bluuebunny/crossref_metadata_2025 huggingface dataset:
The arsonomethyl analogue of 3-phosphoglycerate
Authors: Adams S R , Sparkes M J , Dixon H B F
Published: August 2015

4-Arsono-2-hydroxybutanoic acid, the analogue of 3-phosphoglycerate in which -CH2-AsO3H2 replaces -O-PO3H2, was synthesized. It proved to be a substrate for phosphoglycerate kinase. Its Michaelis constant was only slightly higher than that of the natural substrate, but its catalytic constant was about 1300 times smaller.
The arsonomethyl group as an analogue of phosphate. An X-ray investigation
Authors: Kamiya K , Cruse W B T , Kennard O
Published: August 2015

The X-ray structure analysis of three compounds of interest as enzyme substrates is reported. They are the hydrated forms of (I) DL-2-amino-4-arsonobutanoic acid [HO–AsO2-–CH2–CH2–CH(NH3+)–CO2H], (II) DL-2-amino-4-phosphonobutanoic acid [HO–PO2-–CH2–CH2–CH(NH3+)–CO2H] and the hydrated barium salt of (III) D-3-phosphoglycerate [HO–PO2-–O–CH2–CH(OH)–CO2-]. The structures were fully refined to R factors of 0.033, 0.053 and 0.046. For the compounds (I) and (II) the charge distribution was directly determined by locating all H atoms. The co-ordination around As and P is approximately tetrahedral, with the valency angle between the two charged O atoms enlarged to 112 degrees in compound (I), 166 degrees in compound (II) and 122 degrees in compound (III). The As-X bond distances are increased relative to P-X to accommodate the increased atomic radius. The analysis establishes that the compounds are structural analogues. Tables of co-ordinates for H atoms, anisotropic thermal parameters, bond lengths and bond angles for the three compounds have been deposited as Supplementary Publication SUP 50122 (5 pages) with the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained directly [see Biochem J. (1983) 209, 5].
Lipid metabolism in microsomal fraction from photosynthetic tissue. Effects of catalase and hydrogen peroxide on oleate desaturation
Authors: Murphy D J , Mukherjee K D , Latzko E
Published: August 2015

On incubation of microsomal fraction from pea (Pisum sativum L.) leaves with ammonium [1-14C]oleate or [1-14C]oleoyl-CoA in the presence of ATP, CoA, Mg2+ and NADH, the major reactions observed were those catalysed by oleoyl-CoA synthetase, oleoyl-CoA thioesterase, oleoyl-CoA:phosphatidylcholine acyltransferase and oleoyl phosphatidylcholine desaturase. The reaction catalysed by oleoyl phosphatidylcholine desaturase was specifically inhibited by H2O2, and this inhibitory effect was overcome by catalase (EC 1.11.1.6).
Occurrence of glycosylation and deglycosylation of exogenously administered ganglioside GM1 in mouse liver
Authors: Ghidoni R , Sonnino S , Chigorno V , Venerando B , Tettamanti G
Published: August 2015

Ganglioside GM1, 3H-labelled at the level of terminal galactose or of sphingosine, was intravenously injected into Swiss albino mice and some steps in its metabolic fate in the liver were investigated. After administration of [3H]sphingosine-labelled GM1 all major liver gangliosides [GM3, GM2, GM1, GD1a-(NeuAc, NeuGl)] became radioactive, the radioactivity residing in all cases on the sphingosine moiety. The specific radioactivity was highest in GM1, which carried about 53% of the radioactivity incorporated into gangliosides, followed by GM2, with 34.5% of incorporated radioactivity, GM3 and GD1a-(NeuAc, NeuGl), both with about 5% of incorporated radioactivity. After administration of [3H]galactose-labelled GM1 the only radioactive gangliosides present in the liver were GM1 and GD1a-(NeuAc, NeuGl), the former carrying about 95% of the total ganglioside-incorporated radioactivity, the latter about 3%. Both gangliosides were radioactive exclusively in the terminal galactose residue. According to these results exogenously administered GM1, after being taken up by the liver, is mainly degraded to GM2 and GM3, a part being, however, sialylated to GD1a-(NeuAc, NeuGl). All this suggests that exogenous GM1 may be involved in the metabolic routes of endogenous liver gangliosides.
Cartilage proteoglycan binding region and link protein. Radioimmunoassays and the detection of masked determinants in aggregates
Authors: Ratcliffe A , Hardingham T
Published: August 2015

Antibodies have been raised in rabbits to the hyaluronate-binding region and link-protein components of aggregated proteoglycans from pig laryngeal cartilage. The anti-(binding region) antibodies did not bind 125I-labelled link protein, nor was 125I-labelled binding region bound by the anti-(link protein) antibodies. The antisera were applied in sensitive inhibition radioimmunoassays to determine binding region and link protein in purified proteoglycan preparations. With intact proteoglycan aggregates, the antigenic sites of link protein, and to a lesser extent binding region, were masked. Heat treatment in the presence of sodium dodecyl sulphate (0.025%, w/v) was found to overcome this masking, thereby allowing the determination of link protein and binding region in aggregated proteoglycan preparations in pure and impure samples.
A study of equilibrium binding of link protein to hyaluronate
Authors: Lyon M , Nieduszynski I A
Published: August 2015

