bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphodiesterase activity is a novel property of alkaline phosphatase from osseous plate
Authors: Rezende A A , Pizauro J M , Ciancaglini P , Leone F A
Published: August 2015

Phosphodiesterase activity is a novel property of the still-enigmatic alkaline phosphatase from osseous plate. Bis-(p-nitrophenyl) phosphate was hydrolysed at both pH 7.5 and 9.4 with an apparent dissociation constant (K0.5) of 1.9 mM and 3.9 mM respectively. The hydrolysis of p-nitrophenyl-5′-thymidine phosphate followed hyberbolic kinetics with a K0.5 of 500 microM. For p-nitrophenyl phenylphosphonate, site-site interactions [Hill coefficient (h) = 1.3] were observed in the range between 0.2 and 100 microM, and K0.5 was 32.8 mM. The hydrolysis of cyclic AMP by the enzyme followed more complex kinetics, showing site-site interactions (h = 1.7) and K0.5 = 300 microM for high-affinity sites. The low-affinity sites, representing 85% of total activity, also showed site-site interactions (h = 3.8) and a K0.5 of about 22 mM. ATP and cyclic AMP were competitive inhibitors of bis-(p-nitrophenyl) phosphatase activity of the enzyme and Ki values (25 mM and 0.6 mM for cyclic AMP and ATP respectively) very close to those of the K0.5 (22 mM and 0.7 mM for cyclic AMP and ATP respectively), determined by direct assay, indicated that a single catalytic site was responsible for the hydrolysis of both substrates. Non-denaturing PAGE of detergent-solubilized enzyme showed coincident bands on the gel for phosphomonohydrolase and phosphodiesterase activities. Additional evidence for a single catalytic site was the similar pKa values (8.5 and 9.7) found for the two ionizing groups participating in the hydrolysis of bis-(p-nitrophenyl) phosphate and p-nitrophenyl phosphate. The alkaline apparent pH optima, the requirement for bivalent metal ions and the inhibition by methylxanthines, amrinone and amiloride demonstrated that rat osseous-plate alkaline phosphatase was a type I phosphodiesterase. Considering that there is still confusion as to which is the physiological substrate for the enzyme, the present results describing a novel property for this enzyme could be of relevance in understanding the mineralization process.
The binding of lysophospholipids to rat liver fatty acid-binding protein and albumin
Authors: Thumser A E , Voysey J E , Wilton D C
Published: August 2015

The binding of lysophospholipids to rat liver fatty acid-binding protein (FABP) and to BSA and human serum albumin was investigated by using competitive displacement fluorescence assays by monitoring the displacement of the fluorescent fatty acid probe 11-(dansylamino)undecanoic acid (DAUDA). In addition, direct binding assays using changes in tryptophan fluorescence were possible with albumin. Liver FABP was able to bind a range of lysophospholipids, oleoyl-lysophosphatidic acid (lysoPA), oleoyl-lysophosphatidylcholine (lysoPC), oleoyl-lysophosphatidylethanolamine (lysoPE) and oleoyl-lysophosphatidylglycerol, with similar affinity and a Kd of about 1 microM. Liver FABP was also able to bind lysophospholipids generated by the action of phospholipase A2 or phospholipase A1 (triacylglycerol lipase) on phospholipid vesicles. A possible physiological role for liver FABP in lysophospholipid metabolism within the cell is discussed. Albumin was shown to bind lysoPA with higher affinity than either lysoPC or lysoPE, and the initial minimal DAUDA displacement by lysoPA indicated that lysoPA was binding to the primary high-affinity fatty acid-binding sites on albumin and that, like oleic acid, about 3 mol of ligand/mol was bound to these sites. Kd values in the microM range were indicated for lysoPC and lysoPE, whereas, by comparison with oleic acid, the Kd for lysoPA was significantly lower and high-affinity binding in the nM range was indicated. Overall, the data suggest that, because of structural similarity, lysoPA binds to albumin in a similar manner to long-chain fatty acids.
Estimation of peroxisomal β-oxidation in rat heart by a direct assay of acyl-CoA oxidase
Authors: Chu C , Mao L F , Schulz H
Published: August 2015

The contribution of peroxisomes to palmitate beta-oxidation in rat heart was estimated by either inhibiting mitochondrial beta-oxidation or measuring the activity of acyl-CoA oxidase. When respiratory inhibitors such as KCN or antimycin plus rotenone, or inhibitors of mitochondrial fatty acid uptake such as 2-tetradecylglycidic acid or 2-bromopalmitate, were used, degrees of inhibitions ranging from 24% to 87% were observed for palmitate beta-oxidation by a rat heart homogenate. Although the oxidation of palmitoyl-L-carnitine by coupled rat heart mitochondria was almost completely (94%) inhibited by KCN, the inhibition by antimycin plus rotenone was incomplete (77%) and was stimulated by L-carnitine. A direct assay of acyl-CoA oxidase, based on the spectrophotometric measurement at 300 nm of 2,4-decadienoyl-CoA formation from 4-trans-decenoyl-CoA, was evaluated with the aim of obtaining reliable values for the activity of this enzyme, which is presumed to catalyse the rate-limiting step of peroxisomal beta-oxidation. Activities determined by use of this assay were much higher than activities obtained by a coupled assay [Small, Burdett and Connock (1985) Biochem. J. 227, 205-210] commonly used to measure the activity of acyl-CoA oxidase. However, both methods yielded the same relative activities with different tissue homogenates. Based on an estimated palmitoyl-CoA oxidase activity of 0.3 nmol/min per mg of protein, the contribution of peroxisomes to palmitate beta-oxidation in a rat heart homogenate would optimally be 4%, and most likely is several-fold lower.
Inhibition of L-type calcium-channel activity by thapsigargin and 2,5-t-butylhydroquinone, but not by cyclopiazonic acid
Authors: Nelson E J , Li C C R , Bangalore R , Benson T , Kass R S , Hinkle P M
Published: August 2015

