bluuebunny/crossref_metadata_2025 huggingface dataset:
Affinity chromatography of human plasma low- and high-density lipoproteins. Elution by selective cleavage of a bond in the spacer
Authors: Wichman A
Published: August 2015

Human plasma low- and high-density lipoproteins were found to bind to Sepharose gels containing coupled cholesterol or cholic acid. The lipoproteins were bound very strongly, and it was not possible to elute them under non-denaturing conditions. The detergents Triton X-100 and sodium dodecyl sulphate eluted the lipoproteins in partly denatured form. Adsorbents were used where the steroid was coupled through a spacer containing a thiol ester bond. It was thus possible to elute bound lipoproteins by selective cleavage of the bond with hydroxylamine. A small proportion of albumin was the only contaminant detected, the amounts depending on which ligand was used. Low- and high-density lipoproteins were separated by gel filtration. They behaved as did the native molecules when analysed by gel filtration, immunodiffusion, immunoelectrophoresis and electrophoresis in polyacrylamide gradient gels. The high capacity and the selectivity of the adsorbents make them suitable for the removal of lipoproteins from protein solutions.
Synthesis of membrane glycoproteins in rat small-intestinal villus cells. Effect of colchicine on the redistribution of l-[1,5,6-3H]fucose-labelled membrane glycoproteins among Golgi, lateral basal and microvillus membranes
Authors: Quaroni A , Kirsch K , Weiser M M
Published: August 2015

To define the role of cytoplasmic microtubules in the biogenesis of plasmalemma glycoproteins of rat small-intestinal villus cells, we studied the effect of colchicine on the incorporation of L-[1,5,6-3H]fucose into Golgi, lateral basal and microvillus membranes. Colchicine was administered intraperitoneally before or after injection of radioactive fucose. The incorporation of radioactivity into Golgi membranes was little affected by colchicine, which did not prevent the redistribution of most of the labelled glycoproteins from the Golgi complex into other parts of the villus cell. The incorporation of labelled glycoproteins into the microvillus membrane was greatly inhibited by colchicine given 2 h or 10 min before the radioactive fucose: all labelled glycoproteins present in this membrane were equally affected. In contrast, the administration of colchicine considerably increased the incorporation of radioactivity into the lateral basal part of the plasmalemma, and prevented the disappearance of most of the labelled glycoproteins from this membrane at late times after fucose injection. These results suggest that cytoplasmic microtubular structures are important for the polarization of the intestinal villus cell and the biogenesis of the microvillus membrane, although playing little or no role in the movement of membrane components from the Golgi complex to the lateral basal part of the plasmalemma.
Activation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in homogenates of developing rat brain
Authors: Maltese W A , Volpe J J
Published: August 2015

The specific activity of 3-hydroxy-3-methylglutaryl-CoA reductase increases when homogenates of developing rat brain are incubated at 37 degrees C or kept on ice. This increase is completely blocked by the addition of F- to the homogenization medium and the assay mixture. The capacity for activation of the reductase is greatest during the early postnatal period and declines as brain maturation proceeds. The data suggest that catalytic modification of the reductase may play a role in the regulation of cholesterol synthesis in the developing brain.
Effects of antimycin A and 2-deoxyglucose on secretion in human platelets. Differential inhibition of the secretion of acid hydrolases and adenine nucleotides
Authors: Holmsen Holm , Robkin Linda , Day H. James
Published: August 2015

1. Shape change, aggregation and secretion of dense-granule constituents in platelets differ in their dependence on cellular energy metabolism. The possibility that such a difference also exists between secretion of dense-granule constituents and acid hydrolases was investigated. 2. Human platelets were incubated with [14C]adenine in plasma, and then washed and resuspended in salt solutions. The effects of incubating the cells with antimycin A and 2-deoxyglucose on the concentrations of [14C]ATP, ADP, AMP, IMP and inosine plus hypoxanthine and on thrombin-induced secretion of ATP plus ADP and acid hydrolases were studied. The metabolic inhibitors only affected 14C-labelled nucleotides, whereas thrombin only liberated unlabelled ATP and ADP. 3. The extent of secretion decreased progressively with time during incubation with the metabolic inhibitors. At any time the secretion of acid hydrolases, β-N-acetylglucosaminidase, β-glucuronidase and β-galactosidase was inhibited to a greater extent than secretion of ATP plus ADP (dense-granule secretion). 4. Incubation with the metabolic inhibitors shifted the log (dose)–response relationship to higher thrombin concentrations, and with a greater shift for acid hydrolase secretion than for dense-granule secretion. 5. Antimycin, when present alone, caused a marked decrease in the rate of acid hydrolase secretion, but had no effect on dense-granule secretion. 6. These results further support the view that acid hydrolase secretion and dense-granule secretion are separate processes with different requirements for ATP energy. Acid hydrolase secretion, but not dense-granule secretion, appears to depend on a simultaneous rapid generation of ATP, which can be accomplished by oxidative, but not by glycolytic, ATP production.
Characterization of cyanide-insensitive respiration in mitochondria and submitochondrial particles of Moniliella tomentosa
Authors: Vanderleyden Jos , Kurth Jochen , Verachtert Hubert
Published: August 2015

