bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein synthesis in chloroplasts. Characteristics and products of protein synthesis in vitro in etioplasts and developing chloroplasts from pea leaves
Authors: Siddell S G , Ellis R J
Published: August 2015

The function of plastid ribosomes in pea (Pisum sativum L.) was investigated by characterizing the products of protein synthesis in vitro in plastids isolated at different stages during the transition from etioplast to chloroplast. Etioplasts and plastids isolated after 24, 48 and 96h of greening in continuous white light, use added ATP to incorporate labelled amino acids into protein. Plastids isolated from greening leaves can also use light as the source of energy for protein synthesis. The labelled polypeptides synthesized in isolated plastids were analysed by electrophoresis in sodium dodecyl sulphate-ureapolyacrylamide gels. Six polypeptides are synthesized in etioplasts with ATP as energy source. Only one of these polypeptides is present in a 150 000g supernatant fraction. This polypeptide has been identified as the large subunit of Fraction I protein (3-phospho-D-glycerate carboxylyase EC 4.1.1.39) by comparing the tryptic ‘map’ of its L-(35S)methionine-labelled peptides with the tryptic ‘map’ of large subunit peptides from Fraction I labelled with L-(35S)methionine in vivo. The same gel pattern of six polypeptides is seen when plastids isolated from greening leaves are incubated with either added ATP or light as the energy source. However, the rates of synthesis of particular polypeptides are different in plastids isolated at different stages of the etioplast to chloroplast transition. The results support the idea that plastid ribosomes synthesize only a small number of proteins, and that the number and molecular weight of these proteins does not alter during the formation of chloroplasts from etioplasts.
The mechanism of inhibition of ribonucleic acid synthesis by 8-hydroxyquinoline and the antibiotic lomofungin.
Authors: Fraser R S , Creanor J
Published: August 2015

RNA synthesis in yeast is rapidly inhibited by 8-hydroxyquinoline and the phenazine antibiotic lomofungin (5-formyl-1-methoxycarbonyl-4,6,8-trihydroxyphenazine). It is shown that lomofungin, like 8-hydroxyquinoline, is a chelating agent for bivalent cations. The mechanism of inhibition of RNA synthesis by lomofungin and 8-hydroxyquinoline was investigated in experiments with isolated Escherichia coli RNA polymerase. The results show that both inhibitors are capable of inhibiting polymerase activity solely by chelating the dissociable cations Mn2+ and Mg2+. Evidence is presented which shows that inhibition may occur in the absence of any direct contact between the RNA polymerase or DNA template and the inhibitor. The possibility that inhibition might also occur by chelation of the Zn2+, which is tightly bound to the polymerase, is discussed: it is concluded that lomofungin or 8-hydroxyquinoline is likely to inhibit the enzyme by removal of Mn2+ and Mg2+ before chelating the Zn2+. On the basis of inhibition by chelation of Mn2+ and Mg2+, explanations are proposed for why lomofungin and 8-hydroxyquinoline inhibit synthesis of ribosomal and polydisperse RNA more than that of 5S RNA and tRNA, and for why protein synthesis is not immediately inhibited in the intact yeast cell.
Purification of multiple forms of the soluble 17α-hydroxy steroid dehydrogenase or rabbit liver.
Authors: Hasnain S , Williamson D G
Published: August 2015

Eight distinct forms of the soluble 17alpha-hydroxy steroid dehydrogenase of rabbit liver were resolved by DEAE-cellulose chromatography and isoelectric focusing. Five of these enzymes were homogeneous as judged by polyacrylamide-gel electrophoresis. Substrate-specificity studies carried out with oestradiol-17alpha and oestradiol-17alpha 3-glucuronide revealed a variation in activity toward these substrates among the different purified enzyme forms. Three forms of the 17alpha-hydroxy steroid dehydrogenase exhibited equal activity toward both oestrogen substrates, whereas three forms of the enzyme displayed a greater activity toward the glucuronide derivative of oestradiol-17alpha. One enzyme in particular is essentially specific for oestradiol-17alpha 3-glucuronide, its activity toward oestradiol-17alpha being only one-thirtieth that observed with the 3-glucuronide derivative.
Preparation of polyribosome aminoacyl-transfer ribonucleic acid from the muscle of chick embryos.
Authors: Nwagwu M
Published: August 2015

A procedure for preparing polyribosome aminoacyl-tRNA free from contamination by supernatant aminoacyl-tRNA and free amino acids is described. Important features of the procedure are the use of acidic buffers to help protect the amino acid-tRNA linkage and the inclusion of sodium dodecyl sulphate, to inhibit ribonuclease activity. The specific radioactivity of polyribosome aminoacyl-tRNA is high within 30s and reaches a maximum in 2 1/2 min, well ahead of polyribosome peptides which, as described by Herrmann et al. (1971), attain maximum specific radioactivity in about 10 min.
The preparation of nylon-tube-supported hexokinase and glucose 6-phosphate dehydrogenase and the use of the co-immobilized enzymes in the automated determination of glucose.
Authors: Morris D L , Campbell J , Hornby W E
Published: August 2015

