M retained its ability to react with antibodies against trypsin, whereas bound trypsin did not.
Stereospecificity of hepatic l-tryptophan 2,3-dioxygenase
Authors: Watanabe Y , Fujiwara M , Yoshida R , Hayaishi O
Published: August 2015

Tryptophan 2,3-dioxygenase [L-tryptophan–oxygen 2,3-oxidoreductase (decyclizing), EC 1.13.11.11] has been reported to act solely on the L-isomer of tryptophan. However, by using a sensitive assay method with D- and L-[ring-2-14C]tryptophan and improved assay conditions, we were able to demonstrate that both the D- and L-stereoisomers of tryptophan were cleaved by the supernatant fraction (30000 g, 30 min) of liver homogenates of several species of mammals, including rat, mouse, rabbit and human. The ratio of activities toward D- and L-tryptophan was species variable, the highest (0.67) in ox liver and the lowest (0.07) in rat liver, the latter being hitherto exclusively used for the study of hepatic tryptophan 2,3-dioxygenase. In the supernatant fraction from mouse liver, the ratio was 0.23 but the specific activity with D-tryptophan was by far the highest of all the species tested. To identify the D-tryptophan cleaving enzyme activity, the enzyme was purified from mouse liver to apparent homogeneity. The specific activities toward D- and L-tryptophan showed a parallel rise with each purification step. The electrophoretically homogeneous protein had specific activities of 0.55 and 2.13 mumol/min per mg of protein at 25 degrees C toward D- and L-tryptophan, respectively. Additional evidence from heat treatment, inhibition and kinetic studies indicated that the same active site of a single enzyme was responsible for both activities. The molecular weight (150000), subunit structure (alpha 2 beta 2) and haem content (1.95 mol/mol) of the purified enzyme from mouse liver were similar to those of rat liver tryptophan 2,3-dioxygenase. The assay conditions employed in the previous studies on the stereospecificity of hepatic tryptophan 2,3-dioxygenase were apparently inadequate for determination of the D-tryptophan cleaving activity. Under the assay conditions in the present study, the purified enzyme from rat liver also acted on D-tryptophan, whereas the pseudomonad enzyme was strictly specific for the L-isomer.
The kinetics of effector binding to phosphofructokinase. The binding of Mg2+-1,N6-ethenoadenosine triphosphate to the catalytic site
Authors: Roberts D , Kellett G L
Published: August 2015

1. The binding of the fluorescent ATP analogue, Mg2+-1,N6-etheno-ATP, to the catalytic site of rabbit skeletal muscle phosphofructokinase has been studied by stopped-flow fluorimetry [Roberts & Kellet (1979) Biochem. J. 183, 349–360]. 2. Binding of Mg2+-1,N6-etheno-ATP to the catalytic site is consistent with a two-step mechanism of the type: (formula: see text); in which the diffusion-controlled binding of ligand, L, is accompanied by prior interconversion of enzyme from one form, E, to another, E. 3. The allosteric activators, phosphate and cyclic AMP, which promote an R-type conformation, appear to stabilize slightly different conformations, R and R' respectively. 4. The binding of Mg2+-1,N6-etheno-ATP to the catalytic site is strongly affected by its binding to the inhibitory site. The rate constant for the displacement of Mg2+-1,N6-ethenol-ATP from the catalytic site, k32, is 470 +/- 35 s-1 for the R' conformation, whereas it is 6.0 +/- 0.09 s-1 for the T conformation induced by binding of Mg2+-1,N6-ethenol-ATP to the inhibitory site.
Preparation of resealed pig lymphocyte plasma-membrane vesicles
Authors: Johnstone A P , Crumpton M J
Published: August 2015

Pig lymphocyte plasma-membrane vesicles that are impermeable to large molecules were prepared by means of centrifugation on a continuous dextran gradient. They were isolated in a yield of 60–80% of the total plasma membrane recovered, and were characterized as being impermeable to macromolecules by their exclusion of inulin (mol.wt. 5200) and Dextran T10 (mol.wt. 10 000).
The effect of iodothyronines on the conversion of thyroxine into 3,3′-5-tri-iodothyronine in isolated rat renal tubules
Authors: Heyma P , Larkins R G , Campbell D G
Published: August 2015

Isolated rat renal tubules prepared by collagenase digestion were used to study the effects of 3,3′,5′-tri-iodothyronine (‘reverse T3’, rT3) and other iodothyronines on the formation of 3,3′,5-tri-iodothyronine (T3) from thyroxine (T4). rT3 inhibited the conversion with a dose response over the concentration range 1.5nM-1.5microM. The inhibition was competitive in nature. Both 3,3′-di-iodothyronine and 3′,5′-di-iodothyronine also inhibited the production of T3 and T4 in isolated rat renal tubules, but tetraiodothyroacetic acid and 3,5-di-iodothyronine were found to have no effect. These experiments demonstrate in an intact cell system that some naturally occurring iodothyronines have significant effects on T4 deiodination.
Inhibition of adenosine triphosphatase, 5-hydroxytryptamine transport and proton-translocation activities of resealed chromaffin-granule ‘ghosts’
Authors: Apps David K. , Pryde James G. , Sutton Raul , Phillips John H.
Published: August 2015

