bluuebunny/crossref_metadata_2025 huggingface dataset:
Increase in hepatic catalase and glycerol phosphate dehydrogenase activities on administration of clofibrate and clofenapate to the rat
Authors: Krishnakantha T. P. , Kurup C. K. Ramakrishna
Published: August 2015

1. Clofenapate (methyl 2-[4-(p-chlorophenyl)phenoxy]-2-methylpropionate) fed to the rat in the diet increased the content of mitochondrial protein in the liver by 50–60%. In this respect it resembled the related compound clofibrate (ethyl α-p-chlorophenoxyisobutyrate), which is widely used as an antihypercholesterolaemic drug. 2. Both compounds when fed to the rat enhanced the activity of α-glycerol phosphate dehydrogenase in the liver mitochondria manyfold, but were without effect on the enzyme in the soluble fraction. 3. On the other hand, the catalase activity in the supernatant fraction increased twofold after administration of the drugs. The mitochondrial catalase activity showed a consistent decrease. 4. It was unlikely that under the influence of the drug the increase in catalase activity took place in the mitochondrial particles and was leached into the cytosol during isolation. 5. The increase in catalase activity in the cytosol under the influence of the drug is best explained on the assumption that peroxisomes which contain this enzyme, and which are known to increase on administration of the drug, were broken during the process of cellular fractionation and released the enzyme into the cytosol. 6. All the above effects shown by both drugs were fully reversed when drugs were withdrawn from the diet. 7. Clofenapate was effective in bringing about the above changes when administered to the animal at one-hundredth the concentration of clofibrate.
Proteoglycans of the knee-joint cartilage of young normal and lame pigs
Authors: Šimůnek Z. , Muir Helen
Published: August 2015

Intensive rearing, and restricted activity, induce rapid growth in pigs, but they often become lame. Groups of normal and lame pigs reared intensively were killed when 10 or 25 weeks old. Although there were no differences in the overall composition of the knee-joint cartilage of lame and sound animals, the proteoglycans in the cartilage of the lame pigs were extracted more easily by a standardized sequential procedure and contained a higher proportion of molecules of smaller size as assessed by gel chromatography on 6% agarose and Sepharose 4B. These increased at the expense of both the larger and mediumsized molecules. Differences were most evident at 10 weeks of age, when there was twice as much of the smaller proteoglycans in the cartilage of lame pigs. Despite these size-differences, the compositions of the proteoglycans in corresponding sequential extracts of cartilage of lame and normal groups were the same, as were the changes in chemical composition that accompany development. Proteoglycans from lame animals may have undergone limited proteolysis, thus decreasing their size without changing their composition detectably. As the differences between normal and lame groups were greater at 10 weeks than at 25 weeks of age, the first weeks after birth (when the greatest changes occur in the proteoglycans and in the cartilage) may be a critical period in the maturation of articular cartilage in this species. At this time, rapid gain in weight produced by intensive rearing may be too great for the immature cartilage to bear.
Amphibian ribosomal ribonucleic acids
Authors: Rogers M. Elizabeth , Klein Gerda
Published: August 2015

1. The larger rRNA molecules of different species of amphibia do not all have the same mobility on polyacrylamide gels, and the difference appears to be one between the Anuran and Urodele amphibia. 2. Alteration of conditions of electrophoresis so as to distinguish between differences of conformation and molecular weight does not affect the results. 3. The mobility on gel electrophoresis of the rRNA precursor in two species of amphibia is similar, although the intermediate processing products differ. 4. It is suggested that the mobility differences observed represent differences of molecular weight, and are an example of the variability of the products of transcription and processing of rRNA genes in two amphibian genera.
Low-molecular-weight solutes released during mild acid hydrolysis of the lipopolysaccharide of Pseudomonas aeruginosa. Identification of ethanolamine triphosphate
Authors: Drewry David T. , Gray George W. , Wilkinson Stephen G.
Published: August 2015

A careful examination of the low-molecular-weight solutes released during mild acid hydrolysis of the lipopolysaccharide of Pseudomonas aeruginosa (N.C.T.C. 1999) revealed the presence of ethanolamine triphosphate. During storage, the compound decomposed to give ethanolamine pyrophosphate, identified in a previous study (Drewry et al., 1971); PPi may be a further decomposition product. Evidence for the attachment of ethanolamine triphosphate to a polysaccharide fraction was obtained, but the possibility that some was attached to the lipid A moiety was not excluded. Basic compounds released during the hydrolysis of lipopolysaccharide included amino acids, polyamines and oligopeptides.
Properties of halophil nicotinamide–adenine dinucleotide phosphate-specific isocitrate dehydrogenase. True Michaelis constants, reaction mechanisms and molecular weights
Authors: Aitken D. M. , Brown A. D.
Published: August 2015

