bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and characterization of the ferredoxin component of 25-hydroxycholecalciferol 1α-hydroxylase
Authors: Kulkoski J A , Ghazarian J G
Published: August 2015

The chick kidney mitochondrial iron–sulphur protein (ferredoxin), a component of the NADPH–cytochrome P-450 reductase functional in the 1 alpha-hydroxylation of 25-hydroxycholecalciferol, was purified to homogeneity by chromatography on DEAE-cellulose, gel filtration on Sephadex G-100 and preparative electrophoresis on polyacrylamide gel. A novel NADPH–cytochrome c reductase assay utilizing crude renal NADPH–ferredoxin reductase was used for the detection of the ferredoxin. A mol. wt. of 53 000 was determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and by Sephadex G-100 gel filtration of the 125I-labelled ferredoxin. The ferredoxin has a sedimentation constant (S 20, w) of 2.66S, an A411/A280 of 0.4, and a molar absorptivity of 7300 cm-1 . M-1. The electron-paramagnetic-resonance spectrum after reduction with Methyl Viologen and dithionite was characteristic of ferredoxins with signals at g = 1.956 and 2.025. Two iron and two labile sulphur atoms per molecule of ferredoxin were released by acid. Ouchterlony immunodiffusion tests by using goat anti-(bovine adrenal ferredoxin) antiserum showed precipitin reactions with the bovine adrenal ferredoxin and the chick renal ferredoxin as antigens, suggesting that the renal ferredoxin shares antigenic determinants(s) with the natural adrenal antigen. Amino acid analysis showed that of the total number of residues per molecule of ferredoxin, glutamic acid and aspartic acid are the most abundant residues, comprising 17 and 15% respectively.
UDP-glucosyltransferase activity of housefly microsomal fraction
Authors: Morello A , Repetto Y
Published: August 2015

Housefly UDP-glucosyltransferase activity towards p-nitrophenol was demonstrated in a system in vitro. The activity is localized in the microsomal fraction, requires UDP-glucose, is slightly stimulated by Mg2+ and is activated optimally over a wide range of detergent concentration. Phenobarbital increases the enzyme(s) activity about 3-fold, with p-nitrophenol as substrate, which differs from the corresponding mammalian glucuronyltransferase.
Characterization of the second prosthetic group of the flavoenzyme NADH–acceptor reductase (component C) of the methane mono-oxygenase from Methylococcus capsulatus (Bath)
Authors: Colby J , Dalton H
Published: August 2015

1. A new two-step purification is described that routinely yields 100mg quantities of component C for biochemical studies. 2. Chemical analyses show component C purified by this procedure to contain 2 g-atoms of iron, 2 mol of acid-labile sulphide (S) and 1 mol of FAD per mol of protein. 3. The Fe-S core of component C was extruded by treating the protein with p-methoxybenzenethiol in hexamethyl phosphoramide/50mM-Tris/HCl buffer, pH 8.5 (4:1, v/v), under anaerobic conditions. The spectral properties of the extruded core suggest that component C contains 1 mol of [2Fe-2S(S-Cys)4] centre per mol of protein. 4. E.p.r. spectroscopy confirms the presence of a Fe-S centre in component C. 5. Component C catalyses the reduction by NADH of ferricyanide, 2,6-dichlorophenol-indophenol or horse heart cytochrome c, with specific activities of 50–230 units/mg of protein. 6. The optimum pH for the NADH-acceptor reductase activity is 8.5–9.0, and the apparent Km values for NADH and NADPH are 0.05mM and 15.5mM respectively. 7. Unlike methane mono-oxygenase activity, NADH-acceptor reductase activity of component C is not inhibited by 8-hydroxyquinoline or by acetylene.
Analysis of histones from the yeast Saccharomyces carlsbergensis
Authors: Pastink A , Berkhout T A , Mager W H , Planta R J
Published: August 2015

Basic chromosomal proteins were isolated from the chromatin of the yeast Saccharomyces carlsbergensis by extraction with H2SO4 and were purified by ion-exchange chromatography. Electrophoresis of the purified fraction on acetic acid/urea gels revealed the presence of four main components. These four proteins were identified as histones H2A, H2B, H3 and H4 on the basis of their amino acid composition, molecular weight and solubility properties, all of which are very similar to the corresponding properties of the various histone proteins from other eukaryotic organisms. A fifth basic protein could be isolated from yeast chromatin by extraction with HClO4. The available evidence indicates this protein to be an H1-type histone. Yeast thus appears to contain a complete set of histone proteins which are strongly homologous to the histones occurring in higher eukaryotes.
Artificially induced active transport of amino acid driven by the efflux of a sugar via a heterologous transport system in de-energized Escherichia coli
Authors: Bentaboulet M , Robin A , Kepes A
Published: August 2015

Consistent with the model of an H+ cotransport, amino acid uptake can be driven by a proton gradient generated by an efflux of sugar when the normal energy sources are suppressed. Heterologous countertransport is completely inhibited by uncouplers unlike homologous countertransport. Positive coupling was obtained with methyl thiogalactoside/proline, methyl thiogalactoside/phenylalanine, gluconate/proline; however, the poor coupling efficiency suggests a more complex sequence of reactions.
Interaction of mitochondrial malate dehydrogenase monomer with phospholipid vesicles
Authors: Webster K A , Patel H V , Freeman K B , Papahadjopoulos D
Published: August 2015

