bluuebunny/crossref_metadata_2025 huggingface dataset:
The 1H nuclear-magnetic-resonance spectroscopy of cobalt(II)-β-lactamase II
Authors: Galdes A , Hill H A , Baldwin G S , Waley S G , Abraham E P
Published: August 2015

The 1H n.m.r. spectra of beta-lactamase II in the presence of Co(II) were studied. Analysis of the spectra suggests that Co(II) binds at the same two metal-binding sites as does Zn(II). The binding of Co(II) at the first site is much weaker than the binding of Zn(II) at this site, whereas the binding of Co(II) at the second site is tighter than the binding of Zn(II). The binding of Co(II) to the mono-zinc(II)-enzyme caused only one marked change in the spectrum, namely a decrease in the intensity of the resonances assigned to the C-2 and C-4 protons of one histidine residue (residue E). However, when the spectra of the apoenzyme and the Co(II)-enzyme were compared, there were many differences. A significant fraction of the protons in the whole molecule are affected by the binding of Co(II) at the first metal-ion-binding site (where the ligands are the enzyme's sole thiol group and three histidine residues). This may be because the first site is internal, or because of a difference in conformation between the apoenzyme and the mono-Co(II)-enzyme. The second site may be located on the surface of the molecule.
The serum competitor of oestrogen–rat α1-foetoprotein interactions. Identification as a mixture of non-esterified fatty acids
Authors: Vallette G , Benassayag C , Savu L , Delorme J , Nunez E A , Doumas J , Maume G , Maume B F
Published: August 2015

The novel endogenous serum ligands of rat alpha 1-foetoprotein previously demonstrated in different mammalian sera were identified by g.l.c.–mass-spectrometric methods as a mixture of non-esterified long-chain and predominantly unsaturated fatty acids. Detailed comparative analyses of these ligands extracted from foetal- and pregnant-rat sera, rat amniotic fluid and foetal human sera are presented. We also show that an important fraction of these ligands remains associated with the rat alpha 1-foetoprotein after purification; analyses are given for the composition of this lipid moiety of the foetoprotein. The physiological relevance of these results is discussed.
Synthesis of biliverdin and bilirubin 1-O-acyl-β-d-glucopyranuronic acids
Authors: Compernolle F
Published: August 2015

Biliverdin and bilirubin mono- and di-beta-glucuronides were prepared by nucleophilic substitution of the 1-O-mesyl derivative of alpha-ethoxyethyl-protected glucuronic acid (compound II) with the tetrabutylammonium salts of biliverdin and bilirubin. Removal of the acetal-protecting groups by mild acid treatment yielded biliverdin glucuronides, which were reduced to bilirubin glucuronides. Depending on reaction conditions the pure beta-anomers or mixtures highly enriched in the beta-anomers were obtained. The biliverdin and bilirubin glucuronides were identical with pigments derived from bile. They were characterized as the IX alpha isomers and the beta-anomers by alkaline hydrolysis, n.m.r. spectroscopy, hydrolysis with beta-glucuronidase and conversion into dipyrrolic azopigments. Model reactions of the 1-O-mesylate (II) with other nucleophiles also were performed, i.e. the acetate anion and various alcohols.
Studies on the immunoglobulin-G Fc-fragment receptor from neonatal rat small intestine
Authors: Wallace K H , Rees A R
Published: August 2015

1. A method for preparing the small-intestinal brush-border membrane of neonatal rats is described in which enzymic methods are used to remove associated polysaccharide and cell nuclei. 2. 125I-labelled IgG (immunoglobulin G) and 125I-labelled IgG Fc fragment have high specific binding and low non-specific binding to brush borders prepared in this way. F(ab)'2 fragment however, does not bind, indicating the existence of a specific receptor for the Fc fragment of IgG. The receptor system is saturable, and the affinity (KA) for the binding of rat IgG was determined by both equilibrium and kinetic methods. 3. The binding of heterologous IgG species (human and bovine) was compared and demonstrated a close similarity between human IgG and rat IgG in their receptor affinities. 4. Kinetic results are presented that are consistent with previously proposed models of ligand-induced receptor aggregation.
Control of proteoglycan synthesis. Studies on the activation of synthesis observed during culture of articular cartilages
Authors: Sandy J D , Brown H L , Lowther D A
Published: August 2015

When slices of adult rabbit articular cartilage were incubated in culture medium, the rate of incorporation of [35S]sulphate or [3H]acetate into glycosaminoglycans increased 4-8 fold during the first 5 days of incubation. Similar changes in biosynthetic activity were observed during culture of adult bovine cartilage. The activation of synthesis was not serum-dependent, but appeared to be a result of the depletion of tissue proteoglycan that occurs under these incubation conditions [Sandy, Brown & Lowther (1978) Biochim. Biophys. Acta 543, 536–544]. Thus, although complete activation was observed in serum-free medium, it was not observed if the cartilage was cultured inside dialysis tubing or in medium containing added proteoglycan subunit. The average molecular size of the proteoglycans synthesized by activated tissue was slightly larger than normal, as determined by chromatography on Sepharose CL-2B, and the average molecular size of the glycosaminoglycans synthesized by activated tissue was markedly increased over the normal. The increase in chain size was accompanied by an increase in the proportion of the chains degraded by chondroitinase ABC; these results are consistent with the preferential synthesis by activated chondrocytes of chondroitin sulphate-rich proteoglycans. The increase in glycosaminoglycan chain size was observed whether the chains were formed on endogenous core protein or on exogenous benzyl-beta-D-zyloside. An approximate 4-fold activation in culture of glycosaminoglycan synthesis on protein core was accompanied by a 1.54-fold increase in the rate of incorporation of [3H]serine into the chondroitin sulphate-linkage region of the proteoglycans. A 2.8-fold activation in culture of glycosaminoglycan synthesis on benzyl-beta-D-zyloside was accompanied by a 1.7-fold increase in the rate of incorporation of [3H]benzyl-beta-D-zyloside into glycosaminoglycans. The activation of glycosaminoglycan synthesis was, however, accompanied by no detectable change in the activity of xylosyltransferase (EC 2.4.2.26) in cell-free extracts. These results are discussed in relation to current ideas on the control of proteoglycan synthesis in cartilage.
Lipogenesis in the brain of suckling rats. Studies on the mechanism of mitochondrial-cytosolic carbon transfer
Authors: Patel Tarun B. , Clark John B.
Published: August 2015

