bluuebunny/crossref_metadata_2025 huggingface dataset:
Cleavage of bacterial flagellin with cyanogen bromide. Chemical and physical properties of the protein fragments
Authors: Parish C. R. , Ada G. L.
Published: August 2015

1. Flagellin, isolated from the flagella of Salmonella adelaide, was shown by various criteria to be a pure protein. It had a molecular weight of about 40000 and contained three methionine, six tyrosine, 11 arginine and 25 lysine residues/mol., of which 11 of the lysine residues were present as ∈-N-methyl-lysine. 2. After treatment of flagellin with cyanogen bromide in formic acid, four main fragments (A, B, C and D) were obtained, with as many as six minor components that represented partial degradation products. The major fragments were estimated by amino acid analysis to have molecular weights of about 18000 for fragment A, 12000 for fragment B, 5500 for fragment C and 4500 for fragment D. Fragments A, B and D, but not fragment C, were recovered pure by gel chromatography as monitored by polyacrylamide-gel electrophoresis. 3. A complex between fragments C and D was also isolated (mol.wt. 10000) after limited oxidation of flagellin by chloramine-t before digestion by cyanogen bromide. After oxidation essentially only two fragments were released from flagellin by cyanogen bromide: the ‘C,D’ complex and a presumed ‘AB’ fragment. 4. The sum of the amino acid analyses of fragments A and B and the ‘C,D’ complex gave residue values that agreed well with the amino acid composition of native flagellin. 5. Fragments A and D contained tyrosine, and ten of the 11 ∈-N-methyl-lysine residues of the molecule were in fragment A. Reaction with [125I]iodide at small extents of substitution showed that, in flagellin, the tyrosine residue of fragment D was more readily substituted than those of fragment A. By contrast, in polymerized flagellin, the tyrosine residues of fragment A were more readily substituted. 6. Treatment of flagellin with carboxypeptidases A and B revealed the C-terminal sequence -Leu-Leu-Leu-Arg. Arginine and leucine were released by carboxypeptidase from the ‘C,D’ complex but not from fragment D, indicating that fragment C was C-terminal. 7. On the basis of the results from amino acid analysis, carboxypeptidase digestion, N-terminal analysis, iodination studies and polyacrylamide-gel electrophoresis, the sequence of fragments in flagellin was considered to be B–A–D–C; in the polymer, fragment A was exposed. It is suggested that methylation of the lysine residues occurred in the organism after flagellin had polymerized.
The structural organization of haem synthesis in rat liver mitochondria
Authors: Jones M. S. , Jones O. T. G.
Published: August 2015

1. Anaerobic conditions are normally necessary for incorporation of iron into haems and only ferrous iron is used. After addition of succinate to an incubation mixture containing intact or ultrasonically treated mitochondria, Fe3+ is used, but only if no inhibitors prevent the transfer of electrons from the mitochondrial respiratory chain to oxygen. 2. A dual-wavelength spectrophotometric assay for ferrochelatase is described that has been used for the continuous assay of incorporation of metal ions into porphyrins. Constants are given for the determination of rates of formation of protohaem and cobalt protoporphyrin, mesohaem, cobalt mesoporphyrin and zinc mesoporphyrin. For cobalt mesoporphyrin formation the Km for Co2+ is 11×10−6m and that for mesoporphyrin is 5×10−6m. 3. An improved method for the separation of inner and outer membranes of mitochondria is described. Mitochondria swollen in hypo-osmotic media were contracted in hyperosmotic potassium chloride solution containing ATP and the outer membranes detached by mild ultrasonic treatment. Sucrose inhibited the ATP-induced contraction and decreased the yield of outer membranes. 4. Ferrochelatase is associated with cytochrome oxidase, which is used as a marker for inner mitochondrial membranes. 5. By using as substrate porphyrin dissolved in phospholipid micelles, ferrochelatase activity of intact mitochondria was shown to be latent, and to be liberated by ultrasonic treatment. 6. No ferrochelatase was detectable in microsomes or soluble cell components.
Isolation and properties of hyaluronic acid from bovine heart valves
Authors: Meyer F. A. , Preston B. N. , Lowther D. A.
Published: August 2015

1. A soluble extract of bovine heart valves was obtained after the tissue had been pulverized at liquid-nitrogen temperatures in a mill. 2. Hyaluronic acid was isolated from the crude extract by sedimentation equilibrium in a caesium chloride density gradient (Franek & Dunstone, 1966). 3. Analysis of the product indicated that it contained 15% of protein and the molar ratio of glucuronic acid to glucosamine was 1·27. 4. Its physicochemical properties, as determined by lightscattering, viscosity and sedimentation studies, suggested that its molecular size and configuration were similar to those of hyaluronic acid isolated from ox synovial fluid (Preston, Davies & Ogston, 1965).
The action of a bacterial agarase on agarose, porphyran and alkali -treated porphyran
Authors: Duckworth M , Turvey J R
Published: August 2015

1. A purified extracellular agarase from a Cytophaga species was used to hydrolyse agarose, porphyran and alkali-treated porphyran. 2. The hydrolysate from agarose was separated by gel filtration into the series of neoagarosaccharides, the predominant member of which was the tetrasaccharide. 3. Enzyme action on alkali-treated porphyran gave neoagarosaccharides and other oligosaccharides containing 6-O-methyl-d-galactose units. From the composition of these oligosaccharides it is deduced that action of the enzyme on a d-galactosidic linkage is four to five times faster than on the 6-O-methyl-d-galactosidic linkage. 4. Enzyme action on native porphyran gives a similar series of oligosaccharides but in smaller yield, much of the polysaccharide being either not degraded or only degraded to a series of large, highly sulphated oligosaccharides. 5. For porphyran, it is concluded that 6-O-methyl ether groups are distributed randomly on half the d-galactose units, but that the 6-sulphate groups on l-galactose units tend to occur in blocks.
Oligonucleotide studies. Optical rotatory dispersion of five homodinucleotides
Authors: Inoue Yasuo , Satoh Kimihiko
Published: August 2015

