bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthesis of haem cytochrome c prosthetic group from δ-aminolaevulinate by the cell sap from rat liver
Authors: De Córdova Carmen Sáez , Cohén Regina , González-Cadavid Néstor F.
Published: August 2015

To determine whether the prosthetic group of cytochrome c is synthesized and linked to the apoprotein in the cytosol or in connexion with the endoplasmic reticulum, we have studied the incorporation in vitro of δ-amino[14C]laevulinate into porphyrin compounds and cytochrome c by the cell sap from rat liver. The radioactive precursor was incorporated into a trichloroacetic acid-precipitable form partially resistant to extractions by acid solvents, suggesting the existence of a fraction covalently linked to protein. The activity was proportional to the amount of protein incubated, did not increase substantially by supplementation with the microsomal fraction and an energy source, and was very low in the pH5 fraction. Addition of increasing amounts of haemin inhibited the incorporation, as with purified δ-aminolaevulinate dehydratase. [14C]Protoporphyrin IX was identified by paper chromatography, together with a shoulder running as protohaem IX. The cell sap in the absence of ribosomes was also able to incorporate radioactivity into purified cytochrome c, and the addition of ribosomes significantly enhanced the activity. The precursors of haem c were synthesized in the soluble system by the known haem-synthetic pathway, as shown by the kinetics of labelling of the coproporphyrin, protoporphyrin and haem fractions, and the activities were concentrated in the precipitate obtained between 40 and 60% saturation with (NH4)2SO4. The presence of ferrochelatase was indicated by the incorporation of55Fe into proto- and haemato-haem identified by paper chromatography. It is concluded that the cell sap from rat liver contains the complete set of enzymes for the synthesis from δ-aminolaevulinate of haem c and its linkage to a small pool of free apoprotein c present in soluble form. This suggests that an ancillary pathway of haem synthesis occurs in the cytosol for at least the formation of the prosthetic group, which is linked post-translationally to that pool of apoprotein c synthesized by free polyribosomes.
The association of the sulphogalactosylglycerolipid of rat brain with myelination
Authors: Pieringer Joanne , Rao G. Subba , Mandel Paul , Pieringer Ronald A.
Published: August 2015

The sulphogalactosylglycerolipid of rat brain is closely associated with the process of myelination, as demonstrated by the following observations. 1. The lipid is barely detectable in rat brain before 10 days of age, accumulates rapidly between age 10 and 25 days, and remains relatively constant in amount (between 0.3 and 0.4μmol per brain) thereafter into adult life. 2. The activity of adenosine 3′-phosphate 5′-sulphatophosphate–galactosyldiacylglycerol sulphotransferase is almost absent before 10 days of age, attains a maximum at age 20 days, and slowly decreases thereafter with increasing age. This developmental pattern correlates well with that of other myelin-specific metabolites. 3. Both the concentration of the sulphogalactosylglycerolipid and the activity of sulphotransferase are greatly decreased in the non-myelinating jimpy mouse. 4. The myelin fraction of rat brain contains most of the sulphogalactosylglycerolipid. The lipid occurs in a diacyl and an alkylacyl form. Determinations of the relative amount of each type in brain showed about a 1:1 mixture in both 21-day-old and adult rats. Rats injected with H235SO4 at 20 days of age lost35S from the diacyl form at a higher rate than from the alkylacyl compound over a 21-day period. These data suggest that the diacyl form has a higher turnover than the alkylacyl derivative. The percentage of the total sulpholipid content of brain contributed by the sulphogalactosylglycerolipid is 16% in 21-day-old rats and 8.4% in adult rats.
Alterations in regulation of insulin biosynthesis in pregnancy and starvation studied in isolated rat islets of Langerhans
Authors: Bone Adrian J. , Howell Simon L.
Published: August 2015

1. Insulin biosynthesis in isolated rat islets of Langerhans was determined by the incorporation of [3H]leucine into newly synthesized islet proteins. Anti-insulin serum covalently coupled to a solid phase (CNBr-activated Sepharose 4B) was used to separate the immunoreactive proinsulin and insulin from other islet proteins. This method was applied to a study of the regulation of insulin biosynthesis in isolated rat islets of Langerhans during pregnancy, and immediately after a period of food deprivation. 2. Islets isolated from pregnant rats showed an increased basal rate of synthesis compared with the non-pregnant controls. In addition, they showed a significant increase in biosynthesis of proinsulin and insulin in comparison with the normal islets over a range of glucose concentrations of 2–20mm. 3. Addition of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine significantly increased the insulin-synthetic response of normal islets over the glucose range 5–20mm, so that their glucose response approached that of islets from pregnant rats. 4. Normal female rates were injected with a long-acting progesterone derivative (hydroxyprogesterone hexanoate), to investigate the role of progesterone on the increased insulin biosynthesis observed in islets in pregnancy. There appeared to be no marked difference in insulin biosynthesis between the islets from the progesterone-injected and control rats in the presence of 2mm- or 6mm-glucose alone. However, in the presence of 4mm- or 6mm-glucose and 3-isobutyl-1-methylxanthine there was a significant increase in insulin biosynthesis in the progesterone-treated animals. 5. Total islet protein biosynthesis was determined by the incorporation of [3H]leucine into trichloroacetic acid-precipitable islet proteins. Islets isolated from normal rats showed a 1.6-fold increase in incorporation over the glucose concentration range 2–20mm, and this value remained unchanged during starvation; however, rates of incorporation were significantly raised in islets isolated from pregnant rats in the presence of 20mm-glucose. 6. Islets from starved and fed control rats were incubated in the presence of increasing concentrations of glucose or glucose+3-isobutyl-1-methylxanthine. The islets isolated from the starved animals showed a diminished insulin-synthetic response to glucose as compared with the controls; this response was partially restored to normal values by elevation of cyclic AMP concentrations by using 3-isobutyl-1-methylxanthine. 7. It is suggested that the alterations in glucose-stimulated insulin biosynthesis observed in islets during pregnancy and after a period of starvation could be attributable, at least in part, to a long-term alteration of the cyclic AMP system, and in pregnancy to a direct or indirect effect of progesterone on β-cell function.
The effect of calcium on lead absorption in rats
Authors: Meredith P A , Moore M R , Goldberg A
Published: August 2015

