bluuebunny/crossref_metadata_2025 huggingface dataset:
The synthesis and hydrolysis of long-chain fatty acyl-coenzyme A thioesters by soluble and microsomal fractions from the brain of the developing rat
Authors: Brophy P J , Vance D E
Published: August 2015

1. The specific activities of long-chain fatty acid-CoA ligase (EC6.2.1.3) and of long-chain fatty acyl-CoA hydrolase (EC3.1.2.2) were measured in soluble and microsomal fractions from rat brain. 2. In the presence of either palmitic acid or stearic acid, the specific activity of the ligase increased during development; the specific activity of this enzyme with arachidic acid or behenic acid was considerably lower. 3. The specific activities of palmitoyl-CoA hydrolase and of stearoyl-CoA hydrolase in the microsomal fraction decreased markedly (75%) between 6 and 20 days after birth; by contrast, the corresponding specific activities in the soluble fraction showed no decline. 4. Stearoyl-CoA hydrolase in the microsomal fraction is inhibited (99%) by bovine serum albumin; this is in contrast with the microsomal fatty acid-chain-elongation system, which is stimulated 3.9-fold by albumin. Inhibition of stearoyl-CoA hydrolase does not stimulate stearoyl-CoA chain elongation. Therefore it does not appear likely that the decline in the specific activity of hydrolase during myelogenesis is responsible for the increased rate of fatty acid chain elongation. 5. It is suggested that the decline in specific activity of the microsomal hydrolase and to a lesser extent the increase in the specific activity of the ligase is directly related to the increased demand for long-chain acyl-CoA esters during myelogenesis as substrates in the biosynthesis of myelin lipids.
The role of free serum tryptophan in the biphasic effect of acute ethanol administration on the concentrations of rat brain tryptophan, 5-hydroxytryptamine and 5-hydroxyindol-3-ylacetic acid
Authors: Badawy A A , Evans M
Published: August 2015

1. Acute administration of ethanol exerts a biphasic effect on the concentrations of rat brain tryptophan, 5-hydroxytryptamine and 5-hydroxyindol-3-ylacetic acid. Both effects are associated with corresponding changes in the availability of circulating free tryptophan. 2. The initial increases in the above concentrations are prevented by ergotamine, are unaltered by allopurinol and are potentiated by theophylline, whereas the later decreases are prevented by both ergotamine and allopurinol. 3. It is suggested that the initial enhancement by ethanol of brain tryptophan metabolism is caused by catecholamine-mediated lipolysis followed by displacement of protein-bound serum tryptophan, whereas the activation of liver tryptophaan pyrrolase, which is produced by the same mechanism, leads to the later decreases in the brain concentrations of tryptophan and its metabolites. 4. The initial effects of ethanol can be reproduced by an equicaloric dose of sucrose, and a comparison of the two treatments alone could therefore be misleading. 5. The effects of ethanol on liver and brain tryptophan metabolism have also been examined in mice, and a comparison of the results with those previously reported suggests that the ethanol effects are strain-dependent.
Calcium metabolism and amylase release in rat parotid acinar cells
Authors: Kanagasuntheram P , Randle P J
Published: August 2015

1. A method is described for the isolation of rat parotid acinar cells by controlled digestion of the gland with trypsin followed by collagenase. As judged by Trypan Blue exclusion, electron microscopy, water, electrolyte and ATP concentrations and release of amylase and lactate dehydrogenase, the cells are morphologically and functionally intact. 2. A method was developed for perifusion of acinar cells by embedding them in Sephadex G-10. Release of amylase was stimulated by adrenaline (0.1-10μM), isoproternol (1 or 10 μM), phenylephrine (1 μM), carbamoylcholine (0.1 or 1 μM), dibutyryl cycle AMP (2 MM), 3-isobutyl-1-methylxanthine (1mM) and ionophore A23187. The effects of phenylephrine, carbamoylcholine and ionophore A23187 required extracellular Ca2+, whereas the effects of adrenaline and isoproterenol did not. 3. The incorporation of 45Ca into parotid cells showed a rapidly equilibrating pool (1-2 min) corresponding to 15% of total Ca2+ and a slowly equilibrating pool (> 3h) of probably a similar dimension. Cholinergic and α-adrenergic effectors and ionophore A23187 and 2,4-dinitrophenol increased the rate of incorporation of 45Ca into a slowly equilibrating pool, whereas β-adrenergic effectors and dibutyryl cyclic AMP were inactive. 4. The efflux of 45Ca from cells into Ca2+-free medium was inhibited by phenylephrine and carbamoylcholine and accelerated by isoproterenol, adrenaline (β-adrenergic effect), dibutyryl cyclic AMP and ionophore A23187. 5. A method was developed for the measurement of exchangeable 45Ca in mitochondria in parotid pieces. Incorporation of 45Ca into mitochondria was decreased by isoproterenol, dibutyryl cyclic AMP or 2,4-dinitrophenol, increased by adrenaline, and not changed significantly by phenylephrine or carbamoylcholine. Release of 45Ca from mitochondria in parotid pieced incubated in a Ca2+-free medium was increased by isoproterenol, adrenaline, dibutyryl cyclic AMP or 2,4-dinitrophenol and unaffected by phenylephrine or carbamoylcholine. 6. These findings are compatible with a role for Ca2+ as a mediator of amylase-secretory responses in rat parotid acinar cells, but no definite conclusions about its role can be drawn in the absence of knowledge of the molecular mechanisms involved, their location, and free Ca2+ concentration in appropriate cell compartment(s).
Transannular dioxygenation of 9,10-dimethyl-1,2-benzanthracene by cytochrome P-450 oxygenase of rat liver
Authors: Chen C , Tu M
Published: August 2015