1. The optical rotatory dispersion (ORD) of five homodinucleotides, ApAp(3′), CpCp(3′), GpGp(3′), IpIp(3′) and UpUp(3′) (where A, C, G, I and U represent adenosine, cytidine, guanosine, inosine and uridine respectively, and p to the left of a nucleoside symbol indicates a 5′-phosphate and to the right it indicates a 3′-phosphate), were measured as a function of pH, ionic strength and Mg2+ concentration. 2. The ORD titrations of ApAp(3′) and CpCp(3′), which were made by measuring the ORD curves at closely spaced pH intervals, exhibit a maximum at approx. pH5·0 and 5·7 for ApAp(3′) and CpCp(3′) respectively in the profile of the magnitude of the first Cotton effect versus pH. The results indicate that the conformational rigidity of these dinucleotides depends on the ionization state of a 3′-terminal phosphate group. 3. ApAp(3′) was shown to exist as an approximately 1:1 equilibrium mixture of the two major ionic species represented by Ap(−1)Ap(−1) and Ap(−1)Ap(−2) at pH6·16, whereas at pH7·5 it exists exclusively as a form of Ap(−1)Ap(−2). 4. To ascertain the effects of the presence of a terminal phosphate group and of the ionization of the secondary phosphate on the conformation of adenylate dimer, we measured the ORD of ApA, ApAp(3′)CH3 and ApAp(2′). The rotatory power of the first Cotton effect in the above series of dinucleotides decreased at 20° in the order ApA> ApAp(3′)CH3≈ApAp(3′)(−1)> ApAp(2′) at pH7> ApAp(3′) at pH7. 5. The pH–rotation profiles were also obtained for ApAp(2′), CpCp(2′) and UpUp(3′), but no corresponding maximum was observed. Although simple nearest-neighbour calculations based on the ORD data of IpIp(3′) and 5′-IMP account for the observed ORD spectrum of polyinosinic acid at low salt concentration, there were large discrepancies between calculated and experimental results of the polyguanylic acid ORD even at low ionic strength. 6. The extent to which the amplitude of the Cotton effects of IpIp(3′) increases with salt concentration, especially by the addition of Mg2+, was much greater than that observed for ApAp(3′). The implication of such salt effects on the ORD is considered.
The effect of age on protein synthesis in mouse liver
Authors: Mainwaring W. I. P.
Published: August 2015

1. A system of microsomes and 105000g supernatant from livers of old mice is less able to promote the incorporation of [14C]phenylalanine into protein than a similar system from livers of young animals. 2. The decrease in [14C]phenylalanine incorporation is attributable to changes in microsomes from old animals rather than in the cell-sap fraction. 3. Decreased synthetic ability is found in various classes of microsomes from older animals, namely unfractionated, light and heavy microsomes, but not in detergent-washed ribonucleoprotein particles. 4. Deletions of certain detergent-soluble microsomal proteins accompany the decreased synthetic ability of microsomes from older animals. 5. Microsomes from old mice are less responsive to a synthetic messenger RNA, polyuridylic acid, and this is partly due to a higher rate of hydrolysis in the presence of cell sap from animals of extreme age. 6. Other more direct evidence, from the priming of a cell-free protein-synthesizing system from bacteria and the examination of ribonucleoprotein particles on sucrose density gradients, suggests that senescence is accompanied by a decrease in messenger RNA content.
Metabolic studies on retinoic acid in the rat
Authors: Sundaresan P. R. , Bhagavan H. N.
Published: August 2015

The nature of metabolites in the urine arising from differentially labelled retinoic acid was investigated after injection of physiological doses into retinol-deficient rats. Distribution of radioactivity after partition of urine into ether-soluble, acidic and water-soluble fractions revealed that there were at least six metabolites in urine. Of these, the major metabolite(s) was one lacking both C-14 and C-15 of retinoic acid. Enzymic or alkaline hydrolysis of acidic and water-soluble fractions did not release any retinoic acid, thus indicating that retinoyl β-glucuronide was not present in urine in significant amounts.
Changes in drug-metabolizing activities in the livers of suckling rats as a result of treatment of the lactating mothers with phenobarbitone and chlorpromazine
Authors: Darby F. J.
Published: August 2015

1. Lactating female rats were treated with phenobarbitone or chlorpromazine. 2. Hepatic microsomal fractions from the suckling offspring were studied to see if changes had occurred in the rates of drug metabolism. 3. Treatment with phenobarbitone significantly increased the cytochrome P-450 concentration, the activity of NADPH–cytochrome c reductase (EC 1.6.2.3) and the rates of metabolism of aniline, ethylmorphine and [2-14C]pentobarbitone. 4. Treatment with chlorpromazine produced only small increases in the cytochrome P-450 concentration and the rate of hydroxylation of aniline.
Rate-determining processes and the number of simultaneously active sites of d-glyceraldehyde 3-phosphate dehydrogenase
Authors: Trentham D. R.
Published: August 2015