The association between bovine and porcine mitochondrial malate dehydrogenase (EC 1.1.1.37) and phospholipid vesicles was investigated. At concentrations at which malate dehydrogenase exists as a dimer, entrapment within the aqueous compartment but not binding of the 14C-labelled enzyme was observed. The dissociated enzyme was labile to moderate heat and to p-chloromercuribenzoate, but in both cases inactivation was decreased by incubation with suspensions of charged phospholipid vesicles. This suggested an interaction between enzyme subunits and phospholipid, and this was confirmed by direct binding measurements and by studies that followed changes in the fluorescein-labelled enzyme. The circular-dichroism spectra of the enzyme indicated a high alpha-helix content, and suggested that a small conformational change occurred when the enzyme dissociated. Fluorescence data also suggested less-rigid molecules after dissociation. A possible mechanism, based on the flexibility of enzyme monomer and its interaction with phospholipids, by which mitochondrial matrix enzymes are specifically localized in cells, is discussed.
Mixed anhydrides of nucleotides and mesitylenecarboxylic acid as new specific inhibitors of mitochondrial adenosien triphosphatase
Authors: Kozlov I A , Shalamberidze M V , Novikova I Y , Sokolova N I , Shabarova Z A
Published: August 2015

Mixed anhydrides of nucleoside triphosphates and mesitylenecarboxylic acid inhibit soluble mitochondrial ATPase (adenosine triphosphatase), but do not inhibit ATPase of submitochondrial particles. Inhibition of soluble mitochondrial ATPase by the mixed anhydride of epsilon-ATP and mesitylenecarboxylic acid is followed by the covalent binding of one nucleotide residue to a molecule of the protein. It is suggested that this covalent binding occurs in the catalytic site of the mitochondrial ATPase. The mixed anhydride of ADP and mesitylenecarboxylic acid inhibits the ATPase activity of submitochondrial particles and has no effect on the activity of soluble mitochondrial ATPase. After separation of the submitochondrial particles from the mixed anhydride of ADP and mesitylenecarboxylic acid, their ATPase activity is restored to its original value (half-time of reactivation 3–4 min). Incubation of submitochondrial particles or soluble mitochondrial ATPase with the mixed anhydride of ADP and mesitylenecarboxylic acid results in AMP formation.
The influence of thyroxine administered in vivo on the transmembrane protonic electrochemical potential difference in rat liver mitochondria
Authors: Shears S B , Bronk J R
Published: August 2015

When mitochondria from normal and thyroxine-treated rats were energized by incubation with succinate, phosphate and MgCl2, it was found that the hormone treatment increased the transmembrane protonic electrochemical potential difference by 16mV and the respiration rate by 46%. Other experiments show these changes to be associated with increases in the intramitochondrial K+ and phosphate concentrations.
Kinetic behaviour of acid phosphatase-albumin co-polymers in homogeneous phase and under gel-immobilized conditions
Authors: Cantarella M , Remy M H , Scardi V , Alfani F , Iorio G , Greco G
Published: August 2015

1. An analysis of the kinetic behaviour of immobilized acid phosphatase (EC 3.1.3.2) layers, gelled on the active surface of an ultrafiltration membrane, was carried out. 2. Two possible forms of such immobilized-enzyme systems were dealt with, namely enzyme-polyalbumin co-gelation through an ultrafiltration process, and enzyme co-polymerization to the same albumin polymers and subsequent gelation. 3. A preliminary analysis was also performed on both the corresponding homogeneous-phase (soluble systems to provide reference kinetics. 4. The main conclusions drawn are: (i) the enzyme-albumin co-polymers show a decrease in specific activity compared with the corresponding free enzyme in both soluble and immobilized forms; (ii) in the homogeneous phase a slight increase in the apparent Michaelis constant was measured for the co-polymerized enzyme compared with the free one, which suggests a decrease in affinity towards substrate; (iii) the activation energy in the immobilized phase is halved, compared with that in the homogeneous phase, which indicates that the combined mass-transfer/reaction step is rate-controlling.
Use of photosensitive hydrophobic probes to label the membrane of the human erythrocyte
Authors: Wells E , Findlay J B
Published: August 2015

Two photosensitive hydrophobic probes, azido [3H]benzene and 1-azido-4-iodo[3H]-benzene, have been compared for their effectiveness in labelling, selectively, the intramembranous domains of lipid and proteins. Both partition preferentially into the lipid bilayer and, upon irradiation, covalently attach to both phospholipids and membrane proteins; the more extrinsic polypeptides have a significantly lower specific radioactivity than that of the intrinsic species. Proteolytic experiments also reveal higher labelling of intramembranous regions of the proteins. Consistently, the iodinated form of the probe showed the greater preference for the non-polar phase and a higher degree of selectivity for labelling hydrophobic regions. The results also suggest that penetration through the annulus of tightly bound lipid surrounding integral proteins occurs readily.
Proteins of the kidney microvillar membrane. The amphipathic form of dipeptidyl peptidase IV
Authors: Macnair D C , Kenny A J
Published: August 2015

Dipeptidyl peptidase IV was solubilized from the microvillar membrane of pig kidney by Triton X-100. The purified enzyme was homogeneous on polyacrylamide-gel electrophoresis and ultracentrifugation, although immunoelectrophoresis indicated that amino-peptidase M was a minor contaminant. A comparison of the detergent-solubilized and proteinase (autolysis)-solubilized forms of the enzyme was undertaken to elucidate the structure and function of the hydrophobic domain that serves to anchor the protein to the membrane. No differences in catalytic properties, nor in sensitivity to inhibition by di-isopropyl phosphorofluoridate were found. On the other hand, several structural differences could be demonstrated. Both forms were about 130,000 subunit mol.wt., but the detergent form appeared to be larger by no more than about 4,000. Electron microscopy showed both forms to be dimers, and gel filtration revealed a difference in the dimeric mol.wt. of about 38 000, mainly attributable to detergent molecules bound to the hydrophobic domain. Papain converted the detergent form into a hydrophilic form that could not be distinguished in properties from the autolysis form. A hydrophobic peptide of about 3500 mol.wt. was identified as a product of papain treatment. The detergent and proteinase forms differed in primary structure. Partial N-terminal amino acid sequences were shown to be different, and the pattern of release of amino acids from the C-terminus by carboxypeptidase Y was essentially similar. The results are consistent with a model in which the protein is anchored to the microvillar membrane by a small hydrophobic domain located within the N-terminal amino acid sequence of the polypeptide chain. The significance of these results in relation to biosynthesis of the enzyme and assembly in the membrane is discussed.
The isolation and properties of a second glycoprotein (LGP-II) from the articular lubricating fraction from bovine synovial fluid
Authors: Swann D A , Mintz G
Published: August 2015

