scholarly journals Dihydroceramide:sphinganine C-4-hydroxylation requires Des2 hydroxylase and the membrane form of cytochrome b5

2006 ◽  
Vol 397 (2) ◽  
pp. 289-295 ◽  
Author(s):  
Ayako Enomoto ◽  
Fumio Omae ◽  
Masao Miyazaki ◽  
Yasunori Kozutsumi ◽  
Toshitsugu Yubisui ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Erythrocyte Membrane ◽  
Cytochrome B5 ◽  
Soluble Form ◽  
Affinity Column ◽  
Vitro Assay ◽  
Membrane Spanning ◽  
Triton X ◽  
Membrane Spanning Domains

Des2 (degenerative spermatocyte 2) is a bifunctional enzyme that produces phytoceramide and ceramide from dihydroceramide. The molecular mechanism involved in C-4-hydroxylation has not been studied in detail. In the present paper, we report that C-4-hydroxylation requires an electron-transfer system that includes cytochrome b5 and that the hydroxylase activity is reconstituted in an in vitro assay with purified recombinant Des2. FLAG-tagged mouse Des2 was expressed in insect Sf9 cells and was purified by solubilization with digitonin and anti-FLAG antibody affinity column chromatography. The activity of dihydroceramide:sphinganine C-4-hydroxylase was reconstituted with the purified FLAG–Des2, mb5 (the membrane form of cytochrome b5) and bovine erythrocyte membrane. The apparent Km and Vmax of Des2 for the substrate N-octanoylsphinganine were 35 μM and 40 nmol·h−1·mg of protein−1 respectively. The Km of the hydroxylase for mb5 was 0.8 μM. Interestingly, mb5 was not replaced with the soluble form of cytochrome b5, which lacks the C-terminal membrane-spanning domain. The erythrocyte membrane was separated into Triton X-100-soluble and -insoluble fractions, and the detergent-soluble fraction was replaced by the soluble or membrane form of b5R (NADH-cytochrome b5 reductase). The Triton-X-100-insoluble fraction contained trypsin-resistant factors. The Des2 protein is found in the endoplasmic reticulum and is assumed to have three membrane-spanning domains. The findings of the present study indicate that the hydroxylation requires complex formation between Des2 and mb5 via their membrane-spanning domains and electron transfer from NADH to the substrate via the reduction of mb5 by b5R.

Characterization of an in vitro assay for import of 3-phosphoglycerate kinase into the glycosomes of Trypanosoma brucei

1990 ◽  
Vol 10 (9) ◽  
pp. 4545-4554
Author(s):  
J M Sommer ◽  
J A Thissen ◽  
M Parsons ◽  
C C Wang
Keyword(s):  
Trypanosoma Brucei ◽  
Protein Import ◽  
Sodium Dodecyl ◽  
Phosphoglycerate Kinase ◽  
Proteinase K ◽  
Vitro Assay ◽  
Protein Core ◽  
Triton X ◽  
Gamma S

