bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of pyruvate dehydrogenase in rat heart. Mechanism of regulation of proportions of dephosphorylated and phosphorylated enzyme by oxidation of fatty acids and ketone bodies and of effects of diabetes: role of coenzyme A, acetyl-coenzyme A and reduced and oxidized nicotinamide-adenine dinucleotide
Authors: Kerbey A L. , Randle P J. , Cooper R H. , Whitehouse S , Pask H T. , Denton R M.
Published: August 2015

The proportion of active (dephosphorylated) pyruvate dehydrogenase in perfused rat heart was decreased by alloxan-diabetes or by perfusion with media containing acetate, n-octanoate or palmitate. The total activity of the dehydrogenase was unchanged. 2. Pyruvate (5 or 25mM) or dichloroacetate (1mM) increased the proportion of active (dephosphorylated) pyruvate dehydrogenase in perfused rat heart, presumably by inhibiting the pyruvate dehydrogenase kinase reaction. Alloxan-diabetes markedly decreased the proportion of active dehydrogenase in hearts perfused with pyruvate or dichloroacetate. 3. The total activity of pyruvate dehydrogenase in mitochondria prepared from rat heart was unchanged by diabetes. Incubation of mitochondria with 2-oxo-glutarate plus malate increased ATP and NADH concentrations and decreased the proportion of active pyruvate dehydrogenase. The decrease in active dehydrogenase was somewhat greater in mitochondria prepared from hearts of diabetic rats than in those from hearts of non-diabetic rats. Pyruvate (0.1-10 mM) or dichloroacetate (4-50 muM) increased the proportion of active dehydrogenase in isolated mitochondria presumably by inhibition of the pyruvate dehydrogenase kinase reaction. They were much less effective in mitochondria from the hearts of diabetic rats than in those of non-diabetic rats. 4. The matrix water space was increased in preparations of mitochondria from hearts of diabetic rats. Dichloroacetate was concentrated in the matrix water of mitochondria of non-diabetic rats (approx. 16-fold at 10 muM); mitochondria from hearts of diabetic rats concentrated dichloroacetate less effectively. 5. The pyruvate dehydrogenase phosphate phosphatase activity of rat hearts and of rat heart mitochondria (approx. 1-2 munit/unit of pyruvate dehydrogenase) was not affected by diabetes. 6. The rate of oxidation of [1-14C]pyruvate by rat heart mitochondria (6.85 nmol/min per mg of protein with 50 muM-pyruvate) was approx. 46% of the Vmax. value of extracted pyruvate dehydrogenase (active form). Palmitoyl-L-carnitine, which increased the ratio of [acetyl-CoA]/[CoA] 16-fold, inhibited oxidation of pyruvate by about 90% without changing the proportion of active pyruvate dehydrogenase.
Insulin-like actions of nickel and other transition-metal ions in rat fat-cells
Authors: Saggerson E. D , Sooranna S R. , Evans C J.
Published: August 2015

NiCl2 (1-6mM) decreased adrenaline and glucagon-stimulated lipolysis in rat fat-cells, and also considerably stimulated [U-14C]glucose incorporation into fat-cell lipids. 2. These insulin-like effects were also observed with CuCl, CuCl2, CoCl2 and (to a lesser extent) with MnCl2. 3. NiCl2 was less effective in mimicking insulin effects on [U-14C]fructose metabolism than on glucose utilization. 4. It is tentatively suggested that these transition-metal ions may mimic actions of insulin at the fat-cell plasma membrane which decrease lipolysis and stimulate glucose transport, but do not mimic certain other effects of the hormone on intracellular metabolic processes. 5. These results are discussed with reference to suggestions that redistributions of cellular Ca2+ are associated with insulin action in fat-cells.
The regulation of carbamoyl phosphate synthase activity in rat liver mitochondria
Authors: McGivan J D. , Bradford N M. , Mendes-Mourão J
Published: August 2015

The rate at which isolated rat liver mitochondria synthesized citrulline with NH4C1 as nitrogen source was markedly dependent on the protein content of the diet. 2. Citrulline synthesis was not rate-limited by substrate concentration, substrate transport or ornithine transcarbamoylase activity under the conditions used. 3. The intramitochondrial content of an activator of carbamoyl phosphate synthase, assumed to be N-acetyl-glutamate, varied markedly with dietary protein content. The variation in the concentration of this activator was sufficient to account for the observed variation in the rates of citrulline synthesis if this synthesis were rate-limited by the activity of carbamoyl phosphate synthase. 4. The rates of urea formation from NH4Cl as nitrogen source in isolated liver cells showed variations in response to diet that closely paralleled the variations in the rates of citrulline synthesis observed in isolated mitochondria. 5. These results are consistent with the postulate that when NH4Cl plus ornithine are present in an excess, the rate of urea synthesis is regulated at the level of carbamoyl phosphate synthase activity.
Growth hormone and drug metabolism. Acute effects on microsomal mixed-function oxidase activities in rat liver
Authors: Wilson J T. , Spelsberg T C.
Published: August 2015

