bluuebunny/crossref_metadata_2025 huggingface dataset:
Microbiological degradation of bile acids. The preparation of hexahydroindane derivatives as substrates for studying cholic acid degradation
Authors: Hayakawa S , Takata T , Fujiwara T , Hashimoto S
Published: August 2015

Relatively large amounts of 3-(3aalpha-hexahydro-7abeta-methyl-1,5-dioxoindan-4alpha-yl)propionic acid (IIb), which is believed to be one of the intermediates involved in the degradation of cholic acid (I), were needed to identify is further degradation products. A simple method for the preparation of this compound was then investigated. Arthrobacter simplex could degrade-3-oxoandrost-4-ene-17beta-carboxylic acid (IIIa) to 3-(1beta-carboxy-3aalpha-hexahydro-7abeta-methyl-5-oxoindan-4alpha-yl)propionic acid (IVa) in good yield, the structure of which was established by partial synthesis. It was therefore expected that, if a similar degradation by this organism occurred with 17alpha-hydroxy-3-oxoandrost-4-ene-17beta-carboxylic acid (IIIb), which is easily obtained by chemical oxidation of commercially availabe 17alpha-hydroxydeoxycorticosterone, the resulting product, 3-(1beta-carboxy-3aalpha-hexahydro-1alpha-hydroxy-7abeta-methyl-5-oxoindan-4alpha-yl)propionic acid (IVb), could be readily converted chemically into the required dioxocarboxylic acid, (IIb). Exposure of compound (IIIb) to A. simplex produced, as expected, compound (IVb) which was then oxidized with NaBiO3 to give a reasonable yield of compound (IIb).
Purification and properties of a cellulase from Aspergillus niger
Authors: Hurst P L , Nielsen J , Sullivan P A , Shepherd M G
Published: August 2015

A cellulolytic enzyme was isolated from a commercial cellulase preparation form Aspergillus niger. A yield of about 50mg of enzyme was obtained per 100g of commerial cellulase. The isolated enzyme was homogeneous in the ultracentrifuge at pH 4.0 and 8.0, and in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis but showed one major and two minor bands in disc gel electrophoresis. No carbohydrate was associated with the protein. Amino acid analysis revealed that the enzyme was rich in acidic and aromatic amino acids. Data from the amino acid composition and dodecyl sulphate/polyacrylamide-gel electrophoresis indicated a molecular weight of 26000. The purified enzyme was active towards CM-cellulose, but no activity towards either cellobiose or p-nitrophenyl beta-D-glucoside was detected under the assay conditions used. The pH optimum for the enzyme was pH 3.8-4.0, and it was stable at 25 degrees C over the range pH 1-9; maximum activity (at pH 4.0) was obtained at 45 degrees C. The cellulase was more stable to heat treatment at pH 8.0 than at 4.0. Kinetic studies gave pK values between 4.2 and 5.3 for groups involved in the enzyme-substrate complex.
A pulse-radiolysis study of the manganese-containing superoxide dismutase from Bacillus stearothermophilus. A kinetic model for the enzyme action
Authors: McAdam M E , Fox R A , Lavelle F , Fielden E M
Published: August 2015

The enzymic reaction mechanism of a manganese-containing superoxide dismutase from Bacillus stearothermophilus was studied by using pulse radiolysis. During catalysis (pH 8.9; 25 degrees C), changes occurring in the kinetics of substrate disappearance and in the visible absorption of the enzyme at 480 nm established that the simple two-step mechanism found for copper- and iron-containing superoxide dismutases is not involved. At a low ratio (less than 15) of substrate concentration to enzyme concentration the decay of O2–is close to exponetial, whereas at much higher ratios (greater than 100) the observed decay is predominantly zero-order. The simplest interpretation of the results invokes a rapid one-electron oxidation-reduction cycle (‘the fast cycle’) and, concurrently, a slower reaction giving a form of the enzyme that is essentially unreactive towards O2– but which undergoes a first-order decay to yield fully active native enzyme (‘the slow cycle’). The fast cycle involves the native enzyme EA and a form of the enzyme EB which can be obtained also by treating the form EA with H2O2. Computer calculations made with such a simple model predict behaviour in excellent agreement with the observed results.
An investigation of the interactions of the allosteric modifiers of pyruvate kinase with the enzyme from Carcinus maenas hepatopancreas
Authors: Giles I G , Poat P C , Munday K A
Published: August 2015

1. Pyruvate kinase purified from the hepatopancrease of Carcinus maenas exhibited sigmoidal saturation kinetics with respect to the substrate phosphoenolpyruvate in the absence of the allosteric activator fructose 1,6-bisphosphate, but normal hyperbolic saturation was seen in the presence of this activator. The activation appears to be the result of a decrease in the s0.5 (phosphoenolpyruvate) and not to a change in Vmax. 2. In the presence of ADP and ATP at a constant nucleotide-pool size the results indicate that phosphoenolpyruvate co-operativity is lost on increasing the [ATP]/[ADP] ratio. 3. Paralleling this change is the observation that the fructose 1,6-bisphosphate activation became less at the [ATP]/[ATP] ratio was increased. This was due to the enzyme exhibiting a near-maximal activity in the absence of activator. 4. L-Alanine inhibited the enzyme, but homotropic co-operative interactions were only seen with a cruder (1000000g supernatant) enzyme preparation. The inhibition by alanine could be overcome by increasing the concentration of either phosphoenolpyruvate or fructose 1,6-bisphosphate, although increasing the L-alanine concentration did not appear to be able to reverse the activation by fructose 1,6-bisphosphate. 5. In the presence of a low concentration of phosphoenolpyruvate, increasing the concentration of the product, ATP, caused an initial increase in enzyme activity, followed by an inhibitory phase. In the presence of either fructose 1,6-bisphosphate or L-alanine only inhibition was seen. 6. The inhibition by ATP could not be completely reversed by fructose 1,6-bisphosphate.
Fragments produced by digestion of human immunoglobulin G subclasses with pepsin in urea
Authors: Parr D M
Published: August 2015