Thapsigargin (TG), 2,5-t-butylhydroquinone (tBHQ) and cyclopiazonic acid (CPA) all inhibit the initial Ca(2+)-response to thyrotropin-releasing hormone (TRH) by depleting intracellular Ca2+ pools sensitive to inositol 1,4,5-trisphosphate (IP3). Treatment of GH3 pituitary cells for 30 min with 5 nM TG, 500 nM tBHQ or 50 nM CPA completely eliminated the TRH-induced spike in intracellular free Ca2+ ([Ca2+]i). Higher concentrations of TG and tBHQ, but not CPA, were also found to inhibit strongly the activity of L-type calcium channels, as measured by the increase in [Ca2+]i or 45Ca2+ influx stimulated by depolarization. TG and tBHQ blocked high-K(+)-stimulated 45Ca2+ uptake, with IC50 values of 10 and 1 microM respectively. Maximal inhibition of L-channel activity was achieved 15-30 min after drug addition. Inhibition by tBHQ was reversible, whereas inhibition by TG was not. TG and CPA did not affect spontaneous [Ca2+]i oscillations when tested at concentrations adequate to deplete the IP3-sensitive Ca2+ pool. However, 20 microM TG and 10 microM tBHQ blocked [Ca2+]i oscillations completely. The effect of drugs on calcium currents was measured directly by using the patch-clamp technique. When added to the external bath, 10 microM CPA caused a sustained increase in the calcium-channel current amplitude over 8 min, 10 microM tBHQ caused a progressive inhibition, and 10 microM TG caused an enhancement followed by a sustained block of the calcium current over 8 min. In summary, CPA depletes IP3-sensitive Ca2+ stores and does not inhibit voltage-operated calcium channels. At sufficiently low concentrations, TG depletes IP3-sensitive stores without inhibiting L-channel activity, but, for tBHQ, inhibition of calcium channels occurs at concentrations close to those needed to block agonist mobilization of intracellular Ca2+.
Rapid heparin-sensitive Ca2+ release following Ca2+-ATPase inhibition in intact HL-60 granulocytes. Evidence for Ins(1,4,5)P3-dependent Ca2+ cycling across the membrane of Ca2+ stores
Authors: Favre C J , Lew D P , Krause K H
Published: August 2015

In many cell types, emptying of intracellular Ca2+ stores after application of inhibitors of the intracellular Ca(2+)-ATPase (e.g. thapsigargin) is astonishingly rapid. It was the aim of this study to elucidate the underlying mechanism. We first compared thapsigargin-induced emptying of intracellular Ca2+ stores in intact and homogenized HL-60 granulocytes. Thapsigargin-induced Ca2+ release was rapid in intact cells (33.9 +/- 4.9% of store content/min), but it was slow in permeabilized or homogenized cells (7.7 +/- 3.9 and 12 +/- 3.8% of store content/min respectively). To study whether the Ins(1,4,5)P3 receptor might be involved in thapsigargin-induced Ca2+ release, we tested the effect of heparin, a competitive Ins(1,4,5)P3 antagonist. In homogenized and permeabilized preparations, heparin did not interfere with thapsigargin-induced Ca2+ release. In contrast, when introduced into intact cells by an endocytosis/osmotic-shock procedure, heparin, but not the inactive de-N-sulphated heparin, decreased the rate of Ca2+ release by approx. 70%. Heparin inhibited Ca2+ release in response to the Ins(1,4,5)P3-generating receptor agonist N-formylmethionyl-leucyl-phenylalanine (f-MLP) (50 nM) and to thapsigargin (50 nM) at comparable concentrations. Heparin inhibition was competitive for f-MLP-induced, but not for thapsigargin-induced, Ca2+ release. In permeabilized cells, the addition of low Ins(1,4,5)P3 concentrations before thapsigargin increased the rate of thapsigargin-induced Ca2+ release 4-fold. Taken together, our results suggest that the rapid Ca(2+)-ATPase-inhibitor-induced Ca2+ release is due to a partial activation of the Ins(1,4,5)P3 receptor in resting cells. This implies Ca2+ cycling across the membrane of Ins(1,4,5)P3-sensitive Ca2+ stores in resting cells.
Modulation of crystal formation by bone phosphoproteins: role of glutamic acid-rich sequences in the nucleation of hydroxyapatite by bone sialoprotein
Authors: Hunter G K , Goldberg H A
Published: August 2015

Bone sialoprotein (BSP) is a bone-specific glycoprotein containing phosphoserine and sulphotyrosine residues and regions of contiguous glutamic acid residues. Recent studies in this laboratory have shown that BSP is capable of nucleating the bone mineral hydroxyapatite in a steady-state agarose gel system. We show here that chemical modification of carboxylate groups abolishes the nucleation activity of BSP, but enzymic dephosphorylation has no effect. Formation of hydroxyapatite is also induced by poly(L-glutamic acid) and poly(D-glutamic acid), but not by poly(L-aspartic acid) or poly(L-lysine). Calreticulin, a muscle protein with short sequences of contiguous glutamic acid residues, also lacks nucleation activity. These findings suggest that the nucleation of hydroxyapatite by BSP involves one or both of the glutamic acid-rich sequences. Based on these findings and others, we propose that polycarboxylate sequences represent a general site for growth-modulating interactions between proteins and biological crystals.
Oxidation of low-density lipoprotein by hypochlorite causes aggregation that is mediated by modification of lysine residues rather than lipid oxidation
Authors: Hazell L J , van den Berg J J M , Stocker R
Published: August 2015

