bluuebunny/crossref_metadata_2025 huggingface dataset:
Steady-state enzyme kinetics with high-affinity substrates or inhibitors. A statistical treatment of dose–response curves
Authors: Henderson Peter J. F.
Published: August 2015

A statistical treatment of steady-state enzyme kinetic measurements is described that allows for depletion of free substrate or free inhibitor concentrations owing to significant binding to the enzyme. Vmax., Km or Ki, enzyme concentration, the concentration of substrate or inhibitor required for a half-maximal effect and standard errors of these parameters can be calculated from dose–response measurements; the concentration of each component of the system may be estimated also. The statistically best values of the parameters are used to convert dose–response curves into convenient linear forms. The method is applied to dose–response measurements of hydroxyquinoline N-oxide inhibition of bacterial respiration and aminopterin inhibition of dihydrofolate reductase. Two FORTRAN programs for this method have been deposited as Supplementary Publication no. SUP 50019 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1973) 131, 5.
The structure of novel C35 pentacyclic terpenes from Acetobacter xylinum
Authors: Förster Hans J. , Biemann Klaus , Haigh W. Geoffrey , Tattrie Neil H. , Colvin J. Ross
Published: August 2015

A novel C35 terpene and its monounsaturated analogue were isolated from cultures of Acetobacter xylinum, together with traces of their C36 homologues. These substances were found to be hopane derivatives substituted by a five-carbon chain bearing four vicinal hydroxyl groups. For the parent hydrocarbon the term bacteriohopane is proposed. The elucidation of the structures utilized high-resolution mass spectrometry of the terpenes, degradation to C32 hydrocarbons and detailed mass-spectrometric comparison of these with C32 hydrocarbons synthesized from known pentacyclic triterpenes. High-resolution mass-spectral data of the terpenes are presented. N.m.r. data are in agreement with the proposed structures, which are further supported by the isolation from the same organism of 22-hydroxyhopane and derivative hopene(s).
Reaction products from N-methyl-N-nitrosourea and deoxyribonucleic acid containing thymidine residues. Synthesis and identification of a new methylation product, O4-methyl-thymidine
Authors: Lawley P. D. , Orr D. J. , Shah S. A. , Farmer P. B. , Jarman M.
Published: August 2015

1. DNA was treated with N-methyl-N-nitrosourea at pH7–8, 37°C, degraded to yield 3- and 7-methylpurines and deoxyribonucleosides and the reaction products were separated by chromatography on ion-exchange resins. The following methods for identification and determination of products were used: with unlabelled N-methyl-N-nitrosourea, u.v. absorption; use of methyl-14C-labelled N-methyl-N-nitrosourea and use of [14C]thymine-labelled DNA. 2. The synthesis of O4-methylthymidine and its identification by u.v. and mass spectroscopy are reported. 3. 3-Methylthymidine and O4-methylthymidine were found as methylation products from N-methyl-N-nitrosourea with thymidine and with DNA, in relatively small yields. Unidentified products containing thymine were found in enzymic digests of N-methyl-N-nitrosourea-treated DNA, which may be phosphotriesters. 4. The possible role of formation of methylthymines in mutagenesis by N-methyl-N-nitrosourea is discussed.
Protein synthesis and loss of viability in rye embryos. The lability of transferase enzymes during senescence
Authors: Roberts B. E. , Osborne D. J.
Published: August 2015

Poor germination and eventual loss of viability of rye grains on storage reflects a decreasing ability of embryos to synthesize protein in vivo. Cell-free protein-synthesizing systems from low viability embryo stocks exhibit lesions in the soluble components of the post-ribosomal supernatant fractions. tRNA and aminoacyl-tRNA synthetases are not impaired. The activity of enzymes concerned with transfer of phenylalanyl-tRNA from the aminoacyl to the peptidyl site on the ribosome is slightly decreased. The transfer enzymes (transferase I) involved in the binding of phenylalanyl-tRNA to the aminoacyl site show a loss of activity that closely reflects the loss of viability and the decline of protein synthesis of the embryo in vivo. The inactivation of labile transferase I components may be a major factor leading to senescence and loss of viability in rye grains.
Kinetics of concomitant transfer and hydrolysis reactions catalysed by the exocellular dd-carboxypeptidase–transpeptidase of Streptomyces R61
Authors: Frère J.-M. , Ghuysen J.-M. , Perkins H. R. , Nieto M.
Published: August 2015

When Ac2-l-Lys-d-Ala-d-Ala and either meso-diaminopimelic acid or Gly-l-Ala are exposed to the exocellular dd-carboxypeptidase–transpeptidase of Streptomyces R61, transpeptidation reactions yielding Ac2-l-Lys-d-Ala-(d)-meso- diaminopimelic acid and Ac2-l-Lys-d-Ala-Gly-l-Ala occur concomitantly with the hydrolysis of the tripeptide into Ac2-l-Lys-d-Ala. The proportion of the enzyme activity which can be channelled in the transpeptidation and the hydrolysis pathways depends upon the pH and the polarity of the environment. Transpeptidation is favoured both by increasing the pH and by decreasing the water content of the reaction mixtures. Kinetics suggest that the reactions proceed through an ordered mechanism in which the acceptor molecule (meso-diaminopimelic acid or Gly-l-Ala) binds first to the enzyme. Both acceptors behave as non-competitive inhibitors of the hydrolysis pathway. Transpeptidation is inhibited by high concentrations of Gly-l-Ala but not by high concentrations of meso-diaminopimelic acid. The occurrence on the enzyme of an additional inhibitory binding site for Gly-l-Ala is suggested.
Purification and some properties of cytoplasmic aspartate aminotransferase from sheep liver
Authors: Campos-Cavieres María , Munn Edward A.
Published: August 2015