Adult male rats were subjected either to sham operation or to hypophysectomy and adrenalectomy and maintained for a total of 10 days before treatment with growth hormone. Results of the early effects of growth hormone on the activities of the mixed-function oxidases in rat liver over a 96h period after growth-hormone treatment are presented. 2. Hypophysectomy and adrenalectomy result in decreased body and liver weight and decreased drug metabolism (mixed-function oxidases). Concentrations of electron-transport-system components are also decreased. 3. In the hypophysectomized/adrenalectomized rats, growth hormone decreases the activities of the liver mixed-function oxidases and the cytochrome P-450 and cytochrome c reductases, as well as decreasing the concentration of cytochrome P-450 compared with that of control rats. Similar but less dramatic results are obtained with sham-operated rats. 4. It is concluded that whereas growth hormone enhances liver growth, including induction of many enzyme activities, it results in a decrease in mixed-function oxidase activity. Apparently, mixed-function oxidase activity decreases in liver when growth (mitogenesis) increases.
The regulation of phenylalanine hydroxylase in rat tissues in vivo. The maintenance of high plasma phenylalanine concentrations in suckling rats: a model for phenylketonuria
Authors: Delvalle J A. , Greengard O
Published: August 2015

Maximum inhibition of phenylalanine hydroxylase activity in the liver (85%) and in the kidney (50%) of suckling rats required the administration of over 9 mumol of p-chlorophenylalanine/10g body weight. Despite the decrease in the total activity from 184 to 34 units per 10g body weight, the injection of as much as 26 mumol of phenylalanine was required for its concentration in plasma to be still considerably elevated 12h later. In rats injected with p-chlorophenylalanine every 48h and with phenylalanine every 24h from 3 to 18 days of age, the hepatic and renal phenylalanine hydroxylase remained inhibited, whereas the activities of three other hepatic enzymes were unchanged. There was about 20% inhibition of brain and body growth, but no interference with the developmental formation of several cerebral enzymes (four dehydrogenases, hexokinase and glutaminase) was detected. In the course of this prolonged treatment, the phenylalanine concentrations in plasma increased gradually; on day 2 and day 8 (measured 12h after the last injection) they were 800 and 1395 nmol/ml respectively; on day 15, 12 and 18h after the usual injection, the values were 2030 and 1030 respectively as opposed to the 96 nmol in untreated rats. This degree of hyperphenylalaninaemia, persisting for 18h per day throughout a critical period of development, fulfils the primary criterion of a suitable animal model for phenylketonuria.
Fb′2, a new peptic fragment of human immunoglobulin G
Authors: Parr D M , Connell G E , Kells D I C , Hofmann T
Published: August 2015

The digestion of a human IgG1 K myeloma protein with pepsin in the presence of 8M-urea was observed to produce a fragment, designated Fb′2, which differed from the products of aqueous peptic digestion and from other characteristic immunoglobulin digestion products. 2. Fragment Fb′s was also found when two other IgG1/K proteins were treated similarly. 3. Sedimentation-equilibrium studies showed the mol.wt. of fragment Fb′2 to be 56800. 4. On reduction, two equivalents of each of three peptides were released from fragment Fb′s; these were characterized by N- and C-terminal determinations and by amino acid sequencing. 5. Fragment Fb′2 was shown to consist of the constant regions of both light chains, from residue Ile-117 to the C-terminus, and the CH1 domains and hinge region of the heavy chains, from residue Val-113 to residue Met-252, with a gap of five residues within the intrachain disulphide loop, between residues Leu-174 and Tyr-180.
Conformational analysis of deoxyribonucleic acid from PM2 bacteriophage. The effect of size on supercoil shape
Authors: Campbell A M
Published: August 2015

Laser light-scattering studies of bacteriophage PM2 DNA showed the molecule to have mol.wt. 5.9 } 10(6) and root-mean -square radius 125 nm at an ionic strength of 0.2 mol/litre. Computer-generated curves compatible with these data were compared with the experimental interference curve for several structural models of the molecules. The data fit best to an asymmetric four-armed planar molecule in which all four arms emerge from or close to the one area of the molecule. This contrasts with the smaller DNA molecules investigated, which have shown a three-armed molecule, whose symmetry varies with primary structure.
Electron-paramagnetic-resonance studies on the molybdenum of nitrate reductase from Escherichia coli K12
Authors: Bray R C , Vincent S P , Lowe D J , Clegg R A , Garland P B
Published: August 2015

Studies on the respiratory nitrate reductase (EC 1.7.99.4) from Escherichia coli K12 by electron-paramagnetic-resonance spectroscopy indicate that its molybdenum centre is comparable with that in other molybdenum-containing enzymes. Two Mo(V) signals may be observed; one shows interaction of Mo(V) with a proton exchangeable with the solvent and has: A (1H) 0.9-1.2mT; g1 = 1.999; g2=1.985; g3 = 1.964; gav. = 1.983. Molybdenum of both signal-giving species may be reduced with dithionite and reoxidized with nitrate.
A comparison of the properties of renin isolated from pig and rat kidney
Authors: Lauritzen M , Damsgaard J J , Rubin I , Lauritzen E
Published: August 2015

