bluuebunny/crossref_metadata_2025 huggingface dataset:
The hormone-binding unit of luteinizing hormone receptor
Authors: Metsikkö M K , Rajaniemi H J
Published: August 2015

Rat ovarian luteinizing hormone/human choriogonadotropin binding sites were labelled with 125I-choriogonadotropin in vivo, and the resulting 125I-choriogonadotropin-receptor complexes were solubilized by Triton X-100 and purified by use of antibodies to choriogonadotropin immobilized to agarose. The purified 125I-choriogonadotropin-receptor complex was treated with glutaraldehyde to crosslink radiolabelled hormone to the receptor. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the crosslinked product revealed a labelled Mr 130 000 major band in addition to the hormone and its alpha-subunit, indicating that a single receptor component was linked to the hormone. Unoccupied binding sites for luteinizing hormone were also solubilized by Triton X-100 from pseudopregnant rat ovaries, and attached to choriogonadotropin-agarose. The agarose gel was washed, and eluted with 0.1 M-sodium acetate, pH 4. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the pH 4 eluate revealed an Mr 90 000 major band which was abolished when ovaries presaturated with choriogonadotropin were used as starting material. These observations suggest that the hormone-binding component of the luteinizing hormone receptor is a polypeptide of Mr 90 000. This polypeptide was isolated and labelled with Na 125I. The labelled polypeptide showed a single band on sucrose density gradient centrifugation and on gel filtration on agarose.
Restriction of bacterial growth by inhibition of polyamine biosynthesis by using monofluoromethylornithine, difluoromethylarginine and dicyclohexylammonium sulphate
Authors: Bitonti A J , McCann P P , Sjoerdsma A
Published: August 2015

Bacterial growth was measurably slowed by a combination of drugs which inhibit polyamine-biosynthetic enzymes. Addition of DL-alpha-monofluoromethylornithine, which was shown to inactivate irreversibly ornithine decarboxylase extracted from Escherichia coli (Ki = 0.36 mM) and Pseudomonas aeruginosa (Ki = 0.30 mM), DL-alpha-difluoromethylarginine and dicyclohexylammonium sulphate to cultures of E. coli or P. aeruginosa resulted in a 40 and 70% increase in generation times (decreased growth rates) respectively, which was completely reversed by the addition of 0.1 mM-putrescine plus 0.1 mM-spermidine to the medium. Decreased intracellular polyamine concentrations correlated with increased generation times; putrescine concentration was decreased by 70% in E. coli and 80% in P. aeruginosa, while spermidine concentration was decreased by 50% in E. coli and 95% in P. aeruginosa. Subsequent investigation of the inactivation of the ornithine decarboxylase by monofluoromethylornithine indicated that it was active-site directed, as the normal substrate ornithine slowed the rate of inhibition. Specific interference with polyamine biosynthesis may be a viable approach to control of some bacterial infections.
Inhibition of acetyl-CoA carboxylase activity in isolated rat adipocytes incubated with glucagon. Interactions with the effects of insulin, adrenaline and adenosine deaminase
Authors: Zammit Victor A. , Corstorphine Clark G.
Published: August 2015

1. Adipocytes isolated from epididymal fat-pads of fed rats were incubated with different concentrations of glucagon, insulin, adrenaline and adenosine deaminase, and the effects of these agents on the ‘initial’ activity of acetyl-CoA carboxylase in the cells were studied. 2. Glucagon (at concentrations between 0.1 and 10nm) inhibited acetyl-CoA carboxylase activity. Maximal inhibition was approx. 70% of the ‘control’ activity in the absence of added hormone, and the concentration of hormone required for half-maximal inhibition was 0.3–0.5nm-glucagon. 3. Incubation of cells with adenosine deaminase resulted in a similar inhibition of acetyl-CoA carboxylase activity. Preincubation of adipocytes with adenosine deaminase did not alter either the sensitivity of carboxylase activity to increasing concentrations of glucagon or the maximal extent of inhibition. 4. Adrenaline inhibited acetyl-CoA carboxylase to the same extent as glucagon. Preincubation of the cells with glucagon did not alter the sensitivity of enzyme activity to adrenaline or the degree of maximal inhibition. 5. Insulin activated the enzyme by 70–80% of ‘control’ activity. Preincubation of the cells with glucagon did not alter the concentration of insulin required to produce half the maximal stimulatory effect (about 12μunits of insulin/ml). The effects of insulin and glucagon appeared to be mediated completely independently, and were approximately quantitatively similar but opposite. These characteristics resulted in the mutual cancellation of the effects of the two hormones when they were both present at equally effective concentrations. 6. The implications of these findings with regard to current concepts about the mechanism of regulation of acetyl-CoA carboxylase and to the regulation of the enzyme in vivo are discussed.
Purification and properties of tryptophan synthase from baker's yeast (Saccharomyces cerevisiae)
Authors: Bailey C J , Turner P D
Published: August 2015

Tryptophan synthase was purified from baker's yeast. The purified enzyme exhibited one band on polyacrylamide-gel electrophoresis, had no detectable N-terminal amino acid and C-terminal alanine. The amino acid composition was close to that predicted by recent studies on the DNA sequence of the structural gene for the enzyme. Kinetic parameters for the following three activities were measured: indole-serine condensation, indolylglycerol phosphate lyase and the overall reaction of serine with 1-(indol-3-yl)glycerol 3-phosphate. The Km for indole was much lower than suggested by previous investigations, and the value of 11 microM was measured by a fluorimetric assay.
Carbohydrate is linked through ethanolamine to the C-terminal amino acid of Trypanosoma brucei variant surface glycoprotein
Authors: Holder A A
Published: August 2015

