bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies on the phosphorylation of the inhibitory subunit of troponin during modification of contraction in perfused rat heart
Authors: England P J
Published: August 2015

1. Rat hearts were perfused with 32Pi, and contractile force was increased by positive inotropic agents (agents that increase contractility). The inhibitory subunit of troponin (troponin I) was then isolated by affinity chromatography in 8M-urea, and its 32P content measured. Incorporation of phosphate into the subunit was calculated on the basis of the [gamma-32P]ATP specific radioactivity in the hearts. 2. When hearts were perfused with 30 nM-DL-isoprenaline (N-isopropylnoradrenaline), there was an increase in contractile force over 30s which was paralleled by an increase in troponin I phosphorylation. When hearts were perfused for 25s with increasing concentrations of isoprenaline from 1 NM to 0.6 muM, there was again a parallel increase in contractile force and troponin I phosphorylation. The maximum phosphorylation observed was 1.5 mol of phosphate/mol of troponin I, which was reached after 25s with 0.1 muM-isoprenaline. 3. Hearts were stimulated with a 15s pulse perfusion of 30nM-DL-isoprenaline. There was an increase in contractile force which was followed by a return to the control value within 50s. Troponin I phosphorylation increased to a plateau value which was reached within 30s, and remained constant for 60s after the isoprenaline pulse. Phosphorylase a and 3′:5′-cyclic AMP concentration showed changes similar to that of the contractile force. There was no change in 3′:5′-cyclic GMP concentration. 4. When hearts stimulated with a 15S pulse of isoprenaline were subsequently perfused with 0.6 muM-acetylcholine, the changes in contractile force, phosphorylase a and 3′:5′-cyclic AMP were very similar to those seen with the 15s pulse of isoprenaline alone. Troponin I phosphorylation increased to a maximum 30s after the end of the isoprenaline pulse, but then rapidly decreased during the subsequent 30s. This decrease was preceded by a 60% increase in the concentration of 3′:5′-cyclic GMP. 5. Hearts were perfused with 0.2 muM-glucagon for periods up to 60s. Contractile force showed little change for the first 30s, but then increased rapidly. This was paralleled by changes in 3′:5′-cyclic AMP concentration. Troponin I phosphorylation increased slowly, but the increase in contractile force had reached a maximum before significant phosphorylation had occurred. 6. It is concluded that under certain conditions, e.g. immediately after β-adrenergic stimulation, there is a good correlation between contractile force and troponin I phosphorylation. However, under other conditions, e.g. when contractile force is decreasing after removal of β-adrenergic stimulation or in the presence of glucagon, contractile force and troponin I phosphorylation are not well correlated. These results suggest that mechanisms for modifying cardiac contractility, other than troponin I phosphorylation, must be present in rat heart.
The degradation of semisynthetic tritiated insulin by perfused mouse livers
Authors: Halban P A , Karakash C , Davies J G , Offord R E
Published: August 2015

Semisynthetic [3H]insulin was stored under various conditions for up to 180 days and the stability of the insulin under these conditions was assessed. A sample that had been stored for 180 days was repurified and shown to be degraded at the same rate as native insulin by perfused mouse livers, even at low physiological concentrations. After perfusion, intact insulin could be separated from degradation products, and the radioactivity associated with the insulin fraction could be used to determine the percentage degradation. The initial rate of degradation of insulin was a linear function of concentration over the range 360pM-1.9nM.
Validation of a simple radiochemical assay measuring hydrolysis of choline-labelled microsomal phosphatidylcholine by phospholipase C. pH-dependence
Authors: Cater B R , Trivedi P , Hallinan T
Published: August 2015

Selective hydrolysis of phosphatidylcholine species, which are selectively radioactively labelled in vivo, does not appear to interfere with a radiochemical assay for hydrolysis of microsomal phosphatidylcholine by C-type phospholipases from Bacillus cereus or Clostridium perfringens. Both phospholipases substantially hydrolysed phosphatidylcholine over the pH range 4.0-10.0.
Intranuclear maturation pathways of rat liver ribosomal ribonucleic acids
Authors: Dabeva M D , Dudov K P , Hadjiolov A A , Emanuilov I , Todorov B N
Published: August 2015

The maturation of pre-rRNA (precursor to rRNA)in liver nuclei is studied by agar/ureagel electrophoresis, kinetics of labelling in vivo with [14C] orotate and electron-microscopic observation of secondary structure of RNA molecules. (1) Processing starts from primary pre-rRNA molecules with average mol. wt. 4.6×10(6)(45S) containing the segments of both 28S and 18S rRNA. These molecules form a heterogeneous peak on electrophoresis. The 28S rRNA segment is homogeneous in its secondary structure. However, the large transcribed spacer segment (presumably at the 5′-end) is heterogeneous in size and secondary structure. A minor early labelled RNA component with mol.wt. about 5.8×10(6) is reproducibly found, but its role as a pre-rRNA species remains to be determined. (2) The following intermediate pre-rRNA species are identified: 3.25×10(6) mol.wt.(41S), a precursor common to both mature rRNA species; 2.60×10(6)(36S) and 2.15×10(6)(32S) precursors to 28S rRNA; 1.05×10(6) (21S) precursor to 18S rRNA. The pre-rRNA molecules in rat liver are identical in size and secondary structure with those observed in other mammalian cells. These results suggest that the endonuclease-cleavage sites along the pre-rRNA chain are identical in all mammalian cells. (3) Labelling kinetics and the simultaneous existence of both 36S and 21S pre-rRNA reveal that processing of primary pre-rRNA in adult rat liver occurs simultaneously by at least two major pathways: (i) 45S → 41S → 32S+21S → 28S+18S rRNA and (ii) 45S → 41S → 36S+18S → 32S → 28S rRNA. The two pathways differ by the temporal sequence of endonuclease attack along the 41 S pre-rRNA chain. A minor fraction (mol.wt.2.9×10(6), 39S) is identified as most likely originating by a direct split of 28S rRNA from 45S pre-rRNA. These results show that in liver considerable flexibility exists in the order of cleavage of pre-rRNA molecules during processing.
Studies on rat ovarian receptors for lutropin (luteinizing hormone). Applicability of radioimmunoassay to measure lutropin bound to receptors
Authors: Muralidhar K , Moudgal N R
Published: August 2015

