bluuebunny/crossref_metadata_2025 huggingface dataset:
Equilibrium and kinetic studies of the aggregation of porphyrins in aqueous solution
Authors: Brown S B , Shillcock M , Jones P
Published: August 2015

An investigation of the behavior of protoporphyrin IX, deuteroporphyrin IX, haematoporphyrin IX and coproporphyrin III in aqueous solution revealed extensive and complex aggregation processes. Protoporphyrin appears to be highly aggregated under all conditions studied. At concentrations below 4 μM, aggregation of deutero-, haemato- and coproporphyrin is probably restricted to dimerization. At approx. 4muM each of these three porphyrins exhibits sharp changes in spectra consistent with a “micellization” process to form large aggregates of unknown size. This critical concentration increases with increasing temperature and pH, but is not very sensitive to variation in ionic strength. Temperature-jump kinetic studies on deuteroporphyrin also imply an initial dimerization process, the rate constants for which are comparable with those for various synthetic porphyrins, followed by a further extensive aggragation. The ability of a particular porphyrin to dimerize appears to parallel that of the corresponding iron(III) complexes (ferrihaems), although it is thought that ferrihaems do not exhibit further aggregation under these conditions.
Activities of citrate synthase and other enzymes of Acetobacter xylinum in situ and in vitro
Authors: Swissa M , Weinhouse H , Benziman M
Published: August 2015

The activities of a number of enzymes, extracted from Acetobacter xylinum, that are involved in carbohydrate metabolism may be accounted for in situ in permeabilized cells. The kinetic properties of citrate synthase and glycerokinase observed in vitro are also retained in situ. So is the regulatory sensitivity of these enzymes. Both in vitro and in situ, (a) citrate synthase, in contrast with the enzyme for other Gram-negative bacteria, is inhibited by ATP and is insensitive to NADH, and (b) glycerokinase is inhibited by fructose diphosphate and the ratio of its activities towards glycerol and dihydroxyacetone is the same.
Identiy of brain alcohol dehydrogenase with liver alcohol dehydrogenase
Authors: Duncan R J , Kline J E , Sokoloff L
Published: August 2015

A method for obtaining electrophoretically homogeneous rat liver alcohol dehydrogenase (EC 1.1.1.1) at a specific activity of 2-2.5 μmol/min per mg of protein is presented. Anti-sera prepared against the purified enzyme inhibit alcohol dehydrogenase by up to 75% and cause precipitation of virtually all the enzyme. The antisera were shown by immunoelectrophoresis of a partially purified liver homogenate to be specifically directed against alcohol dehydrogenase and were used to demonstrate that the alcohol dehydrogenases of rat brain and liver share common antigens. The total activity of alcohol dehydrogenase in rat brain homogenates is normally quite low, with as much as 10% of the total activity attributable to the activity in the blood contained within the brain; in cases of severe liver damage (induced experimentally with carbon tetrachloride) this contribution may rise to as much as 60%.
Similarities in the polypeptide composition of glyoxysomal and endoplasmic-reticulum membranes from castor-bean endosperm
Authors: Bowden L , Lord J. M
Published: August 2015

Microsomal fractions, glyoxysomes and mitochondria were isolated from homogenates of germinating castor-bean (Ricinus communis) endosperm by sucrose-density-gradient centrifugation. Washed membrane preparations from these cellular fractions were examined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. At corresponding developmental stages the endoplasmic-reticulum and glyoxysomal membranes were strikingly similar in polypeptide composition, at least 16 polypeptides being present in membranes isolated from 3-day-old tissue. Supplying [35S]methionine to intact endosperm tissue resulted in the labelling of all membrane polypeptides, the specific radioactivity in the endoplasmic reticulum being greater than for equivalent polypeptides of the glyoxysomal membrane. Washing these membranes with sodium deoxycholate solution extensively solubilized protein components, with the exception of a predominant polypeptide of mol.wt. 55000. Mitochondrial membrane preparations differed from those of the endoplasmic reticulum and glyoxysomes in polypeptide molecular-weight distribution and the [35S]methionine-labelling pattern. The similarity in polypeptide composition between endoplasmic-reticulum and glyoxysomal membranes is discussed in relation to glyoxysome biogenesis.
The cellular origin of glyoxysomal proteins in germinating castor-bean endosperm
Authors: Bowden L , Lord J. M
Published: August 2015

The capacity of castor-bean endosperm tissue to incorporate [35S]methionine into proteins of the total particulate fraction increased during the first 3 days of germination and subsequently declined. At the onset of germination 66% of the incorporated 35S was found in the separated endoplasmic-reticulum fraction, with the remainder in mitochondria, whereas at later developmental stages an increasing proportion of 35S was recovered in glyoxysomes. The kinetics of [35S]methionine incorporation into the major organelle fractions of 3-day-old endosperm tissue showed that the endoplasmic reticulum was immediately labelled, whereas a lag period preceded the labelling of mitochondria and glyoxysomes. When kinetic experiments were interrupted by the addition of an excess of unlabelled methionine, incorporation of [35S]methionine into the endoplasmic reticulum rapidly ceased, but incorporation into mitochondia and glyoxysomes continued for a further 1h. Examination of isolated organelle membranes during this period showed that the addition of unlabelled methionine resulted in a stimulated incorporation of [35S]no methionine into the endoplasmic-reticulum membrane for 30 min, after which time the 35S content of this fraction declined, whereas that of the glyoxysomal membranes continued to increase slowly. The 35S-labelling kinetics of organelles and fractions derived therefrom are discussed in relation to the role of the endoplasmic reticulum in protein synthesis during glyoxysome biogenesis.
Renin substrate in granules from rat kidney cortex
Authors: Morris B J. , Johnston C I.
Published: August 2015