1. A procedure for the purification of the cytoplasmic isoenzyme of aspartate aminotransferase from sheep liver is described. 2. The purified isoenzyme shows a single component in the ultracentrifuge at pH7.6 and forms a single protein band on agar-gel electrophoresis at pH6.3 or 8.6, as well as when stained for protein or activity after polyacrylamide-gel or cellulose acetate electrophoresis at pH8.8. 3. Immunoelectrophoresis on agar gel yields only one precipitin arc associated with the protein band, with rabbit antiserum to the purified isoenzyme. By immunodiffusion, cross-reaction was detected between the cytoplasmic isoenzymes from sheep liver and pig heart, but not between the cytoplasmic and mitochondrial sheep liver isoenzymes. 4. The s20,w of the enzyme is 5.69S and the molecular weight determined by sedimentation equilibrium is 88900; 19313 molecules of oxaloacetate were formed/min per molecule of enzyme at pH7.4 and 25°C. 5. The amino acid composition of the isoenzyme is presented. It has about 790 residues per molecule. 6. The holoenzyme has a maximum of absorption at 362nm at pH7.6 and 25°C. 7. A value of 2.1 was found for the coenzyme/enzyme molar ratio. 8. The purified enzyme revealed two bands of activity on polyacrylamide-gel electrophoresis at pH7.4 and an extra, faster, band in some circumstances. These bands occurred even when dithiothreitol was present throughout the isolation procedure. 9. Three main bands were obtained by electrofocusing on polyacrylamide plates with pI values 5.75, 5.56 and 5.35. 10. Structural similarities with cytoplasmic isoenzymes from other organs are discussed.
The interaction of 3-deoxy-3-fluoro-d-glucose with the hexose-transport system of the human erythrocyte
Authors: Riley G. J. , Taylor N. F.
Published: August 2015

1. By using an optical method the kinetic parameters of hexose transport across the human erythrocyte membrane were determined for several sugars. The series of half-saturated constants is as follows: 3-deoxy-3-fluoro-d-glucose = 3-O-methyl-d-glucose <d-glucose<d-mannose <3-deoxy-d-glucose<d-galactose<l-arabinose. 2. Estimations of the dissociation energy of the 3-deoxy-3-fluoro-d-glucose–carrier and d-glucose–carrier complexes suggest that the binding of the two sugars to the transport system is equivalent. 3. Incubation of the erythrocytes with 3-deoxy-3-fluoro-d-glucose results in a small but significant release of F−anion. Cells treated in this way lose their ability to transport glucose.
The thermal denaturation of human oxyhaemoglobins A, A2, C and S
Authors: Kinderlerer J. , Lehmann H. , Tipton K. F.
Published: August 2015

1. The time-courses of thermal denaturation of human oxyhaemoglobins A, A2, C and S at 45°C were studied by following the increase in protein fluorescence. Haemoglobins S and C were less stable than haemoglobin A, whereas haemoglobin A2 was considerably more stable. 2. The time-courses of denaturation did not follow first-order kinetics and could be fitted most simply to a co-operative scheme in which the partial denaturation of the α chain preceded that of the β chain. 3. The denaturation of these haemoglobins was studied as a function of temperature by using optical rotatory dispersion. Haemoglobin A2 was again more stable than the others. The addition of small quantities of haemoglobin A2 had a disproportionate effect on the stability of haemoglobin C. 4. The thermodynamic parameters of the denaturation process were calculated.
Uptake and release of bilirubin by skin
Authors: Kapoor Chiranjiv L. , Murti Coimbatore R. Krishna , Bajpai Prakash C.
Published: August 2015

1. Skin epithelium of albino rat, mouse and guinea pig was shown to accumulate bilirubin from a medium containing free or bound bilirubin. 2. The Km values for bound bilirubin were 2.22×10-3, 1.33×10-3 and 9.5×10-4m for rat, mouse and guinea pig respectively and the corresponding Km values for free bilirubin were 5.2×10-4, 4.0×10-4, 1.8×10-4m; the Vmax. values of bound and free bilirubin were unchanged. 3. The uptake showed saturation kinetics. Bound bilirubin was released together with serum proteins. Free bilirubin bound to skin was not released into the medium. 4. Freezing and thawing of skin epithelium did not cause any significant lowering of the uptake of bilirubin but heat-denatured skin epidermis took up only 50% of the bound bilirubin or free bilirubin taken up by control unheated skin epithelium. 5. The uptake of free and bound bilirubin was prevented by HgCl2 but not by sodium arsenate, NaCN, NaF, cycloheximide, 2,4-dinitrophenol or iodoacetate. 6. Most of the free bilirubin was bound to the lipids or lipoprotein fraction of skin epithelium and could be extracted by solvents. 7. Rat skin showed the highest accumulation and efflux of bilirubin.
Studies on bone matrix glycoproteins. Incorporation of [1-14C]glucosamine and plasma [14C]glycoprotein into rabbit cortical bone
Authors: Triffitt James T. , Owen Maureen
Published: August 2015

The radioactively labelled constituents present in bone matrix were compared 12 days after injection of either [14C]glucosamine or plasma [14C]glycoprotein. Both precursors are utilized in the synthesis of organic matrix by bone tissue. Cortical bone from animals injected with [14C]glucosamine contains radioactivity derived from glucosamine and plasma glycoproteins and all glycoprotein fractions are labelled. Plasma [14C]glycoprotein labels the less acidic glycoproteins to a greater extent than the more acidic components. An antibody has been raised against the less-acidic-glycoprotein fraction of bone. The latter contains a glycoprotein of α-mobility that appears to be concentrated specifically in bone tissue and which is present also in plasma. This α-glycoprotein accounts for a large proportion of the components labelled and retained in bone matrix after [14C]glucosamine injection.
The effect of trimethoprim on macromolecular synthesis in Escherichia coli. Ribosome maturation in RCstr and RCrel strains
Authors: Midgley J. E. M. , Smith R. J.
Published: August 2015

