Behavior and structure of the band 3 anion transport site: a /sup 35/Cl and /sup 37/Cl NMR study

Thesis/Dissertation · Tue Jan 01 04:00:00 EST 1985

OSTI ID:7108300

Falke, J J

The present thesis focuses on band 3, the most heavily used ion transport protein in typical vertebrate systems. The strategy employed involves /sup 35/Cl NMR, which is shown to be a sensitive probe of (1) Cl/sup -/ migration from solution to a binding site, (2) Cl/sup -/ binding to the site, and (3) the sidedness of a membrane-bound site. Both low-affinity (K/sub D >> 0.5 M) and high-affinity (K/sub D/ = 80 +/- 20 mM) sites are observed on both surfaces of isolated red cell membranes. The latter sites are identified as band 3 transport sites by their affinity for a variety of anions. The transport sites on both sides on both sides of the membrane together behave like a homogeneous population under varying (pCl/sup -/), (Br/sup -/), or pH. These results support the ping-pong model for the transport cycle. The rates of Cl/sup -/ binding and dissociation at the inward- and outward-facing sites are shown by /sup 35/Cl and /sup 37/Cl NMR to exceed 10/sup 5/ events sec/sup -1/ site/sup -1/. Three classes of inhibitors are found: (1) niflumic acid has no effect on Cl/sup -/ migration or binding to the transport site - this inhibitor slows the translocation of bound Cl/sup -/ during both influx and efflux; (2) 1.2-cyclohexanedione (CHD) slows the migration of Cl/sup -/ between the transport site and solution; and (3) phenylglyoxal (PG) modifies an essential transport site arginine. The transport site remains intact following a variety of stripping and proteolytic treatments. Together these results indicate that the transport site is buried in the membrane, where access to solution is provided by a channel that can be blocked by CHD. All essential transport site and channel residues lie on the papain-generated fragments. A model involving a sliding hydrophobic barrier is presented for the ion translocation event.

• Research Organization: California Inst. of Tech., Pasadena (USA)
• OSTI ID: 7108300
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BODY FLUIDS
CELL CONSTITUENTS
CELL MEMBRANES
CHLORINE 35
CHLORINE 37
CHLORINE ISOTOPES
ERYTHROCYTES
ISOTOPES
LIGHT NUCLEI
MAGNETIC RESONANCE
MATERIALS
MEMBRANE TRANSPORT
MEMBRANES
NUCLEAR MAGNETIC RESONANCE
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
PROTEINS
RESONANCE
STABLE ISOTOPES
STRUCTURAL MODELS