bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased expression of the inositol 1,4,5-trisphosphate receptor in human leukaemic (HL-60) cells differentiated with retinoic acid or dimethyl sulphoxide
Authors: Bradford P G , Autieri M
Published: August 2015

The Ins(1,4,5)P3 receptor was examined in human promyelocytic leukaemic cells (HL-60) and in HL-60 cells differentiated towards granulocytes with either retinoic acid (RA) or dimethyl sulphoxide (Me2SO). HL-60 cell membranes enriched in marker enzyme activities of the endoplasmic reticulum and the plasma membrane possess a high-affinity binding site for [3H]Ins(1,4,5)P3 (KD = 22 nM). Electrotransfer studies indicate that Ins(1,4,[32P]5)P3 binds specifically to a 260 kDa protein of HL-60 cell membranes. This Ins(1,4,5)P3-binding protein selectively binds Ca(2+)-mobilizing inositol phosphates and other inositol phosphates which also bind to the purified InsP3 receptor, suggesting that the Ins(1,4,5)P3-binding protein of HL-60 cell membranes is the InsP3 receptor. When HL-60 cells are incubated with 1 microM-RA or with 1.25% Me2SO the cells differentiate within 5-7 days into cells resembling neutrophils in both structure and function. Treated cells cease to proliferate, acquire the ability to reduce Nitro Blue Tetrazolium dye, and undergo morphological changes typical of differentiated granulocytes. Concomitant with HL-60 cell differentiation, the maximal [3H]Ins(1,4,5)P3 binding in membranes increases 3-4-fold, with no change in KD. The results suggest that there is an absolute increase in the level of the InsP3 receptor during HL-60 cell differentiation and that the expression of this signal-transducing protein may be specifically regulated by differentiation factors.
Effect of complement-protein-C3b density on the binding of complement factor H to surface-bound C3b
Authors: Koistinen V
Published: August 2015

Various amounts of the activation fragment C3b of the complement (C) protein C3 were coupled to Sepharose 4B by catalysis with the C3 convertase of the alternative pathway of C. The binding of radioactively labelled C proteins B and H (= factor H) to the C3b-carrying particles was assayed. It was found that the relative binding of H, but not of B, fell rapidly with decreasing densities of solid-phase C3b, suggesting a sigmoidal relationship between C3b density and binding of H. To study the phenomenon in more detail, preformed C3b was coupled to activated thiopropyl-Sepharose 6B at various densities. By using this model system, it was shown that the binding of H/unit amount of C3b was positively correlated to C3b density up to a C3b concentration of about 0.5 mg/ml of gel, whereas binding of B was independent of C3b density. The results show that accumulation of high densities of C3b on a surface creates high-affinity binding sites for H. Because H has recently been shown to form dimers in solution, the interaction of dimeric H with neighbouring C3b molecules is a likely explanation for the phenomenon. The C3b density effect may be a regulatory mechanism keeping the activation of the alternative pathway of C on activating surface within reasonable limits.
Methanol oxidation and assimilation in Hansenula polymorpha. An analysis by 13C n.m.r. in vivo
Authors: Jones J G , Bellion E
Published: August 2015

The metabolism of methanol was monitored in whole cells of the methylotrophic yeast Hansenula polymorpha by using [13C]methanol and n.m.r. in vivo. The main products observed under normal conditions were trehalose and glycerol, whereas cells that were starved before exposure to [13C]methanol also accumulated glutamate, glutamine and alanine; formate was also more prominent in spectra from starved cells. Cells exposed to high methanol concentration together with high oxygenation oxidized methanol extensively, leading to formaldehyde accumulation; label was not found in any subsequent metabolic products, indicating possible cell inactivation. [13C]Formate was incorporated into metabolic products in glucose-grown cells exposed to 150 mM-methanol for 3 h, but not in cells starved for 3 h, in which it was oxidized. At 21 degrees C such 3 h-starved cells showed a slower metabolism of [13C]methanol compared with those at 37 degrees C, and also converted methanol into formate rather than into assimilation products. The labelling pattern in trehalose from starved cells at 37 degrees C was consistent with methanol assimilation via the pentose phosphate pathway. Lack of appearance of labelled formaldehyde and formate during metabolism under normal conditions suggests that the linear oxidation pathway is not a major contributor to methanol oxidation; their appearance in extreme conditions suggests instead a more likely role in detoxification.
Conversion of citrate synthase into citryl-CoA lyase as a result of mutation of the active-site aspartic acid residue to glutamic acid
Authors: Man W J , Li Y , O'Connor C D , Wilton D C
Published: August 2015

The active-site aspartic acid residue, Asp-362, of Escherichia coli citrate synthase was changed by site-directed mutagenesis to Glu-362, Asn-362 or Gly-362. Only very low catalytic activity could be detected with the Asp→Asn and Asp→Gly mutations. The Asp→Glu mutation produced an enzyme that expressed about 0.8% of the overall catalytic rate, and the hydrolysis step in the reaction, monitored as citryl-CoA hydrolysis, was inhibited to a similar extent. However, the condensation reaction, measured in the reverse direction as citryl-CoA cleavage to oxaloacetate and acetyl-CoA, was not affected by the mutation, and this citryl-CoA lyase activity was the major catalytic activity of the mutant enzyme. This high condensation activity in an enzyme in which the subsequent hydrolysis step was about 98% inhibited permitted considerable exchange of the methyl protons of acetyl-CoA during catalysis by the mutant enzyme. The Km for oxaloacetate was not significantly altered in the D362E mutant enzyme, whereas the Km for acetyl-CoA was about 5 times lower. A mechanism is proposed in which Asp-362 is involved in the hydrolysis reaction of this enzyme, and not as a base in the deprotonation of acetyl-CoA as recently suggested by others. [Karpusas, Branchaud & Remington (1990) Biochemistry 29, 2213-2219; Alter, Casazza, Zhi, Nemeth, Srere & Evans, (1990) Biochemistry 29, 7557-7563].
Impact of tumour necrosis factor-α and interferon-γ on tetrahydrobiopterin synthesis in murine fibroblasts and macrophages
Authors: Werner E R , Werner-Felmayer G , Fuchs D , Hausen A , Reibnegger G , Yim J J , Wachter H
Published: August 2015