When Escherichia coli was inhibited with trimethoprim in media supplemented with nucleotide bases, glycine and methionine, both RCstr and RCrel strains continued to accumulate RNA at rates very close to those in growing controls. The effects of trimethoprim on protein synthesis were studied by using as an experimental basis the rate of maturation of ribosomal particles from RNA-rich precursors. 1. In RCstr cultures given nucleotide bases but no amino acids, RNA accumulation was inhibited because of amino acid lack. However, maturation of ribosomes from their precursors was more severely inhibited than was the synthesis of rRNA. The restraints on protein synthesis were more severe at the level of translation than the transcription of operons specific for the formation of ribosomal proteins. The kinetic delay time in the passage of rRNA from RNA-rich intermediates to the final ribosome products was therefore increased some three- to four-fold. 2. In RCrel cultures in the same conditions, trimethoprim inhibition stopped ribosomal particle synthesis, but rRNA-rich precursors accumulated. 3. If glycine+methionine were also added to inhibited RCstr cultures, RNA accumulation resumed at a high rate. However, ribosomal maturation was still considerably disturbed because of a disproportionate response of the cells in the formation of protein and RNA. 4. With RCrel cultures, addition of the amino acids caused a large increase in the rate of ribosome maturation, though the degree of disproportionation between the rates of rRNA and ribosomal protein synthesis was now identical with that found in RCstr strains. 5. When inhibited RCrel cultures were supplemented, there was still a severe inhibition of protein synthesis at the level of chain initiation, but inaccuracies in the process of polypeptide chain elongation were greatly decreased. This suggests that the effects of the RCrel mutation on the fidelity of protein synthesis in bacteria are not directed at the point of chain initiation.
The synthesis of pteroylpolyglutamates by sheep liver enzymes in vitro
Authors: Gawthorne Jeffrey M. , Smith Richard M.
Published: August 2015

1. Sephadex G-15 was used to separate pteroylmonoglutamates from corresponding polyglutamate derivatives. 2. Pteroylpolyglutamates were formed when 5-formyltetrahydro[2-14C]pteroylglutamic acid, 5-[methyl-14C]tetrahydropteroylglutamic acid or tetrahydro[2-14C]pteroylglutamic acid was incubated at pH8.4 with ATP, MgCl2, KCl, l-glutamic acid and sheep liver cytosol. The γ-glutamyl side chain appeared to be lengthened by the stepwise addition of single glutamate moieties. 3. The subcellular distribution of pteroylpolyglutamates paralleled that of pteroylpolyglutamate synthetase activity, and followed the order cytosol>‘nuclear’ fraction>microsomal fraction>mitochondria.
Activation of volatile fatty acids in bovine liver and rumen epithelium. Evidence for control by autoregulation
Authors: Ash R. , Baird G. D.
Published: August 2015

1. The total capacities of homogenates of bovine liver and rumen epithelium to activate acetate, propionate and butyrate were determined. 2. Activating capacities were assayed by measuring the rate of formation of the corresponding CoA esters. The methods used for determining the concentrations of the CoA esters allowed the CoA esters of acetate, propionate and butyrate to be distinguished. It was thus possible to investigate the effect of the presence of a second volatile fatty acid on the rate at which a given volatile fatty acid was activated. 3. The propionate-activating capacity in rumen epithelium was decreased by about 87% in the presence of butyrate, the acetate-activating capacity in liver was decreased by about 55% in the presence of either propionate or butyrate, and the butyrate-activating capacity in liver was decreased by about 40–50% in the presence of propionate. 4. All three activating capacities in liver appeared to be located in the mitochondrial matrix and membrane. The three activating capacities had similar locations to each other in rumen epithelium as well, although in this case activity was more evenly divided between the mitochondria and the cytoplasm. 5. The relative activating capacities towards the volatile fatty acids in the two tissues, together with the ability of one volatile fatty acid to inhibit the activation of another volatile fatty acid, appear to ensure that butyrate is mainly metabolized in the rumen epithelium and that propionate is metabolized in the liver.
A possible role of adenylate cyclase in the longterm dietary regulation of insulin secretion from rat islets of Langerhans
Authors: Howell Simon L. , Green Irene C. , Montague William
Published: August 2015

1. Adenylate cyclase activity and patterns of insulin release in response to various concentrations of glucose were determined in islets of Langerhans isolated from starving, fed, or glucose-loaded rats. 2. Basal and glucagon-stimulated activities of adenylate cyclase were lower in islets from starved than from fed rats. The minimum glucose concentration required for stimulation of insulin secretion was higher, whereas the maximum secretory response to glucose was lower, in islets from starved than from fed rats. 3. Adenylate cyclase activity in islets of Langerhans obtained from fed rats loaded with glucose by intermittent intravenous or intraperitoneal injections over 5h was significantly higher than that seen in islets from normal fed rats. Islets obtained from glucose-loaded rats required a lower glucose concentration for stimulation of insulin secretion and attained a higher maximal response to glucose stimulation than those derived from fed rats. 4. Incubation in vitro of islets isolated from normal fed rats, for periods of 1 to 24h in the presence of high concentrations of glucose resulted in an activation of adenylate cyclase that occurred progressively from 2 to 7h and which was maintained during 24h of incubation. The increase of adenylate cyclase activity in isolated islets incubated for 4h in the presence of glucose was not prevented by addition of cycloheximide or actinomycin D. Galactose or 2-deoxyglucose was ineffective in increasing adenylate cyclase activity, and pyruvate (20mm) was less effective than glucose. 5. It is suggested that glucose or a glucose metabolite may exert long-term effects on islet cell adenylate cyclase.
Biosynthesis of immunoglobulin A (IgA). Secretion and addition of carbohydrate to monomer and polymer forms of a mouse myeloma protein
Authors: Corte E. Della , Parkhouse R. M. E.
Published: August 2015

