bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel dye-linked formaldehyde dehydrogenase with some properties indicating the presence of a protein-bound redox-active quinone cofactor
Authors: Klein C R , Kesseler F P , Perrei C , Frank J , Duine J A , Schwartz A C
Published: August 2015

Dye-linked formaldehyde dehydrogenase from methylamine-grown Hyphomicrobium zavarzinii ZV 580, a tetramer of M(r) 210,000 with subunits of M(r) 54,000, was purified to homogeneity in five steps with 10% yield. The enzyme shows optimal affinity for, and activity with, formaldehyde (Km 67 microM) compared with other aldehydes. Pyridoxal phosphate, pyrroloquinoline quinone and other cofactors that would give the enzyme a distinctive absorption spectrum are absent. Slight changes are observed in the spectrum at 300-550 nm on oxidation of the enzyme with Wurster's Blue (WB) and reduction with formaldehyde. Titration of the native reduced enzyme with WB accounts for 2 mol of electrons per mol of tetrameric enzyme. The circumstantial evidence supporting the presence of a redox-active quinone cofactor bound to the polypeptide chain comprises a signal at g = 2.0049 in the X-band e.p.r. spectrum of the enzyme oxidized with WB, which disappears on reduction with formaldehyde, and a positive reaction of the native as well as the denatured and dialysed enzyme in the redox-cycling assay with glycinate and NitroBlue Tetrazolium (quinone staining). The oxidized enzyme is inhibited by equimolar amounts of phenylhydrazine, which is also a reductant. Hydrazone formation was absent with completely inhibited enzyme, according to photometric evidence. Likewise, the glycinate-dependent reduction of NitroBlue Tetrazolium was not affected by the inhibitor. It is concluded that an oxidation product of the hydrazine is the actual inhibitor which reacts with an amino acid residue of the active site rather than with the prospective quinone cofactor.
Cloning and characterization of a cDNA representing a putative complement-regulatory plasma protein from barred sand bass (Parablax neblifer)
Authors: Dahmen A , Kaidoh T , Zipfel P F , Gigli I
Published: August 2015

It has been demonstrated previously that plasma from a number of vertebrate species including the phylogenetically old barred sand bass possesses molecules that cleave the alpha'-chain of the activated third (C3b) and fourth (C4b) components of the human complement system. A specific protease and a cofactor protein were identified to be responsible for this cleavage. The cofactor activity in sand bass correlated with a 110 kDa polypeptide chain of a 360 kDa plasma protein. The evolutionary conservation was probed at the cDNA level and subsequently a cDNA clone of barred sand bass was isolated that represents a protein with structural similarity to mammalian complement-regulatory proteins. The cDNA (SB1) was identified by immunoscreening of a sand bass liver expression library using affinity-purified IgG antibodies raised against the isolated 110 kDa material. The cDNA is 3397 bp in size and the open reading frame represents a protein of 1053 amino acid residues with a hydrophobic signal peptide indicative of a secreted protein. The calculated mass of the mature protein (SBP1) is 115.2 kDa which is in good agreement with the molecular mass of 110 kDa determined for the sand bass serum protein. Similarly to mammalian complement-regulatory proteins, the protein deduced from the sand bass cDNA is organized into short consensus repeats (SCR). It consists of 17 SCRs, of which SCRs 2, 12 and 16 exhibit significant homology to SCRs 2, 15 and 19 of human factor H, and SCRs 11, 12 and 13 have homology to SCRs 1, 2 and 3 of human C4b-binding protein. For the first time a complete cDNA representing a putative complement-regulatory protein which is structurally related to mammalian complement proteins has been isolated from a bony fish.
Reaction of peroxynitrite with oxyhaemoglobin: interference with photometrical determination of nitric oxide
Authors: Schmidt K , Klatt P , Mayer B
Published: August 2015

A frequently applied photometrical assay of NO is based on the reaction of NO with oxyhaemoglobin. This study shows that peroxynitrite induces spectral changes of oxyhaemoglobin identical with those elicited by NO. Like a variety of other agents, peroxynitrite did not interfere with NO measurements using a Clark-type electrode, demonstrating that electrochemical detection has an advantage over the oxyhaemoglobin method for specific determination of NO.
The catalytic subunit of phosphatidylinositol 3-kinase is a substrate for the activated platelet-derived growth factor receptor, but not for middle-T antigen-pp60c-src complexes
Authors: Roche S , Dhand R , Waterfield M D , Courtneidge S A
Published: August 2015

The interaction of phosphatidylinositol 3-kinase (PI 3-K) with polyoma-virus middle-T antigen-pp60c-src (mT:cSrc) complexes and with the platelet-derived growth factor (PDGF) receptor has been investigated. Firstly, we undertook reconstitution studies, using proteins derived from a baculovirus expression system. The p110 catalytic subunit of the PI 3-K associated with tyrosine kinases only when complexed with the p85 alpha regulatory subunit. Both p85 alpha and p110 were substrates of the PDGF receptor. In contrast, only the p85 alpha subunit was detectably phosphorylated when PI 3-K was associated with mT:cSrc. Secondly, we studied PI 3-K in mammalian cells. In mT-antigen-transformed NIH-3T3 cells neither p85 alpha nor p110 was phosphorylated on tyrosine residues in vivo, even though p85 alpha was a substrate in kinase assays in vitro. In quiescent NIH-3T3 cells, PI 3-K showed detectable activity in vitro; PDGF stimulation resulted in a rapid and transient association of PI 3-K with the receptor, which was correlated with a transient increase in intrinsic P13-K activity (approx. 2-fold). The activated PDGF receptor phosphorylated p110 in vitro, at one major site. In vivo, PDGF stimulation induced tyrosine phosphorylation of p110 that persisted for at least 1 h after stimulation. Immunodepletion of the PDGF receptor from stimulated cell lysates showed that p110 was released from the receptor in a tyrosine-phosphorylated form. From these results we conclude that (i) the mT:cSrc complex and the PDGF receptor differ in their association with PI 3-K activity, (ii) PDGF receptor appears to activate PI 3-K in vivo both by relocation of the enzyme and by stimulation of its intrinsic activity, and (iii) tyrosine phosphorylation of the p110 subunit by the PDGF receptor may play a role in PI 3-K regulation in some circumstances.
Measurement of pentose phosphate-pathway activity in a single incubation with [1,6-13C2,6,6-2H2]glucose
Authors: Ross B D , Kingsley P B , Ben-Yoseph O
Published: August 2015

