Detection of NADPH-diaphorase activity in Paramecium primaurelia.
Recently, we showed that Paramecium primaurelia synthesizes molecules functionally related to the cholinergic system and involved in modulating cell-cell interactions leading to the sexual process of conjugation. It is known that nitric oxide (NO) plays a role in regulating the release of transmitter molecules, such as acetylcholine, and that the NO biosynthetic enzyme, nitric oxide synthase (NOS), shows nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity. In this work, we detected the presence of NADPH-d activity in P. primaurelia. We characterized this activity histochemically by examining its specificity for beta-NADPH and alpha-NADH co-substrates, and sensitivity both to variations in chemico-physical parameters and to inhibitors of enzymes showing NADPH-d activity. Molecules immunologically related to NOS were recognized by the anti-rat brain NOS (bNOS) antibody. Moreover, bNOS immunoreactivity and NADPH-d activity sites were found to be co-localized. The non-denaturing electrophoresis, followed by exposure to beta-NADPH or alpha-NADH co-substrates, revealed the presence of a band of apparent molecular mass of about 124 kDa or a band of apparent molecular mass of about 175 kDa, respectively. In immunoblot experiments, the bNOS antibody recognized a single band of apparent molecular mass of about 123 kDa.
Amaroli
Andrea
A
Dipartimento per lo Studio del Territorio e delle sue Risorse, University of Genoa, Genoa, Italy.
Ognibene
Marzia
M
Trielli
Francesca
F
Trombino
Sonya
S
Falugi
Carla
C
Delmonte Corrado
Maria Umberta
MU
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006651:Histocytochemistry; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007150:Immunohistochemistry; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009252:NADPH Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D017830:Octoxynol / Q000737:chemistry; D010247:Paramecium / Q000201:enzymology*; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D009243:NAD; D009249:NADP; D017830:Octoxynol; D009252:NADPH Dehydrogenase