bluuebunny/crossref_metadata_2025 huggingface dataset:
Production and properties of antisera to dexamethasone–protein conjugates
Authors: Dumasia M. C. , Chapman D. I. , Moss M. S. , O'Connor C.
Published: August 2015

Antibodies to dexamethasone 21-hemisuccinate conjugated to bovine serum albumin were produced in rabbits. Antisera diluted 1:3000 bound 50% of 90pg of [1,2-3H]dexamethasone. The cross-reaction of the antisera with other synthetic and natural corticosteroids was measured.
The deoxyribonucleic acid modification enzyme of bacteriophage P1. Subunit structure
Authors: Brockes Jeremy P.
Published: August 2015

The bacteriophage P1 modification enzyme was purified 1400-fold from induced lysogens of a thermoinducible mutant of bacteriophage P1. The most purified fraction, when analysed by polyacrylamide-gel electrophoresis in sodium dodecyl sulphate, showed two principal stained bands. The two bands co-sedimented in a glycerol gradient with the modification activity, at a rate which, when compared with the rate of sedimentation of marker proteins, corresponds to a sedimentation coefficient in water of 6S. The mobilities of the bands on sodium dodecyl sulphate–polyacrylamide-gel electrophoresis corresponded to polypeptides of molecular weight 70000 and 45000 and they were present in equimolar amounts. It was concluded that the 6S species of the enzyme is a dimer of unlike subunits.
Studies on the high-sulphur proteins of reduced Merino wool. Amino acid sequence of protein SCMKB-IIIA3
Authors: Swart Louis S. , Haylett Thomas
Published: August 2015

The complete amino acid sequences of wool protein SCMKB-IIIA3 (131 residues) and a minor component SCMKB-IIIA3A (130 residues) have been determined. The proteins are mutually homologous and have free threonine as the N-terminal residue and carboxymethylcysteine as the C-terminus. The peptides used for the sequence work were obtained by trypsin, thermolysin, pepsin and chymotrypsin digestions and were fractionated by chromatography on DEAE-cellulose, gel filtration on Sephadex G-25 and G-50, paper chromatography and electrophoresis. The Edman degradation method (employing both the Beckman Sequencer and a non-automatic procedure) was used to obtain the sequences of the peptides.
Multiple forms of acetylcholinesterase from pig brain
Authors: McIntosh Christopher H. S. , Plummer David T.
Published: August 2015

1. A number of methods of solubilization of pig brain acetylcholinesterase (EC 3.1.1.7) were studied. The multiple enzymic forms of the resultant preparations were examined by polyacrylamide-gel electrophoresis. 2. Butanol extraction, Nagarase treatment and ultrasonication proved unsuitable as preparatory methods, but detergent treatment (Triton X-100, Triton X-100–KCl and lysolecithin) gave good yields. 3. Separation of soluble enzyme in three systems of polyacrylamide-gel electrophoresis were compared and the relative advantages are discussed. 4. By using a 6% (w/v) gel and continuous buffer system two forms of acetylcholinesterase were detected in Triton X-100-solubilized enzyme, but the incorporation of a sample and spacer gel and a discontinuous buffer system resolved this into four components. The forms of the soluble enzyme extracted by different methods differed in mobility. 5. With gradient polyacrylamide-gel electrophoresis between two and six forms were detected, depending on the method used for extraction. The average molecular weights of the five forms most frequently found were 60000, 130000, 198000, 266000 and 350000. 6. Treatment of the Triton X-100-extracted enzyme with 2.5m-urea altered the pattern and evidence of dissociation was observed. 7. The results are discussed in the light of present theories on the molecular structure of acetylcholinesterase.
The interaction of inositol hexaphosphate with methaemoglobin
Authors: Kilmartin J. V.
Published: August 2015

1. Inositol hexaphosphate causes the shape of the oxidation–reduction equilibrium curve to become hyberbolic at acid pH values. 2. Inositol hexaphosphate also causes a decrease in the alkaline oxidation Bohr effect at these same pH values. 3. These results support the idea that inositol hexaphosphate causes methaemoglobin to take up the deoxyhaemoglobin quaternary structure at pH6.5.
Comparison of the sedimentation and gel-filtration behaviour of human apotransferrin and its copper and iron complexes
Authors: Charlwood Peter A.
Published: August 2015

Equilibrium-dialysis experiments showed that Tris or citrate in the solution prevented copper from occupying completely the specific metal-binding sites on human transferrin. Differential measurements of sedimentation velocity under conditions where two atoms of copper per molecule of protein were bound showed an increase in s020,w, relative to that of the apoprotein, practically the same as that produced by two atoms of iron. Gel-filtration experiments made under the same conditions to investigate the effect of copper binding on the Stokes radius of the protein showed merely that it lost most of the metal as it passed down the column.
Biosynthesis of ergotamine by Claviceps purpurea (Fr.) Tul
Authors: Bassett R. A. , Chain E. B. , Corbett K.
Published: August 2015

