bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of mitochondrial biogenesis. Occurrence of non-functioning components of the mitochondrial respiratory chain in Saccharomyces cerevisiae grown in the presence of proteinase inhibitors: evidence for proteolytic control over assembly of the respiratory chain
Authors: Galkin A V , Tsoi T V , Luzikov V N
Published: August 2015

Yeast was grown in glucose- or galactose-containing media without or with proteinase inhibitors, phenylmethanesulphonyl fluoride and pepstatin. Culture growth was practically not affected by these compounds. Yeast growth on glucose in the presence of either phenylmethanesulphonyl fluoride or pepstatin entails accumulation of cytochromes c, c1, b and aa3 to a 25–30% excess above the control by the stationary phase, while cell respiration is unaffected. During growth on galactose the maximal cytochrome content (per unit weight of biomass) is reached in the mid-exponential phase and then decreases by 30–40% towards the stationary phase, while cell respiration remains constant. Addition of phenylmethanesulphonyl fluoride or pepstatin in the mid-exponential phase blocks the decrease in cytochrome levels and has no effect on cell respiration. Mitochondrial populations isolated from stationary-phase control and phenylmethanesulphonyl fluoride-grown cells glucose cultures display identical succinate oxidase and partial-respiratory-chain activities, despite the differences in cytochrome contents. However, the activities of individual respiratory complexes measured after maximal activation are nearly proportional to the amounts of corresponding components. The same situation holds true for mitochondrial populations from mid-exponential-phase, stationary-phase control and stationary-phase inhibitor-grown cells of galactose cultures. The findings suggest that the ‘surplus’ respiratory-chain components do not participate in electron flow because of the lack of interaction with adjacent carriers.
Studies on the assembly of the rat lens capsule. Biosynthesis of a cross-linked collagenous component of high molecular weight
Authors: Heathcote J G , Bailey A J , Grant M E
Published: August 2015

1. Intact rat lenses in tissue culture synthesize hydroxy[3H]proline-containing polypeptides of apparent mol.wt. approx. 180000, which become assembled into aggregates of higher molecular weight with time. 2. Both the 180000-mol.wt. species and the aggregates are components of the deoxycholate-insoluble base-membrane matrix. 3. Formation of the high-molecular-weight aggregate is accompanied by the biosynthesis of the reducible hydroxylysine-derived cross-link hydroxylysino-5-oxo-norleucine. 4. Hydroxylysino-5-oxonorleucine and dehydrohydroxylysinonorleucine are the major reducible cross-links present in intact foetal and 1-month-old calf lens capsules.
The influence of fluoride administration on the structure of proteoglycans in the developing rat incisor
Authors: Smalley J W , Embery G
Published: August 2015

1. 35S-labelled chondroitin 4-sulphate proteoglycan was isolated from the mineralized elements of the developing incisor teeth of Harvard rats receiving intraperitoneal administration of Na235SO4. 2. The chondroitin 4-sulphate proteoglycan underwent a decrease in molecular size in fluorotic teeth as judged by gel filtration on Sepharose 2B. 3. When examined by anion-exchange chromatography on DEAE cellulose-52, the proteoglycan from fluorotic teeth resolved into four peaks in comparison with the material from non-fluorotic teeth, which exhibited only a single major peak. 4. Both the single peak from non-fluoridated teeth and the four peaks from the fluorotic teeth were further resolved on cellulose acetate electrophoresis. 5. Isolated chondroitin 4-sulphate chains obtained from fluorotic teeth also were of smaller molecular size as judged by gel filtration on Sephadex G-150. 6. Some possible influences of fluoride on the metabolism of these connective-tissue components in the developing rat incisor are discussed.
The effect of glucagon treatment and starvation of virgin and lactating rats on the rates of oxidation of octanoyl-l-carnitine and octanoate by isolated liver mitochondria
Authors: Zammit Victor A.
Published: August 2015

1. Oxygen-consumption rates owing to oxidation of octanoate or octanoylcarnitine by isolated mitochondria from livers of fed, starved and glucagon-treated virgin or 12-day-lactating animals were measured under State-3 and State-4 conditions, in the presence or absence of l-malate and inhibitors of tricarboxylic acid-cycle activity (malonate and fluorocitrate). 2. Mitochondria from fed lactating animals had a slightly lower rate of octanoylcarnitine oxidation than did those of fed virgin animals, whereas the rates of octanoate oxidation were unaffected. 3. Starvation of virgin animals for 24h or 48h resulted in a large (70–100%) increase in mitochondrial octanoylcarnitine oxidation; rates of octanoate oxidation were either unaffected (24 and 48h starvation in the absence of malonate and fluorocitrate) or diminished by 30% (48h starvation in the presence of inhibitors). In lactating animals, 24h starvation resulted in a smaller increase in the rate of octanoylcarnitine oxidation than that obtained for mitochondria from virgin rats. 4. Glucagon treatment (by intra-abdominal injection) of fed virgin and lactating rats increased the rate of mitochondrial oxidation of both octanoylcarnitine and octanoate. Injection of glucagon into 48h-starved virgin rats did not increase further the already elevated rate of octanoylcarnitine oxidation, but reversed the inhibition of octanoate β-oxidation observed for these mitochondria in the presence of malonate and fluorocitrate. 5. It is suggested that glucagon activates octanoylcarnitine oxidation by increasing the activity of the carnitine/acylcarnitine transport system [Parvin & Pande (1979) J. Biol. Chem.254, 5423–5429] and that the increase in octanoate oxidation by mitochondria from glucagon-treated animals is caused by the increased rate of ATP synthesis in these mitochondria. 6. The results are discussed in relation to the increased capacity of the liver to oxidize long-chain fatty acids and carnitine esters of medium-chain fatty acids under conditions characterized by increased ketogenesis.
High affinity of α-foetoprotein for arachidonate and other fatty acids
Authors: Carlsson R N K , Estes T , Degroot J , Holden J T , Ruoslahti E
Published: August 2015