Tumour necrosis factor-alpha causes an up to 30-fold induction of GTP cyclohydrolase I (EC 3.5.4.16) activity in murine dermal fibroblasts in a dose-dependent manner. Owing to the high constitutive activities of 6-pyruvoyltetrahydropterin synthase and sepiapterin reductase (EC 1.1.1.153), this potentiates biosynthesis of tetrahydrobiopterin. Murine macrophages already contain high activities of GTP cyclohydrolase I when unstimulated, and this is further augmented up to 4-fold by tumour necrosis factor-alpha/interferon-gamma. In Western blots an antiserum to murine liver GTP cyclohydrolase I does not stain cell extracts with high enzyme activities, suggesting that the cytokine induced peripheral form of GTP cyclohydrolase I might differ from the liver form.
Glycosyl-phosphatidylinositol-anchored membrane proteins can be distinguished from transmembrane polypeptide-anchored proteins by differential solubilization and temperature-induced phase separation in Triton X-114
Authors: Hooper N M , Bashir A
Published: August 2015

Treatment of kidney microvillar membranes with the non-ionic detergent Triton X-114 at 0 degrees C, followed by low-speed centrifugation, generated a detergent-insoluble pellet and a detergent-soluble supernatant. The supernatant was further fractionated by phase separation at 30 degrees C into a detergent-rich phase and a detergent-depleted or aqueous phase. Those ectoenzymes with a covalently attached glycosyl-phosphatidylinositol (G-PI) membrane anchor were recovered predominantly (greater than 73%) in the detergent-insoluble pellet. In contrast, those ectoenzymes anchored by a single membrane-spanning polypeptide were recovered predominantly (greater than 62%) in the detergent-rich phase. Removal of the hydrophobic membrane-anchoring domain from either class of ectoenzyme resulted in the proteins being recovered predominantly (greater than 70%) in the aqueous phase. This technique was also applied to other membrane types, including pig and human erythrocyte ghosts, where, in both cases, the G-PI-anchored acetylcholinesterase partitioned predominantly (greater than 69%) into the detergent-insoluble pellet. When the microvillar membranes were subjected only to differential solubilization with Triton X-114 at 0 degrees C, the G-PI-anchored ectoenzymes were recovered predominantly (greater than 63%) in the detergent-insoluble pellet, whereas the transmembrane-polypeptide-anchored ectoenzymes were recovered predominantly (greater than 95%) in the detergent-solubilized supernatant. Thus differential solubilization and temperature-induced phase separation in Triton X-114 distinguished between G-PI-anchored membrane proteins, transmembrane-polypeptide-anchored proteins and soluble, hydrophilic proteins. This technique may be more useful and reliable than susceptibility to release by phospholipases as a means of identifying a G-PI anchor on an unpurified membrane protein.
Possible role of histone acetylation and histone H1° replacement for the initiation of replication in regenerating rat liver
Authors: Weiss G , Talasz H , Puschendorf B
Published: August 2015

The role of histone acetylation and DNA synthesis has been investigated extensively in the regenerating rat liver system in the presence and absence of the cyclophosphamide derivative mafosfamide. We demonstrate a mafosfamide-induced inhibition of maximum histone acetyltransferase activity followed by a second elevation of enzyme activity and an accompanying total suppression of DNA synthesis for 7-8 h. The maximum of histone acetyltransferase activity, in parallel with an elevated acetylation in vivo, the consecutive replacement of histone H1(0) amd initiation of replication occur sequentially in the presence and absence of mafosfamide, but with a temporary delay of 7-8 h. Our data indicate that modifications of histone acetyltransferase (EC 2.3.1.48) activity do not significantly influence the acetylation patterns of histones H3 and H4. The mafosfamide-induced change of histone acetyltransferase activity and acetylation in vivo, the shift of histone H1(0) exchange and the consecutive transition of initiation of replication suggest that these three events might be functionally related.
Identification by protein microsequencing of a proteinase-V8-cleavage site in a folding intermediate of chick muscle creatine kinase
Authors: Morris G E , Jackson P J
Published: August 2015

We have identified by protein microsequencing a glutamic acid residue (Glu-166) in a folding intermediate of chick muscle creatine kinase that is very sensitive to cleavage by staphylococcal proteinase V8. Most other glutamic acid residues, including Glu-168, are already partly protected from proteolytic attack at this stage. After the final stages of protein refolding, when enzyme activity is recovered, Glu-166 is also resistant to proteolysis.
Characterization of ligand binding and processing by gastrin-releasing peptide receptors in a small-cell lung cancer cell line
Authors: Cardona C , Bleehen N M , Reeve J G
Published: August 2015