The C-terminal amino acid of the variant surface glycoprotein from Trypanosoma brucei is glycosylated. For two variant proteins that terminate in an aspartic acid and a serine residue respectively, it was shown that the sugar side chain is linked through ethanolamine to the alpha-carboxy group of the amino acid.
Inhibition of pepsin by analogues of pepsinogen-(1–12)-peptide with substitutions in the 4–7 sequence region
Authors: Dunn B M , Lewitt M , Pham C
Published: August 2015

Derivatives of the 1-12 sequence of pig pepsinogen were prepared by solid-phase peptide synthesis. The three derivatives contain substitutions in the 4-7 region of the 1-12 sequence. Glycine was used to replace the hydrophobic residues -Val-Pro-Leu-Val- in pairs. After cleavage and purification, the synthetic peptides were compared with a synthetic peptide of the native sequence, prepared at the same time, with respect to their ability to inhibit the pepsin-catalysed clotting of milk. Inhibitory potency, determined from plots of percentage inhibition versus concentration of synthetic peptide, is inversely correlated with the substitution of glycine residues for the hydrophobic residues. Therefore the equilibrium inhibition of pepsin by these peptides is dominated by the hydrophobic nature of the 4-7 sequence region.
Electron transfer flavoprotein from pig liver mitochondria. A simple purification and re-evaluation of some of the molecular properties
Authors: Husain M , Steenkamp D J
Published: August 2015

Electron transfer flavoprotein (ETF) from pig liver mitochondria has been purified to homogeneity by a three-step procedure with approx. 10-fold higher yields than previously reported. The purified ETF exhibits an absorption coefficient for the bound FAD of 13.5 mM-1.cm-1 at 436 nm and an isoelectric point of 6.75. Gel filtration, sodium dodecyl sulphate gel electrophoresis and flavin analysis indicate that pig liver ETF is a dimer, composed of non-identical subunits (Mr 38 000 and 32 000) with only one FAD/dimer. Anaerobic reduction by dithionite produces anionic flavin semiquinone as a stable intermediate and establishes the flavin to be the only redox-active chromophore in ETF.
Pseudomonas cytochrome C-551 peroxidase. A purification procedure and study of CO-binding kinetics
Authors: Foote N , Thompson A C , Barber D , Greenwood C
Published: August 2015

A procedure is described for the purification of cytochrome c peroxidase from Pseudomonas aeruginosa involving extraction by sonication, followed by acid precipitation and chromatography on only two types of gel. The final preparation had a purity ratio A407/A280 of 4.2, and was found to be essentially pure by isoelectric focusing. The enzyme was shown to be unstable during degassing under vacuum except in the presence of detergent. The kinetics of CO binding to dithionite-reduced peroxidase were studied with stopped-flow and flash-photolysis techniques, and the results obtained between pH 5 and 7 suggest the existence of two forms of dithionite-reduced enzyme in slow equilibrium.
Inhibition of microsomal oxidation of alcohols and of hydroxyl-radical-scavenging agents by the iron-chelating agent desferrioxamine
Authors: Cederbaum A I , Dicker E
Published: August 2015

Rat liver microsomes (microsomal fractions) catalyse the oxidation of straight-chain aliphatic alcohols and of hydroxyl-radical-scavenging agents during NADPH-dependent electron transfer. The iron-chelating agent desferrioxamine, which blocks the generation of hydroxyl radicals in other systems, was found to inhibit the following microsomal reactions: production of formaldehyde from either dimethyl sulphoxide or 2-methylpropan-2-ol (t-butylalcohol); generation of ethylene from 4-oxothiomethylbutyric acid; release of 14CO2 from [I-14C]benzoate; production of acetaldehyde from ethanol or butanal (butyraldehyde) from butan-1-ol. Desferrioxamine also blocked the increase in the oxidation of all these substrates produced by the addition of iron-EDTA to the microsomes. Desferrioxamine had no effect on a typical mixed-function-oxidase activity, the N-demethylation of aminopyrine, nor on the peroxidatic activity of catalase/H2O2 with ethanol. H2O2 appears to be the precursor of the oxidizing radical responsible for the oxidation of the alcohols and the other hydroxyl-radical scavengers. Chelation of microsomal iron by desferrioxamine most likely decreases the generation of hydroxyl radicals, which results in an inhibition of the oxidation of the alcohols and the hydroxyl-radical scavengers. Whereas desferrioxamine inhibited the oxidation of 2-methylpropan-2-ol, dimethyl sulphoxide, 4-oxothiomethylbutyrate and benzoate by more than 90%, the oxidation of ethanol and butanol could not be decreased by more than 45-60%. Higher concentrations of desferrioxamine were required to block the metabolism of the primary alcohols than to inhibit the metabolism of the other substrates. The desferrioxamine-insensitive rate of oxidation of ethanol was not inhibited by competitive hydroxyl-radical scavengers. These results suggest that primary alcohols may be oxidized by two pathways in microsomes, one dependent on the interaction of the alcohols with hydroxyl radicals (desferrioxamine-sensitive), the other which appears to be independent of these radicals (desferrioxamine-insensitive).
The effect of starvation on the control of phosphofructokinase activity in the epithelial cells of the rat small intestine
Authors: Jamal A , Kellett G L
Published: August 2015