9,10-Dimethyl-1,2-benzanthracene is oxygenated by rat liver microsomal cytochrome P-450 oxygenase to its 9,10-epidioxide. This transannular 1,4-peroxide is converted further into the diol by the microsomal preparation and NADPH. These two products constitute the majority of the metabolites found under the conditions described.
Action of human cathepsin G on the oxidized B chain of insulin
Authors: Blow A M J , Barrett A J
Published: August 2015

The specificity of cathepsin G, a serine neutral proteinase from human neutrophil leucotyes, was determine dby its action on the insulin B chain. The most susceptible bonds were Phe-24-Phe-25, Leu-15-Tyr-16 and Tyr-16-Leu-17. Other bonds hydrolysed were Leu-6-Cys(O3H)-7, Leu-11-Val-12, Leu-17-Val-18 and Phe-25-Tyr-26. These results suggest that the specificity of cathespin G is closer to that of pig chymotrypsin C than ox Chymotrypsin A. Tables listing amino acid composition, N-terminal residue, and yields of isolated peptides have been deposited as Supplementary Publication SUP 50 075 (8 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7B2, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1977) 161,1.
Glycosidases from the culture medium of Physarum polycephalum
Authors: Kilpatrick D C , Stirling J L
Published: August 2015

Eight exo-glycosidase activities were detected in the axenic culture medium of the myxomycete, Physarum polycephalum. The secretion of each enzyme examined followed the growth curve and continued during the stationary phase after the cessation of growth. Two or more forms of each enzyme were detected after electrophoretic separation. The beta-N-acetyl-D-hexosaminidase activity was readily separated into its two electrophoretic forms, X and Y, which were purified 145- and 306-fold respectively. These beta-N-acetyl-D-hexosaminidases had several similar characteristics. Evidence is presented that the major electrophoretic form of alpha-D-galactosidase is heterogeneous. The possible functions of extracellular glycosidases in teir occurrence and properties.
Partial amino acid sequence of rat pre-prolactin
Authors: Maurer R A , Gorski J , McKean D J
Published: August 2015

Rat pituitary mRNA was used to direct the cell-free synthesis of pre-prolactin labelled with [4,5-3H]leucine and either [35S] methioninc or [35S] cystine. Sequence analysis of the labelled protein indicates that pre-prolactin has 29 amino acid residues joined to the N-terminus of the prolactin sequence. Leucine residues were found at positions 13, 14, 15, 16, 21 and 22, methionine residues at positions 1, 17 and 18, and a cysteine residue at position 24 of the precursor sequence, and this partial sequence shows considerable similarity with other precursors that have been sequenced.
Structural investigation of loose connective tissue by using a series of dextran fractions as non-interacting macromolecular probes
Authors: Meyer F A , Koblentz M , Silberberg A
Published: August 2015

The ability of the uncharged open-coil dextran molecules to penetrate tissue space, without coil-shape change, was utilized to probe (by partitioning experiments) the structural arrangement of the collagen-fibre network and the proteoglycan system. Hyaluronidase digests most of the proteoglycans away and enables the respective contributions to the exclusion volume to be evaluated by using a series of different-molecular-weight dextrans. It appears that the major part of the exclusion volume is due to the collagen-fibril as a rod and the dextran coil as an impenetrable sphere. The additional exclusion due to the proteoglycans could be accounted for by a set of points (regions of high proteoglycan-segment density) over which the dextran coild cannot pass. These points are an average of 50 nm apart and are indicative of local extensive entanglement of high-molecular-weight proteoglycans with each other. Reasons are given why these entanglements could not act as cross-links in long-term elastic loading of the tissue.
Properties of rat liver microsomal stearoyl-coenzyme A desaturase
Authors: Jeffcoat R , Brawn P R , Safford R , James A T
Published: August 2015

1. Rat liver microsomal stearoyl-CoA desaturase activity was shown to be stimulated by both bovine serum albumin and a basic cytoplasmic protein from rat liver. 2. Partially purified desaturase is unaffected by either of these two proteins. 3. Bovine serum albumin appears to exert its effect on the crude system by protecting the desaturase substrate, stearoly-CoA, from the action of endogenous thiolesterases. 4. By using partially purified enzyme preparations, it was possible to establish the substate specificity of the delta9-fatty acyl-CoA desaturase with the C14, C15, C16, C17, C18 and C19 fatty acyl-CoA substrates. Maximum enzyme activity was shown with stearoyl-CoA decreasing with both palmitoyl-CoA and nonadecanoyl-CoA, as reported previously for free fatty acids. 5. Both cytochrome b5 and NADH-cytochrome b5 reductase (EC 1.6.2.2) are required for these studies and a method is described for the purification of homogeneous preparations of detergent-isolated cytochrome b5 from rat liver. 6. From amino acid analyses, a comparison was made of the hydrophobicity of the membrane portion of cytochrome b5 with the hydrophobicity reported for stearoyl-CoA desaturase. The close resemblance of the two values suggested that unlike cytochrome b5 and its reductase, the stearoyl-CoA desaturase may be largely buried in the endoplasmic reticulum.
Inhibition of lipogenesis by halothane in isolated rat liver cells
Authors: Mapes J P
Published: August 2015

