scholarly journals Isolation and structural characterization of the Leishmania donovani kinetoplastid membrane protein-11, a major immunoreactive membrane glycoprotein

1995 ◽  
Vol 305 (1) ◽  
pp. 307-313 ◽  
Author(s):  
A Jardim ◽  
V Funk ◽  
R M Caprioli ◽  
R W Olafson
Keyword(s):  
Membrane Protein ◽  
Cell Surface ◽  
Leishmania Donovani ◽  
Surface Membrane ◽  
Immunoblot Analysis ◽  
Post Translational Modifications ◽  
Sds Page ◽  
Membrane Molecule

A novel membrane molecule, previously observed to be co-isolated with lipophosphoglycan and called lipophosphoglycan-associated protein, has been detected in Leishmania donovani promastigotes and amastigotes. This kinetoplastid membrane protein (KMP-11) has been purified by preparative SDS/PAGE after organic solvent extraction of promastigote membranes. Isoelectric-focusing experiments indicated that this was an acidic protein with an isoelectric point of 4.8. Immunoblot analysis of subcellular fractions, together with 125I-labelling experiments, showed this molecule to be associated with the promastigote cell surface membrane. KMP-11 was expressed at a copy number similar to that of lipophosphoglycan (1 x 10(6)-2 x 10(6) molecules per cell), making this glycoprotein one of the major features on the parasite cell surface. The primary structure, less a blocked N-terminal region, was determined by automated Edman degradation of peptides derived from CNBr or enzymic fragmentation. Several post-translational modifications were also found during these studies, including an O-linked oligosaccharide and an NG-monomethylarginine functionality which was verified by m.s. Finally, a set of sequential synthetic peptides was made based on the established partial sequence allowing structural determination of two distinct antibody-binding sites for the monoclonal antibodies L98 and L157.

Characterization of Glycoprotein Fraction from Carp Pituitaries Isolated Using Concanavalin A as the Affinity Ligand

1998 ◽  
Vol 63 (3) ◽  
pp. 434-440 ◽  
Author(s):  
Irena Hulová ◽  
Jana Barthová ◽  
Helena Ryšlavá ◽  
Václav Kašička
Keyword(s):  
Concanavalin A ◽  
Reversed Phase ◽  
Amino Acid Sequences ◽  
Gel Chromatography ◽  
Affinity Ligand ◽  
Sds Page ◽  
Terminal Amino ◽  
Α And Β Subunits

Glycoproteins that have affinity to Concanavalin A were isolated from the acetone-dried pituitaries of common carp (Cyprinus carpio L.). Two fractions of glycoproteins were separated using gel chromatography on Superdex 75HR. The fraction with lower molecular weight (30 000) corresponding to the carp gonadotropin cGtH II was composed of two subunits as determined using SDS-PAGE. This protein fraction was further divided into four components using reversed-phase HPLC. Two fractions were pure α and β subunits of cGtH II as follows from immunodetection and from determination of N-terminal amino acid sequences. The other two were a mixture of α and β subunits as was also revealed by N-terminal analysis. Capillary electrophoresis was also used for characterization of isolated glycoproteins.

scholarly journals Kv2.1 cell surface clusters are insertion platforms for ion channel delivery to the plasma membrane

2012 ◽  
Vol 23 (15) ◽  
pp. 2917-2929 ◽  
Author(s):  
Emily Deutsch ◽  
Aubrey V. Weigel ◽  
Elizabeth J. Akin ◽  
Phil Fox ◽  
Gentry Hansen ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Cell Surface ◽  
Ion Channel ◽  
Protein Trafficking ◽  
Hippocampal Neurons ◽  
Surface Membrane ◽  
Cell Types ◽  
Homogeneous Surface ◽  
Hek Cells ◽  
Membrane Protein Trafficking

Voltage-gated K+ (Kv) channels regulate membrane potential in many cell types. Although the channel surface density and location must be well controlled, little is known about Kv channel delivery and retrieval on the cell surface. The Kv2.1 channel localizes to micron-sized clusters in neurons and transfected human embryonic kidney (HEK) cells, where it is nonconducting. Because Kv2.1 is postulated to be involved in soluble N-ethylmaleimide–sensitive factor attachment protein receptor–mediated membrane fusion, we examined the hypothesis that these surface clusters are specialized platforms involved in membrane protein trafficking. Total internal reflection–based fluorescence recovery after photobleaching studies and quantum dot imaging of single Kv2.1 channels revealed that Kv2.1-containing vesicles deliver cargo at the Kv2.1 surface clusters in both transfected HEK cells and hippocampal neurons. More than 85% of cytoplasmic and recycling Kv2.1 channels was delivered to the cell surface at the cluster perimeter in both cell types. At least 85% of recycling Kv1.4, which, unlike Kv2.1, has a homogeneous surface distribution, is also delivered here. Actin depolymerization resulted in Kv2.1 exocytosis at cluster-free surface membrane. These results indicate that one nonconducting function of Kv2.1 is to form microdomains involved in membrane protein trafficking. This study is the first to identify stable cell surface platforms involved in ion channel trafficking.

scholarly journals Proteases of germinating winged-bean (Psophocarpus tetragonolobus) seeds: purification and characterization of an acidic protease

1996 ◽  
Vol 313 (2) ◽  
pp. 423-429 ◽  
Author(s):  
Rajamma USHA ◽  
Manoranjan SINGH
Keyword(s):  
Physiological Role ◽  
Immunoblot Analysis ◽  
Protein Band ◽  
Winged Bean ◽  
Psophocarpus Tetragonolobus ◽  
Ph Optimum ◽  
Apparent Homogeneity ◽  
Sds Page ◽  
Storage Protein Mobilization