1. Studies on the incorporation of [3-14C]pyruvate and d-3-hydroxy[3-14C]butyrate into the brain lipid fraction by brain homogenates of the suckling (7-day-old) rat have been carried out. 2. Whereas approximately twice as much CO2 was evolved from pyruvate compared with 3-hydroxybutyrate metabolism, similar amounts of the radioactivity of these two precursors were incorporated into the lipid fraction. Furthermore, in both cases the incorporation into lipid was almost tripled when glucose (10mm) or NADPH (2.5mm) was added to the incubation media. 3. If 5mm-(—)-hydroxycitrate, an ATP–citrate lyase inhibitor, was added to the incubation the incorporation of carbon from pyruvate was inhibited to 39% of the control and from 3-hydroxybutyrate to 73% of the control, whereas CO2 production from both precursors was not affected. 4. The incorporation from pyruvate or 3-hydroxybutyrate into lipids was not affected by the presence of 10mm-glutamate in the medium (to encourage N-acetylaspartate production). However, incorporation from pyruvate was inhibited by 21% in the presence of 5mm-amino-oxyacetate (a transaminase inhibitor) and by 83% in the presence of both hydroxycitrate (5mm) and amino-oxyacetate. 5. Incorporation from 3-hydroxybutyrate into brain lipids was inhibited by 20% by amino-oxyacetate alone, but by 55% in the presence of both hydroxycitrate and amino-oxyacetate. 6. It is concluded that the mechanism of carbon transfer from pyruvate into lipids across the mitochondrial membrane in the suckling rat brain is mainly via citrate and N-acetylaspartate. 3-Hydroxybutyrate, in addition to using these routes, may also be incorporated via acetoacetate formation and transport to the cytosol.
Dephosphorylation and activation of exogenous glycogen synthase by adipose-tissue phosphatase
Authors: Brown Joan Heller , Eichner Ronald D. , Thompson Barbara , Mayer Steven
Published: August 2015

Exogenous purified rabbit skeletal-muscle glycogen synthase was used as a substrate for adipose-tissue phosphoprotein phosphatase from fed and starved rats in order to (1) compare the relationship between phosphate released from, and the kinetic changes imparted to, the substrate and (2) ascertain if decreases in adipose-tissue phosphatase activity account for the apparent decreased activation of endogenous glycogen synthase from starved as compared with fed rats. Muscle glycogen synthase was phosphorylated with [γ-32P]ATP and cyclic AMP-dependent protein kinase alone, or in combination with a cyclic AMP-independent protein kinase, to 1.7 or 3mol of phosphate per subunit. Adipose-tissue phosphatase activity determined with phosphorylated skeletal-muscle glycogen synthase as substrate was decreased by 35–60% as a consequence of starvation. This decrease in phosphatase activity had little effect on the capacity of adipose-tissue extracts to activate exogenous glycogen synthase (i.e. to increase the glucose 6-phosphate-independent enzyme activity), although there were marked differences in the activation profiles for the two exogenous substrates. Glycogen synthase phosphorylated to 1.7mol of phosphate per subunit was activated rapidly by adipose-tissue extracts from either fed or starved rats, and activation paralleled enzyme dephosphorylation. Glycogen synthase phosphorylated to 3mol of phosphate per subunit was activated more slowly and after a lag period, since release of the first mol of phosphate did not increase the glucose 6-phosphate-independent activity of the enzyme. These patterns of enzyme activation were similar to those observed for the endogenous adipose-tissue glycogen synthase(s): the glucose 6-phosphate-independent activity of the endogenous enzyme from fed rats increased rapidly during incubation, whereas that of starved rats, like that of the more highly phosphorylated muscle enzyme, increased only very slowly after a lag period. The observations made here suggest that (1) changes in glucose 6-phosphate-independent glycogen synthase activity are at best only a qualitative measure of phosphoprotein phosphatase activity and (2) the decrease in glycogen synthase phosphatase activity during starvation is not sufficient to explain the differential glycogen synthase activation in adipose tissue from fed and starved rats. However, alterations in the phosphorylation state of glycogen synthase combined with decreased activity of phosphoprotein phosphatase, both as a consequence of starvation, could explain the apparent markedly decreased enzyme activation.
The effects of cutting or of stretching skeletal muscle in vitro on the rates of protein synthesis and degradation
Authors: Seider M J , Kapp R , Chen C P , Booth F W
Published: August 2015

Rates of protein synthesis were significantly lower in the cut soleus and extensor digitorum longus muscles than in their uncut counterparts. Rates of protein degradation were significantly higher in cut soleus muscles, but not in cut extensor digitorum longus muscles as compared with their uncut controls. Concentrations of ATP and phosphocreatine were significantly lower in cut soleus and extensor digitorum longus muscles after incubation in vitro in contrast with respective control uncut muscles. These data indicate that cutting of muscle fibres alters rates of protein synthesis and degradation, in addition to altering concentrations of high-energy phosphates. Since these findings stressed the importance of using intact muscles to study protein metabolism, additional studies were made on intact muscles in vitro. Stretched soleus muscles had higher concentrations of high-energy phosphates at the end of an incubation period than did unstretched muscles. However, the length of the soleus, extensor digitorum longus and diaphragm muscles during incubation did not affect rates of protein degradation.
Effects of proteinase inhibitors on adenylate cyclase
Authors: McIlroy P J , Richert N D , Ryan R J
Published: August 2015

