bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthesis and separation by thin-layer chromatography of bilirubin-IX isomers. Their identification as tetrapyrroles and dipyrrolic ethyl anthranilate azo derivatives
Authors: Blanckaert N , Heirwegh K. P. M. , Compernolle F
Published: August 2015

Procedures for the synthesis, separation and determination of structure of the bilirubin-IX isomers are described. 1. The four biliverdin-IX isomers were prepared by oxidative cleavage of haemin and were separated as their dimethyl esters. The individual esters were reduced with NaBH4, and the bilirubin esters obtained were subjected to alkaline hydrolysis yielding the corresponding bilirubin-IX isomers. 2. The bilirubin-IX isomers were structurally characterized (a) at the tetrapyrrolic stage by mass spectrometry of their trimethylsilyl derivatives and (b) by formation and structural analysis of their dipyrrolic ethyl anthranilate azo derivatives. 3. The absorption spectrum of bilirubin-IX α differed strikingly from the spectra of the other isomers. The presence of a pronounced shoulder around 453 nm in the spectrum of bilirubin-IXβ allows easy differentiation from bilirubin-IXdelta. Methylation of the carboxyl groups largely eliminates the spectral differences between the IXα- and non-α isomers. 4. The bilirubin-IX isomers are conveniently separated by t.l.c. Detection and unequivocal identification is possible on a micro-scale by (a) t.l.c. with respect to reference compounds and (b) subsequent formation and t.l.c. of the more stable ethyl anthranilate azopigments. 5. Pronounced differences in polarity, i.e. solvent distribution, between the bilirubin-IX isomers indicate that a re-evaluation of conclusions reached previously with regard to the presence in, or absence from, biological fluids of some isomers and their relative amounts is needed.
The isolation and characterization of bilirubin diglucuronide, the major bilirubin conjugate in dog and human bile
Authors: Gordon E R , Goresky C A , Chang T H , Perlin A S
Published: August 2015

The chemical structure of the major conjugate of bilirubin was unequivocally elucidated by structural analysis. The conjugated bilirubins were first separated from the lipid components of human duodenal aspirates or dog gall-bladder bile, and then resolved by t.l.c. into a series of tetrapyrroles. The major tetrapyrrole was then converted into its more stable dipyrrolic azo derivative for further analysis. The conjugated moiety of the azopigment was characterized after methanolysis with sodium methoxide. This reaction yields two types of product, those soluble in water and those soluble in organic solvents. The organic-soluble fraction was shown by t.l.c. and mass spectrometry to contain the methyl esters of the dipyrrolic azo derivatives of bilirubin. The water-soluble materials were analysed by enzymic procedures, t.l.c., n.m.r. spectrometry and combined g.l.c. and mass spectrometry. This analysis showed that the only water-soluble product resulting from the methanolysis was glucuronic acid. The structure was identical with that of pure standards, on both mass spectrometry and n.m.r. spectroscopy. No contaminating moieties were found. Quantitative measurement indicated that the glucuronic acid had been released in a 1:1 molar ratio with the resulting methyl esters of the dipyrrolic azo derivatives of bilirubin. This unequivocally establishes bilirubin diglucuronide as the major pigment present in bile. Past problems with identification of bilirubin diglucuronide were shown to originate from procedures which resulted in incomplete separation and isolation of the azopigments of the conjugated bilirubins, owing to contamination by biliary lipids.
The rate of reaction of superoxide radical ion with oxyhaemoglobin and methaemoglobin
Authors: Sutton H C , Roberts P B , Winterbourn C C
Published: August 2015

Superoxide radical ions (O2-) produced by the radiolytic reduction of oxygenated formate solutions and by the xanthine oxidase-catalysed oxidation of xanthine were shown to oxidize the haem groups in oxyhaemoglobin and reduce those in methaemoglobin as in reactions (1) and (2): (see articles) Reaction (1) is suppressed by reaction (8) when [O2-]exceeds 10 muM, but consumes all the O2- generated in oxyhaemoglobin solutions when [oxyhaemoglobin] greater than 160 muM and [O2-]less than 1 nM at pH 7. The yield of reaction (2) is also maximal in methaemoglobin solutions under similar conditions, but less than one haem group is reduced per O2- radical. From studies of (a) the yield of reactions (1) and (2) at variable [haemoglobin] and rates of production of O2-, (b) their suppression by superoxide dismutase, and (c) equilibria observed with mixtures of oxyhaemoglobin and methaemoglobin, it is shown that k1/k2=0.7 +/- 0.2 and k1 = (4 +/- 1) × 10(3) M-1-S-1 At pH7, and k1 and k2 decrease with increasing pH. Concentrations and rate constants are expressed in terms of haem-group concentrations. Concentrations of superoxide dismutase observed in normal erythrocytes are sufficient to suppress reactions (1) and (2), and hence prevent the formation of excessive methaemoglobin.
Autophagy-related changes of arylsulphatases A and B in rat liver lysosomes
Authors: Sanghavi P , Koenig H
Published: August 2015

