Dp. Mehta et al., CHARACTERIZATION, SUBCELLULAR-LOCALIZATION, AND DEVELOPMENTAL REGULATION OF A CYSTEINE PROTEINASE FROM DICTYOSTELIUM-DISCOIDEUM, Archives of biochemistry and biophysics, 321(1), 1995, pp. 191-198
Previous studies showed that vegetative cells of Dictyostelium discoid
eum make a cysteine proteinase called proteinase-1, which contains mul
tiple residues of GlcNAc-1-P linked directly to peptidyl serines. As a
prelude to understanding the function of this novel carbohydrate modi
fication, we purified and extensively characterized this proteinase in
terms of its enzymatic activity, subcellular localization, and develo
pmental regulation, The purified enzyme has an apparent molecular weig
ht of 38 kDa in heat-denatured, reducing SDS/PAGE and 55 kDa under non
reducing conditions. Native gel electrophoresis and isoelectric focusi
ng revealed two protein bands with equal activity and having pI values
of 2.5 and 2.6. Even more complex patterns are found in non-heat-dena
tured SDS/PAGE gels, However, partial amino acid sequencing of the pur
ified protein gave predominantly a single sequence. The enzyme is inhi
bited by trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane, Na-p-
tosyl-L-lysine chloromethyl ketone, N-tosyl-L-phenylalanine chlorometh
yl ketone, and leupeptin, has a pH optimum of 5.0, and cofractionates
with lysosomal enzymes in bacterially grown cells. It appears to compr
ise about 90% of the total cysteine proteinase activity in cells at a
time when the cells have just finished clearing the bacterial lawn, Pr
ior to this point and after the onset of development, its level is 2-
to 20-fold lower. This remarkably fine regulation parallels the develo
pmental regulation of other cysteine proteinases in Dictyostelium. Bas
ed on these results it appears that proteinase-1 may be primarily used
for specialized proteolysis just before the onset of development rath
er than for simply digesting the bacteria for food. (C) 1995 Academic
Press, Inc.