1. The effect of depriving rats of food for 48 h on the specific activity of phosphofructokinase in the epithelial cells of the small intestine and on the regulatory properties of the enzyme displayed in crude (particle-free) mucosal extracts was studied. 2. The specific activity of phosphofructokinase, measured under optimal conditions at pH8, in the mucosa of fed rats showed a negative aboral gradient along the intestine, decreasing from 15.2 +/- 1.2 units (mumol/min)/g wet wt. in the proximal jejunum to 4.6 +/- 1.2 units/g wet wt. in the terminal ileum. 3. After starvation, the gradient was diminished, but not abolished; the diminution in gradient was due almost exclusively to a decrease in the specific activity of phosphofructokinase in the proximal jejunum by about 30%, there being no change in the terminal ileum. 4. In fed rats, the susceptibility of phosphofructokinase to inhibition by ATP, when assayed in crude mucosal extracts under suboptimal conditions, was independent of length along the small intestine; the ratio of the activity observed at pH 7.0 in the presence of 0.5 mM-fructose 6-phosphate and 2.5 mM-ATP to the optimal activity at pH 8, v0.5/V, was 0.36 +/- 0.05 in the proximal jejunum and 0.42 +/- 0.07 in the terminal ileum. 5. After starvation, the susceptibility of phosphofructokinase to inhibition by ATP was increased and was again found to be independent of length along the small intestine: after starvation, v0.5/V was 0.19 +/- 0.04 and 0.20 +/- 0.07 for the proximal jejunum and the terminal ileum respectively. 6. Re-feeding of previously starved rats on a high-carbohydrate diet overnight for 16 h restored both the specific activities of phosphofructokinase and its susceptibility to inhibition by ATP to normal values for fed rats. 7. The data support the idea that the specific activities and the regulatory properties of phosphofructokinase in the epithelial cells of rat small intestine are mediated by distinct humoral factors. 8. The changes in glucose utilization rate of the jejunum when rats are starved can in principle be accounted for by a combination of changes in the specific activity and in the regulatory properties of mucosal phosphofructokinase.
Alteration of sarcoplasmic reticulum after denervation of chicken pectoralis muscle
Authors: Tate C A , Bick R J , Myers T D , Pitts B J R , Van Winkle W B , Entman M L
Published: August 2015

To determine the neural influence on the function of the sarcoplasmic reticulum (SR) of fast-twitch skeletal muscle, the superior pectoralis muscle of adult chicken was denervated, and the SR was isolated at 20 days post-denervation. The isolated SR was probably derived from the longitudinal SR and was relatively free of contaminants. The protein profile of the SR was quantitatively changed after denervation with an increase in the M55 and 30000-mol.wt. proteins relative to the Ca2+-ATPase. Ca2+-dependent ATPase activity and phosphoenzyme formation were lower in the denervated-muscle SR; however, the enzyme catalytic-centre activity was similar to the control value. The decrease in Ca2+-ATPase activity in denervated-muscle SR was accompanied by a lower Ca2+ accumulation so that the relationship between Ca2+ accumulation and Ca2+-dependent ATPase activity was well maintained in the SR from denervated muscle. The data imply that denervation may result in a diminution of functional Ca2+ pump sites. Evidence is presented, though, which suggests that denervation affects a single class of Ca2+-binding sites of the Ca2+-ATPase, resulting in a lower affinity for Ca2+.
Biosynthesis of intestinal microvillar proteins. Pulse-chase labelling studies on maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV
Authors: Danielsen E M , Sjöström H , Norén O
Published: August 2015

The biogenesis of three intestinal microvillar enzymes, maltase-glucoamylase (EC 3.2.1.20), aminopeptidase A (aspartate aminopeptidase, EC 3.4.11.7) and dipeptidyl peptidase IV (EC 3.4.14.5), was studied by pulse-chase labelling of pig small-intestinal explants kept in organ culture. The earliest detectable forms of the enzymes were polypeptides of Mr 225000, 140000 and 115000 respectively. These were found to represent the enzymes in a ‘high-mannose’ state of glycosylation, as judged by their susceptibility to treatment with endo-beta-N-acetylglucosaminidase H (EC 3.2.1.96). After about 40-60 min of chase, maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV were further modified to yield the mature polypeptides of Mr 245000, 170000 and 137000 respectively, which were expressed at the microvillar membrane after 60-90 min of chase. The fact that the enzymes before reaching the microvillar membrane were found in a Ca2+-precipitated membrane fraction (intracellular and basolateral membranes), but not in soluble form, indicates that during biogenesis maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV are transported and assembled in a membrane-bound state.
Relationships between protein phosphorylation and electron transport in the reconstituted chloroplast system
Authors: Horton P , Foyer C
Published: August 2015

Phosphorylation of the light-harvesting chlorophyll protein (LHCP) by the thylakoid protein kinase has been examined in the reconstituted chloroplast system. The level of phosphorylation by [32P]Pi was maximal at high light intensity and in the absence of 3-phosphoglycerate; dephosphorylation resulted from a subsequent decrease in light intensity or from the addition of 3-phosphoglycerate. Addition of ribose 5-phosphate, which acts as an ATP ‘sink’, also caused dephosphorylation. It is concluded that the degree of phosphorylation is dependent on the redox state and energy state of the system, thereby providing a mechanism for adapting light harvesting to the demands of carbon assimilation.
Rat mast cells permeabilized with Sendai virus secrete histamine in response to Ca2+ buffered in the micromolar range
Authors: Gomperts B D , Baldwin J M , Micklem K J
Published: August 2015