High-yielding strains of Claviceps purpurea (Fr.) Tul, grown on a defined medium, have been used for a study of the biosynthesis of the peptide ergot alkaloid, ergotamine. l-[U-14C]tryptophan, dl-[2-14C]mevalonic acid lactone, sodium [2-14C]acetate, sodium [14C]formate and the methyl group of l-[methyl-14C]methionine were efficiently incorporated into the peptide alkaloids and specifically labelled the ergoline moiety of ergotamine. These results are the same as previously found for the biosynthesis of other ergot alkaloids. Time-course incubation experiments demonstrated that l-[U-14C]phenylalanine, l-[U-14C]proline and l-[U-14C]alanine were incorporated into the peptide ergot alkaloids. Chemical degradation of the radioactive alkaloid derived from additional precursor incubation experiments showed that phenylalanine and proline function as the most efficient precursors, and specifically label the constitutive side-chain phenylalanyl and prolyl moieties of the alkaloid. The evidence obtained from l-[U-14C]alanine-incorporation experiments was inconclusive. However, degradation of ergotamine isolated after incubation with dl-[1-14C]alanine, showed that the carboxyl group of the labelled amino acid was specifically incorporated into the α-hydroxy-α-amino acid residue of the alkaloid. This, in conjunction with the l-[U-14C]alanine-incorporation results, showed conclusively that all three carbon atoms of alanine were incorporated as a biosynthetic unit into the α-hydroxy-α-amino acid moiety of ergotamine.
The uptake of γ-aminobutyrate by organotypic cultures of chick spinal cord
Authors: Tunnicliff G. , Cho Y. D. , Blackwell Natalie , Martin R. O. , Wood J. D.
Published: August 2015

1. Explants of spinal cord from 10-day chick embryos maintained for up to 16 days in culture rapidly accumulated γ-amino[3H]butyrate when incubated at 25°C or 36°C in a medium containing 50nm-γ-aminobutyrate. The mechanism of the uptake process has many of the properties of an active-transport system: it is Na+-dependent, temperature-sensitive, inhibited by ouabain, and displays saturation kinetics. The apparent Km for γ-aminobutyrate is 1.7×10−5m, and Vmax. is 33pmol/min per g. 2. The rate of accumulation of γ-amino[3H]butyrate in cultures between the ages of 3 and 16 days was remarkably constant and was not related to the morphological maturity of the spinal-cord explants. 3. The present demonstration in spinal-cord explants of an active transport system for γ-aminobutyrate, already established for non-cultured nervous tissue, means that nervous-tissue culture can provide a convenient model for studying uptake processes in the central nervous system.
Effects of infection with Diplococcus pneumoniae on synthesis of ribonucleic acids in rat liver
Authors: Thompson William L. , Wannemacher Robert W.
Published: August 2015

Rats infected with virulent Diplococcus pneumoniae developed a significant increase in the rate of incorporation of labelled orotate into hepatic RNA when compared with pair-fed controls inoculated with heat-killed organisms. The finding was readily detected in rats raised on either a low-protein diet (6% casein) or a diet containing adequate amounts of protein (18% casein). The increase in hepatic RNA synthesis was observed by 12h and was maximal by 16–20h after inoculation with the D. pneumoniae. Most of the infection-related increase in RNA synthesis was associated with the bound ribosomal RNA fraction of the liver. A small but less significant increase was observed in the synthesis of free ribosomal RNA. The increased synthesis of RNA in the liver of infected rats resulted in a marked elevation of the liver RNA/DNA ratio, the major increase being observed in concentration of bound ribosomal RNA fraction. When followed sequentially, the infection-related increase in synthesis of hepatic RNA was preceded by a flux of amino acids into liver and was followed by elevated synthesis rates of serum globulin proteins. These findings suggest that the infectious process was able to regulate hepatic RNA synthesis by altering the rate of transcription of new RNA. This mechanism was stimulated even in rats that had been severely depleted of body protein and amino acids by feeding them on a low-protein diet. The infection-related stimulation of liver RNA and protein synthesis thus appeared to take place at the expense of other body proteins.
A study of some enzymes of glycerolipid biosynthesis in rat liver after subtotal hepatectomy
Authors: Mangiapane E. Heather , Lloyd-Davies Katherine A. , Brindley David N.
Published: August 2015

1. The accumulation of triglyceride in the liver remnant after subtotal hepatectomy (removal of 82% of the liver) exceeded that described for partial hepatectomy (removal of 70% of the liver). 2. Palmitoyl-CoA synthetase, glycerol phosphate acyltransferase and diglyceride acyltransferase activities were measured in the microsomal fraction, and phosphatidate phosphohydrolase activity was measured in the particle-free supernatant fraction, prepared from the liver remnant at various times after subtotal hepatectomy. 3. The only enzyme showing a significant change in specific activity was phosphatidate phosphohydrolase. The specific activity was approximately fivefold that of the control value at 6h after operation and threefold that of the control at 10, 16 and 24h after operation. A smaller increase in the specific activity of the enzyme in sham-operated animals occurred only at 6h after operation. 4. However, at this time the total phosphohydrolase activity of the remaining liver in the sham-operated rats was approximately threefold that in hepatectomized rats. 5. Injection of actinomycin D prevented the increase in activity of phosphatidate phosphohydrolase but did not prevent the accumulation of triglyceride.
Induction and suppression of the key enzymes of glycolysis and gluconeogenesis in isolated perfused rat liver in response to glucose, fructose and lactate
Authors: Wimhurst Janet M. , Manchester K. L.
Published: August 2015