The total arylsulphatase activity and the relative activities of lysosomal arylsulphatases A and B were measured in the liver of control rats and rats subjected to treatments that provoke hepatic autophagocytosis. The total liver arylsulphatase activities were increased in starved and starved glucagon-treated rats, but not in sham-operated and hepatectomized rats. Arylsulphatases A and B in the mitochondrial-lysosomal (M-L) fraction were separated by polyacrylamide-gel electrophoresis at pH 8.8; they were made visible by incubating the gels with p-nitrocatechol sulphate as substrate, and measured by quantitative densitometry. In untreated controls, arylsulphatases A and B comprised 41.4 +/- 0.5% and 58.6 +/- 0.5% of the total arylsulphatase activity respectively; the arylsulphatase A/arylsulphatase B activity ratio was 0.71. All experimental treatments produced a significant decrease in the percentage of lysosomal arylsulphatase present as the A form and an increase in that present as the B form, and the activity ratio of arylsulphatase A/arylsulphatase B declined. The magnitude of these changes increased in the following direction: starvation for 24h=sham hepatectomy less than glucagon + starvation less than subtotal hepatectomy. These results indicate that the arylsulphatase A/arylsulphatase B activity ratio in liver lysosomes of normal rats is maintained within rather narrow limits, and this ratio declines during enhanced autophagocytosis. These findings, together with observations that suggest that arylsulphatase B may be a partially degraded form of arylsulphatase A, are consistent with the view that the A form is more rapidly converted into the B form during autophagy, owing to the digestive activity of the other lysosomal hydrolases present in autophagic vacuoles.
Quantitative analysis of metabolism of hepatic triglyceride in ethanol-treated rats
Authors: Abrams M A , Cooper C
Published: August 2015

An acute intraperitoneal injection of ethanol (0.7 or 2.1g/kg body wt.) causes the reversible, dose-dependent accumulation of hepatic triglyceride in rats. By using a pulse of [14C]palmitate injected into a tail vein, it was found that ethanol (2.1g/kg)had no effect on the flux of unesterified fatty acid of serum (4.3mumol/min per 100g body wt.). However, either dose increased the fraction of the total flux going to liver from 0.16 to0.27 as rapidly as could be measured (30s), and it remained elevated until all ethanol had been cleared from the blood. The fraction of the total radioactivity in lipids of liver that was in triglyceride increased linearly for 1 h from 30 to 50% and there was a simultaneous decrease in phospholipid from 60 to 40%. The rate of synthesis of hepatic triglyceride derived directly from unesterified fatty acid of serum was calculated by using the flux rate of unesterified fatty acid in serum, the fractional hepatic uptake of this flux, and the percentage of liver fatty acid esterified to triglyceride. This contribution is related to the total synthetic rate of hepatic triglyceride (rate of accumulation+rate of release) to determine quantitatively how much of the developing fatty liver is attributable to increased uptake of unesterfied fatty acid of serum. At the higher dose of ethanol, about half of the accumulating triglyceride is derived from this source, whereas with the lower dose of ethanol it can account for all of the build-up.
Stimulation of prolactin synthesis and of adenosine 3':5'-cyclic phosphate formation by prostaglandins and thyroliberin in cultured rat pituitary cells
Authors: Gautvik K M , Kriz M
Published: August 2015

1. The effects of prostaglandins E2 and F2alpha on prolactin synthesis were examined in a clonal strain of rat pituitary tumour cells, and compared with those of thyroliberin. 2. The prostaglandins and thyroliberin gave a dose-related and time-dependent stimulation of prolactin synthesis. The maximal effects (about twofold increases) were observed after 54h of treatment with 25nM-prostaglandin E2 and 2.5nM-prostaglandin F2alpha. A similar stimulation of prolactin synthesis was observed after 250nM-thyroliberin. The combined treatment with prostaglandins and thyroliberin did not increase prolactin synthesis over and above that obtained with each compound alone. 3. After removal of prostaglandins E2 and F2alpha there was a complete reversal of prolactin synthesis to pre-stimulation values 18h later (t1/2less than or equal to 9h). The rapid reversible effect of prostaglandins was in contrast with that of thyroliberin, where prolactin synthesis returned to control values with a t1/2 of about 42 h. 4. Prostaglandin E2 (5mum) and thyroliberin (5mum) increased cellular concentrations of cyclic AMP eight- and four-fold respectively. Maximal effects were observed after 2-5min of incubation. The increases in cyclic AMP were biphasic; normal values were obtained 60 min after the start of incubation with prostaglandin E2 or thyroliberin. 5. The dose/response curve showed that prostaglandin E2 caused maximal increase of cyclic AMP at 50nM. Concentrations of prostagland in E2 that caused half-maximal stimulation of cyclic AMP accumulation and of prolactin synthesis were 4 and 5nM respectively. 6. Combined treatment with prostaglandin E2 and thyroliberin in concentrations that separately caused maximal cyclic AMP increases did not result in a further increase in this cyclic nucleotide. 7. These results are consistent with a role of cyclic AMP in mediating the effects or prostaglandins and thyroliberin on prolactin synthesis. However, if cyclic AMP is involved as a common intracellular mediator of prolactin synthesis, it cannot alone explain all the effects of prostaglandins and thyroliberin in this cell system.
Hydrogen-ion titration studies on erythrocyte membranes
Authors: Hallam C , Wrigglesworth J M
Published: August 2015

1. H+ titration was used to detect the presence of ionizable groups on human erythrocyte plasma membranes. Between pH2.9 and 11.3, two significant peaks of H+ association/dissociation occur in the differential from of the titration curve, one at pH3. 1. And the other at pH10.3. 2. After disruption of membrane structure by exposure to high pH or by the addition of sodium dodecyl sulphate, maxima of H+ association/dissociation were seen at pH3.1,4.3,6.5,10.3 and 10.7. 3. Spectrophotometric assay and selective chemical treatments were used to identify several of the titratable residues. 4. The degree of eleectrostatic interaction between titratable charged groups was investigated by comparing the titration characteristics of the membranes before and after modification of membrane structure.
Reduction of cytochromes by nitrite in electron-transport particles from Nitrobacter winogradskyi: proposal of a mechanism for H+ translocation
Authors: Cobley J G
Published: August 2015