1. Halothane at clinically effective concentrations [2.5 and 4% (v/v) of the gas phase of the incubation flask] was found to inhibit significantly lipogenesis from endogenous substrates, e.g., glycogen, or from added lactate plus pyruvate. This was accompanied by a decrease in the ratio of the free [NAD+]/[NADH] of the mitochondrion and the cytoplasm, as shown by the [3-hydroxybutyrate]/[acetoacetate] ratio and the [lactate]/[pyruvate] ratio. 2. Acetoacetate or pyruvate decreased the inhibitory effect of halothane and restored lipogenesis to control rates. They were reduced rapidly by 3-hydroxybutyrate dehydrogenase or lactate dehydrogenase respectively, with the concomitant oxidation of NADH and the generation of NAD+. 3. These results suggest that the mechanism by which halothane inhibits lipogenesis from glycogen or lactate is by inhibition of the oxidation of NADH; this results in inhibition of flux of carbon through pyruvate dehydrogenase and a shortage of acetyl-CoA for fatty acid synthesis. Thus when NADH acceptors are added in the presence of halothane, the concentration of mitochondrial NAD+ is raised so that the flux of carbon through pyruvate dehydrogenase increases and lipogenesis is restored.
The formation of menaquinone-4 (vitamin K) and its oxide in some marine invertebrates
Authors: Burt V T , Bee E , Pennock J F
Published: August 2015

Three echinoderms, a crustacean, a mollusc and a coelenterate were injected with either [2-14C]mevalonate and unlabelled menadione (2-methyl-1,4-naphthaquinone) or 2-[14C]methyl-1,4-naphthaquinone. In each case the label was incorporated into menaquinone-4.[2-14C]Mevalonate was incorporated into menaquinone-4 in the crab (Carcinus maenas) only in the presence of menadione, the organism presumably being unable to synthesize the naphthaquinone nucleus. The starfish Asterias rubens and C. maenas incorporated the label into a compound which was identified chromatographically as 2,3-epoxymenaquinone-4. The significance of menaquinone-4 and these animals is discussed.
Measurement of the absolute rates of cholesterol biosynthesis in isolated rat liver cells
Authors: Gibbons G F , Pullinger C R
Published: August 2015

Triparanol [2-(4-chlorophenyl)-1-(4-diethylaminoethoxyphenyl)-1-p-tolylethanol] at a concentration of 2 micronm has no effect on the overall conversion of [2=14C]acetate into C27 sterols by isolated liver cells. In the presence of triparanol, however, the formation of radioactive cholesterol is inhibited by 85-90% and the balance of radioactivity appears in the C27 sterol desmosterol (cholesta-5,24-dien-3beta-ol). The very small weights of desmosterol which accumulate under these conditions were, as a routine, quantitatively converted into the heptafluorobutyrate 3-enol ester of cholesta-4,24-dien-3-one. This derivative has a high electron-capturing capability, a property that enables extremely small quantities (less than 0.25pmol) of the material to be accurately measured by gas chromatography with electron-capture detection. Measurements of the mass and specific radioactivity of the newly biosynthesized desmosterol formed in the presence of triparanol provides an accurate assessment of the amount of cholesterol that would be synthesized by the liver cells in the absence of the drug.
Kinetic studies on the enzymes conjugating bile acids with taurine and glycine in bovine liver
Authors: Vessey D A , Crissey M H , Zakim D
Published: August 2015

Synthesis of glyco- or tauro-cholate from choloyl-CoA and the respective amino acid is shown to be catalysed by the soluble fraction of liver cells. Kinetic evidence supports the conclusion that there are separate enzymes for the synthesis of glycocholate and taurocholate. The kinetic parameters of these enzymes were determined.
The molecular size and shape of liver glycogen
Authors: Geddes R , Harvey J D , Wills P R
Published: August 2015

The molecular-weight distribution of liver glycogen has been established from the analysis of sedimentation rates of fractions separated on sucrose density gradients and from the direct measurement of the diffusion coefficients of these fractions by laser-intensity-fluctuation spectroscopy. Hydrodynamic studies indicated that all fractions of glycogen of mol.wt.exceeding 25x10(6) had about 1.1 g of water per g of polysaccharide associated with them. The hydration and hydrodynamic behaviour of all fractions of mol.wt. exceeding 25x10(6) was similar, whereas smaller fractions behaved anomalously, indicating a substantially different overall structure.
Evidence that latent collagenases are enzyme-inhibitor complexes
Authors: Sellers A , Cartwright E , Murphy G , Reynolds J J
Published: August 2015

Specific collagenase from the culture media of various rabbit tissues and cells exists in active and latent forms. Latent collagenase is most effectively activated with 4-aminophenylmercuric acetate, a thiol-blocking reagent, strongly suggesting that latent forms are enzyme-inhibitor complexes. A collagenase inhibitor from bone cultures, which may be closely related to the inhibitor of such latent enzyme complexes, was partially characterized.
Phosphorylation of synaptic-membrane proteins from ox cerebral cortex in vitro. Preparation of fractions enriched in phosphorylated proteins by using extraction with detergents and urea, and gel filtration
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(s) that transfer 32P from [gamma-32P]ATP to hydroxyl groups of serine and threonine residues in membrane-protein substrates. In the present work, labelled membrane fragments were partitioned into soluble and insoluble fractions with Triton X-100, Nonidet P. 40, sodium deoxycholate and urea, and the distribution of 32P-labelled protein in the fractions was determined by polyacrylamide-gel electrophoresis and radioautography. A high percentage of phosphorylated protein sustrates remained insoluble, including those whose phosphorylation was most highly stimulated by cyclic AMP. Whole membrane fragments and samples prepared by detergent extraction were fractionated on Sepharose 6B columns in the presence of low concentrations of sodium dodecyl sulphate and pooled fractions were analysed by polyacrylamide-gel electrophoresis and radioautography. Phosphorylated proteins were fractionated on the basis of their molecular weight, but homogeneous protein was not obtained. The results are discussed in relation to the techniques used and the results obtained in other laboratories.
Physical and binding properties of large fragments of human serum albumin
Authors: Geisow M J , Beaven G H
Published: August 2015