Link protein was extracted from bovine femoral-head cartilage, radiolabelled while in the proteoglycan-aggregate stage, and then purified by density-gradient centrifugation and gel chromatography. The purity of the preparation was assessed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and two species with approx. mol.wts. 45000 and 48000 were observed. Sedimentation-velocity experiments were performed in 0.5 M-guanidinium chloride/5 mM-phosphate, pH 7.4, and yielded an SO20, w of 4.75S. The proportion of link protein unable to interact with hyaluronate was determined by chromatography on Sepharose CL-4B. The binding of link protein to high-molecular-weight hyaluronate was studied by frontal-gel chromatography on Sepharose CL-4B in 0.5 M-guanidinium chloride/5 mM-phosphate/0.1% bovine serum albumin, pH 7.4. Experiments were performed at 10, 17 and 25 degrees C and the results were treated as described by Scatchard [(1949) Ann. N.Y. Acad. Sci. 51, 660-672]. Dissociation constants of approx. (1-4) X 10(-8) M were obtained. The length of hyaluronate occupied per link-protein molecule was determined to be six to seven disaccharides.
Isolation and characterization of an endo-β-galactosidase from Bacteroides fragilis
Authors: Scudder P , Uemura K , Dolby J , Fukuda M N , Feizi T
Published: August 2015

Six strains of Bacteroides fragilis were examined and all found to produce endo-beta-galactosidase, an enzyme that hydrolyses internal β-galactosidic linkages of oligosaccharides belonging to the poly-N-acetyl-lactosamine series, with the common structure GlcNAc β 1 leads to 3Gal β 1 leads to 4GlcNAc/Glc. The enzyme was produced without the addition of an inducer such as keratan sulphate. It was purified 7000-fold from the culture supernatant and obtained with a yield 4-10-fold greater than from sources described previously. The specificity of the enzyme towards bovine corneal keratan sulphate, milk oligosaccharides and the glycolipids lacto-N-neotetraosylceramide and lacto-N-tetraosylceramide closely resembled that of the endo-beta-galactosidase isolated from Escherichia freundii. A novel observation was that both enzymes hydrolysed the type 2 sequence, Gal β 1 leads to 4GlcNAc β 1 leads to 3Gal β 1 leads to 4Glc, at about twice the rate of the type 1 isomer, Gal β 1 leads to 3GlcNAc β 1 leads to 3Gal β 1 leads to 4Glc. Because of the ease of purification of the enzyme and high yield in the absence of contaminating glycosidases and proteinases, Bacteroides fragilis is a valuable source of endo-beta-galactosidase for the structural analysis of carbohydrate chains.
Low-temperature resonance-Raman spectra of Japanese-lacquer-tree (Rhus vernicifera) laccase, type-2-copper-depleted laccase and H2O2-treated type-2-copper-depleted laccase
Authors: Musci G , Desideri A , Morpurgo L , Garnier-Suillerot A , Tosi L
Published: August 2015

Resonance-Raman spectra of Japanese-lacquer-tree (Rhus vernicifera) laccase, type-2-copper-depleted laccase and the latter form treated with H2O2 were measured in liquid and frozen solution, on excitation into the 600 nm absorption band. Significant changes in intensity and/or frequency of the bands lying in the 370-430 cm-1 region were observed on freezing, indicating local structural rearrangements taking place at the blue copper site. These findings corroborate previous suggestions based on e.p.r. measurements and redox data [Morpurgo, Calabrese, Desideri & Rotilio (1981) Biochem. J. 193, 639-642]. They show the strong dependence of the physical properties of blue copper centres on local symmetry. Some conclusions on the origin of the Raman bands are also drawn.
Purification and partial characterization of a rat retina alcohol dehydrogenase active with ethanol and retinol
Authors: Julià P , Farrés J , Parés X
Published: August 2015

Homogeneous alcohol dehydrogenase (ADH) from rat retina was obtained by chromatography on DEAE-Sepharose and AMP-hexane-Sepharose. The enzyme is a dimer of Mr congruent to 80000 and oxidizes ethanol using NAD+ as a cofactor. Careful activity determinations demonstrate unambiguously that rat retina ADH is active with retinol as a substrate. This result opens the question about the role of retina ADH in the visual cycle.
The fatty acid composition of 1,2-diacylglycerol and polyphosphoinositides from human erythrocyte membranes
Authors: Allan D , Cockcroft S
Published: August 2015

The fatty acid compositions of 1,2-diacylglycerol and polyphosphoinositides have been determined in human erythrocyte membranes that have been incubated in the presence or in the absence of Ca2+. The results show that the diacylglycerol that is formed in Ca2+-treated membranes has a fatty acid composition closely similar to that of the inositides, consistent with previous indications that Ca2+ stimulates the activity of a polyphosphoinositide phosphodiesterase in the membranes. In contrast with some previous results, it appears that these plasma-membrane inositides and their derived diacylglycerols are rich in stearic acid and arachidonic acid.
Effects of bleomycin and methylprednisolone on the biosynthesis of oligosaccharide-lipids and glycoproteins in lung
Authors: Mohapatra N , Lynn W S , Bhattacharyya S N
Published: August 2015

Tissues from rabbit lung were found to incorporate radioactivity from [3H]mannose into the oligosaccharide of a polar lipid soluble in chloroform/methanol/water (10:10:3, by vol.). Only one oligosaccharide-lipid was formed and the composition of the radiolabelled carbohydrate moiety was Glc3Man9(GlcNAc)2. An antitumour antibiotic, bleomycin, and an anti-inflammatory steroid, methylprednisolone, partially inhibited the incorporation of [3H]mannose into oligosaccharide-lipids and in addition resulted in the production of two new components, Man5(GlcNAc)2 and Man2(GlcNAc)2. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the glycoproteins synthesized from the oligosaccharide-lipid intermediates showed labelled components with Mr greater than 200000, 130000, 80000 and 62000. The addition of the drugs resulted in the same pattern with the addition of a new component of Mr 36000. Endoglucosaminidase H-treatment of the glycopeptides isolated from the Pronase-digested glycoproteins indicated that the oligosaccharide units in the glycoproteins were of the high-mannose-type.
Structural and circular-dichroism studies on the interaction between human C̅1-esterase inhibitor and C̅1s
Authors: Nilsson T , Sjöholm I , Wiman B
Published: August 2015