1. A novel component in the respiratory chain of Nitrobacter winogradskyi was identified. This component absorbs maximally at 552.5 nm when in its reduced form, has an Eo' (pH7.0) value of-110mV and undergoes reduction by a mechanism involving the transfer of a single electron. 2. Degrees of reduction of cytochromes c and a1 in electron-transport (ET) particles were monitored during the course of NO2- oxidation, and the effects of ADP together with Pi, oligomycin and of carbonyl cyanide phenylhydrazone were determined. 3. The influences of ionophorous antibiotics, NH4Cl and cyclohexylamine hydrochloride on the reductions of cytochromes c and a1 by NO2- indicate that the flow of reducing equivalents from cytochrome a1 (+350mV) to cytochrome c (+270mV) is facilitated by deltapsi, the electrical component of the protonmotive force. 4. Cytochromes c and a1 in ET particles are reduced by the non-physiological reductant KBH4 in a manner similar to that observed with the physiological reductant NO2-. 5. To account both for the observed cytochrome reductions and for the translocation of H+ ions which accompanies NO2- oxidation, a mechanism is proposed which involves the transfer of a hydride equivalent (H+ plus 2e) inward across the membrane of the ET particle in response to deltapsi.
Enzymic hydrolysis of myelin basic protein and other proteins in central nervous system and lymphoid tissues from normal and demyelinating rats
Authors: Buletza G F , Smith M E
Published: August 2015

Proteolytic activity of central-nervous-system tissue of the normal rat was examined over the pH range 2-9 with casein, haemoglobin and myelin basic protein as substrates. With casein as a substrate, brain and spinal cord homogenates showed very similar activity profiles with increasing pH, with the main peaks of proteolytic activity at pH 3-4 and 5-6. When haemoglobin was used, one broad main peak of activity from pH 3 to 5 was demonstrated. There was no optimum pH, however, for proteolytic activity with myelin basic protein as a substrate, and considerable hydrolysis were observed from pH 3.5 up to pH8. Proteolytic activity at the various pH values was compared by using homogenates of spinal cords from rats with acute experimental allergic encephalomyelitis and those from rats injected with Freund's adjuvant alone. The profiles of activity were similar with peaks at pH 3.5 and 5.5 with casein as a substrate, but the specific activity was significantly higher at most pH values in the spinal-cord homogenates from rats with experimental allergic encephalomyelitis. Similarly the spinal-cord homogenates from these latter rats contained much more proteolytic activity toward myelin basic protein throughout the pH range than was present in the control spinal cords. Homogenates from lymph nodes of rats with experimental allergic encephalomyelitis and from those of the controls contained two to three times as much proteolytic activity as that of the central-nervous-system tissue and had a different proteolytic activity profile form that of the central-nervous system, with higher activity at the neutral than at acid pH. The results are discussed with regard to the probability that inflammatory cells such as lymphocytes may be the cause of the increased proteolytic activity in the central nervous system of animals with experimental allergic encephalomyelitis, and that enzymes from these cells possess the capability of digesting myelin basic protein.
Effect of haematocrit value and pO2 on the redox state and metabolism of the perfused liver
Authors: Mayes P A , Felts J M
Published: August 2015

1. The haematocrit value and pO2 of blood perfusing the isolated liver were varied. Provided O2 content of the blood was not rate-limiting, O2 consumption was related to haemotocrit value rather than O2 saturation or pO2. 2. Hypoxia caused the blood-glucose concentration and ketogenesis to increase and the output of very-low-density (d less than 1.006) lipoproteins to decrease. 3. A decrease in pO2 caused an increase in both the (lactate)/(pyruvate) and (3-hydroxybutyrate)/(acetoacetate) and a decrease in (ATP)/(ADP) ratios, independently of O2 consumption. 4. The more reduced redox state was associated with a shift in the balance between the oxidation and esterification of free fatty acids in favour of oxidation. 5. Acetoacetate may be an important hydrogen acceptor during hypoxia of the liver.
A novel manual method for protein-sequence analysis
Authors: Chang J Y , Creaser E H
Published: August 2015

A novel manual method for protein-sequence analysis is described. Three peptides, the hexapeptide (Leu-TRP-Met-Arg-Phe-Ala), insulin A chain and glucagon were used to test this technique. Peptides (1 or 2 nmol) were hydrolysed with acid and their qualitative amino acid compositions were confirmed by reacting with 4-NN-dimethylaminoazobenzene-4'-sulphonylchloride and 4-NN-dimethylaminoazobenzene 4'-isothiocyanate. Sequence determination of 20-200 nmol of peptide was then performed by the combined use of phenyl isothiocyanate and 4-NN-dimethylaminoazobenzene 4'-isothiocyanate, a new procedure that is analogous to the dansyl-Edman method with the replacement of dansyl chloride by 4-NN-dimethylaminoazobenzene 4'-isothiocyanate as the N-terminal residue determination reagent. On t.l.c. this new N-terminal reagent gave brightly coloured 4-NN-dimethylaminoazobenzene-4-thiohydantoins of amino acids and showed the following advantages: (1) the detection sensitivity is in the pmol range; (2) u.v. observation is not required; (3) there is no destruction of acid-labile amino acids; (4) two-dimensional t.l.c. separation is adequate to identify 24 amino acids, except leucine and isoleucine (this pair of amino acids can be resolved by using 4-NN-dimethylaminoazobenzene-4'-sulphonyl chloride); (5) the determination of a new N-terminal residue (from coupling to t.l.c. identification) takes only 3 h; (6) the colour difference beteen isothiocyanate, thiocarbamoyl and thiohydantoin derivatives facilitates the identifications.
Isolation and properties of a small manganese-ion-stimulated bacterial alkaline phosphatase
Authors: Fitt P S , Peterkin P I
Published: August 2015

