bluuebunny/crossref_metadata_2025 huggingface dataset:
The cellular location and specificity of bacterial cytochrome c peroxidases
Authors: Goodhew C F , Wilson I B , Hunter D J , Pettigrew G W
Published: August 2015

The locations of cytochrome c peroxidase and catalase activities in the two Gram-negative bacteria Pseudomonas stutzeri (N.C.I.B. 9721) and Paracoccus denitrificans (N.C.I.B. 8944) were investigated by the production of spheroplasts. In both species the cytochrome c peroxidase was predominantly periplasmic: 92% of total activity in Ps. stutzeri and 98% of nonmembrane-bound activity in Pa. denitrificans were found in this cellular compartment. In contrast, the catalase was mostly in the cytoplasmic fraction. Purification of the Pa. denitrificans cytochrome c peroxidase showed it to be the haem c-containing polypeptide of Mr 42,000 that has already been described by Bosma, Braster, Stouthamer & Van Versefeld [(1987) Eur. J. Biochem. 165, 665-670] but was not identified by them as a peroxidase. The visible-absorption spectra of the enzyme closely resemble those of cytochrome c peroxidase from Pseudomonas aeruginosa but the donor specificity is different, with the Pa. denitrificans enzyme preferring the basic mitochondrial cytochromes c to the acidic cytochromes c-551 and reacting well with the Pa. denitrificans cytochrome c-550.
Expression of epidermal-growth-factor receptor in the K562 cell line by transfection. Altered receptor biochemistry
Authors: Allen H , Hsuan J , Clark S , Maziarz R , Waterfield M D , Flavell R A , Haley J
Published: August 2015

The epidermal-growth-factor (EGF) receptor was expressed in the human erythroleukaemic cell line K562 by transfection of the receptor cDNA. EGF-receptor biochemistry appears altered in the K562 transfectants. Autophosphorylation of the K562 receptor is not stimulated substantially by EGF. Tyrosine kinase activity of the receptor is high in the absence of EGF, whereas receptor affinity for EGF is low. K562 cells are shown to lack mRNA for transforming growth factor alpha (TGF alpha). Therefore autocrine stimulation of the K562 receptor, at least by TGF alpha, does not explain the observed receptor biochemistry. The K562 receptor is phosphorylated at a single major site in intact cells, a threonine residue that may be Thr-669. Possible mechanisms of regulation of the EGF receptor in the K562 transfectants are discussed.
Electron-transport components of the 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine Δ12-desaturase (Δ12-desaturase) in microsomal preparations from developing safflower (Carthamus tinctorius L.) cotyledons
Authors: Smith M A , Cross A R , Jones O T G , Griffiths W T , Stymne S , Stobart K
Published: August 2015

The major cytochrome in microsomal membrane preparations from developing seeds of safflower (Carthamus tinctorius, var High Linoleate), has a reduced-minus-oxidized difference spectrum characteristic of a b-type cytochrome, and was identified from its midpoint-potential (E'7.2) value as cytochrome b5. Cytochromes P-450 and P-420 were also present. The cytochrome b5 content of microsomal preparations from a number of oilseed species was found to be in the order of 200-300 pmol/mg of protein. The cytochrome b5 was reduced in the membrane preparations by NADH, demonstrating the presence of an NADH: cytochrome b5 reductase; NADPH was a less effective donor. Microsomal membranes catalysed the NAD(P)H-dependent conversion of radioactive oleate into linoleate, indicating acyl-CoA: lysophosphatidylcholine acyltransferase and 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine delta 12-desaturase (delta 12-desaturase) activity. Desaturation of oleate to linoleate was unaffected by CO, but inhibited by CN-. The addition of oleoyl-CoA to the NADH-reduced membranes resulted in the CN(-)-sensitive partial re-oxidation of cytochrome b5, indicating that electrons from NADH were transferred to the site of desaturation via this cytochrome. The delta 12-desaturase in safflower, therefore, is CN(-)-sensitive and appears to require cytochrome b5 and NADH: cytochrome b5 reductase for activity.
Oxidative degradation of rat mast-cell heparin proteoglycan
Authors: Metcalfe D D , Thompson H L , Klebanoff S J , Henderson W R
Published: August 2015

The susceptibility of rat mast-cell heparin to oxidative degradation was examined. Heparin as a component of intact mast-cell granules (MCG) was degraded following ingestion by normal human neutrophils. In contrast, neutrophils from patients with chronic granulomatous disease (CGD), which do not respond to stimulation with respiratory-burst activity, exhibited a greatly diminished ability to degrade phagocytosed MCG heparin. MCG-associated heparin also was cleaved by H2O2 plus Fe2+ (Fenton's reagent). Isolated heparin proteoglycan (average Mr approx. 750,000) was rapidly cleaved to smaller molecules similar in size to commercial pig heparin upon exposure to Fenton's reagent. This cleavage was inhibited by catalase and by the hydroxyl-radical (OH.)-scavenger mannitol, but not by superoxide dismutase (SOD). The cleavage products retained approx. 26% of the anticoagulant activity of the native molecule. The heparin proteoglycan was also cleaved by acetaldehyde/xanthine oxidase/FeSO4, a system that generates superoxide (O2.-), H2O2 and OH.. Whereas the cleavage at relatively high iron ion concentrations was inhibited by catalase and mannitol but not by SOD, at lower iron ion concentrations the cleavage was inhibited by catalase, mannitol and SOD. These findings suggest the involvement of OH., which at high Fe2+ concentrations is generated by Fenton's reagent (H2O2 plus Fe2+), and at low iron ion concentrations is generated by the iron-ion-catalysed interaction between O2.- and H2O2 (Haber-Weiss reaction). These studies suggest that oxygen radicals generated by activated phagocytes may contribute to the degradation in vivo of both solubilized and granule-associated proteoglycan heparin.
One of the major sulphated proteins secreted by rat hepatocytes contains low-sulphated chondroitin sulphate
Authors: Sjöberg E M , Fries E
Published: August 2015