Triethyloxonium tetrafluoroborate was used to O-alkylate nylon-tube thus producing the imidate salt of the nylon which was further made to react with 1,6-diaminohexane. 2. Hexokinase (EC 2.7.1.1) and glucose 6-phosphate dehydrogenase (EC 1.1.1.49) were immobilized on the amino-substituted nylon tube through glutaraldeyde and bisimidates. 3. The effect of varying the conditions of O-alkylation and the amount of enzyme immobilized on the activity of nylon tube-hexokinase derivatives was determined. 4. The effect of varying the amount of enzyme immobilized on the activity of nylon-tube-glucose 6-phosphate dehydrogenase derivatives was determined. 5. The thermal stability of nylon-tube-hexokinase and nylon-tube-glucose 6-phosphate dehydrogenase derivatives was studied. 6. Different ratios of hexokinase and glucose 6-phosphate dehydrogenase were co-immobilized on nylon tube, and the rate of conversion of glucose into 6-phosphogluconolactone was compared with the individual activities of the immobilized enzymes. 7. Hexokinase and glucose 6-phosphate dehydrogenase co-immobilized on nylon tube were used in the automated analysis of glucose.
Isolation and protein composition of membranes of neurosecretory vesicles and plasma membranes from the neural lobe of the bovine pituitary gland
Authors: Vilhardt H , Baker R V , Hope D B
Published: August 2015

1. Homogenates of neural lobes of bovine pituitary glands were fractionated by differential ultracentrifugation. 2. Neurosecretory vesicles were isolated by sucrose-gradient ultracentrifugation and membranes were obtained after hypo-osmotic lysis of the particles. 3. A method is described for the isolation of a preparation of purified neuronal plasma membranes by using a fraction enriched in nerve endings as a starting material. 4. The purity of the subcellular fractions was estimated by enzyme assays and by examination with the electron microscope. 5. On the basis of the results it was estimated that neuronal plasma membranes constitute more than 30% of the protein of the nerve endings and neurosecretory vesicles more than 45% of the total amount of protein in the homogenate. 6. The proteins of membranes of neurosecretory vesicles and of plasma membranes were solubilized by means of sodium dodecyl sulphate. Polyacrylamide-gel electrophoresis of such preparations showed that both membranes contained a large number of proteins, including three glycoproteins.
The synthesis and secretion of cartilage procollagen
Authors: Harwood R , Bhalla A K , Grant M E , Jackson D S
Published: August 2015

1. Isolation of free and membrane-bound ribosomes from embryonic chick sternal-cartilage cells labelled for 4min with [14C]proline and their subsequent analysis for hydroxy[14C]proline indicated that cartilage procollagen biosynthesis occurs on bound ribosomes. 2. Nascent procollagen polypeptides on bound ribosomes isolated from cells labelled with [14C]lysine were found to contain hydroxy[14C]lysine indicating that hydroxylation of lysine commences while the growing chains are still attached to the ribosomes. 3. Analysis of bound ribosomes labelled with either [14C]proline or [14C]lysine on sucrose density gradients indicated that cartilage procollagen is synthesized on large polyribosomes in the range 250-400S. 4. Microsomal preparations isolated from cells pulse-labelled for 4 min with [14C]proline were used to determine the direction of release of nascent procollagen polypeptides. Puromycin induced the vectorial release of nascent procollagen polypeptides into the microsomal vesicles suggesting that the first step in the secretion of procollagen polypeptides is their transfer from the ribosomes through the membrane of the endoplasmic reticulum into the cisternal space. 5. The procollagen polypeptides secreted by cartilage cells were shown to be linked by inter-chain disulphide bonds. 6. Examination of the state of aggregation of pro-α chains in subcellular fractions isolated from cartilage cells labelled with [14C]proline for various periods of time have provided data on the timing and location of inter-chain disulphide-bond formation. This process commences in the rough endoplasmic reticulum after the release of completed pro-α chains from membrane-bound ribosomes. Pro-α chains isolated from fractions of smooth endoplasmic reticulum were virtually all present as disulphide-bonded aggregates, suggesting that either disulphide bonding is completed in this cellular compartment, or that procollagen needs to be in a disulphide-bonded form to be transferred to this region of the endoplasmic reticulum. 7. Comparison of these results with previously published data on disulphide bonding in tendon cells suggest that the rate of inter-chain disulphide-bond formation is significantly slower in cartilage cells.
Effect of a single oral dose of methanol, ethanol and propan-2-ol on the hepatic microsomal metabolism of foreign compounds in the rat
Authors: Powis G
Published: August 2015

Methanol and ethanol administered to rats as a single oral dose increased aniline hydroxylation by the hepatic microsomal fraction by a maximum of 169 and 66% respectively, whereas aminopyrine demethylation was inhibited by 51 and 61%. The concentration of microsomal cytochrome P-450, and the activities of NADPH-cytochrome c reductase and NADPH-cytochrome P-450 reductase were unchanged. Propan-2-ol, administered as a single oral dose, increased microsomal aniline hydroxylation by 165% and increased aminopyrine demethylation by 83%. The concentration of cytochrome P-450 was unchanged whereas NADPH-cytochrome c reductase and NADPH-cytochrome P-450 reductase were both increased by 38%. Methanol, ethanol and propan-2-ol administration resulted in a decreased type I spectral change but had no effect on the reverse type I spectral change. Methanol administration decreased the type II spectral change whereas ethanol and propan-2-ol had no effect. Cycloheximide blocked the increases in aniline hydroxylation and aminopyrine demethylation but could not completely prevent the decreases in aminopyrine demethylation. The increases in aniline hydroxylation were due to an increase in V, but Km was unchanged. The ability of acetone to enhance and compound SKF 525A to inhibit microsomal aniline hydroxylation was decreased by the administration of all three alcohols. The decrease in the metabolism of aminopyrine may result from a decrease in the binding to the type I site with a consequent failure of aminopyrine to stimulate the reduction of cytochrome P-450. Methanol administration may lead to an increase in aniline hydroxylation because of a failure of aniline to inhibit cytochrome P-450 reduction.
Colchicine binding to bovine anterior pituitary slices and inhibition of growth-hormone release
Authors: Sheterline P , Schofield J G , Mira F
Published: August 2015