A high-molecular-weight glycoprotein (LGP-I) was shown [Swann, Sotman, Dixon & Brooks (1977) Biochem. J. 161, 473–485] to be the major constituent in the articular lubricating fraction from bovine synovial fluid. In addition to the LGP-I component, a second glycoprotein (LGP-II) was also present. After fractionation of bovine synovial fluid by sequential sedimentation in CsCl density gradients, the LGP-I and LGP-II components were separated by gel-permeation chromatography. The LGP-II component was then purified by chromatography on DEAE Bio-Gel A and Bio-Gel P-150. The molecular weight of the LGP-II component was 48,800 calculated from sedimentation-equilibrium measurements. Amino acids represented 53% (w/w) and carbohydrate constituents 36% (w/w) of the molecule. Glutamic acid and lysine (144 and 100 residues/1000 residues) were the major amino acids. Glucosamine, mannose, galactose and N-acetylneuraminic acid [representing 8.0, 6.6, 9.5 and 11.9% (w/w) respectively] were the only carbohydrate constituents detected. Immunodiffusion analysis showed that LGP-II component did not form a detectable precipitin line with antiserum to bovine serum. It appears likely, therefore, that this glycoprotein is synthesized by the joint tissues and is not derived from serum.
Conjugated and unconjugated bilirubins in humans and rhesus monkeys. Structural identity of bilirubins from biles and meconiums of newborn humans and rhesus monkeys
Authors: Blumenthal S G , Taggart D B , Rasmussen R D , Ikeda R M , Ruebner B H , Bergstrom D E , Hanson F W
Published: August 2015

1. Bilirubin-IXalpha monoglucuronide was the predominant bilirubin in biles and meconiums of newborn humans and rhesus monkeys. Rhesus-monkey baby biles contained slightly more diglucuronide than did human baby biles. 2. Bilrubin-IXalpha glucoside, bilirubin-IXalpha xyloside and bilirubin-IXbeta were also constituents of human and rhesus-monkey baby biles and meconiums. Bilirubin-IXalpha glucuronide glucoside was present in human and rhesus-monkey baby biles but not in meconiums. The identity of the bilirubins was confirmed by u.v.-visible and mass spectroscopy of the azodipyrroles obtained by treating the bilirubins with diazotized ethyl anthranilate. The resulting azodipyrroles were identical with the corresponding azodipyrroles obtained from human adult biles and also from reduced isomers of biliverdin. 3. Bilirubin-IXbeta was present in much higher proportions in the extracts of meconiums than in the extracts of biles from the same babies. 4. Oxidation of bilirubins to biliverdins occurs in utero to a small but undetermined extent. The resulting green pigments were present in meconiums collected from the lower small and large intestines of newborn babies and rhesus monkeys. 5. Butanol extracted most of the bilirubins present in biles. This modified method proved to be quick and easy. Little hydrolysis of bilirubins took place during extraction or separation by t.l.c.
Purification and characterization of a phospholipase A2 from the venom of the coral snake, Micrurus fulvius microgalbineus (Brown and Smith)
Authors: Possani L D , Alagòn A C , Fletcher P L , Varela M J , Juliá J Z
Published: August 2015

A phospholipase A2 was purified from the Mexican coral snake Micrurus fulvius microgalbieus (Brown and Smith). Gel filtration of the soluble crude venom on Sephadex g-50 resolved five fractions, of which fraction II had 98% of the total phospholipase activity. This fraction was rechromatographed on a CM-cellulose column that resolved eight fractions, four of which had an important phospholipase activity. The first fraction (II-1) was homogeneous by polyacrylamide-gel electrophoresis and displayed a phospholipase specific activity of 920 units/mg of protein. The apparent molecular weight as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was approx. 14000. The amino acid analysis revealed the presence of 119 amino acid residues, with 12 half-cystines. the N-terminal sequence was shown to be Ser-Leu-Leu-Asx-Phe-Lys-Asx-Met-Ile-Glu-Ser-Thr..., which is homologous with that of phospholipases from other snake venoms.
Enzyme-substrate and enzyme-inhibitor complexes of triose phosphate isomerase studied by 31P nuclear magnetic resonance
Authors: Campbell I D , Jones R B , Kiener P A , Waley S G
Published: August 2015