Mitochondria and submitochondrial particles of the osmophilic yeast-like fungus Moniliella tomentosa may respire by means of two pathways: a normal cytochrome pathway, sensitive to cyanide and antimycin A, and an alternative pathway, which is insensitive to these inhibitors but is specifically inhibited by salicylhydroxamic acid. The affinities of both oxidases for succinate and NADH as substrates, for O2 as terminal electron acceptor, and for AMP as stimulator of the alternative oxidase were determined. 1. Submitochondrial particles of M. tomentosa may also respire by means of a cyanide-sensitive and/or cyanide-insensitive system. 2. The activities of both oxidases as compared with the total activity are roughly the same in submitochondrial particles as in the original mitochondria. 3. The terminal oxidase of the cyanide-insensitive pathway requires a 10-fold higher O2 concentration for saturation than does cytochrome c oxidase. 4. The apparent Km for succinate is about 3 times higher for the alternative than for the normal oxidase when measured in mitochondria, and 4–10 times higher when measured in submitochondrial particles. The apparent Km for NADH is roughly the same for both oxidases. 5. The apparent Km values of both oxidases for succinate are always lower in submitochondrial particles than in mitochondria. 6. The apparent Km for AMP, acting as a stimulator of the alternative oxidase, is the same (25μm) in mitochondria as in sub-mitochondrial particles. These results are discussed in the light of the structure and localization of the components of the alternative oxidase.
Uptake of L-tri-iodothyronine by isolated rat liver cells. A process partially inhibited by metabolic inhibitors; attempts to distinguish between uptake and binding to intracellular proteins
Authors: Eckel J , Rao G S , Rao M L , Breuer H
Published: August 2015

1. Rat liver cells obtained by dispersion with collagenase were used to investigate the mode of entry of L-tri-iodothyronine into the cell. 2. The hormone was taken up very rapidly at 23 degrees C; the linear phase of uptake lasted for up to approx. 20 s. 3. A plot of the initial rates of uptake against different concentrations of L-tri-iodothyronine yielded a sigmoidal curve. The Eadie–Hofstee plot (v/[S]2 versus v) yielded two straight lines. The uptake component with an apparent Kt value of 86 +/- 15 pM was designated as system I, and the second uptake component with an apparent Kt of 726 +/- 11 pM as system II. The Hill plot for system I was not linear; the apparent Hill coefficient for system II was calculated to be 2.1.4. Uptake of L-tri-iodothyronine by system I was higher at pH 6.4 than at pH 7.4; system II was relatively insensitive to changes in the pH of the external medium. 5. Both systems exhibited a transition temperature at about 16 degrees C in the Arrhenius plot. The activation energies of the two systems below and above 16 degrees C were 72.8 and 47.7 and 54.4 and 33.1 J/mol respectively. 6. Inhibitors of cellular energy reduced the uptake by system I to a larger extent than that by system II. 7. Replacement of Na+ in the external medium by either K+ or choline led to uptake that followed normal Michaelis–Menten kinetics. 8. Thiol-group-blocking agents reduced the uptake of the hormone by both systems. 9. Treatment of liver cells with beta-glucosidase, Pronase and neuraminidase led to a decrease in the uptake of L-tri-iodothyronine by system I, whereas uptake by system II was decreased after treatment with phospholipase A2, beta-galactosidase. Pronase and neuraminidase. 10. The stereoisomer D-tri-iodothyronine (100–3000 pM) did not affect system I, but uptake by system II decreased with increasing concentration of D-tri-iodothyronine. Reverse L-tri-iodothyronine (2–100 pM) and L-thyroxine (100–3000 pM) did not influence uptake by either system. 11. Under identical conditions of incubation, the uptake of L-tri-iodothyronine was 3.7 times higher than binding to cytosol proteins. The binding was insensitive to metabolic inhibitors. The results suggest that cytosol proteins are not directly involved in the uptake of L-tri-iodothyronine. 12. Plasma-membrane vesicles also take up the hormone rapidly at 23 degrees C. Increasing the osmolarity of the external medium led to a decrease in the uptake of L-tri-iodothyronine by vesicles. 13. Uptake as a function of L-tri-iodothyronine concentration exhibited a sigmoidal curve. The Eadie–Hofstee plot showed two uptake components with apparent Kt values of 96.8 and 1581 pM. 14. The results of our study are consistent with a carrier-mediated translocation of the hormone into the cell.
Protein phosphorylation in human peripheral blood lymphocytes. Subcellular distribution and partial characterization of adenosine 3′:5′-cyclic monophosphate-dependent protein kinase
Authors: Chaplin D D , Wedner H J , Parker C W
Published: August 2015

Cytoplasmic and membrane fractions prepared from human peripheral-blood lymphocytes both contained cyclic AMP-dependent protein kinase activity and endogenous protein kinase substrates. Protein kinase activity in the particulate fractions was not eluted with 0.25 M-NaCl, suggesting that it was not derived from non-specifically absorbed soluble cytoplasmic protein kinase. Nor was the particulate protein kinase activity eluted by treatment with cyclic AMP, suggesting that the catalytic subunit is membrane-bound and arguing against cyclic AMP-induced translocation of particulate activity. Cyclic AMP-dependent protein-phosphorylating activity in the cytoplasmic fraction was highly sensitive to inhibition by Mn2+, and was co-eluted from DEAE-cellulose primarily with type-I rabbit skeletal-muscle kinase. Cyclic AMP-dependent phosphorylating activity in the plasma-membrane fractions was stimulated at low [Mn2+] and inhibited only at high [Mn2+]. When solubilized with Nonidet P-40, plasma-membrane protein kinase was co-eluted from DEAE-cellulose with type-II rabbit muscle kinase. These differences, together with the strong association of the particulate kinases with the particulate fraction, suggest the possibility of compartmentalized protein phosphorylation in intact lymphocytes.
Isolation and partial characterization of high-density lipoprotein HDL1 from rat plasma by gradient centrifugation
Authors: Lusk L T , Walker L F , DuBien L H , Getz G S
Published: August 2015