Peroxidation of low-density lipoprotein (LDL) lipid is generally thought to represent the initial step in a series of modification reactions that ultimately transform the protein moiety of the lipoprotein into a form recognized by receptors different from those that bind native LDL. Uptake of LDL via these alternative receptors can lead to the formation of lipid-laden cells, which are typical for the early stages of atherogenesis. We have studied the oxidative modification of LDL by hypochlorite (-OCl), a powerful oxidant produced from H2O2 and chloride via the action of myeloperoxidase which is released from activated neutrophils and monocytes. Exposure of LDL to reagent or enzymically generated -OCl at 4 or 37 degrees C resulted in immediate and preferential oxidation of amino acid residues of apolipoprotein B-100, the single protein associated with LDL. Lysine residues quantitatively represented the major target and, like tryptophan, were oxidized to approximately the same extent with reagent or enzymically generated -OCl. In contrast, LDL lipid oxidation was less favoured than protein oxidation, as judged by the amounts of lipid hydroperoxides, chlorohydrins, cholesterol or fatty acid oxidation products formed. Treatment with -OCl caused aggregation of LDL, as shown by an increased turbidity of the oxidized LDL solution and elution from a size-exclusion h.p.l.c. column of high-molecular-mass LDL complexes. Chemical modification of lysine residues before oxidation with -OCl prevented aggregation, while it enhanced the extent of lipid peroxidation. Treatment of LDL with -OCl also caused the formation of carbonyl groups and release of ammonia; both these modifications were inhibited by lysine-residue modification before oxidation. These results demonstrate that aggregation reactions are dependent on initial lysine oxidation by -OCl, followed by deamination and carbonyl formation, but do not involve lipid (per)oxidation. We propose that the observed -OCl-mediated aggregation of LDL is caused, at least in part, by cross-linking of apoproteins by Schiff-base formation independently of lipid peroxidation.
Recruitment of mitochondrial cyclophilin to the mitochondrial inner membrane under conditions of oxidative stress that enhance the opening of a calcium-sensitive non-specific channel
Authors: Connern C P , Halestrap A P
Published: August 2015

Binding of mitochondrial matrix cyclophilin (CyP) to the rat liver mitochondrial membranes was detected by SDS/PAGE and Western blotting with suitable antipeptide antibodies. Binding was not affected by prior exposure of mitochondria to Ca2+, adenine nucleotides or inhibitors of the adenine nucleotide translocase, but was greatly increased by t-butyl hydroperoxide (tBH), phenylarsine oxide or diamide. These all sensitized the opening of the non-specific mitochondrial pore to [Ca2+], and the effect of tBH was shown to be maintained after washing away the tBH, consistent with it being caused by the enhanced CyP binding. The bound CyP did not demonstrate peptidyl-prolyl cis-trans isomerase activity. CyP-binding was prevented by 5 microM cyclosporin A, but not reversed by cyclosporin treatment of the membranes. The effect of tBH on binding was concentration-dependent and maximal within 30 s.
The ether lipid 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine induces expression of fos and jun proto-oncogenes and activates AP-1 transcription factor in human leukaemic cells
Authors: Mollinedo F , Gajate C , Modolell M
Published: August 2015

The ether lipid analogue 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) has been recently shown to induce apoptosis in the human leukaemic HL-60 and U937 myeloid cell lines [Mollinedo, Martinez-Dalmau and Modolell (1993) Biochem. Biophys. Res. Commun. 192, 603-609]. We have found that ET-18-OCH3 is also able to promote apoptosis in the human leukaemic Jurkat T lymphoid cell line. This lymphoid cell line as well as the two myeloid HL-60 and U937 cell lines incorporated significant amounts of exogenously added radiolabelled ET-18-OCH3. Addition of ET-18-OCH3 to these human leukaemic cells induced an increase in the steady-state mRNA levels of fos and jun proto-oncogenes, components of the transcription factor AP-1. These increases in fos and jun mRNA levels were associated with the activation of the AP-1 transcription factor after addition of ET-18-OCH3 to human leukaemic cells, as assessed by an enhanced binding activity of transcription factor AP-1 to its cognate DNA sequence as well as by stimulation of transcription from an AP-1 enhancer element. These data demonstrate that the ether lipid ET-18-OCH3 can affect gene expression by inducing expression of fos and jun proto-oncogenes and by modulating the activity of transcription factor AP-1.
Arylamine N-acetyltransferase in Balb/c mice: identification of a novel mouse isoenzyme by cloning and expression in vitro
Authors: Kelly S L , Sim E
Published: August 2015

Three genes encoding arylamine N-acetyltransferase were identified in Balb/c mice. All three genes were cloned from genomic DNA, sequenced and expressed in a bacterial expression system. Two of the genes corresponded to Nat-1 and Nat-2 which have been previously identified in A/J and C57B1/6 strains of mice (Martell et al., 1991). The new gene, designated Nat-3, can be distinguished from the other mouse Nat genes both by specific amplification using PCR and by restriction-endonuclease digestion. The products of all three genes are demonstrated to catalyse acetylation of aminofluorene and anisidine following expression in Escherichia coli.
13C-n.m.r. of the cyanylated β-lactoglobulins: evidence that Cys-121 provides the thiol group of β-lactoglobulins A and B
Authors: Phelan P , Malthouse J P
Published: August 2015

The thiol groups of beta-lactoglobulins A and B have been cyanylated using [13C]KCN. The samples of [cyanato-13C]-cyanylated-beta-lactoglobulins A and B which we prepared had signals at 109.7 p.p.m. and 114.4 p.p.m. We conclude that the thiocyanate carbon having a chemical shift of 109.7 p.p.m. is in an apolar environment similar to a cyclohexane solvent, whereas the thiocyanate carbon having a chemical shift of 114.4 p.p.m. is in a polar environment similar to water. The signals with chemical shifts of 109.7 p.p.m. are assigned to the thiocyanate carbons of the native [cyanato-13C]cyanylated-beta-lactoglobulins A and B. We deduce that the signal at 114.4 p.p.m. is due to an irreversibly denatured/unfolded species produced by alkaline denaturation, which is caused by intramolecular thiol/disulphide exchange occurring during our cyanylation procedure. We propose that Cys-119 is cyanylated in the irreversibly denatured species and Cys-121 is cyanylated in the native [cyanato-13C]cyanylated-beta-lactoglobulins A and B. We suggest that the same intramolecular thiol-disulphide exchange reactions occurred when McKenzie and co-workers [McKenzie, Ralston and Shaw (1972) Biochemistry 11, 4539-4547] alkylated beta-lactoglobulins with iodoacetamide. Therefore the one mol of thiol/mol of monomer in the native beta-lactoglobulins is due to the thiol of Cys-121 and is not due to an equimolar mixture of Cys-119 and Cys-121 as they suggested.
Relative specificities of a series of β-lactam-recognizing enzymes towards the side-chains of penicillins and of acyclic thioldepsipeptides
Authors: Xu Y , Soto G , Adachi H , van der Linden M P , Keck W , Pratt R F
Published: August 2015

