bluuebunny/crossref_metadata_2025 huggingface dataset:
Two independently regulated Ca2+ entry mechanisms coexist in Jurkat T cells during T cell receptor antigen activation
Authors: Chow S C , Kass G E , Orrenius S
Published: August 2015

Receptor-mediated Ca2+ influx was studied in the human leukaemic T cell line, Jurkat. Stimulation of these cells through the T cell antigen-receptor complex with OKT3 (an antibody against the CD3 molecules of the T cell antigen-receptor complex), or inhibition of the endoplasmic reticular Ca(2+)-ATPase with thapsigargin, resulted in Ca2+ mobilization from intracellular stores and the activation of Ca2+ and Mn2+ entry. The rates of thapsigargin-induced Ca2+ and Mn2+ entry in Jurkat cells were 76% and 64% respectively of those observed after treatment of these cells with OKT3. The combined addition of thapsigargin plus OKT3 to Jurkat cells produced an enhanced effect on the sustained increase in the cytosolic free Ca2+ concentration that was greater than that obtained by addition of thapsigargin or OKT3 alone. The rates of Ca2+ and Mn2+ entry were increased to 119% and 112% respectively of the OKT3-induced rates. Taken together, these results suggest that the inositol 1,4,5-trisphosphate-sensitive Ca(2+)-pool-dependent bivalent cation entry only accounts for 57% and 52% respectively of the total OKT3-dependent Ca2+ and Mn2+ entry, and that the rest is mediated by second messenger(s). Thus two separate pathways coexist in regulating Ca2+ entry in Jurkat cells during activation mediated through the T cell receptor.
Properties of the pyridoxaldimine form of glutamate semialdehyde aminotransferase (glutamate-1-semialdehyde 2,1-aminomutase) and analysis of its role as an intermediate in the formation of aminolaevulinate
Authors: Tyacke R J , Harwood J L , John R A
Published: August 2015

Glutamate semialdehyde aminotransferase (glutamate-1-semialdehyde 2,1-aminomutase; EC 5.4.3.8) was converted into its pyridoxaldimine form by exhaustive replacement of endogenous pyridoxamine phosphate with pyridoxal phosphate. The isomerization of glutamate 1-semialdehyde to 5-aminolaevulinate by this form of the enzyme followed an accelerating time course which indicated that the enzyme initially had no activity but was converted into the active pyridoxamine phosphate form in an exponential process characterized by a rate constant (k) of 0.027 s-1. The pyridoxaldimine form of the enzyme was converted rapidly into the pyridoxamine form by (S)-4-aminohex-5-enoate and much more slowly by 4-aminobutyrate. The steady-state velocity of the enzyme increased in a markedly non-linear fashion with increasing enzyme concentration, indicating that the extent of dissociation of an intermediate in the reaction to free diaminovalerate and the pyridoxaldimine form of the enzyme depends upon the concentration of the enzyme.
Purification and properties of the uroporphyrinogen decarboxylase from Rhodobacter sphaeroides
Authors: Jones R M , Jordan P M
Published: August 2015

Uroporphyrinogen decarboxylase (EC 4.1.1.37) was purified 600-fold from Rhodobacter sphaeroides grown anaerobically in the light. The enzyme, under both denaturing and non-denaturing conditions, is a monomer of M(r) 41,000. The Km values are 1.8 microM and 6.0 microM for the conversion of uroporphyrinogen I and III to coproporphyrinogen I and III respectively. The enzyme is susceptible to inhibition by both uroporphyrinogen and uroporphyrin. The pH optimum is 6.8 and the isoelectric point is 4.4. The importance of cysteine and arginine residues is implicated from studies with inhibitors. The sequence of the first 29 amino acids of the N-terminus shows a high degree of similarity to the primary structures of other uroporphyrinogen decarboxylases. Studies on the order of decarboxylation of the four acetic acid side chains of uroporphyrinogen III suggest that at high substrate levels a random route is preferred.
Definition of the EGTA-independent interface involved in the serum gelsolin-actin complex
Authors: Feinberg J , Capony J P , Benyamin Y , Roustan C
Published: August 2015

The gelsolin-actin complex in the presence of Ca2+ revealed at least three interacting sites on the gelsolin molecule located in the S1, S2-3, and S4-6 domains. In the presence of EGTA, the N-terminal domain of gelsolin is known to be involved. However, the corresponding site on the surface of actin is poorly defined. The present result locates the Ca(2+)-independent plasma gelsolin-binding site on the actin surface. Natural and synthetic actin peptides were tested for their possible interaction with gelsolin and monitored by fluorescence anisotropy measurements and e.l.i.s.a. The interface was thus located within the 360-372 actin sequence near the C-terminal extremity. In addition, we used a chymotryptic digest of gelsolin and determined that its N-terminal domain (S1) was implicated in this interface. We conclude that the interaction of the 41-126 region of plasma gelsolin is the counterpart of the 360-372 sequence in subdomain 1 of actin.
Stereoselectivity of Ins(1,3,4,5)P4 recognition sites: implications for the mechanism of the Ins(1,3,4,5)P4-induced Ca2+ mobilization
Authors: Wilcox R A , Challiss R A , Baudin G , Vasella A , Potter B V , Nahorski S R
Published: August 2015

