bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of monoferric fragments obtained by tryptic cleavage of bovine transferrin
Authors: Brock J H , Arzabe F R , Richardson N E , Deverson E V
Published: August 2015

1. The electrophoretically fast (F) and slow (S) fragments obtained by tryptic cleavage of bovine iron-saturated transferrin differed in carbohydrate content and peptide ‘maps’. 2. A fragment capable of binding one Fe3+ ion per molecule was isolated after brief tryptic digestion of bovine apotransferrin and shown closely to resemble the S fragment obtained from the iron-saturated protein. 3. Fragments F and S are probably derived from the N- and C-terminal halves of the transferrin molecule respectively. 4. Bovine transferrin could donate iron to rabbit reticulocytes, but the monoferric fragments possessed little iron-donating ability.
A simple sensitive method, applicable to small polypeptides, for the determination of proteins in polyacrylamide gels after isoelectric focusing
Authors: Storring P L , Tiplady R J
Published: August 2015

A simple method is described for the determination of polypeptides and proteins in polyacrylamide gels after isoelectric focusing. Precipitates formed in trichloroacetic acid, under controlled conditions, are quantified densitometrically by measuring the proportion of light scattered. The procedure is of particular value in its applicability to smaller polypeptides, with mol.wts. of 3000-6000, which are not fixed adequately in gels by other procedures currently in use. The working range, over which polypeptide concentration is proportional to the effective absorbance, is approx. 1-30 microgram per component.
Glucuronic acid conjugates of bilirubin-IXα in normal bile compared with post-obstructive bile. Transformation of the 1-O-acylglucuronide into 2-, 3-, and 4-O-acylglucuronides
Authors: Compernolle Frans , Van Hees Gustaaf P. , Blanckaert Norbert , Heirwegh Karel P. M.
Published: August 2015

Structures have been determined for bilirubin-IXα conjugates in freshly collected bile of normal rats, dogs and man and in post-obstructive bile of man and rats. The originally secreted conjugate has been characterized as azopigment (I), i.e. a 1-O-acyl-β-d-glucopyranuronic acid glycoside. Conversion of the acetylated methyl ester of azopigment (I) into methyl 2,3,4-tri-O-acetyl-1-bromo-1-deoxy-β-d-glucopyranuronate (V) indicates the pyranose ring structure for the carbohydrate and a C-1 attachment for the bilirubin-IXα acyl group. Alternative procedures for deconjugation of azopigment (I) and its derivatives are also described. In post-obstructive bile, the 1-O-acylglucuronide is converted into 2-, 3- and 4-O-acylglucuronides via sequential intramolecular migrations of the bilirubin acyl group. The following approach was utilized. (1) The tetrapyrrole conjugates were cleaved to dipyrrolic aniline and ethyl anthranilate azopigments, and the azopigments were separated as the acids or methyl esters. (2) The isomeric methyl esters were characterized by mass spectral analysis of the acetates and silyl ethers. (3) The free glycosidic function was demonstrated by 1-oxime and 1-methoxime derivative formation. (4) The position of the dipyrrolic O-acyl group was determined for the methyl esters by protecting the free hydroxyl groups of the glucuronic acid moieties as the acetals formed with ethyl vinyl ether and by further conversion of the carbohydrates into partially methylated alditol acetates. These were analysed by using g.l.c.–mass spectrometry. The relevance of the present results with regard to previous reports on disaccharidic conjugates is discussed. Details of procedures for the formation of chemical derivatives for g.l.c. and mass spectrometry have been deposited as Supplementary Publication SUP 50081 (15 pages) at the British Library Lending Division, Boston Spa, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978), 169, 5.
Pyruvate-to-ethanol pathway in Entamoeba histolytica
Authors: Lo H S , Reeves R E
Published: August 2015

The pyruvate-to-ethanol pathway in Entamoeba histolytica is unusual when compared with most investigated organisms. Pyruvate decarboxylase (EC 4.1.1.1), a key enzyme for ethanol production, is not found. Pyruvate is converted into acetyl-CoA and CO2 by the enzyme pyruvate synthase (EC 1.2.7.1), which has been demonstrated previously in this parasitic amoeba. Acetyl-CoA is reduced to acetaldehyde and CoA by the enzyme aldehyde dehydrogenase (acylating) (EC 1.2.1.10) at an enzyme activity of 9 units per g of fresh cells with NADH as a reductant. Acetaldehyde is further reduced by either a previously identified NADP+-linked alcohol dehydrogenase or by a newly found NAD+-linked alcohol dehydrogenase at an enzyme activity of 136 units per g of fresh cells. Ethanol is identified as the product of soluble enzymes of amoeba acting on pyruvate or acetyl-CoA. This result is confirmed by radioactive isotopic, spectrophotometric and gas-chromatographic methods.
A simple procedure for the isolation and purification of protamine messenger ribonucleic acid from trout testis
Authors: Gedamu L , Iatrou K , Dixon G H
Published: August 2015

