bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of membrane-anchored heparin-binding epidermal growth factor-like growth factor and CD9 on macrophages
Authors: OUCHI Noriyuki , KIHARA Shinji , YAMASHITA Shizuya , HIGASHIYAMA Shigeki , NAKAGAWA Tsutomu , SHIMOMURA Iichiro , FUNAHASHI Tohru , KAMEDA-TAKEMURA Kaoru , KAWATA Sumio , TANIGUCHI Naoyuki , MATSUZAWA Yuji
Published: August 2015

Heparin-binding epidermal-growth-factor-like growth factor (HB-EGF) is a potent mitogen for smooth-muscle cells (SMCs) belonging to the EGF family. We have previously determined that HB-EGF is expressed in macrophages and SMCs of human atherosclerotic lesions and that its membrane-anchored precursor, proHB-EGF, also has a juxtacrine mitogenic activity which is markedly enhanced by CD9, a surface marker of lymphohaemopoietic cells. Therefore, when both proHB-EGF and CD9 are expressed on macrophages, they may strongly promote the development of atherosclerosis. In the present study we have investigated the changes in proHB-EGF and CD9 in THP-1 cells during differentiation into macrophages and by the addition of oxidized low-density lipoproteins (OxLDL) and assessed juxtacrine growth activity of THP-1 macrophages for human aortic SMCs. HB-EGF and CD9 at both the mRNA and the protein level were up-regulated after differentiation into macrophages, and further expression of HB-EGF was induced by the addition of OxLDL or lysophosphatidylcholine. Juxtacrine induction by formalin-fixed growth was suppressed to control levels by an inhibitor of HB-EGF and was partially decreased by anti-CD9 antibodies. These results suggest that co-expression of proHB-EGF and CD9 on macrophages plays an important role in the development of atherosclerosis by a juxtacrine mechanism.
Expression of the Schwanniomyces occidentalis SWA2 amylase in Saccharomyces cerevisiae: role of N-glycosylation on activity, stability and secretion
Authors: YÁÑEZ Esther , CARMONA A. Teresa , TIEMBLO Mercedes , JIMÉNEZ Antonio , FERNÁNDEZ-LOBATO María
Published: August 2015

The role of N-linked glycosylation on the biological activity of Schwanniomyces occidentalis SWA2 α-amylase, as expressed in Saccharomyces cerevisiae, was analysed by site-directed mutagenesis of the two potential N-glycosylation sites, Asn-134 and Asn-229. These residues were replaced by Ala or Gly individually or in various combinations and the effects on the activity, secretion and thermal stability of the enzyme were studied. Any Asn-229 substitution caused a drastic decrease in activity levels of the extracellular enzyme. In contrast, substitutions of Asn-134 had little or no effect. The use of antibodies showed that α-amylase was secreted in all the mutants tested, although those containing substitutions at Asn-229 seemed to have a lower rate of synthesis and/or higher degradation than the wild-type strain. α-Amylases with substitution at Asn-229 had a 2 kDa lower molecular mass than the wild-type protein, as did the wild-type protein itself after treatment with endoglycosidase F. These findings indicate that Asn-229 is the single glycosylated residue in SWA2. Thermostability analysis of both purified wild-type (T50 = 50 °C, where T50 is the temperature resulting in 50% loss of activity) and mutant enzymes indicated that removal of carbohydrate from the 229 position results in a decrease of approx. 3 °C in the T50 of the enzyme. The Gly-229 mutation does not change the apparent affinity of the enzyme for starch (Km) but decreases to 1/22 its apparent catalytic efficiency (kcat/Km). These results therefore indicate that glycosylation at the 229 position has an important role in the extracellular activity levels, kinetics and stability of the Sw. occidentalis SWA2 α-amylase in both its wild-type and mutant forms.
Soluble form of complement C3b/C4b receptor (CR1) results from a proteolytic cleavage in the C-terminal region of CR1 transmembrane domain
Authors: HAMER Isabelle , PACCAUD Jean-Pierre , BELIN Dominique , MAEDER Christine , CARPENTIER Jean-Louis
Published: August 2015

The complement C3b/C4b receptor (CR1) is an integral protein, anchored in the plasma membrane through a hydrophobic domain of 25 amino acids, but is also found in the plasma in soluble form (sCR1). A recombinant, soluble form of CR1 has been demonstrated to reduce complement-dependent tissue injury in animal models of ischaemia/reperfusion. In view of the important pathophysiological relevance of sCR1, we have investigated the mechanisms governing CR1 release by using various mutated and chimaeric receptors transiently expressed in COS cells. Pulse-chase experiments revealed that (1) sCR1 is produced by a proteolytic process, (2) the cleavage site lies within the C-terminus of CR1 transmembrane domain, (3) the proteolytic process involves a fully glycosylated CR1 form and (4) this process takes place in late secretory vesicles or at the plasma membrane.
Multiple forms of DNA polymerase from the thermo-acidophilic eubacterium Bacillus acidocaldarius: purification, biochemical characterization and possible biological role
Authors: FALCO Mariarosaria DE , GRIPPO Pasquale , ROSSI Mosè , ORLANDO Pierangelo
Published: August 2015