In the presence of low extracellular Ca2+, Sendai virus generates permeability lesions in the membrane of rat mast cells. This causes leakage of intracellular phosphorylated metabolites and lactate dehydrogenase; it also permits uptake of normally impermeant aqueous solutes such as the complex of Ca2+ with N-hydroxyethylethylenediaminetriacetic acid. We have used this system to buffer the concentration of cytosol Ca2+ in the micromolar range and thus to cause release of the contents of the secretory granules such as histamine and beta-N-acetylglucosaminidase. We argue that such release occurs by a normal exocytotic secretory mechanism for the following reasons. (1) While leakage of cytosol components is progressively inhibited by Ca2+ in the range 1-10 microM, release of histamine is dependent on Ca2+ in this range. Higher concentrations of Ca2+ are inhibitory to both permeabilization and histamine release. (2) While leakage of phosphorylated metabolites is enhanced in metabolically inhibited cells and the leakage of lactate dehydrogenase is insensitive to metabolic inhibition, the release of histamine and beta-N-acetylglucosaminidase is strictly dependent on an intact metabolic process.
Regulation of cholesterol synthesis in the liver and mammary gland of the lactating rat
Authors: Gibbons G F , Pullinger C R , Munday M R , Williamson D H
Published: August 2015

The activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase; EC 1.1.1.34) in the lactating mammary gland of rats killed between 10:00 and 14:30 h was 2-3 times that in the livers of the same animals. In contrast, after injection of 3H2O in vivo, the rate of appearance of 3H in the cholesterol of the gland was much lower than that in the liver. In the mammary gland of virgin and non-lactating animals, the activity of HMG-CoA reductase was less than 10% of that of the lactating gland. The activity of HMG-CoA reductase in the lactating mammary gland was significantly (P less than 0.005) lower at midnight than at mid-day, and appeared to show an inverse relationship to the activity of the liver enzyme. However, there was no corresponding change in the incorporation of 3H into the gland cholesterol. Withdrawal of food for 6h had no effect on the activity of HMG-CoA reductase in the lactating mammary gland, but resulted in a significant decrease (P less than 0.005) in that of the liver. Starvation of lactating rats for 24h produced a significant decrease (P less than 0.005) in the activity of the enzyme in both organs. There was also a significant decline in the rate at which 3H2O was incorporated in vivo into the cholesterol of both organs (liver, P less than 0.05; gland, P less than 0.005). Giving a high-fat palatable diet together with chow to lactating animals led to a decline in HMG-CoA reductase activity in the mammary gland, but not in liver. This decrease in the gland was not accompanied by a corresponding decline in the apparent rate of cholesterol synthesis.
Defective utilization of haem in selenium-deficient rat liver
Authors: Correia M A , Burk R F
Published: August 2015

We have previously suggested that an inherent defect in hepatic haem utilization was responsible for the rapid stimulation of hepatic microsomal haem oxygenase activity observed in selenium-deficient rats given phenobarbital, a well known inducer of haem formation. To test this hypothesis, hepatic haem content was deliberately raised in selenium-deficient rats by administration of either tryptophan or allylisopropylacetamide, or by injecting haem itself. We now report that selenium-deficient rats are apparently relatively less efficient in utilizing hepatic haem than normal controls. The findings detailed in the present paper thus indicate that stimulation of hepatic microsomal haem oxygenase activity is indeed a manifestation of abnormal haem utilization in selenium deficiency. This suggests a novel role for selenium in hepatic haem metabolism.
Effects of glucose-containing peritoneal-dialysis solutions on rates of lipogenesis in vivo in the liver, brown and white adipose tissue of chronic uraemic rats
Authors: Klim R A , Williamson D H
Published: August 2015

Chronic uraemic rats had decreased food intake, and this was accompanied by decreased weight of the epididymal fat-pads and interscapular brown adipose tissue. Normal rats whose food intake was restricted to an amount similar to that of the uraemic rats showed similar decreases in weight of the adipose-tissue depots. In addition, the food-restricted rats had decreased liver weight compared with normal or uraemic rats. The basal rate of lipogenesis was decreased in liver and epididymal fat-pads of food-restricted and uraemic rats and in interscapular brown adipose tissue of uraemic rats. Administration of a low-glucose-containing (1.36%) peritoneal-dialysis solution slightly increased lipogenesis in liver of uraemic rats, but had no significant effect in epididymal fat-pads. For brown fat, the rate of lipogenesis was increased in normal, food-restricted and uraemic groups, but the values for the last group were 4-5-fold lower than for the food-restricted or control groups. A high-glucose-containing (3.86%) peritoneal-dialysis solution gave similar rates of lipogenesis in liver, epididymal fat-pads and brown fat of all three groups, but for brown fat moderately uraemic rats showed a considerably lower rate of lipogenesis than did mildly uraemic rats. The basal plasma insulin concentration was lower in the food-restricted (50%) and uraemic (70%) groups than in the control group. The low-glucose peritoneal-dialysis solution increased plasma insulin to control values in the food-restricted rats, but had no significant effect on plasma insulin in the uraemic rats, despite a significant increase in blood glucose in this group. It is concluded that there is an impairment of the lipogenic response to intraperitoneal glucose loads in interscapular brown adipose tissue of uraemic rats, and that this is not due to the accompanying decrease in food intake. The hypoinsulinaemia may be an important factor. The possible relevance of this finding to the obesity observed in some uraemic patients treated by peritoneal dialysis with glucose-containing solutions is discussed.
Tryptophan and glucose metabolism in rat liver cells. The effects of dl-6-chlorotryptophan, 4-chloro-3-hydroxyanthranilate and pyrazinamide
Authors: Cook J S , Pogson C I
Published: August 2015

