bluuebunny/crossref_metadata_2025 huggingface dataset:
Stimulation of collagen galactosyltransferase and glucosyltransferase activities by lysophosphatidylcholine
Authors: Anttinen H
Published: August 2015

Lysophosphatidylcholine stimulated the activities of collagen galactosyl- and glucosyl-transferases in chick-embryo extract and its particulate fractions in vitro, whereas essentially no stimulation was noted in the high-speed supernatant, where the enzymes are soluble and membrane-free. The stimulatory effect of lysophosphatidylcholine was masked by 0.1% Triton X-100. In kinetic experiments lysophosphatidylcholine raised the maximum velocities with respect to the substrates and co-substrates, whereas no changes were observed in the apparant Km values. Phospholipase A preincubation of the chick-embryo extract resulted in stimulation of both transferase activities, probably gy generating lysophosphatides from endogenous phospholipids. No stimulation by lysophosphatidylcholine was found when tested with 500-fold-purified glycosyltransferase. The results suggest that collagen glycosyltransferases must be associated with the membrane structures of the cell in order to be stimulated by lysophosphatidylcholine. Lysophosphatidylcholine could have some regulatory significance in vivo, since its concentration in the cell is comparable with that which produced marked stimulation in vitro.
A new method for the assay of tissue. S-adenosylhomocysteine and S-adenosylmethione. Effect of pyridoxine deficiency on the metabolism of S-adenosylhomocysteine, S-adenosylmethionine and polyamines in rat liver
Authors: Eloranta T O , Kajander E O , Raina A M
Published: August 2015

The hepatic synthesis and accumulation of S-adenosylhomocysteine, S-adenosylmethionine and polyamines were studied in normal and vitamin B-6-deficient male albino rats. A method involving a single chromatography on a phosphocellulose column was developed for the determination of S-adenosylhomocysteine and S-adenosylmethionine from tissue samples. Feeding the rat with pyridoxine-deficient diet for 3 or 6 weeks resulted in a four- to five-fold increase in the concentration of S-adenosylhomocysteine, whereas that of S-adenosylmethionine was only slighly elevated. The concentration of putrescine was decreased to half, that of spermidine was somewhat decreased and that of spermine remained fairly constant. The activities of L-ornithine decarboxylase, S-adenosyl-L-methionine decarboxylase, L-methionine adenosyltransferase and S-adenosyl-L-homocysteine hydrolase were moderately increased. S-Adenosylmethionine decarboxylase showed no requirement for pyridoxal 5′-phosphate. The major effect of pyridoxine deficiency of S-adenosylmethionine metabolism seems to be a block in the utilization of S-adenosylhomocysteine, resulting in the accumulation of this metabolite to a concentration that may inhibit biological methylation reactions.
One-carbon metabolism in Neurospora crassa wild-type and in mutants partially deficient in serine hydroxymethyltransferase
Authors: Cossins E A , Chan P Y , Combepine G
Published: August 2015

1. The concentrations of folate-dependent enzymes in Neurospora crassa Lindegren A wild type (FGSC no. 853), Ser-l mutant, strain H605a (FGSC no. 118), and for mutant, strain C-24 (FGSC no. 9), were compared during exponential growth on defined minimal media. Both mutants were partially lacking in serine hydroxymethyltransferase, but contained higher concentrations of 10-formyltetrahydrofolate synthetase than did the wild type. Mycelia of the mutants contained higher concentrations of these enzymes when growth media were supplemented with 1mM-glycine. In the wild-type, this glycine supplement also increased the specific activities of 5,10-methylenetetrahydrofolate dehydrogenase and 5,10-methylenetetrahydrofolate reductase. 5. During growth, total folate and polyglutamyl folate concentrations were greatest in the wild-type. Methylfolates were not detected in mutant Ser-l, and were only present in the for mutant after growth in glycine-supplemented media. Exogenous glycine increased folate concentration threefold in the wild type, mainly owing to increases in unsubstituted polyglutamyl derivatives. 3. Feeding experiments using 14C-labelled substrates showed that C1 units were generated from formate, glycine and serine in the wild type. Greater incorporation of 14C occurred when mycelia were cultured in glycine-supplemented media. Formate and serine were precursors of C1 units in the mutants, but the ability to cleave glycine was slight or lacking.
Re-activation of the peptidyltransferase centre of rabbit reticulocyte ribosomes after inactivation by exposure to low concentrations of magnesium ion
Authors: Cox R A , Greenwell P , Hirst W
Published: August 2015

1. The larger subrivosomal particles of rabbit reticulocytes retained full activity in the puromycin reaction and in poly(U)-directed polyphenylalanine synthesis after 4h at 0° C when buffered 0.5M-NH4Cl/10-30mM-MgCl2 was the solvent. 2. Activity in the puromycin reaction was diminished to approx 10% after 15-30 min at 0° C when the concentration of MgCl2 was lowered to 2mM. 3. Activity was not restored when the concentration of MgCl2 was raised from 2mM to 10-30 mM at 0° C. However, activity was recovered as measured by both assay systems when the ribosome fraction was heated to 37° C at the higher concentrations of MgCl2. 4. Recovery of activity was noted during the course of the polyphenylalanine synthesis in 50 mM-KCl/5mM-MgCl2/25mM-Tris/HCl, pH 7.6, at 37° C. Re-activation was slow at 20° C and below. 5. No more than about 5% of the protein moiety of the subparticle was lost in 0.5M-NH4Cl on decreasing MgCl2 concentration from 10mM to 2mM. No proteins were detected in the supernatant fractions by gel electrophoresis after ribosomes were separated by differential centrifugation. The supernatant fraction was not essential for the recovery of activity. However, at higher (e.g. 1M) concentrations of NH4Cl, proteins were split from the subparticle. 6. The loss and regain of activity found on lowering and restoring the concentration of MgCl2 at 0.5M-NH4Cl appears to arise from a conformational change that does not seem to be associated with a loss and regain of particular proteins. 7. A 2% decrease in E260 was noticed when the concentration of Mg2+ was restored, and the change in the spectrum indicated a net increase of approx. 100A-U base-pairs per subribosomal particle. 8. When the concentration of Mg2+ was restored, s20,w of the subparticle remained at 52± 1S until the sample was incubated at 37° C when s20,w increased to 56 ± 1S compared with the value of 58 ± 1S for the subparticle as originally isolated.
Ribonucleic acid synthesis in rabbit erythroid cells
Authors: Hunt J A
Published: August 2015

