The synthesis of ATP by glycolytic enzymes in the postsynaptic density and the effect of endogenously generated nitric oxide ( glycolysis )

The major contribution of this paper is the finding of a glycolytic source of ATP in the isolated postsynaptic density (PSD). The enzymes involved in the generation of ATP are glyceraldehyde-3-phosphate dehydrogenase (G3PD) and phosphoglycerate kinase (PGK). Lactate dehydrogenase (LDH) is available for the regeneration of NAD1, as well as aldolase for the regeneration of glyceraldehyde-3-phosphate (G3P). The ATP was shown to be used by the PSD Ca21ycalmodulin-dependent protein kinase and can probably be used by two other PSD kinases, protein kinase A and protein kinase C. We confirmed by immunocytochemistry the presence of G3PD in the PSD and its binding to actin. Also present in the PSD is NO synthase, the source of NO. NO increases the binding of NAD, a G3PD cofactor, to G3PD and inhibits its activity as also found by others. The increased NAD binding resulted in an increase in G3PD binding to actin. We confirmed the autophosphorylation of G3PD by ATP, and further found that this procedure also increased the binding of G3PD to actin. ATP and NO are connected in that the formation of NO from NOS at the PSD resulted, in the presence of NAD, in a decrease of ATP formation in the PSD. In the discussion, we raise the possible roles of G3PD and of ATP in protein synthesis at the PSD, the regulation by NO, as well as the overall regulatory role of the PSD complex in synaptic transmission. The experiments in this paper are a continuation of our work on postsynaptic density (PSD) and an elaboration of the possible role of the PSD in synaptic function. The PSD organelle is a dense concentration of proteins attached to the cellular surface of the postsynaptic membrane in dendritic spine heads; it contains receptors for some, and possibly all, of the neurotransmitters, plus channels for Ca21, K1, and possibly other relevant ions, plus three protein kinases and their substrates (for reviews, see refs. 1 and 2). The particular set of experiments described here were generated by three papers: first and second, the discovery by Walsh and Kuruc (3) by using gel electrophoresis and Rogalski-Wilk and Cohen (4) by using specific antibody that glyceraldehyde-3-phosphate dehydrogenase (G3PD) is found in the isolated PSD and that it is bound to the PSD protein, actin (4); and third, the finding by Aoki et al. (5) using immunocytochemistry that NO synthase (NOS), the enzyme producing NO from L-arginine, is also present in the PSD as well as other neuronal sites. Furthermore, a linkage between G3PD and NOS was shown by the publication in 1992 of three papers wherein NO was found to stimulate a so-called ADP ribosylation of G3PD in brain (6), platelets (7), and erythrocytes (8) (see ref. 9), though later papers (10, 11) indicated that NO stimulates the linkage of NAD to purified G3PD, probably to a cysteine residue in G3PD (12), and not the ADP ribosylation per se. In this paper, we confirm the presence of the G3PD in the PSD, show that its response to NO is similar to that found from other sources, and indicate factors that regulate G3PD binding to actin. Furthermore, we demonstrate that G3PD and other glycolytic enzymes are present in the PSDs and are capable of glycolytic metabolism and synthesis of ATP. MATERIALS AND METHODS Materials. [g-32P]ATP (6000 Ciymmol; 1 Ci 5 37 GBq), [adenylate-32P]NAD (30 Ciymmol), and [32P]orthophosphate (32Pi) (2000 Ciymmol) were purchased from Amersham. The following compounds were products of Sigma: G-actin; porcine muscle G3PD; sodium nitroprusside (SNP), a source of NO (13); calmodulin (CaM); L-arginine; N-L-arginine-methyl ester (N-LAME), specific inhibitor of NOS (14); microcystinLR, a potent inhibitor of protein phosphatase type 1 and type 2A (15); and leupeptin, inhibitor of Ca21-activated proteases (16, 17). S,S9-dinitrosodithiol (SND), another source of NO (18), was obtained from Calbiochem. Affinity purified rabbit anti-G3PD antibodies were prepared and characterized (19) by injecting purified human red cell G3PD into rabbits. LDH, hexokinase and phosphoglycerate kinase (PGK) were products of Worthington. Other chemicals were of reagent grade from commercial sources. Electrophoresis. In the following experiments, SDSyPAGE was carried out according to ref. 20. All experiments were performed three times. Inter-experimental variations in relative band intensities were less than 10%. Subcellular Fractions. Adult porcine brain, obtained from a local slaughter house, was dissected, and the cerebral cortex was employed for the studies illustrated in the figures. In some cases, rat cerebral cortex was also used. Synaptosome, synaptic membrane, PSD fractions (21), and a crude synaptic vesicle (CSV) fraction (22) were all isolated as described, with the CSV fraction supplied by R. S. Cohen (University of Illinois, Chicago). Determination of the Activities of Glycolytic Enzymes in the Isolated PSD Fractions. The activities of G3PD (23), lactate dehydrogenase (LDH) (24), PGK (25), and hexokinase (26) were all determined as described. Enzyme levels in the PSD fractions were estimated by comparing the PSD enzyme activities to those of the purified enzymes and converting to specific units or amounts (units or mg per mg PSD). NO-Stimulated [Adenylate-32P]NAD Incorporation into G3PD in Subcellular Fractions Isolated from Adult Porcine The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1997 by The National Academy of Sciences 0027-8424y97y9413273-6$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: CaM, calmodulin; G3P, glyceraldehyde-3 phosphate; G3PD, glyceraldehyde-3-phosphate dehydrogenase; LDH, lactate dehydrogenase; PGK, phosphoglycerate kinase; NOS, NO synthase; PSD, postsynaptic density; SNP, sodium nitroprusside; N-LAME, N-L-arginine-methyl ester; SND, S,S9-dinitrosodithiol; mPSDp, major PSD protein. ¶To whom reprint requests should be addressed at: The Rockefeller University, 1230 York Avenue, New York, NY 10021.