Potato and rabbit muscle phosphorylases: comparative studies on the structure,function and regulation of regulatory and nonregulatory enzymes

Summary Phosphorylases (EC 2.4.1.1) from potato and rabbit muscle are similar in many of their structural and kinetic properties, despite differences in regulation of their enzyme activity. Rabbit muscle phosphorylase is subject to both allosteric and covalent controls, while potato phosphorylase is an active species without any regulatory mechanism. Both phosphorylases are composed of subunits of approximately 100 000 molecular weight, and contain a firmly bound pyridoxal 5-phosphate. Their actions follow a rapid equilibrium random Bi Bi mechanism. From the sequence comparison between the two phosphorylases, high homologies of widely distributed regions have been found, suggesting that they may have evolved from the same ancestral protein. By contrast, the sequences of the N-terminal region are remarkably different from each other. Since this region of the muscle enzyme forms the phosphorylatable and AMP-binding sites as well as the subunit-subunit contact region, these results provide the structural basis for the difference in the regulatory properties between potato and rabbit muscle phosphorylases. Judged from CD spectra, the surface structures of the potato enzyme might be significantly different from that of the muscle enzyme. Indeed, the subunit-subunit interaction in the potato enzyme is tighter than that in the muscle enzyme, and the susceptibility of the two enzymes toward modification reagents and proteolytic enzymes are different. Despite these differences, the structural and functional features of the cofactor, pyridoxal phosphate, site are surprisingly well conserved in these phosphorylases. X-ray crystallographic studies on rabbit muscle phosphorylase have shown that glucose-1-phosphate and orthophosphate bind to a common region close to the 5-phosphate of the cofactor. The muscle enzyme has a glycogen storage site for binding of the enzyme to saccharide substrate, which is located away from the cofactor site. We have obtained, in our reconstitution studies, evidence for binding of saccharide directly to the cofactor site of potato phosphorylase. This difference in the topography of the functional sites explains the previously known different specificities for saccharide substrates in the two phosphorylases. Based on a combination of these and other studies, it is now clear that the 5-phosphate group of pyridoxal phosphate plays a direct role in the catalysis of this enzyme. Information now available on the reaction mechanism of phosphorylase is briefly described.     

Altered regulation of cell cycle machinery involved in interleukin-1-induced G(1) and G(2) phase growth arrest of A375S2 human melanoma cells

Interleukin-1 (IL-1) inhibits the growth of A375S2 human melanoma cells by arresting them at G(1) and G(2) phases of the cell cycle. The arrests are preceded by a rapid decrease in kinase activities of cyclin E-Cdk2 and cyclin B1-Cdc2, which are critical for G(1)-S and G(2)-M progression, respectively. IL-1 quickly enhances the protein expression of the CDK inhibitor p21(cip1). The induced p21 binds preferentially to cyclin E-Cdk2, and the increase in p21 binding parallels the decrease in cyclin E-Cdk2 activity. Thus, p21 is likely to be responsible for the inhibition of cyclin E-Cdk2 activity and G(1) arrest. Coinciding with the decrease in cyclin B1-Cdc2 activity, there is an increase in tyrosine phosphorylation of Cdc2, suggesting that an increase in the inactive Tyr-15-phosphorylated form of Cdc2 is involved in the decrease in cyclin B1-Cdc2 activity and G(2) arrest. Furthermore, we found that IL-1 causes rapid dephosphorylation of p107, but not of pRb or p130, while the total protein levels of p130 are increased. Thus, IL-1 may exert its growth-arresting effects via p107 and p130 pathways rather than through pRb.     

In-Situ Observation of Membrane Protein Folding during Cell-Free Expression

Proper insertion, folding and assembly of functional proteins in biological membranes are key processes to warrant activity of a living cell. Here, we present a novel approach to trace folding and insertion of a nascent membrane protein leaving the ribosome and penetrating the bilayer. Surface Enhanced IR Absorption Spectroscopy selectively monitored insertion and folding of membrane proteins during cell-free expression in a label-free and non-invasive manner. Protein synthesis was performed in an optical cell containing a prism covered with a thin gold film with nanodiscs on top, providing an artificial lipid bilayer for folding. In a pilot experiment, the folding pathway of bacteriorhodopsin via various secondary and tertiary structures was visualized. Thus, a methodology is established with which the folding reaction of other more complex membrane proteins can be observed during protein biosynthesis (in situ and in operando) at molecular resolution.     