1. Measurements were made of the activities of the four key enzymes involved in gluconeogenesis, pyruvate carboxylase (EC 6.4.1.1), phosphoenolpyruvate carboxylase (EC 4.1.1.32), fructose 1,6-diphosphatase (EC 3.1.3.11) and glucose 6-phosphatase (EC 3.1.3.9), of serine dehydratase (EC 4.2.1.13) and of the four enzymes unique to glycolysis, glucokinase (EC 2.7.1.2), hexokinase (EC 2.7.1.1), phosphofructokinase (EC 2.7.1.11) and pyruvate kinase (EC 2.7.1.40), in livers from starved rats perfused with glucose, fructose or lactate. Changes in perfusate concentrations of glucose, fructose, lactate, pyruvate, urea and amino acid were monitored for each perfusion. 2. Addition of 15mm-glucose at the start of perfusion decreased the activity of pyruvate carboxylase. Constant infusion of glucose to maintain the concentration also decreased the activities of phosphoenolpyruvate carboxylase, fructose 1,6-diphosphatase and serine dehydratase. Addition of 2.2mm-glucose initially to give a perfusate sugar concentration similar to the blood sugar concentration of starved animals had no effect on the activities of the enzymes compared with zero-time controls. 3. Addition of 15mm-fructose initially decreased glucokinase activity. Constant infusion of fructose decreased activities of glucokinase, phosphofructokinase, pyruvate carboxylase, phosphoenolpyruvate carboxylase, glucose 6-phosphatase and serine dehydratase. 4. Addition of 7mm-lactate initially elevated the activity of pyruvate carboxylase, as also did constant infusion; maintenance of a perfusate lactate concentration of 18mm induced both pyruvate carboxylase and phosphoenolpyruvate carboxylase activities. 5. Addition of cycloheximide had no effect on the activities of the enzymes after 4h of perfusion at either low or high concentrations of glucose or at high lactate concentration. Cycloheximide also prevented the loss or induction of pyruvate carboxylase and phosphoenolpyruvate carboxylase activities with high substrate concentrations. 6. Significant amounts of glycogen were deposited in all perfusions, except for those containing cycloheximide at the lowest glucose concentration. Lipid was found to increase only in the experiments with high fructose concentrations. 7. Perfusion with either fructose or glucose decreased the rates of ureogenesis; addition of cycloheximide increased urea efflux from the liver.
Ribonucleic acid synthesis in chloroplasts
Authors: Hartley M. R. , Ellis R. J.
Published: August 2015

Chloroplasts isolated from young spinach leaves incorporate [3H]uridine into RNA. This incorporation shows an absolute requirement for light and does not occur in lysed chloroplasts. Fractionation by polyacrylamide-gel electrophoresis of the RNA synthesized in vitro reveals a major discrete product of molecular weight 2.7×106 and two minor products of molecular weight 1.2×106 and 0.47×106. These discrete products are super-imposed on a background of polydisperse RNA. The incorporation of 32Pi into chloroplast rRNA species (mol.wt. 1.05×106 and 0.56×106) in excised spinach leaves proceeds after a distinct lag period compared with the incorporation into cytoplasmic rRNA species (mol.wt. 1.34×106 and 0.7×106). Incorporation of 32Pi into chloroplast RNA species of molecular weight 2.7×106, 1.2×106, 0.65×106 and 0.47×106 proceeds without such a time-lag. The kinetics of labelling of the individual RNA components is consistent with the rapidly labelled RNA species of molecular weight 1.2×106 and 0.65×106 being precursors to the more slowly labelled rRNA species of molecular weight 1.05×106 and 0.56×106 respectively.
The mode of action of adenosine 3′:5′-cyclic monophosphate in mammalian islets of Langerhans. Effects of insulin secretagogues on islet-cell protein kinase activity
Authors: Montague W. , Howell S. L.
Published: August 2015

1. Protein kinase activity was measured in islets of Langerhans that had been incubated in the presence of agents known to affect insulin release. 2. Glucagon, theophylline, caffeine and 3-isobutyl-1-methylxanthine, agents that raise cyclic AMP concentrations in islet cells and stimulate insulin release, increased protein kinase activity. Adrenaline and diazoxide, agents that decrease cyclic AMP concentrations and inhibit insulin secretion, decreased the activity. 3. The increase in protein kinase activity produced by different concentrations of 3-isobutyl-1-methylxanthine was apparently related to the increase in intracellular concentrations of cyclic AMP. 4. The sulphonylureas, tolbutamide and glibenclamide, agents that increase insulin release, also increased the protein kinase activity; however, leucine, arginine and xylitol, which also stimulate insulin release, were without effect on the kinase activity. 5. Increasing the glucose concentration of the incubation medium from 2 to 20mm had no effect on protein kinase activity. Further, the ability of 3-isobutyl-1-methylxanthine to increase the protein kinase activity was not affected by the glucose concentration of the incubation medium. 6. These results suggest that agents which affect insulin secretion by altering cyclic AMP concentrations may exert their effects on hormone release by altering the activity of a cyclic AMP-dependent protein kinase in islet cells.
Effect of phenylpyruvate on enzymes involved in fatty acid synthesis in rat brain
Authors: Land John M. , Clark John B.
Published: August 2015

1. The activities of, and the effects of phenylpyruvate on, citrate synthase (EC 4.1.3.7), acetyl-CoA carboxylase (EC 6.4.1.2) and fatty acid synthetase derived from the brains of 14-day-old and adult rats were investigated. 2. The brain citrate synthase from 14-day-old rats had a Km for oxaloacetate of 2.38μm and for acetyl-CoA of 16.9μm, and a Vmax. of 838nmol of acetyl-CoA incorporation/min per mg of mitochondrial protein. From adult rat brain this enzyme had a Km for oxaloacetate of 2.5μm and for acetyl-CoA of 16.6μm and a Vmax. of 1070nmol of acetyl-CoA incorporated/min per mg of mitochondrial protein. Phenylpyruvate inhibited the enzyme from adult and young rat brains in a competitive fashion with respect to acetyl-CoA, with a Ki of 700μm. 3. The brain acetyl-CoA carboxylase from 14-day-old rats had a Km for acetyl-CoA of 21μm and a Vmax. of 0.248nmol/min per mg of protein, and from adult rats a Km for acetyl-CoA of 21μm and a Vmax. of 0.173nmol/min per mg of protein. The enzyme from young and adult rats required citrate (Ka=3mm) for activation and were inhibited non-competitively by phenylpyruvate, with a Ki of 10mm. 4. The brain fatty acid synthetase from 14-day-old rats had a Km for acetyl-CoA of 7.58μm and a Vmax. of 1.1 nmol of malonyl-CoA incorporated/min per mg of protein, and from adult rats a Km for acetyl-CoA of 4.9μm and a Vmax. of 0.48nmol of malonyl-CoA incorporated/min per mg of protein. Phenylpyruvate acted as a competitive inhibitor with respect to acetyl-CoA with a Ki of 250μm for the enzyme from 14-day-old rats. 5. These results are discussed with respect to phenylketonuria, and it is suggested that the inhibition of the brain fatty acid synthetase and possibly the citrate synthetase by phenylpyruvate could explain the defective myelination characteristic of this condition.
Regulation of uptake of purines, pyrimidines and amino acids by Candida utilis
Authors: Jones R. W. , Wild D. G.
Published: August 2015

