Mitocryptide-2, a neutrophil-activating cryptide, is a specific endogenous agonist for formyl-peptide receptor-like 1

Peptides simultaneously produced during maturation and degradation of peptidergic hormones and functional proteins have recently become a great interest because they display unpredictably different biological roles than the parent proteins. Namely, we discovered two novel functional cryptic peptides, mitocryptide-1 (MCT-1) and mitocryptide-2 (MCT-2), hidden in mitochondrial cytochrome c oxidase and cytochrome b, that efficiently induced neutrophilic migration and activation at nanomolar concentrations. We named these functional “cryptic” peptides hidden in protein structures as “cryptides.” In this study, we investigated the receptor molecules and cellular signaling mechanisms for neutrophil-activating N-formylated cryptide MCT-2. In order to identify the receptor molecules, we established HEK-293 cells stably expressing either formyl-peptide receptor (FPR) or its homologue FPR-like 1 (FPRL1), because neutrophilic cells express these receptor molecules which recognize N-formylated peptides. We observed that MCT-2 directly bound to FPRL1 and promoted an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), and neither interacted with nor activated FPR, demonstrating that MCT-2 is a specific agonist for FPRL1. Moreover, MCT-2 induced not only [Ca²⁺]_i increase and phosphorylation of extracellular signal-regulated protein kinases 1 and 2, but also β-hexosaminidase release in neutrophilic/granulocytic cells differentiated from HL-60 cells. Such signaling events were diminished by pretreatment with pertussis toxin, indicating that MCT-2-promoted neutrophilic function is a consequence of G_i- or G_o-type G protein-dependent intracellular signaling events via FPRL1 activation. These findings suggest that MCT-2, a cryptide derived from mitochondrial cytochrome b, is a specific endogenous agonist for FPRL1 which is proposed to play key roles in inflammatory responses but whose physiological agonists are equivocal.     

Phosphofructokinase from porcine heart, liver and erythrocytes

1. Phosphofructokinase from porcine heart, liver and erythrocytes were purified by affinity chromatographies on Cibacron Blue Sepharose and N6-ATP agarose. 2. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the heart and liver enzymes consist of only one kind of subunit, namely, M and L type subunits, respectively, whereas the erythrocyte enzyme comprises of three kinds of subunits, M, L and C types. 3. Some kinetic and regulatory properties of the enzymes were also measured.     

Aluminum: a pH-dependent inhibitor of NADP-isocitrate dehydrogenase from porcine heart

Aluminum showed a pH-dependent inhibitory effect on NADP-isocitrate dehydrogenase from porcine heart. Aluminum ions (Al³⁺) acted as a partial competitive inhibitor of the enzyme with respect to the substratethreo-Ds-isocitrate and inhibited the enzyme non-competitively with respect to NADP at pH 6.85. Fractional velocity plot analysis showed theK _i of the enzyme for aluminum ions to be 0.88m. When pH was elevated to 8.0, aluminum ions, which occur as a form of the Al(OH)₄ ^– anion, acted as partial uncompetitive and non-competitive inhibitors of the enzyme with respect to the substrates isocitrate and NADP, respectively. TheK _i of the enzyme was determined to be 5.64 m at pH 8.0 by fractional velocity plot analysis. The inhibition of NADP-isocitrate dehydrogenase by two forms of aluminum ions may explain aluminum toxicity in various tissues and organs.     

Structure of the neutrophil-activating protein from Helicobacter pylori

Helicobacter pylori is a major human pathogen associated with severe gastroduodenal diseases, including ulcers and cancers. An H.pylori protein that is highly immunogenic in humans and mice has been identified recently. This protein has been termed HP-NAP, due to its ability of activating neutrophils. In order to achieve a molecular understanding of its unique immunogenic and pro-inflammatory properties, we have determined its three-dimensional structure. Its quaternary structure is similar to that of the dodecameric bacterial ferritins (Dps-like family), but it has a different surface potential charge distribution. This is due to the presence of a large number of positively charged residues, which could well account for its unique ability in activating human leukocytes.     