The effects of a number of proteinase inhibitors on rat ovarian and rat hepatic adenylate cyclase preparations were examined. N alpha-tosylarginine methyl ester, 7-amino-1-chloro-3-L-tosylamidoheptan-2-one, 1-chloro-4-phenyl-3-L-tosylamidobutan-2-one, 1-chloro-4-methyl-3-L-tosylamidopentan-2-one and other low-molecular-weight proteinase inhibitors blocked hormonally stimulated adenylate cyclase from either source with hepatic preparations requiring higher concentrations. Addition of nucleotides (ATP, GTP, GDP, CTP or ITP) to inhibited ovarian preparations did not reverse inhibition, nor did dithiothreitol reverse phenylmethanesulphonyl fluoride-inhibited ovarian adenylate cyclase. The kinetics of the inhibition of rat ovarian adenylate cyclase were examined by following the production of cyclic AMP after the addition of inhibitors to membrane preparations preincubated under assay conditions with human choriogonadotropin, guanosine 5′-[beta gamma-imido]triphosphate of NaF. 7-Amino-1-chloro-3-L-tosylamidoheptan-2-one, 1-chloro-4-phenyl-3-L-tosylamidobutan-2-one and 1-chloro-4-methyl-3-L-tosylamidopentan-2-one had two effects on human-choriogonadotropin-stimulated adenylate cyclase. At low concentrations (less than or equal to 0.2 mM) there was an irreversible inhibition of hormonally-stimulated cyclase with maximum first-order inhibitory rate constants of 0.05–0.08 min-1. At higher concentrations the irreversible effect persisted, but, in addition, there was a marked decrease in the cyclase initial velocity to 25–50% of that of control values. N alpha-tosylarginine methyl ester had similar effects; at low concentrations (less than or equal to 2 mM) it inhibited irreversibly, and at higher concentrations it decreased the initial velocity (50% at 10 mM). At high concentrations (greater than 3 mM) N alpha-tosylarginine methyl ester also inhibited NaF- and guanosine 5′-[beta gamma-imidol]-triphosphate-stimulated cyclase but in a reversible manner. 7-Amino-1-chloro-3-L-tosylamidoheptan-2-one inhibited NaF-stimulated adenylate cyclase in two ways, as for human-choriogonadotropin-stimulated adenylate cyclase, but required 10–20-fold higher concentrations. The low-concentration irreversible effect can be explained by a continual inactive in equilibrium active conversion of adenylate cyclase during hormonal stimulation in which the inactive to active conversion is blocked by the inhibitors. The high-concentration effect is a direct one on the active catalytic moiety of the enzyme.
Kinetic measurements of the biliary excretion of metabolized compounds
Authors: Powell G M , Jones J G , Curtis C G
Published: August 2015

1. Rats received constant infusions of bromosulphophthalein or [carboxy-14C]cholic acid at a range of concentrations. 2. Kinetic parameters describing the biliary excretion of the compounds were determined. 3. The biliary excretion of both compounds could be described by the same kinetic parameters already calculated for phenolphthalein disulphate, which is excreted in the bile unchanged.
Chemiluminescence of lipid vesicles supplemented with cytochrome c and hydroperoxide
Authors: Cadenas E , Boveris A , Chance B
Published: August 2015

The increase in light emission of hydroperoxide-supplemented cytochrome c observed on addition of lipid vesicles was related to the degree of unsaturation of the fatty acids of the phospholipids: dipalmitoyl phosphatidylcholine was without effect, whereas dioleoyl phosphatidylcholine and soya-bean phosphatidylcholine enhanced chemiluminescence 2- and 3-fold respectively. Effects on light-emission were similar to those on O2 uptake. The chemiluminescence of the present system was sensitive to cyanide and to the radical trap 2,5-di-t-butylquinol, indicating a catlytic activity of cytochrome c and the presence of free-radical species respectively. Lipid-vesicle enhanced chemiluminescence showed different kinetic behaviours, apparently depending on unsaturation: three phases are described for soya-bean phosphatidylcholine, whereas only one phase was present in mixtures containing dipalmitoyl and dioleoyl phospholipids. Chemiluminescence of lipid vesicles supplemented with cytochrome c and hydroperoxide showed similar kinetic patterns with H2O2 and primary (ethyl) and tertiary (t-butyl and cumene) hydroperoxides. Participation of singlet molecular oxygen, mainly on the phase III of chemiluminescence, is suggested by the increase of light-emission by 1,4-diazabicyclo[2.2.2]-octane as well as by data from spectral analysis.
Hormonal regulation of protein synthesis in the rat epididymis. Characterization of androgen-dependent and testicular fluid-dependent proteins
Authors: Jones R , Brown C R , Von Glós K I , Parker M G
Published: August 2015

