bluuebunny/crossref_metadata_2025 huggingface dataset:
The isolation and properties of the protein calliphorin
Authors: Munn E. A. , Feinstein A. , Greville G. D.
Published: August 2015

1. A procedure for the isolation of the protein calliphorin from larvae and pupae of the blowfly Calliphora erythrocephala is described. 2. The calliphorin preparation shows a single component in the ultracentrifuge at pH6.3 and gives a single band when stained for protein after agar-gel or starch-gel electrophoresis at pH6.3 or 8.6. Immunoelectrophoresis yields only one arc, associated with the stained spot, to a rabbit antiserum known to react with 13 other soluble components of Calliphora pupae. 3. Calliphorin has s020,w 19.4S, D020,w 3.25×10−7cm2·s−1and f/f0 1.22, indicating a molecular weight of 528000 and a compact symmetrical structure. The molecular weight determined by the meniscus-depletion sedimentation-equilibrium method is 529000. 4. In 6.2m-guanidine hydrochloride calliphorin dissociates into six components each with a molecular weight of about 87000. Calliphorin reversibly dissociates into components with sedimentation coefficients of about 7S as the pH is raised progressively above pH6.5. 5. Calliphorin has an unusually high tyrosine and phenylalanine content (442 and 400mol/mol of protein respectively), a relatively high methionine content (162mol/mol of protein) and very little cystine or cysteine (18mol/mol of protein). The E280/E250 ratio is 3.2. The pure protein contains 0.4–0.5% carbohydrate. 6. When examined in the electron microscope by the negative staining technique the protein is seen to consist of particles which are right prisms, being 105Å wide and 65Å high, rectangular in side view and curvilinear equilateral triangles in surface view.
Metabolic effects of propionate in normal and vitamin B12-deficient rats
Authors: Williams D. L. , Spray G. H. , Hems R. , Williamson D. H.
Published: August 2015

1. Administration of propionate caused a twofold increase in the concentrations of lactate and pyruvate in the blood of vitamin B12-deficient rats, whereas there was a slight decrease in lactate and a 50% increase in pyruvate in normal rats. 2. Concentrations of total ketone bodies in the blood of normal rats were not significantly altered by propionate administration but the [3-hydroxybutyrate]/[acetoacetate] ratio decreased from 3.0 to 2.0. In the vitamin B12-deficient rats there was a 40% decrease in total ketone bodies and a change in the ratio from 3.4 to 1.2. 3. The changes in the concentration of ketone bodies in freeze-clamped liver preparations were similar in pattern to those observed in blood. 4. Propionate administration caused a decrease in the concentration of acetyl-CoA in the livers of both groups of animals, but the absolute decrease was greater in the vitamin B12-deficient group. The decrease in the concentration of CoA was similar in both groups. 5. As in blood, there were threefold increases in the concentrations of lactate and pyruvate in the livers of the vitamin B12-deficient rats after propionate administration, whereas there was no significant change in the concentrations of these metabolites in the normal rats. 6. There was a 50% inhibition of glucose synthesis in perfused livers from vitamin B12-deficient rats when lactate and propionate were substrates as compared with lactate alone. 7. It is concluded that the conversion of lactate into glucose is inhibited in vitamin B12-deficient rats after propionate administration, and that this effect is due to inhibition of the pyruvate carboxylase step resulting from a decrease in acetyl-CoA concentration and a postulated increase in methylmalonyl-CoA concentration.
The effect of hypophysectomy and growth hormone replacement on albumin synthesis and catabolism in the rat
Authors: Kernoff L. M. , Pimstone B. L. , Solomon J. , Brock J. F.
Published: August 2015

Albumin synthesis and catabolism were respectively measured by McFarlane's (1963) sodium [14C]carbonate method and I-labelled albumin in hypophysectomized rats, both untreated and treated with growth hormone. Hypophysectomy resulted in a decrease in both albumin synthesis and catabolic rates. These changes as shown by pair-feeding experiments could not be ascribed to decreased food intake alone. Growth hormone was shown to partially restore both albumin synthesis and catabolic rates. It is proposed that growth hormone stimulates albumin synthesis and that its effect on albumin catabolism is secondary to changes in the mass of the intravascular albumin pool.
Metabolism of perhydroanthracenes in the rabbit
Authors: Robertson J. S. , Dunstan P. J.
Published: August 2015

1. The metabolism in rabbits of several perhydroanthracenes was investigated. All compounds were to different degrees excreted in the urine as glucuronides. 2. The aglycones of the glucuronides were found to be racemic secondary alcohols, having the hydroxyl group at a β-methylene carbon. Where determinable, the hydroxyl group was shown to have an equatorial configuration. 3. These results suggested that hydroxylation was enzyme mediated, and, when compared with the results from studies on other alicyclic hydrocarbons, that the same enzyme system participated generally in the hydroxylation of such hydrocarbons. 4. A model for the enzyme active site is proposed to accord with the pattern of hydroxylation observed.
Bovine pepsinogens and pepsins. The sequence around a reactive aspartyl residue
Authors: Meitner Patricia A.
Published: August 2015

The specific inhibitor, N-diazoacetylnorleucine methyl ester reacts stoicheiometrically with bovine pepsin resulting in a simultaneous loss of all enzymic activity. A peptide containing a modified aspartyl group was isolated from bovine pepsin labelled with 14C-labelled inhibitor. The aspartic acid residue is presumed to be part of the active centre and is in the same heptapeptide sequence as in porcine pepsin: Ile-Val-Asp-Thr-Gly-Thr-Ser.
The substrate specificity of acid α-glucosidase from rabbit muscle
Authors: Palmer T. N.
Published: August 2015

