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Z Jia

Publications and source records attributed to Z Jia.

At least 19 recordsLinked to original sources
PubMed1998

A fluorescence study of ligand-induced conformational changes in cytosolic fructose-1,6-bisphosphatase from germinating castor oil seeds.

The intrinsic fluorescence of homogeneous castor oil seed cytosolic fructose-1,6-bisphosphatase (FBPasec) was used as an indicator of conformational changes due to ligand binding. Binding of the substrate and the inhibitor fructose-2,6-bisphosphate (F-2,6-P2) was quantitatively compared to their respective kinetic effects on enzymatic activity. There are two distinct types of substrate interaction with FBPasec, corresponding to catalytic and inhibitory binding, respectively. Inhibitory substrate binding shares several characteristics with F-2,6-P2 binding which indicates that both ligands bind at the same site. However, F-2,6-P2 does not prevent fluorescence transitions attributed to catalytic substrate binding. The marked synergistic inhibition of FBPasec by AMP and F-2,6-P2 appears to arise via AMP's promotion of F-2,6-P2 binding. Based on the X-ray crystal structure of porcine kidney FBPase our modelling studies suggest the existence of a distinct F-1,6-P2/F-2,6-P2 inhibitory binding site which partially overlaps with the enzyme's catalytic site. We propose that a pronounced allosteric transition mediated by AMP binding increases access of F-1,6-P2 and F-2,6-P2 to this common inhibitory binding site.

Adenosine Monophosphate↗
PubMed1998

Aldehyde reductase: the role of C-terminal residues in defining substrate and cofactor specificities.

The only major structural difference between aldehyde reductase, a primarily NADPH-dependent aldo-keto reductase, and aldose reductase, a dually coenzyme-specific (NADPH/NADH) member of the same superfamily, is an additional eight amino acid residues in the substrate/inhibitor binding site (C-terminal region) of aldehyde reductase. On the premise that this segment defines the substrate specificity of the enzyme, a mutant of aldehyde reductase lacking residues 306-313 was constructed. In contrast to wild-type enzyme, the mutant enzyme reduced a narrower range of aldehydes and the new substrate specificity was not similar to aldose reductase as might have been predicted. A major change in coenzyme specificity was observed, however, the mutant enzyme being distinctly NADH preferring(Km, NADH = 35 microM, compared to <5 mM for wild-type and Km, NADPH = 670 microM, compared to 35 microM for wild type). Upon analyzing coordinates of aldehyde and aldose reductase, we found that deletion of residues 306-313 may have created a truncated enzyme that retained the three-dimensional structural features of the enzyme's C-terminal segment. The change in substrate specificity could be explained by the new alignment of amino acids. The reversal of coenzyme specificity appeared to be due to a significant backbone shift initiated by the formation of a strong hydrogen bond between Tyr319 and Val300. A similar bond exists in aldose reductase (Tyr309-Ala299). It appears, therefore, that as far as coenzyme specificity is concerned, deletion of residues 306-313 has converted aldehyde reductase into an aldose reductase-like enzyme.

Aldehyde Reductase↗
PubMed1998

Purification, crystallization and preliminary X-ray analysis of the Escherichia coli phytase.

A recombinant form of Escherichia coli phytase, which hydrolyzes phytic acid into phosphate and myo-inositol, has been expressed, purified and crystallized. Crystals have been obtained by the method of bulk crystallization in 10 mM sodium acetate buffer (pH 4.5) without using a conventional precipitant. The enzyme crystallized in space group P21, with unit-cell dimensions a = 74.9, b = 72.2, c = 82.4 A, and beta = 92.0 degrees. Crystals diffract to at least 2.2 A at a rotating-anode X-ray source and a 2.3 A resolution data set has been collected, giving completeness of 98.0% and an Rsym of 0.072. Assuming there are two phytase molecules in the asymmetric unit, the solvent content is calculated to be 42.1%. A self-rotation function shows a clear twofold non-crystallographic symmetry relating two molecules of E. coli phytase in the asymmetric unit.

6-Phytase↗
PubMed1998

Structure of type III antifreeze protein at 277 K.

