Affinity labelling of the Ca(2+)-activated neutral proteinase (calpain) in intact human platelets.
Two irreversible calpain inhibitors, benzyloxycarbonyl (Cbz)-Leu-Leu-Tyr-Ch2F and Cbz-Leu-Leu-Tyr-CHN2, were shown earlier [Anagli, Hagmann and Shaw (1991) Biochem. J. 274, 497-502] to penetrate intact platelets and to inactivate calpain. This permitted an evaluation of certain functions attributed to this proteinase. For example, in platelets pretreated with these inhibitors, talin and actin-binding protein were protected from subsequent degradation when the Ca2+ level was raised. On the other hand, additional properties of stimulated platelets attributed to calpain remained unaffected by this treatment, and such hypotheses may be dismissed. Radioiodinated inhibitors permitted confirmation of the labelling of calpain by the procedures used. Although Cbz-Leu-Leu-Tyr-CHN2 is more effective in vitro than the corresponding fluoromethyl ketone, we now show that the latter penetrates more readily. These two inhibitors, and two additional ones, t-butyloxycarbonyl-Val-Lys(Cbz)-Leu-Tyr- CHN2 and Cbz-Leu-Tyr-CH2F, have been radioiodinated to permit a comparison of their intracellular labelling patterns in activated platelets. Calpain is the major target of all four inhibitors. Although they are closely related peptide structures, variations with respect to the labelling of additional proteins were observed. These were minor in the case of the peptidyl diazomethyl ketones, but were major in the case of the fluoromethyl ketones. However, in contrast to calpain, this labelling was neither time-dependent nor Ca(2+)-dependent. Radiolabelling and cellular fractionation studies were used to localize active calpain during platelet activation. Calpain appears to be activated in the cytosol and translocated to the membrane or cytoskeletal sites.
Anagli
J
J
Friedrich Miescher-Institut, Basel, Switzerland.
Hagmann
J
J
Shaw
E
E
D000345:Affinity Labels / Q000737:chemistry; D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000201:enzymology*; D015153:Blotting, Western; D002154:Calpain / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000737:chemistry; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007457:Iodine Radioisotopes; D008969:Molecular Sequence Data; D015394:Molecular Structure; D015539:Platelet Activation
D000345:Affinity Labels; D007457:Iodine Radioisotopes; D002154:Calpain