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Soy protein isolate (SPI) and wheat gluten (WG) are widely used in commercial food applications in Asia for their nutritional value and functional properties. However, individually each exhibits poor gelation. In this study, we examined the microbial transglutaminase (MTGase)-induced gelation properties of SPI and WG mixtures with high intensity ultrasonic pretreatment. Ultrasonic treatment reduced the particle size of SPI/WG molecules, which led to improvements in surface hydrophobicity (Ho) and free sulfhydryl (SH) group content. However, MTGase crosslinking facilitated the formation of disulfide bonds, markedly decreasing the content of free SH groups. Ultrasonic treatment improved the gel strength, water holding capacity, and storage modulus and resulted in denser and more homogeneous networks of MTGase-induced SPI/WG gels. In addition, ultrasonic treatment changed the secondary structure of the gel samples as determined by Fourier transform infrared spectroscopic analysis, with a reduction in α-helices and β-turns and an increase in β-sheets and random coils. Thus, ultrasound is useful in facilitating the gelation properties of MTGase-induced SPI/WG gels and might expand their utilization in the food protein gelation industry.

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Phosphodiesterase-5 (PDE5) is the target for sildenafil, vardenafil, and tadalafil, which are drugs for treatment of erectile dysfunction and pulmonary hypertension. We report here the crystal structures of a fully active catalytic domain of unliganded PDE5A1 and its complexes with sildenafil or icarisid II. These structures together with the PDE5A1-isobutyl-1-methylxanthine complex show that the H-loop (residues 660 -683) at the active site of PDE5A1 has four different conformations and migrates 7-35 Å upon inhibitor binding. In addition, the conformation of sildenafil reported herein differs significantly from those in the previous structures of chimerically hybridized or almost inactive PDE5. Mutagenesis and kinetic analyses confirm that the H-loop is particularly important for substrate recognition and that invariant Gly 659 , which immediately precedes the H-loop, is critical for optimal substrate affinity and catalytic activity.Cyclic nucleotide phosphodiesterases (PDEs) 3 are key enzymes controlling cellular concentrations of second messengers cAMP and cGMP by hydrolyzing them to 5Ј-AMP and 5Ј-GMP, respectively. The human genome encodes 21 PDE genes that are categorized into 11 families (1-9). Alternative mRNA splicing of the PDE genes produces over 60 PDE isoforms that distribute in various cellular compartments and control physiological processes. PDE molecules contain a conserved catalytic domain and variable regulatory regions. However, each PDE family possesses a characteristic pattern of substrate specificity and inhibitor selectivity (6).Inhibitors of PDEs have been widely studied as therapeutics as follows: cardiotonics, vasodilators, smooth muscle relaxants, antidepressants, antithrombotics, antiasthmatics, and agents for improving cognitive functions such as learning and memory (10 -17). Some of the most successful examples of these drugs are the PDE5 inhibitors sildenafil (Viagra), vardenafil (Levitra), and tadalafil (Cialis) that have been used for treatment of male erectile dysfunction (15). Sildenafil has also recently been approved (Revatio) for treatment of pulmonary hypertension (18). However, reported side effects of these medications such as headache and visual disturbance suggest a need for further study of the molecular basis of the selectivity of PDE5 inhibitors (19). Two co-crystal structures of the catalytic domain of PDE5 with sildenafil showed differences in the conformation of the inhibitor bound to the catalytic site (20 -22). However, it remains unknown whether these conformations are biologically relevant because the PDE5 enzyme used in the studies is either almost inactive (20) or a chimeric hybrid of the PDE5 catalytic domain with replacement of a PDE4 segment (the H-loop) (21,22). In addition, the crystal structure of the catalytic domain of PDE5A1 in complex with the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) showed that the conformation of the H-loop at the active site is different from that in PDE4 (23) and in other published PDE5 structures (20 -22...

PDE4 (phosphodiesterase-4)-selective inhibitors have attracted much attention as potential therapeutics for the treatment of both depression and major inflammatory diseases, but their practical application has been compromised by side effects. A possible cause for the side effects is that current PDE4-selective inhibitors similarly inhibit isoforms from all four PDE4 subfamilies. The development of PDE4 subfamily-selective inhibitors has been hampered by a lack of structural information. In the present study, we rectify this by providing the crystal structures of the catalytic domains of PDE4A, PDE4B and PDE4D in complex with the PDE4 inhibitor NVP {4-[8-(3-nitrophenyl)-[1,7]naphthyridin-6-yl]benzoic acid} as well as the unliganded PDE4C structure. NVP binds in the same conformation to the deep cAMP substrate pocket and interacts with the same residues in each instance. However, detailed structural comparison reveals significant conformational differences. Although the active sites of PDE4B and PDE4D are mostly comparable, PDE4A shows significant displacements of the residues next to the invariant glutamine residue that is critical for substrate and inhibitor binding. PDE4C appears to be more distal from other PDE4 subfamilies, with certain key residues being disordered. Our analyses provide the first structural basis for the development of PDE4 subfamily-selective inhibitors.