Preparation of milligram quantities of purified poly(A)+ (polyadenylated) protamine mRNA from trout testis tissue was accomplished by a simple procedure using gentle conditions. This involves chromatography of the total nucleic acids isolated by dissociation of polyribosomes with 25 mM-EDTA to release messenger ribonucleoprotein particles and deproteinization of the total postmitochondrial supernatant with 0.5% sodium dodecyl sulphate in 0.25 M-NaCl by binding it to a DEAE-cellulose column. Total RNA was bound under these conditions, and low-molecular-weight RNA, lacking 18S and 28S RNA, could be eluted with 0.5 M-NaCl and chromatographed on oligo(dT)-cellulose columns to select for poly(A)+ RNA. Further purification of both the unbound poly(A)- RNA and the bound poly(A)+ mRNA on sucrose density gradients showed that both 18S and 28S rRNA were absent, being removed during the DEAE-cellulose chromatography step. Poly(A)- RNA sedimented in the 4S region whereas the bound poly(A)+ RNA fraction showed a main peak at 6S [poly(A+) protamine mRNA] and a shoulder in the 3-4S region. Analysis of the main peak and the shoulder on a second gradient showed that most of the main peak sedimented at 6S, whereas the shoulder sedimented slower than 4S. The identity of the poly(A)+ protamine mRNA was established by the following criteria: (1) purified protamine mRNA migrated as a set of four bands on urea/polyacrylamide-gel electrophoresis; (2) analysis of the polypeptides synthesized in the wheat-germ extract by starch-gel electrophoresis showed a single band of radioactivity which co-migrated exactly with the carrier trout testis protamine standard; and (3) chromatography of the polypeptide products on CM-cellulose (CM-52) showed the presence of three or four radioactively labelled protamine components that were co-eluted with the unlabelled trout testis protamine components added as carrier. The availability of large quantities of purified protamine mRNA should now permit a more thorough analysis of its physical and chemical properties.
Separation of oestrone UDP-glucuronyltransferase and p-nitrophenol UDP-glucuronyltransferase activities
Authors: Tukey R H , Billings R E , Tephly T R
Published: August 2015

Rabbit liver UDP-glucuronyltransferase activity was resolved into two separate fractions on DEAE-cellulose, one containing most of the transferase activity toward oestrone and the other most of the activity toward p-nitrophenol. These two activities were completely separated by rechromatography of each fraction on a second DEAE-cellulose column.
Isolation and hydroxylysine glycoside content of some cyanogen bromide-cleaved fragments of collagen from bovine corneal stroma
Authors: Panjwani N A , Harding J J
Published: August 2015

Six CNBr-cleaved fragments of insoluble collagen from bovine cornea were isolated, characterized and examined for hydroxylysine glycosides. Thus the general distribution of most of the glycoside along the collagen molecule was determined. Collagen from bovine stroma is almost entirely type I. This work forms a basis for the pinpointing of glycoside-attachment sites along the collagen molecules of bovine cornea.
Kinetic studies with the use of proton-magnetic-resonance spectroscopy of the specific α-deuteration of amino acids by Escherichia coli aspartate aminotransferase
Authors: Gout E , Chesne S , Beguin C G , Pelmont J
Published: August 2015

Escherichia coli aspartate aminotransferase was exposed to aspartate or phenylalanine without oxo acid in buffered 2H2O. The alpha-hydrogen of the amino acids underwent first-order exchange with respect to both substrate and enzyme. P.m.r. spectroscopy gave consistent reaction-rate constants. The deuterium-exchange rate was only moderately increased by addition of oxo acids and was of the same order as the transamination rate. No beta-deuteration was observed. The C(alpha)-H-bond-breaking step is discussed as a part of the entire transamination mechanism.
The effect of lutropin on specific protein synthesis in tumour Leydig cells and in Leydig cells from immature rats
Authors: Janszen Felix H. A. , Cooke Brian A. , Van Driel Maria J. A. , Van Der Molen Henk J.
Published: August 2015

The amount of35S incorporated into the various proteins after separation by electrophoresis on sodium dodecyl sulphate/polyacrylamide gels was used as an estimate of their synthesis in the Leydig cells. Increased synthesis of proteins with apparent mol.wts. 27000 and 29000 was observed 3h after addition of lutropin to tumour Leydig cells. Incubation of Leydig cells from immature rats with lutropin (100ng/ml) for 2h or longer resulted in increased synthesis of proteins with apparent mol.wts. 11000, 21000, 27000 and 29000. At higher concentrations (≥100ng/ml) of lutropin there was a decrease in the synthesis of a protein with apparent mol.wt. 13000. The amount of lutropin required for the stimulation of protein synthesis in both types of Leydig cells was similar to that needed for stimulation of steroidogenesis. Lutropin-stimulated specific protein synthesis was not due to increased concentrations of testosterone, however, because (1) addition of testosterone to the cells had no effect on the synthesis of the proteins, and (2) inhibition of steroidogenesis with elipten phosphate (an inhibitor of the cholesterol side-chain-cleavage enzyme complex) did not abolish the effect of lutropin. The stimulation of specific protein synthesis was also not due to contaminating follitropin in the lutropin preparation. Addition of actinomycin D to the cells at the start of the incubation prevented the effect of lutropin on specific protein synthesis, indicating that mRNA synthesis may be needed for this effect of lutropin. Incubation of the cells with cycloheximide for 30min after labelling of the proteins did not result in a detectable decrease in the amounts of the lutropin-induced proteins, indicating that their half-life is longer than 30min.
Fractionation of microsomal membranes on the basis of their surface properties
Authors: Ohlsson R , Jergil B , Walter H
Published: August 2015