The complex formed between the enzyme triose phosphate isomerase (EC 5.3.1.1.), from rabbit and chicken muscle, and its substrate dihydroxyacetone phosphate was studied by 31P n.m.r. Two other enzyme-ligant complexes examined were those formed by glycerol 3-phosphate (a substrate analogue) and by 2-phosphoglycollate (potential transition-state analogue). Separate resonances were observed in the 31P n.m.r. spectrum for free and bound 2-phosphoglycollate, and this sets an upper limit to the rate constant for dissociation of the enzyme-inhibitor complex; the linewidth of the resonance assigned to the bound inhibitor provided further kinetic information. The position of this resonance did not vary with pH but remained close to that of the fully ionized form of the free 2-phosphoglycollate. It is the fully ionized form of this ligand that binds to the enzyme. The proton uptake that accompanies binding shows protonation of a group on the enzyme. On the basis of chemical and crystallographic information [Hartman (1971) Biochemistry 10, 146–154; Miller & Waley (1971) Biochem. J. 123, 163–170; De la Mare, Coulson, Knowles, Priddle & Offord 1972) Biochem. J. 129, 321–331; Phillips, Rivers, Sternberg, Thornton & Wilson (1977) Biochem. Soc. Trans. 5, 642–647] this group is believed to be glutamate-165. On the other hand, the position of the resonance of D-glycerol 3 phosphate (sn-glycerol 1-phosphate) in the enzyme-ligand complex changes with pH, and both monoanion and dianon of the ligand bind, although dianion binds better. The substrate, dihydroxyacetone phosphate, behaves essentially like glycerol 3-phosphate. The experiments with dihydroxy-acetone phosphate and triose phosphate isomerase have to be carried out at 1 degree C because at 37 degrees C there is conversion into methyl glyoxal and orthophosphate. The mechanismof the enzymic reaction and the reasons for rate-enhancement are considered, and aspects of the pH-dependence are discussed in an Appendix.
Spectrophotometric studies on the interaction between triose phosphate isomerase and inhibitors
Authors: Jones R B , Waley S G
Published: August 2015

The binding of ligands to chicken muscle triose phosphate isomerase was studied. Changes in u.v. absorbance of the enzyme were used to measure binding, and the dissociation constant was determined over a range of pH values. The ligands were 2-phosphoglycollate and rac-glycerol 3-phosphate (only the D-isomer, sn-glycerol 1-phosphate, binds appreciably). Non-linear regression was used to fit calculated curves to the experimental points and hence to compare different models. Both active sites in the dimeric enzyme probably bound 2-phosphoglycollate, without any interaction between the sites. The results of crystallographic analysis [phillips, Rivers, Sternberg, Thornton & Wilson (1977) Biochem. Soc Trans. 5, 642–647], and experiments on the 1H, 13C and 31P n.m.r. of enzyme or 2-phosphoglycollate were combined with the present results to provide the basis for a model in which binding depends on glutamic acid-165 being protonated and on the ligant being fully ionized; additionally, binding affects the ionization of one histidine residue (probably histidine-100). The binding of the glycerol 3-phosphate, on the other hand, was independent of pH over the range pH 6.5–8.5 but decreased at lower pH values. This is explained on a model in which the binding of the monoanion of the ligand is markedly affected by the protonation of a residue in the enzyme, but the binding of the dianion is only slightly affected by this ionization.
Metabolism and insulin-releasing capabilities of glucosamine and N-acetylglucosamine in isolated rat islets
Authors: Zawalich W S , Dye E S , Matschinsky F M
Published: August 2015

The ability of glucosamine and N-acetylglucosamine to stimulate insulin secretion from perifused rat islets and the suitability of these hexoses to be metabolized in a static incubation was studied under various conditions. N-Acetylglucosamine alone stimulated insulin release with a threshold of 10 mM, with half-maximal effect at approx. 16 mM, and maximally at 20 mM. With higher concentrations stimulation was slightly diminished. Release caused by 20 mM-N-acetylglucosamine was unaffected by 30 mM-mannoheptulose, but was blocked by 2-deoxyglucose or iodoacetate (1 mM). At moderate concentrations, (2.75–20 mM), the metabolism of N-acetyl[1-3H]glucosamine was similar to that of [1-3H]glucose and secretion rates paralleled the corresponding rates of metabolism with these hexoses. Glucosamine (27.5 mM) alone weakly stimulated insulin secretion, which was unaltered by 30 mM-mannoheptulose but blocked by 2-deoxyglucose or iodoacetate. A lower rate of [1-3H]glucosamine metabolism appeared to account for its weaker stimulatory efficacy. Insulin release caused by 27.5 mM-glucosamine or 27.5 mM-N-acetylglucosamine in the presence of basal (2.75 mM) glucose was accurately predicted based on the summed metabolic rates of these compounds. The data strengthen the theory proposing that metabolites or cofactors generated during metabolism are essential for triggering insulin secretion.
Hepatocyte differentiation in culture. Appearance of tyrosine aminotransferase
Authors: Yeoh G C , Bennett F A , Oliver I T
Published: August 2015

Liver of rat foetuses from 14 to 19 days of gestation and cultured hepatocytes derived from foetuses of 14 or 15 days gestation show a limited capacity to transaminate tyrosine. This low tyrosine transamination activity can be ascribed to aspartate aminotransferase. Definitive tyrosine aminotransferase can be demonstrated in 1-day-old cultures of hepatocytes taken from 19-day foetuses, but not from 15-day foetuses. However, after 3 days of culture hepatocytes from 15-day foetuses are able to synthesize tyrosine aminotransferase. Induction studies reveal that dexamethasone is capable of increasing tyrosine aminotransferase activity once it is detectable in culture.
Steroidogenesis in isolated adrenocortical cells. Correlation with receptor-bound adenosine 3′:5′-cyclic monophosphate
Authors: Podesta Ernesto J. , Milani Alfred , Steffen Hans , Neher Robert
Published: August 2015

