bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and comparative properties of isoenzymes of nicotinamide–adenine dinucleotide phosphate–isocitrate dehydrogenase from rat heart and liver
Authors: Islam M. , Bell Joyce L. , Baron D. N.
Published: August 2015

1. Rat liver and heart major isoenzymes of NADP–isocitrate dehydrogenase have each been purified about 100-fold by a combination of ammonium sulphate fractionation and chromatography on ion-exchange cellulose and their properties compared. 2. The properties were similar in respect of pH, inhibition by Hg2+and Michaelis constants for isocitrate and NADP. 3. Some of the properties of the isoenzymes were different. 4. The heart isoenzyme was activated about 210% by 0.8m-ammonium sulphate whereas the liver isoenzyme was unaffected. The heart isoenzyme showed greater sensitivity to inactivation by heat (30°C for 30min), whereas the liver isoenzyme was more sensitive to inactivation by p-chloromercuribenzoate and by Cu2+. 5. The Michaelis constants with 3-acetylpyridine–adenine dinucleotide phosphate showed a twofold difference between liver and heart isoenzyme. 6. The differential sensitivity to heat and its mainly non-cytoplasmic location may be an explanation of the failure of plasma isocitrate dehydrogenase activity to increase after a myocardial infarction.
Binding of rat liver and hepatoma polyribosomes to stripped rough endoplasmic reticulum in vitro. Biological or an artifact?
Authors: Hochberg A. A. , Stratman F. W. , Zahlten Rainer N. , Morris H. P. , Lardy H. A.
Published: August 2015

Exposed thiol groups do not appear to be related to the binding of 32P-labelled polyribosomes to stripped rough endoplasmic reticulum in vitro. Treating stripped rough endoplasmic reticulum with GSSG did not diminish binding of polyribosomes, suggesting that binding in vitro has no correlation with the inhibition of protein synthesis in vitro reported by Kosower et al. (1971). Thiol reagents, which are known to dissociate ribosomes, did not significantly decrease binding of 32P-labelled polyribosomes to stripped rough endoplasmic reticulum. Denaturing the protein of 32P-labelled polyribosomes or stripped rough endoplasmic reticulum of liver or hepatoma with heat, trichloroacetic acid, or HClO4 did not alter the binding in vitro. Therefore, the practice of measuring the binding of 32P-labelled polyribosomes to stripped rough endoplasmic reticulum in vitro (Shires et al., 1971b) is an unsuitable indicator of biological significance in the intact cell.
Spin–spin interaction between molybdenum and one of the iron–sulphur systems of xanthine oxidase and its relevance to the enzymic mechanism
Authors: Lowe David J. , Lynden-Bell Ruth M. , Bray Robert C.
Published: August 2015

1. Electron-paramagnetic-resonance (e.p.r.) studies at 9 and 35GHz at helium temperatures have given new information relating to the structure and mechanism of action of xanthine oxidase. 2. As reported by others, the enzyme gives two types of e.p.r. signal attributed to iron–sulphur systems. The first has gav.=1.95. Parameters of the second are determined as g1 2.12, g2 2.007 and g3 1.91, with gav.=2.01. This species seems to have a slightly higher redox potential than the former one. 3. Temperature-dependent changes in the form of Mov e.p.r. signals from the enzyme, observed under certain conditions, are shown to be due to weak spin–spin interaction between Mov and gav.=1.95 Fe/S. The phenomenon has been studied most fully for the Slow Mov signal. Here, the spectral change takes the form of an additional approximately isotropic 11G splitting, detected below about 45°K only. Samples without Fe/S reduced showed no such changes of spectrum. 4. Similar spectral changes were observed in the Rapid Mov signals, obtained in rapid-freezing experiments, but only in samples corresponding to relatively long reaction times with the substrate. It is suggested therefore that the phenomenon may provide a means of distinguishing enzyme centres with Mo only reduced from those in which both Mo and Fe/S are reduced. 5. Additional rapid-freezing data tending to support a two- rather than a one-electron transfer of reducing equivalents from substrates to xanthine oxidase are reported.
The distribution of surface antigens during fractionation of mouse liver plasma membranes
Authors: Gurd James W. , Evans W. H. , Perkins Harold R.
Published: August 2015

1. Antiserum to purified mouse liver plasma membranes was prepared and the partially purified γ-globulin antibody fraction was iodinated with 125I. The reaction of the 125I-labelled γ-globulin antibody with isolated mouse liver plasma membranes was studied. 2. The γglobulin antibody bound specifically to mouse liver plasma membranes and there was little reaction with mouse liver intracellular membranes or with surface-membrane fractions from either rat liver or pig lymphocytes. 3. ‘Light’ and ‘heavy’ mouse liver plasma-membrane subfractions bound similar amounts of γ-globulin antibody, and this is consistent with a surface origin for the light fraction. 5. Plasma membranes were fractionated by sequential extraction with 50mm-NaHCO3–Na2CO3 buffer, pH10.2, containing 10mm-EDTA and aq. 33% (v/v) pyridine. The alkali-soluble and -insoluble fractions and the pyridine-soluble and -insoluble fractions all reacted with the antiserum, and the cross-reactivity among the various fractions and with the total plasma membranes was investigated. 5. The results are discussed in terms of the arrangement of the antigenic determinants within the membrane.
The formation of cholest-5-ene-3β,26-diol as an intermediate in the conversion of cholesterol into bile acids by liver mitochondria
Authors: Mitropoulos K. A. , Avery M. D. , Myant N. B. , Gibbons G. F.
Published: August 2015