Partition in dextran-poly(ethylene glycol) aqueous-phase systems can be used for both membrane subfractionation and gaining information on membrane surface properties [H. Walter (1977) in Methods of Cell Separation (Catsimpoolas, N., ed.), vol. 1, pp. 307-354, Plenum, New York]. Smooth, light rough and heavy rough rat liver microsome (obtained by sucrose-density-gradient centrifugation) were subjected to countercurrent distribution in such a system. Smooth microsomal membranes had the highest, heavy rough microsomal membranes the lowest and light rough microsomal membranes an intermediate partition coefficient. The separation is based primarily on hydrophobic differences in the membrane surfaces of the three preparations and is thus due to microsomal properties not previously utilized in their fractionation. The method permits additional subfractionations of microsomes.
The availability of inorganic sulphate in blood for sulphate conjugation of drugs in rat liver in vivo. (35S)Sulphate incorporation into harmol sulphate
Authors: Mulder G J , Scholtens E
Published: August 2015

1. When Na235SO4 is injected intravenously in rats, it is immediately available for sulphate conjugation of the phenolic drug harmol (7-hydroxyl-1-methyl-9H-pyrido[3,4-b]indole) in the liver. This was established by following the time course of the biliary excretion of the sulphate conjugate of harmol, and the incorporation of [35S]sulphate into harmol sulphate. 2. During the 10min immediately after injection of Na235SO4 re-distribution of [35S]sulphate took place, which resulted in a rapid initial decrease in the plasma concentration of [35S]sulphate; a concomitant decrease in the amount of [35S]sulphate incorporated into harmol sulphate was observed, indicating that the co-substrate of sulphation, adenosine 3′-phosphate 5′-sulphatophosphate, equilibrates rapidly with [35S]sulphate in plasma. 3. The results suggest that the pool size of adenosine 3′-phosphate 5′-sulphatophosphate is very small; therefore the specific radioactivity of [35S]sulphate in plasma determines the specific radioactivity incorporated into sulphate esters at any time.
Characterization of the mutant-unc D-gene product in a strain of Escherichia coli K12. An altered β-subunit of the magnesium ion-stimulated adenosine triphosphatase
Authors: Fayle D R H , Downie J A , Cox G B , Gibson F , Radik J
Published: August 2015

Membranes from a mutant strain of Escherichia coli K12 carrying the uncD409 allele were washed in low-ionic-strength buffers in the presence or absence of the proteinase inhibitor p-aminobenzamidine. Unlike membranes from a normal strain, those from strain AN463 (uncD409) did not become proton-permeable, as judged by NADH-induced atebrinfluorescence quenching, when the membranes were washed in the absence of p-aminobenzamide. Furthermore, ATP-dependent atebrin-fluorscence quenching in such washed membranes could not be reconstituted by the addition of solubilized Mg2+-stimulated adenosine triphosphatase preparations. The examination by two-dimensional polyacrylamide-gel electrophoresis of the polypeptide composition of the washed membranes from strain AN463 (uncD409) indicated the presence of a polypeptide of similar molecular weight to the normal beta-subunit of the Mg2+-stimulated adenosine triphosphatase, but with an altered isoelectric point. Both the normal and abnormal beta-subunits were identified in membranes prepared from a partial diploid strain carrying both the unc+ and uncD409 alleles. It is concluded that the uncD gene codes for the beta-subunit of the Mg2+-stimulated adenosine triphosphatase.
Some transport properties of resealed washed human erythrocyte membranes
Authors: Mawby W J , Findlay J B C
Published: August 2015

A comparison was made between the phosphate- and glucose-transport systems of intact erythrocytes and resealed washed membranes. Glucose transport exhibits identical properties in both cases, but the phosphate-transport system does not appear to have survived the membrane isolation procedure unaltered. Evidence is presented to support the suggestion that some form of structural perturbation has occurred to the protein mediator of phosphate exchange.
The nature of the allosteric interactions of ribonuclease and its ligands
Authors: Walker E J , Ralston G B , Darvey I G
Published: August 2015

The allosteric model for ribonuclease activity by Walker, Ralston & Darvey [(1975) Biochem.J. 147, 425–433; (1976) Biochem.J. 153, 329–337] involves the binding of a large number of molecules of substrate or substrate analogue to a series of allosteric sites on the enzyme. In the present paper, the nature of these allosteric interactions is investigated. The effects of ionic strength pH carbamoylation of lysine to homocitrulline and of deamidation of glutamine and asparagine on plots of velocity versus substrate concentration are examined and evidence is presented that the allosteric transition involves an electrostatic interaction between the negatively charged substrate molecules and the cationic groups on the enzyme.
Preparation of allosteric ribonuclease
Authors: Walker E J , Ralston G B , Darvey I G
Published: August 2015

A method for the preparation of allosteric ribonuclease from bovine pancreas is described. The effects of freeze-drying ribonuclease from acid and alkaline solutions on plots of velocity versus substrate concentration for the hydrolysis of 2′:3′-cyclic CMP are examined. Comparison of these plots with the plots obtained with severeal commercial enzyme preparations indicates that the conformation of the enzyme is dependent on the method of preparation. Aging experiments demonstrate that further conformational changes occur at different rates, depending on the methods of storage. Results suggest that the allosteric behaviour of ribonuclease has not always been observed with commercial preparations, owing to variations in methods of preparation and storage of the enzyme.
Mechanism of oxyhaemoglobin breakdown on reaction with acetylphenylhydrazine
Authors: French J K , Winterbourn C C , Carrell R W
Published: August 2015