The reaction between complement factor C1s and C1-esterase inhibitor has been investigated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, N-terminal amino acid analysis and c.d. studies. It is confirmed that a very stable stoichiometric 1:1 complex with a molecular weight of about 180000 is formed, involving the light chain of C1s. On the sodium dodecyl sulphate/polyacrylamide gels a small peptide with a molecular weight of about 5000 can be seen, which may be released from the C-terminal portion of the inhibitor moiety in a manner analogous to that occurring in other similar proteinase-inhibitor reactions. By N-terminal amino acid analysis, a newly formed threonine residue is found in the complex, suggesting that the inhibitor peptide chain is cleaved in the complex between C1s and C1-esterase inhibitor. The stabilizing bond may therefore be an ester bond. C.d. studies of the native C1-esterase inhibitor indicated the presence of about 38% alpha-helix, about 24% beta-structure and about 38% unordered structure. By gradual cleavage of the disulphide bridges under non-denaturating conditions, gradual changes in the c.d. spectra occurred, suggesting loss of ordered secondary structures. The c.d. spectra of the complex between C1s and C1-esterase inhibitor indicate that tryptophan residues are affected by the complex-formation.
Rat liver glutathione S-transferases. A study of the structure of the basic YbYb-containing enzymes
Authors: Hayes J D
Published: August 2015

A purification scheme was devised that resulted in the resolution of a number of basic glutathione S-transferases from rat liver, three of which contained two subunits of molecular mass 23500 Da (i.e. Yb monomers). These were identified as transferases D, C and A by their elution positions from CM-cellulose and their specific activities towards a variety of substrates. Hybridization, immunotitration and peptide ‘mapping’ experiments demonstrated that transferases D, C and A comprise Yb2Yb2, Yb1Yb2 and Yb1Yb1 subunits.
Study of hyaluronic acid flexibility by electric birefringence
Authors: Trimm H H , Jennings B R
Published: August 2015

Transient-electric-birefringence experiments were conducted on four samples of hyaluronic acid over the molecular-mass (M) range 5 X 10(4)-4 X 10(6) in dilute aqueous solution. The geometrical, optical and electrical characteristics were monitored via the rotary relaxation times, optical-polarizability antisotropies and electrical polarizabilities respectively. Each indicates the molecular conformation to be consistent with some degree of rigidity at low M but that this does not persist at high M. The molecules do not become true random coils, but are best characterized in terms of a persistence length of 20 nm or 20 disaccharide units.
Ionization of tyrosine and lysine residues in native and modified horse cytochrome c
Authors: Boswell A P , Moore G R , Williams R J P , Harris D E , Wallace C J A , Bocieck S , Welti D
Published: August 2015

1H-n.m.r. and 13C-n.m.r. spectroscopy of horse cytochrome c and 1H-n.m.r. spectroscopy of the lysine-modified proteins N epsilon-acetimidyl-, N epsilon-amidino-, N epsilon-trifluoroacetyl- and N epsilon-maleyl-cytochrome c have shown that, although the lysine modifications do not greatly perturb the protein structure at pH7 and 27 degrees C, at higher temperature or at alkaline pH some parts of the structure are markedly perturbed. At pH7 and 27 degrees C the region of the protein about Ile-57 is affected in all the modified proteins, though not all to the same degree. N epsilon-Maleylation most seriously affects the protein structure, and the fully maleylated protein is readily unfolded. At 27 degrees C all four of the tyrosine residues of native horse cytochrome c have pKa values above 11, but in N epsilon-acetimidyl-cytochrome c the pKa of one tyrosine residue is 10.2.
Studies of the acetyl-CoA-binding site of rat liver spermidine/spermine N1-acetyltransferase
Authors: Della Ragione F , Erwin B G , Pegg A E
Published: August 2015

Rat liver spermidine/spermine N1-acetyltransferase was found to be strongly inhibited by the dyes Cibacron F3GA, Coomassie Brilliant Blue and Congo Red. Inhibition was competitive with respect to acetyl-CoA and Ki values of 0.7 microM and 52 microM were determined for Cibacron F3GA and Coomassie Brilliant Blue respectively. The enzyme was strongly retained by columns of Affi-Gel Blue, which contains Cibacron F3GA linked to agarose. It was not eluted from this adsorbent in the presence of 10 mM-spermidine/0.5 M-NaCl/50 mM-Tris/HCl, pH 7.5, but was released by 1 mM-CoA in 10 mM-spermidine/50 mM-Tris/HCl, pH 7.5. These results are consistent with the presence in the enzyme of a dinucleotide fold that binds acetyl CoA and has a high affinity for Cibacron F3GA. The spermidine/spermine N1-acetyltransferase was irreversibly inactivated by exposure to butane-2,3-dione in sodium borate, pH 7.8, or by exposure to phenylglyoxal or camphorquinone-10-sulphonic acid. All of these reagents are known to interact with arginine residues in proteins under the conditions in which they inactivated the acetyltransferase. Inactivation was prevented by the presence of acetyl-CoA or CoA, but to a lesser extent by 3′-dephospho-CoA and not at all by NAD or adenosine. This protection suggests that an arginine residue at the active site is involved in the binding of the acetyl-CoA substrate. Treatment of the assay mixture but not the spermidine N1-acetyltransferase with alkaline phosphatase prevented the reaction taking place. This suggests that the apparent loss of enzyme activity in response to alkaline phosphatase reported by Matsui, Otani, Kamei & Morisawa [(1982) FEBS Lett. 150, 211-213] is due to dephosphorylation of the acetyl-CoA substrate and that the 3′-phosphate group is essential for activity.
Investigation of the catalytic site of actinidin by using benzofuroxan as a reactivity probe with selectivity for the thiolate-imidazolium ion-pair systems of cysteine proteinases. Evidence that the reaction of the ion-pair of actinidin (pKI 3.0, pKII 9.6) is modulated by the state of ionization of a group associated with a molecular pKa of 5.5
Authors: Salih E , Brocklehurst K
Published: August 2015