Kinetic studies on the synthesis of RNA in mature bone-marrow erythroid cells from rabbits were made by measuring the incorporation of [2-3H]adenosine into the ATP pool and RNA over periods up to 8h. By use of equations to fit the pool specific radioactivity and an equation using the same type of pool to generate the rate of linear DNA synthesis, good agreement between the pool parameters is found, provided that the ATP pool is measured in whole cell extracts, and assuming that the dATP and ATP pools equilibrate rapidly. RNA-synthesis rates were measured by using curve fits to equations developed by using the pool specific-radioactivity curves. The rate of synthesis of poly(A)-containing RNA varied in three experiments from 90 to 220mol/min per cell, with half-life of nuclear processing of 12-22 min with a mean of 16 min. Ribosomal RNA is synthesized at a rate of 70-200 mol/min per cell with an average half-life of nuclear processing of 37 min for the 18S RNA and 214 min for the 28S RNA. When the stable rRNA components are subtracted from the nRNA synthesis, the rate of nRNA synthesis is between 2 and 6fg/min per cell with an average half-life of degradation of 27 min. The rate of synthesis of poly(A)-containing RNA is 1.5-3.5% of the RNA-synthesis rates. These rates are compared with the RNA-synthesis rates found in L cells and concentrations of globin mRNA found in various erythroid-cell preparations.
Effects of dicyclohexylcarbodi-imide on proton translocation coupled to fumarate reduction in anaerobically grown cells of Escherichia coli K-12
Authors: Gutowski S J , Rosenberg H
Published: August 2015

The addition of dicyclohexylcarbodi-imide to anaerobic cells of Escherichia coli K12 decreases both the observed extent of proton translocation coupled to fumarate reduction by endogenous substrates and the t1/2 of proton re-entry after such translocation, but does not affect fumarate uptake. Dicyclohexylcarbodi-imide also inhibits fumarate reductase activity in cell extracts.
Amino components of the lipopolysaccharide from Pseudomonas aeruginosa N.C.T.C. 8505. Presence of 2,4-diamino-2,4,6-trideoxy-d-glucose
Authors: Wilkinson S G
Published: August 2015

Configurations were determined for previously identified amino components of the lipopolysaccharide from Pseudomonas aeruginosa N.C.T.C. 8505. Glucosamine and galactosamine belong to the D-series, and alanine and aminogalacturonic acid to the L-series. An additional amino component was identified as 2,4-diamino-2,4,6-trideoxy-D-glucose. This compound may be a characteristic component of the O-specific chain in lipopolysaccharides of strains of Pseudomonas aeruginosa belonging to Habs sero-group 3.
Biospecific-elution chromatography with ‘imphilytes’ as stationary phases
Authors: Yon R J
Published: August 2015

Six out of seven enzymes tested (four of them nicotinamide nucleotide-dependent dehydrogenases) showed differences in chromatographic behaviour in the presence and absence of their biospecific ligands, when chromatographed on immobilized amphipathic ampholytes (‘imphilytes’) as stationary phases. Some enzymes were adsorbed more tightly, others less tightly, in the presence of ligands. These results have implications for enzyme purification in general, and for some types of affinity chromatography in particular.
l-serine dehydratase from Arthrobacter globiformis
Authors: Gannon F , Bridgeland E S , Jones K M
Published: August 2015

1. L-Serine dehydratase (EC 4.2.1.13) was purified 970-fold from glycine-grown Arthrobacter globiformis to a final specific activity of 660micronmol of pyruvate formed/min per mg of protein. 2. The enzyme is specific for L-serine; D-serine, L-threonine and L-cysteine are not attacked. 3. The time-course of pyruvate formation by the purified enzyme, in common with enzyme in crude extracts and throughout the purification, is non-linear. The reaction rate increases progressively for several minutes before becoming constant. The enzyme is activated by preincubation with L-serine and a linear time-course is then obtained. 4. The substrate-saturation curve for L-serine is sigmoid. The value of [S]0.5 varies with protein concentration, from 6.5mM at 23microng/ml to 20mM at 0.23microng/ml. The Hill coefficient remains constant at 2.9.5 The enzyme shows a non-specific requirement for a univalent or bivalent cation. Half-maximal activity is produced by 1.0mM-MgCl2 or by 22.5mM-KCl. 6. L-Cysteine and D-serine act as competitive inhibitors of L-serine dehydratase, with Ki values of 1.2 and 4.9mM respectively. L-Cysteine, at higher concentrations, also causes a slowly developing irreversible inhibition of the enzyme. 7. Inhibition by HgCl2 (5micronM)can be partially reversed in its initial phase by 1mM-L-cysteine, but after 10 min it becomes irreversible. 8. In contrast with the situation in all cell-free preparations, toluene-treated cells of A. globiformis form pyruvate from L-serine at a constant rate from the initiation of the reaction, show a hyperbolic substrate-saturation curve with an apparent Km of 7mM and do not require a cation for activity.
Evaluation of benzofuroxan as a chromophoric oxidizing agent for thiol groups by using its reactions with papain, ficin, bromelain and low-molecular-weight thiols
Authors: Shipton M , Stuchbury T , Brocklehurst K
Published: August 2015