Measurement of receptor-bound unlabelled physiologically active lutropin (luteinizing hormone, LH) was possible by a modified radioimmunoassay. The conventional radioimmunoassay conducted at 4° C was inadequate, whereas the modified assay performed at 37° C could measure receptor-bound lutropin. The radioimmunoassay at 37° C takes only 36h for completion compared with 5-7 days at 4° C. The sensitivity and range of dose-response curves are, however, unaltered. The validity of the technique was established by a number of criteria.
Studies on rat ovarian receptors for lutropin (luteinizing hormone). Interaction with β-subunit of sheep lutropin
Authors: Muralidhar K , Moudgal N R
Published: August 2015

By using radioimmunoassay, the interaction of sheep lutropin (luteinizing hormone, LH) β-subunit with rat ovarian receptors was investigated. The binding of β-subunit was specific, although of much lower order than that of lutropin. Sheep lutropin β-subunit effectively inhibited the binding of human choriogonadotropin (chorionic gonadotropin, gCG) to the ovary, showing that both occupy the same sites. The binding of sheep lutropin β-subunit to ovary was not followed by any detectable increase in cyclic AMP. The ovarian response to lutropin in terms of cyclic AMP production was inhibited in the presence of free β-subunit. The α-subunit of lutropin, when used at concentrations where contamination with whole lutropin was negligible, enhanced the degree of binding of β-subunit; this did not lead to increased cyclic AMP in the tissue. Surprisingly, the binding of β-subunit in vitro was drastically decreased by the prior removal of all endogenous rat lutropin bound to receptors. The implications of these data are discussed in the light of the reported biological activity of the β-subunit.
Carcass glycogen as a potential source of glucose during short-term starvation
Authors: Sugden M C , Sharples S C , Randle P J
Published: August 2015

In small rats deprived of food for 19h (or 43h), 36% (or 39%) of the glycogen that disappeared was lost from the carcass and 64% (or 61%) from liver. Carcass glycogen is potentially a substantial source of glucose during short-term starvation via the Cori cycle.
Preparation and characterization of armadillo submandibular glycoproteins
Authors: Wu A M , Pigman W
Published: August 2015

The nine-banded armadillo (Dasypus novemcinctus mexicanus Peters) was chosen for this study so that a comparison could be made of the salivary mucus glycoproteins of an ancient mammalian species with those derived from previously studied, more highly evolved species. Two mucus glycoproteins, armadillo submandibular glycoprotein A and armadillo submandibular glycoprotein B, were prepared from the armadillo submandibular gland by a modification of the method of Tettamanti & Pigman (1968) (Arch. Biochem. Biophys. 124, 41-50). The composition of glycoprotein A is the simplest one among the known mucus glycoproteins. Six amino acids constitute 98.5 mol/100mol of the protein of glycoprotein A and 82 mol/100 mol of that of glycoprotein B. These are serine and threonine (which make up 40-50% of the molar amino acid composition), glutamic acid, glycine alanine and valine. Proline is absent from glycoprotein A and comprises only 2.3% of glycoprotein B. For both glycoproteins, the protein content, as determined by the method of Lowry, Rosebrough, Farr & Randall (1951) (J. Biol. Chem 193, 265-275), with bovine serum albumin as standard, was nearly 60% higher than when determined by the sum of the amino acids. The ratios of total mol of amino acid/total mol of carbohydrate are 1:0.63 for glycoprotein A and 1:0.68 for glycoprotein B, N-Acetylneuraminic acid and N-acetylgalactosamine, in a molar ratio of about 0.35:1.00, are the principal carbohydrates present in both glycoproteins. Neutral sugars seem to be absent from glycoprotein A, but galactose and fucose are present in glycoprotein B. The carbohydrate side chains in glycoprotein A are composed of about two-thirds monosaccharide and one-third disaccharide residues, whereas those of glycoprotein B are more complex. For both glycoproteins, essentially all of the N-acetylgalactosamine was attached O-glycosidically to the hydroxyamino acid residues of the protein core. The linkage of N-acetylneuraminic acid glycoprotein A was extremely sensitive to dilute acid and neuraminidase. Glycoprotein B has chemical properties similar to those of glycoprotein A. However, whereas glycoprotein A was susceptible to both Clostridium perfringens and Vibrio cholerae neuraminidases, only the latter enzyme had an effect on glycoprotein B at pH 4.75. Both glycoproteins were homogeneous by cellulose acetate electrophoresis and ultracentrifugal analyses. The apparent mol.wts. of glycoprotein A and glycoprotein B were 7.8 X 10(4) and 3.1 X 10(4) respectively.
Labelling of egg phosphatidylcholine vesicles and myelin membrane with a photoreactive lipophilic reagent
Authors: Abu-Salah K M , Findlay J B C
Published: August 2015

The preparation and isolation of [3H]phenyl azide, a photosensitive non-polar probe, is reported. The reagent partitions into the lipid bilayer of egg phosphatidylcholine vesicles and bovine myelin membranes. On photoactivation to generate the nitrene grouping, as much as 90% of the covalently attached label is associated with the fatty acyl residues of the constituent phospholipid molecules. The remainder is found in the polar head groups. The cholesterol component of myelin membranes is also heavily labelled. These results suggest that such reagents may be used to probe the hydrophobic regions of natural membranes.
Biochemical and physiochemical characterization of pepsin-solubilized type-II collagen from bovine articular cartilage
Authors: Herbage D , Bouillet J , Bernengo J C
Published: August 2015