1. An alkaline phosphatase was partially purified from extracts of Halobacterium cutirubrum. 2. The enzyme has a mol.wt. of 15 500 and is therefore less than one-quarter of the size of other known bacterial alkaline phosphatases. 3. It is stimulated up to ten-fold by Mn2+, but not by Ca2+ or Mg2+. 4. The activities with and without Mn2+ cannot be separated by gel filtration and have similar restricted substrate specificities. 5. The only substrates for the enzyme that have so far been found are p-nitrophenyl phosphate, 5'-dATP, 5'-dTMP and 5'-dTTP.
Isolation of albumin from whole human plasma and fractionation of albumin-depleted plasma
Authors: Travis J , Bowen J , Tewksbury D , Johnson D , Pannell R
Published: August 2015

The dye Cibacron Blue F-3-GA was conjugated to Sepharose to provide an affinity column for serum albumin. Passage of whole human plasma through a column of Cibacron Blue-Sepharose results in the removal of approx. 98% of the albumin. The latter can be quantitatively recovered by desorption with NaSCN. Albumin-depleted plasma can be readily resolved into discrete fractions by a combination of conventional biochemical techniques. In particular, the resolution of plasma proteins with properties similar to those of native human plasma albumin can readily be accomplished by ion-exchange chromatography of the Sepharose-dye-treated plasma on DEAE-cellulose.
The β-glucosidase in the gut contents of the snail Achatina achatina
Authors: Umezurike G M
Published: August 2015

1. The enzyme beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) from the gut contents of active Achatina achatina exists in two molecular forms, beta-glucosidase C (mol.wt. about 82000) and D (mol.wt. about 41000). 2. Only the lower-molecular-weight species was found in the gut contents of aestivating snails or in extracts from their digestive glands and washed gut walls. 3. On re-activation of some aestivating snails, betion of ATP and Mg2+ to the isolated gut contents or to extracts from washed gut walls led to the formation of higher-molecular-weight forms of the enzyme, beta-glucosidase A (mol.wt. about 329000) and beta-glucosidase B (mol.wt. about 165000). 5. All these forms of the enzyme have similar pH optimum (pH 5.0-5.6). 6. The Michaelis constants (Km) and heat stability of the enzyme increased with increasing molecular complexity.
Studies on partially reduced mammalian cytochrome oxidase reactions with ferrocytochrome c
Authors: Greenwood C , Brittain T
Published: August 2015

The kinetics of the electron-transfer process which occurs between ferrocytochrome c and partially reduced mammalian cytochrome oxidase were studied by the rapid spectrophotometric techniques of stopped flow and temperature jump. Stopped-flow experiments showed initial very fast extinction changes at 605 nm and at 563 nm, indicating the simultaneous reduction of cytochrome a and oxidation of ferrocytochrome c. During this ‘burst’ phase, say the first 50 ms after mixing, it was invariably found that more cytochrome c had been oxidized than cytochrome a had been reduced. This discrepancy in electron equivalents may be accounted for by the rapid reduction of another redox site in the enzyme, possibly that associated with the extinction changes observed at 830 nm. During the incubation period in which the partially reduced oxidase was prepared, the rate of reduction of cytochrome a by ferrocytochrome c, at constant reactant concentrations, decreased with time. Temperature-jump experiments showed the presence of two relaxation processes. The faster of the two phases was assigned to the electron-transfer reaction between cytochrome c and cytochrome a. A study of the concentration-dependence of the reciprocal relaxation time for this phase yielded a rate constant of 9 X 10(6)M-1-s-1 for the electron transfer from cytochrome c to cytochrome a, and a value of 8.5 X 10(6)M-1-s-1 for the reverse reaction. The equilibrium constant for the electron-transfer reaction is therefore close to unity. The slower phase has been interpreted as signalling the transfer of electrons between cytochrome a and another redox site within the oxidase molecule.
Induction of benzo[a]pyrene Mono-oxygenase in liver cell culture by the photochemical generation of active oxygen species. Evidence for the involvement of singlet oxygen and the formation of a stable inducing intermediate
Authors: Paine A J
Published: August 2015

1. The photochemical generation of excited states of oxygen in liver cell culture by the mild ilumination of culture medium containing riboflavin, results in stimulation of benzo[a]pyrene 3-mono-oxygenase, a cytochrome P-450-linked mono-oxygenase. 2. The same large increase in mono-oxygenase activity was found when medium containing riboflavin was illuminated in the absence of cells and then stored in the dark for 24h before contact with the cells. From this it may be inferred that stimulation is due to the formation of a stable inducer in the culture medium. Further experiments indicate that the stable inducer is due to the photo-oxidation of an amino acid. 3. Evidence that singlet oxygen is responsible for initiating the stimulation of the mono-oxygenase is based on the use of molecules that scavenge particular active oxygen species. Of all the scavengers tested, only those that scavenge single oxygen inhibited the stimulation. 4. A hypothesis is developed to relate the stimulation of the mono-oxygenase by singlet oxygen in cultured cells to the regulation of the cytochrome P-450 enzyme system in vivo. It is suggested that single oxygen generation within cells may be a common factor linking the many structurally diverse inducers of the enzyme system.
Adenylate cyclase, guanylate cyclase and cyclic nucleotide phosphodiesterases of guinea-pig cardiac sarcolemma
Authors: St Louis P J , Sulakhe P V
Published: August 2015