True values of Michaelis constants of the NADP+-specific isocitrate dehydrogenase from Halobacterium salinarium were not very different from those of the apparent constants reported by Aitken et al. (1970). The true constants were affected by salt in a similar manner to that of the apparent constants obtained with NADP+ at fixed concentrations of 1.0–0.2mm and threo-ds-(+)-isocitrate at fixed concentrations of 2.0–0.125mm. The response of apparent Vmax. to salt concentration was highly dependent on fixed substrate concentration in solutions of sodium chloride but much less so in solutions of potassium chloride. At several levels the results emphasize the difficulty of generalizing about the salt relations of a halophil enzyme without adequate attention to substrate concentration. The enzyme has at least two different reaction mechanisms depending on salt concentration. In its ‘physiological’ form (i.e. in 1.0m-potassium chloride), and also in 1.0m-sodium chloride, the reaction mechanism is ordered with NADP+ the first substrate added and NADPH the last product released. In 0.25m-sodium chloride, however, the mechanism is different and is probably non-sequential. In 4.0m-sodium chloride with low concentrations of either fixed substrate, there was evidence of a co-operative action of the variable substrate. The evidence suggests that salt participates in the reaction mechanism in two ways: one is the reversible addition to the enzyme in a manner analogous to that of a substrate; the other is dead-end complex-formation. The relative contributions of these two types of reaction determine whether salt activates or inhibits the enzyme. In addition, the inhibition caused by high concentrations of sodium chloride is more complex than the corresponding inhibition by potassium chloride. Gel-filtration experiments indicated that at very low salt concentrations the enzyme has an apparent molecular weight of about 70800. In ‘physiological’ concentrations of potassium chloride the enzyme appears to be a dimer (mol.wt. 122000–135000) and, in 1.0–4.0m-sodium chloride, it behaves as a trimer or tetramer (mol.wt. 224000–251000). A preliminary method of purifying the enzyme is described.
Deoxyribonucleic acid synthesis in mammalian systems. Permealysed Ehrlich ascites cells in vitro first label Okazaki-type low-molecular-weight deoxyribonucleic acid and then high-molecular-weight deoxyribonucleic acid
Authors: Burgoyne Leigh A.
Published: August 2015

During the evaluation of a method of preparing permealysed Ehrlich ascites cells, shortterm labelling experiments were carried out with d[3H]TTP. In the first minute the bulk of the label appeared as low-molecular-weight pieces of DNA. Subsequently the label appeared in DNA of much higher molecular weight. A brief description of the preparation procedure and the properties of the product is provided. Evidence is presented to show that the nucleotide was incorporated directly without intermediate conversion into dTMP or thymidine.
Immunological properties of N-acetyl-β-d-glucosaminidase of normal human liver and of GM2-gangliosidosis liver
Authors: Carroll M. , Robinson D.
Published: August 2015

Antisera were raised to a partially purified preparation of human liver hexosaminidase and to highly purified preparations of hexosaminidase isoenzymes A and B. All the antisera precipitated the enzyme in an enzymically active form, which could be located on immunodiffusion and immunoelectrophoretic gels by using a histochemical substrate. The antisera to the purified isoenzymes were shown to react with hexosaminidase from human liver, kidney, brain and spleen, but did not cross-react with human liver β-glucosidase, β-galactosidase, α-mannosidase, β-xylosidase, arylsulphatase or acid phosphatase. Hexosaminidases A and B were immunologically identical. The immunological properties of the hexosaminidases from livers of patients with three types of GM2-gangliosidoses were closely similar. No evidence could be found for cross-reacting material in enzyme-deficient states.
The effect of a lipid-rich diet on the properties and composition of lipoprotein particles from the Golgi apparatus of guinea-pig liver
Authors: Chapman M. J. , Mills G. L. , Taylaur C. E.
Published: August 2015

1. A cell fraction rich in Golgi apparatus was isolated from the livers of guinea pigs fed on a lipid-rich diet (1.6% cholesterol, 15% corn oil). 2. The Golgi cisternae and secretory vesicles contained electron-dense particles which were tentatively identified as VLD (very-low-density) and LD (low-density) lipoproteins. Particles of moderate electron density, 150–500nm in diameter, were seen associated with membranous elements of the Golgi-apparatus cell fraction. Disruption of this cell fraction permitted the release of these three species of particles, which were separated into particulate lipid, and VLD and LD lipoproteins. 3. The large particles of moderate electron density, isolated as particulate lipid, were distinct from both species of Golgi particles in their chemical composition and in possessing an immunochemically unreactive apolipoprotein(s). Morphological observations suggest that the particulate lipid arose from cytoplasmic lipid droplets which were present as contaminants of the Golgi-rich fraction. 4. The chemical and immunochemical results are consistent with the suggestion that the Golgi LD particles are precursors of the VLD particles, into which they may be transformed by the addition of both triglyceride and cholesteryl ester. The present results provide further support for the proposal that the Golgi VLD particles are precursors of the serum VLD lipoproteins in the guinea pig. 5. Hepatic Golgi VLD particles isolated from guinea pigs fed on the lipid-rich diet contained significantly higher molar amounts (relative to protein) of both cholesteryl ester and triglyceride than similar particles from animals fed on a normal diet. These results suggest that the type of Golgi VLD particle produced from the LD particle is a direct consequence of the amount and composition of the dietary lipid. 6. Hepatic Golgi LD particles isolated from guinea pigs fed on different diets were similar in chemical composition and contained approx. 50% by weight of phospholipid. We conclude that the Golgi LD particle is normally present in the Golgi-apparatus cell fraction from guinea-pig liver, and may represent the end product of lipoprotein biosynthesis in the smooth endoplasmic reticulum. 7. The serum LD lipoproteins and Golgi LD particles were quite distinct in chemical composition. However, these two lipoprotein species were immunochemically identical and exhibited a similar range of flotation rate. It appears unlikely that the Golgi LD particles are secreted as the precursors of the serum LD lipoproteins.
The amino acid sequence of cytochrome c from Allium porrum L. (leek)
Authors: Brown R. H. , Boulter D.
Published: August 2015