1. Acid α-glucosidase was purified 3500-fold from rabbit muscle. 2. The enzyme was activated by cations, the degree of activation varying with the substrate. Enzyme action on glycogen was most strongly activated and activation was apparently of a non-competitive type. With rabbit liver glycogen as substrate, the relative Vmax. increased 15-fold, accompanied by an increase in Km from 8.3 to 68.6mm-chain end over the cation range 2–200mm-Na+ at pH4.5. Action on maltose was only moderately activated (1.3-fold, non-competitively) and action on maltotriose was marginally and competitively inhibited. 3. The pH optimum at 2mm-Na+ was 4.5 (maltose) and 5.1 (glycogen). Cation activation of enzyme action on glycogen was markedly pH-dependent. At 200mm-Na+, the pH optimum was 4.8 and activity was maximally stimulated in the range pH4.5–3.3. 4. Glucosidase action on maltosaccharides was associated with pronounced substrate inhibition at concentrations exceeding 5mm. Of the maltosaccharides tested, the enzyme showed a preference for p-nitrophenyl α-maltoside (Km 1.2mm) and maltotriose (Km 1.8mm). The extrapolated Km for enzyme action on maltose was 3.7mm. 5. The macromolecular polysaccharide substrate glycogen differed from linear maltosaccharide substrates in the kinetics of its interaction with the enzyme. Activity was markedly dependent on pH, cation concentration and polysaccharide structure. There was no substrate inhibition. 6. The enzyme exhibited constitutive α-1,6-glucanohydrolase activity. The Km for panose was 20mm. 7. The enzyme catalysed the total conversion of glycogen into glucose. The hydrolysis of α-1,6-linkages was apparently rate-limiting during the hydrolysis of glycogen. 8. Enzyme action on glycogen and maltose released the α-anomer of d-glucose. 9. The results are discussed in terms of the physiological role of acid α-glucosidase in lysosomal glycogen catabolism.
The crystalline yolk-platelet proteins and their soluble plasma precursor in an amphibian, Xenopus laevis
Authors: Redshaw M. R. , Follett B. K.
Published: August 2015

A single lipophosphoprotein complex, vitellogenin, was isolated and purified from the plasma of oestrogen-stimulated female toads by preparative ultracentrifugation and chromatography on TEAE-cellulose (triethylaminoethylcellulose). The protein contains 12% lipid, 1.5% phosphorus, 1.6% calcium and smaller amounts of carbohydrates and biliverdin. In amino acid composition it is identical with total yolk-platelet protein. The platelet protein, however, is fractionated on TEAE-cellulose into two components, a high-molecular-weight lipovitellin and a smaller phosvitin. Analyses of the soluble plasma vitellogenin suggest that it is a complex of two phosvitin molecules covalently bound to one lipovitellin dimer, and that it is the immediate precursor of the yolk proteins, into which it is converted by a molecular rearrangement. Uptake of vitellogenin from the plasma into the growing oocyte, and its subsequent crystallization as a yolk platelet, appear to be enhanced by gonadotrophic hormones.
The amino acid sequence of cytochrome c from Abutilon theophrasti Medic. and Gossypium barbadense L. (cotton)
Authors: Thompson E. W. , Notton B. A. , Richardson M. , Boulter D.
Published: August 2015

The amino acid sequences of Abutilon and Gossypium cytochromes c were determined on 1μmol of protein. The molecules consist of 111 residues and are homologous with other mitochondrial plant cytochromes c. Experimental details are given in a supplementary paper that has been deposited as Supplementary Publication SUP 50005 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1971), 121, 7.
Synthesis of phosphoenolpyruvate from propionate in sheep liver
Authors: Smith R. M. , Osborne-White W. S.
Published: August 2015

1. Utilization of propionate by sheep liver mitochondria was stimulated equally by pyruvate or α-oxoglutarate, with formation predominantly of malate. Pyruvate increased conversion of propionate carbon into citrate, whereas α-oxoglutarate increased formation of phosphoenolpyruvate. The fraction of metabolized propionate converted into phosphoenolpyruvate was about 17% in the presence or absence of α-oxoglutarate and about 7% in the presence of pyruvate. Pyruvate consumption was inhibited by 80% by 5mm-propionate. 2. Compared with rat liver, sheep liver was characterized by very high activities of phosphoenolpyruvate carboxykinase and moderately high activities of aconitase in the mitochondria and by low activities of ‘malic’ enzyme, pyruvate kinase and lactate dehydrogenase in the cytosol. Activities of phosphoenolpyruvate carboxy-kinase were similar in liver cytosol from rats and sheep. Activities of malate dehydrogenase and NADP-linked isocitrate dehydrogenase in sheep liver were about half those in rat liver. 3. The phosphate–dicarboxylate antiport was active in sheep liver mitochondria, but compared with rat liver mitochondria the citrate–malate antiport showed only low activity and mitochondrial aconitase was relatively inaccessible to external citrate. The rate of swelling of mitochondria induced by phosphate in solutions of ammonium malate was inversely related to the concentration of malate. 4. The results are discussed in relation to gluconeogenesis from propionate in sheep liver. It is proposed that propionate is converted into malate by the mitochondria and the malate is converted into phosphoenolpyruvate by enzymes in the cytosol. In this way sufficient NADH would be generated in the cytosol to convert the phosphoenolpyruvate into glucose.
The effect of noradrenaline on glyceride synthesis and oxidative metabolism in vitro in the brown fat of newborn rabbits
Authors: Knight B. L. , Myant N. B.
Published: August 2015