Solubilization of collagen from bovine articular with pepsin requires the preliminary extraction of proteoglycans from the ground substance. Biochemical and physiochemical properties of this pepsin-solubilized collagen are independent of the pretreatment (extraction with 1.5M-CaCl2, 5M-guanidinium chloride or 0.2M-NaOH) and of the age range (2-4-year-old and 2-month-old animals). Characterization of the de-natured components, of the CNBr peptides and of the amino acid and cross-link composition shows that the collagen of the hyaline cartilage is all type II. Electrical birefringence measurements showed the presence of tropocollagen molecules (length 280nm) and molecules whose length is slightly less than twice that of the tropocollagen molecules. This latter molecule may be a dimer composed of two monomers linked by intermolecular head-to-tail bonds and whose theoretical length (530nm), according to the quarter-stagger theory, is in good agreement with our measured values (510-530nm). We have verified that the beta-components of this collagen are formed of two alpha-chains linked by the stable intermolecular bond, dehydrodihydroxylysinonorleucine. These dimeric molecules are absent from solutions of skin collagen whose beta-components possess only aldol-type intramolecular cross-links. Although reconstituted fibres from solutions of skin and cartilage collagen are similar, the segment-long spacing crystallites formed with pepsin-solubilized cartilage collagen present a symmetrical and dimeric form corresponding to the lateral aggregation of two monomers with an overlap (90nm) of the C-terminal ends.
Purification and properties of the methane mono-oxygenase enzyme system from Methylosinus trichosporium OB3b
Authors: Tonge G M , Harrison D E F , Higgins I J
Published: August 2015

1. A three-component enzyme system that catalyses the oxidation of methane to methanol has been highly purified from Methylosinus trichosporium. 2. The components are (i) a soluble CO-binding cytochrome c, (ii) a copper-containing protein and (iii) a small protein; the mol. wts. are 13 000, 47 000 and 9400 respectively. The cytochrome component cannot be replaced by similar cytochrome purified from Pseudomonas extorquens or by horse heart cytochrome c. 3. The stoicheiometry suggests a mono-oxygenase mechanism and the specific activity with methane as substrate is 6 micronmol/min per mg of protein. 4. Other substrates rapidly oxidized are ethane, n-propane, n-butane and CO. Dimethyl ether is not a substrate. 5. The purified enzyme system utilizes ascorbate or, in the presence of partially purified M. trichosporium methanol dehydrogenase, methanol as electron donor but not NADH or NADPH. 6. Activity is highly sensitive to low concentrations of a variety of chelating agents, cyanide, 2-mercaptoethanol and dithiothreitol. 7. Activity is highly pH-dependent (optimum 6.9-7.0) and no component of the enzyme is stable to freezing. 8. The soluble CO-binding cytochrome c shows oxidase acitivity and the relationship between this and the oxygenase activity is discussed.
Mechanism of ‘L’-type pyruvate kinase from rabbit liver. Evidence against phosphoenzyme formation
Authors: Dann L G , Britton H G
Published: August 2015

The ‘L’-type pyruvate kinase from rabbit liver does not catalyse exchange between phosphoenol[1-14C]pyruvate and pyruvate at either pH 8.5 or 6.2. Spectrophotometric experiments at pH 8.5 and 6;2 and gel-filtration experiments with [32P]phosphoenolpyruvate at pH 8,5 also fail to demonstrate phosphoenzyme formation. It is concluded that it is very unlikely that the enzyme has a phosphoenzyme mechanism.
Macromolecular distribution near the limits of density-gradient columns. Some applications to the separation and fractionation of glycoproteins
Authors: Creeth M J , Horton J R
Published: August 2015

1. Expressions are derived for the distribution at density-gradient equilibrium of macromolecules whose densities are (a) close to the values characterizing the solution limits or (b) outside the span of the gradient. 2. Density-distribution predicted by the expressions agree with those obtained by rigorous methods. 3. The distribution equations are applied to hypothetical mixtures of proteins and glycoproteins in commonly used density-gradient media to simulate separation and fractionation conditions. 4. It is shown that CsBr, although less efficient than CsCl for fractionation, is nevertheless adequate for most purposes; in analytical experiments it may often have advantages over CsCl. Limitations on the use of LiBr are explored. 5. An expression is derived which allows the variance of the partial specific volume of the macromolecular component to be determined from the variance of the buoyant density. It is shown that the relative resolving powers of different salts is expressed by their values of the quantity (formula: see text). 6. The equations are applied to a well-characterized glycoprotein preparation at equilibrium in CsCl and in Cs2SO4:it is shown that the much wider distribution in CsCl than in Cs2SO4 is explicable in terms of the variance in buoyant density and the solvation properties of the salts. 7. Limitations of the expressions arise when dispersity in density is represented by a low apparent molecular weight; realistic simulations can then only be obtained when the component is fully banded.
Affinity-chromatographic isolation and some properties of troponin C from different muscle types
Authors: Head J F , Weeks R A , Perry S V
Published: August 2015

