Inhibition of adenylate cyclase activity during the reversible shape change in human platelets.

Washed platelets in the absence of physiological activators possess gravity inducible shape change, which is paralleled by an increase of phosphatidic acid (PA), polyphosphoinositides (PPI) and an inhibition of PGE1 and Gpp(NH)p- stimulated adenylate cyclase (AC) activity. Incubation of platelets at 37 degrees C for 1 hr decreases (32P)PPI and restores their response to low doses of thrombin (0.015 U/ml). Simultaneously an increase of PGE1- and Gpp(NH)p- stimulation of AC is observed. The relaxation of the platelets influences predominantly the cAMP levels without significantly affecting the dissociation constants of the stimulators. Forskolin-induced activation of AC is the same in stimulated and relaxed platelets. It is suggested that the initial increase of PA inhibits the coupling of regulatory and receptor proteins to AC and has no effect on the catalytic unit.

Reversible shape change in platelets is regulated by phosphatidylinositol metabolism.

Reversible platelet activation was studied after mechanical activation by low speed centrifugation (600 xg). Immediately following centrifugation platelets exhibited no shape change response to low doses of thrombin, collagen and ADP. After incubation at 37 degrees C a time-dependent recovery of the shape change response was observed. This was accompanied by a 50% decrease of 32P-incorporation into phosphatidic acid (PA) phosphatidylinositol-monophosphate (PIP), but not PIP2, relative to levels observed immediately after centrifugation. After 60 minutes platelet relaxation was complete: [32]PA and [32]PIP reached lowest levels and the shape change response to low doses of thrombin was completely restored.

Interaction of the cytoskeletal component vinculin with bilayer structures analyzed with a photoactivatable phospholipid.

The cytoskeletal component vinculin has been proposed to act as an actin-plasma membrane linker. In order to demonstrate a possible direct interaction of vinculin with bilayers, photolabeling with a phospholipid generating a highly reactive carbene was used. This phosphatidylcholine analogue (1-palmitoyl-2-[10-[4-[(trifluoromethyl)diazirinyl]phenyl]-[3H] 9-oxaundecanoyl]-sn-glycero-3-phosphocholine), with the photoactivatable diazirine group on its apolar portion, has been shown to label selectively membrane-embedded domains of membrane proteins. Vinculin is significantly labeled upon incubation and photolysis with liposomes containing trace amounts of this photoactivatable phospholipid, but only when the liposomes also contain acidic phospholipids. Labeling of vinculin is markedly increased (5-17-fold) by all acidic phospholipids tested so far (30%, w/w), compared to labeling in neutral phospholipids. Labeling is high at low ionic strength, but significant vinculin labeling can still be observed at physiological salt concentrations and acidic phospholipid content of the membrane. Our results provide evidence that vinculin inserts into the hydrophobic part of the bilayer by interacting with acidic phospholipids. A similar interaction may be of importance in vivo.

Venom exonuclease. IV. Intrinsic fluorescence of the exonuclease from Crotalus adamanteus venom.

The intrinsic fluorescence of the exonuclease isolated from Crotalus adamanteus venom, was studied. The position of its maximum at 335 nm and half-width of the emission band 55 nm (lambda exc. 295 nm) suggested the existence of at least two types of tryptophan residues in the enzyme molecule. Differential analysis of the fluorescence spectra obtained by excitation at 280 and 295 nm revealed about 12.5% contribution of the tyrosine fluorescence in the overall emission excited at 280 nm. The environment of the tryptophan residues in the exonuclease was studied by quenching of their fluorescence with various ionic (NO3-, NO2-, I-, Br- and Cs+) and non-ionic agents (acrylamide, chloroform-methanol). On this basis, fractions of inner (non-polar) and surface tryptophan residues located in charged and neutral regions of the enzyme molecule were evaluated. More than half of the residues (60%) was found in the inner part of the exonuclease while most of its surface tryptophans--in a neutral region(s).