The effects of Ca2+ on lead absorption as PbCl2 and 203PbCl2 were studied in rats. 1. Doubling of dietary calcium with Ca3(PO4)2 significantly decreased lead absorption as assessed by 203Pb retention, tissue lead concentration, urinary excretion of delta-aminolaevulinate and increased activities of delta-aminolaevulinate dehydratase and ferrochelatase. 2. Similar effects on lead absorption were shown by the Ca2+ salts, Cl-, CO32-, PO43-, SO42-, gluconate and glycerophosphate. 3. CaCl2 and calcium glycerophosphate were found to be most effective in decreasing lead absorption when administered immediately before lead dosage. 4. A negative exponential relationship was found between CaCl2 concentration and 203Pb absorption at 120h. The result suggest that, above 4 mmol of administered calcium, residual lead absorption is unaffected by increasing gastrointestinal calcium concentrations. 5. Increased systemic calcium had no effect on lead retention. 6. Calcium in the concentrations found in domestic hard-water supplies significantly decreased absorption of a solution of 203Pb dissolved in it compared with absorption of 203Pb dissolved in soft or distilled water. 7. Milk and skimmed milk were found to have no effect on 203Pb absorption in rats.
Ribonucleic acid synthesis in vitro in primary spermatocytes isolated from rat testis
Authors: Grootegoed J. Anton , Grollé-Hey Anne H. , Rommerts Focko F. G. , Van Der Molen Henk J.
Published: August 2015

The incorporation of [3H]uridine into RNA was studied quantitatively (by incorporation of [3H]uridine into acid-precipitable material) and qualitatively (by phenol extraction and electrophoretic separation of RNA in polyacrylamide gels) in preparations enriched in primary spermatocytes, obtained from testes of rats 26 or 32 days old. The rate of incorporation of [3H]uridine into RNA of isolated spermatocytes was constant during the first 8h of incubation, after which it decreased, but the decreased rate of incorporation was not reflected in a marked change in electrophoretic profiles of labelled RNA. In isolated spermatocytes, [3H]uridine was incorporated mainly into heterogeneous RNA with a low electrophoretic mobility. Most of this RNA was labile, as shown when further RNA synthesis was inhibited with actinomycin D. Spermatocytes in vivo also synthesized heterogeneous RNA with a low electrophoretic mobility. A low rate of incorporation of [3H]uridine into rRNA of isolated spermatocytes was observed. The cleavage of 32S precursor rRNA to 28S rRNA was probably retarded in spermatocytes in vitro as well as in vivo. RNA synthesis by preparations enriched in early spermatids or Sertoli cells was qualitatatively different from RNA synthesis by the spermatocyte preparations. It is concluded that isolated primary spermatocytes maintain a specific pattern of RNA synthesis, which resembles RNA synthesis in spermatocytes in vivo. Therefore isolated spermatocytes of the rat can be used for studying the possible regulation of RNA synthesis during the meiotic prophase.
Ornithine decarboxylase and polyamines in liver and kidneys of rats on cyclical regimen of protein-free and protein-containing diets. Relationship to deoxyribonucleic acid synthesis in liver
Authors: Farwell Donald C. , Miguez Jose B. , Herbst Edward J.
Published: August 2015

1. The activity of ornithine decarboxylase in the liver and kidneys of rats maintained on a cyclical regimen of protein-free and protein-containing diets was investigated. There was a daily activation of the enzyme in response to the feeding of protein after 3 days feeding of protein-free diet. 2. The activation of ornithine decarboxylase in the liver and kidneys of rats re-fed on protein was demonstrable throughout 16 cycles of alternating 3-day periods of protein-free and protein-containing diets. The magnitude of the activation in the kidneys diminished from 20-fold stimulation in the first cycle to 5-fold stimulation (compared with animals fed with protein-free diet) in the later cycles of protein re-feeding. The activation of the enzyme in liver was decreased from 20-fold stimulation in the first cycle to approx. 10-fold stimulation in later cycles. 3. The concentration of spermidine was increased by approx. 50% in the liver of animals during cycling from protein-free to protein-containing diets. Spermine was unchanged, and putrescine was maintained at a low concentration approx. one-fifth to one-tenth that of spermidine after protein re-feeding. 4. The incorporation of [3H]thymidine into liver DNA was increased 10-fold in animals re-fed with protein compared with animals receiving protein-free diets. 5. The activation of ornithine decarboxylase by re-feeding of protein was inhibited 90% by the injection of propane-1,3-diamine during re-feeding. The stimulation of DNA synthesis was inhibited 60% by multiple injections of propane-1,3-diamine during the re-feeding of protein.
Biosynthesis and release of glycoproteins by human skin fibroblasts in culture
Authors: Sear Christopher H. J. , Grant Michael E. , Jackson David S.
Published: August 2015