1. Benzofuroxan (benzofurazan 1-oxide, benzo-2-oxa-1,3-diazole N-oxide) was evaluated as a specific chromophoric oxidizing agent for thiol groups. 2. Aliphatic thiol groups both in low-molecular-weight molecules and in the enzymes papain (EC 3.4.22.2), ficin (EC 3.4.22.3) and bromelain (EC 3.4.22.4) readily reduce benzofuroxan to o-benzoquinone dixime; potential competing reactions of amino groups are negligibly slow. 3. The fate of the thiol depends on its structure: a mechanism is proposed in which the thiol and benzofuroxan form an adduct which, if steric factors permit, reacts with another molecule of thiol to form a disulphide; when the thiol is located in the active site of a thiol proteinase and steric factors preclude enzyme dinner formation, the adduct reacts instead with water or HO- to form a sulphenic acid; attack on the sulphur atom of the adduct by either a sulphur or oxygen nucleophile releases o-benzoquinone dioxine. 4. Benzofuroxan contains n o proton-binding sites with pKa values in the range 3-10 and probably none in the range 0-14; o-benzoquinone dioxine undergoes a one-proton ionization with pKa=6.75.5. o-benzoquinone dioxime absorbs strongly at wavelengths greater than 410nm, where absorption by benzofuroxan, proteins and simple thiol compounds is negligible; 416 nm is an isosbestic point (epsilon 416 = 5110 litre. mol-1-cm-1); epsilon430=3740+[1460/(1+[H+]/Ka)] where pKa=6.75. 6. The possibility of acid-base catalysis of the oxidation by active-centre histidine residues of the thiol proteinases is discussed.
Enzymic assays for isomers of 2,6-diaminopimelic acid in walls of Bacillus cereus and Bacillus megaterium
Authors: Day A , White P J
Published: August 2015

An enzymic assay for individual isomers (meso-, LL- and DD-) of 2,6-diaminopimelate was developed. The enzyme 2,6-diaminopimelate decarboxylase specifically attacked meso-diaminopimelate and was used to measure this isomer manometrically. The meso- and LL-isomers were measured together manometrically in a coupled assay with diaminopimelate decarboxylase and diaminopimelate epimerase (which converts LL-diaminopimelate into meso-diaminopimelate). The DD-isomer was not attacked by either enzyme and was measured, as residual diaminopimelate after the coupled assay, by a colorimetric method, which was also used to measure total diaminopimelate before enzymic treatments. The coupled enzymes were also used to prepare pure DD-isomer from chemically synthesized diaminopimelate. A mixture of diaminopimelate isomers was present in walls of four strains of Bacillus megaterium [in each about 75% (w/w) meso-, 18% LL- and 7% DD-] and in walls of two strains of Bacillus cereus (about 85% meso-, 8% LL- and 7% DD-). One strain of B. cereus contained at least 95% meso-diaminopimelate, with only traces of LL- and DD-isomers. Peptidoglycan from Escherichia coli was assayed as containing at least 95% meso-isomer. The proportion of isomers in the wall of a strain of B. megaterium remained constant after growth in a variety of different media.
The oxidation-reduction potential of the reaction-centre chlorophyll (P700) in Photosystem I. Evidence for multiple components in electron-paramagnetic-resonance signal 1 at low temperature
Authors: Evans M C W , Sihra C K , Slabas A R
Published: August 2015

The oxidation-reduction potential of the reaction-centre chlorophyll of Photosystem I (P700) in spinach chloroplasts was determined by using the ability of the reaction centre to photoreduce the bound ferredoxin and to photo-oxidize P700 on illumination at 20K as an indicator of the oxidation state of P700. This procedure shows that P700 is oxidized with Em (pH8.0)(mid-point redox potential at pH8.0)congruent to +375mV. Further oxidation of the chloroplast preparations by high concentrations of K3Fe(CN)6(10mM) in the presence of mediating dyes leads to the appearance of a large radical signal with an apparent Em congruent to +470mVA second, light-inducible, radical also appears over the same potential range. We propose that these signals are due to bulk chlorophyll oxidation and not, as was previously thought [Knaff & Malkin (1973) Arch. Biochem. Biophys. 159, 555-562], to reaction-centre oxidation. A number of optical techniques were used to determine Em of P700. Dual-wavelength spectroscopy (697-720nm) indicates Em congruent to +460-+480mV. The spectrum of the sample during the titration showed a large contribution to the signal by bulk chlorophyll oxidation, in agreement with the electron-paramagnetic-resonance results and those of Ke, Sugahara & Shaw [(1975) Biochim. Biophys. Acta 408, 12-25]. The light-induced absorbance change at 435 nm, usually attributed to P700, showed a potential dependence similar to that of bulk chlorophyll oxidation. Determination of Em of P700 on the basis of the appearance of the P700 signal in oxidized-versus-reduced difference spectra showed Em (pH8.0) congruent to +360mV. Measurements of the effect of potential on the irreversible photo-oxidation of P700 at 77K showed that P700 became oxidized in this potential range. We conclude that the reaction-centre chlorophyll of Photosystem I has Em (pH8.0) congruent to +375mV.
A phosphodiesterase in rat kidney cortex that hydrolyses glycerylphosphorylinositol
Authors: Dawson R M C , Hemington N
Published: August 2015