Because several groups have recently questioned a mediating role for cyclic AMP in adrenocortical steroidogenesis, we analysed the problem in more detail by measuring three different cyclic AMP pools in cells isolated from decapsulated rat adrenals. Extra-cellular, total intracellular and bound intracellular cyclic AMP were determined by radioimmunoassay in comparison with corticosterone production induced by low corticotropin concentrations. The increase in extracellular and total intracellular cyclic AMP with low corticotropin concentrations was dependent on the presence of a phosphodiesterase inhibitor and short incubation times. Bound intracellular cyclic AMP was less dependent on these two parameters. In unstimulated cells cyclic AMP bound to its receptor represents only a small fraction of the total intracellular cyclic AMP. After stimulation by a concentration of corticotropin around the threshold for corticosterone production, an increase in bound cyclic AMP was observed which correlated very well with steroidogenesis both temporally and with respect to corticotropin concentration. This finding was complemented by measuring a concomitant decrease in free receptor sites. Full occupancy of the receptors was not necessary for maximal steroidogenesis. Binding kinetics of cyclic [3H]AMP in concentrations equivalent to the intracellular cyclic AMP concentration suggest the presence of at least three different intracellular cyclic AMP pools. These observations are in agreement with a possible role for cyclic AMP as a mediator of acute steroidogenesis induced by low corticotropin concentrations.
The effects of calcium ions and adenine nucleotides on the activity of pig heart 2-oxoglutarate dehydrogenase complex
Authors: McCormack J G , Denton R M
Published: August 2015

1. The effects of Ca2+ (mainly by using EGTA buffers), pH, ATP and ADP on the activity of the 2-oxoglutarate dehydrogenase complex from pig heart were explored. 2. Ca2+ (about 30 micrometer) resulted in a decrease in the apparent Km for 2-oxoglutarate from 2.1 to 0.16 mM (at pH 7) without altering the maximal velocity. At 0.1 mM-oxoglutarate there was a 4–5-fold activation by Ca2+, with an apparent Km for Ca2+ of 1.2 micrometer. A similar activation was also observed with Sr2+ (Km 15.1 micrometer), but not wised markedly from pH 7.4 TO 6.6. The effects of Ca2+ remained evident over this pH range. 4. In the presence of Mg2+, ATP resulted in a marked increase in the apparent Km for oxoglutarate, whereas ADP greatly decreased thisp arameter. The concentrations of adenine nucleotide required for half-maximal effects were about 10 micrometer in each case. 5. The effects of the adenine nucleotides and Ca2+ on the apparent Km for oxoglutarate appeared to be essentially independent of each other, reversible, and demonstrable in the presence of end product inhibition by NADH and obtained. 6. Effects similar to those described above were also observed on the activity of 2-oxoglutarate dehydrogenase from rat heart and brown adipose tissue. 7. We discuss the mechanisms controlling this enzyme's activity and compare these regulatory features with those of NAD-isocitrate dehydrogenase and the pyruvate dehydrogenase system, which are also sensitive to Ca2+ and adenine nucleotides.
Tyrosine aminotransferase induction in hepatocytes cultured from rat foetuses treated with dexamethasone in utero
Authors: Yeoh G C T , Arbuckle T , Oliver I T
Published: August 2015

1. The administration of dexamethasone to foetal rats in utero does not result in the appearance of specific tyrosine aminotransferase activity even after 24 h. 2. When foetal hepatocytes are cultured in vitro from animals treated in utero with dexamethasone, significantly higher activities of specific tyrosine aminotransferase are found than in untreated controls. 3. Dexamethasone in vitro induces specific tyrosine aminotransferase in cells cultured from control animals and the effect is maximal at 10 nM in the culture medium. 4. Actinomycin D at 0.2 microgram/ml in the culture medium completely prevents the induction of activity in vitro. 5. In cultures established from animals treated with dexamethasone in utero, the increase in specific tyrosine aminotransferase activity over the control cultures is only marginally decreased in the presence of actinomycin D. 6. The results can be interpreted to mean that dexamethasone in utero stimulates the transcription of enzyme-specific mRNA, which is not rranslated until a translational block in the foetal liver is removed by the conditions of culture in vitro.
Cytosolic location of an endo-N-acetyl-β-D-glucosaminidase activity in rat liver and kidney
Authors: Pierce R J , Spik G , Montreuil J
Published: August 2015

Endo-N-acetyl-beta-D-glucosaminidase activity towards an oligomannosidic type glycoamino acid substrate was found in the soluble fraction of rat liver and kidney. No evidence for a lysosomal form of the activity was found.
Immunochemical characterization of monoamine oxidase from human liver, placenta, platelets and brain cortex
Authors: Russell S M , Davey J , Mayer R J
Published: August 2015

1. Antiserum raised to purified human liver monoamine oxidase was used to characterize the monoamine oxidase from human liver, brain cortex, placenta and platelets. 2. Antibodies to monoamine oxidase were purified by adsorption with a mitochondrial preparation. 3. Monoamine oxidase was present in liver particle-free supernatant as measured by enzyme activity and immunodiffusion. 4. Multiple precipitin lines were obtained on immunodiffusion analysis against the purified liver enzyme. It is proposed that this is due to either aggregation or to differential lipid binding. 5. The results suggest that the functionally different enzymes found in liver, brain cortex, platelets and placenta are immunochemically related and may be identical.
Flavin-dependent substrate photo-oxidation as a chemical model of dehydrogenase action
Authors: Haas W , Hemmerich P
Published: August 2015

