bluuebunny/crossref_metadata_2025 huggingface dataset:
The reaction of rabbit muscle creatine kinase with diethyl pyrocarbonate
Authors: Clarke D E , Price N C
Published: August 2015

The reaction of rabbit muscle creatine kinase with diethyl pyrocarbonate was studied. It was found that up to five of the sixteen histidine groups per enzyme subunit could be modified, and under the conditions employed, there was no evidence for formation of the disubstituted derivative of histidine. Evidence was obtained for small but significant amounts of modification of lysine and cysteine groups; tyrosine groups were not modified. Modification of the enzyme led to inactivation; this could be protected against by inclusion of substrates or, more effectively, by inclusion of the combination MgADP plus creatine plus nitrate, which is thought to produce a ‘transition-stage-analogue’ complex. Analysis of data on the rates of inactivation and the stoicheiometry of modification suggested that there was one essential histidine group per enzyme subunit, modification of which led to inactivation.
Crystallization of the Fv fragment of mouse myeloma protein M315
Authors: Aschaffenburg R , Phillips D C , Rose D R , Sutton B J , Dower S K , Dwek R A
Published: August 2015

The Fv fragment of mouse myeloma protein M313 was crystallized from poly(ethylene glycol) solution in the form of monoclinic crystals, space group C2 and unit cell dimensions a = 5.96 nm (59.6 A), b = 5.66 nm (56.6 A), c = 13.79 nm (13.9 A) and beta = 99.7 degrees. Some unusual effects of poly(ethylene glycol)on protein crystals were noted and are discussed.
Purification and properties of nitrogenase in ethylene glycol at sub-zero temperatures
Authors: Tsopanakis A D , Bray R C , Smith B E
Published: August 2015

Both the protein components Kp1 and Kp2 of nitrogenase from Klebsiella pneumoniae were found to be stable in aq. 50% (v/v) ethylene glycol at +30 degrees C or below. At -20 degrees C in this medium their sensitivities to O2 were diminished somewhat. Though purification could be carried out at -20 degrees C, the product had the same specific activity and was obtained in the same yield as when the purification was carried out by standard procedures. This suggests that such procedures yield enzyme undamaged in the course of the purification by O2, thermal denaturation or proteolytic digestion.
Multiplicity of molecules carrying blood-group-I antigen on erythrocyte membranes
Authors: Childs R A , Feizi T , Tonegawa Y
Published: August 2015

A human serum containing a monoclonal anti-(blood-group I) antibody was used to investigate the distribution of blood-group-I antigen on erythrocyte membrane components. Sodium dodecyl sulphate/polyacrylamide-gel-electrophoresis profiles of immuneprecipitates by using 3H-labelled (by the galactose oxidase/NaB3H4 method) and 125I-labelled solubilized stroma were compared. Different radioactive profiles were revealed by the two radiolabelling methods. In the immunoprecipitates the predominant 125I radioactivity within the gel had the electrophoretic mobility of Band-3 protein (apparent mol.wt. 90 000–100 000), whereas the 3H radioactivity revealed a diffusely migrating component(s) (apparent mol.wt. range 40 000–70 000) in addition to radioactivity compatible with glycolipids at the dye front. The diffusely migrating 3H-labelled component was shown to have a similar electrophoretic mobility to a subpopulation of erythrocyte poly(glycosyl)ceramides with blood-group-I activity.
Interaction of di-iodinated 125I-labelled α-bungarotoxin and reversible cholinergic ligands with intact synaptic acetylcholine receptors on isolated skeletal-muscle fibres from the rat
Authors: Darveniza P , Morgan-Hughes J A , Thompson E J
Published: August 2015

1. Intact synaptic acetylcholine receptors on freshly isolated rat skeletal-muscle fibres were characterized by their interaction with di-iodinated 125I-labelled alpha-bungarotoxin, acetylcholine and other cholinergic ligands at room temperature (22 deggrees C). 2. The time course and concentration dependence of 125I-labelled alpha-bungarotoxin association conformed to a bimolecular mechanism. In time-course experiments with different concentrations of 125I-labelled alpha-bungarotoxin (1.4–200 nM) the bimolecular-association rate constant, k + 1, was (2.27 +/- 0.49) × 10(4)M-1.S-1 (mean +/- S.D., N = 10). In concentration-dependence experiments, k + 1 was 2.10 × 10(4)M-1.S-1 and 1.74 × 10(4) M-1.S-1 with 10 and 135 min incubations respectively. In association experiments the first-order rate constant was proportional to the 125I-labelled alpha-bungarotoxin concentration. 125I-Labelled alpha-bungarotoxin dissociation was first order with a dissociation constant, k-1, less than or equal to 3 × 10(-6)S(-1) (half-life greater than or equal to 60 h.) The results indicated a single class of high-affinity toxin-binding sites at the end-plate with an equilibrium dissociation constant, Kd, equal to or less than 100 pM. The number of toxin-binding sites was (3.62 +/- 0.46) × 10(7) (mean +/- S.D., n = 22) per rat end-plate. 3. The apparent inhibitor dissociation constants, Ki, for reversible cholinergic ligands were determined by studying their effect at equilibrium on the rate of 125I-labelled alpha-bungarotoxin binding. There was heterogeneity of binding sites for cholinergic ligands, which were independent and non-interacting with antagonists. In contrast agonist affinity decreased with increasing receptor occupancy. Cholinergic ligands in excess inhibited over 90% of 125I-labelled alpha-bungarotoxin binding. 4. Cholinergic ligand binding was accompanied by an increase in entropy, which was greater for the agonist carbachol (delta So = +0.46 kJ.mol-1.K-1) than the antagonist tubocurarine (delta So = +0.26 kJ.mol-1.K-1). 5. The entropy and affinity changes that accompanied agonist binding suggested that agonists induced significant conformational changes in intact acetylcholine receptors. 6. The affinity and specificity of 125I-labelled alpha-bungarotoxin and tubocurarine binding to synaptic acetylcholine receptors from slow and fast muscle fibres were the same. 7. The study of binding only requires milligram amounts of tissue and may have application to other neurobiological studies and to the study of human neuromuscular disorders.
The binding of ferric iron by ferritin
Authors: Treffry A , Harrison P M
Published: August 2015