Two DNA polymerase isoenzymes, called DpA and DpB on the basis of their elution order from DEAE cellulose, were purified to homogeneity from the thermo-acidophilic eubacterium Bacillus acidocaldarius. The enzymes are weakly acidophilic proteins constituted by a single subunit of 117 and 103 kDa respectively. DpA and DpB differ in thermostability, in thermophilicity, in sensitivity to assay conditions and in resistance to sulphydryl-group blocking agents such as N-ethylmaleimide and p-hydroxymercuriobenzoate. They differ also in synthetic template-primer utilization, in the apparent Km for dNTPs and in processivity. In particular, DpA utilizes more effic iently synthetic templates-primers such as poly(dA)·poly(dT), poly(dT)·(rA)12-18 and poly(rA)·(dT)12-18 and presents a greater tendency to accept dNTP analogues modified in the sugar or in the base ring, such as cytosine β-D-arabinofuranoside 5ʹ-triphosphate, 2ʹ,3ʹ-dideoxyribonucleosides 5ʹ-triphosphate, butylphenyl-dGTP and digoxigenin-conjugated dUTP. In addition, DpA presents an exonuclease activity that preferentially hydrolyses DNA in the 5ʹ-3ʹ direction, whereas DpB lacks this activity. The possible biological role of the enzymes is discussed.
Identification of cardiac oxidoreductase(s) involved in the metabolism of the lipid peroxidation-derived aldehyde-4-hydroxynonenal
Authors: SRIVASTAVA Sanjay , CHANDRA Animesh , ANSARI H. Naseem , SRIVASTAVA K. Satish , BHATNAGAR Aruni
Published: August 2015

The aim of this study was to identify the cardiac oxidoreductases involved in the metabolism of 4-hydroxy-2-trans-nonenal (HNE), an α,β unsaturated aldehyde generated during the peroxidation of ω-6 polyunsaturated fatty acids. In homogenates of bovine, human and rat ventricles the primary pyridine coenzyme-linked metabolism of HNE was associated with NADPH oxidation. The NADPH-dependent enzyme catalysing HNE reduction was purified to homogeneity from bovine heart. The purified enzyme displayed kinetic and immunological properties identical with the polyol pathway enzyme aldose reductase (AR), and catalysed the reduction of HNE to its alcohol 1,4-dihydroxynonene (DHN), with a Km of 7±2 μM. In the presence of NADP the enzyme did not catalyse the oxidation of DHN. During catalysis, HNE did not cause inactivation of AR. Nevertheless when the apoenzyme was incubated with HNE a dissociable complex was formed between the enzyme and HNE, followed by irreversible loss of activity. Inactivation of the enzyme by HNE was prevented by NADP. Partial modification of the enzyme with HNE led to a 17-fold increase in the KHNEm and Kglyceraldehydem, and the HNE-modified enzyme had a 500-fold higher IC50 for sorbinil than for the reduced enzyme, whereas the IC50 for tolrestat increased 25-fold. Incubation of the enzyme with radiolabelled HNE resulted in the incorporation of 2 mol of the aldehyde per mol of the enzyme. Sequence analysis of the radiolabelled peptides revealed modification of Cys-298 and Cys-187. The amino acid sequence of the HNE-modified peptides confirmed that the HNE-reducing cardiac enzyme is AR and not a related protein such as the fibroblast-growth-factor-regulated protein FR-1 or the mouse vas deferens protein MVDP. These results indicate that AR represents the only major oxidoreductase in the heart capable of utilizing HNE. The high affinity of the enzyme for HNE, the lack of inactivation during catalysis, and the lack of significant alcohol dehydrogenase activity of the protein suggests that AR-mediated catalysis of HNE is unlikely to be limited by substrate/product inhibition. Thus AR might constitute an antioxidative enzyme involved in myocardial protection against endogenous and exogenous cytotoxic aldehydes and against oxidative stress.
Characterization and sugar-binding properties of arcelin-1, an insecticidal lectin-like protein isolated from kidney bean (Phaseolus vulgaris L. cv. RAZ-2) seeds
Authors: FABRE Christine , CAUSSE Henri , MOUREY Lionel , KONINKX Jos , RIVIÈRE Michel , HENDRIKS Henno , PUZO Germain , SAMAMA Jean-Pierre , ROUGÉ Pierre
Published: August 2015

Arcelin-1 is a lectin-like protein found in the seeds of wild varieties of the kidney bean (Phaseolus vulgaris). This protein displays insecticidal properties, but the mechanism of action is as yet unknown. In the present study we investigated the biochemical and biophysical properties of arcelin-1 from Phaseolus vulgaris cv. RAZ-2. Native arcelin-1 is a dimeric glycoprotein of 60 kDa, built from the non-covalent association of two identical monomers. This dimer resists dissociation by chaotropic agents and is highly resistant to proteolytic enzymes. Each subunit contains 10% (w/w) neutral sugars which belong to the high-mannose and complex-type glycans attached to three glycosylation sites. No interaction of the protein with simple sugars could be detected, but arcelin-1 displays an intrinsic specificity in binding complex glycans. Arcelin-1 therefore differs from the closely related phytohaemagglutinin lectins and α-amylase inhibitor in several respects: oligomerization states, sugar-binding affinities and the type and number of glycan chains. These features may be related to the toxicity of arcelin-1.
Selectivity of post-translational modification in biotinylated proteins: the carboxy carrier protein of the acetyl-CoA carboxylase of Escherichia coli
Authors: RECHE Pedro , LI Yi-Li , FULLER Christopher , EICHHORN Knut , PERHAM N. Richard
Published: August 2015