1. Confluent human skin fibroblasts maintained in a chemically defined medium incorporate l-[1-3H]fucose in a linear manner with time into non-diffusible macromolecules for up to 48h. Chromatographic analysis demonstrated that virtually all the macromolecule-associated3H was present as [3H]fucose. 2. Equilibrium CsCl-density-gradient centrifugation established that [3H]fucose-labelled macromolecules released into the medium were predominantly glycoproteins. Confirmation of this finding was provided by molecular-size analyses of the [3H]fucose-labelled material before and after trypsin digestion. 3. The [3H]fucose-labelled glycoproteins released into fibroblast culture medium were analysed by gel-filtration chromatography and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. These techniques demonstrated that the major fucosylated glycoprotein had an apparent mol.wt. of 230000–250000; several minor labelled species were also detected. 4. Dual-labelling experiments with [3H]fucose and14C-labelled amino acids indicated that the major fucosylated glycoprotein was synthesized de novo by cultured fibroblasts. The non-collagenous nature of this glycoprotein was established by three independent methods. 5. Gel-filtration analysis before and after reduction with dithiothreitol showed that the major glycoprotein occurs as a disulphide-bonded dimer when analysed under denaturing conditions. Further experiments demonstrated that this glycoprotein was the predominant labelled species released into the medium when fibroblasts were incubated with [35S]cysteine. 6. The relationship between the major fucosylated glycoprotein and a glycoprotein, or group of glycoproteins, variously known as fibronectin, LETS protein, cell-surface protein etc., is discussed.
The respiratory chain of a newly isolated Methylomonas Pl1
Authors: Drabikowska A K
Published: August 2015

1. Whole cells of Methylomonas Pl1 contained ubiquinone, identified as ubiquinone-8. No naphthaquinone was detected. Ubiquinone was located predominantly in the particulate fraction, which also contained most of the NADH oxidase activity. 2. Aerobic incubation of cells with formaldehyde or methanol resulted in about 20% reduction of ubiquinone, irrespective of the presence or absence of dinitrophenol. On inhibition of the respiration by cyanide, ubiquinone became partly reduced by endogenous substrates (15—25%), and a further reduction occurred only in the presence of formaldehyde (up to 60%). When endogenous substrates were completely exhausted, then 44 and 23% of ubiquinone was reduced by formaldehyde or methanol respectively. 3. The difference spectra at room and liquid-N2 temperatures revealed the presence of cytochrome b and two cytochromes c (c-552.5 and c-549) all tightly bound to the membrane. Cytochrome c-552.5 was also found in the soluble fraction. 4. Redox changes of cytochromes b and c, with methanol or formaldehyde as substrates, respond to the aerobic and anaerobic states of the cell and to KCN inhibition in a manner characteristic of the electron carriers of the respiratory chain. 5. The merging point for electron transport from NADH dehydrogenase and formaldehyde dehydrogenase is suggested to be at the level of ubiquinone.
Midpoint redox potentials of plant and algal ferredoxins
Authors: Cammack R , Rao K K , Bargeron C P , Hutson K G , Andrew P W , Rogers L J
Published: August 2015

Midpoint potentials of plant-type ferredoxins from a range of sources were measured by redox titrations combined with electron-paramagnetic-resonance spectroscopy. For ferredoxins from higher plants, green algae and most red algae, the midpoint potentials (at pH 8.0) were between —390 and —425 mV. Values for the major ferredoxin fractions from blue-green algae were less negative (between —325 and —390 mV). In addition, Spirulina maxima and Nostoc strain MAC contain second minor ferredoxin components with a different potential, —305 mV (the highest so far measured for a plant-algal ferrodoxin) for Spirulina ferrodoxin II, and —455 mV (the lowest so far measured for a plant-algal ferredoxin) for Nostoc strain MAC ferredoxin II. However, two ferredoxins extracted from a variety of the higher plant Pisum sativum (pea) had midpoint potentials that were only slightly different from each other. These values are discussed in terms of possible roles for the ferredoxins in addition to their involvement in photosynthetic electron transport.
Incorporation of d-glucose-, l-alanine- and phosphate-transport systems from rat renal brush-border membranes into liposomes
Authors: Kinne R , Faust R G
Published: August 2015

An extract of soluble proteins was prepared from a rat kidney brush-border membranes by Triton X-100 solubilization followed by centrifugation for 1 h at 100000g. Its protein composition was markedly different from that of the brush-border membranes. Proteoliposomes were formed by co-sonication of the Triton X-100-free extract with a naturally occurring mixture of phospholipids extracted from rat kidney. These proteoliposomes were shown to contain Na+-stimulated D-glucose-, L-alanine- and phosphate-transport systems.
Biliary excretion of some anionic derivatives of diethylstilboestrol and phenolphthalein in the guinea pig
Authors: Barford P A , Olavesen A H , Curtis C G , Powell G M
Published: August 2015

The metabolic fates and modes of excretion of diethylstilboestrol mono[35S]sulphate and diethylstilboestrol di[35S]sulphate were studied in the guinea pig. Comparative studies were also made with [G-3H]diethylstilboestrol and phenolphthalein di[35S]sulphate. Diethylstiboesterol di[35S]sulphate was extensively eliminated in the bile unchanged. After administration of diethylstilboestrol mono[35S]sulphate, extensive biliary elimination of radioactivity was also recorded. Radioactive components were identified as diethylstilboestrol disulphate, diethylstilboestrol monosulphate monoglucuronide and unchanged diethylstilboestrol monosulphate. When [G-3H]diethylstilboestrol was administered, 3H-labelled diethylstilboestrol monoglucuronide, diethylstilboestrol monosulphate monoglucuronide and diethylstilboestrol disulphate appeared in the bile. Phenolphthalein di[35S]sulphate was excreted unchanged in bile. These findings are discussed in relation to studies carried out in the rat [Barford, Olavesen, Curtis & Powell (1977) Biochem. J. 164, 423–430] and species differences are related to differences in enzyme activities in rat and guinea-pig liver.
The pathway of glutamate metabolism in rat brain mitochondria
Authors: Dennis Steven C. , Clark John B.
Published: August 2015

