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
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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
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