The reaction of oxyhaemoglobin and acetylphenylhydrazine, which results in haemoglobin denaturation and precipitation, was found to be influenced by H202 and superoxide (O2-.) generated during the reaction. By analysing the different haemoglobin oxidation products, it was found that by influencing the rate at which oxyhaemoglobin was oxidized, H2O2 accelerated the overall haemoglobin breakdown, and O2-. inhibited it. By adding GSH (reduced glutathione) or ascorbate, it was possible to slow down the rates of both oxyhaemoglobin oxidation and O2-. production, and the overall rate of haemoglobin breakdown. These results are compatible with a mechanism involving production of the acetylphenylhydrazyl free radical, and with GSH, ascorbate and O2-. acting as radical scavengers and preventing its further reactions. The reaction produced choleglobin, as well as acetylphenyldiazine and methaemoglobin, which combined to form a haemichrome. The haemichrome was less stable and precipitated first. It was also less stable than the haemichrome formed by direct reaction of acetylphenyldiazine with methaemoglobin, and it is proposed that this is because the methaemoglobin produced from oxyhaemoglobin and acetylphenylhydrazine was modified by the free radicals and H2O2 produced in the reaction.
Studies on the time course and rate-limiting steps in the activation of adenylate cyclase in rat liver by cholera toxin
Authors: Fischer J , Kohler T R , Lipson L G , Flores J , Witkum P A , Sharp G W G
Published: August 2015

Cholera toxin stimulates adenylate cyclase in rat liver after intravenous injection. The stimulation follows a short latent period of 10min, and maximum stimulation was attained at 120min. Half-maximal stimulation was achieved at 35min. In contrast with this lengthy time course in the intact cell, adenylate cyclase in broken-cell preparations of rat liver in vitro were maximally stimulated by cholera toxin (in the presence of NAD+) in 20min with half-maximal stimulation in 8min. Binding of cholera toxin to cell membranes by the B subunits is followed by translocation of the A subunit into the cell or cell membrane, and separation of the A1 polypeptide chain from the A2 chain by disulphide-bond reduction, and finally activation of adenylate cyclase by the A1 chain and NAD+. As the binding of cholera toxin is rapid, two possible rate-limiting steps could be the determinants of the long time course of action. These are translocation of the A1 chain from the outside of the cell membrane to its site of action (this includes the time required for separation from the whole toxin) or the availability of NAD+ for activation. When NAD+ concentrations in rat liver were elevated 4-fold, by the administration of nicotinamide, no change in the rate of activation of adenylate cyclase by cholera toxin was observed. Thus the intracellular concentration of NAD+ is not rate-limiting and the major rate-limiting determinant in intact cells must be between the time of toxin binding to the cell membrane and the appearance of subunit A1 at the enzyme site.
Interaction between synthetic analogues of quinoxaline antibiotics and nucleic acids. Changes in mechanism and specificity related to structural alterations
Authors: Lee J S , Waring M J
Published: August 2015

The interaction with DNA of six chemically synthesized derivatives of the quinoxaline antibiotics was investigated. Five of the compounds bound only weakly to DNA or not at all; for these substances spectrophotometric measurements, sedimentation studies with closed circular duplex bacteriophage-PM2 DNA and thermal-denaturation profiles were used to determine limits fot the binding constants. No interaction could be detected with two products of degradation of echinomycin (quinomycin A), one of which, echinomycinic acid dimethyl ester, had the lactone linkages opened, whereas the other retained an intact octapeptide ring but had a broken cross-bridge. The other compounds studied were des-N-tetramethyl-triostin A (‘TANDEM’) and its derivatives. A derivative of ‘TANDEM’ IN WHICH benzyloxycarbonyl moieties replace both quinoxaline chromophores had binding constants to nucelic acids in the range 10(2)–10(3)-1, whereas no interaction could be detected for a benzyloxycarbonyl derivative that, in addition, had the cross-bridge broken. The derivative of ‘TANDEM’ with L-serine in place of D-serine in both positions showed no detectable interaction with Clostridium perfringens DNA, whereas the binding constant to poly(dA-dT) was approx 2 × 10(3)M-1. ‘TANDEM’ itself bound strongly to DNA, and the bathochromic and hypochromic shifts in its u.v.-absorption spectrum in the presence of DNA were similar to those seen with echinomycin. From the effect on the sedimentation coefficient of closed circular duplex bacteriophage-PM2 DNA the mechanism of binding was shown to involve bifunctional intercalation, typical of the naturally occurring quinoxaline antibiotics. Solvent-partition analysis was used to determine binding constants for the interaction between ‘TANDEM’ and a variety of natural and synthetic DNA species. The pattern of specificity thus revealed differed markedly from that previously found with the naturally occurring quinoxaline antibiotics. Most striking was the evident large preference for (A + T)-rich DNA species, in complete contrast with echinomycin and triostin A. The highest binding constant was found for poly(dA-dT), the interaction with which appeared highly co-operative in character. The conformations adopted by those quinoxaline compounds that bind strongly to DNA were examined withe aid of molecular models on the basis of results derived from n.m.r. and computer studies. It appears that the observed patterns of base-sequence specificity are determined, at least in part, by the structure and conformation of the sulphur-containing cross-bridge.
Preparation of chromatin containing ribosomal deoxyribonucleic acid from the macronucleus of Tetrahymena pyriformis
Authors: Jones R W
Published: August 2015