It was previously shown that digestion of human IgG1/kappa myeloma proteins with pepsin in the presence of 8 M-urea produces fragments which differ from other proteolytic fragments of IgG, including those produced by peptic digestion in aqueous buffers. The two large urea/pepsin fragments each consist of three peptides, and together account for all of the constant region of the light chains and most of the constant region of the heavy chains. Myeloma proteins of subclasses IgG2, IgG3 and IgG4 with kappa light chains were digested with pepsin in 8 M-urea, and the resulting fragments compared with those produced from IgG1/kappa proteins. Gel filtration, starch- and polyacrylamide-gel electrophoresis and sequence analysis have shown that the peptides from each subclass are analogous with those from IgG1. A brief investigation of the products of urea/pepsin digestion of myeloma proteins with lambda light chains has shown that in these proteins light-chain cleavage occurs at residue leucine-182, instead of or as well as at residue 117, where cleavage takes place in kappa chains. Comparison of sequences around sites of urea/pepsin cleavage has shown that pepsin has quite restricted specificity under these conditions.
The aminoacylation of transfer ribonucleic acid. Recognition of methionine by Escherichia coli methionyl-transfer ribonucleic acid synthetase
Authors: Old J M , Jones D S
Published: August 2015

The mechanism of the recognition of methionine by Escherichia coli methionyl-tRNA synthetase was examined by a kinetic study of the recognition of methionine analogues in the ATP-PPi exchange reaction and the tRNA-aminoacylation reaction. The results show that the recognition mechanism consists of three parts: (1) the recognition of the size, shape and chemical nature of the amino acid side chain at the methionine-binding stage of the reaction; (2) the recognition of the length of the side chain at the stage of aminoacyl-adenylate complex-formation; (3) the recognition of the sulphur atom in the side chain at the stage of methionyl-tRNA formation. It is proposed that the sulphur atom interacts with the enzyme to induce a conformational change. A model of the active site incorporating the mechanism of methionine recognition is presented.
The structure of L-lactate oxidase from Mycobacterium smegmatis
Authors: Sullivan P A , Soon C Y , Schreurs W J , Cutfield J F , Shepherd M G
Published: August 2015

1. An improved purification was developed for L-lactate oxidase from Mycobacterium smegmatis. 2. The mol.wt. of the native enzyme by a sedimentation-equilibrium analysis was 345 000, and other ultracentrifuge methods gave values in the range 345 000-350 000. 3. An amino acid analysis, determinations of protein and flavin, a sedimentation-velocity analysis and an approach to equilibrium analysis gave values for the subunit mol.wt. in the range 43 500-47 000. 4. It was concluded that L-lactate oxidase contains eight subunits of mol.wt. 43 500. 5. Cross-linking of the subunits with dimethyl suberimidate and electron-microscopy studies were consistent with an octameric structure.
The high-performance liquid-chromatographic analysis of ficaprenol and dolichol
Authors: Keenan R W , Rice N , Quock R
Published: August 2015

A high-performance liquid-chromatographic method was devised which is capable of resolving the p-nitrobenzoyl derivatives of polyprenols containing 35-110 or more carbon atoms. This procedure was used for the determination of ficaprenol and pig liver dolichol composition and can be applied to mixtures of polyprenols as an analytical or preparative technique.
Semisynthetic analogues of insulin. The use of N-substituted derivatives of methionine as acid-stable protecting groups
Authors: Saunders Derek J. , Offord Robin
Published: August 2015

1. We describe the use of benzyloxycarbonylmethionine and ethoxycarbonylmethionine for the selective protection of the amino groups of glycine-A1 and lysine-B29 of pig insulin. We have used the Edman method to remove residues from the N-terminal and of the B-chain of the NA1NB29-di-protected derivatives. The benzyloxycarbonyl group shows slight but noticeable lability in the acid-cleavage step, but the ethoxycarbonyl group remained intact even after five cycles of degradation. 2. We have prepared the following truncated forms of insulin via the di(ethoxycarbonylmethionyl) derivative: des-PheB1-insulin;des-(PheB1-ValB2)-insulin; des-(PheB1-ValB2-AsnB3)-insulin;des- (PheB1-ValB2-AsnB3-GlnB4)-insulin; des-(PheB1-ValB2-AsnB3 -GlnB4-HisB5)-insulin. 3. Insulin was re-synthesized from the di-protected des-PheB1-insulin by reaction with an active ester of t-butoxycarbonyl-l-phenylalanine. The product after deprotection crystallized, and the immunoreactivity of the crystalline material was identical with that of the native protein. 4. We have prepared the following analogues of insulin in a similar manner: [l-AlaB1]insulin; [l-ValB1]insulin; [l-TyrB1]insulin; [m-F-l-PheB1]insulin; [o-F-l-PheB1]-insulin; [o-F-l-PheB2]des-PheB1-insulin. All had between 34 and 62% of the activity of insulin in the fat-cell test. 5. We have also investigated the use of the benzyol, toluene-p-sulphonyl, p-nitrobenzyloxycarbonyl and 2,4-dinitrophenyl groups for the N-protection of the methionine active esters. Each should have had some particular advantage over the benzyloxycarbonyl and ethoxycarbonyl groups, but all proved in practice to have disadvantages that more than outweighed anything in their favour.
Effect of lipophilic character on the biological activity of synthetic angiotensin peptides
Authors: Nouailhetas V L A , Nakaie C R , Juliano L , Paiva A C M
Published: August 2015