The amino acid sequence of leek cytochrome c was determined with 0.4μmol of protein. The sequence was deduced solely from a chymotryptic digest. The cytochrome was homologous with other plant cytochromes c of mitochondrial origin. Leek cytochrome c has an N-acetylated ‘tail’ as compared with mammalian cytochrome c, and two residues of ε-N-trimethyl-lysine. Unlike other plant cytochromes c, leek cytochrome c has glutamic acid or glutamine in position 11, leucine in position 20 and alanine in position 51. 4-Hydroxyproline partially substitutes for proline in position 79. Experimental details are given in a supplementary paper that has been deposited as Supplementary Publication SUP 50012, at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973) 131, 5.
Protein kinase activity in commercially available crystalline yeast alcohol dehydrogenase
Authors: Kemp B. E. , Froscio M. , Murray A. W.
Published: August 2015

Commercially available crystalline yeast alcohol dehydrogenase contained protein kinase activity. Casein and phosvitin were readily phosphorylated, but whole calf thymus histone was not. The protein kinase activity was inhibited by KCl, was not stimulated by cyclic AMP and could be separated from the alcohol dehydrogenase activity by sucrose density centrifugation.
Phospholipase A1 of human blood platelets (Short Communication)
Authors: Smith J. B. , Silver M. J. , Webster G. R.
Published: August 2015

Substantial phospholipase A1 activity has been demonstrated in human blood platelets, and a rapid method for its measurement is described. The enzyme requires taurocholate for full activity and in these conditions the pH optimum is 4.8. The phospholipase activity is released from platelets by incubation with thrombin.
Wheat-germ aspartate transcarbamoylase. The effect of ligands on the inactivation of the enzyme by trypsin and denaturing agents
Authors: Yon Robert J.
Published: August 2015

In the absence of added ligands aspartate transcarbamoylase (EC 2.1.3.2) from wheat germ is inactivated fairly rapidly by trypsin, by heat (60°C), by highly alkaline conditions (pH11.3) and by sodium dodecyl sulphate. Addition of UMP alone, at low concentrations, decreases the rate of inactivation by each of these agents significantly. Carbamoyl phosphate alone does not alter the rate of inactivation by trypsin and by the detergent, but it antagonizes the effect of UMP in protecting the enzyme against these agents. These results have been interpreted to mean that two conformational states are reversibly accessible to the enzyme, namely an easily inactivated state favoured in the presence of carbamoyl phosphate and a more resistant state favoured in the presence of UMP. In the absence of ligands the enzyme is in the easily inactivated conformation. At very high concentrations l-aspartate also protects the enzyme but to a smaller extent than UMP. Some implications of these results are discussed.
Human cathepsin B1. Inhibition by α2-macroglobulin and other serum proteins
Authors: Starkey Phyllis M. , Barrett Alan J.
Published: August 2015

1. Normal human serum was found to inhibit human cathepsin B1. 2. The major inhibitor present in serum was purified and identified as α2-macroglobulin. 3. α2-Macroglobulin was found to bind cathepsin B1 in an approximately 1:1 molar ratio. When bound, the enzyme retained about 50% of its proteolytic activity, and up to 80% of its activity against α-N-benzoyl-dl-arginine 2-naphthylamide. 4. Pretreatment of α2-macroglobulin with cathepsin B1 inactivated by exposure to pH8.5 or iodoacetic acid, in large molar excess, did not prevent the subsequent binding of active enzyme. Active enzyme, once bound, was not protected from inhibition by 1-chloro-4-phenyl-3-tosylamido-l-butan-2-one. 5. Cathepsin B1 was also inhibited by human immunoglobulin G, at high concentration. 6. Because it had been suggested that haptoglobin is responsible for the inhibition of ‘cathepsin B’ by serum, a method was devised for the selective removal of haptoglobin from mixtures of serum proteins by adsorption on haemoglobin covalently linked to Sepharose. No evidence was obtained that haptoglobin has any inhibitory activity against the enzyme.
Choline acetyltransferase in the nettle Urtica dioica L
Authors: Barlow Richard B. , Dixon Robert O. D.
Published: August 2015

Extracts of acetone-dried powders prepared from nettle leaves were shown to catalyse the synthesis of acetylcholine. The specific activity of the enzyme in these extracts is of the same order as that of extracts from mammalian sources, such as ox brain, and the effects of temperature and pH are similar to those reported for mammalian choline acetyltransferase. Synthesis is not restricted to the younger leaves but appears to be continuous up to senescence.
The utilization of fructose by Escherichia coli. Properties of a mutant defective in fructose 1-phosphate kinase activity
Authors: Ferenci T. , Kornberg H. L.
Published: August 2015

1. The isolation and properties of a mutant of Escherichia coli devoid of fructose 1-phosphate kinase activity are described. 2. This mutant grew in media containing any one of a variety of substances, including hexoses, hexose 6-phosphates, sugar acids and glucogenic substrates, at rates not significantly different from those at which the parent organism grew on these substrates. However, only the parent grew on fructose or fructose 1-phosphate. 3. Fructose and fructose 1-phosphate inhibit the growth of the mutant, but not of its parent, on other carbon sources. 4. Even though not previously exposed to fructose, the mutant took up [14C]fructose rapidly but to only a small extent: [14C]fructose 1-phosphate was identified as the predominant labelled product. In contrast, the equally rapid but more extensive uptake of [14C]fructose by the parent organism required prior growth in the presence of fructose.
Regulation by cytidine nucleotides of the acylation of sn-[14C]glycerol 3-phosphate. Regional and subcellular distribution of the enzymes responsible for phosphatidic acid synthesis de novo in the central nervous system of the rat
Authors: Possmayer F. , Meiners B. , Mudd J. B.
Published: August 2015