The isotopically substituted molecule D-[1,6-13C2,6,6-2H2]glucose is introduced for measuring the relative activities of the pentose phosphate pathway (PPP) and glycolysis in a single incubation. PPP activity in cultured cells was determined by gas chromatography/mass spectrometric analysis of lactate produced by cells incubated with [1,6-13C2,6,6-2H2]glucose. Two other isotopes, [1,5,6-13C3]glucose and [6-13C,1,6,6-2H3]glucose, were not satisfactory for measurements of this activity. This method has four advantages over the traditional one in which 14CO2 production from [1-14C]glucose and [6-14C]glucose is compared: (1) repeated measurements can be made on a single set of cells, (2) only a single incubation is required, (3) extensive CO2 production by Krebs-cycle activity does not interfere with the measurements and (4) it is not necessary to measure the amount of glucose consumed in order to calculate relative activities of the PPP and glycolysis. Preliminary observation indicates that rat brain PPP activity may be measured in vivo with [1,6-13C2,6,6-2H2]glucose when combined with microdialysis techniques.
Identification of two elements involved in regulating expression of the murine leukaemia inhibitory factor gene
Authors: Hsu L W , Heath J K
Published: August 2015

Mouse leukaemia inhibitory factor (LIF) is a polyfunctional cytokine which exhibits multiple functions in vitro and in vivo. Two forms of LIF cDNA, differing at their 5′ ends, have been described encoding either diffusible (D-LIF) or matrix-associated (M-LIF) forms of the protein [Rathjen, Toth, Willis, Heath and Smith (1990) (Cell 62, 1105-1114]. The present report describes the DNA sequence and functional characterization of the murine LIF gene and its surrounding transcriptional regulatory elements. Transient transfection of constructs containing the LIF gene and various amounts of 5′-non-coding sequence failed to give detectable levels of expression, suggesting the presence of inhibitory sequences within the LIF gene. Stable cell lines were produced by transfection of experimental constructs containing various lengths of 5′-non-coding sequence of the LIF gene, or the heterologous phosphoglycerate kinase promoter, linked to an LIF/neomycin-resistance-hybrid-coding sequence. The frequency of recovery of stable clones indicated that sequences located in the first intron between the transcriptional start sites for D-LIF and M-LIF act to suppress expression of the gene in most genomic locations. This region is rich in GC residues and has been shown to be hypomethylated in vitro [Kaspar, Dvorak and Bartunek (1993) FEBS Lett. 319, 159-162]. Analysis of the LIF/neomycin-resistance transgene expression in these stable cell clones demonstrated that transcripts containing the M-LIF or D-LIF exons required the presence of sequences located between -1200 and -3200 in the LIF gene. In the absence of these sequences, transcription is initiated elsewhere within the first intron. These sequences can be replaced by the heterologous phosphoglycerate kinase promoter. Deletion of the GC-rich region between the D-LIF and M-LIF transcriptional start sites results in the appearance of transcripts that do not splice out the first intron of the LIF gene. These may result from gene or promoter trapping of the LIF gene. Sequence analysis of the region between -1200 and -3200 revealed a number of minimal steroid-response elements, regions of similarity to DNAase I-hypersensitive sites in the uteroglobin gene and a region of alternating purine/pyrimidine sequence. This study therefore defines two important regulatory regions in the LIF gene: a GC-rich region in the first intron and a distal ‘enhancer’ located between -3200 and -1200.
Replacement of both tryptophan residues at 388 and 412 completely abolished cytochalasin B photolabelling of the GLUT1 glucose transporter
Authors: Inukai K , Asano T , Katagiri H , Anai M , Funaki M , Ishihara H , Tsukuda K , Kikuchi M , Yazaki Y , Oka Y
Published: August 2015

A mutated GLUT1 glucose transporter, a Trp-388, 412 mutant whose tryptophans 388 and 412 were both replaced by leucines, was constructed by site-directed mutagenesis and expressed in Chinese hamster ovary cells. Glucose transport activity was decreased to approx. 30% in the Trp-388, 412 mutant compared with that in the wild type, a similar decrease in transport activity had been observed previously in the Trp-388 mutant and the Trp-412 mutant which had leucine at 388 and 412 respectively. Cytochalasin B labelling of the Trp-388 mutant was only decreased rather than abolished, a result similar to that obtained previously for the Trp-412 mutant. Cytochalasin B labelling was finally abolished completely in the Trp-388, 412 mutant, while cytochalasin B binding to this mutant was decreased to approx. 30% of that of the wild-type GLUT1 at the concentration used for photolabelling. This level of binding is thought to be adequate to detect labelling, assuming that the labelling efficiency of these transporters is similar. These findings suggest that cytochalasin B binds to the transmembrane domain of the glucose transporter in the vicinity of helix 10-11, and is inserted covalently by photoactivation at either the 388 or the 412 site.
Limited proteolysis of phospholipase C-γ 1 indicates stable association of X and Y domains with enhanced catalytic activity
Authors: Fernald A W , Jones G A , Carpenter G
Published: August 2015