1. The activities of the enzymes involved in the metabolism of cyclic nucleotides were studied in sarcolemma prepared front guinea-pig heart ventricle; the enzyme activities reported here were linear under the assay conditions. 2. Adenylate cyclase was maximally activated by 3mM-NaF; NaF increased the Km for ATP (from 0.042 to 0.19 mM) but decreased the Ka for Mg2+ (from 2.33 to 0.9 mM). In the presence of saturating Mg2+ (15 mM), Mn2+ enhanced adenylate cyclase, whereas Co2+ was inhibitory. beta-Adrenergic amines (10-50 muM) stimulated adenylate cyclase (38+/-2%). When added to the assay mixture, guanyl nucleotides (GTP and its analogue, guanylyl imidophosphate) stimulated basal enzyme activity and enhanced the stimulation by isoproterenol. By contrast, preincubation of sarcolemma with guanylyl imidodiphosphate stimulated the formation of an ‘activated’ form of the enzyme, which did not reveal increased hormonal sensitivity. 3. The guanylate cyclase present in the membranes as well as in the Triton X-100-solubilized extract of membranes exhibited a Ka for Mn 2+ of 0.3 mM; Mn2+ in excess of GTP was required for maximal activity. Solubilized guanylate cyclase was activated by Mg2+ only in the presence of low Mn2+ concentrations; Ca2+ was inhibitory both in the absence and presence of low Mn2+. Acetylcholine as well as carbamolycholine stimulated membrane-bound guanylate cyclase. 4. Cylic nucleotide phosphodiesterase activities of sarcolemma exhibited both high-and low-Km forms with cyclic AMP and with cyclic GMP as substrate. Ca2+ ions increased the Vmax. of the cyclic GMP-dependent enzyme.
Testosterone and 6-N,2'-O-dibutyryladenosine 3′:5′-cyclic monophosphate stimulate protein and lysosomal enzyme secretion in rat seminal vesicle
Authors: Koenig H , Lu C Y , Bakay R
Published: August 2015

Rat seminal-vesicle secretion was studied in vitro in a slice-incubation system. Seminal-vesicle slices were preincubated with 32Pi for 15 min, rinsed, and incubated in an isotope-free ‘chase’ medium for up to 4h. Gland slices spontaneously discharged protein, three lysosomal hydrolases and trichloroacetic acid-insoluble 32P into the medium in a time- and temperature-dependent manner. Testosterone (10 muM) and dibutyryl cyclic AMP (1 mM) stimulated the discharge of protein, acid hydrolases and trichloroacetic acid-insoluble 32P, and also stimulated the incorporation of 32Pi into trichloroacetic acid-insoluble components. The acid phosphatase and beta-N-acetylhexosaminidase isoenzymes were separated by isoelectric focusing. These hydrolases were secreted into the medium as acidic isoenzymes, presumably contained within primary lysosomes, whereas they occurred largely as less acidic and basic isoenzymes in the glandular tissue.
Glucosamine metabolism in regenerating rat liver
Authors: Akamatsu N , Nakajima H , Miyata S
Published: August 2015

1. Glycoprotein synthesis was investigated with [1-14C]glucosamine in vivo. [14C]Glucosamine was administered intravenously 24h after hepatectomy to rats. 2. Incorporation into the acid-soluble fraction was maximum at 15 min after injection both in sham-operated and hepatectomized rats. 3. Enhancement of incorporation into UDP-N-acetylhexosamine in regenerating liver was observed. However, its specific activity was lower, because of a greater enhancement of synthesis de novo of the amino sugar. 4. In the liver acid-insoluble fraction, maximum incorporation of [14C]glucosamine was at 30 min in sham-operated rats and 2 h in hepatectomized rats respectively. 5. In sham-operated rats, incorporation into the plasma acid-insoluble fraction followed that of the liver acid-insoluble fraction, but hepatectomy resulted in a rapid enchancement of incorporation into plasma. 6. It is concluded that synthesis of liver glycoproteins is stimulated after partial hepatectomy and that glycoproteins synthesized are released rapidly into the plasma.
Isolation and characterization of an active three-chain molecular species of bovine thrombin
Authors: Giglio J R , Rossi A , Leone F A , Chiericeto G , Say J C
Published: August 2015

Partially purified bovine prothrombin was activated in half-saturated trisodium citrate seeded with thrombin, and the resulting thrombin was chromatographed on Amerblite IRC-50, followed by rechromatography on DEAE-Sephadex A-50. Five fractions, possessing both esterase and clotting activities, were partially isolated, but fraction VI was shown to be a pure three-chain active species with threonine, isoleucine and lysine, in 1:1:1 molar proportions as N-termini. The amino acid composition and C-termini of fraction VI were determinied. The molecular weights of the isolated chains, as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, were 7300, 12000 and 19500 respectively. These data, when taken together with the amino acid sequence of the two-chain thrombin reported by Magnusson et al. (1975) [in Prothrombin and related Coagulation Factors, (Hember, H. C. & Veltkamp, J. J., eds.), pp. 25-46, Leiden University Press, Leiden], indicated that proteolysis occurred at the Arg(78)-Lys(79) peptide bond of the B chain of a precursor molecular species, thus converting this two-chain species into the three-chain active form described here.
The magnesium-ion-dependent adenosine triphosphatase of bovine cardiac Myosin and its subfragment-1
Authors: Taylor R S , Weeds A G
Published: August 2015