1. The regional and subcellular distribution of the incorporation of sn-[14C]glycerol 3-phosphate into rat brain lipids in vitro was investigated and compared with the relative specific activity of various chemical and enzyme markers. The similarity between the subcellular distribution of this incorporation and of NADPH-cytochrome c reductase activity indicated that the synthesis of phosphatidic acid via this route correlated with the presence of endoplasmic reticulum. 2. Experiments in which various amounts of the microsomal fraction were added to fixed amounts of nuclear, myelin, nerve-ending and mitochondrial preparations clearly demonstrated that the endoplasmic-reticulum contamination of these fractions was entirely responsible for the incorporation of sn-[14C]glycerol 3-phosphate. 3. The presence of CMP or CTP inhibited the incorporation of sn-[14C]glycerol 3-phosphate into the whole homogenate. Similar effects were observed with individual fractions, except for the mitochondria. With the mitochondrial fraction the effect of these cytidine nucleotides varied with the preparation, stimulating in some preparations and inhibiting with other preparations. The presence of CDP-choline stimulated the incorporation into the whole homogenate and to a lesser extent into the subcellular fractions. 4. These results indicate that the various organelles of the central nervous system are more dependent on endoplasmic reticulum for the production of glycerolipids de novo than has previously been appreciated.
Androgen dynamics in vitro in the human prostate gland. Effect of cyproterone and cyproterone acetate
Authors: Giorgi Eleonora P. , Shirley I. M. , Grant J. K. , Stewart Joan C.
Published: August 2015

Hyperplastic and adenocarcinomatous human prostatic tissue was superfused in vitro with radioactively labelled androst-4-ene-3,17-dione, testosterone and 5α-dihydrotestosterone (17β-hydroxy-5α-androstan-3-one), with and without addition of the anti-androgens cyproterone and cyproterone acetate. Cyproterone competitively inhibited the entry of the androgens into the majority of the tissues, whereas cyproterone acetate increased this entry. These findings indicated that transport of androstenedione, testosterone and 5α-dihydrotestosterone into prostatic tissue is performed by a specific mechanism, possibly involving a carrier situated in the cell membrane. The extent of metabolism of the three androgens was also modified: formation of 5α-dihydrotestosterone from testosterone, and of the latter from androstenedione, was decreased by cyproterone and increased by the acetate. Acetate was more effective than cyproterone in decreasing the `uptake' of the perfused androgens by the tissue; at the same time, it increased the androgen clearance from the tissue. As cyproterone acetate is the more potent of the two anti-androgens, the possibility that these findings in vitro are related to the different anti-androgenic potency exhibited by the two compounds in vivo is discussed. `Uptake' of the two anti-androgens and the response to their action on androgen dynamics were similar in adenocarcinomatous and hyperplastic glands.
Developmental changes in microheterogeneity of foetal plasma glycoproteins of mice
Authors: Gustine David L. , Zimmerman Ernest F.
Published: August 2015

Changes in microheterogeneity of foetal plasma glycoproteins during development of mouse embryos were investigated. Analysis of foetal plasma by polyacrylamide-gel electrophoresis indicated three major zones of proteins: (1) transferrins, (2) α-foetoproteins and (3) albumin. Three transferrins (Tr1, Tr2, Tr3) and five α-foetoproteins (Fp1, Fp2, Fp3, Fp4, Fp5) were resolved. Evidence for the presence of transferrins was the binding of 59Fe to the three electrophoretic variants. By day 15.5 of gestation, there was a marked increase in the more-acidic components (Tr3, Fp4, Fp5) and a decrease in the less-acidic ones (Tr1, Tr2, Fp1, Fp2, Fp3). Treatment of foetal plasma with neuraminidase at this time of development converted the more acidic components into Tr1 and Tr2 and Fp1, Fp2 and Fp3. Furthermore, it was shown that early in development (day 12.5) only the less-acidic components of transferrin and α-foetoprotein were synthesized; at the later time in development (day 14.5) new synthesis of the acidic components of both groups occurred. That these more-acidic components of α-foetoprotein (Fp4, Fp5) were in fact electrophoretic variants of the less-acidic α-foetoproteins was shown by the immunoprecipitation of labelled Fp4 and Fp5 with anti-Fp1, anti-Fp2 and anti-Fp3. From these results it is postulated that the plasma glycoproteins that are synthesized later in development contain increased amounts of sialic acid and that the observed changes in microheterogeneity of these proteins represent regulation of glycoprotein biosynthesis at the level of carbohydrate attachment.
Interrelationship of glutathione–cystine transhydrogenase and glutathione reductase in developing rat intestine
Authors: States Beatrice , Segal Stanton
Published: August 2015