Transient kinetic studies of the reversible oxidative phosphorylation of d-glyceraldehyde 3-phosphate catalysed by d-glyceraldehyde 3-phosphate dehydrogenase show that all four sites of the tetrameric lobster enzyme are simultaneously active, apparently with equal reactivity. The rate-determining step of the oxidative phosphorylation is NADH release at high pH and phosphorolysis of the acyl-enzyme at low pH. For the reverse reaction the rate-determining step is a process associated with NADH binding, probably a conformation change, at high pH and d-glyceraldehyde 3-phosphate release at low pH. NADH has previously been shown to be a competitive inhibitor of the enzyme with respect to d-glyceraldehyde 3-phosphate and vice versa. This is consistent with the mechanism deduced from transient experiments given the additional proviso that 1-arseno-3-phosphoglycerate has a half-life of about 1min or longer at pH7. The dissociation constants of d-glyceraldehyde 3-phosphate and 1,3-diphosphoglycerate to the NAD+-bound enzyme are too large to measure but are nevertheless consistent with the low Km values of these substrates.
Some new aspects of the metabolism of phenacetin in the rat
Authors: Nery R.
Published: August 2015

1. Four new metabolites of phenacetin in the urine of the rat are described; these are (i) N-acetyl-S-ethylcysteine, (ii) quinol, (iii) acetamide and (iv) probably N-acetyl-S-2-(4-ethoxyacetanilido)cysteine S-oxide. 2. Metabolites (i), (iii) and (iv) were characterized and estimated by g.l.c., by t.l.c., by paper chromatography, by chemical reactions or by radioactive techniques after administration to rats of [ethyl-14C]phenacetin and [acetyl-3H]phenacetin; metabolite (ii), which was excreted mainly as conjugates of sulphuric acid and glucosiduronic acid, was measured by paper chromatography and characteristic colour reactions after enzymic and chemical hydrolysis of the conjugates. 3. Small amounts of azoxy-4-[ethyl-14C]ethoxybenzene and an unknown metabolite were also found in the urine of rats after administration of [ethyl-14C]phenacetin. 4. The likely mechanisms and some biological implications of these metabolic reactions are discussed.
Protein phosphokinase reactions in mammalian testis. Stimulatory effects of adenosine 3′:5′-cyclic monophosphate on the phosphorylation of basic proteins
Authors: Reddi A. H. , Ewing L. L. , Williams-Ashman H. G.
Published: August 2015

The soluble fraction of rat testis homogenates is rich in enzymes catalysing the phosphorylation by ATP of many different proteins. The phosphorylation of certain histones, and also of a homogeneous basic protein from guinea-pig seminal-vesicle secretion, is markedly enhanced by 3′:5′-cyclic AMP. Various factors affecting these reactions as catalysed by partially purified testicular enzyme preparations are described, and their possible physiological significance is discussed.
Bacterial metabolism of 4-chlorophenoxyacetate
Authors: Evans W. C. , Smith B. S. W. , Moss P. , Fernley H. N.
Published: August 2015

1. A pseudomonad capable of utilizing 4-chlorophenoxyacetate (CPA) as sole source of organic carbon was isolated from soil. 2. The organism was grown in liquid culture and the following compounds were isolated and identified in culture extracts: 4-chloro-2-hydroxyphenoxyacetate, 4-chlorocatechol, β-chloromuconate probably the cis–trans isomer and γ-carboxymethylene-Δαβ-butenolide. 3. Cells grown on 4-chlorophenoxyacetate were able to metabolize 4-chloro-2-hydroxyphenoxyacetate, 4-chlorocatechol and γ-carboxymethylene-Δαβ-butenolide without a lag period. They were not adapted to 4-chlorophenol, or to either culture isolated or synthetic β-chloromuconate, possibly because of stereospecificity towards the cis–cis isomer. 4. On the basis of isolation and induction evidence, the following metabolic pathway is proposed for the breakdown of 4-chlorophenoxyacetate by this organism: 4-chlorophenoxyacetate → 4-chloro-2-hydroxyphenoxyacetate → 4-chlorocatechol → cis–cis-β-chloromuconate → γ-carboxymethylene-Δαβ-butenolide → maleylacetate and fumarylacetate → fumarate and acetate.
Bacterial metabolism of 4-chloro-2-methylphenoxyacetate. Formation of glyoxylate by side-chain cleavage
Authors: Gamar Y. , Gaunt J. K.
Published: August 2015

Crude extracts of Pseudomonas sp. grown on 4-chloro-2-methylphenoxyacetate as sole source of carbon were shown to oxidize 4-chloro-2-methylphenoxyacetate to 5-chloro-o-cresol and glyoxylate. A labelled 2,4-dinitrophenylhydrazone was isolated from an incubation mixture in which 4-chloro-2-methylphenoxy[carboxy-14C]acetate was used. The hydrazone was shown to behave identically on thin-layer chromatograms with the authentic 2,4-dinitrophenylhydrazone of glyoxylate. Radioactivity assay showed that 82% of the side chain of 4-chloro-2-methylphenoxyacetate was recovered as glyoxylate.
Radiation-target molecular weights of urease and of l-glutamate dehydrogenase, and their relevance to the size of the functional subunits
Authors: Blum Elizabeth , Alper Tikvah
Published: August 2015

Activities of the oligomeric enzymes urease and l-glutamate dehydrogenase were measured after exposure as dry preparations to various doses of electron radiation. Inactivation curves were exponential. ‘Target sizes’ deduced from these were small compared with the molecular weights of the whole enzyme molecules, but accorded well with independent estimates of the sizes of functional subunits. It was concluded that, when these were in associated from, there could be no transfer of absorbed energy between subunits.
Studies on an extract of rat testicular microsomal fraction that catalyses the transformation of progesterone into 17-hydroxyprogesterone and androgens
Authors: Ford Henry C. , O'Donnell Vincent J.
Published: August 2015