Purification and partial biochemical characterization of a human monocyte-derived, neutrophil-activating peptide that lacks interleukin 1 activity

A novel monocyte-derived neutrophil-activating peptide (MONAP) produced by lipopolysaccharide- and phorbol myristate acetate-stimulated human peripheral blood monocytes was purified by sequential ion exchange-high performance liquid chromatography (HPLC), size exclusion HPLC, and reversed phase HPLC. Biologic activities of the purified cytokine were monitored by either an enzyme release assay or a chemotaxis assay, using peripheral human neutrophils. Purified MONAP was found to be homogeneous, giving a single peak on size-exclusion HPLC, reversed-phase HPLC, as well as a single 10-kDa band on silver-stained polyacrylamide gels. Purified MONAP stimulate human neutrophil chemotaxis at an estimated molarity of 5 x 10(-11) M. Half-maximal enzyme release of cytochalasin B pretreated neutrophils occurred at 2 to 3 x 10(-10) M, whereas superoxide anion production elicited by various concentrations of MONAP was found to be low. Isolated human peripheral monocytes, as well as human eosinophils, showed no chemotactic response to MONAP, indicating neutrophil specificity. MONAP activity was separated from thymocyte-stimulating activity by reversed-phase HPLC, indicating nonidentity with interleukin (IL)-1. This was further supported by heat resistance of MONAP, which is in contrast to the heat sensitivity of IL-1. In addition, IL-1 obtained as a by-product during isolation of MONAP did not stimulate human neutrophil chemotaxis.     

Discovery of porcine microRNAs and profiling from skeletal muscle tissues during development

MiRNAs (microRNAs) play critical roles in many important biological processes such as growth and development in mammals. In this study, we identified hundreds of porcine miRNA candidates through in silico prediction and analyzed their expression in developing skeletal muscle using microarray. Microarray screening using RNA samples prepared from a 33-day whole embryo and an extra embryo membrane validated 296 of the predicted candidates. Comparative expression profiling across samples of longissimus muscle collected from 33-day and 65-day post-gestation fetuses, as well as adult pigs, identified 140 differentially expressed miRNAs amongst the age groups investigated. The differentially expressed miRNAs showed seven distinctive types of expression patterns, suggesting possible involvement in certain biological processes. Five of the differentially expressed miRNAs were validated using real-time PCR. In silico analysis of the miRNA-mRNA interaction sites suggested that the potential mRNA targets of the differentially expressed miRNAs may play important roles in muscle growth and development.     

Activation by spermine of citrate synthase from porcine heart

Spermine activated citrate synthase from porcine heart by decreasing the Km value for the substrate oxaloacetate without affecting the maximal velocity. Spermine markedly increased the maximal velocity of the saturation function with respect to acetyl-CoA as the substrate under conditions of intracellular concentrations of oxaloacetate, but the enzyme was not activated by spermine under conditions of higher concentrations of oxaloacetate. The concentration of spermine required for 50% activation of the enzyme was about 50 microM. Spermidine showed only a little activation, while putrescine caused no activation. Spermine, which contributes to an activation of Ca2(+)-sensitive dehydrogenases of the citric acid cycle by enhancing Ca2+ uptake into mitochondria, can activate citrate synthase directly, and is responsible for the stimulation of oxidative metabolism in mitochondria.     

Characterization of an S-type lectin purified from porcine heart

We have purified a carbohydrate-binding protein from porcine heart by affinity chromatography on asialofetuin-Sepharose and have characterized this protein with respect to its size, amino acid composition, partial amino acid sequence, and carbohydrate-binding specificity. Porcine heart lectin (PHL) has a subunit molecular mass of 14,700 and is immunologically cross-reactive with a polyclonal antibody raised against a lectin isolated from calf heart. The amino acid composition of PHL is similar to that of lectins that have been isolated from calf heart, bovine brain, and rat lung. Moreover, the primary sequences of four tryptic fragments (52 amino acids total) derived from PHL are closely related to sequences previously determined for 10 other vertebrate-derived lectins. The ability of PHL to agglutinate rabbit erythrocytes was inhibited only by oligosaccharides containing terminal beta-galactosyl residues. These data indicate that PHL is a vertebrate "S-type" lectin and provide further evidence that the structures and carbohydrate-binding specificities of these lectins are highly conserved across diverse vertebrate genera.     