Liver cells pre-incubated with 1 mM-DL-6-chlorotryptophan are less sensitive to tryptophan-mediated inhibition of gluconeogenesis; this effect is apparent both at physiological (0.1 mM) and higher (0.5 mM) concentrations of tryptophan. 4-Chloro-3-hydroxyanthranilate (1-100 microM) has effects similar to those of DL-6-chlorotryptophan. The effects of both compounds are consistent with a decrease in quinolinate formation, a consequence of inhibition of 3-hydroxyanthranilate oxidase. Pyrazinamide (0.25-5.0 mM) significantly decreased flux through the glutarate pathway and potentiated tryptophan-mediated inhibition of gluconeogenesis; these changes were apparent at physiological concentrations of tryptophan. The effects of pyrazinamide are consistent with an increase in quinolinate formation resulting from inhibition of picolinate carboxylase.
Interactions between retinyl phosphate and bivalent cations
Authors: Shidoji Y , Silverman-Jones C , Noji S , De Luca L M
Published: August 2015

In the presence of Mn(II) ions, the u.v. absorption spectrum of retinyl phosphate (Ret-P) solubilized in Triton X-100 micelles, phosphatidylcholine liposomes or rat liver microsomes exhibited a shift from the maximum of 330 nm to 287 nm. The effect of Mn(II) was reversed by adding EDTA or phosphate buffer. The same spectral change was found in the presence of poly-L-lysine in place of Mn(II) ions. The e.s.r. spectrum of Mn(II) in the presence or in the absence of Ret-P clearly showed that approx. 75% of the initial concentration of Mn(II) ions is bound to Ret-P when the molar ratio of Ret-P to Mn(II) ions is 4:1; no such binding occurred in the presence of retinol or retinoic acid. The appearance of two isosbestic points at 303 and 368 nm, in the presence of Mn(II) ions, suggests the existence of an equilibrium between an Mn(II)-bound monomer and an Mn(II)-bound dimer of Ret-P in Triton X-100 micelles. The same effect on the u.v.-absorption spectrum of Ret-P was also induced by Co(II), Cr(II), Zn(II) and Fe(II), but not by Mg2+ or Cu(II). The formation of the ‘metachromatic complex’ between Ret-P and Mn(II) or Co(II) inhibited the synthesis of retinyl phosphate mannose (Ret-P-Man) from exogenous and endogenous Ret-P and guanosine diphosphate [14C]mannose when bovine serum albumin was added after the metal ion. However, the order of addition did not influence Ret-P-Man synthesis in incubations containing MgCl2, which does not form the metachromatic complex with Ret-P. These results suggest that the bioavailability of proteins, polyamines and metal ions may control the extent to which Ret-P can be mannosylated in the intact membrane.
The lipid fluidity of rat liver membrane subfractions
Authors: Whetton Anthony D. , Houslay Miles D. , Dodd Nicholas J. F. , Evans W. Howard
Published: August 2015

1. The lipid fluidity of three major rat liver plasma-membrane subfractions, as well as Golgi apparatus and endocytic fractions, was assessed with a fatty acid spin probe by using e.s.r. techniques. 2. The sinusoidal (blood-facing) plasma-membrane subfraction was the most fluid of the three plasma-membrane regions. Fractions originating from the bile-canalicular and contiguous (lateral) regions were most rigid. Endocytic fractions isolated (endosomes and diacytosomes) were of a similar fluidity to fractions originating from the sinusoidal plasma-membrane region. By far the most fluid fractions examined were derived from the Golgi-apparatus complex. 3. The three plasma-membrane subfractions each showed a different response to the bilayer-fluidizing effect of benzyl alcohol. 4. Arrhenius-type plots of the order parameter S and outer hyperfine splitting, 2T∥, identified lipid-phase separations in the plasma-membrane subfractions.
On the mechanism of the modulation in vitro of acyl-CoA:cholesterol acyltransferase by progesterone
Authors: Synouri-Vrettakou S , Mitropoulos K A
Published: August 2015

The assay of acyl-CoA:cholesterol acyltransferase (ACAT) in the presence of progesterone resulted in a lower enzyme activity and this inhibition was dependent on the concentration of steroid in the assay mixture. The incubation at 37 degrees C of rat liver microsomal fraction followed by the re-isolation of treated microsomal vesicles and the assay of ACAT resulted in a pre-incubation-time-dependent increase in the activity of the enzyme. This rate of increase was inhibited by the presence of progesterone in the pre-incubation mixture. The incubation of the microsomal fraction in the presence of cholesterol/phosphatidylcholine liposomes, followed by the re-isolation of the treated microsomal vesicles and assay of ACAT, resulted in time-dependent and liposomal cholesterol-concentration-dependent transfer of cholesterol to microsomal vesicles and in an increase in the activity of ACAT. The presence of progesterone during pre-incubation had no effect on the rate of transfer of liposomal cholesterol to the microsomal vesicles. However, progesterone decreased the rate of change in ACAT activity. This effect can be attributed to progesterone associated with treated microsomal vesicles and present during the enzyme assay. Consistent with this, the presence of progesterone has no effect on the size of the non-esterified cholesterol pool that acts as substrate for ACAT. The size of the ACAT substrate pool was modulated in vitro or in vivo and ACAT activity was assayed in the presence of various concentrations of progesterone. The data suggest that the interaction of the steroid with ACAT is at a site other than the catalytic site and that changes in the size of the substrate pool are associated with an increase in ACAT activity, but do not result in changes in the conformation of the enzyme or in co-operative transitions of the enzyme.
The proteolytic digestion of ox neurofilaments with trypsin and α-chymotrypsin
Authors: Chin T K , Eagles P A M , Maggs A
Published: August 2015