1. The effect of noradrenaline on the synthesis of glyceride from [U-14C]glucose and on gas exchange in the brown fat of newborn rabbits in vitro was investigated. 2. The specific radioactivity of l-glycerol 3-phosphate was lower than that of lactate, presumably because glycerol derived from glyceride was rephosphorylated by glycerokinase. 3. In the basal state more than 25% of the total respiration was due to pyruvate oxidation. Noradrenaline stimulated glyceride synthesis and total respiration without changing the proportion of the total respiration due to pyruvate oxidation. 4. The extra ADP released by noradrenaline stimulation of glyceride synthesis could not have supported more than 2% of the observed increase in substrate oxidation if mitochondria from brown-fat-cells remain fully coupled in the stimulated state, but could have supported about one-third of the observed increase if they become uncoupled in the presence of noradrenaline.
Pteroylglutamate derivatives in Pisum sativum L. Biosynthesis of cotyledonary tetrahydropteroylglutamates during germination
Authors: Roos A. J. , Cossins E. A.
Published: August 2015

1. The concentrations of pteroylglutamate derivatives in the cotyledons of pea (Pisum sativum L.) seedlings were determined by microbiological assay by using Lactobacillus casei (A.T.C.C. 7469), Streptococcus faecalis (A.T.C.C. 8043) and Pediococcus cerevisiae (A.T.C.C. 8081). During germination the pteroylglutamate content of the cotyledons increased rapidly from 0.2μg/g dry wt. to 4.0μg/g dry wt., the maximum values being reached approx. 120h after imbibition. 2. Approx. 50% of the pteroylglutamate pool of 3-day-old seedlings was accounted for by highly conjugated derivatives. The concentrations of such derivatives were greatest when precautions were taken to inactivate endogenous enzymes before extraction of the tissues. 3. Individual derivatives present in the tissue extracts were separated by chromatography on DEAE-cellulose. Synthesis of the major derivative, 5-methyltetrahydropteroylmonoglutamate, was inhibited by administration of 0.1mm-aminopterin and -amethopterin solutions during imbibition. Under these conditions pteroylglutamic acid accumulated in the tissues. 4. Feeding experiments employing [2-14C]pteroylglutamic acid and 5[14C]-methyltetrahydro-pteroylmonoglutamic acid revealed that both compounds were incorporated into conjugated and unconjugated derivatives of the pteroylglutamate pool.
The localization of 1,25-dihydroxycholecalciferol in bone cell nuclei of rachitic chicks
Authors: Weber J. C. , Pons V. , Kodicek E.
Published: August 2015

1. A simple technique has been developed to obtain subcellular fractions of chick bone. The method yielded 60–70% of total DNA in the nuclear debris fraction and 80–90% of total 14C recovered in bone after a dose of radioactive vitamin D. 2. After a dose of [4-14C,1,2-3H2]cholecalciferol (0.5μg) was given to vitamin D-deficient chicks, the time-course of total 14C radioactivity in the epiphysis, metaphysis and diaphysis of proximal tibiae was measured. The maximum concentrations were reached at 6h, corresponding to a similar peak of radioactivity in blood, decreasing until 24h and indicating the dependence on the circulating 14C and on the blood supply of the three bone components. 3. The 14C radioactivity of cholecalciferol and 25-hydroxycholecalciferol (expressed per mg of DNA) followed the pattern of incorporation of total 14C radioactivity in all three bone components. The more polar metabolite fraction reached a peak of radioactivity at 6–9h and maintained its concentration over the 24h period studied in all anatomical bone components. 4. After a dose of [4-14C,1-3H]cholecalciferol (0.5μg) was given to vitamin D-deficient chicks, the subcellular distribution was studied. At 24h after dosing, the nuclear fraction contained 27% and the supernatant fraction had 67% of total 14C recovered in the bone filtrate. When the 14C in the residual bone fragments was included, the nuclear fraction contained up to 35% of the total radioactivity in the bone. 5. The subcellular distribution pattern of individual vitamin D metabolites indicated that the purified nuclear fraction concentrated the polar metabolite, which lost 3H at C-1, so that 77% of the radioactivity could be accounted for by 1,25-dihydroxycholecalciferol. The supernatant fraction contained smaller amounts of 1,25-dihydroxycholecalciferol (9%), with 66% of 25-hydroxycholecalciferol forming the major metabolite, corresponding to its concentration found in blood at 24h. 6. The preferential accumulation of 1,25-dihydroxycholecalciferol in the nuclear fraction and the overall pattern of other metabolites, found previously in intestinal tissue, suggests a similar mechanism of action in bone to that postulated for the intestinal cell in calcium translocation.
The identification of the site of action of NN′-dicyclohexylcarbodi-imide as a proteolipid in mitochondrial membranes
Authors: Cattell K. J. , Lindop C. R. , Knight I. G. , Beechey R. B.
Published: August 2015