1. On isoelectric focusing, renin from rat kidneys showed three activity peaks with pI values at pH 5.0, 5.2 and 5.4 after a purification procedure involving differential centrifugation, acidification, chromatography on Sephadex G-75 and dialysis. 2. The preparation (purified 140-fold) was compared with a crude kidney extract in the absence and presence of 3 M-urea by isoelectric focusing. The pattern of activity distribution was confirmed by these experiments and the content of isoenzymes in the three groups calculated. 3. Pig renin was prepared and compared with rat renin with regard to molecular weight, acid activation, behaviour on isoelectric focusing, immunogenicity and substrate affinity. 4. Extracts of rat kidney contained multiple forms of renin with mol.wt. between 39000and 42000, whereas active pig renin had an approximate mol.wt. of 40000. Acidification of rat renal extracts did not increase the activity of renin, indicating the absence of an inactive form of renin in rat kidneys, whereas pig renin was activated by this procedure. Pig renin has isoelectric points at pH 4.6, 4.8, 5.05 and 5.2, significantly lower than for rat renin. The isoenzymes from the two species had no antigenicity in common, as shown by crossed immunoelectrophoresis or rocket immunoelectrophoresis. 5. The Michaelis constants for pig and rat renin were in the same range, 1 × 10(-6) M, when rat renin substrate was used. The relative content of rat isoenzyme with pI in the pH ranges 4.9-5.1, 5.1-5.3 and 5.3-5.5 was approx. 20, 27 and 53% respectively. Purified pig renin prepared in two different ways had isoenzymes with pI in the pH regions 4.5-4.7, 4.7-4.9, 4.9-5.05 and 5.05-5.20 in the approximate proportions 14, 24, 28 and 29%.
Is a Schiff base involved in the mechanism of the Δ4-3-oxo steroid 5α- or 5β-reductases from mammalian liver?
Authors: Wilton D C
Published: August 2015

The saturation of the delta4-double bond of delta4-3-oxo steroids by the mammalian 5alpha- or 5beta-reductases is by an enzyme mechanism that does not involve a Schiff-base intermediate.
Molecular forms and activities of glycosidases in cultures of amniotic-fluid cells
Authors: Hultberg B , Lindsten J , Sjöblad S
Published: August 2015

Ion-exchange chromatography of gel filtration demonstrated the presence of different molecular forms of nine lysosomal enzymes in cultured amniotic-fluid cells. The patterns of molecular forms were similar to those known from skin fibroblasts and liver tissue. During cultivation total enzyme activities fluctuated with the number of passages, without any consistent trend of increase or decrease, and without correlation to the dominating cell type in the culture.
Ascorbic acid, metal ions and the superoxide radical
Authors: Halliwell B , Foyer C H
Published: August 2015

1. No evidence could be found for production of the superoxide radical, O2-, during autoxidation of ascorbic acid at alkaline pH values. Indeed, ascorbate may be important in protection against O2- genat-d in vivo. 2. Oxidation of ascorbate at pH 10.2 was stimulated by metal ions. Stimulation by Fe2+ was abolished by superoxide dismutase, probably because of generation of O2- during reduction of O2 by Fe2+, followed by reaction of O2- with ascorbate. EDTA changed the mechanism of Fe2+-stimulated ascorbate oxidation. 3. Stimulation of ascorbate oxidation by Cu2+ was also decreased by superoxide dismutase, but this appears to be an artifact, since apoenzyme or bovine serum albumin showed similar effects.
Abnormal carbohydrate composition of the major penetrating membrane protein of En(a-) human erythrocytes
Authors: Tanner M J , Jenkins R E , Anstee D J , Clamp J R
Published: August 2015

The major penetrating membrane glycoprotein (band 3) was isolated from En(a-) and normal human erythrocytes. The two proteins differed only in carbohydrate composition. Band 3 from En(a-) erythrocytes contained greater amounts of galactose and N-acetyl-glucosamine. The loss of the sialoglycoprotein sialotetrasaccharides in the En(a-) cell is not compensated by the appearance of these units in band 3 of En(a-) erythrocytes.
Influence of the adrenal glucocorticoids on the stimulation of synthesis of hepatic ribonucleic acid and plasma acute-phase globulins by leucocytic endogenous mediator
Authors: Thompson W L , Abeles F B , Beall F A , Dinterman R E , Wannemacher R W
Published: August 2015

An injection of unpurified leucocytice endogenous mediator into rats results in an increased incorporation of [6(-14)C]orotate into hepatic RNA, an increase in the concentration of RNA associated with the bound ribosomal fraction of liver, and increases in the concentrations in serum of acute-phase proteins such as alpha2-macrofoetoprotein and haptoglobin. If given 3 days after adrenalectomy or 7 days after hypophysectomy, leucocyte factor did not induce the increase in RNA synthesis or alpha2-macrofoetoprotein concentrations but did stimulate an increase in serum haptoglobin. When hypophysectomized or adrenalectomized rats received daily subcutaneous injections of 0.5mg of cortisol, leucocyte factor again induced a significant increase in the synthesis of hepatic RNA and an increase in the concentration of serum alpha2-macrofoetoprotein. These observations suggest that leucocyte factor can regulate acute-phase-protein synthesis at several different sites, one or more of which requires permissive action of the glucocorticoid hormones. Futher, leucocyte factor will stimulate an increase rate of incorporation of orotate into hepatic ribosomes when added in vitro in the presence of cortisol to a liver-perfusion system. Thus the stimulatory effect of leucocyte factor may be directy on liver but may require the presence of other hormones to stimulate the incorporation of orotate into RNA.
The effects of denervation on protein turnover of rat skeletal muscle
Authors: Goldspink D F
Published: August 2015