Biotin-dependent enzymes contain a biotinyl-lysine residue in a conserved sequence motif, MKM, located in a surface hairpin turn in one of the two β-sheets that make up the domain. A sub-gene encoding the 82-residue C-terminal biotinyl domain from the biotin carboxy carrier protein of acetyl-CoA carboxylase from Escherichia coli as a fusion protein with glutathione S-transferase was created and over-expressed in E. coli. The biotinyl domain was readily released by cleavage with thrombin. Five mutant domains were created in which the conserved MKM motif was systematically replaced: by MAK and KAM, in which the target lysine is moved one place; by KKM and MKK, in which a second potential site for biotinylation is introduced; and by DKA, the motif found in the correspondingly conserved site of lipoylation in the structurally related lipoyl domains of 2-oxo acid dehydrogenase multienzyme complexes. No biotinylation of the MAK or KAM mutants was observed in vivo or by purified biotinyl protein ligase in vitro; in the KKM and MKK mutants, only one lysine residue, presumed to be that in its native position in the hairpin turn, was found to be biotinylated in vivo and in vitro. The DKA mutant was not biotinylated in vivo, but was partly lipoylated and octanoylated. It was also a poor substrate for lipoylation in vitro catalysed by the E. coli lipoyl protein ligase encoded by the lplA gene. The flanking sequence in the MKM motif is important, but not crucial, and appears to have been conserved in part to be compatible with the subsequent carboxylation reactions of biotin-dependent enzymes. The DKA motif, displayed in the hairpin loop, is sufficient to address lipoylation in E. coli but probably by a pathway different from that mediated by the lplA-dependent ligase. The recognition of the structurally homologous lipoyl and biotinyl domains by the appropriate ligase evidently has a major structural component to it, notably the positioning of the target lysine residue in the exposed hairpin loop, but there appear to be additional recognition sites elsewhere on the domains.
Suppressive effect of insulin on the synthesis of sucrase–isomaltase complex in small intestinal epithelial cells, and abnormal increase in the complex under diabetic conditions
Authors: TAKENOSHITA Makoto , YAMAJI Ryoichi , INUI Hiroshi , MIYATAKE Kazutaka , NAKANO Yoshihisa
Published: August 2015

An abnormally high level of the sucrase-isomaltase (SI) complex in the small intestine of rats with streptozotocin-induced insulin-dependent diabetes mellitus (IDDM) was normalized in 11 h by the administration of insulin, in addition to normalization of the blood glucose level. Phlorizin, an inhibitor of renal glucose reabsorption, also caused normalization of the blood glucose level in the IDDM rats; however, the level of the SI complex was barely changed. When mucosa explants were cultured in a medium, the SI complex synthesized during the cultivation was accumulated as its precursor protein without maturation, owing to the absence of pancreatic proteases, and the amount of the precursor protein that accumulated in the explants was decreased by the addition of insulin into the medium. Further, the mRNA level of the SI complex in the explants incubated with insulin was obviously lower than that in the absence of insulin. These results indicate that insulin has a suppressive effect on the synthesis of the SI complex, presumably by decreasing the transcriptional level of the gene encoding the complex, in small-intestinal epithelial cells. Thus the synthesis of the SI complex might exceed normal levels in the epithelial cells as a direct result of the depletion of insulin under IDDM conditions.
Identification of a novel mouse hepatic 52 kDa protein that interacts with the cAMP reponse element of the rat angiotensinogen gene
Authors: WU Jie , JIANG Qin , CHEN Xing , WU Xiao-Hua , CHAN S. D. John
Published: August 2015

To identify the nuclear protein(s) that interact with the putative cAMP response element (CRE) of the rat angiotensinogen (ANG) gene (i.e. nt 806-779 upstream of the transcriptional start site), mouse liver nuclear proteins were prepared for the present studies. The DNase 1 footprinting protection analysis revealed that nt -799/-788 in the 5ʹ-flanking region of the rat ANG gene are protected by the mouse liver nuclear protein. Gel mobility-shift assays revealed that the addition of the unlabelled DNA fragment, ANG nt -806/-779 competed effectively with the binding of the labelled ANG nt -806/-779 to the mouse liver nuclear proteins but the addition of unlabelled mutants of ANG nt -806/-779 were only weakly effective in competing with the labelled ANG nt -806/-779. The addition of unlabelled CRE of the somatostatin (SOM) gene and the CRE of the tyrosine aminotransferase (TAT) gene was also ineffective in competing with the labelled ANG nt -806/-779. Southwestern blot analysis revealed that the labelled ANG nt -806/-779 interacted with two mouse liver nuclear proteins with apparent molecular masses of 52 and 43 kDa, whereas the labelled SOM-CRE, TAT-CRE and the CRE of the phosphoenolpyruvate carboxykinase (PEPCK) gene interacted with one molecular species of 43 kDa. The binding of the labelled ANG nt -806/-779 to the 52 kDa protein was effectively competed for by the addition of unlabelled ANG nt -806/-779 but not by unlabelled SOM-CRE, TAT-CRE and PEPCK-CRE. Finally, Western blot analysis revealed that polyclonal antibodies against the CRE-binding protein (CREB) interacted with the mouse liver nuclear 43 kDa protein but not with the 52 kDa protein. These studies demonstrate that the CRE of the rat ANG gene (ANG nt -806/-779) interacts with the 43 kDa CREB and a novel 52 kDa protein from mouse liver. The novel 52 kDa protein is immunologically distinct from the 43 kDa CREB. These studies suggest that the 52 kDa protein might have a role in the expression of the hepatic ANG gene.
Human insulin production from a novel mini-proinsulin which has high receptor-binding activity
Authors: CHANG Seung-Gu , KIM Dae-Young , CHOI Ki-Doo , SHIN Jae-Min , SHIN Hang-Cheol
Published: August 2015

To increase the folding efficiency of the insulin precursor and the production yield of insulin, we have designed a mini-proinsulin (M2PI) having the central C-peptide region replaced with a sequence forming a reverse turn. The mini-proinsulin was fused at the N-terminus to a 21-residue fusion partner containing a His10 tag for affinity purification. The gene for the fusion protein was inserted downstream of the T7 promoter of the expression plasmid pET-3a, and the fusion proteins were produced as inclusion bodies in the Escherichia coli cytoplasm at levels up to 25% of the total cell protein. The protein was sulphonated, cleaved by CNBr and the M2PI mini-proinsulin was purified using ion-exchange chromatography. The refolding yield of M2PI was 20-40% better than that of proinsulin studied at the same molar concentrations, indicating that the short turn-forming sequence is more effective in the refolding process than the much longer C-peptide. Native human insulin was succesfully generated by subsequent enzymic conversion of mini-proinsulin. The mini-proinsulin exhibited high receptor-binding activity, about 50% as potent as insulin, suggesting that this single-chained mini-proinsulin may provide a foundation in understanding the receptor-bound structure of insulin as well as the role of C-peptide in the folding and activity of proinsulin.
Harpin and hydrogen peroxide both initiate programmed cell death but have differential effects on defence gene expression in Arabidopsis suspension cultures
Authors: DESIKAN Radhika , REYNOLDS Adele , HANCOCK T. John , NEILL J. Steven
Published: August 2015