1. The formation of a complex between troponin I and troponin C that is stable in 6M-urea and dependent on Ca2+ was demonstrated in extracts of vertebrate striated and smooth muscles. 2. A method using troponin I coupled to Sepharose is described for the rapid isolation of troponin C from striated and smooth muscles of vertebrates. 3. Troponin C of rabbit cardiac muscle differs significantly in amino acid composition from troponin C of skeletal muscle. The primary structures of troponin C of red and white skeletal muscle are very similar. 4. The troponin C-like protein isolated from rabbit uterus muscle has a slightly different amino acid composition, but possess many similar properties to the forms of troponin C isolated from other muscle types. 5. The electrophoretic mobilities of the I-troponin C complexes formed from components isolated from different muscle types are determined by the troponin I component.
Studies in vitro of the effects of adenosine 3′:5′-cyclic monophosphate on the phosphorylation of nuclear proteins in isolated rat heart nuclei
Authors: Akhtar R A , Itzhaki S
Published: August 2015

Isolated rat heart nuclei were prepared by homogenization and sucrose-density-gradient centrifugation. The protein/DNA ratio of these nuclei was 3.1:1 (w/w), and the histones/non-histone proteins/DNA proportions were 1.4:1.6:1 (by wt.). Non-histone proteins were fractionated into six major groups by elution on a quaternized anion-exchanger (QAE-Sephadex A-50 column with increasing concentrations of NaCl in 5M-urea/0.01 M-Tris/HCl buffer (pH8.3). When isolated nuclei were incubated in a medium containing [gamma-32P]ATP, a differential distribution of 32P was observed in the six fractions of nonhistone proteins. The fractions eluted from the Sephadex column with 0.35M- and 0.6M-NaCl contained contained 80% of the total radioactivity incorporated into the non-histone proteins. This incorporation into the 0.35M- and 0.6M-NaCl fractions was increased by 66 and 112% respectively in the presence of cyclic AMP. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of these two particular fractions showed a selective increase in labelling of five protein bands in the presence of cyclic AMP.
Modification of pig heart lactate dehydrogenase with methyl methanethiosulphonate to produce an enzyme with altered catalytic activity
Authors: Bloxham D P , Wilton D C
Published: August 2015

Methyl methanethiosulphonate was used to produce a modification of the essential thiol group in lactate dehydrogenase which leaves the enzyme catalytically active. Methyl methanethiosulphonate produced a progressive inhibition of enzyme activity, with 2mM-pyruvate and 0.14mM-NADH as substrates, which ceased once the enzyme had lost 70-90% of its activity. In contrast, with 10mM-lactate and 0.4mM-NAD+ as substrates the enzyme was virtually completely inhibited. The observed inhibition was critically dependent on the chosen substrate concentration, since methanethiolation with methyl methanethiosulphonate resulted in a large decrease in affinity for pyruvate. At 0.14mM-NADH, methanethiolation increased the apparent KmPyr from from 40micronM for the control enzyme to 12mM for the modified enzyme. Steady-state kinetics showed that there was not a statistically significant change in either KmNADH or KsNADH. At saturating NADH and pyruvate concentrations, the Vmax. was virtually unaffected for the methanethiolated enzyme. However, a decrease in Vmax. was observed when the modified enzyme was incubated in dilute solution. The modification of lactate dehydrogenase by methyl methanethiosulphonate involved the active site, since inhibition was completely prevented by substrate-analogue pairs such as NADH and oxamate or NAD+ and oxalate. The formation of complexes between methanethiolated lactate dehydrogenase and substrates or substrate analogues can also be shown by re-activation experiments. The methanethiolated enzyme was re-activated in a time-dependent reaction by dithiothreitol and this was prevented by oxamate, by NADH and by NADH plus oxamate in increasing order of effectiveness. The results of this work are interpreted in terms of a role for the essential thiol group in the binding of substrates.
Substrate specificity of amino acid transport in sheep erythrocytes
Authors: Young J D , Ellory J C
Published: August 2015

The specificity of amino acid transport in normal (high-glutathione) sheep erythrocytes was investigated by studying the interaction of various neutral and dibasic amino acids in both competition and exchange experiments. Apparent Ki values were obtained for amino acids as inhibitors of L-alanine influx. Amino acids previously found to be transported by high-glutathione cells at fast rates (L-cysteine, L-alpha-amino-n-butyrate) were the most effective inhibitors. D-Alanine and D-alpha-amino-n-butyrate were without effect. Of the remaining amino acids studied, only L-norvaline, L-valine, L-norleucine, L-serine and L-2,4-diamino-n-butyrate significantly inhibited L-alanine uptake. L-Alanine efflux from pre-loaded cells was markedly stimulated by extracellular L-alanine. Those amino acids that inhibited L-alanine influx also stimulated L-alanine efflux. In addition, D-alanine, D-alpha-amino-n-biutyrate, L-threonine, L-asparagine, L-alpha, beta-diaminoproprionate, L-ornithine, L-lysine and S-2-aminoethyl-L-cysteine also significantly stimulated L-alanine efflux. L-Lysine uptake was inhibited by L-alanine but not by D-alanine, and the inhibitory potency of L-alanine was not influenced by the replacement of Na+ in the incubation medium with choline. L-Lysine efflux from pre-loaded cells was stimulated by L-alanine but not by D-alanine. It is concluded that these cells possess a highly selective stero-specific amino acid-transport system. Although the optimum substrates are small neutral amino acids, this system also has a significant affinity for dibasic amino acids.
An oligomycin-resistant adenosine triphosphatase and its effects on cellular growth, mitochondrial oxidative phosphorylation and respiratory proton translocation in Saccharomyces cerevisiae
Authors: Somlo M , Reid R A , Krupa M
Published: August 2015