In an attempt to understand more of the subtle differences between bacterial beta-lactamases and DD-peptidases, comparisons have been made between the specificities of these enzymes towards the phenylacetyl side chain, generally thought to be favoured by beta-lactamases, and the NN'-diacetyl-L-lysyl side chain, widely employed in low-molecular-mass substrates of DD-peptidases. These comparisons were carried out with both a penicillin and an acyclic thioldepsipeptide reaction nucleus and employing a range of both beta-lactamases and DD-peptidases. Rather contrary to general expectations, a general preference for reaction of both groups of enzymes with penicillins rather than thioldepsipeptides was observed and for the phenylacetyl rather than the NN'-diacetyl-L-lysyl side chain. Quantitative comparisons suggested that the side chains of penicillins may be bound in relatively similar sites in all of the enzymes whereas the side chains of thioldepsipeptides are more heterogeneously bound, both with respect to each other and to the comparable side chains of penicillins.
Wortmannin inhibits the effects of insulin and serum on the activities of glycogen synthase kinase-3 and mitogen-activated protein kinase
Authors: Welsh G I , Foulstone E J , Young S W , Tavaré J M , Proud C G
Published: August 2015

We have previously shown that insulin causes inactivation of glycogen synthase kinase-3 (GSK-3) in Chinese hamster ovary cells over-expressing the human insulin receptor (CHO.T cells). We now show that serum and phorbol ester also cause rapid inactivation of GSK-3, both in CHO.T cells and in the nontransfected parental cell line, CHO.K1 cells. Rapamycin was without effect on the inactivation of GSK-3 by insulin, serum or phorbol ester, indicating that the p70 S6 kinase pathway is not involved. In contrast, wortmannin, a potent inhibitor of phosphatidylinositol 3-kinase, blocked the effects of both insulin and serum on GSK-3 activity, and also substantially reduced the activation of both p90 S6 kinase (by insulin) and mitogen-activated protein (MAP) kinase (by insulin and serum). These findings imply (i) that GSK-3 activity is regulated by a cascade involving MAP kinase and p90 S6 kinase and (ii) that wortmannin affects an early step in the MAP kinase pathway. One can infer from this that GSK-3 may be an important regulatory enzyme for the control of several biosynthetic pathways, key enzymes in which are regulated by GSK-3-mediated phosphorylation. Wortmannin had a smaller effect on the activation of MAP kinase by phorbol ester, indicating that phorbol esters may stimulate MAP kinase by a different or additional mechanism to that employed by insulin or serum. Wortmannin had very little effect on the inactivation of GSK-3 by phorbol ester: possible reasons for this are discussed.
Chemical modification of Penicillium 1,2-α-d-mannosidase by water-soluble carbodi-imide: identification of a catalytically important aspartic acid residue
Authors: Yoshida T , Maeda K , Kobayashi M , Ichishima E
Published: August 2015

1,2-alpha-D-Mannosidase from Penicillium citrinum was inactivated by chemical modification with 1-ethyl-3-(3-dimethylamino-propyl)carbodi-imide (EDC). Most of the activity was lost after modification in the absence of a nucleophile, glycine ethyl ester. 1-Deoxymannojirimycin (dMM), a competitive inhibitor of the enzyme, showed partial protection against the inactivation. After the modification by EDC without the presence of a nucleophile, proteolytic digests of the enzyme were analysed by reversed-phase h.p.l.c. and a unique peptide was shown to decrease when dMM was present during the modification. The peptide was absent from the digests of unmodified enzyme. The amino acid sequence of the peptide (A; Ile-Gly-Pro) was identical in part with that of the adjacent peptide (B; Ile-Gly-Pro-Asp-Ser-Trp-Gly-Trp-Asp-Pro-Lys). When cholecystokinin tetrapeptide (Trp-Met-Asp-Phe-NH2) was modified by EDC alone, the modified peptide could be separated from unmodified peptide by reversed-phase h.p.i.c., and Edman degradation was stopped before the modified aspartic acid residue. This suggested that, in the enzyme, peptide A was derived from peptide B by the modification. Consequently, Asp-4 in peptide B was assumed to be masked by dMM during the modification, and to be involved in the interaction of the enzyme with its substrate.
Characterization of monoclonal antibodies against Naja naja oxiana neurotoxin I
Authors: Stiles B G , Sexton F W , Guest S B , Olson M A , Hack D C
Published: August 2015