The ligand-binding properties of the gastrin-releasing peptide (GRP) receptor and the cellular processing of GRP have been studied in the small-cell lung cancer (SCLC) cell line COR-L42. Scatchard analysis of GRP receptor expression indicated a single class of high-affinity receptors (Kd 1.5 nM) and approx. 6700 receptors/cell. GRP bound to its receptor with a Ki of 2.4 nM. The bombesin-related peptides neuromedin B (NMB) and phyllolitorin also bound to GRP receptors with Ki values of 22.7 and 59.1 nM respectively. Binding of 125I-GRP to COR-L42 cells increased rapidly at 37 degrees, achieved a maximum at 10 min and declined rapidly thereafter. At 4 degrees C, maximum binding was achieved at 30 min and the subsequent decline in cell-associated radioactivity was slower than that seen at 37 degrees C. Acid/salt extraction, to separate surface-bound ligand from internalized GRP, indicated that after receptor binding 125I-GRP was rapidly internalized. To determine the pathway of 125I-GRP degradation, binding studies were carried out with the lysosomotropic agent chloroquine (5 mM), and with phosphoramidon (10 microM), an inhibitor of the membrane-bound enzyme (EC 3.4.24.11). Both agents markedly inhibited the degradation of GRP, indicating that this process involves a lysosomal pathway and a phosphoramidon-sensitive pathway, possibly involving the EC 3.4.24.11 enzyme. GRP receptor down-regulation was observed following a 10 min exposure to 100 nM-GRP. With longer pretreatment times the number of binding sites recovered to 80% of control values. Treatment with 5 mM-chloroquine plus GRP or cycloheximide (10 micrograms/ml) plus GRP demonstrated that the majority of GRP receptors are recycled. NMB and phyllolitorin pretreatment did not influence the subsequent binding of 125I-GRP, suggesting that these peptides do not down-regulate GRP receptors.
Purification of an Arg-Gly-Asp selective matrix receptor from brain synaptic plasma membranes
Authors: Bahr B A , Lynch G
Published: August 2015

Brain synaptic plasma membranes specifically associated with matrix protein monolayers containing the Arg-Gly-Asp sequence recognized by integrin-type adhesion receptors. Experiments using fibronectin affinity chromatography to identify the synaptosomal receptors responsible for this interaction led to the purification of a 55 kDa Arg-Gly-Asp recognition protein that is labelled by antibodies against the alpha 5 beta 1 integrin.
Chlorine-containing compounds produced during Dictyostelium development. Detection by labelling with 36Cl
Authors: Kay R R , Taylor G W , Jermyn K A , Traynor D
Published: August 2015

DIF-1 [Differentiation-Inducing Factor 1; 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one] is a novel chlorinated signal molecule that induces stalk-cell differentiation during development of Dictyostelium discoideum. Here we introduce the use of the radioisotope 36Cl to label DIF-1 and other low-Mr chlorinated compounds produced during development. H.p.l.c. and t.l.c. were used to resolve the labelled compounds. We find the following. (1) At least 14 dialysable 36Cl-labelled compounds are released into the medium by cells labelled continuously through development with Na36Cl. (2) The compounds can be classified into two major groups according to their times of accumulation in development. The early group of compounds starts accumulating at the end of aggregation, co-ordinately with DIF-1; the late group is only made at the end of development, by mature fruiting bodies. There may also be an intermediate group made during culmination. (3) The early group of compounds has been identified as comprising DIF-1 and seven of its metabolites by co-chromatography with the authentic compounds. These metabolites had previously only been recognized in suspensions of living cells incubated with exogenous DIF-1. Their detection here, from cells undergoing normal development, suggests that endogenous DIF-1 is metabolized in normal development in much the same way as is DIF-1 added to cells in suspension. (4) The intermediate and late groups of compounds are not obvious DIF-1 metabolites. They may have some role unconnected with DIF signalling. (5) A group of 36Cl-labelled late compounds remain cell-associated after washing of the fruiting bodies, and these are greatly enriched in stalk, compared with spore, cells. (6) Other slime-mould species were labelled with 36Cl. All three tested, namely D. mucoroides, D. vinaceo-fuscum and P. violaceum, also produced chloro compounds. D. mucoroides produced DIF-1 by the criterion of h.p.l.c. co-elution with authentic DIF-1. A developmentally regulated metabolism of chlorinated compounds may therefore be widespread amongst slime moulds. To our knowledge, labelling with 36Cl in vivo has not been reported before and provides a powerful general method for investigating chlorinated compounds in diverse organisms.
Identification of a 80 kDa calmodulin-binding protein as a new Ca2+/calmodulin-dependent kinase by renaturation blotting assay (RBA)
Authors: Kato M , Hagiwara M , Hidaka H
Published: August 2015

We surveyed rabbit brain cytosol for a new Ca2+/calmodulin (CaM)-dependent kinase. The renaturation blotting assay (RBA) exploits the ability of blotted SDS-denatured proteins to regain enzymic activity after guanidine treatment. Using RBA, we found that the eluate of rabbit brain cytosol from a CaM affinity column contains at least four electrophoretically distinct protein kinase bands which were autophosphorylated in a Ca2+/CaM-dependent manner. The 49 kDa band and the 60 kDa band were alpha and beta subunit of CaM kinase II, and the 42 kDa band was presumed to be CaM kinase I, but the 80 kDa band could not be attributed to any reported Ca2+/CaM-dependent protein kinases. The 80 kDa protein kinase was isolated by three-step chromatography. We examined the phosphorylation of exogenous substrates by 80 kDa protein kinase, and histone IIIs and myosin light chain were phosphorylated in a Ca2+/CaM-dependent manner. W-7, a specific inhibitor for calmodulin, inhibited this kinase activity, but KN-62, a specific inhibitor for CaM kinase II, had no effect on this protein kinase activity. Autoradiography using boiled rabbit brain homogenate as substrate showed three intrinsic substrates (80 kDa, 60 kDa and 42 kDa), which were phosphorylated in a Ca2+/CaM-dependent manner. These findings suggest that a new Ca2+/CaM-dependent protein kinase could be identified by the RBA.
Wheat and barley allergens associated with baker's asthma. Glycosylated subunits of the α-amylase-inhibitor family have enhanced IgE-binding capacity
Authors: Sanchez-Monge R , Gomez L , Barber D , Lopez-Otin C , Armentia A , Salcedo G
Published: August 2015

