bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential long-term effects of d-chloramphenicol on the biogenesis of mitochondria in normal and regenerating rat liver
Authors: González-Cadavid Néstor F. , Bello Elba Marina Avila , Ramírez José Luis
Published: August 2015

1. Normal and partially hepatectomized rats (150g) were injected daily with d-chloramphenicol (20mg) for a period of 4 weeks, in order to investigate whether defective mitochondria could be induced in vivo in higher organisms as in yeast, and to measure the degree of inhibition of the mitochondrial function thus obtained. 2. The antibiotic did not affect growth and increased the amount of liver protein without changing the mitochondrial yield. 3. The respiration of isolated mitochondria from regenerated liver (regeneration completed) with succinate, α-oxo-glutarate, isocitrate and malate, was decreased in the chloramphenicol-treated rats, whereas in normal liver the antibiotic increased the mitochondrial oxygen consumption with succinate and did not significantly change the respiration with other substrates. 4. Mitochondrial cytochromes and respiratory enzymes were also decreased in amount in regenerated liver from the treated rats and enhanced in normal liver. 5. The protein specific radioactivities of most mitochondrial and microsomal subfractions, 30min after an injection of [14C]leucine, were decreased in regenerated liver under the action of chloramphenicol. Conversely, the incorporation of [14C]leucine into proteins of most subfractions in incubations of liver slices was enhanced in the case of normal rats treated with the antibiotic. 6. It is concluded that in regenerated liver chloramphenicol induces functionally defective mitochondria by inhibiting their biogenesis, whereas in normal liver the stimulation of respiration and protein synthesis is probably a secondary detoxication response.
Partial purification and kinetics of oestriol 16α-glucuronyltransferase from the cytosol fraction of human liver
Authors: Rao Govind S. , Rao Marie Luise , Breuer Heinz
Published: August 2015

An enzyme that conjugates the 16α-hydroxyl group of oestriol with glucuronic acid was found in the cytosol fraction of human liver. The enzymic activity could not be sedimented when the cytosol fraction was centrifuged at 158000gav. for 120min. The oestriol 16α-glucuronyltransferase was purified 100-fold by 0–30% saturation of the cytosol fraction with ammonium sulphate followed by filtration of the precipitate through Sephadex G-200. The activity was eluted at the void volume. The product of the reaction, oestriol 16α-monoglucuronide, was identified by paper chromatography and by crystallization of radioactive product to constant specific radioactivity. The optimum temperature was 37°C, and the activation energy was calculated to be 11.1kcal/mol. The apparent Michaelis–Menten constants for oestriol and UDP-glucuronic acid were 13.3 and 100μm respectively. Cu2+, Zn2+ and Hg2+ inhibited, whereas Mg2+, Mn2+ and Fe2+ stimulated the enzyme. Substrate-specificity studies indicated that the amount of oestradiol-17β, oestradiol-17α and oestrone conjugated was not more than about 5% of that found for oestriol. Oestriol 16α-monoglucuronide, a product of the reaction, did not inhibit the 16α-oestriol glucuronyltransferase; in contrast, UDP, another product of the reaction, inhibited the enzyme competitively with respect to UDP-glucuronic acid as the substrate, and non-competitively with respect to oestriol as the substrate. ATP and UDP-N-acetylglucosamine did not affect the oestriol 16α-glucuronyltransferase. 17-Epioestriol acted as a competitive inhibitor and 16-epioestriol as a non-competitive inhibitor of the glucuronidation of oestriol. 5α-Pregnane-3α,20α-diol also inhibited the enzyme non-competitively. It is most likely that the oestriol 16α-glucuronyltransferase described here is bound to the membranes of the endoplasmic reticulum.
The recombination of dimers of immunoglobulin peptide chains
Authors: Stevenson G. T. , Dorrington K. J.
Published: August 2015

1. Both the γ and light peptide chains of human pooled and myeloma immunoglobulin G can be prepared as non-aggregating dimers at pH5.4 in 4mm-sodium acetate buffer. The dimeric state is maintained by non-covalent bonds, since the formation of interchain disulphide bonds was prevented by alkylation of the thiol groups. In the case of the light chains there is some evidence that the dimers are in equilibrium with a small amount of monomer. 2. When such dimers of the γ and light chains are mixed at pH5.4 in 4mm-sodium acetate buffer they combine rapidly, yielding a product that resembles the original immunoglobulin G in its physicochemical and antigenic properties. However, the original optical rotatory dispersion spectrum was regained only with the homogeneous myeloma protein. The recombined pooled immunoglobulin G had a spectrum slightly different from the original, suggesting that at least some of the recombinant molecules had not regained native conformations. 3. Dimers of γ chains stabilized by interchain disulphide bonds were able to recombine with light chains. However, light chains stabilized in the dimeric state by interchain disulphide bonds would not combine with γ chains. 4. The chains of rabbit immunoglobulin G behave similarly to the human chains in this system, apart from the alkylated light chains showing clearer evidence of monomeric components.
The reversible reaction of protein amino groups with exo-cis-3,6-endoxo-Δ4-tetrahydrophthalic anhydride. The reaction with lysozyme
Authors: Riley M. , Perham R. N.
Published: August 2015

1. The reaction of exo-cis-3,6-endoxo-Δ4-tetrahydrophthalic anhydride with amino groups of model compounds and lysozyme is described. 2. Reaction with the ∈-amino group of Nα-acetyl-l-lysine amide gives rise to two diastereoisomeric products; at acid pH the free amino group is liberated with anchimeric assistance by the neighbouring protonated carboxyl group with a half-time of 4–5h at pH3.0 and 25°C. 3. The amino groups of lysozyme can be completely blocked, with total loss of enzymic activity. Dialysis at pH3.0 results in complete recovery of the native primary and tertiary structure of lysozyme and complete return of catalytic activity. 4. The specificity of reaction of this and other anhydrides with amino groups in proteins is discussed.
Purification and some properties of a non-haem iron protein from the bacteroids of soya-bean (Glycine max Merr.) nodules
Authors: Koch Burton , Wong Peter , Russell Sterling A. , Howard Robert , Evans Harold J.
Published: August 2015

