PubMed Health⌕ Search

Biomedical subjects

M Popov

Publications and source records attributed to M Popov.

At least 19 recordsLinked to original sources
PubMed2000

Processing of N-linked oligosaccharide depends on its location in the anion exchanger, AE1, membrane glycoprotein.

The human erythrocyte anion exchanger (AE)1 (Band 3) contains a single complex N-linked oligosaccharide that is attached to Asn(642) in the fourth extracellular loop of this polytopic membrane protein, while other isoforms (AE2, AE3 and trout AE1) are N-glycosylated on the preceding extracellular loop. Human AE1 expressed in transfected human embryonic kidney (HEK)-293 or COS-7 cells contained a high-mannose oligosaccharide. The lack of oligosaccharide processing was not due to retention of AE1 in the endoplasmic reticulum since biotinylation assays showed that approx. 30% of the protein was expressed at the cell surface. Moving the N-glycosylation site to the preceding extracellular loop in an AE1 glycosylation mutant (N555) resulted in processing of the oligosaccharide and production of a complex form of AE1. A double N-glycosylation mutant (N555/N642) contained both a high-mannose and a complex oligosaccharide chain. The complex form of the N555 mutant could be biotinylated showing that this form of the glycoprotein was at the cell surface. Pulse-chase experiments showed that the N555 mutant was efficiently converted from a high-mannose to a complex oligosaccharide with a half-time of approx. 4 h, which reflected the time course of trafficking of AE1 from the endoplasmic reticulum to the plasma membrane. The turnover of the complex form of the N555 mutant occurred with a half-life of approx. 15 h. The results show that the oligosaccharide attached to the endogenous site in extracellular loop 4 in human AE1 is not processed in HEK-293 or COS-7 cells, while the oligosaccharide attached to the preceding loop is converted into the complex form.

Animals↗
PubMed2000

Resolution of glycoproteins by a lectin gel-shift assay.

Gel-shift assays previously described in the literature are based on protein-protein or protein-DNA interactions. We show that carbohydrate-lectin interactions can be successfully used to alter the electrophoretic mobility of glycosylated, but not nonglycosylated, protein species in SDS-polyacrylamide gels. We were able to separate the two closely migrating mono- (95 kDa) and nonglycosylated (92 kDa) forms of a polytopic membrane protein, anion exchanger 1 (AE1), synthesized by cell-free translation or in transfected HEK293 cells. Concanavalin A was selected as the lectin due to the high mannose content of the oligosaccharide chain on AE1. Concanavalin A was either added to the samples prior to loading or copolymerized in a top layer of the separating gel, the latter being the method of choice. The presence of concanavalin A resulted in slower mobility of the monoglycosylated protein while the mobility of the nonglycosylated form was not altered. The shift in mobility was dependent on concentration of concanavalin A and the length of separating gel containing copolymerized concanavalin A. When a diglycosylated mutant of AE1 was tested, good separation was achieved at lower concentrations of concanavalin A. This lectin gel-shift assay allows the separation of N-glycosylated and nonglycosylated forms of the protein.

Animals↗
PubMed1999

Calnexin interaction with N-glycosylation mutants of a polytopic membrane glycoprotein, the human erythrocyte anion exchanger 1 (band 3).

The interaction of the endoplasmic reticulum chaperone calnexin with N-glycosylation mutants of a polytopic membrane glycoprotein, the human erythrocyte anion exchanger (AE1), was characterized by cell-free translation and in transfected HEK293 cells, followed by co-immunoprecipitation using anti-calnexin antibody. AE1 contains 12-14 transmembrane segments and has a single site of N-glycosylation at Asn-642 in the fourth extracytosolic loop. This site was mutated (N642D) to create a nonglycosylated protein. Calnexin showed a preferential interaction with N-glycosylated AE1 relative to nonglycosylated AE1 both in vitro and in vivo. This interaction could be blocked by inhibition of glucosidases I and II with castanospermine. Calnexin had access to novel N-glycosylated sites created in other extracytosolic loops in AE1 by site-directed or insertional mutagenesis. The interaction with AE1 was enhanced when multiple sites were introduced into the same loop or into two different loops. An association of calnexin with truncated versions of N-glycosylated AE1 was detected after release of the nascent chains from ribosomes with puromycin. The results show that the interaction of calnexin with the polytopic membrane glycoprotein AE1 was dependent on the presence but not the location of the oligosaccharide. Furthermore, calnexin was associated with AE1 after release of AE1 from the translocation machinery.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1999

Transmembrane folding of the human erythrocyte anion exchanger (AE1, Band 3) determined by scanning and insertional N-glycosylation mutagenesis.