The effects of denervation on muscle weight, rates of protein synthesis and breakdown, and RNA concentraitons were studied in the soleus and extensor digitorum longus muscle. Althrough the soleus underwent a true atrophy after section of the sciatic nerve, the extensor digitorum longus continued to grow, albeit at a lower rate than innervated controls. At 24h after nerve section protein breakdown was increased in both muscle types when compared with internal controls, and remained so throughout the 10 days studied. The possibility that this increased catabolism might arise from conformational changes of proteins after denervation was not substantiated, as myofibrillar or soluble proteins of denervated and control tissues were equally susceptible to degradation in vitro by three proteinases. Tyrosine uptake into the denervated extensor digitorum longus was decreased throughout the 10 days studied, whereas two phases of increased transport of the amino acid were found in the soleus. Significant decreases in rates of protein synthesis were found 1 and 2 days after denervation and results are presented that suggest that these changes may result from a decrease in ribosomal involvement in the translation process. These initial decreases were not maintained and the rate of protein synthesis was in fact increased when compared with controls, at 7 and 10 days. The increased synthetic rates of the 7-day denervated tissues were reflected as proportional increases in both myofibrillar and soluble proteins. It is suggested that the increase in synthesis at this time may result from an increase in both the abailability and active involvement of ribosomes, and that these anabolic trends may be caused by spontaneous fibrillation and/or the amount of passive stretching of the denervated muscles.
The route of secretion of procollagen. The influence of αα′-bipyridyl, colchicine and antimycin A on the secretory process in embryonic-chick tendon and cartilage cells
Authors: Harwood R , Grant M E , Jackson D S
Published: August 2015

I. Embryonic-chick tendon cells were pulse-labelled for 4 min with [14C]proline and the 14C-labelled polypeptides were chased with unlabelled proline for up to 30 min. Isolation of subcellular fractions during the chase period and their subsequent analysis for bacterial collagenase-susceptible 14C-labelled peptides demonstrated the transfer of procollagen polypeptides from rough to smooth microsomal fractions and thence to the extracellular medium. Parallel analyses of Golgi-enriched fractions indicated the involvement of this organelle in the secretory pathway of procollagen. Sodium dodecylsulphate/polyacrylamide-gel electrophoresis of the 14C-labelled polypeptides present in the Golgi-enriched fractions demonstrated that the procollagen polypeptides were all present as disulphide-linked pro-gamma components. 2. When similar kinetic studies of the intracellular transport of procollagen were conducted with embryonic-chick cartilage cells almost identical results were obtained, but the rate of translocation of cartilage procollagen was significantly slower than that observed for tendon procollagen. 3. When hydroxylation of procollagen polypeptides was inhibited by alphaalpha′-bipyridyl, the nascent polypeptides accumulated in the rough microsomal fraction. 4. When cells were pulse-labelled for 4min with [14C)proline and the label was chased in the presence of colchicine, secretion of procollagen was inhibited and an intracellular accumulation of procollagen 14C-labelled polypeptides was observed in the Golgi-enriched fractions. 5. The energy-dependence of the intracellular transport of procollagen was demonstrated in experiments in which antimycin A was found to inhibit the transfer of procollagen polypeptides from rough to smooth endoplasmic reticulum. 6. It is concluded that procollagen follows the classical route of secretion taken by other extracellular proteins.
Role of ubiquinone in the mitochondrial generation of hydrogen peroxide
Authors: Boveris A , Cadenas E , Stoppani A O M
Published: August 2015

Antimycin-inhibited bovine heart submitochondrial particles generate O2- and H2O2 with succinate as electron donor. H2O2 generation involves the action of the mitochondrial superoxide dismutase, in accordance with the McCord & Fridovich [(1969) j. biol. Chem. 244, 6049-6055] reaction mechanism. Removal of ubiquinone by acetone treatment decreases the ability of mitochondrial preparations to generate O2- and H2O2, whereas supplementation of the depleted membranes with ubiquinone enhances the peroxide-generating activity in the reconstituted membranes. Addition of superoxide dismutase to ubiquinone-reconstituted membranes is essential in order to obtain maximal rates of H2O2 generation since the acetone treatment of the membranes apparently inactivates (or removes) the mitochondrial superoxide dismutase. Parallel measurements of H2O2 production, succinate dehydrogenase and succinate-cytochrome c reductase activities show that peroxide generation by ubiquinone-supplemented membranes is a monotonous function of the reducible ubiquinone content, whereas the other two measured activities reach saturation at relatively low concentrations of reducible quinone. Alkaline treatment of submitochondrial particles causes a significant decrease in succinate dehydrogenase activity and succinate-dependent H2O2 production, which contrasts with the increase of peroxide production by the same particles with NADH as electron donor. Solubilized succinate dehydrogenase generates H2O2 at a much lower rate than the parent submitochondrial particles. It is postulated that ubisemiquinone (and ubiquinol) are chiefly responsible for the succinate-dependent peroxide production by the mitochondrial inner membrane.
Lipogenesis from ketone bodies in rat brain. Evidence for conversion of acetoacetate into acetyl-coenzyme A in the cytosol
Authors: Patel M S , Owen O E
Published: August 2015

The metabolism of acetoacetate via a proposed cytosolic pathway in brain of 1-week-old rats was investigated. (-)-Hydroxycitrate, an inhibitor of ATP citrate lyase, markedly inhibited the incorporation of carbon from labelled glucose and 3-hydroxybutyrate into cerebral lipids, but had no effect on the incorporation of labelled acetate and acetoacetate into brain lipids. Similarly, n-butylmalonate and benzene-1,2,3-tricarboxylate inhibited the incorporation of labelled 3-hydroxybutyrate but not of acetoacetate into cerebral lipids. These inhibitors had no effect on the oxidation to 14CO2 of the labelled substrates used. (-)-Hydroxycitrate decreased the incorporation of 3H from 3H2O into cerebral lipids by slices metabolizing either glucose or 3-hydroxybutyrate, but not in the presence of acetoacetate. (-)-Hydroxycitrate also differentially inhibited the incorporation of [2-14C]-leucine and [U-14C]leucine into cerebral lipids. The data show that, although the acetyl moiety of acetyl-CoA generated in brain mitochondria is largely translocated as citrate from these organelles to the cytosol, a cytosolic pathway exists by which acetoacetate is converted directly into acetyl-COA in this cellular compartment.
Studies on J-chain biosynthesis in tumours producing immunoglobulins in NZB mice
Authors: Kaji H
Published: August 2015