Phospholipase C-gamma 1 (PLC-gamma 1) was treated with Staphylococcus aureus V8 protease (V8) and the digestion products were analysed with site-specific antibodies. V8 treatment generated three immunodetectable PLC-gamma 1 fragments of 120, 97, and 39 kDa. The 39 kDa fragment is derived from the C-terminus of PLC-gamma 1 and includes the conserved Y domain present in all PLC isoenzymes. The 120 and 97 kDa fragments are derived from the N-terminus of PLC-gamma 1, possess the conserved X domain common to all PLC isoenzymes, and the src-homology domains unique to PLC-gamma 1 and -gamma 2. It is likely that the 97 kDa fragment is a V8 product of the 120 kDa fragment. As the C-terminal 39 kDa fragment, and either of the N-terminal 120 or 97 kDa fragments, were precipitable with antibody specific to a sequence present in only the 39 kDa fragment, the data indicate co-precipitation of separate polypeptide chains that remain associated after V8 proteolysis. Importantly, V8 treatment increased the activity of PLC-gamma 1 and did not alter the calcium requirement. The influence of other modulators of PLC-gamma 1 activity, however, was lost following V8 treatment. These results suggest the stable association of the X and Y domains within PLC-gamma 1, and demonstrate that proteolysis in the region of PLC-gamma 1 that is subject to tyrosine phosphorylation can enhance catalytic activity.
Role of the 5′-untranslated region of mRNA in the synthesis of S-adenosylmethionine decarboxylase and its regulation by spermine
Authors: Shantz L M , Viswanath R , Pegg A E
Published: August 2015

S-Adenosylmethionine decarboxylase (AdoMetDC), a rate-limiting enzyme in polyamine biosynthesis, is regulated by polyamines at the levels of both transcription and translation. Two unusual features of AdoMetDC mRNA are a long (320 nt) 5′-untranslated region (5′UTR), which is thought to contain extensive secondary structure, and a short (15 nt) open reading frame (ORF) within the 5′UTR. We have studied the effects of altering these elements on both the expression of AdoMetDC and its regulation by n-butyl-1,3-diaminopropane (BDAP), a spermine synthase inhibitor. Human AdoMetDC cDNAs containing alterations in the 5′UTR, as well as chimaeric constructs in which the AdoMetDC 5′UTR was inserted ahead of the luciferase-coding region, were transfected into COS-7 cells. Construct pSAM320, which contains all of the 5′UTR, the AdoMetDC protein-coding region and the 3′UTR, was expressed poorly (2-fold over the endogenous activity). Deletion of virtually the entire 5′UTR, leaving nt -12 to -1, increased expression 59-fold, suggesting that 5′UTR acts as a negative regulator. The same effect was seen when the 27 nt at the extreme 5′ end were removed (pSAM293, 47-fold increase), or when the internal ORF which is present in this region was destroyed by changing the ATG to CGA (pSAM320-ATG, 38-fold increase). The expression and regulation of pSAM44 (made by deleting nt -288 to -12), which has very little predicted secondary strucutre, was very similar to that of pSAM320 indicating that the terminal 27 nt including the internal ORF rather than extensive secondary structure may be responsible for the low basal levels of AdoMetDC expression. These results, confirmed using luciferase constructs, suggest that the negative effect on expression is predominantly due to the internal ORF. Depletion of spermine by BDAP increased the expression from pSAM320 more than 5-fold without affecting AdoMetDC mRNA levels. Expression from pSAM293 was unchanged by spermine depletion, whereas that from pSAM320-ATG was increased 2.5-fold. These results indicate the presence of a spermine response element in the first 27 nt of the 5′UTR that may include but is not entirely due to the internal ORF.
Gene dissection demonstrates that the Escherichia coli cysG gene encodes a multifunctional protein
Authors: Warren M J , Bolt E L , Roessner C A , Scott A I , Spencer J B , Woodcock S C
Published: August 2015

The C-terminus of the Escherichia coli CysG protein, consisting of amino acids 202-457, was expressed as a recombinant protein using gene dissection methodology. Analysis of the activity of this truncated protein, termed CysGA, revealed that it was able to methylate uroporphyrinogen III in the same S-adenosyl-L-methionine (SAM)-dependent manner as the complete CysG protein. However, this truncated protein was not able to complement E. coli cysG cells, thereby suggesting that the first 201 amino acids of the CysG protein had an enzymic activity associated with the conversion of dihydrosirohydrochlorin into sirohaem. Analysis of the N-terminus of the CysG protein revealed the presence of a putative pyridine dinucleotide binding site. When the purified CysG protein was incubated with NADP+, uroporphyrinogen III and SAM the enzyme was found to catalyse a coenzyme-mediated dehydrogenation to form sirohydrochlorin. The CysGA protein on the other hand showed no such coenzyme-dependent activity. Analysis of the porphyrinoid material isolated from strains harbouring plasmids containing the complete and truncated cysG genes suggested that the CysG protein was also involved in ferrochelation. The evidence presented in this paper suggests that the CysG protein is a multifunctional protein involved in SAM-dependent methylation, pyridine dinucleotide dependent dehydrogenation and ferrochelation.
The mammalian transferrin-independent iron transport system may involve a surface ferrireductase activity
Authors: Jordan I , Kaplan J
Published: August 2015