Mutations at the OLI 1 or OLI 2 loci of mitochondria DNA in Saccharomyces cerevisiae are associated with a diminished growth rate in nutritionally suboptimal cultures supplemented with an oxidizable carbon source. In the case of mutant OR146(OLI1) there is a 35% loss of mitochondrial protein during fractionation in vitro, suggesting that the mutationally altered adenosine triphosphatase(ATPase) confers some instability on the mitochondrial membrane. The possibility is discussed that this reflects an unstable mitchondrial population in vivo, leading the observed growth deficiency. Mitochondria from mutant OR146 at the OLI 1 locus show a relatively oligomycin-resistant State-3 respiration, but the same ADP/O and respiratory-control quotients as the isonuclear wild-type. A slightly lowered Qo2 with NADH-linked substrates was observed and is discussed. For both strains the apparent H+/O ratios were close to 4 with pyruvate, ethanol and alpha-oxoglutarate, but consistently lower with succinate and citrate. For each substrate a characteristic t 1/2 (time for half-decay of the transmembrane pH differential) range was found, consistent with the view that the substrates effecitvely carry the protons back across the membrane. As expected, H+/O ratios were independent of t 1/2 for all substrates, with the exception of alpha-oxoglutarate in the case of the wild-type, where an inverse correlation was found. The lack of this correlation in the case of the mutant was the only apparent difference in the translocation parameters observed. A hypothesis relating this to the functioning of the oligomycin-resistant ATPase is proposed.
Comparative study of nuclear binding sites for oestradiol in rat testicular and uterine tissue. Determination of low amounts of specific binding sites by an [3H] oestradiol-exchange method
Authors: de Boer W , de Vries J , Mulder E , van der Molen H J
Published: August 2015

1. An [3H]oestradiol-exchange method was developed for the determination of oestradiol-receptor complexes in the nuclear fraction of immature rat testicular tissue. This method permits the determination of nuclear oestradiol-receptor sites in the presence of a relatively large amount of non-specific oestradiol binding present in testicular nuclei. After incubation of nuclei for 60min at 20 degrees C in the presence of [3H]oestradiol with or without a 1000-fold excess of non-radioactive diethylstilboestrol, specific binding can be determined quantitatively in the KCl-extractabe fraction, which contains 40% of the total receptor population. 2. The amount of receptor-bound steroid present in the 0.4m-KCl extract of testicular neclei remained constant during incubation at 20 degrees C. For uterine nuclei incubated with [3H]oestradiol at 37 degrees C a shift of specifically bound [3H]oestradiol occurred from the KCl-soluble fraction to the KCl-insoluble fraction. 3. In intact rat testis, about 20% of the total receptor concentration was present in its nuclear form. Hypophysectomy 5 days before measurement resulted in a twofold decrease in the amount of receptor, which was present mainly in the cytosol. After injection of choriogonadotropin to intact animals, the total receptor concentration increased threefold. 4. This nuclear exchange method might be useful for determination of occupied specific receptor sites in tissues with relatively low contents of specific receptors.
Effect of thyroliberin on the concentration of adenosine 3′:5′-phosphate and on the activity of adenosine 3′:5′-phosphate-dependent protein kinase in prolactin-producing cells in culture
Authors: Gautvik K M , Walaas E , Walaas O
Published: August 2015

1. The effects of thyroliberin were studied in cultured rat pituitary-tumour cells that synthesize and secrete prolactin (the GH4C1 cell strain). 2. Prolactin and cyclic AMP were measured by radioimmunological methods, and a cyclic AMP-dependent protein kinase was characterized by using histone as substrate. 3. Prolactin release was studied after 5-60min of treatment, and synthesis after 48h of treatment with thyroliberin. One-half maximum stimulation of release and synthesis were observed at 0.25 and at 4nM respectively. 4. Cyclic AMP was temporarily increased in cell suspensions after treatment with thyroliberin, and one-half maximum stimulation was observed at 25nM. 5. Dibutyryl cyclic AMP increased prolactin release and synthesis, one-half maximum effects being obtained at 20 micronM. 6. A cyclic AMP-dependent protein kinase, which was one-half maximally stimulated at 30 nM-cyclic AMP, was demonstrated. 7. An increase in the activity ratio (-cyclic AMP/+cyclic AMP) of the cyclic AMP-dependent protein kinase was observed after treatment with thyroliberin. Total protein kinase activity in the presence of cyclic AMP was unaltered. The time-course of enzyme activation was similar to that of cyclic AMP formation and corresponded to the time when prolactin release was first observed. 8. It is concluded that thyroliberin induces cyclic AMP formation, resulting in the activation of a cyclic AMP-dependent protein kinase.
The release of alanine by rat diaphragm muscle in vitro
Authors: Snell K , Duff D A
Published: August 2015

Alanine release by rat diaphragm muscle in vitro is stimulated by glutamate, valine, leucine and glucose. The stimulation by glutamate and valine (but not leucine) is inhibited by 3-mercaptopicolinate. These results suggest a metabolic route involving phosphoenolpyruvate carboxykinase which directs amino acid carbon skeletons towards pyruvate synthesis for alanine formation.
Effect of acetoacetate on glucose metabolism in the soleus and extensor digitorum longus muscles of the rat
Authors: Maizels E Z , Ruderman N B , Goodman M N , Lau D
Published: August 2015