Ins(1,3,4,5)P4 was able to mobilize the entire Ins(1,4,5)P3-sensitive intracellular Ca2+ store in saponin-permeabilized SH-SY5Y human neuroblastoma cells in a concentration-dependent manner, yielding an EC50 value of 2.05 +/- 0.45 microM, compared with 0.14 +/- 0.03 microM for Ins(1,4,5)P3. However, L-Ins(1,3,4,5)P4 [= D-Ins(1,3,5,6)P4] failed to cause mobilization of intracellular Ca2+ at concentrations up to 100 microM. Binding studies using pig cerebellar membranes as a source of both Ins(1,4,5)P3/Ins(1,3,4,5)P4-specific binding sites have revealed a marked contrast in their stereospecificity requirements. Ins(1,4,5)P3-receptors from pig cerebella exhibited stringent stereospecificity, L-Ins(1,4,5)P3 and L-Ins(1,3,4,5)P4 were > 1000-fold weaker, whereas Ins(1,3,4,5)P4 (IC50 762 +/- 15 nM) was only about 40-fold weaker than D-Ins(1,4,5)P3 (IC50 20.7 +/- 9.7 nM) at displacing specific [3H]Ins(1,4,5)P3 binding from an apparently homogeneous Ins(1,4,5)P3 receptor population. In contrast, the Ins(1,3,4,5)P4-binding site exhibited poor stereoselectivity. Ins(1,3,4,5)P4 produced a biphasic displacement of specific [32P]Ins(1,3,4,5)P4 binding, with two-site analysis revealing KD values for high- and low-affinity sites of 2.1 +/- 0.5 nM and 918 +/- 161 nM respectively. L-Ins(1,3,4,5)P4 also produced a biphasic displacement of specific [32P]Ins(1,3,4,5)P4 binding which was less than 10-fold weaker than with D-Ins(1,3,4,5)P4 (IC50 values for the high- and low-affinity sites of 17.2 +/- 3.7 nM and 3010 +/- 542 nM respectively). Therefore, although L-Ins(1,3,4,5)P4 appears to be a high-affinity Ins(1,3,4,5)P4-binding-site ligand in pig cerebellum, it is a very weak agonist at the Ca(2+)-mobilizing receptors of permeabilized SH-SY5Y cells. We suggest that the ability of D-Ins(1,3,4,5)P4 to access intracellular Ca2+ stores may derive from specific interaction with the Ins(1,4,5)P3- and not the Ins(1,3,4,5)P4-receptor population.
Clarification of the pH-dependent kinetic behaviour of papain by using reactivity probes and analysis of alkylation and catalysed acylation reactions in terms of multihydronic state models: implications for electrostatics calculations and interpretation of the consequences of site-specific mutations such as Asp-158-Asn and Asp-158-Glu
Authors: Mellor G W , Patel M , Thomas E W , Brocklehurst K
Published: August 2015

1. The complex behaviour of papain (EC 3.4.22.2) in acidic media has been investigated by (a) stopped-flow reactivity probe kinetics using 4,4′-dipyrimidyl disulphide (I) and 2,2′-dipyridyl disulphide (II) as thiol-specific time-dependent inhibitors with markedly different susceptibilities to activation by hydronation (protonation) and (b) using the multitasking application program SKETCHER for the rapid evaluation of pH-dependent kinetic data by means of interactive manipulation of calculated curves. 2. The substantially lower basicity of (I) (pKa 0.91) than that of (II) (pKa 2.45) combined with retention of high reactivity permitted the pKa for the formation of the (Cys-25)-S-/(His-159)-Im+H ion-pair state of papain to be determined kinetically as 3.4, a value close to that (3.3) deduced by potentiometric difference titration [Lewis, Johnson and Shafer (1976) Biochemistry 15, 5009-5017] and lower than the value (approx. 4) often reported from pH-dependent kinetic studies. The higher values are now known to arise from inadequate data analysis that does not take account of other overlapping kinetically influential ionizations. 3. Re-evaluation of the extensive sets of pH-kcat/Km data for the hydrolysis of nine substrates by papain reported by Polgár and Halász (1978) (Eur. J. Biochem. 88, 513-521) by making use of SKETCHER, the known pKa value (3.4) from the reaction with compound (I) and two additional kinetically influential pKa values deduced from the reaction with compound (II) now permits the identification of the pH-dependent events in reactions of papain with inhibitors and substrates. 4. A major conclusion is that, whereas in reactions of simple alkylating agents and compound (I) full nucleophilic character of (Cys-25)-S-/(His-159)-Im+H is provided by hydronic dissociation with pKa 3.3-3.4, in catalysis relatively little catalytic competence is produced consequent upon ion-pair formation. Substantial catalytic competence requires further hydronic dissociation with pKa approx. 4, and for cationic substrates further enhancement is produced by hydronic dissociation with pKa approx. 5. 5. The present work, together with the kinetic analysis of reactions of papain in alkaline media reported by Mellor, Thomas, Topham and Brocklehurst [Biochem. J. (1993) 290, 289-296], defines the kinetically influential ionizations of papain as 3.4, 4.0, 5.0, 8.3 and 10.0 of which 3.4 and 8.3 relate to the formation and subsequent dehydronation of the ion-pair state.(ABSTRACT TRUNCATED AT 400 WORDS)
Ricin induces the production of tumour necrosis factor-α and interleukin-1 β by human peripheral-blood mononuclear cells
Authors: Licastro F , Morini M C , Bolognesi A , Stirpe F
Published: August 2015

Ricin induced the release of tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 beta by human peripheral-blood mononuclear cells in a dose- and time-dependent manner. The inhibition induced by ricin upon the phytohaemagglutinin (PHA)-driven lymphocyte proliferation was greater in cultures of mononuclear cells than that observed in monocyte-free cultures of lymphocytes, and was decreased after addition of an anti-TNF-alpha antibody to PHA-activated cultures. Low levels of TNF-alpha were detected in plasma from rats poisoned with ricin. These results suggest that ricin induced the release of macrophage-derived cytokines which may have a role in the pathogenesis of ricin toxic effects.
Modulation of the proton-translocation stoichiometry of H+-ATP synthases in two phototrophic prokaryotes by external pH
Authors: Krenn B E , Van Walraven H S , Scholts M J C , Kraayenhof R
Published: August 2015