Fatty acid analysis of purified bovine alpha-foetoprotein showed it to contain 2.7 mol of fatty acid/mol of alpha-foetoprotein. Purified alpha-foetoprotein focused at isoelectric point 4.8. Removal of bound ligands from alpha-foetoprotein by charcoal treatment changed its isoelectric point to 5.2. This change could be reversed by addition of exogenous fatty acids to the defatted alpha-foetoprotein. Albumin isolated from the same foetal calf serum source as alpha-foetoprotein contained 1.4 mol of fatty acid/mol of protein. alpha-Foetoprotein and albumin contained comparable amounts of fatty acids with 14 to 18 carbon atoms, but alpha-foetoprotein contained 16 times as much of the long-chain polyunsaturated fatty acids as albumin. alpha-Foetoprotein was found to have slightly higher affinity for palmitate and linoleate and severalfold higher affinity for arachidonate than albumin. These findings suggest that alpha-foetoprotein may play a role in the foetal metabolism of the long-chain polyunsaturated fatty acids.
Nucleoside transport in human erythrocytes. Apparent molecular weight of the nitrobenzylthioinosine-binding complex estimated by radiation-inactivation analysis
Authors: Jarvis S M , Young J D , Ellory J C
Published: August 2015

Nitrobenzylthioninosine, a potent nucleoside-transport inhibitor, binds specifically to functional nucleoside transport sites. Irradiation of freeze-dried human erythrocyte membranes with high-energy electrons was used to estimate the apparent molecular weight of the nitrobenzylthioninosine-binding complex in situ. The nitrobenzylthioinosine-binding complex had an apparent mol.wt. of 122000.
Phosphate utilization and constitutive synthesis of phosphatases in Thermoactinomyces vulgaris Tsilinsky
Authors: Sinha U , Singh V P
Published: August 2015

Thermoactinomyces vulgaris utilized both organic and inorganic phosphates with equal efficiency for its growth. The specific activities of the thermophilic acid and alkaline phosphatases were found to be maximum at 1 mM concentration of each phosphate source. All the phosphatase isoenzymes (three alkaline and one acidic) were observed irrespective of the substrate source and concentration, suggesting constitutive synthesis of the enzymes. During growth and differentiation, both acid and alkaline phosphatases exhibited uniformly stable patterns of isoenzymes with slight variations in their specific activities.
Evidence that in chick embryos destruction of hepatic microsomal cytochrome P-450 haem is a general mechanism of induction of δ-aminolaevulinate synthase by porphyria-causing drugs
Authors: Lim L K , Srivastava G , Brooker J D , May B K , Elliott W H
Published: August 2015

A variety of prophyrinogenic compounds were tested for their effect in ovo on chick-embryo liver microsomal cytochrome P-450 haem concentration and mitochondrial delta-aminolaevulinate synthase activity. With all drugs tested, there was a 30—50% decrease in cytochrome P-450 haem concentration within 1 h of treatment, and this was closely followed by an increase in delta-aminolaevulinate synthase activity. The relationship was independent of the extent of enzyme induction and is consistent with the proposal that drug-mediated destruction of cytochrome P-450 haem is the primary mechanism of induction of delta-aminolaevulinate synthase. After induction, synthesis of delta-aminolaevulinate synthase could be maintained by inhibiting further haem synthesis. These studies suggest that induction of porphyria is a combination of two distinct processes: (a) induction of delta-aminolaevulinate synthase synthesis by destruction of cytochrome P-450 haem and consequent depletion of cellular free haem; (b) maintenance of continued delta-aminolaevulinate synthase synthesis by preventing replenishment of cellular haem either by inhibiting haem synthesis and/or by promoting continuous removal of newly synthesized haem.
Distribution of secretory component in hepatocytes and its mode of transfer into bile
Authors: Mullock Barbara M. , Hinton Richard H. , Dobrota Miloslav , Peppard Jane , Orlans Eva
Published: August 2015

Immunoglobin A in bile and other external secretions is mostly bound to a glycoprotein known as secretory component. This glycoprotein is not synthesized by the same cells as immunoglobulin A and is not found in blood. We now report the mechanism by which secretory component reaches the bile and describe its function in immunoglobulin A transport across the hepatocyte. Fractionation of rat liver homogenates by zonal centrifugation was followed by measurement of the amounts of secretory component in the various fractions by rocket immunoelectrophoresis. Secretory component was found in two fractions. One of these was identified as containing Golgi vesicles from its isopycnic density and appearance in the electron microscope; the other contained principally fragments of the plasma membrane of the sinusoidal face of the hepatocyte, as shown by its particle size and content of marker enzymes. Only the latter fraction bound 125I-labelled immunoglobulin A added in vitro. At 5min after intravenous injection of [14C]fucose, the secretory component in the Golgi fraction was labelled, but not that in the plasma membrane. The secretory component in the sinusoidal plasma membrane did, however, become labelled before the first labelled secretory component appeared in bile, about 30min after injection. We suggest that fucose is added to the newly synthesized secretory component in the Golgi apparatus. The secretory component then passes, with the other newly secreted glycoproteins, to the sinusoidal plasma membrane. There it remains bound but exposed to the blood and able to bind any polymeric immunoglobulin A present in serum. The secretory component then moves across the hepatocyte to the bile-canalicular face in association with the endocytic-shuttle vesicles which carry immunoglobulin A. Hence there is a lag before newly synthesized secretory component appears in bile.
Changes in some cytoplasmic enzymes from red cells fractionated into age groups by centrifugation in Ficoll/Triosil gradients. Comparison of normal humans and patients with Duchenne muscular dystrophy
Authors: Galbraith D A , Watts D C
Published: August 2015