When isolated hepatocytes are incubated with 35SO4(2-), a specific set of secretory proteins is labelled. One of these proteins is electrophoretically heterogeneous, with an apparent molecular mass of 35-45 kDa [Marcks von Würtemberg & Fries (1989) Biochemistry 28, 4088-4093]. Here we report that treatment with chondroitinase ABC converted the broad electrophoretic band of this protein, with a 50-60% loss of radioactivity, into a relatively homogeneous band with a molecular mass of 28 kDa. Size determination by gel chromatography of the protein's oligosaccharide chain (released by alkali treatment) indicated that it contained about 40 hexose units. Similar analysis of the enzyme-resistant oligosaccharide chain remaining linked to the protein after chondroitinase ABC treatment indicated a size of between six and eight hexose units. These observations suggest that the protein's oligosaccharide chain carries only three or four sulphate groups, of which one or two are located close to the polypeptide chain. Consistent with this hypothesis, the free oligosaccharide behaved like a low-sulphated glycosaminoglycan upon ion-exchange chromatography.
Effect of denervation on the expression of glycogen phosphorylase in mouse skeletal muscle
Authors: Leyland D M , Turner P C , Beynon R J
Published: August 2015

After sciatectomy of the left hind-limb of C57BL/J mice, a denervation-induced muscular atrophy ensued and was accompanied by a decrease in the specific activity of glycogen phosphorylase to approx. 25% of control values. The cofactor of phosphorylase, pyridoxal 5′-phosphate, was used as a specific label in the determination of the degradation rate of the enzyme following nerve section. After a delay of 3-4 days, phosphorylase was degraded approx, twice as rapidly in the denervated gastrocnemius (0.20 day-1) as in the control muscle (0.12 day-1). The effect of denervation on phosphorylase mRNA was measured by quantitative Northern-blot analysis using a rat skeletal-muscle phosphorylase cDNA probe. After an initial rapid decline, phosphorylase mRNA levels stabilized in denervated muscle at 50% of the value measured in the contralateral control muscle.
Sensitivity of inhibition of rat liver mitochondrial outer-membrane carnitine palmitoyltransferase by malonyl-CoA to chemical- and temperature-induced changes in membrane fluidity
Authors: Kolodziej M P , Zammit V A
Published: August 2015

We have tested the possibility that alterations in the fluidity of the outer membrane of rat liver mitochondria could result in changes in the sensitivity of overt carnitine palmitoyltransferase (CPT I) to malonyl-CoA [Zammit (1986) Biochem. Soc. Trans. 14. 676-679]. The sensitivity of CPT I to malonyl-CoA inhibition was measured by using highly purified mitochondrial outer membranes prepared from fed or 48 h-starved rats in the presence and absence of agents that increase membrane fluidity by perturbing membrane lipid order [benzyl alcohol, isoamyl alcohol (3-methylbutan-l-ol) and 2-(2-methoxyethoxy)ethyl-8-(cis-2-n-octylpropyl)octanoate (A2C)]. All these agents resulted in marked decreases in the ability of malonyl-CoA to inhibit CPT I. This effect was accompanied by a modest increase in the absolute activity of CPT I in the absence of malonyl-CoA when the short-chain alcohols were used, but not when A2C was used, suggesting that the effect of increased membrane fluidity to decrease the malonyl-CoA sensitivity of CPT I may occur independently from other actions that may affect more directly the active site of the enzyme. In confirmation of the potential importance of fluidity changes, we showed that a marked increase in sensitivity of CPT I to malonyl-CoA could be produced when assays were performed at lower temperatures than those normally employed. These observations are discussed in the context of the slowness of the changes in CPT I sensitivity to malonyl-CoA inhibition that are induced by physiological perturbations.
Effect of pH on the oxidation pathway of α-methyldopa catalysed by tyrosinase
Authors: Serna Rodríguez P , Rodríguez López J N , Tudela J , Varón R , García Cánovas F
Published: August 2015

This paper deals with the quantitative description of the effect of pH on the oxidation pathway of alpha-methyldopa. Tyrosinase catalyses the oxidation by molecular oxygen of alpha-methyldopa to o-alpha-methyldopaquinone, which evolves non-enzymically through a branched pathway with cyclization or hydroxylation reactions. The intermediates of the hydroxylation branch have been identified, and the corresponding rate constants have been determined. These compounds, which have been detected in melanosomes and in tumour cells, have great cytotoxic power and could have physiological significance in acidic media.
Comparison of extracellular ATP and UTP signalling in rat renal mesangial cells. No indications for the involvement of separate purino- and pyrimidino-ceptors
Authors: Pfeilschifter J
Published: August 2015

Extracellular ATP and UTP caused a rapid formation of InsP3, with similar kinetics and dose-dependences. ITP also displayed strong agonistic properties in terms of InsP3 production, whereas CTP was almost inactive. Pretreatment of the cells with pertussis toxin attenuated ATP- and UTP-stimulated InsP3 generation to a comparable extent, indicating that both nucleotides couple to phospholipase C by a pertussis-toxin-sensitive G-protein. Short-term (15 min) treatment of the cells with phorbol 12-myristate 13-acetate (PMA) produced a dose-dependent inhibition of ATP- and UTP-induced InsP3 formation. Furthermore, down-regulation of protein kinase C by long-term (24 h) exposure of the cells to PMA resulted in a comparable potentiation of phosphoinositide hydrolysis by both nucleotides. Preincubation of mesangial cells with ATP or UTP caused a pronounced cross-desensitization of subsequent nucleotide-stimulated InsP3 production. ATP and UTP displayed no additivity in terms of InsP3 formation, when used at maximally effective concentrations. In contrast, the peptide hormone angiotensin II interacted in an additive manner with either nucleotide in stimulating phosphoinositide hydrolysis. Reactive Blue 2, a putative P2y-purinoceptor antagonist, caused a rightward shift of both the ATP and UTP dose-response curves. However, since 2-methylthio-ATP was only a partial agonist in stimulating InsP3 formation, the mesangial-cell ATP receptor appears to be different from a classic P2y-receptor. In summary, these results provide no evidence for separate purino- and pyrimidino-ceptors on mesangial cells. In contrast, ATP and UTP may use a common nucleotide receptor for transducing their signals in mesangial cells.
Lysocardiolipin formation and reacylation in isolated rat liver mitochondria
Authors: Schlame M , Rüstow B
Published: August 2015