1. Glutathione reductase and glutathione–cystine transhydrogenase activity in supernatant fractions of whole homogenates and homogenates of mucosal and muscular layers were determined in developing rat intestine after determination of the optimum conditions for assay of the two enzymes. In jejunum from adult rat, the Km values for GSSG reductase and GSH–cystine transhydrogenase activities were 0.25mm-GSSG and 0.23mm-cystine respectively. 2. The two activities could be differentiated by stability studies since GSSG reductase was stable at 60°C for 10min and could be stored at 4°C for 24h without loss of activity. GSH–cystine transhydrogenase, on the other hand, was denatured at 60°C and completely inactive after 24h storage at 4°C. 3. Based on calculations of total activities, both enzymes increased from the eighteenth day until the animals were young adults. 4. Total GSSG reductase activity increased at a greater rate with age than total GSH–cystine transhydrogenase activity as evidenced by activity ratios for GSH–cystine transhydrogenase/GSSG reductase of 0.44 and 0.12 in ileum from suckling and adult rats respectively, and 0.31 and 0.24 in jejunum from suckling and adult rats respectively. 5. In mucosa from adult rats GSSG reductase was more active in the ileum than in the jejunum, whereas GSH–cystine transhydrogenase activity was higher in the jejunum. 6. GSH–cystine transhydrogenase was active only in the muscle cells of the ileum of 7-day-old rats but became localized primarily in the mucosal layer in the adult rat. However, GSSG reductase activity was distributed evenly between the two layers throughout the intestine.
Glucose metabolism in the newborn rat. Temporal studies in vivo
Authors: Snell Keith , Walker Deryck G.
Published: August 2015

1. The concentrations of plasma d-glucose, l-lactate, free fatty acids and ketone bodies and of liver glycogen were measured in caesarian-delivered newborn rats at time-intervals up to 4h after delivery. Glucose and lactate concentrations decreased markedly during the first hours after delivery, but there was a delay of 60–90min before significant glycogen mobilization occurred. 2. The specific radioactivity of plasma d-glucose was measured as a function of time for up to 75min after the intraperitoneal injection of d-[6-14C]glucose and d-[6-3H]glucose into caesarian-delivered rats at 0, 1 and 2h after delivery. Calculations revealed that there was an appreciable rate of glucose formation at all ages studied, but immediately after delivery this was exceeded by the rate of glucose utilization. Around 2h post partum the rate of glucose utilization decreased dramatically and this coincided with a reversal of the immediately postnatal hypoglycaemia. 3. The specific radioactivity of plasma l-lactate and the incorporation of 14C into plasma d-glucose and liver glycogen was measured as a function of time after the intraperitoneal injection of l-[U-14C]lactate into rats immediately after delivery. The logarithm of the specific radioactivity of plasma l-[U-14C]lactate decreased linearly with time for at least 60min after injection and the calculated rate of lactate utilization exceeded the rate of lactate formation. 4. 14C incorporation into plasma d-glucose was maximal from 30–60min after injection of l-[U-14C]lactate and the amount incorporated at 60min was 23% of that present in plasma lactate. Although 14C was also incorporated into liver glycogen the amount was always less than 3% of that present in plasma glucose. 5. The results are discussed in relationship to the adaptation of the newly born rat to the extra-uterine environment and the possible involvement of gluconeogenesis at this time before feeding is established.
The purification and properties of extracellular glycosidases of the cellular slime mould Dictyostelium discoideum
Authors: Every D. , Ashworth J. M.
Published: August 2015

The purification of β-N-acetylhexosaminidase, α-glucosidase, α-mannosidase and β-glucosidase from the spent growth medium of Dictyostelium discoideum strain Ax-2 myxamoebae is described. β-N-Acetylhexosaminidase and α-glucosidase were obtained in high yield and as homogeneous preparations whereas the α-mannosidase preparation consisted of two electrophoretically distinct isoenzymes. The physical, chemical and kinetic properties of these enzymes are described.
The redox potentials of the two-iron plant algal ferredoxins. An electrostatic model
Authors: Kassner R. J. , Yang W.
Published: August 2015

The two-iron–sulphur co-ordination centre in plant and algal ferredoxins is considered as a collection of charged ions whose net negative charge is twice that of the one-iron–sulphur protein rubredoxin. Calculation of the electrostatic free-energy changes for reduction of the two types of proteins indicates that the redox potential of the two-iron–sulphur proteins should be more negative than that of the one-iron–sulphur protein and that in biological systems the ferredoxins should function as one-electron transfer proteins.
Subunit interactions in horse spleen apoferritin. Dissociation by extremes of pH
Authors: Crichton R. R. , Bryce Charles F. A.
Published: August 2015

1. The dissociation of horse spleen apoferritin as a function of pH was analysed by sedimentation-velocity techniques. The oligomer is stable in the range pH2.8–10.6. Between pH2.8 and 1.6 and 10.6 and 13.0 both oligomer and subunits can be detected. At pH values between 1.6 and 1.0 the subunit is the only species observed, although below pH1.0 aggregation of the subunits to a particle sedimenting much faster than the oligomer occurs. 2. When apoferritin is first dissociated into subunits at low pH values and then dialysed into buffers of pH1.5–5.0, the subunit reassociates to oligomer in the pH range 3.1–4.3. 3. U.v.-difference spectroscopy was used to study conformational changes occurring during the dissociation process. The difference spectrum in acid can be accounted for by the transfer of four to five tyrosine residues/subunit from the interior of the protein into the solvent. This process is reversed on reassociation, but shows the same hysteresis as found by sedimentation techniques. The difference spectrum in alkali is more complex, but is consistent with the deprotonation of tyrosine residues, which appear to have rather high pK values. 4. In addition to the involvement of tyrosine residues in the conformational change at low pH values, spectral evidence is presented that one tryptophan residue/subunit also changes its environment before dissociation and subsequent to reassociation. 5. Analysis of the dissociation and reassociation of apoferritin at low pH values suggests that this is a co-operative process involving protonation and deprotonation of at least two carboxyl functions of rather low intrinsic pK. The dissociation at alkaline pH values does not appear to be co-operative. 6. Of the five tyrosine residues/subunit only one can be nitrated with tetranitromethane. Guanidination of lysine residues results in the modification of seven out of a total of nine residues/subunit. Nine out of the ten arginine residues/subunit react with cyclohexanedione.
Polypyrroles formed from porphobilinogen and amines by uroporphyrinogen synthetase of Rhodopseudomonas spheroides
Authors: Davies Richard C. , Neuberger Albert
Published: August 2015

