+ Translate
+ Most Popular
Advantages and disadvantages of bordeaux mixture and of lime-sulphur used on apples in the growing season
Observations on the Umaria marine bed
10 years of hearing conservation in the Royal Air Force
Chocolate crumb - dairy ingredient for milk chocolate
Effect of daily gelatin ingestion on human scalp hair
Comparison of rice bran and maize bran as feeds for growing and fattening pigs
The composition of pampas-grass (Cortaderia argentea.)
The Accraian Series:
The mechanism of the Liebermann-Burchard reaction of sterols and triterpenes and their esters
Cerebrovascular Doppler ultrasound studies (cv-Doppler)
Toria: PT-303 - first national variety
Hair growth promoting activity of tridax procumbens
Productivity of Pekin x Khaki Campbell ducks
A stable cytosolic expression of VH antibody fragment directed against PVY NIa protein in transgenic potato plant confers partial protection against the virus
Solar treatment of wheat loose smut
Swimmers itch in the Lake of Garda
Bactofugation and the Bactotherm process
The effects of prefrontal lobotomy on aggressive behavior in dogs
Visual rating scales for screening whorl-stage corn for resistance to fall armyworm
Breakdown of seamounts at the trench axis, viewed from gravity anomaly
Kooken; pennsylvania's toughest cave
Recovery of new dinosaur and other fossils from the Early Cretaceous Arundel Clay facies (Potomac Group) of central Maryland, U.S.A
Zubor horny (Bison bonasus) v prirodnych podmienkach Slovensku
The extended Widal test in the diagnosis of fevers due to Salmonella infection
Hair of the american mastodon indicates an adaptation to a semi aquatic habitat

Topological analysis of H+,K(+)-ATPase using in vitro translation

Topological analysis of H+,K(+)-ATPase using in vitro translation

Journal of Biological Chemistry 269(24): 16909-16919

ISSN/ISBN: 0021-9258

PMID: 8207013

The membrane topology of the alpha subunit of the H+,K(+)-ATPase was investigated by using in vitro transcription/translation of DNA sequences encoding fusion proteins that contained possible membrane-spanning segments. The vectors consisted of DNA sequences encoding (a) either the first 101 (M0 vectors) or the first 139 (M1 vectors) amino acids of the N-terminal region of the alpha subunit of the ATPase, (b) a variable region, and then (c) the C-terminal 177 amino acids of the C-terminal region of the beta subunit, with five N-linked glycosylation sites. The variable region of the fusion protein contained the cDNA sequences representing the possible eight or 10 membrane-spanning segments either alone or in various combinations. Transcription/translation was performed in the presence of [35S]methionine using a coupled reticulocyte lysate in the absence and presence of microsomes. The fusion protein was identified by autoradiography following separation using SDS-polyacrylamide gel electrophoresis. Glycosylation of a translated sequence corresponded to membrane insertion and translocation of the C-terminal beta sequence. This method allowed analysis of signal anchor sequences using the M0 vector. The presence of a stop transfer sequence in the variable segment of the M1 vector resulted in inhibition of translocation of the C-terminal beta sequence. The sequences for the first four membrane segments could act as either signal anchor or stop transfer sequences. Therefore, this region of the alpha subunit has four membrane-spanning segments that are co-inserted with translation. The sequence corresponding to membrane segment M8 acted as a stop transfer sequence. The sequence corresponding to membrane segment M9 acted as a signal anchor sequence, and that corresponding to membrane segment M10 acted as a stop transfer sequence. The sequences representing the fifth, sixth, and seventh (M5, M6, and M7) membrane segments were unable to co-insert into the membrane. These data verify the first four and the eight membrane-spanning segments of the alpha subunit of the gastric H+,K(+)-ATPase and provide evidence for translational insertion of an additional pair of membrane-spanning segments, M9 and M10. It appears that insertion of membrane segments M5, M6, and M7 is determined differently from the other membrane-spanning segments. In combination with other methods, this in vitro transcription/translation method is useful for defining the membrane topology of the P type ATPases.

Please choose payment method:

(PDF emailed within 1 workday: $29.90)

Accession: 018187234

Download citation: RISBibTeXText

Related references

In vitro transcription/translation analysis for the identification of translation-terminating mutations. Methods in Molecular Biology 92: 127-144, 1998

Analysis of translation initiation using a chloroplast in vitro translation system. Plant and Cell Physiology 48: S84-S84, 2007

In vitro translation of messenger rna and analysis of translation products. Soreq, H (Ed ) Ibro (International Brain Research Organisation) Handbook Series: Methods in The Neurosciences, Vol 7 Molecular Biology Approach to The Neurosciences Xv+253p John Wiley And Sons: Chichester, England; New York, N Y , Usa Illus Paper 187-194, 1984

An in vitro translation investigation of the membrane topology of the novel SERCA CA-2+ ATPase of the slow twitch muscle. Molecular Biology of the Cell 5(Suppl ): 190A, 1994

In vitro translation of mRNA for arginine esterase, the major secretory protein of dog prostate, and in vitro processing of the translation product. Canadian Journal of Biochemistry and Cell Biology 63(7): 705-710, 1985

Sequences mediating the translation of mouse S16 ribosomal protein mRNA during myoblast differentiation and in vitro and possible control points for the in vitro translation. Genes and Development 5(9): 1723-1736, 1991

A new in vitro translation system for non-radioactive assay from tobacco chloroplasts: effect of pre-mRNA processing on translation in vitro. Plant Journal: for Cell and Molecular Biology 49(2): 367-376, 2007

Methodology for the analysis of transcription and translation in transcription-coupled-to-translation systems in vitro. Methods 86: 51-59, 2015

Topological analysis of Hin-catalysed DNA recombination in vivo and in vitro. Molecular Microbiology 51(4): 1143-1154, 2004

Effects of ginsenosides from stems and leaves on activities of Na+, K+-ATPase, Ca2+ -ATPase and Mg2+ -ATPase in rabbit cerebrum in vitro. Zhongguo Yao Li Xue Bao 9(6): 486-489, 1988

Dependence of the adenovirus tripartite leader on the p220 subunit of eukaryotic initiation factor 4F during in vitro translation. Effect of p220 cleavage by foot-and-mouth-disease-virus L-protease on in vitro translation. European Journal of Biochemistry 207(2): 471-477, 1992

Topological fragment index for the analysis of molecular substructures and their topological environment in active compounds. Journal of Chemical Information and Modeling 49(2): 162-168, 2009

Quantitative Analysis of Weak Antilocalization Effect of Topological Surface States in Topological Insulator BiSbTeSe 2. Nano Letters 19(4): 2450-2455, 2019

Expression of Na,K-ATPase beta-subunit in transformed MDCK cells increases the translation of the Na,K-ATPase alpha-subunit. Annals of the new York Academy of Sciences 986: 652-654, 2003

In vitro analysis of translation enhancers. Methods in Molecular Biology 451: 113-124, 2008