[Ile5]angiotensin II (angiotensin) derivatives bearing acetyl, propionyl, butyryl, pentanoyl or hexanoyl moieties at the N-terminal amino group were synthesized. The myotropic effects in vitro (on guinea-pig ileum and rat uterus) of desamino-angiotensin and of the above compounds did not correlate with their partition coefficients in butan-1-ol/acetic acid/water. The pressor effects in vivo in rats showed a negative correlation with the partition coefficients, discouraging further attempts to raise the pressor potency of angiotensin analogues by increasing their hydrophobicity. The half-times for onset and reversal of the biological responses also did not correlate with partition coefficients, but reversal was retarded by the presence of a free amino group. It is concluded that partition between aqueous medium and the lipophilic receptor environment is not a limiting factor for angiotensin activity.
4,4′-dimethylcholesta-7,9,14-trienol is an intermediate in the demethylation of dihydroagnosterol
Authors: Tavares I A F , Munday K A , Wilton D C
Published: August 2015

1. 4,4′-Dimethylcholesta-7,9,14-trienol is an intermediate in the metabolism of dihydroagnosterol to cholesterol by rat liver homogenate. 2. This triene is reduced by a rat liver microsomal preparation in the presence of NADPH to give 4,4′-dimethylcholesta-7,9-dienol under anaerobic conditions. 3. Reduction of the triene in the presence of [4-3H2]NADPH resulted in the incorporation of 3H into the product. 4. Under aerobic conditions the triene is converted into cholesterol by a rat liver homogenate.
Decreased liver cytochrome P-450 in rats caused by norethindrone or ethynyloestradiol
Authors: White I N H , Muller-Eberhard U
Published: August 2015

1. 19-Nor-17alpha-pregna-1,3,5(10)-trien-20-yne-3,17-diol (ethynyloestradiol) or 17beta-hydroxy-19-nor-17alpha-pregn-4-en-20-yn-3-one (norethindrone) but not 17alpha-ethyl-17beta-hydroxy-19-norandrost-4-en-3-one (norethandrolone) caused a time-dependent loss of cytochrome P-450 when incubated in vitro with rat liver microsomal fractions and NADPH-generating systems. 2. The enzyme system catalysing the norethindrone-mediated loss of cytochrome P-450 had many characteristics of the microsomal mixed-function oxidases. It required NADPH and air, and was inhibited by Co. However, it was unaffected by 1 mM-compound SKF 525A. 3. In microsomal fractions from phenobarbitone-pretreated rats the norethindrone-mediated loss of cytochrome P-450 was increased relative to controls. The norethindrone-mediated cytochrome P-450 loss was less pronounced when the animals were pretreated with 3beta-hydroxy-pregn-5-en-2-one 16alpha-carbonitrile (pregnenolone 16alpha-carbonitrile). Pretreatment with 3-methylcholanthrene rendered the animals resistant to the norethindrone effect. 4. Administration in vivo [100mg/kg, intraperitoneally] of norethindrone or ethinyl oestradiol also produced a time-dependent loss of liver cytochrome P-450. Norethandrolone had a similar, though much less-marked, effect. All three steroids lead to an induction of 5-aminolaevulinate synthase and an accumulation of porphyrins in the liver. 5. The loss of cytochrome P-450 and the accumulation of porphyrins in the liver 2 h after the administration of norethindrone to female rats was similar to that seen in males. 6. Rats pretreated with phenobarbitone and given norethindrone or ethynyloestradiol (100mg/kg, intraperitoneally) formed green pigments in their livers. These had characteristics similar to the green pigments produced in the livers of rats after the administration of 2-allyl-2-isopropylacetamide. No green pigments could be extracted from the livers of control rats or those given norethandrolone, oestradiol or progesterone.
Enzymes metabolizing Δ1-pyrroline-5-carboxylate in rat tissues
Authors: Herzfeld A , Mezl V A , Knox W E
Published: August 2015

The direction and capacity for the metabolism of delta1-pyrroline-5-carboxylate in a number of rat tissues ere investigated by measuring the activities of delta1-pyrroline-5-carboxylate reductase, delta1-pyrroline-5-carboxylate dehydrogenase and proline oxidase. Each of these enzymes catalyzed unidirectional reactions in which delta1-pyrroline-5-carboxylate was either the substrate or product. Delta1-Pyrroline-5-carboxylate reductase activities that were much higher than any previously reported were obtained by avoiding its inactivation in the cold. delta1-Pyrroline-5-carboxylate dehydrogenase, previously said to act on both D- and L-isomers of delta1-pyrroline-5-carboxylate, acted only on the L-isomer. Proline oxidase could not be measured in two adult tissues, in which an inhibitor appeared after birth. The activity of delta1-pyrroline-5-carboxylate reductase significantly paralleled that of ornithine aminotransferase in 23 tissues, showing a widespread potential for proline synthesis from ornithine. An independently distributed potential in fewer tissues for proline degradation to alpha-oxoglutarate was shown by the significantly similar tissue distributions of proline oxidase. Delta1-pyrroline-5-carboxylate dehydrogenase and glutamate dehydrogenase. Reverse metabolism of glutamate or proline to ornithine would be atypical in rat tissues with these distributions of unidirectional enzyme reactions.
Permeability of the mitochondrial membrane to bicarbonate ions
Authors: Selwyn M J , Walker H A
Published: August 2015