The uptake of [ring C-methoxyl-3H]colchicine into bovine anterior pituitary slices was studied. The data suggest that more than one site exists for the binding of colchicine. At low concentrations colchicine binds to saturable trypsin-sensitive site(s), with a dissociation constant of 3.1 +/- 0.69 mug. The binding capacity of these sites is 8.58 +/- 0.60 pmol of colchicine/mg of wet pituitary. At higher colchicine concentrations binding occurs predominantly to sites which exhibit non-saturation kinetics. Subcellular fractionation of colchicine-labelled slices shows that 90% of the saturable sites are present in the fraction containing cytosol, where the binding protein has a molecular weight of about 11.9 x 10(4) and constitutes 0.7% of the protein present. The nuclear fraction contains 10% of the saturable sites, and the mitochondria and granule fraction contain only non-saturable sites. The rate of colchicine uptake was studied at 0.84 mm- and 2mum-colchicine. At both concentrations the colchicine space exceeded the total tissue water within 10 min. Equilibration with the saturable binding sites was complete in 120 min at 2mum-colchicine. A concentration of colchicine (13.4 mum) which would give 81% maximum binding was found to decrease the length of observable microtubules in tissue fixed at 37 degrees C in glutaraldehyde by 83 +/- 4%. The colchicine-binding protein could be partially purified by using a standard procedure for isolation of brain tubulin. Colchicine inhibits the release of growth hormone in the presence of 3-isobutyl-1-methylxanthine (0.1 mm), but does not alter basal release. The concentration-dependence of colchicine inhibition is similar to that of colchicine binding, but maximum inhibition is only 35%.
Enzymological aspects of the pathways for trimethylamine oxidation and C1 assimilation in obligate methylotrophs and restricted facultative methylotrophs
Authors: Colby J , Zatman L J
Published: August 2015

Extracts of trimethylamine-grown W6A and W3A1 (type M restricted facultative methylotrophs) contain trimethylamine dehydrogenase whereas similar extracts of Bacillus PM6 and Bacillus S2A1 (type L restricted facultative methylotrophs) contain trimethylamine mono-oxygenase and trimethylamine N-oxide demethylase but no trimethylamine dehydrogenase. Extracts of the restricted facultatives and of the obligate methylotroph C2A1 contain hexulose phosphate synthase-hexulose phosphate isomerase activity; hydroxypyruvate reductase was not detected. Neither the restricted facultatives nor the obligates 4B6 and C2A1 contain all the enzymes of the hexulose phosphate cycle of formaldehyde assimilation as originally proposed by Kemp & Quayle (1967). Organisms PM6 and S2A1 lack transaldolase and use a modified cycle involving sedoheptulose 1,7-diphosphate and sedoheptulose diphosphatase. The obligates 4B6 and C2A1, and the type M organisms W6A and W3A1, use a different modification of the assimilatory hexulose phosphate cycle involving the Entner-Doudoroff-pathway enzymes phosphogluconate dehydratase and phospho-2-keto-3-deoxygluconate aldolase. The lack of fructose diphosphate aldolase and hexose diphosphatase in these organisms may be a partial explanation of their restricted growth-substrate range. Enzymological evidence suggests that all the obligates and the restricted facultatives use a dissimilatory hexulose phosphate cycle to accomplish the complete oxidation of formaldehyde to CO2 and water.
Regulation of carbohydrate metabolism in lymphoid tissue. Quantitative aspects of [U-14C]glucose oxidation by rat spleen slices
Authors: Suter D , Weidemann M J
Published: August 2015

When washed spleen slices from fed rats are incubated with 3 mm-[U-14C]glucose, the rate of glucose utilization (46.2 mumol/h per g dry wt.) is sufficient to account, theoretically, for 80% of the O2 consumption. Measurement of net lactate production, however, and the fate of the radioactive carbon, indicates that the contribution of glucose to the respiratory fuel of the tissue is only 25-30% whereas 60-70% of the glucose utilized is converted into lactate. At saturating glucose concentrations (above 5 mm) its contribution to the respiratory fuel of the slice is increased to a maximum value of 34-39%. Only 2% of the glucose utilized is metabolized via the oxidative steps of the pentose phosphate pathway. Starvation for 72 h marginally increases both the rate of glucose utilization (by 21%) and its net contribution to the respiratory fuel (by 29%). Insulin, glucagon, adrenaline and adenosine 3′:5′-cyclic monophosphate have no significant effect on either the rate of glucose utilization or on the pattern of radioactive isotope distribution. The uptake of glucose is increased by only 20%, whereas the production of lactate doubles when slices are incubated under anaerobic conditions. In assessing the suitability of spleen slices for metabolic studies, the only serious major perturbation, compared with the freeze-clamped organ, is an elevated mitochondrial [NAD+]/[NADH] ratio (connected with increased endogenous NH3 production) that is partially restored to normal values on incubation with glucose. Equal proportions of erythrocytes and leucocytes are found in the washed spleen slice. Metabolic contributions of the constituent cell populations in the washed slice are calculated and it is concluded that lymphocytes account for the major part of the glycolytic metabolism (80-90%), whereas the contribution of erythrocytes is insignificant.
The complexity of satellite deoxyribonucleic acid in a higher plant
Authors: Sinclair J , Wells R , Deumling B , Ingle J
Published: August 2015