Benzofuroxan reacts with the catalytic-site thiol group of actinidin (EC 3.4.22.14, the cysteine proteinase from Actinidia chinensis) to produce stoicheiometric amounts of the chromophoric reduction product, o-benzoquinone dioxime, and of a catalytically inactive derivative of actinidin that is devoid of thiol and that is assumed to contain, initially at least, the sulphenic acid of cysteine-25. A similar result applies also to papain (EC 3.4.22.2). The rate of o-benzoquinone dioxime formation is neither increased by inclusion of 2-mercaptoethanol or hydroxylamine in the reaction mixture nor decreased by changing the solvent from H2O to 2H2O. The change of solvent was shown to be without effect also on the rate of reaction of benzofuroxan with papain. These results suggest that the reactions of benzofuroxan with both actinidin and papain involve rate-determining attack of the catalytic-site thiol group to produce an intermediate adduct that then reacts rapidly with water to form enzyme sulphenic acid and o-benzoquinone dioxime. The pH-dependence of the second-order rate constant for the reaction of benzofuroxan with actinidin was determined in the pH range 4.3-10.2. In marked contrast with the analogous reaction of papain (reported by Shipton & Brocklehurst [(1977) Biochem. J. 167, 799-810]) the pH-k profile for the actinidin reaction clearly contains a sigmoidal component with pKa 5.5, in which k increases with decreasing pH. These data together with the molecular pKa values for S-/ImH+ ion-pair formation and decomposition (3.0 and 9.6) suggest that the combined nucleophilic-electrophilic reactivity of the ion-pair of actinidin might be controlled by the state of ionization of another ionizing group, associated with the molecular pKa of 5.5. The pH-dependence of k for the reaction of actinidin with benzofuroxan at 25 degrees C at I 0.1 in aqueous buffers containing 6.7% (v/v) ethanol is probably adequately described by: k = k1/(1 + [H+]/KI + KII/[H+]) + k2/(1 + [H+]/KII + KIII/ [H+] + k3/(1 + [H+]/KIII) in which kI = 2.55 M -1 X s -1, k2 = 1.35 M -1, k3 = 0.93 M -1 X s -1, pKI = 3.0, pKII = 5.5 and pKIII = 9.6. By contrast, the analogous reaction of papain may be described by the same equation but with kI = 0, k2 = 2.2 M -1 X s -1, k3 = 1.3 M -1 X s -1, pKII = 3.6 and pKIII = 9.0.
The role of Mg2+ and Mn2+ in the enzyme-catalysed activation of nitrogenase Fe protein from Rhodospirillum rubrum
Authors: Guth J H , Burris R H
Published: August 2015

The activation of the Fe protein of nitrogenase (Rr2) from glutamate-grown Rhodospirillum rubrum by activating enzyme (AE) was investigated. AE is confirmed to have Mr about 20 000 and is shown to operate catalytically. There is a role in activation for metal-ion-ATP, which can be met by either MnATP or MgATP. There is also a site of action for free metal ions. This site prefers Mn2+ (apparent Kd approx. 20 microM) over Mg2+ (apparent Kd approx. 20 mM) by a factor of 1000-fold. Non-activated Rr2 does not contain this binding site. MnATP is an inhibitor of C2H2 reduction, and excess Mg2+ inhibits both AE activity and C2H2 reduction, when each is studied independently under otherwise optimal conditions. The activity of AE is increased in normal reaction mixtures (in which AE activity and nitrogenase activity occur simultaneously) by Mg2+ concentrations in excess of ATP concentrations; this occurs because the excess Mg2+ prevents ATP from chelating the free Mn2+ necessary for optimal AE activity.
Methyl esterification of glutamic acid residues of methyl-accepting chemotaxis proteins in Bacillus subtilis
Authors: Ahlgren J A , Ordal G W
Published: August 2015

The amino acid residue modified in the reversible methylation of Bacillus subtilis methyl-accepting chemotaxis proteins was identified as glutamic acid; methylation results in the formation of glutamate 5-methyl ester. Identification was made by comparing the behaviour of a 3H-labelled compound isolated from proteolytically hydrolysed methyl-accepting chemotaxis proteins labelled in vivo with that of authentic methylated amino acids by chromatographic and electrophoretic techniques. Also, the isolated compound on mild alkaline hydrolysis shows behaviour identical with that of authentic glutamate 5-methyl ester. [3H]Methanol released by mild alkaline hydrolysis was made to react with 3,5-dinitrobenzyl chloride to form [3H]methyl 3,5-dinitrobenzoate, which was identified by reverse-phase high-pressure liquid chromatography.
Cyclic AMP mediates β-adrenergic-induced increases in N-linked protein glycosylation in rat parotid acinar cells
Authors: Kousvelari E E , Grant S R , Banerjee D K , Newby M J , Baum B J
Published: August 2015

beta-Adrenergic stimulation of rat parotid cells by isoprenaline (isoproterenol) results in 2-3-fold increases in [3H]mannose incorporation into N-linked oligosaccharides. This occurs without perceptible lag and is linear with time for 60 min after agonist addition. Concomitantly, isoprenaline markedly increases cellular cyclic AMP. Examination of individual proteins by sodium dodecyl sulphate/polyacrylamide-gradient-gel electrophoresis reveals that glycosylation changes are primarily associated with four secretory proteins, of approx. Mr 17000, 32000, 38000 and 220000. Beta-Adrenoreceptor activation additionally elicits a slight increase in parotid protein synthesis. The greatest increase in [14C]leucine incorporation is that into another secretory protein (Mr approx. 24000). Exposure of cells to dibutyryl cyclic AMP yields results comparable with those after isoprenaline treatment. Forskolin, which increases parotid-cell cyclic AMP, also causes similar effects. Conversely, dibutyryl cyclic GMP shows no such response. The data are consistent with the notion that beta-adrenergic stimulation of N-linked protein glycosylation in rat parotid cells is mediated by cyclic AMP.
Modification of hydroxymethylbilane synthase (porphobilinogen deaminase) by pyridoxal 5′-phosphate. Demonstration of an essential lysine residue
Authors: Hart G J , Leeper F J , Battersby A R
Published: August 2015