Cell suspensions of mouse plasma-cell tumour MOPC 315 secreting predominantly IgA (immunoglobulin A) monomer and dimer were incubated with radioactive leucine, mannose, galactose and fucose for various periods of time. The amounts of secreted and intracellular immunoglobulins were measured by co-precipitation with specific antibody, and the molecular species present were assessed by electrophoresis in polyacrylamide gels. Analysis of the secreted myeloma protein demonstrated that monomer and dimer IgA molecules are identical with respect to carbohydrate composition and rate of secretion. Within the cell, the myeloma protein is almost entirely accounted for by monomer units which either leave the cell as such or are polymerized with the addition of J chain close to the time of secretion. The results support the concept of a stepwise addition of carbohydrate residues to IgA immunoglobulin during the process of secretion. Similar patterns of carbohydrate assembly were found for the monomer or dimer molecules. Mannose residues are added at an early stage, whereas fucose is added close to the time of secretion. Galactose is also added early, but some may also be incorporated at a later stage. Control of IgA polymerization is considered unlikely to reflect regulation at the level of carbohydrate addition, and it is suggested that the critical controlling factor is the J chain.
Stimulation of ribonucleic acid polymerase activity in vitro by prostatic steroid–protein receptor complexes
Authors: Davies P. , Griffiths K.
Published: August 2015

A system has been developed which allows the stimulation in vitro of prostatic RNA polymerase by prostatic 5α-dihydrotestosterone–protein receptor complexes prepared from the tissues of castrated rats. The reconstitution in vitro of such a system necessitates the purification of several subcellular components. Two 5α-dihydrotestosterone–receptor complexes are located in the prostatic soluble supernatant fraction, separable by selective ammonium sulphate fractionation, and one complex can be isolated from the nuclear fraction. In the presence of all these complexes, stimulation of RNA polymerase in intact nuclei and nucleoli was observed. The complexes also increased the activity of the enzyme solubilized from whole nuclei. Greater stimulation of this system was noted in the presence of prostatic chromatin as template, as compared with that observed with calf thymus DNA or liver chromatin as template. The effects of the complexes on subnuclear forms of RNA polymerase, of nucleolar and extranucleolar origin, are also described. RNA polymerase solubilized from nucleoli is more susceptible to stimulation by the 5α-dihydrotestosterone–receptor complexes than is the ‘nucleoplasmic’ enzyme. Stimulation occurs less readily in the presence of Mn2+and at high ionic strength than in the presence of Mg2+and at low ionic strength. Preliminary experiments show that prostatic nucleolar RNA polymerase transcribes prostatic chromatin poorly as compared with the nucleoplasmic enzyme. The observations reported indicate an involvement of non-histone proteins associated with DNA in the process by which stimulation of enzyme activity by the 5α-dihydrotestosterone–receptor complexes is achieved. The implications of these findings in the mechanism of steroid hormone action is considered.
Effects of clofibrate on the metabolism of progesterone and oestradiol in rat liver microsomal fraction
Authors: Einarsson Kurt , Gustafsson Jan-Åke , Hellström Kjell
Published: August 2015

1. The substrate conversion of [4-14C]progesterone and [4-14C]oestradiol during incubation with the liver microsomal fraction from both control and clofibrate-treated rats amounted to about 10–15 and 20–25% respectively. 2. The metabolites of progesterone formed by preparations from control rats were hydroxylated in the 16α-position (14%), the 6β-position (12%) and the 2α-position (7%). Of the products formed from oestradiol 12% were recovered as a 16α-hydroxylated derivative whereas 5% had a 6β- and 2% a 6α-hydroxyl group. 3. Clofibrate affected the microsomal metabolism of both progesterone and oestradiol. It induced 7α-hydroxylation of both compounds, metabolic conversions not found in control rats. The 6β-hydroxylation of progesterone and the 6α-hydroxylation of oestradiol were enhanced by a factor of 2 and 3.5 respectively. The 2α-hydroxylation, and the 20α- and 20β-hydroxy steroid reduction of progesterone were significantly decreased as were the 16α- and the 6β-hydroxylation of oestradiol.
Dietary protein intake and skeletal-muscle protein metabolism in rats. Studies with the ammonium chloride-wash fraction from crude polyribosomes of well-nourished and protein-depleted rats
Authors: Alexis S. Daniel , Young Vernon R.
Published: August 2015