Glycosomes are microbody organelles found in kinetoplastida, where they serve to compartmentalize the enzymes of the glycolytic pathway. In order to identify the mechanism by which these enzymes are targeted to the glycosome, we have modified the in vitro import assay developed by Dovey et al. (Proc. Natl. Acad. Sci. USA 85:2598-2602, 1988). This assay measures the uptake of in vitro-translated Trypanosoma brucei glycosomal 3-phosphoglycerate kinase (gPGK) by purified glycosomes. Up to 50% of the total 35S-gPGK in the glycosomal fraction was resistant to extraction by 3 M urea or treatment with proteinase K (500 micrograms/ml). The glycosome-associated 35S-gPGK could be chemically cross-linked to the endogenous glycosomal proteins to form a sodium dodecyl sulfate-resistant complex, suggesting that it is close to the intraglycosomal protein matrix. Deoxycholate solubilized the glycosome and thereby rendered the glycosome-associated 35S-gPGK fully susceptible to proteinase K. However, the glycosome-associated 35S-gPGK was not digested by proteinase K in the presence of Triton X-100, which cannot dissolve the glycosomal protein core. The 35S-gPGK synthesized in vitro was able to bind directly to protein cores, where it became resistant to urea extraction and proteinase K digestion. However, the 35S-gPGK-protein core complex exhibited a much higher density than the 35S-gPGK-glycosome complex and was readily separable in sucrose gradients. Thus, in our in vitro import assay, the 35S-gPGK appeared to associate with intact glycosomes, possibly reflecting import of protein into the organelle. Complete denaturation of the 35S-gPGK in 8 M urea prior to the assay enhanced the efficiency of its association with glycosomes. Native gPGK did not compete with the association of in vitro-translated gPGK unless it was denatured. The assay exhibited time and temperature dependence, but it did not require externally added ATP and was not inhibited by the nonhydrolyzable analogs adenosine-5'-(beta,gamma-imido)-triphosphate and gamma-S-ATP. However, the presence of 20 to 30 microM ATP inside the glycosome may fulfill the requirement for protein import.

scholarly journals Molecular cloning, characterization, subcellular localization and dynamics of p23, the mammalian KDEL receptor.

1993 ◽  
Vol 120 (2) ◽  
pp. 325-338 ◽  
Author(s):  
B L Tang ◽  
S H Wong ◽  
X L Qi ◽  
S H Low ◽  
W Hong
Keyword(s):  
Golgi Apparatus ◽  
Brefeldin A ◽  
Cell Types ◽  
In Vitro Translation ◽  
Tubular Structures ◽  
Human Sequence ◽  
Intermediate Compartment ◽  
Membrane Spanning ◽  
Membrane Spanning Domains

We have isolated a cDNA clone (mERD2) for the mammalian (bovine) homologue of the yeast ERD2 gene, which codes for the yeast HDEL receptor. The deduced amino acid sequence bears extensive homology to its yeast counterpart and is almost identical to a previously described human sequence. The sequence predicts a very hydrophobic protein with multiple membrane spanning domains, as confirmed by analysis of the in vitro translation product. The protein encoded by mERD2 (p23) has widespread occurrence, being present in all the cell types examined. p23 was localized to the cis-side of the Golgi apparatus and to a spotty intermediate compartment which mediates ER to Golgi transport. A majority of the intracellular staining could be accumulated in the intermediate compartment by a low temperature (15 degrees C) or brefeldin A. During recovery from these treatments, the spotty intermediate compartment staining of p23 was shifted to the perinuclear staining of the Golgi apparatus and tubular structures marked by p23 were observed. These tubular structures may serve to mediate transport between the intermediate compartment and the Golgi apparatus.

Na+/H+Exchanger NHE3 Has 11 Membrane Spanning Domains and a Cleaved Signal Peptide:  Topology Analysis Using In Vitro Transcription/Translation†

Biochemistry ◽  
2000 ◽  
Vol 39 (27) ◽  
pp. 8102-8112 ◽  
Author(s):  
Mirza Zizak ◽  
Megan E. Cavet ◽  
Denis Bayle ◽  
Chung-Ming Tse ◽  
Stefan Hallen ◽  
...  
Keyword(s):  
Signal Peptide ◽  
In Vitro Transcription ◽  
Topology Analysis ◽  
Membrane Spanning ◽  
Membrane Spanning Domains

scholarly journals Microtubule translocation properties of intact and proteolytically digested dyneins from Tetrahymena cilia.