Programmed cell death is increasingly viewed as a key component of the hypersensitive disease resistance response of plants. The generation of reactive oxygen species (ROS) such as H2O2 triggers a cell death programme in Arabidopsis suspension cultures following challenge with the bacterial elicitor harpin. Both harpin and exogenous H2O2 initiate a cell death pathway that requires gene expression, and also act as signalling molecules to induce the expression of plant defence genes encoding enzymes such as phenylalanine ammonia-lyase (PAL), glutathione S-transferase (GST) and anthranilate synthase (ASA1), an enzyme of phytoalexin biosynthesis in Arabidopsis. H2O2 induces the expression of PAL1 and GST but not that of ASA1. Harpin initiates two signalling pathways, one leading to increased ROS generation and expression of PAL1 and GST mRNA, and another leading to increased GST and ASA1 expression, independent of H2O2.
Molecular cloning of a murine homologue of membrane cofactor protein (CD46): preferential expression in testicular germ cells
Authors: TSUJIMURA Akira , SHIDA Kyoko , KITAMURA Masaya , NOMURA Midori , TAKEDA Junji , TANAKA Hiromitsu , MATSUMOTO Misako , MATSUMIYA Kiyomi , OKUYAMA Akihiko , NISHIMUNE Yoshitake , OKABE Masaru , SEYA Tsukasa
Published: August 2015

Human membrane cofactor protein (MCP, CD46) has been suggested, although no convincing evidence has been proposed, to be a fertilization-associated protein, in addition to its primary functions as a complement regulator and a measles virus receptor. We have cloned a cDNA encoding the murine homologue of MCP. This cDNA showed 45% identity in deduced protein sequence and 62% identity in nucleotide sequence with human MCP. Its ectodomains were four short consensus repeats and a serine/threonine-rich domain, and it appeared to be a type 1 membrane protein with a 23-amino acid transmembrane domain and a short cytoplasmic tail. The protein expressed on Chinese hamster ovary cell transfectants was 47 kDa on SDS/PAGE immunoblotting, ~ 6 kDa larger than the murine testis MCP. It served as a cofactor for factor I-mediated inactivation of the complement protein C3b in a homologous system and, to a lesser extent, in a human system. Strikingly, the major message of murine MCP was 1.5 kb and was expressed predominantly in the testis. It was not detected in mice defective in spermatogenesis or with immature germ cells (until 23 days old). Thus, murine MCP may be a sperm-dominant protein the message of which is expressed selectively in spermatids during germ-cell differentiation.
Identification and localization of myosin phosphatase in human platelets
Authors: MURÁNYI Andrea , ERDDI Ferenc , ITO Masaaki , GERGELY Pál , HARTSHORNE J. David
Published: August 2015

Type 1 (PP1) and type 2A (PP2A) phosphatase activity was measured in three subcellular fractions of human platelets. About 80% of the activity was in the high-speed supernatant. Western blots showed that the catalytic subunit of PP1 (PP1c), including α- and δ-isoforms, was present in each fraction, but the level of the catalytic subunit of PP2A was very low in the low-speed pellet (cytoskeletal fraction). Various antibodies detected a subunit similar to the 130 kDa subunit (M130) of myosin phosphatase (MP) of smooth muscle in the low- and the high-speed pellets of human platelets. PP1c and associated proteins were isolated by microcystin-Sepharose. Many proteins were separated from each fraction, including myosin, actin and PP1c. M130 was separated only from the low-speed and the high-speed pellets. Kinase activities were detected in the unbound fractions, and fractions from the low- and high-speed pellets phosphorylated M130 and myosin respectively. Treatment of platelets with calyculin A increased the phosphorylation level of many proteins, including myosin heavy- and light-chains, and caused association of cytoskeletal proteins with the low-speed pellet. No marked change in the distribution of PP1c and M130 was detected. These results suggest that the MP in human platelets is composed of PP1c plus a subunit similar to M130 of the smooth muscle phosphatase.
Signalling pathways of an insulin-mimetic phosphoinositolglycan–peptide in muscle and adipose tissue
Authors: KESSLER Alexandra , MÜLLER Günter , WIED Susanne , CRECELIUS Anna , ECKEL Jürgen
Published: August 2015

A novel phosphoinositolglycan-peptide (PIG-P) from the yeast Saccharomyces cerevisiae potently mimicks insulin action on glucose transport and metabolism in rat muscle and adipose tissue. The aim of the present study was to elucidate the cellular signalling pathways of this insulin-mimetic compound. Rapid onset and reversibility of PIG-P action on glucose transport were observed in isolated adipocytes with a half-time of transport stimulation of 6-8 min (insulin less than 5 min). Combined treatment with PIG-P and insulin indicated additive stimulation of glucose transport at submaximal concentrations and non-additive action of both agents at maximal doses. The tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) was markedly increased in response to PIG-P in rat cardiomyocytes without any effect on the tyrosine phosphorylation of the insulin receptor β-subunit. PIG-P action in these cells was accompanied by phosphorylation/dephosphorylation of several proteins with molecular masses of 15-30 kDa, a response not detected with insulin. Downstream signalling of IRS-1 was then analysed by monitoring IRS-1-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity in cardiomyocytes. A stable (2 and 15 min incubation with PIG-P) 7-fold stimulation corresponding to about 50% of insulin action could be detected. Increased tyrosine phosphorylation of IRS-1 and enhanced PI 3-kinase activity in response to PIG-P independent of the insulin receptor was also observed in isolated adipocytes. Involvement of PI 3-kinase in PIG-P action was subsequently confirmed by the dose-dependent inhibition of PIG-P-activated glucose transport in rat diaphragm and adipocytes by the PI 3-kinase inhibitors wortmannin and LY294002. These data suggest divergent upstream signalling by insulin and PIG-P involving phosphoproteins not affected by insulin. However, PIG-P and insulin action converge at the level of IRS-1 inducing insulin-independent PI 3-kinase-mediated signalling to glucose transport.
Reconstitution of Cu2+-depleted bovine serum amine oxidase with Co2+
Authors: AGOSTINELLI Enzo , MATTEIS Giovanna DE , MONDOVÌ Bruno , MORPURGO Laura
Published: August 2015

