Purification and biochemical characterization of Eumiliin from Euphorbia milii var. hislopii latex.
A protease, which we designate Eumiliin, was isolated from the latex of Euphorbia milii var. hislopii by a combination of ion-exchange chromatographic steps using DEAE-Sephacel and gel-filtration with Sephadex G-75. Eumiliin is a monomeric protein with an apparent molecular mass of 30 kDa by SDS-PAGE under reducing conditions and gave one main peak at 29,814 KDa in MALDI-TOF/TOF mass spectrometry. Eumiliin has caseinolytic and fibrinogenolytic activities, but no hemorrhagic or defibrinating activities. The enzyme readily hydrolyzes the Aalpha-chain of fibrinogen and, more slowly, the Bbeta-chain. Its fibrinogenolytic activity is inhibited by beta-mercaptoethanol and leupeptin. In contrast, EDTA and benzamidine did not affect the activity of Eumiliin. The caseinolytic activity of Eumiliin had a pH optimum of 8.0 and was stable in solution at up to 40 degrees C; activity was completely lost at >or=80 degrees C. Intraplantar injection of Eumiliin (1-25 microg/paw) caused a dose- and time-dependent hyperalgesia, which peaked 1-5h after enzyme injection. Intraplantar injection of Eumiliin (1-25 microg/paw) also caused an oedematogenic response that was maximal after 1h. Morphological analyses indicated that Eumiliin induced an intense myonecrosis, with visible leukocyte infiltrate and damaged muscle cells 24h after injection.
Fonseca
K C
KC
Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia, Uberlândia, MG, Brazil.
Morais
N C G
NC
Queiroz
M R
MR
Silva
M C
MC
Gomes
M S
MS
Costa
J O
JO
Mamede
C C N
CC
Torres
F S
FS
Penha-Silva
N
N
Beletti
M E
ME
Canabrava
H A N
HA
Oliveira
F
F
D000818:Animals; D002364:Caseins / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D028481:Euphorbia / Q000737:chemistry*; D005340:Fibrinogen / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D002364:Caseins; D005340:Fibrinogen; D010447:Peptide Hydrolases; C551121:eumiliin, Euphorbia milii