Investigation of biosynthesis of J chain in plasmacytomas induced in NZB mice revealed that this protein was not only synthesized in the cells that produce polymer immunoglobulin A but also in those that produce immunoglobulin G monomer. It was also found that protein similar to J chain of BALB/c-mice was associated with polymer immunoglobulin A but not with immunoglobulin G of NZB mouse myeloma proteins.
The specificity of a 7 α-hydroxy steroid dehydrogenase from Escherichia coli
Authors: Haslewood E S , Haslewood G A D
Published: August 2015

1. Thirty-eight steroids were tested as substrates for a 7 alpha-hydroxy steroid dehydrogenase preparation from a strain of Escherichia coli; an improved method of making the crude enzyme is described. 2. Steroids having a 7 alpha-hydroxyl group in the molecule were substrates except (a) when the 5 beta-cholan-24-oic acid side chain was shortened to less than four carbon atoms and (b) in certain cases when sulphate ester groups were present in the molecule. 3. For testing with the enzyme, a new specimen of 7 alpha-hydroxy-3,12-dioxo-5 beta-cholan-24-oic acid was made, which had properties different from those previously described.
The kinetics of formation of horseradish peroxidase compound I by reaction with peroxobenzoic acids. pH and peroxo acid substituent effects
Authors: Davies D M , Jones P , Mantle D
Published: August 2015

1. The kinetics of formation of horseradish peroxidase Compound I were studied by using peroxobenzoic acid and ten substituted peroxobenzoic acids as substrates. Kinetic data for the formation of Compound I with H2O2 and for the reaction of deuteroferrihaem with H2O2 and peroxobenzoic acids, to form a peroxidatically active intermediate, are included for comparison. 2. The observed second-order rate constants for the formation of Compound I with peroxobenzoic acids decrease with increasing pH, in the range pH 5-10, in contrast with pH-independence of the reaction with H2O2. The results imply that the formation of Compound I involves a reaction between the enzyme and un-ionized hydroperoxide molecules. 3. The maximal rate constants for Compound I formation with unhindered peroxobenzoic acids exceed that for H2O2. Peroxobenzoic acids with bulky ortho substituents show marked adverse steric effects. The pattern of substituent effects does not agree with expectations for an electrophilic oxidation of the enzyme by peroxoacid molecules in aqueous solution, but is in agreement with that expected for a reaction involving nucleophilic attack by peroxo anions. 4. Possible reaction mechanisms are considered by which the apparent conflict between the pH-effect and substituent-effect data may be resolved. A model in which it is postulated that a negatively charged ‘electrostatic gate’ controls access of substrate to the active site and may also activate substrate within the active site, provides the most satisfactory explanation for both the present results and data from the literature.
Studies on sequencing of peptides from the carboxyl terminus by using the thiocyanate method
Authors: Rangarajan M , Darbre A
Published: August 2015

We report on our experience in applying the thiocyanate method developed by Stark (1968) (Biochemistry 7, 1796-1807) to the sequencing of short peptides from the carboxyl end in free solution. Yields fell to very low levels after three cycles of degradation. The method was time-consuming because of the filtration and freeze-drying stages involved. To overcome these problems, peptides were attached to modified polystyrene polymers for sequential degradation in the solid phase, and a maximum of six amino acids was determined. Also, ribonuclease was attached to active-ester glass beads and sequential degradation was carried out to determine six amino acids at the C-terminal end of this protein.
Methods for fitting equations with two or more non-linear parameters
Authors: Nimmo I A , Atkins G L
Published: August 2015

1. Descriptions are given of two ways for fitting non-linear equations by least-squares criteria to experimental data. One depends on solving a set of non-linear simultaneous equations, and the other on Taylor's theorem. 2. It is shown that better parameter estimates result when an equation with two or more non-linear parameters is fitted to all the sets of data simultaneously than when it is fitted to each set in turn.
Phosphorylation of synaptic-membrane proteins from ox cerebral cortex in vitro. Partition of substrates and protein kinase activities with triton X-100
Authors: Dunkley P R , Holmes H , Rodnight R
Published: August 2015

Synaptic-membrane fragments from ox cerebral cortex contain basal and cyclic AMP-stimulated protein kinase activity catalysing the phosphorylation of endogenous substrates. Extraction of membrane fragments with Triton X-100 solubilized less than 20% of the kinase activity and left the major part of the endogenous substrates in the insoluble fraction.
The separation and sequencing of permethylated peptides by mass spectrometry directly coupled to gas-liquid chromatography
Authors: Priddle J D , Rose K , Offord R E
Published: August 2015

The use of g.l.c. coupled to mass spectrometry to separate and sequence permethylated acetyl- and trifluoroacetyl-peptides in a single operation is described. Both electron impact and chemical ionization were used to induce fragmentation, and the latter was found to be more sensitive. Chromatographic retention data are presented which suggest that peptide derivatives of molecular weight of at least 750 are accessible to the technique. The application of our methods to the determination of the primary sequence of proteins is discussed.
The effect of insulin and glucocorticoids on the synthesis and degradation of phosphoenolpyruvate carboxykinase (GTP) in rat adipose tissue cultured in vitro
Authors: Meyuhas O , Reshef L , Ballard F J , Hanson R W
Published: August 2015