1. Highly purified resealed chromaffin-granule ‘ghosts’ were assayed for ATPase and ATP-driven H+-translocation and 5-hydroxytryptamine-uptake activities, and for 5-hydroxytryptamine uptake driven by an imposed transmembrane H+-gradient. The effects of several inhibitors on these activities were studied. 2. Dicyclohexylcarbodi-imide inhibits all of these activities, but not in parallel; at low concentrations it decreases the permeability of the membrane to protons. 3. 4-Chloro-7-nitrobenzofuran (Nbf-Cl) and silicotungstate inhibit ATP-dependent activities, without effect on 5-hydroxytryptamine uptake driven by an imposed H+-gradient. 4. Tributyltin chloride inhibits all of the activities. 5. Treatment of the ‘ghosts’ with low concentrations of urea inhibits 5-hydroxytryptamine uptake and ATP-dependent generation of a transmembrane H+-gradient, without inhibiting ATPase activity. 6. Nbf-Cl and silicotungstate are without effect on the rate of leakage of 5-hydroxytryptamine from preloaded ‘ghosts’, whereas dicyclohexylcarbodi-imide and tributyltin chloride accelerate the rate of leakage. 7. Treatment of the membranes with 14C-labelled Nbf-Cl labels several proteins; membranes treated with dicyclohexyl[14C]carbodi-imide are labelled predominantly in a protein of low molecular weight, which may be analogous to the mitochondrial H+-conducting proteolipid. 8. It is concluded that Nbf-Cl and silicotungstate inhibit the H+-translocating ATPase of the granule membrane; that dicyclohexylcarbodi-imide inhibits the ATPase, and inhibits 5-hydroxytryptamine accumulation by accelerating leakage of the amine; and that the effects of tributyltin chloride are due to inhibition of the ATPase, and collapse of the transmembrane H+-gradient through OH−-anion exchange.
Nucleoside transport in human and sheep erythrocytes. Evidence that nitrobenzylthioinosine binds specifically to functional nucleoside-transport sites
Authors: Jarvis S M , Young J D
Published: August 2015

Nitrobenzyl[35S]thioinosine binding and nitro[3H]benzylthioinosine binding to nucleoside-permeable and nucleoside-impermeable sheep erythrocyte membranes was investigated, and compared with that found for human erythrocytes. High-affinity nitrobenzylthioinosine-binding sites (apparent KD congruent to 1 nM) were present on human and nucleoside-permeable but not nucleoside-impermeable sheep erythrocyte membranes (8400 and 18 sites/cell for human and sheep nucleoside-permeable sheep erythrocytes was displaced by nitrobenzylthioguanosine and dipyridamole. Uridine, inosine and adenosine inhibited binding. The smaller number of nitrobenzylthioinosine sites on nucleoside-permeable cells compared with human erythrocytes corresponded to a considerably lower Vmax. for uridine influx in these cells (0.53 × 10(-20) mol/cell per s at 25 degrees C compared with 254 × 10(-20) mol/cell per s). It is suggested that high-affinity nitrobenzylthioinosine binding represents a specific interaction with functional nucleoside-transport sites. The uridine-translocation capacity for each transport site at 25 degrees C is 180 molecules/site per s for both nucleoside-permeable sheep cells and human erythrocytes (assuming a 1:1 interaction between nitrobenzylthioinosine and the nucleoside-transport system).
Cerebral ribosomal protein phosphorylation in experimental hyperphenylalaninaemia
Authors: Roberts S , Morelos B S
Published: August 2015

Investigations were carried out on the effects of phenylalanine loading on ribosomal protein phosphorylation in cerebral cortices of infant rats. Administration of L-phenylalanine intraperitoneally, in doses of 1 or 2 mg/g body wt., resulted within 30 min in a significant decrease in incorporation of radioactivity from intracisternally administered [32P]Pi into constitutive ribosomal proteins of the cerebral 40S subunit. This phenomenon was not accompanied by significant variations in 32P uptake into the cerebral cytosol. Incorporation of radioactivity into ribosomal proteins of the cerebral 60S subunit exhibited only minor variations under these circumstances. Alterations in the phosphorylation state of cerebral 40S ribosomal proteins induced by phenylalanine loading involved principally the S6 protein, which exists in multiple states of phosphorylation. The proportions of the more highly phosphorylated congeners of this protein were markedly decreased, as detected by two-dimensional electrophoretograms and autoradiographs of the cerebral 40S ribosomal proteins. Phenylalanine loading also altered the relative extent of phosphorylation of the S6 protein in cerebral polyribosomes and monoribosomes. In control animals, the specific radioactivity of 40S proteins in cerebral polyribosomes was five to ten times that of 40S proteins in the monoribosome population. At 1 h after phenylalanine administration, the specific radioactivities of 40S proteins in the two ribosome populations tended to approach equality. These alterations in ribosomal protein phosphorylation were accompanied by a decrease in the proportion of polyribosomes in purified ribosome preparations isolated from cerebral cortices of phenylalanine-treated infant rats. In animals given the higher dose of phenylalanine (2 mg/g body wt.), subsequent administration of a mixture of seven neutral amino acids, which resulted in partial recovery of polyribosomes, also tended to reverse the changes in ribosomal protein phosphorylation. Variations in the activities of ribonuclease enzymes in the cerebral cytosol were also observed under these conditions. Administration of phenylalanine increased the activities of cerebral ribonucleases, whereas subsequent treatment with the amino acid mixture partly reversed this effect. The results suggest that alterations in cerebral ribosomal protein phosphorylation, ribosome aggregation and ribosome function are interrelated in experimental hyperphenylalaninaemia.
The site of cartilage matrix degradation
Authors: Dingle J T , Dingle T T
Published: August 2015

1. A ‘cavity’ organ culture method was developed, which allowed the insertion within bovine nasal cartilage of 10 microliter of 35S-labelled proteoglycan aggregate. 2. Stimulation of the tissue with retinol or catabolin led to degradation of both the cartilage matrix and the proteoglycan within the cavity. 3. Proteoglycan aggregate trapped in polyacrylamide beads was not broken down in the cavities of living cartilage, except under autolytic conditions. 4. It is suggested that degradation of proteoglycan aggregate by chondrocytes occurs in a pericellular region.