Equilibrium-dialysis experiments with 59Fe-labelled Fe(III) chelate solutions show that ferritin is capable of binding a limited number of Fe(III) atoms. Some of this Fe(III) is readily removed, but up to about 200 Fe(III) atoms/molecule remain bound after extensive washing. Some exchange of labelled Fe(III) with endogenous unlabelled ferritin Fe occurs during prolonged dialysis against 59Fe(III)-citrate, but there is a net binding of Fe(III). Bound Fe(III) resembles endogenous Fe(III) in several respects. It appears to be attached to the micelle and not to the protein component of ferritin. Although the physiological mechanism of Fe incorporation into ferritin is unknown, our experiments suggest the possibility that some iron finds its way into ferritin as Fe(III) chelate.
Delineation of fucosyltransferase activities with thiol reagents
Authors: Kessel D , Chou T H
Published: August 2015

The thiol reagent dithiothreitol inhibits the activity of a core GDP-fucose-N-acetylglucosaminide alpha-6-fucosyltransferase in plasma and blood-cell homogenates, while promoting the activity of alpha-2- and alpha-3-fucosyltransferases. The latter enzymes catalyse transfer of fucose on to terminal galactose and subterminal N-acetylglucosamine residues respectively. A thiol-blocking reagent N-ethylmaleimide does not affect the activity of the alpha-6-fucosyltransferase, but inhibits the other two enzymes. These results indicate the presence of a critical disulphide linkage in the alpha-6-fucosyltransferase, and provide a means of delineation of different fucosyltransferases.
The effect of inhibitors on the oxygen kinetics of terminal oxidases of Acanthamoeba castellanii
Authors: Lloyd D , Edwards S , Kristensen B , Degn H
Published: August 2015

1. Respiration of growing cultures of Acanthamoeba castellanii is inhibited less than 60% by azide (35 mM); the respiration of early-exponential-phase cultures differs from that of late-exponential-phase cultures in being stimulated by up to 120% by low concentrations (less than 1 mM) of this inhibitor. Azide (0.5 mM) plus 1 mM-salicylhydroxamic acid gives 80% inhibition of respiration in early- or late-exponential-phase cultures. 2. Lineweaver-Burk plots of 1/v against 1/[O2] for growing and stationary-phase cultures give values of less than 1 muM for the apparent Km for oxygen. 3. These values are not significantly altered when determined in the presence of 1 mM-salicylhydroxamic acid. 4. Higher values (greater than 7 muM) for apparent Km values for oxygen were obtained in the presence of azide, which gives non-linear Lineweaver-Burk plots. 5. Competitive inhibition of respiration by CO occurs with Ki 2.4 muM. 6. The results are discussed in terms of the presence of three terminal oxidases in this organism, namely two oxidases with high affinities for oxygen (cytochrome c oxidase of the main phosphorylating electron-transport chain and the salicylhydroxamic acid-sensitive oxidase) and a third oxidase with a low affinity for oxygen, sensitive to inhibition by cyanide but not by azide or salicylhydroxamic acid. The relative contributions to oxygen utilization by these oxidases change during the growth of a batch culture.
Studies on the mechanism of hepatic microsomal N-oxide formation. N-oxidation of NN-dimethylaniline by a reconstituted rabbit liver microsomal cytochrome P-448 enzyme system
Authors: Hlavica P , Hülsmann S
Published: August 2015

The N-oxidation of NN-dimethylaniline was studied by using a reconstituted rabbit liver microsomal enzyme system consisting of highly purified cytochrome P-448, NADPH-cytochrome c reductase and lipid factor. Both cytochrome P-448 and NADPH-cytochrome c reductase were required for optimum N-oxygenating activity; the catalytic capacity of the reductase fraction for supporting N-oxide formation varied with the isolation procedure applied. Addition of microsomal lipids to the assay media stimulated N-oxidation of the arylamine. N-Oxide formation appeared to be not generally controlled by electron transfer from cytochrome b5 to cytochrome P-448. The present work confirms that cytochrome P-448 can mediate about 44% of the rabbit liver microsomal N-oxidation of NN-dimethylaniline, thus reinforcing the existence of at least two distinct tertiary amine N-oxidases, i.e. haemoprotein and flavoprotein oxidase, in liver microsomal fractions.
The mechanism of hepatic iron uptake from native and denatured transferrin and its subcellular metabolism in the liver cell
Authors: Milsom J P , Batey R G
Published: August 2015