1. A procedure is described for the fractionation into age classes of human red cells on a Ficoll/Triosil discontinuous density gradient. The fractionated cells appear minimally affected by the procedure. 2. Individual blood samples show a normal cell distribution, but the mean cell density may vary slightly. To allow for this in comparing samples, cell ages are expressed in terms of the mean and standard deviations from the mean. 3. Reticulocyte number decays exponentially in the gradient. By back extrapolation, a standard deviation can be computed corresponding to the theoretical age at which the sample contains 100% reticulocytes. This value can be used to calculate the theoretical activity of an enzyme in the reticulocytes and the enzyme half-life. 4. Glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and glutathione reductase activities in unfractionated and age-fractionated cells were investigated in adults, children and patients with Duchenne muscular dystrophy. 5. The half-life for adult glucose 6-phosphate dehydrogenase (48 days) compares with that found by other workers. In children the half-life is 24 days, although the initial activity is essentially unaltered. 6. The half-life of 6-phosphogluconate dehydrogenase was found to be 117 days; the activity of glutathione reductase did not alter throughout the life of the cell. There was no difference between adults and children for these enzymes. The same results were obtained with dystrophic patients as for control children for all three enzymes.
Properties of a terminal deoxyribonucleotidyltransferase isolated from wheat germ
Authors: Brodniewicz-Proba T , Buchowicz J
Published: August 2015

An enzyme able to catalyse the polymerization of deoxyribonucleotides in a template-independent manner was isolated from dry wheat germ. This activity is associated with a soluble protein which is homogeneous with respect to the molecular weight (approx. 500000) and, under denaturing conditions, dissociates into product of two size classes, 67000 and 45000 daltons respectively. The enzyme-catalysed polymerization can be primed by oligo- as well as poly-deoxyribonucleotides, and is highly efficient (234 nmol/h per mg of finally purified protein) when only one of the four deoxyribonucleoside 5′-triphosphates is present in the incubation mixture. An extension of the 3′-hydroxy termini of polydeoxyribonucleotide chains for approx. 40 nucleotide residues was achieved when non-denatured DNA and [3H]dTTP were used as the primer and substrate respectively. It is concluded that the enzyme isolated from wheat germ shares catalytic properties with the terminal deoxynucleotidyltransferase of mammalian thymus. Unlike that transferase, however, the plant enzyme prefers non-denatured to single-stranded DNA as primer and requires both Mg2+ and Mn2+ ions for maximal activity.
The nature of the sulphur atom liberated from xanthine oxidase by cyanide. Evidence from e.p.r. spectroscopy after 33S substitution
Authors: Malthouse J P , Bray R C
Published: August 2015

Active xanthine oxidase was labelled specifically with 33S in the cyanide-labile site of the molybdenum centre. The Very Rapid molybdenum (V) e.p.r. signal, generated from this, shows strong coupling of 33S to molybdenum, providing unambiguous evidence that, at least in the signal-giving species, this sulphur atom is a ligand of molybdenum. The structure of the signal-giving species is discussed.
Effect of ligand binding on the conformation of human plasma vitamin D binding protein (group-specific component)
Authors: Surarit R , Svasti J
Published: August 2015

Several techniques have been used to demonstrate that the binding of specific ligands to human plasma vitamin D binding protein induces a change in protein conformation. Apoprotein and holoprotein show circular dichroism spectra of similar form in the peptide region with double minima at 207 and 218 nm. The minimum mean residue ellipticity of apoprotein (20.6 × 10(3) degrees.cm2.dmol-1) is decreased by about 8% after vitamin D3 binding, suggesting a small change in the backbone conformation. Spectrofluorimetric studies showed that 25-hydroxycholecalciferol causes a saturable enhancement of intrinsic fluorescence of human vitamin D binding protein and alters the pH profile of protein fluorescence, suggesting that there are alterations in the local environment of tryptophan residue(s) after ligand binding. Furthermore, in the presence of 25-hydroxycholecalciferol, the rate of chemical modification of the amino groups in human vitamin D binding protein is decreased and the susceptibility of intact vitamin D binding protein to proteolytic degradation is reduced, suggesting that some surface sites in the vitamin D binding protein molecule are less accessible to external agents. In addition, although the absorbance of vitamin made if difficult to interpret the ultraviolet spectra of holoprotein and apoprotein, the presence of vitamin D binding protein appears to stabilize the vitamin in an aqueous environment, a phenomenon that may be of physiological importance.
The presence of lysophosphatidylcholine in chromaffin granules
Authors: Arthur Gilbert , Sheltawy Ayman
Published: August 2015