Three large fragments of human serum albumin were produced by peptic digestion of the native protein [Geisow & Beaven (1977) Biochem. J. 161, 619-625]. Fragment P44 represents residues 1-386 and fragments P29 and P31 represent residues 49-307 and residues 308-584 respectively of the albumin molecule. The large N-terminal fragment P44 has a similar percentage of alpha-helix to stored defatted albumin, although the alpha-helix content of all the fragments is significantly less than that of freshly prepared albumin. The fragment P44 appears to account for all the binding of the hydrophobic probe 8-anilinonaphthalene-1-sulphonate to albumin. N-Acetyl-L-tryptophan binds to this fragment and displaces one of the bound molecules of 8-anilinonaphthalene-1-sulphonate. Bilirubin binds to fragments P44 and P29, and the complexes show similar circular-dichroism spectra to that of the complex between bilirubin and whole albumin. These results are in agreement with affinity-labeling work on albumin with reactive ligands where substitution occurs in the N-terminal region of the molecule. The sharp conformational transitional transition in albumin which is observed between pH4 and 3.5 was absent from the fragments. This isomerization, usually called the N-F transition, probably occurs in intact albumin as a result of the unfolding or separation of the C-terminal third of the protein from the remainder of the molecule.
Binding of triethyltin to cat haemoglobin and modification of the binding sites by diethyl pyrocarbonate
Authors: Elliott B M , Aldridge W N
Published: August 2015

Cat haemoglobin binds 2 mol of triethyltin/mol of haemoglobin. Pretreatment of the haemoglobin with diethyl pyrocarbonate at pH6.0 prevents binding to one site only, whereas photo-oxidation with Methylene Blue removes both sites. Pretreatment of rat haemoglobin with diethyl pyrocarbonate also leads to the loss of one binding site. The possibility is discussed that the two binding sites for triethyltin on both cat and rat haemoglobin have a different chemical nature.
Regional differences in ribonuclease content of rat and mouse kidney
Authors: Liu Dai Kee , Matrisian Paul E.
Published: August 2015

Kidney cortex, red medulla and white medulla were separated into nuclei, mitochondria, microsomal and 105000g supernatant fractions. Assay of RNAase (ribonuclease) activity at pH7.8 revealed that, for each subcellular fraction, activity was much greater in cortex than in red or white medulla; this was true for both free RNAase and total (free plus latent) RNAase. For example, the free RNAase activity in the 105000g supernatant of cortex was 5 and 8 times higher than in red and white medulla respectively. No latent RNAase activity was found in any particulate fraction. Latent supernatant RNAase activities (suggesting presence of bound RNAase inhibitor) were similar in cortex and medulla. The cortex supernatant contained minimal free RNAase inhibitor, whereas that of the red and white medulla showed about one-third and one-tenth respectively of the inhibitor activity measured in liver. Adrenalectomy did not change RNAase activity in any fraction nor the content of free RNAase inhibitor in the kidney supernatant, but did decrease the liver RNAase inhibitor content by 40%. In supernatants from mouse kidney, both free and total RNAase activities of both cortex and red medulla were similar to those of rat red medulla. Mouse cortex contained appreciably higher amounts of free RNAase inhibitor than rat cortex. The difference between the rat and mouse cortical RNAase activity and inhibitor content may help explain the relative ease with which satisfactory renal polyribosome profiles were obtained from mouse kidneys. Our results, as well as those of Kline & Liberti [(1973) Biochem. Biophys. Res. Commun.52, 1271–1277], showing that renal red and white medulla are more active than cortex in protein synthesis, are consistent with the hypothesis that the RNAase–RNAase-inhibitor system may participate in the regulation of protein synthesis.
Diabetes and the control of pyruvate dehydrogenase in rat heart mitochondria by concentration ratios of adenosine triphosphate/adenosine diphosphate, of reduced/oxidized nicotinamide-adenine dinucleotide and of acetyl-coenzyme A/coenzyme A.
Authors: Kerbey A L , Radcliffe P M , Randle P J
Published: August 2015

1. The proportion of active (dephosphorylated) pyruvate dehydrogenase in rat heart mitochondria was correlated with total concentration ratios of ATP/ADP, NADH/NAD+ and acetyl-CoA/CoA. These metabolites were measured with ATP-dependent and NADH-dependent luciferases. 2. Increase in the concentration ratio of NADH/NAD+ at constant [ATP]/[ADP] and [acetyl-CoA]/[CoA] was associated with increased phosphorylation and inactivation of pyruvate dehydrogenase. This was based on comparison between mitochondria incubated with 0.4mM- or 1mM-succinate and mitochondria incubated with 0.4mM-succinate+/-rotenone. 3. Increase in the concentration ratio acetyl-CoA/CoA at constant [ATP]/[ADP] and [NADH][NAD+] was associated with increased phosphorylation and inactivation of pyruvate dehydrogenase. This was based on comparison between incubations in 50 micrometer-palmitotoyl-L-carnitine and in 250 micrometer-2-oxoglutarate +50 micrometer-L-malate. 4. These findings are consistent with activation of the pyruvate dehydrogenase kinase reaction by high ratios of [NADH]/[NAD+] and of [acetyl-CoA]/[CoA]. 5. Comparison between mitochondria from hearts of diabetic and non-diabetic rats shows that phosphorylation and inactivation of pyruvate dehydrogenase is enhanced in alloxan-diabetes by some factor other than concentration ratios of ATP/ADP, NADH/NAD+ or acetyl-CoA/CoA.
Effects of di-isopropyl phosphorofluoridate on rat liver microsomal and lysosomal β-glucuronidase
Authors: Mandell Brian , Stahl Philip
Published: August 2015