Osmotic-swelling techniques show that HCO3- enters mitochondria by an electrogenic process, effectively HCO3- uniport, under non-energized conditions. This mode of translocation accounts for previous reports of non-entry of HCO3- in experiments with energy-linked Ca2+ uptake. The effects of HCO3- on mitochondrial respiration are reported and discussed.
Preferential utilization in vitro of iron bound to diferric transferrin by rabbit reticulocytes
Authors: Zapolski E J , Princiotto J V
Published: August 2015

59Fe uptake by rabbit reticulocytes from human transferrin-bound iron was studied by using transferrin solutions (35, 50, 65, 80 and 100% saturated with iron) whose only common characteristic was their content of diferric transferrin. During the early incubation period, 59Fe uptake from each preparation by reticulocytes was identical despite wide variations in amounts of total transferrin, total iron, monoferric transferrin and apotransferrin in solution. During the later phase of incubation, rate of uptake declined and was proportional to each solution's monoferric transferrin content. Uptake was also studied in a comparative experiment which used two identical, partially saturated transferrin preparations, one uniformly 59Fe-labelled and the other tracer-labelled with [59Fe]diferric transferrin. In both experiments, iron uptake by reticulocytes corresponded to utilization of a ferric ion from diferric transferrin before utilization of iron from monoferric transferrin.
Collagen synthesis in the muscle of developing chick embryos
Authors: Nwagwu M , Reid S A
Published: August 2015

Radioactive protein was prepared from the leg muscle of chick embryos, 11, 14, 16 and 17 days old, each injected with radioactive proline and incubated for 30, 60 or 90 min afterwards. The radioactive protein was incubated with collagenase purified by chromatography on a Sephadex G-100 column. Under this condition, only collagen is digested into products soluble in trichloroacetic acid. The relative rate of collagen synthesis was determined by comparing the amount of radioactivity released into the supernatant fraction and that in the residue, by the method of Diegelmann & Peterkofsky [(1972) Dev. Biol. 28, 443–453]. The results show that the rate of collagen synthesis remains at approx. 10% of the rate of synthesis of other non-collagenous proteins during the development of chick embryonic muscle from 11 to 17 days. This suggests that the synthesis of collagen and that of other proteins are co-ordinately regulated at these stages of development.
The catabolism of intravenously injected heparan N-[35S]sulphate in the rat
Authors: Perry Martin A. , Powell Gillian M. , Wusteman Frederick S. , Curtis C. Gerald
Published: August 2015

The metabolic fate of heparan N-[35S]sulphate was studied in rats. Heparan sulphate was obtained from either bovine aorta or lung and labelled with 35S by desulphation and subsequent resulphation in vitro. Experiments in which heparan N-[35S]sulphate was administered intravenously to either free-range or wholly anaesthetized rats with ureter cannulae established that substantial desulphation occurs in vivo, with elimination of inorganic [35S]sulphate in urine. Oligosaccharides labelled with 35S, possible intermediates in heparan sulphate degradation, could not be detected in urine or blood. The general distribution of radioactivity after administration of heparan N-[35S]sulphate, as demonstrated by whole-body radioautography, suggested that desulphation was not restricted to one organ in particular. Support for this view was obtained in experiments in which heparan N-[35S]sulphate was administered to animals after the removal of kidneys, liver, spleen, pancreas or gastrointestinal tract. In all cases inorganic [35S]sulphate was still produced. The ability of rats of desulphate heparan N-[35S]sulphate was progressively impaired by increasing concentrations of heparin administered simultaneously. It was concluded that heparan sulphate is metabolized at a number of sites in the body by a sequence of degradative events leading to the formation of inorganic sulphate. It is also concluded that at least some of these events are common to heparan sulphate and heparin.
Cortisol induction of pulmonary maturation in the rabbit foetus. Its effects on enzymes related to phospholipid biosynthesis and on marker enzymes for subcellular organelles
Authors: Possmayer Fred , Duwe Gail , Metcalfe Roy , Stewart-DeHaan P. Jill , Wong Ching , Heras Jorge Las , Harding Paul G. R.
Published: August 2015

1. Cortisol treatment of rabbit foetuses in utero at 24 days gestation produced a significant decrease in the lung-weight to body-weight ratio compared with littermate controls by day 26. Histological examination revealed that the alveoli of the treated lungs were more open, the walls were thinner and the osmiophilic bodies were more numerous. 2. Cortisol treatment as described above produced significant increases (P<0.05) in the rates of incorporation of [14C]choline into phosphatidylcholine and of [14C]ethanolamine into phosphatidylethanolamine in vitro compared with littermate controls. This indicates that glucocorticoids produce an overall increase in phospholipid metabolism rather than a specific increase in phosphatidylcholine production. 3. The addition of 1,2-diacyl-sn-glycerols from egg phosphatidylcholine produced a 10-fold increase in the activity of choline phosphotransferase and a 3-fold increase in the activity of ethanolamine phosphotransferase in rabbit lung homogenates. The addition of 1,2-dipalmitoyl-sn-glycerol did not affect these activities. These results demonstrate that in the presence of exogenous 1,2-dipalmitoyl-sn-glycerol, the activities of these enzymes are dependent on the presence of endogenous 1,2-diacylglycerols. 4. Cortisol administration had no significant effect on the activity of choline phosphotransferase or ethanolamine phosphotransferase with endogenous or exogenously added diacylglycerols. The activities of other endoplasmic-reticulum enzymes (sn-glycerol 3-phosphate phosphatidyltransferase, sn-glycerol 3-phosphate acyltransferase and NADPH–cytochrome c reductase) were not significantly altered by the hormone administration. Oestrone sulphate sulphohydrolase activity was significantly decreased (P<0.05) by cortisol injection, but this effect varied with the foetuses from different does. 5. Cortisol administration had no effect on the activities of mitochondrial (monoamine oxidase, succinate dehydrogenase), plasma-membrane (5′-nucleotidase) or lysosomal (acid phosphatase, N-acetyl-β-d-glucosaminidase) enzymes. The activity of membrane-associated phosphatidate phosphohydrolase, an enzyme associated with the osmiophilic granules of the type-II alveolar cells, was increased in the lungs of treated foetuses, but the difference was not significant (0.10>P>0.05).
Effect of buffer composition on binding of adenosine 3′:5′-cyclic monophosphate by bovine adrenal extracts
Authors: Linkola J , Fyhrquist F , Weber T H , Puutula L
Published: August 2015