1. The pathway of glutamate metabolism in non-synaptic rat brain mitochondria was investigated by measuring glutamate, aspartate and ammonia concentrations and oxygen uptakes in mitochondria metabolizing glutamate or glutamine under various conditions. 2. Brain mitochondria metabolizing 10mm-glutamate in the absence of malate produce aspartate at 15nmol/min per mg of protein, but no detectable ammonia. If amino-oxyacetate is added, the aspartate production is decreased by 80% and ammonia production is now observed at a rate of 6.3nmol/min per mg of protein. 3. Brain mitochondria metabolizing glutamate at various concentrations (0–10mm) in the presence of 2.5mm-malate produce aspartate at rates that are almost stoicheiometric with glutamate disappearance, with no detectable ammonia production. In the presence of amino-oxyacetate, although the rate of aspartate production is decreased by 75%, ammonia production is only just detectable (0.3nmol/min per mg of protein). 4. Brain mitochondria metabolizing 10mm-glutamine and 2.5mm-malate in States 3 and 4 were studied by using glutamine as a source of intramitochondrial glutamate without the involvement of mitochondrial translocases. The ammonia production due to the oxidative deamination of glutamate produced from the glutamine was estimated as 1nmol/min per mg of protein in State 3 and 3nmol/min per mg of protein in State 4. 5. Brain mitochondria metabolizing 10mm-glutamine in the presence of 1mm-amino-oxyacetate under State-3 conditions in the presence or absence of 2.5mm-malate showed no detectable aspartate production. In both cases, however, over the first 5min, ammonia production from the oxidative deamination of glutamate was 21–27nmol/min per mg of protein, but then decreased to approx. 1–1.5nmol/min per mg. 6. It is concluded that the oxidative deamination of glutamate by glutamate dehydrogenase is not a major route of metabolism of glutamate from either exogenous or endogenous (glutamine) sources in rat brain mitochondria.
Biosynthesis of a hypermodified nucleotide in Saccharomyces carlsbergensis 17S and HeLa-cell 18S ribosomal ribonucleic acid
Authors: Brand R C , Klootwijk J , Planta R J , Maden B E H
Published: August 2015

The biosynthesis of a hypermodified nucleotide, similar to or identical with 3-(3-amino-3-carboxypropyl)-1-methylpseudouridine monophosphate, present in Saccharomyces carlsbergensis 17S and HeLa-cell 18S rRNA, was investigated with respect to the sequence of reactions required for synthesis and their timing in ribosome maturation. In both yeast and HeLa cells methylation precedes attachment of the 3-amino-3-carboxypropyl group. In yeast the methylated precursor nucleotide was tentatively characterized as 1-methylpseudouridine. This precursor nucleotide was demonstrated in both 37S and most of the cytoplasmic 18S pre-rRNA (rRNA precursor) molecules. The synthesis of the hypermodified nucleotide is completed just before the final cleavage of 18S pre-rRNA to give 17S rRNA, so that the final addition of the 3-amino-3-carboxypropyl group is a cytoplasmic event. Comparable experiments with HeLa cells indicated that formation of 1-methylpseudouridine occurs at the level of 45S RNA and addition of the 3-amino-3-carboxypropyl group occurs in the cytoplasm on newly synthesized 18S RNA.
The interaction of α-N-(p-toluenesulphonyl)-p-guanidino-L-phenylalanine methyl ester with thrombin and trypsin
Authors: Klausner Y S , Rigbi M , Ticho T , De Jong P J , Neginsky E J , Rinott Y
Published: August 2015

The syntheses are described of p-guanidino-L-phenylalanine and some of its derivatives. alpha-N-(p-Toluenesulphonyl)-p-guanidino-L-phenylalanine methyl ester is an excellent substrate of bovine trypsin (EC 3.4.21.4) (Km 57 micron; kcat. 320s-1 at pH 7.4-8.0) and a very poor substrate of human thrombin (EC 3.4.21.5) (Km 190 micron, kcat. 0.2s-1) and bovine chymotrypsin (EC 3.4.21.1). The ester inhibits thrombin clotting activity. It also inhibits the amidase and esterase activities of human thrombin, this inhibition being of the mixed type. The inhibition constant, K1, of the order of 1 micron, increases with increasing inhibitor concentration. This suggests that the enzyme binds the inhibitor at multiple sites. The importance of the residue at the P1 position [notation of Berger & Schechter (1970) Philos. Trans. R. Soc. London Ser. B 257, 249-264] in determining the selectivity of a substrate or quasi-substrate among trypsin-like enzymes is borne out. p-Guanidino-L-phenylalanine may have a use in the synthesis of selective peptide inhibitors of thrombin.
Reversible inhibitors of penicillinases
Authors: Kiener P A , Waley S G
Published: August 2015