Liver mitochondrial cardiolipin (CL) is distinguished from other phospholipids by the presence of linoleoyl in almost all molecular species, and the biosynthesis of these species is not yet understood. The present study was carried out in order to test the hypothesis that the linoleoyl proportion of CL may be specifically enriched by a deacylation-reacylation cycle. Incorporation of [14C]glycerol 3-phosphate into the metabolites of the CL pathway was accompanied by formation of 14C-labelled monolyso- and dilyso-CL. Labelling of dilyso-CL was increased or decreased by stimulation or inhibition respectively of mitochondrial phospholipase A2. These data suggest a rapid deacylation of newly formed [14C]CL by phospholipase A2, whereas endogenous mitochondrial CL was very resistant to hydrolytic degradation. Unlike dilyso-CL, monolyso-CL could be reacylated by [14C]linoleoyl residues. [14C]Linoleoyl incorporation into CL was also observed when exogenous CL was added instead of monolyso-CL, thus indicating the concerted action of de- and re-acylation. Although 1-palmitoyl-2-[14C]linoleoyl-phosphatidylcholine was a suitable acyl donor under experimental conditions, the reaction was not a transacylation but required splitting of [14C]linoleic acid from phosphatidylcholine and formation of [14C]linoleoyl-CoA as an intermediate. The [14C]linoleoyl was mainly bound to the sn-2(2") position of CL, and a small portion (about 20%) to the sn-1(1") position. It is concluded that a cycle, comprising CL deacylation and monolyso-CL reacylation by linoleoyl-CoA, provides a potential mechanism for the remodelling of molecular species of newly formed CL.
The preferential uptake of very-low-density lipoprotein cholesteryl ester by rat liver in vivo
Authors: Holder J C , Zammit V A , Robinson D S
Published: August 2015

The removal from the blood and the uptake by the liver of injected very-low-density lipoprotein (VLDL) preparations that had been radiolabelled in their apoprotein and cholesteryl ester moieties was studied in lactating rats. Radiolabelled cholesteryl ester was removed from the blood and taken up by the liver more rapidly than sucrose-radiolabelled apoprotein. Near-maximum cholesteryl ester uptake by the liver occurred within 5 min of the injection of the VLDL. At this time, apoprotein B uptake by the liver was only about 25% of the maximum. Maximum uptake of the injected VLDL apoprotein B label was not achieved until at least 15 min after injection, by which time the total uptakes of cholesteryl ester and apoprotein B label were very similar. The results suggest that preferential uptake of the lipoprotein cholesteryl ester by the liver occurred before endocytosis of the entire lipoprotein complex. The fate of the injected VLDL cholesteryl ester after its uptake by the liver was also monitored. Radiolabel associated with the hepatic cholesteryl ester fraction fell steadily from its early maximum level, the rate of fall being faster and more extensive when the fatty acid, rather than the cholesterol, moiety of the ester was labelled. By 30 min after the injection of VLDL containing [3H]cholesteryl ester, over one-third of the injected label was already present as [3H]cholesterol in the liver. When VLDL containing cholesteryl [14C]oleate was injected, a substantial proportion (about 25%) of the injected radiolabelled fatty acid appeared in the hepatic triacylglycerol fraction within 60 min: very little was present in the plasma triacylglycerol fraction at this time.
Regulation of skeletal-muscle AMP deaminase. Evidence for a highly pH-dependent inhibition by ATP of the homogeneous derivative of the rabbit enzyme yielded by limited proteolysis
Authors: Ranieri-Raggi M , Raggi A
Published: August 2015

Limited proteolysis of rabbit skeletal-muscle AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) with trypsin results in conversion of the enzyme into a species which over the pH range 6.5-7.1 exhibits hyperbolic kinetics at low K+ concentration even in the absence of ADP, but shows a 20% decrease in activity at saturating substrate concentration. Analysis by sedimentation-equilibrium techniques reveals the proteolysed enzyme to be homogeneous and to have a molecular mass of 222,000 Da, indicative of a trimeric structure with a subunit molecular mass of 72,000 Da, in contrast with the tetrameric structure of the native enzyme, composed of four 79,000-Da subunits. These observations suggest a role of the 7,000-Da fragment which is removed by proteolysis in the maintenance of the three-dimensional structure of the subunit that causes the enzyme at low K+ concentration to show homotropic positive co-operativity. Study of the influence of pH, isolated from that of K+, on the kinetics of AMP deaminase reveals a highly pH-dependent inhibitory effect by ATP which is completely absent at acid pH values and abruptly manifests itself just above neutrality. This phenomenon may have significance in the metabolism of exercising muscle, in connection with the pH-dependent interaction of AMP deaminase with the thick filament.
Non-selective cation channels in basolateral-membrane vesicles from pars recta of rabbit kidney proximal tubule
Authors: Blokkebak-Poulsen J , Sheikh M I , Jacobsen C
Published: August 2015