Uptake of uracil by Candida utilis is increased by addition of leucine to a minimal medium in which organisms are growing. This response requires protein synthesis and has kinetics consistent with the induction of additional uracil transport by the amino acid or a derivative. Consequently, the contribution of exogenous radioactive uracil to the pyrimidine nucleotide pools increases so that RNA made after the amino acid is added is of greater specific radioactivity. Some other amino acids are as effective as leucine in increasing the incorporation of uracil into RNA. Growth with leucine present also increases to different extents the initial rates of uptake of adenine, cytosine, uridine, lysine, histidine, threonine, phenylalanine, aspartic acid and leucine itself. The action of leucine on lysine transport appears to involve induction. These effects are not restricted to leucine; growth with aspartic acid or phenylalanine in the medium gives similar results. Lysine, on the other hand, is without action on the uptake of leucine, aspartic acid, phenylalanine, threonine or uracil but decreases the initial rates of uptake of both histidine and lysine. We suggest that lysine represses its own transport. Similarly, there is a specific decrease in uracil uptake caused by growth with this pyrimidine. Thus in C. utilis there are complex interrelationships in the uptake of nitrogen-containing compounds.
Acceleration of gluconeogenesis from lactate by lysine (Short Communication)
Authors: Cornell Neal W. , Lund Patricia , Hems Reginald , Krebs Hans A.
Published: August 2015

l-Lysine (2mm) causes an increase (mean 60%) in the rate of gluconeogenesis from lactate in isolated liver cells. The effect is of a catalytic nature. No other amino acid has the same effect, though ornithine is slightly active. The effect is additional to the stimulatory effects of oleate and of dibutyryl cyclic AMP.
Inhibition of the biosynthesis of sterols by phenyl and phenolic compounds in rat liver
Authors: Ranganathan S. , Ramasarma T.
Published: August 2015

The presence of mitochondria increased the incorporation of [2-14C]mevalonate into sterols in a cell-free system from rat liver. Various phenyl and phenolic compounds inhibited the incorporation of mevalonate when added in vitro. p-Hydroxycinnamate, a metabolite of tyrosine, was the most powerful inhibitor among the compounds tested. Catechol, resorcinol and quinol were inhibitory at high concentrations. Organic acids lacking an aromatic ring were not inhibitory. Two hypocholesterolaemic drugs, Clofibrate (α-p-chlorophenoxyisobutyrate) and Clofenapate [α,4-(p-chlorophenyl)phenoxyisobutyrate], which are known to affect some step before the formation of mevalonate in the biosynthesis of cholesterol in vivo, showed inhibition at a step beyond the formation of mevalonate in vitro. The presence of the aromatic ring and the carboxyl group in a molecule appears to be necessary for the inhibition.
Effect of light on enzymes of phenylpropanoid metabolism and hispidin biosynthesis in Polyporus hispidus
Authors: Nambudiri A. M. D. , Vance C. P. , Towers G. H. N.
Published: August 2015

The effects of light on growth, pigmentation and the activities of enzymes involved in the deamination of phenylalanine and tyrosine and in the biosynthesis of hispidin were examined in Polyporus hispidus. Evidence is presented for the stimulation of phenylalanine ammonia-lyase activity by light. Tyrosine ammonia-lyase activity and aminotransferase activities for phenylalanine and tyrosine were higher in the dark. Tracer studies showed that conversion of cinnamate into p-coumarate is enhanced by light. p-Coumaric acid hydroxylase, catalysing the conversion of p-coumarate into caffeate, could be detected only in cultures exposed to light. These results suggest that the cinnamate pathway for the metabolism of phenylalanine, leading to hispidin synthesis, is regulated by light in P. hispidus.
Carotenoid biosynthesis in a Flavobacterium sp.: stereochemistry of hydrogen elimination in the desaturation of phytoene to lycopene, rubixanthin and zeaxanthin (Short Communication)
Authors: McDermott John C. B. , Britton George , Goodwin T. W.
Published: August 2015

[2-14C,(2R)-2-3H1]- and [2-14C,(2S)-2-3H1]-Mevalonates were rapidly incorporated into phytoene, lycopene, rubixanthin and zeaxanthin in a Flavobacterium system obtained by disruption of the bacterial cells by shaking with glass beads. Four hydrogen atoms arising from the 2-pro-S-hydrogen atoms of mevalonate were lost in the desaturation of phytoene to lycopene, rubixanthin and zeaxanthin. The desaturation of phytoene involves trans-elimination of hydrogen in the introduction of the double bonds at C-7, C-11, C-7′ and C-11′.
Thermolability of 28S ribosomal ribonucleic acid from the liver of Crotalus durissus terrificus (Ophidia, Reptilia)
Authors: Giorgini J. F. , De Lucca F. L.
Published: August 2015