Hepatic iron uptake and metabolism were studied by subcellular fractionation of rat liver homogenates after injection of rats with a purified preparation of either native or denatured rat transferrin labelled with 125I and 59Fe. (1) With native transferrin, hepatic 125I content was maximal 5 min after injection and then fell. Hepatic 59Fe content reached maximum by 16 h after injection and remained constant for 14 days. Neither label appeared in the mitochondrial or lysosomal fractions. 59Fe appeared first in the supernatant and, with time, was detectable as ferritin in fractions sedimented with increasingly lower g forces. (2) With denatured transferrin, hepatic content of both 125I and 59Fe reached maximum by 30 min. Both appeared initially in the lysosomal fraction. With time, they passed into the supernatant and 59Fe became incorporated into ferritin. The study suggests that hepatic iron uptake from native transferrin does not involve endocytosis. However, endocytosis of denatured transferrin does occur. After the uptake process, iron is gradually incorporated into ferritin molecules, which subsequently polymerize; there is no incorporation into other structures over 14 days.
Rapid effects of isoprenaline, glucagon, pacing and potassium arrest on post-heparin lipoprotein lipase activity in the perfused rat heart
Authors: Simpson J
Published: August 2015

The amount of lipoprotein lipase activity released by heparin into the perfusion medium of isolated rat hearts could be increased within 60s by isoprenaline, glucagon or pacing. Potassium arrest and propranolol inhibited the effects of isoprenaline and pacing respectively.
Transport of glutamine by rat kidney brush-border membrane vesicles
Authors: McFarlane-Anderson N , Alleyne G A O
Published: August 2015

Transport of glutamine by brush-border vesicles prepared from the renal cortex was studied. The transport system had both Na+-dependent and Na+-independent components. The presence of Na+ in the incubation resulted in an ‘overshoot’ at 30s at which time the rates of transport were approx. 8 times the values obtained in the absence of Na+. Variation of the glutamine concentration showed that the system obeyed Michaelis-Menten kinetics with Km and Vmax. values for the Na+-dependent system of 0.86 mM and 9.6 nmol/min per mg of protein respectively. Vesicles obtained from chronically acidotic rats showed similar kinetic characteristics. The Km and Vmax. values for the Na+-dependent system were 0.76 mM and 9.6 nmol/min per mg of protein respectively. There was increased uptake of glutamine by vesicles from acidotic rats and this increase was associated with increased activity of gamma-glutamyltransferase in these preparations. Vesicles from acidotic rats, however, showed no increase in glucose transport and no increase in the activity of maltase, another brush-border enzyme.
Studies on the isolation and properties of renin granules from the rat kidney cortex
Authors: Sagnella G A , Peart W S
Published: August 2015

The present study was undertaken to isolate and investigate some physicochemical properties of renin granules from the rat kidney cortex. Two preparations of subcellular organelles were used: a primary-granule fraction, which allowed the properties of lysosomes to be compared simultaneously with those of renin granules, and a semi-purified preparation of the latter. The specific activity of renin in the primary-granule preparations was about 4-fold higher than in the original homogenate; that of the semi-purified renin-granule preparation was about 18-fold higher than in the homogenate, and consisted mainly of electron-dense granules but some mitochondria were also observed. Renin and acid phosphatase release from the primary-granule preparation was increased by lowering osmolality, by a low-molecular-weight solute (glucose) and by Triton X-100 or digitonin. Enzyme release was decreased by lowering the incubation temperature (4 degrees C) or the presence of CaCl2. Renin release from the partially purified granule preparation was not affected by cyclic AMP, cyclic GMP and ATP.
Studies on enzymes of collagen biosynthesis and the synthesis of hydroxyproline in macrophages and mast cells
Authors: Myllylä Raili , Seppä Heikki
Published: August 2015

The activities of four intracellular enzymes of collagen biosynthesis were assayed in freshly isolated rat peritoneal macrophages and mast cells and compared with the same enzymes in freshly isolated chick-embryo tendon cells. The macrophages were found to contain activities of all four enzymes, those of prolyl and lysyl hydroxylase being 7 and 12% respectively of those in the tendon cells when expressed per cell or 3 and 4% when expressed per unit of soluble cell protein. The corresponding values for hydroxylysyl galactosyltransferase and galactosylhydroxylysyl glucosyltransferase activities were about 82 and 68% or 32 and 24% respectively. When the macrophages were incubated in suspension with [14C]proline, they synthesized a small but significant amount of non-diffusible hydroxy[14C]proline. The synthesis per cell was only about 0.1% of that formed by the tendon cells, and its distribution between the cells and the medium also differed from that in the tendon cells. The hydroxy[14C]proline synthesized by the macrophages may be present in the Clq subcomponent of the complement, but its amount was too small to allow any characterization of the protein. All four enzyme activities, and in particular the two hydroxylysyl glycosyltransferase activities, seem to be present in macrophages in a large excess compared with the very low rate of synthesis of hydroxy-proline-containing polypeptide chains. The mast cell extract was found to inhibit all four enzyme activities, but even when corrected for this inhibition, prolyl and lysyl hydroxylase activities in the mast cells were less than 0.08% and the two hydroxylysyl glycosyltransferase activities less than 1% of those in the tendon cells. The intracellular enzyme pattern of collagen biosynthesis in the mast cells is thus completely or virtually completely repressed.
Biosynthesis of skin collagens in normal and diabetic mice
Authors: Kern P , Moczar M , Robert L
Published: August 2015

Synthesis of collagens in vitro was studied on minced mouse skins incubated with [3H]-proline in organ-culture conditions. A comparative study was carried out on genetically diabetic mice (KK strain) and control mice (Swiss strain). After incubation, neutral-salt-soluble and acid-soluble collagens were extracted. The insoluble dermis was digested by pepsin and type I and type III collagens separated by differential precipitation in neutral salt solutions. Type I and Type III collagens were characterized by ion-exchange and molecular-sieve chromatography, amino acid analysis and by the characterization of CNBr peptides. In diabetic-mouse skin, the relative proportion of type III collagen was significantly higher than in control-mouse skin. The incorporation of radioactively labelled proline into hydroxyproline of type III collagen was significantly faster in diabetic-mouse skin than in control-mouse skin. No significant modifications in the total collagen content of the skin or of their rates of synthesis were observed between the two strains. Alteration in the ratio of type III to type I collagen in the diabetic-mouse skin can be interpreted as a sign of alteration of the regulation of collagen biosynthesis and may be related to the structural alterations observed in the diabetic intercellular matrix.
Sequential changes in rat liver nuclear tri-iodothyronine receptors and mitochondrial α-glycerophosphate dehydrogenase activity after administeration of tri-iodothyronine
Authors: Nakamura Hirotoshi , Hamada Satoshi , Imura Hiroo
Published: August 2015