The stoichiometry between proton translocation and ATP synthesis/hydrolysis was studied in two different photosynthetic prokaryotes, the thermophilic cyanobacterium Synechococcus 6716 and the purple bacterium Rhodospirillum rubrum. The H+/ATP ratio was determined by acid-base transitions as a function of the external pH. The H+/ATP ratio of the Synechococcus 6716 ATP synthase was found to increase with increasing pH. In contrast, in R. rubrum this ratio decreased with increasing pH. These results were qualitatively supported by experiments using the fluorescence probe 9-aminoacridine. The degree of coupling between the H+ flux and the ATP synthesis/hydrolysis reaction is apparently modulated by the conditions under which the proton pump has to work. Such modulation of the H+/ATP ratio may be of physiological significance for an organism, for example when ATP synthesis is necessary at low proton-electrochemical potential difference (delta mu H+ levels). The different pH dependencies of the H+/ATP ratios in these organisms are considered in relation to the differences in the charged amino acids that are present in the F0 subunits a and c.
Effect of cell volume on Acridine Orange fluorescence in hepatocytes
Authors: Völkl H , Friedrich F , Häussinger D , Lang F
Published: August 2015

Hepatic proteolysis is inhibited by cell swelling following a variety of experimental manoeuvres, such as reduction of extracellular osmolarity, concentrative uptake of amino acids, or blockade of K+ channels by barium. On the other hand, proteolysis is known to be accomplished by pH-sensitive lysosomal proteases. Accordingly, NH3/NH4+ inhibits proteolysis by intralysosomal alkalinization. The present study has been performed to test for an effect of cell volume on the pH of acidic intracellular compartments, as assessed by Acridine Orange fluorescence at > 520 nm (F > 520). F > 520 is enhanced by NH3/NH4+ (2 and 20 mmol/l respectively), by glutamine (2 mmol/l), by the K(+)-channel blocker barium (10 mmol/l) and by reduction of extracellular osmolarity (by 20 and 80 mosmol/l respectively). The observations point to release of Acridine Orange from acidic cellular compartments, which is indicative of alkalinization of these compartments during cell swelling. This effect may contribute to the regulation of proteolysis.
Substrate affinity of the protein tyrosine kinase pp60c-src is increased on thrombin stimulation of human platelets
Authors: Liebenhoff U , Brockmeier D , Presek P
Published: August 2015

Human blood platelets contain high levels of non-receptor protein tyrosine kinases of the Src family, particularly pp60c-src, suggesting an important role for these enzymes in platelet physiology. Indeed, in response to various agonists of platelet function, a number of proteins become phosphorylated at tyrosine residues. However, no enzymic activation of an Src-related tyrosine kinase has yet been shown in platelets. In searching for the kinase(s) responsible, we found that all agonists tested that directly or indirectly activate protein kinase C in platelets (phorbol 12-myristate, 13-acetate, thrombin, vasopressin, collagen, calcium ionophore A23187) increased the overall activity of pp60c-src determined by IgG phosphorylation in an immunocomplex assay in the presence of low ATP concentrations. On the other hand, elevation of cyclic AMP directly by forskolin or indirectly by prostaglandin E1, or elevation of cyclic GMP by sodium nitroprusside did not significantly affect the activity of the enzyme. To substantiate the differences in enzyme activity, we determined Km and Vmax, values of pp60c-src from resting and thrombin-stimulated platelets. Thrombin treatment increased substrate affinity of pp60c-src as indicated by a 2- to 3-fold decrease in the Km values for ATP and the exogenous protein substrate casein. Vmax. values were only slightly altered under the assay conditions used. To further rule out modifications of pp60c-src in phosphorylation as a probable cause of the changed substrate affinity, we analysed tryptic phosphopeptides of immunoprecipitated, 32P-labelled pp60c-src of unstimulated and stimulated platelets. The platelet agonists listed above induced an increase in pp60c-src phosphorylation at Ser-12, which is the amino acid phosphorylated by protein kinase C. Surprisingly, we found that elevation of cyclic AMP did not affect 32P labelling of pp60c-src. On the basis of our data, we suggest that phosphorylation at Ser-12 might be one of the signal-triggering events that cause the increase in substrate affinity of pp60c-src.
Ligand-induced conformational changes modify proteolytic cleavage of the adipocyte insulin-sensitive glucose transporter
Authors: Yano Y , May J M
Published: August 2015

The transport conformation of the human erythrocyte glucose transporter (GLUT1) modifies rates of proteolytic cleavage of this protein by a variety of enzymes. We investigated the effects of ligand-induced conformational change on the susceptibility to enzymic cleavage of the insulin-sensitive rat adipocyte glucose transporter (GLUT4). A GLUT4-enriched slow sedimenting microsomal fraction was prepared from basal adipocytes and subjected to PAGE and immunoblotting. The GLUT4 protein was detected in these immunoblots with a C-terminal-specific antiserum as an M(r)-46,000-50,000 doublet. GLUT1 protein was not detected by a GLUT1-specific antiserum in these membranes. Tryptic digestion caused loss of the GLUT4 signal in immunoblots in a time- and concentration-dependent fashion. Low-M(r) membrane-bound fragments were not observed in electrophoretic gels, whether detection was attempted by immunoblotting or by counting radioactivity in gel slices following photolabelling with [3H]cytochalasin B. Transport-specific ligands known to induce an outward-facing conformation in the human erythrocyte GLUT1 protein retarded cleavage of the GLUT4 protein by submaximal concentrations of trypsin, whereas ligands known to induce an inward-facing conformation increased the extent of cleavage. The transported substrate D-glucose retarded tryptic cleavage of GLUT4. This result contrasts with the known behaviour of GLUT1, in which D-glucose accelerates cleavage. Cleavage of GLUT4 by thermolysin was also retarded by the outward-binding analogue 4,6-O-ethylidene glucose. These results show that the conformational sensitivity to proteolysis of GLUT4 mirrors that of GLUT1, except that the glucose-loaded GLUT4 has a different steady-state configuration, which may reflect underlying kinetic differences between the two proteins.
Diurnal patterns of cardiac and hepatic pyruvate dehydrogenase complex activity in gold-thioglucose-obese mice
Authors: Bryson J M , Cooney G J , Wensley V R , Blair S C , Caterson I D
Published: August 2015