As a model of flavin-dependent biological dehydrogenation, flavin-sensitized photodehydrogenation and photodecarboxylation were studied by variation of substrate, flavin, pH and solvent. Evidence for the following rules is given. (1) When the reactive site of a photosubstrate is an alpha-carbon atom of the type CH-CO2-, decarboxylation is preferred over dehydrogenation, whereas the reverse is true for the neutral CH-CO2H. (2) Consequently these reactions do not exhibit a measurable isotope effect with C2H-CO2-, in contrast with the findings by Penzer, Radda, Taylor & Taylor [(1970) Vitam. Horm. (N.Y.) 28, 441–466], which could not be reproduced. When the substate does not contain a carboxylate group, isotope effects occur, in verification of previous reports, e.g. for benzyl alcohol C6H5-C2H20H. (3) The mechanism of flavin-sensitized substrate photodecarboxylation is assumed to consist in a primary carbanion fixation at the flavin nucleus (position 4a, 5 or 8) with concomitant liberation of CO2. This step is followed by rapid fragmentation of the adduct CH-Fl-red., provided that the substrate contains a functional and electron-donating group X, e.g. X = OH, OCH3 or NH2 (but not NH3+ !) in X CH-CO2-. (4) The minimal requirement for flavin-sensitized C-H dehydrogenation is the presence of a hydroxyl group. For example, methanol as substrate and solvent is dehydrogenated at pH sufficiently alkaline for detection of the presence of the active species CH3O-, whereas at more acidic pH substrate dehydrogenation is competing with flavin autophotolysis, which depends on the substituents in the flavin nucleus.
High-molecular-weight nuclear polyadenylate-containing ribonucleic acid isolated from the lactating guniea-pig mammary gland contains milk-protein messenger ribonucleic acid sequences
Authors: Bathurst I C , Craig R K , Campbell P N
Published: August 2015

1. Steady-state poly(A)-containing nuclear RNA was isolated from the lactating guinea-pig mammary gland and analysed by sucrose-gradient centrifugation under denaturing conditions. 2. Nucleic acid-hybridization studies demonstrated the presence of small amounts of high-molecular-weight RNA species containing milk-protein mRNA sequences sedimenting at 25S. These were not found in the post-nuclear supernatant, where milk-protein sequences sedimented only between 12S and 15S; these were also the predominant species in the nuclear fraction. 3. The results are consistent with the transcription of milk-protein genes as high-molecular-weight precursor RNA species, 3-4 times as large as the active mRNA species isolated from the post-nuclear fraction.
Structural aspects of the dye-linked alcohol dehydrogenase of Rhodopseudomonas acidophila
Authors: Bamforth C W , Quayle J R
Published: August 2015

1. A dye-linked alcohol dehydrogenase was purified 60-fold from extracts of Rhodopseudomonas acidophila 10050 grown aerobically on ethanol. 2. The properties of this enzyme were identical with those of the alcohol dehydrogenase synthesized by this organism during growth on methanol anaerobically in the light, and they are judged to be the same enzyme. 3. The enzyme gave a single protein band, coincident with alcohol dehydrogenase activity, during electrophoresis on polyacrylamide gel. 4. The amino acid composition, ioselectric point, u.v. and visible absorption spectra of the enzyme were determined and compared with those of other similar enzymes. 5. The presence of 0.7–1.0 g-atom of non-haem, acidlabile iron/mol of enzyme was shown by atomic absorption spectrophotometry and colorimetric assay. The iron could not be dissociated from the enzyme by dialysis against chelating agents. 6. E.p.r. spectroscopy of the enzyme did not indicate any redox function for the iron during alcohol dehydrogenation, but showed a signal at g = 2.00 consistent with the presence of a protein-bound organic free radical. 8. Antisera were raised against alcohol (methanol) dehydrogenases purified from Rhodopseudomonas acidophila, Paracoccus denitrificans and Methylophilus methylotrophus. 9. The antiserum to the Rhodopseudomonas acidophila enzyme cross-reacted with neither of the two other antisera, nor with crude extracts of methanol-grown Hyphomicrobium X and Pseudomonas AM1, thus emphasizing its singular biochemical properties.
The temperature-dependence of adenylate cyclase from baker's yeast
Authors: Londesborough J , Varimo K
Published: August 2015

The Michaelis constant of membrane-bound adenylate cyclase increased from 1.1 to 1.8 mM between 7 and 38 degrees C (delta H = 13 kJ/mol). Over this temperature range, the maximum velocity increased 10-fold, and the Arrhenius plot was nearly linear, with an average delta H* of 51 kJ/mol. The temperature-dependence of the reaction rate at 2 mM-ATP was examined in more detail: for Lubrol-dispersed enzyme, Arrhenius plots were nearly linear with average delta H* values of 45 and 68 kJ/mol, respectively, for untreated and gel-filtered enzymes; for membrane-bound enzyme, delta H changed from 40 kJ/mol above about 21 degrees C to 62 kJ/mol below 21 degrees C, but this behaviour does not necessarily indicate an abrupt, lipid-induced, transition in the reaction mechanism.
Biochemical characterization of guanidinium chloride-soluble dentine collagen from lathyritic-rat incisors
Authors: Wohllebe M , Carmichael D J
Published: August 2015

alpha- and beta-Chains were isolated by sequential ion-exchange and gel-filtration chromatography of guanidinium chloride-soluble dentine collagen obtained from Tris/NaCl-extracted EDTA-demineralized lathyritic-rat incisors. The alpha-chains were identified as alpha 1 I and alpha 2 by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and amino acid analysis of the intact chains and their CNBr peptides. The dentine alpha-chains exhibited higher lysine hydroxylation and phosphate content, but lower hydroxylysine glycosylation, than alpha-chains from skin. Increased lysine hydroxylation was observed in the helical sequences. The alpha 1 I/alpha 2 ratio was approx. 3:1, and was presumably due to the presence of (alpha 1 I)3 molecules along with (alpha 1 I)2 alpha 2 molecules as shown recently for neutral-salt-soluble dentine collagen [Wohllebe & Carmichael (1978) Eur. J. Biochem. 92, 183–188]. In the borohydride-reduced beta 11- and beta 12-chains from guanidinium chloride-soluble dentine collagen, the reduced cross-links hydroxylysinohydroxynorleucine and hydroxylysinonorleucine were present. A higher proportion of hydroxylysinonorleucine in the reduced beta 12-chain probably reflects differences in extent of hydroxylation of specific lysine residues of the alpha 1 I- and alpha 2-chains.
Identification of two lithocholic acid-binding proteins. Separation of ligandin from glutathione S-transferase B
Authors: Hayes J D , Strange R C , Percy-Robb I W
Published: August 2015