1989 ◽  
Vol 108 (6) ◽  
pp. 2327-2334 ◽  
Author(s):  
R D Vale ◽  
Y Y Toyoshima
Keyword(s):  
Force Production ◽  
In Vitro Assay ◽  
Regulatory Function ◽  
Vitro Assay ◽  
Ciliary Beating ◽  
Glass Surfaces ◽  
Triton X ◽  
Gamma S ◽  
Motile Activity

Tetrahymena cilia contain a three-headed 22S (outer arm) dynein and a single-headed 14S dynein. In this study, we have employed an in vitro assay of microtubule translocation along dynein-coated glass surfaces to characterize the motile properties of 14S dynein, 22S dynein, and proteolytic fragments of 22S dynein. Microtubule translocation produced by intact 22S dynein and 14S dynein differ in a number of respects including (a) the maximal velocities of movement; (b) the ability of 22S dynein but not 14S dynein to utilize ATP gamma S to induce movement; (c) the optimal pH and ionic conditions for movement; and (d) the effects of Triton X-100 on the velocity of movement. These results indicate that 22S and 14S dyneins have distinct microtubule translocating properties and suggest that these dyneins may have specialized roles in ciliary beating. We have also explored the function of the multiple ATPase heads of 22S dynein by preparing one- and two-headed proteolytic fragments of this three-headed molecule and examining their motile activity in vitro. Unlike the single-headed 14S dynein, the single-headed fragment of 22S dynein did not induce movement, even though it was capable of binding to microtubules. The two-headed fragment, on the other hand, translocated microtubules at velocities similar to those measured for intact 22S dynein (10 microns/sec). This finding indicates that the intact three-headed structure of 22S dynein is not essential for generating microtubule movement, which raises the possibility that multiple heads may serve some regulatory function or may be required for maximal force production in the beating cilium.

scholarly journals Virus-mediated release of endosomal content in vitro: different behavior of adenovirus and rhinovirus serotype 2.

1995 ◽  
Vol 131 (1) ◽  
pp. 111-123 ◽  
Author(s):  
E Prchla ◽  
C Plank ◽  
E Wagner ◽  
D Blaas ◽  
R Fuchs
Keyword(s):  
Vitro Assay ◽  
Forming Mechanism ◽  
Endosomal Membrane ◽  
Influenza Virus Hemagglutinin ◽  
Late Endosomes ◽  
Nonenveloped Virus ◽  
Capsid Protein Vp1 ◽  
Membrane Spanning

Endosomal penetration by nonenveloped viruses might be accomplished by either local breakdown of the endosomal membrane (e.g., adenovirus) or formation of a membrane-spanning pore by capsid proteins. Uncoating of the nonenveloped virus human rhinovirus serotype 2 (HRV2) has been shown to occur from late endosomes and to be entirely dependent on the acidic pH in this compartment (Prchla, E., E. Kuechler, D. Blaas, and R. Fuchs. 1994. J. Virol. 68: 3713-3723). To investigate further the mechanism of uncoating of HRV2, an in vitro assay was established to test viruses or virus-derived peptides for their capacity to release cointernalized biotin-dextran of different molecular mass (10 and 70 kD) from isolated endosomes. The suitability of the assay was demonstrated by use of a fusogenic peptide derived from influenza virus hemagglutinin (GALA-INF3). Whereas adenovirus induced a low pH-dependent release of up to 46% of the internalized biotin-dextran and did not show any significant size selectivity (as expected for endosome disruption), HRV2 mediated release of 27% of the 10 kD dextran and only traces of the 70-kD dextran. Similarly, GALA-INF3-induced release of biotin-dextran was also size dependent. The potential role of the capsid protein VP1 in HRV2 uncoating in vivo was also substantiated in our in vitro system using an amphipathic, NH2-terminal peptide of VP1. Taken together, these data favor the model of a specific pore-forming mechanism for HRV2 uncoating which is in contrast to the membrane-disrupting mechanism of adenovirus.

scholarly journals Characterization of an in vitro assay for import of 3-phosphoglycerate kinase into the glycosomes of Trypanosoma brucei.