Studies on aconitase species from Saccharomyces cerevisiae, porcine and bovine heart, obtained by a modified isolation method

Aconitase (citrate (isocitrate) hydro-lyase, EC 4.2.1.3) was isolated from Saccharomyces cerevisiae, porcine and bovine heart by a simplified method including affinity chromatography on Blue Dextran-Sepharose. Partial characterisation reveals that the aconitase species are all similar due to molecule size, amino acid composition, isoelectric point and enzymatic activity. Aconitase appears as a single polypeptide chain with a small carbohydrate content. A molecular weight of 79000 +/- 2000 and a Svedberg constant of s20,w = 4.75 +/- 0.2 S indicate a compact structure of aconitase. Due to different properties among the yeast aconitase species concerning isoelectric point and enzymatic activity a coherence between net charge of the protein and redox state of the Fe-S cluster can be expected.     

Fucosyl-GM1a, an endoglycoceramidase-resistant ganglioside of porcine brain

The use of bovine brain has been prohibited in many countries because of the world-wide prevalence of mad cow disease, and thus porcine brain is expected to be a new source for the preparation of gangliosides. Here, we report the presence of a ganglioside in porcine brain which is strongly resistant to hydrolysis by endoglycoceramidase, an enzyme capable of cleaving the glycosidic linkage between oligosaccharides and ceramides of various glycosphingolipids. Five major gangliosides (designated PBG-1, 2, 3, 4, 5) were extracted from porcine brain by Folch's partition, followed by mild alkaline hydrolysis and PBA column chromatography. We found that PBG-2, but not the others, was strongly resistant to hydrolysis by the enzyme. After the purification of PBG-2 with Q-Sepharose, Silica gel 60 and Prosep-PB chromatographies, the structure of PBG-2 was determined by GC, GC-MS, FAB-MS and NMR spectroscopy as Fucalpha1-2Galbeta1-3GalNAcbeta1-4(NeuAcalpha2-3)Galbeta1-4Glcbeta1-1'Cer (fucosyl-GM1a). The ceramide was mainly composed of C18:0 and C20:0 fatty acids and d18:1 and d20:1 sphingoid bases. The apparent kcat/Km for fucosyl-GM1a was found to be 30 times lower than that for GM1a, indicating that terminal fucosylation makes GM1a resistant to hydrolysis by the enzyme. This report indicates the usefulness of endoglycoceramidase to prepare fucosyl-GM1a from porcine brain.     

Isolation and morphology of single Purkinje cells from the porcine heart

Purkinje cells were isolated from both ventricles of young adult domestic pigs and examined by transmitted light or laser scanning confocal microscopy. Purkinje cells in free running Purkinje fibres were organised in multicellular strands where individual cells were tightly connected end-to-end and closely side-to-side. After isolation, single cells gradually lost the elongated appearance and became more rounded, but the cell membrane remained smooth and undamaged. The contractile material was not very dense and was seen most clearly in the submembraneous area. Staining of the cell membrane with the lipophilic fluorescent (lye di-8-ANNEPS, and visualization with confocal microscopy, confirmed that the cell surface membrane was smooth without blebs. This staining also showed that Purkinje cells had no transversal tubules. We reconstructed the three-dimensional geometry of the Purkinje cells and determined the cell size. The average values were 62 +/- 9 microm for length, 32 +/- 3 microm for width, and 41 +/- 4 microm for depth (n = 7). Calculated cross-section area and volume were 1047 +/- 167 microm2 and 47 +/- 14 pl. Compared to ventricular cells, the morphology of the Purkinje cells reflects their specific role in impulse conduction.     