1. The effect of acetoacetate on glucose metabolism was compared in the soleus, a slow-twitch red muscle, and the extensor digitorum longus, a muscle composed of 50% fast-twitch red and 50% white fibres. 2. When incubated for 2h in a medium containing 5 mM-glucose and 0.1 unit of insulin/ml, rates of glucose uptake, lactate release and glucose oxidation in the soleus were 19.6, 18.6 and 1.47 micronmol/h per g respectively. Acetoacetate (1.7 mM) diminished all three rates by 25-50%; however, it increased glucose conversion into glycogen. In addition, it caused increases in tissue glucose, glucose 6-phosphate and fructose 6-phosphate, suggesting inhibition of phosphofructokinase. The concentrations of citrate, an inhibitor of phosphofructokinase, and of malate were also increased. 3. Rates of glucose uptake and lactate release in the extensor digitorum longus were 50-80% of those in the soleus. Acetoacetate caused moderate increases in tissue glucose 6-phosphate and possibly citrate, but it did not decrease glucose uptake or lactate release. 4. The rate of glycolysis in the soleus was approximately five times that previously observed in the perfused rat hindquarter, a muscle preparation in which acetoacetate inhibits glucose oxidation, but does not alter glucose uptake or glycolysis. A similar rate of glycolysis was observed when the soleus was incubated with a glucose-free medium. Under these conditions, tissue malate and the lactate/pyruvate ratio in the medium were decreased, and acetoacetate did not decrease lactate release or increase tissue citrate or glucose 6-phosphate. An intermediate rate of glycolysis, which was not decreased by acetoacetate, was observed when the soleus was incubated with glucose, but not insulin. 5. The data suggest that acetoacetate glucose inhibits uptake and glycolysis in red muscle under conditions that resemble mild to moderate exercise. They also suggest that the accumulation of citrate in these circumstances is linked to the rate of glycolysis, possibly through the generation of cytosolic NADH and malate formation.
A kinetic study of the soluble 5′-nucleotidase of rat liver
Authors: van den Berghe G , van Pottelsberghe C , Hers H G
Published: August 2015

1. The kinetic properties of the 5′-nucleotidase (EC 3.1.3.5) present in the cytosol of rat liver were investigated in relation to the conversion of adenine nucleotides into uric acid, with particular reference to the stimulation of this process by fructose. The enzyme was assayed by the release of Pi and by a new and more sensitive radiochemical procedure. 2. When IMP was used as substrate, the partially purified enzyme displayed almost hyperbolic kinetics (h = 1.1) with S0.5 = 1.2 mM. Similar kinetics were observed with GMP and other nucleoside 5′-monophosphates, except AMP. 3. Vmax. of the enzyme for AMP was about the same as for IMP, but the kinetics were sigmoidal (h = 1.6) with S 0.5 = 10 mM. 4. The hydrolysis of IMP was inhibited competitively by GMP. IMP, at concentrations up to 0.5 mM, had a paradoxical stimulatory action on the hydrolysis of 2-5 mM-AMP and was inhibitory at higher concentrations. 5. The activity of the enzyme towards AMP and IMP was stimulated by ATP and GTP, and inhibited by Pi. Activators and inhibitor approximately cancelled each others' effects. At pH 7.4, the enzymic activity with 0.2 mM-AMP was undetectable under physiological conditions. 6. It is concluded that, in the liver cell, AMP is not hydrolysed by the soluble 5′-nucleotidase, but that its degradation requires prior deamination to IMP.
Assay of flavin nucleotides in pancreatic islets by a differential fluorimetric technique
Authors: Brolin S E , Agren A
Published: August 2015

Differences in fluorescence intensity between FMN and FAD at high and low pH values were utilized to determine the concentration of each nucleotide. Amounts down to 1 pmol were measurable. The FMN and FAD contents of pancreatic islets of obese-hyperglycaemic mice (gene symbol ob/ob) were compared with those of the exocrine pancreas, the liver and heart muscle. As expected, the FAD content of each tissue was greater than that of FMN. The contents of both nucleotides were significantly higher in the islets than in the exocrine part of the pancreas, but lower than in the heart muscle and liver. Possible relationships between the flavin content of the islets and their capacity for oxidative phosphorylation are briefly discussed.
Some properties of aldehyde dehydrogenase from sheep liver mitochondria
Authors: Hart G J , Dickinson F M
Published: August 2015

Aldehyde dehydrogenase from sheep liver mitochondria was purified to homogeneity as judged by electrophoresis on polyacrylamide gels, and by sedimentation-equilibrium experiments in the analytical ultracentrifuge. The enzyme has a molecular weight of 198000 and a subunit size of 48000, indicating that the molecule is a tetramer. Fluorescence and spectrophotometric titrations indicate that each subunit can bind 1 molecule of NADH. Enzymic activity is completely blocked by reaction of 4mol of 5,5′-dithiobis-(2-nitrobenzoate)/mol of enzyme. Excess of disulfiram or iodoacetamide decreases activity to only 50% of the control value, and only two thiol groups per molecule are apparently modified by these reagents.
Purification and properties of a new testosterone 17β-dehydrogenase (NADP+) from guinea-pig liver
Authors: Kaguera E , Toki S
Published: August 2015

As a result of studies of guinea-pig live testosterone 17beta-dehydrogenase (NADP+) (EC 1.1.1.64), a new testosterone 17beta-dehydrogenase was discovered. The new enzyme was purified to a single homogeneous protein from the 105 000 g-supernatant fraction of guinea-pig liver by (NH4)2SO4 fractional precipitation and two gel-filtration stages, DEAE-cellulose column chromatography and hydroxyapatite column chromatography. It was characterized by many properties. The enzyme has almost the same properties as the classical testosterone 17beta-dehydrogenase (NADP+) (EC 1.1.1.64), with respect to cofactor requirement, pH optima for dehydrogenation, effect of phosphate ion on the NAD+-dependent reaction and molecular weight, but characteristic differences were observed in substrate-specificity between the two dehydrogenases. With various androstane derivatives, the configuration of the A/B-ring junction was closely connected with enzyme activity. 5alpha-Androstanes, such as 5alpha-androstane-3alpha,17beta-diol, 5alpha-androstane-3beta,17beta-diol and 17beta-hydroxy-5alpha-androstan-3-one, and 5beta-congeners, such as 5beta-androstane-3alpha,17beta-diol, 5beta-androstane-3beta,17beta-diol and 17beta-hydroxy-5beta-androstan-3-one, served as substrates for both the EC 1.1.1.64 enzyme and the new enzyme. The EC 1.1.1.64 enzyme oxidized testosterone more rapidly than did the new enzyme. These comparisons were based on the relative activities, apparent Km values and apparent Vmax values.
Characterization of serum lipoproteins of the shark Centrophorus squamosus
Authors: Mills G L , Taylaur C E , Chapman M J , Forster G R
Published: August 2015