1. Protein synthesis has been investigated in different regions of the rat epididymis by measuring incorporation of [35S]methionine in tissue minces incubated in vitro followed by analysis of labelled proteins on polyacrylamide gels containing sodium dodecyl sulphate. Rates of synthesis were highest in the proximal cauda > distal cauda > initial segment > ductuli efferentes > corpus > distal caput > proximal caput. One protein (mol.wt. 23 000) characterized the initial segment, three proteins (mol.wts. 18 500, 19 000 and 32 000) the caput and one protein (mol.wt. 47 000) the cauda. 2. After castration, [35S]methionine incorporation in all regions of the epididymis was reduced to < 10% of that in normal animals but could be restored to control levels within 5 days by testosterone treatment. Other steroids (corticosterone, oestrogen or progesterone) were ineffective. 3. The synthesis of the 18 500, 19 000, and 32 000 mol.wt. proteins in the caput and the 47 000 mol.wt. protein in the cauda were preferentially regulated by androgens, whilst the synthesis of 23 000 and approx. 80 000 mol.wt. proteins in the initial segment was dependent upon factors present in testicular fluid. 4. The androgen-dependent and testicular fluid-dependent proteins were major components of epididymal secretion. Purification and characterization of the 18 500, 19 000, 23 000 and 32 000 mol.wt. proteins showed them to be acidic glycoproteins with a carbohydrate content of 7.6-13.2%. The 47 000 mol.wt. protein, on the other hand, is highly basic. 5. A possible role for these proteins in the acquisition of motility, fertilizing capacity and storage of spermatozoa in the epididymis is discussed.
Insulin action and binding in isolated hepatocytes from fasted, streptozotocin-diabetic, and older, spontaneously obese rats
Authors: Cech James M. , Freeman Richard B. , Caro Jose F. , Amatruda John M.
Published: August 2015

Insulin binding and basal and insulin-stimulated uptake of α-aminoisobutyric acid were measured in isolated hepatocytes from young control rats as well as from older spontaneously obese, 72h-starved, and nonketotic streptozotocin-diabetic rats. Isolated hepatocytes from older spontaneously obese rats are similar to those from younger smaller rats in size, maximal insulin responsiveness, the dose–response relationship for insulin-stimulated aminoisobutyrate uptake, and the number and affinity of insulin receptors. Hepatocytes from 72h-fasted rats have similar numbers of insulin receptors per cell as cells from young control animals, but are significantly smaller, have an enhanced basal rate of aminoisobutyrate uptake, and are insulin resistant with regard to maximal insulin-stimulated uptake of aminoisobutyrate at 0.1mm-aminoisobutyrate. Because of the decreased maximal response to insulin, the concentration of insulin that elicits a half-maximal response of aminoisobutyrate uptake is decreased. Hepatocytes from diabetic animals, like those from starved rats, have significantly greater basal rates of aminoisobutyrate uptake; whereas the maximal absolute insulin response is the same as control cells, the percentage response is smaller. These cells bind significantly more insulin than do control cells. The increase in insulin binding is reflected in a shift to the left of the dose–response curve for insulin-stimulated uptake of aminoisobutyrate. These studies indicate that there is no insulin resistance with regard to uptake of aminoisobutyrate in hepatocytes from older obese rats. Furthermore, the insulin resistance observed in hepatocytes from starved rats occurs despite an increase in the number of receptors per unit surface area and cannot be explained by alterations in the interaction between insulin and its receptor. The enhanced insulin binding per unit surface area, however, is reflected in the shift to the left of the dose–response curve for insulin. This is also true for hepatocytes from diabetic animals, in which insulin binding per cell is increased.
Zinc-, copper- and cadmium-binding protein in Ehrlich ascites tumour cells
Authors: Koch J , Wielgus S , Shankara B , Saryan L A , Shaw C F , Petering D H
Published: August 2015

The properties of Ehrlich ascites tumour cells exposed in vivo to cadmium were investigated as a function of the zinc status of the host animals. Tumour-cell growth was inhibited by cadmium in both zinc-sufficient and zinc-deficient animals. However, cells in zinc-sufficient tumours accumulate much less cadmium than those in deficient tumours. The subcellular distributions of cadmium and zinc do not depend on zinc status. Cadmium and zinc are bound to a low-molecular-weight protein with properties similar to metallothionein. Without exposure to cadmium, a zinc- and copper-binding protein is still present that behaves like a metallothionein. This protein can rapidly bind cadmium added to Ehrlich cells in vitro. It is shown that the zinc- and copper-binding protein contains free thiol groups. Ehrlich cells isolated from cadmium-treated animals are viable and show normal incorporation of uridine into RNA, but the cellular uptake of thymidine and its incorporation into DNA are inhibited.
The presence and longitudinal distribution of the glutathione S-transferase in rat epididymis and vas deferens
Authors: Hales Barbara F. , Hachey Christiane , Robaire Bernard
Published: August 2015

The presence of the glutathione S-transferases, enzymes that catalyse the conjugation of glutathione with a variety of compounds, is reported here, for the first time, in the mammalian epididymis–vas deferens. These glutathione S-transferases, approx. 50% of those from rat liver on a per-mg-of-protein basis, are resolved by isoelectric focusing into six peaks, each with a characteristic isoelectric point and substrate specificity. By these same criteria, the first three peaks (pI 8.9, 8.2 and 7.8) can be identified as transferases B, A and C respectively. The fifth peak (pI7.2) may correspond to transferase M; the fourth (pI7.5) and sixth (pI7.0) peaks do not correspond to previously described transferases. The distribution of transferase activity towards any one substrate studied differs in sequential sections of the epididymis and vas deferens; in addition, the longitudinal-distribution pattern differs for each of the three substrates studied. Isoelectric focusing of the cytosol fractions of the different sections further substantiates these observations. The potential significance of these enzymes and of their distribution in terms of epididymal function, maturation of spermatozoa, is discussed.
The human complement system: assembly of the classical pathway C3 convertase
Authors: Kerr M A
Published: August 2015