The dynamics of the induction of nuclear tri-iodothyronine receptors and mitochondrial α-glycerophosphate dehydrogenase were studied in rat liver after a single injection of tri-iodothyronine. The maximal binding capacity (Cmax.) and association constant (Ka) of the nuclear receptors were determined by Scatchard analyses with and without correction for the endogenous tri-iodothyronine measured by radioimmunoassay. The administration of tri-iodothyronine induced sequential increases in the concentration of nuclear receptors and α-glycerophosphate dehydrogenase activity in the liver. The nuclear-receptor concentration was increased to 2.5 times that in the hypothyroid rat 1 day after the administration of hormone, and then decreased, with a half-life of about 2 days. α-Glycerophosphate dehydrogenase activity changed in parallel with the nuclear-receptor concentration, showing a delayed response. The total amount of non-histone protein in the liver was significantly increased 3 days after the administration. It seems likely therefore that the tri-iodothyronine-induced increase in nuclear-receptor concentration is responsible, at least in part, for the induction of this enzyme. The possibility is also suggested that nuclear receptors may be one of the non-histone proteins selectively synthesized at an early stage of the hormonal stimulation. Throughout the time course, the Ka values of the nuclear receptors for tri-iodothyronine remained unchanged, when corrected for endogenous tri-iodothyronine bound to the non-histone proteins, although they were apparently changed when the correction was not made. The results obtained provide further evidence for hormonal modulation of the nuclear receptors which is closely linked with the hormonal effect.
Increase in fatty acid synthetase content of 3T3-L cells undergoing spontaneous and chemically induced differentiation to adipocytes
Authors: Ahmad P M , Russell T R , Ahmad F
Published: August 2015

3T3-L fibroblasts differentiate into adipose cells when maintained in a non-growing state. The specific activity of fatty acid synthetase of differentiated cells was 25–30-fold higher than that present in 3T3-L fibroblasts or in 3T3-C2 cells that possess an extremely low incidence of differentiation to adipocytes. The results of immunochemical analysis indicate that the increased specific activity of fatty acid synthetase in the differentiated cells is due to an increase in the cellular content of this enzyme. The rate of conversion of adipose cells was accelerated by brief exposure of confluent non-growing cultures of 3T3-L cells to 3-isobutyl-1-methylxanthine. This was accompanied by an increase in the specificity activity of fatty acid synthetase, which was also shown to be due to an increase in the cellular content of this enzyme. The continuous presence of 3-isobutyl-1-methylxanthine in the culture medium was not required to elicit the morphological and biochemical changes in 3T3-L cells that occurred many days after the removal of the inducer but earlier than the onset of spontanous differentiation.
Metabolism of glucose in hyper- and hypo-thyroid rats in vivo. Minor role of endogenous insulin in thyroid-dependent changes in glucose turnover
Authors: Okajima F , Ui M
Published: August 2015

1. Rates and rate coefficients of glucose utilization and replacement (glucose turnover) as well as its recycling were determined in rats by using [U-14C]- and [2-3H]-, [3-3H]- or [6-3H]-glucose. 2. In euthyroid rats, the blood concentration of glucose was 1.5 times and its turnover rate was 2 times as high in the fed state as in the starved state; consequently the rate coefficient, a measure of the capacity of rats to utilize blood glucose, was also higher in the former than in the latter. 3. Induction of mild diabetes by streptozotocin exerted little influence on the content and turnover of blood glucose in the starved state, whereas it caused hyperglycaemia and a decrease in the rate coefficient after feeding. 4. Induction of hyperthyroidism caused increases in rates and rate coefficients of glucose turnover to substantially the same extent whether or not the plasma concentration of insulin was lowered by treatment with streptozotocin or injection with anti-insulin serum. 5. It is concluded that thyroid hormones are capable of enhancing glucose turnover in the starved state independently of endogenous insulin, which plays a significant role in increasing glucose utilization in the fed state.
Composition and spectral properties of a cytochrome-enriched fraction obtained from chloroplasts by digitonin treatment
Authors: Cox R P
Published: August 2015

A cytochrome-enriched fraction obtained from chloroplasts after treatment with the detergent digitonin contained cytochromes f, b-559LP and b-563 in the approximate proportions 1:1:2, close to those observed in unfractionated chloroplasts. The spectrum of cytochrome b-563 at temperature of liquid N2 showed a single asymmetrical alpha-band with a maximum at 561 nm.
An active-site-directed adenosine triphosphate analogue binds to the β-subunits of factor F1 mitochondrial adenosine triphosphatase with its triphosphate moiety
Authors: Drutsa V L , Kozlov I A , Milgrom Y M , Shabarova Z A , Sokolova N I
Published: August 2015

