Functional characterization of the transmembrane segment VII of the NHE1 isoform of the Na+/H+ exchangerThis paper is one of a selection of papers published in this Special Issue, entitled The Cellular and Molecular Basis of Cardiovascular Dysfunction, Dhalla 70th Birthday Tribute.

2007 ◽  
Vol 85 (3-4) ◽  
pp. 319-325 ◽  
Author(s):  
Jie Ding ◽  
Raymond W.P. Ng ◽  
Larry Fliegel
Keyword(s):  
Amino Acids ◽  
70Th Birthday ◽  
Functional Characterization ◽  
Integral Membrane Protein ◽  
Transmembrane Segment ◽  
Transmembrane Segments ◽  
Pore Lining ◽  
Positively Charged ◽  
Selection Of

The Na+/H+ exchanger isoform 1 is an integral membrane protein that regulates intracellular pH. It extrudes 1 intracellular H+ in exchange for 1 extracellular Na+. It has 2 large domains, an N-terminal membrane domain of 12 transmembrane segments and an intracellular C-terminal regulatory domain. We characterized the cysteine accessibility of amino acids of the critical transmembrane segment TM VII. Residues Leu 255, Leu 258, Glu 262, Leu 265, Asn 266, Asp 267, Val 269, Val 272, and Leu 273 were all mutated to cysteine residues in the cysteineless NHE1 isoform. Mutation of amino acids E262, N266, and D267 caused severe defects in activity and targeting of the intact full length protein. The balance of the active mutants were examined for sensitivity to the sulfhydryl reactive reagents, positively charged MTSET ((2- (trimethylammonium)ethyl)methanethiosulfonate) and negatively charged MTSES ((2-sulfonatoethyl)methanethiosulfonate). Leu 255 and Leu 258 were sensitive to MTSET but not to MTSES. The results suggest that these amino acids are pore-lining residues. We present a model of TM VII that shows that residues Leu 255, Leu 258, Glu 262, Asn 266, and Asp 267 lie near the same face of TM VII, lining the ion transduction pore.

scholarly journals Human xylosyltransferase I and N-terminal truncated forms: functional characterization of the core enzyme

2006 ◽  
Vol 394 (1) ◽  
pp. 163-171 ◽  
Author(s):  
Sandra Müller ◽  
Jennifer Disse ◽  
Manuela Schöttler ◽  
Sylvia Schön ◽  
Christian Prante ◽  
...  
Keyword(s):  
Amino Acids ◽  
Catalytic Activity ◽  
Binding Capacity ◽  
Functional Characterization ◽  
Terminal Region ◽  
Binding Motif ◽  
Heparin Binding ◽  
Loss Of Function ◽  
The Impact

Human XT-I (xylosyltransferase I; EC 2.4.2.26) initiates the biosynthesis of the glycosaminoglycan linkage region and is a diagnostic marker of an enhanced proteoglycan biosynthesis. In the present study, we have investigated mutant enzymes of human XT-I and assessed the impact of the N-terminal region on the enzymatic activity. Soluble mutant enzymes of human XT-I with deletions at the N-terminal domain were expressed in insect cells and analysed for catalytic activity. As many as 260 amino acids could be truncated at the N-terminal region of the enzyme without affecting its catalytic activity. However, truncation of 266, 272 and 273 amino acids resulted in a 70, 90 and >98% loss in catalytic activity. Interestingly, deletion of the single 12 amino acid motif G261KEAISALSRAK272 leads to a loss-of-function XT-I mutant. This is in agreement with our findings analysing the importance of the Cys residues where we have shown that C276A mutation resulted in a nearly inactive XT-I enzyme. Moreover, we investigated the location of the heparin-binding site of human XT-I using the truncated mutants. Heparin binding was observed to be slightly altered in mutants lacking 289 or 568 amino acids, but deletion of the potential heparin-binding motif P721KKVFKI727 did not lead to a loss of heparin binding capacity. The effect of heparin or UDP on the XT-I activity of all mutants was not significantly different from that of the wild-type. Our study demonstrates that over 80% of the nucleotide sequence of the XT-I-cDNA is necessary for expressing a recombinant enzyme with full catalytic activity.