1. Subcellular fractions of rat kidney cortex generated angiotensin I continuously over 2h when incubated at 37degreesC with rat renin, indicating the presence of renin substrate within cells in the renal cortex. 2. Renin substrate was located in highest specific concentration in particulate fractions. The particles containing renin substrate had a sedimentation velocity slightly lower than mitochondria and renin granules but greater than the microsomal fraction. 3. Isopycnic gradient centrifugation indicated a density of 1.190g/ml for the particles containing renin substrate, compared with 1.201 for renin granules, 1.177 for mitochondria, and 1.170 and 1.230 for lysosomes in the heavy-granule fraction. 4. In the liver, renin substrate was also found in particles, but these had a lower sedimentation rate than those from the kidney. 5. The molecular weights of renin substrate in kidney and liver granules and rat plasma were similar, namely 61000-62000. 6. On the basis of these biochemical findings, a mechanism for the intrarenal production of angiotensin, incorporating a subcellular reaction scheme, is proposed.
Effect of succinylation (3-carboxypropionylation) on the conformation and immunological activity of ovalbumin
Authors: Kidwai S A , Ansari A A , Salahuddin A
Published: August 2015

The epsilon-amino groups of ovalbumin were modified with succinic anhydride; as many as 16 lysine residues were succinylated (3-carboxypropionylated). The five succinylated derivatives thus prepared were homogeneous with respect to the extent of chemical modification as shown by electrophoretic and immunological data. Succinylation of the amino groups altered electrophoretic mobility and isoionic pH of ovalbumin in the expected direction. U.v.-absorption and fluorescence spectra suggested changes in the microenvironment of the chromophores in the modified proteins. The difference-spectral results showed greater exposure of tyrosine and tryptophan residues in the succinylated ovalbumin. Increase in susceptibility to tryptic digestion, Stokes radius and intrinsic viscosity of native ovalbumin, which was observed on successive increase in the chemical modification, demonstrated a conformational change that was proportional to the extent of modification. The loss of immunological reactivity caused by chemical modification also indicated a conformational change in succinylated ovalbumin. The fact that the intrinsic viscosity of maximally modified ovalbumin was less than one-third of that for the completely denatured protein in 6M-guanidinium chloride suggested that the modified protein contained significant residual native structure. The latter presumably accommodates some antigenic determinants accounting for 37% residual immunological activity observed with maximally succinylated ovalbumin.
The concerted inactivation of Escherichia coli uridine diphosphate galactose 4-epimerase by sugar nucleotide together with a free sugar
Authors: Blackburn P , Ferdinand W
Published: August 2015

1. The combined effect of the sugar nucleotides UDP-D-fucose or UDP-D-glucuronic acid together with the free sugars D-fucose or L-arabinose is the inactivation of the Escherichia coli enzyme UDP-galactose 4-epimerase (EC 5.1.3.2). The sugar nucleotide or the free sugar alone or the sugar nucleotide plus 5'-Ump do not inactivate the enzyme. 2. The inactivation of the enzyme by its substrate UDP-D-glucose was not affected by the presence of free sugar. 3. In all cases the inactivation observed follows pseudo-first-order kinetics. 4. A comparison of various sugar nucleotides indicates that the hydroxymethyl group at position 6 of the sugar moiety of the natural substrates is important for substrate binding.
Human cathepsin G. Catalytic and immunological properties
Authors: Starkey P M. , Barrett A J.
Published: August 2015

1. The specificity of cathepsin G, a neutral proteinase from human spleen, was examined by use of low-molecular-weight substrates. The enzyme was found to hydrolyse several synthetic substrates also hydrolysed by chymotrypsin, but with different kinetic constants. 2. Maximal activity against benzoyl-DL-phenylalanine 2-naphthol ester and azo-casein was in the range pH 7.5-8.0. 3. The sensitivity of cathepsin G to the action of potential inhibitors was determined, and compared with those of bovine chymotrypsin and subtilisin. Cathepsin G showed the characteristics of a serine proteinase, but was less affected by the chloromethyl ketone of tosylphenylalanine than was chymotrypsin. 4. A rabbit anti-(human cathepsin G) serum was raised, and precipitin lines formed in agarose gel were stained for activity of the enzyme. 5. Cathepsin G was shown to be immunologically identical with the chymotrypsin-like enzyme of the azurophil granules of the neutrophil granulocytes.
The computation of hyperbolic dependences in enzyme kinetics
Authors: Airas R. K
Published: August 2015

A computer program aimed at analysing results following Michaelis-Menten kinetics can be used unmodified in the treatment of other kinetic results provided that the kinetic equations in these cases can be written in the form of the Michaelis-Menten equation. A list is presented of the parameters to be set instead of substrate concentration and reaction rate, and of constants replacing Km and V, if such a program is applied in analysing enzyme inhibitions, activations and pH-dependences.
[Nitrotyrosyl]cytochrome c. Studies of the effect of iron binding, protein denaturants and oxidation-reduction potentials
Authors: Do Nascimento A J
Published: August 2015

