(NO) is responsible for nitrergic neurotransmission in the gut, and its release is dependent on its de novo synthesis by neuronal nitric oxide synthase (nNOS). The magnitude of NO synthesis and release during neurotransmission may be related to the fraction of catalytically active nNOS out of a larger pool of inactive nNOS in the nerve terminals. The purpose of the present study was to identify catalytically active and inactive pools of nNOS in the varicosities from mouse gut. Enteric varicosities were confirmed as nitrergic by colocalization of nNOS with the nerve varicosity marker synaptophysin. Low-temperature SDS-PAGE of these varicosity extracts showed 320-, 250-, and 155-kDa bands when blotted with anti-nNOS1422-1433 and 320-and 155-kDa bands when blotted with anti-nNOS1-20 antibodies, respectively. The 320-and 155-kDa bands represent dimers and monomers of nNOS␣; the 250-and 135-kDa bands represent dimers and monomers of nNOS␤. Immunoprecipitation with calmodulin (CaM) showed that a portion of nNOS␣ dimer was bound with CaM. On the other hand, a portion of nNOS␣ dimer, nNOS␤ dimer, and all monomers lacked CaM binding. The CaM-lacking nNOS fractions reacted with anti-serine 847-phospho-nNOS. In vitro assays of NO production revealed that only the CaM-bound dimeric nNOS␣ was catalytically active; all other forms were inactive. We suggest that the amount of catalytically active nNOS␣ dimers may be regulated by serine 847 phosphorylation and equilibrium between dimers and monomers of nNOS␣. nitric oxide; isoforms of nNOS; serine 847-phosphorylated nNOS; enteric nerve varicosities; calmodulin-bound nNOS NITRIC OXIDE (NO) generated by neuronal nitric oxide synthase (nNOS) is responsible for nitrergic inhibitory neurotransmission in the gut (4,15,25). However, regulation of nitrergic neurotransmission is not well understood. The classical neurotransmitters, acetylcholine and catecholamines, are preformed and stored in secretory granules in the nerve terminals. The secretory granules exist as a large "reserve" pool and a smaller "readily releasable" pool; the latter is docked on the varicosity membrane. During nerve stimulation, propagation of an action potential in the nerve terminal causes influx of calcium into the terminal, resulting in a quantal release of the transmitter from the releasable pool (21). Regulation of the readily releasable pool of the secretory granules serves as an important determinant of the amount of the neurotransmitters released with each episode of nerve stimulation. On the other hand, NO is a highly diffusible gas and it is not preformed nor stored in secretory granules. nNOS localized to membranes of neural dendrites and motor nerve terminals are the tentative sites of NO generation during retrograde and anterograde nitrergic neurotransmission, respectively (5, 11). It is possible that only a specific fraction of nNOS in the nerve terminal with catalytic activity participates in NO production by the action of calcium influx upon nerve stimulation. We hypothesized that, analogous to t...

Sustained neurotransmitter release at synapses during high-frequency synaptic activity involves the mobilization of synaptic vesicles (SVs) from the tightly clustered reserve pool (RP). Synapsin I (Syn I), a brain-specific peripheral membrane protein that undergoes activity-dependent cycles of SV association and dissociation, is implicated in RP organization via its ability to cluster SVs. Although Syn I has affinity for phospholipids, the mechanism for the reversible association of synapsin with SV membranes remains enigmatic. Here, we show that rat Syn I is able to sense membrane curvature via an evolutionary conserved amphipathic lipid packing sensor motif (ALPS). Deletion or mutational inactivation of the ALPS impairs the ability of Syn I to associate with highly curved membranes and with SVs. Furthermore, a Syn I mutant lacking ALPS displays defects in its ability to undergo activity-induced cycles of dispersion and reclustering in neurons and fails to induce vesicle clustering in vitro. Our data suggest a crucial role for ALPS-mediated sensing of membrane curvature in regulating synapsin function.

Alternative methods of oxygen supply are of crucial importance, especially in viscous fermentations and shear‐sensitive fermentations. A method of oxygen supply that completely eliminates the gas–liquid transport resistance has been presented. The method involves a need‐based liquid‐phase decomposition of hydrogen peroxide to provide the necessary oxygen. When Xanthomonas campestris was cultivated (viscous cultivation) using this method of oxygen supply, dissolved oxygen (DO) levels were maintained above the setpoint of 50% throughout the cultivation, whereas the conventional cultivation was able to meet culture oxygen demand only for about 6 h in a 72‐h fermentation. Furthermore, the maximum specific growth rate and xanthan yields in the novel cultivation were 89% and 169%, respectively, of those obtained in conventional cultivation. A mathematical model was also developed to simulate and predict results in fermentations employing the presented methodology. In addition, studies with HOCl pretreatments indicated that monofunctional catalase may be responsible for the decomposition of H2O2 supplied externally to cells; HOCl pretreatments also increased the tolerance of cells to H2O2. The decomposition kinetics of externally supplied H2O2 was Michaelis–Menten in nature with vmax = 1.196 × 10−6M s−1 and Km = 0.21 mM. The catalase concentration was estimated to be 3.4 × 10−10 mol/g of cells. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:714–723, 1988.

This investigation demonstrates the presence and binding of the protein LC8 (described as "protein inhibitor of nNOS" or PIN in some reports) to different components of neuronal nitric oxide synthase (nNOS) in nitrergic varicosities of mice gut. Whole varicosity extracts showed three (320-, 250-, and 155-kDa) nNOS bands with anti-nNOS(1422-1433) antibody and a 10-kDa band with anti-LC8 antibody. The LC8 immunoprecipitate (IP) showed three nNOS bands, suggesting that LC8 was bound with all three forms of nNOS but dissociated from them during SDS-PAGE. Studies using LC8 IP and supernatant and probed with anti-CaM showed that LC8 was not associated with CaM-bound 320-kDa nNOS but was present in the CaM-lacking fraction. Probing these fractions with anti-serine847-P-nNOS showed that 320-kDa serine847-phosphorylated-nNOS consisted of LC8-bound and LC8-lacking components. Subsequent studies with varicosity membrane and cytosolic fractions separately showed that membrane contained CaM-bound and CaM-lacking, serine847-phosphorylated 320-kDa nNOS; both these fractions lacked LC8. On the other hand, the cytosolic fraction contained CaM-lacking, serine847-phosphorylated 320-kDa, 250-kDa, and 155-kDa nNOS bands that were all associated with LC8. These studies, along with in vitro nitric oxide assays, show that in gut nitrergic nerve varicosities 1) all cytosolic serine847-phosphorylated nNOS was catalytically inactive and bound with LC8, and 2) membrane-associated nNOS consisted of catalytically active, CaM-bound and catalytically inactive, CaM-lacking, serine847-phosphorylated nNOSalpha dimers, both of which lacked LC8. These results suggest that LC8 may dissociate from the 320-kDa nNOSalpha dimer upon binding to membrane, thus supporting the view that LC8 may transport nNOSalpha dimer to the varicosity membrane for participation in nitrergic neurotransmission.

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