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Proteome analysis plays a key role in the elucidation of the functions and applications for numerous proteins. For proteome analyses, various microplate-and microarray-based techniques have been developed by a number of researchers. Their intent was to immobilize proteins on the surface of a solid substrate in a site-directed manner while retaining structure and native biological function. In this review, we focus on recent advances in immobilization methodology for proteins/enzymes on a surface, including those using the affinity peptides screened by random peptide library systems. We also discuss applications of the affinity peptide-mediated immobilization method in fields related to proteome analysis, particularly our recent work concerning immunoassay and protein-protein interaction analysis.

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Fructose-1,6-bisphosphatase (FBPase) is one of the key enzymes in gluconeogenesis. Although FBPase activity has been detected in several hyperthermophiles, no orthologs corresponding to the classical FBPases from bacteria and eukaryotes have been identified in their genomes. An inositol monophosphatase (IMPase) from Methanococcus jannaschii which displayed both FBPase and IMPase activities and a structurally novel FBPase (Fbp Tk ) from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 have been proposed as the "missing" FBPase. For this study, using T. kodakaraensis, we took a genetic approach to elucidate which candidate is the major gluconeogenic enzyme in vivo. The IMPase/FBPase ortholog in T. kodakaraensis, Imp Tk , was confirmed to possess high FBPase activity along with IMPase activity, as in the case of other orthologs. We therefore constructed ⌬fbp and ⌬imp strains by applying a gene disruption system recently developed for T. kodakaraensis and investigated their phenotypes. The ⌬fbp strain could not grow under gluconeogenic conditions while glycolytic growth was unimpaired, and the disruption resulted in the complete abolishment of intracellular FBPase activity. Evidently, fbp Tk is an indispensable gene for gluconeogenesis and is responsible for almost all intracellular FBPase activity. In contrast, the endogenous imp Tk gene could not complement the defect of the fbp deletion, and its disruption did not lead to any detectable phenotypic changes under the conditions examined. These facts indicated that imp Tk is irrelevant to gluconeogenesis, despite the high FBPase activity of its protein product, probably due to insufficient transcription. Our results provide strong evidence that the true FBPase for gluconeogenesis in T. kodakaraensis is the Fbp Tk ortholog, not the IMPase/FBPase ortholog.

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Temperature scanning Fourier transform infrared, TS-FTIR, spectroscopy of various amorphous sugar matrixes was conducted to investigate the relationship between the glass transition temperature, T(g), of an amorphous sugar matrix and the nature of the hydrogen bonds in the matrix. An amorphous sugar matrix was prepared by air-drying an aqueous solution of sugar, and the degree of formation of hydrogen bonds in the matrix was evaluated at different temperatures using the peak positions of the IR band corresponding to the O-H stretching vibration at around 3400 cm(-1). The T(g) value increased with increasing peak position of the O-H stretching vibration at T(g) and were correlated reasonably well with the magnitude of the peak shift by the temperature increase (from 25 degrees C) to the T(g) value. This demonstrates that the amorphous sugar matrix, in which the segments are fixed by fewer hydrogen bonds, has a higher thermal resistance. The glycosidic linkage largely contributes to the restriction of the segments, pyranose ring, rather than a hydrogen bond. As the degree of polymerization of pyranose rings increases, the degree of hydrogen bond formation needed to hold the matrix in a fixed position decreases. However, the magnitude of the restriction of pyranose rings by a glycosidic linkage changes depending on the type: the restrictions imposed by alpha-1,1 and -1,6 glycosidic linkages are the tightest and most flexible of all of the types of glycosidic linkages, respectively.

Fructose-1,6-bisphosphatase (FBPase) is one of the key enzymes of the gluconeogenic pathway. Although enzyme activity had been detected in Archaea, the corresponding gene had not been identified until a presumable inositol monophosphatase gene from Methanococcus jannaschii was found to encode a protein with both inositol monophosphatase and FBPase activities. Here we display that a gene from the hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1, which does not correspond to the inositol monophosphatase gene from M. jannaschii, displays high FBPase activity. The FBPase from strain KOD1 was partially purified, its Nterminal amino acid sequence was determined, and the gene (Tk-fbp) was cloned. Tk-fbp encoded a protein of 375 amino acid residues with a molecular mass of 41,658 Da. The recombinant Tk-Fbp was purified and characterized. Tk-Fbp catalyzed the conversion of fructose 1,6-bisphosphate to fructose 6-phosphate following Michaelis-Menten kinetics with a K m value of 100 M toward fructose 1,6-bisphosphate, and a k cat value of 17 s ؊1 subunit ؊1 at 95°C. Unlike the inositol monophosphatase from M. jannaschii, Tk-Fbp displayed strict substrate specificity for fructose 1,6-bisphosphate. Activity was enhanced by Mg 2؉ and dithioerythritol, and was slightly inhibited by fructose 2,6-bisphosphate. AMP did not inhibit the enzyme activity. We examined whether expression of Tk-fbp was regulated at the transcription level. High levels of Tk-fbp transcripts were detected in cells grown on pyruvate or amino acids, whereas no transcription was detected when starch was present in the medium. Orthologue genes corresponding to Tk-fbp with high similarity are present in all the complete genome sequences of thermophilic Archaea, including M. jannaschii, Pyrococcus furiosus, Sulfolobus solfataricus, and Archaeoglobus fulgidus, but are yet to be assigned any function. Taking into account the high FBPase activity of the protein, the strict substrate specificity, and its sugar-repressed gene expression, we propose that Tk-Fbp may represent the bona fide FBPase in Archaea.Glycolysis and gluconeogenesis are pathways involved in the degradation and synthesis of intracellular sugars, respectively.