The supernatant obtained by centrifugation of Triton N-101-treated freeze-dried rat testicular microsomal fraction at 105000gav. for 2h transformed progesterone into testosterone via 17-hydroxypregn-4-ene-3,20-dione and androst-4-ene-3,17-dione. Hydroxylation at C-17 of 3β-hydroxypregn-5-en-20-one and deoxycorticosterone was not observed. Non-haem iron protein, cytochrome P-450 and material with NADPH dehydrogenase activity were precipitated by 40% saturation of the supernatant with ammonium sulphate; however, it was not possible to establish the participation of these substances in the 17α-hydroxylase and side-chain-cleavage activities also present in the precipitate. The results of gel-filtration chromatography indicated that the Triton N-101 extract consisted primarily of a suspension of small particles of microsomes and that the progesterone 17-hydroxylase and the 17-hydroxypregn-4-ene-3,20-dione side-chain-cleavage enzyme were not in true solution.
The compartmentation of non-esterified and esterified cholesterol in the superovulated rat ovary
Authors: Flint A. P. F. , Armstrong D. T.
Published: August 2015

1. The specific radioactivities of non-esterified and esterified cholesterol, progesterone and 20α-hydroxypregn-4-en-3-one were determined in slices of superovulated rat ovary after incubation with [1-14C]acetate in vitro for various times. The specific radioactivities of progesterone and 20α-hydroxypregn-4-en-3-one were equal, and (during the fourth hour of incubation) exceeded those of the non-esterified cholesterol and the esterified cholesterol by factors of 2.8 and 7.6 respectively. 2. After separation of homogenates of superovulated rat ovary slices previously incubated with [14C]acetate into subcellular fractions by differential centrifugation, the specific radioactivities of non-esterified cholesterol in the cytosol, mitochondria, lipid-containing storage granules and microsomal fraction were 1220, 1510, 1420 and 4020d.p.m./μmol respectively; the corresponding values for the specific radioactivity of the esterified cholesterol were 600, 700, 730 and 760d.p.m./μmol. The specific radioactivities of progesterone and 20α-hydroxypregn-4-en-3-one were equal in all fractions; the corresponding mean specific radioactivity of progesterone+20α-hydroxypregn-4-en-3-one was 6150d.p.m./μmol. 3. By using glutamate dehydrogenase and cytochrome (a+a3) as mitochondrial markers, the presence of cholesterol side-chain cleavage enzyme was demonstrated in microsomal fraction free of mitochondrial contamination. 4. The specific radioactivities of ovarian non-esterified and esterified cholesterol, progesterone and 20α-hydroxypregn-4-en-3-one were determined up to 8h after the intravenous injection of [4-14C]cholesterol into superovulated rats. At all times the specific radioactivities of progesterone and 20α-hydroxypregn-4-en-3-one were equal to the specific radioactivity of non-esterified cholesterol and exceeded, by up to 3.3-fold, that of the esterified cholesterol. 5. It is concluded that non-esterified cholesterol formed from [14C]acetate in the endoplasmic reticulum equilibrates slowly with non-esterified cholesterol in other subcellular fractions, and is preferentially converted into steroids. Such a mechanism presupposes the operation of a microsomal cholesterol side-chain cleavage enzyme using non-esterified cholesterol as its substrate. Unrelated evidence is presented in support of the existence of such an enzyme. The results are discussed in the light of other biochemical and electron-microscopic findings relating to the compartmentation of cholesterol in steroidogenic tissues.
The effects of salicylate on the activity of rat liver tryptophan pyrrolase in vitro and in vivo
Authors: Badawy A. A.-B. , Smith M. J. H.
Published: August 2015

1. Salicylate, in concentrations of 0.05mm and above, inhibits the basal activity of tryptophan pyrrolase in homogenates of rat liver and the activity induced by cortisol but not that induced by tryptophan. The inhibition is abolished by adding haematin to the reaction mixtures. 2. The intraperitoneal injection of 400mg of sodium salicylate/kg in the rat causes a decrease in the tryptophan pyrrolase activity in the liver at 30min, the activity is restored to normal at 2h, increases to sixfold after 5h and returns to the basal value at 12h. The activation of the enzyme by salicylate is prevented by the administration of cycloheximide but not by pretreatment with actinomycin D. The effects of the combined injection of salicylate and cortisol are additive, whereas those of salicylate plus tryptophan are not. The injection of salicylate causes a progressive increase in the holo-/apo-enzyme ratio and an increased content of tryptophan in the liver over a period of 3h. 3. It is suggested that salicylate inhibits tryptophan pyrrolase activity in vitro and in vivo by interacting with iron protoporphyrins and causes a later enhancement of the enzyme activity in vivo by a mechanism involving the release of tryptophan from its binding sites on circulating albumin and on other proteins.
Studies on the high-sulphur proteins of reduced Merino wool. Amino acid sequence of protein SCMBK-IIIB3
Authors: Haylett T. , Swart L. S. , Parris D.
Published: August 2015

The complete amino acid sequence of wool protein SCMKB-IIIB3 was determined. The peptides used for the sequence work were obtained by peptic and thermolysin digestions and were fractionated by chromatography on DEAE-cellulose, paper chromatography and electrophoresis. The peptides were analysed by dansyl–Edman degradation, mass spectrometry and tritium-labelling of C-terminal residues. The protein consists of 98 residues and has acetylalanine as N-terminal residue and carboxymethylcysteine as C-terminus. It is homologous with protein SCMKB-IIIB2 (Haylett & Swart, 1969). A salient feature of the sequence of protein SCMKB-IIIB3 is three consecutive cysteine residues.
A simulation study of brain compartments. Metabolism of glutamate and related substances in mouse brain
Authors: Van Den Berg C. J. , Garfinkel D.
Published: August 2015

A computer model of the metabolism of glutamate, glutamine, γ-aminobutyrate, and the tricarboxylic acid cycle in mouse brain has been constructed in terms of 39 reactions among 19 substances or groups of substances (permitting manipulation of 30 independent variables). The model is divided into two compartments, in conformity with previous models based on indirect evidence, and it is found that this compartmentation is indeed the same as that indicated directly with specifically 14C-labelled acetate and glucose. The movement of materials between the large and small compartments has been studied; glutamine appears to flow from the small to the large compartment, γ-aminobutyrate in the reverse direction.
The synthesis and decay of histone fractions and of deoxyribonucleic acid in the developing avian brain
Authors: Bondy Stephen C.
Published: August 2015