The diurnal pattern of the activity of the pyruvate dehydrogenase complex (PDHC) was studied in the heart and liver of gold-thioglucose (GTG)-obese mice and age-matched controls. The diurnal pattern of lipogenesis was also measured in the liver. Both lean and obese mice had one main eating period, from 20:00 to 24:00 h. Eating produced no change in serum glucose of control mice but there was a significant rise in serum insulin and triacylglycerols. There was also a 3-fold increase in cardiac PDHC activity and a 3-fold increase in hepatic lipogenesis in the control mice, but little change in hepatic PDHC activity. GTG-obese mice were hyperglycaemic, hyperinsulinaemic and hypertriglyceridaemic at all times studied, with significant increases in these parameters being seen in response to eating. Eating produced little change in cardiac PDHC activity, but there was a 5-fold increase in hepatic PDHC activity, paralleled by a 10-fold increase in hepatic lipogenesis. Hepatic PDHC activity was significantly higher in GTG-obese mice at all times except 16:00 h. The simultaneous rise of hepatic PDHC activity, lipogenesis and serum triacylglycerols in GTG-obese mice suggests an increased utilization of glucose for lipogenesis. The lack of change in heart PDHC activity in GTG-obese mice over 24 h suggests that a general decrease in PDHC activity may contribute to the development of the glucose intolerance and insulin resistance of obesity and non-insulin-dependent diabetes. However, it appears that a different level of metabolic control allows hepatic PDHC activity of the same obese animals to increase in response to hyperinsulinaemia and contribute to the higher rates of lipogenesis seen in obese mice.
Intact transmembrane isoforms of the neural cell adhesion molecule are released from the plasma membrane
Authors: Olsen M , Krog L , Edvardsen K , Skovgaard L T , Bock E
Published: August 2015

Three soluble neural cell adhesion molecule (NCAM) polypeptide classes of M(r) values 190,000 (NCAM-s1), 135,000 (NCAM-s2) and 115,000-110,000 (NCAM-s3) have been demonstrated in rat brain and cerebrospinal fluid [Krog, Olsen, Dalseg, Roth and Bock (1992) J. Neurochem. 59, 838-847]. NCAM-s3 is known to arise from released glycosylphosphatidylinositol (GPI)-linked NCAM [He, Finne and Goridis (1987) J. Cell. Biol. 105, 2489-2500] as well as from extracellularly cleaved transmembrane NCAM isoforms [Nybroe, Linnemann and Bock (1989) J. Neurochem. 53, 1372-1378]. In this study the origin of NCAM-s1 and NCAM-s2 and the function of soluble NCAM forms were investigated. It was shown that all three soluble forms could be released from brain membranes with M(r) values identical to the three major membrane-associated forms: the large transmembrane 190,000-M(r) form (NCAM-A), the smaller transmembrane 135,000-M(r) form (NCAM-B) and the GPI-anchored 115,000-110,000-M(r) form (NCAM-C). A polyclonal antibody, directed against transmembrane and cytoplasmic epitopes common to NCAM-A and NCAM-B, was shown to react with NCAM-s1 and NCAM-s2. Furthermore, NCAM-B was shown to be shed in a presumably intact soluble form from membranes of cells transfected with this isoform. Thus, NCAM-s1 and NCAM-s2 probably represent intact released transmembrane NCAM-A and NCAM-B. The soluble transmembrane forms are likely to exist in vivo, as NCAM-s1 and NCAM-s2 were readily demonstrated in cerebrospinal fluid. By density-gradient centrifugation it was shown that shed transmembrane NCAM-B was present in fractions of high, as well as low, density, indicating that a fraction of the shed NCAM is associated with minor plasma membrane fragments. Finally, it was shown that isolated soluble NCAM inhibited cell binding to an immobilized NCAM substratum, attributing a pivotal role to soluble NCAM in vivo as a modulator of NCAM-mediated cell behaviour.
Dual phosphorylation and autophosphorylation in mitogen-activated protein (MAP) kinase activation
Authors: Her J H , Lakhani S , Zu K , Vila J , Dent P , Sturgill T W , Weber M J
Published: August 2015

p42mapk [mitogen activated protein (MAP) kinase; extracellular signal-regulated protein kinase (ERK)] is a serine/threonine-specific protein kinase that is activated by dual tyrosine and threonine phosphorylation in response to diverse agonists. Both the tyrosine and threonine phosphorylations are necessary for full enzymic activity. A MAP kinase activator recently purified and cloned has been shown to be a protein kinase (MAP kinase kinase) that is able to induce the dual phosphorylation of MAP kinase on both the regulatory tyrosine and threonine sites in vitro. In the present paper we have utilized MAP kinase mutants altered in the sites of regulatory phosphorylation to show, both in vivo and in vitro, that phosphorylation of the tyrosine and the threonine can occur independently of one another, with no required order of phosphorylation. We also utilized kinase-defective variants of MAP kinase with mutations in either the ATP-binding loop or the catalytic loop, and obtained data suggesting that the activity or structure of the catalytic loop of MAP kinase plays an important role in its own dual phosphorylation.
Affinity labelling of smooth-muscle myosin light-chain kinase with 5′-[p-(fluorosulphonyl)benzoyl]adenosine
Authors: Komatsu H , Ikebe M
Published: August 2015