Reversible competitive inhibitors of a penicillinase, beta-lactamase 1 from Bacillus cereus, were studied. These represent the first inhibitors of a penicillinase that lack the beta-lactam ring. The products of the enzymic reaction, namely penicilloic acids, are inhibitors; their decarboxylation products, the penilloic acids, are also inhibitors, and have somewhat lower Ki values. Inhibitors have been prepared from benzylpenicillin, phenoxymethyl-penicillin, methicillin (2,6-dimethoxybenzamidopenicillanic acid) and 3-hydroxy-4-nitrobenzamidopenicillanic acid. Decarboxylation of the penicilloic acids from benzyl-penicillin, or from phenoxymethylpenicillin, leads to epimerization (at C-5) of the penilloic acid. Nuclear-magnetic resonance spectroscopy at a frequency of 270 MHz can distinguish the epimers. Other competitive inhibitors studied were boric acid, benzene boronic acid and m-aminobenzeneboronic acid. Boric acid itself was the best inhibitor of beta-lactamase I so far found.
Recognition of individual Escherichia coli transfer ribonucleic acids by 1-adenine-specific methyltransferase from rat liver
Authors: Kraus J
Published: August 2015

Purified 1-adenine-specific tRNA methyltransferase from rat liver preferentially methylated Escherichia coli tRNA species containing the target adenylate residue in a G-T-psi-C-G-A-A-U-C sequence. The results of methylation of various tRNA species are discussed.
Ouabain binding to phospholipid-dependent adenosine triphosphatase
Authors: Goodman S L , Wheeler K P
Published: August 2015

The role of phospholipid in the binding of ouabain to the (Na+ + K+)-dependent adenosine triphosphatase was studied. Enzyme preparations obtained from rabbit kidney were treated with Lubrol WX to remove the phospholipid component essential for ATPase activity. Reconstituted enzyme samples were prepared by the addition of phosphatidylserine and sedimentation of an enzymically active lipid-protein complex. The binding of ouabain to both kinds of preparations was measured under equilibrium conditions with the use of 3H-labelled ouabain and initial ouabain concentrations in the range 0.01-1 micrometer. The main findings were: (i) (Mg2+ + Pi) promoted binding of significant quantities of ouabain only to the reconstituted enzyme; (ii) the absence of added Na+, (Mg2+ + ATP) similarly promoted binding only to the reconstituted samples; (iii) the addition of Na+ in the presence of (Mg2+ + ATP) increased the amount of ouabain bound to the reconstituted enzyme when the ouabain concentration was below about 0.1 micrometer, but it had no effect when the ouabain concentration was about 1 micrometer; (iv) (Mg2+ + ATP) induced ouabain binding to the depleted enzyme only when Na+ was also added; (v) the amount of ouabain bound to both depleted and reconstituted enzymes was the same in the presence of (Mg2+ + ATP + Na+); (vi) the reconstituted enzyme appeared to have a greater affinity for Na+ than did the depleted enzyme.
Phosphorylation of additional sites on pyruvate dehydrogenase inhibits its re-activation by pyruvate dehydrogenase phosphate phosphatase
Authors: Sugden P H , Hutson N J , Kerbey A L , Randle P J
Published: August 2015

The phosphorylation of sites additional to an inactivating site inhibits the formation of active pig heart pyruvate dehydrogenase complex from inactive pyruvate dehydrogenase phosphate complex by pig heart pyruvate dehydrogenase phosphate phosphatase.
Physical measurements of the liver glucocorticoid receptor
Authors: Litwack G , Cake M H , Filler R , Taylor K
Published: August 2015

Physical measurements were made on the cytosolic form of the liver [3H]dexamethasone receptor. These include a Stokes radius of 3.5 nm, determined by gel filtration, and sedimentation coefficients of 5.1 and 7-8S, by sucrose-density-gradient centrifugation. From these measurements, the following physical properties were calculated: apparent mol. wt. 78000 (the 5.1 S form); D app. 6.1 × 10(-7) cm2-S-1; f/fo 1.25; axial ratio 4.7; these indicate a globular protein. Measurements of sedimentation coefficient of cytosol steroid-receptor complexes previously subjected to various activating conditions gave different values and lead to the conclusion that the mechanism of activation in vitro enabling the steroid-receptor complex to bind to DNA is more complex than simple disaggregation to a uniform size.
Characterization of colchicine-binding activity in Dictyostelium discoideum
Authors: Cappuccinelli P , Hames B D
Published: August 2015

A colchicine-binding component was detected in vegetative amoebae of Dictyostelium discoideum by using a Millipore-filter assay. The colchicine-binding activity is temperature-and time-dependent, maximum binding occurring at 22-35 degrees C after 60 min incubation. Further increases in temperature are without effect on the extent of binding, but bound colchicine is released with increased time of incubation. Furthermore, colchicine-binding activity itself decreased in the high-speed supernatant from D. discoideum, with half the activity being lost in approx. 2.5h. Several lines of evidence, including the saturation kinetics of colchicine binding, enhancement of colchicine binding by tartrate, insensitivity to lumicolchicine, precipitation of the binding protein by vinblastine and behaviour of the binding protein on DEAE-cellulose and Sephadex resins, suggest that the colchicine-binding protein may be tubulin.
Comparison of the metabolism of chylomicrons and chylomicron remnants by the perfused liver
Authors: Gardner R S , Mayes P A
Published: August 2015

1. The hepatic metabolism of chylomicrons and chylomicron remnants was compared after adding approximately equal numbers of each lipoprotein particle to the perfusate of isolated livers. 2. At least 40% of the added remnants were metabolized by the liver compared with less than 3% for chylomicrons. 3. There was significantly more net removal of labelled remnants than of chylomicrons by the liver. 4. A greater proportion of labelled cholesterol than of labelled triacylglycerol fatty acids was transferred to the liver from each lipoprotein. 5. Cholesteryl esters of remnants were hydrolysed to triacylglycerol fatty lipoprotein. 5. Cholesteryl esters of remnants were hydrolysed to triacylglycerol fatty acids of remnants were oxidized to CO2 more extensively than those of chylomicrons. 6. There was greater oxidation of remnant glycerolipic [(1(-14)C]oleate than of glycerolipid [1(-14)C]palmitate. 7. A large fraction of the fatty acids of remnants, but not of chylomicrons, was transferred to phospholipids, which were released by the liver in a lipoprotein of relative density less than 1.006. 8. Label from remnants, but not from chylomicrons, was found in lipoproteins of relative density greater than 1.006, which were not released during perfusion but could be flushed out from the liver at the end of perfusion.
Studies on the mitochondrially bound form of rat brain creatine kinase
Authors: Booth Robert F. G. , Clark John B.
Published: August 2015

