Reconstitution of N1-methylnicotinamide and p-aminohippuric acid transport in phospholipid vesicles with a protein fraction isolated from dog kidney membranes

P. D. Holohan, N. I. Pessah, Isaac N Pessah, C. R. Ross

Research output: Contribution to journalArticle

8 Scopus citations

Abstract

N1-methylnicotinamide (NMN) and p-aminohippurate (PAH) transport can be reconstituted in phospholipid vesicles by incorporting the solubilized membrane proteins into artifical phospholipid membranes. After solubilization with the non-ionic detergent, Lubrol WX, the protein solution was added to a phospholipid solution (prepared by dissolving the phospholipids in the detergents) and the phospholipid:protein vesicles were allowed to form during dialysis. Transport in these vesicles was studied with radioactively labeled NMN or PAH; the transport for NMN was 173 to 394 pmoles/mg of protein/5 min and for PAH was 263 to 505 pmoles/mg of protein/5 min. Transport into control vesicles (without protein) was less than 10% of these values. The transport of NMN and PAH displayed properties of facilitated diffusion: equilibrium was reached within 5 min; pH dependence (optimum for NMN was 7.4, for PAH, 7.0); protein and lipid concentration dependence; saturability (K(m) estimated from 1/2 saturation was approximately 1 mM for both NMN and PAH); an specificity, in that known competitors of these transport systems inhibited uptake. In addition, it appears as if specific phospholipids are required in that only sphingomyelin and phosphatidylcholine functioned in reconstituting transport. Using reconstitution as the mode of assaying the proteins through an isolation procedure after solubilization, we achieved a 45 fold purification of the NMN and PAH transport proteins. Interestingly, although our data clearly show that the two transport systems are direct entities, the two activities copurify. Based upon these results we propose that we have accomplished a partial purification of the 'carrier' proteins involved in the renal secretion of organic compounds.

Original languageEnglish (US)
Pages (from-to)343-356
Number of pages14
JournalMolecular Pharmacology
Volume16
Issue number1
StatePublished - 1979
Externally publishedYes

ASJC Scopus subject areas

  • Pharmacology

Fingerprint Dive into the research topics of 'Reconstitution of N<sup>1</sup>-methylnicotinamide and p-aminohippuric acid transport in phospholipid vesicles with a protein fraction isolated from dog kidney membranes'. Together they form a unique fingerprint.

  • Cite this