1. The turnover of cerebral histones and DNA after injection of [4,5-3H]leucine or [methyl-3-3H]thymidine, respectively, was studied in the developing chick. 2. Chromatin was prepared from chick nuclei that had been purified by centrifugation through 1.9m-sucrose. 3. Nuclear proteins were fractionated into three major histone classes, F1 (lysine-rich), F2(b) (slightly lysine-rich) and [F3+F2(a)] (arginine-rich), and a non-histone protein residue. 4. The proportions of the histone classes remained constant throughout the period of development studied. 5. All histone fractions decayed at a similar rate, initially with a half-life of around 5 days, later with a half-life of 19 days. 6. Non-histone proteins from chromatin decayed in a heterogeneous manner with a wide range of half-lives. 7. Short-term labelling studies showed that all histone fractions were synthesized at the same rate. 8. Some non-histone proteins were very rapidly synthesized relative to histones. 9. DNA had a longer half-life than any histone fraction studied. A biphasic exponential decay curve with half-lives of 23 and 50 days was found. 10. It was concluded that the turnover of histones can occur independently of that of DNA and that different histone classes have similar rates of synthesis and decay.
A role for phospholipids in the binding and metabolism of drugs by hepatic microsomes. Use of the fluorescent hydrophobic probe 1-anilinonaphthalene-8-sulphonate
Authors: Eling T. E. , DiAugustine R. P.
Published: August 2015

1. The pretreatment of rat liver microsomes with phospholipase C or D decreased the N-demethylation of (+)-benzphetamine. The hydroxylation of aniline was essentially unchanged by pretreatment of microsomes with phospholipase C. 2. Some components of the microsomal mixed-function oxidase system were impaired by phospholipases. 3. The fluorescence of 1-anilinonaphthalene-8-sulphonate (ANS) was greatly enhanced by microsomes. Phospholipase C or D markedly decreased ANS–microsome fluorescence. Quantum yield of ANS–microsome fluorescence appeared to be related directly to phospholipid content of microsomes. 4. Most of the drugs studied enhanced ANS–microsome fluorescence. Warfarin, however, displaced ANS fluorescence competitively from microsomes. The latter effect was postulated as being due to warfarin competing with ANS for the cationic site on microsomal phosphatidylcholine. 5. ANS fluorescence was also increased by the presence of phospholipid micelles. The fluorescence of ANS–phosphatidylcholine micelles was modified by warfarin and (+)-benzphetamine in a manner similar to that observed with microsomes. Warfarin decrease of fluorescence was absent when ANS was bound to phosphatidic acid, which lacks a cationic site. 6. Trypsin pretreatment of microsomes did not modify ANS–microsome fluorescence, including drug-induced changes. 7. It was postulated that phospholipids have a permissive role in the metabolism of most drugs by hepatic microsomes and that the ANS probe might reflect interactions of compounds with microsomal membrane phospholipids.
The transport of citrate and other tricarboxylic acids in two species of Pseudomonas
Authors: Lawford H. G. , Williams G. R.
Published: August 2015

When cells of Pseudomonas are grown on citrate as the sole carbon source they oxidize citrate and isocitrate rapidly. Fluorocitrate inhibits the oxidation of citrate. Fluorocitrate-treated cells accumulate [6-14C]citrate, as shown by a rapid Millipore-filtration technique. In the absence of fluorocitrate most of the [6-14C]-citrate is lost in the form of14CO2. The isolation of a pseudomonad characterized by its ability to grow on tricarballylate as a sole carbon source has facilitated the study of the tricarboxylate-carrier specificity. Cells grown on citrate will exchange radioactive citrate for unlabelled citrate or isocitrate but not for cis-aconitate, trans-aconitate or tricarballylate. Cells grown on tricarballylate will exchange radioactive citrate for unlabelled citrate, cis-aconitate or tricarballylate, but not for isocitrate or trans-aconitate. The properties of the exchange system involved are compared with those of the related system in mitochondria.
Changes in the liver mitochondrial oxidation of succinate during cold-exposure
Authors: Aithal H. N. , Ramasarma T.
Published: August 2015

1. Exposure of rats to low environmental temperature resulted in increased activities of several hepatic oxidative-enzyme systems. 2. Simultaneous with increase in liver ubiquinone in cold-exposed rats, the ubiquinone-dependent succinate-neotetrazolium chloride reductase activity also increased. Such an increase could also be obtained by enriching liver with ubiquinone by feeding with an exogenous source. 3. Succinate–neotetrazolium chloride reductase activity could be increased by preincubation of mitochondria with succinate and the mechanism of this activation appears to be different from that obtained on addition of ubiquinone. 4. Succinate–neotetrazolium chloride reductase activity was found to be more labile than succinate dehydrogenase on freezing and thawing and storage, and the presence of succinate gave protection against this loss in hepatic mitochondria obtained from both normal and cold-exposed animals.
Studies on gastric mucoproteins. The isolation and characterization of the mucoprotein of the water-soluble mucus from pig cardiac gastric mucosa
Authors: Snary D. , Allen A.
Published: August 2015