When hydroxymethylbilane synthase (porphobilinogen deaminase) from Euglena gracilis is incubated with pyridoxal 5′-phosphate at pH 7.0 and 0 degree C, it rapidly loses part of its activity. The proportion of activity that remains decreases as the concentration of the modifier increases up to approx. 2mM, above which no further significant inactivation occurs. Dialysis of the partly inactivated enzyme restores its activity, whereas reduction with NaBH4 makes the inactivation permanent. The maximum inactivation achievable from one cycle of the treatment with pyridoxal 5′-phosphate, then with borohydride, is 53 +/- 5%; taking this modified enzyme through second and third cycles causes further loss of activity. The enzyme from Rhodopseudomonas spheroides behaves similarly, but there are quantitative differences. Spectroscopic evidence indicates that the inactivation procedure modifies lysine residues, and labelling studies show that epsilon-N-pyridoxyl-L-lysine is a product when permanently inactivated enzyme is completely hydrolysed. Several lysine residues per molecule of the E. gracilis enzyme are modified by the treatment with pyridoxal 5′-phosphate and borohydride, but only one appears to be essential for enzymic activity, since porphobilinogen protects the enzyme against inactivation and then one fewer lysine residue per molecule of enzyme is affected. It is suggested that, during the biosynthesis of hydroxymethylbilane, the first porphobilinogen unit is covalently bound to the enzyme through the epsilon-amino group of the essential lysine.
Mode of action of a bactericidal protein induced in the haemolymph of Sarcophaga peregrina (flesh-fly) larvae
Authors: Okada M , Natori S
Published: August 2015

The mode of action of a bactericidal protein (sarcotoxin I) purified from the haemolymph of Sarcophaga peregrina (flesh-fly) larvae was studied, focusing attention on its effect on the function of the membrane of Escherichia coli. Sarcotoxin I almost completely blocked the uptakes of tetraphenylphosphonium ion and proline, which are known to be driven by a membrane potential, indicating that this protein disrupts the membrane potential of bacteria.
Differential patterns of arabinosylation by membranes of suspension-cultured cells of Phaseolus vulgaris (French bean) after subculture or elicitation
Authors: Bolwell G P
Published: August 2015

Suspension-cultured cells of Phaseolus vulgaris (French bean) incorporated [1-3H] arabinose in vivo into high-Mr polymers that could be separated into glycoprotein and polysaccharide. Microsomal membranes from suspension-cultured cells of beans incorporated arabinose from UDP-beta-L-arabinose in vitro into both polysaccharide and glycoprotein. The enzyme involved in arabinan synthesis, arabinan synthase, appeared to be immunologically distinct from the protein:arabinosyltransferase system. Both these activities are inducible, but behave differently with either plant-growth-regulator or fungal-elicitor treatments. After subculture of cells entering the stationary growth phase the arabinan synthase activity reaches much higher values than does that of the protein transferase system during the initial period of cell division and growth, whereas after elicitation at the same growth stage, all the increased incorporation of arabinose occurs into glycoprotein of Mr higher than 200 000 and to a greater extent into a specific glycoprotein of Mr 42 500. Preliminary characterization of these glycoproteins prepared under non-reducing conditions and after acid and alkaline hydrolysis suggests that the high-Mr glycoprotein material is similar to arabinogalactan protein, whereas the lower-Mr material may be a hydroxyproline-rich protein existing as a dimer and that specifically increases during the hypersensitive response of the cells to the fungal elicitor from Colletotrichum lindemuthianum.
The reversibility of cytosolic dehydrogenase reactions in hepatocytes from starved and fed rats. Effect of fructose
Authors: Vind C , Grunnet N
Published: August 2015

The metabolism of [2-3H]lactate was studied in isolated hepatocytes from fed and starved rats metabolizing ethanol and lactate in the absence and presence of fructose. The yields of 3H in ethanol, water, glucose and glycerol were determined. The rate of ethanol oxidation (3 mumol/min per g wet wt.) was the same for fed and starved rats with and without fructose. From the detritiation of labelled lactate and the labelling pattern of ethanol and glucose, we calculated the rate of reoxidation of NADH catalysed by lactate dehydrogenase, alcohol dehydrogenase and triosephosphate dehydrogenase. The calculated flux of reducing equivalents from NADH to pyruvate was of the same order of magnitude as previously found with [3H]ethanol or [3H]xylitol as the labelled substrate [Vind & Grunnet (1982) Biochim. Biophys. Acta 720, 295-302]. The results suggest that the cytoplasm can be regarded as a single compartment with respect to NAD(H). The rate of reduction of acetaldehyde and pyruvate was correlated with the concentration of these metabolites and NADH, and was highest in fed rats and during fructose metabolism. The rate of reoxidation of NADH catalysed by lactate dehydrogenase was only a few per cent of the maximal activity of the enzymes, but the rate of reoxidation of NADH catalysed by alcohol dehydrogenase was equal to or higher than the maximal activity as measured in vitro, suggesting that the dissociation of enzyme-bound NAD+ as well as NADH may be rate-limiting steps in the alcohol dehydrogenase reaction.
Cyclic nucleotides and their relationship to complement-component-C2 synthesis by human monocytes
Authors: Lappin D , Riches D W H , Damerau B , Whaley K
Published: August 2015

