RESUMO
The analytical performance for paper spray (PS) using a new insert sample approach based on paper with paraffin barriers (PS-PB) is presented. The paraffin barrier is made using a simple, fast and cheap method based on the stamping of paraffin onto a paper surface. Typical operation conditions of paper spray such as the solvent volume applied on the paper surface, and the paper substrate type are evaluated. A paper substrate with paraffin barriers shows better performance on analysis of a range of typical analytes when compared to the conventional PS-MS using normal paper (PS-NP) and PS-MS using paper with two rounded corners (PS-RC). PS-PB was applied to detect sugars and their inhibitors in sugarcane bagasse liquors from a second generation ethanol process. Moreover, the PS-PB proved to be excellent, showing results for the quantification of glucose in hydrolysis liquors with excellent linearity (R(2) = 0.99), limits of detection (2.77 mmol L(-1)) and quantification (9.27 mmol L(-1)). The results are better than for PS-NP and PS-RC. The PS-PB was also excellent in performance when compared with the HPLC-UV method for glucose quantification on hydrolysis of liquor samples.
Assuntos
Espectrometria de Massas/métodos , Papel , Parafina , Glucose/análise , Solventes/química , Propriedades de SuperfícieRESUMO
Preference for specific protein substrates together with differential sensitivity to activators and inhibitors has allowed classification of serine/threonine protein phosphatases (PPs) into four major types designated types 1, 2A, 2B and 2C (PP1, PP2A, PP2B and PP2C, respectively). Comparison of sequences within their catalytic domains has indicated that PP1, PP2A and PP2B are members of the same gene family named PPP. On the other hand, the type 2C enzyme does not share sequence homology with the PPP members and thus represents another gene family, known as PPM. In this report we briefly summarize some of our studies about the role of serine/threonine phosphatases in growth and differentiation of three different eukaryotic models: Blastocladiella emersonii, Neurospora crassa and Dictyostelium discoideum. Our observations suggest that PP2C is the major phosphatase responsible for dephosphorylation of amidotransferase, an enzyme that controls cell wall synthesis during Blastocladiella emersonii zoospore germination. We also report the existence of a novel acid- and thermo-stable protein purified from Neurospora crassa mycelia, which specifically inhibits the PP1 activity of this fungus and mammals. Finally, we comment on our recent results demonstrating that Dictyostelium discoideum expresses a gene that codes for PP1, although this activity has never been demonstrated biochemically in this organism.
Assuntos
Blastocladiella/enzimologia , Dictyostelium/enzimologia , Neurospora crassa/enzimologia , Fosfotreonina/metabolismo , Animais , Especificidade por SubstratoRESUMO
Preference for specific protein substrates together with differential sensitivity to activators and inhibitors has allowed classification of serine/threonine protein phosphatases (PPs) into four major types designated types 1, 2A, 2B and 2C (PP1, PP2A, PP2B and PP2C, respectively). Comparison of sequences within their catalytic domains has indicated that PP1, PP2A and PP2B are members of the same gene family named PPP. On the other hand, the type 2C enzyme does not share sequence homology with the PPP members and thus represents another gene family, known as PPM. In this report we briefly summarize some of our studies about the role of serine/threonine phosphatases in growth and differentiation of three different eukaryotic models: Blastocladiella emersonii, Neurospora crassa and Dictyostelium discoideum. Our observations suggest that PP2C is the major phosphatase responsible for dephosphorylation of amidotransferase, an enzyme that controls cell wall synthesis during Blastocladiella emersonii zoospore germination. We also report the existence of a novel acid- and thermo-stable protein purified from Neurospora crassa mycelia, which specifically inhibits the PP1 activity of this fungus and mammals. Finally, we comment on our recent results demonstrating that Dictyostelium discoideum expresses a gene that codes for PP1, although this activity has never been demonstrated biochemically in this organism
Assuntos
Blastocladiella/enzimologia , Dictyostelium/enzimologia , Células Eucarióticas/enzimologia , Neurospora crassa/enzimologia , Fosfotreonina/metabolismo , Germinação/fisiologia , Especificidade por SubstratoRESUMO
Extracts of the aquatic fungus Blastocladiella emersonii were found to contain protein phosphatases type 1, type 2A, and type 2C with properties analogous to those found in mammalian tissues. The activities of all three protein phosphatases are developmentally regulated, increasing during sporulation, with maximum level in zoospores. Protein phosphatases 2A and 2C, present in zoospore extracts, catalyze the dephosphorylation of L-glutamine:fructose-6-phosphate amidotransferase (EC 2.6.1.16, amidotransferase), a key regulatory enzyme in hexosamine biosynthesis. The protein phosphatase inhibitor okadaic acid induces encystment and inhibits germ tube formation but does not affect the synthesis of the chitinous cell wall. These results strongly suggest that phosphatase 2C is responsible for the dephosphorylation of amidotransferase in vivo. This dephosphorylation is inhibited by uridine-5'-diphospho-N-acetylglucosamine, the end product of hexosamine synthesis and the substrate for chitin synthesis. This result demonstrates a dual role of uridine-5'-diphospho-N-acetylglucosamine by inhibiting the activity of the phosphorylated form of amidotransferase and by preventing its dephosphorylation by protein phosphatases.