1. Uroporphyrinogen I synthetase of Rhodopseudomonas spheroides was purified more than 200-fold from the soluble protein of broken bacterial cells. The enzyme had molecular weight 36000, an isoelectric point of 4.46 and migrated as a single active protein band on disc-gel electrophoresis at pH7.5 and 8.9. 2. The enzyme consumed porphobilinogen and formed uroporphyrinogen at pH8.2 without the accumulation of intermediates. In the presence of hydroxylamine, ammonia or methoxyamine the production of porphyrinogen was inhibited and the enzyme formed open-chain polypyrroles instead. 3. These polypyrroles behaved like uroporphyrinogen on Sephadex G-25; they were colourless and had unsubstituted α-pyrrolic positions. The inhibitory amines were incorporated into the molecules. 4. The polypyrroles formed porphyrins non-enzymically and the cyclization reaction was accompanied by the release of the inhibitory amine. Exchange of the amino function of the original porphobilinogen in the polypyrrole was complete with hydroxylamine and almost complete with methoxyamine, both ammonia and methoxyamine being present in the polypyrrolic material. 5. The behaviour, properties and composition of the radioactive hydroxylamine derivative were consistent with a tetrapyrrolic structure, probably a pyrrylmethane, that was not cyclized, rather than with di-, tri- or penta-pyrrolic structures. No monopyrrolic or dipyrrolic Ehrlich-positive material was released on cyclization. The ammonia and methoxyamine derivatives had properties similar to the hydroxylamine derivative. 6. Another modified pyrrole was detected only in experiments with hydroxylamine. It differed from both porphobilinogen and known dipyrroles and appeared to be a monopyrrole. 7. The participation of positively charged reaction centres in the enzymic mechanism, particularly in the cyclization step, is discussed.
The labelling of proteins to high specific radioactivities by conjugation to a 125I-containing acylating agent. Application to the radioimmunoassay
Authors: Bolton A. E. , Hunter W. M.
Published: August 2015

1. A new method is described for labelling proteins to high specific radioactivities with 125I. The protein is treated with a 125I-labelled acylating agent, iodinated 3-(4-hydroxyphenyl)propionic acid N-hydroxysuccinimide ester, which reacts with free amino groups in the protein molecule to attach the 125I-labelled groups by amide bonds. 2. Three protein hormones have been labelled by this method, human growth hormone, human thyroid-stimulating hormone and human luteinizing hormone. Specific radioactivities of up to 170, 120 and 55μCi/μg respectively have been obtained for these hormones. 3. The immunoreactivity of these labelled hormones has been investigated by using a radioimmunoassay system specific for each hormone. These preparations have also been compared with and found to be equal or superior to labelled hormones prepared by chemical substitution of 125I into tyrosine residues of the proteins by using the chloramine-t-oxidation procedure. 4. With some antisera the immunoreactivity of the antigen was diminished by the introduction of a single I atom into the tyrosyl groups, whereas antigen containing a single 125I-labelled 3-(4-hydroxyphenyl)propionamide group showed the same immunoreactivity as the unmodified antigen.
Determination of asparagine, glutamine and pyrrolidonecarboxylic acid in total enzymic hydrolysates of peptides and glycopeptides by gas–liquid chromatography
Authors: Hediger Hedy , Stevens Richard L. , Brandenberger Hans , Schmid Karl
Published: August 2015

A new procedure for the qualitative and quantitative determination of asparagine, glutamine and pyrrolidonecarboxylic acid in total enzymic hydrolysates of peptides and glycopeptides based on g.l.c. has been developed. Under the conditions of esterification and trifluoroacetylation N-trifluoroacetylaspartic acid mono-n-butyl ester was formed from asparagine and N-trifluoroacetylglutamic acid mono-n-butyl ester from both glutamine and pyrrolidonecarboxylic acid. To distinguish between the latter two compounds, the esterification was carried out at room temperature yielding 30% of esterified pyrrolidonecarboxylic acid but less than 1% of esterified glutamine. In extending the g.l.c. of amino acids, the previously unknown positions in the g.l.c. elution pattern of the following amino acids could also be reproducibly determined: carboxymethylcysteine, homoserine, hydroxylysine and ∈-methyl-lysine. Further, certain glycopeptides were investigated and the artifacts due to their carbohydrate moieties were determined.
Kinetic studies on the acid hydrolysis of the methyl ketoside of unsubstituted and O-acetylated N-acetylneuraminic acid
Authors: Neuberger A. , Ratcliffe Wendy A.
Published: August 2015