The reaction of the mixed anhydride of [3H]ATP and mesitylenecarboxylic acid and soluble mitochondrial adenosine triphosphatase is accompanied by the covalent binding of one molecule of the inhibitor to a molecule of the enzyme and results in the inhibition of adenosine triphosphatase activity by more than 90%. The electrophoresis of adenosine triphosphatase modified by reaction with the mixed anhydride of [3H]ATP and mesitylenecarboxylic acid in polyacrylamide gel in the presence of sodium dodecyl sulphate showed that the inhibitor is bound to the beta-subunit of the enzyme. The results suggest that ATP may also bind to the beta-subunit of the adenosine triphosphatase with its triphosphate moiety.
Incorporation of exogenous precursors into uridine and ribonucleic acid. Nucleotide compartmentation in the renal cortex in vivo
Authors: Cortes Pedro , Levin Nathan W. , Dumler Francis , Venkatachalam Kumarapuram K. , Verghese Chacko P. , Bernstein Jay
Published: August 2015

The possibility of compartmentation of UTP in vivo was investigated in the renal cortex of unanaesthetized rats. In addition, liver and spleen were studied in order to compare tissues with different utilization of precursors for pyrimidine nucleotide synthesis. After continuous 2h infusions of [3H]uridine or [3H]orotate, their incorporation into UTP, UDP-sugars and RNA was quantified. Rates of RNA synthesis were calculated by dividing the incorporation of precursor into RNA by the average specific radioactivity of the UTP pool. Although similar RNA-synthesis rates might have been expected with the two precursors, higher rates were found with uridine than with orotate. The relative incorporation into UDP-sugars of these precursors was also different. Similar results were obtained in the liver. In the spleen, equal amounts of both precursors were incorporated into UTP, but [3H]orotate incorporation did not lead to labelling of RNA. To evaluate the heterogeneity of cells with respect to the metabolism of pyrimidines, precursor incorporation was studied in isolated glomeruli and by radioautography. Incorporation into glomeruli was qualitatively similar to but quantitatively different from results in the renal cortex. Although there is obvious tissue heterogeneity, compartmentation of UTP pools is the most credible explanation for the results obtained with the renal cortex and liver. Consequently RNA and UDP-sugars may originate from two different UTP pools. Tissue heterogeneity is the likely explanation for the results obtained in the spleen. Studies of synthesis of pyrimidine and RNA, particularly in relation to growth and regeneration, must take into consideration the precursor used, the apparent existence of UTP compartmentation and the degree of cellular heterogeneity.
Uridine kinase activities in developing, adult and neoplastic rat tissues
Authors: Herzfeld A , Raper S M
Published: August 2015

Uridine kinase activities were found chiefly in the soluble fractions of rat tissues. In normal adults the activities ranged from 13 munits/g in skeletal muscle to 178 munits/g in colon. Enzyme activities in several rat neoplasms were significantly higher (e.g. in a fibrosarcoma, mammary carcinoma, renal carcinoma, pancreatic carcinoma and lymphocytic lymphoma, but not in a fast-growing Morris hepatoma). The activities were not related to tumour growth rates or sizes. In normal foetal liver, lung, brain, heart and kidney, uridine kinase concentrations equalled or exceeded those in the adult homologous tissue, but maximal activities in liver were reached 3–5 days post partum. In suckling rats the intestinal activity decreased substantially immediately after birth and normally did not rise again until late in the third postnatal week. Premature upsurges could be evoked by an injection of cortisol or by starvation of the pups overnight. Pancreatic activity was absent from 1-day-old rats, and only about 5% of the adult activity was reached by day 20; adult activities were attained rapidly after weaning. In pancreas, precocious formation or uridine kinase was elicited by overnight starvation of 2-week-old rats.
Importance of mammalian nuclear-envelope nucleoside triphosphatase in nucleo-cytoplasmic transport of ribonucleoproteins
Authors: Agutter P S , McCaldin B , McArdle H J
Published: August 2015

The nucleoside triphosphate-stimulated efflux of RNA from isolated nuclei was studied under a range of conditions, and the effects of these conditions on the process were compared with the properties of the nucleoside triphosphatase located in the pore complex. A marked similarity between the rate of efflux and the rate of nucleoside triphosphate hydrolysis was apparent, in terms of substrate specificity, sensitivity to treatment with insolubilized trypsin, kinetics and the effects of increased ionic strength and of many inhibitors. These results are taken, in view of earlier evidence, to suggest that the activity of the nucleoside triphosphatase is a prerequisite for nucleo-cytoplasmic RNA transport in vivo. There are some indications that the nuclear-envelope lipid is also involved in regulating the efflux process.
Casein turnover in rabbit mammary explants in organ culture
Authors: Al-Sarraj Khalidah , Newbury John , White David A. , Mayer R. John
Published: August 2015

1. Explants of mammary gland from mid-pregnant rabbits were cultured in medium 199 containing insulin, prolactin and cortisol, and specific anti-casein immunoglobulin G was used to measure the amount, rate of synthesis and rate of degradation of casein in the explants in the presence of hormones and after removal of hormones from previously stimulated tissue. 2. The amount of casein in particle-free supernatants prepared from mammary explants was measured by ‘rocket’ immunoelectrophoresis. 3. The rate of incorporation of l-[4,5-3H]leucine into casein was measured after isolation of the casein by immunoadsorbent chromatography and polyacrylamide-gel electrophoresis in the presence of urea and sodium dodecyl sulphate. 4. Casein accumulates in mammary explants in the presence of insulin, prolactin and cortisol, but not in the absence of hormones. Removal of hormones after 24h in culture results in a decrease in the rate of accumulation of casein in the explants. 5. Casein-synthetic rate increases in mammary explants in the presence of insulin, prolactin and cortisol, but not in the absence of hormones. Removal of hormones after 24h in culture results in continued casein synthesis at approx. 30% of the rate in the presence of hormones. The synthetic rate does not decrease to values observed in explants cultured throughout in the absence of hormones. 6. Casein is not degraded in mammary explants during a phase of rapid casein accumulation (36–72h) in the presence of hormones. Furthermore casein is not degraded when hormones are removed from the tissue after between 36 and 72h in culture. 7. Casein is glycosylated in mammary explants; the extent of glycosylation parallels the rate of synthesis. The glycosylated protein is rapidly secreted from the tissue. 8. The results are consistent with the notion that after hormonal stimulation mammary explants from mid-pregnant rabbits synthesize, glycosylate and rapidly secrete casein. Removal of hormones decreases the synthetic rate of casein, but does not cause the accumulation of a pool of degradable casein in the lobuloalveolar cells.
Comparison of glyoxalase I purified from yeast (Saccharomyces cerevisiae) with the enzyme from mammalian sources
Authors: Marmstål Ewa , Aronsson Anne-Charlotte , Mannervik Bengt
Published: August 2015