1. A phosphodiesterase, active at an alkaline pH, is present in the outer cortex of rat kidney and hydrolyses glycerylphosphorylinositol into glycerol and phosphorylinositol. Some inositol cyclic phosphate can also be formed indicating that the enzyme can act as a cyclizing phosphotransferase. 2. The enzyme is stimulated by Ca2+(2-3mM) whereas Mg2+ is inhibitory. 3. The activity is markedly stimulated by low concentrations of thiol reagents (1-2mM) such as cysteine or dithiothreitol. 4. The properties of the enzyme have been compared with glycerylphosphinicocholine diesterase (EC 3.1.4.2), which is also present in the isolated enzyme complex, and it is concluded that the enzymes have separate identities.
Prenatal induction of sucrase activity in rat jejunum
Authors: Celano P , Jumawan J , Horowitz C , Lau H , Koldovsky O
Published: August 2015

Daily application of cortisone acetate (10mg/100g body wt.) or L-tri-iodothyronine (20 microng/100g body wt.) to female rats in the last (third) week of pregnancy elicits a precocious appearance of jejunal sucrase in their foetuses.
Effects of adenosine 3′:5′-cyclic monophosphate and serum on synthesis of hyaluronic acid in confluent rat fibroblasts
Authors: Tomida M , Koyama H , Ono T
Published: August 2015

A small amount of hyaluronic acid is synthesized in confluent cultures of rat fibroblasts, which have a high content of cyclic AMP. Addition of calf serum caused a rapid decrease in the cellular cyclic AMP content and large increases in hyaluronic acid synthetase activity and hyaluronic acid production. Addition of cyclic AMP also caused a marked increase in hyaluronic acid synthetase activity within 2h and then increased hyaluronic acid production. The effects of cyclic AMP and serum on hyaluronic acid synthesis were additive. Prostaglandin E2, which increased the cyclic AMP by stimulating adenylate cyclase, was as effective as cyclic AMP in increasing hyaluronic acid synthetase activity, but AMP was far less effective than cyclic AMP. These results indicate that cyclic AMP itself stimulates the mucopolysaccharide synthesis and that the effect of serum is not due to a decrease in cyclic AMP in the cells.
Glucose turnover in the post-absorptive rat and the effects of halothane anaesthesia
Authors: Heath D F , Frayn K N , Rose J G
Published: August 2015

1. Rates and rate coefficients of glucose utilization and replacement in post-absorptive rats, either conscious or under halothane anaesthesia, were determined in a thermoneutral environment by using [5-3H]- and [U-14C]glucose. Label was not injected into rats under halothane until about 0.5h after anaesthesia was initiated. 2. Comparison with the results for 24h-starved rats in the preceding paper [Heath et al. (1977) Biochem. J. 162, 643-651] showed that insulin concentrations were considerably higher but rate coefficients for glucose utilization were little altered in post-absorptive rats. Sensitivity to insulin was thus considerably increased by a 24h period of starvation in the rat. 3. Fractional recycling of glucose carbon in post-absorptive rats was under one-half of that in starved rats, reflecting the larger contribution of liver glycogenolysis to glucose production in the former. 4. In post-absorptive rats halothane decreased the mean rate of glucose utilization by about 17%. This decrease was associated with an increase in mean plasma insulin concentration, showing that halothane decreased sensitivity to insulin. 5. Recycling was slightly increased by halothane, indicating that the contribution of liver glycogen to the total glucogenic rate was decreased, probably because liver glycogen concentration were about 40% lower throughout the rate determinations in halothane. 6. Comparison of our results with earlier work shows that during and shortly after induction of halothane anaesthesia glucose turnover must have been greatly increased whereas from about 0.5h after induction it was decreased.
A glycopeptide isolated from human gastric juice
Authors: Goso K , Hotta K
Published: August 2015

A glycopeptide containing 69% carbohydrate was isolated from human gastric juice. The complex was found to be homogeneous and to have mol.wt. 9600. The glycopeptide consisted of a protein core to which were linked, by O-glycosidic linkages to threonine and N-glycosidic linkages, carbohydrate side chains composed of N-acetylgalactosamine, N-acetylglucosamine, galactose, mannose, fucose and sialic acid, in the proportions 2:10:7:4:12:1.
Preliminary characterization of two thymus-dependent xenoantigens from mouse lymphocytes
Authors: Trowbridge I S , Nilsen-Hamilton M , Hamilton R T , Bevan M J
Published: August 2015

Preliminary characterization of two mouse thymus-dependent (T) lymphocyte xenoantigens, T25 and T200, which are selectively labelled by lactoperoxidase-catalysed iodination of T-cells, is described. Both molecules are membrane-bound glycoproteins. Fractionation of membrane vesicles prepared from BW5147 lymphoma cells by sedimentation through sucrose density gradients show that antigens T25 and T200 are in fractions enriched with plasma membrane. Moreover antigen T200 is partially degraded when viable cells are treated briefly with low concentrations of trypsin. Both molecules are efficiently solubilized in buffers containing sodium deoxycholate or Nonidet P-40, as measured by failure to sediment at 100000g for 60min. However, gel filtration on Sepharose 6B showed the presence of aggregated material in Nonidet P-40 extracts which was not found in deoxycholate-solubilized membranes. After solubilization in detergent, antigens T25 and T200 bind to, and may be specifically eluted from, columns of pea lectin--Sepharose or concanavalin A--Sepharose. Both molecules are heterogeneous when examined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. As judged by its binding to columns of pea lectin, at least part of the heterogeneity of mouse thymocyte antigen T25 resides in its carbohydrate moiety.
Chemical and physical characterization of a phosphoprotein, Protein C, from human saliva and comparison with a related protein A
Authors: Bennick A
Published: August 2015