1. Epididymal adipose tissue from the rat was maintained in culture for periods of up to 96h. 2. After an initial decrease in protein synthesis during the first 24h of culture, the adipose tissue recovered its capacity to synthesize and accumulate proteins of a relatively large size. 3. The activity of phosphoenolpyruvate carboxykinase decreased in a parallel manner, but increased again after 24h of incubation of the tissue in culture, to a value twice that noted in the tissue in vivo. This increase in enzyme activity was due to an increase in its rate of synthesis. 4. Both insulin and dexamethasone (9alpha-fluoro-16alpha-methyl-11beta,17,-21-trihydroxypregna-1,4-diene-3,20-dione) inhibited phosphoenolpyruvate carboxykinase synthesis, but dexamethasone also decreased total protein synthesis. 5. The half-life of phosphoenolpyruvate carboxykinase in adipose tissue cultured in vitro was 5-7h and was not altered by insulin or dexamethasone. 6. It is concluded that both insulin and glucocroticoids lower the activity of phosphoenolpyruvate carboxykinase in rat adipose tissue by decreasing its rate of synthesis.
Androgen-dependent synthesis of basic secretory proteins by the rat seminal vesicle
Authors: Higgins S J , Burchell J M , Mainwaring W I P
Published: August 2015

1. Two basic proteins were purified from secretions of rat seminal vesicles by using Sephadex G-200 chromatography and polyacrylamide-gel electrophoresis under denaturing conditions. 2. It is not certain that these two proteins are distinct species and not subunits of a larger protein, but their properties are similar. Highly basic (pI = 9.7), they migrate to the cathode at high pH and their amino acid composition shows them to be rich in basic residues and serine. Threonine and hydrophobic residues are few. Both proteins are glycoproteins and have mol.wts. of 17000 and 18500. 3. Together these two proteins account for 25-30% of the protein synthesized by the vesicles, but they are absent from other tissues. 4. Changes in androgen status of the animal markedly affect these proteins. After castration, a progressive decrease in the basic proteins is observed and the synthesis of the two proteins as measured by [35S]methionine incorporation in vitro is is decreased. Testosterone administration in vivo rapidly restores their rates of synthesis. 5. These effects on specific protein synthesis are also observed for total cellular protein, and it is suggested that testosterone acts generally on the total protein-synthetic capacity of the cell and not specifically on individual proteins. Proliferative responses in the secretory epithelium may also be involved. 6. The extreme steroid specificity of the induction process suggests that the synthesis of these basic proteins is mediated by the androgen-receptor system. 7. The biological function of these proteins is not clear, but they do not appear to be involved in the formation of the copulatory plug.
Studies on the relative biopotencies and intestinal absorption of different apo-β-carotenoids in rats and chickens
Authors: Sharma R V , Mathur S N , Ganguly J
Published: August 2015

1. The biopotencies relative to beta-carotene of several apocarotenoids, such as 8′-, 10′- and 12′-apo-beta-carotenal and methyl 8′-apo-beta-carotenoate, were investigated in rats, on a molar basis, by both curative-growth assay and liver-storage tests. 2. In the curative-growth assays, on a molar basis the biopotencies of 8′-, 10′- and 12′-apo-beta-carotenal and methyl 8′-apo-beta-carotenoate were 72, 78, 72 and 53% respectively, whereas on a weight basis the corresponding values were 93, 111, 111 and 63%, with respect to beta-carotene taken as 100%. In terms of yield of vitamin A, these values were much lower in the liver-storage tests. 3. When 8′-apo-beta-carotenal was fed, the unchanged aldehyde together with small amounts of the corresponding alcohol and larger proportions of the acid rapidly appeared in the tissues of both rats and chickens. The 8′-apocarotenol, 8′-apocarotenoic acid and its methyl ester were absorbed unchanged. The free acid disappeared most rapidly from the tissues, but its methyl ester persisted in the tissues longest. 4. On the basis of these observations it is suggested that most of an apocarotenal is oxidized to the corresponding acid, which, in turn, is mostly degraded to retinoic acid, with small proportions of it being attacked by the dioxygenase system giving retinal.
Role of protein degradation in the growth of livers after a nutritional shift
Authors: Conde R D , Scornik O A
Published: August 2015

Fractional rates of synthesis and degradation of liver porteins were estimated during the rapid restoration of liver mass observed in protein-depleted mice when they are fed with an adequate diet. 1. Net protein gain was fastest 12h after the nutritional shift, when it reached a rate of 48% per day. 2. The RNA/protein ratio in livers of protein-depleted animals was essentially the same as in normal livers; it increased by a maximum of 13% 12h after the nutritional shift. 3. Rates of protein synthesis in vivo were measured by the incorporation into liver protein of massive amounts of L-[1-14C]leucine. In protein-depleted animals, the rate of synthesis per mg of RNA was 72% of that in normal livers. Normal rates were recovered within 12h of the nutritional shift. 4. The fraction of newly synthesized protein retained by the liver was studied after they were pulse-labelled by the intravenous injection of radioactive leucine, and, 5 min later, pactamycin (an inhibitor of the initiation of protein synthesis); 3h later the livers in both experimental situations retained 58% of the newly synthesized protein. 5. Fractional rates of protein degradation were estimated either from the difference between the synthesis of stable liver proteins and the net protein increase, or by the disappearance of radioactivity from the liver protein previously labelled by the administration to the mice of NaH14CO3. Both procedures demonstrated a large decrease in the rate of protein degradation during liver growth.
The effect of intralysosomal sucrose storage on the turnover of hamster fibroblast lysosomal and Golgi-apparatus enzymes
Authors: Warburton M J , Wynn C H
Published: August 2015