5′-(p-(Fluorosulphonyl)[14C]benzoyl)adenosine (FSBA) was synthesized and used as a probe to study the ATP-binding site of smooth-muscle myosin light-chain kinase (MLCK). FSBA modified both free MLCK and calmodulin/MLCK complex, resulting in inactivation of the kinase activity. Nearly complete protection of the calmodulin/MLCK complex against FSBA modification was obtained by addition of excess ATP whereas MLCK activity alone was lost in a dose-dependent manner even in the presence of excess ATP. These results suggest that FSBA modified ATP-binding sites and ATP-independent sites, and the latter sites are protected by calmodulin binding. The results also suggest that the ATP-binding site is accessible to the nucleotide substrate regardless of calmodulin binding. The FSBA-labelled MLCK was completely proteolysed by alpha-chymotrypsin, and the 14C-labelled peptides were isolated and sequenced. The sequence of the labelled peptide was Ala-Gly-X-Phe, where X is the labelled residue. The sequence was compared with the known MLCK sequence, and the labelled residue was identified as lysine-548, which is located downstream of the GXGXXG motif conserved among ATP-utilizing enzymes.
The developmental profile of lactoferrin in mouse epididymis
Authors: Yu L C , Chen Y H
Published: August 2015

A sandwich e.l.i.s.a. method was developed to examine the distribution of lactoferrin in mouse reproductive tract. The lactoferrin concentration was found to be much higher in oviduct, uterus, vagina, vas deferens or epididymis than in serum, but the concentration in ovary, testis, seminal vesicle, prostate or coagulating gland was comparable with that in serum. The existence of lactoferrin in male sexual organs was confirmed by Western-blot analyses for tissue proteins. Lactoferrin in male sexual organs was shown to have a molecular mass similar to that of the deglycosylated form of lactoferrin purified from mouse uterine luminal fluid. Northern-blot analyses for total RNA prepared from male sexual organs indicated that only epididymis contained the lactoferrin mRNA. The lactoferrin mRNA was found in the prepubertal period and increased with the growth of epididymis. The mRNA level in prepubertal epididymis could be stimulated by 17 beta-oestradiol, but was not influenced by testosterone.
Metabolism of endogenous nutrients in islets of Goto-Kakizaki (GK) rats
Authors: Sener A , Malaisse-Lagae F , Östenson C G , Malaisse W J
Published: August 2015

The metabolism of endogenous nutrients was examined in pancreatic islets of control and Goto-Kakizaki (GK) rats. At the ultrastructural level, no glycogen was found in the islet cells of GK rats, a situation similar to that prevailing in normal islets. Likewise, by measuring the output of L-lactate from islets first incubated at 16.7 mM D-glucose and then at 2.8 mM D-glucose, no evidence of glycogenolysis was found in the islets of GK rats. The production of NH4+ and that of 14CO2 from islets prelabelled with either L-[U-14C]glutamine or [U-14C]palmitate were higher, however, in GK than in control rats. The changes in NH4+ and 14CO2 production evoked by D-glucose, by a non-metabolized analogue of L-leucine (2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid; BCH) and by 3-phenylpyruvate were qualitatively comparable in control and GK rats. The secretory response to these three secretagogues was severely decreased in the islets of GK rats. This coincided with an impaired enhancing action of D-glucose on the conversion of [2-3H]glycerol into 3HOH. It is concluded that the catabolism of endogenous amino and fatty acids in islets is greater in GK than in control rats, especially at low D-glucose concentration. This may account, in part at least, for the altered secretory response to BCH and 3-phenylpyruvate. For glucose-induced insulin release, however, an impaired acceleration of the glycerol phosphate shuttle apparently also participates in the secretory defect.
13C n.m.r. isotopomer and computer-simulation studies of the non-oxidative pentose phosphate pathway of human erythrocytes
Authors: Berthon H A , Bubb W A , Kuchel P W
Published: August 2015

13C double-quantum filtered correlation spectroscopy (DQF-COSY) provides a novel method for the detection of reactions involving carbon-bond scissions. We report the use of this technique to investigate isotopic exchange reactions of the non-oxidative pentose phosphate pathway in human erythrocytes. These exchange reactions resulted in the formation of a range of isotopic isomers (isotopomers) of glucose 6-phosphate after incubation of a mixture of universally 13C-labelled and unlabelled glucose 6-phosphate with fructose 1,6-bisphosphate and haemolysates. These isotopomers were detected in the coupling patterns of cross-peaks within the DQF-COSY spectrum of the deproteinized sample. A computer model which fully describes the reactions of the non-oxidative pentose phosphate pathway in human erythrocytes has previously been constructed and tested with 31P n.m.r. time-course data in our laboratory. This model was refined using 13C n.m.r. time-course data and extended to include the range of isotopomers which may be formed experimentally by the reactions of the non-oxidative pentose phosphate pathway. The isotopomer ratios obtained experimentally from the DQF-COSY spectrum were consistent with simulations generated by this model.
Reaction of proteasomes with peptidylchloromethanes and peptidyldiazomethanes
Authors: Savory P J , Djaballah H , Angliker H , Shaw E , Rivett A J
Published: August 2015