1. The development of the total rat brain creatine kinase was studied in brain homogenates. Until approx. 14–15 days after birth, the activity remains less than one-third that of the adult activity (207±6 units/g wet wt. s.d.; n=3). Over the next 10 days the activity increases markedly to the adult value and thereafter remains essentially constant. 2. In the adult brain, approx. 5% (11.9±2.2 units/g wet wt. s.d.; n=5) of the total creatine kinase is associated with the mitochondrial fraction. This creatine kinase could not be solubilized by sodium acetate solutions of up to 0.8m concentration, whereas 66% of the hexokinase associated with brain mitochondria was released under these conditions. 3. Rat brain mitochondria incubated in the presence of various concentrations of creatine (1, 5 and 10mm) and ADP (100μm) synthesized phosphocreatine at rates of approx. 4.5, 11 and 17.5nmol/min per mg of mitochondrial protein. Atractyloside (50μm) or oligomycin (1.5μg/mg of mitochondrial protein) completely inhibited the synthesis of phosphocreatine. 4. The apparent Km and Vmax. values of the mitochondrially bound rat brain creatine kinase were determined in both directions. The Vmax. in the direction of phosphocreatine synthesis is 237nmol/min per mg of mitochondrial protein, with an apparent Km for creatine of 1.67mm and for MgATP2−of 0.1mm, and in the reverse direction Vmax. is 489nmol/min per mg of mitochondrial protein, with an apparent Km for phosphocreatine of 0.4mm and for MgADP−of 27μm. 5. The results are discussed with reference to the role that the mitochondrially bound creatine kinase may play in the development of brain energy metabolism.
Androgenic regulation of elongation of polyribonucleotide chains on rat ventral-prostate chromatin
Authors: Thomas P , Davies P , Griffiths K
Published: August 2015

The kinetics of polyribonucleotide-chain elongation by rat ventral-prostate RNA polymerase B with homologous chromatin as a template were investigated. Chain elongation was measured under conditions wherein all initiation had occurred, no reinitiation took place and the reaction rate was constant. The kinetic behaviour of prostate RNA polymerase B was consistent with a mathematical model formulated for the multisubstrate enzyme. The addition of each nucleoside triphosphate was independent of the other three. The overall rate of chain elongation was lower when prostate chromatin from castrated rats was used than with prostate chromatin from normal rats. The inclusion of dihydrotestosterone-receptor complexes stimulated the rate of elongation. Androgenic effects did not appear to be directed towards the addition of individual nucleoside triphosphates, but probably towards one of the other major events in RNA-chain elongation, i.e., unwinding of DNA or movement of the enzyme along the template.
Effect of potassium deficiency on mouse kidney lysosomal enzymes
Authors: Cleveland C E , Swank R T
Published: August 2015

Mice of inbred strains A/J, C57BL/6J and C57BL/6J beige were kept on a K+-deficient diet for up to 40 days to determine the magnitude and mechanism of changes in tissue lysosomal enzymes. From days 10 to 40 glucuronidase activity increased 3-fold in kidney of K+-deficient mice, but there was little effect on beta-galactosidase or acid phosphatase activity. Similar increases in kidney glucuronidase activity occurred in inbred strains known to have genetically altered control of the synthesis (A/J) and secretion (C57BL/6J beige) of glucuronidase in kidney proximal-tubule cells. Deprivation of K+ did not affect glucuronidase activity in liver, spleen, lung and brain, but there was a 2-3-FOld increase in glucuronidase activity in heart in the C57BL/6J and C57BL/6J beige strains. As shown by specific antibody titration, increased glucuronidase activity in kidney of K+-deficient mice was accompanied by accumulation of enzyme molecules. Likewise in kidney of deficient mice there was an increased rate of synthesis of glucuronidase as measured by incorporation of labelled leucine into immunoprecipitable glucuronidase. In kidney of K+-deficient mice the elevated glucuronidase activity was found in both collecting-tubule and interstitial cells of the medulla. It is probable therefore that a significant fraction of the increased kidney lysosomal synthesis and enzyme activity is due to infiltrating cells.
The formation of lipid-linked sugars as intermediates in glycoprotein synthesis in rabbit mammary gland
Authors: Speake Brian K. , White David A.
Published: August 2015

