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41.
Down-regulation and inactivation of neutral endopeptidase 24.11 (enkephalinase) in human neutrophils 总被引:2,自引:0,他引:2
E G Erd?s B Wagner C B Harbury R G Painter R A Skidgel X G Fa 《The Journal of biological chemistry》1989,264(24):14519-14523
Neutral endopeptidase (EC 3.4.24.11, NEP) is an integral membrane protein of human neutrophils. NEP is identical with the common acute lymphoblastic leukemia antigen (CALLA) of leukemic cells. The expression of NEP on the surface of neutrophils is down-regulated by endocytosis which can be induced by phorbol 12-myristate 13-acetate (PMA) at 37 degrees C. The activity of the enzyme on the surface of intact cells decreases by 76% within 5 min. The activity can be recovered, however, if the cells are lysed within 5 min of the endocytosis. After 30 min, only 32% of the NEP activity is present in the neutrophil lysates. The loss of activity is presumably due to proteolytic inactivation. Diacylglycerol and monoclonal antibody to CALLA/NEP also induce internalization of NEP. PMA induces endocytosis even at 4 degrees C, but NEP is not inactivated at that temperature. The disappearance of NEP activity after adding PMA was inhibited by various agents. Among the most active were the phospholipase inhibitor 4-bromophenacyl bromide and a combination of the serine protease and cathepsin inhibitors, diisopropylfluorophosphate and N-ethylmaleimide. The employment of fluorescent monoclonal antibody confirmed the down-regulation and internalization of NEP antigen on the neutrophils. Since NEP inactivates chemotactic peptides and thereby affects chemotaxis of neutrophils (Painter, R. G., Dukes, R., Sullivan, J., Carter, R., Erd?s, E. G., and Johnson, A. R. (1988) J. Biol. Chem. 263, 9456-9461), the down-regulation of NEP activity on the cell membrane may modulate the function of these cells in inflammation. 相似文献
42.
Hydrolysis of substance p and neurotensin by converting enzyme and neutral endopeptidase 总被引:24,自引:0,他引:24
Angiotensin I converting enzyme (ACE) and neutral endopeptidase ("enkephalinase"; NEP), were purified to homogeneity from human kidney. NEP cleaved substance P (SP) at Gln6-Phe7,-Phe8, and Gly9-Leu10 and neurotensin (NT) at Pro10-Tyr11 and Tyr11-Ile12. NEP hydrolyzed 0.1 mM SP, NT and their C-terminal fragments at the following rates (mumol/min/mg): SP1-11 = 7.8, SP4-11 = 11.7, SP5-11 = 15.4, SP6-11 = 15.6, SP8-11 = 6.7, NT1-13 = 2.9, and NT8-13 = 4.0. Purified ACE rapidly inactivated SP as measured in bioassay. HPLC analysis showed that ACE cleaved SP at Phe8-Gly9 and Gly9-Leu10 to release C-terminal tri- and dipeptide (ratio = 4:1). The hydrolysis was Cl- dependent and inhibited by captopril. ACE released mainly C-terminal tripeptide from SP methyl ester, but only dipeptide from SP free acid. Modification of arginine residues in ACE with cyclohexanedione or butanedione similarly inhibited hydrolysis of SP, bradykinin and Bz-Gly-Phe-Arg (80-93%) indicating an active site arginine is required for hydrolysis of SP. ACE hydrolyzed NT at Tyr11-Ile12 to release Ile12-Leu13. SP, NT and their derivatives (0.1 mM) were cleaved by ACE at the following rates (mumol/min/mg): SP1-11 = 1.2, SP methyl ester = 0.7, SP free acid = 8.5, SP4-11 = 2.4, SP5-11 = 0.9, SP6-11 = 1.4, SP8-11 = 0, NT1-13 = 0.2, and NT8-13 = 1.3. Peptide substrates were used as inhibitors of ACE (substrate = FA-Phe-Gly-Gly) and NEP (substrate = Leu5-enkephalin).(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
43.
Carboxypeptidase M, a glycosylphosphatidylinositol-anchored membrane glycoprotein, is highly expressed in Madin-Darby canine kidney (MDCK) cells, where it was previously shown that the glycosylphosphatidylinositol anchor and N-linked carbohydrate are apical targeting signals. Here, we show that carboxypeptidase M has an unusual, non-polarized distribution, with up to 44% on the basolateral domain of polarized MDCK cells grown on semipermeable inserts. Alkaline phosphatase, as well as five other glycosylphosphatidylinositol-anchored proteins, and transmembrane gamma-glutamyl transpeptidase exhibited the expected apical localization. Basolateral carboxypeptidase M was readily released by exogenous phosphatidylinositol-specific phospholipase C, showing it is glycosylphosphatidylinositol-anchored, whereas apical carboxypeptidase M was more resistant to release. In contrast, the spontaneous release of carboxypeptidase M into the medium was much higher on the apical than the basolateral domain. In pulse-chase studies, newly synthesized carboxypeptidase M arrived in equal amounts within 30 min on both domains, indicating direct sorting. After 4-8 h of chase, the steady-state distribution was attained, possibly due to transcytosis from the basolateral to the apical domain. These data suggest the presence of a unique basolateral targeting signal in carboxypeptidase M that competes with its apical targeting signals, resulting in a non-polarized distribution in MDCK cells. 相似文献
44.