Mammalian cells accumulate iron from ferric citrate or ferric nitrilotriacetate through the activity of a transferrin-independent iron transport system [Sturrock, Alexander, Lamb, Craven and Kaplan (1990) J. Biol. Chem. 265, 3139-3145]. The uptake system might recognize and transport ferric-anion complexes, or cells may reduce ferric iron at the surface and then transport ferrous iron. To distinguish between these possibilities we exposed cells to either [59Fe]ferric citrate or ferric [14C]citrate and determined whether accumulation of iron was accompanied by the obligatory accumulation of citrate. In HeLa cells and human skin fibroblasts the rate of accumulation of iron was three to five times greater than that of citrate. Incubation of fibroblasts with ferric citrate or ferric ammonium citrate resulted in an enhanced accumulation of iron and citrate; the molar ratio of accumulation approaching unity. A similar rate of citrate accumulation, however, was observed when ferric citrate-incubated cells were exposed to [14C]citrate alone. Further studies demonstrated the independence of iron and citrate accumulation: addition of unlabelled citrate to cells decreased the uptake of labelled citrate without affecting the accumulation of 59Fe; iron uptake was decreased by the addition of ferrous chelators whereas the uptake of citrate was unaffected; reduction of ferric iron by ascorbate increased the uptake of iron but had no effect on the uptake of citrate. When HeLa cells were depleted of calcium, iron uptake decreased, but there was little effect on citrate uptake. These results indicate that transport of iron does not require the obligatory transport of citrate and vice versa. The mammalian transferrin-independent iron transport system appears functionally similar to iron transport systems in both the bacterial and plant kingdoms which require the activities of both a surface reductase and a ferrous metal transporter.
Preparation and characterization of novel substrates of insulin proteinase (EC 3.4.99.45)
Authors: Werlen R C , Offord R E , Rose K
Published: August 2015

The specificity of insulin proteinase (EC 3.4.99.45) has been difficult to categorize using only its natural substrates. By exploiting the fact that two substrates competing for the same enzyme inhibit one another, we have found some new substrates of the insulin proteinase from porcine muscle. Two of these substrates, a tryptic fragment of BSA and a fragment of cytochrome c, have been shown to be cleaved at a single site. The albumin fragment, as well as another fragment of cytochrome c., have susceptibilities (Vmax/Km) comparable with that of insulin. In a second aspect of the study, the porcine-muscle enzyme was shown to be related to other members of its superfamily in that it was immunoprecipitated by a monoclonal antibody raised against the insulin-degrading enzyme from human red blood cells and has the same cleavage sites on insulin as has the rat skeletal-muscle insulin proteinase. We note, however, a possible discrepancy between our results and those of another group regarding the subunit size (110 kDa) of the immunoprecipitated material.
Oxidized mucus proteinase inhibitor: a fairly potent neutrophil elastase inhibitor
Authors: Boudier C , Bieth J G
Published: August 2015

N-chlorosuccinimide oxidizes one of the methionine residues of mucus proteinase inhibitor with a second-order rate constant of 1.5 M-1.s-1. Cyanogen bromide cleavage and NH2-terminal sequencing show that the modified residue is methionine-73, the P′1 component of the inhibitor's active centre. Oxidation of the inhibitor decreases its neutrophil elastase inhibitory capacity but does not fully abolish it. The kinetic parameters describing the elastase-oxidized inhibitor interaction are: association rate constant kass. = 2.6 x 10(5) M-1.s-1, dissociation rate constant kdiss. = 2.9 x 10(-3) s-1 and equilibrium dissociation constant Ki = 1.1 x 10(-8) M. Comparison with the native inhibitor indicates that oxidation decreases kass. by a factor of 18.8 and increases kdiss. by a factor of 6.4, and therefore leads to a 120-fold increase in Ki. Yet, the oxidized inhibitor may still act as a potent elastase inhibitor in the upper respiratory tract where its concentration is 500-fold higher than Ki, i.e. where the elastase inhibition is pseudo-irreversible. Experiments in vitro with fibrous human lung elastin, the most important natural substrate of elastase, support this view: 1.35 microM elastase is fully inhibited by 5-6 microM oxidized inhibitor whether the enzyme-inhibitor complex is formed in the presence or absence of elastin and whether elastase is pre-adsorbed on elastin or not.
Thermal stability of methanol dehydrogenase is altered by the replacement of enzyme-bound Ca2+ with Sr2+
Authors: Harris T K , Davidson V L
Published: August 2015

Methanol dehydrogenase (MEDH) possesses tightly bound Ca2+ in addition to its pyrroloquinoline quinone prosthetic group. Ca2+ was replaced with Sr2+ by growing the host bacterium, Paracoccus denitrificans, in media in which Ca2+ was replaced with Sr2+. At temperatures in the transition region for stability, the rate constants for inactivation of MEDH purified from these cells (Sr-MEDH) were 2-fold lower than those for MEDH. However, Arrhenius plots yielded an activation energy (Ea) of 699 kJ (167 kcal)/mol for MEDH compared with 640 kJ (153 kcal)/mol for Sr-MEDH. Further analysis by transition-state theory yielded values for the activation enthalpy (delta H*) and activation entropy (delta S*) of 696 kJ (166 kcal)/mol and 1.73 kJ (414 cal)/mol per K for MEDH and 637 kJ (152 kcal)/mol and 1.55 kJ (371 cal)/mol per K for Sr-MEDH. The higher rate of inactivation of MEDH than Sr-MEDH at higher temperatures is a consequence of a more favourable net gain in entropy. This positive entropy contribution increases at high temperatures, and reduces the more favourable stability obtained from the enthalpy contribution for the free energy (delta G*) of inactivation. The differences in these thermodynamic data are discussed in relation to the recently determined crystal structure of MEDH as well as 1H electron-nuclear double resonance studies of the influence of Sr2+ substitution on the structure of the pyrroloquinoline quinone-derived radical in MEDH.
Site-directed mutagenesis of proposed active-site residues of penicillin-binding protein 5 from Escherichia coli
Authors: van der Linden M P G , de Haan L , Dideberg O , Keck W
Published: August 2015

