The H,K-ATPase abd Na,K-ATPase are heterodimeric P-type ATPases consisting of an a-subunit that traverses the membrane several times with most of its mass cytoplasmically disposed and a b-subunit that traverses the membrane just once with most of its mass lumenally disposed. There has been some argument regarding the topology and specific number of a-subunit transmembrane segments, varying from 7-12. Previous approaches involve proteolysis followed by laborious transmembrane peptide identification using Edman sequencing or regio-specific antibodies. Due to the large number of peptides, defining topology is a complex problem. Here we utilize Matrix Assisted Laser Desorption Ionization mass spectrometry (MALDI-MS) to identify cytoplasmically oriented regions of the gastric H,K-ATPase. H,K-ATPase-enriched cytoplasmic-side-out vesicles isolated from rabbit stomach were trypsinized and released peptides and analyzed by MALDI-MS to obtain the masses of cytoplasmic peptides. Tryptic peptides were also separated by RP-HPLC and the fractions subjected to MALDI-MS and PSD analysis. Using this approach we were ble to identify cytoplasmically oriented regions in the a-subunit from Met1-Arg92, Ser165-Arg280,Val351-Lys785,Ala838--Lys851 and Phe997-Tyr1035. Thus, both the N- and C-terminus of the a-subunit were confirmed to be cytoplasmic and Asn226 and Asn731 were not glycosylated. Our current observations with trypsin are consistent with the 10 transmembrane segment hypothesis of the a-subunit. Analysis with chymotrypsin appears to further defines the topology in the 950-1016 region of the H,K-ATPase. Complete analysis of the tryptic and chymotryptic released peptides, as well as labeling with membrane-sided reagents will be performed to arrive at a topological model of the H,K-ATPase.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR001614-19
Application #
6308866
Study Section
Project Start
2000-03-01
Project End
2002-02-28
Budget Start
1998-10-01
Budget End
1999-09-30
Support Year
19
Fiscal Year
2000
Total Cost
$9,880
Indirect Cost
Name
University of California San Francisco
Department
Type
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143
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Sahoo, Pabitra K; Smith, Deanna S; Perrone-Bizzozero, Nora et al. (2018) Axonal mRNA transport and translation at a glance. J Cell Sci 131:
Tran, Vy M; Wade, Anna; McKinney, Andrew et al. (2017) Heparan Sulfate Glycosaminoglycans in Glioblastoma Promote Tumor Invasion. Mol Cancer Res 15:1623-1633
Liu, Tzu-Yu; Huang, Hector H; Wheeler, Diamond et al. (2017) Time-Resolved Proteomics Extends Ribosome Profiling-Based Measurements of Protein Synthesis Dynamics. Cell Syst 4:636-644.e9
Bikle, Daniel D (2016) Extraskeletal actions of vitamin D. Ann N Y Acad Sci 1376:29-52
Twiss, Jeffery L; Fainzilber, Mike (2016) Neuroproteomics: How Many Angels can be Identified in an Extract from the Head of a Pin? Mol Cell Proteomics 15:341-3
Cil, Onur; Phuan, Puay-Wah; Lee, Sujin et al. (2016) CFTR activator increases intestinal fluid secretion and normalizes stool output in a mouse model of constipation. Cell Mol Gastroenterol Hepatol 2:317-327
Posch, Christian; Sanlorenzo, Martina; Vujic, Igor et al. (2016) Phosphoproteomic Analyses of NRAS(G12) and NRAS(Q61) Mutant Melanocytes Reveal Increased CK2? Kinase Levels in NRAS(Q61) Mutant Cells. J Invest Dermatol 136:2041-2048
Julien, Olivier; Zhuang, Min; Wiita, Arun P et al. (2016) Quantitative MS-based enzymology of caspases reveals distinct protein substrate specificities, hierarchies, and cellular roles. Proc Natl Acad Sci U S A 113:E2001-10

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