The assembly of the classical pathway C3 convertase in the fluid phase has been studied. The enzyme is assembled from C2 and C4 on cleavage of these proteins by C1s. Once assembled, the enzyme activity decays rapidly. Kinetic evidence has been obtained that this decay is even more rapid than previously suggested (kdecay is 2.0 min-1 at 37 degrees C). As a result, optimal C3 convertase activity is only observed with high C1s levels, which result in rapid rates of cleavage of C2 and increased rates of formation of the C3 convertase. Using high concentrations of C1s at lower temperatures (22 degrees C) in the presence of excess substrate we have demonstrated kinetically that the enzyme comprises an equimolar complex of C4b and cleaved C2. We have obtained direct evidence from gel-filtration experiments for the role of C2a as the catalytic subunit of the enzyme. C2b appears to mediate the interaction between C4 (or C4b) and C2 at pH 8.5 and at low ionic strength where the interactions can easily be detected. It may therefore be important in the assembly of the enzyme, though it is not involved in the catalytic activity. The decay of the C3 convertase reflects the release of C2a from the C4b x (C2b) x C2a complex, and the stabilizing effect of iodine on the C3 convertase is therefore apparently one of stabilizing the C4b-C2z interaction, which is otherwise weak. C1s is not a part of the C3 convertase enzyme.
Solubilization of UDP-glucose-specific starch synthetase in sweet-potato (Ipomoea batatas) starch granules by the urea/pullulanase method
Authors: Sasaki T , Kainuma K
Published: August 2015

The ADP-glucose- or UDP-glucose-specific starch synthetase bound to sweet-potato (Ipomoea batatas) starch granules is localized in the granules, and the UDP-glucose-specific enzyme was solubulized by urea/pullulanase treatment of the starch granules.
Human cathepsin B. Application of the substrate N-benzyloxycarbonyl-l-arginyl-l-arginine 2-naphthylamide to a study of the inhibition by leupeptin
Authors: Knight C G
Published: August 2015

1. The kinetic parameters Kcat. and Km were determined for the hydrolysis of some arginine naphthylamides by human cathepsin B. 2. A new and efficient synthesis of Z-Arg-Arg-NNap (benzyloxycarbonyl-L-arginyl-L-arginine 2-naphthylamide) was developed. 3. Z-Arg-Arg-NNap was a specific and sensitive substrate for cathepsin B, and was used for kinetic studies. 4. Values of kcat. were maximal in the pH range 5.4–6.2, and depended on a single ionizing group of pKa 4.4. 5. Leupeptin was a purely competitive inhibitor of human cathepsin B. 6. The effect of pH on the apparent inhibitor constant, Ki (app.), was determined. Ki (app.) was pH-independent in the range pH 4.3–6.0, with the mean value 7 × 10(-9) M.
Interactions of basic polypeptides and proteins with calmodulin
Authors: Itano T , Itano R , Penniston J T
Published: August 2015

Low concentrations (less than 10 microgram/ml) of a number of highly basic polypeptides inhibit the calmodulin-stimulated cyclic nucleotide phosphodiesterase. Inhibitory compounds include synthetic polypeptides [polylysine (D and L) and polyarginine] and basic proteins (protamine, histones H1, H2A, H2B, H3 and H4 and myelin basic protein). Polylysine of mol.wt. about 2000 or higher was inhibitory, but pentalysine did not inhibit. Other basic proteins and compounds did not inhibit, including bradykinin, spermine and putrescine. In mixtures of calmodulin and basic protein, complexes were formed whether Ca2+ was present or not. This was true for polylysine, myelin basic protein and histone H2B. These interactions suggest that the inhibition of the phosphodiesterase is due to interaction of these basic proteins with calmodulin. The wide variety of basic polypeptides and proteins that affect the calmodulin stimulation of phosphodiesterase indicates that these interactions are not specific.
Size heterogeneity of rat pituitary prolactin
Authors: Wallis M , Daniels M , Ellis S A
Published: August 2015

The occurrence of multiple forms of rat prolactin with different molecular weights (size heterogeneity) was studied with anterior pituitary extracts, purified rat prolactin and 125I-labelled rat prolactin. In each case, three main forms of the hormone were detected by gel filtration on Sephadex G-100: a major one (80–90%) corresponding to monomeric prolactin (mol.wt. 22000–25000), a peak (8–20%) that could be a dimer (mol.wt. 45000–50000) and a small quantity (1–5%) of a component of much greater molecular weight. On freezing and thawing of 125I-labelled rat prolactin, there was little interconversion of monomer and ‘dimer’ peaks, but both were converted substantially to very high-molecular-weight material. All three peaks of 125I-labelled rat prolactin could be precipitated by anti-(rat prolactin) serum and all three gave similar patterns of radioactive peptides after digestion with chymotrypsin followed by high-voltage paper electrophoresis. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the monomer peak of 125I-labelled prolactin migrated as a single component of mol.wt. 22000, the very high-molecular-weight peak largely dissociated to a component running in the same position as the monomer, and the ‘dimer’ peak migrated partly as a component of mol.wt. 45000 and partly as a component migrating with monomeric prolactin. No treatment was found that could dissociate the ‘dimer’ peak completely to monomeric prolactin.
Formation of super-reduced Chromatium high-potential iron–sulphur protein in aqueous solution by pulse radiolysis
Authors: Butler J , Sykes A G , Buxton G V , Harrington P C , Wilkins R G
Published: August 2015

Both the oxidized and reduced forms of Hipip (high-potential iron–sulphur protein) are reduced (approx. 30% yields) by eaq.- in a single-stage process, rate constants 1.7 × 10(10) and 1.8 × 10(10) M-1 . s-1 respectively, at 25 degrees C, pH 7.0 (5 mM-phosphate). Super-reduced Hipip, which is formed in the latter case, has a spectrum which closely resembles that of reduced ferredoxin, i.e. Fe4S4 (SR)4(3-) clusters. The spectrum is stable over 2 s periods investigated. Super-reduced Hipip is reoxidized with O2, rate constant 4.8 × 10(6) M-1 . s-1 at 25 degrees C.
Incorporation of l-[3H]fucose and d-[3H]glucosamine into cell-surface-associated glycoconjugates in epidermis of cultured pig skin slices
Authors: King I A , Tabiowo A , Williams R H
Published: August 2015