The isolation of a highly purified phosphoprotein, previously named protein C, from human parotid saliva is described. A chemical and physical characterization of protein C was undertaken and the properties of protein C were compared with those of a related protein A. The content of glycine, proline and dicarboxylicamino acids accounts for 83% of the total resideus of protein C and it contains 2.0 mol of P/mol of protein, most likely as phosphoserine. The protein also contains 1.2% glucose, but no hexosamine. The N-terminus is blocked and the proposed C-terminal sequence is -Ser(Gly, Pro)Gln. The molecular weight determined from ultracentrifugation is 16300. Circular dichroism and nuclear magnetic resonance fail to demonstrate the presence of polyproline structure, and there are no conformational changes under a variety of conditions. With specific antisera to protein C the protein can be detected in submandibular as well as in parotid saliva, but there is only reaction of partial identity of proteins A and C. It is proposed that at least part of the difference between proteins A and C is due to the presence of an additional length of peptide at the C-terminus of protein C.
The allosteric mechanism of bovine liver glutamate dehydrogenase. Evidence from circular-dichroism studies for a conformational change in the ternary complex enzyme-(oxidized nicotinamide-adenine dinucleotide)-glutarate
Authors: Chen S S , Engel P C , Bayley P M
Published: August 2015

1. Computer averaging of multiple scans was used to refine the circular dichroism spectrum of bovine liver glutamate dehydrogenase, revealing well-defined structure in the aromatic region. 2. The circular dichroism of NAD+ bound to glutamate dehydrogenase is strongly negative at 260nm, probably owing to immobilization of the adenosine moiety. Loss of the characteristic adenine-nicotinamide interaction suggests that the coenzyme is bound in an unstacked conformation. 3. Glutarate and succinate, substrate analogues that are both inhibitors competitive with glutamate, do not significantly perturb the circular-dichroism spectrum of the enzyme in the absence of NAD+. 4. In the presence of NAD+, 150nM-succinate decreases the negative circular dichroism corresponding to bound coenzyme, but does not affect the protein circular dichroism. However, ISOmM-glutarate causes profound alternations of the circular-dichroism spectra of the bound NAD+ and of the enzyme, indicative of a protein conformational change. This direct evidence of conformational change specifically promoted by C5 dicarboxylates confirms the previous inference from protection studies. 5. The conformational change is discussed in relation to the allosteric mechanism of glutamate dehydrogenase.
Inactivation of human α1-proteinase inhibitor by thiol proteinases
Authors: Johnson D , Travis J
Published: August 2015

Human plasma alpha1 proteinase inhibitor is the body's principal modulator of serine proteinases (such as those released from phagocytic cells). Cysteine-active-site proteinases, which are not inhibited, have now been found to inactivate this important inhibitor by proteolytic cleavage of a scissile peptide bond. Papain carries out this inactivation catalytically, whereas cathepsin B1 acts stoicheiometrically. Thus thiol proteinases could easily disrupt the delicately regulated balance between serine proteinases and alpha1 proteinase inhibitor.
Effect of prolonged ethanol ingestion on hepatic lipogenesis and related enzyme activities
Authors: Savolainen Markku J. , Hiltunen J. Kalervo , Hassinen Ilmo E.
Published: August 2015

1. Hepatic lipogenesis in vivo and the activities of enzymes associated with fatty acid synthesis in the liver were studied in rats fed for 21 days on liquid diets containing ethanol. 2. The ethanol-fed rats developed a moderate hepatic triacylglycerol accumulation during this period. When carbohydrate was replaced by ethanol in the diet, the rate of fatty acid synthesis was slower in the ethanol-fed rats on low-, medium- and high-fat diets than in the appropriate controls. However, when the fat/carbohydrate ratio was kept the same in the ethanol-fed and control rats, ethanol had no influence on the rate of fatty acid synthesis. 3. Glucose 6-phosphate dehydrogenase activity was lower in the ethanol-fed group. ‘Malic’ enzyme activity did not change during the ethanol treatment when the fat/carbohydrate ratio was kept unchanged. 4. The ATP citrate lyase activity was lower in the ethanol-fed rats on all diets, whereas acetyl-CoA synthetase activity was independent of the composition of the control diet, but was lower in the ethanol-fed rats, in which the concentration of the active form of pyruvate dehydrogenase was also lower. 5. It is concluded that hepatic fatty acid synthesis does not play any major role in ethanol-induced triacylglycerol accumulation. Careful design of the diets is necessary to reveal the specific effects of ethanol on the enzymes associated with lipogenesis.
Characterization of the major diazo-positive pigments in bile of homozygous Gunn rats
Authors: Blanckaert Norbert , Fevery Johan , Heirwegh Karel P. M. , Compernolle Frans
Published: August 2015

Bilinoid pigments in bile of homozygous Gunn rats (jj) were analysed either after formation of dipyrrolic ethyl anthranilate azo derivatives or as the unmodified parent tetrapyrroles. 1. T.l.c. of the azo derivatives revealed seven major unconjugated components which were structurally characterized by chemical tests, spectrophotometry and mass spectrometry. In addition, two minor components were identified as azodipyrrole (A+B)-glucoside and azodipyrrole (A+B)-β-d-glucuronide. 2. Extraction and t.l.c. of the tetrapyrrolic pigments showed 13 major yellow diazo-positive bands. Four of them, accounting for 59% of total diazo-positive material, were identified as unconjugated bilirubin-IXα, -IXβ, -IXγ and -IXδ. A fifth band (16%) was characterized as a mixture of two isomeric monohydroxyl derivatives and another band (8%) as a dihydroxyl derivative of bilirubin-IXα. 3. Although unconjugated bilirubin-IXα constitutes one-third of total diazo-positive material in bile of our strain of Gunn rats, the daily amount excreted represented only about 3–4% of daily bilirubin production. 4. Phototherapy caused a 2.2-fold increase in the biliary output of diazo-positive bilinoids, but did not affect markedly their composition. However, an additional diazo-negative pigment, accounting for one-third of total yellow colour, was observed but was not identified. Mass-spectral data on two dipyrrolic azopigments have been deposited as Supplementary Publication SUP 50076 (3 pages) with the British Library Lending Division, Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1977) 161, 1.
Effect of a nutritional shift on the degradation of abnormal proteins in the mouse liver. Decreased degradation during rapid liver growth
Authors: Amils R , Conde R D , Scornik O A
Published: August 2015