Structure and function of the NHE1 isoform of the Na+/H+ exchanger

2002 ◽  
Vol 80 (5) ◽  
pp. 499-508 ◽  
Author(s):  
Emily Slepkov ◽  
Larry Fliegel
Keyword(s):  
Amino Acids ◽  
Intracellular Ph ◽  
Ph Regulation ◽  
Integral Membrane Protein ◽  
Amino Acid Residues ◽  
Transmembrane Helices ◽  
Cytoskeletal Organization ◽  
Transmembrane Segments ◽  
And Function ◽  
Extracellular Sodium

The Na+/H+ exchanger is a ubiquitous, integral membrane protein involved in pH regulation. It removes intracellular acid, exchanging a proton for an extracellular sodium ion. There are seven known isoforms of this protein that are the products of distinct genes. The first isoform discovered (NHE1) is ubiquitously distributed throughout the plasma membrane of virtually all tissues. It plays many different physiological roles in mammals, including important functions in regulation of intracellular pH, in heart disease, and in cytoskeletal organization. The first 500 amino acids of the protein are believed to consist of 12 transmembrane helices, a membrane-associated segment, and two reentrant loops. A C-terminal regulatory domain of approximately 315 amino acids regulates the protein and mediates cyto skel etal interactions. Studies are underway to determine the amino acid residues important in NHE1 function. At present, it is clear that transmembrane segment IV is important in NHE1 function and that transmembrane segments VII and IX are also involved in transport. Further experiments are required to elucidate the mechanism of transport and regulation of this multifunctional protein.Key words: cation transport, intracellular pH, membrane proteins, Na+/H+ exchanger.

scholarly journals Functional characterization of constituent enzyme fractions of mycobacillin synthetase

1985 ◽  
Vol 230 (3) ◽  
pp. 785-789 ◽  
Author(s):  
S K Ghosh ◽  
S Majumder ◽  
N K Mukhopadhyay ◽  
S K Bose
Keyword(s):  
Amino Acids ◽  
Functional Characterization ◽  
Enzyme Fraction ◽  
Sequential Activation

The enzyme fraction A, a constituent enzyme of the three-fraction enzyme mycobacillin synthetase, independently and sequentially activated five amino acids starting from L-proline, producing the pentapeptide Pro(Asp1,Glu1,Tyr1)Asp. The fractions B and C were unable to function independently. However, the fraction B synthesized the nonapeptide Pro(Asp3,Glu1,Tyr2,Ser1)Leu, sequentially activating the pentapeptide and next four amino acids, whereas the fraction C synthesized mycobacillin by the sequential activation of the nonapeptide and the remaining four amino acids. The pH optima of the above enzymes are almost identical (pH 7.8), but their Km values are a little different.

scholarly journals Proline residues in transmembrane segment IV are critical for activity, expression and targeting of the Na+/H+ exchanger isoform 1

2004 ◽  
Vol 379 (1) ◽  
pp. 31-38 ◽  
Author(s):  
Emily R. SLEPKOV ◽  
Signy CHOW ◽  
M. Joanne LEMIEUX ◽  
Larry FLIEGEL
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Mammalian Cells ◽  
Integral Membrane Protein ◽  
Amide Hydrogen ◽  
Wild Type ◽  
Transmembrane Segment ◽  
Mutant Proteins ◽  
Transmembrane Segments ◽  
Induced Changes

NHE1 (Na+/H+ exchanger isoform 1) is a ubiquitously expressed integral membrane protein that regulates intracellular pH in mammalian cells. Proline residues within transmembrane segments have unusual properties, acting as helix breakers and increasing flexibility of membrane segments, since they lack an amide hydrogen. We examined the importance of three conserved proline residues in TM IV (transmembrane segment IV) of NHE1. Pro167 and Pro168 were mutated to Gly, Ala or Cys, and Pro178 was mutated to Ala. Pro168 and Pro178 mutant proteins were expressed at levels similar to wild-type NHE1 and were targeted to the plasma membrane. However, the mutants P167G (Pro167→Gly), P167A and P167C were expressed at lower levels compared with wild-type NHE1, and a significant portion of P167G and P167C were retained intracellularly, possibly indicating induced changes in the structure of TM IV. P167G, P167C, P168A and P168C mutations abolished NHE activity, and P167A and P168G mutations caused markedly decreased activity. In contrast, the activity of the P178A mutant was not significantly different from that of wild-type NHE1. The results indicate that both Pro167 and Pro168 in TM IV of NHE1 are required for normal NHE activity. In addition, mutation of Pro167 affects the expression and membrane targeting of the exchanger. Thus both Pro167 and Pro168 are strictly required for NHE function and may play critical roles in the structure of TM IV of the NHE.

scholarly journals Purification and Characterization of the Bacterial UDP-GlcNAc:Undecaprenyl-Phosphate GlcNAc-1-Phosphate Transferase WecA