1. Electron microscope autoradiography indicated that L-[3H]fucose and D-[3H]glucosamine were both incorporated into cell-surface-associated glycoconjugates in the epidermis of cultured pig skin slices. 2. Acid hydrolysis and paper chromatography of skin homogenates confirmed that there was little metabolic conversion of the labeled precursors to other sugars. 3. Epidermis was separated from dermis using CaCl2, and was extracted with 8 M-urea/5% (w/v) sodium dodecyl sulphate and was then analysed by gel electrophoresis. The major component labelled with D-[3H]glucosamine had an apparent molecular weight in excess of 200 000. This material was not labelled with L-[3H]fucose. Lower molecular-weight components were labelled to a similar extent with both L-[3H]fucose and D-[3H]glucosamine. 4. The high molecular-weight material labelled with D-[3H]glucosamine was released into the medium when the epidermal cells were dispersed with trypsin, indicating that it was either surface-associated or was extracellular. It was also labelled with D-[14C]glucuronic acid, 35SO4(2-) and to a small extent with 14C-labelled amino acids indicating that it contained glycosaminoglycans derived from epidermal proteoglycans. This was confirmed by the fact that it was degraded by testicular hyaluronoglucosidase. It was not present in isolated membranes but was recovered in the soluble fraction from epidermal homogenates. It is therefore only very loosely bound at the cell surface or is present in the extracellular spaces. 5. Membrane-bound [3H]glycoproteins were identified after differential centrifugation of epidermal homogenates. The radioactivity profiles of membrane glycoproteins were similar whether L-[3H]fucose or D-[3H]glucosamine were used and both consisted of a major heterogeneous peak in the apparent mol.wt. range 70 000–150 000. [3H]Glycoproteins in this molecular-weight range were also major components of a plasma-membrane-enriched fraction. These glycoproteins were probably bound to the membrane by hydrophobic interactions, since they were only solubilized by treatment with detergent or organic solvent. They contained terminal sialic acid residues, since they were degraded by neuraminidase.
Mechanism of formation of the A/B cis ring junction of ecdysteroids in Polypodium vulgare
Authors: Davies T G , Lockley W J S , Boid R , Rees H H , Goodwin T W
Published: August 2015

1. The fates of the alpha-, 4 alpha- and 4 beta-hydrogen atoms of cholesterol during formation of the A/B cis ring junction of ecdysteroids was investigated by administration of [4-14C, 3 alpha-3H], [4-14C, 4 alpha-3H]- and [4-14C, 4 beta-3H]cholesterol species to the fern, Polypodium vulgare, and isolation of the 20-hydroxyecdysone formed in each case. 2. The 3H was retained in the ecdysteroid formed from each substrate. 3. Location of the 3H in the 20-hydroxyecdysone indicated that migration of 3H from the 3 alpha- and 4 beta-positions to C-4 and C-5, respectively, had occurred, whereas the 4 alpha-3H atom was retained at C-4. 4. A possible mechanism for the formation of the A/B cis ring junction of ecdysteroids in P. vulgare is presented.
The effect of fluroxene [(2,2,2-trifluoroethoxy)ethane] on haem biosynthesis and degradation
Authors: Ziman Melanie R. , Bradshaw Jean J. , Ivanetich Kathryn M.
Published: August 2015

Acute fluroxene treatment of male Wistar rats decreases the amounts of hepatic microsomal cytochrome P-450 and haem, increases the activities of hepatic δ-aminolaevulinate synthase and haem oxygenase, and increases the amounts of haem precursors (δ-aminolaevulinate and porphobilinogen) in the urine. All of the above effects of fluroxene are enhanced by pretreatment of the experimental animals with 3-methylcholanthrene and phenobarbital. The amounts of porphyrins in the urine and faeces were generally unaffected by acute fluroxene treatment of uninduced or 3-methylcholanthrene- or phenobarbital-induced Wistar rats. 2,2,2-Trifluoroethyl ethyl ether, the saturated analogue of fluroxene, did not affect the amounts of hepatic cytochrome P-450 and haem, the amounts of any of the haem precursors in the urine or faeces, or the activity of hepatic haem oxygenase in phenobarbital-induced male Wistar rats. The amounts of hepatic cytochrome P-450 and haem and of the haem precursors in urine and faeces, and the activity of δ-aminolaevulinate synthase, were generally not altered by acute fluroxene treatment of uninduced male Long–Evans rats. Chronic treatment of Wistar rats with fluroxene resulted in small increases in the amounts of δ-aminolaevulinate and porphyrins in urine. The amounts of porphobilinogen in urine were elevated up to 2000%, whereas the amounts of the porphyrins in faeces were generally unaffected. After chronic fluroxene treatment, the activity of δ-aminolaevulinate synthase was increased, whereas the activity of uroporphyrinogen synthase was decreased. It is concluded that acute fluroxene treatment may affect haem biosynthesis and degradation by a mechanism similar to allylisopropylacetamide, namely by stimulating an atypical cytochrome P-450-dependent pathway for haem degradation. The effects of chronic fluroxene treatment on haem biosynthesis may be a consequence of this mechanism or a result of the inhibition by fluroxene of uroporphyrinogen synthase. Chronic fluroxene treatment of male rats affects the haem biosynthetic pathway in a manner similar to that seen in human genetic acute intermittent porphyria.
Components involved in virally mediated membrane fusion and permeability changes
Authors: Wyke A M , Impraim C C , Knutton S , Pasternak C A
Published: August 2015