Discovery of a eugenol oxidase from Rhodococcus sp. strain RHA1

A gene encoding a eugenol oxidase was identified in the genome from Rhodococcus sp. strain RHA1. The bacterial FAD-containing oxidase shares 45% amino acid sequence identity with vanillyl alcohol oxidase from the fungus Penicillium simplicissimum. Eugenol oxidase could be expressed at high levels in Escherichia coli, which allowed purification of 160 mg of eugenol oxidase from 1 L of culture. Gel permeation experiments and macromolecular MS revealed that the enzyme forms homodimers. Eugenol oxidase is partly expressed in the apo form, but can be fully flavinylated by the addition of FAD. Cofactor incorporation involves the formation of a covalent protein-FAD linkage, which is formed autocatalytically. Modeling using the vanillyl alcohol oxidase structure indicates that the FAD cofactor is tethered to His390 in eugenol oxidase. The model also provides a structural explanation for the observation that eugenol oxidase is dimeric whereas vanillyl alcohol oxidase is octameric. The bacterial oxidase efficiently oxidizes eugenol into coniferyl alcohol (KM=1.0 microM, kcat=3.1 s-1). Vanillyl alcohol and 5-indanol are also readily accepted as substrates, whereas other phenolic compounds (vanillylamine, 4-ethylguaiacol) are converted with relatively poor catalytic efficiencies. The catalytic efficiencies with the identified substrates are strikingly different when compared with vanillyl alcohol oxidase. The ability to efficiently convert eugenol may facilitate biotechnological valorization of this natural aromatic compound.     

Purification and amino acid sequencing of NAF, a novel neutrophil-activating factor produced by monocytes

Human blood mononuclear cells were cultured for 24 h in the presence of LPS (100 ng per 5 x 10(6) cells), and a monocyte-derived neutrophil-activating factor (NAF) was purified to apparent homogeneity from the conditioned media. The purification consisted of ammonium sulphate precipitation, gel filtration, chromatography on phosphocellulose followed by hydroxylapatite, and reversed-phase HPLC on C4 and CN-propyl columns. Amino acid sequence analysis (32 of 50 presumed residues) shows that NAF is a novel peptide with little homology to known ones. Crude and pure NAF stimulated human neutrophils to release granule enzymes and to produce superoxide and H2O2.     

Structural characterization of the dihydropyridine receptor-linked calcium channel from porcine heart.

Ca(2+)-channel was purified 230-fold from digitonin extracts of the porcine cardiac sarcolemmal membranes by means of a four-step procedure. Two antibodies, a site-directed antibody against the sequence 1691-1707 of the rabbit cardiac alpha 1 subunit (anti-CCP5) and a monoclonal antibody directed to rabbit skeletal muscle alpha 2 delta subunit-complex (MCC-1), effectively immunoprecipitated the 125I-labeled cardiac Ca(2+)-channel complex in 0.2% digitonin. SDS-PAGE analysis of the immunoprecipitates under reducing conditions revealed that the cardiac channel is mainly composed of two large polypeptides of 190 and 150 kDa, and five smaller polypeptides of 60, 55, 35, 30, and 25 kDa. An additional polypeptide of either 79 or 55 kDa is crosslinked with the 190 kDa component to form 250-270 kDa (approximately 270 kDa) to the extent of 15-20% through disulfide bond(s). The 190 kDa component (alpha 1) is responsible for photoaffinity labeling with [3H]diazepine, since minor photolabeled approximately 270 kDa was converged to the major labeled 190 kDa component when electrophoresed under reducing conditions. The 150 kDa component (alpha 2) was derived by reduction of disulfide bonds from another 190 kDa component of glycopolypeptide which was separated from the channel complex in 1% Triton X-100 and capable of binding to WGA-Sepharose. The four smaller components of 60, 35, 30, and 25 kDa were not covalently associated with the large components through disulfide bonds, whereas the 55 kDa polypeptide was suggested to be a mixture of two kinds of peptides with respect to the disulfide bond: one was crosslinked with alpha 1 through disulfide linkage and the other was not covalently associated with any other component.(ABSTRACT TRUNCATED AT 250 WORDS)