Limited proteolysis of maize NADP-malic enzyme.
The incubation of maize malic enzyme at 37 degrees C with trypsin at a ratio of 150:1 of malic enzyme to trypsin caused rapid and complete inactivation of enzyme activity. The inactivation was caused by fairly specific cleavage of the enzyme monomer (62 kDa) into 40 kDa and 20 kDa fragments. The intensity of 40 kDa band increased with the time of treatment of enzyme with trypsin from 2 to 30 min. Substrates, especially NADP (25 microM) provided almost total protection against trypsin inactivation of the enzyme activity. The studies carried out with various other endoproteases indicated that endoprotease Lys-C was most effective in inactivating malic enzyme activity. The kinetic properties of the truncated enzyme have been studied. The Km value for malate in case of native and modified enzyme was found to be identical. Km NADP for the modified enzyme was slightly higher indicating that after proteolysis the enzyme affinity for NADP had decreased. Limited proteolysis with trypsin did not show any appreciable change in fluorescence properties of the modified enzyme. Binding of NADPH to the enzyme was not affected after modification.
Molecular Biology and Agricultural Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India.
S
S
Pinto
S R
SR
Rao
A S
AS
Bhagwat
D001671:Biochemistry / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008274:Magnesium / Q000737:chemistry; D008291:Malate Dehydrogenase / Q000737:chemistry*; D008956:Models, Chemical; D010447:Peptide Hydrolases / Q000737:chemistry; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry; D059748:Proteolysis; D013379:Substrate Specificity; D014357:Trypsin / Q000737:chemistry; D014361:Trypsin Inhibitors / Q000494:pharmacology; D003313:Zea mays / Q000201:enzymology*
D011506:Proteins; D014361:Trypsin Inhibitors; D008291:Malate Dehydrogenase; C031917:malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+); D010447:Peptide Hydrolases; D014357:Trypsin; D008274:Magnesium; D008239:Lysine