Purified satellite DNA from melon (Cucumis melo) was shown to contain at least two components from thermal-denaturation and renaturation studies. Two components were separated after partial renaturation, a fast-renaturing fraction similar in complexity to mouse satellite DNA, and one with 6000 times greater complexity. Both components renatured very accurately, indicating a minimum of sequence divergence. Centrifugation of the purified satellite DNA in Ag+/Cs2SO4 gradients resolved two major and several minor fractions. The two major fractions were only slightly enriched for fast- or slow-renaturing sequences.
The isolation of collagen-associated proteoglycan from bovine nasal cartilage and its preferential interaction with α2 chains of type I collagen
Authors: Lee-Own V , Anderson J C
Published: August 2015

A collagen complex from bovine nasal cartilage was prepared by extraction of the tissue with 3M-MgCl2 solutions, by using two different procedures. When it was compared with calf skin acid-soluble tropocollagen by polyacrylamide-gel electrophoresis, the 3M-MgCl2-soluble cartilage collagen in the complex appeared to be predominantly type I in nature, consisting of both α1 and α2 chains. The soluble cartilage collagens were digested with purified bacterial collagenase, and the soluble digests were fractionated on Sepharose 4B. Hydroxyproline-free proteoglycan was isolated in the excluded volume of the column eluate, and this was found to be an aggregate which could be dissociated to link proteins and proteoglycan subunit by equilibrium-density-gradient centrifugation in a CsCl-4M-guanidinium chloride gradient. Interaction with calf skin-soluble tropocollagen was studied by CM-cellulose chromatography. The link-protein system did not interact, but proteoglycan from the bottom of the gradient did interact. In addition, when proteoglycan subunit was allowed to interact with collagen, there was a preferential binding to the α2 and β12 components, and this effect was also observed with the proteoglycan material obtained from the collagenase digests of 3M-MgCl2-soluble cartilage collagen complexes. However, specificity for α2 and β12 chains was not exhibited by chondroitin sulphate glycosaminoglycan, and it is therefore concluded that preference for α2 and β12 chains is a function of the intact proteoglycan structure.
Rat haemoglobin heterogeneity. Two structurally distinct α chains and functional behaviour of selected components
Authors: Garrick L M , Sharma V S , McDonald M J , Ranney H M
Published: August 2015

Six haemoglobins were separated analytically from haemolysates of adult Wistar rats (Rattus norvegicus) by cellulose acetate electrophoresis and preparatively by DEAE-cellulose chromatography. The globin chains were separated from unfractionated haemolysates by CM-cellulose chromatography by using a non-linear formic acid-pyridine gradient followed by CM-cellulose chromatography in 8M-urea by using a gradient of increasing Na+ concentration in phosphate buffer, pH 6.7. Two α chains and three non-α chains were identified. Chains isolated from purified haemoglobins were correlated with chains isolated from unfractionated haemolysates by electrophoresis on urea-starch gels to make presumptive assignments of the subunit composition of the six haemoglobin tetramers. Partial amino acid sequences were determined for the major and minor α chains. The oxygen equilibria of two of the major haemoglobin components and of the unfractionated haemolysate were examined at pH 7.5 and 8.0. The two purified haemoglobins exhibited similar oxygen affinities; the haemolysate, however, had a lower oxygen affinity than either of the two purified haemoglobins. Both the haemolysate and the two haemoglobins showed an alkaline Bohr effect larger than that of human haemoglobin A.
Properties of protein-chlorophyll complexes from pea (Pisum sativum L.) leaves. The organization of chlorophyll
Authors: Scott B , Gregory R P F.
Published: August 2015

Chlorophyll-protein-detergent complexes were prepared from pea chloroplasts by using sodium dodecylbenzenesulphonate and polyacrylamide-gel electrophoresis. Circular-dichroism spectra showed that complex CPI has a dimeric arrangement of chlorophyll a, with additional weaker interactions. Ellipticities were determined for both complexes and for purified chlorophylls in solution, and it is argued that the circular dichroism of complex CPII is derived from chlorophyll-protein interaction rather than from interaction between chlorophylls a and b. The detergent could be removed from the complexes by using urea and gel filtration, leaving the chlorophyll-protein in solution, although in each case a diminished ellipticity indicated some loss of organization. Three-peaked circular-dichroism spectra of chloroplast fragments before and after addition of detergent were compared with a curve obtained by summing graphically the spectra of complexes CPI, CPII and the free-pigment fraction. There was good correspondence at 650 nm, and the longer-wavelength peaks agreed in form and magnitude, but with discrepancies in position. It was concluded that complexes CPI and CPII pre-exist in the original material, but that there is an environmental effect which is destroyed when the complexes are extracted.
The active site of penicillinase from Staphylococcus aureus PC1. Isolation of a specific covalent complex with the substrate quinacillin
Authors: Virden R , Bristow A F , Pain R H
Published: August 2015

1. The penicillinase-catalysed hydrolysis of quinacillin was quenched by addition of 5 m-guanidinium chloride or 1% (w/v) sodium dodecyl sulphate, and the quenched reaction mixture was dialysed exhaustively against solutions of the denaturant. 2. Irreversibly bound quinacillin was shown by titration with HgCl2 to be covalently attached to the protein by the β-lactam carboxyl group. 3. The derivative was found to be stable over the pH range 3.5-8.5. 4. Chymotryptic hydrolysis of the product and subsequent fractionation showed that quinacillin was bound to one or possibly two peptides.
Studies of the acetylcholinesterase from houseflies (Musca domestica L.) resistant and susceptible to organophosphorus insecticides
Authors: Devonshire A L
Published: August 2015