The kinetics of the Mg2+-dependent ATPase (adenosine triphosphatase) activity of bovine cardiac myosin and its papain subfragment-1 were studied by using steady-state and pre-steady-state techniques, and results were compared with published values for the corresponding processes in the ATPase mechanism of rabbit skeletal-muscle myosin subfragment-1. The catalytic-centreactivity for cardiac subfragment-1 is 0.019s-1, which is less than one-third of that determined for the rabbit protein. The ATP-induced isomerization process, measured from enhancement of protein fluorescence on substrate binding, is similarly decreased in rate, as is also the isomerization process associated with ADP release. However, the equilibrium constant for ATP cleavage, measured by quenched-flow by using [γ-32P]ATP, shows little difference in the two species. Other experiments were carried out to investigate the rate of association of actin with subfragment-1 by light-scattering changes and also the rate of dissociation of the complex by ATP. The dissociation rate increases with increasing substrate concentration, to a maximum at high ATP concentrations, with a rate constant of about 2000s-1. It appears that isomerization processes which may involve conformational changes have substantially lower rate constants for the cardiac proteins, whereas equilibrium constants for substrate binding and cleavage are not significantly different. These differences may be related to the functional properties of these myosins in their different muscle types. Kinetic heterogeneity has been detected in both steady-state and transient processes, and this is discussed in relation to the apparent chemical homogeneity of cardiac myosin.
Isolation and characterization of two sequence-specific endonucleases from Anabaena variabilis
Authors: Murray K , Hughes S G , Brown J S , Bruce S A
Published: August 2015

Two endonucleases, AvaI and AvaII, were isolated from Anabaena variabilis on the basis of their ability to make a limited number of breaks at specific points in bacteriophage λ DNA. Neither enzyme has cofactor requirements beyond Mg2+. Endonuclease AvaI makes eight breaks in the phage λ chromosome at which the 5'-terminal sequence is pPy-C-G-N. AvaII endonuclease cuts phage λ DNA more extensively, yielding fragments with the 5'-terminal sequence G-T-C-N or G-A-C-N. Neither enzyme generates cohesive ends.
Thiol-protein disulphide oxidoreductases. Assay of microsomal membrane-bound glutathione-insulin transhydrogenase and comparison with protein disulphide-isomerase
Authors: Ibbetson A L , Freedman R B
Published: August 2015

1. Inhibition of endogenous microsomal NADPH oxidase by CO enables membrane-bound glutathione-insulin transhydrogenase (EC 1.8.4.2) to be assayed conveniently by a linked assay involving NADPH and glutathione reductase (EC 1.6.4.2). 2. The specific activity of the enzyme in rat liver microsomal preparations is of the order of 1 nmol of oxidized glutathione formed/min per mg of membrane protein. 3. The specific activity of the enzyme is comparable in rough and smooth microsomal fractions, and the activity is not affected by treatment with EDTA and the removal of ribosomes from rough microsomal fractions. 4. Membrane-bound glutathione-insulin transhydrogenase is not affected by concentrations of deoxycholate up to 0.5%, whereas protein disulphide-isomerase (EC 5.3.4.1) is drastically inhibited. 5. On these grounds it is concluded that, in rat liver microsomal fractions, glutathione-insulin transhydrogenase and protein disulphide-isomerase activities are not both catalysed by a single enzyme species.
The aminoacylation of transfer ribonucleic acid. Inhibitory effects of some amino acid analogues with altered side chains
Authors: Old J M , Jones D S
Published: August 2015

Several amino acid analogues that are able to replace amino acid residues in binding positions of the biologically active C-terminal tetrapeptide amide sequence, Trp-Met-Asp-PheNH2, of the gastrins were examined for their ability to inhibit the aminoacylation of tRNA in an Escherichia coli and rat liver system. Although in both systems the amino acid side chains are involved in the recognition process, the structural requirements of the side chain in the two systems are not comparable. Analogues of methionine and phenylalanine behaved similarly in the E. coli and rat liver systems, whereas analogues of tryptophan behaved differently. From the results it is possible to suggest structural features of the amino acid side chains which are required for recognition by the aminoacyl-tRNA synthetases.
Chemical synthesis of all-trans-β-retinoyl phosphate
Authors: Frot-Coutaz J P , de Luca L M
Published: August 2015

all-trans-β-Retinoic acid is phosphorylated to retinoyl phosphate by bis(triethylamine) phosphate with yields of 10-15%. The product is soluble in methanol and is eluted from DEAE-cellulose acetate at a concentration of 0.1M-ammonium acetate in 99% (v/v) methanol. Its phosphate/retinoic acid molar ratio is 1. Retinoyl phosphate has an absorption maximum at 360nm in methanol, whereas retinoic acid has a maximum at 350 nm. The compound is hydrolysed at pH2 and pH13 for 20 min at 37 degrees C, but is relatively stable under the same conditions at pH4, 6, 8 and 10. Retinoyl phosphate (RF 0.1) can be separated from retinyl phosphate (RF 0.2) by chromatography on thin layers of silica gel in chloroform/methanol/water (60:25:4, by vol.).
Uridine diphosphate xylosyltransferase activity in cartilage from manganese-deficient chicks
Authors: McNatt M L , Fiser F M , Elders M J , Kilgore B S , Smith W G , Hughes E R
Published: August 2015

The glycosaminoglycan content of cartilage is decreased in manganese deficiency in the chick (perosis). The activity of xylosyltransferase, the first enzyme in the biosynthetic pathway of sulphated glycosaminoglycans, was studied in the epiphysial cartilage of 4-week-old chicks which had been maintained since hatching on a manganese-deficient diet. Enzymic activity was measured by the incorporation of [14C]xylose from UDP-[14C]xylose into trichloroacetic acid precipitates. Optimal conditions for the xylosyltransferase assay were established and shown to be the same for both control and manganese-deficient cartilage. Assay of the enzyme by using an exogenous xylose acceptor showed no difference in xylosyltransferase activity between control and manganese-deficient tissue. Further, the extent of xylose incorporation was greater in manganese-deficient than in control cartilage preparations, suggesting an increase in xylose-acceptor sites on the endogenous acceptor protein in the deficient cartilage. 35S turnover in the manganese-deficient cartilage was also increased. The data suggest that the decreased glycosaminoglycan content in manganese-deficient cartilage is due to decreased xylosylation of the acceptor protein plus increased degradation of glycosaminoglycan.
Subcellular localization of oestrogen-induced uterine peroxidase
Authors: Lyttle C R , Jellinck P H
Published: August 2015