The characteristics of 86Rb+ fluxes through conductive channels in basolateral-membrane vesicles isolated from pars recta of rabbit kidney proximal tubule were investigated. In RbCl-, KCl- and NaCl-loaded vesicles a transient and almost equal accumulation of 86Rb+ was observed. The uptakes of 86Rb+ were inhibited to the same extent by 10 mM-BaCl2 in all loadings. The accumulation was driven by an electrical diffusion potential. The 86Rb+ flux was dependent on intravesicular Ca2+. Increasing concentrations of Ca2+ gradually decreased the 86Rb+ uptake. At 10 microM-Ca2+ the radioisotope flux was below 20% of control. The vesicles containing the channel showed very low selectivity among the univalent cations K+, Rb+, Li+, Na+ and choline+.
The phosphoprotein that regulates platelet Ca2+ transport is located on the plasma membrane, controls membrane-associated Ca2+-ATPase and is not glycoprotein Ib β-subunit
Authors: Darnanville A , Bredoux R , Clemetson K J , Kieffer N , Bourdeau N , Levy-Toledano S , Caen J P , Enouf J
Published: August 2015

The localization and identity of the human platelet 24 kDa cyclic AMP (cAMP)-dependent phosphoprotein, previously reported to regulate Ca2+ transport, was investigated. It was found to be located on plasma membranes after isolation of these membranes from microsomes. Thus cAMP-dependent regulation of Ca2+ transport was associated with the plasma membrane fraction. Time course studies showed that the catalytic subunit of cAMP-dependent protein kinase (c-sub) induced a maximal 2-fold stimulation of Ca2+ uptake by the plasma membrane vesicles. This stimulation was dose-dependent up to 15 micrograms of c-sub/ml. The increase in Ca2+ uptake also depended upon the outside Ca2+ concentration, and was maximal at 1 microM. As regards the identity of the phosphoprotein, it was clearly distinct from the beta-subunit of glycoprotein Ib, as after electrophoresis under reduced conditions it appeared as a 24 kDa protein, but under non-reduced conditions it appeared as a 22 kDa and not as a 170 kDa protein. Nevertheless, glycoprotein Ib was certainly present, because it was detected with two polyclonal antibodies raised against its two subunits. Furthermore, the 24 kDa phosphoprotein was also present in membranes isolated from platelets obtained from patients with Bernard Soulier Syndrome; these membranes contain no glycoprotein Ib.
Group B streptococci inactivate complement component C5a by enzymic cleavage at the C-terminus
Authors: Bohnsack J F , Mollison K W , Buko A M , Ashworth J C , Hill H R
Published: August 2015

Incubation of recombinant human C5a (rC5a) with the 7360 strain of group B streptococci (GBS) destroyed the ability of rC5a to stimulate chemotaxis or adherence of purified human polymorphonuclear leucocytes (PMNs). Treatment of 125I-labelled rC5a with GBS 7360 correspondingly decreased rC5a binding to human PMNs. This also resulted in an approx. 600 Da decrease in the molecular mass of rC5a as determined by SDS/PAGE. Incubation of rC5a with the GBS strain GW, which only minimally altered the ability of rC5a to activate human PMNs, did not affect rC5a binding to PMNs and did not alter the molecular mass of rC5a on SDS/PAGE. Plasma-desorption m.s. of rC5a inactivated by GBS 7360 showed that the GBS cleaved the rC5a between histidine-67 and lysine-68 near the C-terminus of rC5a. This mechanism of inactivation of C5a by proteolytic cleavage at the C-terminus of C5a is consistent with the known critical role of the C-terminus in C5a activation of human PMNs. This C5a-cleaving proteinase activity may contribute to the pathophysiology of GBS infections.
Heterogeneous regulation of constitutive thrombomodulin or inducible tissue-factor activities on the surface of human saphenous-vein endothelial cells in culture following stimulation by interleukin-1, tumour necrosis factor, thrombin or phorbol ester
Authors: Archipoff G , Beretz A , Freyssinet J M , Klein-Soyer C , Brisson C , Cazenave J P
Published: August 2015

Thrombomodulin and tissue-factor activities were measured on the surface of confluent human saphenous-vein endothelial cells (HSVEC) cultivated in 96-multiwell plates. Thrombomodulin activity was measured in the presence of purified human thrombin (2.2 nM) and protein C (65 nM). Tissue-factor activity was measured with purified human Factor VII (5 nM) and Factor X (400 nM). Generated activated protein C and Factor Xa released in the supernatant were assayed with chromogenic substrates. Resting cells exhibited significant thrombomodulin activity, but no detectable tissue-factor activity. After 4 h of preincubation with tumour necrosis factor (TNF, 22-2200 pM), interleukin-1 (IL-1, 5.7-570 nM) or phorbol myristate acetate (PMA, 1.61-161 nM) there was an increase in tissue-factor activity and a concomitant decrease in thrombomodulin activity. However, the extent of both responses varied according to the nature of the stimulus. Thrombin (0.44-44 nM) also induced an increase in tissue-factor activity, but had no effect on thrombomodulin activity. Kinetic studies showed that for all stimuli the increase in tissue factor was transient, reaching a maximum after 4-8 h of preincubation with the stimulating agent and returning to normal values after 24 h. IL-1 and TNF induced a time-dependent decrease in thrombomodulin, by respectively 47% and 67% of control values after 24 h. However, PMA induced only a transient down-regulation of thrombomodulin, full activity being recovered after 18 h. Hence this simultaneous assay system, using intact HSVEC and purified human coagulation factors, enabled us to observe that the regulation of thrombin generation could be diversely affected by various substances known to stimulate the endothelium. This suggests that the simultaneous and opposite modulation of these proteins does not represent an unified response of the endothelial cells to procoagulant stimuli. These results also confirm the absence of effect of thrombin on the expression of thrombomodulin on the cell surface.
Use of ‘solid-state’ promoters in the electrochemistry of cytochrome c at a gold electrode
Authors: Santucci R , Faraoni A , Campanella L , Tranchida G , Brunori M
Published: August 2015