Glyoxalase I from yeast (Saccharomyces cerevisiae) purified by affinity chromatography on S-hexylglutathione–Sepharose 6B was characterized and compared with the enzyme from rat liver, pig erythrocytes and human erythrocytes. The molecular weight of glyoxalase I from yeast was, like the enzyme from Rhodospirillum rubrum and Escherichia coli, significantly less (approx. 32000) than that of the enzyme from mammals (approx. 46000). The yeast enzyme is a monomer, whereas the mammalian enzymes are composed of two very similar or identical subunits. The enzymes contain 1Zn atom per subunit. The isoelectric points (at 4°C) for the yeast and mammalian enzymes are at pH7.0 and 4.8 respectively; tryptic-peptide ‘maps’ display corresponding dissimilarities in structure. These and some additional data indicate that the microbial and the mammalian enzymes may have separate evolutionary origins. The similarities demonstrated in mechanistic and kinetic properties, on the other hand, indicate convergent evolution. The kcat. and Km values for the yeast enzyme were both higher than those for the enzyme from the mammalian sources with the hemimercaptal adduct of methylglyoxal or phenylglyoxal as the varied substrate and free glutathione at a constant and physiological concentration (2mm). Glyoxalase I from all sources investigated had a kcat./Km value near 107s−1·m−1, which is close to the theoretical diffusion-controlled rate of enzyme–substrate association. The initial-velocity data show non-Michaelian rate saturation and apparent non-linear inhibition by free glutathione for both yeast and mammalian enzyme. This rate behaviour may have physiological importance, since it counteracts the effects of fluctuations in total glutathione concentrations on the glyoxalase I-dependent metabolism of 2-oxoaldehydes.
The exchange of histidine C-2 protons in superoxide dismutases. A novel method for assigning histidine-metal ligands in proteins
Authors: Cass A E G , Hill H A O , Bannister J V , Bannister W H , Hasemann V , Johansen J T
Published: August 2015

The rates of exchange of the C-2 protons of histidine residues in copper-zinc superoxide dismutase are substantially decreased by metal ion binding. This observation was used to distinguish between ligand and non ligand histidine residues in bovine and yeast copper-zinc superoxide dismutases; the effect was shown to depend only on metal ion co-ordination and not as a consequence of concomitant changes in protein structure. Selective deuteration of the zinc-only proteins at pH (uncorrected pH-meter reading) 8.2 and 50 degrees C resulted in the distinction between copper and zinc ligand resonances in the 1H n.m.r. spectrum of the enzymes. This method is proposed as a generally applicable technique for identifying histidine residues as ligands in metalloproteins.
Inhibition by trioxalen (psoralen) plus near-ultraviolet light of the induction of ornithine decarboxylase in Chinese-hamster cells
Authors: Heimer Y M , Riklis E
Published: August 2015

Trioxalen (trimethylpsoralen) plus near-u.v. light, a potent inhibitor of DNA and RNA synthesis, inhibits the induction of ornithine decarboxylase in stationary-phase V79 fibroblasts. It does not affect the translation of pre-existing mRNA. The method, in view of its high degree of specificity and precise timing, is a better choice for inhibiting RNA synthesis than the commonly used chemical inhibitors and precursor analogues.
Wheat-germ aspartate transcarbamoylase. Purification and cold-lability
Authors: Grayson J E , Yon R J , Butterworth P J
Published: August 2015

1. Aspartate transcarbamoylase was purified approx. 3000-fold from wheat (Triticum vulgare) germ in 15-20% yield. The product has a specific activity of 14 mumol/min per mg of protein and is approx. 90% pure. The purification scheme includes the use of biospecific “imphilyte” chromatography as described by Yon [Biochem. J. (1977) 161, 233-237]. The enzyme was passed successively through columns of CPAD [N-(3-carboxypropionyl)aminodecyl]-Sepharose in the absence and presence respectively of the ligands UMP and L-aspartate. In the second passage the enzyme was specifically displaced away from impurities with which it co-migrated in the first passage. These two steps contributed a factor of 80 to the overall purification. 2. The enzyme is slowly inactivated on dilution at 0 degrees C and pH 7.0, the inactivation being partially reversible. A detailed investigation of the temperature- and pH-dependence of the cold-inactivation suggested that it was initiated by the perturbation of the pKa values of groups with a moderately high and positive heat of ionization, which were tentatively identified as histidine residues. These findings support a new concept of cold-lability proposed by Bock, Gilbert & Frieden [Biochem. Biophys. Res. Commun. (1975) 66, 564-569].
Haemoglobin from the tadpole shrimp, Lepidurus apus lubbocki Characterization of the molecule and determination of the number of polypeptide chains
Authors: Ilan E , Daniel E
Published: August 2015