A non-haem iron protein was isolated from an extract of soya-bean nodule bacteroids by a procedure including protamine sulphate and heat precipitation followed by chromatography on DEAE-cellulose. The purified protein contains non-haem iron and acid-labile sulphur and exhibits a spectrum with a rather broad absorption shoulder in the region 380–440nm and a more prominent peak at 280nm. From sedimentation-velocity measurements an apparent s20,w value of 1.3S was calculated. The protein functions as an electron carrier between the reducing system of illuminated chloroplast fragments and nitrogenase from nodule bacteroids, but it failed to function as a cofactor for the photochemical reduction of NADP in the presence of spinach chloroplasts. Also, it is inactive as a cofactor in the enzymic degradation of pyruvate to acetyl phosphate and CO2 in the presence of a ferredoxin-free extract of Clostridium pasteurianum. Repeated freezing, storage and thawing of the non-haem iron protein resulted in a marked loss of activity in the photochemical acetylene-reduction assay. A major portion of the activity that was lost was restored as a result of treatment with sodium sulphide, mercaptoethanol and ferrous ammonium sulphate.
The penetration of serum albumin into phospholipid monolayers of different fatty acid chain length and interfacial charge
Authors: Quinn P. , Dawson R. M. C.
Published: August 2015

1. The highest surface pressure of phosphatidylcholine monolayers allowing penetration of delipidated serum albumin decreased in the order dibehenoyl>distearoyl>dipalmitoyl=dimyristoyl. This pressure was not related to the area occupied or to the space available between the phospholipid molecules at the interface. 2. Penetration of albumin into yeast phosphatidylcholine monolayers was increased by adding a small percentage of long-chain anions (phosphatidic acid, dicetylphosphoric acid) to the film but only when the protein was below its isoelectric point (i.e. positively charged). 3. Stearylamine added to phosphatidylcholine monolayers had no effect on albumin penetration even when the protein was oppositely charged to that of the phospholipid/water interface. 4. The results are discussed in relation to the activation of certain phospholipases by anionic amphipathic substances.
Aspects of carnitine ester metabolism in sheep liver
Authors: Snoswell A. M. , Henderson G. D.
Published: August 2015

1. Carnitine acetyltransferase (EC 2.3.1.7) activity in sheep liver mitochondria was 76nmol/min per mg of protein, in contrast with 1.7 for rat liver mitochondria. The activity in bovine liver mitochondria was comparable with that of sheep liver mitochondria. Carnitine palmitoyltransferase activity was the same in both sheep and rat liver mitochondria. 2. The [free carnitine]/[acetylcarnitine] ratio in sheep liver ranged from 6:1 for animals fed ad libitum on lucerne to approx. 1:1 for animals grazed on open pastures. This change in ratio appeared to reflect the ratio of propionic acid to acetic acid produced in the rumen of the sheep under the two dietary conditions. 3. In sheep starved for 7 days the [free carnitine]/[acetylcarnitine] ratio in the liver was 0.46:1. The increase in acetylcarnitine on starvation was not at the expense of free carnitine, as the amounts of free carnitine and total acid-soluble carnitine rose approximately fivefold on starvation. An even more dramatic increase in total acid-soluble carnitine of the liver was seen in an alloxan-diabetic sheep. 4. The [free CoA]/[acetyl-CoA] ratio in the liver ranged from 1:1 in the sheep fed on lucerne to 0.34:1 for animals starved for 7 days. 5. The importance of carnitine acetyltransferase in sheep liver and its role in relieving `acetyl pressure' on the CoA system is discussed.
Homologies in amino acid composition and structure of histone F2al isolated from human leukaemic cells
Authors: Desai Laxman S. , Foley George E.
Published: August 2015

Histones F2al extracted from normal and neoplastic cells possess similar amino acid compositions. Tryptic and chymotryptic peptides of the F2al histones have identical chromato-electrophoretic RF values. It is concluded that histones F2al from various sources have similar overall structures. The observed differences in the ratios of ∈-N-monomethyl- and di-∈-N-methyl-lysine in the histones from normal and neoplastic cells may be of significance with respect to gene regulation.
Effect of growth hormone on fibrinogen synthesis
Authors: Jeejeebhoy K. N. , Bruce-Robertson A. , Sodtke U. , Foley M.
Published: August 2015

A method described by McFarlane (1963a) for the measurement of the absolute rate of synthesis of liver-made plasma proteins was used to show that, within a few hours of giving bovine growth hormone to rats, fibrinogen synthesis increased significantly without a change in albumin synthesis.
Metabolism of β-ionone. Isolation, characterization and identification of the metabolites in the urine of rabbits
Authors: Ide Hiroyuki , Toki Satoshi
Published: August 2015

1. Rabbits dosed orally with β-ionone excreted in the urine unchanged β-ionone, 3-oxo-β-ionone, 3-oxo-β-ionol, dihydro-3-oxo-β-ionol and 3-hydroxy-β-ionol. 2. Excretion products were isolated as 2,4-dinitrophenylhydrazone derivatives (β-ionone, 3-oxo-β-ionone, 3-oxo-β-ionol and dihydro-3-oxo-β-ionol) and as p-nitrobenzoate derivatives (3-oxo-β-ionol, dihydro-3-oxo-β-ionol and 3-hydroxy-β-ionol), which were characterized and identified by comparison with the synthetic authentic compounds. 3. The glucuronides of 3-oxo-β-ionol and dihydro-3-oxo-β-ionol were also detected in the urine. The latter compound was isolated as free glucuronide, sodium salt and 2,4-dinitrophenylhydrazone.
Hyperfine structure of [57Fe]iron in the Mössbauer spectrum of the high-potential iron protein from Chromatium
Authors: Evans M. C. W. , Hall D. O. , Johnson C. E.
Published: August 2015