1. The effect of overloading of hamster fibroblast lysosomes with sucrose on the turnover of lysosomal and Golgi-apparatus enzymes was studied. Arylsulphatase B and UDP-galactose-N-acetylglucosamine galactosyltransferase were chosen as appropriate marker enzymes. The relative contributions of changes in the rates of synthesis and degradation to the increased activities of these enzymes after uptake of sucrose were examined by isotopic-labelling experiments. The effects of sucrose uptake on the degradation of total cellular protein and of the cytoplasmic enzyme, alkaline ribonuclease, were determined for comparative purposes. 2. The rates of enzyme synthesis in the presence and absence of sucrose were compared by pulse-labelling the cells with 14C-labelled amino acids, followed by isolation of purified enzymes and determination of their radioactivity. Sucrose uptake produced increases of 270% and 90% respectively in the rates of synthesis of arylsulphatase B and galactosyltransferase, whereas the rate of synthesis of alkaline ribonuclease was not affected. 3. The rates of degration of the enzymes were estimated by measuring the decay with time of the radioactivity of purified enzymes from prelabelled cells. In the absence of sucrose, the apparent half-lives of arylsulphatase B and galactosyltransferase were about 30 days and 40h respectively. After uptake of sucrose, the half-life of arylsulphatase B decreased to about 10 days and that of galactosyltransferase to 10h. Neither the half-life of alkaline ribonuclease (4 days) nor the rate of degradation of total cellular protein was affected by the uptake of sucrose. 4. These results indicate that the hyperactivity of lysosmal and Golgi-apparatus enzymes after the uptake of sucrose is accompanied by increases in the rates of both synthesis and degradation, and that the increased rates of degradation are insufficient to prevent accumulation of the excess of enzymes synthesized; also that the effects of sucrose uptake are restricted to the vacuolar apparatus.
The composition of an unusual precursor of 50 S ribosomes in a mutant of Escherichia coli
Authors: Markey F , Sims P F , Wild D G
Published: August 2015

Escherichia coli strain 15–28 is a mutant which during exponential growth contains large amounts of a ‘47S’ ribonucleoprotein precursor to 50S ribosomes. The ‘47S particles’ are more sensitive to ribonuclease than are 50S ribosomes. The 23 S RNA of 47S particles may be slightly undermethylated, but cannot be distinguished from the 23S RNA of 50S ribosomes by sedimentation or electrophoresis. Isolated particles have 10–15% less protein than do 50S ribosomes; proteins L16, L28 and L33 are absent. Comparison with precursor particles studied by other workers in wild-type strains of E. coli suggests that the assembly of 50S ribosomes in strain 15–28 is atypical.
Liposomal retention of a modified anti-inflammatory steroid
Authors: Shaw I H , Knight C G , Dingle J T
Published: August 2015

In studies with synthetic lecithins, dipalmitoylphosphatidylcholine was found to be the preferred form for liposomal retention of cortisol esters at 37 degrees C. Cortisol palmitate was retained longer than cortisol octanoate, whereas unesterified cortisol escaped readily from liposomes. Such a liposome composition may allow the controlled release of modified anti-inflammatory agents, particularly when used for intra-articular administration.
Leucine and d-3-hydroxybutyrate as lipid precursors in developing rat spinal cord and liver
Authors: Ramsey R B
Published: August 2015

By using a labelled ketone body, D-3-hydroxy[3-14C]butyrate, or a ketone-body precursor, L-[4,5-3H]leucine, preferential labelling of spinal-cord cholesterol was demonstrated. In contrast, the phospholipid fraction was more heavily labelled in the liver. In both tissues phosphatidylcholine was the major labelled complex lipid. Incorporation ob both substrates into total lipid, on a tissue-weight basis, was appreciably greater for spinal cord.
Altered polyamine metabolism in the PRO/Re strain of inbred mice
Authors: Manen C A , Blake R L , Russell D H
Published: August 2015

The PRO/Re strain of inbred mice are characterized by abnormally high concentrations of proline in both blood (hyperprolinaemia) and urine (prolinuria). They excrete increased amounts of polyamines in their urine. Male PRO/Re mice excreted putrescine at 175% and spermidine at 300% the amount of male C57BL/6J controls. Female PRO/Re mice excreted putrescine at 115% and spermidine at 150% of the amount in the urine of female controls. Examination of the enzymes involved in polyamine biosynthesis revealed that ornithine decarboxylase, the initial enzyme in the polyamine-biosynthetic pathway, was increased by 150% in the kidneys and by 100% in the liver of male PRO/Re mice. There was no significant difference between PRO/Re and C57BL/6J male mice for either putrescine- or spermidine-stimulated S-adenosylmethionine decarboxylase activity. Female PRO/Re mice showed no significant difference from female C57BL/6J mice for any of the enzymes examined. When the concentrations of the polyamines in the tissues of the PRO/Re mice were determined, spermidine and spermine concentrations in the kidneys of the male PRO/Re mice were twice those of the controls. Spermidine concentration in the livers of both male and female PRO/Re mice was approx. 130% that of the controls. Polyamine concentrations in the brains were similar in controls and mutants. The increased polyamine biosynthesis and excretion in the PRO/Re mutant mice may be a mechanism to decrease the extent of proline accumulation.
Binding of diethylstilboestrol to deoxyribonucleic acid by rat liver microsomal fractions in vitro and in mouse foetal cells in culture
Authors: Blackburn G M , Thompson M H , King H W S
Published: August 2015