Instability of 28S rRNA of Crotalus durissus terrificus liver was observed during hotphenol extraction: purified 28S rRNA is converted into an 18S RNA component by heat treatment. It was also found that ‘6S’ and ‘8S’ low-molecular-weight RNA species were released during the thermal conversion. This conversion and the release of the low-molecular-weight species were also induced by 8m-urea and 80% (v/v) dimethyl sulphoxide at 0°C. Evidence is presented that this phenomenon is an irreversible process and results from the rupture of hydrogen bonds. The 18S RNA product was shown to be homogeneous by polyacrylamide-gel electrophoresis and by sucrose-density-gradient centrifugation. The base composition of the 18S RNA products obtained by heat, urea or dimethyl sulphoxide treatments was similar. The C+G content of the 18S RNA product was different from that of the native 18S rRNA, but similar to that of 28S rRNA.
High total histone/deoxyribonucleic acid ratios for rat liver nuclei
Authors: Chanda Subir K. , Ickowicz Regina , Dounce Alexander L.
Published: August 2015

The ratios of total histone to DNA for rat liver nuclei isolated by four methods as well as for calf liver nuclei isolated by one method were determined by obtaining the ratios of the total areas of the electrophoretic histone peaks for the liver nuclei to the corresponding total area given by a known amount of standard calf thymus histone. Ratios of total histone to DNA of approx. 2 for rat liver nuclei isolated at pH3.8 or 5.8 and for calf liver nuclei isolated at pH3.8 were confirmed twice by the above procedure and also by direct measurement, by the method of Lowry et al. (1951), of histone extracted in 0.2m-H2SO4. The histones of calf thymus, calf liver and rat liver were characterized by their amino acid compositions and by polyacrylamide-gel electrophoresis.
A comparative study of ribonuclease hydrolysis of rat brain-cortex and liver membrane-bound ribosomes
Authors: Simpkins H. , Panko E. , Tay S.
Published: August 2015

Because it has been proposed that the ribosome–membrane interaction is different in endoplasmic reticulum derived from a non-secretory and secretory cell we undertook a study to determine whether attachment of the ribosome to the membrane involved ribosomal RNA and if the rRNA in ribosomes derived from the two classes of cell possessed an altered susceptibility to RNAase (ribonuclease) hydrolysis. We found that brain ribosomes appeared to possess more regions accessible to nuclease attack, independent of whether a sequence-dependent RNAase (T1) or a sterically hindered RNAase bound to Enzite polymer was employed. These results were independent of whether the ribosomes were membrane-bound or detached from the endoplasmic reticulum membranes, but at high RNAase concentration these differences became negligible. No conclusions, however, could be drawn as to whether ribosomal RNA is involved in the attachment of the ribosome to the endoplasmic reticulum membrane, because of the presence of endogeneous membrane-associated RNAases. Analysis of the rRNA fragments by polyacrylamide-gel electrophoresis suggests that the sites available for attack by low concentrations of nuclease in bound-ribosomes derived from brain cortex are different from those of liver.
N-Acetyl-β-d-hexosaminidase component A. Different forms in human tissues and fluids
Authors: Ikonne J. U. , Ellis R. B.
Published: August 2015

1. Hexosaminidase A of human serum was resolved into two components, a minor form with properties identical with those of the single hexosaminidase A component of human liver, and a major form with significantly different properties. 2. The major serum hexosaminidase A form was eluted from a DEAE-cellulose column at a lower salt concentration than that required to elute the liver form. 3. A multiple-pass technique was used to elute the major serum enzyme A from a Sephadex G-150 column before that of liver enzyme A. 4. Clostridium perfringens neuraminidase converted the major component of serum hexosaminidase A into a form that was held less tightly by DEAE-cellulose, but the minor component of the A enzyme of serum, and the A enzyme of liver were not affected. 5. The hexosaminidase A from tears was similar to the A enzyme from serum, whereas those from several human tissues and from urine and lymph were similar to the liver form. 6. The A enzyme from serum may be derived from the A enzyme from liver by glycosylation before secretion.
Separation and partial characterization of two types of alkaline phosphatase from Serratia marcescens (Short Communication)
Authors: Bhatti A. R.
Published: August 2015

The purification and separation of two electrophoretically distinct and chemically different alkaline phosphatases from Serratia marcescens is described.
Purification and some properties of rabbit antiovalbumin
Authors: Ansari Aftab A. , Salahuddin A.
Published: August 2015

Unlike previous reports that the ovalbumin–anti-ovalbumin complex did not dissociate completely in acid media, our results showed complete dissociation of the complex both in 1.2m-acetic acid, pH2.3, and in KCl–HCl, pH2.2, I 0.06. Thus Sephadex chromatography of the solution obtained by dissolving the antigen–antibody precipitate in these media repeatedly gave two peaks corresponding to anti-ovalbumin and ovalbumin. Further, gel-diffusion and immunoelectrophoresis experiments showed that the phosphate groups of ovalbumin are not involved in the antigenic sites. The antibody thus purified was more easily precipitated than previous preparations. The molecular weight and Stokes radius of the antibody were calculated from its gel-filtration behaviour and were found to be 148000 and 4.8nm respectively. The molecular weight determined by sodium dodecyl sulphate–polyacrylamide gel electrophoresis was essentially similar (about 0.7% lower).
The amino acid sequence of cytochrome c′ from Alcaligenes sp. N.C.I.B. 11015
Authors: Ambler R. P.
Published: August 2015

The amino acid sequence of the cytochrome c′ from Alcaligenes sp. N.C.I.B. 11015 (Iwasaki's ‘Pseudomonas denitrificans’) has been determined. This organism is the only non-photosynthetic bacterium in which the protein has been found. The protein consists of a single polypeptide chain of 127 residues, with a single haem covalently attached to two cysteines. Unlike normal cytochromes c, the haem attachment site is very close to the C-terminus. The amino acid sequence around the haem attachment site is very similar to that of Chromatium vinosum D cytochrome c′. Detailed evidence for the amino acid sequence of the protein has been deposited as Supplementary Publication SUP 50022 at the British Library (Lending Division), (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5.
Some characteristics of the coagulation Factor Xa purified from human serum
Authors: Berre Åse Gladhaug , Østerud Bjarne , Christensen Terje B. , Holm Turid , Prydz Hans
Published: August 2015