Mitochondrial membranes were incubated with NN′-dicyclohexyl[14C]carbodi-imide, which irreversibly inhibited the partial reactions of oxidative phosphorylation by 95–100%. Solutions of the membranes were analysed on polyacrylamide gels. Of the radioactivity recovered from the gels 90% was shown to be associated with a single protein of molecular weight about 10000. The radioactive protein and associated phospholipid was solubilized from the membrane by extraction with chloroform–methanol mixtures and was concentrated 50-fold by solvent fractionation and adsorption chromatography on Sephadex LH-20. Several protein–radioactivity peaks were obtained by Sephadex LH-20 chromatography. However, 90–100% of the radioactivity in each peak was shown to be associated with a single protein similar to the major radioactive protein observed in electrophoretograms of the membrane solutions. It is concluded that dicyclohexylcarbodi-imide inhibits mitochondrial oxidative phosphorylation by reacting covalently with a group on this chloroform–methanol-soluble protein. The possible role of this protein in oxidative phosphorylation is discussed.
Calcium binding by human erythrocyte membranes. Significance of carboxyl, amino and thiol groups
Authors: Forstner Janet , Manery J. F.
Published: August 2015

1. The role of the ionized carboxyl groups of proteins of the erythrocyte membrane as Ca2+ receptor sites was investigated. A water-soluble carbodi-imide [1-cyclohexyl-3-(2-morpholinoethyl)carbodi-imide methotoluene-p-sulphonate], referred to as carbodi-imide reagent, and glycine methyl ester were used to modify the free carboxyl groups of the membrane. The degree of modification was estimated from amino acid analyses, which showed the amount of glycine incorporated. As the concentration of carbodi-imide reagent was raised (0.1–0.4m) incorporation of glycine increased and Ca2+ binding decreased by about 77%. At 0.4m-carbodi-imide reagent all of the binding of Ca2+ to protein was abolished and it was estimated that about 37% of the side-chain carboxyl groups of aspartic acid plus glutamic acid had been blocked by glycine. 2. Acetylation of all of the free amino groups was achieved by incubating the erythrocyte ‘ghosts’ at pH10.3 with acetic anhydride (10–15mg/10mg of ‘ghost’ protein). Acetylation increased by 1.5-fold the capacity of the ‘ghost’ to bind Ca2+, indicating that the remaining carboxyl groups of aspartic acid and glutamic acid were made available for Ca2+ binding by this procedure. These findings support the concept that in normal ‘ghosts’, at pH7.4, Ca2+ binding to free carboxyl groups is partially hindered by the presence of charged amino groups. 3. Treatment of ‘ghosts’ with N-acetylneuraminidase, which removed 94% of sialic acid residues, and treatment with 1mm-p-chloromercuribenzoate did not alter Ca2+ binding. The major effect of 5.8mm-p-chloromercuribenzoate upon ‘ghosts’ was to cause a solubilization of a calcium–membrane complex, which included about one-third of the ‘ghosts’ protein. The molar ratio of Ca2+: protein in the solubilized material was the same as that in the intact (untreated) ‘ghosts’.
The C4-pathway of photosynthesis. Evidence for an intermediate pool of carbon dioxide and the identity of the donor C4-dicarboxylic acid
Authors: Hatch M. D.
Published: August 2015

1. Leaves were exposed to 14CO2 under steady-state conditions for photosynthesis. The kinetics of entry or loss of label in pools of CO2 and other compounds was examined during the period of the pulse and a ‘chase’ with 12CO2. 2. With maize the kinetics of labelling of the major CO2 pool and of depletion of label during a ‘chase’ was consistent with this pool being derived from the C-4 of malate and being the precursor of the C-1 of 3-phosphoglycerate. 3. Similar results were obtained for Amaranthus leaves except that the C-4 of aspartate rather than malate was apparently the primary source of CO2. 4. The size and turnover time of the CO2 and C4 acid pools was calculated. These results provided the basis for estimating the concentration of CO2 in the bundle-sheath cells or chloroplasts assuming the pool was largely restricted to one or other of these compartments. 5. These findings are considered in relation to current schemes for the C4-pathway and the operation of a CO2 concentrating mechanism to serve ribulose diphosphate carboxylase.
Microbial oxidation of amines. Partial purification of a mixed-function secondary-amine oxidase system from Pseudomonas aminovorans that contains an enzymically active cytochrome-P-420-type haemoprotein
Authors: Eady R. R. , Jarman T. R. , Large P. J.
Published: August 2015

1. Crude extracts of Pseudomonas aminovorans grown on methylamine, di-methylamine, trimethylamine or trimethylamine N-oxide contain an enzyme or enzyme system catalysing the NADH- or NADPH- and oxygen-dependent oxidation of dimethylamine to methylamine and formaldehyde. 2. The enzyme has been partially purified about five-fold. It is unstable, but can be stabilized by addition of 5% (v/v) ethanol. 3. The partially purified enzyme will utilize either NADH (Km 6.5μm) or NADPH (Km 13.2μm): The following secondary amines have been shown to be substrates: dimethylamine, ethylmethylamine, diethylamine, methyl-n-propylamine, ethyl-n-propylamine, n-butylmethylamine and N-methylethanolamine. The Km values and comparative reaction rates for each substrate have been determined. Where the alkyl groups are different, the aldehyde products are derived from both groups. 4. The enzyme system has a pH optimum of 6.8 and is inhibited by mercurials, thiol compounds, cyanide and carbon monoxide. 5. The partially purified preparation had a spectral maximum at 412nm with shoulders at 427 and 550nm. Reduction with dithionite or NAD(P)H bleached the 412nm peak, and the shoulder at 427nm became a peak. Additional peaks appeared at 550 and 580–588nm. Reduction of a preparation bubbled with carbon monoxide enhanced and sharpened the Soret peak and caused it to shift to 422nm. 6. Analysis of the preparation showed the presence of flavin, acid-extractable iron and non-acid-extractable iron in the proportion 1.1:1.9:1. On reduction with dithionite or NADPH the preparation showed an electron-paramagnetic-resonance signal at around g=1.946.
Properties of the sarcolemmal calcium ion-stimulated adenosine triphosphatase of hamster skeletal muscle
Authors: McNamara Dennis B. , Sulakhe Prakash V. , Dhalla Naranjan S.
Published: August 2015