The magnetic hyperfine structure observed in the 57Fe Mössbauer spectra of the high-potential iron protein from Chromatium shows that the iron atoms are inequivalent in pairs, with hyperfine fields of 121 and 90kG.
Bilirubin conjugates of human bile. Nuclear-magnetic-resonance, infrared and optical spectra of model compounds
Authors: Kuenzle Clive C.
Published: August 2015

N.m.r., i.r. and optical spectra of model compounds were recorded. These were to help in elucidating the structures of the phenylazo derivatives of bilirubin conjugates isolated from human bile. Model compounds included commercial and human bile bilirubin, mesobilirubin, bilirubin dimethyl ester, dimethoxybilirubin dimethyl ester and the corresponding phenylazo derivatives. The phenylazo derivative of vinylneoxanthobilirubinic acid was also investigated. All compounds were of the type IXα, and no other isomer could be detected with the spectroscopic methods employed. The compounds crystallize as the lactams, except for dimethoxybilirubin dimethyl ester and its phenylazo derivative, which are held in the lactim ether configuration. With all other compounds no tautomeric forms other than the lactams could be detected, although small proportions of bilirubin must exist as the lactim. Bilirubin does not form a betaine, a structure that has been proposed by von Dobeneck & Brunner (1965) to explain the bathochromic shift of its optical spectrum as compared with the expected position of the absorption maximum at 420nm. However, this shift to 453nm can be explained on the basis of internal hydrogen bonds occurring between the carboxylic protons and the pyrrole rings of bilirubin, as proposed by Fog & Jellum (1963), and new evidence for such a bonding has been accumulated. The bilirubin sulphate described by Watson (1958), which is formed by treatment of bilirubin with concentrated sulphuric acid and acetic anhydride, was also investigated. The main product of this reaction was isolated as its phenylazo derivative, and was shown to be 3,18-di(ethylidene sulphate)-2,7,13,17-tetramethylbiladiene-ac-8,12-dipropionic acid. The reaction leading to this compound is an addition of sulphuric acid to the vinyl side chains of bilirubin according to Markownikoff's rule.
Spectrophotometric studies of acyl-coenzyme A synthetases of rat liver mitochondria
Authors: Garland P. B. , Yates D. W. , Haddock B. A.
Published: August 2015

1. Deca-2,4,6,8-tetraenoic acid is a substrate for both ATP-specific (EC 6.2.1.2 or 3) and GTP-specific (EC 6.2.1.–) acyl-CoA synthetases of rat liver mitochondria. The enzymic synthesis of decatetraenoyl-CoA results in new spectral characteristics. The difference spectrum for the acyl-CoA minus free acid has a maximum at 376nm with εmM 34. Isosbestic points are at 345nm and 440nm. 2. The acylation of CoA by decatetraenoate in mitochondrial suspensions can be continuously measured with a dual-wavelength spectrophotometer. 3. By using this technique, three distinct types of acyl-CoA synthetase activity were demonstrated in rat liver mitochondria. One of these utilized added CoA and ATP, required added Mg2+ and corresponded to a previously described `external' acyl-CoA synthetase. The other two acyl-CoA synthetase activities utilized intramitochondrial CoA and did not require added Mg2+. Of these two `internal' acyl-CoA synthetases, one was insensitive to uncoupling agents, was inhibited by phosphate or arsenate, and corresponded to the GTP-specific enzyme. The other corresponded to the ATP-specific enzyme. 4. Atractylate inhibited the activity of the two internal acyl-CoA synthetases only when the energy source was added ATP. 5. The amount of intramitochondrial CoA acylated by decatetraenoate was independent of whether the internal ATP-specific or GTP-specific acyl-CoA synthetase was active. It is concluded that these two internal acyl-CoA synthetases have access to the same intramitochondrial pool of CoA. 6. The amount of intramitochondrial CoA that could be acylated with decatetraenoate was decreased by the addition of palmitoyl-dl-carnitine, 2-oxoglutarate, or pyruvate. These observations indicated that pyruvate dehydrogenase (EC 1.2.4.1), oxoglutarate dehydrogenase (EC 1.2.4.2), carnitine palmitoyltransferase (EC 2.3.1.–), citrate synthase (EC 4.1.3.7), and succinyl-CoA synthetase (EC 6.2.1.4) all have access to the same intramitochondrial pool of CoA as do the two internal acyl-CoA synthetases.
The stereochemistry of hydrogen transfer from reduced nicotinamide–adenine dinucleotide phosphate in the reduction of ethylenic linkages during cholesterol biosynthesis
Authors: Wilton D. C. , Watkinson I. A. , Akhtar M.
Published: August 2015

It is shown that during the saturation of steroid carbon–carbon double bonds at Δ24,25 and Δ14,15 the `hydride ion' originates from the 4B side of the NADPH.
Properties and substrate specificities of the phenylalanyl-transfer-ribonucleic acid synthetases of Aesculus species
Authors: Anderson J. W. , Fowden L.
Published: August 2015