The binding of cyclic [3H] AMP by bovine adrenal extracts was studied in different buffer systems; 50 mM-Tris/HCl, pH 7.5, buffer containing 4mM-EDTA (disodium salt) offered advantages such as low non-specific interference and high binding affinity. 2-Mercaptoethanol decreased, but EDTA and theophylline increased, the specificity of cyclic [3H]AMP binding as expressed by the displacement of cyclic [3H]AMP by a 1000-fold excess of unlabelled cyclic GMP. The study indicated heterogeneity of the binding material. Mg2+-dependent binding may be eliminated in Tris/EDTA buffer. The heterogeneity and the specificity of intracellular cyclic AMP binders and their dependence in environmental factors should be considered in the evaluation of cyclic AMP binding and second-messenger systems.
The effects of inhibition of fatty acid oxidation in suckling newborn rats
Authors: Pégorier J P , Ferré P , Girard J
Published: August 2015

Inhibition of fatty acid oxidation with pent-4-enoate in suckling newborn rats caused a fall in blood [glucose] and blood [ketone bodies] and inhibition of gluconeogenesis from lactate. Glucose utilization was not increased in newborn rats injected with pent-4-enoate. Active fatty acid oxidation appears to be essential to support gluconeogenesis and to maintain normal blood [glucose] in suckling newborn rats.
Effect of lutropin on phosphorylation of endogenous proteins in testis Leydig cells. Correlation with testosterone production
Authors: Cooke Brian A. , Lindh Monica L. , Janszen Felix H. A.
Published: August 2015

The effect of lutropin on phosphorylation of endogenous proteins in testis Leydig cells was investigated, by incubating purified Leydig cells with lutropin and [32P]Pi followed by sodium dodecyl sulphate/polyacrylamide-slab gel electrophoresis of the [32P]phosphoproteins. The radioactivity of the proteins was quantified by densitometry of the radio-autograms obtained. The following results were obtained. 1. Lutropin increased the amount of32 P incorporated into three proteins (A, B and C) with apparent mol.wts. of 14300, 57000 and 77600 respectively. 2. The increase in incorporation of32P into these proteins was detectable within 5min, reaching a maximum in approx. 20min. 3. The32P incorporated into protein B (but not proteins A and C) was significantly increased with 0.1 and 1.0ng of lutropin/ml. Incorporation of32P into all three proteins was significantly increased with 10ng of lutropin/ml, reaching a maximum with 100ng/ml. 4. Testosterone production was significantly increased with 1ng of lutropin/ml, and between 10 and 1000ng/ml the degree of stimulation of testosterone production and incorporation of32P into proteins A, B and C was similar. 5. Cyclic AMP production was significantly increased with 10ng of lutropin/ml and had not reached a maximum with 1000ng/ml. 6. In Leydig cells isolated from hypophysectomized rats 3h after injection of choriogonadotropin in vivo, phosphoproteins with the same molecular weights as proteins A, B and C were found. No further increases in incorporation of32P into these proteins were obtained when lutropin was added to the Leydig cells in vitro. 7. Dibutyryl cyclic AMP (but not follitropin or testosterone) also stimulated the incorporation of32P into proteins A, B and C in Leydig cells.
Accelerated hepatic haem catabolism in the selenium-deficient rat
Authors: Burk R F , Correia M A
Published: August 2015

1. Hepatic microsomal cytochrome P-450 concentrations are lower in selenium-deficient rats treated with phenobarbital for 4 days than in similarly treated control rats. 2. No defect in haem synthesis was found on the basis of measurements of delta-aminolaevulinate synthase (EC 2.3.1.37), delta-aminolaevulinate dehydratase (EC 4.2.1.24) and ferrochelatase (EC 4.99.1.1) activities, and urinary excretion of delta-aminolaevulinate, porphobilinogen, uroporphyrin and coproporphyrin. 3. No defect in apo-(cytochrome P-450) separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 4. An increase in haem catabolism was found. An 8-fold increase in hepatic microsomal haem oxygenase (EC 1.14.99.3) activity occurred in selenium-deficient rats after phenobarbital treatment, compared with a less than 2-fold increase in control rats. Also excretion of 14CO in the breath after administration of delta-amino[5-14C]laevulinate was greater by phenobarbital-treated selenium-deficient rats than by similarly treated controls. 5. These studies demonstrate that the defective induction of cytochrome P-450 by phenobarbital in selenium-deficient rats is accompanied by increased haem catabolism. This could be due to increased breakdown of cytochrome P-450 or to catabolism of haem before it attaches to the apo-cytochrome. The role of selenium in stabilizing cytochrome P-450 and/or in protecting haem from breakdown remains to be determined.
Stimulation of steroid glucuronidation by uridine diphosphate N-acetylglucosamine and by microsomal disruption
Authors: Wilkinson J , Hallinan T
Published: August 2015