Two major classes of protease are shown to occur in germinating winged-bean (Psophocarpus tetragonolobus) seeds, by assaying extracts at pH 8.0 and pH 5.1 with [14C]gelatin as substrate. At pH 8.0, the activity profile of the enzyme shows a steady rise throughout the period of germination, whereas the activity at the acidic pH is very low up to day 5 and then increases sharply reaching a peak on day 11, followed by an equally sharp decline. The winged-bean acidic protease (WbAP) has been purified to apparent homogeneity, as attested by a single protein band on both PAGE and SDS/PAGE. WbAP is a monomeric enzyme with a molecular mass of 35 kDa and a pH optimum of 6.0. It is a thiol protease that does not belong to the papain family and it has tightly bound Ca2+ as shown by 45Ca2+-exchange studies. Besides gelatin and casein, it hydrolyses a 29 kDa winged-bean protein, indicating a prospective physiological role for it in storage-protein mobilization. Immunoblot analysis shows that it occurs only in the seeds and sprouting tubers of this plant and also that it is synthesized in developing seeds just before desiccation. It appears that the newly synthesized enzyme is inactive, and activation takes place around day 6 of germination. However, neither the mechanism of activation nor the signal that triggers it is clearly understood.

scholarly journals Isolation and partial characterization of antibody- and globin-enriched complexes from membranes of dense human erythrocytes

1991 ◽  
Vol 278 (1) ◽  
pp. 57-62 ◽  
Author(s):  
R Kannan ◽  
J Yuan ◽  
P S Low
Keyword(s):  
Membrane Protein ◽  
Cell Surface ◽  
Integral Membrane Protein ◽  
Protein Band ◽  
Reticuloendothelial System ◽  
Band 3 ◽  
Isolation And Characterization ◽  
Protein Clusters ◽  
Associated Proteins

In previous studies we have described a process whereby an erythrocyte in biochemical distress can initiate its own removal by macrophages of the reticuloendothelial system. This process involves the clustering of the integral membrane protein band 3 by denatured haemoglobin and the subsequent recognition of the exofacial poles of clustered band 3 and associated proteins by autologous antibodies. To determine whether this clearance pathway might mediate normal cell turnover, the fraction of normal erythrocytes containing the 0.5% densest cells, which are known to be destined for immediate removal, was isolated and characterized biochemically. This densest fraction was found to contain 6 times more membrane-bound globin (haemichromes) and 10 times more surface-bound autologous IgG than the other fractions containing cells of lower density. To determine whether the autologous IgG was physically associated with the haemichrome-stabilized membrane protein clusters, a procedure was developed for isolation and characterization of the microscopic aggregates. The isolated aggregates were found to contain a disulphide-cross-linked mixture of several membrane proteins, predominantly haemichromes, spectrin and band 3. Although the aggregates constituted only 0.09% of the total membrane protein, they still contained approximately 55% of the total cell-surface IgG. Since in control studies anti-(blood group A) antibodies, which are distributed randomly over the surface of type A cells, could not be recovered in the aggregate, we conclude that the autologous cell-surface IgGs were physically associated with the membrane protein clusters when they were co-isolated with them in our procedure. Thus the 640-fold enrichment of autologous IgG in the aggregates compared with regions of the membrane devoid of tightly clustered protein suggests that sites of integral protein clustering either are non-specifically sticky to IgG or are viewed as foreign or ‘non-self’ by the immune system and aggressively opsonized with IgG.

scholarly journals Structural Characterization of the Lactoferrin Receptor from Neisseria meningitidis

1999 ◽  
Vol 181 (14) ◽  
pp. 4417-4419 ◽  
Author(s):  
Thorsten Prinz ◽  
Markus Meyer ◽  
Annika Pettersson ◽  
Jan Tommassen
Keyword(s):  
Membrane Protein ◽  
Cell Surface ◽  
Neisseria Meningitidis ◽  
Outer Membrane ◽  
Structural Characterization ◽  
Outer Membrane Protein ◽  
Lactoferrin Receptor ◽  
Topology Model

ABSTRACT The meningococcal lactoferrin receptor is composed of the integral outer membrane protein LbpA and the peripheral lipoprotein LbpB. Homooligomeric complexes of LbpA and heterooligomers consisting of LbpA and LbpB were identified. Furthermore, five cell surface-exposed loops of LbpA were identified, which partially confirms a previously proposed topology model.

Identification and partial characterization of an extracellular acid phosphatase activity of Leishmania donovani promastigotes

1982 ◽  
Vol 2 (1) ◽  
pp. 76-81
Author(s):  
M Gottlieb ◽  
D M Dwyer
Keyword(s):  
Acid Phosphatase ◽  
Leishmania Donovani ◽  
Surface Membrane ◽  
Extracellular Enzyme ◽  
Growth Cycle ◽  
Growth Media ◽  
Ph Optimum ◽  
Membrane Bound ◽  
Extracellular Acid Phosphatase

An extracellular acid phosphatase was detected in the growth media of Leishmania donovani promastigotes. The enzyme was released at all stages of the growth cycle and in amounts which accounted for 90% of the total amount of this enzyme in the culture. The exoenzyme exhibited a pH optimum of 4.5 to 5.0 and was active with a variety of organic phosphates. The enzymatic activity was excluded from Sephacryl S-300 and was retained by ultrafilters with nominal molecular weight cutoffs of up to 300,000. The results of comparative studies indicated that the extracellular enzyme was distinct from a surface membrane-bound acid phosphatase of L. donovani promastigotes which has been previously described.

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