1. The incorporation of d-[1-14C]mannose, d-[2-3H]mannose and N-acetyl-d-[1-14C]-glucosamine into glycoproteins and lipid-linked intermediates of mammary explants obtained from lactating rabbits was studied. The amount of radioactivity incorporated into lipid-linked intermediates was very low compared with the incorporation into protein. Most of the radioactivity incorporated into the chloroform/methanol-soluble fraction was present as neutral lipid. Radioactivity from d-[2-3H]mannose was incorporated mainly into the fatty acid moiety, whereas radioactivity from d-[1-14C]mannose and N-acetyl-d-[1-14C]glucosamine was present in the glycerol moiety of triacylglycerol. 2. The labelled lipid-linked intermediate that was soluble in chloroform/methanol/water (10:10:3, by vol.) was partially characterized and was found to exhibit properties characteristic of an oligosaccharide linked to lipid via a pyrophosphate bridge. It migrated largely as a single zone of radioactivity on t.l.c. and was eluted from a column of DEAE-cellulose acetate as a single peak by 50mm-ammonium acetate. 3. The oligosaccharide moiety was released from the lipid by mild acid hydrolysis. The size of the oligosaccharide was estimated by paper chromatography to be 10 or 11 monosaccharide units. 4. d-[1-14C]Mannose was incorporated largely into glycopeptides with molecular weights in the range 40000–80000, as determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Label from N-acetyl-d-[1-14C]glucosamine was incorporated into a glycopeptide with an electrophoretic mobility identical with that of rabbit casein (mol.wt. 32000) as well as into glycopeptides of higher molecular weight. 5. Approx. 50% of the total radioactivity in the protein labelled from N-acetyl-d-[1-14C]glucosamine was present as galactosamine, a component of the carbohydrate portion of rabbit casein. No labelled galactosamine was present in the lipid-linked oligosaccharide labelled from N-acetyl-d-[1-14C]glucosamine. It thus appears that the lipid-linked oligosaccharide is not involved in the glycosylation of casein.
Studies on secretion and endocytosis of macromolecules by cultivated skin fibroblasts. Effects of anti-microtubular agents on secretion and endocytosis of lysosomal hydrolases and of sulphated glycosaminoglycans
Authors: von Figura K , Kresse H , Meinhard U , Holtfrerich D
Published: August 2015

Fibroblasts were incubated in the presence of the anti-microtubular drugs colchicine, vinblastine and vincristine. In concentrations between 10nm and 1 mM these drugs stimulated the secretion of beta-N-acetylglucosaminidase, alpha-N-acetylglucosaminidase and beta-glucuronidase, but not of beta-galactosidase. The endocytosis of beta-N-acetylhexosaminidase and alpha-N-acetylglucosaminidase, but not of beta-glucuronidase, was inhibited at drug concentrations higher than 0.1 micrometer. Formation, secretion and association with the cell membrane of sulphated proteoglycans were not affected by anti-microtubular drugs. Endocytosis of sulphated proteoglycans and their subsequent degradation was inhibited by drug concentrations above 0.1 micrometer. The inhibition of intracellular glycosaminoglycan degradation led to a moderate storage of these compounds. These results suggest that microtubules participate in the control of secretion and endocytosis of lysosomal enzymes, and in the endocytosis and degradation of lysosomal substrates such as sulphated proteoglycans.
Biochemical aspects of cardiac muscle differentiation. Determination of deoxyribonucleic acid template availability and 3′-hydroxyl termini in nuclei and chromatin by using exogenous deoxyribonucleic acid polymerases
Authors: Claycomb William C.
Published: August 2015

Experiments were designed to determine whether DNA synthesis ceases in terminally differentiating cardiac muscle of the rat because the activity of the putative replicative DNA polymerase (DNA polymerase α) is lost or whether the activity of this enzyme is lost because DNA synthesis ceases. DNA-template availability and 3′-hydroxyl termini in nuclei and chromatin, isolated from cardiac muscle at various times during the developmental period in which DNA synthesis and the activity of DNA polymerase α are decreasing, were measured by using Escherichia coli DNA polymerase I, Micrococcus luteus DNA polymerase and DNA polymerase α under optimal conditions. Density-shift experiments with bromodeoxyuridine triphosphate and isopycnic analysis indicate that DNA chains being replicated semi-conservatively in vivo continue to be elongated in isolated nuclei by exogenous DNA polymerases. DNA template and 3′-hydroxyl termini available to exogenously added DNA polymerases do not change as cardiac muscle differentiates and the rate of DNA synthesis decreases and ceases in vivo. Template availability and 3′-hydroxyl termini are also not changed in nuclei isolated from cardiac muscle in which DNA synthesis had been inhibited by administration of isoproterenol and theophylline to newborn rats. DNA-template availability and 3′-hydroxyl termini, however, were substantially increased in nuclei and chromatin from cardiac muscle of adult rats. This increase is not due to elevated deoxyribonuclease activity in nuclei and chromatin of the adult. Electron microscopy indicates that this increase is also not due to dispersal of the chromatin or disruption of nuclear morphology. Density-shift experiments and isopycnic analysis of DNA from cardiac muscle of the adult show that it is more fragmented than DNA from cardiac-muscle cells that are, or have recently ceased, dividing. These studies indicate that DNA synthesis ceases in terminally differentiating cardiac muscle because the activity of a replicative DNA polymerase is lost, rather than the activity of this enzyme being lost because DNA synthesis ceases.
Deacylation of acetyl-coenzyme A and acetylcarnitine by liver preparations
Authors: Snoswell A M , Tubbs P K
Published: August 2015

The breakdown of acetylcarnitine catalysed by extracts of rat and sheep liver was completely abolished by Sephadex G-25 gel filtration, whereas the hydrolysis of acetyl-CoA was unaffected. Acetyl-CoA and CoA acted catalytically in restoring the ability of Sephadex-treated extracts to break down acetylcarnitine, which was therefore not due to an acetylcarnitine hydrolase but to the sequential action of carnitine acetyltransferase and acetyl-CoA hydrolase. Some 75% of the acetyl-CoA hydrolase activity of sheep liver was localized in the mitochondrial fraction. Two distinct acetyl-CoA hydrolases were partially purified from extracts of sheep liver mitochondria. Both enzymes hydrolysed other short-chain acyl-CoA compounds and succinyl-CoA (3-carboxypropionyl-CoA), but with one acetyl-CoA was the preferred substrate.
Characterization of the papain active centre by using two-protonic-state electrophiles as reactivity probes. Evidence for nucleophilic reactivity in the un-interrupted cysteine-25-histidine-159 interactive system
Authors: Shipton M , Brochlehurst K
Published: August 2015

