Different sensitivity to trypsin of the human platelet plasma and intracellular membrane Ca2+ pumps.
The Ca2+ pumps associated with human platelet plasma and intracellular membranes have been further characterized by their sensitivity to trypsin. (a) Tryptic degradation of the Ca2+-ATPases has been followed by immunoblotting. It resulted in fragmentation into peptides of 80, 55, 35, and 24 kDa for both enzymes. Subcomplete hydrolysis obtained with a ratio of trypsin/membrane protein of 0.05-0.1 for the two Ca2+ pumps resulted in the total disappearance of the 100-, 80-, and 35-kDa fragments. However, maximum degradation was reached within 1 min for the intracellular enzyme but needed 5 min of incubation for the plasma membrane enzyme. (b) This effect of trypsin has been correlated with its effect on both the Ca2+-ATPase activities. The plasma membrane enzyme showed a maximum inhibition of 50-60% which was obtained using a trypsin/protein ratio of 0.1 and 5 min of incubation. A much higher trypsin sensitivity was observed for the intracellular enzyme because the maximum inhibition reached 80% after only 1 min of incubation. (c) Finally, the two Ca2+ transport systems studied showed different trypsin reactivities; the Ca2+ uptake by the plasma membrane vesicles was inhibited by 20-25%, and this maximum inhibition was observed after 5 min of incubation with trypsin. In contrast, the Ca2+ transport associated with the intracellular membrane vesicles was difficult to detect after trypsin treatment. Taken together, the results show that the two Ca2+ pumps can be distinguished by their trypsin sensitivity.
Enouf
J
J
U-150 Institut National de la Santé et de la Recherche Médicale, Hôpital Lariboisière, Paris, France.
Lompré
A M
AM
Bredoux
R
R
Bourdeau
N
N
de La Bastie
D
D
Levy-Toledano
S
S
D000818:Animals; D001692:Biological Transport; D001792:Blood Platelets / Q000201:enzymology*; D002118:Calcium / Q000097:blood*; D000252:Calcium-Transporting ATPases / Q000097:blood*; D002462:Cell Membrane / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007118:Immunoassay; D007425:Intracellular Membranes / Q000201:enzymology; D007473:Ion Channels / Q000378:metabolism*; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D010446:Peptide Fragments / Q000378:metabolism; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D014357:Trypsin / Q000378:metabolism*
D007473:Ion Channels; D010446:Peptide Fragments; D014357:Trypsin; D000252:Calcium-Transporting ATPases; D002118:Calcium