1. Blood serum from the shark Centrophorus squamosus (Bonnaterre) was shown to contain VLD (very-low-density), LD (low-density) and HD (high-density) lipoproteins. 2. In shape, size and general physical properties, these lipoproteins were very similar to those described for other animals. The VLD lipoproteins were the major components of the mixture, and HD lipoproteins were present at the lowest amount. 3. In addition to the usual lipid components, the shark lipoproteins also contain substantial amounts of hydrocarbon, probably mainly squalene, and monoalkyldiacylglycerols. Only trace amounts of wax ester were detected. 4. The protein moiety of the VLD and LD lipoproteins contained a component which, in its solubility and electrophoretic properties, molecular weight and amino acid composition, resembled the B apolipoprotein of man and other mammals. This accounted for a large part of the total shark apolipoprotein. 5. There were also present smaller amounts of proteins which were soluble in 8M-urea. In their electrophoretic mobility on basic polyacrylamide gel, some of these were like the A and C apoproteins of man. 6. The electrophoretic distribution of the soluble proteins from the VLD and LD lipoproteins resembled that in higher mammals, but in the HD lipoproteins the similarity was less.
Purification and properties of urease from bovine rumen
Authors: Mahadevan S , Sauer F D , Erfle J D
Published: August 2015

Urease (urea amidohydrolase, EC 3.5.1.5) was extracted from the mixed rumen bacterial fraction of bovine rumen contents and purified 60-fold by (NH4)2SO4 precipitation, calcium phosphate-gel adsorption and chromatography on hydroxyapatite. The purified enzyme had maximum activity at pH 8.0. The molecular weight was estimated to be 120000-130000. The Km for urea was 8.3 X 10(-4) M+/-1.7 X 10(-4) M. The maximum velocity was 3.2+/-0.25 mmol of urea hydrolysed/h per mg of protein. The enzyme was stabilized by 50 mM-dithiothreitol. The enzyme was not inhibited by high concentrations of EDTA or phosphate but was inhibited by Mn2+, Mg2+, Ba2+, Hg2+, Cu2+, Zn2+, Cd2+, Ni2+ and Co2+. p-Chloromercuribenzenesulfphonate and N-ethylmaleimide inhibited the enzyme almost completely at 0.1 mM. Hydroxyurea and acetohydroxamate reversibly inhibited the enzyme. Polyacrylamide-gel electrophoresis showed that the mixed rumen bacteria produce ureases which have identical molecular weights and electrophoretic mobility. No multiple forms of urease were detected.
Studies on sex-organ development. Changes in nuclear and chromatin composition and genomic activity during spermatogenesis in the maturing rooster testis
Authors: Mezquita C , Teng C S
Published: August 2015

We developed a technique to separate nuclei of rooster testis by centrifugation through a discontinuous sucrose density gradient and by sedimentation at unit gravity. Four different major fractions obtained from testicular nuclei and one from the vas deferens were characterized according to their velocity of sedimentation, morphology and DNA content. The ratios (w/w) of basic proteins, non-histone proteins and RNA to DNA decreased during spermiogenesis both in nuclei and chromatin. Changes in the electrophoretic patterns of histones and non-histone proteins were detected especially in the elongated spermatids. The lack of uptake of [3H]uridine in elongating and elongated spermatids and in spermatozoa was demonstrated by radioautography and by the detection of labelled RNA extracted from different fractions of nuclei. Template activity for RNA synthesis and the binding of actinomycin D by testicular nuclei reached a peak in the elongated spermatid stage, when the histones are replaced by the protamine.
Sites and specificity of the reaction of bipyridylium compounds with anaerobic respiratory enzymes of Escherichia coli. Effects of permeability barriers imposed by the cytoplasmic membrane
Authors: Jones Robert W. , Garland Peter B.
Published: August 2015

The ability of the oxidized and singly reduced species of several bipyridylium cations to cross the cytoplasmic membrane of Escherichia coli was studied to locate the sites of reaction of the dyes with anaerobic respiratory enzymes. Benzyl Viologen radical crossed the membrane rapidly, whereas the oxidized species did not. The oxidized or radical species of Methyl Viologen, Morfamquat or Diquat did not rapidly cross the membrane. It was also shown that the dithionite anion does not cross the cytoplasmic membrane of E. coli. Diquat radical donates electrons to the nitrate reductase pathway at the periplasmic aspect of the membrane, whereas Benzyl Viologen radical reacted directly with nitrate reductase itself (EC 1.7.99.4) at the cytoplasmic aspect of the membrane. Thus the pathway of electron transfer in the nitrate reductase pathway is transmembranous. Formate hydrogenlyase (EC 1.2.1.2) and an uncharacterized nitrite reductase activity react with bipyridylium dyes at the periplasmic aspect of the membrane. Fumarate reductase (succinate dehydrogenase; EC 1.3.99.1) reacts with bipyridylium radicals, and formate dehydrogenase (cytochrome) (EC 1.2.2.1) with ferricyanide, at the cytoplasmic aspect of the membrane. The differing charge and membrane permeation of oxidized and radical species of bipyridylium dyes greatly complicate their use as potentiometric mediators in suspensions of cells or membrane vesicles.
Net activity of phospholipase A2 in brain and the lack of stimulation of the phospholipase A2-acylation stem
Authors: Rowe C E
Published: August 2015