Seven monoclonal antibodies (mAbs) were developed against neurotoxin I (NT-1), a protein from central Asian cobra (Naja naja oxiana) venom which binds specifically to nicotinic acetylcholine receptor (AchR). All of the mAbs cross-reacted with another long-chain post-synaptic neurotoxin, Bungarus multicinctus alpha-bungarotoxin (alpha-BT), but not Naja naja kaouthia alpha-cobratoxin, in an enzyme-linked immunosorbent assay (e.l.i.s.a.). Short-chain post-synaptic neurotoxins like Naja naja atra cobrotoxin, Laticauda semifasciata erabutoxin b, or N. n. oxiana neurotoxin II did not cross-react with the NT-1 mAbs, but an antigen(s) found in Dendroaspis polylepis, Acanthophis antarcticus and Pseudechis australis venoms was immunoreactive. The e.l.i.s.a. readings for dithiothreitol-reduced NT-1 and NT-1 mAbs ranged from 13 to 27% of those for native toxin but reduced alpha-BT was not immunoreactive. Synthetic NT-1 peptides were used in epitope-mapping studies and two, non-contiguous regions (Cys15-Tyr23 and Lys25-Gly33 or Pro17-Lys25 and Asp29-Lys37) were recognized by the NT-1 mAbs. The NT-1 mAbs individually inhibited 31-71% of alpha-BT binding to AchR in vitro and afforded a slight protective effect in vivo with a toxin: antibody mole ratio of 1:1.5. This report is the first to describe mAbs which recognize and protect against a heterologous, long-chain, post-synaptic neurotoxin from snake venom.
The non-phospholipase A2 subunit of β-bungarotoxin plays an important role in the phospholipase A2-independent neurotoxic effect: characterization of three isotoxins with a common phospholipase A2 subunit
Authors: Chu C C , Chu S T , Chen S W , Chen Y H
Published: August 2015

Three isotoxins (SP I-III) of the beta-bungarotoxin family were purified to homogeneity via a series of isolation procedures including a final step of h.p.l.c. on an SP column washed with a linear gradient of 0.2-0.6 M sodium acetate at pH 7.4. Their proportions varied greatly with the batch of venom. Each isotoxin was demonstrated by SDS/PAGE to contain a phospholipase A2 subunit and a non-phospholipase A2 subunit. The three proteins were reductively alkylated with 4-vinylpyridine and the alkylated derivatives of the two subunits of each isotoxin were separated. N-Terminal sequence analysis of the alkylated derivatives revealed that the three isotoxins probably share a common phospholipase A2 subunit but differ in their non-phospholipase A2 subunits. The non-phospholipase A2 subunits of SP II and SP III were identical with those of beta 2- and beta 1-toxin respectively, except that there was an additional valine inserted between Thr-18 and Val-19 in beta 2-toxin and Pro-18 and Val-19 in beta 1-toxin. The non-phospholipase A2 subunit of SP I differed greatly from that of SP III but was almost identical with that of SP II, except that Lys-14 and Ala-29 in SP II were replaced by Arg-14 and Glu-29 in SP I. Analysis of the effect of CaCl2 on protein fluorescence showed the existence of a low- and a high-affinity site on the different domains of each isotoxin for Ca2+ binding. The three isotoxins showed no great difference in their ability to bind Ca2+ on both the high- and low-affinity site. They had slightly different phospholipase A2 activities but differed to a great extent with respect to their neurotoxic effects. LD50 values increased in the order SP I > SP II > SP III. In contrast, the ability to inhibit the indirectly evoked contraction of chick biventer cervicis muscle was in the order SP III > SP II > SP I.
Phosphinic peptide analogues as potent inhibitors of Corynebacterium rathayii bacterial collagenase
Authors: Yiotakis A , Lecoq A , Nicolaou A , Labadie J , Dive V
Published: August 2015

Pseudo-substrate analogues of collagenase from Corynebacterium rathayii, in which the scissile peptide bond is replaced by a phosphinic moiety, were synthesized and evaluated as inhibitors of this enzyme. The phosphinic tetrapeptide, Z-Phe-psi(PO2CH2)-Gly-Pro-Nle (1), was found to be a potent inhibitor of collagenase with a Ki value of 8 nM. Increasing the length of the phosphinic-containing inhibitors from tetra- to hepta-peptide size further improves the potency of these compounds. The heptapeptide analogue, Z-Phe-Gly-Pro-Phe-psi(PO2CH2)-Gly-Pro-Nle-OMe, with a Ki value of 0.6 nM, is the most potent inhibitor reported to date for bacterial collagenases. A comparison between the phosphinic analogue Z-Phe-psi(PO2CH2)-Gly-Pro-Nle (1) and the phosphonamide peptide Z-Phe-psi(PO2NH)-Gly-Pro-Nle (2) shows that for bacterial collagenase the replacement of a CH2 by an NH group results only in a modest increase in affinity from Ki = 8 nM for compound 1 to Ki = 6 nM for compound 2. Most of the phosphorus-containing inhibitors of this series are slow- or slow-tight-binding inhibitors with second-order rate constants for association and dissociation varying respectively for the kon values from 1 x 10(3) to 26 x 10(3) M-1.s-1 and for the koff values from 3 x 10(-4) to 2 x 10(-5) s-1. Interestingly, the lower affinity of the molecule containing a D residue in the P1 position of the inhibitor, compared with the molecule with an L residue in this position, is mainly the consequence of a lower rate constant for association of these D stereoisomers with the enzyme. This study demonstrates that phosphinic peptide analogues are potent inhibitors of a bacterial collagenase. The development of new phosphinic peptides should lead to the discovery of potent inhibitors of other zinc metalloproteases. Details of how the analogues were synthesized are given in Supplementary Publication SUP 50176 (14 pages), which has been deposited with the British Library Document Supply Centre, Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, from whom copies can be obtained on the terms indicated in Biochem. J. (1994) 297, 9.
The intracellular distribution of inositol polyphosphates in HL60 promyeloid cells
Authors: Stuart J A , Anderson K L , French P J , Kirk C J , Michell R H
Published: August 2015