1. Crude polyribosomes from skeletal muscle of the hind leg of rats fed on a low-protein diet for 10 days are less active in cell-free protein synthesis than are polyribosomes obtained from well-nourished control rats. 2. The polyribosomes were salt-washed (0.5m-NH4Cl) and the wash extract was examined for its amino acid incorporating activity and for EF (elongation factor) 1 and EF 2 activities. 3. Compared with preparations from control rats, the salt-wash fraction from protein-depleted rats was less active and showed lower EF 1 and EF 2 activity. 4. The ribosomes were rendered equal in activity by salt-washing, but no inhibitor was detected in the salt wash. 5. Differences in the incorporating activity of crude polyribosomes from the diet groups persisted in the presence of saturating amounts of partially purified EF 1 and EF 2. 6. It is concluded that the lowered protein-synthetic activity of crude polyribosomes caused by restricted protein intake is not causally related to the lower activities of EF 1 and EF 2 in the polyribosome preparations. 7. The possible nature of the change in crude polyribosome activity due to low-protein feeding is discussed.
Synthesis of mitochondrial protein in the flight muscles of the Colorado beetle. Differentiation in vivo between extra- and intra-mitochondrial contributions to the amino acid incorporation into mitochondrial protein
Authors: Bartelink A. K. M. , De Kort C. A. D.
Published: August 2015

By using cycloheximide, an inhibitor of cytoplasmic protein synthesis, conditions were investigated to estimate in vivo the extra- and intra-mitochondrial contributions to the synthesis of organelle protein in the flight muscles of Colorado beetles. With 4-day-old beetles about 15% of the [14C]leucine incorporation into mitochondrial protein is resistant to the action of cycloheximide. The incorporation into cytosol protein is inhibited by more than 99.5% with cycloheximide. During the first hour after precursor administration the incorporation into mitochondrial protein proceeds, in both the presence and the absence of cycloheximide, at a more-or-less linear rate with time. The cycloheximide-resistant amino acid incorporation is sensitive to the inhibitor of mitochondrial protein synthesis, chloramphenicol. The uncertainties inherent in the use of cycloheximide were discussed in arriving at the conclusion that about 15% of the mitochondrial protein is formed inside the organelle.
The photochemical activities and electron carriers of developing barley leaves
Authors: Plesničar Marijana , Bendall Derek S.
Published: August 2015

The development of photochemical activities in isolated barley plastids during illumination of dark-grown plants has been studied and compared with the behaviour of plastocyanin, cytochromes f, b-559LP, b-563 and b-559HP and pigments P546 (C550) and P700. Electron-transport activity dependent on Photosystem 1 and cyclic photophosphorylation dependent on N-methylphenazonium methosulphate (phenazine methosulphate) were very active relative to the chlorophyll content after only a few minutes of illumination of etiolated leaves, and then rapidly declined during the first few hours of greening. By contrast, Photosystem 2 activity (measured with ferricyanide as electron acceptor) and non-cyclic photophosphorylation were not detectable during the first 2½h of greening, but then increased in total amount in parallel with chlorophyll. The behaviour of the electron carriers suggested their association with either Photosystem 1 or 2 respectively. In the first group were plastocyanin, cytochrome f and cytochrome b-563, whose concentrations in the leaf did not change during greening, and cytochrome b-559LP whose concentration fell to one-half its original value, and in the second group were cytochrome b-559HP and pigment P546, the concentrations of which closely followed the activities of Photosystem 2. Pigment P700 could not be detected during the first hour, during which time some other form of chlorophyll may take its place in the reaction centre of Photosystem 1. The plastids started to develop grana at about the time that Photosystem 2 activity became detectable.
A model for the passage of the neurohypophysial hormones and their related proteins through the rat neurohypophysis
Authors: Burford Graham D. , Clifford Peter , Jones C. W. , Pickering Brian T.
Published: August 2015

The change in the radioactivity of vasopressin–neurophysin in the rat neurohypophysis after an intracisternal injection of [35S]cysteine was fitted to several mathematical models. The data fitted best a model in which there is a linear input of radioactive protein into one pool of the neurohypophysis, from which it is either released by an exponential process or transferred to a second pool from which it is released by a second exponential process with a rate constant much lower than the first. This model is compatible with the existence of a ‘readily releasable’ pool first postulated by Sachs et al. (1967). Data for the change in radioactivity of vasopressin also gave a good fit in this model. Calculation of the rate constants suggested that the first pool represented about 2% of the total hormone.
The metabolism of nitrilotriacetate by a pseudomonad
Authors: Cripps Roger E. , Noble Ann S.
Published: August 2015

1. An organism that grows on nitrilotriacetate as sole source of carbon and energy was isolated in pure culture and was identified as a pseudomonad. 2. Cell-free extracts of the nitrilotriacetate-grown pseudomonad contain an enzyme that catalyses the NADH-and O2-dependent oxidation of nitrilotriacetate to iminodiacetate and glyoxalate. This enzyme is absent from extracts of glucose-grown cells. 3. Compared with growth on glucose, growth on nitrilotriacetate results in increased activities of enzymes of glycine and serine metabolism, namely serine hydroxymethyltransferase, glycine decarboxylase, serine–oxaloacetate aminotransferase and hydroxypyruvate reductase. 4. Cell-free extracts of the nitrilotriacetate-grown organism contain the enzyme glyoxalate carboligase and, when supplemented with NADH, Mg2+and thiamin pyrophosphate, can catalyse the anaerobic conversion of glyoxalate into glycerate. 5. These results are incorporated in a scheme which shows the oxidative metabolism of nitrilotriacetate by the successive removal of C2 units to form 2mol of glyoxalate and 1mol of glycine per mol of nitrilotriacetate degraded. The glyoxalate and glycine are then both metabolized to glycerate by separate pathways, via tartronic semialdehyde and serine respectively. The role of this scheme in the growth of the organism on nitrilotriacetate is discussed.
Criteria for identifying a myelin-like fraction from developing brain (Short Communication)
Authors: Agrawal Harish C. , Trotter John L. , Mitchell Raymond F. , Burton Robert M.
Published: August 2015