1990 ◽  
Vol 10 (9) ◽  
pp. 4545-4554 ◽  
Author(s):  
J M Sommer ◽  
J A Thissen ◽  
M Parsons ◽  
C C Wang
Keyword(s):  
Trypanosoma Brucei ◽  
Protein Import ◽  
Sodium Dodecyl ◽  
Phosphoglycerate Kinase ◽  
Proteinase K ◽  
Vitro Assay ◽  
Protein Core ◽  
Triton X ◽  
Gamma S

Glycosomes are microbody organelles found in kinetoplastida, where they serve to compartmentalize the enzymes of the glycolytic pathway. In order to identify the mechanism by which these enzymes are targeted to the glycosome, we have modified the in vitro import assay developed by Dovey et al. (Proc. Natl. Acad. Sci. USA 85:2598-2602, 1988). This assay measures the uptake of in vitro-translated Trypanosoma brucei glycosomal 3-phosphoglycerate kinase (gPGK) by purified glycosomes. Up to 50% of the total 35S-gPGK in the glycosomal fraction was resistant to extraction by 3 M urea or treatment with proteinase K (500 micrograms/ml). The glycosome-associated 35S-gPGK could be chemically cross-linked to the endogenous glycosomal proteins to form a sodium dodecyl sulfate-resistant complex, suggesting that it is close to the intraglycosomal protein matrix. Deoxycholate solubilized the glycosome and thereby rendered the glycosome-associated 35S-gPGK fully susceptible to proteinase K. However, the glycosome-associated 35S-gPGK was not digested by proteinase K in the presence of Triton X-100, which cannot dissolve the glycosomal protein core. The 35S-gPGK synthesized in vitro was able to bind directly to protein cores, where it became resistant to urea extraction and proteinase K digestion. However, the 35S-gPGK-protein core complex exhibited a much higher density than the 35S-gPGK-glycosome complex and was readily separable in sucrose gradients. Thus, in our in vitro import assay, the 35S-gPGK appeared to associate with intact glycosomes, possibly reflecting import of protein into the organelle. Complete denaturation of the 35S-gPGK in 8 M urea prior to the assay enhanced the efficiency of its association with glycosomes. Native gPGK did not compete with the association of in vitro-translated gPGK unless it was denatured. The assay exhibited time and temperature dependence, but it did not require externally added ATP and was not inhibited by the nonhydrolyzable analogs adenosine-5'-(beta,gamma-imido)-triphosphate and gamma-S-ATP. However, the presence of 20 to 30 microM ATP inside the glycosome may fulfill the requirement for protein import.

Improved in vitro assay for determining the mucin adherence of bacteria sensitive to Triton X-100 treatment

2015 ◽  
Vol 60 (5) ◽  
pp. 435-442 ◽  
Author(s):  
Varvara Tsilia ◽  
Pieter Van den Abbeele ◽  
Tom Van de Wiele
Keyword(s):  
In Vitro Assay ◽  
Vitro Assay ◽  
Triton X

Mammalian multidrug-resistance proteins (MRPs)

2011 ◽  
Vol 50 ◽  
pp. 179-207 ◽  
Author(s):  
Andrew J. Slot ◽  
Steven V. Molinski ◽  
Susan P.C. Cole
Keyword(s):  
Multidrug Resistance ◽  
Gene Knockout ◽  
Multidrug Resistance Proteins ◽  
Resistance Proteins ◽  
Gene Knockout Mice ◽  
Membrane Spanning ◽  
Endogenous Metabolites ◽  
Membrane Spanning Domains