Acetylcholinesterase from the heads of insecticide-resistant and -susceptible houseflies (Musca domestica L.) was studied in vitro. The enzymes could not be distinguished electrophoretically, and their behaviour on polyacrylamide-disc-gel electrophoresis was influenced by the presence of Triton X-100 in both the homogenate and the gels. In the absence of detergent, the acetylcholinesterase was heterogeneous, but behaved as a single enzyme when it was present. By analogy with studies of acetylcholinesterase from other sources, these observations were attributed to aggregation of the enzyme when not bound by membranes. The enzyme from resistant flies was more slowly inhibited than the susceptible enzyme, bimolecular rate constants (ki) differing by approx. 4-20-fold for a range of organophosphorus compounds. The kinetics of inhibition of acetylcholinesterase were consistent with the results of electrophoresis, i.e. they corresponded to those of a single enzyme in the presence of Triton X-100, but a mixture of enzymes in its absence. The susceptibility of the more sensitive components in these mixtures was determined.
Determination of 2-acetamido-2-deoxy-D-galactose and mechanism of formation of chromogens
Authors: Serafini-Cessi F
Published: August 2015

A method for the colorimetric determination of 2-acetamido-2-deoxy-D-galactose was developed. The procedure is based on the high reactivity of the aldehyde group of this amidosugar with pentane-2,4-dione in anhydrous alkaline conditions. The product of reaction was crystallized and the structure 1-C-acetonyl-2-acetamido-2-deoxy-D-galactitol was deduced from chemical evidence. When the N-acetyl group of this compound is split off by hydrolysis, the formation of pyrrole groups ensues by condensation of the free amino group with the carbonyl group of the chain at C-1. 2-Methylpyrrole was isolated by steam distillation after mild alkaline hydrolysis and estimated by reaction with p-dimethylaminobenzaldehyde. A more complex pyrrole is formed during acid hydrolysis under the conditions used in the direct Ehrlich reaction.
Salmonella typhimurium HfrA, a mutant in which adenosine triphosphate can drive amino acid transport but not energy-dependent nicotinamide nucleotide transhydrogenation
Authors: Kay W W , Bragg P D
Published: August 2015

In contrast with wild-type Salmonella typhimurium LT2, strain HfrA did not have ATP-driven energy-dependent transhydrogenase activity, although ATP-dependent quenching of atebrin fluorescence was normal. Respiration-dependent and energy-independent transhydrogenase, and Ca2+-activated ATPase (adenosine triphosphatase) activities were similar in both strains. Purified ATPases from the two strains had similar specific activities, similar subunit polypeptides, and were equally effective in restoring energy-dependent transhydrogenase activities to membrane particles of strain LT2 from which the ATPase had been stripped. The purified ATPases from both strains could restore respiration-dependent but not ATP-dependent transhydrogenation to stripped particles of strain HfrA. Both strains grew aerobically equally well on salts media containing glucose, malate, succinate, citrate, acetate, pyruvate, fumarate, lactate or aspartate as substrates. Growth on glucose under anaerobic conditions was similar. Strains LT2 and HfrA were equally effective in the accumulation under both aerobic and anaerobic conditions of the amino acids proline, phenylalanine, histidine, lysine, isoleucine and aspartic acid. Inhibition of amino acid accumulation by KCN and dicyclohexylcarbodi-imide occurred to the same extent in both strains. The complete inhibition by dicyclohexylcarbodi-imide of amino acid uptake under anaerobic conditions suggested that ATP could drive amino acid uptake in both strains. The ability of strain HfrA to carry out ATP-dependent transport or quenching of atebrin fluorescence but not ATP-dependent transhydrogenation is different from the wild-type strain and from any previously described energy-coupling mutant. It is difficult to reconcile the properties of this mutant with the chemiosmotic hypothesis.
Calcitonin-sensitive adenylate cyclase in rat renal tubular membranes
Authors: Loreau N , Lepreux C , Ardaillou R
Published: August 2015

1. Renal tubular membranes from rat kidneys were prepared, and adenylate cyclase activity was measured under basal conditions, after stimulation by NaF or salmon calcitonin. Apparent Km value of the enzyme for hormone-linked receptor was close to 1 × 10(-8) M. 2. The system was sensitive to temperature and pH. pH was found to act both on affinity for salmon calcitonin-linked receptor and maximum stimulation, suggesting an effect of pH on hormone-receptor binding and on a subsequent step. 3. KCl was without effect areas whereas CoCl and CaCl2 above 100 muM and MnCl2 above 1 muM inhibited F--and salmon calcitonin-sensitive adenylate cyclase activities. The Ca2+ inhibition of the response reflected a fall in maximum stimulation and not a loss of affinity of salmon calcitonin-linked receptor for the enzyme. 4. The measurement of salmon calcitonin-sensitive adenylate cyclase activity as a function of ATP concentration showed that the hormone increases the maximum velocity of the adenylate cyclase. GTP, ITP and XTP at 200 muM did not modify basal, salmon calcitonin- and parathyroid hormone-sensitive adenylate cyclase activities. 5. Basal, salmon calcitonin- and F--sensitive adenylate cyclase activities decreased at Mg2+ concentrations below 10 mM. High concentrations of Mg2+ (100 mM) led to an inhibition of the F--stimulated enzyme. 6. Salmon calcitonin-linked receptor had a greater affinity for adenylate cyclase than human or porcine calcitonin-linked receptors. There was no additive effect of these three calcitonin peptides whereas parathyroid hormone added to salmon calcitonin increased adenylate cyclase activity, thus showing that both hormones bound to different membrane receptors. Human calcitonin fragments had no effect on adenylate cyclase activity. 7. Salmon calcitonin-stimulated adenylate cyclase activity decreased with the preincubation time. This was due to progressive degradation of the hormone and not to the rate of binding to membrane receptors.
A study of the interrelationship between the triacylglycerol and protein components of very-low-density lipoproteins using the perfused rat liver
Authors: Petersburg S J , Madeley A , Robinson D S
Published: August 2015