Multiple criteria must be used to relate a membrane fraction (‘myelin-like’) to myelin. These reported in this communication are electron microscopy, marker enzymes, sodium dodecyl sulphate–polyacrylamide-gel electrophoretic patterns and lipid composition.
A low-molecular-weight protein cross-reacting with human liver N-acetyl-β-d-glucosaminidase
Authors: Carroll Mark , Robinson Donald
Published: August 2015

Antisera were raised to preparations of hexosaminidase isoenzymes A and B purified from human liver. Protein that cross-reacted with the liver hexosaminidase was detected by an antibody-consumption method. A cross-reacting protein with a low molecular weight (20000) was partially characterized and purified from control human liver. This protein is also present in the liver of patients with Tay–Sachs disease or with Sandhoff's disease. Hexosaminidases A and B gave an immunological reaction of partial identity with the low-molecular-weight protein. The possible identity of the low-molecular-weight cross-reacting protein as a subunit of hexosaminidase is discussed.
A study of the reaction of protoporphyrin IX with human globin
Authors: Treffry Amyra , Ainsworth Stanley
Published: August 2015

The present paper reports an investigation of the reaction of protoporphyrin IX with globin prepared from the HbA0 component of human blood. The porphyringlobin produced is always heterogeneous; however, when globin is used immediately after preparation, its affinity for porphyrin is higher and the product less heterogeneous than when the globin has been frozen or freeze-dried. The affinity of globin for haemin is less affected by its history. With freshly prepared globin, reconstitution at room temperature provides a different distribution of porphyringlobin species than reconstitution at 4°C. Further changes in the species distribution of cold-reconstituted samples may be observed by gel electrophoresis when the samples are aged for 24h at room temperature. Chromatographic separation of such porphyringlobin samples on CM-Sephadex generally revealed five species with two in predominating amounts. It was consistently observed that over a period of 18 days, the faster moving of the two main components decreased in amount whereas the slower-moving component correspondingly increased. However, when the main components are separated, they remain homogeneous over the same length of time. The effect of light on porphyringlobin was also investigated. It was shown that porphyringlobin is photo-oxidized: as a result the porphyrin is destroyed together with most of the histidine, methionine and all of the tryptophan residues of the protein.
Observations on the use of solid-phase-coupled antibodies in the radioimmunoassay of human placental lactogen
Authors: Gardner Jacqueline , Bailey Gordon , Chard Timothy
Published: August 2015

A detailed comparative assessment was made of the use of solid-phase-coupled antibodies in radioimmunoassay, by using an assay for human placental lactogen as a model system. The major advantages of the solid-phase technique are: (1) in common with the use of a second antibody, it is universally applicable; (2) separation can be carried out rapidly; (3) in contrast with some other techniques, the separation of antibody-bound and free antigen is virtually complete. The disadvantages when compared with other procedures are: (1) a considerable proportion of the antibody may be lost during the initial coupling reaction; (2) the tubes must be continuously mixed during incubation, and much effort is expended in removing and replacing the caps; (3) there is a decrease in the apparent affinity constant of the antibody after coupling, which is reflected in a lower sensitivity of the assay system. It is concluded that solid-phase antibodies are of greatest value in those systems in which the supply of antiserum is abundant, and in which the achievement of high sensitivity is not a requirement.
The binding of sodium dodecyl sulphate to bovine serum albumin at high binding ratios
Authors: Allen Geoffrey
Published: August 2015

The extent of binding of sodium dodecyl sulphate to bovine serum albumin at high binding ratios was investigated by gel filtration. The weight ratio of bound sodium dodecyl sulphate to bovine serum albumin increases with the NaCl concentration, and, except at low salt concentrations, with the concentration of sodium dodecyl sulphate. In the presence of 1.0g of sodium dodecyl sulphate/l, the binding ratio varied from 1.0 (at 0.04m-Na+) to 2.2 (at 0.44m-Na+). In the presence of 0.24m-Na+, the binding ratio increased with sodium dodecyl sulphate concentration, from 0.9 (0.2g of sodium dodecyl sulphate/l) to 2.0 (5g of sodium dodecyl sulphate/l), at 26°C, in a dilute sodium phosphate buffer. No significant dependence of the binding ratio upon temperature in the range 26–45°C was observed. These results differ from those of Reynolds & Tanford (1970a) obtained by equilibrium dialysis.
Accumulation of lanthanum by rat liver mitochondria
Authors: Reed Ken C. , Bygrave Fyfe L.
Published: August 2015

The interaction of La3+ with rat liver mitochondria was examined with a wide variety of techniques permitting measurement of respiratory and structural responses. It is concluded that La3+ is accumulated by mitochondria in a process that is at least partially energy-dependent, and is bound with quite high affinity to membrane-associated sites both external and internal to the inner membrane. The relative insensitivity of the accumulation process to respiratory inhibitors and to the permeant anion acetate has interesting implications for the mechanism of active cation transport.
Anti-anabolic effects of adenosine 3′:5′-cyclic monophosphate. Inhibition of protein synthesis
Authors: Sellers Anthony , Bloxham David P. , Munday Kenneth A. , Akhtar Muhammad
Published: August 2015

1. Evidence is presented that cyclic AMP inhibits the incorporation of l-[4,5-3H]leucine into protein in a cell-free system from rat liver. This inhibition occurs after aminoacyl-tRNA formation. 2. Microsomal fractions, isolated after the incubation of postmitochondrial supernatant with cyclic AMP and ATP, show a diminished ability to synthesize protein. Both cyclic AMP and ATP are required for this effect. 3. A possible physiological role for the anti-anabolic action of cyclic AMP is discussed in terms of the control of gluconeogenesis.
Formation of acetoacetate from 3-hydroxy-3-methylglutarate by rat liver and isolation of a mitochondrial coenzyme A-transferase activity involved
Authors: Deana R. , Meneghello R. , Manzi L. , Gregolin C.
Published: August 2015