Alignment of the amino acid sequence of penicillin-binding protein 5 (PBP5) with the sequences of other members of the family of active-site-serine penicillin-interacting enzymes predicted the residues playing a role in the catalytic mechanism of PBP5. Apart from the active-site (Ser44), Lys47, Ser110-Gly-Asn, Asp175 and Lys213-Thr-Gly were identified as the residues making up the conserved boxes of this protein family. To determine the role of these residues, they were replaced using site-directed mutagenesis. The mutant proteins were assayed for their penicillin-binding capacity and DD-carboxypeptidase activity. The Ser44Cys and the Ser44Gly mutants showed a complete loss of both penicillin-binding capacity and DD-carboxypeptidase activity. The Lys47Arg mutant also lost its DD-carboxypeptidase activity but was able to bind and hydrolyse penicillin, albeit at a considerably reduced rate. Mutants in the Ser110-Gly-Asn fingerprint were affected in both acylation and deacylation upon reaction with penicillin and lost their DD-carboxypeptidase activity with the exception of Asn112Ser and Asn112Thr. The Asp175Asn mutant showed wild-type penicillin-binding but a complete loss of DD-carboxypeptidase activity. Mutants of Lys213 lost both penicillin-binding and DD-carboxypeptidase activity except for Lys213His, which still bound penicillin with a k+2/K' of 0.2% of the wild-type value. Mutation of His216 and Thr217 also had a strong effect on DD-carboxypeptidase activity. Thr217Ser and Thr217Ala showed augmented hydrolysis rates for the penicillin acyl-enzyme. This study reveals the residues in the conserved fingerprints to be very important for both DD-carboxypeptidase activity and penicillin-binding, and confirms them to play crucial roles in catalysis.
Binding of surfactant protein A to the lipid A moiety of bacterial lipopolysaccharides
Authors: Van Iwaarden J F , Pikaar J C , Storm J , Brouwer E , Verhoef J , Oosting R S , van Golde L M G , van Strijp J A G
Published: August 2015

Surfactant protein A (SP-A) enhances the phagocytosis of opsonized and non-opsonized bacteria by alveolar macrophages, but it is not known with which component of the bacterial surface it associates. We investigated the interaction of SP-A with lipopolysaccharides (LPS), which are important biologically active constituents of the outer membranes of Gram-negative bacteria. Flow cytometry was used to study the binding of fluorescein isothiocyanate-labelled SP-A either to LPS of various chain lengths coupled to magnetic beads or to Gram-negative bacteria. The binding of SP-A to LPS-coated beads was saturable, both time- and concentration-dependent, and required both Ca2+ and Na+. SP-A bound to the lipid A moiety of LPS and to LPS from either the Re-mutant of Salmonella minnesota or the J5-mutant of Escherichia coli. In contrast, it did not bind to O111 LPS of E. coli, suggesting that SP-A binds only to rough LPS. The binding of SP-A to LPS was not affected by mannan and heparin or by deglycosylation of the SP-A, indicating that the carbohydrate-binding domain and the carbohydrate moiety of SP-A are not involved in its interaction with LPS. We also observed saturable and concentration-dependent binding of SP-A to the live J5 mutant of whole E. coli, but not to its O111 mutant. In addition, Re LPS aggregated in the presence of SP-A, Ca2+ and Na+. We conclude that SP-A associates with LPS via the lipid A moiety of rough LPS and may be involved in the anti-bacterial defences of the lung.
The role of tryptophan 97 of cytochrome P450 BM3 from Bacillus megaterium in catalytic function. Evidence against the ‘covalent switching’ hypothesis of P-450 electron transfer
Authors: Munro A W , Malarkey K , McKnight J , Thomson A J , Kelly S M , Price N C , Lindsay J G , Coggins J R , Miles J S
Published: August 2015

The ‘Covalent Switching’ hypothesis suggests that a strongly conserved tryptophan residue acts as a mediator of electron-transfer flow between redox partners in cytochrome P-450 systems [Baldwin, Morris and Richards (1991) Proc. R. Soc. London B 245, 43-51]. We have investigated the effect of alteration of the conserved tryptophan (Trp-97) in cytochrome P-450 BM3 (P-450 102) from Bacillus megaterium. Replacement of Trp-97 with Ala, Phe or Tyr results in a decrease in the natural haem content and alters the resting spin state of the remaining haem in the purified mutant enzymes. However, kinetic analyses indicate that the mutant enzymes retain high levels of catalytic activity. C.d. and e.p.r. spectroscopy also reveal little alteration in secondary structure or change in the pattern of haem ligation. These findings cast doubt on the covalent switching mechanism of intermolecular electron flow in the P-450s, but indicate that this residue plays a role in the association of the haem prosthetic group.
Kinetics of an enzyme reaction in which both the enzyme-substrate complex and the product are unstable or only the product is unstable
Authors: Garrido-del Solo C , García-Cánovas F , Havsteen B H , Valero E , Varón R
Published: August 2015