Lysophosphatidylcholine is thought to be a characteristic component of the chromaffin granules in adrenal glands. By the use of a t.l.c. system that resolves minor phospholipids satisfactorily, this subcellular location was confirmed in the present study in bovine glands. However, phospholipid degradation was demonstrated in homogenates of the adrenal medulla and cortex under conditions similar to those of subcellular fractionation (incubation at 4°C for 90min). Phosphatidylethanolamine and cardiolipin were hydrolysed, but the concentration of lysophosphatidylcholine did not change, indicating that the latter was present in the medulla before this treatment. Attempts were made to decrease the time between death of the animal and the extraction of lipids. Lysophosphatidylcholine was easily demonstrable in lipid extracts of the dissected medulla and even in those of the whole bovine gland. For practical reasons it is not possible to decrease further the time lapse before extraction in the case of this animal. Adrenal glands were obtained from anaesthetized and untreated rabbits. These were frozen immediately in liquid N2 and the lipids were extracted. In a control experiment, the glands from rabbit were dissected and treated in the same manner as with those of ox, and then the lipids were extracted. No lysophosphatidylcholine was detected in the extracts from glands frozen in liquid N2 but lysophosphatidylcholine was observed in the controls. These results suggest that lysophosphatidylcholine is not a component of chromaffin granules, but is produced if the period between death of the animal and lipid extraction is unduly prolonged. To discover whether lysophosphatidylcholine affected the permeability barrier properties of chromaffin granules, sonicated liposomes of egg phosphatidylcholine alone or with lysophosphatidylcholine (15mol/100mol) were prepared. Both types were shown by electron microscopy to be largely made up of single bilayer vesicles. The exchange diffusion of [14C]dopamine was measured across their membranes. Both types of liposomes had similar capture volumes (0.5μl/μmol of phospholipid), and the activation energies of the exchange diffusion of dopamine were also similar (31kJ/mol). These results indicate that the presence of this proportion of lysophosphatidylcholine in chromaffin-granule membranes is not likely to influence their barrier properties towards catecholamines.
Nearest-neighbour analysis of myoglobin antigenic sites. Nearest-neighbour residues whose replacement can alter the environment of binding-site residue(s) and thus change their characteristics and binding capability
Authors: Kazim A L , Atassi M Z
Published: August 2015

By using the known antigenic structure of sperm-whale myoglobin previously determined in this laboratory and the X-ray co-ordinates for the myoglobin molecule, we have calculated the nearest-atom distances between each of the residues of the antigenic sites and all the other amino acids of the myoglobin molecule. These calculations have enabled us to identify the nearest-neighbour residues to each of the residues in the five antigenic sites, and which thus describe the immediate molecular environment of the sites. The influences of chemical changes or replacements in these environmental residues on the binding capacity of an antigenic site, when considered together with replacements directly in the antigenic sites, are expected to account for the major effects and will be extremely useful in explaining the cross-reactions of myoglobins from various species. However, it is stressed that the analysis has limitations due to the qualitative estimates of the effects, the influences of substitutions of once-removed or even at more distant locations (especially when they are cumulative) and finally the influences of any conformational re-adjustments when these occur as a result of the replacement(s).
Interaction of dinitrophenyl-pepstatins with human cathepsin D and with anti-dinitrophenyl antibody. Development of potential reagents for the localization in vivo of active proteinases at sites of tissue injury
Authors: Knight C G , Hornebeck W , Matthews I T W , Hembry R M , Dingle J T
Published: August 2015

Extracellular cathepsin D has been observed by various cytochemical methods at sites of tissue injury. However, the role of this enzyme in connective tissue matrix degradation is uncertain because there are no histochemical methods for determining whether or not the cathepsin D is active at such sites in living tissues. We considered that the combined use of a labelled tight-binding inhibitor with immunoprecipitation of the enzymes might overcome this problem. We have explored the application of derivatives of the inhibitor pepstatin, as only active cathepsin D binds pepstatin tightly. A series of N-pepstatinyl-N'-dinitrophenyl-alpha, omega-diaminoalkanes were synthesized with alkyl-chain lengths of two, four and six carbon atoms. These compounds were tight-binding inhibitors of human cathepsin D. In fluorescence-quenching titrations the dinitrophenyl groups were also fully available to bind high-affinity anti-dinitrophenyl antibody. It was shown by immunodiffusion in gels and by gel permeation chromatography that N-pepstatinyl-N'-dinitrophenyl-1,6-diaminohexane was a bifunction inhibitor able to bind cathepsin D and anti-dinitrophenyl antibody at the same time.
Methylation of nuclear DNA in Physarum polycephalum
Authors: Whittaker P A , Hardman N
Published: August 2015

The restriction endonucleases HpaII and HhaI, whose action is inhibited by the presence of methylated base analogues at the recognition sequences in the DNA substrate, were used to investigate the distribution of 5-methylcytosine in nuclear DNA from Physarum polycephalum. Physarum DNA is digested into two fractions by these enzymes: a low-molecular-weight (M–) compartment comprising 80% of the DNA, and a high-molecular-weight (M+) compartment containing 20% of the DNA. The DNA fraction showing resistance to digestion by restriction endonuclease HpaII is cleaved by its isoschizomer MspI, indicating that methylated endonuclease-HpaII-specific sites are present in M + DNA. Additional properties of sequences in the M+ compartment were investigated.
Sequence of residues 400–403 of bovine serum albumin
Authors: Reed R G , Putnam F W , Peters T
Published: August 2015

A large tryptic peptide of bovine serum albumin (residues 377–582) was subjected to 32 cycles of Edman degradation to determine the sequence of the last remaining unknown segment of this protein. Residues 400–403 were identified as gly-Phe-Gln-Asn. Amide assignments were also made at positions 388 (glutamine), 389 (asparagine), 391 (aspartic acid) and 392 (glutamine).
Theoretical analysis of the consequences of cyclic nucleotide phosphodiesterase negative co-operativity. Amplification and positive co-operativity of cyclic AMP accumulation
Authors: Erneux C , Boeynaems J M , Dumont J E
Published: August 2015

