Volume 51: 151-158, 2002


Purification and Properties of Ornithine Carbamoyltransferase from Loggerhead Turtle Liver


E. BELLOCCO1, C. DI SALVO1, G. LAGANA1, U. LEUZZI1, E. TELLONE1, A. KOTYK2, A. GALTIERI1

1Department of Organic and Biological Chemistry, University of Messina, Messina, Italy, and 2Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic



Received June 5, 2001
Accepted August 7, 2001


Summary
Ornithine carbamoyltransferase has been purified from the liver of the loggerhead turtle Caretta caretta by a single-step procedure using chromatography on an affinity column to which the transition-state analogue, d-N-(phosphonoacetyl)- L-ornithine (d-PALO), was covalently bound. The procedure employed yielded an enzyme which was purified 373-fold and was judged to be homogeneous by nondenaturing and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme showed a specific activity of 224. The molar mass of the C. caretta enzyme was approximately 112 kDa, the single band obtained by SDS-PAGE indicated a subunit molar mass of 39.5 kDa; hence, the enzyme is a trimer of identical subunits. It catalyzes an ordered sequential mechanism in which carbamoyl phosphate binds first, followed by L-ornithine. The Michaelis constants were 0.858 mM for L-ornithine and 0.22 mM for carbamoyl phosphate, the dissociation constant of the enzyme-carbamoyl phosphate complex was 0.50 mM.


Key words
Loggerhead turtle · Caretta caretta · Ornithine carbamoyltransferase · Enzyme kinetics · Enzyme thermostability

Reprint requests
Prof. Dr. A. Kotyk, DrSc., Institute of Physiology, Academy of Sciences of the Czech Republic, Vídeòská 1083, 142 20 Prague 4, Czech Republic. Fax: 420-2-44472284. E-mail:
kotyk@biomed.cas.cz

PHYSIOLOGICAL RESEARCH
© 2002 by the Institute of Physiology, Czech Academy of Sciences

ISSN 0862 - 8408

Issue 2