A kinetic analysis of the Michaelis-Menten mechanism has been made for the case in which both the enzyme-substrate complex and the product are unstable or only the product is unstable, either spontaneously or as the result of the addition of a reagent. This analysis allows the derivation of equations which under conditions of limiting enzyme concentration relate the concentration of all of the species to the time. A kinetic data analysis is suggested, which leads to the evaluation of the kinetic parameters involved in the reaction. The analysis is based on the equation which describes the formation of products with time and one's experimental progress curves. We demonstrate the method numerically by computer simulation of the reaction with added experimental errors and experimentally by the use of data from the kinetic study of the action of tyrosinase on dopamine.
Intracellular adenosine 3′,5′-phosphate formation is essential for down-regulation of surface adenosine 3′,5′-phosphate receptors in Dictyostelium
Authors: Van Haastert P J M
Published: August 2015

Dictyostelium discoideum cells contain cell surface cyclic AMP (cAMP) receptors that bind cAMP as a first messenger and intracellular cAMP receptors that bind cAMP as a second messenger. Prolonged incubation of Dictyostelium cells with cAMP induces a sequential process of phosphorylation, sequestration and down-regulation of the surface receptors. The role of intracellular cAMP in down-regulation of surface receptors was investigated. Down-regulation of receptors does not occur under conditions that specifically inhibit the formation of intracellular cAMP (the drug caffeine or mutant cells lacking adenylate cyclase) or conditions that inhibit the function of intracellular cAMP (mutants lacking protein kinase A activity). Cell-permeable non-hydrolysable cAMP derivatives were used to investigate further the requirement of intracellular cAMP for down-regulation. The Sp isomer of 6-thioethylpurineriboside 3′,5′-phosphorothioate (6SEth-cPuMPS) does not bind to the surface receptor, enters the cell and has relative high affinity for protein kinase A. 6SEth-cPuMPS alone has no effect on down-regulation. However, together with an agonist of the surface receptor, the analogue induces down-regulation in caffeine-treated wild-type cells and in mutant cells lacking adenylate cyclase, but not in mutant cells lacking protein kinase A. These results indicate that intracellular cAMP formation and activation of protein kinase A are essential for down-regulation of the surface cAMP receptor.
Regulation of T-cell-receptor-stimulated bivalent-cation entry in Jurkat E6 cells: role of protein kinase C
Authors: Conroy L A , Merritt J E , Hallam T J
Published: August 2015

Stimulation of Jurkat E6 cells with anti-CD3 antibody results in a characteristic rise in [Ca2+]i which is due to both the release of Ca2+ from intracellular stores and the entry of external Ca2+. Individual components of the [Ca2+]i increase were investigated by measuring intracellular Ca2+ release in the absence of external Ca2+ and determining influx of bivalent cations by following the entry of Mn2+. The increase in [Ca2+]i induced by anti-CD3 antibody in the presence or absence of extracellular Ca2+ could be inhibited by the non-selective kinase inhibitor staurosporine, which also inhibits anti-CD3-stimulated phospholipase C activity. Staurosporine also inhibits the influx of bivalent cations induced by anti-CD3 antibody, but not that induced by depletion of intracellular Ca2+ stores using thapsigargin. The effect of staurosporine was compared with that of Ro 31-8425, a potent and selective inhibitor of protein kinase C (PKC). Ro 31-8425, at concentrations up to 10 microM, has no inhibitory effect on the anti-CD3 antibody-induced [Ca2+]i increase or phospholipase C activity. These studies are consistent with the concept that augmentation of [Ca2+]i by stimulated T-cell receptors requires activation of a kinase, probably a tyrosine kinase such as p56lck, ZAP-70 or p59fyn, and is independent of PKC. Phorbol esters inhibit the anti-CD3-stimulated [Ca2+]i increase and phospholipase C activity, showing that this can be negatively regulated by PKC. A small potentiation of the anti-CD3 antibody-induced [Ca2+]i rise in the presence of extracellular Ca2+ was detected in the presence of Ro 31-8425; this suggests that T-cell-receptor ligation can also limit the increase in [Ca2+]i via PKC activation.
Purification and molecular characterization of the NAD+-dependent acetaldehyde/alcohol dehydrogenase from Entamoeba histolytica
Authors: Bruchhaus I , Tannich E
Published: August 2015

A bifunctional 95 kDa polypeptide (EhADH2) harbouring acetaldehyde dehydrogenase and alcohol dehydrogenase activities was purified to homogeneity from trophozoite extracts of the protozoan parasite Entamoeba histolytica. Kinetic studies revealed that the enzyme utilizes NAD+ rather than NADP+ as cofactor. Km values for acetyl-CoA, acetaldehyde and ethanol were found to be 0.015, 0.15 and 80 mM respectively in the presence of 0.2 mM NAD+. The primary structure of EhADH2 as deduced from respective amoebic DNA sequences showed striking similarity to the trifunctional AdhE protein of Escherichia coli and the bifunctional AAD protein of Clostridium acetobutylicum. Alignment with a number of aldehyde dehydrogenases and alcohol dehydrogenases from various species suggested that the two catalytic functions of EhADH2 are located on separate parts of the molecule. By cross-linking experiments and electron-microscopic analysis, native EhADH2 was found to be organized in a homopolymeric fashion consisting of more than 20 associated promoters which form rods about 50-120 nm in length.
A 13C isotopomer n.m.r. method for monitoring incomplete β-oxidation of fatty acids in intact tissue
Authors: Gavva S R , Wiethoff A J , Zhao P , Malloy C R , Sherry A D
Published: August 2015