Glucuronidation of oestrone and of oestradiol in microsomal fractions was markedly and significantly stimulated by UDP-N-acetylglucosamine and by ultrasonication: Triton X-100 also stimulated. This is consistent with compartmentation of UDP-glucuronyl-transferase. Stimulation by UDP-N-acetylglucosamine may be physiologically significant.
Immunological evaluation of repeated administration of erythrocyte-entrapped protein to C3H/HeJ mice
Authors: Fiddler M B , Hudson L D S , Desnick R J
Published: August 2015

The immunogenicity of heterologous protein entrapped in autologous erythrocytes was evaluated in a mouse system. Multiple intravenous administrations of an erythrocyte-entrapped bovine beta-glucuronidase preparation resulted in little, if any, immunological response to the entrapped protein or erythrocyte carrier. Erythrocyte entrapment also protected protein from circulating antibody elicited by prior subcutaneous sensitization with unentrapped enzyme in adjuvant. Thus erythrocyte entrapment may protect potentially immunogenic agents from immunological surveillance.
Enhancement of the anti-anabolic response to adenosine 3′:5′-cyclic monophosphate during development. The inhibition of hepatic protein synthesis
Authors: Klaipongpan A , Bloxham D P , Akhtar M
Published: August 2015

Cyclic AMP causes an age-dependent inhibition of protein synthesis in rat liver. The onset of inhibition is about 10—12 days after birth. The developmental response to cyclic AMP is associated with a change in the microsomal component of the protein-synthesizing system.
The biosynthesis of brain gangliosides. Separation of membranes with different ratios of ganglioside sialylating activity to gangliosides
Authors: Landa C A , Maccioni H J F , Arce A , Caputto R
Published: August 2015

Brain subcellular fractions were analysed for ganglioside-sialylating activity by measuring the incorporation of N-[3H]acetylneuraminic acid from CMP-N-[3H]acetylneuraminic acid into endogenous ganglioside acceptors (endogenous incorporation) and into exogenous lactosyceramide (haematoside synthetase activity). The ratios of endogenous incorporation to gangliosides and of haematoside synthetase to gangliosides for the synaptosomal and mitochondrial fractions from a washed crude mitochondrial fraction were lower than those obtained for other membrane fractions. The differences appear to reflect intrinsic characteristics of each membrane fraction. The results of labelling in vitro and the time course of labelling of gangliosides of the different subcellular fractions in vivo after injection of N-[3H]acetylmannosamine are consistent with the possibility of a subcellular site for synthesis of gangliosides different from that of ganglioside deposition.
Demonstration of the phosphorylation of acetyl-coenzyme A carboxylase within intact rat epididymal fat-cells
Authors: Brownsey R W , Hughes W A , Denton R M
Published: August 2015

Intact rat epididymal fat-cells were incubated with 32Pi and the intracellular proteins separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. One of the phosphorylated proteins has the same RF value as [14C]biotin-labelled acetyl-CoA carboxylase purified from fat-cells and is specifically precipitated after incubation with antiserum raised against acetyl-CoA carboxylase. No significant changes in the extent of phosphorylation of acetyl-CoA carboxylase were detected after exposure of the cells to insulin.
Affinity chromatography of thiol-containing purines and ribonucleic acid
Authors: Melvin W T , Keir H M
Published: August 2015

Evidence is presented for the specific interaction of 6-mercaptopurine with mercurated cellulose. Following from this, a new method is described for the affinity chromatography of thiol-containing molecules and of RNA containing incorporated 6-thioguanosine on columns of mercurated cellulose. This technique may find application in the study of RNA metabolism and gene expression.
Lysosomes and membrane recycling. A hypothesis
Authors: Dean R T
Published: August 2015

A mechanism for intralysosomal membrane recycling is proposed. After invagination of the lysosomal membrane during autophagy, intralysosomal vesicles are formed. It is suggested that membrane can bleb out from these internal vesicles, probably in the form of very small vesicles, and return to the external lysosomal membrane by membrane fusion. This mechanism would conserve lysosomal membrane during autophay, and is analogous to current models of plasma-membrane recycling. Its relationship to turnover of lysosomal-membrane proteins and other proteins is discussed.
Use of flux ratio measurements for the determination of the order of addition of substrates and products in enzyme reactions
Authors: Britton H G , Dann L G
Published: August 2015

1. Methods of determining the order of addition of substrates and dissociation of products by using flux ratios are investigated. Where an enzyme obeys hyperbolic steady-state velocity kinetics it is concluded that it may be particularly useful to compare the measured flux ratios with those calculated from the steady-state velocity parameters. 2. An expression is derived relating the relative contribution of the two pathways in a branched pathway to the flux ratios. 3. The relationship of equilibrium-reaction-rate measurements [Boyer & Silverstein (1963) Acta Chem. Scand. 17, Suppl. 1, S195] to the flux ratios is considered. Equilibrium-reaction rates are shown to be affected both by the addition of substrates and dissociation of products. Methods of analysing the data to distinguish between these events are discussed. 4. Methods of measurement of flux ratios are described, and it is concluded that the non-equilibrium steady-state method is preferable to measurements at chemical equilibrium. 5. The relative significance of flux ratio measurements and steady-state velocity inhibition data is discussed. It is concluded that flux ratios, when taken in conjunction with the inhibition data, provide the least ambiguous information about mechanism.
Localization of the ribonucleotide sites in rat liver mitochondrial deoxyribonucleic acid
Authors: Lonsdale D M , Jones I G
Published: August 2015