The lipoproteins isolated from rat plasma by flotation in the density range 1.019-1.063 g/ml were further characterized. Using rate zonal ultracentrifugation, we isolated two lipoproteins in almost equal proportions from this density range. Similar isolations may be accomplished with density gradients in a swinging-bucket rotor. On isopycnic-density-gradient ultracentrifugation one component banded at rho = 1.031 g/ml and the other at rho = 1.054 g/ml. More that 98% of the apoprotein of the lighter component was B protein, and hence this particle is LD (low-density) lipoprotein. Of the apoproteins of the rho = 1.054 g/ml particles, designated lipoprotein HDL1, over 60% was arginine-rich peptide, and the remainder was A-I, A-IV and C peptides. The molecular weight of these lipoproteins determined by agarose column chromatography was 2.36 × 10(6) for LD lipoprotein and 1.30 × 10(6) for lipoprotein HDL1. On electron microscopy the radius of LD lipoprotein was 14.0 nm and that of lipoprotein HDL1 was 10.0 nm, in contrast with molecular radii of 10.4 nm and 8.4 nm respectively determined from the gel-permeation-chromatography data. The lipid and phospholipid composition of both particles was determined. Lipoprotein HDL1 was notable for both the concentration of its esterified cholesterol, which was similar to that of LD lipoprotein, and the low triacylglycerol content, resembling that of HD lipoprotein. The possible origin of lipoprotein HDL1 is discussed.
Studies on the mechanism of lanosterol 14 α-demethylation. A requirement for two distinct types of mixed-function-oxidase systems
Authors: Gibbons F G , Pullinger C R , Mitropoulos K A
Published: August 2015

Carbon monoxide inhibited the removal of C-32 of dihydrolanosterol (I), but not of its metabolites 5 alpha-lanost-8-ene-3 beta,32-diol (II) and 3 beta-hydroxy-5 alpha-lanost-8-en-32-al (III). It appears therefore that cytochrome P-450 is a component of the enzyme system required to initiate oxidation of the 14 alpha-methyl group, but not of that responsible for the subsequent oxidation steps required for elimination of C-32 as formic acid. Non-radioactive compounds (II) and (III), when added to cell-free systems actively converting dihydrolanosterol into cholesterol, inhibited 14 alpha-demethylation measured by the rate of formation of labelled cholesterol from dihydro[1,7,15,22,26,30-14C]lanosterol or of labelled formic acid from dihydro[32-14C]lanosterol. However, neither compound (II) nor compound (III) accumulated radioactive label under these conditions. These observations could be attributed partly to inhibition of the initial oxidation of the 14 alpha-methyl group by compounds (II) and (III).
Spectroscopic forms of carbonmonoxi-cytochrome oxidase
Authors: de Fonseka K , Chance B
Published: August 2015

A systematic study of the errors of low-temperature recording of kinetics of the cytochrome oxidase-CO reaction had identified the classic devitrification process of Keilin & Hartree [(1950) Nature (London)165, 504-505]. The methodology described here minimizes this effect, and the computation methods afford appropriate ways of detecting a residual effect. Thus it has been possible to identify that absorption difference spectra and kinetics of the reaction of fully reduced or half-reduced cytochrome oxidase with CO indicate only one spectroscopic form of the respective carbonmonoxi-cytochrome oxidase.
The anion-transport protein of the human erythrocyte membrane. Studies on fragments produced by pepsin digestion
Authors: Tanner M J A , Williams D G , Kyle D
Published: August 2015

We have studied the fragmentation by pepsin in 1 M-acetic acid of the erythrocyte anion-transport protein in erythrocyte membranes. The location of the fragments obtained was determined by radioiodinating the protein with the use of lactoperoxidase, and identifying the labelled peptides obtained in peptide “maps” of thermolysin digests of the fragments. Three of the fragments were found to be related overlapping products, and shared a common C-terminus. The major site of pepsin cleavage leading to the C-termini of these fragments was shown to be close to the major site of extracellular cleavage of the protein by proteinases active at a neutral pH. Another two fragments were isolated and shown to be derived from the C-terminal portion of the protein. No well-defined large radioactive fragments of the protein were solubilized from the membrane by pepsin in 1 M-acetic acid, the bulk of the radioactivity attributable to the anion transport protein being recovered in very small fragments that could not be resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Our results suggest that the polypeptide chain of the anion-transport protein emerges at the extracellular face of the membrane 8000-13000 daltons on the N-terminal side of the major site of extracellular cleavage of the protein by proteinases that are active at a neutral pH.
Reversal of part of the aldehyde dehydrogenase reaction pathway during the hydrolysis of an ester
Authors: Duncan R J S
Published: August 2015

An aldehyde dehydrogenase from rabbit liver, a homogeneous protein on three distinct polyacrylamide-gel systems, has an associated 4-nitrophenyl esterase activity. At pH 7.0 in the presence of 80 micrometer-NADH and 800 micrometer-4-nitrophenyl acetate the enzyme produces NAD+ and a stoicheiometric amount of an aldehyde, as well as hydrolysing the ester. On this and other evidence it is proposed that ester hydrolysis occurs at the usual active site of the enzyme.
Proteoglycans from adult human gingival epithelium
Authors: Wiebkin O W , Bartold P M , Thonard J C
Published: August 2015

Proteoglycans extracted from human gingival epithelium appear to contain a proportion of molecules that will interact with hyaluronic acid to form macromolecular aggregates. In contrast, proteoglycans from underlying connective tissue behaved differently. The interactions of hyaluronic acid with proteoglycans from either epithelium or cartilage may be similar, but not necessarily identical.
Quantitative N-terminal analysis of fibrinogen-fibrin-related antigen [FR antigen] from human plasma
Authors: Harris J U , Johnson A J , Merskey C , Wang M T , Robinson D
Published: August 2015