Two different Cu2+-depleted derivatives of bovine serum amine oxidase (BSAO) have recently been prepared, which contain about 0.5 mol/dimer of phenylhydrazine-reactive topa quinone (TPQ) cofactor and, depending on the reagents used, about 0.2 or 0.7 residual Cu2+/dimer [Agostinelli, De Matteis, Sinibaldi, Mondovì and Morpurgo (1997) Biochem. J. 324, 497-501]. The benzylamine oxidase activity of both derivatives was < 5% and increased up to ≈ 20% on incorporation of Co2+, irrespective of the residual Cu2+ content, which was unaffected by the treatment according to atomic absorption and ESR spectroscopy. The residual Cu2+ ions appeared to be distributed one per dimer and to be bound to inactive subunits, whereas Co2+ was bound to active subunits. The change in the active site had an appreciable influence on the kinetic behaviour. With several amines, the kinetic parameters, Km and kc, measured for Co2+-BSAO were different from those for native BSAO. This excludes the possibility that the catalytic activity was due to residual Cu2+. Furthermore, Co2+ restored to nearly native level the intensity of the TPQ 480 nm band and the reactions with phenylhydrazine or benzylhydrazine, which had been slowed down or abolished, respectively, in Cu2+-depleted samples. The CD spectrum, measured for the derivative with low Cu2+ content, was compatible with Co2+ binding to the copper site. The amine oxidase activity of the Co2+ derivative, which cannot form a semiquinone radical as an intermediate of the catalytic reaction, strongly suggests that the Cu+-semiquinone is not an obligatory intermediate of BSAO catalytic pathway.
Characterization of mini-protein S, a recombinant variant of protein S that lacks the sex hormone binding globulin-like domain
Authors: WIJNEN Merel VAN , STAM G. Jeanette , CHANG T. G. Glenn , MEIJERS C. M. Joost , REITSMA H. Pieter , BERTINA M. Rogier , BOUMA N. Bonno
Published: August 2015

Protein S is a vitamin K-dependent glycoprotein involved in the regulation of the anticoagulant activity of activated protein C (APC). Also, an anticoagulant role for protein S, independent of APC, has been described. Protein S has a unique C-terminal sex hormone binding globulin (SHBG)-like domain that represents about half of the molecule. To define the role of this domain in APC cofactor activity and in binding to C4b-binding protein (C4BP), we have constructed a recombinant protein S molecule of N-terminal residues 1-242 that lacks the SHBG domain (mini-protein S). A panel of monoclonal antibodies directed against the N-terminal region of protein S recognized plasma-derived protein S, wild-type recombinant protein S and mini-protein S with similar affinities, whereas a monoclonal antibody that recognizes an epitope in the SHBG domain did not detect mini-protein S. Mini-protein S did not bind to C4BP in a solid-phase binding assay, and the cofactor activity of mini-protein S was not inhibited by preincubation with C4BP. In a plasma coagulation assay, the cofactor activity of mini-protein S was lower than wild-type or plasma-derived preparations. In contrast, no difference in APC cofactor activities was observed when the preparations were tested in purified systems that monitor the APC-mediated degradation of factors Va or VIIIa. In conclusion, we constructed a protein S molecule that fails to bind C4BP and still displays cofactor activity for APC. This confirms the role of the C-terminal SHBG region in C4BP binding and demonstrates that N-terminal residues 1-242 are sufficient for the expression of APC cofactor activity in a system using purified components. In plasma, however, the C-terminal SHBG region plays a role in the expression of optimal APC cofactor activity.
Lysosomal sulphate transport is dependent upon sulphydryl groups
Authors: CHOU Hsu-Fang , PASSAGE Merry , JONAS J. Adam
Published: August 2015

Using thiol blocking agents, we examined the role of sulphydryl groups for function of the lysosomal sulphate transport system. Monothiol binding reagents, p-hydroxymercuribenzoic acid (p-HMB) and p-chloromercuribenzene sulphonic acid (p-CMBS), dithiol binding reagents such as CuCl2, the alkylating agent, N-ethylmaleimide (NEM), and NADH all inhibited lysosomal sulphate transport. The inhibitory effects of NEM and Cu2+ were not additive, suggesting that they both act upon the same critical sulphydryl group(s). Unlike the case for NEM, the inhibitory effects of Cu2+ were reversed by the reducing agent, dithiothreitol. Exposure to NEM resulted in a seven-fold increase in Km to 867 μM versus a control value of 126 μM and a modest decrease in Vmax to 99 pmol per unit β-hexosaminidase per 30 s versus a control value of 129 pmol per unit β-hexosaminidase per 30 s. Similar although somewhat less dramatic results were obtained using Cu2+ with an increase of Km to 448 μM and a Vmax of 77 pmol per unit β-hexosaminidase per 30 s. The sulphate transport activity of detergent solubilized lysosomal membranes could be bound to a p-chloromercuribenzoic acid (p-CMB)-Sepharose sulphydryl affinity resin and eluted with mercaptoethanol. Sulphydryl groups thus appear to play a role in sulphate transport through effects on substrate affinity. Sulphydryl-binding appears to be a strategy that may be useful for purification of the transporter.
The polypeptide backbone of recombinant human zona pellucida glycoprotein-3 initiates acrosomal exocytosis in human spermatozoa in vitro
Authors: CHAPMAN R. Neil , KESSOPOULOU Efrossini , ANDREWS D. Paul , HORNBY P. David , BARRATT L. R. Christopher
Published: August 2015