1. Gel filtration of the water-soluble radioactive mucus produced three radioactive fractions, fraction A excluded on Sepharose 4B, fraction B included on Sepharose 4B but excluded on Sephadex G-200, and fraction C included on Sephadex G-200. 2. The specific radioactivities of fractions A and B were the same, with fraction C a little lower, whether the material was labelled with 14C-labelled carbohydrate or with 3H-labelled protein prepared by incubation of mucosal scrapings in vitro with [U-14C]glucose or [G-3H]threonine respectively. 3. Fractions A and B had an analysis of protein 22%, hexose 28%, hexosamine 28%, fucose 10% and sialic acid 1%; fraction C had an analysis closely similar to this, except that it contained about 10% of a protein contaminant. 4. All three fractions had closely similar A and H blood-group activities. 5. Ultracentrifuge studies showed fractions A, B and C were polydisperse with s025,w values of 18.7S, 4.9S and 3.9S respectively. 6. The unfractionated water-soluble mucus contained only two peaks, fraction A 18.7S and a peak of 4.4S, which was a combination of fractions B and C. 7. The radioactive mucoprotein accounted for 85% by weight of the soluble mucus and the results show that it consisted of two distinct fractions A and B–C, which were chemically, biosynthetically and immunologically very similar.
The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Reactivation of phospholipase A-inactivated enzyme by phospholipids and detergents
Authors: Attwood D. , Graham A. B. , Wood G. C.
Published: August 2015

Specific degradation of the phospholipid membrane of guinea-pig liver microsomal fraction with phospholipase A inactivated glucuronyltransferase. The inactivation was reversed by phosphatidylcholine and mixed microsomal phospholipid micelles at concentrations similar to those present in intact microsomal preparations. The other commonly occurring phospholipids did not reactivate phospholipase A-treated enzyme. Since the mixed microsomal phospholipids consisted mainly of phosphatidylcholine, it is concluded that the reactivation by phospholipids is phosphatidylcholine-specific. Reactivation was also achieved by low concentrations of the cationic detergents cetylpyridinium chloride and cetyltrimethylammonium bromide. Higher concentrations of these detergents inactivated the glucuronyltransferase activity of intact and phospholipase A-treated microsomal fractions. Anionic detergents were potent inactivators of the glucuronyltransferase activity of untreated and phospholipase A-treated microsomal fractions, whereas non-ionic detergents had little effect on the activity of either preparation. Measurements of the zeta-potentials of the micellar species used in this study showed that no obvious relationship existed between the zeta-potentials and the ability to reactivate glucuronyltransferase. However, high positive or negative zeta-potentials were correlated with the ability of the amphipathic compound to inactivate glucuronyltransferase.
Hurler's, Hunter's and Morquio's syndromes. A biochemical study in the light of current views of the underlying defects
Authors: Dean M. F. , Muir Helen , Ewins R. J. F.
Published: August 2015

Glycosaminoglycans were isolated from the urine of three patients with Hurler's, Hunter's and Morquio's syndromes and also from the liver and spleen of the case of Hurler's syndrome by a procedure avoiding further degradation. A method of determining the proportions of dermatan sulphate, heparan sulphate and chondroitin sulphate in each preparation is described. The relative proportions of these glycosaminoglycans in the urine and organs of the case of Hurler's syndrome were very similar. Glycosaminoglycans from the organs were of much lower molecular weight than normal, consisting of single chains of molecular weight about 5000 together with multiples of up to four such chains attached to peptide moieties. The linkage region normally attaching glycosaminoglycan chains to protein in whole protein–polysaccharides of connective tissue was degraded progressively towards serine. The total output and relative proportions of abnormal glycosaminoglycans in the urine were compared in two brothers with Hunter's syndrome examined on two occasions 4 years apart. At comparable ages they excreted about the same amount, and the relative proportions of each glycosaminoglycan remained essentially constant. The composition and chromatographic behaviour of the glycosaminoglycan in the urine from the case of Morquio's syndrome indicated that it consisted of material containing about one-third keratan sulphate and two-thirds chondroitin sulphate as part of the same molecule, as in proteoglycans of cartilage. The total output of glycosaminoglycans, although higher than normal, was considerably less than in other types of Mucopolysaccharidoses.
The urinary excretion of orally administered pteroyl-l-glutamic acid by the rat
Authors: Blair J. A. , Dransfield E.
Published: August 2015

1. The urinary excretion of folates after oral administration of [2-14C]pteroyl-l-glutamic acid was studied by assaying the radioactivity in the urine and in materials purified and characterized by t.l.c. 2. Radioactivity excreted was 6.8, 5.9 and 30.7% of the oral dose in the first 24h after doses of 3.1, 32 and 320μg/kg respectively. 3. Extensive decomposition of urinary folates to pteroyl-l-glutamic acid was prevented by antioxidants or collection of urine frozen. 4. At the three dosages, two major and one minor radioactive compounds were isolated. One of the major metabolites was 5-methyltetrahydropteroylglutamic acid. The others were unidentified but were not pteroylglutamic acid, 7,8-dihydro-, 5,6,7,8-tetrahydro-, 5- or 10-formyl-tetrahydro-, 5,10-methylidyne-tetrahydro-, 5-formimidoyl-tetrahydro-, 5,10-methylene-tetrahydro-, 5-methyltetrahydro-pteroylglutamic acid, nor any decomposition products of these compounds formed during isolation. Labelled unconjugated pteridines were absent. 5. Labelled pteroyl-l-glutamic acid was displaced by oral administration of unlabelled pteroyl-l-glutamic acid (1.6mg/kg) when given 3.5h after, but not when given 24h after the labelled dose. 6. The results show that orally administered [2-14C]pteroyl-l-glutamic acid is absorbed without metabolism and is then metabolized into naturally occurring tetrahydro-folates. 7. These findings are discussed with reference to previous work.
A system for the study in situ of the pentose phosphate pathway in rabbit liver
Authors: Williams J. F. , Rienits K. G. , Clark M. G.
Published: August 2015