The direct electrochemistry of cytochrome c at a gold electrode was investigated by cyclic voltammetry using, as promoters, microperoxidase (the haem-undecapeptide obtained by hydrolysis of cytochrome c), Fe(III)-protoporphyrin IX or protoporphyrin-IX, all entrapped in a cellulose triacetate membrane. The results indicate that these immobilized systems strongly enhance the rate of electron transfer between the protein in solution and the electrode surface, and thus behave as ‘solid-state’ promoters, though with differing efficiencies. These results are of interest because they raise the possibility of engineering an efficient and versatile promoter active also at inert electrode surfaces.
Extracellular ATP causes lysis of mouse thymocytes and activates a plasma membrane ion channel
Authors: Pizzo P , Zanovello P , Bronte V , Di Virgilio F
Published: August 2015

Extracellular ATP (ATPo) caused a concentration-dependent lysis of mouse thymocytes. Lysis, as judged by release of the cytosolic enzyme lactate dehydrogenase, was preceded by depolarization of the plasma membrane and by Ca2+ influx. Both Na+ uptake (which sustained plasma membrane depolarization) and Ca2+ influx showed (1) the same dependence on the ATPo concentration; (2) the same nucleotide specificity; and (3) the same Hill coefficient. However, whereas the rise in the cytosolic free Ca2+ concentration ([Ca2+]i) was fully inhibited by the known Ca2+ blocker verapamil, plasma membrane depolarization was enhanced under these conditions. Plasma membrane depolarization was greater and was shifted to lower ATPo concentrations in the absence of extracellular Ca2+ (Ca2+o), whereas the rise in [Ca2+]i was greater in Na(+)-free media. Plasma membrane depolarization also occurred in Na(+)-free choline- or methylglucamine-containing media, and was potentiated by chelation of free divalent ions with EDTA, supporting previous reports pointing to ATP4-as the active species. Among a number of purine and pyrimidine nucleotides, only adenosine 5′-[gamma-thio]triphosphate and ADP were partially effective. Furthermore, ethidium bromide (Mr 380), Lucifer Yellow (Mr 463) and Eosin Yellowish (Mr 692) did not permeate through the ATPo-activated channel. These findings suggest that lytic effects of ATPo in mouse thymocytes depend on the activation of a membrane channel with low selectivity for cations and an Mr cut-off of 200.
Evidence for shared epitopes within the ‘naked’ protein domains of human mucus glycoproteins. A study performed by using polyclonal antibodies and electron microscopy
Authors: Sheehan J K , Boot-Handford R P , Chantler E , Carlstedt I , Thornton D J
Published: August 2015

Polyclonal antibodies were raised in rabbits towards reduced subunits of human cervical mucus glycoproteins. The reduced subunits almost completely inhibited the antiserum, whereas the intact mucins and the heavily glycosylated fragments obtained after digestion of reduced subunits with trypsin (T-domains) caused only partial inhibition. Periodate oxidation of intact mucins, reduced subunits and T-domains caused no effect on the antibody response, and fragments obtained by more extensive proteolysis of the reduced subunits (P-domains) showed no inhibitory activity. By using electron microscopy, antibodies from T-domain-adsorbed antisera were revealed as bound to cervical mucin reduced subunits, either directly or with colloidal gold-Protein A. Binding sites (100-150 nm apart) were observed at the ends and at internal positions of the reduced subunits. We conclude that the antibodies do not recognize carbohydrate structures but are directed to two kinds of protein epitopes, one shared by whole mucins, reduced subunits and T-domains, and the other specific to the reduced subunit fragment. The latter epitopes are ‘cryptic’ and are probably shielded within folded protein domains stabilized by disulphide bonds. Human bronchial, cervical, gastric and salivary mucus glycoproteins share some of these cryptic epitopes.
An acetylation method for the quantification of membrane lipids, including phospholipids, polyphosphoinositides and cholesterol
Authors: Stein J M , Smith G A , Luzio J P
Published: August 2015

A method for the quantification of membrane lipids by acetylation with [3H]acetic anhydride is described. A standard lipid, labelled with 14C or 32P, is added to the sample and is simultaneously acetylated. The 3H/14C or 3H/32P ratios obtained in the acetylated lipids are proportional to the initial lipid concentration. The method has been used to quantify cholesterol, phosphatidylethanolamine, phosphatidylinositol, sphingomyelin, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in amounts between 2 and 10 nmol.
Structure and biological activities of a new mastoparan isolated from the venom of the hornet Vespa basalis
Authors: Ho C L , Hwang L L
Published: August 2015

By gel filtration on a Fractogel TSK HW 50 column followed by cation-exchange chromatography on CM-Trisacryl M, a tetradecapeptide amide, designated ‘mastoparan B’, was purified from the venom of the hornet Vespa basalis. Its amino acid sequence was determined as: Leu-Lys-Leu-Lys-Ser-Ile-Val-Ser-Trp-Ala-Lys-Lys-Val-Leu-NH2 and its molecular mass was measured to be 1611 Da by fast-atom-bombardment mass spectrometry. In addition to having a common structure of vespid mastoparans, the peptide shows a less hydrophobic sequence at positions 1, 2, 5, 8 and 9. The peptide caused liberation of histamine from rat peritoneal mast cells and induced oedema in the rat paw. However, the latter effect was inhibited by ‘anti-serotonin’ (anti-5-hydroxytryptamine) (cyproheptadine), but not by antihistamine (chlorpheniramine). The peptide also possesses a potent haemolytic activity which acts in synergy with the lethal protein of the venom, suggesting the possible involvement of mastoparan B in the lethal effect of Vespa basalis venom.
Maintenance of structural and functional characteristics of skeletal-muscle mitochondria and sarcoplasmic-reticular membranes after chronic ethanol treatment
Authors: Cardellach F , Taraschi T F , Ellingson J S , Stubbs C D , Rubin E , Hoek J B
Published: August 2015

