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Characterization, subcellular localization, and developmental regulation of a cysteine proteinase from Dictyostelium discoideum.
Arch Biochem Biophys. 1995 Aug 01; 321(1):191-8.AB

Abstract

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.

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Authors+Show Affiliations

Mehta DP
La Jolla Cancer Research Foundation, California 92037, USA.
Etchison JR
No affiliation info available
Freeze HH
No affiliation info available

MeSH

Amino Acid SequenceAnimalsCell FractionationChromatography, AffinityCysteine EndopeptidasesDictyosteliumElectrophoresis, Polyacrylamide GelGene Expression Regulation, EnzymologicGene Expression Regulation, FungalKineticsMolecular Sequence DataMolecular WeightPeptide FragmentsProtease InhibitorsProtozoan ProteinsSequence Homology, Amino AcidSubcellular FractionsSubstrate Specificity

Pub Type(s)

Comparative Study
Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

7639520

Citation

Mehta, D P., et al. "Characterization, Subcellular Localization, and Developmental Regulation of a Cysteine Proteinase From Dictyostelium Discoideum." Archives of Biochemistry and Biophysics, vol. 321, no. 1, 1995, pp. 191-8.
Mehta DP, Etchison JR, Freeze HH. Characterization, subcellular localization, and developmental regulation of a cysteine proteinase from Dictyostelium discoideum. Arch Biochem Biophys. 1995;321(1):191-8.
Mehta, D. P., Etchison, J. R., & Freeze, H. H. (1995). Characterization, subcellular localization, and developmental regulation of a cysteine proteinase from Dictyostelium discoideum. Archives of Biochemistry and Biophysics, 321(1), 191-8.
Mehta DP, Etchison JR, Freeze HH. Characterization, Subcellular Localization, and Developmental Regulation of a Cysteine Proteinase From Dictyostelium Discoideum. Arch Biochem Biophys. 1995 Aug 1;321(1):191-8. PubMed PMID: 7639520.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Characterization, subcellular localization, and developmental regulation of a cysteine proteinase from Dictyostelium discoideum. AU - Mehta,D P, AU - Etchison,J R, AU - Freeze,H H, PY - 1995/8/1/pubmed PY - 1995/8/1/medline PY - 1995/8/1/entrez SP - 191 EP - 8 JF - Archives of biochemistry and biophysics JO - Arch Biochem Biophys VL - 321 IS - 1 N2 - 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. SN - 0003-9861 UR - https://www.unboundmedicine.com/medline/citation/7639520/Characterization_subcellular_localization_and_developmental_regulation_of_a_cysteine_proteinase_from_Dictyostelium_discoideum_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0003-9861(85)71385-9 DB - PRIME DP - Unbound Medicine ER -