1. When [14C]cholesterol was incubated with rat liver mitochondria, radioactive 26-hydroxycholesterol, 3β-hydroxychol-5-enoic acid and other bile acids were isolated from the incubation mixture. 2. In the absence of added 26-hydroxycholesterol, the specific radioactivity of the 26-hydroxycholesterol formed from [14C]cholesterol during the incubation was higher than that of the 3β-hydroxychol-5-enoic acid. Addition of increasing amounts of 26-hydroxycholesterol led to a progressive fall in the specific radioactivity, and to a progressive increase in the mass, of the 3β-hydroxychol-5-enoic acid recovered at the end of the incubation. 3. It is concluded that 26-hydroxycholesterol is an intermediate in the formation of 3β-hydroxychol-5-enoic acid from cholesterol. 4. Comparison of the specific radioactivity of the 26-hydroxycholesterol formed in the incubation mixture with that of the added [14C]cholesterol indicates that endogenous cholesterol in mitochondria is accessible to cholesterol 26-hydroxylase.
Sporulation in Bacillus subtilis. Properties and time of synthesis of alkali-soluble protein of the spore coat
Authors: Wood D. A.
Published: August 2015

An alkali-extractable protein fraction comprising 6% of the dry weight of the spore can be removed from spores of Bacillus subtilis 168. Three different extraction procedures each yield at least one similar protein. Extracted protein behaved as a single species on ion-exchange chromatography or gel filtration, but two polypeptides were found on electrophoresis. Comparison of molecular weights on electrophoresis and by sucrosegradient analysis suggests that the protein(s) undergo aggregation into multimers. Extracted spores remain viable, but are altered in density and lysozyme sensitivity and they aggregate together. Electron microscopy of extracted spores shows that loss of material seems to occur from the outer coat layers. Extraction therefore probably removes a specific fraction of the spore-coat protein, but without impairment to the spore protoplast. This protein can first be detected immunologically 4h after the initiation of sporulation, and the synthesis of this protein is sensitive to chloramphenicol, actinomycin D and rifamycin. Labelling experiments also show that the protein begins to be synthesized early in sporulation. Examination of the ability of asporogenous mutants to produce cross-reacting material indicates that some event in stage II of sporulation determines its production.
A catalytic mechanism for the enzyme benzylamine oxidase from pig plasma
Authors: Taylor C. E. , Taylor R. S. , Rasmussen C. , Knowles P. F.
Published: August 2015

Initial-velocity and product-inhibition studies on the enzyme benzylamine oxidase from pig plasma indicate that the order of substrate addition and product release is benzylamine on, ammonia off, oxygen on, hydrogen peroxide off, benzaldehyde off. Ammonia, but not benzaldehyde, is released under strictly anaerobic conditions which provides independent evidence for this order. Benzyl alcohol is a substrate for the enzyme. A chemical mechanism consistent with all the data is proposed.
The amino acid sequence of protein SCMK-B2A from the high-sulphur fraction of wool keratin
Authors: Elleman T. C.
Published: August 2015

1. The amino acid sequence of protein SCMK-B2A, a reduced and S-carboxymethylated protein from the high-sulphur fraction of wool, has been determined. 2. This protein of 171 amino acid residues displays both a high degree of internal homology and extensive external homology with other members of the SCMK-B2 group of proteins. 3. Evidence is presented which suggests that the SCMK-B2 group of proteins are produced by separate non-allelic genes.
The effects of univalent anions on catecholamine fluxes and adenosine triphosphatase activity in storage vesicles from the adrenal medulla
Authors: Taugner G.
Published: August 2015

1. Influx and efflux of catecholamine and adenosine triphosphatase activity were studied in storage vesicles of bovine adrenal medulla. 2. In the absence of ATP the influx of catecholamine was slow and was not influenced by various anions, whereas the efflux increased in the sequence of anions given by the lyotrophic series. 3. In the presence of ATP the efflux was enhanced compared with that in the absence of ATP; the anion-dependent sequence, however, in which the efflux increased was the same as in the absence of ATP. 4. The ATP-dependent catecholamine influx and the adenosine triphosphatase activity are correlated. The sequence in which anions affect adenosine triphosphatase activity and catecholamine influx, however, is completely different from the lyotrophic anion series. 5. No correlation was found between adenosine triphosphatase activity and the efflux of catecholamine.
The biosynthesis of (+)-α-pinene in Pinus species
Authors: Banthorpe Derek V. , Le Patourel Geoffrey N. J.
Published: August 2015

1. The degradation of (+)-α-pinene biosynthesized from 3RS-[2-14C]mevalonate by Pinus radiata or Pinus nigra revealed an asymmetrical labelling pattern whereby the moiety derived from isopentenyl pyrophosphate contained at least 90% of the incorporated tracer. This pattern differed both in asymmetry and position of labelling from previous results obtained with P. nigra, but is consistent with the generally accepted hypothetical mechanism for the biosynthesis of the pinane skeleton. 2. (+)-α-Pinene biosynthesized in Pinus attenuata and in the previously named two species from 3RS-[2-14C,(4R)-4-3H1]mevalonate and its (4S)-isomer retained all the 4R hydrogen atoms (within the experimental error) but lost all the 4S hydrogen atoms of the precursor. This stereospecificity of hydrogen loss is the same as that previously found for the formation of geraniol and nerol in other plant species, and the result may be reasonably inferred to be general for monoterpenes.
The characterization of C-phycocyanin from an extremely halo-tolerant blue–green alga, Coccochloris elabens
Authors: Kao Oranda H. W. , Berns Donald S. , Town William R.
Published: August 2015

