Characterization, subcellular localization, and developmental regulation of a cysteine proteinase from Dictyostelium discoideum.
Previous studies showed that vegetative cells of Dictyostelium discoideum make a cysteine proteinase called proteinase-1, which contains multiple residues of GlcNAc-1-P linked directly to peptidyl serines. As a prelude to understanding the function of this novel carbohydrate modification, we purified and extensively characterized this proteinase in terms of its enzymatic activity, subcellular localization, and developmental regulation. The purified enzyme has an apparent molecular weight of 38 kDa in heat-denatured, reducing SDS/PAGE and 55 kDa under nonreducing conditions. Native gel electrophoresis and isoelectric focusing 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-denatured SDS/PAGE gels. However, partial amino acid sequencing of the purified protein gave predominantly a single sequence. The enzyme is inhibited by trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane, Na-p-tosyl-L-lysine chloromethyl ketone, N-tosyl-L-phenylalanine chloromethyl ketone, and leupeptin, has a pH optimum of 5.0, and cofractionates with lysosomal enzymes in bacterially grown cells. It appears to comprise about 90% of the total cysteine proteinase activity in cells at a time when the cells have just finished clearing the bacterial lawn. Prior to this point and after the onset of development, its level is 2- to 20-fold lower. This remarkably fine regulation parallels the developmental regulation of other cysteine proteinases in Dictyostelium. Based on these results it appears that proteinase-1 may be primarily used for specialized proteolysis just before the onset of development rather than for simply digesting the bacteria for food.
Mehta
D P
DP
La Jolla Cancer Research Foundation, California 92037, USA.
Etchison
J R
JR
Freeze
H H
HH
D000595:Amino Acid Sequence; D000818:Animals; D002458:Cell Fractionation; D002846:Chromatography, Affinity; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004023:Dictyostelium / Q000201:enzymology* / Q000254:growth & development / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011480:Protease Inhibitors / Q000494:pharmacology; D015800:Protozoan Proteins; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity
D010446:Peptide Fragments; D011480:Protease Inhibitors; D015800:Protozoan Proteins; D003546:Cysteine Endopeptidases; C095028:cprG protein, Dictyostelium