iPr2P-F (di-isopropyl phosphorofluoridate) administration to rats produces a liver-dependent specific elevation of plasma β-glucuronidase activity. The response is unaffected by puromycin pretreatment. By using subcellular-fractionation techniques, the rise in plasma β-glucuronidase activity was correlated temporally with a fall in liver microsomal β-glucuronidase activity. After iPr2P-F treatment, liver microsomal membranes are depleted of β-glucuronidase but slowly return to normal over 1 week. On the other hand, liver lysosomal β-glucuronidase activity is high at early time points (less than 60min) after iPr2P-F administration but decreases to below control values; this lasts for a few days. The response to iPr2P-F was demonstrated in isolated hepatocytes prepared from iPr2P-F-treated rats. In such preparations, microsomal β-glucuronidase is lost rapidly, followed by a specific decrease in hepatocyte lysosomal β-glucuronidase. The results suggest that a pool of microsomal β-glucuronidase serves as precursor to plasma β-glucuronidase in iPr2P-F-treated rats, and further, that microsomal β-glucuronidase may serve as precursor to lysosomal β-glucuronidase.
Kinetic evidence for a ‘mnemonical’ mechanism for rat liver glucokinase
Authors: Storer A C , Cornish-Bowden A
Published: August 2015

Inhibition studies of glucokinase were carried out with the products of the reaction, glucose 6-phosphate and MgADP-, as well as with ADP3-, Mg2+ and ATP4-. The results of these, together with those of kinetic studies of the uninhibited reaction described previously [Storer & Cornish-Bowden (1976) Biochem. J. 159, 7-14], indicate that the enzyme obeys a ‘mnemonical’ mechanism. This implies that the co-operativity observed with glucose as substrate arises because glucose binds differentially to two forms of the free enzyme that are not in equilibrium under steady-state conditions. The mechanism predicts the decrease in glucose co-operativity observed at low concentrations of MgATP2-. The product-inhibition results suggest that glucose 6-phosphate is released first and that it is possibly displaced by MgATP2- in a concerted reaction.
The soluble methane mono-oxygenase of Methylococcus capsulatus (Bath). Its ability to oxygenate n-alkanes, n-alkenes, ethers, and alicyclic, aromatic and heterocyclic compounds
Authors: Colby J , Stirling D I , Dalton H
Published: August 2015

1. Methane mono-oxygenase of Methylococcus capsulatus (Bath) catalyses the oxidation of various substituted methane derivatives including methanol. 2. It is a very non-specific oxygenase and, in some of its catalytic properties, apparently resembles the analogous enzyme from Methylomonas methanica but differs from those found in Methylosinus trichosporium and Methylomonas albus. 3. CO is oxidized to CO2. 4. C1-C8 n-alkanes are hydroxylated, yielding mixtures of the corresponding 1- and 2-alcohols; no 3- or 4-alcohols are formed. 5. Terminal alkenes yield the corresponding 1,2-epoxides. cis- or trans-but-2-ene are each oxidized to a mixture of 2,3-epoxybutane and but-2-en-1-ol with retention of the cis or trans configuration in both products; 2-butanone is also formed from cis-but-2-ene only. 6. Dimethyl ether is oxidized. Diethyl ether undergoes sub-terminal oxidation, yielding ethanol and ethanal in equimolar amounts. 7. Methane mono-oxygenase also hydroxylates cyclic alkanes and aromatic compounds. However, styrene yields only styrene epoxide and pyridine yields only pyridine N-oxide. 8. Of those compounds tested, only NADPH can replace NADH as electron donor.
The effects of amphiphilic cationic drugs and inorganic cations on the activity of phosphatidate phosphohydrolase
Authors: Bowley M , Cooling J , Burditt S L , Brindley D N
Published: August 2015

1. Phosphatidate phosphohydrolase from the particle-free supernatant of rat liver was assayed by using emulsions of phosphatidate as substrate. 2. The inhibition of the phosphohydrolase by chlorpromazine was of a competitive type with respect to phosphatidate. The potency of various amphiphilic cationic drugs as inhibitors of this reaction was related to their partition coefficients into a phosphatidate emulsion. 3. The effect of chlorpromazine on the phosphohydrolase activity was complementary rather than antagonistic towards Mg2+. Chlorpromazine stimulated the phosphohydrolase activity in the absence of added Mg2+ and was able to replace the requirement for Mg2+. However, at optimum concentrations of Mg2+, chlorpromazine inhibited the reaction, as did Ca2+. The phosphohydrolase activity was also stimulated by Co2+ and to a lesser extent by Mn2+, Fe2+, Fe3+, Ca2+, spermine and spermidine when Mg2+ was not added to the assays. 4. It is concluded that the inhibition of phosphatidate phosphohydrolase by amphiphilic cations can largely be explained by the interaction of these compounds with phosphatidate, which changes the physical properties of the lipid, making it less available for conversion into diacylglycerol. 5. The implications of these results to the effects of amphiphilic cations in redirecting glycerolipid synthesis at the level of phosphatidate are discussed.
The isolation and characterization of corticotropin from the pituitary gland of the ostrich Struthio camelus
Authors: Naudé R J , Oelofsen W
Published: August 2015