Most tissues contain multiple forms of cyclic nucleotide phosphodiesterases (3′:5′-cyclic-nucleotide 5′ nucleotidohydrolase, EC 3.1.4.17). Consequently, in most, if not in all, tissues, substrate-velocity curves deviate from Michaelian kinetics and exhibit an apparent negative co-operativity. We have studied the possible theoretical consequences of this property on the quantitative features of cyclic AMP accumulation in response to activation of adenylate cyclase. Negative co-operativity of phosphodiesterases tends to generate a “positively co-operative” cyclic AMP accumulation curve. It amplifies the stimulation of cyclic AMP accumulation as compared with the stimulation of cyclic AMP synthesis. It enhances the sensitivity of cyclic AMP accumulation to slight variation of phosphodiesterase maximal velocity. It tends to shift the cyclic AMP accumulation curve to higher concentrations of stimulator as compared with the adenylate cyclase activation curve. This accounts for much of the data in the literature of hormonal effects on phosphodiesterase activity. It shows that the characteristics of cyclic nucleotide phosphodiesterases are as important as those of adenylate cyclase in determining the response of the system.
Tryptophan pyrrolase in haem regulation. Experiments with administered haematin and the relationship between the haem saturation of tryptophan pyrrolase and the activity of 5-aminolaevulinate synthase in rat liver
Authors: Welch A N , Badawy A A B
Published: August 2015

1. Administration of haematin to rats decreases 5-aminolaevulinate synthase activity in whole liver homogenates. 2. An inverse relationship between this decrease and the increase in saturation of apo-(tryptophan pyrrolase) with haem is observed during the initial phase of treatment with haematin. 3. Significant changes in both functions are caused by a 1 mg/kg dose of haematin, whereas the maximum effects are achieved by the 5 mg/kg dose. 4. Prevention by allopurinol of the conjugation of exogenously administered haematin with apo-(tryptophan pyrrolase) renders this haem available for further repression of 5 aminolaevulinate synthase. 5. The various aspects of the relationship between synthase activity and the haem saturation of tryptophan pyrrolase are discussed.
Inhibition of rat brain tryptophan metabolism by ethanol withdrawal and possible involvement of the enhanced liver tryptophan pyrrolase activity
Authors: Badawy A A B , Punjani N F , Evans C M , Evans M
Published: August 2015

1. Chronic ethanol administration to rats was previously shown to enhance brain 5-hydroxytryptamine synthesis by increasing the availability of circulating tryptophan to the brain secondarily to the NAD(P)H-mediated inhibition of liver tryptophan pyrrolase activity. 2. At 24h after ethanol withdrawal, all the above effects were observed because liver [NAD(P)H] was still increased. By contrast, all aspects of liver and brain tryptophan metabolism were normal at 12 days after withdrawal. 3. At 7–9 days after withdrawal, brain 5-hydroxytryptamine synthesis was decreased, as was tryptophan availability to the brain. Liver tryptophan pyrrolase activity at these time-intervals was maximally enhanced. 4. Administration of nicotinamide during the withdrawal phase not only abolished the withdrawal-induced enhancement of tryptophan pyrrolase activity on day 8, but also maintained the inhibition previously caused by ethanol. Under these conditions, the withdrawal-induced decreases in brain 5-hydroxytryptamine synthesis and tryptophan availability to the brain were abolished, and both functions were enhanced. Nicotinamide alone exerted similar effects in control rats. 5. It is suggested that ethanol withdrawal inhibits brain 5-hydroxytryptamine synthesis by decreasing tryptophan availability to the brain secondarily to the enhanced liver tryptophan pyrrolase activity. 6. The results are discussed in relation to the possible involvement of 5-hydroxytryptamine in dependence on ethanol and other drugs.
Use of a novel rapid preparation of fat-cell plasma membranes employing Percoll to investigate the effects of insulin and adrenaline on membrane protein phosphorylation within intact fat-cells
Authors: Belsham G J , Denton R M , Tanner M J A
Published: August 2015

1. A rapid method was developed for the preparation of plasma membranes from either isolated rat fat-cells or intact epididymal fat-pads with the use of density-gradient centrifugation in the presence of Percoll. On the basis of 5′-nucleotidase activity, the yield of plasma membranes was about 50% and purification over 10-fold. Activities of marker enzymes indicated that contamination by mitochondria and microsomal fraction was small. 2. Incorporation of 32Pi into proteins associated with plasma membranes within isolated fat-cells was investigated. Four major bands of labelled phosphoproteins were separated by sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis; the apparent subunit mol.wts. were 67 000, 61 000, 26 000 and 20 000. None of these phosphoprotein bands corresponded to periodate/Schiff-staining glycoproteins. The extent of phosphorylation of the 61 000 mol.wt phosphoprotein band was increased by about 30 and 60% after exposure of fat-cells for 15 min to insulin or adrenaline respectively.
General characteristics of normal and stress-enhanced protein degradation in Lemna minor (duckweed)
Authors: Cooke R J , Davies D D
Published: August 2015