The time courses of changes in cyclic nucleotide levels in monocytes have been studied. Histamine and prostaglandin E2 (PGE2) produced a rapid rise in cyclic AMP (peak 15 min) levels, which returned to normal within 4h, whereas cholera toxin, NaF and phosphodiesterase inhibitors produced slow sustained rises lasting over 24h. With the exception of isobutylmethylxanthine (10 mumol X 1(-1), none of these reagents altered cyclic GMP levels. alpha 1-Adrenergic and nicotinic cholinergic receptor-ligand interactions and imidazole produced rapid and relatively short-lived falls in cyclic AMP, and rises in cyclic GMP. In contrast, prostaglandin synthetase inhibitors produced delayed but more sustained falls in cyclic AMP but no rises in cyclic GMP. Agents that increased cyclic AMP decreased complement-component-C2 production, and those that decreased cyclic AMP increased C2 production. Agents that increased cyclic GMP alone (ascorbate, nitroprusside and prostaglandin F2 alpha) did not affect C2 production. Antigen-antibody complexes that stimulate C2 synthesis produced falls in cyclic AMP and rises in cyclic GMP similar to those produced by adrenergic and cholinergic ligands. Serum-treated complexes and anaphylatoxins, which inhibited C2 production, were associated with changes in cyclic AMP similar to those produced by histamine and PGE2. These data suggest that there are two transmembrane signals involved in the regulation of C2 production by monocytes. The inhibitory signal is adenylyl cyclase activation. The stimulatory signal is not so obvious, but may be Ca2+ influx, since the time courses of changes in cyclic nucleotides produced by agents that stimulate C2 synthesis are identical, and alpha 1-adrenergic agonists cause the formation of Ca2+ channels.
Nucleotide sequence encoding the flavoprotein and hydrophobic subunits of the succinate dehydrogenase of Escherichia coli
Authors: Wood D , Darlison M G , Wilde R J , Guest J R
Published: August 2015

The nucleotide sequence of a 3614 base-pair segment of DNA containing the sdhA gene, encoding the flavoprotein subunit of succinate dehydrogenase of Escherichia coli, and two genes sdhC and sdhD, encoding small hydrophobic subunits, has been determined. Together with the iron-sulphur protein gene (sdhB) these genes form an operon (sdhCDAB) situated between the citrate synthase gene (gltA) and the 2-oxoglutarate dehydrogenase complex genes (sucAB): gltA-sdhCDAB-sucAB. Transcription of the gltA and sdhCDAB gene appears to diverge from a single intergenic region that contains two pairs of potential promoter sequences and two putative CRP (cyclic AMP receptor protein)-binding sites. The sdhA structural gene comprises 1761 base-pairs (587 codons, excluding the initiation codon, AUG) and it encodes a polypeptide of Mr 64268 that is strikingly homologous with the flavoprotein subunit of fumarate reductase (frdA gene product). The FAD-binding region, including the histidine residue at the FAD-attachment site, has been identified by its homology with other flavoproteins and with the flavopeptide of the bovine heart mitochondrial succinate dehydrogenase. Potential active-site cysteine and histidine residues have also been indicated by the comparisons. The sdhC (384 base-pairs) and sdhD (342 base-pairs) structural genes encode two strongly hydrophobic proteins of Mr 14167 and 12792 respectively. These proteins resemble in size and composition, but not sequence, the membrane anchor proteins of fumarate reductase (the frdC and frdD gene products).
A transient increase in diacylglycerols is associated with the action of vasopressin on hepatocytes
Authors: Hughes B P , Rye K A , Pickford L B , Barritt G J , Chalmers A H
Published: August 2015

Vasopressin induced a transient increase of 50% in the total concentration of diacylglycerols (determined by g.l.c.) in isolated hepatocytes. The increase was maximal at 0.25 min, and the concentration of diacylglycerols in cells treated with vasopressin had returned to the basal value by 4 min. No change in the concentration of diacylglycerols was observed after the treatment of cells with glucagon. The dependency of this effect on the concentration of vasopressin was similar to that of the effect of the hormone on 45Ca2+ efflux measured at 0.1 mM extracellular Ca2+. Vasopressin increased the proportion of arachidonic acid and stearic acid and decreased the proportion of oleic acid present in the diacylglycerols. In hepatocytes prelabelled with [14C]arachidonic acid, vasopressin increased the amount of [14C]diacylglycerol. The effects of vasopressin on the total concentration of diacylglycerols and [14C]diacylglycerol were mimicked by an exogenous phospholipid phosphodiesterase (phospholipase C) from Clostridium perfringens. The results are consistent with the conclusion that the transient increase in diacylglycerols induced by vasopressin is caused by the rapid hydrolysis of both the phosphoinositides and one or more other phospholipids.
Oxygen uptake associated with Sendai-virus-stimulated chemiluminescence in rat thymocytes contains a significant non-mitochondrial component
Authors: Kolbuch-Braddon M E , Peterhans E , Stocker R , Weidemann M J
Published: August 2015

Sendai virus (150 haemagglutinating units/10(6) cells) stimulates rat thymocytes incubated in medium containing 5 mM-glucose at 37 degrees C to produce luminol-dependent chemiluminescence and a simultaneous increase in O2 consumption of 40%. Stimulation of thymocytes with Sendai virus is accompanied by reduction of exogenous acetylated ferricytochrome c, which is inhibited by superoxide dismutase, and the quantitative conversion of ferricyanide to ferrocyanide, which is not. Replacement of air in the gas space with N2 inhibits the chemiluminescent response by 97% but does not prevent the virus-stimulated reduction of ferricyanide. The non-permeant ferricyanide anion (2 mM) also inhibits the chemiluminescent response to Sendai virus, its accompanying ‘extra’ O2 uptake and the reduction of acetylated ferricytochrome c without affecting the basal respiration of the cells. Thymocytes in which the basal O2 consumption has been stimulated maximally with dinitrophenol (10 microM) or inhibited completely with antimycin A (0.1 microM) respond to Sendai virus with an additional increment of ferricyanide-inhibitable O2 consumption. The chemiluminescent response to virus is not inhibited by concentrations of antimycin A that block the basal respiration completely. We suggest that a portion of the increased O2 uptake induced by Sendai virus is involved in the non-mitochondrial reduction of O2 to O2- at the cell surface where the non-permeant ferricyanide anion inhibits O2-. formation by acting as an alternative high-affinity electron acceptor to O2.
Studies on the glutathione S-transferase activity associated with rat liver mitochondria
Authors: Ryle C M , Mantle T J
Published: August 2015