1.2,2′-Dipyridyl disulphide (2-Py-S-S-2-Py) and n-propyl 2-pyridyl disulphide (propyl-S-S-2-Py) were used as two-protonic-state reactivity probes to investigate the active centre of papain (EC 3.4.22.2).2. The existence of a striking rate optimum at pH approx. 4 in the reaction of papain not only with the symmetrical probe but also with the unsymmetrical probe is shown to constitute compelling evidence that the thiolate ion component of the cysteine-25-histidine-159 interactive system of papain possesses appreciable nucleophilic character. It is not a necessary requirement that the probe reagent should engage the imidazolium ion of histidine-159 in hydrogen-bonding for the sulphur atom of the interactive system to display nucleophilic character. The single proton-binding site of propyl-S-S-2-Py cannot simultaneously interrupt the active-centre ion pair and provide for rate enhancement as the pH is lowered towards 4. The possible implication of this for the mechanism of papain-catalysed hydrolysis is discussed. 3. The suspected difference in the active centres of papain and ficin (EC 3.4.22.3), which could be a lack in ficin of a carboxy group conformationally equivalent to that of aspartic acid-158 of papain is confirmed. The reactivity of the papain thiol group towards both probe reagents is controlled by two ionizations with pKa close to 4 that are positively co-operative. 4. In the reaction of papain with 2-Py-S-S-2-Py. the reactivity appears to be controlled also by an addition ionization with pKa approx. 5. Possible origins of this additional ionization are discussed. K. The spectral and ionization characteristics of propyl-S-S-2-Py are reported. 6. The reagent reacts rapidly with thiol groups at the sulphur atom distal from the pyridyl ring to provide, at pH values below 9, stoicheiometric release of 2-thiopyridone. This property, together with the ability of the reagent markedly to increase its electrophilicity consequent on protonation, suggests alkyl-2-pyridyl disulphides in general as valuable two-protonic-state reactivity probes with exceptional specificity for thiol groups.
Bile salts of the green turtle Chelonia mydas (L.)
Authors: Haslewood G A D , Ikawa S , Tökés L , Wong D
Published: August 2015

1. Bile salts of the green turtle Chelonia mydas (L.) were analysed as completely as possible. 2. They consist of taurine conjugates of 3 alpha, 7 alpha, 12 alpha, 22 xi-tetrahydroxy-5 beta-cholestan-26-oic acid (tetrahydroxysterocholanic acid) and 3 alpha 12 alpha, 22 xi-trihydroxy-5 beta-cholestan-26-oic acid, with minor amounts of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5beta-cholan-24-oic acid (cholic acid), 3alpha, 12 alpha-dihydroxy-5beta-cholan-24-oic acid (deoxycholic acid) and possibly other bile acids. 3. Cholic acid and deoxycholic acid represent the first known examples of bile acids common to chelonians and other animal forms: they may indicate independent evolution in chelonians to C24 bile acids. 4. The discovery of a 7-deoxy C27 bile acid is the first evidence that C27 bile acids or their conjugates have an enterohepatic circulation.
What happens when data are fitted to the wrong equation?
Authors: Ellis K J , Duggleby R G
Published: August 2015

In many problems of data analysis it is necessary to fit the data to a mathematical equation. Random errors of measurement will be responsible for deviations between the data and the equation, but superimposed on this there may be deviations that result from the equation being an inadequate description of the system from which the data were obtained. Plots of the residual (i.e. the difference between the experimental and calculated values of the dependent variable) against each of the experimental variables have been previously used to detect a misfit between the data and the equation. In the present paper, we show that the shape of the residual plots may be used as a guide in choosing a more appropriate equation. In addition, residual plots give useful information on the error structure of the data, and hence the weighting factors that should be used in the analysis.
The proton exchange of the pro-S hydrogen atom at C-1 in dihydroxyacetone phosphate and d-fructose 1,6-bisphosphate catalysed by class-I and class-II aldolases
Authors: Galdes A , Hill H A O
Published: August 2015

The efficacy of class-I and class-II aldolases in catalysing the C-1 proton exchange in fructose 1,6-bisphosphate and dihydroxyacetone phosphate was investigated. The rate of this reaction was at least two orders of magnitude slower in class-II than in the class-I aldolases. It is suggested that this difference reflects the formation of different intermediates in the reactions catalysed by the two classes of aldolase.
Extracellular manganese-stimulated deoxyribonuclease as a marker event in sporulation of Bacillus subtilis
Authors: Akrigg A , Mandelstam J
Published: August 2015

A considerable amount of Mn2+-stimulated DNAase (deoxyribonuclease) activity is released by Bacillus subtilis 168 during sporulation in a glucose-deficient medium; much smaller amounts are released during starvation for phosphate or nitrogen. Protein synthesis is required. Two forms of evidence are presented that production of the DNAase is associated with events late in stage II of sporulation. 19 Thymidine starvation, which inhibits the biochemical events associated with sporulation, also inhibits release of the DNAase. 2. Several asporogenous mutants blocked at stage II or earlier and unable to produce alkaline phosphatase (a stage-II event) do not produce the enzyme. Mutants blocked towards the end of stage II or later produce both enzymes. During sporulation of the wild-type strain, the DNAase appears about 1 h after alkaline phosphatase. The results suggest that production of the DNAase is controlled by a still-undiscovered stage-II genetic locus.