Static measurements of the reaction of ligand binding were done by conventional spectrophotometry. The ligand-binding reactions with nitrated cytochrome c were performed with imidazole, iminazole, CO and NO. The stoicheiometry was found to be 1:1, and the stability constants for the complexes formed between the nitrated cytochrome c and the ligands are: 2.58 × 10(4) M-1 (imidazole); 1.01 × 10(2) M-1 (iminazole); 3.6 × 10(4) M-1 (CO); 2.74 × 10(4) M-1 (NO). It was found that the electrometric potentials at pH 7.0 and 25degreesC of [aminotyrosyl]cytochrome c are E'o form II = 0.115 V and E'o form I = 0.260 V, where forms I and II are two species of protein co-existing in the protein solution. The isoelectric point for the oxidized form of [nitrotyrosyl]cytochrome c was 10.05, at 4degreesC.
Mode of interaction between β-lactam antibiotics and the exocellular DD-carboxypeptidase–transpeptidase from Streptomyces R39.
Authors: Fuad N , Frère J M , Ghuysen J M , Duez C , Iwatsubo M
Published: August 2015

The exocellular DD-carboxypeptidase-transpeptidase of Streptomyces R39 is inhibited by beta-lactam antibiotics according to the same general scheme of reaction as the exocellular DD-carboxypeptidase-transpeptidase of Streptomyces R61. However, the values for the kinetic constants involved in the reaction are very different for the two enzymes and provide an explanation for the observation that the R39 enzyme is more sensitive to beta-lactam antibiotics than the R61 enzyme. Further, particular beta-lactams influence the kinetic constants to different extents depending on the source of the enzyme, so that a physical basis for the spectrum of antibiotic activity against particular enzyme systems is provided.
Modification of brain deoxyribonucleic acid base content with maturation in normal and malnourished rats
Authors: Penn N W
Published: August 2015

The mol percentage of 5-hydroxymethylcytosine is 2.2 times greater in the adult than in 2-day-old rat brain DNA. The concentration of 5-hydroxymethylcytosine falls in corresponding liver DNA preparations. This normal increase in brain 5-hydroxymethylcytosine is abolished in rats placed on an 8%-protein diet 5 days after birth.
Effect of elongation factor 2 and of adenosine diphosphate-ribosylated elongation factor 2 on translocation
Authors: Montanaro L , Sperti S , Testoni G , Mattioli A
Published: August 2015

1. The effect of elongation factor 2 (EF 2) and of adenosine diphosphate-ribosylated elongation factor 2 (ADP-ribosyl-EF 2) on the shift of endogenous peptidyl-tRNA from the A to the P site of rat liver ribosomes (measured by the peptidyl-puromycin reaction) and on the release of deacylated tRNA (measured by aminoacylation) was investigated. 2. Limiting amounts of EF2, pre-bound or added to ribosomes, catalyse the shift of peptidyl-tRNA in the presence of GPT; when the enzyme is added in substrate amounts GMP-P(CH2)P [guanosine (beta, gamma-methylene)triphosphate] can partially replace GTP. ADP-ribosyl-EF 2 has no effect on the shift of peptidyl-tRNA when present in catalytic amounts, but becomes almost as effective as EF 2 when added in substrate amounts together with GTP; GMP-P(CH2)P cannot replace GTP. 3. The release of deacylated tRNA is induced only by substrate amounts of added EF 2 and also occurs in the absence of guanine nucleotides. In this reaction ADP-ribosyl-EF 2 is only 25% as effective as EF 2 in the absence of added nucleotide, but becomes 60-80% as effective in the presence of GTP or GMP-P(CH2)P. 4. The results obtained on protein-synthesizing systems are consistent with the hypothesis that ADP-ribosyl-EF 2 can operate a single round of translocation followed by binding of aminoacyl-tRNA and peptide-bond formation. 5. From the data obtained with the native enzyme it is concluded that the two moments of translocation require different conditions of interaction of EF 2 with ribosomes; it is suggested that the shift of peptidyl-tRNA is catalysed by EF 2 pre-bound to ribosomes, and that the release of tRNA is induced by a second molecule of interacting EF 2. The hydrolysis of GTP would be required for the release of pre-bound EF 2 from ribosomes. 5. The inhibition of the utilization of limiting amounts of EF 2 on ADP-ribosylation is very likely the consequence of a concomitant decrease in the rate of association and dissociation of the enzyme from ribosomes.
Production of 1,2-diacylglycerol and phosphatidate in human erythrocytes treated with calcium ions and ionophore A23187
Authors: Allan D , Watts R , Michell R H
Published: August 2015

1. When the ionophore A23187 and Ca2+ were added to normal human erythrocytes, the incorporation of 32P into phosphatidate was enhanced within 1 min, but there was only slight labelling of other phospholipids. 2. Labelling of phosphatidate in these cells did not continue to increase after about 20min at 37°C; by this time, radioactivity in phosphatidate was about ten times higher inionophore A23187-treated cells than in controls. A net synthesis of phosphatidate was measured in response to the increase in intracellular Ca2+ concentration; the content of this phospholipid in the cell was increased by approximately ∼50%. 3. In the presence of 2.5 mM-Ca2+ a maximum effect was seen with about 0.5 μg of ionophore/ml. 4. The concentration of Ca2+ giving half-maximal labelling of phosphatidate in the presence of 10 μg of ionophore A23187/ml was about 10 μM. 5. A rapid decrease of ATP content in the cell occurred in ionophore-treated cells. 6. Labelling of phosphatidate appeared to be secondary to the production of 1,2-diacylglycerol in the cells; accumulation of 1,2-diacylglycerol was only seen after about 15 min. After 60 min, the 1,2-diacylglycerol content of the cells was five to seven times that of untreated control cells. 7. The change in the shape of erythrocytes treated with Ca2+ and ionophore appeared to be related to accumulation of 1,2-diacylglycerol. 8. The source of 1,2-diacylglycerol has not been definitely identified, but its fatty acid compositon was similar to that of phosphatidylcholine. However, it has an unusually high content of hexadecenoic acid, a fatty acid not common in the major erythrocyte phospholipids. 9. Accumulation of 1,2-diacyglycerol also occurred in energy-starved cells, even in the absence of calcium; in this case it appeared to be produced by phosphatidate breakdown.
Isolation of a Golgi-apparatus-enriched fraction from leukaemic cells
Authors: Warley A , Cook G M
Published: August 2015