A method is described that enables a chromatin fraction containing ribosomal DNA (DNA containing sequences coding for rRNA) to be prepared from the macronuclei of growing or stationary cultures of Tetrahymena pyriformis. This material is obtained in yields of between 25 and 75% of the theoretical maximum. The DNA in this fraction was identified as ribosomal DNA on the basis of its density and molecular weight, and it appears not to be appreciably contaminated by other DNA. The method relies on the approximate assumption that ribosomal DNA is the smallest species of DNA in chromatin in the nucleus, and avoids the use of mechanical force, or enzyme action, to fractionate chromatin.
The role of zinc in the stabilization of the dimeric form of bacterial α-amylase
Authors: Tellam R , Winzor D J , Nichol L W
Published: August 2015

Bacterial alpha-amylase was shown by equilibrium and velocity-sedimentation studies to be a monomer-dimer equilibrium system in 0.10M-NaCl/0.015M-calcium acetate/0.010M-EDTA, pH7.0; an association constant of 2.4 × 10(3)M-1 is obtained. Studies of the binding of Zn2+ to alpha-amylase in 0.10M-NaCl/0.005M-calcium acetate, pH7.0, yielded binding curves that exhibit dependence on the concentration of alpha-amylase (Zn2+-free) used in the equilibrium-dialysis experiments. Results are described very satisfactorily by a reaction scheme in which Zn2+ binds exclusively to the dimer of the above monomer–dimer system with an association constant of 1.0 × 10(6)M-1. The present results refute the earlier scheme for dimer stabilization by Zn2+ in which the metal ion formed a cross-link between two non polymerizing monomer units.
Diffusional increase and decrease in half-maximal-activity substrate concentrations with two-substrate enzymic reactions
Authors: Engasser J M , Hisland P
Published: August 2015

Diffusional effects on two-substrate enzymic reactions mainly depend on the relative affinities of the enzyme for its two substrates. With two substrates of widely different affinities, diffusional limitations increase and decrease the half-maximal-activity concentration of the high-and low-affinity substrate respectively.
Evidence for an essential arginine recognition site on adenosine 3′:5′-cyclic monophosphate-dependent protein kinase of rabbit skeletal muscle
Authors: Matsuo M , Huang C H , Huang L C
Published: August 2015

On the basis of the chemical and structural features of the amino acid sequences in the vicinities of phosphorylatable hydroxyamino acid residues in several of the well-known protein substrates for skeletal-muscle cyclic AMP-dependent protein kinase, it is hypothesized that the phosphorylatable residue at position i and arginine residue at position i-3 of these protein substrates are located on a peptide turn on the hydrophilic protein surface. It is further hypothesized that there is an arginine-recognition site near the active centre on the protein kinase. This site is essential for the function of cyclic AMP-dependent protein kinase, for, not only does it recognize specifically the exposed arginine residue of the protein substrate, but, more importantly, via the interaction with arginine-(i′3), it may help to steer the topologically adjacent serine-i into proper orientation on the nearby active centre for phosphorylation. Model-building and kinetic data that provide support for the proposed hypotheses are presented.
Characterization of a region of the primary sequence of troponin C involved in calcium ion-dependent interaction with troponin I
Authors: Weeks R A , Perry S V
Published: August 2015

1. The CNBr digest of troponin C from rabbit fast skeletal muscle was shown to possess many of the functional properties of the whole troponin C molecule. 2. A peptide corresponding to residues 83-134 was isolated, which forms a Ca(2+-dependent complex with troponin I and neutralizes the inhibition by troponin I of the Mg(2+-stimulated adenosine triphosphatase of desensitized actomyosin. 3. The peptide inhibits the phosphorylation of fast-skeletal-muscle, but not cardiac-muscle, troponin I, by 3′ :5′-cyclic AMP-dependent protein kinase. In this property it was as effective as whole skeletal-muscle troponin C when compared on a molar basis. 4. Biological activity was also present in other fractions obtained from the CNBr digest. 5. By gel filtration and affinity chromatography of the whole CNBr digest of troponin C, two peptides, one of which was identified as representing residues 83-134, were shown to form Ca(2+-dependent complexes with troponin I. 6. The significance of these findings for the mechanism of interaction of troponin C and troponin I is discussed.
Purification and properties of retinoic acid-binding protein from chick-embryo skin
Authors: Sani B P , Banerjee C K
Published: August 2015

Retinoic acid-binding protein, which is considered to mediate the biological function of retinoic acid in epithelial differentiation and in the possible control of tumorigenesis, was reproducibly purified from chick-embryo skin by using DEAE-Sephadex and Sephadex G-100 column chromatography and isoelectric focusing. About 1mg of protein was isolated from 60g of skin. The purified protein-ligand complex was found to be homogeneous by electrophoresis on polyacrylamide gels. The binding protein has mol.wt. 17800 and pI 4.5. The binding of [3H]retinoic acid to the protein was completely inhibited by mercury compounds. The inhibition is reversible on treatment without dithithreitol; about 50% of the retinoic acid-binding capacity of the mercury-compound-treated protein is restored by chromatography on Sephadex G-25. iodoacetamide treatment of the protein irreversibly inhibits about 50% of retinoic acid binding.
The interaction of purified acetylcholinesterase from pig brain with liposomes
Authors: Reavill C A , Wooster M S , Plummer D T
Published: August 2015