1. Coagulation Factor X was purified from human serum to apparent homogeneity in disc gel electrophoresis, sodium dodecyl sulphate–polacrylamide-gel electrophoresis, immunoelectrophoresis and analytical ultracentrifugation. The method used was a modification of that described by Gladhaug & Prydz (1970). 2. The method permits the isolation of an activated form of Factor X (Xa) which has a molecular weight of about 25000. 3. Factor Xa is a glycoprotein containing about 14% carbohydrate. A preliminary report of the amino acid composition is given.
Biosynthetic pathway of desmosines in elastin
Authors: Francis Gillian , John Rhys , Thomas John
Published: August 2015

1. Elastins purified by various methods from the ligamentum nuchae and the lungs of cattle of various ages were analysed for amino acid compositions and lysine-derived cross-links. 2. In fully mature elastins from adults the main cross-links were desmosine, isodesmosine and lysinonorleucine and the so-called aldol-condensation product. Trace amounts of merodesmosine were also found. 3. During the normal maturation of elastins the amounts of desmosine, isodesmosine and lysinonorleucine increased, whereas the aldol-condensation product and intact lysine residues decreased and merodesmosine remained the same. 4. Elastins from young animals contained significant amounts of dehydromerodesmosine whereas elastins from adults contained virtually nil. Evidence is presented which suggests that the biosynthetic pathway of desmosine and isodesmosine proceeds via the aldol-condensation product and dehydromerodesmosine.
Steroid-binding properties and stabilization of cytoplasmic glucocorticoid receptors from rat thymus cells
Authors: Bell Philip A. , Munck Allan
Published: August 2015

1. A competitive binding assay was adapted for determination of the specific binding of glucocorticoids to cytoplasmic receptors from rat thymus cells. The steroid–receptor complexes prepared by incubation of a cytoplasmic fraction from rat thymus cells with [1,2-3H2]cortisol or with [1,2,4-3H3]triamcinolone acetonide had rates of dissociation at 37°C similar to those from intact cells. 2. The cytoplasmic receptor was unstable at 3°C, but the rate of inactivation was decreased in the presence of 2.5mm-EDTA. The steroid–receptor complex was stable. 3. Rate constants for association and for dissociation, and association constants, were determined for the interactions of cortisol, cortexolone, dexamethasone and triamcinolone acetonide with the cytoplasmic receptor at 3°C. Differences in the association constants for different steroids could largely be accounted for by the differences in the rate constants for dissociation, but the rate constants for association did not vary greatly; the implications of these findings for the nature of the steroid-binding site are discussed. 4. A cytoplasmic fraction prepared from cells which had been incubated at 37°C under anaerobic conditions bound much less [1,2-3H2]cortisol than did a fraction from aerobic cells, but the binding capacity was restored after exposure of the anaerobic cells to O2. 5. The specific binding of [1,2-3H2]-cortisol to intact thymus cells incubated aerobically was not affected by the presence of 0.1mm-cycloheximide, nor did this concentration of cycloheximide inhibit the recovery of specific binding observed when anaerobic cells were transferred to an aerobic atmosphere. 6. The energy dependence of specific binding of cortisol to the receptor is discussed with reference to possible mechanisms.
The fusion of erythrocytes by fatty acids, esters, retinol and α-tocopherol
Authors: Ahkong Q. F. , Fisher D. , Tampion W. , Lucy J. A.
Published: August 2015

1. The ability of a number of carboxylic acids, their esters, retinol and α-tocopherol to induce fusion of hen erythrocytes in vitro was investigated. 2. Some 30 different fat-soluble substances (100μg/ml) were found to cause the formation of multinucleated erythrocytes with a suspension of 3×108 erythrocytes/ml. The most effective agents induced fusion within 5–10min at 37°C; some substances required about 1h. 3. Inclusion of Dextran 60C in the test medium minimized colloid osmotic lysis caused by exogenous lipids that induce cell fusion. 4. Cell swelling, followed by cell adhesion, was then seen to precede cell fusion. 5. Fusion occurred with C10–C14 saturated carboxylic acids, with unsaturated, longer-chain carboxylic acids and their mono-esters; retinol, and to a lesser extent α-tocopherol, also caused cell fusion. 6. C6–C9, C15, C16 and C18 saturated carboxylic acids did not induce fusion within 4h; glyceryl dioleate was only weakly active, and glyceryl trioleate was inactive in the test system. 7. Fusion was facilitated by a high ratio of chemical agents to cell number and by incubation between pH5 and 6. It was inhibited by EDTA and by serum albumin. 8. Glyceryl mono-oleate caused both a similar fusion of several species of mammalian erythrocyte and the interspecific fusion of human and chicken erythrocytes. 9. The term ‘fusogenic’ is proposed to describe chemical, viral and physical agents that cause membranes to fuse. 10. The biochemical mechanisms involved and the possible biological significance of membrane fusion by fusogenic lipids are discussed.
Identification and measurement of the folates in sheep liver
Authors: Osborne-White William S. , Smith Richard M.
Published: August 2015