Certain observations reported previously from this laboratory have not proved reproducible. These are (1) the relatively rapid hydrolysis of added phosphatidylcholine by phospholipase A2 of tissue from the cerebral cortex of the guinea pig and (2) the stimulation by 10 micron-noradrenaline and by 1.0nM-cyclic AMP of the phospholipase A2-acylation system of isolated synaptic membranes.
Turnover of myelin proteins in mouse brain in vivo
Authors: Lajtha A , Toth J , Fujimoto K , Agrawal H C
Published: August 2015

The incorporation of tyrosine into proteins was measured after the subcutaneous implantation of a pellet of [14C]tyrosine in mice. This method keeps the specific radioactivity of free tyrosine fairly constant and makes it possible to follow incorporation up to a 10-day period. At the end of 10 days most of the protein-bound tyrosine was replaced (i.e. most protein turned over) in lung, liver, heart, kidney and spleen; about half was replaced in brain, one-quarter in muscle. The rate of protein turnover in myelin was approx. 40% of that of whole brain proteins; at 10 days one-fifth of the myelin proteins were replaced. All protein components of myelin measured were in a dynamic state; incorporation decreased in the following order, Wolfgram greater than DM-20 greater than basic greater than proteolipid proteins. The incorporation of tyrosine into each protein fraction was greater in the 0-5-day than in the 5-10-day period, indicating heterogeneity of metabolic rates. The results show that after myelination at least a portion of each protein component of myelin is undergoing significant metabolic turnover. In the adult, myelin components are not stable, but turnover is heterogeneous, and each protein may be compartmentalized. Turnover can be influenced by a variety of factors.
A pulse-radiolysis study of the manganese-containing superoxide dismutase from Bacillus stearothermophilus
Authors: McAdam M E , Levelle F , Fox R A , Fielden E M
Published: August 2015

In the preceding paper the mechanism of catalysis of the manganese-containing superoxide dismutase from Bacillus stearothermophilus was shown to involve a ‘fast cycle’ and a ‘slow cycle’ [McAdam, Fox, Lavelle & Fielden, 1977 (Biochem. J. 165, 71-79)]. Further properties of the enzyme was considered in the present paper. Pulse-radiolysis studies, under conditions of low substrate concentration to (i.e. when the fast cycle predominates), showed that enzyme activity decreases as pH increases (6.5-10.2). Activity was unaffected by the addition of H2O2 or NaN3 but slightly decreased by KCN. Both H2O2 and the reducing radical anion CO2– caused a decrease in A480 of the native enzyme. The rate of the fast catalytic cycle was independent of temperature (5-55 degrees C), and as temperature increases the slow cycle becomes relatively more important. Arrhenius parameters of the rate contants were estimated. The possible identity of the various forms of the enzyme is considered.
An evaluation of ways of using equilibrium dialysis to quantify the binding of ligand to macromolecule
Authors: Nimmo I A , Atkins G L , Strange R C , Percy-Robb I W
Published: August 2015

1. The effect of systematic error (loss of ligand, complex or macromolecule) on three of the experimental designs by which equilibrium dialysis may be used to quantify the interaction of ligand and macromolecule is examined theoretically, and the design that is least sensitive to systematic error is identified. 2. Thirteen methods for fitting the binding isotherm to experimental data are compared by using them to analyse simulated data containing random error, and the most reliable method is identified.
Purification and some properties of arylsulphatases A and B from rabbit kidney cortex
Authors: Helwig J J , Farooqui A A , Bollack C , Mandel P
Published: August 2015

Arylsulphatases A and B (EC 3.1.6.1) of rabbit kidney cortex were purified 5250- and 7720-fold respectively by a multiple-column-chromatography method. The specific activity toward 4-nitrocatechol sulphate was 42mumol/min per mg for arylsulphatase A and 62 mumol/min per mg for arylsulphatase B. Each enzyme migrated as a single band on polyacrylamide-gel electrophoresis, and the enzyme activity corresponded to the band of protein on the gel. The rate of hydrolysis of ascorbic acid 2-sulphate by arylsulphatase A was three times that for cerebroside 3-sulphate. Arylsulphatase B hydrolysed UDP-N–acetylgalactosamine 4-sulphate and glucosamine 4,6-disulphate, but not galactosamine 6-sulphate.
Chromous ion reduction of mammalian cytochrome oxidase and some of its derivatives
Authors: Greenwood C , Brittain T , Brunori M , Wilson M T
Published: August 2015

The reduction of cytochrome c oxidase by Cr2+, followed by means of stopped-flow spectrophotometry, exhibits two phases: the faster Cr2+-concentration-dependent reaction has an initial rate constant of 1.1 × 10(4)M-1-S-1, but reaches a rate limit at high concentration of reductant; the slower phase is concentration-independent with a rate of 0.3S-1. The activation energies of the fast and the slow processes are 35 and 71 kJ/mol respectively. The reduction kinetics of the mixed-valence CO complex and the cyanide-inhibited enzyme were compared with those of the fully oxidized forms: both the liganded species have a fast phase identical with that found in the oxidized oxidase. A comparison of the kinetic difference spectra obtained for the fast phase of reduction of oxidized oxidase with those obtained on reduction of the liganded species suggests that the rapid phase arises from the reduction ofhaem a, and the slow phase from the reduction of haem a3.
The apparent molecular weights of human intestinal aminopeptidase, enterokinase and maltase in native duodenal fluid
Authors: Magee A I , Grant D A W , Hermon-Taylor J
Published: August 2015

The apparent molecular weights of human intestinal aminopeptidase, enterokinase and maltase in native duodenal fluid were estimated by gel chromatography on Sephadex G-200 under different conditions of operational buffer and temperature. No evidence for environmentally induced changes in molecular form was found.