A surgical procedure for the isolation of the liver from the systemic circulation of the anaesthetized rabbit is described. The technique allowed the metabolism in situ of intraportally infused substrates to be followed for periods up to 5min, free from the contaminating influences of metabolism by other body tissues. Details of the procedures necessary to achieve the uniform infusion, homogeneous distribution and containment of 14C-labelled glucose substrates in the liver by haemostasis are described. Changes in pO2, pCO2, pH and the concentrations of NADP+, NADPH and glucose during each minute interval of the total 5min period of metabolism are given. Reactant ratios of the lactate dehydrogenase system and the adenine nucleotide system have been calculated from the concentrations of the pertinent metabolites for the same period of metabolism. Glucose production by rabbit liver in situ proceeded at the rate of 1.08μmol/min per g wet wt. of liver during the 5min metabolic interval. The presence of the oxidative reactions of the pentose phosphate pathway of glucose metabolism was inferred from the quotient oxidation of [1-14C]glucose/oxidation of [6-14C]glucose=1.8.
The amino acid sequence of a human ϰ light chain
Authors: Milstein Celia P. , Deverson E. V.
Published: August 2015

The amino acid sequence of the light chain of the myeloma protein Dee was studied. The light chain is of the κ type and of subgroup I. The variable part contains some substitutions that are unique and also some that have been observed already in other κI chains (repeated variants). Based on these repeated variants a subdivision of the κI subgroup is proposed.
A study of the formation of fluorescent derivatives of 4-hydroxyphenethylamine (tyramine), 4-hydroxy-3-methoxyphenethylamine (3-methoxytyramine) and related compounds by reaction with hydrazine in the presence of nitrous acid
Authors: Scott P. H.
Published: August 2015

A reaction is described that allows the preparation of fluorescent derivatives of a group of related compounds with the basic 4-hydroxyphenethylamine structure. Examination of the reaction shows that it takes place in two stages, which can be considered separately. (1) Reaction of hydrazine with nitrous acid: this is instantaneous at room temperature and involves the reaction of 1 mol of hydrazine with 2 mol of nitrous acid. (2) Reaction with 4-hydroxyphenethylamine compounds: this occurs slowly at room temperature but the rate of reaction is significantly increased at higher temperatures. Ammonium sulphamate is added to remove excess of nitrous acid, found to be detrimental to the reaction. Examination of reagent concentrations necessary for maximum fluoresence yield demonstrated the need for a 40-fold molar excess of the reagent formed in the first stage. The derivatives fluoresce in alkaline solution, the fluorescence of derivatives of 4-hydroxy compounds being stable for over 1h at room temperature, those of 4-hydroxy-3-methoxy compounds being slightly less stable. The derivatives were distinguishable from their parent compounds by t.l.c.
Effect of development on the activity of microsomal drug-metabolizing enzymes in rat liver
Authors: Basu T. K. , Dickerson J. W. T. , Parke D. V. W.
Published: August 2015

1. The activity of rat liver microsomal drug-metabolizing enzymes was determined at various ages between 6 and 100 days post natum. The enzymes studied were: aromatic hydroxylases by using as substrate biphenyl, which is metabolized by oxidation to 2- and 4-hydroxybiphenyl; nitroreductase by using p-nitrobenzoate as substrate, which is metabolized by reduction to p-aminobenzoate; glucuronyl synthetase by using 4-methylumbelliferone as the substrate, which is conjugated to give 4-methylumbelliferone glucuronide, and cytochrome P-450, which is regarded as the major terminal mixed-function oxidase in hepatic microsomal hydroxylations. 2. The activity of biphenyl 2-hydroxylase reached a peak at 21 days, biphenyl 4-hydroxylase and 4-methyl glucuronyl transferase at 24 days, cytochrome P-450 at 31 days, and p-nitrobenzoate reductase at 38 days of age. After the peak activity had been reached, the activity of each enzyme decreased with age, and in the case of biphenyl 2-hydroxylase the activity fell to a negligible value at 52 days of age. 3. Neither the addition of Triton X-100 to the incubation medium nor the treatment of the animals with phenobarbital resulted in any increase in the activity of biphenyl 2-hydroxylase at 52 days of age. 4. The activity of biphenyl 2-hydroxylase was threefold higher in rats fed on a synthetic diet than in rats fed on a commercial stock diet. 5. These findings are discussed.
Polysaccharides in germination. Xyloglucans (‘amyloids’) from the cotyledons of white mustard
Authors: Gould Sheila E. B. , Rees D. A. , Wight N. J.
Published: August 2015

Two xyloglucan fractions have been isolated from the cotyledons of resting white-mustard seeds, the first by extraction with hot EDTA, and the second by subsequent extraction with alkali or lithium thiocyanate. Although both appear to have the ‘amyloid’ type of structure in which chains of (1→4)-linked β-d-glucopyranose residues carry d-xylose-rich side chains through position 6, these side chains are rather different in structure in the two polysaccharide fractions, and the second or ‘insoluble’ xyloglucan has fewer of them. The side chains in both polysaccharides are also different from those in other seed amyloids, especially in having xylose linked through positions 3 and 4 (instead of through position 2 as usual) and in containing fucose residues. Both polysaccharides show the characteristic blue ‘amyloid’ colour with iodine in the presence of sodium sulphate, and it is suggested that this arises by the interaction of iodine molecules and possibly iodide ions within the interstices between aggregated xyloglucan chains. ‘Soluble’ xyloglucan is metabolized during germination and is presumed to have a reserve function. ‘Insoluble’ xyloglucan is metabolized less completely over the period studied but its lack of turnover during cell-wall differentiation indicates that it also is a reserve. These and other β-(1→4)-linked reserve polysaccharides of seeds might also have a structural function which is of particular value for the survival of the dormant seed.
Factors affecting the activity of oestradiol hydroxylase in rat liver microsomal subfractions
Authors: Brown B. J. , Jellinck P. H.
Published: August 2015