C-Phycocyanin was isolated and purified from a uni-algal culture of an extremely halo-tolerant blue–green alga, Coccochloris elabens. This alga can be grown under laboratory conditions in 25% (w/v) NaCl. Purified halophile phycocyanin was characterized by amino acid analysis and the measurement of sedimentation velocity, fluorescence polarization and immunodiffusion as a function of protein concentration, pH and ionic strength. The results were compared with those of studies of phycocyanin isolated from Plectonema calothricoides and from several other sources. The states of aggregation previously characterized as being present in other C-phycocyanins, monomer, trimer and hexamer, were present in halophile phycocyanin and were characterized as antigenically related to all C-phycocyanins tested. The equilibrium between 3S monomer and 11S hexamer at low concentrations in halophile phycocyanin was quantitatively similar to that for other phycocyanins. The effect of pH and ionic strength on the 6S (trimer) and 11S (hexamer) aggregation of halophile phycocyanin was markedly salt-dependent and the relative amount of each aggregate in the presence of 2m-NaCl was like that of C-phycocyanin from mesophiles, in the absence of additional salt. In antigenic relationship and aggregation properties, the phycocyanin from C. elabens appeared to be most closely related to that isolated from the thermophilic blue–green alga, Synechococcus lividus. Amino acid content of the halophile phycocyanin indicated the presence of a significantly larger number of acidic residues than that found in mesophiles. Explanations of the properties of the halophile protein require consideration of a strong contribution of hydrophobic forces and utilize both charge-shielding and salting-out effects.
The preparation and analysis of a new trinucleotide from yeast ribonucleic acid, containing two adjacent 2′-O-methylpentose residues
Authors: Trim A. R. , Parker J. E.
Published: August 2015

Quantities of the order of 10mg of three alkali-stable trinucleotides were prepared from yeast ribonucleic acid. Analyses showed that they were of the type N1m-N2m-N3p, where m signifies 2′-O-methylation of the pentose residue. One, Am-Um-Gp, is newly described.
Terminal sequence studies of high-molecular-weight ribonucleic acid. The 3′-termini of rabbit globin messenger ribonucleic acid
Authors: Hunt J. A.
Published: August 2015

Haemoglobin mRNA isolated from EDTA-treated polyribosomes has an apparent molecular weight of 120000–180000 estimated by condensation with 3H-labelled isoniazid after periodate oxidation. Analysis of the ribonuclease digests of isoniazid-labelled RNA by paper electrophoresis and column chromatography enables the amount of contaminating 18S, 7S, 5S and 4S RNA to be estimated, and a corrected molecular weight of globin mRNA as the acid is 161000 or 500 nucleotides in length. This molecule contains two groups of 3′-terminal sequences in equal yield; G-Y-A6 and G-Y-A7 in the ratio 3:2, and G-N9–16-Y-A2 and G-N9–16-Y-N3 in the ratio 3:2. The significance of these sequences is discussed in relation to the poly(A) content of globin mRNA, the specificity of the sequences, and possible function in processing and biosynthesis of mRNA.
Interaction between polyuridylic acid and rabbit globin messenger ribonucleic acid
Authors: Hunt J. A.
Published: August 2015

Poly(U) binds to globin mRNA in 0.1m-NaCl. Studies with ribonuclease digestion of this complex suggest that there are polyadenylate-rich sequences in the mRNA containing about 30–40 adenylate residues. The sequences appear to be homogenous and of approximately the same length for both α- and β-globin mRNA. They are most likely located at the 3′ terminus of the molecule.
A sequence of seventy-three nucleotides from the coliphage R17 genome
Authors: Rensing Ulrich F. E.
Published: August 2015

1. A sequence of 73 nucleotides of the RNA genome from coliphage R17 was determined. It can be read through in only one translational frame. The fragment is not part of the coatprotein cistron (Min Jou et al., 1972), nor does it come from the untranslated sequences described previously (Steitz, 1969; Nichols, 1970; Cory et al., 1970; de Wachter et al., 1971; Contreras et al., 1971; Cory et al., 1972). It contains two sequences of 23 and 24 nucleotides, 22 of which are identical. This kind of reiteration is the first one found in bacteriophage nucleic acid. 2. Improved conditions were found and tested for blocking oligonucleotides with carbodi-imide and cleaving by ribonuclease A at cytidylate residues. 3. A synthetic medium is described which allows labelling in vivo with 32P to give specific radioactivities higher than those obtained in the procedures used previously.
Selective extraction of polymers from cell walls of Gram-positive bacteria (Short Communication)
Authors: Pavlik Jiri G. , Rogers Howard J.
Published: August 2015

Brief heating of Bacillus Licheniformis cell walls at 100°C in aqueous buffers of pH3.0–4.0 removes some polymers but not others from the mucopeptides. For example, relatively undegraded teichuronic acid can be extracted at 100°C in 20min at pH3.0 whereas the teichoic acids are not removed. Similar specificity can be shown with walls from three other species of micro-organism.
The use of 3-bromopropionic acid for the determination of protein thiol groups
Authors: Bradbury A. F. , Smyth D. G.
Published: August 2015

S-Carboxymethylcysteine, formed by the reaction of iodoacetic acid with cysteine, was found to undergo intramolecular cyclization to yield 3-oxo-(2H,3H,5H,6H-1,4-thiazine)-5-carboxylic acid. The cyclization was studied under various conditions and the product was isolated and characterized. S-Carboxyethylcysteine, formed by the reaction of 3-bromopropionic acid with cysteine, did not undergo the cyclization reaction. The use of 3-bromopropionic acid was examined as an alternative to iodoacetic acid for the protection and determination of protein thiol groups.
Evidence for the amino acid sequence of porcine pancreatic elastase
Authors: Shotton David M. , Hartley Brian S.
Published: August 2015

