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Dipeptidyl peptidase IV-related protein (DPPX) was found to be preferentially expressed in the brain tissue. We isolated two rat cDNA clones encoding DPPX-S and DPPX-L from a brain cDNA library, of which DPPX-L had a longer sequence at the NH2 terminus. The biosynthesis of DPPXs was examined in both in vitro and in vivo systems. In the cell-free translation system, DPPX-S and DPPX-L were synthesized as 93-kDa and 97-kDa forms, respectively, which are in good agreement with the molecular masses estimated from their primary structure. In COS-1 cells transfected with the cDNAs, DPPX-S and DPPX-L were initially synthesized as 113-kDa and 117-kDa forms, respectively, with high-mannose type oligosaccharides, which were then converted to 115-kDa and 120-kDa forms, mostly with the complex-type sugar chains. Immunofluorescence-micro-scopic observations confirmed that both DPPXs were expressed on the cell surface. DPPXs were found to have no enzyme activity of DPPIV, even when they were mutated to have the consensus active-site sequence Gly-X-Ser-X-Gly for serine proteases. Immu-noblot analysis of samples prepared from various rat tissues demonstrated that DPPX-S, but not DPPX-L, was detectable only in the brain tissue. These results indicate that, of the two isoforms, DPPX-S is preferentially expressed in the brain tissue as the surface glycoprotein without protease activity, although its function remains unknown at present. [ABSTRACT FROM AUTHOR]

A cDNA encoding a 5′-nucleotidase was identified by screening a λgt10 cDNA library from the electric lobe of Discopyge ommata using a cDNA probe containing the complete open reading frame coding for the rat liver enzyme. Nucleotide sequence analysis defines an open reading frame of 577 amino acids, corresponding to a calculated molecular mass of 63833 Da. The N-terminus of the mature protein, as determined by direct protein sequencing, is preceded by 29 amino acid residues comprising a signal peptide. The C-terminus contains a stretch of hydrophobic amino adds, considered to be cleaved on post-translational modification and exchanged for glycosylphosphatidylinositol as a membrane anchor. The predicted protein contains four potential N-linked glycosylation sites. Electric ray 5′-nucleotidase shares 61 % amino acid identity with the enzymes from rat liver and human placenta, and about 23% with bacterial proteins possessing 5′-nucleotidase activity and also additional enzyme activities like UDP-glucose hydrolase. Polyclonal antibodies raised against 5′-nucleotidase from mammalian sources or the electric ray electric organ reveal mutual cross-reactivity. Interestingly, there are 5–7 domains highly conserved in procaryotes and vertebrates in enzymes exhibiting 5′-nucleotidase, 3′-nucleotidase or phosphodiesterase activity. 5′-nucleotidase isolated from Torpedo electric organ hydrolyzes UDP-glucose at 8% of the rate of AMP hydrolysis. The possible phylogenetic origin of vertebrate 5′-nucleotidase from multifunctional nucleotide hydrolases is discussed. [ABSTRACT FROM AUTHOR]

Mammalian serine racemase is a brain-enriched enzyme that converts L- into D-serine in the nervous system. D-Serine is an endogenous co-agonist at the ‘glycine site’ of N-methyl D-aspartate (NMDA) receptors that is required for the receptor/channel opening. Factors regulating the synthesis of D-serine have implications for the NMDA receptor transmission, but little is known on the signals and events affecting serine racemase levels. We found that serine racemase interacts with the Golgin subfamily A member 3 (Golga3) protein in yeast two-hybrid screening. The interaction was confirmed in vitro with the recombinant proteins in co-transfected HEK293 cells and in vivo by co-immunoprecipitation studies from brain homogenates. Golga3 and serine racemase co-localized at the cytosol, perinuclear Golgi region, and neuronal and glial cell processes in primary cultures. Golga3 significantly increased serine racemase steady-state levels in co-transfected HEK293 cells and primary astrocyte cultures. This observation led us to investigate mechanisms regulating serine racemase levels. We found that serine racemase is degraded through the ubiquitin-proteasomal system in a Golga3-modulated manner. Golga3 decreased the ubiquitylation of serine racemase both in vitro and in vivo and significantly increased the protein half-life in pulse-chase experiments. Our results suggest that the ubiquitin system is a main regulator of serine racemase and n-serine levels. Modulation of serine racemase degradation, such as that promoted by Golga3, provides a new mechanism for regulating brain D-serine levels and NMDA receptor activity. [ABSTRACT FROM AUTHOR]

GCP170, a member of the golgin family associated with the cytoplasmic face of the Golgi membrane, was found to have a Golgi localization signal at the NH[sub 2]terminal region (positions 137-237). Using this domain as bait in the yeast two-hybrid screening system, we identified a novel protein that interacted with GCP170. The 2.0-kilobase mRNA encoding a 137-amino acid protein of 16 kDa designated GCP16 was ubiquitously expressed. Immunofluorescence microscopy showed that GCP16 was co-localized with GCP170 and giantin in the Golgi region. Despite the absence of a hydrophobic domain sufficient for participating in membrane localization, GCP16 was found to be tightly associated with membranes like an integral membrane protein. Labeling experiments with [³H]palmitic acid and mutational analysis demonstrated that GCP16 was acylated at Cys[sup 69] and Cys[sup 72], accounting for its tight association with the membrane. A mutant without potential acylation sites (C69A/C72A) was no longer localized to the Golgi, indicating that the acylation is prerequisite for the Golgi localization of GCP16. Although the mutant GCP16, even when overexpressed, had no effect on protein transport, overexpression of the wild type GCP16 caused an inhibitory effect on protein transport from the Golgi to the cell surface. Taken together, these results indicate that GCP16 is the acylated membrane protein, associated with GCP170, and possibly involved in vesicular transport from the Golgi to the cell surface. [ABSTRACT FROM AUTHOR]