1. Oestradiol hydroxylase activity, as measured by the formation of water-soluble products, was significantly higher in the smooth endoplasmic reticulum of rat liver than in the rough membrane. 2. The isolated membrane fractions retained their activity for at least 6 months if stored at -30°C and were more stable in tris–HCl than in sodium phosphate buffer. 3. The stability of the oestradiol-hydroxylating system was inversely related to lipid peroxidation and was decreased by phospholipases and deoxycholate, which damage the reticular membranes. Ribonuclease had no effect on this system. 4. Added polyribosomes did not influence the metabolism of oestradiol in the smooth membranes but some inhibition in the yield of water-soluble metabolites was produced by corticosterone. 5. The effect of spermine on microsomal hydroxylation was investigated. It is proposed that this polyamine acts either by direct activation of the enzyme complex or by inhibition of the lipid peroxidase pathway in a linked system rather than by stabilization of the reticular membranes.
Oxidative phosphorylation. The relation between the specific binding of trimethlytin and triethyltin to mitochondria and their effects on various mitochondrial functions
Authors: Aldridge W. N. , Street B. W.
Published: August 2015

1. A binding site (site 1) is present in mitochondria with affinity for trimethyltin and triethyltin adequate for a site to which they could be attached when the processes of energy conservation are inhibited. 2. The quantitative relationships between the binding of trimethyltin and triethyltin to site 1 and their effects on various mitochondrial functions have been examined. 3. ATP synthesis linked to the oxidation of pyruvate, succinate and intramitochondrial substrate, ATP synthesis and oxygen uptake (succinate or pyruvate as substrate) stimulated by uncoupling agents are all inhibited by trimethyltin and triethyltin; when inhibition is less than 50% the ratio (percentage inhibition)/(percentage of binding site 1 complexed) is approx. 10:1. 4. ATP synthesis linked to the oxidation of reduced cytochrome c (ascorbate+NNN′N′-tetramethyl-p-phenylenediamine), ATP hydrolysis and oxygen uptake in the presence of low concentrations of trimethyltin and triethyltin approach zero activity as the proportion of binding site 1 complexed approaches 100%. 5. Possible interpretations of these findings are discussed with reference to published arrangements for coupling of electron transport to ATP synthesis and also to our present knowledge of the chemical and biological specificity of trialkyltin compounds.
Lipid metabolism by rat lung in vitro. Effect of starvation and re-feeding on utilization of [U-14C]glucose by lung slices
Authors: Scholz Richard W. , Rhoades Rodney A.
Published: August 2015

1. The incorporation of [U-14C]glucose into several lipid components of lung and liver slices, and the activities of glucose 6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), ‘malic’ enzyme (EC 1.1.1.40) and NADP–isocitrate dehydrogenase (EC 1.1.1.42) of the cell cytosol were examined in normal, starved and re-fed rats. 2. Lipogenesis and the activities of these enzymes in liver were decreased markedly in rats starved for 72h. Re-feeding starved rats on a fat-free diet for 72h resulted in the well documented hyperlipogenic response in liver, particularly in its ability to convert glucose into neutral lipid, and increased activities of glucose 6-phosphate dehydrogenase, ‘malic’ enzyme and 6-phosphogluconate dehydrogenase to values approx. 700, 470 and 250% of controls respectively. 3. Approx. 70% of the total label in lung lipids was present in the phospholipid fraction. Hydrolysis of lung phospholipids revealed that lipogenesis from glucose was considerable, with approx. 40% of the total phospholipid radioactivity present in the fatty acid fraction. 4. Incorporation of glucose into total lung lipids was decreased by approx. 40% in lung slices of starved rats and was returned to control values on re-feeding. Although phospholipid synthesis from glucose was decreased in lung slices of starved rats, the decrease proportionally was greater for the fatty acid fraction (approx. 50%) as compared with the glycerol fraction (approx. 25%). 5. The activities of lung glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and NADP–isocitrate dehydrogenase were not affected by the dietary alterations. ‘Malic’ enzyme activity was not detected in lung cytosol preparations. 6. The results are discussed in relation to the surface-active lining layer (surfactant) of the lung.
α-Crystallin. Fractionation of subunits and sequence studies on an isolated polypeptide
Authors: Augusteyn Robert C. , Spector Abraham
Published: August 2015

α-Crystallin was carboxymethylated with radioactive iodoacetic acid in the presence of 7.6m-urea and then separated into six major fractions by chromatography on DEAE-cellulose in 7m-urea. Based on the amino acid compositions, specific radioactivities and sodium dodecyl sulphate–gel electrophoresis of the fractions, it was concluded that α-crystallin contains at least four different subunits: DU1A and DU1B, containing no cysteine; a third component represented by DU2B and DU3 containing one cysteine one cysteine residue per subunit; and DU4, which probably contains two residues of cysteine per subunit. Subunit DU1A was shown to be of sufficient purity for sequence studies. Cyanogen bromide cleavage yielded two peptides, CB-1 and CB-2, in approximately equal amounts as expected. The sum of the molecular weights and amino acid compositions of the peptides were both in excellent agreement with the results obtained for subunit DU1A. The amino acid sequence of the first sixteen residues of peptide CB-1 is: Ser-Leu-Thr-Lys-Asp-Phe-Asp-Glu-Val-Asn-Ile-Asp-Val-Ser-His-Phe-. The sequence of the first seventeen residues of peptide CB-2 is: Asp-Ile-Ala-Ile-Ser-His-Pro-Trp-Ile-Arg-Pro-Ser-Phe-Phe-Glu-Phe-His-. The N-terminal sequence of subunit DU1A was shown to be N-acetylmethionine followed by peptide CB-2.