The preparation and purification of tryptic peptides from aminoethylated Dip-elastase and [14C]carboxymethylated Dip-elastase, and of peptic peptides from native elastase is described. A summary of the results of chemical studies used to elucidate the amino acid sequence of these peptides is presented. Full details are given in a supplementary paper that has been deposited as Supplementary Publication SUP 50016 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973), 131, 1–20. These results, together with those from previously published papers, are used to establish the complete amino acid sequence of elastase, which is a single polypeptide chain of 240 residues, molecular weight 25900, containing four disulphide bridges.
Interaction of quinacrine mustard with calf thymus histone fractions
Authors: Selander Ritva-Kajsa , De La Chapelle Albert
Published: August 2015

1. Interactions of histone fractions with quinacrine mustard were investigated by fluorimetry and spectrophotometry and the results were interpreted with the aid of thinlayer chromatography. 2. Characteristic differences were found between the various histone fractions of calf thymus. The conditions that favoured histone conformational changes and aggregation, also favoured interaction between histones and the dye; low concentrations of SO42− brought about more interaction than did Cl−; urea, guanidinium and iodide ions were inhibitory to binding. 3. Changes in the physical state of all the quinacrine mustard–protein complexes occurred as a function of ionic strength and pH. The most salt-dependent interaction was found in the arginine-rich histone fraction. 4. The interaction of the calf thymus histone fractions with quinacrine mustard was compared with the interaction of bovine plasma albumin and protamine with quinacrine mustard. 5. The relationship between dye-binding and the aggregation of histone fractions was discussed.
Evidence for defective transfer ribonucleic acid in polymyopathic hamsters and its inhibitory effect on protein synthesis
Authors: Bester André J. , Gevers Wieland
Published: August 2015

1. Different reaction steps involved in protein synthesis were studied in skeletal muscles from control and myopathic hamsters. 2. There was no difference between partially purified aminoacyl-tRNA synthetases from myopathic and control animals in yield or catalytic activity, as tested with exogenous deacylated tRNA. 3. However, isolated deacylated tRNA from myopathic muscle was aminoacylated by these synthetases to a lesser extent than that derived from control muscle. 4. Addition of deacylated tRNA isolated from control muscle improved the performance of pH5 enzymes from myopathic muscle in polypeptide synthesis on homologous polyribosomes; tRNA isolated from myopathic animals did not. 5. Preparation of extracts from both types of animals in the presence of the ribonuclease-absorbent bentonite led to an increased capacity of endogenous tRNA to accept amino acids in pH5 enzymes prepared from normal and abnormal tissue, but the difference between the two systems remained the same. 6. Total tRNA nucleotidyltransferase activity, tested with twice-pyrophosphorolysed rat liver tRNA, was identical in both extracts. 7. Added tRNA nucleotidyltransferase incorporated more AMP and CMP into endogenous tRNA with the pH5 enzyme from myopathic muscle than with that from control muscle. 8. Preincubation of deacylated tRNA from myopathic muscle with ATP, CTP and tRNA nucleotidyltransferase more than doubled its subsequent aminoacyl-acceptor activity, and halved the extent of the defect relative to aminoacylation of control tRNA similarly treated. Endogenous tRNA in pH5 enzyme preparations behaved likewise. 9. It is suggested that a 3′-exonuclease in myopathic muscles attacks tRNA molecules in such a way that some of them remain substrates for tRNA nucleotidyltransferase, which may incorporate into RNA not only AMP and CMP, but also GMP. 10. Cell-free protein synthesis in preparations from myopathic hamster muscles is limited by the supply of intact tRNA molecules.
Biosynthesis of a mycobacterial lipopolysaccharide. Incorporation of [14C]acyl groups by whole cells in vivo
Authors: Narumi Koyu , Keller John M. , Ballou Clinton E.
Published: August 2015

1. Mycobacterium phlei (A.T.C.C. 356) cells were incubated with 14C-labelled short-chain fatty acids and the 6-O-methylglucose-containing lipopolysaccharides that became esterified with radioactive acyl groups were isolated. The pattern of labelling of these lipopolysaccharides with the different acyl groups, the effects of different conditions on labelling patterns, and the kinetics of the turnover of 14C-labelled acyl groups were studied. 2. The labelling patterns are summarized as follows. [1-14C]Acetate was incorporated into all of the acyl groups. [1-14C]Propionate led to labelling of propionate and succinate, while [1-14C]isobutyrate was incorporated mostly as such, along with a trace amount in iso-octanoate. 3. Under the conditions of the experiments, [1-14C]acetate was rapidly incorporated into succinyl (3-carboxypropionyl) and octanoyl groups, whereas the acetyl groups themselves were labelled more slowly. Radioactivity in propionyl and succinyl groups, originating from [1-14C]propionate, attained maximum values and then gradually decreased in both. Incorporation of [1-14C]isobutyrate proceeded slowly but reached a plateau and remained constant. While n-butyrate is not a normal constituent of methyl-glucose-containing lipopolysaccharides, it was incorporated as such when n-[1-14C]-butyrate was supplied in the medium. 4. [1-14C]Acetyl groups were readily displaced by unlabelled acetate. On the other hand, the specific radioactivity of the succinyl group continued to increase during a 3h incubation with unlabelled succinate. Propionyl and succinyl groups, labelled by [1-14C]propionate, were displaced slowly by unlabelled propionate or succcinate. The isobutyryl group of the lipopolysaccharides did not turn over, in contrast to the results obtained with the other acyl substituents.
The effect of acute and prolonged ethanol treatment on the concentrations of coenzyme A, carnitine and their derivatives in rat liver
Authors: Kondrup Jens , Grunnet Niels
Published: August 2015