1. A sarcolemmal fraction was isolated from hamster hind-leg skeletal muscles by successive treatment with lithium bromide and potassium chloride. The membranous fraction was observed to contain a highly active Ca2+-stimulated ATPase (adenosine triphosphatase), a Mg2+-stimulated ATPase, and an Na++K+-stimulated Mg2+-dependent ouabain-sensitive ATPase. 2. The Ca2+-stimulated ATPase activity was pH-dependent, the optimum being pH7.6. 3. Optimum activation of this enzyme was obtained with 3–4mm-Ca2+ when 4mm-ATP was present as a substrate, and was not influenced by Na+, K+ or ouabain, whereas 2,4-dinitrophenol, sodium azide, oligomycin, sodium fluoride and ethanedioxybis(ethylamine)tetra-acetate were inhibitory. 4. The Ca2+-stimulated ATPase was markedly inhibited by thiol-blocking reagents, and cysteine was able to reverse this inhibition. 5. Various bivalent cations stimulated ATP hydrolysis by the sarcolemmal fraction in the following decreasing order of potency: Mg2+, Ca2+, Mn2+, Co2+, Sr2+, Ba2+, Zn2+, Cu2+.
Inhibition by oxalomalate of rat liver mitochondrial and extramitochondrial aconitate hydratase
Authors: Adinolfi A. , Guarriera-Bobyleva V. , Olezza S. , Ruffo A.
Published: August 2015

1. The effect of oxalomalate on the oxidation of citrate and cis-aconitate in rat liver mitochondria, and on the activity of mitochondrial and cytoplasmic aconitate hydratase, has been investigated. 2. Oxalomalate that was added to intact rat liver mitochondria at high concentrations (2mm) produced complete inhibition of citrate and cis-aconitate oxidation, but lower concentrations (0.1–0.25mm) inhibited oxidation of citrate more than that of cis-aconitate. 3. Aconitate hydratase that was either extracted from mitochondria or soluble in the cytoplasm, was strongly inhibited by low concentrations of oxalomalate (0.01–0.2mm), the mitochondrial enzyme being more sensitive than the soluble one. 4. Oxalomalate, when added together with citrate, produced competitive inhibition; the Ki values calculated were 1×10−6m for the mitochondrial and 2.5×10−6m for the cytoplasmic enzyme. 5. With both the enzymic preparations oxalomalate added together with the substrates inhibited the initial rate of the reaction citrate→cis-aconitate more than that of the reaction isocitrate→cis-aconitate. 6. After 2min of preincubation of the inhibitor with either of the enzymic preparations the inhibition increased tenfold and became irreversible; under these conditions both the reactions were inhibited to the same extent. 7. The inhibition by oxalomalate of aconitate hydratase appeared to be similar in many respects to that produced by fluorocitrate on the same enzyme.
The effects of certain glycols, substituted glycols and related organic solvents on the thermal stability of soluble collagen
Authors: Hart G. J. , Russell A. E. , Cooper D. R.
Published: August 2015

The effects of a number of related diols, substituted diols and glycerol on the thermal stability of acid-soluble calf skin collagen were investigated. Thermal transition temperatures were determined by optical rotation measurement. Short-chain diols with terminal hydroxyl groups, i.e. ethylene glycol and propane-1,3-diol, stabilized the protein at all accessible concentrations. Stabilization was also observed with glycerol and diethylene glycol. Higher homologues in the diol series produced various effects, as did hydroxyl-group positional isomerism. Monoalkyl substitution of diols progressively lowered the denaturation temperature of collagen. Results are discussed in relation to possible mechanisms of perturbant action.
The composition of peptidochitodextrins from sarcophagid puparial cases
Authors: Lipke H. , Geoghegan T.
Published: August 2015

1. N-Bromosuccinimide cleaved proteins and pigments from fly puparia, increasing the chitin:protein ratio from 0.5 to 1.5. The product afforded subfractions (ratio 5:1) of molecular weights of 1200 and 1600 devoid of aromatic residues and N-terminal β-alanine, direct aryl links between polysaccharide chains being discounted. 2. The chitin–protein complex decreased in molecular weight when treated with Pronase, which suggested polypeptide bridges within the native chitin micelle. The limit dextrins generated by chitinase were mixtures of unsubstituted dextrins and peptidylated oligosaccharides, with the former predominating. 3. Peptidochitodextrins of similar molecular weight but markedly different solubility were prepared, which were indistinguishable with respect to amino acid, glucosamine, acetyl, X-ray or infrared characteristics. It is suggested that physical interactions contribute to the stability of the integument in addition to the covalent bonds that form during sclerotization.
Lysine biosynthesis in Saccharomyces. Conversion of α-aminoadipate into α-aminoadipic δ-semialdehyde
Authors: Sinha Asru K. , Bhattacharjee J. K.
Published: August 2015