Fibrinogen-fibrin-related antigen (FR antigen) was isolated from as little as 1 ml of human plasma by immuno-affinity chromatography with agarose-bound antibody to human fibrinogen. N-terminal analysis was performed to determine the nature and extent of proteolytic degradation of the FR antigen in patients with disseminated intravascular coagulation and in normal subjects. Thrombin cleavage of the A- and B-peptides from fibrinogen in vitro was monitored by the appearance of N-terminal glycine, and an increase in glycine was shown in the FR antigen of patients with disseminated intravascular coagulation. As plasmin progressively degraded fibrinogen, increases in N-terminal alanine, aspartic acid and lysine were observed, corresponding to the known plasmin-cleavage points of fibrinogen; increases in these N-terminal amino acids were also found in the patients' FR antigen. Thrombin treatment in vitro was used to remove fibrinopeptide A (N-terminal alanine) from the samples and to reflect specifically the N-terminal alanine at the plasmin-cleavage point (Arg-42-Ala-43) of the B beta-chain on assay; this alanine was increased progressively in the FR antigen of a patient during urokinase therapy, and was high in other patients when the FR antigen was examined by this procedure.
A study of the copper-binding proteins in liver and kidney tissue of neonatal normal and mottled mutant mice
Authors: Port A E , Hunt D M
Published: August 2015

The Cu2+-binding proteins from liver and kidney tissue of 7–8-day-old brindled (Mobr) mice and their normal littermates were compared. (1) Separation over Bio-Gel P-10 showed that the differences in the Cu2+ content of mutant tissues were largely associated with a low-molecular-weight protein fraction (mol.wt. 14 500). (2) Further purification of this low-molecular-weight fraction by anion-exchange chromatography revealed four subfractions. The Cu2+ content of each subfraction reflected the Cu2+ status of the tissue of origin; the Cu2+ contents of the mutant kidney subfractions were elevated and those of the mutant liver were depressed compared with normal. In contrast, the protein contents of the subfractions were less variable and did not reflect the differing Cu2+ contents. (3) Amino acid analysis of the four subfractions from CuCl2-treated mutant and normal animals revealed clos similarities. The proteins showed high glycine, glutamic acid, serine, alanine and lysine contents and a rather variable cysteine content. Differences were apparent in the normal liver subfractions, which showed a higher cysteine content and lower glutamic acid content than did either the mutant liver or normal and mutant kidney subfractions. These observations, together with the recorded presence of aromatic amino acids, indicated that these proteins are not thioneins.
Hydrophobic proteins of lamellated osmiophilic bodies isolated from pig lung
Authors: Phizackerley P J R , Town M H , Newman G E
Published: August 2015

1. In addition to proteins that are insoluble in organic solvents, lamellated bodies isolated from pig lung and surfactant prepared from bronchopulmonary lavage fluid contain another group of proteins that are extracted together with lipid into the organic phase. 2. These hydrophobic proteins constitute about 40% of the total protein of lamellated bodies and about 13% of the total protein of surfactant isolated from lavage fluid, whereas less than 1% of the total protein of pig lung microsomal fraction and mitochondria is extracted by organic solvents. 3. The hydrophobic proteins of lamellated bodies were separated into four fractions and freed from phospholipid by chromatographic procedures. Their apparent molecular weights vary between 11 500 and 16 500, they contain 72–79% of hydrophobic residues and 16–22% of sulphur-containing amino-acids, and leucine is the major N-terminal amino acid in each case.
Developmental changes in the content of oestrogen receptors in the hypothalamus of the female rat
Authors: White J O , Hall C , Lim L
Published: August 2015

Hypothalamic cytosol and nuclear oestrogen receptors are present at birth. A 2-fold increase in cytoplasmic receptor content occurs by the second week, whereas the first significant and equivalent increase in nuclear receptor occurs in the fourth week. The latter reflects reported increases in oestradiol availability thought to lead to complete feminine sexual differentiation. The presence of nuclear receptors in the newborn suggests a requirement for oestrogenic stimulation in early development.
The effect of reduction of perfusion rate on lactate and oxygen uptake, glucose output and energy supply in the isolated perfused liver of starved rats
Authors: Iles R A , Baron P G , Cohen R D
Published: August 2015

1. Lactate and O2 uptake and glucose output were studied in isolated livers from starved rats at perfusate flow rates varying from 100 to 7% of “normal” (11.25-0.75 ml/min per 100 g body wt.). 2. With moderate diminution of flow rate, lactate and oxygen uptake fell more slowly than would be expected if uptake purely depended on substrate supply. 3. Use of a mathematical model suggests that the intrinsic capacity of the liver for lactate uptake is unaffected until the flow rate falls below 25% of “normal”. 4. Some lactate uptake was always observed even at 7% of the “normal” flow rate. 5. At flow rates below 33% of the “normal”, lactate was increasingly metabolized by pathways other than gluconeogenesis, which became a progressively less important consumer of available O2. 6. ATP content decreased with diminution of flow rate, but substantially less markedly than did lactate uptake and glucose output. 7. Intracellular pH fell from a mean value of 7.25 at “normal” flow rate to 7.03 at 7% of the “normal” flow rate.
Regulation of p-nitroanisole O-demethylation in perfused rat liver. Adenine nucleotide inhibition of NADP+-dependent dehydrogenases and NADPH-cytochrome c reductase
Authors: Kauffman F C , Evans R K , Reinke L A , Thurman R G
Published: August 2015