An n.m.r. method is presented for monitoring the extent to which fatty acids undergo beta-oxidation without release of shorter-chain intermediates. It is based upon a 13C isotopomer analysis of glutamate from tissue presented with a mixture of [2,4,6,8-13C]octanoate and [1,2,3,4-13C]octanoate. The method does not require steady-state metabolic or isotopic conditions, so it may be applied during a variety of metabolic circumstances, including perfused tissue under stress and in vivo. We have tested the method in perfused rat hearts during anoxia, a model where previous work has shown that beta-oxidation of palmitate is incomplete and shorter-chain intermediates are released [Rabinowitz and Hercker (1974) Arch. Biochem. Biophys. 161, 621-627]. Indeed, n.m.r. spectra of freeze-clamped, acid-extracted tissue show that octanoate undergoes complete beta-oxidation in control normoxic rat hearts, but not in anoxic hearts. Complete beta-oxidation of octanoate was observed under a number of other metabolic conditions in perfused rat hearts, including low-pressure-induced ischaemia, KCl arrest and in the presence of high concentrations of competing substrates. We also demonstrate that the technique is applicable in intact tissue by taking direct measurements in perfused rat hearts using a recently published [13C]homonuclear decoupling technique and in in vivo heart and liver removed from rats after an intravenous infusion of a mixture of [2,4,6,8-13C]octanoate and [1,2,3,4-13C]octanoate.
Regulation of cytosolic calcium and insulin secretion by galanin and ATP receptors: interactions of pertussis-toxin-sensitive and -insensitive signalling pathways
Authors: Lang J , Boulay F , Parker P , Gierschik P , Wollheim C B
Published: August 2015

In a previous study it was found that the expression of the exogenous fMet-Leu-Phe-receptor (NFPR) in the insulin-secreting cell line RINm5F mediates inhibition of hormone release and additionally raises cytosolic calcium concentration ([Ca2+]i) by activating phospholipase C (PLC) in a pertussis-toxin (PTX)-sensitive manner. We investigated whether an endogenous receptor could elicit similar effects and examined the interaction with PTX-insensitive signalling pathways. The hormone galanin inhibited insulin release at subnanomolar concentrations and increased [Ca2+]i, mainly by a PTX-sensitive mechanism with an EC50 (50 nM) comparable with that for hyperpolarization of membrane potential. The effect of galanin or fMet-Leu-Phe on [Ca2+]i was inhibited by pre-activation of the P2-receptor by ATP, which mobilizes calcium in a PTX-insensitive fashion. Simultaneous activation of the P2- and peptide receptors caused additive increases in [Ca2+]i saturating at a calcium concentration corresponding to the optimal ATP response. This suggests a specific convergence of PTX-sensitive and -insensitive pathways. In contrast, galanin and FMLP inhibited the insulin secretion induced by ATP (1-100 microM), but only when added prior to the nucleotide. In permeabilized cells, FMLP added after the calcium stimulus still inhibited secretion, indicating that the inefficacy observed in intact cells was not due to the rapid ATP-evoked rise in [Ca2+]i. Thus, (i) insulin-secreting cells possess an endogenous PTX-sensitive pathway mobilizing [Ca2+]i, (ii) inhibitory hormones preferentially activate different effectors depending on the agonist concentration and (iii) activation of NFPR or galanin receptor reveals an unusual dissociation between [Ca2+]i rises and insulin secretion, pointing towards an overriding inhibitory control of exocytosis.
Protein kinase C phosphorylation of rat liver S-adenosylmethionine synthetase: dissociation and production of an active monomer
Authors: Pajares M A , Durán C , Corrales F , Mato J M
Published: August 2015

The regulation of rat liver S-adenosylmethionine synthetase (AdoMet synthetase), a key enzyme in methionine metabolism, by protein kinase C (PKC) phosphorylation has been studied. Both enzyme forms, tetramer and dimer, are phosphorylated by this kinase in the same residue, Thr-342, of the sequence. Phosphorylation of the dimer leads to its dissociation, with production of a fully-active monomer. The kinetics of the monomer have been studied, and a KmMet of 931.9 microM, a KmATP of 708 microM and a Vmax of 66.8 nmol/min/mg have been calculated. Alkaline phosphatase treatment of both enzyme forms (tetramer and dimer) produces a reduction in their activity with no change in the oligomeric state. On the other hand, PKC phosphorylation of the alkaline phosphatase-treated AdoMet synthetase forms leads to the dissociation of the dimer to produce a monomer. Rephosphorylation occurs again in the same residue, Thr-342, of the sequence. The significance of AdoMet synthetase regulation by PKC phosphorylation is further discussed.
Insulin receptor substrate 1 is phosphorylated by the serine kinase activity of phosphatidylinositol 3-kinase
Authors: Tanti J F , Grémeaux T , Van Obberghen E , Le Marchand-Brustel Y
Published: August 2015

Insulin receptor substrate (IRS) 1, which is tyrosine phosphorylated in response to insulin, presents multiple serine/threonine phosphorylation sites. To search for a serine kinase activity towards IRS 1, immunoprecipitates from basal or stimulated 3T3-L1 adipocytes were used in an in vitro kinase assay. When IRS 1 was isolated from insulin-treated cells, serine phosphorylation of IRS 1 occurred, which we attribute to the kinase activity of the phosphatidylinositol 3-kinase (PI3-kinase). Importantly, in an in vitro reconstitution assay, an excess of the PI3-kinase subunit prevents this phosphorylation. Together, our results suggest that following insulin stimulation, PI3-kinase associates with IRS 1, allowing for its serine phosphorylation. This phosphorylation event could play a role in the modulation of insulin signalling.
A low-affinity, low-molecular-mass endothelin-A receptor in neonatal rat heart
Authors: Woodcock E A , Land S L , Andrews R K , Linsenmeyer M , Woodcock D M
Published: August 2015