1. A Golgi-apparatus-enriched fraction was isolated from acute leukaemic lymphoblasts of AKR mice by using an homogenate stabilized with 1 mM-glutaraldehyde. 2. The isolated fraction, which was shown morphologically to be enriched in dictyosomes, possessed between 44- and 76-fold increase in specific activity, compared with the tumour homogenate, of UDP-galactose-glycoprotein galactosyltransferase and between 3- and 10.5-fold increase in relative specific activity of UDP-N-acetygalactosamine-polypeptide N-acetylgalactosaminyltransferase. 3. Plasma membranes isolated from the leukaemic lymphoblasts also possessed glycoprotein galactosyltransferase activity, though in contrast with Golgi-apparatus-enriched material had no detectable polypeptide N-acetygalactosaminyltransferase. 4. The difficulties associated with maintaining the morphological integrity of the Golgi apparatus in subcellular fractionation are discussed.
The purification and biological properties of pancreatic big glucagon
Authors: O'Connor K J , Lazarus N R
Published: August 2015

1. Big glucagon was present in extracts of ox, dog, rat and turkey pancreas, representing 10-15% of the glucagon immunoreactivity, and was shown to be of islet origin by its presence in extracts of isolated pigeon islets. 2. Big glucagon was homogeneous by immunoassay after polyacrylamide-gel electrophoresis and was more electronegative than little glucagon. 3. Big glucagon was purified from bovine pancreas, and its apparent molecular weight was estimated by gel filtration as 8200±9%. 4. Limited tryptic proteolysis of the molecule produced an immunoreactive component slightly smaller than little glucagon. 5. Linear dilution curves were obtained with mammalian big glucagons by using both enteroglucagon cross-reacting and ‘little-glucagon-carboxyl-end-specific’ antisera. 6. The half-times for the disappearance of the immunoreactivity of big and little glucagon that had been injected into the rat circulation were 6.9 and 3.2min respectively. 7. Big glucagon was approximately one-sixth as effective as little glucagon in displacing radioactive little glucagon from its liver membrane receptor. 8. Big glucagon was equipotent on a molar basis with little glucagon in the stimulation of the mouse islet adenylate cyclase, an indicator of insulinogenic activity. 9. On a molar basis, big glucagon inhibited basal liver adenylate cyclase activity to the same extent that little glucagon stimulated the enzyme. 10. Big glucagon was without effect on blood glucose concentration in the rat in doses up to 5μg/kg. 11. Big glucagon was equipotent, on a molar basis, with little glucagon in stimulating lipolysis in isolated chicken fat-cells.
Effects of clofibrate on some microsomal hydroxylations involved in the formation and metabolism of bile acids in rat liver
Authors: Angelin B O , Björkhem I , Einarsson K
Published: August 2015

1. The liver microsomal metabolism of [4-14C]cholesterol, endogenous cholesterol, 7 α-hydroxy-4-[6 β-3H]cholesten-3-one, 5-β-[7 β-3H]cholestane-3 α, 7 α-diol and [3H]lithocholic acid was studdied in control and clofibrate (ethyl p-chlorophenoxyisobutyrate)-treated rats. 2. The extent of 7 α-hydroxylation of exogenous [414C]cholesterol and endogenous cholesterol, the latter determined with a mass fragmentographic technique, was the same in the two groups of rats. The extent of 12 α-hydroxylation of 7 α-hydroxy-4-cholesten-3-one and 5 β-cholestane-3 α, 7 α-diol was increased by about 60 and 120% respectively by clofibrate treatment. The 26-hydroxylation of 5 β-cholestane-3 α, 7 α-diol was not significantly affected by clofibrate. The 6 β-hydroxylation of lithocholic acid was about 80% higher in the clofibrate-treated animals than in the controls. 3. The results are discussed in the context of present knowledge about the liver microsomal hydroxylating system and bile acid formation in patients with hypercholesterolaemia, treated with clofibrate.
Bacterial catabolism of threonine. Threonine degradation initiated by L-threonine-NAD+ oxidoreductase
Authors: Bell S C , Turner J M
Published: August 2015