Human gamete interaction is of fundamental biological importance, yet the molecular interactions between spermatozoa and the zona pellucida are poorly understood. Surprisingly, the role of the polypeptide backbone of zona pellucida glycoprotein 3 (ZP3), the putative ligand for spermatozoa activation, has been largely overlooked. Purified recombinant human ZP3 was expressed in Escherichia coli as a C-terminal fusion to the dimeric glutathione S-transferase (GST) from Schistosoma japonicum and was shown to induce acrosomal exocytosis in live, capacitated human spermatozoa. The level of exocytosis is comparable with that obtained using purified, glycosylated, recombinant human ZP3 [van Duin, M., Polman, J. E. M., DeBreet, I. T. M., Van Ginneken, K., Bunschoten, H., Grootenhuis, A., Brindle, J. and Aitken, R. J. (1994). Biol Reprod. 51, 607-617]. These data imply that the polypeptide chain of human ZP3 contributes to recognition of spermatozoa during acrosomal exocytosis in vitro.
A variant of the bovine noradrenaline transporter reveals the importance of the C-terminal region for correct targeting to the membrane and functional expression
Authors: BURTON D. Lucille , KIPPENBERGER G. Andree , LINGEN Bettina , BRÜSS Michael , BÖNISCH Heinz , CHRISTIE L. David
Published: August 2015

We have characterized a cDNA clone which encodes a variant (bNAT2) of the bovine noradrenaline transporter. This cDNA differs from the previously identified bovine noradrenaline transporter (bNAT1) in the sequence encoding part of the cytoplasmic-facing C-terminus and the 3ʹ-untranslated region. The bNAT1 and bNAT2 cDNA clones are encoded by a 5.8 and 3.6 kb mRNA species respectively. The bNAT1 and bNAT2 proteins, which are identical apart from their C-terminal 31 and 18 residues, were stably expressed in HEK293 cells. Cells expressing bNAT1 showed a high level of desipramine-sensitive [3H]noradrenaline uptake activity, whereas no activity was present in bNAT2 cells. The bNAT1 and bNAT2 proteins were present as major 80 and 50 kDa species respectively. Cells expressing bNAT1 showed strong immunostaining of the plasma membrane, whereas bNAT2 was present in the endoplasmic reticulum/Golgi region. Treatment of membrane samples from bNAT1 cells with peptide N-glycosidase F resulted in the formation of a predominantly 50 kDa species, but little effect was observed after similar treatment of bNAT2 cell membranes. These results indicate that bNAT2 is retained in the endoplasmic reticulum and that the glycosylation of this variant differs from that of bNAT1. The characterization of bNAT2 and its comparison with bNAT1 highlight the importance of the cytoplasmic-facing C-terminus for the intracellular trafficking of neurotransmitter transporters.
Increased intracellular sequestration of the insulin-regulated aminopeptidase upon differentiation of 3T3-L1 cells
Authors: ROSS A. Stuart , KELLER R. Susanna , LIENHARD E. Gustav
Published: August 2015

In fat and muscle cells, the glucose transporter GLUT4 is sequestered in an intracellular compartment under basal conditions and redistributes markedly to the plasma membrane in response to insulin. Recently, we characterized a membrane aminopeptidase, designated IRAP (insulin-regulated aminopeptidase), that colocalizes with intracellular GLUT4 and similarly redistributes markedly to the plasma membrane in response to insulin in adipocytes. In contrast to GLUT4, IRAP is also expressed in 3T3-L1 fibroblasts, and this finding provided an opportunity to compare its subcellular distribution in fibroblasts and adipocytes. The relative amount of IRAP at the cell surface was measured by a cell surface biotinylation method. The portion of total IRAP at the cell surface in unstimulated adipocytes was 30% of that in unstimulated fibroblasts. Upon insulin treatment the portion of IRAP at the cell surface was the same in fibroblasts and adipocytes, and was increased 1.8-fold in fibroblasts and 8-fold in adipocytes. A similar analysis of the distribution of the transferrin receptor (TfR), the paradigm for recycling plasma membrane receptors, revealed that the portions of the TfR at the cell surface in both the basal and insulin-treated states were almost unchanged upon differentiation, and that insulin caused an increase of about 1.6-fold in the amount of TfR at the cell surface. These results show that enhanced intracellular sequestration of IRAP occurs during adipogenesis, and that this effect underlies the larger insulin-elicited fold increase of IRAP at the cell surface in adipocytes.
Active sites residues of beef liver carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase (CPT-II)
Authors: NIC a'BHÁIRD Nóirin , YANKOVSKAYA Victoria , RAMSAY R. Rona
Published: August 2015

The carnitine acyltransferases which catalyse the reversible transfer of fatty acyl groups between carnitine and coenzyme A have been proposed to contain a catalytic histidine. Here, the chemical reactivity of active site groups has been used to demonstrate differences between the active sites of beef liver carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase-II (CPT-II). Treatment of CPT-II with the histidine-selective reagent, diethyl pyrocarbonate (DEPC), resulted in simple linear pseudo-first-order kinetics. The reversal of the inhibition by hydroxylamine and the pKa (7.1) of the modified residue indicated that the residue was a histidine. The order of the inactivation kinetics showed that 1 mol of histidine was modified per mol of CPT-II.
The role of calmodulin-binding sites in the regulation of the Drosophila TRPL cation channel expressed in Xenopus laevis oocytes by Ca2+, inositol 1,4,5-trisphosphate and GTP-binding proteins
Authors: LAN Ling , BRERETON Helen , BARRITT J. Greg
Published: August 2015