The effect of long-term ethanol intake on the structural and functional characteristics of rat skeletal-muscle mitochondria and sarcoplasmic reticulum was investigated. Functionally, skeletal-muscle mitochondria were characterized by a high respiratory control index and ADP/O ratio and a high State-3 respiration rate with different substrates. These parameters were not significantly different in preparations from control and ethanol-fed rats, except for a small increase in the rate of oxidation of alpha-oxoglutarate/malate in the latter. In submitochondrial particles from the two groups of animals there was no significant difference in cytochrome content, ATPase activity or the activity of respiratory-chain complexes. Mitochondrial membranes from untreated and ethanol-fed rats showed no difference in the baseline e.s.r. order parameter, and both preparations were equally sensitive to disordering by ethanol in vitro. Similarly, sarcoplasmic-reticulum preparations were not significantly affected by long-term ethanol feeding with respect to Ca2(+)-ATPase activity or in baseline order parameter and susceptibility to membrane disordering by ethanol in vitro. These membranes were also equally sensitive to degradation by exogenous phospholipase A2. Ethanol feeding did not alter the class composition of mitochondrial or sarcoplasmic-reticulum membrane phospholipids, nor the acyl composition of individual phospholipid classes. Specifically, the changes in acyl composition that characteristically occur in liver microsomal phosphatidylinositol and liver mitochondrial cardiolipin were not observed in the corresponding phospholipids from skeletal-muscle membranes. In experiments where membrane preparations from liver and skeletal muscle from the same ethanol-fed animals were compared, the liver membranes developed membrane tolerance, with the muscle membranes retaining normal sensitivity to disordering effects by ethanol. It is concluded that: (a) different tissues from the same animals differ in their susceptibility to ethanol; (b) the tissue-specific lack of development of membrane tolerance correlates with a lack of chemical changes in the phospholipids and with a retention of normal function of mitochondria and sarcoplasmic reticulum; (c) effects of chronic ethanol intake on muscle function are not due to a defect in the mitochondrial energy supply.
Albino mutants of Streptomyces glaucescens tyrosinase
Authors: Jackman M P , Hajnal A , Lerch K
Published: August 2015

Site-directed mutagenesis was used to determine the functional role of several residues of Streptomyces glaucescens tyrosinase. Replacement of His-37, -53, -193 or -215 by glutamine yields albino phenotypes, as determined by expression on melanin-indicator plates. The purified mutant proteins display no detectable oxy-enzyme and increased Cu lability at the binuclear active site. The carbonyl derivatives of H189Q and H193Q luminesce, with lambda max. displaced more than 25 nm to a longer wavelength compared with native tyrosinase. The remaining histidine mutants display no detectable luminescence. The results are consistent with these histidine residues (together with His-62 and His-189 reported earlier) acting as Cu ligands in the Streptomyces glaucescens enzyme. Conservative substitution of the invariant Asn-190 by glutamine also gives an albino phenotype, no detectable oxy-enzyme and labilization of active-site Cu. The luminescence spectrum of carbonyl-N190Q, however, closely resembles that of the native enzyme under conditions promoting double Cu occupancy of the catalytic site. A critical role for Asn-190 in active-site hydrogen-bonding interactions is proposed.
Characterization of inositol 1,4,5-trisphosphate- and inositol 1,3,4,5-tetrakisphosphate-binding sites in rat cerebellum
Authors: Challiss R A J , Willcocks A L , Mulloy B , Potter B V L , Nahorski S R
Published: August 2015

1. The properties of specific Ins(1,4,5)P3- and Ins(1,3,4,5)P4-binding sites have been compared in a crude ‘P2’ cerebellar membrane fraction. 2. A homogeneous population of [3H]Ins(1,4,5)P3-binding sites was present (KD 23.1 +/- 3.6 nM) at high density (Bmax. 11.9 +/- 1.8 pmol/mg of protein); whereas data obtained for [32P]Ins(1,3,4,5)P4 specific binding were best fitted to a two-site model, the high-affinity binding component (KD 2.6 +/- 0.7 nM) constituted 64.2 +/- 4.3% of the total population and was present at relatively low density (Bmax. 187 +/- 27 fmol/mg of protein). 3. The two high-affinity inositol polyphosphate-binding sites exhibited markedly different pH optima for radioligand binding, allowing the two sites to be independently investigated. At pH 8.0, [3H]Ins(1,4,5)P3 binding was maximal, whereas [32P]Ins(1,3,4,5)P4 specific binding was very low; conversely, at pH 5.0, [32P]Ins(1,3,4,5)P4 binding was maximal, whereas [3H]Ins(1,4,5)P3 binding was undetectably low. 4. Both inositol polyphosphate-binding sites exhibited marked positional and stereo-specificity. Of the analogues studied, only phosphorothioate substitution to form inositol 1,4,5-trisphosphorothioate was tolerated at the Ins(1,4,5)P3-binding site, with only a 2-3-fold loss of binding activity. Addition of a glyceroyl moiety at the 1-phosphate position or addition of further phosphate substituents at the 3- or 6-positions caused dramatic losses in displacing activity. Similarly, complete phosphorothioate substitution of Ins(1,3,4,5)P4 caused an approx. 6-fold loss of binding activity at the [32P]Ins(1,3,4,5)P4-binding site, whereas Ins(1,4,5,6)P4, Ins(1,3,4,6)P4, Ins(1,4,5)P3 and Ins(1,3,4,5,6)P5 were bound at least 100-fold weaker at this site. Therefore, only the phosphorothioate derivatives retained high affinity and selectivity for the two inositol polyphosphate-binding sites. 5. Heparin and pentosan polysulphate were potent but non-selective inhibitors at Ins(1,4,5)P3- and Ins(1,3,4,5)P4-binding sites. N-Desulphation (with or without N-reacetylation) of heparin decreased inhibitory activity at the Ins(1,4,5)P3-, but not at the Ins(1,3,4,5)P4-binding site; however, the selectivity of this effect was only about 4-fold. O- and N-desulphated N-reacetylated heparin was essentially inactive at both sites. 6. The results are discussed with respect to the separate identities of the inositol polyphosphate-binding sites.
Angiotensin II-induced phosphatidylcholine hydrolysis in cultured vascular smooth-muscle cells. Regulation and localization
Authors: Lassègue B , Alexander R W , Clark M , Griendling K K
Published: August 2015