The human erythrocyte anion exchanger (AE1, Band 3) contains up to 14 transmembrane segments, with a single site of N-glycosylation at Asn642 in extracellular (EC) loop 4. Scanning and insertional N-glycosylation mutagenesis were used to determine the folding pattern of AE1 in the membrane. Full-length AE1, when expressed in transfected human embryonic kidney (HEK)-293 or COS-7 cells, retained a high-mannose oligosaccharide structure. Scanning N-glycosylation mutagenesis of EC loop 4 showed that N-glycosylation acceptor sites (Asn-Xaa-Ser/Thr) spaced 12 residues from the ends of adjacent transmembrane segments could be N-glycosylated. An acceptor site introduced at position 743 in intracellular (IC) loop 5 that could be N-glycosylated in a cell-free translation system was not N-glycosylated in transfected cells. Mutations designed to disrupt the folding of this loop enhanced the level of N-glycosylation at Asn743 in vitro. The results suggest that this loop might be transiently exposed to the lumen of the endoplasmic reticulum during biosynthesis but normally folds rapidly, precluding N-glycosylation. EC loop 4 insertions into positions 428, 484, 754 and 854 in EC loops 1, 2, 6 and 7 respectively were efficiently N-glycosylated, showing that these regions were extracellular. EC loop 4 insertions into positions 731 or 785 were poorly N-glycosylated, which was inconsistent with an extracellular disposition for these regions of AE1. Insertion of EC loop 4 into positions 599 and 820 in IC loops 3 and 6 respectively were not N-glycosylated in cells, which was consistent with a cytosolic disposition for these loops. Inhibitor-affinity chromatography with 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonate (SITS)-Affi-Gel was used to assess whether the AE1 mutants were in a native state. Mutants with insertions at positions 428, 484, 599, 731 and 785 showed impaired inhibitor binding, whereas insertions at positions 754, 820 and 854 retained binding. The results indicate that the folding of the C-terminal region of AE1 is more complex than originally proposed and that this region of the transporter might have a dynamic aspect.

Amino Acid Sequence↗
PubMed1997

Mapping the ends of transmembrane segments in a polytopic membrane protein. Scanning N-glycosylation mutagenesis of extracytosolic loops in the anion exchanger, band 3.

Band 3, the anion exchanger of human erythrocytes, contains up to 14 transmembrane (TM) segments and has a single endogenous site of N-glycosylation at Asn642 in extracellular (EC) loop 4. The requirements for N-glycosylation of EC loops and the topology of this polytopic membrane protein were determined by scanning N-glycosylation mutagenesis and cell-free translation in a reticulocyte lysate supplemented with microsomal membranes. The endogenous and novel acceptor sites located near the middle of the 35 residue EC loop 4 were efficiently N-glycosylated; however, no N-glycosylation occurred at sites located within sharply defined regions close to the adjacent TM segments. Acceptor sites located in the center of EC loop 3, which contains 25 residues, were poorly N-glycosylated. Expansion of this loop with a 4-residue insert containing an acceptor site increased N-glycosylation. Acceptor sites located in short (<10 residues) loops (putative EC loops 1, 2, 6, and 7) were not N-glycosylated; however, insertion of EC loop 4 into EC loops 1, 2, or 7, but not 6, resulted in efficient N-glycosylation. Acceptor sites in putative intracellular (IC) loop 5 exhibited a similar pattern of N-glycosylation as EC loop 4, indicating a lumenal disposition during biosynthesis. To be efficiently N-glycosylated, EC loops in polytopic membrane proteins must be larger than 25 residues in size, with acceptor sites located greater than 12 residues away from the preceding TM segment and greater than 14 residues away from the following TM segment. Application of this requirement allowed a significant refinement of the topology of Band 3 including a more accurate mapping of the ends of TM segments. The strict distance dependence for N-glycosylation of loops suggests that TM segments in polytopic membrane proteins are held quite precisely within the translocation machinery during the N-glycosylation process.

Amino Acid Sequence↗
PubMed1993

[The role and importance of intraoperative cholangiography in modern biliary surgery].

UNLABELLED: The aim of the study is to show that routine use of intraoperative cholangiography may significantly reduce postoperative complications after cholecystectomy. We made a prospective study on 200 cholecystectomized patients in the period IX 1988-III 1989. Patients are divided in two groups: A--control group consisting of 100 cholecystectomized patients without intraoperative cholangiography and B--also consisting of 100 patients with routinely used intraoperative cholangiography. Results are given in the following table where the complications are separated by the time of appearance, specific signs and symptoms. CONCLUSION: Routine use of intraoperative cholangiography significantly reduces the number of complications (A:B = 19:9) which is statistically significant (chi-square test. p < 0.05).

Adult↗
PubMed1992

[Wounds from modern small arms and international humanitarian law].

The article cites the concept, basic standards and principles of international humane law concerning the banning or restriction of some types of conventional weapons. The authors describe international methods for appreciation of destructive effects of wounding missiles. On the basis of literature and their own experiments the authors make a conclusion that actually it would be impossible to formulate a straight definition between "human" and "inhumane" bullets, as well as to establish a criterion which could determine the admissible or inadmissible limits for gunshot injuries.

Firearms↗
PubMed1983

[Intensification and effectiveness of scientific research in hygiene (several problems of information supply)].

The nature of the concept "intensification" and the notions effect and effectiveness, associated with it are discussed. The nature of the separate kinds effect, obtained in hygienic science are determined - primary (health) and secondary (derivative). Effectiveness is determined on that base - as juxtaposition of the kinds of effects obtained with the resources spent in the field of hygienic science. Studies were carried out on the increased intensity of utilization of information resources as factor for the improvement of effectiveness in hygienic science. The place and role of information - retrieval systems are determined. Numerous concrete information systems are discussed as well as some of the factors in the evaluation of their effectiveness.

Bulgaria↗