1. Formation of acetoacetate from 3-hydroxy-3-methylglutarate was observed in the perfused rat liver. Production of 3.5μmol of acetoacetate/h per g of tissue was obtained. 2. Formation of acetoacetate was catalysed mainly by the mitochondrial fraction of the homogenized liver, at a rate of 62nmol/h per mg of protein. 3. Experiments with hydroxy-[3-14C]methylglutarate demonstrated that the acetoacetate formed was derived mainly from this compound. 4. A mitochondrial transferase activity catalysing the transfer of a CoA molecule from succinyl-CoA (3-carboxypropionyl-CoA) to hydroxymethylglutarate was shown. The Km value for hydroxymethylglutarate was 5×10−3m.
Half-life and rate of synthesis of globin messenger ribonucleic acid. Determination of half-life of messenger ribonucleic acid and its relative synthetic rate in erythroid cells
Authors: Hunt John A.
Published: August 2015

The specific radioactivity of mouse globin mRNA in blood reticulocytes was measured after injection of [3H]uridine into anaemic mice up to 60h before collection of reticulocytes. From these data, the decay of the acid-soluble nucleotide pool in the marrow and the relative marrow-cell composition it is possible to build models that allow the cell life-times and half-life of mRNA in the erythroid cells of the marrow to be calculated. Best fit of models to these data favour a model with either one or two cell divisions from the onset of mRNA synthesis. The single-cell-division model has cell times of 20, 13 and 7h respectively for the basophilic erythroblast, polychromatophilic erythroblast and reticulocyte. The two-cell-division model has cell times of 12, 12, 12 and 7h for the basophilic erythroblast 1 and 2, polychromatophilic erythroblast and reticulocyte respectively. Both models have an mRNA half-life of 17h and a constant rate of mRNA synthesis until enucleation at the reticulocyte stage, when synthesis stops. A declining rate of mRNA synthesis can be accommodated in a two-cell-division model, when synthesis halves at each cell division and cell times are essentially the same as above, but mRNA half-life is either 9h in the basophilic and polychromatophilic erythroblasts and 17h in the later cells, or 10h in the basophilic erythroblasts and polychromatophilic erythroblasts and 14.5h in later cells. In all cases it is clear that mRNA synthesis occurs over a time-period of only 30–36h and that mRNA cannot be pre-synthesized in precursor erythroid cells.
The enzymology of adenosine triphosphate sulphurylase from spinach leaf tissue. Kinetic studies and a proposed reaction mechanism
Authors: Shaw W. H. , Anderson J. W.
Published: August 2015

1. Sulphate-dependent PPi–ATP exchange, catalysed by purified spinach leaf ATP sulphurylase, was correlated with the concentration of MgATP2− and MgP2O72−; ATP sulphurylase activity was not correlated with the concentration of free Mg2+. 2. Sulphate-dependent PPi–ATP exchange was independent of PPi concentration, but dependent on the concentration of ATP and sulphate. The rate of sulphate-dependent PPi–ATP exchange was quantitatively defined by the rate equation applicable to the initial rate of a bireactant sequential mechanism under steady-state conditions. 3. Chlorate, nitrate and ADP inhibited the exchange reaction. The inhibition by chlorate and nitrate was uncompetitive with respect to ATP and competitive with respect to sulphate. The inhibition by ADP was competitive with respect to ATP and non-competitive with respect to sulphate. 4. ATP sulphurylase catalysed the synthesis of [32P]ATP from [32P]PPi and adenosine 5′-sulphatophosphate in the absence of sulphate; some properties of the reaction are described. Enzyme activity was dependent on the concentration of PPi and adenosine 5′-sulphatophosphate. 5. The synthesis of ATP from PPi and adenosine 5′-sulphatophosphate was inhibited by sulphate and ATP. The inhibition by sulphate was non-competitive with respect to PPi and adenosine 5′-sulphatophosphate; the inhibition by ATP was competitive with respect to adenosine 5′-sulphatophosphate and non-competitive with respect to PPi. It was concluded that the reaction catalysed by spinach leaf ATP sulphurylase was ordered; expressing the order in the forward direction, MgATP2− was the first product to react with the enzyme and MgP2O72− was the first product released. 6. The expected exchange reaction between sulphate and adenosine 5′-sulphatophosphate could not be demonstrated.
Useful agents for the study of glutathione metabolism in erythrocytes. Organic hydroperoxides
Authors: Srivastava Satish K. , Awasthi Yogesh C. , Beutler Ernest
Published: August 2015

t-Butyl hydroperoxide and cumene hydroperoxide, both known to be substrates for glutathione peroxidase, were used to oxidize erythrocyte GSH. Addition of concentrations of hydroperoxides equimolar with respect to GSH in the erythrocytes or whole blood quantitatively oxidizes GSH in the erythrocytes with a half-time of 4.5s at 37°C and about three times as long at 4°C. In the presence of glucose, normal erythrocytes regenerate all the GSH in about 25min. However, glucose 6-phosphate dehydrogenase-deficient erythrocytes failed to regenerate GSH. Treatment of erythrocytes with hydroperoxides does not affect erythrocyte survival in rabbits. Oxidation of erythrocyte GSH with equimolar concentrations of hydroperoxides does not lead to formation of mixed disulphides of haemoglobin and GSH. The hydroperoxides do not affect erythrocyte glycolytic and hexose monophosphate-shunt-pathway enzymes. Previous studies on transport of GSSG from erythrocytes were confirmed by using t-butyl hydroperoxide to oxidize erythrocyte GSH.
Uptake and release of ferritin iron. Surface effects and exchange within the crystalline core
Authors: Hoy Terence G. , Harrison Pauline M. , Shabbir Mohammad
Published: August 2015