The multicatalytic endopeptidase complex (proteasome) has multiple distinct peptidase activities. These activities have often been referred to as ‘chymotrypsin-like’, ‘trypsin-like’ and ‘peptidylglutamyl-peptide hydrolase’ activities according to the type of residue in the P1 position, although it is now clear that mammalian proteasomes have at least five distinct catalytic sites. In the present study, potential affinity-labelling reagents (peptidylchloromethanes, peptidyldiazomethanes, a peptidylfluoromethane and peptidylsulphonium salts) containing hydrophobic, basic or acidic amino acid residues in the P1 position have been tested for inhibition of the different activities of the rat liver proteinase complex. The results show that individual peptidase activities of proteasomes can be inhibited by a variety of peptidylchloromethanes and peptidyldiazomethanes. Although the rate of inactivation of proteasomes by even the most effective peptidylchloromethanes and peptidyldiazomethanes are often quite slow (k(obs)/[I] in the range 0.1-10 M-1 x s-1) compared with the reaction of similar compounds with some other proteinases, the results provide useful information concerning the specificity of the distinct catalytic centres of proteasomes, and some selective affinity-labelling reagents have been identified. Tyr-Gly-Arg-chloromethane was found to be a useful inhibitor of trypsin-like activity. Inhibition of the other peptidase activities was often incomplete, even after repeated addition of inhibitor, and it proved to be difficult to predict the effect of different reagents. For example, Cbz-Tyr-Ala-Glu-chloromethane was found to inhibit ‘chymotrypsin-like’ activity (assayed with Ala-Ala-Phe-7-amino-4-methylcoumarin or succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin), while the best inhibitors of ‘peptidylglutamyl-peptide hydrolase’ activities (assayed with benzyloxycarbonyl-Leu-Leu-Glu beta-naphthylamide) were peptidyldiazomethanes containing hydrophobic amino acid residues. These results suggest that the original nomenclature of proteasome activities is misleading, because the residue in the P1 position is not the only determinant of specificity.
Effects of histone acetylation, ubiquitination and variants on nucleosome stability
Authors: Li W , Nagaraja S , Delcuve G P , Hendzel M J , Davie J R
Published: August 2015

The properties of the nucleosomes of a salt-soluble, transcriptionally active gene-enriched fraction of chicken erythrocyte chromatin were evaluated by hydroxyapatite dissociation chromatography. We have demonstrated previously that the salt-soluble, transcriptionally active gene-enriched polynucleosomes are enriched in dynamically acetylated and ubiquitinated histones, and in an atypical U-shaped nucleosome that possessed about 20% less protein than a typical nucleosome. Further, newly synthesized histones H2A and H2B exchange preferentially with the nucleosomal histones H2A and H2B of this salt-soluble chromatin fraction. Analysis of the histones eluting from the hydroxyapatite-bound chromatin demonstrated that hyperacetylated and ubiquitinated (u), including multi-ubiquitinated, H2A-H2B.1 dimers dissociated at lower concentrations of NaCl than unmodified dimers or dimers with histone variants H2A.Z and/or H2B.2. Cross-linking studies revealed that at least 50% of uH2B.1 was paired with uH2A. uH2A-uH2B.1 dimers dissociated at lower NaCl concentrations than H2A-uH2B.1 dimers. Hyperacetylated histone (H3-H4)2 tetramers also eluted at lower concentrations of NaCl than unmodified tetramers. Our results support the idea that acetylation and ubiquitination of histones H2A and H2B.1 increase the lability of H2A-H2B.1 dimers in transcriptionally active nucleosomes. In contrast, our observations suggest that histone variants H2A.Z and H2B.2. stabilize the association of the H2A-H2B dimer in nucleosomes. The elevated lability of the H2A-H2B dimer may facilitate processes such as the exchange of these dimers with newly synthesized histones, the elongation process of transcription and transcription factor binding.
A calmodulin-stimulated Ca2+ pump in plasma-membrane vesicles from Trypanosoma brucei; selective inhibition by pentamidine
Authors: Benaim G , Lopez-Estraño C , Docampo R , Moreno S N J
Published: August 2015

Despite previous reports [McLaughlin (1985) Mol. Biochem. Parasitol. 15, 189-201; Ghosh, Ray, Sarkar and Bhaduri (1990) J. Biol. Chem. 265, 11345-11351; Mazumder, Mukherjee, Ghosh, Ray and Bhaduri (1992) J. Biol. Chem. 267, 18440-18446] that the plasma membrane of different trypanosomatids only contains Ca(2+)-ATPase that does not show any demonstrable dependence on Mg2+, a high-affinity (Ca(2+)-Mg2+)-ATPase was demonstrated in the plasma membrane of Trypanosoma brucei. The enzyme became saturated with micromolar amounts of Ca2+, reaching a Vmax. of 3.45 +/- 0.66 nmol of ATP/min per mg of protein. The Km,app. for Ca2+ was 0.52 +/- 0.03 microM. This was decreased to 0.23 +/- 0.05 microM, and the Vmax. was increased to 6.36 +/- 0.22 nmol of ATP/min per mg of protein (about 85%), when calmodulin was present. T. brucei plasma-membrane vesicles accumulated Ca2+ on addition of ATP only when Mg2+ was present, and released it to addition of the Ca2+ ionophore A23187. In addition, this Ca2+ transport was stimulated by calmodulin. Addition of NaCl to Ca(2+)-loaded T. brucei plasma-membrane vesicles did not result in Ca2+ release, thus suggesting the absence of a Na+/Ca2+ exchanger in these parasites. Therefore the (Ca(2+)-Mg2+)-ATPase would be the only mechanism so far described that is responsible for the long-term fine tuning of the intracellular Ca2+ concentration of these parasites. The trypanocidal drug pentamidine inhibited the T. brucei plasma-membrane (Ca(2+)-Mg2+)-ATPase and Ca2+ transport at concentrations that had no effect on the Ca(2+)-ATPase activity of human or pig erythrocytes. In this latter case, pentamidine behaved as a weak calmodulin antagonist, since it inhibited the stimulation of the erythrocyte Ca(2+)-ATPase by calmodulin.
Molecular characterization of hNRP, a cDNA encoding a human nucleosome-assembly-protein-I-related gene product involved in the induction of cell proliferation
Authors: Simon H U , Mills G B , Kozlowski M , Hogg D , Branch D , Ishimi Y , Siminovitch K A
Published: August 2015