In cultured vascular smooth-muscle cells (VSMC), angiotensin II (AngII) induces a biphasic, sustained increase in diacylglycerol (DG) of unclear origin. To determine whether hydrolysis of phosphatidylcholine (PC) is a possible source of DG, we labelled cellular PC with [3H]choline, and measured the formation of intra- and extra-cellular [3H]choline and [3H]phosphocholine after stimulation with AngII. AngII induced a concentration-dependent release of choline from VSMC that was significant at 2 min and was sustained over 20 min. In contrast, accumulation of choline inside the cells was very slight. AngII also increased the formation of [3H]myristate-labelled phosphatidic acid, and, in the presence of ethanol, of [3H]phosphatidylethanol, characteristic of a phospholipase D (PLD) activity. Extracellular release of choline was partially inhibited by removal of extracellular Ca2+ (54 +/- 9% inhibition at 10 min) or inhibition of receptor processing by phenylarsine oxide (79 +/- 8% inhibition at 20 min). The protein kinase C activator phorbol myristate acetate also stimulated a large release of choline after a 5 min lag, which was unaffected by the Ca2+ ionophore ionomycin, but was additive with AngII stimulation. Down-regulation of protein kinase C by a 24 h incubation with phorbol dibutyrate (200 nM) decreased basal choline release, but had no effect on AngII stimulation. We conclude that AngII induces a major PC hydrolysis, probably mainly via PLD activation. This reaction is partially dependent on Ca2+ and is independent of protein kinase C, and appears to be mediated by cellular processing of the receptor-agonist complex. Our results are consistent with a preferential hydrolysis of PC from the external leaflet of the plasmalemma, and raise the possibility that PC hydrolysis occurs in specialized ‘signalling domains’ in VSMC.
Characterization of high-affinity ryanodine-binding sites of rat liver endoplasmic reticulum. Differences between liver and skeletal muscle
Authors: Shoshan-Barmatz V , Pressley T A , Higham S , Kraus-Friedmann N
Published: August 2015

In this study, the binding of [3H]ryanodine to liver microsomal subfractions was investigated. The specific binding of [3H]ryanodine, as determined both by vacuum filtration and by ultracentrifugation, is to a single class of high-affinity binding sites with a Kd of 10 +/- 2.5 nM and density of 500 +/- 100 and 1200 +/- 200 fmol/mg of protein by the filtration and centrifugation methods respectively. [3H]Ryanodine binding reached equilibrium in about 1 min and 2 min at 36 degrees C and 24 degrees C respectively, and the half-time of dissociation at 37 degrees C was approx. 15 s. The binding of [3H]ryanodine is Ca(2+)-independent: it is slightly stimulated by NaCl, Mg2+, ATP and InsP3 but strongly inhibited by caffeine, diltiazem and sodium dantrolene. Thus the binding of ryanodine to endoplasmic reticulum membranes shares some of the characteristics of its binding to the sarcoplasmic reticulum but also differs from it in several important properties, such as its Ca(2+)-independence, its rapid association and dissociation, and its inhibition by caffeine. The structural similarities between the skeletal muscle and liver binding sites were further explored by employing in vitro DNA amplification techniques, using the known sequence of the skeletal muscle receptor as reference point. The data obtained with this method indicate that the liver does not process mRNA for the skeletal muscle ryanodine receptor.
Purification of recombinant human prostromelysin. Studies on heat activation to give high-Mr and low-Mr active forms, and a comparison of recombinant with natural stromelysin activities
Authors: Koklitis P A , Murphy G , Sutton C , Angal S
Published: August 2015

Recombinant human prostromelysin was purified in a single step using Procion Red-Sepharose chromatography. The purified prostromelysin was self-activated to high-Mr (45,000) and low-Mr (28,000) forms by incubation at 55 degrees C without the addition of extraneous activators. The two forms of stromelysin were subsequently separated, again using Procion Red-Sepharose. Both of the heat-activated recombinant forms demonstrated similar specific activities (for the macromolecular substrates casein, gelatin, elastin, proteoglycan and type IV collagen) when compared with either heat- or trypsin-activated natural stromelysin. The heat-activated recombinant stromelysins both showed similar abilities to potentiate activation of human procollagenase when compared with trypsin-activated natural stromelysin.
Purification and characterization of human brain prolyl endopeptidase
Authors: Kalwant S , Porter A G
Published: August 2015

Prolyl endopeptidase (EC 3.4.21.26) was purified from human brain by a series of column-chromatographic steps using DEAE-cellulose DE-52, hydroxyapatite, phenyl-Sepharose, Sephacryl S-200 and f.p.l.c. (Mono Q). The enzyme was purified by a factor of 943 and was homogeneous in a SDS/polyacrylamide gel as judged by Coomassie Blue staining. The Mr estimated by SDS/PAGE is 79,600, and under native conditions on Sephacryl S-200 it is 85,600. Therefore the enzyme exists as a monomer. With benzyloxycarbonylglycylproline p-nitroanilide as substrate, the optimum pH of the enzyme is 6.8, and with the substrate concentration between 0.059 mM and 0.37 mM the Km is 9.0 x 10(-4) M. The pI of the enzyme is 4.75. The enzyme is classified as a serine proteinase, as it is strongly inhibited by di-isopropyl fluorophosphate. However, other serine proteinase inhibitors do not inhibit the enzyme significantly, suggesting that the active site of prolyl endopeptidase differs from that of classical serine proteinases such as trypsin. Polyclonal antibodies were raised against purified human brain prolyl endopeptidase in rabbits. Western-blot analysis, enzyme-inhibition assays, antibody binding and immunoprecipitation experiments indicated that the polyclonal antibodies are both specific and inhibitory to the enzyme activity.
Human leucocyte aspartylglucosaminidase. Evidence for two different subunits in a more complex native structure
Authors: Halila R , Baumann M , Ikonen E , Enomaa N , Peltonen L
Published: August 2015