1. HL60 promyeloid cells contain high intracellular concentrations of inositol polyphosphates, notably inositol 1,3,4,5,6-pentakisphosphate (InsP5) and inositol hexakisphosphate (InsP6). To determine their intracellular location(s), we studied the release of inositol (poly)phosphates, of ATP, and of cytosolic and granule-enclosed enzymes from cells permeabilized by four different methods. 2. When cells were treated with digitonin, all of the inositol phosphates were released in parallel with the cytosolic constituents. Most of the InsP5 and InsP6 was released before significant permeabilization of azurophil granules. 3. Similar results were obtained from cells preloaded with ethylene glycol and permeabilized by osmotic lysis. 4. Electroporation at approximately 500 V/cm caused rapid release of free inositol. Higher field strengths provoked release of most of the ATP, InsP5 and InsP6, but only slight release of the intracellular enzymes. Multiple discharges released approximately 80-90% of total InsP5 and InsP6. In the absence of bivalent-cation chelators, InsP5 and InsP6 were released less readily than ATP. 5. Treatment of cells with Staphylococcus aureus alpha-toxin caused quantitative release of inositol and ATP, without release of intracellular enzymes. However, inositol phosphates were released much less readily than inositol or ATP. Even after prolonged incubation with a high concentration of alpha-toxin, only approximately 50-70% of InsP2, InsP3 and InsP4 and < or = 20% of InsP5 and InsP6 were released, indicating that the high charge or large hydrated radius of InsP5 and InsP6 might limit their release through small toxin-induced pores. 6. These results indicate that most intracellular inositol metabolites are either in, or in rapid exchange with, the cytosolic compartment of HL60 cells. However, they leave open the possibility that a small proportion of cellular InsP5 and InsP6 (< or = 10-20%) might be in some intracellular bound form.
Vesicle-micelle structural transition of phosphatidylcholine bilayers and Triton X-100
Authors: De la Maza A , Parra J L
Published: August 2015

The structural transition stages induced by the interaction of the non-ionic surfactant Triton X-100 on phosphatidylcholine unilamellar vesicles were studied by means of static and dynamic light-scattering, transmission-electron-microscopy (t.e.m.) and permeability changes. A linear correlation was observed between the effective surfactant/lipid molar ratios (Re) (‘three-stage’ model proposed for the vesicle solubilization) and the surfactant concentration throughout the process. However, this correlation was not noted for the partition coefficients of the surfactant between the bilayer and the aqueous medium (K). Thus a sharp initial K increase was observed until a maximum value was achieved for permeability alterations of 50% (initial step of bilayer saturation). Further surfactant additions resulted in a fall in the K values until 100% of bilayer permeability. Additional amounts of surfactant led to an increase in K until bilayer solubilization. Hence, a preferential incorporation of surfactant molecules into liposomes governs the initial interaction steps, leading to the initial stage of bilayer saturation with a free surfactant concentration that was lower than its critical micelle concentration (c.m.c.). Additional amounts of surfactant increased the free surfactant until the c.m.c. was reached, after which solubilization started to occur. Thus the initial step of bilayer saturation was achieved for a smaller surfactant concentration than that for the Resat, although this concentration was the minimum needed for solubilization to start. Large unilamellar vesicles began to form as the surfactant exceeded 15 mol% (50% bilayer permeability), the maximum vesicle growth being attained for 22 mol% (400 nm). Thereafter, static light-scattering started to decrease gradually, this fall being more pronounced after 40 mol%. The t.e.m. picture for 40 mol% (Resat.) showed unilamellar vesicles, although with traces of smaller structures. From 50 mol% the size distribution curves began to show a bimodal distribution. The t.e.m. pictures for 50-64 mol% revealed tubular structures, together with open bilayer fragments. Thereafter, increasing amounts of surfactant (65-69 mol%) led to planar multilayered structures which gradually tended to form concentric and helicoidal conformations. The scattered intensity decreased to a low constant value at more than 71-72 mol%. However, the surfactant concentration for the Re(sol) (72.6 mol %) still presented traces of aggregated structures, albeit with mono-modal size-distribution curves (particle size of 50 nm). This vesicle size corresponded to the liposome solubilization via mixed-micelle formation.
Binding characteristics of scavenger receptors on liver endothelial and Kupffer cells for modified low-density lipoproteins
Authors: De Rijke Y B , Biessen E A L , Vogelezang C J M , van Berkel T J C
Published: August 2015

Previous studies showed that both endothelial and Kupffer cells contain specific recognition sites of oxidized low-density lipoprotein (OxLDL), in addition to recognition sites which recognize OxLDL and acetylated LDL (AcLDL). We have determined the binding characteristics of the recognition sites for OxLDL on Kupffer cells and endothelial cells (OxLDL-specific binding-site) in comparison to the recognition site for AcLDL on endothelial cells, which recognizes both AcLDL and OxLDL (Ac/OxLDL binding site). The capacity of Kupffer cells to bind OxLDL (Bmax. = 779 ng of 125I-OxLDL/mg of cell protein; Kd = 6 micrograms/ml) was comparable to the binding-capacity of endothelial cells (Bmax. = 803 ng of 125I-OxLDL/mg of cell protein; Kd = 5 micrograms/ml). The effect of net charge of modified LDL on its affinity for the recognition sites on Kupffer and endothelial cells was evaluated using competition studies. The affinity of AcLDL for the Ac/OxLDL binding site was greatly increased from 460 micrograms/ml to 4 micrograms/ml with increasing extent of modification and thus net charge. The Ac/OxLDL binding-site on endothelial cells also displayed an increased affinity towards LDL with an increasing degree of oxidation. The affinity of OxLDL for the Ac/OxLDL binding-site appeared to be about 4-fold higher than that of AcLDL with a similar extent of modification. At higher degrees of oxidation of LDL, the affinity for the OxLDL-specific site on endothelial and Kupffer cells was also strongly enhanced; the OxLDL-specific binding-site possesses a higher affinity for mildly oxidized LDL as compared with the Ac/OxLDL binding-site. It is concluded that recognition of OxLDL by both the OxLDL-specific binding-site and the Ac/OxLDL binding-site on liver endothelial and Kupffer cells depends on the net negative charge of modified LDL. The similarity in binding pattern of these binding sites makes it likely that the newly described 95 kD OxLDL binding protein on Kupffer cells [Y. B. De Rijke and Th. J. C. van Berkel, J. Biol. Chem. (1994), 269, 824-827] contains a recognition site with similar structural elements as described earlier for scavenger receptors.
Non-specific binding of advanced-glycosylation end-products to macrophages outweighs specific receptor-mediated interactions
Authors: Shaw S M , Crabbe M J C
Published: August 2015