1. Intact F glycoprotein is required to induce permeability changes in Lettrée cells or in erythrocytes. Some HN glycoproteins may also be required. Permeability changes thus offer a simple, accurate and rapid means of assaying the integrity of F glycoprotein in certain viral preparations. 2. The ‘1-day’ virus (which contains intact F glycoprotein but which differs morphologically from ‘3 day’ virus) does not cause permeability changes; it can be rendered active by various physical treatments. It is concluded that the environment in which F glycoprotein is embedded is a determining factor for permeability changes. 3. The entry of fluorescently labelled peptides into cells made permeable by virus has been measured. Peptides having a molecular weight in excess of 1000 enter poorly, suggesting a ‘pore’ size of approx. 1 nm in diameter. 4. Two novel assay methods concerned with virus—cell fusion are described. The first measures the fluorescence enhancement that occurs when anthroylstearate is transferred from anthroylstearate-labelled virus to cells. The second measures the giant-cell formation that occurs when partially fused erythrocytes are exposed to hypo-osmotic treatment. The ‘1-day’ virus is active in these assays. In contrast with permeability changes, virus—cell fusion is insensitive to changes in external Ca2+-concentration. 5. The results are compatible with a model [Knutton & Pasternak (1979) Trends Biochem. Sci. 4, 220—223; Impraim, Foster, Micklem & Pasternak (1980) Biochem. J. 186, 847—860] in which virus—cell fusion is a prerequisite for permeability changes, and in which permeability changes are the cause of haemolysis and giant-cell (polykaryon) formation.
Role of adrenaline and cyclic AMP in appearance of tyrosine aminotransferase in perinatal rat liver
Authors: Ghisalberti A V , Steele J G , Cake M H , McGrath M C , Oliver I T
Published: August 2015

1. Adrenaline increased hepatic tyrosine aminotransferase activity when injected into foetal rats or 2-day-old rats. 2. The inhibition of the postnatal increase in tyrosine aminotransferase activity which occurred in adrenalectomized newborn rats rapidly overcome by injection of adrenaline or dibutyryl cyclic AMP. 3. The effects of adrenaline or dibutyryl cyclic AMP on the tyrosine aminotransferase activity in foetal, adrenalectomized newborn and 2-day-old rats could be partially or completely blocked by prior treatment with actinomycin D. 4. Dibutyryl cyclic AMP induced tyrosine aminotransferase activity in hepatocytes cultured from 15-day foetal rats in glucocorticoid-free medium. 5. Actinomycin D at 0.2 microgram/ml in the culture medium completely prevented the induction of tyrosine aminotransferase activity by dibutyryl cyclic AMP in cultured cells. 6. The results suggest that adrenaline and cyclic AMP stimulate a transcriptional event during induction of tyrosine aminotransferase in perinatal liver.
Asymmetric distribution of cytochrome P-450 and NADPH–cytochrome P-450 (cytochrome c) reductase in vesicles from smooth endoplasmic reticulum of rat liver
Authors: Cooper Michael B. , Craft John A. , Estall Margaret R. , Rabin Brian R.
Published: August 2015

1. The topography of cytochrome P-450 in vesicles from smooth endoplasmic reticulum of rat liver has been examined. Approx. 50% of the cytochrome is directly accessible to the action of trypsin in intact vesicles whereas the remainder is inaccessible and partitioned between luminal-facing or phospholipid-embedded loci. Analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis reveals three major species of the cytochrome. Of these, the variant with a mol.wt. of 52000 is induced by phenobarbitone and this species is susceptible to trypsin. 2. After trypsin treatment of smooth membrane, some NADPH–cytochrome P-450 (cytochrome c) reductase activity remains and this remaining activity is enhanced by treatment with 0.05% deoxycholate, which renders the membranes permeable to macromolecules. In non-trypsin-treated control membranes the reductase activity is increased to a similar extent. These observations suggest an asymmetric distribution of NADPH–cytochrome P-450 (cytochrome c) reductase in the membrane. 3. As compared with dithionite, NADPH reduces only 44% of the cytochrome P-450 present in intact membranes. After tryptic digestion, none of the remaining cytochrome P-450 is reducible by NADPH. 4. In the presence of both a superoxide-generating system (xanthine plus xanthine oxidase) and NADPH, all the cytochrome P-450 in intact membrane (as judged by dithionite reducibility) is reduced. The cytochrome P-450 remaining after trypsin treatment of smooth vesicles cannot be reduced by this method. 5. The superoxide-dependent reduction of cytochrome P-450 is prevented by treatment of the membranes with mersalyl, which inhibits NADPH–cytochrome P-450 (cytochrome c) reductase. Thus the effect of superoxide may involve NADPH–cytochrome P-450 reductase and cytosolically orientated membrane factor(s).
Desaturation of linoleic acid from exogenous lipids by isolated chloroplasts
Authors: Jones A V , Harwood J L
Published: August 2015

When [14C]diacylgalactosylglycerol was added to isolated pea or lettuce chloroplasts linolenate synthesis was seen. The desaturation of [14C]linoleate in diacylgalactosylglycerol to [14C]linolenate was stimulated by the addition of a soluble protein fraction containing lipid-exchange activity. Other [14C]acyl lipids were ineffective, except that [14C]phosphatidylcholine in the presence of UDP-galactose and sn-glycerol 3-phosphate could also supply [14C]linoleate for desaturation. These results are consistent with a role of diacylgalactosylglycerol in linolenate synthesis, as indirectly suggested by labelling experiments.
Interrelationship between anionic and cationic forms of glutathione S-transferases of human liver
Authors: Awasthi Y C , Dao D D , Saneto R P
Published: August 2015