Brief digestion of ox neurofilaments with trypsin liberates fragments that are soluble and have molecular weights ranging from 164 000 to 97 000. Peptide fingerprinting indicates that these regions, termed the tryptic head-regions, arise from the 205 000- and 158 000-mol.wt. components of the triplet. The remains of the parent polypeptides sediment with normal filaments and have been termed tail-regions. Digestion of neurofilaments with chymotrypsin also liberates soluble fragments (chymotryptic head-regions) but these have mol.wts. 171 000 and 119 000, though they too originate from the higher-molecular-weight triplet polypeptides. Tryptic and chymotryptic head-regions have extensive homology, and a low (less than or equal to 20%) helix content. Electron microscopy shows that chymotryptic digestion rapidly reduces the length of filaments, probably because this enzyme preferentially attacks the 72 000-mol.wt. polypeptide. In contrast, brief digestion with trypsin does not reduce filament length even though more than 90% of the two higher-molecular-weight components have been cleaved. These results indicate that the backbone of native filaments is formed from the 72 000-mol.wt. polypeptide together with the tail-regions from the 205 000- and 158 000-mol.wt. polypeptides. The corresponding head-regions of these components, which can represent nearly 75% of each molecule, are not necessary for preserving the backbone of native neurofilaments and are therefore good candidates for being the side arms that connect these filaments in nerve cells.
Bovine lens aldehyde dehydrogenase. Purification and preliminary characterization
Authors: Ting H H , Crabbe M J C
Published: August 2015

Cytoplasmic aldehyde dehydrogenase from bovine lens was purified to apparent homogeneity by using ion-exchange and affinity chromatography. Sedimentation-equilibrium ultracentrifugation, gel-filtration chromatography and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis show that the enzyme is a dimer of Mr 114000, with subunits of Mr 57000. The enzyme does not dissociate into monomers in the presence of Ca2+ or Mg2+. The enzyme has a pI of 5.0, an activation energy of 35.1kJ/mmol and a pK value of 8.6 with acetaldehyde as substrate. The enzyme is a prolate ellipsoid with a Stokes radius of 4nm. Progesterone, deoxycorticosterone and chlorpropamide inhibited enzyme activity, and this inhibition may play a role in cataract formation in patients maintained on systemic corticosteroids and in tablet-dependent diabetics.
The presence of polysaccharide in normal human gastric mucus
Authors: Clamp J R , Cooper B , Creeth J M , Ene D , Barrett J , Gough M
Published: August 2015

Polysaccharide material was found in the proteolysis glycopolypeptide fraction from normal human gastric mucus. The polysaccharide was identified by carbohydrate and amino acid analyses, by elemental analysis and from its behaviour on density-gradient ultracentrifugation. The polysaccharide is polydisperse with a weight-average molecular mass of 300 000 Da. Over 85% of the polysaccharide consists of galactose, and this represents 26% of all the galactose present in the fractions after beta-elimination with reduction of the glycopolypeptide material.
Azidocalmodulin derivatives. Activation of, and binding to, three target proteins: aorta myosin light-chain kinase, erythrocyte (Mg2+ + Ca2+)-dependent ATPase and cardiac sarcoplasmic-reticulum kinase
Authors: Molla A , Hincke M T , Katz S , Lazaro R
Published: August 2015

Different azidocalmodulin derivatives were synthesized by modification of either one carboxylic acid group or one or several arginine residues and their binding and activation capacity investigated in three target enzyme systems. The systems studied were smooth-muscle myosin light-chain kinase, cardiac sarcoplasmic-reticulum kinase and erythrocyte (Mg2+ + Ca2+)-dependent ATPase. The results indicated that the activation ability of each calmodulin derivative was different depending on the system studied. Binding studies carried out by the displacement of 125I-calmodulin indicated that the monosubstitutions did not greatly alter the apparent Kd of calmodulin for the enzymes but that the modification of four arginine residues caused a 4-8-fold increase in the apparent Kd in all systems. These results have shown that azidocalmodulin derivatives may have different degrees of usefulness in the study of calmodulin target proteins in different systems, with the behaviour of the derivatives not predictable on the basis of the nature (soluble or membrane-bound) or the type (ATPase or kinase) of enzyme system to be investigated. However, the monosubstituted calmodulin and, in particular, the carboxylic acid-group-modified derivative (where the modification was statistically dispersed over the protein chain) are good candidates for photolabelling calmodulin-binding proteins.
Guinea-pig kidney β-N-acetylgalactosaminyltransferase towards Tamm-Horsfall glycoprotein. Requirement of sialic acid in the acceptor for transferase activity
Authors: Serafini-Cessi F , Dall'Olio F
Published: August 2015

A beta-N-acetylgalactosaminyltransferase that preferentially transferred N-acetylgalactosamine to Sd(a-) Tamm-Horsfall glycoprotein was found in guinea-pig kidney microsomal preparations. This enzyme was kidney-specific and was able to transfer the sugar to other glycoproteins, such as fetuin and alpha 1-acidic glycoprotein. The presence of sialic acid in the acceptors was essential for the transferase activity when either glycoproteins or their Pronase glycopeptides were used as acceptors. Two glycopeptides (Tamm-Horsfall glycopeptides I and II) with a different carbohydrate composition were separated by DEAE-Sephacel chromatography from Pronase-digested Tamm-Horsfall glycoprotein. The amount of N-acetylgalactosamine transferred to glycopeptides by the enzyme correlated with their degree of sialylation. Enzymic digestion of N-[14C]acetylgalactosamine-labelled Tamm-Horsfall glycopeptide II showed that the transferred sugar was susceptible to beta-N-hexosaminidase. The amount of sugar cleaved by beta-hexosaminidase was strongly increased when the labelled Tamm-Horsfall glycopeptide II was pretreated with mild acid hydrolysis, a procedure that removed the sialic acid residues. Alkaline borohydride treatment of the labelled Tamm-Horsfall glycopeptide II did not release radioactivity, thus indicating that enzymic glycosylation took place at the N-asparagine-linked oligosaccharide units of Tamm-Horsfall glycoprotein.
Determination of Michaelis parameters from differentials of progress curves
Authors: Petersen L C
Published: August 2015