1. CoA, acetyl-CoA, long-chain acyl-CoA, carnitine, acetylcarnitine and long-chain acylcarnitine were measured in rat liver under various conditions. 2. Starvation caused an increase in the contents of these intermediates, except that of carnitine. 3. A single dose of ethanol had no effect on CoA content, whereas those of acetyl-CoA, acetylcarnitine and carnitine were increased and those of long-chain acyl-CoA and acylcarnitine were decreased. 4. Four weeks' adaptation to ethanol consumption did not change the effect of ethanol administration on these metabolites. 5. It is suggested that ethanol directly increases hepatic fatty acid synthesis and esterification. It is also suggested that this change is reversible and limited to the period of ethanol oxidation. 6. It is demonstrated that ethanol-induced triglyceride accumulation is not related to carnitine deficiency.
The degradation of l-histidine, imidazolyl-l-lactate and imidazolylpropionate by Pseudomonas testosteroni
Authors: Coote J. G. , Hassall H.
Published: August 2015

1. Imidazol-5-ylpropionate and imidazol-5-yl-lactate are degraded by Pseudomonas testosteroni via inducible pathways. 2. Growth on either compound as the sole source of carbon results in the induction of the enzymes for histidine catabolism. 3. The pathway of histidine degradation in this organism, a non-fluorescent Pseudomonad, is shown to be the same as that operating in Pseudomonas fluorescens and Pseudomonas putida. It consists of the successive formation of urocanate, imidazol-4-on-5-ylpropionate, N-formimino-l-glutamate, N-formyl-l-glutamate and glutamate. 4. Whole cells of P. testosteroni accumulate urocanate in the reaction mixture when incubated with imidazolylpropionate, but only after an adaptive lag period which is removed by previous growth on imidazolylpropionate as the source of carbon. 5. Imidazolyl-lactate is oxidized to imidazolylpyruvate, which then gives rise to histidine by specific transamination with l-glutamate. 6. Cells grown on histidine, urocanate or imidazolylpropionate are also able to degrade imidazolyllactate. 7. Mutants lacking urocanase are unable to grow on imidazolylpropionate, imidazolyl-lactate, histidine or urocanate. One with impaired histidase activity cannot utilize histidine or imidazolyl-lactate, but grows normally on imidazolylpropionate or urocanate. A mutant unable to grow on imidazolylpropionate is indistinguishable from the wild-type with respect to growth on histidine, imidazolyl-lactate or urocanate. 8. Thus it is established that imidazolyl-lactate is metabolized via histidine whereas imidazolylpropionate enters the histidine degradation pathway after conversion into urocanate.
Protein kinase activity in membrane preparations from ox brain. Stimulation of intrinsic activity by adenosine 3′:5′-cyclic monophosphate
Authors: Weller Malcolm , Rodnight Richard
Published: August 2015

1. Properties of the stimulation by cyclic AMP of the intrinsic protein kinase activity of membrane fragments from ox brain were studied. 2. Stimulation of activity declined from about 100% at 1min to less than 20% at 10min. The time-course was explained by the observation that cyclic AMP did not stimulate turnover of protein-bound serine phosphate once the membrane protein was fully phosphorylated. 3. Cyclic AMP accelerated the activity of a component of the basal activity rather than activating a different kinase. 4. The pH optimum for both the stimulated and basal activities was 7.2–7.4. NaCl (100mm) and KCl (10–100mm) inhibited the stimulated activity but did not affect the basal activity. 5. Strychnine and theophylline inhibited both activites equally, but the stimulated activity was more sensitive to inhibition by adenosine, bicuculline, vinblastin, veratrine, N-ethylmaleimide and cysteine. 6. No firm evidence for a role for endogenous cyclic AMP in the basal activity was found, but the possibility was not excluded. 7. Some 90% of both the stimulated and basal activities remained in an insoluble form after treatment of the membrane fragments with Triton X-100 (0.5%).
The heterogeneous distribution of acid hydrolases within a homogeneous population of cultured mammalian cells
Authors: Milsom Judith P. , Wynn C. H.
Published: August 2015

1. Chinese-hamster ovary fibroblasts were cultured to provide a homogeneous cell population. Homogenates obtained from these cells were fractionated by centrifugation techniques and the resulting fractions were analysed for protein and for enzymes representative of certain subcellular particles. 2. Unlike those in rat liver homogenates, the mitochondrial and lysosomal populations proved impossible to separate by differential centrifugation owing to the similarity of their sedimentation properties. Their resolution was possible by using isopycnic centrifugation in a continuous sucrose density gradient. 3. The mitochondrial population equilibrated at a density of 1.17g·cm−3 as in rat liver homogenates. However, the lysosomal population equilibrated at a lower rather than a higher density position than the mitochondria and the probable reasons for this are discussed. 4. The lysosomal population subdivided into two groups characterized by differences in acid hydrolase content and equilibrium densities. The fraction with a density of 1.15g·cm−3 contained the majority of arylsulphatases A and B, of cathepsin and of β-acetylglucosaminidase activities, whereas that with a density of 1.09g·cm−3 contained the majority of the acid phosphatase and acid ribonuclease activities. The probable division of the lysosomal population of a single cell into a number of distinguishable subgroups is suggested.
The proton-translocating nicotinamide–adenine dinucleotide (phosphate) transhydrogenase of rat liver mitochondria
Authors: Moyle Jennifer , Mitchell Peter
Published: August 2015