The roles of calmodulin-binding sites in the regulation by Ca2+, inositol 1,4,5-trisphosphate (InsP3) and GTP-binding regulatory proteins (G-proteins) of the Drosophila melanogaster TRPL (transient-receptor-potential-like) non-specific Ca2+ channel were investigated. Wild-type TRPL protein and two mutant forms, TRPL (W713G) and TRPL (W814G), in which a key tryptophan residue in each of the two putative calmodulin-binding sites (Sites 1 and 2, respectively) was replaced by glycine, were expressed heterologously in Xenopuslaevis oocytes. Immunofluorescence studies indicated that the expressed TRPL, TRPL (W713G) and TRPL (W814G) proteins are located at the plasma membrane. TRPL oocytes (oocytes injected with trpl cRNA) and TRPL (W814G) oocytes [oocytes injected with trpl (W814G) cRNA] exhibited substantially greater rates of basal (constitutive) Ca2+ inflow (measured using fluo-3 and the Ca2+ add-back protocol) than mock-injected oocytes (mock oocytes). In TRPL (W713G) oocytes, this difference was abolished. In TRPL and TRPL (W814G) [oocytes injected with trpl (W713G) cRNA], but not in TRPL (W713G) oocytes, basal Ca2+ inflow was inhibited by W13, an inhibitor of calmodulin action. Calmodulin (3 μM intracellular) inhibited basal Ca2+ inflow in TRPL but not in TRPL (W713G) or TRPL (W814G) oocytes. Staurosporin, an inhibitor of protein kinase C (PKC), inhibited, while PMA (an activator of PKC) stimulated, basal Ca2+ inflow in TRPL oocytes. In oocytes incubated in the presence of PMA (to suppress Ca2+ inflow through endogenous receptor-activated Ca2+ channels), the InsP3-induced stimulation of Ca2+ inflow through TRPL channels was more clearly evident than in oocytes incubated in the absence of PMA. InsP3 caused a significant stimulation of Mn2+ inflow in TRPL but not in mock oocytes. Rates of InsP3-stimulated Ca2+ inflow through the TRPL, TRPL (W713G) and TRPL (W814G) channels were similar. The ability of GTPγS to stimulate Ca2+ inflow through TRPL channels was inhibited by 50% in TRPL (W713G) oocytes but was unaffected in TRPL (W814G) oocytes. It is concluded that, in the environment of the Xenopus oocyte, the Drosophila TRPL channel is activated by (a) interaction with Ca2+/calmodulin at calmodulin-binding Site 1; (b) PKC; (c) InsP3 in a process that does not involve Ca2+ and calmodulin; and (d) a trimeric G-protein(s) through both a Ca2+/calmodulin-dependent and a Ca2+/calmodulin-independent mechanism.
Influence of lipid on the structure and phosphorylation of protein kinase C α substrate peptides
Authors: VINTON B. Betsy , WERTZ L. Stacey , JACOB Jaison , STEERE Joanna , GRISHAM M. Charles , CAFISO S. David , SANDO J. Julianne
Published: August 2015

The structure and phosphorylation of two protein kinase C (PKC) α substrate peptides were investigated in varying lipid systems using enzyme activity assays and circular dichroism (CD) spectroscopy. The α-peptide, which exhibits the typical PKC α substrate motif and is based on the pseudosubstrate region of PKC α, was phosphorylated to a similar extent in bovine brain phosphatidylserine vesicles or diheptanoylphosphatidylcholine (PC7) micelles (both with 5 mol % 1,2-dioleoyl-sn-glycerol), whereas neuromodulin (NM)-peptide, which does not exhibit this motif by virtue of its primary structure, was phosphorylated to a much lesser extent in the PC7 micellar system. CD spectra of the peptides indicated that NM-peptide underwent a dramatic structural change in the presence of dimyristoylphosphatidylserine (DMPS) vesicles, whereas spectra acquired in PC7 micelles were similar to those acquired in buffer alone. No significant structural change was observed in the α-peptide in the presence of either lipid. PKC activity assays conducted with a series of NM-peptides successively substituted with nitroxide spin labels at each residue position suggested that several residues distal to the phosphorylation site are necessary for substrate recognition. The effect of these substitutions is not consistent with the binding of the NM-peptide to PKC in an extended structure, but is consistent with the binding of this peptide in a helical conformation. Furthermore, the docking of a helical NM-peptide to the substrate binding site of PKC suggests that the interaction is energetically feasible. These results suggest that PKC may recognize some non-linear substrate motifs and that lipid binding may convert a protein into a better PKC substrate.
Characterization of the kainate-binding domain of the glutamate receptor GluR-6 subunit
Authors: KEINÄNEN Kari , JOUPPILA Annukka , KUUSINEN Arja
Published: August 2015

Recombinant fragments of the kainate-selective glutamate recep-tor subunit GluR-6 were expressed in insect cells and analysed for [3H]kainate binding activity in order to characterize the structural determinants responsible for ligand recognition. Deletion of the N-terminal ~ 400 amino-acid-residue segment and the C-terminal ~ 90 residues resulted in a membrane-bound core fragment which displayed pharmacologically native-like [3H]kainate binding properties. Further replacement of the membrane-embedded segments M1-M3 by a hydrophilic linker peptide gave rise to a soluble polypeptide which was accumulated in the culture medium. When bound to chelating Sepharose beads via a C-terminal histidine tag, the soluble fragment showed low-affinity binding of [3H]kainate, which was displaced in a concentration-dependent manner by unlabelled domoic acid, L-glutamate and 6-cyano-7-nitroquinoxaline-2,3-dione. Our results indicate that the kainate-binding site is formed exclusively by the two discontinuous extracellular segments (S1 and S2) which are homologous to bacterial amino-acid-binding proteins. Ligand binding characteristics of soluble S1-S2 chimaeras between the GluR-6 and GluR-D subunits showed that, whereas both S1 and S2 segments contribute to agonist-selectivity, the N-terminal one-third of the GluR-D S2 segment is sufficient to confer α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-binding capacity to the chimaeric ligand-binding domain.
Characterization of human involucrin promoter distal regulatory region transcriptional activator elements–a role for Sp1 and AP1 binding sites
Authors: BANKS Eric B. , CRISH James F. , WELTER Jean F. , ECKERT Richard L.
Published: August 2015