High and low rates of very-low-density-lipoprotein triacylglycerol release from the perfused rat liver were achieved by using livers taken respectively from animals that had been given fructose for 48h or from animals that had been starved for 18h. 2. The higher rates of very-low-density-lipoprotein triacylglycerol release by the livers of the fructose-fed rats were associated with higher rates of very-low-density-lipoprotein protein release. 3. When the livers were perfused in the presence of [3H]leucine, radioactivity was incorporated into the very-low-density-lipoprotein apoproteins. The higher rates of very-low-density-lipoprotein triacylgycerol and protein release by the livers of fructose-fed rats were associated with a greater total incorporation of radioactivity into those apoproteins that entered the running gel during polyacrylamide-gel electrophoresis. However, the distribution of radioactivity among the various apoproteins was not significantly changed by the dietary treatments used.
Amino acid biosynthesis in mixed rumen cultures
Authors: Sauer F D , Erfle J D , Mahadevan S
Published: August 2015

Mixed rumen micro-organisms, maintained in continuous culture readily incorporated labelled HCO3- and acetate into amino acids. Labelled propionate, in contrast, was utilized only for isoleucine biosynthesis, but failed to label other amino acids to any significant extent. Evidence was obtained showing that in these mixed, i.e. symbiotic, cultures foward tricarboxylic acid-cycle reactions only proceed to 2-oxoglutarate. 14C distribution in amino acids clearly shows that 2-oxoglutarate is not oxidized further by tricarboxylic acid-cycle enzymes. Instead, acetate is carboxylated to pyruvate which is then carboxylated to oxaloacetate. Oxaloacetate equilibrates with fumarate and thereby carbon atoms 1 and 4 as well as carbon atoms 2 and 3 are randomized. Evidence was also obtained for the carboxylation of propionate to 2-oxobutyrate, isovalerate to 4-methyl-2-oxopentanoate, phenylacetate and hydroxyphentlacetate to the corresponding phenyl- and hydroxyphenyl-pyruvic acids and succinate to 2-oxoglutarate. Of the amino acid precursors investigated, only 3-hydroxypyruvate, the precursor of serine, appeared to be synthesized via an oxidative step, i.e. 3-phosphoglyceric acid to 3-phosphohydroxypyruvic acid. Most 2-oxo precursors of amino acids in these organisms appear to be formed via reductive carboxylation of the precursor acid.
Stimulation of protein synthesis in cultured heart muscle cells by glucose
Authors: David M , Avi-Dor Y
Published: August 2015

Glucose stimulated the rate of incorporation of [3H]leucine into HCLO4-insoluble fraction of cultured rat heart muscle cells under both aerobic and anaerobic conditions. In the aerobic system the incorporation proceeded at a constant rate during 3h of incubation with and without glucose whereas in the anaeorbic system the incorporation ceased after approx. 60 min and could be renewed only by the addition of glucose. No correlation was found to exist between the above effect of glucose on protein synthesis and glucose-dependent changes in the intracellular ATP concentration. The extent of the stimulation of protein synthesis was related to the concentration of glucose. The effect of glucose was suppressed by cycloheximide but was not affected by actinomycin D. Glucose had no effect on the rate of transport of α-aminoisobutyric acid. Mannose also stimulated [3H]leucine incorporation. Substances that did not produce lactate were ineffective. Iodoacetate inhibited the stimulatory effect of glucose, but pyruvate, which by itself had no apprecialbe stimulatory action, relieved the inhibition induced by iodoacetate. There was no concomitant change in the concentration of ATP when iodoacetate inhibition was reversed by pyruvate. L-Lactate or other intermediates of energy metabolism could not relieve the inhibitory effect of iodoacetate.
The purification and properties of a β-N-acetylhexosaminidase from Trichomonas foetus
Authors: Edwards R G , Thomas P , Westwood J H
Published: August 2015

A β-N-acetylhexosaminidase was purified 800-fold from extracts of Trichomonas foetus by affinity chromatography on a column of N-(epsilon-aminohexanoyl)-2-acetamido-2-deoxy-β-D-glucopyranosylamine bound to CNBr-activated Sepharose. The enzyme has a dual specificity for the p-nitrophenyl β-D-glycosides of N-acetylglucosamine and N-acetyl-galactosamine. The parent sugars are both competitive inhibitors. The enzyme has a mol. wt. approx. 150000 and a pH optimum of 6.2. It is suggested that the same active site catalyses both activities and that no part is played by the 4-hydroxyl group in substrate binding, but it is involved in determining the catalytic rate.
Immunochemical studies with a specific antiserum to rabbit fibroblast collagenase
Authors: Werb Z , Reynolds J J
Published: August 2015

1. Antisera were raised against the collagenase from rabbit synovial fibroblasts and characterized by immunoprecipitation and immunoinhibition reactions. 2. Immunoglobulins from the antisera were potent inhibitors of the action of rabbit collagenase on both reconstituted collagen fibrils and collagen in solution. 3. The antibody-binding fragment, Fab′, produced by digesting the IgG (immunoglobulin G) with pepsin, inhibited collagenase activity just as well as whole IgG. 4. A specific antiserum to the rabbit collagenase was raised by a multi-step procedure. An initial antiserum was made by injecting partially purified collagenase as a complex with sheep α2-macroglobulin into a sheep. The non-specific antiserum so obtained was used to produce precipitin lines with the purified enzyme, and these lines were used as antigen for the production of the specific antiserum. 5. An IgG preparation from the specific antiserum was a specific and potent inhibitor of the rabbit synovial fibroblast collagenase. Neutral metallo-proteinase activity secreted by the rabbit fibroblasts was not inhibited by the antibody to the rabbit collagenase. 6. Criteria for determination of the specificity of antisera are discussed.
Isolation of membrane-bound renal kallikrein and kininase
Authors: Ward P E , Gedney C D , Dowben R M , Erdös E G
Published: August 2015