1. The NAD(P) transhydrogenase activity of the soluble fraction of sonicated rat liver mitochondrial preparations was greater than the NAD-linked isocitrate dehydrogenase activity, and the NAD-linked and NADP-linked isocitrate dehydrogenase activities were not additive. The NAD-linked isocitrate dehydrogenase activity was destroyed by an endogenous autolytic system or by added nucleotide pyrophosphatase, and was restored by a catalytic amount of NADP. 2. We concluded that the isocitrate dehydrogenase of rat liver mitochondria was exclusively NADP-specific, and that the oxoglutarate/isocitrate couple could therefore be used unequivocally as redox reactant for NADP in experiments designed to operate only the NAD(P) transhydrogenase (or loop 0) segment of the respiratory chain in intact mitochondria. 3. During oxidation of isocitrate by acetoacetate in intact, anaerobic, mitochondria via the rhein-sensitive, but rotenone- and arsenite-insensitive, NAD(P) transhydrogenase, measurements of the rates of carbonyl cyanide p-trifluoromethoxyphenylhydrazone-sensitive and carbonyl cyanide p-trifluoromethoxyphenylhydrazone-insensitive pH change in the presence of various oxoglutarate/isocitrate concentration ratios gave an →H+/2e− quotient of 1.94±0.12 for outward proton translocation by the NAD(P) transhydrogenase. 4. Measurements with a K+-sensitive electrode confirmed that the electrogenicity of the NAD(P) transhydrogenase reaction corresponded to the translocation of one positive charge per acid equivalent. 5. Sluggish reversal of the NAD(P) transhydrogenase reaction resulted in a significant inward proton translocation. 6. The possibility that isocitrate might normally be oxidized via loop 0 at a redox potential of −450mV, or even more negative, is discussed, and implies that a P/O quotient of 4 for isocitrate oxidation might be expected.
Changes in enzyme activities and distributions during glucose de-repression and respiratory adaptation of anaerobically grown Saccharomyces carlsbergensis
Authors: Cartledge T. G. , Lloyd D.
Published: August 2015

1. During anaerobic glucose de-repression the respiration rate of whole cells of Saccharomyces carlsbergensis remained constant and was insensitive to antimycin A but was inhibited by 30% by KCN. Aeration of cells for 1 h led to increased respiration rate which was inhibited by 80% by antimycin A or KCN. 2. Homogenates were prepared from sphaeroplasts of anaerobically grown, glucose de-repressed cells and the distribution of marker enzymes was investigated after zonal centrifugation on sucrose gradients containing MgCl2. These homogenates contained no detectable cytochrome c oxidase or catalase activity. The complex density distributions of NADH– and NADPH–cytochrome c oxidoreductases and adenosine triphosphatase(s) [ATPase(s)] were very different from those of anaerobically grown, glucose-repressed cells. 3. The specific activity of total ATPase was lowered and sensitivity to oligomycin decreased from 58 to 7% during de-repression. 4. Cytochrome c oxidase and catalase activities were detectable in homogenates of cells after 10min aeration. Zonal centrifugation indicated complex, broad sedimentable distributions of all enzyme activities assayed; the peaks of activity were at 1.27g/ml. 5. Centrifugation of homogenates of cells adapted for 30min and 3 h indicated a shift of density of the major sedimentable peak from 1.25g/ml (30min) to 1.235g/ml (3 h). After 30min adaptation a minor zone of oligomycin-sensitive ATPase and 15% of the total cytochrome c oxidase activities were detected at ρ=1.12g/l; these particles together with those of higher density containing cytochrome c oxidase, ATPase and NADH–cytochrome c oxidoreductase activities were all sedimented at 105g-min. 6. Electron microscopy indicated that the mitochondria-like structures of anaerobically grown, glucose-de-repressed cells were similar to those of repressed cells. After 10min of respiratory adaptation highly organized mitochondria were evident which resembled the condensed forms of mitochondria of aerobically grown, glucose-de-repressed cells. High-density zonal fractions of homogenates of cells after adaptation also contained numerous electron-dense vesicles 0.05–0.2μm in diameter. 7. The possibility that the `promitochondria' of anaerobically grown cells may not be the direct structural precursors of fully functional mitochondria is discussed.
The site at which 4-iodoacetamidosalicylate reacts with glutamate dehydrogenases
Authors: Holbrook J. John , Roberts P. Ann , Wallis Robert B.
Published: August 2015

1. Bovine, porcine and chicken liver glutamate dehydrogenases were irreversibly inhibited by a tenfold excess of radioactive 4-iodoacetamidosalicylic acid at pH7.5. 2. Inhibition was accompanied by the covalent incorporation of 1.1 mol of labelled inhibitor/mol of polypeptide chain. Acid hydrolysis yielded Nε-carboxymethyl-lysine as the sole labelled amino acid. No labelled S-carboxymethylcysteine was recovered from the bovine or porcine enzymes. 3. The labelled bovine enzyme was hydrolysed with trypsin. The radioactivity was found at lysine-126 in a peptide comprising residues 119–130 of the sequence. 4. The amino acid compositions of the tryptic peptides containing labelled lysine from the porcine and chicken enzymes were similar to that of the bovine peptide.
Synthesis, characterization and properties of uridine 5′-(α-d-apio-d-furanosyl pyrophosphate)
Authors: Kindel P. K. , Watson R. R.
Published: August 2015