The distribution of oestrogen-induced peroxidase in the resuspended 8000g pellet of rat uterine homogenates was examined by centrifugation in a sucrose density gradient. Within 10h of treatment with oestradiol, peroxidase activity was found in a region devoid of catalase or urate oxidase (peroxisomal markers) which did not overlap the fractions containing succinate dehydrogenase (mitochondrial marker) or acid phosphatase (lysosomal marker). The induced uterine enzyme was localized in reticular membrane-bound vesicles with isopycnic density of 1.28g/ml from which it could be released by treatment with detergent.
Acute effects in vivo of anti-insulin serum on rates of fatty acid synthesis and activities of acetyl-coenzyme A carboxylase and pyruvate dehydrogenase in liver and epididymal adipose tissue of fed rats
Authors: Stansbie D , Brownsey R W , Crettaz M , Denton R M
Published: August 2015

Plasma insulin concentrations in fed rats were altered acutely by administration of glucose or anti-insulin serum. Rates of fatty acid synthesis in adipose tissue and liver were estimated from the incorporation of 3H from 3H2O. In the adipose tissue dehydrogenase and acetyl-CoA carboxylase were evident. In liver, although changes in rates of fatty acid synthesis were found, the initial activity of pyruvate dehydrogenase did not alter, but small parallel changes in acetyl-CoA carboxylase activity were observed.
Metabolic effect of α-and the β-adrenergic stimulation of rat submaxillary gland in vitro
Authors: Thompson M P , Williamson D H
Published: August 2015

1. Incubation of submaxillary-gland slices with isoproterenol, a β-adrenergic agonist, stimulated glucose removal by 41% and decreased tissue [glucose 6-phosphate] by 50%. Propranolol blocked these effects of isoproterenol. 2. Phenylephrine, an α-adrenergic agonist, stimulated glucose removal by 35% and decreased tissue [glucose 6-phosphate] by 75%. In addition, phenylephrine also completely overcame the inhibition of pyruvate removal caused by acetoacetate metabolism and decreased tissue [atp] by 45%. Phentolamine blocked the effects of phenylephrine. 3. In contrast with β-adrenergic stimulation, α-adrenergic stimulation required exogenous Ca2+. 4. These results explain the different metabolic responses of the submaxillary gland to adrenaline in the presence and absence of exogenous Ca2+.
A proposed sequence of hormones controlling the induction of luteal 20α-hydroxy steroid dehydrogenase and progesterone withdrawal in the late-pregnant rat
Authors: Smith D H , Kuhn N J
Published: August 2015

1. The previously reported induction of luteal 20α-hydroxy steroid dehydrogenase by administration of aminoglutethimide to late-pregnant rats was shown to be unaffected by prior removal of the foetuses. Aminoglutethimide therefore does not act via the foetuses in this context. 2. The ability of injected oestrogen to prevent the above induction was lost by delaying the injection for 12h after aminoglutethimide, although the increase in enzyme activity begins only after 24h. 3. Induction of 20α-hydroxy steroid dehydrogenase by foetoplacental removal on day 18 of pregnancy was inhibited by human choriogonadotropin, lutropin (luteinizing hormone) and pregnant-mare serum gonadotropin, but not by somatotropin (growth hormone), thyrotropin or follitropin (follicle-stimulating hormone) 4. Indomethacin blocked the normal induction of 20α-hydroxy steroid dehydrogenase in late pregnancy and that caused by aminoglutethimide. It partially blocked that caused by human choriogonadotropin given on days 19-20 and that caused by 2-bromo-α-ergocryptine on days 5-6, but failed to block that caused by human choriogonadotropin on days 15-16 or by foetoplacental removal on day 18 of pregnancy. 5. These findings, and the control of progesterone synthesis in late pregnancy, are interpreted in terms of a sequence of hormonal or enzymic syntheses, each of which is inhibited by the product of the preceding synthesis.
Purification and some properties of a medium-chain acyl-thioester hydrolase from lactating-rabbit mammary gland which terminates chain elongation in fatty acid synthesis
Authors: Knudsen J , Clark S , Dils R
Published: August 2015

1. An acyl-thioester hydrolase was isolated from the cytosol of lactating-rabbit mammary gland. The purified enzyme terminates fatty acid synthesis at medium-chain (C8:0-C12:0) acids when it is incubated with fatty acid synthetase and rate-limiting concentrations of malonyl-CoA. These acids are characteristic products of the lactating gland. 2. The mol.wt. of the enzyme is 29000±500 (mean±S.D. of three independent preparations), as estimated by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. 3. The enzyme also hydrolyses acyl-CoA esters of chain lengths C10:0-C16:0 when these are used as model substrates. The greatest activity was towards dodecanoyl-CoA, and the three preparations had specific activities of 305, 1130 and 2010 nmol of dodecanoyl-CoA hydrolysed/min per mg of protein when 56muM substrate was used. 4. The way in which this enzyme controls the synthesis of medium-chain fatty acids by fatty acid synthetase is briefly discussed.
The glycerol phosphate, dihydroxyacetone phosphate and monoacylglycerol pathways of glycerolipid synthesis in rat adipose-tissue homogenates
Authors: Dodds P F , Gurr M I , Brindley D N
Published: August 2015