Human liver glutathione S-transferases (GSH S-transferases) were fractionated into cationic and anionic proteins. During fractionation with (NH4)2SO4 the anionic GSH S-transferases are concentrated in the 65%-saturated-(NH4)2SO4 fraction, whereas the cationic GSH S-transferases separate in the 80%-saturated-(NH4)2SO4 fraction. From the 65%-saturated-(NH4)2SO4 fraction two new anionic GSH S-transferases, omega and psi, were purified to homogeneity by using ion-exchange chromatography on DEAE-cellulose, Sephadex G-200 gel filtration, affinity chromatography on GSH bound to epoxy-activated Sepharose and isoelectric focusing. By a similar procedure, cationic GSH S-transferases were purified from the 80%-saturated-(NH4)2SO4 fraction. Isoelectric points of GSH S-transferases omega and psi are 4.6 and 5.4 respectively. GSH S-transferase omega is the major anionic GSH S-transferase of human liver, whereas GSH S-transferase psi is present only in traces. The subunit mol.wt. of GSH S-transferase omega is about 22500, whereas that of cationic GSH S-transferases is about 24500. Kinetic and structural properties as well as the amino acid composition of GSH S-transferase omega are described. The antibodies raised against cationic GSH S-transferases cross-react with GSH S-transferase omega. There are significant differences between the catalytic properties of GSH S-transferase omega and the cationic GSH S-transferases. GSH peroxidase II activity is displayed by all five cationic GSH S-transferases, whereas both anionic GSH S-transferases do not display this activity.
Interrelationship between cationic and anionic forms of glutathione S-transferases of bovine ocular lens
Authors: Saneto R P , Awasthi Y C , Srivastava S K
Published: August 2015

Since the eye is constantly exposed to potentially damaging chemical compounds present in the atmosphere and vascular system, we investigated the physiological role of glutathione S-transferase (GSH S-transferase) in detoxification mechanisms operative in the ocular lens. We have purified an anionic and a cationic GSH S-transferase from the bovine lens to homogeneity through a combination of gel filtration, ion-exchange and affinity chromatography. The anionic (pI 5.6) and cationic (pI 7.4) S-transferases were found to have distinct kinetic parameters (apparent Km and Vmax. pH optimum and energy of activation). However, both species were demonstrated to have similar molecular weights and amino acid compositions. Double-immunodiffusion and immunotitration studies showed that both lens S-transferases were immunologically similar. The very close similarity in amino acid compositions and immunological properties strongly indicates that these two transferases either originate from the same gene or at least share common antigenic determinants and originate from similar genes. The bovine lens GSH S-transferases had no glutathione peroxidase activity with either t-butyl hydroperoxide or cumene hydroperoxide as substrate. However, the antibody raised against the homogeneous anionic glutathione S-transferase from the bovine lens was found to precipitate both glutathione S-transferase and glutathione peroxidase activities out of solution in the supernatant of a crude bovine liver homogenate.
The relationship between glycosylation and glycoprotein metabolism of mouse neuroblastoma N18 cells
Authors: Milenkovic A G , Johnson T C
Published: August 2015

Two inhibitors of glycosylation, glucosamine and tunicamycin, were utilized to examine the effect of glycosylation inhibition in mouse neuroblastoma N18 cells on the degradation of membrane glycoproteins synthesized before addition of the inhibitor. Treatment with 10 mM-glucosamine resulted in inhibition of glycosylation after 2h, as measured by [3H]fucose incorporation into acid-insoluble macromolecules, and in a decreased rate of glycoprotein degradation. However, these results were difficult to interpret since glucosamine also significantly inhibited protein synthesis, which in itself could cause the alteration in glycoprotein degradation [Hudson & Johnson (1977) Biochim. Biophys. Acta 497, 567-577]. N18 cells treated with 5 microgram of tunicamycin/ml, a more specific inhibitor of glycosylation, showed a small decrease in protein synthesis relative to its effect on glycosylation, which was inhibited by 85%. Tunicamycin-treated cells also showed a marked decrease in glycoprotein degradation in experiments with intact cells. The inhibition of glycoprotein degradation by tunicamycin was shown to be independent of alterations in cyclic AMP concentration. Polyacrylamide-gel electrophoresis of isolated membranes from N18 cells, double-labelled with [14C]fucose and [3H]fucose, revealed heterogeneous turnover rates for specific plasma-membrane glycoproteins. Comparisons of polyacrylamide gels of isolated plasma membranes from [3H]fucose-labelled control cells and [14C]fucose-labelled tunicamycin-treated cells revealed that both rapidly and slowly metabolized, although not all, membrane glycoproteins became resistant to degradation after glycosylation inhibition.
The use of various immobilized-triazine affinity dyes for the purification of 6-phosphogluconate dehydrogenase from Bacillus stearothermophilus
Authors: Qadri F , Dean P D G
Published: August 2015

1. 6-Phosphogluconate dehydrogenase from Bacillus stearothermophilus was purified approximately 260-fold on triazine-immobilized dye columns to a final specific activity of 54 mumol of NADP+ reduced/min per mg of protein and an overall yield of 62%. 2. An investigation of the capacities of different triazine dyes that inhibit 6-phosphogluconate dehydrogenase was carried out. Cibacron Blue F3G-A and Procion Red HE-3B strongly inhibited the enzyme in free solution and were therefore chosen as the ligands in the purification scheme. 3. KCl was found to be the most suitable agent for eluting 6-phosphogluconate dehydrogenase from Procion Red HE-3B-Sepharose 6B. NADP+ could specifically elute 6-phosphogluconate dehydrogenase from Cibacron Blue F3G-A-Sepharose 6B. 4. A study of the effect of temperature on the binding of pure 6-phosphogluconate dehydrogenase to both Cibacron Blue-Sepharose and Procion Red-Sepharose showed that the binding increased with an increase in temperature.