Perfusion of rat livers with 10 mM-fructose or pretreatment of the rat with 6-aminonicotinamide (70 mg/kg) 6 h before perfusion decreased intracellular ATP concentrations and increased the rate of p-nitroanisole O-demethylation. This increase was accompanied by a decrease in the free [NADP+]/[NADPH] ratio calculated from concentrations of substrates assumed to be in near-equilibrium with isocitrate dehydrogenase. After pretreatment with 6-aminonicotinamide the [NADP+]/[NADPH] ratio also declined. Reduction of NADP+ during mixed-function oxidation may be explained by inhibition of of one or more NADPH-generating enzymes. Glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase and “malic” enzyme, partially purified from livers of phenobarbital-treated rats, were inhibited by ATP and ADP. Inhibitor constants of ATP for the four dehydrogenases varied considerably, ranging from 9 micrometer for “malic” enzyme to 1.85 mM for glucose 6-phosphate dehydrogenase. NADPH-cytochrome c reductase was also inhibited by ATP (Ki 2.8 mM) and by ADP (Ki 0.9 mM), but not by AMP. Concentrations of ATP and ADP that inhibited glucose 6-phosphate dehydrogenase and the reductase were comparable with concentrations in the intact liver. Thus agents that lower intracellular ATP may accelerate rates of mixed-function oxidation by a concerted mechanism involving deinhibition of NADPH-cytochrome c reductase and one or more NADPH-generating enzymes.
Postnatal development of uridine diphosphate glucuronyltransferase activity towards bilirubin and 2-aminophenol in human liver
Authors: Onishi S , Kawade N , Itoh S , Isobe K , Sugiyama S
Published: August 2015

UDP-glucuronyltransferase activities towards 2-aminophenol and bilirubin were studied in a total of 70 human subjects, including premature and full-term newborn babies, infants, children and adults. These two activities have been reported in rat to develop latefoetally and neonatally respectively, but in man they both develop neonatally. There is a linear relationship between the logarithm of each liver transferase activity and the logarithm of the number of days after birth during the first 3 months of life, after which each activity remains constant.
Lipopolysaccharides of R mutants isolated from Vibria cholerae
Authors: Hisatsune K , Kondo S
Published: August 2015

The chemical and serological properties of lipopolysaccharides isolated from the S form and from the R form of cholera vibrios were compared. It was found that the S-R mutation of cholera vibrios involves total elimination of the two component amino sugars of S-form lipopolysaccharides, i.e. quinovosamine and perosamine. This elimination resulted in the loss of O-specificity of S-form lipopolysaccharides and concomitant appearance of strong serological cross-reactivity, in the passive-haemolysis-inhibition test, among R-form lipopolysaccharides regardless of the serotypes (Inaba and Ogawa) of their S parent strains.
Cholic acid binding by glutathione S-transferases from rat liver cytosol
Authors: Hayes J D , Strange R C , Percy-Robb I W
Published: August 2015

Cholic acid-binding activity in cytosol from rat livers appears to be mainly associated with enzymes having glutathione S-transferase activity; at least four of the enzymes in this group can bind the bile acid. Examination of the subunit compositions of different glutathione S-transferases indicated that cholic acid binding and the ability to conjugate reduced glutathione with 1,2-dichloro-4-nitrobenzene may be ascribed to different subunits.
The amino acid sequence of ferredoxin from Brassica napus (rape)
Authors: Takruri I , Boulter D
Published: August 2015

The amino acid sequence of the ferredoxin of Brassica napus was determined by using a Beckman 890C sequencer in combination with the characterization of peptides obtained by tryptic and chymotryptic digestion of the protein; some peptides were subdigested with thermolysin. The molecule consists of a single polypeptide chain of 96 amino acid residues and has an unblocked N-terminus. The primary structure shows considerable similarity with other plant-type ferredoxins.
Sequence of the N-terminal half of Bacillus amyloliquefaciens α-amylase
Authors: Chung H , Friedberg F
Published: August 2015

Bacillus amyloliquefaciens alpha-amylase (1,4-alpha-D-glucan glucanohydrolase. EC 3.2.1.1), which is commercially supplied as ‘Bacillus subtilis alpha-amylase’ does not cross-react immunologically with B. subtilis alpha-amylase. This enzyme (from B. amyloliquefaciens) was cleaved by treatment with CNBr into seven fragments. Peptide A was selected for sequence determination. It is the longest one, containing 185 amino acids (i.e. approx. 50% of the total molecule) and connects to the hexapeptide of the N-terminus. Its primary structure was aligned by use of various proteolytic enzymes. The sequence of amino acids 181-184 is identical with that of amino acids 14-17 of the alpha-amylase isolated from B. subtilis (except that amino acid 183 is asparagine rather than aspartic acid).
Precolicin E1, the major gene product of plasmid-ColE1 deoxyribonucleic acid in vitro
Authors: Watson D H
Published: August 2015