Haemoglobin from the tadpole shrimp, Lepidurus apus lubbocki, was found to have a sedimentation coefficient (s020,w) of 19.3 +/- 0.2 S and a molecular weight, as determined by sedimentation equilibrium, of 798000 +/- 20000. The amino acid composition showed the lack of cysteine and cystine residues. A haem content of 3.55 +/- 0.03% was determined, corresponding to a minimal mol.wt. of 17400 +/- 200. The pH-independence in the range pH 5-11 of the sedimentation coefficient indicates a relatively high stability of the native molecule. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis gave one band with mobility corresponding to a mol.wt. of 34000 +/- 1500. The molecular weight of the polypeptide chain was determined to be 32800 +/- 800 by sedimentation equilibrium in 6 M-guanidinium chloride and 0.1 M-2-mercaptoethanol. The findings indicate that Lepidurus haemoglobin is composed of 24 identical polypeptide chains, carrying two haem groups each.
Purification of fibronectin from human plasma by affinity chromatography under non-denaturing conditions
Authors: Vuento M , Vaheri A
Published: August 2015

Fibronectin was purified from human plasma by affinity chromatography under nondenaturing conditions. The method was based on the previously known binding of fibronectin to gelatin. The novel features of our method are the use of arginine in the elution of fibronectin from immobilized gelatin [Vuento & Vaheri (1978) Biochem. J. 175, 333-336] and the use of arginine-agarose as second affinity step. The purified protein was homogeneous as judged by polyacrylamide-gel electrophoresis, analytical ultracentrifugation and two-dimensional immunoelectrophoresis. The yield was 60%. We propose that the method would be useful in preparation of fibronectin for studies on its biological activities, where it is important that the protein is obtained in a native state.
The kinetics of effector binding to phosphofructokinase. The allosteric conformational transition induced by 1,N6-ethenoadenosine triphosphate
Authors: Roberts D , Kellett G L
Published: August 2015

1. The fluorescent ATP analogue 1,N6-etheno-ATP is a good substrate and an efficient allosteric inhibitor of rabbit skeletal-muscle phosphofructokinase. 2. Fluorescence energy transfer occurs between bound 1,N6-etheno-ATP and phosphofructokinase. 1,N6-Etheno-ATP fluorescence is enhanced, intrinsic protein fluorescence is quenched, and the excitation spectrum of 1,N6-etheno-ATP fluorescence is characteristic of protein absorption. 3. The binding reaction of 1,N6-etheno-ATP observed by stopped-flow fluorimetry is biphasic. The fast phase results from binding to the catalytic site alone. The slow phase results from the allosteric transition of the R conformation into the T conformation induced by the binding of 1,N6-etheno-ATP to the regulatory site. 4. The fluorescence signal that allows the transition of the R conformation into the T conformation to be observed does not arise from 1,N6-etheno-ATP bound to the regulatory site. It arises instead from 1,N6-etheno-ATP bound to the catalytic site as a consequence of changes at the catalytic site caused by the transition of the R conformation into the T conformation. 5. In the presence of excess of Mg2+, the affinity of 1,N6-etheno-ATP for the regulatory site is very much greater in the T state than in the R state.
Decarboxylation of porphyrinogens by rat liver uroporphyrinogen decarboxylase
Authors: Smith A G , Francis J E
Published: August 2015

The decarboxylations of uroporphyrinogens I and III and of heptacarboxylic, hexacarboxylic and pentacarboxylic porphyrinogens III by rat liver uroporphyrinogen decarboxylase were compared, and the results suggest that the removal of the first carboxy group from uroporphyrinogen III is a more rapid step than that from isomer I or the other substrates investigated.
Mechanism of formation, spectrum and reactivity of half-reduced eight-iron Clostridium pasteurianum ferredoxin in pulse-radiolysis studies and the non-co-operativity of the four-iron clusters
Authors: Butler J , Henderson R A , Armstrong F A , Sykes A G
Published: August 2015

Reduction of fully oxidized Clostridium pasteurianum 8-Feox.,ox. ferredoxin by using pulse-radiolysis techniques yields the half-reduced species 8-Feox.,red. ferredoxin. The subsequent oxidation of 8-Feox.,red. ferredoxin with Co(NH3)5Cl2+ was studied. From a comparison with stopped-flow studies on the 2:1 Co(NH3)5Cl2+ oxidation of 8-Fered.,red. ferredoxin to the 8-Feox.,ox. form it is concluded that there is no redox co-operativity between the two 4-Fe centres in these reactions.
Isolation and characterization of binding proteins for retinol from the cytosol, nucleosol and chromatin of the oviduct magnum of laying hens
Authors: Rao M R S , Prasad V R , Padmanaban G , Ganguly J
Published: August 2015

Protein fractions that bind retinol were isolated from the cytosol, nucleosol and chromatin of the oviduct magnum of laying hens. The proteins isolated from the three sources showed similar elution profiles on chromatography through Sephadex G-75 and G-50 columns, and comparable mobility during electrophoresis on sodium dodecyl sulphate/polyacrylamide gels. Their molecular weights were calculated to be around 14500. When oviducts from vitamin A-depleted and vitamin A-repleted immature chicks given oestrogen injections for 6 consecutive days were incubated with [3H]retinyl acetate, uptake of the radioactivity in the nuclei of the vitamin A-depleted tissue was severalfold higher than that in the nuclei from the vitamin A-repleted tissue.
Partial purification and characterization of deoxyguanosine kinase from pig skin
Authors: Green F J , Lewis R A
Published: August 2015