Human leucocyte aspartylglucosaminidase (AGA: 1-aspartamido-beta-N-acetylglucosamine amidohydrolase, EC 3.5.1.26) was purified to homogeneity by using affinity chromatography, gel filtration, chromatofocusing and reverse-phase h.p.l.c. As shown by SDS/PAGE, the homogeneous purified enzyme preparation consists of four polypeptide chains with molecular masses of 25, 24, 18 and 17 kDa. In the native polyacrylamide gel these polypeptides migrate as one active enzyme complex, and by gel filtration the peak of enzyme activity can be detected in a position of about 65 kDa. Digestion with endoproteinase Lys-C or endoproteinase Asp-N, followed by peptide analysis with reverse-phase h.p.l.c., reveals an identical peptide pattern for the 24 and 25 kDa bands as well as for the 17 and 18 kDa bands. This treatment further demonstrated a totally different peptide pattern for the 24/25 kDa versus the 17/18 kDa subunit. The N-terminal sequences of the 17 kDa and the 18 kDa peptides were identical, as determined by Edman degradation. The N-termini of the 24 kDa and the 25 kDa peptides were blocked. The enzyme was partly resistant to endoglycosidases H and F, but N-glycosidase F transformed the 24/25 kDa band into one 23 kDa band and the 17/18 kDa band into one 16 kDa band. Also, immunological data obtained with antisera produced against these subunits showed that AGA consists of two non-identical polypeptides.
The regulatory domain of protein kinase C-ε restricts the catalytic-domain-specificity
Authors: Pears C , Schaap D , Parker P J
Published: August 2015

Protein kinase C (PKC) consists of a family of closely related enzymes that can be divided into two subfamilies (alpha, beta and gamma and delta, epsilon and zeta) on the basis of primary sequence. Functional differences have also been described; thus PKC-alpha, PKC-beta and PKC-gamma readily phosphorylate histone IIIS in vitro, whereas PKC-epsilon will not employ this substrate efficiently. We have previously demonstrated, however, that proteolytic cleavage of PKC-epsilon generates a constitutive kinase activity that is an efficient histone IIIS kinase [Schaap, Hsuan, Totty & Parker (1990) Eur. J. Biochem. 191, 431-435]. In order to investigate the structural basis for this switch in specificity, we have constructed a chimaeric protein containing the regulatory domain of PKC-epsilon fused to the catalytic domain of PKC-gamma. When this is expressed in COS1 cells the chimaeric kinase shows a substrate-specificity similar to that of PKC-epsilon rather than to that of PKC-gamma. This demonstrates a role for the regulatory domain in substrate selection of PKC-epsilon.
Hypomethylation of the decorin proteoglycan gene in human colon cancer
Authors: Adany R , Iozzo R V
Published: August 2015

We have previously reported that the connective tissue stroma of human colon carcinoma contains elevated amounts of decorin, a small proteoglycan involved in the regulation of matrix formation and cell proliferation. These biochemical changes were correlated with increased mRNA levels and general hypomethylation of the decorin gene in human colon cancer DNA. In this report we use a quantitative polymerase chain reaction method coupled with digestion of the DNA template by methylation-sensitive restriction endonucleases to investigate in detail the location of hypomethylated sites in decorin gene. We demonstrate that a specific site in the 3′ region of the gene, encompassing codons 360-361, is specifically hypomethylated in both colon carcinoma and benign polyp. In contrast, three HpaII sites, clustered in the 5′ untranslated region, show full methylation in normal and neoplastic DNA. The lack of such changes in ulcerative colitis DNA suggests that chronic inflammation alone is not sufficient to alter cytosine methylation in the decorin gene. These results suggest the possibility that the 3′ region of the decorin-coding sequence may be involved in the control of decorin gene expression.
Human sputum cathepsin B degrades proteoglycan, is inhibited by α2-macroglobulin and is modulated by neutrophil elastase cleavage of cathepsin B precursor and cystatin C
Authors: Buttle D J , Abrahamson M , Burnett D , Mort J S , Barrett A J , Dando P M , Hill S L
Published: August 2015

The high-Mr alkali-stable form of cathepsin B was purified from purulent human sputum. It was shown to solubilize proteoglycan monomer entrapped in polyacrylamide at a rate comparable with that of human lysosomal cathepsin B. Like the enzyme from lysosomes, sputum cathepsin B was bound by human alpha 2-macroglobulin, which inhibited its action on proteoglycan. Cystatin C in purulent sputum was shown to be the N-terminally truncated form generated by neutrophil elastase cleavage, and sputum cathepsin B was only weakly inhibited by recombinant cystatin C that had been cleaved by neutrophil elastase in vitro. Addition of neutrophil elastase to mucoid sputum led to a 5-fold increase in cathepsin B activity concomitant with a lowering in Mr of the cysteine proteinase from 40,000 to 37,000, i.e. the size of the active enzyme purified from purulent sputum. It is concluded that the high-Mr form of cathepsin B present in purulent sputum is a functional proteinase, unlike similar forms of the enzyme secreted by mammary gland in organ culture. The activity of cathepsin B in sputum is modulated by neutrophil elastase, by a combination of inhibitor inactivation and zymogen activation.