The general features of protein degradation in Lemna minor were studied by using a double-isotope technique. In common with several animal systems, there are correlations between the relative rate of protein degradation on the one hand and molecular weight, charge and carbohydrate content on the other. Large proteins, acidic proteins and non-glycosylated proteins are degraded relatively more rapidly than small or basic proteins, or glycoproteins. The correlations with size and carbohydrate content are explicable on the basis of differential susceptibility to Pronase, whereas the charge correlation cannot be explained on the basis. In addition, acidic proteins are not generally of higher molecular weight than neutral or basic proteins. The correlations are found in fronds growing in normal complete medium and in fronds transferred to medium lacking nitrate of made 50% (v/v) with respect to deuterium oxide, both of which are conditions that induce a large increase in protein breakdown in Lemna. Thus basal protein degradation and enhanced degradation do not appear to differ fundamentally in their general characteristics. The results are discussed in relation to the reported features of normal and enhanced proteolysis in animal tissues and to the possible mechanism of protein degradation in Lemna.
The role of glycogen synthase phosphatase in the glucocorticoid-induced deposition of glycogen in foetal rat liver
Authors: Vanstapel F , Doperé F , Stalmans W
Published: August 2015

1. The mechanism that underlies the induction of glycogen synthesis in the foetal rat liver by glucocorticoids was reinvestigated in conditions where the accumulation of glycogen is either precociously induced with dexamethasone or inhibited by steroid deprivation. It appears that glucocorticoids act as the physiological trigger for glycogen synthesis by inducing both glycogen synthase (a known effect) and its activating enzyme, glycogen synthase phosphatase. 2. The activity of glycogen synthase phosphatase in adult liver stems from the interaction of two protein components [Doperé, Vanstapel & Stalmans (1980) Eur. J. Biochem. 104, 137–146]. Two independent experimental approaches indicate that the cytosolic ‘S-component’ is already well developed in the foetal liver before the onset of glycogen synthesis. The manifold glucocorticoid-dependent increase in synthase phosphatase activity during late gestation must be attributed to the specific development of the glycogen-bound ‘G-component’.
Substrate specificity and mode of action of the cellulases from the thermophilic fungus Thermoascus aurantiacus
Authors: Shepherd M G , Tong C C , Cole A L
Published: August 2015

The substrate specificities of three cellulases and a beta-glucosidase purified from Thermoascus aurantiacus were examined. All three cellulases partially degraded native cellulose. Cellulase I, but not cellulase II and cellulase III, readily hydrolyzed the mixed beta-1,3; beta-1,6-polysaccharides such as carboxymethyl-pachyman, yeast glucan and laminarin. Both cellulase I and the beta-glucosidase degraded xylan, and it is proposed that the xylanase activity is an inherent feature of these two enzymes. Lichenin (beta-1,4; beta-1,3) was degraded by all three cellulases. Cellulase II cannot degrade carboxymethyl-cellulose, and with filter paper as substrate the end product was cellobiose, which indicates that cellulase II is an exo-beta-1,4-glucan cellobiosylhydrolase. Degradation of cellulose (filter paper) can be catalysed independently by each of the three cellulases; there was no synergistic effect between any of the cellulases, and cellobiose was the principal product of degradation. The mode of action of one cellulase (cellulase III) was examined by using reduced cellulodextrins. The central linkages of the cellulodextrins were the preferred points of cleavage, which, with the rapid decrease in viscosity of carboxymethyl-cellulose, confirmed that cellulase III was an endocellulase. The rate of hydrolysis increased with chain length of the reduced cellulodextrins, and these kinetic data indicated that the specificity region of cellulase III was five or six glucose units in length.
Changes in the characteristics and distribution of ferritin in iron-loaded cell cultures
Authors: Hoy T G , Jacobs A
Published: August 2015

When Chang liver cells are grown in an iron-rich medium for up to 20 weeks, iron loading up to 50 times the normal cellular iron content may be obtained, although ferritin increases only to about 10 times normal. Ferritin has been isolated from such cells, and the isoferritin pattern found on elution from DEAE-Sephadex A-50 by increasing chloride concentrations has been used as a basis for studying changes in the properties of ferritin under conditions of cellular loading. A consistent shift of peak ferritin-elution position to higher chloride concentrations (lower pI) occurs when cells are loaded with ferric nitrilotriacetate for increasing lengths of time. A change in immunoreactivity also takes place on loading, the ratio of ferritin reacting with heart and spleen ferritin antibodies increasing at any particular value of pI. Cells were pulse-labelled with [59Fe]ferric nitrilotriacetate and [3H]leucine followed by non-radioactive iron in the same form. During the 72 h after the synthesis of new protein and its incorporation of iron, there is a slight acid shift in its isoelectric point. This effect is seen in both normal and loaded cells, with the whole spectrum being shifted towards lower pI in the loaded state. These findings suggest that the shift to more acidic ferritins on iron loading and the associated changes in antigenicity may be unrelated to subunit composition.
The covalent-binding reaction of complement component C3
Authors: Sim R B , Twose T M , Paterson D S , Sim E
Published: August 2015