Subfamily C of the human ABC (ATP-binding cassette) superfamily contains nine proteins that are often referred to as the MRPs (multidrug-resistance proteins). The ‘short’ MRP/ABCC transporters (MRP4, MRP5, MRP8 and ABCC12) have a typical ABC structure with four domains comprising two membrane-spanning domains (MSD1 and MSD2) each followed by a nucleotide-binding domain (NBD1 and NBD2). The ‘long’ MRP/ABCCs (MRP1, MRP2, MRP3, ABCC6 and MRP7) have five domains with the extra domain, MSD0, at the N-terminus. The proteins encoded by the ABCC6 and ABCC12 genes are not known to transport drugs and are therefore referred to as ABCC6 and ABCC12 (rather than MRP6 and MRP9) respectively. A large number of molecules are transported across the plasma membrane by the MRPs. Many are organic anions derived from exogenous sources such as conjugated drug metabolites. Others are endogenous metabolites such as the cysteinyl leukotrienes and prostaglandins which have important signalling functions in the cell. Some MRPs share a degree of overlap in substrate specificity (at least in vitro), but differences in transport kinetics are often substantial. In some cases, the in vivo substrates for some MRPs have been discovered aided by studies in gene-knockout mice. However, the molecules that are transported in vivo by others, including MRP5, MRP7, ABCC6 and ABCC12, still remain unknown. Important differences in the tissue distribution of the MRPs and their membrane localization (apical in contrast with basolateral) in polarized cells also exist. Together, these differences are responsible for the unique pharmacological and physiological functions of each of the nine ABCC transporters known as the MRPs.

scholarly journals Identification of a soluble form of a ligand for the lymphocyte homing receptor.

1992 ◽  
Vol 176 (5) ◽  
pp. 1415-1419 ◽  
Author(s):  
M Brustein ◽  
G Kraal ◽  
R E Mebius ◽  
S R Watson
Keyword(s):  
Lymph Nodes ◽  
Chimeric Protein ◽  
Normal Serum ◽  
Soluble Form ◽  
Lymphoid Tissues ◽  
Mesenteric Lymph Nodes ◽  
High Endothelial Venules ◽  
Vitro Assay ◽  
Homing Receptor

Lymphocytes are engaged in constant trafficking from the blood into secondary lymphoid tissues, such as peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN), and Peyer's patches (PP). The initial step in this process is the binding of lymphocytes to high endothelial venules (HEV), and in the case of trafficking of cells to the PLN, it is required that they bear the L-selectin surface receptor. Using a chimeric protein, combining the extracellular domains of L-selectin with a human immunoglobulin (Ig) G1 Fc region (L-selectin-IgG), we have probed the expression of ligands for this receptor on HEV and in cell lysates. Two sulfated glycoproteins of 50 and 90 kD have been identified in lysates from PLN and MLN, but not PP. Here we show that the 50-kD molecule is secreted in organ cultures in vitro and is present in the blood of normal animals. Indeed, normal serum inhibits lymphocyte binding to HEV by approximately 50% in an in vitro assay. This inhibitory activity can be removed by passage of the serum over an L-selectin-IgG column and has a molecular mass of approximately 50 kD. We speculate on the possible reasons for secretion of a homing receptor ligand.

Splicing of a retained intron within ROMK K+ channel RNA generates a novel set of isoforms in rat kidney

1999 ◽  
Vol 276 (3) ◽  
pp. C585-C592 ◽  
Author(s):  
A. H. Beesley ◽  
B. Ortega ◽  
S. J. White
Keyword(s):  
Fluorescent Protein ◽  
Mdck Cells ◽  
Rat Kidney ◽  
K Channel ◽  
Major Pathway ◽  
Membrane Spanning ◽  
Green Fluorescent ◽  
Membrane Spanning Domains ◽  
Darby Canine Kidney

The renal outer medulla K+ channel (ROMK) family of K+ channels may constitute a major pathway for K+ secretion in the distal nephron. To date, four main isoforms of this gene have been identified in the rat that differ only in their NH2-terminal amino acids and that share a common “core exon” that determines the remaining protein sequence. Using RT-PCR, we have identified a new set of ROMK isoforms in rat kidney that are generated by the deletion of a region within the ROMK core sequence that is identifiable as a typical mammalian intron. This splicing event was shown to be reproducible in vitro by detection of deleted ROMK mRNA in Madin-Darby canine kidney (MDCK) cells stably transfected with the gene for ROMK2. Translation of the deletion variant of ROMK2 was confirmed in vitro and visualized in MDCK cells following transient transfection with an enhanced green fluorescent protein tag. The deletion in this core region is predicted to generate hydrophilic proteins that are approximately one-third of the size of native ROMK and lack membrane-spanning domains.

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