The reduction of α-aminoadipate to α-aminoadipic δ-semialdehyde by a cell-free extract of Saccharomyces is shown to be a three-step process. First the amino acid reacts with ATP to form an adenylyl derivative. Then the adenylyl derivative of α-aminoadipate is reduced in the presence of NADPH. In the third step the reduced adenylyl derivative of the amino acid is cleaved to form α-aminoadipic δ-semialdehyde. The presence of Mg2+is necessary for the first and second steps. The third step does not need any cofactors. The product of the first step was isolated by chromatography after incubating the cell-free extract of Saccharomyces with α-aminoadipate, ATP and Mg2+. The isolated product was identified as an adenylyl derivative of α-aminoadipate and could be converted into α-aminoadipic δ-semialdehyde under the stated experimental conditions. The product of the second step was too unstable to be identified.
The pathway of glutamine and glutamate oxidation in isolated mitochondria from mammalian cells
Authors: Kovačević Zoran
Published: August 2015

1. Pyruvate strongly inhibited aspartate production by mitochondria isolated from Ehrlich ascites-tumour cells, and rat kidney and liver respiring in the presence of glutamine or glutamate; the production of14CO2 from l-[U-14C]glutamine was not inhibited though that from l-[U-14C]glutamate was inhibited by more than 50%. 2. Inhibition of aspartate production during glutamine oxidation by intact Ehrlich ascites-tumour cells in the presence of glucose was not accompanied by inhibition of CO2 production. 3. The addition of amino-oxyacetate, which almost completely suppressed aspartate production, did not inhibit the respiration of the mitochondria in the presence of glutamine, though the respiration in the presence of glutamate was inhibited. 4. Glutamate stimulated the respiration of kidney mitochondria in the presence of glutamine, but the production of aspartate was the same as that in the presence of glutamate alone. 5. The results suggest that the oxidation of glutamate produced by the activity of mitochondrial glutaminase can proceed almost completely through the glutamate dehydrogenase pathway if the transamination pathway is inhibited. This indicates that the oxidation of glutamate is not limited by a high [NADPH]/[NADP+] ratio. 6. It is suggested that under physiological conditions the transamination pathway is a less favourable route for the oxidation of glutamate (produced by hydrolysis of glutamine) in Ehrlich ascites-tumour cells, and perhaps also kidney, than the glutamate dehydrogenase pathway, as the production of acetyl-CoA strongly inhibits the first mechanism. The predominance of the transamination pathway in the oxidation of glutamate by isolated mitochondria can be explained by a restricted permeability of the inner mitochondrial membrane to glutamate and by a more favourable location of glutamate–oxaloacetate transaminase compared with that of glutamate dehydrogenase.
An assessment of methanolysis and other factors used in the analysis of carbohydrate-containing materials
Authors: Chambers R. E. , Clamp J. R.
Published: August 2015

The stability of monosaccharides in methanolic hydrochloric acid of different strengths and at different temperatures was determined. They are generally stable for 24h in methanolic 1m- and 2m-hydrochloric acid at both 85°C and 100°C, but undergo considerable destruction in methanolic 4m- and 6m-hydrochloric acid at 100°C. Analysis of glycopeptides and oligosaccharides of known composition showed that release of carbohydrate was complete within 3h in methanolic 1m-hydrochloric acid at 85°C. Removal of methanolic hydrochloric acid by rotary evaporation resulted in considerable losses of monosaccharides, which could be prevented by prior neutralization. Methanolysis caused extensive de-N-acetylation of acetamidohexoses, so that a re-N-acetylation step is necessary in the analytical procedure. The addition of acetic anhydride for this purpose also prevented loss of internal standard by adsorption on the insoluble silver salts used in neutralization. Several trimethylsilylating agents were studied and suitable conditions are recommended. The effects on the analytical system of water and some common organic and inorganic contaminants are assessed.
Glucose and fructose metabolism in Zymomonas anaerobia
Authors: McGill D. J. , Dawes E. A.
Published: August 2015

Isotopic and enzymic evidence indicates that Zymomonas anaerobia ferments glucose via the Entner–Doudoroff pathway. The molar growth yields with glucose (5.89) and fructose (5.0) are lower than those for the related organism Zymomonas mobilis and the observed linear growth suggests that energetically uncoupled growth occurs. A survey of enzymes of carbohydrate metabolism revealed the presence of weak phosphofructokinase and fructose 1,6-diphosphate aldolase activities but phosphoketolase, transketolase and transaldolase were not detected. Fermentation balances for glucose and fructose are reported; acetaldehyde accumulated in both fermentations, to a greater extent with fructose which also yielded glycerol and dihydroxyacetone as minor products.
Oxidative phosphorylation. The effect of anions on the inhibition by triethyltin of various mitochondrial functions, and the relationship between this inhibition and binding of triethyltin
Authors: Rose M. S. , Aldridge W. N.
Published: August 2015

1. The binding of triethyltin to rat liver mitochondria is unaffected by the nature of the predominant anion in the incubation medium. 2. With chloride, bromide or iodide as the predominant anion, ATP synthesis linked to the oxidation of pyruvate or succinate and ATP hydrolysis stimulated by 2,4-dinitrophenol are much more sensitive to triethyltin than they are when nitrate or isethionate is the predominant anion. 3. When nitrate or isethionate is the predominant anion, oxygen uptake stimulated by 2,4-dinitrophenol is not inhibited by triethyltin. 4. In the presence of nitrate or isethionate anions, inhibition of ATP synthesis is directly related to the binding of triethyltin to mitochondria. 5. The relationship of the above effects to the anion–hydroxide ion exchange mediated by triethyltin and the relevance of this to published arrangements for coupling of electron transport to ATP synthesis are discussed.
Relationships between carnitine and coenzyme A esters in tissues of normal and alloxan-diabetic sheep
Authors: Snoswell A. M. , Koundakjian Patricia P.
Published: August 2015