1. The intravenous injection of puromycin to mice 0.5 min after administration of radioactive leucine resulted in the release of labelled ribosome-bound nascent protein chains with the next 0.5 min. 2. During the subsequent 13 min, 40% of the liver protein radioactivity disappeared. The rate of this process was already maximal 0.5 min after the injection of puromycin, with no apparent lag. 3. Evidence is presented that this phenomenon represents the selective degradation of puromycinyl-peptides: (a) the magnitude of this fraction corresponded to the calculated proportion of protein radioactivity in nascent chains at the time of the puromycin effect; (b) the size distribution of the proteins disappearing between 2 and 14 min was smaller than that of those retained at 14 min; and (c) when the injection of puromycin was delayed for 5 min, or when the leucine pulse was interrupted by the injection of cycloheximide (rather than puromycin), the fraction disappearing within 14 min was much smaller. 4. The degradation of puromycinyl-peptides was much slower in the rapidly growing livers of animals recovering from a protein depletion than in the protein-depleted controls. It is concluded that the large decrease in the overall rates of total liver protein degradation previously described during liver growth is a general phenomenon, also affecting the rate of scavenging of abnormal proteins.
Enhanced plasma persistence of therapeutic enzymes by coupling to soluble dextran
Authors: Sherwood R F , Baird J K , Atkinson T , Wiblin C N , Rutter D A , Ellwood D C
Published: August 2015

Conjugation of carboxypeptidase G and arginase, two enzymes of therapeutic interest, to a soluble dextran significantly enhanced plasma persistence in normal and tumour-bearing mice. A prolonged decrease in arginine concentrations in plasma of tumour-bearing mice was demonstrated by using the dextran-linked arginase. Gel filtration of dextran-enzyme conjugate showed that enzyme activity co-chromatographed as a single peak with carbohydrate, and enzyme was shown to be covalently linked to the dextran.
Inhibition of binding of corticotropin-(1-24)-tetracosapeptide (Synacthen) to membrane receptors of adrenal cortex by cortisol
Authors: Latner A L , Cook D B , Solanki K U
Published: August 2015

The binding of Synacthen to partially purified bovine adrenocortical plasma membranes was shown to be inhibited by cortisol. The findings suggest that cortisol is involved in a peripheral feedback mechanism for the control of its release.
Bacterial catabolism of threonine. Threonine degradation initiated by l-threonine hydro-lyase (deaminating) in a species of Corynebacterium
Authors: Bell Stephen C. , Turner John M.
Published: August 2015

1. Three bacterial isolates capable of growth on l-threonine medium only when supplemented with branched-chain amino acids, and possessing high l-threonine dehydratase activity, were examined to elucidate the catabolic route for the amino acid. 2. Growth, manometric, radiotracer and enzymic experiments indicated that l-threonine was catabolized by initial deamination to 2-oxobutyrate and thence to propionate. No evidence was obtained for the involvement of l-threonine 3-dehydrogenase or l-threonine aldolase in threonine catabolism. 3. l-Threonine dehydratase of Corynebacterium sp. F5 (N.C.I.B. 11102) was partially purified and its kinetic properties were examined. The enzyme exhibited a sigmoid kinetic response to substrate concentration. The concentration of substrate giving half the maximum velocity, [S0.5], was 40mm and the Hill coefficient (h) was 2.0. l-Isoleucine inhibited enzyme activity markedly, causing 50% inhibition at 60μm, but did not affect the Hill constant. At the fixed l-threonine concentration of 10mm, the effect of l-valine was biphasic, progressive activation occurring at concentrations up to 2mm-l-valine, but was abolished by higher concentrations. Substrate-saturation plots for the l-valine-activated enzyme exhibited normal Michaelis–Menten kinetics with a Hill coefficient (h) of 1.0. The kinetic properties of the enzyme were thus similar to those of the ‘biosynthetic‘ isoenzyme from Rhodopseudomonas spheroides rather than those of the enteric bacteria. 4. The synthesis of l-threonine dehydratase was constitutive and was not subject to multivalent repression by l-isoleucine or other branched-chain amino acids either singly or in combination. 5. The catabolism of l-threonine, apparently initiated by a ‘biosynthetic’ l-threonine dehydratase in the isolates studied, depended on the concomitant catabolism of branched-chain amino acids. The biochemical basis of this dependence appeared to lie in the further catabolism of 2-oxobutyrate by enzymes which required branched-chain 2-oxo acids for their induction.
A spectrophotometric procedure for rapid and sensitive measurements of β-oxidation. Demonstration of factors that can be rate-limiting for β-oxidation
Authors: Osmundsen H , Bremer J
Published: August 2015