1. Two lithocholic acid-binding proteins in rat liver cytosol, previously shown to have glutathione S-transferase activity, were resolved by CM-Sephadex chromatography. 2. Phenobarbitone administration resulted in induction of both binding proteins. 3. The two proteins had distinct subunit compositions indicating that they are dimers with mol.wts. 44 000 and 47 000. 4. The two lithocholic acid-binding proteins were identified by comparing their elution volumes from CM-Sephadex with those of purified ligandin and glutathione S-transferase B prepared by published procedures. Ligandin and glutathione S-transferase B were eluted separately, as single peaks of enzyme activity, at volumes equivalent to the two lithocholic acid-binding proteins. 5. Peptide ‘mapping’ revealed structural differences between the two proteins.
Reinvestigation of the reaction of chymotrypsin with N-furylacryloyltryptophan derivatives at acidic pH
Authors: Fink A L , Feldman R , Zehnder J
Published: August 2015

The reaction of alpha-chymotrypsin with N alpha-3-(2-furyl)acryloyl-L-tryptophan methyl ester (FA-Trp-OMe) and amide has been investigated in aqueous and dimethylsulphoxide cryosolvent solutions from pH2 to 7 and over a wide temperature range. Previous reports have suggested that an intermediate preceding the acyl-enzyme can be detected spectrophotometrically in the reaction with methyl esters of FA-Trp and FA-Tyr at low pH [Yu & Viswanatha (1969) Eur. J. Biochem. 11, 347–352), and that this intermediate is an oxazolinone [Coletti-Previero et al. (1970) FEBS Lett. 11, 213–217]. We show that the previous interpretations of the time-dependent spectral changes were incorrect, and that the only detected intermediate is the acyl-enzyme. This may be isolated by gel filtration at pH less than 2.5, 1 degree C, owing to its relative stability. The pH-dependence of the rates of acylation and deacylation from pH 8.5 to 2.0 are consistent with a single ionization of pK congruent to 7.0 in both aqueous and cryosolvent solutions.
The oxidation-reduction potentials of cytochrome o + c4 and cytochrome o purified from Azotobacter vinelandii
Authors: Yang T , O'Keefe D , Chance B
Published: August 2015

Oxidation-reduction titrations of Azotobacter vinelandii cytochrome o + c4 and cytochrome o were performed with simultaneous potential and absorbance measurements under anaerobic conditions. Cytochrome c4 has a midpoint potential (Em, 7.4) of 260mV and purified cytochrome o has an Em, 7.4 of −18mV. Little change in the midpoint potential of cytochrome o was observed when titrated in the pH range 6.2–9.8.
The catalytically active form of histidinol dehydrogenase from Salmonella typhimurium
Authors: Bürger E , Görisch H , Lingens F
Published: August 2015

The active-enzyme-sedimentation procedure was used to identify the catalytically competent form of histidinol dehydrogenase (EC 1.1.1.23) isolated from Salmonella typhimurium. At pH 9.4 the active species has a sedimentation coefficient S20,W of 5.4S, indicating that the dimer with a mol.wt. of approx. 83 000 is the enzymically active form.
Changes in the sensitivity of chick fibroblasts to Ricinus lectin (RCA I) toxicity in relation to the stage of embryo development
Authors: Bernard B , Aubery M , Bourrillon R
Published: August 2015

The toxic effect of Ricinus lectin RCA I, as estimated by the inhibition of [3H]leucine incorporation, was investigated on chick-embryo fibroblasts at different stages of development. There appeared to be a differential susceptibility of chick-embryo fibroblasts to lectin RCA I. Fibroblasts from 16-day embryos were the most sensitive to its toxic effect in terms of both concentration and time, and cells from 8-day embryos were the least sensitive. This differential sensitivity to the toxic effect of lectin RCA I was closely related to the binding of the lectin: fibroblasts from 16-day embryos had more binding sites (1.5 × 10(7)/cell) with a high affinity than did 12-day (0.45 × 10(7)/cell) or 8-day embryos (0.2 × 10(7)/cell). Studies on the specificity and the removal of bound lectin RCA I by D-galactose indicated that the lectin binding was necessary but not sufficient in itself to cause the toxic effect and that the lectin needed to enter the cells in order to be toxic. The amount of lectin RCA I needed to induce a 50-60% toxicity enters fibroblasts of 16-day embryos more rapidly than those of 12- and 8-day embryos.
Effect of growth conditions on the involvement of cytochrome c in electron transport, proton translocation and ATP synthesis in the facultative methylotroph Pseudomonas AM1
Authors: Keevil C. William , Anthony Christopher
Published: August 2015

