Binding of dynein light chain (PIN) to neuronal nitric oxide synthase in the absence of inhibition.
PIN, an 89-amino-acid polypeptide found in a rat hippocampal cDNA library using the yeast two-hybrid system and various neuronal nitric oxide synthase (nNOS) fragments as bait, was reported to be an inhibitor of nNOS (Science 274, 774-778, 1996). PIN reportedly inhibited nNOS selectively and did not interact with either the endothelial or inducible nitric oxide synthase isoforms. Inhibition was attributed to the ability of PIN to dissociate the catalytically active nNOS homodimer. PIN is a dynein light chain (J. Biol. Chem. 271, 19358-19366, 1996), which suggested that PIN may serve as an axonal transport protein for nNOS. We have synthesized a rat PIN cDNA by recursive polymerase chain reaction and have expressed the protein in Escherichia coli. Recombinant PIN is a folded dimeric, mostly alpha-helical protein with a single deeply buried tryptophan residue. We have also expressed and purified the nNOS fragment to which PIN reportedly binds (residues 163-245). This recombinant peptide has a disordered secondary structure. Gel-filtration experiments show that PIN binds to both the full-length nNOS and nNOS fragment. However, PIN neither inhibits nNOS activity nor dissociates the nNOS dimer into monomeric species. PIN thus possibly functions as a dynein light chain involved in nNOS axonal transport but is not an inhibitor of the enzyme. Our results agree with the proposal (Cell 82, 743-752, 1995) that the PIN recognition sequence in nNOS both lies outside the catalytic core and is not part of the monomer-monomer contact region.
Rodríguez-Crespo
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Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense, Madrid, 28040, Spain.
Straub
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W
Gavilanes
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Ortiz de Montellano
P R
PR
D000818:Animals; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D029721:Drosophila Proteins; D004398:Dyneins / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
D002352:Carrier Proteins; D029721:Drosophila Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; D004398:Dyneins