First differentials of progress curves are easily obtainable in many enzyme assay systems. Such curves may be more readily applicable to kinetic analysis than are the usual progress curves. The theory for this approach is developed, and simple graphical procedures for the determination of Michaelis parameters are indicated. By using an electronic differentiator device the application of the method is demonstrated on the kinetics of three different serine proteinases with various synthetic substrates. Whenever the steady-state concentration of an intermediate of the reaction is proportional to the rate, the transition of this intermediate in substrate-depletion experiments may be analysed in similar terms. This is demonstrated with cytochrome c oxidase kinetics. A number of other possible applications are discussed.
A homogeneous immunoassay for cyclic nucleotides based on chemiluminescence energy transfer
Authors: Campbell A K , Patel A
Published: August 2015

A chemiluminescent derivative of cyclic AMP, aminobutylethylisoluminol succinyl cyclic AMP (ABEI-scAMP), was synthesized in order to develop a homogeneous immunoassay based on non-radiative energy transfer. ABEI-scAMP was chemiluminescent (5.1 × 10(18) luminescent counts X mol-1 at pH 13), pure (greater than 95%) stable and immunologically active. A conventional immunoassay was established using ABEI-scAMP and a solid-phase anti-(cyclic AMP) immunoglobulin G which could detect cyclic AMP at least down to 25fmol. A homogeneous immunoassay for cyclic AMP was established by measuring the shift in wavelength from 460 to 525nm which occurred when ABEI-scAMP was bound to fluorescein-labelled anti-(cyclic AMP) immunoglobulin G. The assay was at least as sensitive as the conventional radioimmunoassay using cyclic [3H]AMP and could measure cyclic AMP over the range 1-1000nM. The homogeneous chemiluminescent energy transfer assay was also able to quantify the association and dissociation of antibody-antigen complexes. Chemiluminescence energy transfer occurred between fluorescein-labelled antibodies and several other ABEI-labelled antigens (Mr values 314-150000) including progesterone, cyclic GMP, complement component C9 and immunoglobulin G. The results provide a homogeneous immunoassay capable of measuring free cyclic AMP under conditions likely to exist inside cells.
Biosynthesis of intestinal microvillar proteins. The effect of swainsonine on post-translational processing of aminopeptidase N
Authors: Danielsen E M , Cowell G M , Norén O , Sjöström H , Dorling P R
Published: August 2015

The post-translational processing of pig small-intestinal aminopeptidase N (EC 3.4.11.2) was studied in organ-cultured mucosal explants. Exposure of the explants to swainsonine, an inhibitor of Golgi mannosidase II, resulted in the formation of a Mr-160000 polypeptide, still sensitive to endo-beta-N-acetylglucosaminidase H. Swainsonine caused only a moderate inhibition of transport of the enzyme through the Golgi complex and the subsequent expression in the microvillar membrane. This may imply that the trimming of the high-mannose core and complex glycosylation of N-linked oligosaccharides is not essential for the transport of aminopeptidase N to its final destination. A different type of processing was observed to take place in the presence of swainsonine, resulting in a considerable increase in apparent Mr (from 140000 to 160000). This processing could not be ascribed to N-linked glycosylation, since treatment of the Mr-160000 polypeptide with endo-beta-N-acetylglucosaminidase H only decreased its apparent Mr by 15000. The susceptibility of the mature Mr-166000 polypeptide, but not the Mr-140000 polypeptide, to mild alkaline hydrolysis suggests that aminopeptidase N becomes glycosylated with O-linked oligosaccharides during its passage through the Golgi complex. Aminopeptidase N was not labelled by [3H]palmitic acid, indicating that the processing of the enzyme does not include acylation.
Mechanism of activation of pyruvate dehydrogenase by mitogens in pig lymphocytes
Authors: Baumgarten E , Brand M D , Pozzan T
Published: August 2015

The activity of pyruvate dehydrogenase in extracts of pig mesenteric lymphocytes was measured under different preincubation conditions. The mitogens concanavalin A and ionophore A23187 both increased pyruvate dehydrogenase activity. In both cases activation required extracellular Ca2+. Digitonin-permeabilized cells required 0.5 microM free Ca2+ for half-maximal activation of pyruvate dehydrogenase. The stimulation by concanavalin A in intact cells was probably not due to changes in effectors of pyruvate dehydrogenase kinase. This evidence suggests that activation of pyruvate dehydrogenase is by Ca2+ activation of pyruvate dehydrogenase phosphatase and supports the view that the cytoplasmic free [Ca2+] rises to something less than 1 microM on stimulation with mitogens.
The stimulation of glycogenolysis and gluconeogenesis in isolated hepatocytes by opioid peptides
Authors: Allan E H , Green I C , Titheradge M A
Published: August 2015

The opioid agonists [leucine]enkephalin, [D-Ala2,D-Leu5]enkephalin and dynorphin-(1-13)-peptide, but not morphine, stimulated the conversion of [2-14C]pyruvate into glucose and glycogenolysis when added directly to isolated hepatocytes. Naloxone produced a small but significant inhibition of both the basal and stimulated rate of incorporation of label into glucose but had no effect on the total glucose output by the cells. The effects of the opioid peptides were mediated by a cyclic AMP-independent mechanism.
The relationship between intracellular pH, the pH gradient and potassium transport in Escherichia coli
Authors: Kroll R G , Booth I R
Published: August 2015