1. Methods are described for the extraction, separation by ion-exchange chromatography and estimation by microbiological assay of the folates in sheep liver. 2. Injection of [2-14C]-pteroylglutamate into a sheep fed on a stock diet led to extensive labelling of chromatographically separable liver folates. About 12% of the label in the liver could not be extracted by the method used. 3. Liver folates were examined in five ewes fed on restricted amounts of a diet of wheaten hay-chaff and gluten and injected weekly with vitamin B12. Chromatographic separation was followed by microbiological assay with Lactobacillus casei, Streptococcus faecalis R. and Pediococcus cerevisiae both before and after treatment of fractions with conjugase (γ-glutamylcarboxypeptidase). Evidence was obtained that the folates present were predominantly polyglutamate forms of tetrahydropteroylglutamate, 5-methyltetrahydropteroylglutamate and 5- (and 10-) formyltetrahydropteroylglutamates. Differences in the responses of the assay organisms permitted quantitative distinction between these three main classes of folates. 4. Methyltetrahydrofolates were eluted in seven successive peaks that were separated by constant increments in the logarithm of eluant [Pi]. A similar relationship existed for seven successive peaks of tetrahydrofolate and may also have existed for each of the two series of formyltetrahydrofolates. 5. Based on these and other observations it is proposed that sheep liver folates consist predominantly of the mono- to hepta-glutamates of each of the reduced pteroates identified. The methods employed allowed quantitative determinations to be made of most of the folates present. The predominant forms were hexaglutamates. 6. Four components active for L. casei were detected that could not be identified. Three of them were polyglutamates.
Inhibition of protein synthesis by N-methyl-N-nitrosourea in vivo
Authors: Kleihues P. , Magee P. N.
Published: August 2015

1. The intraperitoneal injection of N-methyl-N-nitrosourea (100mg/kg) caused a partial inhibition of protein synthesis in several organs of the rat, the maximum effect occurring after 2–3h. 2. In the liver the inhibition of protein synthesis was paralleled by a marked disaggregation of polyribosomes and an increase in ribosome monomers and ribosomal subunits. No significant breakdown of polyribosomes was found in adult rat brains although N-methyl-N-nitrosourea inhibited cerebral and hepatic protein synthesis to a similar extent. In weanling rats N-methyl-N-nitrosourea caused a shift in the cerebral polyribosome profile similar to but less marked than that in rat liver. 3. Reaction of polyribosomal RNA with N-[14C]methyl-N-nitrosourea in vitro did not lead to a disaggregation of polyribosomes although the amounts of 7-methylguanine produced were up to twenty times higher than those found after administration of sublethal doses in vivo. 4. It was concluded that changes in the polyribosome profile induced by N-methyl-N-nitrosourea may reflect the mechanism of inhibition of protein synthesis rather than being a direct consequence of the methylation of polyribosomal mRNA.
Lysine metabolism in mammals
Authors: Fellows Florence C. I. , Lewis Maxwell H. R.
Published: August 2015

The enzymes involved in the initial degradative steps of lysine metabolism, lysine–2-oxoglutarate reductase and saccharopine dehydrogenase, were studied and their activities in different mammals compared. Values obtained in human, rat, pig, dog, cat, ox and sheep liver indicated that in vitro, appreciable degradation of lysine to saccharopine (4–6nmol/min per mg of protein) occurred. The specific activity of saccharopine dehydrogenase in most species studied was higher than that of lysine–oxoglutarate reductase. The rate of production of glutamate from saccharopine in each animal species was investigated and related to the rate of production of α-aminoadipate. The rate of formation of lysine from saccharopine, catalysed by saccharopine oxidoreductase, was examined and correlated with the dietary intake of lysine in each species studied.
Biosynthetic studies on aromatic carotenoids. Biosynthesis of chlorobactene
Authors: Moshier S. E. , Chapman D. J.
Published: August 2015

1. The incorporation of [2-14C]mevalonic acid by Chloropseudomonas ethylica strain 2K into chlorobactene was studied. 2. Oxidative degradation of chlorobactene of constant specific radioactivity produced labelled benzenecarboxylic acids and indicated that the benzene ring originates from mevalonic acid. 3. Decarboxylation studies demonstrated a stereospecific methyl migration in the formation of the 1,2,5-trimethylphenyl group of chlorobactene. The migrating methyl group was derived from the C-3′ position of mevalonic acid.
Carotenoid biosynthesis in Rhodopseudomonas spheroides. S-Adenosylmethionine as the methylating agent in the biosynthesis of spheroidene and spheroidenone
Authors: Singh Raj K. , Britton George , Goodwin Trevor W.
Published: August 2015

[methyl-14C]Methionine and S-adenosyl[methyl-14C]methionine were incorporated into the methoxycarotenoids spheroidene and spheroidenone by Rhodopseudomonas spheroides. The incorporation was greatly enhanced in the presence of lysozyme. On degradation of labelled spheroidene by hydriodic acid, the 14C label was recovered in methyl iodide. Degradation of spheroidenone by reduction and allylic dehydration and demethylation of the reduction product gave a mixture of unlabelled carotenoid hydrocarbons, including 3,4-didehydrolycopene and 3,4-didehydro-7′,8′-dihydrolycopene. The label from [methyl-14C]methionine and S-adenosyl[methyl-14C]methionine was located specifically in the methoxy group of spheroidene and spheroidenone. The biosynthesis of methoxycarotenoids in Rps. spheroides involves methylation of the tertiary hydroxyl groups of intermediates with S-adenosylmethionine.
Influence of mitochondria on phospholipid synthesis in preparations from rat liver
Authors: Roberts J. B. , Bygrave F. L.
Published: August 2015

