Extraction of Phospholipids from Human Erythrocyte Membranes by Hemoglobin Oxidation Products.

This investigation examines oxidation conditions under which hemoglobin extracts membrane phospholipid from erythrocytes and model membranes. In erythrocytes made echinocytic with exogenous phospholipid, addition of hemoglobin oxidized with hydrogen peroxide (H2O2) or Vitamin C (conditions that result in the formation of significant quantities of choleglobin), but not ferricyanide (which produces predominantly methemoglobin), induced dose-dependent shape reversion to less echinocytic forms, consistent with extraction of phospholipids from the exofacial side of the membrane. Erythrocytes preloaded with radiolabeled phosphatidylcholine or NBD-labeled phosphatidylcholine, phosphatidylglycerol or phosphatidic acid, exhibited greatest extraction of radiolabel or fluorescence signal with exogenous hemoglobin oxidized via H2O2 or Vitamin C, but not ferricyanide.

Substrate specificities and activities of AZAP family Arf GAPs in vivo.

The ADP-ribosylation factor (Arf) GTPases are important regulators of vesicular transport in eukaryotic cells. Like other GTPases, the Arfs require guanine nucleotide exchange factors to facilitate GTP loading and GTPase-activating proteins (GAPs) to promote GTP hydrolysis. Whereas there are only six mammalian Arfs, the human genome encodes over 20 proteins containing Arf GAP domains.

ARAP2 effects on the actin cytoskeleton are dependent on Arf6-specific GTPase-activating-protein activity and binding to RhoA-GTP.

ARAP2 is a protein that contains both ArfGAP and RhoGAP domains. We found that it is a phosphatidylinositol (3,4,5)-trisphosphate-dependent Arf6 GAP that binds RhoA-GTP but lacks RhoGAP activity. In agreement with the hypothesis that ARAP2 mediates effects of RhoA, endogenous ARAP2 associated with focal adhesions (FAs) and reduction of ARAP2 expression, by RNAi, resulted in fewer FAs and actin stress fibers (SFs).