Diethylstillboestrol, a synthetic and carcinogenic hormone, binds to DNA as a result of incubation with a liver microsomal preparation in vitro and on incubation with primary mouse foetal cells in culture. Enzymic digestion of DNA samples thus prepared gives several covalent deoxyribonucleoside-diethylstilboestrol products from the microsomal system. One of these is produced in small but significant yield in the tissue-culture system.
Alternative products in the reaction of 2-nitro-5-thiocyanatobenzoic acid with thiol groups
Authors: Price N C
Published: August 2015

2-Nitro-5-thiocyanatobenzoic acid has been proposed as a reagent for converting thiol groups in proteins into their S-cyano derivatives. Evidence was obtained for formation of both the S-cyano derivative and the mixed disulphide derivative. Formation of the S-cyano derivative can be promoted by addition of excess of CN-to the reaction mixture.
The action of snake venom, phospholipase A and trypsin on purified myelin in vitro
Authors: Banik N L , Gohil K , Davison A N
Published: August 2015

1. Purified myelin was incubated with snake venom or phospholipase A in the presence of or absence of trypsin at 37°C, pH7.4, for different times. 2. Analysis of the myelin pellet obtained after centrifugation of the myelin sample incubated with snake venom or phospholipase A alone showed conversion of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine into their corresponding lyso compounds. No significant loss of myelin protein was observed in these samples. 3. A marked digestion of basic proteins and proteolipid protein was observed from the myelin pellet when trypsin was present in the incubation mixture. 4. The digestion of basic protein and particularly of proteolipid from myelin suggest that phospholipases may make protein more exposed to proteolytic enzyme for its digestion. 5. The relevance of the co-operative effect of phospholipases and proteinases as a model system of the mechanism of myelin breakdown in degenerative brain diseases is discussed.
The transport of vitamin D in the serum of primates
Authors: Bouillon R , van Baelen H , de Moor P
Published: August 2015

“Transcalciferin” (the serum transport protein for cholecalciferol and related substances) of two New World monkeys, Cebus apella and Cebus albifrons, was found to be immunologically identical with the transcalciferin of other monkeys and partially with that of man. In contrast with the α-globulin mobility of the transcalciferin of other primates, the transcalciferin of cebus monkey has the electrophoretic mobility of albumin. Most of the serum 25-hydroxycholecalciferol was precipitable with isolated monospecific anti-(human transcalciferin) γ-globulins but not with anti-(human albumin) γ-globulins. These results indicate that the transport of 25-hydroxycholecalciferol in the cebus monkey is not due to albumin itself but to transcalciferin with the electrophoretic mobility of albumin. Similar variants of transcalciferin also exist in man.
The semisynthesis of portions of hen's-egg lysozyme by fragment condensation
Authors: Rees A R , Offord R E
Published: August 2015

1. We have evaluated several methods for coupling protected tryptic peptides and have selected one that is satisfactory in terms of efficiency and degree of racemization. 2. We report the use of the chosen method (which must be slightly modified for each application in the light of the result of micro-scale pilot experiments) for the preparation of five fragments of hen's-egg lysozyme ranging in length from 31 to 51 residues. 3. We report methods for the complete deprotection of these fragments. They have been characterized by end-group determination, amino acid analysis and tryptic digestion.
Rat α2 acute-phase macroglobulin. Isolation and physicochemical properties
Authors: Gauthier F , Mouray H
Published: August 2015

1. Rat α2 acute-phase macroglobulin was isolated from turpentine-injected rats by Sephadex G-200 chromatography and ion-exchange chromatography on DEAE-cellulose. This method, since it does not include (NH4)2SO4 treatment, allows the study of the physicochemical as well as the biological properties of the molecule. 2. The purity of the preparation was demonstrated by ultracentrifugation, polyacrylamide-gel electrophoresis, fused “rocket” immunoelectrophoresis as well as double immunodiffusion. 3. The rat α2 acute-phase macroglobulin was characterized in terms of its main physical and chemical properties. Its isoelctric point was determined by isoelectrofocusing to be 4.55; s020,w was 18.4S and E1%/1cm at 278 nm was 6.8. The mol.wt. was determined by light-scattering to be 770000. 4. The amino acid content was compared with that of rat α1 macroglobulin and was found very similar. The carbohydrate composition of α2 acute-phase macroglobulin was determined to be: hexose, 4.25%; glucosamine, 3.4%; sialic acid, 2%; fucose, 0.2%. From these results it was concluded that α2 acute-phase macroglobulin, although a typical acute-phase reactant, possesses the characteristic physicochemical properties of α macroglobulins.
Conversion of (±/-)-synephrine into p-hydroxyphenylacetaldehyde by Arthrobacter synephrinum. A novel enzymic reaction
Authors: Veeraswamy M , Devi N A , Kutty R K , Rao P V S
Published: August 2015

A partically purified enzyme from Arthrobacter synephrinum was found to catalyse the conversion of (±)-synphrine into p-hydroxyphrenylacetaldehyde and methylamine. The enzyme is highly specific for synephrine and is distinctly different from monoamine oxidase.