The capacity of E. coli cells to regulate intracellular pH (pHi) during net potassium uptake has been investigated. The data show: (a) that cells sense their intracellular pH; (b) that the pH gradient (delta pH) exerts a feedback regulation on pHi; (c) that a mechanism of regulation of pHi exists which may be independent of Na+ [Zilberstein, Agmon, Schuldiner & Padan (1982) J. Biol. Chem. 257, 3687-3691]; and (d) that cells have a limited capacity to raise their intracellular pH in the absence of net K+ transport.
Modification of the interactions of myosin with actin and 5′-adenylyl imidodiphosphate by substitution of ethylene glycol for water
Authors: Marston S B , Tregear R T
Published: August 2015

We studied the effect of replacing water by ethylene glycol as solvent on the properties of skeletal muscle myosin, myosin subfragment-1 (S1) and heavy meromyosin. Ethylene glycol (50%, v/v) had no detectable effect on the affinity of myosin or actomyosin for the substrate analogue 5′-adenylyl imidodiphosphate (AMPPNP). However, the rate constants for formation and dissociation of the myosin X MgAMPPNP complex were reduced 200-fold; the logarithm of the dissociation rate was roughly proportional to the fractional concentration of ethylene glycol. Nucleotide dissociation was accelerated at least 300-fold by pure actin but remained slow with regulated actin in the absence of Ca2+. Ethylene glycol substitution reduced the affinity of S1 and the S1 X MgAMPPNP complex for actin equally (100-fold at 50% ethylene glycol). These results show that ethylene glycol has specific effects on myosin's enzymic mechanism, which can account for its effect on the tension and stiffness of glycerinated muscle fibres.
The purification and properties of cathepsin L from rabbit liver
Authors: Mason R W , Taylor M A J , Etherington D J
Published: August 2015

Cathepsin L was purified from rabbit liver by a method involving whole-tissue homogenization, pH precipitation, ammonium sulphate fractionation and chromatography on CM-Sephadex C-50, phenyl-Sepharose and Sephadex G-75. Pure enzyme was obtained without the necessity of laborious subcellular fractionation techniques. The Mr of the enzyme was determined to be 29 000 by gel filtration, and affinity for concanavalin A-Sepharose indicated that it was a glycoprotein. A novel technique for detection of enzyme activity in agarose isoelectrofocusing gels showed that the enzyme existed in multiple isoenzymic forms with pI values ranging from 5.0 to 5.9. The enzyme catalysed the hydrolysis of azocasein, collagen and Z-Phe-Arg-NMec (where Z and NMec indicate benzyloxycarbonyl and N-methylcoumarin derivative respectively) optimally at pH 5.2, 3.3 and 6.0 respectively. In addition, cathepsin L was found to degrade benzoyl-Phe-Val-Arg-NMec and 3-carboxypropionyl-Ala-Phe-Lys-NMec. However, cathepsin B also cleaved all of these substrates. One major difference between these two enzymes was in their Michaelis constants for Z-Phe-Arg-NMec; cathepsin B had Km 75 microM whereas that of cathepsin L was 0.7 microM. Cathepsin L was inhibited by all of the usual chemical inhibitors of thiol proteinases as well as the more specific inhibitors Z-Phe-Phe-CHN2, Z-Phe-Ala-CHN2, compound E-64 and compound Ep-475. Active-site titration with compound E-64 showed that the purified sample contained 80% active protein, which had kcat. 20s-1 for the substrate Z-Phe-Arg-NMec. Antibodies were raised to active cathepsin L, and these did not cross-react with cathepsin B, thus demonstrating that these two enzymes are immunologically distinct.
Accessibility of DNA-epoxycellulose to sequence-specific DNA-binding proteins
Authors: Lazo P A
Published: August 2015

Phage lambda DNA was covalently coupled to epoxy-activated cellulose to form a stable DNA-cellulose matrix for affinity chromatography of sequence-specific DNA-binding proteins. The accessibility of three specific six-base sequences, GGATCC (BamHI), GAATTC (EcoRI) and AAGCTT (HindIII) was studied quantitatively and qualitatively by restriction analysis followed by labelling of their recessed ends. All sites are randomly accessible. The site accessibility is variable, BamHI greater than HindIII greater than EcoRI, and within the range 20-100% depending on base composition and internal structure of the sequence. DNA-epoxycellulose, because of its high efficiency of coupling, capacity, stability and accessibility, can be of great help in the isolation and characterization of sequence-specific DNA-binding proteins.
Effect of a protein-free diet on muscle protein turnover and nitrogen conservation in euthyroid and hyperthyroid rats
Authors: Carter W J , van der Weijden Benjamin W S , Faas F H
Published: August 2015

Although protein turnover in skeletal muscle is increased in hyperthyroidism and decreased in hypothyroidism, a deficient protein intake tends to increase serum T3 (tri-iodothyronine) while decreasing muscle protein turnover. To determine whether this diet-induced decrease in protein turnover can occur independent of thyroid status, we have examined muscle protein turnover and nitrogen conservation in hyperthyroid rats fed on a protein-free diet. After inducing hyperthyroidism by giving 20 micrograms of T3/100g body wt. daily for 7 days, groups of euthyroid and hyperthyroid animals were divided into subgroups fed on basal and protein-free diets. Muscle protein turnover was measured by N tau-methylhistidine excretion and [14C]tyrosine infusion. Urinary nitrogen output of euthyroid and hyperthyroid animals fed on the protein-free diet was also measured. Although hyperthyroidism increased the baseline rates of muscle protein synthesis and degradation, it did not prevent a decrease in these values in response to protein depletion. Furthermore, hyperthyroid rats showed greatly decreased nitrogen excretion in response to the protein-free diet, although not to values for euthyroid rats. These findings suggest that protein depletion made the experimental animals less responsive to the protein-catabolic effects of T3.