1. Isolates representing seven bacterial genera capable of growth on L-threonine medium, and possessing high L-threonine 3-dehydrogenase activity, were examined to elucidate the catabolic route. 2. The results of growth, manometric and enzymic experiments indicated the catabolism of L-threonine by cleavage to acetyl-CoA plus glycine, the glycine being further metabolized via L-serine to pyruvate, in all cases. No evidence was obtained of a role for aminoacetone in threonine catabolism or for the metabolism of glycine by the glycerate pathway. 3. The properties of a number of key enzymes in L-threonine catabolism were investigated. The inducibly formed L-threonine 3-dehydrogenase, purified from Corynebacterium sp. B6 to a specific activity of about 30-35 mumol of product formed/min per mg of protein, exhibited a sigmoid kinetic response to substrate concentration. The half-saturating concentration of substrate, [S]0.5, was 20mM and the Hill constant (h) was 1.50. The Km for NAD+ was 0.8mM. The properties of the enzyme were studied in cell-free extracts of other bacteria. 4. New assays for 2-amino-3-oxobutyrate-CoA ligase were devised. The Km for CoA was determined for the first time and found to be 0.14mM at pH8, for the enzyme from Corynebacterium sp. B6. Evidence was obtained for the efficient linkage of the dehydrogenase and ligase enzymes. Cell-free extracts all possessed high activities of the inducibly formed ligase. 5. L-Serine hydroxymethyltransferase was formed constitutively by all isolates, whereas formation of the ‘glycine-cleavage system’ was generally induced by growth on L-threonine or glycine. The coenzyme requirements of both enzymes were established, and their linked activity in the production of L-serine from glycine was demonstrated by using extracts of Corynebacterium sp. B6. 6. L-Serine dehydratase, purified from Corynebacterium sp. B6 to a specific activity of about 4mumol of product formed/min per mg of protein, was found to exhibit sigmoid kinetics with an [S]0.5 of about 20mM and h identical to 1.4. Similar results were obtained with enzyme preparations from all isolates. The enzyme required Mg2+ for maximum activity, was different from the L-threonine dehydratase also detectable in extracts, and was induced by growth on L-threonine or glycine.
Spectroscopic studies of the binding of bilirubin by ligandin and aminoazo-dye-binding protein A
Authors: Tipping E , Ketterer B , Christodoulides L , Enderby G
Published: August 2015

Ligandin and aminoazo-dye-binding protein A both bind bilirubin at a single site. Quantitative studies of the interactions using difference spectrophotometry show that at pH 7.0, protein A binds the tetrapyrrole with an association constant (K) greater than or equal to 2 × 10(7) litre/mol, whereas binding by ligandin is slightly weaker (K = 7 × 10(6) litre/mol) at this pH. The protein-bilirubin complexes give rise to absorption and fluorescence spectra quite different from those of unbound bilirubin and also to large Cotton effects. It appears that on binding to both proteins, the ligand is forced into a rigid twisted configuration in a hydrophobic environment. Ligandin and protein A resemble serum albumin in their interactions with bilirubin.
Purification and properties of pantohenase from Pseudomonas fluorescens
Authors: Airas R K , Hietanen E A , Nurmikko V T
Published: August 2015

Pantothenase (EC 3.5.1.22) from Pseudomonas fluorescens UK-1 was purified to homogeneity as judged by disc-gel electrophoresis and isoelectric focusing. The purification procedure consisted of four steps: DEAE-Sephadex chromatography, (NH4)2SO4 precipitation, hydroxyapatite chromatography and preparative polyacrylamide-gel electrophoresis. Gel filtration on Ultrogel AcA 34 was used to determine the molecular weight, and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis to study the subunit molecular weight. The enzyme appeared to be composed of two subunits with mol.wts. of approx. 50000 each. The total mol.wt. of the enzyme was thus about 100000. The isoelectric point was 4.7 at 10 degrees C.
Isolation of (copper, zinc)-thioneins from the livers of copper-injected rats
Authors: Bremner I , Young B W
Published: August 2015

The mixed copper- and zinc-binding proteins, with mol.wt. of approx. 12000, induced in rat liver after injection of copper were isolated and characterized as metallothioneins. Three separate forms were obtained, with 7-11% total metal in the protein, equivalent to 2.6-1.6 cysteine residues/metal atom.
An apparent lack of stereospecificity in the reaction catalysed by deoxyribose 5-phosphate aldolase due to methyl-group rotation and enolization before product release
Authors: Corina D L , Wilton D C
Published: August 2015

In the reaction catalysed by deoxyribose 5-phosphate aldolase (2-deoxy-D-ribose 5-phosphate acetaldehyde-lyase, EC 4.1.2.4) from Salmonella typhimurium, almost complete equilibration of the methyl-group protons of the product, acetaldehyde, occurs before its release from the enzyme surface. This phenomenon does not allow the stereo-chemical course of the reaction to be determined by using hydrogen-isotope labelling of the methyl group to generate a chiral centre.
The properties of uridine diphosphate glucuronyltransferase(s) which catalyse the synthesis of steroid glucuronides in microsomal fractions from guinea-pig liver
Authors: Zakim D , Vessey D A
Published: August 2015

The properties of the UDP-glucuronyltransferase(s) of guinea-pig liver that catalyse the synthesis of steroid glucuronides were examined. There are many similarities between apparently different substrate-specific forms of these enzymes in that all are activated by bivalent metal ions, and all contain at least 2 thiol groups important for enzyme activity. On the other hand, there are significant differences between the enzymes conjugating steroids and those conjugating non-steroids. Only the latter are activated by UDP-N-acetylglucosamine, which enhances their relatively poor affinity for UDP-glucuronic acid. The steroid-conjugating forms of UDP-glucuronyltransferase are not activated by UDP-N-acetylglucosamine and have relatively high apparent affinities for UDP-glucuronic acid. The rate of glucuronidation of testosterone was inhibited by treatment with phospholipase A. Treatment with cholate or Triton X-100 did not enhance the rates of glucuronidation of any steroid tested. The data indicate several similarities between different forms of UDP-glucuronyltransferase, suggesting that there is a large family of related proteins. At the same time there are important differences in the parameters that modulate the rates of different glucuronidation reactions.
The binding of echinomycin to deoxyribonucleic acid
Authors: Wakelin L P G , Waring M J
Published: August 2015