Human involucrin (hINV) is an important precursor of the keratinocyte cornified envelope that is specifically expressed in the suprabasal layers of stratifying epithelia. Previous truncation and mutagenesis experiments have shown that an activator protein 1 (Ap1) site, AP1–5, located 2100 bp upstream of the transcription start site, is required for optimal promoter activity. These previous studies suggest that AP1–5 is part of a distal regulatory region spanning nucleotides -2473 to -2088. In the present report, we study the distal regulatory region (DRR), which surrounds AP1–5. Our studies show that this region contains weak and strong activator elements spanning nucleotides -2473/-2216 and -2140/-2088, respectively. The strong activator element contains AP1–5 and an adjacent specificity protein 1 (Sp1) site. The AP1–5 site is absolutely required for DRR activity, as its mutation reduces transcription to basal levels. Mutagenesis studies of the AP1–5 and Sp1 sites in the presence or absence of the weak activator element indicate that the Sp1 site and the weak activator element synergistically activate the AP1–5 site-dependent transcription. The cooperation between the Sp1 and AP1–5 sites is also observed in the context of the full-length promoter. Gel mobility shift and supershift studies show that Sp1, but not Sp2, Sp3 or Sp4 binds to the Sp1 site. When the Sp1 site is mutated or the distance between the AP1–5 and Sp1 site is increased, the binding of AP1 factors to AP1–5 is markedly reduced. Surprisingly, gel shift studies suggest that activation does not require the formation of a stable AP1/Sp1/DNA ternary complex. These studies suggest that the AP1–5 site is absolutely required for transcriptional activation, that the weak activator element and Sp1 sites serve to enhance this activation, and that the Sp1 site is required for optimal AP1 factor binding at the AP1–5 site.
Activation of calcineurin and smooth muscle myosin light chain kinase by Met-to-Leu mutants of calmodulin
Authors: EDWARDS Robert A. , WALSH Michael P. , SUTHERLAND Cindy , VOGEL Hans J.
Published: August 2015

The effects of replacement of each of the individual Met in calmodulin (CaM) with Leu on the activation of two CaM target enzymes [smooth muscle myosin light chain kinase (smMLCK) and calcineurin (CN)] were investigated. The KD and Pmax (percentage maximal activation) values for activation of both enzymes by M76L-CaM were indistinguishable from wild-type (wt)-CaM, which is consistent with the location of Met-76 in the central linker that is not involved in target protein interaction. The other eight Met in CaM are exposed in the hydrophobic surfaces that are involved in target-enzymes binding, and in general equivalent effects are observed for substitutions of Leu for Met residues in homologous positions in the two CaM domains. However, the importance of the interaction of specific Met residues with the target enzyme depends on the particular enzyme. Leu substitution at Met-36 or Met-109 reduced the affinity of MLCK for the mutant and the maximal activation of CN. MLCK had a higher KD for M51L-CaM whereas M124L-CaM activated the kinase to only 68% of maximal activity induced by wt-CaM; these mutants were indistinguishable from wt-CaM in activation of CN. M71L- and M144L-CaMs behaved like wt-CaM in activation of MLCK, but activated the phosphatase to only about 80% of maximal activity induced by wt-CAM. M72L-CaM exhibited an increased affinity for MLCK compared to wt-CaM and slightly decreased maximal activation, whereas M145L-CaM exhibited maximal activation significantly greater than that due to wt-CaM; these mutants behaved like wt-CaM with respect to CN activation. Finally, a mutant CaM in which all four C-terminal Met were replaced by Leu (M4-CT-L4-CaM) had similar affinities for MLCK and CN as wt-CaM but maximal activation of these enzymes by this mutant was only 60–70% of that achieved with wt-CaM. These results imply that, in addition to removing the autoinhibitory domain from the active site of the target enzyme, CaM must induce a conformational change in the active site itself.
Mitsugumin29, a novel synaptophysin family member from the triad junction in skeletal muscle
Authors: TAKESHIMA Hiroshi , SHIMUTA Misa , KOMAZAKI Shinji , OHMI Kazuhiro , NISHI Miyuki , IINO Masamitsu , MIYATA Atsuro , KANGAWA Kenji
Published: August 2015

In skeletal muscle, excitation–contraction (E–C) coupling requires the conversion of the depolarization signal of the invaginated surface membrane, namely the transverse (T-) tubule, to Ca2+ release from the sarcoplasmic reticulum (SR). Signal transduction occurs at the junctional complex between the T-tubule and SR, designated as the triad junction, which contains two components essential for E–C coupling, namely the dihydropyridine receptor as the T-tubular voltage sensor and the ryanodine receptor as the SR Ca2+-release channel. However, functional expression of the two receptors seemed to constitute neither the signal-transduction system nor the junction between the surface and intracellular membranes in cultured cells, suggesting that some as-yet-unidentified molecules participate in both the machinery. In addition, the molecular basis of the formation of the triad junction is totally unknown. It is therefore important to examine the components localized to the triad junction. Here we report the identification using monoclonal antibody and primary structure by cDNA cloning of mitsugumin29, a novel transmembrane protein from the triad junction in skeletal muscle. This protein is homologous in amino acid sequence and shares characteristic structural features with the members of the synaptophysin family. The subcellular distribution and protein structure suggest that mitsugumin29 is involved in communication between the T-tubular and junctional SR membranes.