Supercoiled rat liver mitochondrial DNA is relaxed by treatment with ribonucleases A, T1 or H. All the supercoiled mitochondrial DNA is sensitive to ribonuclease H and ribonuclease A, but only 35% of the supercoiled population is sensitive to ribonuclease T1. Removal of the ribonucleotides with calf thymus ribonuclease H, followed by denaturation of the mitochondrial DNA and analysis of the single-strand fragment lengths in the electron microscope, showed that the ribonucleotides were randomly located on both strands of the DNA. Endonuclease-S1 digestion of mitochondrial DNA after removal of the ribonucleotides reveals that no unique fragments are produced and ribonucleotides are randomly distributed with respect to one another. The average number of ribonucleotide sites per molecule was estimated to be between 8 and 13. Two possible mechanisms for the origin of ribonucleotide sites are discussed.
Chemical and enzymic studies on the characterization of intermediates during the removal of the 14α-methyl group in cholesterol biosynthesis. The use of 32-functionalized lanostane derivatives
Authors: Akhtar M , Alexander K , Boar R B , McGhie J F , Barton D H R
Published: August 2015

By using cell-free preparations of rat liver it was shown that the removal of the 14alpha-methyl group (C-32) of steroids containing either a delta7(8) or a delta8(9) double bond is attended exclusively by the formation of the corresponding 7,14- and 8,14-dienes respectively (structures of types III and VIII). Cumulative evidence from a variety of experimental approaches had led to the deduction that delta8(14)-steroids are not involved as intermediates on the major pathway of cholesterol biosynthesis. The metabolism of [32-3H]lanost-7-ene-3beta,32-diol (structure of type I) results in the formation of radioactive formic acid, no labelled formaldehyde being formed. By using appropriately labelled species of the compound (I) it was found that the release of formic acid and the formation of 4,4-dimethylcholesta-7,14-dien-3beta-ol (strurcture of type III) were closely linked processes, and that in the conversion of compound (I) into compound (III), 3-beta-hydroxylanost-7-en-32-al (II) is an obligatory intermediate. Both the conversion of lanost-7-ene-3beta,32-diol (I) into 3beta-hydroxylanost-7-en-32-al (II) and the further metabolism of the latter (II) to 4,4-dimethylcholesta-7,14-dien-3beta-ol (III) exhibited a requirement for NADPH and O2. This suggests that the oxidation of the 32-hydroxy group of compound (I) to the aldehyde group of compound (II) does not occur by the conventional alcohol dehydrogenase type of reaction, but may proceed by a novel mechanism involving the intermediacy of a gem-diol. A detailed overall pathway for the 14alpha-demethylation in cholesterol biosynthesis is considered, and proposals about the mechanism of individual steps in the pathway are made.
The dye-linked alcohol dehydrogenase of Rhodopseudomonas acidophila. Comparison with dye-linked methanol dehydrogenases
Authors: Bamforth C W , Quayle J R
Published: August 2015

1. A dye-linked alcohol dehydrogenase was purified 20-fold from extracts of Rhodopseudomonas acidophila 10050 grown anaerobically in the light on methanol/HCO3-. 2. The enzyme resembled many previously reported methanol dehydrogenases from other methylotrophic organisms in coupling to phenazine methosulphate, requiring ammonia as an activator, possessing a pH optimum of 9 and a mol.wt. of approx. 116000. In many other respects the enzyme showed singular properties. 3. The stability of the enzyme under various conditions of temperature and pH was studied. 4. Primary aliphatic amines containing up to nine carbon atoms (the longest tested) were better activators than ammonia. 5. A wide range of primary alcohols and aldehydes served as substrates, with apparent Km values ranging from 57 mM for methanol to 6 micron for ethanol. 6. O2 was an inhibitor competitive with respect to the alcohol substrate. In the presence of O2, apparent Km values of 145 mM were recorded for methanol. 6. Cyanide and alphaalpha'-bipyridine were inhibitors competitive with respect to the amine activator. 7. The properties of the enzyme from Rhodopseudomonas acidophila are compared with those of similar enzymes from other organisms, and implications of the salient differences are discussed.
Comparison of guinea-pig serum lipoproteins after iodination by two different methods
Authors: Weech P K , McTaggart F , Mills G L
Published: August 2015

1. Guinea-pig low-density lipoproteins were isolated by ultracentrifugation and iodinated either by the IC1 method or by the chloramine-T procedure. 2. The efficiency of labelling by both methods was essentially the same. 3. When the two products were compared by ultracentrifugation, gel chromatography and immunodiffusion analysis, no significant difference in their properties was detected. 4. When they were compared by gradient-gel electrophoresis, aggregates were found in the product of the IC1 method, but not in the lipoprotein iodinated by the chloramine-T process. 5. Both products were metabolized by the guinea pig with essentially the same fractional catabolic rate. 6. The fractional catabolic rate of lipoprotein iodinated by the chloramine-T method was not significantly different from that of lipoprotein biologically labelled in the protein moiety with [75Se]selenomethionine. 7. It is concluded that the products of both methods of iodination are almost equally acceptable, provided that the optimum conditions for the chloramine-T reaction are carefully established.
Regulation of the synthesis of lutropin-induced protein in rat testis Leydig cells
Authors: Janszen Felix H. A. , Cooke Brian A. , Van Driel Maria J. A. , Van Der Molen Henk J.
Published: August 2015

