Resolution of two native monomeric 90kDa nitrate reductase active proteins from Shewanella gelidimarina and the sequence of two napA genes.

The reduction of nitrate to nitrite in the bacterial periplasm occurs in the 90kDa NapA subunit of the periplasmic nitrate reductase (NAP) system. Most Shewanella genomes contain two nap operons: napEDABC and napDAGHB, which is an unusual feature of this genus. Two native, monomeric, 90kDa nitrate reductase active proteins were resolved by hydrophobic interaction chromatography from aerobic cultures of Shewanella gelidimarina replete with reduced nitrogen compounds. The 90kDa protein obtained in higher yield was characterized as NapA by electronic absorption and electron paramagnetic resonance spectroscopies and was identified by LC/MS/MS and MALDI-TOF/TOF MS as NapA from the napEDABC-type operon. The other 90kDa protein, which was unstable and produced in low yields, was posited as NapA from the napDAGHB-type operon. Two napA genes have been sequenced from the napEDABC-type and napDAGHB-type operons of S. gelidimarina. Native NAP from S. putrefaciens was resolved as one NapA monomer and one NapAB heterodimer. Two amino acid substitutions in NapA correlated with the isolation of NAP as a NapA monomer or a NapAB heterodimer. The resolution of native, redox-active NapA isoforms in Shewanella provides new insight into the respiratory versatility of this genus, which has implications in bioremediation and the assembly of microbial fuel cells.

Site-directed mutagenesis in the study of membrane transporters.

One of the major goals in membrane transporter research is to understand how transporter proteins work at the molecular level. Ideally, this research would be carried out with a detailed knowledge of the three-dimensional structure of the protein. However, in the absence of atomic resolution structures for many membrane transporters other molecular tools need to be employed. In vitro site-directed mutagenesis is one method that has the capacity to provide both structural information and identification of the role of individual residues and/or regions of a protein that are involved in function.

Extracellular loops 2 and 4 of GLYT2 are required for N-arachidonylglycine inhibition of glycine transport.

Concentrations of extracellular glycine in the central nervous system are regulated by Na(+)/Cl(-)-dependent glycine transporters, GLYT1 and GLYT2. N-Arachidonylglycine (NAGly) is an endogenous inhibitor of GLYT2 with little or no effect on GLYT1 and is analgesic in rat models of neuropathic and inflammatory pain. Understanding the molecular basis of NAGly interactions with GLYT2 may allow for the development of novel therapeutics. In this study, chimeric transporters were used to determine the structural basis for differences in NAGly sensitivity between GLYT1 and GLYT2 and also the actions of a series of related N-arachidonyl amino acids. Extracellular loops 2 and 4 of GLYT2 are important in the selective inhibition of GLYT2 by NAGly and by the related compounds N-arachidonyl-gamma-aminobutyric acid and N-arachidonyl-d-alanine, whereas only the extracellular loop 4 of GLYT2 is required for N-arachidonyl-l-alanine inhibition of transport. These observations suggest that the structure of the head group of these compounds is important in determining how they interact with extracellular loops 2 and 4 of GLYT2. Site-directed mutagenesis of GLYT2 EL4 residues was used to identify the key residues Arg(531), Lys(532), and Ile(545) that contribute to the differences in NAGly sensitivity.

Cocaine- and amphetamine-regulated transcript (CART) peptides modulate the locomotor and motivational properties of psychostimulants.

Drug addiction results from a subversion of neural circuits that control motivation. Although the hedonic and addictive properties of psychostimulants and drugs of abuse are predominantly attributed to dopamine and glutamate, it is appreciated that other signaling molecules in the brain are important. This study suggests that cocaine- and amphetamine-regulated transcript (CART) peptides modulate the locomotor and motivational properties of psychostimulants. The behavioral effects of cocaine and amphetamine were examined in Carttm1Amgen knockout (Cart KO) and wild-type (WT) mice. Acute amphetamine administration increased in locomotor activity in WT mice, but this response was attenuated in Cart KO mice. Repeated amphetamine produced locomotor sensitization in WT mice but hardly any in Cart KO mice. Amphetamine elicited conditioned place preference in both genotypes, but amphetamine's potency was reduced in the Cart KO mice. Intravenous cocaine self-administration was observed in both genotypes, but Cart KO mice consumed less cocaine and responded less for cocaine than WT mice. The behavioral effects of psychostimulants were reduced in the mutant Cart KO mice. By contrast, open field activity and sucrose preference of drug-naive mice WT and Cart KO mice were not significantly different. The attenuated effects of amphetamine and cocaine in Cart KO mice suggest a positive neuromodulatory role for CART peptides in the locomotor and motivational properties of psychostimulants and implicate CART peptides in psychostimulant addiction.

Obesity and diabetes in transgenic mice expressing proSAAS.

ProSAAS is a neuroendocrine peptide precursor that potently inhibits prohormone convertase 1 in vitro. To explore the function of proSAAS and its derived peptides, transgenic mice were created which express proSAAS using the beta-actin promoter. The body weight of transgenic mice was normal until approximately 10-12 weeks, and then increased 30-50% over wild-type littermates. Adult transgenic mice had a fat mass approximately twice that of wild-type mice, and fasting blood glucose levels were slightly elevated. In the pituitary, the levels of several fully processed peptides in transgenic mice were not reduced compared with wild-type mice, indicating that the proSAAS transgene did not affect prohormone convertase 1 activity in this tissue. Because the inhibitory potency of proSAAS-derived peptides towards prohormone convertase 1 is much greater in the absence of carboxypeptidase E activity, the proSAAS transgene was also expressed in carboxypeptidase E-deficient Cpe (fat/fat) mice. Although the transgenic mice were born in the expected frequency, 21 of 22 proSAAS transgenic Cpe (fat/fat) mice died between 11 and 26 weeks of age, presumably due to greatly elevated blood glucose. The levels of several pituitary peptides were significantly reduced in the proSAAS transgenic Cpe (fat/fat) mice relative to non-transgenic Cpe (fat/fat) mice, suggesting that the transgene inhibited prohormone convertase 1 in these mice. Taken together, these results are consistent with a role for proSAAS-derived peptides as neuropeptides that influence body weight independently of their function as inhibitors of prohormone convertase 1.