Echinomycin is a peptide antibiotic which binds strongly to double-helical DNA up to a limit of approximately one molecule per five base-pairs. There is no detectable interaction with rRNA and only extremely feeble non-specific interaction with poly(rA)-poly(rU). Heat denaturation of DNA greatly decreases the binding, and similarly limited interaction is observed with naturally occurring single-stranded DNA. Association constants for binding to nine double-helical DNA species from different sources are presented; they vary by a factor of approximately 10, but are not simply related to the gross base composition. The interaction with DNA is ionic-strength-dependent, the binding constant falling by a factor of 4 when the ionic strength is raised from 0.01 to 0.10mol/litre. From the effect of temperature on the association constant for calf thymus DNA, the enthalpy of interaction is calculated to be about -13kJ/mol (-3kcal/mol). Binding of echinomycin persists in CsCl gradients and the buoyant density of nicked bacteriophage PM2 DNA is decreased by 25 mg/ml. Echinomycin interacts strongly with certain synthetic poly-deoxynucleotides, the binding constant decreasing in the order poly(dG)-poly(dC) greater than poly(dG-dC) greater than poly(dA-dT). For the latter two polymers the number of base-pairs occluded per bound antibiotic molecule is calculated to be three, whereas for poly(dG)-poly(dC) it is estimated to be four to five. Poly(dA)-poly(dT) and poly(dI)-poly(dC) interact only very weakly with the antibiotic. Poly(dI-dC) interacts to a slightly greater extent, but the binding curve is quite unlike that seen with the three strongly binding synthetic polynucleotides. Echinomycin affects the supercoiling of closed circular duplex bacteriophage PM2 DNA in the characteristic fashion of intercalating drugs. At low ionic strength the unwinding angle is almost twice that of ethidium. Likewise the extension of the helix, determined from changes in the viscosity of rod-like sonicated DNA fragments, is nearly double that expected for a simple (monofunctional) intercalation process. On this basis the interaction process is characterized as bifunctional intercalation. At higher ionic strength the unwinding angle relative to that of ethidium and the helix extension per bound echinomycin molecule fall, indicating a smooth progression towards more nearly monofunctional intercalation. Two simpler compounds which act as analogues of the quinoxaline chromophores of echinomycin, quinoxaline-2-carboxamide and the trypanocidal drug Bayer 7602, interact with DNA very much more weakly than does echinomycin, showing that the peptide portion of the antibiotic plays an essential role in determining the strength and specificity of the interaction.
The metabolism of steroids in the fatty liver induced by orotic acid feeding
Authors: Carrella M , Björkhem I , Gustafsson J A , Einarsson K , Hellström K
Published: August 2015

1. The metabolism of 4-[4-14C]androstene-3,17-dione, 4-[4-14C]pregnene-3,20-dione, 5alpha-[4-14C]androstane-3alpha,17beta-diol, [4-14C]cholesterol, 7alpha-hydroxy-4-[6beta-3H]cholesten-3-one, 5beta-[7beta-3H]cholestane-3alpha,7alpha-diol and [3H]lithocholic acid was studied in the microsomal fraction of livers from control and orotic acid-treated male rats. 2. As a result of the treatment the orotic acid-fed rats had fatty livers and subnormal concentrations of cholesterol and triglycerides in serum. 3. The 6beta- and 7alpha-hydroxylation of 4-androstene3,17-dione, and the 2alpha-, 2beta- and 18-hydroxylation of 5alpha-androstane-3alpha,17beta-diol, and the 5alpha-reduction of 4-androstene-3,17-dione and 4-pregnene-3,20-dione were decreased by 40-50% in orotic acid-fed rats. Other oxidative and reductive reactions of the steroid hormones were not significantly affected. 4. The 12alpha-hydroxylation of 7alpha-hydroxy-4-cholesten-3-one was decreased by about 50%, whereas the 7alpha-hydroxylation of cholesterol and the 26-hydroxylation of 5beta-cholestane-3alpha,7alpha-diol were not significantly decreased. The 6beta-hydroxylation of lithocholic acid was stimulated by 40%. 5. The results are discussed in relation to present knowledge of the heapatic drug-metabolizing enzymes and to the recent findings of an abnormal bile acid metabolism in liver disease.
Differences between newly formed and recycled free small ribosome subunits in liver cytoplasm
Authors: Hinton R H , Mullock B M
Published: August 2015

Heterogeneity among the free small ribosomal subunits of rat liver was studied. Newly made free small subunits differ from the bulk of the free subunits in the following ways. (1) The newly made free small subunits sediment slightly faster than most free small subunits and form a band at slightly lower densities. (2) Mature free small subunits, prepared under conditions which minimize the amount of loosely bound protein, readily dimerize in solutions of low ionic strength. Little dimerization is found with newly formed subunits. This permits the separation of fractions enriched in newly formed subunits. (3) The fraction enriched in newly formed subunits contains a distinctive additional protein as compared the most of the free small subunits.
Arachidonic acid release from diacyl phosphatidylethanolamine by human platelet membranes
Authors: Jesse R L , Cohen P
Published: August 2015

