Modification of a Ribonuclease from Rhizopus Sp. with 1-Cyclohexyl-3-(2-Morpholinyl-(4)-Ethyl)Carbodiimide p-Toluenesulfonate

The carboxyl group in a ribonuclease from Rhizopus sp. (RNase Rh) was modified by a water-soluble carbodiimide, 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide p-toluenesulfonate (CMC). From the relation between the extent of modification and the enzymatic activity, it was concluded that at lea...

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Bibliographic Details
Published in:Journal of biochemistry (Tokyo) 1985-07, Vol.98 (1), p.125-132
Main Authors: SANDA, Akihiro, TAKIZAWA, Yoshio, IWAMA, Masanori, IRIE, Masachika
Format: Article
Language:English
Subjects:
Adenosine Monophosphate - metabolism
Amino Acids, Dicarboxylic
Analytical, structural and metabolic biochemistry
Binding Sites
Biological and medical sciences
Carbodiimides
Circular Dichroism
CME-Carbodiimide - analogs & derivatives
cyanamide
Cytidine - pharmacology
Enzymes and enzyme inhibitors
Fundamental and applied biological sciences. Psychology
Hydrogen-Ion Concentration
Hydrolases
Kinetics
Peptide Fragments - analysis
Protein Conformation
Rhizopus
Rhizopus - enzymology
ribonuclease
Ribonucleases - antagonists & inhibitors
Ribonucleases - metabolism
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Summary:The carboxyl group in a ribonuclease from Rhizopus sp. (RNase Rh) was modified by a water-soluble carbodiimide, 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide p-toluenesulfonate (CMC). From the relation between the extent of modification and the enzymatic activity, it was concluded that at least the modification of two carboxyl groups seemed to induce the loss in enzymatic activity. In the presence of 1 M cytidine, RNase Rh activity was protected from the CMC modification. Under conditions in which the enzyme was inactivated to 20% activity, about 70% of the enzymatic activity was retained in the presence of cytidine. The inactivation of the RNase Rh pre-treated with CMC in the presence of cytidine with [14C]CMC indicated that the RNase Rh lost its enzymatic activity with the in-corporation of about one [14C]CMC Therefore, it could be concluded that one carboxyl group is involved in the active site of RNase Rh. The binding of the CMC-modified RNase Rh with 2′-AMP was studied spectro photometrically. The affinity of the modified RNase Rh towards 2′-AMP decreased markedly upon CMC modification.
ISSN:0021-924X
1756-2651
DOI:10.1093/oxfordjournals.jbchem.a135250