Fractions highly enriched in plasma membrane, endoplasmic reticulum or brush border were prepared from homogenized rat kidney cortex. Kallikrein was concentrated in the plasma-membrane fraction, but not in the brush border of the proximal tubules. Kininase II or angiotensin I-converting enzyme was localized in the brush-border membrane. It is suggested that kallikrein in the urine may originate from the plasma membrane of the distal tubules and the conversion of angiotensin I and the inactivation of bradykinin may occur on the lumen membrane of the proximal tubular cells.
Creatine kinase equilibrium and lactate content compared with muscle pH in tissue samples obtained after isometric exercise
Authors: Sahlin Kent , Harris Roger C. , Hultman Eric
Published: August 2015

Muscle biopsies taken from the musculus quadriceps femoris of man were analysed for pH, ATP, ADP, AMP, creatine phosphate, creatine, lactate and pyruvate. Biopsies were taken at rest, after circulatory occlusion and after isometric contraction. Muscle pH decreased from 7.09 at rest to 6.56 after isometric exercise to fatigue. Decrease in muscle pH was linearly related to accumulation of lactate plus pyruvate. An increase of 22μmol of lactate plus pyruvate per g of muscle resulted in a fall of 0.5pH unit. The apparent equilibrium constant of the creatine kinase reaction (apparent KCK) increased after isometric contraction and a linear relationship between log(apparent KCK) and muscle pH was obtained. The low content of creatine phosphate in muscle after contraction as analysed from needle-biopsy samples is believed to be a consequence of an altered equilibrium state of the creatine kinase reaction. This in turn is attributed mainly to a change in intracellular pH.
The kinetics of incorporation in vivo of [14C]acetate and [14C]carbon dioxide into the fatty acids of glycerolipids in developing leaves
Authors: Slack C. R. , Roughan P. G.
Published: August 2015

1. The patterns of incorporation of 14C into glycerolipid fatty acids of developing maize leaf lamina from supplied [1-14C]acetate and from 14CO2 during steady-state photosynthesis were similar. Oleate of phosphatidylcholine and palmitate of phosphatidylglycerol attained linear rates of labelling more rapidly than did other fatty acids, particularly the linoleate and linolenate of monogalactosyl diacylglycerol. 2. After the transfer of lamina from labelled to unlabelled acetate, there was a decrease in labelled oleate and linoleate of phosphatidylcholine and a concomitant increase in the amount of radioactivity in the linoleate and linolenate of monogalactosyl diacylglycerol. 3. The rapidly labelled phospholipids, phosphatidylcholine and phosphatidylglycerol, were shown by differential and sucrose-density-gradient centrifugation to be associated with different organelles, the former being mainly in a low-density membrane fraction, probably microsomal, and the latter mainly in chloroplasts. 4. During a 48h period after supplying spinach leaves with [14C]acetate, radioactivity was lost from the oleate of phosphatidylcholine present in fractions sedimented at 12000g and 105000g, and accumulated in the linolenate of monogalactosyl diacylglycerol of the chloroplast. 5. It is proposed that the phosphatidylcholine of some non-plastid membranes is intimately involved in the process of oleate desaturation and that this lipid serves as a donor of unsaturated C18 fatty acids to other lipids, principally monogalactosyl diacylglycerol, of the chloroplasts.
The action of tributyltin on energy coupling in coupling-factor-deficient submitochondrial particles
Authors: Dawson Alan P. , Selwyn Michael J.
Published: August 2015

1. Tributyltin at concentrations of approx. 1nmol/mg of protein induces respiratory control and lessens the protein permeability of coupling-factor-deficient submitochondrial particles. 2. At these concentrations or lower, it increases the P/O ratio of the particles to a small extent and inhibits the adenosine triphosphatase activity without greatly increasing its sensitivity to uncoupling agents. 3. It fails to stimulate ATP-driven reversed electron transport or transhydrogenase, but stimulates the transhydrogenase driven by aerobic succinate oxidation. 4. The results indicate that, unlike oligomycin, tributyltin does not discriminate between damaged and intact ATP-synthesizing complexes. 5. The relationship between the oligomycin- and tributyltin-binding sites is discussed.
Biochemical evidence for the reversed polarity of the outer membrane of the bacterial forespore
Authors: Wilkinson B J , Deans J A , Ellar D J
Published: August 2015

Measurement of certain membrane-bound enzymic activities was used to study the orientation of the outer membrane of the double-membraned forespore of Bacillus megaterium KM. 2. Adenosine triphosphatase, NADH dehydrogenase and L-malate intact protoplasts, but were readily detected in intact stage II or IV forespores, consistent with reversed polarity of the outer forespore membrane relative to the mother-cell plasma membrane. 3. Measurement of NADH oxidase activity revealed that intact stage III forespores had the same high affinity for NADH as protoplast membrane preparations and protoplast lystates, consistent with ready access of NADH to oxidation sites on the outer forespores membrane. 4. Forespores and protoplasts showed osmometric behaviour in solutions of non-permanent solutes consistent with the presence of an intact permeability barrier in these structures.
Substrate-binding ability of Escherichia coli ribonucleic acid polymerase in relation to its protein composition
Authors: Rognes A , Abraham K A
Published: August 2015