The stoicheiometry of proton translocation, the amounts of cytochromes firmly bound to membranes, and cell yields with respect to succinate and O2 have been measured in the facultative methylotroph Pseudomonas AM1 and in the mutant lacking cytochrome c (mutant PCT76) during carbon-limited growth and carbon-excess growth. →H+/O ratios during endogenous respiration of about 4 were measured in wild-type bacteria grown in carbon-excess conditions, and in the mutant in all growth conditions. During methanol- or succinate-limited growth of wild-type bacteria the →H+/O ratio increased to about 6. Cell yields with respect to succinate and O2 were higher in wild-type than in the mutant lacking cytochrome c by an amount suggesting loss in the mutant of 30% of the ATP-generating capacity of wild-type bacteria. During carbon-limited growth on methanol or succinate some cytochrome c was tightly bound to bacterial membranes, whereas none was tightly bound in bacteria grown in batch-culture or in NH4+-limited conditions. It is proposed that the role of cytochrome c in Pseudomonas AM1 depends on growth conditions and hence on the ‘needs’ of the bacteria. During growth in carbon-excess conditions it is only required for methanol oxidation, mediating between methanol dehydrogenase and cytochrome a/a3. In these conditions oxidation of NADH and succinate by way of cytochrome b and cytochrome a/a3 occurs without the mediation of cytochrome c. This is the only route for oxidation of NADH and succinate in the cytochrome c-deficient mutant in all growth conditions. During carbon-limited growth the cytochrome c becomes bound to the membrane in such a way that it can mediate between cytochromes b and a/a3, hence becoming involved in proton translocation and ATP synthesis during NADH and succinate oxidation. An alternative possibility is that in wild-type bacteria the cytochrome c is always involved in electron transport, but that its involvement in measurable proton translocation only occurs in carbon-limited conditions.
The mechanism of transmembrane ΔμH+ generation in mitochondria by cytochrome c oxidase
Authors: Lorusso M , Capuano F , Boffoli D , Stefanelli R , Papa S
Published: August 2015

In rat liver mitochondria treated with rotenone, N-ethylmaleimide or oligomycin the expected alkalinization caused by proton consumption for aerobic oxidation of ferrocyanide was delayed with respect to ferrocyanide oxidation, unless carbonyl cyanide p-trifluoromethoxyphenylhydrazone was present. 2. When valinomycin or valinomycin plus antimycin were also present, ferricyanide, produced by oxidation of ferrocyanide, was re-reduced by hydrogenated endogenous reductants. Under these circumstances the expected net proton consumption caused by ferrocyanide oxidation was preceded by transient acidification. It is shown that re-reduction of formed ferricyanide and proton release derive from rotenone- and antimycin-resistant oxidation of endogenous reductants through the proton-translocating segments of the respiratory chain on the substrate side of cytochrome c. The number of protons released per electron flowing to ferricyanide varied, depending on the experimental conditions, from 3.6 to 1.5. 3. The antimycin-insensitive re-reduction of ferricyanide and proton release from mitochondria were strongly depressed by 2-n-heptyl-4-hydroxyquinoline N-oxide. This shows that the ferricyanide formed accepts electrons passing through the protonmotive segments of the respiratory chain at the level of cytochrome c and/or redox components of the cytochrome b-c1 complex situated on the oxygen side of the antimycin-inhibition site. Dibromothymoquinone depressed and duroquinol enhanced, in the presence of antimycin, the proton-release process induced by ferrocyanide respiration. Both quinones enhanced the rate of scalar proton production associated with ferrocyanide respiration, but lowered the number of protons released per electron flowing to the ferricyanide formed. 4. Net proton consumption caused by aerobic oxidation of exogenous ferrocytochrome c by antimycin-supplemented bovine heart mitochondria was preceded by scalar proton release, which was included in the stoicheiometry of 1 proton consumed per mol of ferrocytochrome c oxidized. This scalar proton production was associated with transition of cytochrome c from the reduced to the oxidized form and not to electron flow along cytochrome c oxidase. 5. It is concluded that cytochrome c oxidase only mediates vectorial electron flow from cytochrome c at the outer side to protons that enter the oxidase from the matrix side of the membrane. In addition to this consumption of protons the oxidase does not mediate vectorial proton translocation.
Precocious development of cytochrome P-450 in neonatal rat liver after glucocorticoid treatment
Authors: Leakey J E A , Fouts J R
Published: August 2015

Intraperitoneal injection of neonatal rats with glucocorticoid hormones causes precocious development of hepatic cytochrome P-450. Glucagon injection fails to stimulate this cytochrome P-450 development. Adult liver cytochrome P-450 is less responsive to glucocorticoid stimulation than is that of neonatal rat liver. Adrenalectomy of prematurely delivered neonatal animals prevents the early postnatal development of cytochrome P-450. Glucocorticoids failed to increase cytochrome P-450 concentrations in foetal rat liver. These findings imply that, although glucocorticoids are mandatory regulatory factors controlling cytochrome P-450 development, they are not themselves the ‘trigger’ initiating onset of that development.
Biosynthesis and turnover of outer-membrane proteins in Escherichia coli ML308-225
Authors: Allen R J , Scott G K
Published: August 2015

Isolated outer membranes and outer-membrane extracts from Escherichia coli ML308-225 in the early-exponential growth phase contain more protein than do corresponding preparations from late-exponential- or stationary-phase bacteria. Isotope-dilution experiments show that this is due to a loss of protein from the membrane during the exponential growth phase. Inhibition of bacterial growth and protein synthesis stabilizes the outer-membrane-protein concentration. Protein synthesis in the absence of bacterial growth results in higher concentrations of protein in the outer membrane.
Mechanism of the stimulation of serine and alanine transport into isolated rat liver cells by bicarbonate ions
Authors: McGivan J D
Published: August 2015

1. Bicarbonate ions stimulate the transport of serine and alanine into isolated hepatocytes. 2. The effect of bicarbonate is to increase the Vmax. of the transport process without changing the apparent Km. 3. The intracellular pH was estimated from the distribution of the weak base methylamine and the weak acid 5,5′-dimethyloxazolidine-2,4-dione (DMO) across the plasma membrane. 4. The addition of bicarbonate to a cell suspension caused the internal pH to become more acid. 5. The initial rate of serine, alanine and glycine transport was a linear function of the initial difference in pH across the membrane. 6. It is concluded that bicarbonate activates the transport of these amino acids primarily by increasing the pH difference across the plasma membrane. 7. It is suggested that the uptake of serine together with Na+ ions occurs in exchange for H+ ions, which are translocated outwards on the same carrier system. Some preliminary evidence consistent with this model is presented.