Deoxyguanosine kinase, which catalyses the phosphorylation of deoxyguanosine to form deoxyguanosine 5′-monophosphate, was purified 1024-fold from extracts to newborn-pig skin. This activity requires the presence of a bivalent cation and a nucleoside triphosphate, which functions as a phosphate donor, ATP being twice as effective as CTP or GTP and 4 times as effective as UTP. The enzyme appears to have a molecular weight of 58500 as determined by Sephadex-column chromatography. Optimal enzymic activity was observed at pH 8.0; however, the enzyme remained active over a broad pH range of 5.5-9.0. Several deoxyribonucleoside and ribonucleoside monophosphates and triphosphates were tested as effectors of catalytic activity. Effective inhibitors were dGMP [Ki(app.) = 7.6 × 10(-5) M] and dGTP [Ki(app.) = 2.1 × 10(-5) M]. Both of these inhibitors acted in a competitive manner. A Km(app.) of 3.2 × 10(-7) M was measured for deoxyguanosine and a Km(app.) of 3.3 mM was determined for MgATP. Of the four major deoxynucleosides tested, this catalytic activity appears to phosphorylate only deoxyguanosine; thus the enzyme is a specific deoxyguanosine kinase.
The purification and characterization of subcomponent C1s of the first component of bovine complement
Authors: Campbell R D , Booth N A , Fothergill J E
Published: August 2015

Bovine C1s, a subcomponent of the first component of complement, was purified in good yield by a combination of euglobulin precipitation and ion-exchange and molecular-sieve chromatography. Approx. 10 mg can be obtained from 3 litres of serum, representing a yield of 11%. The C1s is obtained in zymogen form, with a mol.wt. of 85000-88000, determined by gel filtration and SDS/polyacrylamide-gel electrophoresis. It is haemolytically active when tested with human C1q and C1r. Activation can be achieved by incubation with human C1r, resulting in cleavage of the C1s chain into two chains of 65000 and 27000 mol.wt. and the generation of an isoleucine N-terminal residue on the smaller chain. Active C1s binds an equimolar amount of di-isopropyl phosphorfluoridate to the smaller chain, which is the C-terminal part in the zymogen. The chains can be separated by ion-exchange in 8 M-urea. All of these characteristics show that bovine C1s is very similar to its human counterpart.
Changes associated with glycolytic-enzyme binding in the equatorial X-ray-diffraction pattern of glycerinated rabbit psoas muscle
Authors: Stewart M , Morton D J , Clarke F M
Published: August 2015

The binding of fructose biphosphate aldolase to the thin filaments of glycerinated rabbit psoas muscle produces a significant change in its low-angle X-ray-diffraction pattern. The intensity of the (11) reflection relative to that of the (10) reflection increases by 26 +/- 3% (mean +/- S.E.M.), which is consistent with the increase in the mass of the thin filaments produced by enzyme binding. A similar effect is found with a mixture of aldolase and glyceraldehyde 3-phosphate dehydrogenase. The significance of the change in intensity is considered with reference to the interpretation of the equatorial patterns obtained from muscles in different physiological states. The magnitude of the increase in the relative intensity of the (11) reflection is lower than that observed between relaxed and contracting muscle and does not bring into question the interpretation linking changes in these patterns to cross-bridge movement. However, the effect due to enzyme binding may be important when making detailed interpretations of these changes. It may also be related to an unusual pattern sometimes observed in cardiac muscle.
Structure of heparan sulphate oligosaccharides and their degradation by exo-enzymes
Authors: Linker A
Published: August 2015

Oligosaccharides obtained from heparan sulphate by nitrous acid degradation were shown to be degraded sequentially by beta-D-glucuronidase or alpha-L-iduronidase followed by alpha D-N-acetylglucosaminidase. Structural analysis of the tetrasaccharide fraction showed the following. (1) N-Acetylglucosamine is preceded by a non-sulphated uronic acid residue that can be either D-glucuronic of L-iduronic acid, but followed by a glucuronic acid residue. (2) The N-acetylglucosamine in the major fraction is sulphated. (3) Very few if any of the uronic acid residues are sulphated (4). The results indicate that the area of the heparan sulphate chain where disaccharides containing N-acetylglucosamine and N-sulphated glucosamine residues alternate is higher in sulphate content than expected and that the sulphate groups are mainly located on the hexosamine units.
Adrenaline and the regulation of acetyl-coenzyme A carboxylase in rat epididymal adipose tissue. Inactivation of the enzyme is associated with phosphorylation and can be reversed on dephosphorylation
Authors: Brownsey R W , Hughes W A , Denton R M
Published: August 2015

1. Exposure of rat epididymal fat-pads or isolated fat-cells to adrenaline results in a decrease in acetyl-CoA carboxylase activity measured both in initial extracts and in extracts incubated with potassium citrate; in addition the concentration of citrate required to give half-maximal activation may also be increased. 2. Incorporation of 32Pi into acetyl-CoA carboxylase within intact fat-cells was investigated and evidence is presented that adrenaline increases the extent of phosphorylation of the enzyme. 3. Dephosphorylation of 32P-labelled acetyl-CoA carboxylase was studied in cell extracts. The rate of release of 32P is increased by 5mM-MgCl2 plus 10–100 microM-Ca2+, whereas it is inhibited by the presence of bivalent metal ion chelators such as EDTA and citrate. 4. The effects of adrenaline on the kinetic properties of acetyl-CoA carboxylase disappear if pad or cell extracts are treated with Mg2+ and Ca2+ under conditions that also lead to dephosphorylation of the enzyme. 5. The results of this study represent convincing evidence that adrenaline inactivates acetyl-CoA carboxylase in adipose-tissue preparations by increasing the degree of phosphorylation of the enzyme.