Functional molecular weight of the lac carrier protein from Escherichia coli as studied by radiation inactivation analysis

Tzipora Goldkorn, G. Rimon, E. S. Kempner, H. R. Kaback

Research output: Contribution to journalArticle

26 Scopus citations

Abstract

Cytoplasmic membrane vesicles prepared from Escherichia coli containing multiple copies of the lac y gene were frozen in liquid nitrogen before or after generation of a proton electrochemical gradient (interior negative and alkaline) and irradiated with a high-energy electron beam at -135°C. Subsequently, the lac carrier protein was extracted into octyl β-D-glucopyranoside, reconstituted into proteoliposomes, and assayed for transport activity. Under all conditions tested, activity decreased as a single exponential function of radiation dosage, allowing straightforward application of target theory for determination of functional molecular mass. When lac carrier activity solubilized from nonenergized vesicles was assayed, the results obtained were consistent with a functional molecular size of 45-50 kDa, a value similar to the size of the protein as determined by other means. Similar values were obtained when the octyl β-D-glucopyranoside extract was irradiated, and the target size observed for D-lactate dehydrogenase was in good agreement with the molecular size of this enzyme. Strikingly, when the same procedures were carried out with vesicles that were energized with appropriate electron donors prior to freezing and irradiation, a functional molecular size of 85-100 kDa was obtained for the lac carrier with no change in the target size of D-lactate dehydrogenase. In contrast, when the vesicles were energized under conditions in which the proton electrochemical gradient was collapsed, the target mass of the lac carrier returned to 45-50 kDa. The results indicate that the functional mass of the lac carrier protein is no greater than a dimer and suggest that the proton electrochemical gradient may cause an alteration in subunit interactions.

Original languageEnglish (US)
Pages (from-to)1021-1025
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume81
Issue number4 I
StatePublished - 1984
Externally publishedYes

ASJC Scopus subject areas

  • General
  • Genetics

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