A 16 kDa protein, designated CM16*, which strongly binds IgE from baker's-asthma patients has been identified as a glycosylated form of the previously reported WTAI-CM16, which is a subunit of the wheat tetrameric alpha-amylase inhibitor. A glycosylated form (CMb*) of BTAI-CMb, the equivalent inhibitor subunit from barley, has been also found to have significantly enhanced IgE-binding capacity. In all, 14 purified members of the alpha-amylase/trypsin-inhibitor family showed very different IgE-binding capacities when tested by a dot-blot assay. The glycosylated components CM16*, CMb* and the previously described non-glycosylated 14.5 kDa allergen from barley (renamed BMAI-1) were found to be the strongest allergens.
Insertion of isolated insulin receptors into placental membrane vesicles
Authors: Christiansen K , Carlsen J
Published: August 2015

Purified human insulin receptors were inserted into placental plasma-membrane vesicles by fusion of membranes with receptor-lysophosphatidylcholine micelles. Scatchard analysis of insulin binding showed that about 10-15% of the added receptors became inserted into the membrane. The receptor number could be increased about 3-fold, corresponding to approx. 5 pmol of receptor/mg of membrane protein. The receptors became firmly bound to the membrane, as they could not be removed by extensive wash. The insertion of exogenous receptors could be demonstrated by immunoblotting. The inserted insulin receptor had the same insulin-binding affinity as the isolated receptor and the endogenous receptor of the membrane. Insulin binding in the presence or absence of Triton X-100 revealed that more than 80% of the exogenous receptors had a right-side-out orientation. Function of the inserted receptors, as observed by insulin-stimulated autophosphorylation, could be demonstrated. About 80% of the added lysophospholipid, corresponding to approx. 160 nmol of lysophospholipid/mg of membrane protein, became integrated into the membrane and was partly metabolized to phospholipid and to non-esterified fatty acid. The method of insertion of isolated insulin receptors using the natural detergent, lysophospholipid, may be a method for insertion of receptors into intact cells, where the lysophospholipid, as in the plasma-membrane vesicles, will be acylated to phospholipid.
Loss of thyroid hormone receptor activity in primary cultured rat hepatocytes is reversed by 2-mercaptoethanol
Authors: Yamamoto N , Inoue A , Takahashi K P , Li Q , Nakamura H , Tagami T , Sasaki S , Imura H , Morisawa S
Published: August 2015

In primary cultures of rat hepatocytes, specific thyroid-hormone-binding activity diminished with time and was hardly detectable at 24 h. In accordance with the loss of 3,5,3′-tri-iodothyronine (T3) binding, responses to the hormone disappeared, as indicated by low induction of the thyroid-hormone-responsive gene S14. In contrast, thyroid hormone receptor proteins were present, as determined by immunostaining with a specific antibody against the receptor. Thus the loss of T3 binding was due to receptor inactivation. After various attempts to restore the T3-binding activity, we found that 2-mercaptoethanol, a reducing agent, when added to the culture medium restored the hormone binding activity in a dose- and time-dependent manner. The observed kinetics and experiments using cycloheximide suggested that mercaptoethanol prevented inactivation of the newly synthesized receptors. Oxidoreductive conditions within cells may have a role in determining the level of activity of thyroid hormone receptors.
Transforming growth factor-α-induced DNA synthesis and c-myc expression in primary rat hepatocyte cultures is modulated by indomethacin
Authors: Skouteris G G , McMenamin M
Published: August 2015

Primary hepatocytes stimulated with epidermal growth factor (EGF) secrete prostaglandins into the culture medium as soon as 1 h after the addition of the EGF. Transforming growth factor-alpha (TGF alpha), a potent hepatocyte mitogen, shares the same receptor with EGF, and its expression is increased after partial hepatectomy. TGF alpha is also secreted in culture. We have observed that TGF alpha induced hepatocyte DNA synthesis (30 h after addition) and at the same time stimulated the production of prostaglandins E2 and F2 alpha by the cultured hepatocytes. Indomethacin at 20-100 microM inhibited the TGF alpha-induced hepatocyte DNA synthesis, and this effect was specifically due to the inhibition of prostaglandin formation. Indomethacin also inhibited a TGF-alpha-induced increase in hepatocyte c-myc expression, indicating that prostaglandins mediate this increase, as previously shown for EGF. TGF alpha increased the expression of the EGF receptor gene, and this was prevented by the presence of an antibody against TGF alpha in the culture medium. We therefore suggest that TGF alpha induces hepatocyte proliferation either through coupling with its receptor (i.e. the EGF receptor) or by subsequent phosphorylation of lipocortin I. This leads to activation of phospholipase. A2, which seems to regulate the metabolism of arachidonic acid and the formation of prostaglandins. Thus hepatocyte proliferation in vitro appears to be controlled by a self-regulatory autocrine pathway involving activation of phospholipase A2 and secretion of prostaglandins and TGF alpha.
Trafficking of glucose transporters in 3T3-L1 cells. Inhibition of trafficking by phenylarsine oxide implicates a slow dissociation of transporters from trafficking proteins
Authors: Yang J , Clark A E , Harrison R , Kozka I J , Holman G D
Published: August 2015