1. The total acid-soluble carnitine concentrations of four tissues from Merino sheep showed a wide variation not reported for other species. The concentrations were 134, 538, 3510 and 12900nmol/g wet wt. for liver, kidney cortex, heart and skeletal muscle (M. biceps femoris) respectively. 2. The concentration of acetyl-CoA was approximately equal to the concentration of free CoA in all four tissues and the concentration of acid-soluble CoA (free CoA plus acetyl-CoA) decreased in the order liver>kidney cortex>heart>skeletal muscle. 3. The total amount of acid-soluble carnitine in skeletal muscle of lambs was 40% of that in the adult sheep, whereas the concentration of acid-soluble CoA was 2.5 times as much. A similar inverse relationship between carnitine and CoA concentrations was observed when different muscles in the adult sheep were compared. 4. Carnitine was confined to the cytosol in all four tissues examined, whereas CoA was equally distributed between the mitochondria and cytosol in liver, approx. 25% was present in the cytosol in kidney cortex and virtually none in this fraction in heart and skeletal muscle. 5. Carnitine acetyltransferase (EC 2.3.1.7) was confined to the mitochondria in all four tissues and at least 90% of the activity was latent. 6. Acetate thiokinase (EC 6.2.1.1) was predominantly (90%) present in the cytosol in liver, but less than 10% was present in this fraction in heart and skeletal muscle. 7. In alloxan-diabetes, the concentration of acetylcarnitine was increased in all four tissues examined, but the total acid-soluble carnitine concentration was increased sevenfold in the liver and twofold in kidney cortex. 8. The concentration of acetyl-CoA was approximately equal to that of free CoA in the four tissues of the alloxan diabetic sheep, but the concentration of acid-soluble CoA in liver increased approximately twofold in alloxan-diabetes. 9. The relationship between CoA and carnitine and the role of carnitine acetyltransferase in the various tissues is discussed. The quantitative importance of carnitine in ruminant metabolism is also emphasized.
The effect of prostaglandins on ox pituitary content of adenosine 3′:5′-cyclic monophosphate and the release of growth hormone
Authors: Cooper R. H. , McPherson M. , Schofield J. G.
Published: August 2015

1. An assay, based on competition between adenosine 3′:5′-cyclic monophosphate (cyclic AMP) and cyclic [3H]AMP for binding to a rabbit skeletal muscle protein, has been used to measure tissue contents of cyclic AMP. The assay has a sensitivity of 0.05pmol of cyclic AMP. Cyclic GMP and cyclic CMP have 0.5%, and cyclic IMP 6.5%, of the ability of cyclic AMP to displace cyclic [3H]AMP from binding protein; AMP, ADP and ATP have no effect. 2. By using this method, the cyclic AMP content of ox pituitary slices exposed to prostaglandin was determined; release of growth hormone was measured by radioimmunoassay. 3. Release of growth hormone was increased by 45min incubation in 1μm-prostaglandin E2 in the absence of theophylline, or in 10nm-prostaglandin E2, 0.1μm-prostaglandin A1 or 1μm-prostaglandin B1 in the presence of 0.5mm-theophylline. 4. Pituitary cyclic AMP content was increased by 10min incubation in 1μm-prostaglandin E2 in the absence of theophylline, or in 0.1μm-prostaglandin E2 in the presence of 0.5mm-theophylline. 5. The maximum increase in cyclic AMP content was observed 10min, and significant changes in growth hormone release 30min, after introduction of prostaglandin E2. 6. The increase in pituitary cyclic AMP content, but not in the rate of release of growth hormone, was observed in the absence of external Ca2+. 7. The stimulation of release of growth hormone by prostaglandin was decreased by preincubation of tissue for 2h in colchicine (100μm) or cytochalasin B (10μg/ml). 8. These results support the suggestion that increased release of growth hormone after treatment with prostaglandin is the result of increased tissue cyclic AMP content, and possibly involves a microfilamentous or microtubular protein.
Intracellular transport of mouse kidney β-glucuronidase induced by gonadotrophin
Authors: Kato Keitaro , Hirohata Itsuyo , Fishman William H. , Tsukamoto Hisao
Published: August 2015

1. The response of renal β-glucuronidase with time to the injection of gonadotrophin was investigated in each submicrosomal fraction of rough and smooth microsomal fractions of mouse kidney homogenate. 2. The increase in β-glucuronidase activity appeared initially in membranes of the rough microsomal fraction, 24h after injection. 3. Afterwards the newly synthesized enzyme appeared in the contents of the rough microsomal fraction and was subsequently found in the smooth microsomal fraction, reaching a maximum concentration in this fraction at 72h. 4. At this juncture, a decrease in the enzyme activity was observed in rough microsomal contents whereas the lysosomal fraction had reached its maximum value. 5. The time-course of the appearance of β-glucuronidase in the submicrosomal fractions after the gonadotrophin stimulation suggests that the newly synthesized enzyme at the site of membrane-bound ribosomes is transferred across the membrane into cisternae of the rough endoplasmic reticulum, and then is transported into lysosomes via the smooth endoplasmic reticulum. 6. The properties of microsomal and lysosomal β-glucuronidases were compared.
Intracellular localization of aldehyde dehydrogenase in rat liver
Authors: Marjanen Leo
Published: August 2015