1. Phenylalanyl-tRNA synthetases have been partially purified from cotyledons of seeds of Aesculus californica, which contains 2-amino-4-methylhex-4-enoic acid, and from four other species of Aesculus that do not contain this amino acid. The A. californica preparation was free from other aminoacyl-tRNA synthetases, and the contaminating synthetase activity in preparations from A. hippocastanum was decreased to acceptable limits by conducting assays of pyrophosphate exchange activity in 0.5m-potassium chloride. 2. The phenylalanyl-tRNA synthetase from each species activated 2-amino-4-methylhex-4-enoic acid with Km 30–40 times that for phenylalanine. The maximum velocity for 2-amino-4-methylhex-4-enoic acid was only 30% of that for phenylalanine with the A. californica enzyme, but the maximum velocities for the two substrates were identical for the other four species. 3. 2-Amino-4-methylhex-4-enoic acid was not found in the protein of A. californica, so discrimination against this amino acid probably occurs in the step of transfer to tRNA, though subcellular localization, or subsequent steps of protein synthesis could be involved. 4. Crotylglycine, methallylglycine, ethallylglycine, 2-aminohex-4,5-dienoic acid, 2-amino-5-methylhex-4-enoic acid, 2-amino-4-methylhex-4-enoic acid, β-(thien-2-yl)alanine, β-(pyrazol-1-yl)alanine, phenylserine and m-fluorophenylalanine were substrates for pyrophosphate exchange catalysed by the phenylalanyl-tRNA synthetases of A. californica or A. hippocastanum. Allylglycine, phenylglycine and 2-amino-4-phenylbutyric acid were inactive.
Biosynthesis of the carbohydrate portion of immunoglobins. Kinetics of synthesis and secretion of [3H]leucine-, [3H]galactose- and [3H]mannose-labelled myeloma protein by two plasma-cell tumours
Authors: Melchers Fritz
Published: August 2015

The kinetics of incorporation of leucine, galactose and mannose into intracellular and secreted myeloma protein, MOPC 21 IgG1 and MOPC 46 κ-type light chain, by cell suspensions of two myeloma plasma-cell tumours, MOPC 21 and MOPC 46, were similar. Radioactive galactose was incorporated to over 90% into galactose residues of intracellular and secreted protein, mannose to over 90% into glucosamine and mannose residues of intracellular protein and to over 90% into glucosamine, mannose and fucose residues of secreted protein, but not into galactose residues. The results show that specific residues in the carbohydrate portion of myeloma proteins can be labelled by specific radioactive monosaccharides, and suggest that fucose residues are added, while myeloma protein is in its final stage of secretion from the plasma cell. The kinetics of incorporation indicate at least three sequential precursor–product relationships between different intracellular forms and the secreted form of myeloma protein.
Effect of streptomycin on lipid composition with particular reference to cyclic depsipeptide biosynthesis in Serratia marcescens and other micro-organisms
Authors: Bermingham Margaret A. C. , Deol B. S. , Still J. L.
Published: August 2015

The addition of low concentrations of streptomycin (5–10μg/ml of medium) to Serratia marcescens caused significant alterations in the lipid composition of this organism, but neither growth nor pigmentation was affected. The acetone-soluble cyclic depsipeptides, which comprise on average 15% of the total lipid, were decreased almost to zero and the total lipid phosphorus was more than doubled in the presence of streptomycin. Most of the phospholipid increase was due to an increase in phosphatidylethanolamine. Cyclic depsipeptides were not leached from the cell in the presence of streptomycin, indicating a definite inhibition of the biosynthetic pathway. The effect of streptomycin on the reported peptidolipids of Rhodopseudomonas spheroides, Halobacterium halobium, Nocardia asteroides and Pseudomonas tabaci was investigated. In the case of the only strictly comparable cellular cyclic depsipeptide (that of N. asteroides) the biosynthesis was strongly inhibited by streptomycin, but cell weight was maintained or even slightly increased. A possible mode and site of action of low concentrations of streptomycin on bacterial lipids is discussed.
Iodinated vancomycin and mucopeptide biosynthesis by cell-free preparations from Micrococcus lysodeikticus
Authors: Bordet Claudette , Perkins H. R.
Published: August 2015

A particulate preparation from Micrococcus lysodeikticus was used to synthesize cell-wall mucopeptide. Radioactive iodinated vancomycin became attached to the preparation simultaneously with a complete inhibition of mucopeptide synthesis. After mucopeptide synthesis had occurred in the absence of antibiotic, the preparation took up more vancomycin, suggesting that new binding sites terminating in acyl-d-alanyl-d-alanine had been produced. The mucopeptide product was divided into a soluble and an insoluble portion, both sensitive to lysozyme. The soluble portion did not combine with vancomycin and hence had presumably lost its terminal d-alanine residues, either by transpeptidation or because of carboxy-peptidase action. The synthesis of both portions was unaffected by the presence of penicillin, but the insoluble part showed increased affinity for vancomycin, thus indicating that penicillin had caused conservation of d-alanyl-d-alanine termini.
Tetrodotoxin-sensitive uptake of ions and water by slices of rat brain in vitro
Authors: Okamoto K. , Quastel J. H.
Published: August 2015

1. Under certain conditions the processes that bring about increased Na+ influx and water uptake into rat brain slices have a tetrodotoxin-sensitive component. 2. Incubation of slices in the absence of glucose, or in the presence of ouabain (0.1mm), protoveratrine (10μm) or electrical stimuli, leads to increased uptakes of water and Na+ that are partially suppressed by tetrodotoxin (3μm). 3. The increased water uptake and Na+ influx due to the presence of 30μm-2,4-dinitrophenol or of 100mm-potassium chloride are unaffected by tetrodotoxin. 4. The additional presence of 27mm-potassium chloride diminishes, or abolishes, the inhibitory effects of tetrodotoxin on the increased uptakes of water and Na+ found on incubation in the absence of glucose or in the presence of protoveratrine. 5. Ouabain increases Na+ influx into caudate nuclei from rat brain and this process is suppressed by tetrodotoxin. Ouabain does not increase water uptake in the caudate nuclei. 6. Diminution of Na+ influx in slices due to tetrodotoxin is not accompanied by an equal efflux of K+. 7. It is concluded that glial-cell membranes are permeable to K+ and Cl− and, as a consequence, when K+ is released from neurons in association with action potentials it, together with water, moves into the glial cells. The major consequence of K+ (and Cl−) permeability of the glial cell is therefore a buffering of the extracellular K+ concentration. 8. l-Glutamate presents an anomalous feature that may indicate that its effects are not restricted to neurons. 9. Calculations are made of the increase of K+ flux into the glia in the presence of ouabain or of protoveratrine or in the absence of glucose or on application of electrical impulses.
Fatty acid utilization during development of the rat
Authors: Lockwood Elizabeth A. , Bailey E.
Published: August 2015