We have compared the rates of insulin stimulation of cell-surface availability of glucose-transporter isoforms (GLUT1 and GLUT4) and the stimulation of 2-deoxy-D-glucose transport in 3T3-L1 cells. The levels of cell-surface transporters have been assessed by using the bismannose compound 2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis(D-mannos -4-yloxy) propyl-2-amine (ATB-BMPA). At 27 degrees C the half-times for the appearance of GLUT1 and GLUT4 at the cell surface were 5.7 and 5.4 min respectively and were slightly shorter than that for the observed stimulation of transport activity (t 1/2 8.6 min). This lag may be due to a slow dissociation of surface transporters from trafficking proteins responsible for translocation. When fully-insulin-stimulated cells were subjected to a low-pH washing procedure to remove insulin at 37 degrees C, the cell-surface levels of GLUT1 and GLUT4 decreased, with half-times of 9.2 and 6.8 min respectively. These times correlated well with decrease in 2-deoxy-D-glucose transport activity that occurred during this washing procedure (t1/2 6.5 min). When fully-insulin-stimulated cells were treated with phenylarsine oxide (PAO), a similar decrease in transport activity occurred (t1/2 9.8 min). However, surface labelling showed that this corresponded with a decrease in GLUT4 only (t1/2 7.8 min). The cell-surface level of GLUT1 remained high throughout the PAO treatment. Light-microsome membranes were isolated from cells which had been cell-surface-labelled with ATB-BMPA. Internalization of both transporter isoforms to this pool occurred when cells were maintained in the presence of insulin for 60 min. In contrast with the surface-labelling results, we have shown that the transfer to the light-microsome pool of both transporters occurred in cells treated with insulin and PAO. These results suggest that both transporters are recycled by fluid-phase endocytosis and exocytosis. PAO may inhibit this recycling at a stage which involves the re-emergence of internalized transporters at the plasma membrane. The GLUT1 transporters that are recycled to the surface in insulin- and PAO-treated cells appear to have low transport activity. This may be because of a failure to dissociate fully from trafficking proteins at the cell surface. GLUT4 transporters appear to have a greater tendency to remain internalized if the normal mechanisms that commit transporters to the cell surface, such as dissociation from trafficking proteins, are uncoupled.
Sphingosine enhances platelet aggregation through an increase in phospholipase C activity by a protein kinase C-independent mechanism
Authors: Hashizume T , Sato T , Fujii T
Published: August 2015

Sphingosine (a potent inhibitor of protein kinase C) at 5-10 microM, which are concentrations lower than those that inhibit this enzyme activity, enhanced the aggregation of rabbit platelets induced by low concentrations of U46619, platelet-activating factor, thrombin and arachidonic acid, whereas H-7 and staurosporine, other protein kinase C inhibitors, failed to do so. Of the sphingosine analogues which also inhibit protein kinase C, psychosine and lyso-GM3 did not show such an enhancing effect. Sphingosine promoted both Ins(1,4,5)P3 formation and an increase in the cytoplasmic free Ca2+ concentration in response to all the agonists used. Furthermore, the hydrolytic action of exogenously added phospholipase C (from Clostridium perfringens) on platelet membrane phospholipids was dose-dependently enhanced by pretreatment of the platelets with sphingosine. These results imply that sphingosine, at relatively low concentrations, brings about hyperaggregability of the platelets by the agonists employed, probably owing to enhancement of the phospholipase C activity. Such an effect appears to be induced by a mechanism independent of protein kinase C inhibition. We suggest that sphingosine might act as a positive modulator for the stimulus-response coupling in the platelets.
Reversible translocation of cytidylyltransferase between cytosol and endoplasmic reticulum occurs within minutes in whole cells
Authors: Tercé F , Record M , Tronchère H , Ribbes G , Chap H
Published: August 2015

Addition of oleic acid to Krebs II cells induced a rapid incorporation of [3H]choline into phosphatidylcholine, since 500 microM of the fatty acid stimulated choline incorporation by 5-fold over the control after 5 min of incubation. In fact, a noticeable increase in phosphatidylcholine labelling could be monitored immediately after 1 min of cell incubation with [3H]choline, at which time 50% of cytosolic cytidylyltransferase activity (EC 2.7.7.15), the regulatory enzyme of phosphatidylcholine synthesis, was translocated on to membranes. Non-esterified [3H]oleic acid content was also increased in the same range of time in the particulate fraction. Subcellular fractionation indicated that endoplasmic reticulum was the unique binding site for cytidylyltransferase even after 1 min of incubation. Also, [3H]oleic acid accumulated mainly in the same internal membrane. Addition of exogenous albumin to cells prelabelled with [3H]oleic acid induced the release of 50% of membrane-bound cytidylyltransferase activity within 1 min, together with a decrease in unesterified oleic acid in the same membrane. Although total depletion of oleic acid was obtained, total release of membrane-bound cytidylyltransferase was not. The remaining minor pool of membrane-bound cytidylyltransferase was not affected by cell incubation with dibutyryl cyclic AMP, suggesting that this pool was neither regulated by fatty acid nor modulated by cyclic-AMP-dependent protein phosphorylation. Addition of [3H]oleic acid directly to an homogenate led to a less specific accumulation of the fatty acid in the endoplasmic reticulum, but cytidylyltransferase remained exclusively associated with this membrane. We concluded that in vivo translocation of cytidylyltransferase provoked by oleic acid concerns one specific pool of the enzyme distinct from the enzyme firmly bound to endoplasmic reticulum, but other factor(s) than fatty acid seem to be required to explain the specificity of endoplasmic reticulum for cytidylyltransferase binding.
The N-terminal domain of antithrombin-III is essential for heparin binding and complex-formation with, but not cleavage by, α-thrombin
Authors: Austin R C , Sheffield W P , Rachubinski R A , Blajchman M A
Published: August 2015