1. A method was developed for synthesizing UDP-apiose [uridine 5′-(α-d-apio-d-furanosyl pyrophosphate)] from UDP-glucuronic acid [uridine 5′-(α-d-glucopyranosyluronic acid pyrophosphate)] in 62% yield with the enzyme UDP-glucuronic acid cyclase. 2. UDP-apiose had the same mobility as uridine 5′-(α-d-xylopyranosyl pyrophosphate) when chromatographed on paper and when subjected to paper electrophoresis at pH5.8. When [3H]UDP-[U-14C]glucuronic acid was used as the substrate for UDP-glucuronic acid cyclase, the 3H/14C ratio in the reaction product was that expected if d-apiose remained attached to the uridine. In separate experiments doubly labelled reaction product was: (a) hydrolysed at pH2 and 100°C for 15min; (b) degraded at pH8.0 and 100°C for 3min; (c) used as a substrate in the enzymic synthesis of [14C]apiin. In each type of experiment the reaction products were isolated and identified and were found to be those expected if [3H]UDP-[U-14C]apiose was the starting compound. 3. Chemical characterization established that the product containing d-[U-14C]apiose and phosphate formed on alkaline degradation of UDP-[U-14C]apiose was α-d-[U-14C]apio-d-furanosyl 1:2-cyclic phosphate. 4. Chemical characterization also established that the product containing d-[U-14C]apiose and phosphate formed on acid hydrolysis of α-d-[U-14C]apio-d-furanosyl 1:2-cyclic phosphate was d-[U-14C]apiose 2-phosphate. 5. The half-life periods for the degradation of UDP-[U-14C]apiose to α-d-[U-14C]apio-d-furanosyl 1:2-cyclic phosphate and UMP at pH8.0 and 80°C, at pH8.0 and 25°C and at pH8.0 and 4°C were 31.6s, 97.2min and 16.5h respectively. The half-life period for the hydrolysis of UDP-[U-14C]-apiose to d-[U-14C]apiose and UDP at pH3.0 and 40°C was 4.67min. After 20 days at pH6.2–6.6 and 4°C, 17% of the starting UDP-[U-14C]apiose was degraded to α-d-[U-14C]apio-d-furanosyl 1:2-cyclic phosphate and UMP and 23% was hydrolysed to d-[U-14C]apiose and UDP. After 120 days at pH6.4 and −20°C 2% of the starting UDP-[U-14C]apiose was degraded and 4% was hydrolysed.
The association between phosphatidylinositol phosphodiesterase activity and a specific subunit of microtubular protein in rat brain
Authors: Quinn P. J.
Published: August 2015

1. Supernatant proteins from rat brain were separated into two fractions containing phosphatidylinositol phosphodiesterase activity by chromatography on DEAE-Sephadex A-50. 2. The first fraction sediments in linear sucrose density gradients in two bands corresponding to molecular weights of 66000 and 36000. There was presumptive evidence that the lighter protein constituted the monomeric form of the enzyme. The second fraction sediments predominantly as a single protein of molecular weight 86000. 3. Treatment of rat brain supernatant with [3H]colchicine abolished the second DEAE-Sephadex peak and removed the lighter protein from the first peak. These proteins emerged in the same position as the protein binding [3H]colchicine at high salt concentration; phospholipase activity was recovered from linear sucrose density gradients in positions corresponding to molecular weights 88000 and 43000, together with an aggregate of molecular weight 140000. Electrophoresis on sodium dodecyl sulphate–urea–polyacrylamide gels of this fraction revealed only three proteins: the α and β-subunits of microtubular protein, of molecular weights 56000 and 52000 respectively, and a protein of molecular weight 38000. 4. A sample of microtubular protein from mouse, labelled in vivo with [3H]proline and 32Pi, was added to rat brain supernatant together with an equal amount of the same microtubular protein treated with cyclic AMP and [γ-32P]ATP and the mixture subsequently characterized by ion-exchange chromatography. Some phospholipase activity characteristic of the second peak from DEAE-Sephadex was associated with one fraction of added microtubular protein. This fraction was identified on the basis of the 3H:32P ratio as the β subunit of the protein treated with ATP and cyclic AMP. The subunit of added microtubular protein untreated with nucleotides was not associated with phospholipase activity.
The purification and properties of Factor X from pig serum and its role in hypercoagulability in vivo
Authors: Dupe R. J. , Howell R. M.
Published: August 2015

The molecular weights or shapes of Factor X preparations determined by gel filtration were dependent on the density of the BaSO4 used for the initial adsorption from serum. One form obtained with BaSO4 of density 2g/ml behaved as if it had a molecular weight of 39000 and possessed preformed clotting activity (Factor Xa), whereas that of the form adsorbed with BaSO4 of density 1g/ml had a molecular weight of 69000 and consisted of inactive Factor X precursor. Thus degradation accompanied by activation seems to occur as a result of surface adsorption on high-density BaSO4 and is associated with an interchange of protein between the two bands observed electrophoretically. The clotting and esterase activities measurable in vitro after complete activation were not matched by a corresponding ability to induce thrombus formation and ‘lethality’ in vivo. The most effective preparations of Factor X in this respect possessed preformed activity, which was enhanced in the presence of phospholipid. Factor X lost activity more rapidly in dilute solution, and its concentration at the surface of phospholipid micelles probably decreases loss by dilution in circulating blood.
Appendix: Structure and conformation of bilirubin. Opposing views that invoke tautomeric equilibria, hydrogen bonding and a betaine may be reconciled by a single resonance hybrid
Authors: Kuenzle C. C. , Weibel M. H. , Pelloni R. R. , Hemmerich P.
Published: August 2015

A novel conformational structure of bilirubin is presented which obtains maximum stabilization through a system of four intramolecular hydrogen bonds. Two hydrogen bonds link oxygen and nitrogen atoms of each end ring to the contralateral carboxyl group. The proposed structure can explain a variety of uncommon features of bilirubin, and reconciles many seemingly contradictory hypotheses by accommodating them in individual structures which are mesomeric forms of one resonance hybrid. In the light of this newly conceived structure the following characteristics of bilirubin are re-evaluated: the stability of the compound, its reaction with diazomethane, the conformational behaviour of its dimethyl ester, its spectral properties, the chirality of the compound when complexed to serum albumin, and the structure of its metal chelates.
Lack of temperature-sensitivity of rat liver pyruvate kinase
Authors: Flory Wayne , Koeppe Roger E.
Published: August 2015