1. Fat-free homogenates from the epididymal fat-pads of rats were used to measure the rate of palmitate esterification with different substrates. The effectiveness of the acyl acceptors decreased in the order glycerol phosphate, dihydroxyacetone phosphate, 2-octadecenyl-glycerol and 2-hexadecylglycerol. 2. Glycerol phosphate and dihydroxyacetone phosphate inhibited their rates of esterification in a mutually competitive manner. 3. The esterification of glycerol phosphate was also inhibited in a partially competitive manner by 2-octadecenylglycerol and to a lesser extent by 2-hexadecylglycerol. However, glycerol phosphate did not inhibit the esterification of 2-octadecenylglycerol. 4. The esterification of dihydroxyacetone phosphate and 2-hexadecylglycerol was more sensitive to inhibition by clofenapate than was that of glycerol phosphate. Norfenfluramine was more effective in inhibiting the esterification of 2-hexadecylglycerol than that of glycerol phosphate or dihydroxyacetone phosphate. 5 It is concluded that rat adipose tissue can synthesize glycerolipids by three independent routes.
Purification and immunochemical characterization of monoamine oxidase from rat and human liver
Authors: Dennick R G , Mayer R J
Published: August 2015

1. Monoamine oxidase from rat and human liver was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. 2. The enzyme activity was extracted from mitochondrial preparations by Triton X-100. The enzyme was purified by (NH4)2SO4 fractionation followed by chromatography on DEAE-cellulose, Sepharose 6B, spheroidal hydroxyapatite, and finally chromatography on diazo-coupled tyramine-Sepharose. 3. Distinct differences occur in the chromatographic behaviour of the two enzymes on both DEAE-cellulose and spheroidal hydroxyapatite. 4. It is unlikely that the purification of the enzymes on tyramine-Sepharose is due to affinity chromatography and reasons for this are discussed. 5. The purified enzymes did not oxidize-5-hydroxytryptamine and the relative activities of the enzymes with benzylamine were increased approx. 1.25-fold compared with the enzyme activities of mitochondrial preparations. 6. Immunotitration of enzyme activity in extracts of mitochondrial preparations from rat liver was carried out with 5-hydroxytryptamine, tyramine and benzylamine. The enzyme activities were completely immunoprecipitated by the same volume of antiserum. Similar results were obtained with the antiserum to the enzyme from human liver.
An addition at the C-terminus of water-buffalo immunoglobin lambda chains
Authors: Svasti J
Published: August 2015

The amino acid sequence of the C-terminal tryptic peptide of pooled water-buffalo immunoglobulin lambda chains was determined as Thr-Val-Lys-Pro-Ser-Glu-Cys-Pro-Ser. This sequence is closely homologous to equivalent sequences from other species, but shows an additional amino acid on the C-terminal side of the interchain half-cystine residue.
Purification and properties of rabbit spermatozoal acrosomal neuraminidase
Authors: Srivastava P N , Abou-Issa H
Published: August 2015

Treatment of rabbit spermatozoa with 50mM-MgCl2 removes the plasma and the outer acrosomal membranes. Subsequent treatment with the detergents Hyamine 2389 and Triton X-100 solubilizes spermatozoal neuraminidase bound to the inner acrosomal membrane. The enzyme was further purified by DEAE-cellulose, Sephadex G-150 and Bio-Gel P-300 column chromato. The enzyme showed a single major band, with the possibility of some minor contaminants, on disc-gel electrophoresis. It had a specific activity of 0.37 micronmal of sialic acid released/min per mg with purified boar Cowper's-gland mucin as the substrate. The enzyme had marked specificity for 2 leads to 6′-linked sialic acid in glycoproteins. The Km of spermatozoal neuraminidase was 1.72 X 10(-6)M with Cowper's-gland mucin, 1.17 X 10(-5)M with fetuin and 8.8 X 10(-4)M with sialyl-lactose as a substrates. The Vmax. was 0.112 micronmol/min per mg with the Cowper's-gland mucin, 0.071 micronmol/min per mg with fetuin and 0.033 micronmol/min per mg with sialyl-lactose as substrate. The enzyme hydrolysed sheep submaxillary-gland mucin as readily as the Cowper's-gland mucin. The optimum of enzyme activity was at pH 5.0 on the Cowper's-gland mucin and at pH4.3 on sialyl-lactose. The enzyme activity was unaffected by 20mM-Na+ and-K+, but was inhibited by 20mM-Ca2+,-Mn2+,-Co2+ and -Cu2+. The enzyme was unstable in dilute solutions, but could be stored indefinitely freeze-dried at --20 degrees C.
Interaction of difluoro-oxaloacetate with aspartate transaminase
Authors: Briley P A , Eisenthal R , Harrison R , Smith G D
Published: August 2015

Diffluoro-oxaloacetate behaves as a competitive inhibitor of 2-oxoglutarate and as an uncompetitive inhibitor with respect to aspartate in steady-state kinetic experiments with cytoplasmic aspartate transaminase. In the presence of high concentrations of aspartate transaminase, difluoro-oxaloacetate is slowly transaminated to difluoro-aspartate, suggesting its use as a kinetic probe to study the reactions of the aminic form of the enzyme.
Cinnabarinate synthase from baboon (Papio ursinus) liver. Identity with catalase
Authors: Savage N , Prinz W
Published: August 2015

The enzyme chinnabarinate synthase was purified from the nuclei of baboon liver. Two purified fractions were obtained that exhibited a typical haem protein absorption spectrum; a probable identity with catalase was demonstrated. It was confirmed that catalase in the presence of Mn2+ produces cinnabarinate from 3-hydroxyanthranilate. Doubt is expressed on the existence of a distinct cinnabarinate synthase enzyme.