The binding of pig brain acetylcholinesterase to artificial phospholipid membranes was investigated at different temperatures. Calculation of the thermodynamic parameters revealed a small negative enthalpy change, but a large negative change in the free energy and a large positive change in the entropy on binding. The large entropy change might be interpreted as being responsible for forming the enzyme-membrane complex and was indicative of hydrophobic interactions between lipid and protein. This conclusion would also favour the hypothesis that the enzyme was an integral protein. Further support for this theory was provided by the study of acetylcholinesterase binding to liposomes containing the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine. Lowering the temperature below the transition temperature or incorporating cholesterol into the liposomes decreased enzyme binding. Both factors could be interpreted as decreasing the fluidity of the hydrocarbon side chains of the phospholipids, causing an increase in bilayer thickness due to closer packing of side chains. This membrane condensation would certainly not favour the binding of integral protein molecules.
The nitrogenase system of Spirillum lipoferum
Authors: Ludden P W , Okon Y , Burris R H
Published: August 2015

The nitrogenase system of Spirillum lipoferum was separated into three components, the normal Mo-Fe and Fe proteins as well as an activating factor for the Fe protein. The rate of activation is increased by Mn2+ or by an excess of Mg2+, and the process requires ATP.
Mechanisms of replenishment of nuclear and rogen receptor in rat ventral prostate
Authors: Van Doorn Edward , Bruchovsky Nicholas
Published: August 2015

1. The concentration of androgen receptor in the nucleus of the prostatic cell is rapidly elevated by the administration in vivo of 2μg of [3H]testosterone to 1-day-castrated rats. From a concentration of 2300 receptors/nucleus at 5min after intravenous injection of hormone, there is an increase to 21000 receptors/nucleus at 60min. At the same time, the amount of binding of androgen in the cytoplasm remains constant at a relatively low value. 2. An identical dose of [3H]testosterone administered to 7-day-castrated rats produces a much smaller change in the concentration of nuclear receptor, from 700 receptors/nucleus at 5min to only 4300 receptors/nucleus at 60min. Thus the reservoir from which nuclear receptor is replenished is considerably smaller in regressed prostatic cells. Again, the amount of binding of androgen in the cytoplasm remains unchanged at a low value over the experimental time course of 60min. 3. In contrast with the scant labelling of cytoplasmic receptor achieved by injecting animals with [3H]testosterone, labelling in vitro, by incubation of tissue slices with radioisotope, indicates that prostate of 1-day-castrated animals actually contains 21400 receptors/cell in the cytoplasmic compartment, and prostate of 7-day-castrated animals 3000 receptors/cell. 4. Owing to the similarity between the concentration of nuclear receptor measured in vivo and the concentration of cytoplasmic receptor measured in vitro, the labelling techniques in vivo and in vitro were used in sequence to demonstrate the movement of most of the cytoplasmic receptor into the nucleus. In the 5–60min interval after the administration of [3H]testosterone to 1-day-castrated rats, a decrease of 17400 receptor molecules in the cytoplasm is exactly mirrored by an increase of 17200 receptor molecules in the nucleus. 5. These results imply that, in prostate of 1-day-castrated rats, nuclear receptor is replenished exclusively by translocation of cytoplasmic receptor. However, in the regressed prostate of 7-day-castrated rats, only about 25% of the nuclear receptor is replenished through translocation of existing cytoplasmic receptor. The remainder is ultimately synthesized during new rounds of cell division induced by hormone.
The effect of lipid peroxidation on the calcium-accumulating ability of the microsomal fraction isolated from chicken breast muscle
Authors: Player T J , Hultin H O
Published: August 2015

The effect of lipid peroxidation on the Ca2+-accumulating and Ca2+-retaining abilities of the microsomal fraction from chicken breast muscle was investigated. At 25 degrees C, enzymic lipid peroxidation did not seriously affect either of these abilities unless ascorbic acid was present, when both were diminished. At 37 degrees C, Ca2+-concentrating ability was decreased further by the effects of heat damage to the membrane. Membrane lipid peroxidation did not affect microsomal adenosine triphosphatase activity unless the microsomal fraction was subsequently washed with albumin. This effect of albumin is possibly due to removal of lipid-breakdown products. Addition of soya-bean phospholipids to the peroxidized vesicles washed with albumin restored adenosine triphosphatase activity, demonstrating a non-specific phospholipid requirement.
Fatty acid biosynthesis in the leaves of barley, wheat and pea
Authors: Wharfe J , Harwood J L
Published: August 2015

1. The incorporation of radioactivity from [1-14C]acetate into the leaf lipids of barley, pea and wheat has been studied in pulse-labelling experiments. 2. There was little increase in the total labelling of lipids after the leaves were transferred to non-radioactive medium. However, there was an increase in the relative labelling of unsaturated fatty acids. In addition, there was an increase in the relative labelling of diacylgalactosylglycerol. 3. The principal radioactively labelled acyl lipids were diacylgalactosylglycerol and phosphatidylcholine. Phosphatidylcholine showed a decreasing proportion of [14C]oleate and an increasing amount of [14C]linoleate with time. Diacylgalactosylglycerol also had decreasing amounts of [14C]oleate but, in addition, had an increasing proportion of [14C]linolenate with time. 4. The absence of significant amounts of [14C]linolenate in phosphatidylcholine appeared to exclude a role for this phospholipid in linoleate desaturation. 5. The specific radioactivities of oleate and linoleate in phosphatidylcholine, diacylgalactosylglycerol and diacylgalabiosylglycerol were very similar in any single experiment. It was concluded that these fatty acids can rapidly exchange between the three intact lipids.
Properties of a cytochrome c-enriched light particulate fraction isolated from the photosynthetic bacterium Rhodopseudomonas spheroides
Authors: Barrett J , Hunter C N , Jones O T G
Published: August 2015