The complement protein C3, when activated by limited proteolysis, forms a short-lived reactive intermediate fragment, ‘nascent’ C3b, which is known to bind covalently to certain surfaces. The characteristics of the covalent binding reaction have been studied by using Sepharose-trypsin as a combined proteolytic activator and binding surface for C3. Binding of C3 to Sepharose-trypsin is saturable, with a maximum of 25-26 molecules of C3b bound per molecule of trypsin. A minimum life-time of about 60 microseconds for the reactive intermediate has been calculated from binding of C3 at saturation. Initial binding efficiencies of over 30% can be obtained at physiological pH and ionic strength. The efficiency of C3 binding to Sepharose-trypsin decreases as pH increases and also shows a slight decline at high ionic strength. The covalent binding of C3 to Sepharose-trypsin can be inhibited by a range of oxygen and nitrogen nucleophiles. Activation of C3 in the presence of radioactive forms of four such nucleophiles, phenylhydrazine, methylamine, glycerol and glucosamine results in apparent covalent incorporation of the nucleophile into the C3d fragment of C3. The quantity of radioactive nucleophile bound can be predicted from the observed potency of the nucleophile as an inhibitor of the binding of C3 to Sepharose-trypsin. The radioactive nucleophiles may be considered as ‘active-site’ labels for C3.
Isomyosins in human type 1 and type 2 skeletal muscle fibres
Authors: Fitzsimons R B , Hoh J F Y
Published: August 2015

Human myosin from different skeletal muscles was analysed in a non-denaturing gel system, and the isoenzyme composition correlated with the histochemical composition of the muscle. Two components (SM1 and SM2) were associated with type 1 (slow-twitch) fibres, and three (FM1, FM2 and FM3) with type 2 (fast-twitch) fibres. Light-chain analysis was performed in sodium dodecyl sulphate/polyacrylamide gels. There are three light chains (LCs1a, LCS1b and LCs2) in type 1 fibres, and three (LCf1, LCf2 and LCf3) in type 2 fibres. LCf1 and LCs1b co-migrate in sodium dodecyl sulphate gels. The ratio of LCf3/LCf2 is correlated with the distribution of the individual fast isoenzymes. These results explain apparent discrepancies in the literature concerning the light-chain distribution of human myosin.
Double-label reductive methylation of tissue proteins for precision two-dimensional polyacrylamide-gel electrophoretic analysis
Authors: Finger J M , Choo K H
Published: August 2015

Reductive methylation has been used to radioactively label crude-extract proteins with 3H or 14C. The procedure achieved good isotope incorporation and resolution of proteins on two-dimensional polyacrylamide gels. It allows high-precision comparison of tissue samples by double-labelling and should facilitate the study of tissue proteins by two-dimensional gel electrophoresis.
Kinetic and equilibrium metal-ion-binding behaviour reflected in a metal-ion-dependent antigenic determinant in bovine prothrombin. Comparison with bovine prothrombin fragment 1
Authors: Madar D A , Hall T J , Hiskey R G , Koehler K A
Published: August 2015

Rabbit anti-(bovine prothrombin fragment 1) antibodies were fractionated by using fragment-1 affinity chromatography in the absence of metal ions, and showed an absolute requirement for the presence of metal ions in their interactions with bovine fragment 1 or prothrombin. These antibodies were employed to evaluate both the rate constants for a protein conformation change and the equilibrium metal-ion binding to isolated bovine fragment 1 and intact prothrombin. The close similarity of the rates obtained for the conformation change in fragment 1 and those observed in prothrombin indicated that the same process is involved in both proteins and that the non-fragment-1 region of the prothrombin has essentially no effect on this process in the fragment-1 region. Equilibrium metal-ion-binding studies indicate that the details of the metal-ion-binding process in fragment 1 and prothrombin are essentially the same. We conclude that the metal-ion-binding behaviour of the fragment-1 domain of intact prothrombin is identical with that of isolated fragment 1.
The effect of tsushimycin on the synthesis of lipid-linked saccharides in aorta
Authors: Elbein A D
Published: August 2015

The antibiotic, tsushimycin, inhibits the formation of dolichyl phosphate mannose, dolichyl phosphate glucose and dolichyl pyrophosphate N-acetylglucosamine in the particulate enzyme preparation from pig aorta. Although this antibiotic also inhibits the incorporation of mannose and glucose into lipid-linked oligosaccharides, these reactions are less sensitive to antibiotic than those involved in the synthesis of lipid-linked monosaccharides. In the presence of tsushimycin, most of the mannose incorporated into lipid-linked oligosaccharides is into one oligosaccharide that has the properties of the heptasaccharide Man5GlcNAc2, whereas in the absence of antibiotic most of the mannose is in larger-sized oligosaccharides. On the other hand, the glucose-labelled lipid-linked oligosaccharides appear to be similar in size in the presence or absence of antibiotic. Tsushimycin also inhibits the formation of lipid-linked monosaccharides by the solubilized enzyme preparation of aorta. Various concentrations of dolichyl phosphate or the detergent, Nonidet P40, had no effect on antibiotic inhibition. Some evidence indicates that tsushimycin binds to the particulate enzyme.
Sequence of a secondary phage λ attachment site located between the pentitol operons of Klebsiella aerogenes
Authors: Loviny T , Neuberger M S , Hartley B S
Published: August 2015

We have determined the nucleotide sequence of a secondary phage lambda attachment site (att) located between the structural genes of the ribitol and D-arabitol catabolic operons of Klebsiella aerogenes. The core region of this secondary attachment site (sequence: GGTTTTTTCGATTAT) shows considerable homology with the 15-base-pair core region common to both the phage att and the primary bacterial att of Escherichia coli K12 (sequence: GCTTTTTTACTAA); however, there is no such clear homology between the sequences flanking the cores of the primary att and this secondary att. Integration of phage lambda into the K. aerogenes secondary att occurred by recombination between the core region of the phage att and an oligo(T.A) stretch located within the K. aerogenes secondary att.
Properties of rat renal phosphate-dependent glutaminase coupled to Sepharose. Evidence that dimerization is essential for activation
Authors: Morehouse R F , Curthoys N P
Published: August 2015

