ADC proteins are degraded by ubiquitin-dependent mechanisms carried out by 26S proteasome, the major degradation pathway for soluble proteins. Experiments carry out with ADC-transgenic cultures of Trypanosoma cruzi incubated with the peptide aldehyde proteasome inhibitor MG-132 show a marked increase of ADC activity probably due to the reduction of the enzyme turnover rate as shown after blocking protein synthesis with cycloheximide.
To ascertain whether the ADC enzyme expressed in transgenic Trypanosoma cruzi has a modified metabolic turnover when attached to the His-tag, cycloheximide is added to parasite cultures transformed with plasmids to stop protein synthesis. ADC half-life inside the parasite is 150 min, the enzyme attached to the His-tail is more stable, with a half-life of about 14 h.
enzyme activities of extracts obtained from ADC or ADCH-transfected parasites are higher in the presence of oxidized glutathione than under reducing conditions with dithiothreitol in the reaction mixture
inactive precursor protein, active recombinant ADC is formed in vivo by a complex of two His-tagged polypeptide chains of 42 and 24 kDa by a subsequent incubation during different times with extracts of Arabidopsis seedlings which contain a proteolytic activity, processing of oat ADC does not require a specific protease
presence of 5 different enzymatic active and immunoreactive molecular forms of ADC with MW of 195000 Da, 115000 Da, 66000 Da, 38000 Da and 23000 Da, gel filtration
presence of 5 different enzymatic active and immunoreactive molecular forms of ADC with MW of 195000 Da, 115000 Da, 66000 Da, 38000 Da and 23000 Da, gel filtration
presence of 5 different enzymatic active and immunoreactive molecular forms of ADC with MW of 195000 Da, 115000 Da, 66000 Da, 38000 Da and 23000 Da, gel filtration
presence of 5 different enzymatic active and immunoreactive molecular forms of ADC with MW of 195000 Da, 115000 Da, 66000 Da, 38000 Da and 23000 Da, gel filtration
presence of 5 different enzymatic active and immunoreactive molecular forms of ADC with MW of 195000 Da, 115000 Da, 66000 Da, 38000 Da and 23000 Da, gel filtration
the full-length 66000 MW arginine decarboxylase polypeptide is synthesized and then cleaved to produce a 42000 MW polypeptide containing the original terminus and a 24000 polypeptide containing the original carboxyl terminus. Both of these are found in the enzyme and held together by disulfide bonds
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
ADC protein attached to the six-His tag at the C-terminal end is purified from parasite extracts by immobilized metal affinity chromatography through a Ni2+-agarose column
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
oat enzyme is expressed in Trypanosoma cruzi epimastigotes after transfection with a C-terminal six-His tag and as untagged enzyme, active ADC is expressed in the parasites and the primary translational product is cleaved into 2 polypeptides through a proteolytic process. When the his-tag is added to the C-terminal end of ADC it causes very significant changes on the metabolic stability and catalytic parameters of the heterologous enzyme expressed in the transformed parasites.