2008 ◽  
Vol 190 (21) ◽  
pp. 7141-7146 ◽  
Author(s):  
Bayan Al-Dabbagh ◽  
Dominique Mengin-Lecreulx ◽  
Ahmed Bouhss
Keyword(s):  
Cell Envelope ◽  
Functional Characterization ◽  
Integral Membrane Protein ◽  
Minimal Length ◽  
Purification And Characterization ◽  
Effects Of Ph ◽  
Undecaprenyl Phosphate ◽  
First Time ◽  
Lipid Substrate

ABSTRACT To date, the structural and functional characterization of proteins belonging to the polyprenyl-phosphate N-acetylhexosamine-1-phosphate transferase superfamily has been relentlessly held back by problems encountered with their overexpression and purification. In the present work and for the first time, the integral membrane protein WecA that catalyzes the transfer of the GlcNAc-1-phosphate moiety from UDP-GlcNAc onto the carrier lipid undecaprenyl phosphate, yielding undecaprenyl-pyrophosphoryl-GlcNAc, the lipid intermediate involved in the synthesis of various bacterial cell envelope components, was overproduced and purified to near homogeneity in milligram quantities. An enzymatic assay was developed, and the kinetic parameters of WecA as well as the effects of pH, salts, cations, detergents, and temperature on the enzyme activity were determined. A minimal length of 35 carbons was required for the lipid substrate, and tunicamycin was shown to inhibit the enzyme at submicromolar concentrations.

scholarly journals Structural and Functional Characterization of Transmembrane Segment IX of the NHE1 Isoform of the Na+/H+Exchanger

2008 ◽  
Vol 283 (32) ◽  
pp. 22018-22030 ◽  
Author(s):  
Tyler Reddy ◽  
Jie Ding ◽  
Xiuju Li ◽  
Brian D. Sykes ◽  
Jan K. Rainey ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Transmembrane Segment

Four novel splice variants of sulfonylurea receptor 1

2002 ◽  
Vol 283 (2) ◽  
pp. C587-C598 ◽  
Author(s):  
Annette Hambrock ◽  
Regina Preisig-Müller ◽  
Ulrich Russ ◽  
Anke Piehl ◽  
Peter J. Hanley ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Splice Variants ◽  
Functional Characterization ◽  
Pcr Analysis ◽  
Sulfonylurea Receptor ◽  
Transmembrane Segments ◽  
Sulfonylurea Receptor 1 ◽  
Green Fluorescent ◽  
Splice Forms

ATP-sensitive K+ (KATP) channels are composed of pore-forming Kir6.x subunits and regulatory sulfonylurea receptor (SUR) subunits. SURs are ATP-binding cassette proteins with two nucleotide-binding folds (NBFs) and binding sites for sulfonylureas, like glibenclamide, and for channel openers. Here we report the identification and functional characterization of four novel splice forms of guinea pig SUR1. Three splice forms originate from alternative splicing of the region coding for NBF1 and lack exons 17 (SUR1Δ17), 19 (SUR1Δ19), or both (SUR1Δ17Δ19). The fourth (SUR1C) is a COOH-terminal SUR1-fragment formed by exons 31–39 containing the last two transmembrane segments and the COOH terminus of SUR1. RT-PCR analysis showed that these splice forms are expressed in several tissues with strong expression of SUR1C in cardiomyocytes. Confocal microscopy using enhanced green fluorescent protein-tagged SUR or Kir6.x did not provide any evidence for involvement of these splice forms in the mitochondrial KATP channel. Only SUR1 and SUR1Δ17 showed high-affinity binding of glibenclamide ( K d≈ 2 nM in the presence of 1 mM ATP) and formed functional KATPchannels upon coexpression with Kir6.2.

scholarly journals Rapid transfer of overexpressed integral membrane protein from the host membrane into soluble lipid nanodiscs without previous purification

2015 ◽  
Vol 396 (8) ◽  
pp. 903-915 ◽  
Author(s):  
Nazhat Shirzad-Wasei ◽  
Jenny van Oostrum ◽  
Petra H.M. Bovee-Geurts ◽  
Lisanne J.A. Kusters ◽  
Giel J.C.G.M. Bosman ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Functional Characterization ◽  
Integral Membrane Protein ◽  
Partial Purification ◽  
Functional Studies ◽  
Host Membrane ◽  
Lipid Nanodiscs ◽  
Membrane Scaffold ◽  
Rapid Transfer