1. A spectrophotometric direct-reading assay for measurements of beta-oxidation by intact mitochondria is described. The procedure relies on the ability of ferricyanide to trap reducing equivalents generated by the acyl-CoA dehydrogenases (EC 1.3.99.3). The reduction of ferricyanide was recorded by using a dual-wavelength spectrophotometer. 2. Oxaloacetate or acetoacetate was used to stimulate the rate of beta-oxidation by rotenone-blocked mitochondria. Although both were effective with rat liver mitochondria, oxaloacetate gave about 75% more stimulation. With heart or kidney mitochondria, only oxaloacetate gave marked stimulation. Acetoacetate had no stimulatory effect with heart mitochondria, but a small stimulatory effect on the rate of beta-oxidation by kidney mitochondria. 3. The stoicheiometry of beta-oxidation-dependent reduction of ferricyanide was examined, and good correlations were found between experimental and theoretical amounts of ferricyanide reduced. 4. Ferricyanide appears as efficient a final electron acceptor as O2. With ferricyanide the rate of beta-oxidation by heart mitochondria can be measured without interference from the oxidation of tricarboxylic acid-cycle intermediates.
Cytochrome c-specific protein methylase III from Neurospora crassa
Authors: Nochumson S , Durban E , Kim S , Paik W K
Published: August 2015

A protein methylase III responsible for specifically methylating the cytochrome c in Neurospora crassa was partially characterized by using unmethylated horse heart cytochrome c as a substrate. This enzyme utilizes S-adenosyl-L-methionine as the methyl donor. An analysis of the distribution of [14C]methyl groups in the peptides obtained by chymotrypsin digestion of the enzymically methylated cytochrome c showed that all of the radioactivity could be recovered within a single peak after chromatography. This indicates that the enzyme methylates a specific amino acid sequence within cytochrome c. On hydrolysis of the radioactive chymotryptic peptide, Me-14C-labelled epsilon -N-mono-methyl-lysine, epsilon-N-dimethyl-lysine and epsilon-N-trimethyl-lysine were identified. The enzyme can easily be extracted from the N. crassa mycelial pads and was purified approx. 30-fold.
Characterization of the extracellular haemoglobins of Artemia salina
Authors: Moens L , Kondo M
Published: August 2015

The following factors were measured for extracellular haemoglobins of Artemia salina: a minimal molecular weight of globin chain per haem group (based on the iron and haem contents), the absorption coefficients, the absorption spectra of various derivatives and the amino acid compositions. These were compared with those of the haemoglobins of other invertebrates. Three Artemia haemoglobins (I, II and III) had similar molecular structures, constructed from two-globin subunits of 122000-130000mol.wt. Since the minimal mol.wt. was determined to be 18000, this suggests that one globin subunit was bound by seven haem groups, and hence one haemoglobin molecule (240000-260000mol.wt.) should contain 14 haem groups. A successful identification of this high-molecular-weight subunit required first the denaturation of haemoglobin in 1% sodium dodecyl sulphate before sodium dodecyl sulphate gel electrophoresis. Denaturation by prolonged incubation (12-36 h) at room temperature in the presence of 0.1% sodium dodecyl sulphate [Bowen, Moise, Waring & Poon (1976) Comp. Biochem. Physiol. B55, 99-103] was accompanied by extensive proteolysis, resulting in low recovery of the stainable protein and heterogeneous gel patterns. Regardless of which electrophoretic system was used, the high-molecular-weight subunit was always present provided that 1% sodium dodecyl sulphate was present during denaturation. These results contrast with those obtained by Bowen et al. (1976). However, preferential cleavage of the globin subunit (alpha) seemed to occur in vitro when standard conditions were used, producing two specific fragments having mol.wts. of 80000 (beta) and 50000 (gamma).
Purification and properties of a ribosomal casein kinase from rabbit reticulocytes
Authors: Issinger O G
Published: August 2015

A casein kinase was isolated and purifed from rabbit reticulocytes. About 90% of the enzyme activity co-sedimented with the ribosomal fraction, whereas about 10% of the enzyme activity was found in the ribosome-free supernatant. Both casein kinases (the ribosome-bound enzyme as well as the free enzyme) showed identical activity and the same molecular weight. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis a single band of about 70 000 mol.wt. was observed. Sucrose-gradient analysis, however, showed that the enzyme activity sedimented with a s20,w of approx. 7.5S. This observation suggested that the casein kinase is a dimer composed of subunits of identical molecular weight. The enzyme utilizes GTP as well as ATP as a phosphoryl donor. It preferentially phosphorylates acidic proteins, in particular the model substrates casein and phosvitin. Casein kinase is cyclic AMP-indepenoent. The Km values for ATP and GTP with phosvitin as a substrate were determined as 1.2 and 8.8 micrometer respectively.
Hormonal control of gluconeogenesis in tubule fragments from renal cortex of fed rats. Effects of α-adrenergic stimuli, glucagon, theophylline and papaverine
Authors: MacDonald David W. R. , Saggerson E. David
Published: August 2015

1. In incubated tubule fragments from renal cortex of fed rats gluconeogenesis from pyruvate was stimulated by adrenaline (1μm optimum) and by the selective α-adrenergic agonists oxymetazoline and amidephrine. The selective β-agonists isoproterenol and salbutamol were ineffective at concentrations up to 10μm. 2. Stimulation of gluconeogenesis by 1μm-adrenaline was almost completely blocked by 10μm-phentolamine (α-antagonist), partially blocked by 10μm-phenoxybenzamine (α-antagonist) and unaffected by 10μm-propranolol (β-antagonist). 3. Adrenaline stimulation of gluconeogenesis was rapid and was sustained for at least 1h. 4. Oxymetazoline (α-agonist) was extremely potent in stimulation of gluconeogenesis. This compound stimulated glucose production from pyruvate, lactate and glutamate, but not from succinate or glycerol. 5. In the absence of Ca2+ oxymetazoline was ineffective, whereas some stimulatory effect of adrenaline on gluconeogenesis was still observed. 6. Glucagon had no effect on gluconeogenesis from pyruvate in the presence of 1.27mm-Ca2+ and inhibited the process in the presence of 0.25mm-Ca2+. Parathyrin (parathyroid hormone) stimulated gluconeogenesis at 1.27mm-Ca2+. 7. In short incubations of tubule fragments glucagon, papaverine and adrenaline significantly increased 3′:5′-cyclic AMP. Adrenaline also slightly decreased 3′:5′-cyclic GMP. Oxymetazoline had no effect on the amount of either cyclic nucleotide. 8. At all concentrations tested, theophylline and papaverine decreased gluconeogenesis from pyruvate. 9. It is concluded that renal gluconeogenesis may be increased by α- but not β-adrenergic stimuli and that this is probably independent of changes in 3′:5′-cyclic AMP or 3′:5′-cyclic GMP. An involvement of Ca2+ in the action of oxymetazoline appears likely, but this is less certain with adrenaline.
The mode of action of 4-methylumbelliferyl β-d-xyloside on the synthesis of chondroitin sulphate in embryonic-chicken sternum
Authors: Gibson Kenneth D. , Segen Barbara J.
Published: August 2015