A consensus linkage map for sugi (Cryptomeria japonica) from two pedigrees, based on microsatellites and expressed sequence tags

A consensus map for sugi (Cryptomeria japonica) was constructed by integrating linkage data from two unrelated third-generation pedigrees, one derived from a full-sib cross and the other by self-pollination of F1 individuals. The progeny segregation data of the first pedigree were derived from cleaved amplified polymorphic sequences, microsatellites, restriction fragment length polymorphisms, and single nucleotide polymorphisms. The data of the second pedigree were derived from cleaved amplified polymorphic sequences, isozyme markers, morphological traits, random amplified polymorphic DNA markers, and restriction fragment length polymorphisms. Linkage analyses were done for the first pedigree with JoinMap 3.0, using its parameter set for progeny derived by cross-pollination, and for the second pedigree with the parameter set for progeny derived from selfing of F1 individuals. The 11 chromosomes of C. japonica are represented in the consensus map. A total of 438 markers were assigned to 11 large linkage groups, 1 small linkage group, and 1 nonintegrated linkage group from the second pedigree; their total length was 1372.2 cM. On average, the consensus map showed 1 marker every 3.0 cM. PCR-based codominant DNA markers such as cleaved amplified polymorphic sequences and microsatellite markers were distributed in all linkage groups and occupied about half of mapped loci. These markers are very useful for integration of different linkage maps, QTL mapping, and comparative mapping for evolutional study, especially for species with a large genome size such as conifers.     

Articulospora sp. produces Art1, an inhibitor of bacterial histidine kinase

A two-component system (TCS) comprising a histidine kinase (HK) sensor and a response regulator (RR) plays important roles in regulating the virulence of many pathogenic bacteria. We used a new screening method to isolate novel inhibitor Art1 against bacterial sensory HK from an acetone extract of solid cultures of Articulospora sp., an aquatic hypomycete. Art1 inhibited the ATP-dependent autophosphorylation of recombinant glutathione S-transferase-fusion protein SasA, a cyanobacterial HK, with an IC50 value of 9.5 microg/ml.     

Ascorbic acid induces a marked conformational change in long duplex DNA.

Ascorbic acid is often regarded as an antioxidant in vivo, where it protects against cancer by scavenging DNA-damaging reactive oxygen species. However, the detailed mechanism of the action of ascorbic acid on genetic DNA is still unclear. We examined the effect of ascorbic acid on the higher-order structure of DNA through real-time observation by fluorescence microscopy. We found that ascorbic acid generates a pearling structure in single giant DNA molecules, with elongated and compact regions coexisting along a molecular chain. Results from electron microscopy and atomic force microscopy indicate that the compact regions assume a loosely packed conformation. A possible mechanism for the induction of this conformational change is discussed in relation to the interplay between the higher-order and second-order structures of DNA.     

Aquaporin subfamily with unusual NPA boxes

Aquaporins have been identified based on highly conserved two asparagine-proline-alanine (NPA) boxes that are important for the formation of a water-permeating pore. Some aquaporin-like sequences, however, have less conserved NPA boxes. Although they have lower homology with conventional aquaporins, they should be included in aquaporin family based on their conserved six transmembrane domains and hydrophobic NPA box-like repeats. They are widely distributed in multicellular organisms. Only SIPs from plants and AQP11/12 from mammals were examined previously and found to be localized inside the cell. Intracellular localization will be a common feature of these aquaporin-like proteins since most of them have positively charged amino acid clusters at the carboxy-termini similar to di-lysine motif (-KKXX) for an endoplasmic reticulum retention signal. Accordingly, they are tentatively named subcellular-aquaporins in this review. Currently, studies on their functions and biological roles are limited. SIPs were shown to function as water channels and the disruption of AQP11 produced neonatally fatal polycystic kidneys. Further works on subcellular-aquaporins will reveal new insights into the roles of aquaporins.     

Evaluation of lipid‐binding properties of the N‐terminal helical segments in human apolipoprotein A‐I using fragment peptides

Although the N‐terminal region in human apolipoprotein (apo) A‐I is thought to stabilize the lipid‐free structure of the protein, its role in lipid binding is unknown. Using synthetic fragment peptides, we examined the lipid‐binding properties of the first 43 residues (1–43) of apoA‐I in comparison with residues 44–65 and 220–241, which have strong lipid affinity in the molecule. Circular dichroism measurements demonstrated that peptides corresponding to each segment have potential propensity to form α‐helical structure in trifluoroethanol. Spectroscopic and thermodynamic measurements revealed that apoA‐I (1–43) peptide has the strong ability to bind to lipid vesicles and to form α‐helical structure comparable to apoA‐I (220–241) peptide. Substitution of Tyr‐18 located at the center of the most hydrophobic region in residues 1–43 with a helix‐breaking proline resulted in the impaired lipid binding, indicating that the α‐helical structure in this region is required to trigger the lipid binding. In contrast, apoA‐I (44–65) peptide exhibited a lower propensity to form α‐helical structure upon binding to lipid, and apoA‐I (44–65/S55P) peptide exhibited diminished, but not completely impaired, lipid binding, suggesting that the central region of residues 44–65 is not pivotally involved in the formation of the α‐helical structure and lipid binding. These results indicate that the most N‐terminal region of apoA‐I molecule, residues 1–43, contributes to the lipid interaction of apoA‐I through the hydrophobic helical residues. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.