PMID- 2804243
OWN - NLM
STAT- MEDLINE
DCOM- 19891221
LR  - 20190828
IS  - 0301-4622 (Print)
IS  - 0301-4622 (Linking)
VI  - 33
IP  - 3
DP  - 1989 Jul
TI  - Mechanism of acridine orange interaction with phospholipids and proteins in renal 
      microvillus vesicles.
PG  - 245-56
AB  - The mechanism of interaction of acridine orange (AO), a fluorescent, weak base, 
      with rabbit kidney brush border membrane vesicles (BBMV) has been studied by 
      absorption, and steady-state and time-resolved fluorescence spectroscopy. 
      Equilibrium binding experiments indicate that AO binds to an apparent single 
      class of sites on BBMV with a dissociation constant of 90 microM and site 
      stoichiometry of 810 nmol/mg protein. The absorption spectra AO indicate that 
      BBMV induces aggregation of AO; experiments with lipid vesicles show that the 
      aggregation requires BBMV membrane proteins. Fluorescence stopped-flow 
      experiments in which 0.15 mg/ml BBMV is mixed with increasing concentrations of 
      AO result in a time course of fluorescence enhancement for [AO] less than 1.5 
      microM, and of fluorescence quenching for [AO] greater than 1.5 microM. Similar 
      stopped-flow experiments with phosphatidylcholine lipid vesicles result only in a 
      fluorescence enhancement time course. These results indicate the presence of two 
      parallel pathways for AO binding to BBMV: one for AO binding to BBMV lipid, the 
      other for AO binding to BBMV protein. Nanosecond lifetime measurements and 
      fluorescence titration experiments confirm the presence of two environments for 
      AO in BBMV. Fluorescence stopped-flow experiments indicate that AO responds to 
      the imposition of an outwardly directed proton gradient by a rapid (less than 0.5 
      s) decrease in fluorescence, corresponding to re-equilibration of AO into the 
      acidic intravesicular compartment, followed by an increase in fluorescence, 
      corresponding to proton flux across the membrane. These findings have been 
      incorporated into a stepwise mechanism for AO interaction with BBMV which have 
      direct implications for the use of AO as a pH indicator in biological systems.
FAU - Holmberg, E G
AU  - Holmberg EG
AD  - Department of Chemistry, State University of New York, Binghamton 13901.
FAU - Verkman, A S
AU  - Verkman AS
FAU - Dix, J A
AU  - Dix JA
LA  - eng
GR  - DK35124/DK/NIDDK NIH HHS/United States
GR  - DK39354/DK/NIDDK NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - Netherlands
TA  - Biophys Chem
JT  - Biophysical chemistry
JID - 0403171
RN  - 0 (Membrane Lipids)
RN  - 0 (Membrane Proteins)
RN  - 0 (Phospholipids)
RN  - F30N4O6XVV (Acridine Orange)
SB  - IM
MH  - Acridine Orange/*metabolism
MH  - Animals
MH  - Hydrogen-Ion Concentration
MH  - In Vitro Techniques
MH  - Kidney Cortex/*metabolism
MH  - Kidney Tubules, Proximal/*metabolism
MH  - Kinetics
MH  - Membrane Lipids/*metabolism
MH  - Membrane Proteins/*metabolism
MH  - Microvilli/*metabolism
MH  - Models, Biological
MH  - Phospholipids/*metabolism
MH  - Protein Binding
MH  - Rabbits
MH  - Spectrophotometry
EDAT- 1989/07/01 00:00
MHDA- 1989/07/01 00:01
CRDT- 1989/07/01 00:00
PHST- 1989/07/01 00:00 [pubmed]
PHST- 1989/07/01 00:01 [medline]
PHST- 1989/07/01 00:00 [entrez]
AID - 0301-4622(89)80026-2 [pii]
AID - 10.1016/0301-4622(89)80026-2 [doi]
PST - ppublish
SO  - Biophys Chem. 1989 Jul;33(3):245-56. doi: 10.1016/0301-4622(89)80026-2.