1. Embryonic-chicken sterna, incubated in medium containing 0.1mm-4-methylumbelliferyl β-d-xyloside (4-methylcoumarin 7-β-d-xyloside), synthesize proteochondroitin sulphate that is significantly undersulphated and shorter than usual [Gibson, Segen & Audhya (1977) Biochem. J.162, 217–233]. 2. Neither the β-d-galactoside nor the β-d-glucuronide of 4-methylumbelliferone, nor 4-methylumbelliferone itself, produced the effects. The only metabolites of 4-methylumbelliferone that were detected in cartilages exposed to 4-methylumbelliferyl β-d-xyloside were unchanged xyloside and chondroitin sulphate covalently attached to 4-methylumbelliferone. 3. Gel filtration of salt extracts of sterna incubated in medium containing the xyloside showed that there were two pools of chondroitin sulphate in the tissue. One pool was identified, on the basis of its elution pattern and the linear kinetics of incorporation of sulphate into it, as proteochondroitin sulphate. Incorporation into the other pool, whose properties suggested that it was methylumbelliferyl-chondroitin sulphate, indicated that it underwent partial turnover. The molecular weight of this chondroitin sulphate was about 19000, and it appeared to be about 70% sulphated. 4. When sterna were incubated in medium containing the xyloside, there was a very large incorporation of sulphate and glucose into glycosaminoglycans that were released into the incubation medium. This contrasts with incubations of sterna in the absence of the xyloside, in which less than 5% of the sulphate incorporated could be recovered from the medium. The glycosaminoglycan released into the medium was 4-methylumbelliferyl-chondroitin sulphate, whose average molecular weight was 7000–8000 and degree of sulphation more than 95%. 5. Incorporation of sulphate into proteochondroitin sulphate was stimulated more than 3-fold by addition of 20% (v/v) human serum and 10nm-l-3,3′,5-tri-iodothyronine. Incorporation into methylumbelliferyl-chondroitin sulphate, in either the tissue or the medium, was not significantly altered. 6. The decrease in chain length and degree of sulphation of proteochondroitin sulphate is explained in terms of competition between peptide-linked primers and methylumbelliferone-containing primers at the intracellular sites of polysaccharidechain elongation and sulphation. The implications of the results for the mechanism of stimulation of proteoglycan synthesis by serum factors are discussed.
Evidence for an electrogenic 3-deoxy-2-oxo-d-gluconate–proton co-transport driven by the protonmotive force in Escherichia coli K12
Authors: Lagarde A
Published: August 2015

Evidence is presented indicating that the carrier-mediated uptake of 3-deoxy-2-oxo-D-gluconate and D-glucuronate in Escherichia coli K12 is driven by the deltapH and deltapsi components of the protonmotive force. 1. Approximately two protons enter the cells with each sugar molecule, independent of the sugar and the strain used. 2. In respiring cells, the magnitude of the pH gradient alone, as measured by distribution of [3H]acetate, appears to be insufficient to account for the chemical gradient of 3-deoxy-2-oxo-D-gluconate that is developed between pH 6.0 and 8.0. 3. If the external pH is varied between 5.5 and 8.0, 3-deoxy-2-oxo-D-gluconate uptake is gradually inhibited by valinomycin plus K+ ions, whereas the inhibition caused by nigericin is concomitantly relieved, thus reflecting the relative contribution of deltapH and deltapsi to the total protonmotive force at each external pH. 4. 3-Deoxy-2-oxo-D-gluconate can be transiently accumulated into isolated membrane vesicles in response to an artificially induced pH gradient. The process is stimulated when the membrane potential is collapsed by valinomycin in the presence of K+ ions.
The phosphorylation potential generated by respiring bovine heart submitochondrial particles
Authors: Ferguson S J , Sorgato M C
Published: August 2015

A phosphorylation potential deltaGp, where deltaGp = deltaGo' + RT2.303 log ([ATP]/([ADP][Pi])), of approx. 44.3 kJ.mol-1 (10.6 kcal.mol-1) was generated by submitochondrial particles that were oxidizing either NADH or succinate. Addition of adenylyl imidodiphosphate, which should suppress adenosine triphosphatase activity of any uncoupled particles, did not raise the phosphorylation potential. Raising the Pi concentration slightly increased the magnitude of the value for [ATP]/[ADP], but this did not fully compensate for the increased Pi concentration, so that the phosphorylation potential decreased slightly as the Pi concentration was raised. The phosphorylation potential developed by submitochondrial particles is lower than that generated by phosphorylating membrane vesicles from some bacteria, and is also less than that developed externally by mitochondria, but is strikingly close to the phosphorylation potential that is generated internally by mitochondria.