Interaction between Escherichia coli RNA polymerase and its substrates, the nucleoside triphosphates, was studied by gel-filtration and dialysis-rate-measurement techniques. 2. The holoenzyme bound variable amounts of ATP and GTP. There was no correlation between substrate-binding ability and enzyme activity of different enzyme preparations. 3. The core enzyme bound a maximum of 0.1 mol of ATP/mol of enzyme. The dissociation constant of this interaction was of the order of 1 × 10(-5)M. The core enzyme did not bind GTP. 4. A protein of mol.wt. 60000, which was eluted in the first fraction during phosphocellulose column chromatography of the holoenzyme, bound appreciable amounts of ATP. The dissociation constant of this interaction was of the order of 3 × 10(-5)-5 × 10(-6)M. 5. Evidence presented shows that this protein, and not the sigma factor, is responsible for the observed variation in the ATP-binding ability of the holoenzyme.
β-amylase-resistant amylose. Effect of urea on the limited hydrolysis of amylose by β-amylase
Authors: Patil N B
Published: August 2015

Amylose prepared from starch dispersed in 10M-urea, pH6.2, was found to be resistant to the action of β-amylase and phosphorylase, though it was degraded by α-amylase. Amylose isolated by conventional methods was similarly refractory after urea treatment, and was hydrolysed by β-amylase to the extent of 32-35%; it had no inhibitory effect towards β-amylase. The physical and chemical properties of the modified amylose were in general comparable with those of normal amylose with a β-amylolysis limit of 94-98%%. Starch and amylopectin were unaffected by urea treatment, i.e. the presence of amylopectin protected amylose against changes induced in it by urea. It is speculated that urea treatment “freezes” amylose molecules in a conformation that renders non-reducing termini inaccessible to the active site of the exo-enzymes. Such changes may limit the degradative action of β-amylase and phosphorylase.
A study of the physicochemical interactions between biliary lipids and chlorpromazine hydrochloride. Bile-salt precipitation as a mechanism of phenothiazine-induced bile secretory failure
Authors: Carey M C , Hirom P C , Small D M
Published: August 2015

Since chlorpromazine hydrochloride [2-chloro-10-(3-dimethylaminopropyl)-phenothiazine hydrochloride] is commonly implicated in causing bile-secretory failure in man and is secreted into bile, we have studied the physicochemical interactions of the drug with the major components of bile in vitro. Chlorpromazine hydrochloride molecules are amphiphilic by virtue of possessing a polar tertiary amine group linked by a short paraffin chain to a tricyclic hydrophobic part. At pH values below the apparent pK (pK'a 7.4) the molecules are water-soluble cationic detergents. We show that bile salts in concentrations above their critical micellar concentrations are precipitated from solution by chlorpromazine hydrochloride as insoluble 1:1 salt complexes. In the case of mixed bile-salt/phosphatidylcholine micellar solutions, however, the degree of precipitation is inhibited by the phospholipid in proportion to its mole fraction. With increases in the concentration of chlorpromazine hydrochloride or bile salt, micellar solubilization of the precipitated complexes results. Sonicated dispersions of the negatively charged phospholipid phosphatidylserine were also precipitated, but dispersions of the zwitterionic phospholipid phosphatidylcholine were not. Chlorpromazine hydrochloride efficiently solubilized these membrane phospholipids as mixed micellar solutions when the drug:phospholipid molar ratio reached 4:1. Polarizing-microscopy and X-ray-diffraction studies revealed that the precipitated complexes were amorphous and potentiometric studies confirmed the presence of a salt bond. Some dissociation of the complex occurred in the case of the most polar bile salt (Ks 0.365). As canalicular bile-salt secretion determines much of bile-water flow, we propose that complexing and precipitation of bile salts by chlorpromazine hydrochloride and its metabolites may be physicochemically related to the reversible bile-secretory failure produced by this drug.
4-NN-dimethylaminoazobenzene 4′-isothiocyanate, a new chromophoric reagent for protein sequence analysis
Authors: Chang J Y , Creaser E H , Bentley K W
Published: August 2015

4-NN-Dimethylaminoazobenzene 4‘-isothiocyanate was synthesized for the purpose of improving the ease and sensitivity of peptide sequence analysis. The method of 4-NN-dimethylaminoazobenzene 4′-isothiocyanate synthesis, the preparation of 24 4-NN-dimethylaminoazobenzene-4′-thiohydantoins of amino acids and their t.l.c. separation are described. All the thiohydantoins, except those of leucine and isoleucine, could be satisfactorily separated by chromatography on a two-dimensional polyamide sheet. The sensitive azo group permits the detection of 4-NN-dimethylaminoazobenzene-4′-thiohydantoins of amino acids as red spots down to pmol amounts directly on the sheet. A simple sensitive method for sequencing dipeptides and the first two or three N-terminal amino acids of proteins is also reported. The colour change of the spots from purple to blue to red after being exposed to HCl vapour, corresponding to the chemical change from 4-NN-dimethylaminoazobenzene-4′ ’ isothiocyanate to the 4-NN-dimethylaminoazobenzene-4′-thiocarbamoyl amino acid derivative to the 4-NN-dimethylaminoazobenzene-4′-thiohydantoin amino acid derivative, reveals a very interesting and valuable feature of this reagent.
Modified methods of dialysis
Authors: Feinberg J G
Published: August 2015

Dialysis modifications are described that are based on the use of a novel rotating dialysis bottle in one- or two-solvent systems. Advantages include complete mixing and rapid equilibrium of diffusing substances, favourable volume changes, economy of solvent and more effective removal of diffusible materials.