Labelling by sup 3 H-N-ethylmaleimide of diamide-oxidized thiol groups in sheep red blood cell (SRBC) membranes

Conference · Mon Mar 11 04:00:00 EST 1991 · FASEB Journal (Federation of American Societies for Experimental Biology); (United States)

OSTI ID:5299694

Lauf, P K ^[1]

---

1. Wright State Univ., Dayton, OH (United States)

Exposure of SRBC to the thiol oxidant diamide activates K:Cl cotransport, reversed upon metabolic restoration of cellular glutathione suggesting redox control of the K:Cl cotransporter, as well as by subsequent exposure to dithiothreitol (DTT). The thiols crucial for activation may be either on the transporter or on a membrane or cytoplasmic regulator. To test this hypothesis, the authors attempted to label with {sup 3}H-N-ethylmaleimide ({sup 3}H-NEM) the thiols protected by diamide oxidation and reduced subsequently by DTT. SRBC were first treated with a diamide concentration activating K:Cl cotransport, followed by a second exposure to unlabeled (cold) NEM to block any non-oxidized thiol, and then hemolyzed to obtain white ghosts. The ghosts were again treated with cold NEM and after reduction by DTT exposed to {sub 3}H-NEM with and without cold NEM. Saturation labelling by {sup 3}H-NEM of diamide protected groups occurred in the range of CTT concentrations inactivating the diamide-stimulated K:Cl cotransport. Saturation labelling with {sup 3}H-NEM occurred at about 25{mu}M NEM suggesting a Ki of less than 10{mu}M NEM. The number of diamide protected thiols was about 5-10,000/cell membrane. At 100{mu}M {sup 3}H-NEM, SRBC not treated with diamide possess at least 100,000 thiols cell and this number is likely to rise by tenfold at higher NEM concentrations. Thus, diamide protected about 1/1,000 of the membrane thiols in both genetically low and high K SRBC, assayed under conditions where K:Cl cotransport is activated in intact cells. Therefore, at least some of the thiols crucial for potential regulation of K:Cl cotransport reside within the plasma membrane.

• OSTI ID: 5299694
• Report Number(s): CONF-9104107--
• Conference Information: Journal Name: FASEB Journal (Federation of American Societies for Experimental Biology); (United States) Journal Volume: 5:4
• Country of Publication: United States
• Language: English

Related Subjects

550501 -- Metabolism-- Tracer Techniques
560300* -- Chemicals Metabolism & Toxicology
59 BASIC BIOLOGICAL SCIENCES
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
ALKALI METALS
ANIMALS
ANTIMITOTIC DRUGS
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BODY FLUIDS
CELL CONSTITUENTS
CELL MEMBRANES
CHEMICAL REACTIONS
CHLORIDES
CHLORINE COMPOUNDS
DOMESTIC ANIMALS
DRUGS
ELEMENTS
ERYTHROCYTES
HALIDES
HALOGEN COMPOUNDS
HYDROGEN COMPOUNDS
IMIDES
ISOTOPE APPLICATIONS
LABELLING
MAMMALS
MATERIALS
MEMBRANE TRANSPORT
MEMBRANES
METABOLISM
METALS
NEM
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
POTASSIUM
RADIOSENSITIZERS
REDOX REACTIONS
RUMINANTS
SHEEP
THIOLS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
VERTEBRATES