Endothelin receptors with endothelin-A (ETa) specificity were present in neonatal rat ventricle. However, in both receptor-binding studies and studies of inositol phosphate accumulation, these receptors had lower affinity for endothelin-1 than ETa receptors on isolated neonatal cardiomyocytes or adult left atria. Receptors in the three myocardial preparations were cross-linked to 125I-endothelin-1 and their molecular masses measured using SDS/PAGE. Receptors on left atria and neonatal cardiomyocytes had the expected molecular mass of 48 kDa, whereas the receptors in neonatal ventricle were smaller (38 kDa). Despite this, neonatal ventricles contained ETa receptor mRNA which was not different in size from that in the isolated cells (4.5 kb). Thus the 38 kDa ETa receptor present in neonatal ventricle appears to be transcribed from full-length ETa receptor mRNA and is possibly formed by processing of the 48 kDa receptor.
Decrease in S-adenosylmethionine synthesis by 6-mercaptopurine and methylmercaptopurine ribonucleoside in Molt F4 human malignant lymphoblasts
Authors: Stet E H , De Abreu R A , Bökkerink J P M , Blom H J , Lambooy L H J , Vogels-Mentink T M , de Graaf-Hess A C , van Raay-Selten B , Trijbels F J M
Published: August 2015

6-Mercaptopurine (6-MP) and methylmercaptopurine ribonucleoside (Me-MPR) are purine anti-metabolites which are both metabolized to methylthio-IMP (Me-tIMP), a strong inhibitor of purine synthesis de novo. Me-MPR is converted directly into Me-tIMP by adenosine kinase. 6-MP is converted into tIMP, and thereafter it is methylated to Me-tIMP by thiopurine methyltransferase, an S-adenosylmethionine (S-Ado-Met)-dependent conversion. S-Ado-Met is formed from methionine and ATP by methionine adenosyltransferase, and is a universal methyl donor, involved in methylation of several macromolecules, e.g. DNA and RNA. Therefore, depletion of S-Ado-Met could result in an altered methylation state of these macromolecules, thereby affecting their functionality, leading to dysregulation of cellular processes and cytotoxicity. In this study the effects of 6-MP and Me-MPR on S-Ado-Met, S-adenosylhomocysteine (S-Ado-Hcy), homocysteine and methionine concentrations are determined. Both drugs cause a decrease in intracellular S-Ado-Met concentrations and an increase in S-Ado-Hcy and methionine concentrations in Molt F4 human malignant lymphoblasts. The effects of both 6-MP and Me-MPR can be ascribed to a decreased conversion of methionine into S-Ado-Met, due to the ATP depletion induced by the inhibition of purine synthesis de novo by Me-tIMP. Both 6-MP and Me-MPR thus affect the methylation state of the cells, and this may result in dysregulation of cellular processes and may be an additional mechanism of cytotoxicity for 6-MP and Me-MPR.
Lithium stimulates accumulation of second-messenger inositol 1,4,5-trisphosphate and other inositol phosphates in mouse pancreatic minilobules without inositol supplementation
Authors: Dixon J F , Hokin L E
Published: August 2015

Previous studies showed that lithium, beginning at therapeutic plasma concentrations in the treatment of manic depression, increased the accumulation of second-messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in cerebral cortex slices of guinea pig and rhesus monkey [Lee, Dixon, Reichman, Moummi, Los and Hokin (1992) Biochem. J. 282, 377-385; Dixon, Lee, Los and Hokin (1992) J. Neurochem. 59, 2332-2335; Dixon, Los and Hokin (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 8358-8362]. These studies have now been extended to a peripheral tissue, mouse pancreatic minilobules. In the presence of carbachol, concentrations of lithium from 1 to 20 mM sharply and progressively increased the accumulation of Ins(1,4,5)P3 and inositol 1,3,4,5-tetrakisphosphate, followed by a decrease. Assay of these inositol polyphosphates by either the prelabelling technique or mass assay gave similar results. Atropine quenching of cholinergically stimulated pancreatic minilobules led to a rapid disappearance of Ins(1,4,5)P3. This disappearance was impeded by lithium. This suggested that the lithium-induced elevation in Ins(1,4,5)P3 was due to inhibition of the 5-phosphatase and, on the basis of the markedly elevated concentrations of inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] and inositol 1,4-bisphosphate in the presence of lithium, probably by feedback inhibition by these latter two compounds. An additional mechanism, i.e. a stimulatory effect of lithium on phospholipase C, cannot, however, be ruled out. The other reaction product of phospholipase C, inositol cyclic 1:2,4,5-trisphosphate, also increased in the presence of lithium. This may also be due to inhibition of the 5-phosphatase, which is the exclusive mechanism for removal of this compound. The effects of lithium on the accumulation of other inositol phosphates paralleled that of Ins(1,4,5)P3, with the exception of inositol 3,4-bisphosphate, which decreased. This was presumably due to the inhibition of Ins(1,3,4)P3 1-phosphatase by lithium. Unlike mouse cerebral cortex slices [Lee, Dixon, Reichman, Moummi, Los and Hokin (1992) Biochem. J. 282, 377-385], inositol supplementation was not required to demonstrate lithium-stimulated Ins(1,4,5)P3 accumulation in mouse pancreatic minilobules. This indicates that inositol depletion sufficient to impair lithium-stimulated Ins(1,4,5)P3 accumulation does not occur in mouse pancreatic minilobules, even though an elevation of cytidine diphosphodiacylglycerol occurred, indicating some inositol depletion due to lithium. Elevation of Ins(1,4,5)P3 by lithium may be a general phenomenon in the central nervous system and peripheral tissues under non-rate-limiting concentrations of inositol.