On binding to murine peritoneal macrophages, maleylated BSA exhibited saturable-binding kinetics, with about 24000 sites/cell. Prolonged incubation of BSA with > 20 mM glucose or 2 months incubation with > or = 0.5 M glucose induced the modified protein to readily bind non-specifically to both cell and tube surfaces. Kinetic studies on the binding of advanced glycated end-products (AGEs) and other modified proteins to macrophages and hepatocytes showed no evidence for specific receptor binding, as neither binding saturation nor cross-competition (homologous or heterologous) was detected. Although there was evidence for uptake of BSA which had been incubated with 0.5 M glucose for 2 months, there was no uptake or degradation of AGEs which had been produced at physiological concentrations of glucose. This has implications for the role of macrophages in the recognition of AGEs, and suggests that the non-specific binding may be important in adhesion of AGEs, particularly in poorly controlled diabetics, and might act as a ‘damage limitation’ mechanism in the potential development of diabetic complications, while low macrophage levels in the blood could seriously potentiate the long-term effects of non-enzymic post-translational protein modifications.
Alternative pathways for phosphatidylcholine synthesis in olive (Olea europaea L.) callus cultures
Authors: Williams M , Harwood J L
Published: August 2015

Olive (Olea europaea L.) callus cultures were incubated with [2-14C]ethanolamine and [Me-14C]choline in order to study phospholipid synthesis. Radioactivity from [Me-14C]choline was shown to be incorporated into the phosphatidylcholine via the CDP-base pathway. [2-14C]Ethanolamine was primarily incorporated into phosphatidylethanolamine, but significant radio-activity was also detected in phosphatidylcholine, indicating the operation of a methylation route. Incubation with [2-14C]ethanolamine indicated that phosphatidylcholine and phosphatidylethanolamine incorporated radiolabel over a similar time course. This led us to investigate the possibility that phosphatidylcholine was being synthesized by a methylation pathway distinct from the direct methylation of phosphatidylethanolamine. There was extensive incorporation of [2-14C]ethanolamine into different components of the aqueous phase of the incubations, within which phospho-base derivatives of ethanolamine were prominent. These intermediates were identified and provided evidence for the operation of an alternative methylation pathway via phosphodimethylethanolamine for the biosynthesis of phosphatidylcholine in olives.
Isolation and characterization of CD47 glycoprotein: a multispanning membrane protein which is the same as integrin-associated protein (IAP) and the ovarian tumour marker OA3
Authors: Mawby W J , Holmes C H , Anstee D J , Spring F A , Tanner M J A
Published: August 2015

The CD47 glycoprotein was isolated from human erythrocytes by immunoprecipitation using monoclonal antibody (mAb) BRIC-125. Enzymic deglycosylation of the protein showed it contained N-linked oligosaccharides, and trypsin proteolysis of the protein in situ in the erythrocyte membrane cleaved it into two portions, one of which was glycosylated. Both the intact protein and the glycosylated fragment had blocked N-termini. Amino acid sequence was obtained from several proteolytic fragments of CD47. Comparison with the sequence database showed the protein to be very similar to or identical with OA3, a multispanning membrane protein. The protein also appears to be the same as the integrin-associated protein, which has a role in cell adhesion in non-erythroid cells. CD47 has six potential N-glycosylation sites, five of which are in an Ig superfamily domain. We show that three of these sites carry N-glycans in erythrocytes. Immunocytochemical staining of human tissues showed that CD47 was broadly distributed on mesenchyme and epithelia at multiple sites. Reactivity was particularly prominent in surface and ductular epithelia, and in the brain. The possible roles of the CD47 glycoprotein are discussed.
Quantification of human serum paraoxonase by enzyme-linked immunoassay: population differences in protein concentrations
Authors: Blatter Garin M C , Abbott C , Messmer S , Mackness M , Durrington P , Pometta D , James R W
Published: August 2015

Paraoxonase is a serum protein bound to high-density lipoproteins (HDLs). The physiological function of the enzyme is unknown, but a role in lipid metabolism has been postulated. To date, studies of the protein have had to rely on measurements of enzyme activity with various substrates. We have developed a highly specific, competitive e.l.i.s.a. using a previously characterized monoclonal antibody. The assay can detect 20 ng of paraoxonase with a working range of 75-600 ng. Intra- and interassay coefficients of variation were 6.5 and 7.9% respectively. Serum concentrations of paraoxonase in healthy subjects from Geneva and Manchester ranged from 25 to 118 micrograms/ml. There were significant differences in mean concentrations between the two groups (Geneva, 79.3 +/- 18.7 micrograms/ml; Manchester, 59.9 +/- 24.1 micrograms/ml: P < 0.001), differences also apparent when subjects were compared according to paraoxonase phenotype. These appeared to be largely a consequence of differences in apolipoprotein A-I concentrations between the two populations, suggesting that HDL particle number may be important in determining serum levels of paraoxonase. Paraoxonase specific activities were also significantly different between the two groups of subjects (Geneva, 2.08 +/- 0.96 units/mg; Manchester, 3.08 +/- 1.73 units/mg: P < 0.001), which may reflect differences in HDL particle composition. The e.l.i.s.a. should furnish the necessary complement to studies of paraoxonase enzymic activity and has already provided evidence for differences with respect to serum levels of the protein both between populations and between phenotypes within populations.
Characterization of the single Ca2+-binding site on the Ca2+-ATPase reconstituted with short- or long-chain phosphatidylcholines
Authors: Starling A P , Khan Y M , East J M , Lee A G
Published: August 2015