At pH9.5 in the presence of 10 mM-Ca2+, human platelet membranes released 22% (167 of 785 nmol) of arachidonic acid that was esterified to phospholipids. With the use of synthetic choline (dinonadecanoyl) and ethanolamine (diheptadecanoyl) phosphoglycerides as internal reference compounds, 115 nmol of the released arachidonic acid was shown to be derived from endogenous breakdown of the phosphatidylethanolamine fraction. Further, the lysophosphatidylethanolamine that was released along with the arachidonic acid was shown virtually to lack fatty aldehydes and to contain a preponderance of fatty acids that have a preference for esterification at the 1-position of naturally occurring phosphatidylethanolamine of human platelets. These findings ruled out plasmalogen phosphatidylethanolamine as the source of the released arachidonic acid. We conclude that diacyl phosphatidylethanolamine was the principal source of arachidonic acid released by human platelet membranes under the conditions described.
Localization of thyroid peroxidase and the site of iodination in rat thyroid gland
Authors: Edwards H H , Morrison M
Published: August 2015

The iodinated protein was localized in thyroid tissue slices by using radioautography. In unfixed tissue, the labelled protein was localized in the colloid, whereas, in tissue that was fixed before the 125I addition, the label was within the follicular cell. This localizes thyroid peroxidase largely on the endoplasmic reticulum of the cell.
Amino acid sequence of a four-iron-four-sulphur ferredoxin isolated from Bacillus stearothermophilus
Authors: Hase T , Ohmiya N , Matsubara H , Mullinger R N , Rao K K , Hall D O
Published: August 2015

1. The primary structure of a 4Fe-4S ferredoxin from Bacillus stearothermophilus was determined and shown to consist of a single polypeptide chain of 81 amino acid residues. The molecular weight of the holoprotein is about 9120. 2. There are only four cysteine residues in the molecule; three of these are located near the N-terminus as a Cys-X-X-Cys-X-X-Cys segment, and the fourth cysteine residue is followed by a proline and located in the C-terminal half. 3. The Fe-S chromophore in B. stearothermophilus ferredoxin was previously well characterized and was shown to consist of a single 4Fe-4S cluster. This ferredoxin sequence establishes for the first time the relative location of the four cysteine residues necessary to bind the 4Fe-4S cluster of a 4Fe ferredoxin, and is in agreement with the criteria for the relative positions of the cysteines proposed from X-ray-crystallographic studies on an 8Fe (two 4Fe-4S clusters) ferredoxin. 4. The sequence of B. stearothermophilus ferredoxin is homologous in many segments to that of other bacterial ferredoxins, the degree of homology being greater towards ferredoxins from Desulfovibrio gigas and photosynthetic bacteria than to Clostridial ferredoxins. 5. The presence of a relatively higher number of glutamic acid and lower number of cysteine residues in the molecule may explain the greater thermal stability and oxygen-insenstivity of this ferredoxin.
A method for the determination of hyaluronate in the presence of other glycosaminoglycans and its application to human intervertebral disc
Authors: Hardingham T E , Adams P
Published: August 2015

A highly sensitive proteoglycan-binding assay was developed for the detection and determination of small amounts of hyaluronate. Application of this assay to human intervertebral-disc tissue showed that the radial distribution of hyaluronate closely followed the distribution of proteoglycans.
Removal of a bound ligand from a macromolecule by gel filtration
Authors: Dixion H B F
Published: August 2015

If a solution of macromolecule of concentration c and volume a, initially saturated with a ligand of dissociation constant K, is gel filtered through a volume v of solution initially free of ligand, the saturation y with which it emerges is given by the equation y-lny-1=Kv/ca.
Circular-dichroism and electron-microscopy studies of human subcomponent C1q before and after limited proteolysis by pepsin
Authors: Brodsky-Doyle B , Leonard K R , Reid K B M.
Published: August 2015

1. A fragment of human subcomponent C1q was prepared by limited proteolysis with pepsin at 37°C for 20 h, and at pH4.4, followed by gel filtration on Sephadex G-200. This fragment was shown to contain all the collagen-like features known to be present in the intact molecule [Reid (1976) Biochem. J.155, 5-17]. 2. Circular-dichroism studies showed the presence of positive bands at 230 and 223 nm in the intact subcomponent C1q and pepsin fragment respectively, compared with a positive band at 220 nm obtained for lathyritic rat skin collagen. These bands were abolished by collagenase treatment, which suggested that there may some collagen-like triple-helical structure in subcomponent C1q and that this structure resides in the pepsin-resistant portion of the molecule. However, the 230 and 223 nm bands had a substantially lower magnitude than that obtained for the unaggregated single fibres of totally triple-helical collagen. 3. Thermal-transition temperatures obtained for subcomponent C1q, the pepsin fragment and the reduced and alkylated pepsin fragment were 48 degrees, 48°and 39°C respectively, compared with a value of 38°C obtained for lathyritic rat skin collagen. 4. Only the unreduced pepsin fragment regained significant amounts (up to 60%) of collagen-like structure, after heat denaturation and cooling, as estimated by circular-dichroism measurements. 5. Electron-microscopy studies of subcomponent C1q and the collagen-like pepsin-resistant fragment of subcomponent C1q showed that the six peripheral globular regions of the molecule were fragmented by pepsin leaving the six collagen-like connecting strands and fibril-like central portion intact.
Human liver glycogen phosphorylase. Kinetic properties and assay in biopsy specimens
Authors: Lederer B , Stalmans W
Published: August 2015