Differential centrifugation of suspensions of French-press-disrupted Rhodopseudomonas spheroides yielded a light particulate fraction that was different in many properties from the bulk membrane fraction. It was enriched in cytochrome c and had a low cytochrome b content. When prepared from photosynthetically grown cells this fraction had a very low specific bacteriochlorophyll content. The cytochrome c of the light particles differed in absorption maxima at 77K from cytochrome c2 attached to membranes; there was pronounced splitting of the alpha-band, as is found in cytochrome c2 free in solution. Potentiometric titration at A552–A540 showed the presence of two components that fitted an n = 1 titration; one component had a midpoint redox potential of +345mV, like cytochrome c2 in solution, and the second had E0′ at pH 7.0 of +110 mV, and they were present in a ratio of approx. 2:3. Difference spectroscopy at 77K showed that the spectra of the two components were very similar. More of a CO-binding component was present in particles from photosynthetically grown cells. Light membranes purified by centrifugation on gradients of 5–60% (w/w) sucrose retained the two c cytochromes; they contained no detectable succinate-cytochrome c reductase or bacteriochlorophyll and very little ubiquinone, but they contained NADH-cytochrome c reductase and some phosphate. Electrophoresis on sodium dodecyl sulphate/polyacrylamide gels showed that the light membranes of aerobically and photosynthetically grown cells were very similar and differed greatly from other membrane fractions of R. spheroides.
Uptake of substrates for milk-fat synthesis by lactating-rabbit mammary gland
Authors: Jones C S , Parker D S
Published: August 2015

1. A cannulation technique is described for measuring arteriovenous differences across the lactating-rabbit mammary gland. 2. Analysis of milk obtained before and after surgery shows no effect of cannulation on milk constituents. 3. Results of blood analysis show significant net changes in the concentrations of glucose, acetate, 3-hydroxybutrate, triacylglycerols and non-esterified fatty acids across the mammary gland. 4. The molar proportions of individual fatty acids in both the triacylglycerol and non-esterified fatty acid fractions did not alter between the arterial and venous samples. 5. The extraction rates are compared with those obtained from other species.
Analytical subcellular fractionation of needle-biopsy specimens from human liver
Authors: Peters T J , Seymour C A
Published: August 2015

1. Fragments (2-20 mg wet wt.) of closed needle-biopsy specimens from human liver were disrupted in iso-osmotic sucrose and subjected to low-speed centrifugation. The supernatant was layered on a linear sucrose-density gradient in the Beaufay small-volume automatic zonal rotor. The following organelles, with equilibrium densities (g/ml) and principal marker enzyme shown in parentheses, were resolved: plasma membrane (1.12-1.14; 5′-nucleotidase); lysosomes (1.15-1.20; N-acetyl-beta-glucosaminidase); mitochondria (1.20; malate dehydrogenase); endoplasmic reticulum (1.17-1.21; neutral alpha-glucosidase); peroxisomes (1.22-1.24; catalase). 2. The distribution of particulate alkaline phosphatase and, to a lesser degree, leucine 2-naphthylamidase followed that of 5′-nucleotidase. gamma-Glutamyltransferase was associated with membranes of significantly higher equilibrium density than was 5′-nucleotidase. 3. The distribution of 12 acid hydrolases was determined in the density-gradient fractions. beta-Glucosidase had a predominantly cytosolic localization, but the other enzymes showed a broad distribution of activity throughout the gradient. Evidence was presented for two populations of lysosomes with equilibrium densities of 1.15 and 1.20 g/ml, but containing differing amounts of each enzyme. Further evidence of lysosomal heterogeneity was demonstrated by studying the distribution of isoenzymes of hexosaminidase and of acid phosphatase. 4. The resolving power of the centrifugation procedure can be further enhanced with membrane perturbants. Digitonin (0.12 mM) selectively disrupted lysosomes, markedly increased the equilibrium density of plasma-membrane components and lowered the density of the endoplasmic reticulum, but did not affect the mitochondria or peroxisomes. Pyrophosphate (15 mM) selectively lowered the equilibrium density of the endoplasmic reticulum.
The purification and some properties of rusticyanin, a blue copper protein involved in iron(II) oxidation from Thiobacillus ferro-oxidans
Authors: Cox J C , Boxer D H
Published: August 2015

The ‘blue’ copper-containing protein rusticyanin was purified to homogeneity from cells of the chemolithotrophic bacterium Thiobacillus ferro-oxidans by (NH4)SO4 fractionation and ion-exchange chromatography. The protein, which is stable at low pH, consists of a single polypeptide chain of mol. wt. 16500 and possesses 0.79 (+/- 0.28)g-atom of Cu/mol. The protein, which does not contain arginine residues, has optical absorbance maxima at 287, 450, 597 and 750 nm and is generally similar to azurin. The isolated protein is reduced directly by Fe2+ with a 1:1 stoicheiometry to Cu. On reduction by Fe2+ the absorption peaks at 450, 597 and 750 nm are abolished, with the appearance of a new absorption band at 320 nm. The results obtained are consistent with rusticyanin being the initial acceptor of electrons from Fe2+ during respiratory iron oxidation.
Calcium ion cycling in rat liver mitochondria
Authors: Ramachandran Chidambaram , Bygrave Fyfe L.
Published: August 2015