Rat liver pyruvate kinase, prepared by Sephadex treatment of a 105g supernatant in phosphate buffer, is quite stable and gives reproducible results when a variety of parameters are altered in the enzyme assay. Incubation of this preparation at 25°C or 0–2°C has no effect on the activation by fructose 1,6-diphosphate or inhibition by ATP or alanine.
Nitrogenase of Klebsiella pneumoniae: evidence for an adenosine triphosphate-induced association of the iron–sulphur protein
Authors: Thorneley R. N. F. , Eady R. R.
Published: August 2015

The effect of various nucleotides on the Fe-containing component of nitrogenase of Klebsiella pneumoniae was investigated by ultracentrifugation and thiol-group reactivity towards 5,5′-dithiobis-(2-nitrobenzoate). In the absence of Na2S2O4, ATP and ADP produced changes in sedimentation behaviour and thiol-group reactivity consistent with association of the protein.
Multiple forms of acetylcholinesterase from human erythrocytes
Authors: Wright David L. , Plummer David T.
Published: August 2015

1. Acetylcholinesterase from human erythrocytes was solubilized with Triton X-100 in strong salt solution and partially purified by (NH4)2SO4 fractionation. This preparation showed three main bands of enzyme activity after electrophoresis on polyacrylamide gel and incubation with either α-naphthyl acetate or acetylthiocholine as enzyme substrate. Two of the multiple forms were completely inhibited by 10μm-eserine and one only partially. Treatment with neuraminidase had no effect on the electrophoretic pattern; therefore sialic acid does not appear to determine or affect the ratios of the acetylcholinesterase multiple forms, unlike those of the serum cholinesterase. 2. Chromatography of the preparation on Sephadex G-200 revealed one major peak of enzyme activity and a suggestion of two minor zones of mol.wt. 546000, 184000 and 93000 (i.e. in the proportion 6:2:1). The main peak was almost completely separated from the Triton X-100 and the overall purification was about 600-fold. Further attempts to purify the enzyme by absorption on calcium phosphate gels were unsuccessful. 3. Electrophoresis of the enzyme preparation on a polyacrylamide gradient for 24h revealed three main bands that corresponded to the three values for molecular weights obtained by column chromatography. After 70h of electrophoresis a further three zones of activity developed making six molecular entities, the molecular weights of which were simple multiples of a monomer, thus resembling the cholinesterase found in serum.
A comparison of the unfolding and dissociation of the large ribosome subunits from Rhodopseudomonas·spheroides N.C.I.B. 8253 and Escherichia coli M.R.E. 600
Authors: Robinson A. , Sykes J.
Published: August 2015

1. The behaviour of the large ribosomal subunit from Rhodopseudomonas spheroides (45S) has been compared with the 50S ribosome from Escherichia coli M.R.E. 600 (and E. coli M.R.E. 162) during unfolding by removal of Mg2+ and detachment of ribosomal proteins by high univalent cation concentrations. The extent to which these processes are reversible with these ribosomes has also been examined. 2. The R. spheroides 45S ribosome unfolds relatively slowly but then gives rise directly to two ribonucleoprotein particles (16.6S and 13.7S); the former contains the intact primary structure of the 16.25S rRNA species and the latter the 15.00S rRNA species of the original ribosome. No detectable protein loss occurs during unfolding. The E. coli ribosome unfolds via a series of discrete intermediates to a single, unfolded ribonucleoprotein unit (19.1S) containing the 23S rRNA and all the protein of the original ribosome. 3. The two unfolded R. spheroides ribonucleoproteins did not recombine when the original conditions were restored but each simply assumed a more compact configuration. Similar treatments reversed the unfolding of the E. coli 50S ribosomes; replacement of Mg2+ caused the refolding of the initial products of unfolding and in the presence of Ni2+ the completely unfolded species (19.1S) again sedimented at the same rate as the original ribosomes (44S). 4. Ribosomal proteins (25%) were dissociated from R. spheroides 45S ribosomes by dialysis against a solution with a Na+/Mg2+ ratio of 250:1. During this process two core particles were formed (21.2S and 14.2S) and the primary structures of the two original rRNA species were conserved. This dissociation was not reversed. With E. coli 50S approximately 15% of the original ribosomal protein was dissociated, a single 37.6S core particle was formed, the 23S rRNA remained intact and the ribosomal proteins would reassociate with the core particle to give a 50S ribosome. 5. The ribonuclease activities in R. spheroides 45S and E. coli M.R.E. 600 and E. coli M.R.E. 162 50S ribosomes are compared. 6. The observations concerning unfolding and dissociation are consistent with previous reports showing the unusual rRNA complement of the mature R. spheroides 45S ribosome and show the dependence of these events upon the rRNA and the importance of protein–protein interactions in the structure of the R. spheroides ribosome.
The primary structure of aspartate aminotransferase from pig heart muscle. Partial sequences determined by digestion with thermolysin and elastase
Authors: Bossa Francesco , Barra Donatella , Carloni Massimo , Fasella Paolo , Riva Francesca , Doonan Shawn , Doonan Hilary J. , Hanford Robin , Vernon Charles A. , Walker John M.
Published: August 2015

Peptides produced by thermolytic digestion of aminoethylated aspartate aminotransferase and of the oxidized enzyme were isolated and their amino acid sequences determined. Digestion by elastase of the carboxymethylated enzyme gave peptides representing approximately 40% of the primary structure. Fragments from these digests overlapped with previously reported sequences of peptides obtained by peptic and tryptic digestion (Doonan et al., 1972), giving ten composite peptides containing 395 amino acid residues. The amino acid composition of these composite peptides agrees well with that of the intact enzyme. Confirmatory results for some of the present data have been deposited as Supplementary Publication 50018 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973) 131, 5.