Normal and mutant forms of human antithrombin-III (AT-III) were synthesized in a cell-free system in order to identify putative functional domains required for heparin binding and complex-formation with alpha-thrombin. Heparin-Sepharose chromatography resulted in the elution of approx. 70% of cell-free-derived normal AT-III-(1-432)-polypeptide as a peak between 0.2 M- and 0.7 M-NaCl. The cell-free-derived normal AT-III also reacted with alpha-thrombin. Approx. 15% of this AT-III formed covalent complexes with alpha-thrombin in 2 min. Unfractionated heparin accelerated the rate of formation of such complexes. Two truncated forms of AT-III (amino acid residues 219-432 and 251-432), containing only the putative thrombin-binding domain, were synthesized independently in this cell-free system. These truncated AT-III polypeptides did not bind heparin and were unable to form stable covalent complexes with alpha-thrombin. However, both of these AT-III polypeptides were cleaved by alpha-thrombin, presumably at the reactive centre Arg-393-Ser-394. The formation of the disulphide bond between Cys-247 and Cys-430 in AT-III-(219-432)-polypeptide had no effect on the results obtained. Mutations in full-length AT-III at Cys-430 had no effect on the ability of AT-III to bind heparin. There was, however, a slight decrease in the formation of stable inhibitory complexes with alpha-thrombin. A cell-free-derived AT-III mutant, devoid of amino acid residues 41-49, which comprise heparin-binding region 1 of AT-III, had slightly decreased heparin binding compared with cell-free-derived normal AT-III-(1-432)-polypeptide. This mutant AT-III polypeptide was unable, however, to form a stable complex with alpha-thrombin. We conclude therefore that the N-terminal domain of AT-III is essential for both heparin binding and complex-formation with alpha-thrombin, but not for the cleavage of AT-III at its reactive centre by alpha-thrombin.
Association of cyclic-AMP-dependent protein kinase with neurofilaments
Authors: Dosemeci A , Pant H C
Published: August 2015

Neurofilament preparations isolated from bovine spinal cord contain cyclic-AMP-dependent protein kinase (PKA) activity. Treatment of this preparation with cyclic AMP, to dissociate the regulatory subunit of the kinase from the catalytic subunit, resulted in retention of the kinase activity but loss of cyclic AMP regulation. This suggests that PKA is associated via its catalytic subunit with the neurofilament preparation. The association of exogenous PKA from bovine heart with the neurofilament preparation and with neurofilaments reconstituted from purified neurofilament proteins was also investigated. Either the free catalytic subunit or combinations of the catalytic and regulatory subunits of PKA were incubated with the preparations, and the degree of association was determined as the level of kinase activity that co-sediments with neurofilaments. The results indicate that the free catalytic subunit of PKA co-sediments with neurofilaments reconstituted from purified proteins. The regulatory subunit of PKA from bovine heart, when pre-mixed with the catalytic subunit, decreased the level of kinase that co-sediments with the neurofilament fraction in a dose-dependent manner. This effect of the regulatory subunit was reversed by inclusion of cyclic AMP in the incubation medium before centrifugation. The above findings suggest that the regulatory subunit, when attached to the catalytic subunit, has an inhibitory effect on its association with neurofilaments, with the implication that the association may be a cyclic-AMP-regulated event.
Kinetic differentiation between enzyme inactivation involving complex-formation with the inactivator and that involving a conformation-change step
Authors: Liu C , Tsou C L
Published: August 2015

It has been suggested that the complexing type of inactivation in which the inactivator binds reversibly with the enzyme before inactivation cannot be differentiated kinetically from that a slow enzyme conformation change is involved as a first step [Rakitzis (1986) J. Theor. Biol. 122, 247-249]. The kinetics of the substrate reaction during modification of enzyme activity previously described [Tsou (1988) Adv. Enzymol. Relat. Areas Mol. Biol. 61, 381-436] have now been applied to this problem and equations derived to show that the slow-conformational-change type can be differentiated from the complexing type by plotting the final concentration of product formed, [P]infinity, against the reciprocal of inactivator concentration. The reaction of hexokinase with 2-chloromercuri-4-nitrophenol has been shown to involve a conformational change of the enzyme before inactivation.
The effect of protein kinase C activation on muscarinic-M3- and K+-evoked release of [3H]noradrenaline and increases in intracellular Ca2+ in human neuroblastoma SH-SY5Y cells
Authors: Murphy N P , McCormack J G , Ball S G , Vaughan P F T
Published: August 2015