1. In the absence of added carnitine pronounced changes occur during development, in the degradation of [U-14C]palmitate to 14C-labelled acid-soluble material (predominantly ketone bodies) and 14CO2 by liver homogenates. The formation of both products by liver homogenates of 5-day-old rats is considerably higher than in foetal livers. This high value is maintained during the suckling period and falls after weaning to adult values which are similar to those of the foetus. Addition of carnitine stimulates production of acid-soluble material throughout development. The effect of added carnitine on CO2 production varies with age, stimulating in foetal and post-weaning age groups but inhibiting during the suckling period. 2. The degradation of [U-14C]palmitate to acid-soluble material and CO2 by heart homogenates also varies during development. Formation of both products is highest 5 days after birth and thereafter decreases steadily to adult values. Addition of carnitine stimulates the production of both CO2 and acid-soluble material at all ages. 3. The activity of carnitine palmitoyltransferase in liver increased after birth to reach maximum activity at 2–5 days of age. Thereafter the activity of the enzyme decreased until day 15 of life and then remained constant until day 30, decreasing after this time to adult values which were about one half of the foetal liver activities.
The enzymic conversion of linoleic acid hydroperoxide by flax-seed hydroperoxide isomerase
Authors: Veldink G. A. , Vliegenthart J. F. G. , Boldingh J.
Published: August 2015

1. The mode of action of flax-seed hydroperoxide isomerase was studied in vitro by using as substrates linoleic acid hydroperoxides formed by soya-bean lipoxygenase. 2. The enzyme converts only 13-hydroperoxyoctadeca-cis-9-trans-11-dienoic acid, whereas the 9-hydroperoxy isomer does not react. 3. The isomerization product was identified by chemical and spectroscopic methods as 13-hydroxy-12-oxo-octadec-cis-9-enoic acid. 4. 12,13-Epoxyoleic acid isomers do not act as intermediates in the isomerization reaction. 5. Suggestions for a functional relationship between hydroperoxide isomerase and lipoxygenase are discussed.
Reactivity of the essential thiol group of lactate dehydrogenase and substrate binding
Authors: Holbrook J. J. , Stinson R. A.
Published: August 2015

1. The preparation of a derivative of pig heart lactate dehydrogenase in which the essential thiol group has been converted into an S-sulpho group is described. The derivative has unchanged s20,w and is catalytically inactive. 2. The rate of reaction of the essential thiol group is controlled by a system with a pK>9. 3. The essential thiol group is protected by NADH against reaction with maleimide. 4. Lactate dehydrogenase in which the essential thiol group has been converted into an S-sulpho group or alkylated with maleimide still binds one molecule of NADH/subunit but with a three- to four-fold diminished affinity. 5. The inhibited enzymes also bind one molecule of NAD+–sulphite complex/subunit but with affinity decreased 103–104-fold. 6. The inhibited enzymes fail to bind C2 and C3 molecules to give the ternary complexes enzyme–NAD+–pyruvate, enzyme–NADH–oxamate and enzyme–NADH–oxalate. The 1:1:1 stoicheiometry of the last-mentioned complex with the native enzyme was established by gel filtration. 7. Structures that account for these results are discussed.
Comparison of immunoadsorbents prepared by coupling sperm-whale myoglobin to a variety of insoluble polymers
Authors: Boegman R. J. , Crumpton M. J.
Published: August 2015

Immunoadsorbents that were potentially suitable for the fractionation of antibodies to myoglobin were prepared by coupling myoglobin to aminoethylcellulose, bromoacetylcellulose, CM-cellulose, an ethylene–maleic anhydride co-polymer and Sepharose 4B. A comparison of the properties of the preparations indicated that Sepharose–myoglobin was superior, as an immunoadsorbent, to the other conjugates. Elution of adsorbed antibodies with m-propionic acid, pH2.5, gave yields of precipitable antibody higher than those achieved with other dissociating solvents. Propionic acid eluted 60–100% of the antibody adsorbed by Sepharose–myoglobin, and 60–90% of the eluted protein was precipitable antibody.
Bacteriolytic enzymes from Staphylococcus aureus. Purification of an endo-β-N-acetylglucosaminidase
Authors: Wadström T. , Hisatsune K.
Published: August 2015

On cultivation of Staphylococcus aureus in a complex liquid medium, bacteriolytic activity is found extracellularly. The maximal amount was found at the end of the exponential growth phase in batch culture, but in continuous culture run under similar conditions the yield was doubled. Isoelectric focusing of dialysed crude culture supernatants showed that the bacteriolytic activity of all four strains studied (M18, 524, Wood 46 and Duncan) was heterogeneous. The most alkaline peak of activity (isoelectric point 9.5±0.1) was assayed against Micrococcus lysodeikticus turbidimetrically. This bacteriolytic activity was purified more than 70-fold after continuous dialysis by adsorption on CM-Sephadex, precipitation with ethanol, heat purification, isoelectric focusing and Sephadex G-100 chromatography. The purified enzyme (isoelectric point 9.6±0.1) was found to give a single band on polyacrylamide-gel and cellulose acetate electrophoresis and was devoid of all 14 staphylococcal enzymes and toxins assayed for. The molecular weight is 70000±5000 as estimated by Sephadex G-100 and G-200 chromatography. The marked instability of the partially and highly purified enzyme was investigated. The mode of action and some properties of this enzyme are given in the following papers (Wadström & Hisatsune, 1970; Wadström, 1970). These results indicate that this extracellular enzyme which is produced by several strains of S. aureus is not a `lysozyme' (endo-β-N-acetylmuramidase) as previously suggested, but an endo-β-N-acetylglucosaminidase.
Kinetic studies with cytosol and mitochondrial phosphoenolpyruvate carboxykinases
Authors: Ballard F. J.
Published: August 2015