1. Distribution of aldehyde dehydrogenase activity in rat liver was studied by measuring the rate of disappearance of acetaldehyde in the presence of each of the subcellular fractions. These were obtained by rough separation of particulate fractions from the soluble portion of the cell, by differential centrifugation, and by isopycnic gradient centrifugation. 2. The maximal rate of acetaldehyde oxidation was 3.7 μmol/min per g, with an apparent Km value below 10-5m. The highest rate of activity was observed in phosphate buffers of high Pi concentration (above 60mm). 3. The activity measured was completely dependent on NAD+. 4. The microsomal fraction and the nuclei were inactive in the assay. Of the total activity 80% was found in the mitochondrial fraction and the remaining 20% in the cytoplasm. 5. The distribution pattern is important from the point of view of acetaldehyde oxidation during ethanol metabolism. The apparent discrepancy of the results obtained by different workers and the localization of acetaldehyde oxidation in vivo is discussed.
Subcellular fractionation by zonal centrifugation of glucose-repressed anaerobically grown Saccharomyces carlsbergensis
Authors: Cartledge T. G. , Lloyd D.
Published: August 2015

1. Homogenates were prepared from sphaeroplasts of anaerobically grown, glucoserepressed Saccharomyces carlsbergensis, and the distributions of marker enzymes investigated after zonal centrifugation on sucrose gradients containing 2mm-MgCl2. 2. These homogenates contained no detectable cytochrome c oxidase, succinate–cytochrome c oxidoreductase, succinate–ferricyanide oxidoreductase, l(+)-lactate–cytochrome c oxidoreductase or catalase. Cytochromes a+a3 and c were not detected. 3. Zonal centrifugation of whole homogenates indicated complex density distributions of the sedimentable portions of NADH– and NADPH–cytochrome c oxidoreductases, adenosine triphosphatases (ATPases), adenosine pyrophosphatase (ADPase), pyrophosphatase and acid p-nitrophenyl phosphatase. Several different ATPases were distinguished on the basis of their sensitivities to oligomycin and ouabain. 4. Differential centrifugation of whole homogenates at 105g-min left 80–90% of the protein, dithionite-reducible cytochrome b, acid hydrolases and pyrophosphatase in a supernatant (S1) together with 65 and 56% of the NADH– and NADPH–cytochrome c oxidoreductases respectively, 25% of the ATPases and 71% of the adenosine monophosphatase. 5. Further analysis of supernatant S1 revealed the presence of a class of small particles containing NADPH–cytochrome c oxidoreductases and ATPases. 6. At least four different populations of large particles were distinguished. 7. Electron microscopy indicated that one of these corresponded to ‘promitochondria’ as described by other workers.
Comparison of the properties of two forms of pyruvate kinase in rat liver and determination of their separate activities during development
Authors: Middleton Marshall C. , Walker Deryck G.
Published: August 2015

1. Two forms of hepatic pyruvate kinase, designated type L and type M, were distinguished on the basis of kinetic, chromatographic, electrophoretic and immunological criteria. They were partially purified and their properties compared with each other and with the purified enzyme from skeletal muscle. 2. In contrast with type L, the type M enzyme showed no marked evidence of co-operative interactions with phosphoenolpyruvate and was not stimulated by fructose diphosphate. 3. The activity profiles of type L and type M enzymes were determined in developing rat liver by utilizing differences in the kinetic properties of the two forms. The high activity of type M enzyme in the early foetal rat decreased in late gestation and immediately after birth to reach a low value, which remained essentially constant for the remainder of the developmental period. The activity of type L enzyme, in contrast, was low in the early foetal and neonatal liver but increased markedly at the onset of weaning. 4. Possible roles of the two forms of hepatic pyruvate kinase in the control of glycolysis and gluconeogenesis are discussed.
The oligosaccharide units of rabbit immunoglobulin G. Asymmetric attachment of C2-oligosaccharide
Authors: Fanger M. W. , Smyth D. G.
Published: August 2015

Two populations of immunoglobulin G (IgG) molecules were isolated from pooled rabbit serum. The first was almost devoid of galactosamine and was obtained in a yield of 7% of the total IgG; the second contained on average a single residue of galactosamine per molecule and was obtained in a yield of 30% of the total IgG. The galactosamine, which is present solely in the C2-oligosaccharide, appeared to be present on one H-chain and not the other in the four-chain structure. Evidence was obtained by the isolation of a glycopeptide linked through a disulphide bridge to a second peptide of the same sequence; the oligosaccharide attached to the first peptide was absent from the complementary peptide. Further evidence was obtained by degradation and analysis of the 5S IgG fragment, which comprises an intact half-molecule coupled through a disulphide bridge to the Fc fragment derived from the opposing half molecule (Goodman, 1965); only the intact H-chain carried the C2-oligosaccharide.
Function of arginase in lactating mammary gland
Authors: Yip Morris C. M. , Knox W. Eugene
Published: August 2015

The potential for a considerable formation of ornithine exists in lactating mammary gland because of its arginase content. Late in lactation arginase reaches an activity in the gland higher than that present in any rat tissue except liver. Occurrence of the urea cycle can be excluded since two enzymes for the further reaction of ornithine in the cycle, carbamoyl phosphate synthetase I and ornithine carbamoyltransferase, are both absent from this tissue. Instead, carbamoyl phosphate synthetase II appears early in lactation, associated with accumulation of aspartate carbamoyltransferase and DNA, consistent with the proposed role of these enzymes in pyrimidine synthesis. The facts require another physiological role for arginase apart from its known function in the urea cycle. Significant activity of ornithine aminotransferase develops in mammary gland in close parallel with the arginase. By this reaction, ornithine can be converted into glutamic semialdehyde and subsequently into proline. The enzymic composition of the lactating mammary gland is therefore appropriate for the major conversion of arginine into proline that is known to occur in the intact gland.