The uptake and subsequent release of iron by apoferritin and ferritin was studied by using labelled iron (59Fe). The experimental results are consistent with predictions arising from a model system developed in the interpretation of previous experiments. In this model, uptake and release of ferritin iron is controlled by the available surface area of the small crystalline particles of hydrous ferric oxide found within the ferritin molecule. Evidence is also presented for the exchange of Fe3+ ions among the various cation sites within these crystallites.
A fluorimetric method for the determination of tryptophan in animal tissues
Authors: Gaitonde M. K.
Published: August 2015

Tryptophan, tryptamine and peptides containing N-terminal tryptophan give two highly fluorescent products on treatment with dithiothreitol and acid ninhydrin reagent 1 or 2. The first fluorescent product (product A) gives an emission at 500nm on activation at 390–400nm and is stable for 20min. The second product (product B), which gives an emission at 530nm on activation at 470nm, is detectable within 1h after the reaction. It gives almost maximum intensity in 4h and is stable for at least 48h. Except lysine, which in equimolar amounts gives less than 1% of a product similar to product B, no other naturally occurring amino compounds give fluorescent products. A procedure is given for the determination of 0.05–34nmol of tryptophan in tissue extracts. By using this procedure rat brain was found to contain 17.56±0.76 (s.e.m.) nmol/g wet wt.
The use of radiolabelled T1 ribonuclease to monitor the efficiency of Edman degradations (Short Communication)
Authors: McKean David J. , Smith George P.
Published: August 2015

A simple radiolabel tracer method is presented for monitoring the efficiency of the first ten cycles of an Edman degradation. This method consists of adding a very small amount of radiolabelled S-carboxymethylated T1 ribonuclease to the protein to be sequenced, and counting the radioactivity of a small sample from each of the degradative cycles.
The isolation of cholest-5-ene-3β,26-diol from human brain
Authors: Smith Andrew G. , Gilbert John D. , Harland W. Arthur , Brooks Charles J. W.
Published: August 2015

Cholest-5-ene-3β,26-diol, isolated from human brain, was further characterized by oxidation to 3-oxocholest-4-en-26-ol and to 3-oxocholest-4-en-26-oic acid. Identification was achieved by comparison (by t.l.c., g.l.c. and g.l.c.–mass spectrometry) with corresponding reference compounds derived from kryptogenin.
The increased rate of ethanol removal from blood of clofibrate-treated rats (Short Communication)
Authors: Hawkins R. A. , Nielsen R. C. , Veech R. L.
Published: August 2015

After ethanol injection similar metabolite changes were found in livers of clofibrate-treated and non-treated rats. The rate of ethanol removal from blood of clofibrate-treated rats was increased by 50%, but this was directly proportional to the increased liver size caused by clofibrate. No other difference needs to be postulated to explain the increased rate of ethanol removal from blood.
Effects of cytochalasin B, colchicine and vincristine on the metabolism of isolated fat-cells
Authors: Loten Ernest G. , Jeanrenaud Bernard
Published: August 2015

1. Colchicine and vincristine only slightly inhibit the metabolism of glucose to CO2 and lipids by isolated fat-cells. 2. Prolonged incubation with these agents causes no further inhibition. 3. Cytochalasin B, however, inhibits glucose metabolism to both CO2 and lipids in fat-cells. 4. However, at a concentration that causes a strong inhibition of glucose metabolism cytochalasin B is without effect on the metabolism of pyruvate, lactate or arginine to these end products. The uptake of labelled α-aminoisobutyrate is likewise not modified. Similarly it does not affect release of glycerol or free fatty acid, or the actions of adrenaline, insulin or caffeine on these parameters. At 10μg/ml it slightly lowers ATP concentrations, an effect that does not occur at 2μg/ml. 5. The transport of fructose into adipocytes by a specific fructose-transport system is also not affected by the agent, but the uptake of 2-deoxyglucose is strongly inhibited. It is concluded that cytochalasin B may specifically inhibit the glucose-transport system of isolated fat-cells. 6. Cytochalasin A has a much weaker action than cytochalasin B on glucose metabolism.
The response of the small intestine to vitamin D. Isolation and properties of chick intestinal polyribosomes
Authors: Emtage J. Spencer , Lawson D. Eric M. , Kodicek Egon
Published: August 2015

Undegraded polyribosome preparations may be obtained from chick intestinal mucosa if ribonuclease activity is strictly controlled. This is best achieved by homogenization of the mucosa directly in rat liver cell-sap. 2. The extent of amino acid incorporation by chick intestinal polyribosomes is greatly influenced by the source of the cell-sap. Sephadex-treated intestinal cell-sap caused impaired incorporation and release of completed polypeptide chains, whereas Sephadex-treated rat liver cell-sap promoted the polymerization of up to 90 amino acids per ribosome. Under optimum conditions 30–35% of the nascent polypeptide chains are completed and released. 3. The preparation of an antiserum against the calcium-binding protein formed in response to vitamin D is described. It is shown that the antiserum is highly specific for calcium-binding protein. 4. This antiserum was used to investigate the ability of chick intestinal polyribosomes to synthesize calciumbinding protein. Only polyribosomes from chicks receiving vitamin D have the ability to synthesize calcium-binding protein. Moreover, the product formed in vitro has the same electrophoretic mobility as calcium-binding protein synthesized in vivo. 5. It is concluded that one of the main functions of vitamin D in the small intestine is to induce the synthesis de novo of calcium-binding protein.