The major proportion of rat liver glutathione S-transferase is cytosolic. Carefully washed mitochondria contain 0.25-0.47% of the cytosolic activity. Subfractionation of washed mitochondria using digitonin treatment revealed that glutathione S-transferase release did not parallel that of any of the mitochondrial marker enzymes. Glutathione S-transferase release paralleled that of lactate dehydrogenase, suggesting that these ‘mitochondrial’ activities are due to loosely bound cytoplasmic forms.
Purification of a neutral proteinase, associated with the actomyosin complex, from uterine myometrium
Authors: Barth R , Hoechst M , Afting E G
Published: August 2015

We have purified and characterized a neutral proteinase activity from pig uterine myometrium. The proteinase co-purified with the actomyosin complex and could only be separated from it by a high concentration of a chaotropic ion, 3M-NaBr. The proteinase was further purified by gel filtration and affinity chromatography. The purified protein showed a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis corresponding to an Mr of 28 000. Gel filtration on Sephadex G-100 in a buffer containing 3M-NaBr gave an Mr of 27 500. Without the addition of the chaotropic Br- ion, the proteinase aggregates to high-Mr forms of more than 10(6)Da. The proteinase has optimum hydrolytic activity with casein as substrate at pH 7.5-8.0. The thiol-group-blocking reagents p-chloromercuribenzoate, p-chloromercuribenzenesulphonate and Hg2+, as well as soya-bean trypsin inhibitor and 4-aminobenzamidine, inhibited the proteinase. Other bivalent cations, chelating agents and the serine-specific reagents 7-amino-1-chloro-3-tosylamido-L-heptan-2-one and phenylmethanesulphonyl fluoride were without any effect on proteinase activity. The proteinase degraded myosin very rapidly at a molar ratio of proteinase to myosin of 1:50, concomitant with the rate of loss of the ATPase activity. Compared with myosin, actin was only a poor substrate and was degraded at a much lower rate, even at a high molar ratio of the proteinase to actin.
Rapid kinetic analysis of the bile-salt-dependent secretion of phospholipid, cholesterol and a plasma-membrane enzyme into bile
Authors: Lowe P J , Barnwell S G , Coleman R
Published: August 2015

Isolated rat livers were perfused under ‘one-pass’ conditions and bile was collected at 1 min intervals. After 1 min pulse, taurocholate appeared in the collected bile within 2 min, peak output occurring 2 min later. In contrast, the increased output of phospholipids and cholesterol was slower, peak output occurring 6-11 min after the original pulse of taurocholate. These results suggest that mixed micelles cannot be formed inside the cell or during passage of bile salts through the membrane, since bile salt and lipids should then parallel each other. The bile salts must therefore be pumped into the lumen and the lipids added subsequently, due to the actions of the bile salts in the canalicular lumen. It is suggested that the biliary lipid is obtained from microdomains of biliary-type lipid in the canaliculus membrane, which are vesiculated and solubilized by the action of bile salts. It is also suggested that this biliary-type lipid is brought continuously to the membrane via vesicle traffic; this traffic is increased during increased bile-salt output, and is a process that can be inhibited by colchicine.
Purification and characterization of chicken erythrocyte ferrochelatase
Authors: Hanson J W , Dailey H A
Published: August 2015

Ferrochelatase (EC 4.99.1.1) was purified 2000-fold to apparent homogeneity from isolated chicken erythrocyte mitochondria. The purified enzyme yields a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with an apparent Mr of 42 000. The enzyme utilizes proto-, meso- and deutero-porphyrin with Km values of 37, 51 and 80 microM respectively. The disubstituted porphyrins 2,4-bisglycol deutero-porphyrin and 2,4-disulphonic deuteroporphyrin were not substrates. Mn2+, Hg2+, Pb2+ and Co2+ were strong inhibitors of the purified enzyme. Palmitic acid and oleic acid stimulated activity, whereas linoleic acid inhibited and phospholipids had variable effects. Chicken ferrochelatase was inhibited by N-ethylmaleimide and iodoacetamide. Inhibition by iodoacetamide was pseudo-first-order, but inhibition by N-ethylmaleimide appeared to be biphasic in nature with an initial high rate followed by a much lower rate of inactivation. The characteristics of the chicken erythrocyte enzyme are compared with those previously reported for mammalian liver ferrochelatase.
Co-operation between plasmin and elastase in elastin degradation by intact murine macrophages
Authors: Chapman H A , Stone O L
Published: August 2015

Intact, thioglycollate-stimulated murine macrophages cultured on an insoluble [3H]-elastin substratum progressively hydrolysed the elastin. Cell lysates had little activity. We compared the effect of various proteinase inhibitors on elastinolysis by either live cells or cell-free, elastase-rich conditioned medium. Only known inhibitors of macrophage elastase blocked the activity of elastase-rich cell-conditioned medium whereas inhibitors of cathepsin B also suppressed intact cell activity. Serum proteinase inhibitors blocked cell-derived soluble elastase activity but not intact cell elastolytic activity. We also observed that plasminogen added to the cell cultures markedly increased elastinolysis by live macrophages or cell-free elastase-rich medium. Purified plasmin alone had no measurable effect on native elastin. Additional experiments indicated that the plasmin enhancement was due to elastin-dependent activation of latent macrophage elastase. These results indicate that live macrophage elastinolysis is a co-operative process involving multiple proteinases, especially a cysteine proteinase(s) and elastase. Plasmin may be a physiological activator of latent macrophage elastase.
Polyamine levels and the activity of their biosynthetic enzymes in human erythrocytes infected with the malarial parasite, Plasmodium falciparum
Authors: Assaraf Y G , Golenser J , Spira D T , Bachrach U
Published: August 2015