1. The two forms of glycogen phosphorylase were purified from human liver, and some kinetic properties were examined in the direction of glycogen synthesis. The b form has a limited catalytic capacity, resembling that of the rabbit liver enzyme. It is characterized by a low affinity for glucose 1-phosphate, which is unaffected by AMP, and a low V, which becomes equal to that of the a form in the presence of the nucleotide. Lyotropic anions stimulate phosphorylase b and inhibit phosphorylase a by modifying the affinity for glucose 1-phosphate. Both enzyme forms are easily saturated with glycogen. 2. These kinetic properties have allowed us to design a simple assay method for total (a + b) phosphorylase in human liver. It requires only 0.5 mg of tissue, and its average efficiency is 90% when the enzyme is predominantly in the b form. 3. The assay of total phosphorylase allows the unequivocal diagnosis of hepatic glycogen-storage disease caused by phosphorylase deficiency. One patient with a complete deficiency is reported. 4. The assay of human liver phosphorylase a is based on the preferential inhibition of the b form by caffeine. The a form displays the same activity when measured by either of the two assays.
Aldolase binding to actin-containing filaments. Formation of paracrystals
Authors: Clarke F M , Morton D J
Published: August 2015

Electron-microscopy observation show that when aldolase binds to F-actin or F-actin-tropomyosin, highly ordered paracrystalline structures are formed consisting of tightly packed filament bundles cross-banded at 36 nm intervals. Morphologically different paracrystalline arrays are formed between aldolase and F-actin-tropomyosin-troponin. The filament bundles are far more extensive and are characterized by a prominent cross-striation at 38nm intervals. It is suggested that this reflects an interaction between troponin and aldolase.
Low-temperature studies on mixed-valence cytochrome oxidase by using magnetic circular dichroism
Authors: Brittain T , Springall J , Greenwood C , Thomson A J
Published: August 2015

The spin states of the haem components of mixed-valence cytochrome oxidase were studied at room temperature and at temperature down to 20K by using magnetic circular dichroism. The room-temperature studies show the presence of a low-spin ferrous haem together with a low-spin ferric haem, which we attribute to heams a3 and a respectively. At temperatures below 100K it appears that the CO of the mixed-valence CO complex may be irreversibly photolysed, and that in this case haems a and a3 assume their high-spin states. Thus in this enzyme haem-haem interactions appear possible at temperatures below 100K.
The relationship between adrogen receptors and the hormonally controlled responses of rat ventral prostate
Authors: Van Doorn E , Craven S , Bruchovsky N
Published: August 2015

1. The administration of dihydrotestosterone to rats orchidectomized 7 days previously stimulated the synthesis of nuclear receptor in prostatic cells several hours in advance of DNA synthesis and mitosis. 2. The synthesis of nuclear receptor is tightly coupled to cell proliferation; consequently, in resting cells, there is no further net synthesis of nuclear receptor above the maximum of approx. 8000 molecules/cell. 3. After orchidectomy a rapid decline in the concentration of free androgen in the nuceus and a slower decline in the concentration of nuclear receptor are observed. 4. Owing to the apparent scarcity of receptor-inactivating factors in the nucleus, and the inverse relationship between amounts of nuclear and cytoplasmic receptors, it is concluded that the nuclear receptor is discharged into the cytoplasm after orchidectomy. 5. The formation of the cytoplasmic receptor is an early event preceding the onset of cellular autolysis. 6. Regressing prostate develops the capacity to eliminate cytoplasmic receptor, and this capacity is retained by the regenerating prostate for at least 14 days. 7. The synthesis of nuclear receptor in early G1 phase may control the entry of cells into the cell cycle and the prolonged retention of receptor in the nucleus may prevent the activation of autophagic processes.
A new approach to assessing collagen turnover by using a micro-assay. A highly efficient and rapid turnover of collagen in rat periodontal tissues
Authors: Sodek J
Published: August 2015

Measurement of [3H]proline incorporation into newly synthesized and mature collagen in connective tissues was used to compare rates and efficiency of collagen turnover. The approach minimizes label-recycling problems. By using a micro-assay to determine hydroxyproline specific radioactivities, a highly efficient and rapid collagen turnover in rat periodontal tissues was demonstrated.
Platelet-aggregating activity of type I and type III collagens from human aorta and chicken skin
Authors: Barnes M J , Gordon J L , MacIntyre D E
Published: August 2015

Human or chicken type III collagen dissolved in 0.1 M-acetic acid was much more potent than type I collagen at inducing platelet aggregation. After incubation in 0.38M-Na2HPO4 to promote fibrillogenesis, the platelet-aggregating activity of both collagen types increased, and type I was then virtually equiactive with type III. Preincubation in cell-free plasma increased the activity of chicken but not that of human collagen. The platelet-aggregating activity of type III collagen did not appear to depend on the integrity of the intrachain disulphide bonds.
Lack of effect of amino acid concentration on protein synthesis in the perfused rat liver
Authors: Peavy D E , Hansen R J
Published: August 2015

The effect of increasing the perfusate concentration of amino acids on the incorporation of labelled valine into protein was followed in perfusions of rat livers lasting for 2h. A fixed amount of labelled and unlabelled valine was added to the perfusate as the other amino acids were increased in multiples of the concentrations normally found in rat plasma. Under these conditions no increase in valine incorporation was observed, which appeared to be in conflict with results published by other workers, However, a different method of labelling from that used here was used in the earlier studies. An increasing amount of a labelled amino acid was added as the concentrations of the unlabelled amino acids were increased in the perfusate. An experiment directly comparing to the two labelling methods produced results that indicated that the apparent increase in liver protein synthesis observed by the other workers could have been due to the method of radioisotope addition. It is therefore concluded that increasing the perfusate concentration of amino acids does not increase amino acid incorporation into liver protein.