The didelphid marsupial, Didelphis aurita, is suggested as an intraguild predator and as key‐species in small mammal assemblages of the Atlantic Forest of Brazil. The field experiments required to test this hypothesis are complex to implement, but the recent revival of regression methods offers a viable alternative. Here we use the dynamic and static regression methods to determine the importance of D. aurita as a competitor and intraguild predator. Capture–recapture data from two localities in the Rio de Janeiro State were used, Garrafão (municipality of Guapimirim), a coastal forest of the Serra do Mar, and Barra de Maricá, a costal sand dune vegetation. Population and microhabitat variables were monitored from April 1997 to April 2003 in Garrafão, and from January 1986 to July 1990 in Barra de Maricá. Microhabitat variables were related to Canopy, Plant, Litter and Rock covers, Obstruction from 0 to 1.5 m, and Number of logs. Exploitation competition was tested by the dynamic method, which models the effects of D. aurita on the per capita growth rate of a species. Interference by predation or competition was tested by the static method, where the abundance of D. aurita at trap stations was regressed against the abundance of other small mammals, after removal of any variation associated with microhabitat factors. Exploitation competition was not detected, but the interference of D. aurita was pervasive, affecting all small mammals studied in the two localities. The clear avoidance of D. aurita by all small mammals tested in two localities of different physiognomies indicates that it functions as an intraguild predator, even if actual predation by D. aurita is an occasional event. 相似文献
45.
We are currently in an interesting phase of plant biotechnology releases, both for the scientists responsible for these innovations who are beginning to see their ideas realized, and for the biotechnology companies that are starting to see a return on their investment. One of the most notable examples, is the introduction of transgenic crops that are engineered to express a Bacillus thuringiensis toxin that confers resistance to insect predation. However, the picture is not altogether positive - there is concern that the introduction of this technology was premature or should not have happened at all, and that the valuable insecticidal properties of Bacillus thuringiensis will be lost. 相似文献
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49.
Amoresano A; Andolfo A; Siciliano RA; Mele A; Coscarella A; De Santis R; Mauro S; Pucci P; Marino G 《Glycobiology》1998,8(8):779-790
MEN 11300 is a hybrid glycoprotein of 297 amino acids obtained by fusion of
the cDNA encoding GM-CSF with the cDNA encoding EPO followed by
transfection of the hybrid gene into CHO cells. The oligonucleotide
construct incorporated a spacing sequence between the two individual cDNAs
which encodes eight amino acids constituting a linker peptide intended to
separate the GM-CSF and EPO moieties. The recombinant MEN 11300 protein was
submitted to a detailed structural characterization including the
verification of the entire amino acid sequence, the assignment of the
disulfide bridges pattern, the identification of the glycosylation sites
and the definition of the glycosidic moiety, including site-specificity.
Partial processing of the C-terminal Arg residue and the occurrence of
N-glycosylation sites at Asn27, Asn155, Asn169, Asn214 were established.
Moreover, O-glycosylation at Ser257 and at the N-terminal region was also
detected. A large heterogeneity was observed in the N-glycans due to the
presence of differently sialylated and fucosylated branched complex type
oligosaccharides whereas O-linked glycans were constituted by GalGalNAc
chains with a different number of sialic acids. The disulfide bridges
pattern was established by direct FABMS analysis of the proteolytic digests
or by ESMS analysis of HPLC purified fractions. Pairing of the eight
cysteine residues resulted in Cys54-Cys96, Cys88-Cys121, Cys138-Cys292, and
Cys160-Cys164. This S-S bridges pattern is identical to that occurring in
the individual natural GM-CSF and EPO, thus showing that the two protein
moieties in MEN 11300 can independently acquire their native
three-dimensional structure.
相似文献
50.
Unusual pattern of bacterial ice nucleation gene evolution 总被引:5,自引:0,他引:5
Edwards AR; Van den Bussche RA; Wichman HA; Orser CS 《Molecular biology and evolution》1994,11(6):911-920
Bacterial ice nucleation activity (INA+ phenotype) can be traced to the
product of a single gene, ina. A remarkably sparse distribution of this
phenotype within three bacterial genera indicates that the ina gene may
have followed an unusual evolutionary path. Southern blot analyses, coupled
with assays for ice-nucleating ability, revealed that within four bacterial
species an ina gene is present in some strains but absent from others.
Results of hybridization experiments using DNA fragments that flank the ina
gene suggested that the genotypic dimorphism of ina may be anomalous. A
phylogenetic analysis of 16S ribosomal RNA gene sequences from a total of
14 ina+ and ina- bacterial strains indicated that the ina+ bacteria are not
monophyletic but instead phylogenetically interspersed among ina- bacteria.
The relationships of ina+ bacteria inferred from ina sequence did not
coincide with those inferred from the 16S data. These results suggest the
possibility of horizontal transfer in the evolution of bacterial ina genes.
相似文献