Coupled transcription and translation of plasmid-ColE1 DNA in vitro under optimized conditions gave one major product. This has an apparent weight of 71 000, the same N-terminal sequence as colicin E1 and was not digested by deoxyribonuclease or ribonuclease. It differed from colicin E1 in its C-terminal residue and amino acid composition. It had lower specific activities in cell killing and in the fluorescence-enhancement in vitro assay of Phillips & Cramer [(1973) Biochemistry 12, 1170-1176] than did colicin E1, but both proteins bound in equimolar amounts to colicin-sensitive and colicin-resistant cells. The product of plasmid-ColE1-DNA-directed protein synthesis was converted into a protein indistinguishable in structure and activity from colicin E1 by incubation in the reaction mixture, after deoxyribonuclease and ribonuclease treatment, for a further 20 h at 37 degrees C. A protein with similar properties to the 71 000-dalton product in vitro was identified in extracts of a ColE1+ colicin-tolerant mutant of Escherichia coli K12. It is concluded that this protein probably represents a pre-form of colicin E1 which may be involved in colicin-E1 secretion or cellular colicin-E1 immunity in colicin-E-producing cells, or both of these processes.
Comparison of bone and osteosarcoma adenylate cyclase. Partial purification of membranes and kinetic properties of enzyme
Authors: Rodan S B , Egan J J , Golub E E , Rodan G A
Published: August 2015

The purpose of this study was to compare the adenylate cyclase of a tumour (rat osteosarcoma) growing in vivo with that of fast-growing embryonic bone. In the tumour the enzyme activity per total protein or DNA (under the same assay conditions) was 6-10-fold lower than in embryonic bone. To characterize this difference, we examined the kinetic properties of the enzyme in partially purified plasma membranes from the two tissues. A purification procedure based on differential centrifugation and discontinuous-sucrose-gradient centrifugation yielded a 10-fold increase in the specific activities of adenylate cyclase and 5'-nucleotidase in bone. The same procedure yielded an enriched membrane preparation from the tumour, but, relative to 5'-nucleotidase, a loss of 30% in adenylate cyclase occurred, which could not be recovered from another fraction. Kinetic analysis revealed that the lower adenylate cyclase activity in the tumour was due to a decrease in Vmax.. There was no significant difference in Ks (approx. 0.15 mM), and in the Km for GTP and p[NH]ppG. There were marked differences, however, in the extent of stimulation by p[NH]ppG, GTP and hormone, which was greater in tumour, and in the K1 for adenosine inhibition, which was 140 microM in bone and 500 microM in tumour. Under maximum stimulatory conditions, the enzyme activity in the tumour approached that in bone. The kinetic differences between bone and tumour enzyme were decreased by detergent solubilization, suggesting that the membrane environment plays a role in the generation of the observed differences.
Influence of the position of the double bond in steroid substrates on the efficiency of the proton-transfer reaction by Pseudomonas testosteroni 3-oxo steroid Δ4–Δ5-isomerase
Authors: Weintraub Hadassa , Baulieu Etienne-Emile , Alfsen Annette
Published: August 2015

Studies of the proton-transfer reaction by Pseudomonas testosteroni 3-oxo steroid Δ4–Δ5-isomerase with Δ5(6)- and Δ5(10)-steroid substrates demonstrate the importance of the position of the double bond for the efficiency of the isomerization process. Thus 3-oxo-Δ5(6)-substrates have markedly high kcat. values, whereas those of 3-oxo-Δ5(10)-substrates are very low and their apparent Km values approach equilibrium dissociation constants. The first step in the isomerization process is: [Formula: see text] which is governed by the k−1/k+1 ratio and is shown to be very similar for the two classes of substrates (3-oxo-Δ5(6)- and -Δ5(10)-steroids). They therefore differ in the steps distal to the initial formation of the Michaelis–Menten complex. The use of the deuterated androst-5(6)-ene-3,17-dione substrate enabled us to calculate individual rate constants k+1 and k−1 as well as to determine the apparent rate-limiting step in the isomerization process. With the deuterated oestr-5(10)-ene-3,17-dione substrate, no significant isotope effect was observed suggesting that a different rate-limiting step may be operative in this isomerization process. Data are presented that indicate that under optimal concentrations of the efficient androst-5(6)-ene-3,17-dione substrate, the forward reaction for ES complex formation (as defined by k+1) is limited only by diffusion and the apparent Km does not approach the equilibrium constant, suggesting that the evolution of this enzyme has proceeded close to ‘catalytic perfection’.
Organization of thiol groups of electric-eel electric-organ sodium-plus-potassium ion-stimulated adenosine triphosphatase studied with bifunctional reagents
Authors: Harris W E , Stahl W L
Published: August 2015

The reactions of three bifunctional thiol-blocking reagents of differing cross-linking spans and two monofunctional thiol-blocking reagents with the Na+ + K+-stimulated ATPase of the electric-eel electric organ were examined. 1,5-Difluoro-2,4-dinitrobenzene with a cross-linking span of 0.3-0.5 nm (3-5 A) and high solubility in non-polar solvent was the most efficient inhibitor of enzyme activity; thus essential thiol groups exist in a non-polar environment and are approx. 0.3-0.5 nm (3-5 A) from their nearest thiol-group neighbours. Ligands promoting phosphorylation of the Na+ + K+-stimulated ATPase decreased the number of thiol groups bridged by 1,5-difluoro-2,4-dinitrobenzene and by 4,4'-difluoro-3,3'-dinitrodiphenyl sulphone [0.7-1.0 nm (7-10 A) span]. Phosphorylation is associated with a conformational change in the enzyme.
Carbon monoxide-reacting haemoproteins in the mitochondrial fraction of Acanthamoeba castellanii
Authors: Edwards S W , Lloyd D
Published: August 2015