Human erythrocytes contain only trace amounts of polyamines and lack active polyamine biosynthetic enzymes. A remarkable increase in polyamine content, and in the activity of ornithine and S-adenosyl-L-methionine decarboxylases, is noted in synchronous cultures of the malarial parasite, Plasmodium falciparum. Polyamine biosynthesis reached peak values during the early trophozoite stage, whereas nucleic acid and protein synthesis occurred later in mature trophozoites. DL-alpha-Difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, did not interfere with merozoite invasion and with ring-form development, but prevented the transformation of trophozoites to schizonts. Concomitantly, the synthesis of proteins and nucleic acids was significantly inhibited. These inhibitory effects could be readily reversed by the diamine putrescine. Macromolecular synthesis and schizogony were normal when 5-10 mM-DL-alpha-difluoromethylornithine and 0.1 mM-putrescine were added to the cultures simultaneously.
Temperature effect of cholesterol association with synaptosomal plasma membranes of rabbit brain
Authors: Deliconstantinos G
Published: August 2015

Association of exogenous cholesterol with rabbit brain synaptosomal plasma membranes follows an exponential path described by the general formula y = a X ebx. The co-operative nature of this association was shown when increasing amounts of unlabelled cholesterol glucoside (up to 0.5 mM) were added to a fixed amount (5 microM) of [14C]cholesterol, when a biphasic curve of the binding of [14C]cholesterol into the membranes was obtained. Arrhenius plots of this association revealed two break points which occur at 25 degrees C and 42 degrees C. The first break apparently corresponds to the transition from the crystalline to the gel phase. The second break may be due to the (continuously) increasing entropy of the system which creates at a certain point difficulties in the binding of cholesterol into the lipid bilayer.
Characteristics of n-octyl β-d-thioglucopyranoside, a new non-ionic detergent useful for membrane biochemistry
Authors: Saito S , Tsuchiya T
Published: August 2015

n-Octyl beta-D-thioglucopyranoside, a new non-ionic detergent, was synthesized. Properties, and applicability to membrane proteins, of this detergent were investigated. The detergent was easily removed by dialysis. The solubilizing power of this detergent for Escherichia coli membrane proteins was similar to that of n-octyl beta-D-glucopyranoside, which has been widely used in membrane biochemistry. No inactivation of proteins was observed after the solubilization. n-Octyl beta-D-thioglucopyranoside was superior to n-octyl beta-D-glucopyranoside in that it was much more stable and could be synthesized at much lower cost.
Effects of prostaglandin I2 and forskolin on the secretion from platelets evoked at basal concentrations of cytoplasmic free calcium by thrombin, collagen, phorbol ester and exogenous diacylglycerol
Authors: Rink T J , Sanchez A
Published: August 2015

Cytoplasmic free calcium ([Ca2+]i) and secretion of ATP were measured in quin2-loaded human platelets. In certain conditions thrombin and collagen cause secretion while [Ca2+]i remains at basal concentrations, a response attributed to activation of protein kinase by diacylglycerol formed by hydrolysis of inositol lipids. This secretion evoked by thrombin could be totally suppressed by prostaglandin I2 or forskolin, as expected from the known ability of cyclic AMP to inhibit phospholipase C. The secretory response evoked by collagen at basal [Ca2+]i and that evoked by exogenous diacylglycerol or phorbol ester, direct activators of protein kinase-C, were much less affected by these inhibitors, suggesting that thrombin and collagen may promote formation of diacylglycerol by different mechanisms.
Characterization of a novel Na+-independent amino acid transporter in horse erythrocytes
Authors: Fincham D A , Mason D K , Young J D
Published: August 2015

Horse erythrocytes are polymorphic with respect to L-alanine permeability. The present investigation compared the specificity, kinetics and cation-dependence of erythrocyte amino acid transport in two groups of thoroughbred horses, those with erythrocyte L-alanine permeabilities in the range 5-15 mumol/h per litre of cells (0.2 mM extracellular L-alanine, 37 degrees C) (transport-negative type) and those with L-alanine permeabilities in the range 450-700 mumol/h per litre of cells (transport-positive type). Transport-positive cells are shown to possess a novel high-affinity, stereospecific, Na+-independent transporter selective for neutral amino acids of intermediate size. This carrier system (provisional designation asc) operates preferentially in an exchange mode and is functionally absent from erythrocytes of transport-negative-type horses.
Amino acid sequence around the active-site serine residue in the acyltransferase domain of goat mammary fatty acid synthetase
Authors: Mikkelsen J , Højrup P , Rasmussen M M , Roepstorff P , Knudsen J
Published: August 2015

Goat mammary fatty acid synthetase was labelled in the acyltransferase domain by formation of O-ester intermediates by incubation with [1-14C]acetyl-CoA and [2-14C]malonyl-CoA. Tryptic-digest and CNBr-cleavage peptides were isolated and purified by high-performance reverse-phase and ion-exchange liquid chromatography. The sequences of the malonyl- and acetyl-labelled peptides were shown to be identical. The results confirm the hypothesis that both acetyl and malonyl groups are transferred to the mammalian fatty acid synthetase complex by the same transferase. The sequence is compared with those of other fatty acid synthetase transferases.