1. Addition of N-ethylmaleimide to rat liver mitochondria respiring with succinate as substrate decreases both the initial rate of Ca2+ transport and the ability of mitochondria to retain Ca2+. As a result, Ca2+ begins to leave the mitochondria soon after it has entered. Half-maximal effects occur at an N-ethylmaleimide concentration of about 100nmol/mg of protein. 2. The efflux of Ca2+ induced by N-ethylmaleimide is not prevented by Mg2+ or by Ruthenium Red at concentrations known to prevent Ca2+ efflux when exogenous phosphate also is present. Swelling of mitochondria does not accompany N-ethylmaleimide-induced Ca2+ efflux. 3. Addition of Ca2+ to rat liver mitochondria in the presence of N-ethylmaleimide produces an immediate decrease in ΔE (membrane potential), which decreases further to only a slight extent over the next 8min. Concomitant with this is an immediate increase and then levelling off of the −59ΔpH (transmembrane pH gradient). 4. Preincubation of rat liver mitochondria with p-chloromercuribenzenesulphonate, which by contrast with N-ethylmaleimide is unable to penetrate the inner mitochondrial membrane, also prevents Ca2+ retention. The ΔE and −59ΔpH respond to Ca2+ addition in a manner similar to that which occurs when N-ethylmaleimide is present. Subsequent addition of mercaptoethanol produces an immediate increase in both ΔE and −59ΔpH. At the same time Ca2+ is rapidly accumulated by the organelles. 5. The above data are interpreted as indicating that under the conditions of Ca2+ efflux seen here, the mitochondria retain their functional integrity. This contrasts with the uncoupling effect of Ca2+ seen in the presence of Pi, which generally leads to a loss of mitochondrial integrity. We suggest that a unique mechanism of Ca2+ cycling is able to take place when mitochondria have been treated with N-ethylmaleimide.
Oxidative degradation of haemoglobin by nitrosobenzene in the erythrocyte
Authors: Hirota K , Itano H A , Vedvick T S
Published: August 2015

Substitutions on the benzene ring of nitrosobenzene did not have the same effect on oxidative haemolysis as substitutions on phenylhydrazine. We previously found that the haemolytic effect of arylhydrazines paralleled their oxidative conversion into ligands of ferrihaemoglobin. In contrast, although most substituted nitrosobenzenes that are ligands of ferrohaemoglobin caused haemolysis and most that are not ligands failed to cause nitrosoarenes appeared to be related more closely to the ease of their reduction to arylhydroxylamines than to their properties as ligands. We propose a mechanism of oxidative degradation whereby the cyclic formation of phenylhydroxylamine from nitrosobenzene within an erythrocyte leads to the accumulation of H2O2, which then reacts with ferrohaemoglobin to initiate the oxidative cleavage of haem. The posulated active intermediate in this reaction is the same as that previously proposed in the oxidative degradation of haemoglobin by phenylhydrzine and in the coupled oxidation of ascorbic acid and haemoglobin.
Measurement and kinetic study of the formation of adrenaline sulphate in vitro
Authors: Wong K P
Published: August 2015

The sulpho-conjugation of [14C]adrenaline form inorganic sulphate and ATP or preformed adenosine 3′-phosphate 5′-sulphatophosphate was demonstrated in the high-speed supernatant prepared from the liver and small intestine of various animals. Hydrolysis with sulphatase indicated the sulphate nature of the conjugate. The overall sulphation reaction has a pH optimum of 9.0. Maximal activity was obtained with a ratio of ATP/Mg2+ of 1 at 4–6mM. Above their optimal concentrations, ATP and Mg2+, separately or in combination, were inhibitory. Dithiothreitol at 3 mM stimulated the reaction by about 30%. The Km for adrenaline, determined by the sulphotransferase reaction and by the three-step (sulphate-activating and sulphotransferase) reactions was 125 micrometer. The rate of synthesis of [14C]-adrenaline sulphate, expressed in pmol/min per mg of protein for the livers of dog, monkey, rat, guinea pig and rabbit were, respectively, 144, 77, 47, 11 and 6. The corresponding values for the small intestines of dog and monkey were 60 and 62. Brain and heart tissues showed no measurable activity.
Effects of p-chlorophenylalanine and α-methylphenylalanine on amino acid uptake and protein synthesis in mouse neuroblastoma cells
Authors: Kelly C J , Johnson T C
Published: August 2015

The phenylalanine analogues p-chlorophenylalanine and alpha-methylphenylalanine were used to inhibit phenylalanine hydroxylase in animal models for phenylketonuria. The present report examines the affects of these analogues on the metabolism of neuroblastoma cells. p-Chlorophenylalanine inhibited growth and was toxic to neuroblastoma cells. Although in vivo this analogue increased cell monoribosomes by 42%, it did not significantly affect poly(U)-directed protein synthesis in vitro. P-Chlorophenylalanine did not compete with phenylalanine or tyrosine for aminoacylation of tRNA and was therefore not substituted for those amino acids in nascent polypeptides. The initial cellular uptake of various large neutral amino acids was inhibited by this analogue but did not affect the flux of amino acids already in the cell; this suggested that an alteration of the cell's amino acid pools was not responsible for the cytotoxicity of the analogues. In contrast with p-chlorophenylalanine, alpha-methylphenylalanine did not exert these direct toxic effects because the administration of alpha-methylphenylalanine in vivo did not affect brain polyribosomes and a comparable concentration of this analogue was neither growth inhibitory nor cytotoxic to neuroblastoma cells in culture. The suitability of each analogue as an inhibitor of phenylalanine hydroxylase in animal models for phenylketonuria is discussed.