We have isolated from a human thymus cDNA library a cDNA clone encoding a potential protein with 54% amino acid similarity to that encoded by a previously identified cDNA for yeast nucleosome assembly protein I (NAP-I). The deduced amino acid sequence for this newly identified cDNA, designated hNRP (human NAP-related protein), contains a potential seven-residue nuclear localization motif, three clusters of highly acidic residues and other structural features found in various proteins implicated in chromatin formation. When expressed as a fusion protein in Escherichia coli, hNRP reacted specifically with a monoclonal antibody raised against human NAP-I. The hNRP transcript was detected in all tissues and cell lines studied, but levels were somewhat increased in rapidly proliferating cells. Moreover, levels of both hNRP mRNA and protein increased rapidly in cultured T-lymphocytes induced to proliferate by incubation with phorbol ester and ionomycin. Phorbol 12-myristate 13-acetate/ionomycin-induced increases in both hNRP mRNA and mitogenesis, as measured by thymidine incorporation, were markedly inhibited, however, in cells treated with an hNRP antisense oligonucleotide. These results demonstrate a correlation between induction of hNRP expression and mitogenesis and taken together with the structural similarities between hNRP and yeast NAP-I suggest that the hNRP gene product participates in DNA replication and thereby plays an important role in the process of cell proliferation.
Phenylarsine oxide and phorbol myristate acetate inhibit the CD3-induced rise of cytosolic Ca2+ in Jurkat cells by refilling internal Ca2+ stores
Authors: Deckert M , Aussel C , Bernard A , Breittmayer J P
Published: August 2015

Phenylarsine oxide (PAO), an inhibitor of tyrosine phosphatases, has been found to inhibit the early elevation in cytosolic Ca2+ concentration ([Ca2+]i), related to the CD3 activation pathway in Jurkat T cells. This inhibition was dose-dependent, consistent with previously reported effects of PAO on tyrosine phosphatases, and reversed by dimercaptopropanol. By contrast, okadaic acid, an inhibitor of serine/threonine phosphatases, had no effect on CD3-induced Ca2+ flux. PAO was compared with phorbol 12-myristate 13-acetate (PMA), which caused a similar, although less potent, inhibition as previously described. The two reagents produced additive inhibition of the CD3-induced [Ca2+]i rise, but did not affect thapsigargin- or ionomycin-driven Ca2+ flux in Jurkat cells. PAO and PMA prevented cells from complete depletion of intracellular Ca2+ stores by an anti-CD3 monoclonal antibody (mAb) and restored, at least partially, the ionomycin-sensitive pool, when added after anti-CD3 mAb. Moreover, the CD3-induced inhibition of phosphatidylserine synthesis, due to depletion of internal Ca2+ stores, is reversed by PAO and PMA. Anti-phosphotyrosine immunoblot analysis show that these effects cannot be accounted for by an inhibition of CD3-induced tyrosine phosphorylations. We propose that PAO and, to a lesser extent, PMA allow the refilling of internal compartments by Ca2+, which consequently abrogates a capacitative entry of external Ca2+.
Isolation of a novel inducible rat heat-shock protein (HSP70) gene and its expression during ischaemia/hypoxia and heat shock
Authors: Mestril R , Chi S H , Sayen M R , Dillmann W H
Published: August 2015

Most of the members of the mammalian heat-shock protein (HSP) gene family have been studied and isolated from human and mouse cells. Few studies have concentrated on the HSPs of rat, a commonly used experimental animal. We have isolated and characterized a novel inducible rat HSP70 gene using an HSP70 cDNA sequence obtained from an ischaemic rat heart cDNA library. The isolated rat HSP70 gene was found to be a functional gene, as indicated by RNAase-protection and Northern-blot analysis. The deduced amino acid sequence of the inducible rat HSP70 exhibits a high degree of similarity to previously isolated mammalian inducible HSP70 gene products. Expression of the inducible HSP70 gene in rat myogenic cells (H9c2) is markedly increased after relatively short periods of hypoxia as well as by heat shock. Two heat-shock elements (HSE) are present in the rat HSP70 promoter. Transient transfection of rat HSP70 promoter/chloramphenicol acetyltransferase constructs into H9c2 cells shows that the presence of either of the two HSEs is sufficient for heat-shock inducibility. In contrast, induction of the rat HSP70/chloramphenicol acetyltransferase constructs by hypoxia is only detectable when both HSEs are present. This leads us to conclude that the induction of HSP70 by hypoxia and heat shock occurs through the same regulatory HSEs but the activation of the inducible HSP70 gene by heat shock is several-fold higher than by hypoxia.
Alkalosis- and ATP-induced increases in the diacyglycerol pool in alveolar type II cells are derived from phosphatidylcholine and phosphatidylinositol
Authors: Sen N , Chander A
Published: August 2015

Alkalosis and ATP increase surfactant secretion in alveolar type II cells, possibly via non-receptor- and receptor-mediated mechanisms respectively. We compared the effects of these two agonists on phosphatidylinositol (PI) and 1,2-diacylglycerol (DAG) pools and on phosphatidylcholine (PC) hydrolysis in alveolar type II cells. Alkalosis, caused by transfer of cells from 5% (control) to 0% CO2 in air, and ATP increased the secretion of surfactant compared with the controls. The stimulated secretion was inhibited by staurosporine, a protein kinase C inhibitor. DAG and PI contents of control cells were 50 +/- 1.1 (mean +/- S.E.M., n = 8) and 14 +/-0.8 nmol/mg phospholipid (n = 7) respectively. The DAG content increased by approximately 50 nmol (100%) within 5 s of treatment with both alkalosis and ATP, returned to control levels by 1 min, and increased again at 5 min by approximately 20 nmol. The PI content decreased maximally by approximately 6 nmol (40%) at 5 s and returned to control levels by 30 s with both alkalosis and ATP, but was unchanged thereafter. Mass-balance analysis of net changes in DAG and PI pools suggests that additional sources, possibly PC, must also contribute to the DAG increase. ATP or alkalosis also increased the hydrolysis of PC. The labelling of phosphocholine was increased (approximately 60%) at as early as 5 s and remained elevated at subsequent time points, whereas labelling of choline was higher only with ATP at 50 s and later, suggesting activation of phospholipase C by both agonists, and of phospholipase D by only ATP. Our studies demonstrate that ATP and alkalosis stimulate rapid hydrolysis of inositol and choline phospholipids to increase the DAG mass in type II cells, and that phospholipase C-stimulated PC hydrolysis is the major pathway for DAG formation.
The region around residue 115 of human bactericidal/permeability-increasing protein is not involved in lipopolysaccharide binding or bactericidal activity. Chemical synthesis and expression of a gene coding for the active domain and characterization of recombinant proteins
Authors: Qi S Y , Li Y , O'Connor C D
Published: August 2015