1. Measurements of Michaelis constants for oxaloacetate in the reaction catalysed by liver phosphoenolpyruvate carboxykinase give values much lower than previously reported. With Mg2+ as bivalent cation, the Michaelis constant was approx. 2.5×10−5m whether the enzyme used was the mitochondrial phosphoenolpyruvate carboxykinase purified from sheep liver or chicken liver or the cytosol enzyme purified from rat liver or sheep liver. 2. When Mn2+ replaced Mg2+ in the reaction a lower Michaelis constant of 9×10−6m was found, but only with the mitochondrial enzymes. 3. With all enzymes malate at high concentration was a competitive inhibitor with respect to oxaloacetate when Mn2+ was the added cation. With Mg2+ the inhibition by malate was competitive with the mitochondrial enzymes and non-competitive with the cytosol enzymes.
Heterogeneity of bile pigment conjugates as revealed by chromatography of their anthranilate azopigments
Authors: Heirwegh K. P. M. , Van Hees G. P. , Leroy P. , Van Roy F. P. , Jansen F. H.
Published: August 2015

1. Azopigments derived from conjugated bile pigments by coupling with the diazonium salt of ethyl anthranilate are analysed conveniently by quantitative t.l.c. or by column chromatography on CM-cellulose. 2. By chromatographic studies combined with a series of chemical tests six groups of azopigments were demonstrable in preparations from bile and from icteric urine of man. Azobilirubin and its β-d-monoglucuronide have hitherto been considered to be the only major derivatives that can be obtained from human bile pigments. In the present work, other azopigments accounted for 30–40% of the total azopigment material, and the amounts of these showed considerable variation among biological fluids. 3. The divergence of the present results from earlier work is probably related to the use of milder diazotization conditions and of chromatographic techniques with a high resolving power. 4. The thin-layer chromatographic systems developed allow rapid and quantitative analysis of azopigments derived from bile pigments.
Studies on energy-linked reactions. Energy-linked reduction of oxidized nicotinamide–adenine dinucleotide by succinate in Escherichia coli
Authors: Sweetman A. J. , Griffiths D. E.
Published: August 2015

1. A method for the preparation of small particles from Escherichia coli is described. 2. These particles catalyse the ATP-driven reduction of NAD+ by succinate. 3. ATP utilized/NADH produced ratios below 2.0 were measured and a stoicheiometry of 1 molecule of ATP utilized per molecule of NADH produced is proposed. 4. The reaction is not inhibited by 2,4-dinitrophenol or by oligomycin but is inhibited by other uncouplers such as pentabromophenol and dicoumarol. 5. The specificity of the energy source, the specificity of the electron acceptor, the effects of pH, Mg2+, Pi and temperature have been studied.
The formation and reduction of the 14,15-double bond in cholesterol biosynthesis
Authors: Watkinson I. A. , Wilton D. C. , Munday K. A. , Akhtar M.
Published: August 2015

It was shown that 100μg quantities of 4,4′-dimethyl[2-3H2]cholesta-8,14-dien-3β-ol (IIIa), tritiated cholesta-8,14-dien-3β-ol, 4,4′-dimethyl[2-3H2]cholesta-7,14-dien-3β-ol, dihydro[2-3H2]lanosterol and [24-3H]lanosterol were converted by a 10000g supernatant of rat liver homogenate into cholesterol in 17%, 54%, 6%, 9.5% and 24% yields respectively. From an incubation of dihydro[3α-3H]lanosterol with a rat liver homogenate in the presence of a trap up to 38% of the radioactivity was found to be associated with a fraction that was unambiguously shown to be 4,4′-dimethylcholesta-8,14-dien-3β-ol. Another related compound, 4,4′-dimethylcholesta-7,14-dien-3β-ol was also shown to be equally effective in its ability to trap compound (IIIa) from an incubation of dihydro[3α-3H]lanosterol. The mechanism of the further conversion of the compound (IIIa) into cholesterol occurred by the reduction of the 14,15-double bond and involved the addition of a hydrogen atom from the medium to C-15 and another from the 4-position of NADPH to C-14. Two possible mechanisms for the removal of the 14α-methyl group in sterol biosynthesis are discussed.
Rate studies of polysaccharide biosynthesis from guanosine diphosphate α-d-glucose and guanosine diphosphate α-d-mannose
Authors: Villemez C. L.
Published: August 2015