The hydrolysis of the model compound 2-O-methyl-4,7,8,9-tetra-O-acetyl-N-acetyl-α-d-neuraminic acid and neuraminidase (Vibrio cholerae) closely resembled that of the O-acetylated sialic acid residues of rabbit Tamm–Horsfall glycoprotein. This confirmed that O-acetylation was responsible for the unusually slow rate of acid hydrolysis of O-acetylated sialic acid residues observed in rabbit Tamm–Horsfall glycoprotein and their resistance to hydrolysis by neuraminidase. The first-order rate constant of hydrolysis of 2-methyl-N-acetyl-α-d-neuraminic acid by 0.05m-H2SO4 was 56-fold greater than that of 2-O-methyl-4,7,8,9-tetra-O-acetyl-N-acetyl -α-d-neuraminic acid. Kinetic studies have shown that in the pH range 1.00–3.30, the observed rate of hydrolysis of 2-methyl-N-acetyl-α-d-neuraminic acid can be attributed to acid-catalysed hydrolysis of the negatively charged CO2− form of the methyl ketoside.
An investigation into the role of malonyl-coenzyme A in isoprenoid biosynthesis
Authors: Higgins M. J. P. , Kekwick R. G. O.
Published: August 2015

1. [14C]Malonyl-CoA was incorporated into isoprenoids by cell-free yeast preparations, by preparations from pigeon and rat liver, and by Hevea brasiliensis latex. 2. In agreement with previous reports the incorporation of acetyl-CoA into isoprenoids was not inhibited by avidin and was not stimulated by HCO3−. In a cell-free yeast preparation addition of HCO3− stimulated the formation of fatty acids from acetyl-CoA and decreased the incorporation into unsaponifiable lipids. 3. The labelling patterns of β-hydroxy-β-methylglutaryl-CoA formed from [2-14C]- and [1,3-14C]-malonyl-CoA in rat and pigeon liver preparations were those that would be expected if malonyl-CoA underwent decarboxylation to acetyl-CoA before incorporation. 4. The labelling pattern of ergosterol formed by cell-free yeast preparations from [2-14C]malonyl-CoA was also consistent with decarboxylation of malonyl-CoA before incorporation. 5. The incorporation of [2-14C]malonyl-CoA into mevalonate by rat liver preparations was related to the malonyl-CoA decarboxylase activity present in the preparation.
Increased liver l-serine–pyruvate aminotransferase activity under gluconeogenic conditions (Short Communication)
Authors: Rowsell Edward V. , Al-Tai Ali H. , Carnie John A. , Rowsell Kathleen V.
Published: August 2015

Rat liver l-serine–pyruvate aminotransferase activity exceeds markedly the normal adult value (a) in the neonatal period, (b) after glucagon injection and (c) after alloxan injection, observations that reinforce the suggestion from comparative findings that the aminotransferase has a role in gluconeogenesis. Some findings, however, argue in favour of l-serine dehydratase as the enzyme of gluconeogenesis from l-serine.
The microbial metabolism of thiophen-2-carboxylate
Authors: Cripps Roger E.
Published: August 2015

1. An organism was isolated by enrichment culture that was capable of using thiophen-2-carboxylate as sole source of carbon, energy and sulphur for growth. 2. Analysis of the cellular protein after growth of the organism on thiophen-2-[14C]carboxylate showed that only glutamate, proline and arginine were labelled. All the radioactivity in the glutamate was confined to C-1. 3. In the presence of 2.1 mm-arsenite, suspensions of the organism converted thiophen-2-[14C]carboxylate into 14C-labelled 2-oxoglutarate which had the same specific radioactivity as the starting material. 4. Cell-free extracts of the organism catalysed the release of 14CO2 from thiophen-2-[14C]carboxylate. This activity was largely dependent on the presence of ATP and CoA and was stimulated by NAD+ and Mg2+. Inclusion of hydroxylamine resulted in the appearance of thiophen-2-carbohydroxamic acid, indicating that the ATP and CoA were involved in the formation of the CoA ester of thiophen-2-carboxylate. 5. High-speed centrifuging of cell-free extracts resulted in supernatants with decreased thiophen-2-carboxylate-degrading activity. Activity was restored by the addition of the high-speed pellet or by Methylene Blue. 6. The metabolism of the CoA ester of thiophen-2-carboxylate by cell-free extracts could be linked to the anaerobic reduction of Methylene Blue. 7. The sulphur atom of the thiophen nucleus was converted into sulphate by growing cultures and resting suspensions of the organism. 8. A degradative pathway is proposed involving the hydroxylation (at C-5) of the CoA ester of thiophen-2-carboxylate followed by further metabolism to 2-oxoglutarate and sulphate.
The distribution of ribosomes between different functional states in livers of fed and starved mice
Authors: Norman Michael , Gamulin Stjepan , Clark Kay
Published: August 2015

1. To investigate the role of ribosome function in regulating protein synthesis, the activity, distribution and functional states of ribosomal particles were investigated in livers of mice fed ad libitum or starved overnight. 2. The distribution of protein-synthesizing activity between polyribosomes of different sizes was analysed after incorporation of radioactive leucine, and the quantitative distribution of ribosomes as native subunits, monomers and polyribosomes was analysed after incorporation of orotic acid. Precursors labelled with 3H or 14C were given separately to fed and starved mice, so that livers from the two groups of animals were processed together. 3. The former experiments showed that starvation has little effect on the distribution of protein-synthesizing activity across polyribosome sedimentation patterns, though the latter experiments showed that the proportion of ribosomes existing as monomers increased from 9.5% to 15.2%, whereas the proportion existing as polyribosomes decreased from 81.4% to 75.6%. Starvation had a negligible effect on the proportion of native subunits, which accounted for 9.1% and 9.2% of the ribosomes in fed and starved mice respectively. 4. The monomeric ribosome fraction was isolated and subjected to ionic conditions which selectively dissociate single ribosomes. Starvation increased the proportion of monomers that dissociated from 59% to 72%, so the monomers that accumulate in livers of starved animals are single ribosomes and not monoribosomes resulting from degradation of polyribosomes. 5. The fate of newly formed ribosomal particles was studied by measuring the specific radioactivity of native subunits, monomers and polyribosomes at different times after injection of radioactively labelled orotic acid. Starvation did not appear to affect equilibration between newly formed particles and polyribosomes, and the radioactivity of polyribosomes in both groups of mice reached about 90% of that in native subunits after 4h. The radioactive labelling of monomers proceeded at a slower rate, especially after starvation. At 4h, the radioactivity of monomers was 64% and 55% that of native subunits in fed and starved mice respectively.
The mitochondrial generation of hydrogen peroxide. General properties and effect of hyperbaric oxygen
Authors: Boveris Alberto , Chance Britton
Published: August 2015