1. Avian corticotropin (ACTH) was purified from both fresh and aged pituitary glands of the ostrich Struthio camelus. 2. The isolation of corticotropin in pure form involved acid/acetone extraction, NaCl fractionation, CM-cellulose chromatography and Sephadex G-50 chromatography. 3. The hormone preparations from fresh and aged glands behaved as single substances on polyacrylamide-gel electrophoresis, and both preparations were found to consist of 39 amino acid residues, in identical molar proportions for the different amino acids. 4. The isoelectric points of the two hormone preparations were estimated to be in the range pH 8.3-8.7, indicating possible differences in amide content, and the N-terminal amino acid of both preparations appeared to be serine. 5. The hormone preparations from fresh and aged glands exhibited similar biological potencies (73 and 77 i.u./mg respectively), as measured by steroidogenesis in vitro. 6. Apart from possible differences in amide content, the corticotropin preparations obtained from fresh and aged glands appear to be indistinguishable.
A fluorimetric study of the role of calcium ions in the stability of thermolysin
Authors: Fontana Angelo , Vita Claudio , Boccu' Enrico , Veronese Francesco M.
Published: August 2015

1. Fluorimetric techniques were used to characterize the environment of tryptophan residues in thermolysin and apo-thermolysin. The apo-thermolysin was obtained by dissolving the enzyme in the presence of 10mm-EDTA, which removed the functional Zn2+ ion and the four Ca2+ ions/molecule from the enzyme. 2. At 25°C in aqueous solution the fluorescence-emission spectrum of the native holoenzyme, on excitation at 290nm, was essentially characteristic of tryptophan, with an emission maximum at 333nm. The emission maximum of the apoenzyme is red-shifted to 338nm and the relative intensity of fluorescence is decreased by 10%, both effects indicating some unfolding of the protein molecule, with the indole groups being transferred to a more hydrophilic environment. 3. Fluorescence quenching studies using KI, N′-methylnicotinamide hydrochloride and acrylamide indicated a more open structure in the apoenzyme, with the tryptophan residues located in a negatively charged environment. 4. The thermal properties of the apoenzyme, as monitored by fluorescence-emission measurements, are dramatically changed with respect to the native holoenzyme. In fact, whereas the native enzyme is heat-stable up to about 80°C, for the apoenzyme a thermal transition is observed near 48°C. The apoenzyme is also unstable to the action of unfolding agents such as urea and guanidinium chloride, much as for other globular proteins from mesophilic organisms. 5. The functional Zn2+ ion does not contribute noticeably to the stability of thermolysin. 6. It is concluded that a major role in the structural stability of thermolysin is played by the Ca2+ ions, which have a bridging function within this disulphide-free protein molecule.
Partial purification, properties and regulation of inosine 5′-phosphate dehydrogenase in normal and malignant rat tissues
Authors: Jackson R C , Morris H P , Weber G
Published: August 2015

IMP dehydrogenase (EC 1.2.1.14) was purified 180-fold from rat liver and from the transplantable rat hepatoma 3924A. The enzymes from the two sources were apparently identical; they exhibited hyperbolic saturation kinetics and an ordered, sequential mechanism, and were subject to inhibition by a number of purine nucleotides. Km values for the substrates, IMP and NAD+, were 12 and 24 micrometer respectively. IMP dehydrogenase activity in a spectrum of rat hepatomas was increased, relative to normal liver, by 2.5—13-fold; these increases correlated with tumour growth rate. Activity in two rat kidney tumours was increased 3-fold relative to that in normal renal cortex; control of activity of this enzyme is apparently altered in neoplastic cells. After partial hepatectomy, IMP dehydrogenase activity began to rise 6 h after operation, reaching a peak of 580% of normal activity by 18 h. Activity in neonatal liver, however, was only slightly higher than that in the adult. Organ-distribution studies showed highest enzyme activities in spleen and thymus. In livers of rats starved for 3 days, where all enzymes, except those involved in gluconeogenesis, showed decreased activity IMP dehydrogenase activity was increased; this change was accompanied by a rise in hepatic GTP concentrations. It is concluded that IMP dehydrogenase is a key enzyme in the regulation of GTP production, and thus involved in regulation of nucleic acid biosynthesis. The increased activity of IMP dehydrogenase in liver of starved rats may be related to the requirements for GTP for gluconeogenesis.
Protein synthesis during right-ventricular hypertrophy after pulmonary-artery stenosis in the dog
Authors: Everett A W , Taylor R R , Sparrow M P
Published: August 2015

The rate of protein synthesis in the heart of normal dogs and those with pulmonary-artery stenosis was measured by a continuous intravenous infusion of [14C]tyrosine. The protein-synthesis rate of both ventricles was the same in normal dogs and averaged 7.5% per day. The right ventricle hypertrophied rapidly after the acute imposition of pulmonary-artery stenosis, the wet weight increasing by 84% after 24 days, with the rate of increase being most rapid over the first 5 days. The left ventricle remained largely unaffected and served as an internal control. During hypertrophy there was an increased incorporation of [14C]tyrosine into protein without a significant change in the specific radioactivity of free tyrosine in the ventricles. After 5 days of stenosis the synthesis rate of the total mixed proteins of the right ventricle had increased to 13.6% per day, compared with 6.2% in the control left ventricle. This increase in synthesis was reflected in both the myofibrillar and sarcoplasmic proteins. After 5 days the protein-synthesis rate decreased, but still remained significantly elevated above that in the control left ventricle by 24 days of stenosis.
Iron-sulphur centres in mitochondria from Arum maculatum spadix with very high rates of cyanide-resistant respiration
Authors: Cammack R , Palmer J M
Published: August 2015