With enzyme preparations from Phaseolus aureus seedlings, the initial rate of 14C-labelled polysaccharide formation from GDP-α-d-[14C]glucose is not increased by additions of GDP-α-d-mannose. However, final incorporation is increased by addition of GDP-α-d-mannose, since the total reaction-time is extended. In contrast, the initial rate of 14C-labelled polysaccharide formation from GDP-α-d-[14C]mannose is increased by all concentrations of GDP-α-d-glucose that are less than that of the GDP-α-d-[14C]mannose. Maximum stimulation of the initial rate occurs at a GDP-α-d-[14C]mannose/GDP-α-d-glucose concentration ratio of about 4:1. However, eventual incorporation from GDP-α-d-[14C]mannose is decreased by the addition of GDP-α-d-glucose, since the reaction rate falls off sharply after about 2min. Reciprocal plots of 14C-labelled polysaccharide formation from GDP-α-d-[14C]mannose result in biphasic graphs. The two straight-line portions of the plot are joined by a curved line in the concentration range between 2–3 and 50μm. Extrapolated Km values for the two linear components are 0.4–1.0 and 700–1500μm. The effect of GDP-α-d-glucose on the kinetics of 14C-labelled polysaccharide formation from GDP-α-d-[14C]mannose is complex, and depends on relative concentrations of the two sugar nucleotides. 14C-labelled polysaccharide formation from GDP-α-d-[14C]glucose also results in biphasic reciprocal plots. One component appears to have Km about 2–3μm, the other about 200–400μm. In this reaction, GDP-α-d-mannose appears to be a competitive inhibitor with Ki 20–30μm. With particulate preparations of P. aureus, GDP-α-d-[14C]glucose appears to be a precursor for the synthesis of one polysaccharide, a glucomannan, the mannose moieties of which are derived from an intermediate existing in the particulate preparation. From the rate results, GDP-α-d-[14C]mannose appears to be a precursor for at least two polysaccharides, one of which is a glucomannan.
A radiochemical renin assay
Authors: Mendelsohn F. A. , Johnston C. I.
Published: August 2015

1. A synthetic 3-([14C]valine)-labelled tetradecapeptide renin substrate was used to measure renin concentration. Renin liberated 14C-labelled angiotensin I, which was separated from the labelled substrate by paper chromatography. The conversion of substrate into angiotensin I was quantitated by liquid-scintillation counting of radioactivity. 2. The rate of conversion of the substrate into angiotensin I was shown to be linearly related to renin concentration and time under suitable conditions. Angiotensin generation measured in this system agrees well with that measured by bioassay. 3. It is suggested that the use of a pure substrate offers advantages that include the standardization of current units of renin measurement.
Bovine pepsinogens and pepsins. A series of zymogens and enzymes that differ in organic phosphate content
Authors: Meitner Patricia A. , Kassell Beatrice
Published: August 2015

Several minor pepsinogens, present in extracts of bovine fundic mucosa obtained from the fourth stomach or abomasum, were separated from the main pepsinogen by chromatography on hydroxyapatite at pH7.3. The major pepsinogen and two of these minor pepsinogens were studied in detail. All three zymogens have N-terminal Ser-Val-, C-terminal -Val-Ala and not more than 1mol of glucose/mol of protein; no significant differences in amino acid composition were found. The pepsinogens differ in their organic phosphate content, which accounts for their chromatographic separation. By activation at 0°C and pH2, a corresponding series of pepsins is formed. These enzymes were separated by hydroxyapatite chromatography at pH5.7. All the pepsins have N-terminal valine, C-terminal alanine and are free from carbohydrate. Again the only difference detected among them is in their organic phosphate content. The pepsins of high phosphate content are converted by an acid phosphatase in vitro into pepsins of low phosphate content.
Differences in the ribosomes prepared from lactating and non-lactating bovine mammary gland
Authors: Herrington M. D. , Hawtrey A. O.
Published: August 2015

1. Ribosomes prepared from bovine lactating mammary gland are able to synthesize protein, whereas similar preparations from non-lactating glands are not. Washing the ribosome suspensions through a medium containing 0.5m-ammonium chloride enhanced their ability to incorporate phenylalanine into polyphenylalanine. 2. Ribosomes isolated from non-lactating bovine mammary gland, in contrast with those from rat liver and lactating mammary gland, contained significant amounts of extraneous nucleases. These enzymes could be removed by washing with a medium A buffer containing 0.5m-ammonium chloride. 3. Only those ribosomes from functionally active tissues were able to bind polyuridylic acid and phenylalanyl-tRNA.
Study of soluble lipoprotein in rat liver mitochondria
Authors: Koppikar S. V. , Fatterpaker P. , Sreenivasan A.
Published: August 2015

1. A water-soluble lipoprotein was isolated and purified from osmotically shocked preparations of rat liver mitochondria by using a technique of Sephadex-sandwich disc electrophoresis. 2. The purified lipoprotein migrates as a distinct sharp zone in high-resolution electrophoretic systems, indicating high degree of purity. 3. The lipoprotein resembles mitochondrial membranes with respect to lipid composition and lipid/protein ratio. 4. The lipoprotein and its apoprotein fraction obtained by delipidization at −18°C to −20°C have common properties with respect to their fluorescence spectra, instability to storage and electrophoretic mobility. 5. The purified lipoprotein has an excitation maximum at 325nm and a fluorescence maximum at 418nm. 6. Storage at 4°C for 4 days or repeated freezing and thawing results in 15–30% decrease in electrophoretic mobility. 7. The patterns of incorporation in vitro of [1-14C]leucine into proteins of the soluble lipoprotein and of mitochondrial membrane of isolated rat liver mitochondria suggest a probable precursor role for the apoprotein in the formation of mitochondrial membrane protein. 8. Lipoprotein preparations isolated from mitochondrial fractions of rat kidney, brain and heart and of chicken and mouse liver resemble closely that obtained from rat liver mitochondria, suggesting that the soluble lipoprotein could be a distinct entity of mitochondrial origin.
Deoxyribonucleic acid synthesis in mammalian nuclei. Incorporation of deoxyribonucleotides and chain-terminating nucleotide analogues
Authors: Waqar M. A. , Burgoyne L. A. , Atkinson M. R.
Published: August 2015