Short-term pretreatment (9 min) with the phorbol ester 12-myristate 13-acetate (PMA) alone had no effect on the basal release of [3H]noradrenaline ([3H]NA), but enhanced K+ (100 mM)-, acetylcholine (0.1 mM)-, carbachol (1 mM)-, muscarine (1 mM)- and arecoline (1 mM)-evoked release by 2.3-, 6.4-, 3.0-, 2.0- and 2.0-fold respectively in SH-SY5Y cells. Maximum effects of PMA were observed after a 10 min preincubation at a concentration of 0.1 microM. There was a 4-fold decrease in the EC50 values (concentration required for 50% of maximal stimulation) observed for carbachol- and acetylcholine-evoked release of [3H]NA in the presence of PMA. The inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate did not alter the K(+)- or carbachol-evoked release of [3H]NA. The enhancement of release in the presence of PMA was more potently inhibited by the protein kinase C inhibitors RO 31-7549 [concentration required for 50% inhibition (IC50) = 0.18 microM/bd and RO 31-8220 (IC50 = 0.56 microM) than by either polymyxin-B or H-7. Furthermore, in the absence of PMA, both K(+)- and carbachol-evoked release was inhibited by these antagonists. Atropine, hexahydro-sila-difenidol and pirenzepine antagonized the PMA-enhanced carbachol-evoked release of [3H]NA, with Ki values of 2.75 +/- 0.25 nM, 2.6 +/- 0.64 nM and 294 +/- 17 nM respectively. These values were consistent with the coupling of an M3 muscarinic receptor to the release of [3H]NA in SH-SY5Y cells. Whereas pretreatment with PMA (5 min) enhanced M3-evoked release of [3H]NA, it decreased the muscarinic-agonist-evoked initial peak (greater than 85%) and plateau phase in intracellular Ca2+. These results suggest that noradrenaline release evoked by muscarinic agonists was triggered not only by relatively small changes in Ca2+ but also by activation of protein kinase C.
Endocytosis and intracellular processing of tissue-type plasminogen activator by rat liver cells in vivo
Authors: Stang E , Krause J , Seydel W , Berg T , Roos N
Published: August 2015

Endocytosis of tissue-type plasminogen activator (t-PA) by different types of rat liver cells was studied in immunocytochemically labelled cryosections as well as in biochemical experiments. For morphological localization of the ligand in different endocytic compartments involved in its catabolism, rat livers were fixed at various times (1-24 min) after injection of t-PA. Late-endosomal and lysosomal compartments were identified by double-labelling the sections with antibodies to the lysosomal proteins glycoprotein Igp 120 and cathepsin D. In liver t-PA was localized in sinusoidal endothelial cells (EC), parenchymal cells (PC) and to some extent in Kupffer cells (KC), indicating that it is internalized and degraded in all three cell types. In specimens fixed 6 min after injection PC, EC and KC were found to contribute to 69, 24 and 7% respectively of total t-PA endocytosed. The transfer from late endosomes to lysosomes was found to be faster in EC than in PC. The morphological findings were supported by studies of the endocytic mechanisms employing isolated perfused livers and primary hepatocytes. The presence of monensin, an inhibitor of lysosomal protein degradation, reduced the amount of t-PA degraded to about 50% of the control values. The catalytic site seems not to be required for the catabolism of t-PA in hepatic cells. The inhibition of t-PA by D-phenylalanyl-L-prolylarginyl-chloromethane did not influence receptor recognition and catabolic processing, as determined in morphological studies using labelled cryosections, in binding studies employing liver cell membranes and primary hepatocytes, as well as in liver-perfusion experiments.
Ubiquitin-RAS peptide extensions as substrates for farnesyl-protein transferase and carboxymethyltransferase
Authors: Yoo Y , Watts S , Rechsteiner M
Published: August 2015

Using oligonucleotide-mediated ‘loop-in’ mutagenesis strategies in M13, a heat-inducible ubiquitin (Ub) gene was extended by sequences coding for the C-terminal 11 amino acids of Ha-RAS. The resulting gene was transformed into AR13 and production of the Ub-peptide extension was induced by heat treatment. After one-step purification, the fusion protein (Ub-cRAS) was used as a substrate for farnesyl-protein transferase. Ub-cRAS was farnesylated on incubation in Xenopus egg extract or rabbit reticulocyte lysate. In contrast, when serine was substituted for the last cysteine in the RAS extension, transfer of the [3H]farnesyl group from [3H] farnesyl pyrophosphate to the modified Ub-cRAS was not observed. Farnesylation of Ub-cRAS permitted us to develop an easy membrane-binding assay for farnesyl-protein transferase enzyme activity. Using this assay, we partially purified the enzyme from rabbit reticulocyte lysate. We also detected methylation of the farnesylated Ub-cRAS terminus in Xenopus egg extract.
Regulation of Na+/Ca2+ exchange in the rat pancreatic B cell
Authors: Plasman P O , Herchuelz A
Published: August 2015

Na+/Ca2+ exchange in the B cell was recently characterized by measuring intracellular-Na(+)-dependent 45Ca2+ uptake in isolated rat pancreatic islet cells. The aim of the present study was to investigate the regulation of this process. Extracellular pH (pHo) and intracellular pH (pHi) markedly affected Na+/Ca2+ exchange. A fall of 0.04 unit in pHi decreased the exchange by 45%, whereas a rise of 0.13 unit increased the uptake by 70%. Mitochondrial poisons (oligomycin, antimycin A and 2,4-dinitrophenol) inhibited reverse Na+/Ca2+ exchange by about 25-50%. The exchanger displayed a low Q10 (temperature coefficient), indicating that it is only indirectly dependent on metabolic energy. The phorbol ester phorbol 12-myristate 13-acetate did not affect Na+/Ca2+ exchange. Likewise, lowering the extracellular K+ concentration did not inhibit 45Ca2+ uptake. In conclusion, the pHi and the metabolic state of the cell may represent important modulatory signals by which insulin secretagogues such as glucose could regulate reverse Na+/Ca2+ exchange in the B cell. The process does not appear to co-transport K+ nor to be influenced by protein kinase C.
Mapping of the epitope/paratope interactions of a monoclonal antibody directed against adenosine 3′,5′-monophosphate
Authors: Nass N , Colling C , Cramer M , Genieser H G , Butt E , Winkler E , Jaenicke L , Jastorff B
Published: August 2015