1. Pigeon heart mitochondria produce H2O2 at a maximal rate of about 20nmol/min per mg of protein. 2. Succinate–glutamate and malate–glutamate are substrates which are able to support maximal H2O2 production rates. With malate–glutamate, H2O2 formation is sensitive to rotenone. Endogenous substrate, octanoate, stearoyl-CoA and palmitoyl-carnitine are by far less efficient substrates. 3. Antimycin A exerts a very pronounced effect in enhancing H2O2 production in pigeon heart mitochondria; 0.26nmol of antimycin A/mg of protein and the addition of an uncoupler are required for maximal H2O2 formation. 4. In the presence of endogenous substrate and of antimycin A, ATP decreases and uncoupler restores the rates of H2O2 formation. 5. Reincorporation of ubiquinone-10 and ubiquinone-3 to ubiquinone-depleted pigeon heart mitochondria gives a system in which H2O2 production is linearly related to the incorporated ubiquinone. 6. The generation of H2O2 by pigeon heart mitochondria in the presence of succinate–glutamate and in metabolic state 4 has an optimum pH value of 7.5. In states 1 and 3u, and in the presence of antimycin A and uncoupler, the optimum pH value is shifted towards more alkaline values. 7. With increase of the partial pressure of O2 to the hyperbaric region the formation of H2O2 is markedly increased in pigeon heart mitochondria and in rat liver mitochondria. With rat liver mitochondria and succinate as substrate in state 4, an increase in the pO2 up to 1.97MPa (19.5atm) increases H2O2 formation 10–15-fold. Similar pO2 profiles were observed when rat liver mitochondria were supplemented either with antimycin A or with antimycin A and uncoupler. No saturation of the system with O2 was observed up to 1.97MPa (19.5atm). By increasing the pO2 to 1.97MPa (19.5atm), H2O2 formation in pigeon heart mitochondria with succinate as substrate increased fourfold in metabolic state 4, with antimycin A added the increase was threefold and with antimycin A and uncoupler it was 2.5-fold. In the last two saturation of the system with oxygen was observed, with an apparent Km of about 71kPa (0.7–0.8atm) and a Vmax. of 12 and 20nmol of H2O2/min per mg of protein. 8. It is postulated that in addition to the well-known flavin reaction, formation of H2O2 may be due to interaction with an energy-dependent component of the respiratory chain at the cytochrome b level.
The apparent stimulation of proteolysis by adenosine triphosphate in tissue homogenates (Short Communication)
Authors: Goldspink David F. , Goldberg Alfred L.
Published: August 2015

Reports that ATP promotes proteolysis in tissue homogenates were reinvestigated. Although ATP increased production of material reacting with ninhydrin or Folin–phenol reagent, ATP did not stimulate protein degradation in such extracts. AMP and adenosine acted similarly to ATP. Deamination of the added nucleotides to produce ammonia caused the increase in ninhydrin-positive material.
Biochemical effects of the porphyrinogenic drug allylisopropylacetamide. A comparative study with phenobarbital
Authors: Rao Manchanahalli R. Satyanarayana , Padmanaban Govindarajan
Published: August 2015

Successive administrations of allylisopropylacetamide, a potent porphyrinogenic drug, increase liver weight, microsomal protein and phospholipid contents. There is an increase in the rate of microsomal protein synthesis in vivo and in vitro. The drug decreases microsomal ribonuclease activity and increases NADPH–cytochrome c reductase activity. Phenobarbital, which has been reported to exhibit all these changes mentioned, is a weaker inducer of δ-aminolaevulinate synthetase and increases the rate of haem synthesis only after a considerable time-lag in fed female rats, when compared with the effects observed with allylisopropylacetamide. Again, phenobarbital does not share the property of allylisopropylacetamide in causing an initial decrease in cytochrome P-450 content. Haematin does not counteract most of the biochemical effects caused by allylisopropylacetamide, although it is quite effective in the case of phenobarbital. Haematin does not inhibit the uptake of [2-14C]allylisopropylacetamide by any of the liver subcellular fractions.
The metabolism of acetamidothiazoles in the rat. 2-Acetamido-4-chloromethylthiazole
Authors: Chatfield D. H. , Hunter W. H.
Published: August 2015

2-Acetamido-4-chloromethylthiazole is metabolized in the rat to (2-acetamido-4-thiazolylmethyl)mercapturic acid and 2-acetamidothiazole-4-carboxylic acid. 2-Acetamido-4-methylthiomethylthiazole and the corresponding sulphoxide and sulphone are also produced as minor metabolites. The identification of the metabolites is described and their formation investigated. Quantitative results on the excretion of the metabolites labelled with 14C are reported.