X-band electron-paramagnetic-resonance spectroscopy at 4.2–77K combined with measurements of oxidation-reduction potential was used to identify iron–sulphur centres in Arum maculatum (cuckoo-pint) mitochondria. In the oxidized state a signal with a derivative maximum at g = 2.02 was assigned to succinate dehydrogenase centre S-3. Unreduced particles showed additional signals at g = 2.04 and 1.98 (at 9.2 GHz), which may be due to a spin-spin interaction. In the reduced state a prominent signal at g = 1.93 and 2.02 was resolved into at least three components that could be assigned to centres S-1 and S-2 of succinate dehydrogenase (midpoint potentials −7 and −240 mV respectively at pH 7.2) and a small amount of centre N-1b (e'o= −240 mV) of NADH-ubiquinone reductase. In addition, changes in line shape around −10 mV indicated the presence of a fourth component in this signal. The latter was more readily reduced by NADH than by succinate, suggesting that it might be associated with the external NADH dehydrogenase. The iron-sulphur centres of NADH-ubiquinone reductase were present in an unusually low concentration, indicating that the alternative, non-phosphorylating, NADH dehydrogenase containing a low number of iron-sulphur centres may be responsible for most of the high rate of oxidation of NADH.
Identification of a non-magnetic iron centre and an iron-storage or -transport material in blue—green algal membranes by Mössbauer spectroscopy
Authors: Evans E H , Carr N G , Rush J D , Johnson C E
Published: August 2015

At least two species of iron were identified in crude membrane preparations of the blue-green algae Chlorogloea fritschii and Anacystis nidulans by Mössbauer spectroscopy. One species was non-magnetic with chemical shift more characteristic of a ferredoxin-like centre than of any known low-spin Fe(II) biological compounds. Studies on cells grown under depleted-iron conditions show the other iron species to be part of the system for iron metabolic transport and/or storage.
Alterations in nicotinamide and adenine nucleotide systems during mixed-function oxidation of p-nitroanisole in perfused livers from normal and phenobarbital-treated rats
Authors: Kauffman Frederick C. , Evans Roxanne K. , Thurman Ronald G.
Published: August 2015

The contents of adenine nucleotides as well as steady-state concentrations of a number of glycolytic, pentose phosphate-pathway and tricarboxylic acid-cycle intermediates were measured in extracts of livers from normal and phenobarbital-treated rats that were perfused with p-nitroanisole. Metabolites were measured in livers that were freeze-clamped during periods of maximal rates of drug metabolism. Treatment of rats with phenobarbital increased rates of p-nitroanisole O-demethylation approx. fivefold. The concentrations of lactate, xylulose 5-phosphate and ribulose 5-phosphate were increased by phenobarbital treatment, whereas that of fructose 1,6-bisphosphate declined. Perfusion of livers with p-nitroanisole produced significant increases in 6-phosphogluconate and ribulose 5-phosphate in livers from phenobarbital-treated rats, but not in livers from control rats. Treatment of rats with phenobarbital caused [NADP+]/[NADPH] to change in the direction of more oxidation, as calculated from measured concentrations of 6-phosphogluconate and ribulose 5-phosphate; however, the [NADP+]/[NADPH] ratio calculated from ‘malic’ enzyme was not changed. Additions of p-nitroanisole produced a reduction of NADP+ as calculated from 6-phosphogluconate dehydrogenase activity, but did not alter the [NADP+]/[NADPH] ratio calculated from substrates assumed to be in equilibrium with ‘malic’ enzyme. Activities of both glucose 6-phosphate dehydrogenase and ‘malic’ enzyme were increased by phenobarbital treatment. NAD+ became more reduced as a result of phenobarbital treatment; however, perfusion of livers with p-nitroanisole did not cause a change in the oxidation–reduction state of this nucleotide. Concentrations of adenine nucleotides in livers were not altered significantly by treatment of rats with phenobarbital; however, a significant decline in the [ATP]/[ADP] ratio occurred during mixed-function oxidation of p-nitroanisole in livers from phenobarbital-treated rats, but not in livers from normal rats. Perfusion of livers with two other substrates for mixed-function oxidation, hexobarbital and aminopyrine, produced an increase in the [NADP+]/[NADPH] ratio calculated from ‘malic’ enzyme. In contrast with livers perfused with p-nitroanisole, there was no significant change in adenine nucleotides in livers exposed to hexobarbital or aminopyrine. Addition of 2,4-dinitrophenol (25μm) to the perfusate containing aminopyrine decreased the [ATP]/[ADP] ratio and tended to prevent the oxidation of NADPH observed with aminopyrine alone. Thus in the presence of an uncoupler of oxidative phosphorylation, NADPH generation may exceed its utilization via mixed-function oxidation.
The effects of ionophores on the fluorescence of the cation 3,3′-dipropyloxadicarbocyanine in the presence of pigeon erythrocytes, erythrocyte ‘ghosts’ or liposomes
Authors: Kimmich G A , Philo R D , Eddy A A
Published: August 2015

1. Pigeon erythrocytes, resealed lysed erythrocytes or liposomes derived from erythrocyte lipids were suspended in solutions containing up to 2 micrometer-3,3′-dipropyloxadicarbocyanine iodide. Gramicidin, valinomycin, nigericin or carbonyl cyanide p-trifluoromethoxy-phenylhydrazone, or combinations of these, were used to induce electrical diffusion potentials dependent on Na+, K+ or protons. In each instance hyperpolarization of the cell membrane lowered the fluorescence of the cell suspension, a process that was completed in about 1 min. Subsequent depolarization caused an increase in fluorescence. 2. Quenching of the fluorescence of the cell suspension appeared to be due to the reversible binding of the dye to the cells. Much larger amounts of dye were bound, both to the intact and to the resealed erythrocytes, than would be expected if partitioning of the dye cation followed the Nernst equation. The dependence of the binding on the extracellular dye concentration was studied in the presence and absence of valinomycin. The results were consistent with the suggestion of Sims, Waggoner, Wang & Hoffman [(1974) Biochemistry 13, 3315-3330] that the dye was bound at both membrane surfaces and that, at low dye concentrations, hyperpolarizing the cells promoted dye binding at the inner membrane surface. 3. The applications of the technique are limited by the circumstance that the direct effect of the electric field on the uptake of the dye into the cells is amplified by a binding process that may be affected by other physiological variables.
Fatty acid biosynthesis by a particulate preparation from germinating pea
Authors: Bolton Paul , Harwood John L.
Published: August 2015