A series of systematically modified cyclic AMP (cAMP) analogues, including newly synthesized benzimidazole ribofuranosyl 3′,5′-monophosphates was used to map the essential molecular interactions between cAMP and the monoclonal antibody 4/2C2 (mab 4/2C2) directed against 2′-O-succinoyl cAMP [Colling, Gilles, Nass, Moka & Jaenicke (1988) Second Messengers Phosphoproteins 12, 123-133]. Its paratope binds the purine base in syn conformation by dipole-dipole interactions and hydrophobic forces and/or stacking interactions. The ribose phosphate moiety is recognized by a combination of charge interactions and H-bonds to the exocyclic and the 5′-oxygen atoms and a hydrophobic interaction at the 2′-position. There is no regioselectivity for the exocyclic oxygen atoms. Compared with the known types of binding, mab 4/2C2 thus shows a new combination of molecular interactions which may be the basis of its strikingly specific recognition and binding of the cyclic adenylates. On this account mab 4/2C2 may become an important tool in studies on cAMP metabolism.
Direct electrochemical studies of hydrogenase and CO dehydrogenase
Authors: Smith E T , Ensign S A , Ludden P W , Feinberg B A
Published: August 2015

The reduction potentials of two relatively high-molecular-mass enzymes were determined directly at an edge pyrolytic graphite electrode by using square-wave voltammetry. The equilibrium reduction potential versus standard hydrogen electrode was determined for Clostridium pasteurianum hydrogenase I (E'0 = -377 +/- 10 mV; molecular mass 60 kDa) and Rhodospirillum rubrum carbon monoxide dehydrogenase (E'0 = -418 +/- 7 mV; molecular mass 62 kDa). The reduction potential of each enzyme was pH-independent, and one electron was transferred per redox centre. The reduction potential was determined to be identical for the CO dehydrogenase, semi-apo-(CO dehydrogenase), and CO dehydrogenase with carbonyl sulphide (COS) or cyanide bound. The electron-transferring efficiency of CO dehydrogenase was affected by two inhibitors, COS and cyanide, as indicated by a diminished analytic current.
The biologically active phospholipid, lysophosphatidic acid, induces phosphatidylcholine breakdown in fibroblasts via activation of phospholipase D. Comparison with the response to endothelin
Authors: van der Bend R L , de Widt J , van Corven E J , Moolenaar W H , van Blitterswijk W J
Published: August 2015

Lysophosphatidic acid (LPA) is a simple phospholipid that possesses hormone- and growth-factor-like properties. LPA initiates its action by inducing GTP-dependent phosphoinositide hydrolysis and inhibiting adenylate cyclase [van Corven, Groenink, Jalink, Eichholtz & Moolenaar (1989) Cell 59, 45-54]. Here we show that LPA stimulates rapid breakdown of phosphatidylcholine (PC) in Rat-1 fibroblasts. LPA-induced PC breakdown occurs through activation of phospholipase D (PLD), as measured by the formation of free choline and phosphatidic acid and by transphosphatidylation in the presence of butan-1-ol. LPA also stimulates generation of diacylglycerol, but there is no detectable formation of phosphocholine, suggesting that a PC-specific phospholipase C (PLC) is not involved. The response to LPA was compared with that to endothelin, a potent inducer of phospholipid hydrolysis but a poor mitogen for Rat-1 cells. Our results indicate that: (1) LPA is less efficient than endothelin in inducing phosphoinositide and PC breakdown; (2) LPA-induced PLD activation is short-lived, levelling off after 2 min, whereas the endothelin-stimulated increase in PLD activity persists for at least 1 h; (3) the effect of LPA on PLD, like that of endothelin, is blocked by long-term pretreatment of the cells with phorbol ester, suggesting that PLD activation occurs through a protein kinase C-dependent mechanism. Furthermore, our results support the notion that there is no simple causal relationship between the degree of agonist-induced phospholipid hydrolysis and the magnitude of the mitogenic response.
A serine proteinase of an archaebacterium, Halobacterium mediterranei. A homologue of eubacterial subtilisins
Authors: Stepanov V M , Rudenskaya G N , Revina L P , Gryaznova Y B , Lysogorskaya E N , IYu Filippova , Ivanova I I
Published: August 2015

A homogeneous serine proteinase secreted by the extreme halophilic bacterium Halobacterium mediterranei 1538 was isolated by affinity chromatography on bacitracin-Sepharose with a yield of 48% (260-fold purification). The enzyme reveals an optimum for pyroglutamyl-Ala-Ala-Leu p-nitroanilide hydrolysis at pH 8.0-8.5 (Km 0.14 mM; k(cat). 36.9 s-1). Its activity increases linearly with NaCl concentration over the range 2-5 M. The substrate specificity of the enzyme is comparable with that of secretory subtilisins, the extent of protein degradation approaching that attained with proteinase K. The enzyme has a molecular mass of 41 kDa and a pI of 7.5. The N-terminal sequence of H. mediterranei serine proteinase reveals a 50% identity with that of Thermoactinomyces vulgaris serine proteinases, indicating that the enzyme belongs to the subtilisin family. Hence the serine proteinase secreted by the halophilic bacterium should be considered as a functional analogue, and a structural homologue, of eubacterial serine proteinases (subtilisins).