Purification of a processing enzyme (VmPE-1) that is involved in post-translational processing of a plant cysteine endopeptidase (SH-EP).
A cysteine endopeptidase, designated SH-EP, occurs in the cotyledons of germinated seeds of Vigna mungo and acts to degrade the seed storage protein in protein storage vacuoles. SH-EP is synthesized on membrane-bound ribosomes as an inactive 45-kDa precursor, which is cotranslationally processed to a 43-kDa intermediate through cleavage of the signal sequence; the 43-kDa intermediate of SH-EP is further processed to the 33-kDa mature enzyme via 39-kDa and 36-kDa intermediates [Mitsuhashi, W. & Minamikawa, T. (1989) Plant Physiol. 89, 274-279]. The present in vitro processing experiments indicated that at least two processing enzymes, designated VmPE-1 and VmPE-2 (V. mungo processing enzymes 1 and 2), were involved in post-translational processing of SH-EP in cotyledons of V. mungo seedlings. VmPE-1 was purified from the cotyledons as a protease that was involved in the processing of the 43-kDa intermediate to the 36-kDa intermediate. The enzyme has a molecular mass of 33 kDa as estimated by SDS/polyacrylamide gel electrophoresis, and showed high similarity to the jackbean asparaginyl endopeptidase in terms of the primary structure and substrate specificity. We discuss the function of VmPE-1 in the processing of SH-EP and related proteases in the cotyledons of germinated seeds.
Okamoto
T
T
Department of Biology, Tokyo Metropolitan University, Japan.
Minamikawa
T
T
D000418:Albumins / Q000378:metabolism; D000595:Amino Acid Sequence; D002852:Chromatography, Ion Exchange; D018548:Cotyledon / Q000201:enzymology / Q000235:genetics; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism; D007122:Immunoelectrophoresis; D007700:Kinetics; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010944:Plants / Q000201:enzymology* / Q000235:genetics; D011499:Protein Processing, Post-Translational; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases
D000418:Albumins; D008565:Membrane Proteins; C031460:azoalbumin; D010450:Endopeptidases; D012697:Serine Endopeptidases; C026515:type I signal peptidase; D003546:Cysteine Endopeptidases; C095010:processing enzyme 1, Vigna mungo