The mechanism of action of lutropin on the stimulation of the synthesis of a specific lutropin-induced protein in rat testis Leydig cells was investigated. Lutropin-induced protein has a mol.wt. of approx. 21000 and is detected by labelling the Leydig-cell proteins with [35S]methionine, followed by separation by polyacrylamide-gel electrophoresis and radioautography of the dried gel. The incorporation of35S into lutropin-induced protein was used as an estimate for the synthesis of the protein. Incubation of Leydig cells with dibutyryl cyclic AMP or cholera toxin also resulted in the stimulation of synthesis of the protein. Synthesis of lutropin-induced protein, when maximally stimulated with 100ng of lutropin/ml, could not be stimulated further by addition of dibutyryl cyclic AMP. Addition of 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, further increased synthesis of the protein in the presence of a submaximal dose of lutropin (10ng/ml) but not in the absence of lutropin or with maximal amounts of lutropin (100 and 1000ng/ml). Actinomycin D prevented the effect of lutropin on the stimulation of lutropin-induced protein synthesis when added immediately or 1h after the start of the incubation, but not when added after 5–6h. This is interpreted as reflecting that, after induction of mRNA coding for lutropin-induced protein, lutropin had no influence on the synthesis of the protein in the presence of actinomycin D. Synthesis of the protein was also stimulated in vivo by injection of choriogonadotropin into rats 1 day after hypophysectomy, and the time course of this stimulation of lutropin-induced protein synthesis in vivo was similar to that obtained by incubating Leydig cells in vitro with lutropin. From these results it is concluded that stimulation of lutropin-induced protein synthesis by lutropin is most probably mediated by cyclic AMP and involves synthesis of mRNA.
Glycerolipid biosynthesis in rat adipose tissue. Influence of adipose-cell size and site of adipose tissue on triacylglycerol formation in lean and obese rats
Authors: Jamdar S C
Published: August 2015

The rates of lipid formation were compared in different fat-depots from lean and obese rats by using [14C]glycerol 3-phosphate, [14C]glucose or [14C]acetate as substrates. In lean animals, subcutaneous adipose tissue showed significantly lower rates of lipid synthesis than did perirenal and gonadal fat-tissue. In obese animals, the rates of lipid synthesis were significantly higher and did not vary from one fat-depot to another. Differences in the rates of lipid formation between lean and obese rats disappeared during dietary restriction of obese animals. The isolated adipocyte preparation did not reflect the true metabolic activity of the adipose organ, since this preparation was mainly derived from smaller adipocytes that were metabolically less active than larger adipocytes. The present study suggests that it is better to use whole tissue preparations to measure lipogenesis and esterification reactions, because these measurements represent the contribution of both larger and smaller adipocytes towards lipid formation.
Detection and separation of lymphocytes with specific surface receptors, by using microparticles
Authors: Ljungsted I , Ekman B , Sjöholm I
Published: August 2015

Horse anti-(human lymphocyte) globulin was immobilized together with fluorescein labelled dextran in spherical microparticles of polyacrylamide (AHLG-particles). The particles had a diameter of 1-5 micrometer and a density of 1.12g/cm3, with globulin exposed on the surface. Human lymphocytes bearing the antigen (thymus-derived lymphocytes) bound the particles, which were easily detected by fluorescence microscopy. In this way, about 58% of circulating human lymphocytes were able to bind AHLG-particles at 23 degrees C. Non-specific binding was low, only 3% when human serum albumin was present in the buffer, and only 4% when non-specific horse globulins were incorporated in the microparticles. The cell-particle complexes could be separated from cells that had not reacted by density-gradient centrifugation in Ficoll/metrizoate. The viability was not changed after the separation procedure. The number of cells binding AHLG-particles corresponded well the the relative amount of T-cells. When the cells binding AHLG-particles were separated from the lymphocytes, the number of T-cells decreased remarkably, indicating that the antibodies bind preferably to the T-cell population. Concanavalin A immobilized in microparticles was sufficiently exposed to initiate the agglutination of the lymphocytes. The agglutination was completely inhibited by preincubating the microparticles with alpha-methyl mannoside.
Oestrogen induction of riboflavin-binding protein in immature chicks. Nature of the secretory protein
Authors: Murthy U S , Adiga P R
Published: August 2015

The riboflavin-binding protein isolated from sera of oestrogen-treated male chicks as well as that synthesized and secreted in vitro by the chicken liver have the same molecular size as that of the egg-yolk protein. Functionally the serum and yolk proteins are similar. This is in contrast with the hormone-induced synthesis, secretion and deposition of phosvitin and lipovitellin in the ovary.
Calcium transport and proton electrochemical potential gradient in mitochondria from guinea-pig cerebral cortex and rat heart
Authors: Nicholls David G.
Published: August 2015

A method is described for the preparation of ‘free’ and ‘synaptosomal’ brain mitochondria from fractions of guinea-pig cerebral cortex respectively depleted and enriched in synaptosomes. Both preparations of mitochondria have a low membrane H+conductance, a high capacity to phosphorylate ADP, and a capacity to accumulate Ca2+ at rates limited by the activity of the respiratory chain. Ca2+ transport by ‘free’ brain mitochondria is compared with that of heart mitochondria. The Ca2+ conductance of ‘free’ brain mitochondria was at least 20 times that for rat heart mitochondria. Ca2+ uptake by brain mitochondria increased the pH gradient and decreased membrane potential, whereas little change occurred during the much slower uptake by heart mitochondria. In the presence of ionophore A23187, dissipative Ca2+ cycling decreased the H+ electrochemical potential gradient of brain mitochondria from 190 to 60mV, but caused only a slight decrease with heart mitochondria, although the ionophore lowered the pH gradient and increased membrane potential. The Ca2+ conductance of ‘free’ brain mitochondria is distinctive in showing a hyperbolic dependency on free Ca2+concentration. In the presence of Ruthenium Red, a rapid Na+ -dependent Ca2+ efflux occurs. The H+ electrochemical potential gradient is maintained during this efflux, and membrane potential increases, with both ‘free’ brain and heart mitochondria. The Na+ requirement for Ca2+ efflux appears not to be related to the high Na+/H+ exchange activity but may represent a direct exchange of Na+ for Ca2+.