On reconstitution of the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum into bilayers of dimyristoleoylphosphatidylcholine [di(C14:1)PC] or dinervonylphosphatidylcholine [di(C24:1)PC] the stoichiometry of Ca2+ binding changes from the usual two Ca2+ ions bound per ATPase molecule to one Ca2+ ion bound per ATPase molecule. For the ATPase in di(C24:1)PC, removal of Ca2+ from the Ca(2+)-bound ATPase results in a decrease in tryptophan fluorescence intensity, as observed for the ATPase in dioleoylphosphatidylcholine [di(C18:1)PC]. For the ATPase in di(C14:1)PC removal of Ca2+ results in no change in tryptophan fluorescence intensity. In the presence of Mg2+, removal of Ca2+ from the ATPase in di(C18:1)PC or di(C24:1)PC results in a decrease in tryptophan fluorescence intensity, but for the ATPase in di(C14:1)PC this results in an increase in intensity. Fluorescence of the ATPase labelled with 4-nitrobenzo-2-oxa-1,3-diazole (NBD) is the same for the ATPase in di(C18:1)PC or di(C24:1)PC, but is markedly greater in di(C14:1)PC, consistent with a 4-fold increase in the E1/E2 equilibrium constant. Addition of Mg2+ to NBD-labelled ATPase in di(C18:1) PC or di(C24:1)PC results in an increase in NBD fluorescence, attributed to stronger binding of Mg2+ to the E1 than to the E2 conformation; addition of Mg2+ had no effect on the fluorescence of the NBD-labelled ATPase in di(C14:1)PC. In the absence of Ca2+, Mg2+ increased the tryptophan fluorescence of the ATPase in di(C14:1)PC, di(C18:3)PC or di(C24:1)PC, with the same binding-constant for Mg2+ in all three lipids. Addition of Mg2+ to the ATPase labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin resulted in a decrease in fluorescence in di(C18:1)PC or di(C24:1)PC but had no effect in di(C14:1)PC. These effects are interpreted in terms of binding of Ca2+ at a single outer Ca2+ binding-site on the ATPase in di(C14:1)PC and di(C24:1)PC, in a conformation in which the inner site is occluded [in di(C14:1)PC] or modified in its affinity for Ca2+ [in di(C24:1)PC]. Thapsigargin binds to the ATPase, reducing its affinity for Ca2+ both in di(C14:1)PC and di(C24:1)PC.
Adenosine stimulates DNA fragmentation in human thymocytes by Ca2+-mediated mechanisms
Authors: Szondy Z
Published: August 2015

Incubation of human thymocytes with an optimum concentration of adenosine and its receptor site agonist, 2-chloroadenosine, induced increases in intracellular cyclic AMP (cAMP) (from a resting 0.6 +/- 0.1 to 4.1 +/- 0.2 pmol/10(7) cells within 5 min) and Ca2+ (from the resting 85 +/- 7 nM to a peak of 210 +/- 25 nM) levels and resulted in internucleosomal DNA fragmentation and cell death (apoptosis). Other adenosine analogues were also effective at inducing DNA fragmentation, the order of potency being 2-p-(carboxyethylphenylethylamino)-5′-carboxyamidoadenosine < 5′-(N-ethylcarboxamide)adenosine < or = cyclopentyladenosine < 2-chloroadenosine (2-CA). 2-CA treatment (with an optimum concentration of 40 microM) selectively depleted a thymocyte subpopulation (15-20% of the total cells) which expressed higher levels of the CD3 molecule and which was found mainly in the CD4+CD8+ double positive immature thymocyte population. DNA fragmentation was prevented by the addition of actinomycin D or cycloheximide to the thymocyte suspension, indicating that this process required both mRNA and protein synthesis. Endonuclease activation and cell killing were dependent on an early, sustained increase in cytosolic Ca2+ concentration, most of which was of extracellular origin and was a result of an adenosine-induced inositol trisphosphate release. Other agents known to elevate intracellular cAMP levels by different mechanisms failed to induce similar DNA fragmentation, but enhanced the effect of adenosine. This suggested a supporting role for cAMP in adenosine-induced DNA fragmentation. Phorbol dibutyrate, a protein kinase. C activator, previously shown to inhibit Ca(2+)-dependent DNA fragmentation and cell killing in human thymocytes [McConkey, Hartzell, Jondal and Orrenius (1989) J. Biol. Chem. 264, 13399-13402], at 60 ng/ml concentration also prevented adenosine-induced DNA fragmentation when added prior to adenosine. This suggested a complex cross-talk between the adenosine-triggered signal transduction cascade and the activation state of protein kinase C in regulating apoptosis of human thymocytes.
Rescue of the complex temperature-sensitive phenotype of Chinese hamster ovary E36ts20 cells by expression of the human ubiquitin-activating enzyme cDNA
Authors: Handley-Gearhart P M , Trausch-Azar J S , Ciechanover A , Schwartz A L
Published: August 2015

The ubiquitin conjugation system is a multi-step pathway in which ubiquitin is activated and conjugated to acceptor proteins, one function of which is to target acceptor proteins for rapid degradation within the cell. The conjugation system is involved in many aspects of cellular functions, including the cell cycle. Several cell-cycle arrest mutant cell lines have been characterized and appear to harbour a mutant ubiquitin-activating enzyme, E1, as their primary defect. One such cell line is ts20, which is derived from Chinese hamster ovary E36 cells. This cell line has been used to characterize some of the potential functions of the ubiquitin conjugation system in vivo, such as its involvement in the maturation of autophagic vacuoles. The present study describes the complete rescue of the complex ts20 phenotype following the expression of the cDNA for human E1. Stable transfectants expressing the human E1 cDNA in the CMVneo expression vector were measured for ubiquitin-conjugation activity, protein degradation and growth in culture at the nonpermissive temperature. This rescue confirms that the phenotype observed in the ts20 cells is due to a defect in the E1 enzyme. Thus, the ts20 cell line will serve as a useful tool to delineate the functions of the ubiquitin system in vivo.