Bactericidal/permeability-increasing protein (BPI) is a potent antimicrobial agent produced by polymorphonuclear leucocytes that specifically interacts with and kills Gram-negative bacteria. An 825 bp gene determining the bactericidal N-terminal domain of human BPI was chemically synthesized and expressed as inclusion bodies in Escherichia coli. The recombinant polypeptide, BPI', was solubilized and conditions under which it folded to give the active protein were determined. Folding was critically dependent on the urea and salt concentrations as well as the pH. BPI' bound with high affinity to Salmonella typhimurium cells (apparent Kd = 36 nM), permeabilized their outer membranes to actinomycin D, specifically activated a synovial fluid phospholipase A2 and showed potent bactericidal activity. In contrast with the native protein, however, it could not be efficiently released from the cell surface by the addition of high concentrations of Mg2+ ions. Pre-incubation of the protein with lipopolysaccharide or trypsin prevented cytotoxicity. However, boiling BPI' immediately before its addition to cells did not block its bactericidal activity, suggesting that it may be able to function even when presented to cells in an unfolded form. A BPI' derivative, containing a 13-residue foreign antigenic determinant genetically inserted between Ala115 and Asp116, was also produced. The derivative was functional in the above assays and bound with high affinity to S. typhimurium (apparent Kd = 74 nM). These results imply that the region defined by these residues is not involved in the lipopolysaccharide-binding or bactericidal activities of BPI. The availability of functional, nonglycosylated recombinant derivatives of BPI should greatly aid detailed studies on its structure, interactions with lipopolysaccharide and mechanism of action.
The magnesium-insertion step of chlorophyll biosynthesis is a two-stage reaction
Authors: Walker C J , Weinstein J D
Published: August 2015

Mg(2+)-chelatase catalyses the first step unique to chlorophyll synthesis, namely the insertion of Mg2+ into protoporphyrin IX. When pea (Pisum sativum L., cv. Spring) chloroplasts are lysed in a buffer lacking Mg2+ and the thylakoids removed by centrifugation, the remaining mixture of light membranes and soluble proteins (LM/S) has high Mg(2+)-chelatase activity. Several lines of evidence are presented to show that the Mg2+ insertion catalysed by this preparation is a two-step reaction consisting of activation followed by Mg2+ chelation. An activated state of Mg(2+)-chelatase is achieved by preincubating LM/S with ATP. The activated state is observed as the elimination of the approx. 6 min lag in the rate of Mg2+ chelation on addition of the porphyrin substrate. The activity of LM/S assayed at low protein concentrations can be greatly enhanced by preincubating at high protein concentrations (12 mg/ml is optimal). This activation effect requires the presence of both LM and S fractions, as well as ATP. Both steps require ATP, but at different concentrations; the first step is optimal at > 0.5 mM (EC50 = 0.3 mM) and the second step is optimal at 0.3 mM (EC50 < 0.2 mM). ATP in the first step could be replaced by ATP[S]; this analogue could not sustain activity in the second step. This activated state was stable for at least 30 min at room temperature, but chilling of preincubated LM/S on ice for 30 min caused an almost complete loss of the activated state.
Ligand size is a major determinant of high-affinity binding of fucose- and galactose-exposing (lipo)proteins by the hepatic fucose receptor
Authors: Biessen E A , Bakkeren H F , Beuting D M , Kuiper J , Van Berkel T J
Published: August 2015

Previous in vivo studies have demonstrated that small galactose-exposing particles are preferentially internalized by the asialoglycoprotein receptor on the parenchymal liver cell and large particles by the galactose-particle receptor on the Kupffer cell. In this study, we have investigated using in vitro binding studies whether the affinity for either receptor is affected by the ligand size. The asialoglycoprotein receptor appeared to bind and process lactosylated proteins irrespective of their size. In contrast, recognition of galactose-exposing proteins by the galactose-particle receptor on the Kupffer cell was strongly dependent on size. The affinity increased 3000-fold with protein sizes increasing from 5 to 15 nm, reaching its maximum at approx. 1 nM for ligands larger than 15 nm. Apparently, the preferential in vivo uptake of large galactose-exposing ligands by Kupffer cells does not result from an inability of the parenchymal liver cells to internalize these ligands, but from the high affinity of large ligands for the galactose-particle receptor and the strategic anatomical localization of the Kupffer cells in the liver. In the preceding paper [Kuiper, Bakkeren, Biessen and Van Berkel (1994) Biochem. J. 299, 285-290] the galactose-particle receptor on the Kupffer cell was suggested to be identical with the fucose receptor. 125I-Lac-LDL-binding studies clearly showed that the galactose-particle receptor exhibited high-affinity binding of fucose-exposing proteins also. The affinity of fucosylated proteins for the galactose-particle receptor was greatly affected by ligand size. The above data strongly support the hypothesis that the galactose-particle receptor is identical with the fucose receptor. The size of neoglycoproteins can be appreciated as a new major determinant of affinity for the fucose receptor.