1. The addition of mitochondria to an incubation system containing the soluble and microsomal fractions of rat liver enhances severalfold the incorporation of each of ethanolamine, phosphorylethanolamine and CDP-ethanolamine into phosphatidylethanolamine. 2. In the presence of microsomal, mitochondrial and soluble fractions, CDP-ethanolamine exhibits the greatest initial rate of incorporation (approx. 6nmol/h per mg of protein), being slightly faster than that of phosphorylethanolamine (approx. 5nmol/h per mg of protein). Incorporation of ethanolamine proceeds very slowly for the first 20min and only after 30min gives rates approaching those of the other two precursors. 3. By using a substrate ‘dilution’ technique it was shown that in the reconstituted system the affinity of each of the enzymes for their respective substrates is very high: 10μm for ethanolamine, 25μm for phosphorylethanolamine and 5μm for CDP-ethanolamine. 4. Isolation of the mitochondrial and microsomal fractions from the medium after incubation together with phosphorylethanolamine showed that about 70% of the total radioactivity was present in the microsomal fraction and about 30% in the mitochondria after only 20min. Similar experiments with ethanolamine as precursor revealed that after 20min only about 15% of the total radioactivity was present in the mitochondria but that after 40min about 30% was present in this fraction. 5. Heating and phospholipase treatment of mitochondria, but not freeze-thawing, eliminated the stimulatory effect of mitochondria on phospholipid synthesis. 6. The reconstituted system exhibits an absolute requirement for Mg2+(2mm gave maximal rates) and is inhibited by very low concentrations of Ca2+(100μm-Ca2+produced half-maximal inhibition with 3mm-Mg2+). Further addition of Mg2+overcame the Ca2+inhibition, suggesting that the inhibitory effect is readily reversible. 7. The concept that modification of the Mg2+/Ca2+ratio is a means of controlling the rate of cellular phospholipid synthesis is introduced.
Effect of methylglyoxal bis(guanylhydrazone) on polyamine metabolism in normal and regenerating rat liver and rat thymus
Authors: Hölttä E. , Hannonen P. , Pispa J. , Jänne J.
Published: August 2015

1. Injections of sublethal doses of methylglyoxal bis(guanylhydrazone), a potent inhibitor of putrescine-activated S-adenosylmethionine decarboxylase in vitro, resulted after a few days in an immense increase in the activity of S-adenosylmethionine decarboxylase in normal and regenerating rat liver and in rat thymus. The increase in the activity of S-adenosylmethionine decarboxylase was at least partly due to a marked lengthening of the half-life of the enzyme. 2. In regenerating liver and thymus there was also a moderate stimulation of the activity of ornithine decarboxylase (EC 4.1.1.17) and a marked accumulation of tissue putrescine. 3. Injection of methylglyoxal bis(guanylhydrazone) into the rat also markedly decreased the activity of diamine oxidase (EC 1.4.3.6) in thymus. 4. In no cases where doses of methylglyoxal bis(guanylhydrazone) close to the LD50 dose for the rat were used was it possible to lower tissue spermidine content to any significant extent. 5. Methylglyoxal bis(guanylhydrazone) seemed to act as a competitive inhibitor for the substrate S-adenosylmethionine and as an uncompetitive inhibitor for the activator putrescine in the decarboxylation of S-adenosylmethionine in vitro. 6. In the diamine oxidase reaction, with putrescine as the substrate, methylglyoxal bis(guanylhydrazone) was a non-competitive inhibitor for putrescine.
Species differences in the metabolism of norephedrine in man, rabbit and rat
Authors: Sinsheimer Joseph E. , Dring L. Graham , Williams R. Tecwyn
Published: August 2015

1. (±)-2-Amino-1-phenyl[1-14C]propan-1-ol ([14C]norephedrine) was administered orally to man, rat and rabbit and the metabolites excreted in the urine were identified and measured. Pronounced species differences in the metabolism of the drug were found. 2. Three male human subjects, receiving 25mg each of [14C]norephedrine hydrochloride, excreted over 90% of the14C in the first day. The main metabolite was the unchanged drug (86% of the dose) and minor metabolites were hippuric acid and 4-hydroxynorephedrine. 3. In rats given 12mg of the drug/kg almost 80% of the14C administered was excreted in the first day. The major metabolites in the urine were the unchanged drug (48% of the dose), 4-hydroxynorephedrine (28%) and trace amounts of side-chain degradation products. 4. Rabbits given 12mg of the drug/kg excreted 85–95% of the dose of14C in the urine in the first 24h after dosing. The major metabolites in the urine were conjugates of 1,2-dihydroxy-1-phenylpropane (31% of the dose) and of 1-hydroxy-1-phenylpropan-2-one (27%) and hippuric acid (20%). The unchanged drug was excreted in relatively small amounts (8%).
Qualitative and quantitative aspects of the biosynthesis of ribonucleic acid and of protein in the liver and the lung of the Syrian golden hamster
Authors: Witschi Hanspeter
Published: August 2015

1. The incorporation of orotic acid and of uridine into total RNA was measured in vivo in liver and lung of the Syrian golden hamster. Specific activities of total acid-soluble UMP were measured in both organs. An estimation of the rate of RNA biosynthesis showed that hamster lung synthesizes RNA at about one-half of the rate of that of hamster liver. 2. The apparent Km and Vmax. values of a few enzymes involved in pyrimidine biosynthesis were measured in the 100000g supernatants of liver and lung. The apparent Km values were very similar in both organs. From the estimated Vmax., it was concluded that hamster lung cells have less capacity to metabolize orotic acid than have liver cells. 3. A time–response and a dose–response study showed that actinomycin D inhibits pulmonary RNA synthesis as efficiently as hepatic RNA synthesis. 4. Protein synthesis, measured as the incorporation of leucine, was inhibited in both organs 30min after a dose of 2mg of cycloheximide/kg. The dose–response patterns were similar in both liver and lung 3h after cycloheximide. 5. It is concluded that RNA and protein synthesis in vivo in hamster lung are very similar to the corresponding reactions in liver. Alterations of RNA and protein synthesis by toxic agents can therefore be evaluated in lung with a similar approach to that used to study the pathological biochemistry of liver.