1. Room-temperature (18 degrees C) CO difference spectra of mitochondrial fractions from the amoeba Acanthamoeba castellanii reveal the presence of at least four CO-reacting haemoproteins. As well as cytochrome a3, other components reacting with CO are: (i) a c-type cytochrome; (ii) a b-type cytochrome; and (iii) another a-type cytochrome. 2. The same components can be identified in low-temperature photodissociation experiments with intact cells or mitochondria. 3. The time of exposure to CO and the nature of the reductant are both important in identifying all the components present, in that the b-type cytochrome is more readily distinguished after longer exposure to CO and more of the c-type cytochrome is detectable when NADH is the reductant 4. Treatment of mitochondria with ultrasound releases two components, identifiable in low-temperature difference spectra as a c-type and a b-type cytochrome; only the latter appears to have any reaction with CO, and the CO-reacting c-type cytochrome is retained in submitochondrial particles. 5. The complexity of the CO-reacting haemoproteins in this organism is compared with the simpler systems found in other eukaryotic organisms.
Inhibition of ornithine decarboxylase of HeLa cells by diamines and polyamines. Effect on cell proliferation
Authors: Branca Andrew A. , Herbst Edward J.
Published: August 2015

1. Ornithine decarboxylase activity is stimulated in high-density HeLa-cell cultures by dilution of or replacement of spent culture medium with fresh medium containing 10% (v/v) horse serum. 2. After stimulation, ornithine decarboxylase activity reaches a peak at 4–6h, then rapidly declines to the low enzyme activity characteristic of quiescent cultures, where it remains during the remainder of the cell cycle. 3. The stimulation of ornithine decarboxylase is eliminated by the addition of 0.5μm-spermine or -spermidine or 10μm-putrescine to the HeLa-cell cultures at the time of re-feeding with fresh medium. Much higher concentrations (1mm) of the non-physiological diamines, 1,3-diamino-propane or 1,3-diamino-2-hydroxypropane, are required to eliminate the stimulation of ornithine decarboxylase in re-fed HeLa-cell cultures. 4. A heat-labile, non-diffusible inhibitor, comparable with the inhibitory protein ornithine decarboxylase antizyme, is induced in HeLa cells by the addition of exogenous diamines or polyamines. 5. Intracellular putrescine is eliminated, intracellular spermidine and spermine are severely decreased and proliferation of HeLa cells is inhibited when cultures are maintained for 48h in the presence of the non-physiological inducer of ornithine decarboxylase antizyme, 1,3-diamino-2-hydroxypropane. Exogenous putrescine, a physiological inducer of the antizyme, does not decrease intracellular polyamines or interfere with proliferation of HeLa cells.
The effect of nitrous oxide inactivation of vitamin B12 on rat hepatic folate. Implications for the methylfolate-trap hypothesis
Authors: Lumb M , Deacon R , Perry J , Chanarin I , Minty B , Halsey M J , Nunn J F
Published: August 2015

Rats exposed to N20 show a decrease in liver folate to about 25% of the initial value after 10 days. There is a transient increase in the amount of 5-methylterrahydropteroylpolyglutamate in the first 24 h, but thereafter the content decreases. The level of 5-methyltetrahydropterolymonoglutamate declines without any transitory increase. The transient accumulation of 5-methyltetrahydropteroylpolyglutamate is due to failure of methionine synthetase. Thereafter the decrease in the amount of methylfolate makes it improbable that trapping of methylfolate is the explanation for failure of folate metabolism in vitamin B12 deficiency.
Tryptophan pyrrolase in haem regulation. The mechanism of the permissive effect of cortisol on the enhancement of 5-aminolaevulinate synthase activity by 2-allyl-2-isopropylacetamide in the adrenalectomized-rat liver
Authors: Morgan C J , Badawy A A
Published: August 2015

The decreased ability of 2-allyl-2-isopropropylacetamide to enhance liver 5-amino-laevulinate synthase activity in the adrenalectomized rat is not associated with a marked depletion of the already low amount of tryptophan pyrrolase haem. Cortisol permits the porphyrogen markedly to enhance synthase activity by rendering it capable of causing a stronger depletion of pyrrolase haem, presumably as result of hormonal induction of pyrrolase synthesis.
An electrophoretic study of endogenous phosphorylation in vitro of the polypeptides of microsomal membrane fractions of mouse liver
Authors: Behar-Bannelier Monique , Murray Robert K.
Published: August 2015

1. The patterns of phosphopolypeptides produced by endogenous phosphorylation in vitro of rough- and smooth-membrane fractions of the microsomal fraction of mouse liver were studied by radioautographic analysis of sodium dodecyl sulphate/polyacrylamide-gel electrophoretograms. 2. A minimum of 17 polypeptides of both rough- and smooth-microsomal-membrane fractions were phosphorylated by using [γ-32P]-ATP as the phosphate donor; only minor differences in phosphorylation pattern between the two membrane fractions were detected. 3. Phosphorylation in vitro by [γ-32P]ATP was markedly stimulated by Mg2+, but not by cyclic AMP, cyclic GMP or Ca2+. The phosphorylation of certain polypeptides was preferentially stimulated by Mg2+. Addition of cyclic AMP resulted in a decrease in the amount of 32P detected in one polypeptide of mol.wt. approx. 56000, present in both the rough- and smooth-membrane fractions. 4. [γ-32P]GTP was found to be a relatively poor donor of 32P as compared with [γ-32P]ATP. However, incubation of rough- and smooth-membrane fractions with this compound resulted in the phosphorylation of one polypeptide of mol.wt. approx. 96000 that was scarcely or not at all phosphorylated by [γ-32P]ATP. 5. Under the conditions of incubation used, appreciable incorporation of 32P from [γ-32P]ATP occurred into products migrating at the front of the electrophoretograms; these products were identified as being principally comprised of 1-phosphatidylinositol 4-phosphate. Incorporation of 32P into this lipid was also markedly stimulated by Mg2+. 6. The overall results show that a considerable number of polypeptides of the rough- and smooth-microsomal-membrane fractions of mouse liver may be phosphorylated in vitro and indicate that the enzymes responsible are principally non-cyclic AMP-dependent protein kinases.
Distance calculation of residues neighbouring to lysozyme antigenic sites. Site-neighbouring residues whose evolutionary substitution can modify the characteristics and binding energy of the sites
Authors: Atassi M Z , Kazim A L
Published: August 2015