In the absence of phosphate, purified rat renal phosphate-dependent glutaminase exists as a catalytically inactive protomer. The addition of phosphate results in both dimerization and activation of the glutaminase. Covalent attachment of the dimeric form of the glutaminase to CNBr-activated Sepharose was achieved with 84% retention of activity. At least 70% of the bound glutaminase activity was expressed even in the absence of added phosphate. In addition, 6-diazo-5-oxo-L-norleucine, which interacts only with the catalytically active form of the glutaminase, inactivates the bound dimeric form of glutaminase at the same rate in either the absence or the presence of added phosphate. Therefore retention of dimeric structure is apparently sufficient to maintain glutaminase activity. In contrast, the coupling of the protomeric form of the enzyme to Sepharose resulted in retention of only 3% of the phosphate-induced glutaminase activity. However, up to 48% of this activity could be reconstituted by addition of soluble glutaminase under conditions that promote dimerization. These results indicate that the monomeric form of the glutaminase has minimal inherent activity and that dimerization is an essential step in the phosphate-induced activation of the glutaminase.
Subcellular localization of low-molecular-weight RNA components in rat liver
Authors: Frederiksen S , Flodgard H , Hellung-Larsen P
Published: August 2015

The subcellular localization of the four major low-molecular-weight RNA components, D, C, A and L, was studied in rat liver cells. The cells were fractionated by a non-aqueous technique into a nuclear and a cytoplasmic fraction. The cytoplasm contained 43% of component D, 57% of component C and more than 80% of component L.
Studies on the mechanism of action of 6-mercaptopurine. Interaction with copper and xanthine oxidase
Authors: Kela U , Vijayvargiya R
Published: August 2015

Interaction between 6-mercaptopurine, Cu2+ and the enzyme xanthine oxidase (EC 1.2.3.2.) was examined. Whereas Cu2+ was found to inhibit the enzyme, 6-mercaptopurine could protect as well as reverse the enzyme inhibition produced by the metal ion. The formation of a complex between 6-mercaptopurine and Cu2+ seems to be responsible for the observed effect. Job's [(1928) Ann. Chem. 9, 113] method has shown the composition of the complex to be 1:1. The apparent stability constant (log K value), as determined by Subhrama Rao & Raghav Rao's [(1955) J. Sci. Chem. Ind. Res. 143, 278], method is found to be 6.74. It is suggested that the formation of a stable complex between 6-mercaptopurine molecules and Cu2+ may be an additional mechanism of action of 6-mercaptopurine, particularly with reference to its anti-inflammatory properties.
Inactivation of rat muscle 5′-adenylate aminohydrolase by tyrosine nitration with tetranitromethane
Authors: Ranieri-Raggi M , Bergamini C , Montali U , Raggi A
Published: August 2015

Reaction of rat muscle AMP deaminase with low molar excess of tetranitromethane results in a rapid loss of free thiol groups and a concomitant decrease in enzyme activity at high, but not at low, AMP concentration. This modification appears to be limited to the same non-essential thiol groups reactive towards specific reagents in non-denaturing conditions. On incubation with higher molar excess of tetranitromethane, a loss of enzyme activity is observed, which correlates with nitration of tyrosine residues. By amino acid analysis, approximately there tyrosine residues per subunit are estimated to be nitrated in the completely inactivated enzyme. The kinetic properties of the partially inactivated AMP deaminase reveal a negative co-operatively behaviour at approximately half saturation. This suggests that modification of tyrosine residues is also responsible for alteration of the binding properties of the hypothesized activating site of AMP deaminase.
Occupancy of sites of phosphorylation in inactive rat heart pyruvate dehydrogenase phosphate in vivo
Authors: Sale G J , Randle P J
Published: August 2015

1. Inactive pyruvate dehydrogenase phosphate complexes were partially purified from hearts of fed, starved or alloxan-diabetic rats by using conditions that prevent phosphorylation or dephosphorylation. 2. Unoccupied sites of phosphorylation were assayed by incorporation of 32P from [gamma-32P]ATP into the complexes. Total sites of phosphorylation were assayed by the same method after complete reactivation, and thus dephosphorylation, of complexes by incubation with pyruvate dehydrogenase phosphate phosphatase. Occupancy is assumed from the difference (total sites–unoccupied sites). Percentage incorporation into individual sites was measured by high-voltage electrophoresis after tryptic digestion. 3. Values (means +/- S.E.M., in nmol of phosphate/unit of inactive complex) for total sites, occupied sites and percentage occupancies, with numbers of observations in parentheses were: fed, 2.1 +/- 0.04, 1.15 +/- 0.04, 54.8 +/- 1.6% (39); starved, 2.05 +/- 0.03, 1.85 +/- 0.03, 90.2 +/- 1.4% (28); alloxan-diabetic, 1.99 +/- 0.03, 1.72 +/- 0.03, 86.4 +/- 1.4% (68%). 4. Values (means +/- S.E.M. for percentage occupancy) for individual sites of phosphorylation in pyruvate dehydrogenase phosphate given in the order sites 1, 2 and 3 were : fed, 100 +/- 2.7, 27.8 +/- 1.6, 33.9 +/- .9; starved, 100 +/- 1.4, 76.2 +/- 2.0, 92.4 +/- 1.5; alloxan-diabetic, 100 +/- 1.2, 64.0 +/- 1.7, 94.6 +/- 1.4. 5. It is concluded that starvation or alloxan-diabetes leads to a 2–3-fold increase in the occupancy of phosphorylation sites 2 and 3 in pyruvate dehydrogenase phosphate in rat heart in vivo.