Abstract Structural and functional characterization of integral membrane proteins in a bilayer environment is strongly hampered by the requirement of detergents for solubilization and subsequent purification, as detergents commonly affect their structure and/or activity. Here, we describe a rapid procedure with minimal exposure to detergent to directly assemble an overexpressed integral membrane protein into soluble lipid nanodiscs prior to purification. This is exemplified with recombinant his-tagged rhodopsin, which is rapidly extracted from its host membrane and directly assembled into membrane scaffold protein (MSP) nanodiscs. We further demonstrate that, even when the MSP was his-tagged as well, partial purification of the rhodopsin-nanodiscs could be achieved exploiting immobilized-metal chromatography. Recoveries of rhodopsin up to 80% were achieved in the purified nanodisc fraction. Over 95% of contaminating membrane protein and his-tagged MSP could be removed from the rhodopsin-nanodiscs using a single Ni2+-affinity chromatography step. This level of purification is amply sufficient for functional studies. We provide evidence that the obtained rhodopsin-nanodisc preparations are fully functional both photochemically and in their ability to bind the cognate G-protein.

Self-Contained Three-Dimensional Bioprinter for Applications in Cardiovascular Research

2019 ◽  
Vol 13 (3) ◽  
Author(s):  
Prabhuti Kharel ◽  
Likitha Somasekhar ◽  
Amy Vecheck ◽  
Kunal Mitra
Keyword(s):  
Functional Characterization ◽  
Three Dimensional ◽  
Environmental Parameters ◽  
3D Bioprinting ◽  
Cardiovascular Research ◽  
Form Complex ◽  
Tissue Constructs ◽  
Long Duration ◽  
Selection Of

Bioprinting is a technique of creating 3D cell-laden structures by accurately dispensing biomaterial to form complex synthetic tissue. The printed constructs aim to mimic the native tissue by preserving the cell functionality and viability within the printed structure. The 3D bioprinting system presented in this paper aims to facilitate the process of 3D bioprinting through its ability to control the environmental parameters within an enclosed printing chamber. This design of the bioprinter targets to eliminate the need for a laminar flow hood, by regulating the necessary environmental conditions important for cell survival, especially during long duration prints. A syringe-based extrusion (SBE) deposition method comprising multiple nozzles is integrated into the system. This allows for a wider selection of biomaterials that can be used for the formation of the extracellular matrix (ECM). Tissue constructs composed of alginate-gelatin hydrogels were mixed with fibrinogen and human endothelial cells which were then characterized and compared using two methodologies: casted and bioprinted. Furthermore, vasculature was incorporated in the bioprinted constructs using sacrificial printing. Structural and functional characterization of the constructs were performed by assessing rheological, mechanical properties, and analyzing live-dead assay measurements.

scholarly journals Characterization of the scrambling domain of the TMEM16 family

2017 ◽  
Vol 114 (24) ◽  
pp. 6274-6279 ◽  
Author(s):  
Sayuri Gyobu ◽  
Kenji Ishihara ◽  
Jun Suzuki ◽  
Katsumori Segawa ◽  
Shigekazu Nagata
Keyword(s):  
Plasma Membranes ◽  
Point Mutations ◽  
Transmembrane Segment ◽  
Stepping Stones ◽  
Transmembrane Segments ◽  
Outer Leaflet ◽  
Intracellular Ca ◽  
A Cell ◽  
Phospholipid Scrambling

The TMEM16 protein family has 10 members, each of which carries 10 transmembrane segments. TMEM16A and 16B are Ca2+-activated Cl− channels. Several other members, including TMEM16F, promote phospholipid scrambling between the inner and outer leaflets of a cell membrane in response to intracellular Ca2+. However, the mechanism by which TMEM16 proteins translocate phospholipids in plasma membranes remains elusive. Here we show that Ca2+-activated, TMEM16F-supported phospholipid scrambling proceeds at 4 °C. Similar to TMEM16F and 16E, seven TMEM16 family members were found to carry a domain (SCRD; scrambling domain) spanning the fourth and fifth transmembrane segments that conferred scrambling ability to TMEM16A. By introducing point mutations into TMEM16F, we found that a lysine in the fourth transmembrane segment of the SCRD as well as an arginine in the third and a glutamic acid in the sixth transmembrane segment were important for exposing phosphatidylserine from the inner to the outer leaflet. However, their role in internalizing phospholipids was limited. Our results suggest that TMEM16 provides a cleft containing hydrophilic “stepping stones” for the outward translocation of phospholipids.

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