1. Fatty acid synthesis was studied in microsomal preparations from germinating pea (Pisum sativum). 2. The preparations synthesized a mixture of saturated fatty acids up to a chain length of C24 from [14C]malonyl-CoA. 3. Whereas hexadecanoic acid was made de novo, octadecanoic acid and icosanoic acid were synthesized by elongation. 4. The products formed during [14C]malonyl-CoA incubation were analysed, and unesterified fatty acids and polar lipids were found to be major products. [14C]Palmitic acid represented a high percentage of the acyl-carrier protein esters, whereas 14C-labelled very-long-chain fatty acids were mainly present as unesterified fatty acids. CoA esters were minor products. 5. The addition of exogenous lipids to the incubation system usually resulted in stimulation of [14C]malonyl-CoA incorporation into fatty acids. The greatest stimulation was obtained with dipalmitoyl phosphatidylcholine. Both exogenous palmitic acid and dipalmitoyl phosphatidylcholine increased the amount of [14C]-stearic acid synthesized, relative to [14C]palmitic acid. Addition of stearic acid increased the amount of [14C]icosanoic acid formed. 6. [14C]Stearic acid was elongated more effectively to icosanoic acid than [14C]stearoyl-CoA, and its conversion was not decreased by addition of unlabelled stearoyl-CoA. 7. Incorporation of [14C]malonyl-CoA into fatty acids was markedly decreased by iodoacetamide and 5,5′-dithiobis-(2-nitrobenzoic acid). Palmitate elongation was sensitive to arsenite addition, and stearate elongation to the presence of Triton X-100 or fluoride. The action of fluoride was not, apparently, due to chelation. 8. The microsomal preparations differed from soluble fractions from germinating pea in (a) synthesizing very-long-chain fatty acids, (b) not utilizing exogenous palmitate–acyl-carrier protein as a substrate for palmitate elongation and (c) having fatty acid synthesis stimulated by the addition of certain complex lipids.
The inhibition of lipid autoxidation by human caeruloplasmin
Authors: Al-Timimi D J , Dormandy T L
Published: August 2015

1. Purified caeruloplasmin was shown to inhibit lipid autoxidation induced by ascorbic acid or inorganic iron in the following systems: (a) an emulsion of linolenic acid in water; (b) an untreated ox brain homogenate in phosphate buffer; (c) a similar homogenate whose susceptibility to autoxidation had been abolished by dialysis or by heating and then restored by the above pro-oxidants. 2. The optimum conditions for this antioxidant activity were studied. 3. Caeruloplasmin did not inhibit autoxidation by u.v. irradiation in dialysed or preheated homogenates. 4. The apoprotein (without copper) had no antioxidant activity, whereas CuSO4 alone was much less effective as an antioxidant. 5. Iron-free transferrin also had some antioxidant activity.
Passive ion permeability of the chromaffin-granule membrane
Authors: Phillips J H
Published: August 2015

‘Ghosts’ of bovine chromaffin granules, in which the complex mixture of proteins and solutes normally found in the granule matrix is replaced by buffered sucrose are osmotically sensitive. They shrink when the osmotic pressure of the suspension medium is increased, and swell if solute entry is facilitated by the addition of ionophores. Swelling in the presence of ionophores has been used to investigate the passive ion permeability of these membranes. They have a very low permeability to K+ ions (of the order of 10(-10) cm/s); their permeability to protons, Na+ and choline ions is too low to be detected by these methods. Their passive permeability to anions decreases in the order: CNS- greater than I- greater than CCl3CO2- greater than Br- greater than Cl- greater than SO4(2)- greater than CH3CO2-, HCO3-, F-, PO4(3)- the permeability to hiocyanate being of the order of 10(-7) cm/s. Coupled proton and anion entry is extremely slow, except for weak acids. Fluoride, unexpectedly, also appears to enter rapidly when proton/K+ exchange is facilitated by nigericin. In the presence of K+ salts, nigericin, like valinomycin, induces lysis of intact granules, an effect that is not dependent on the presence of a permeant anion, but is dependent on the pH gradient across the membrane.
NAD(P)H dehydrogenase and its role in the vitamin K (2-methyl-3-phytyl-1,4-naphthaquinone)-dependent carboxylation reaction
Authors: Wallin R , Gebhardt O , Prydz H
Published: August 2015

A simple three-step method was established for the purification of NAD(P)H dehydrogenase (quinone) (‘DT-diaphorase’, EC 1.6.99.2) from rat liver by affinity chromatography with a recovery of above 50%. The final enzyme preparation was purified about 750-fold and was electrophoretically homogeneous. Gel filtration showed that the enzyme had a mol.wt. of about 55 000, and one molecule of FAD was found per 55 000 mol.wt. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis gave a mol.wt. of about 27 000. Two N-terminal amino acids, asparagine/aspartic acid and glutamine/glutamic acid, were found in about equal yield, suggesting the presence of two non-identical polypeptide chains in the enzyme. NAD(P)H dehydrogenase was selectively removed by this affinity-chromatographic method from a microsomal carboxylation system. The system, which was solubilized by detergent and is dependent on vitamin K (2-methyl-3-phytyl-1,4-naphthaquinone or analogues with other side chains), lost its activity on the removal of the enzyme. The activity can be completely restored to the system by adding purified cytoplasmic NAD(P)H dehydrogenase or by using the quinol form of vitamin K1 (2-methyl-3-phytyl-1,4-naphthaquinol).