Purification of the recombinant human serotonin N-acetyltransferase (EC 2.3.1.87): further characterization of and comparison with AANAT from other species.
Melatonin is synthesized by a series of enzymes, the penultimate one, serotonin N-acetyltransferase, catalyzing the limiting reaction. In the present study, we compared the recombinant serotonin N-acetyltransferases from rat, ovine, and human. The human protein is particularly difficult to purify because it interacts strongly with a putative chaperone protein from bacteria whereas the rat and sheep enzymes, which interact less strongly with this protein, have been purified close to homogeneity. We identified the contaminating protein as GroEL, the bacterial equivalent of Hsp60. We present numerous catalytic activities (substrate and cosubstrate specificities as well as inhibitor specificities) measured on the three species enzymes from which we deduced that the presence of the chaperone might partly explain the differences between the various species enzyme characteristics, beside the inter-species ones resulting from sequence differences. Despite several trials reported in the literature, a purification to homogeneity of the human (recombinant) enzyme has never been described. We present a new purification method, by using an original denaturation/renaturation process in which the enzyme is immobilized on an affinity chromatography column. The enzyme is then eluted in an active and pure form (i.e., absence of chaperone). The up-scaled system permitted us to perform the necessary experiments for the measurement of more accurate affinities of human serotonin N-acetyltransferase towards its main natural substrates, showing that only the activity of the enzyme towards phenylethylamine was modified.
Pharmacologie Moléculaire et Cellulaire, Institut de Recherches SERVIER, 125, chemin de Ronde 78290 Croissy-sur-Seine, France.
Gilles
G
Ferry
Caroline
C
Ubeaud
Claire
C
Dauly
Julien
J
Mozo
Sophie
S
Guillard
Sylvie
S
Berger
Sandrine
S
Jimenez
Catherine
C
Scoul
Gregory
G
Leclerc
Saïd
S
Yous
Philippe
P
Delagrange
Jean A
JA
Boutin
D000595:Amino Acid Sequence; D000818:Animals; D047068:Arylalkylamine N-Acetyltransferase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D017124:Conserved Sequence; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011489:Protein Denaturation; D020673:Protein Renaturation; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000378:metabolism; D016415:Sequence Alignment; D012756:Sheep; D013379:Substrate Specificity
D011994:Recombinant Proteins; C478879:AANAT protein, human; D047068:Arylalkylamine N-Acetyltransferase