By using the antigenic structure of lysozyme determined in this laboratory and the X-ray co-ordinates we have calculated the closest-atom distances between each of the residues in the three antigenic sites and all the other amino acids of the lysozyme molecule. These calculations enabled us to identify the nearest neighbours to each of the site residues. Thus the immediate environment of each site residue is described. For the three antigenic sites there is a total of 71 neighbouring residues. The effects of evolutionary amino acid substitutions in site-neighbouring residues on the binding capacity of protein binding sites in general and on protein antigenic sites in particular are discussed. These, together with the direct replacements in site residues, will acount for the major effects. However, the limitations of this treatment are stressed. The smaller effects on antigenic sites of replacements at once-removed and even at more distant locations, which, when they become cumulative, could be considerable, are brought to attention, together with any influences of conformational readjustments that can take place as a result of evolutionary amino acid replacements.
An improved procedure for the preparation of alkyl sulphate esters suitable for the study of secondary alkylsulphohydrolase enzymes
Authors: White G F , Lillis V , Shaw D J
Published: August 2015

A simple, rapid and convenient method for the synthesis of secondary alkyl sulphate esters is described in which the sodium alkoxide of the parent alcohol is sulphated by using triethylamine-SO3 complex. The procedure gives relatively good yields, even for the sulphation of long-chain alcohols and those in which the hydroxy group is remote from the terminal carbon atoms. Positional isomerization, arising from the migration of the hydroxy group along the carbon chain, is absent, and resolved enantiomers of alcohols react with complete retention of configuration.
Incorporation of 5-substituted uracil derivatives into nucleic acids. The isolation and γ-radiation-sensitivity of bacteriophage T3 containing the thymine analogue 5-vinyluracil
Authors: Chelton E T J , Duggan M , Hunston R N , Jones A S , O'Leary M K , Overton D J , Walker R T
Published: August 2015

Bacteriophage T3 was produced in a form that contained 32% of its normal DNA thymine residues replaced with 5-vinyluracil residues by infecting a thymine-requiring strain of Escherichia coli with phage T3 in a medium containing 5-vinyluracil. When 2′-deoxy-5-vinyluridine was added to the medium instead, no incorporation was observed into the phage DNA, and the presence of the deoxyribonucleoside severely decreased the number of viable phage particles produced. The analogue-containing phage, although initially viable, rapidly lost viability when stored, but it was no more sensitive than was normal phage T3 to the effect of gamma-radiation.
Drug-induced conversion of liver haem into modified porphyrins. Evidence for two classes of products
Authors: De Matteis F , Gibbs A H
Published: August 2015

A porphyrin with inhibitory activity towards protohaem ferro-lyase (EC 4.99.1.1) was isolated from the liver of mice given either griseofulvin or isogriseofulvin. This porphyrin resembles closely in chromatographic and spectral properties the inhibitory pigment isolated after treatment with 3,5-diethoxycarbonyl-1,4-dihydrocollidine, but differs from other green pigments that do not inhibit protohaem ferro-lyase. A hypothesis is proposed to account for the differences in properties between the two groups of pigments and for the mechanism of inhibition of the enzyme.
Comparison of the α-mannosidases in fibroblast cultures from patients with mannosidosis and mucolipidosis II and from controls
Authors: Halley D J , Winchester B G , Burditt L J , d'Azzo A , Robinson D , Galjaard H
Published: August 2015

The intracellular and extracellular acidic alpha-mannosidase in cultures of fibroblasts from mucolipidosis-II patients has normal kinetics. The extracellular activity in cultures of cells from mannosidosis patients is normal, but a mutant enzyme is associated with the cell surface and intracellular fraction. The results support the involvement of membrane cycling and recognition markers in lysosomal enzyme localization.
Interaction of wheat monomeric and dimeric protein inhibitors with α-amylase from yellow mealworm (Tenebrio molitor L. larva)
Authors: Buonocore V , Gramenzi F , Pace W , Petrucci T , Poerio E , Silano V
Published: August 2015

The highly purified alpha-amylase from Tenebrio molitor L. larva (yellow mealworm) reversibly combines with two closely related homogeneous glycoprotein inhibitors, one dimeric (termed ‘inhibitor 0.19’) and one monomeric (termed ‘inhibitor 0.28’), from wheat flour. As established by means of difference spectroscopy and kinetic studies, molar combining ratios for the amylase–inhibitor-0.19 and amylase-inhibitor-0.28 complexes were 1:1 and 1:2 respectively. Two amylase–inhibitor-0.19 complexes with slightly different retention volumes on Bio-Gel P-300 and only one amylase–inhibitor-0.28 complex were observed. Dissociation constants of the amylase–inhibitor-0.19 and amylase–inhibitor-0.28 complexes were 0.85 nM and 0.13 nM respectively. A strong tendency of both complexes to precipitate under an ultracentrifugal field was observed; the minimum molecular weight calculated for the two complexes under such conditions was approx. 95 000. The two complexes showed difference spectra indicating involvement of structurally related or identical tryptophyl side chains in the binding of inhibitors 0.28 and 0.19 to the amylase. A model summarizing the main features of the inhibition of the insect amylase by the two wheat protein inhibitors is proposed.