The properties of a nuclear preparation from rat liver and thymus are described. (1) Nearest-neighbour analysis after incorporation of 32P-labelled nucleotide residues from dATP, dCTP, dGTP, dTTP and arabinofuranosyl analogues of CTP and ATP shows template-dependent DNA synthesis. (2) Where primer termini are limiting, incorporation of arabinofuranosyl analogues of AMP and CMP residues proceeds to a limit indicating that both of these analogues are DNA chain terminators. (3) No large differences have been found between the priming potentialities or the intrinsic DNA polymerase activities of nuclei from resting or regenerating liver and the relationship of this DNA synthesis in vitro to DNA replication or repair in vivo is briefly discussed.
The interaction of concanavalin A with blood-group-substance glycoproteins from human secretions
Authors: Clarke A. E. , Denborough M. A.
Published: August 2015

1. Gastric juice, saliva and ovarian-cyst fluid were fractionated into glycoprotein components by centrifuging to equilibrium in a caesium chloride density gradient. 2. The glycoprotein fractions from the gastric juice of two group O non-secretors, two group O secretors and three group A secretors all formed insoluble complexes with concanavalin A. 3. Fractions showing maximum interaction with concanavalin A had maximum blood-group activity measured by the haemagglutination-inhibition technique. The sulphate content of the gastric glycoproteins was unrelated to the capacity to interact with concanavalin A. 4. No interaction was found between concanavalin A and the glycoprotein fractions from any of the saliva or ovarian-cyst-fluid samples tested, implying that there is a structural difference in blood-group-substance glycoproteins in gastric juice when compared with those in saliva and ovarian-cyst fluid. 5. The protein components of each of the secretions tested, gastric juice, saliva and ovarian-cyst fluid, interacted with concanavalin A.
The kinetic mechanism of 3-hydroxy-3-methylglutaryl-coenzyme A synthase from baker's yeast
Authors: Middleton B.
Published: August 2015

1. The effect of independent variation of both acetyl-CoA and acetoacetyl-CoA on the initial velocity at pH8.0 and pH8.9 gives results compatible with a sequential mechanism involving a modified enzyme tentatively identified as an acetyl-enzyme, resulting from the reaction with acetyl-CoA in the first step of a Ping Pong (Cleland, 1963a) reaction. 2. Acetoacetyl-CoA gives marked substrate inhibition that is competitive with acetyl-CoA. This suggests formation of a dead-end complex with the unacetylated enzyme and is in accord with the inhibition pattern given by 3-oxohexanoyl-CoA, an inactive analogue of acetoacetyl-CoA. 3. The inhibition pattern given by products of the reaction is compatible with the above mechanism. CoA gives mixed inhibition with respect to both substrates, whereas dl-3-hydroxy-3-methylglutaryl-CoA competes with acetyl-CoA but gives uncompetitive inhibition with respect to acetoacetyl-CoA. 4. 3-Hydroxy-3-methylglutaryl-CoA analogues lacking the 3-hydroxyl group are found to compete, like 3-hydroxy-3-methylglutaryl-CoA, with acetyl-CoA but have Ki values ninefold higher, indicating the importance of the 3-hydroxyl group in the interaction. 5. A comparison of inhibition by CoA and desulpho-CoA at pH8.0 and pH8.9 shows that at the higher pH value a kinetically significant reversal of the formation of acetyl-enzyme can occur. 6. Acetyl-CoA homologues do not act as substrates and compete only with acetyl-CoA. A study of the variation of Ki with acyl-chain length suggests the presence near the active centre of a hydrophobic region. 7. These results are discussed in terms of a kinetic mechanism in which there is only one CoA-binding site the specificity of which is altered by acetylation of the enzyme. 8. The rate of 3-hydroxy-3-methylglutaryl-CoA synthesis in yeast is calculated from the kinetic constants determined for purified 3-hydroxy-3-methylglutaryl-CoA synthase and from estimates of the physiological substrate concentrations. The rate of synthesis of 12nmol of 3-hydroxy-3-methylglutaryl-CoA/min per g wet wt. of yeast is still greater than the rate of utilization in spite of the extremely low (calculated) acetoacetyl-CoA concentration (1.8nm).
The effect of iodination of particular tyrosine residues on the hormonal activity of insulin
Authors: Garratt C. J. , Harrison D. M. , Wicks Margaret
Published: August 2015

Insulin dissolved in aqueous or methanolic buffer was iodinated to give preparations containing an average of between one and five iodine atoms per insulin monomer. The resultant preparations were fragmented in various ways and the ratio of tyrosine to monoiodotyrosine and di-iodotyrosine was determined in each fragment. This has allowed the distribution of iodine between the combined A-chain tyrosine residues and the individual B-chain tyrosine residues to be determined. The hormonal activity of each of these iodinated insulin preparations was measured from their effect on the production of 14CO2 from [1-14C]glucose by isolated adipose cells. The results were interpreted as meaning that the iodination of tyrosine residue A19 or B16 leads to the inactivation of insulin. Speculations are made about the nature of an interaction between insulin and a receptor site on the target tissue.
Immunochemistry of cartilage proteoglycan. Immunodiffusion and gel-electrophoretic studies
Authors: Keiser H , Shulman H J. , Sandson J I.
Published: August 2015

Cartilage proteoglycan is thought to be composed of subunits, core proteins with covalently attached sulphated polysaccharide side chains, which form aggregates by non-covalent association with a link protein. The new technique of non-disruptive extraction followed by fractionation in caesium chloride gradients provides a useful means of preparing relatively pure proteoglycan aggregate, subunit and link fractions. Immunological studies of these fractions led to the identification of an antigen associated with the proteoglycan subunit which was common to several species and to the demonstration of additional species-specific antigens in aggregate and link fractions derived from bovine nasal cartilage. Polyacrylamide-gel electrophoresis with sodium dodecyl sulphate of bovine proteoglycan aggregate and link fractions gave two protein bands in the gels and a protein–polysaccharide band at the origin; subunit fractions gave only the band at the origin. These results are consistent with the current concept of cartilage proteoglycan structure.