Fish antifreeze proteins (AFP's) depress the freezing point of blood and other body fluids below that of the surrounding seawater by binding to and inhibiting the growth of seed ice crystals. The high-resolution crystal structure of type III AFP, determined at room temperature, reveals a remarkably flat surface containing most of the ice-binding residues [Jia et al. (1996). Nature (London), 384, 285-288]. Since AFP's function at temperatures close to 273 K, it is important to know whether the structure determined at room temperature undergoes any change at much lower temperature. Therefore, type III AFP has been crystallized at 277 K and its structure determined. Although crystallization conditions at 277 K were similar to those at approximately 295 K, crystal growth took much longer at the lower temperature. Crystals grown at the two temperatures were isomorphous. Initial crystals appeared within 40-50 d and grew to their final size in about 8-12 months, instead of a couple of days at approximately 295 K. The type III antifreeze protein structure from crystals grown at 277 K was essentially the same as that determined at approximately 295 K, with the exception of some minor changes in side-chain conformation. The result is an indication that temperature has a minimal effect on the structure of type III AFP, thus lending increased physiological validity to the room-temperature structure which was used for the initial ice-binding modelling.

Animals↗
PubMed1998

Inhibition of protein tyrosine phosphatases PTP1B and CD45 by sulfotyrosyl peptides.

Sulfotyrosyl peptides corresponding to the known high-affinity substrate phosphotyrosyl peptide sequences in casein and the autophosphorylation sites of insulin receptor and EGF receptor were investigated as inhibitors of protein tyrosine phosphatases PTP1B and CD45. These peptides inhibit both PTP1B and CD45 in the micromolar range competitively and reversibly. The elements required for inhibition were investigated by truncation and substitution of these peptides. Acidic residues N-terminal to the sulfotyrosyl residues are essential for high-affinity binding to PTP1B. The recognition elements required for inhibition of PTP1B and CD45 are different and this suggests the possibility of identifying selective active-site-directed inhibitors for these enzymes.

Binding Sites↗
PubMed1998

Affinity selection from peptide libraries to determine substrate specificity of protein tyrosine phosphatases.

Affinity selection from peptide libraries is a powerful tool that has been used for determining the sequence specificities of a number of enzymes and protein binding domains, including protein kinases, src homology 2 domains, and PDZ domains. We have extended this approach to protein tyrosine phosphatases using peptide libraries containing a nonhydrolyzable phosphotyrosine analog, difluorophosphonomethylphenylalanine. A size-exclusion method is used to separate enzyme-peptide complexes from free peptide, providing several advantages over the traditional immobilized protein affinity column approach. In addition, the feasibility of using mass spectrometric detection to quantitate peptides rapidly and reproducibly is demonstrated as an alternative to quantitation by peptide sequencing. The validity of this analysis is demonstrated by synthesizing individual peptides and comparing their affinity for enzyme with the predictions from the affinity selection process. As a model for these studies the protein tyrosine phosphatase PTP1B is used, providing additional insights into the sequence specificity of this enzyme. In particular, a selection for aromatic amino acids at the pY - 1 position (immediately N-terminal to the phosphotyrosine), as well as a broad pY + 1 selectivity, is observed in addition to the general preference for acidic residues N-terminal to the phosphotyrosine. The approach described here should prove applicable to protein tyrosine phosphatases in general as well as for the study of nonpeptidyl combinatorial libraries.

Amino Acid Sequence↗
PubMed1998

The effects of steric mutations on the structure of type III antifreeze protein and its interaction with ice.

The interaction of proteins with ice is poorly understood and difficult to study, partly because ice is transitory and can present many binding surfaces, and partly because structures have been determined for only two ice-binding proteins. This paper focuses on one of these, a 66-residue antifreeze protein (AFP) from eel pout. The high resolution X-ray structure of this fish AFP demonstrated that the proposed ice-binding surface is remarkably flat for such a small protein. The residues on the planar surface thought to be involved in ice binding are restrained by hydrogen bonds or by tight packing of their side-chains. To probe the requirement for a flat binding surface, a conserved alanine in the center of the AFP planar surface was substituted with larger residues. Six alanine replacement mutants (Ala16 > Cys, Thr, Met, Arg, His and Tyr), designed to disrupt the planarity of the surface and sterically block binding to ice, were characterized by X-ray crystallography and compared with the wild-type AFP. In each case, the detail provided by these crystal structures has helped explain the effects of the mutation on antifreeze activity. The substitutions, Ala16 > His and Ala16 > Tyr, were large enough to shield Gln44, one of the putative ice-binding residues, contributing to their very low thermal hysteresis activity. In addition to sterically hindering the putative ice-binding site, the bulkier residues also caused shifts in the putative ice-binding residues owing to the tight packing of side-chains on the planar surface. This unexpected consequence of the mutations helps account for the severely reduced antifreeze activity. One explanation for residual antifreeze activity in some of the mutants lies in the possibility that AFPs have a role in shaping the site on the ice to which they bind. Thus, side-chain dislocations might be partially accommodated by ice that can freeze around them. It is evident that the disruption of the planarity, by introducing larger residues at the center of the proposed ice-binding site, is not the only factor responsible for the loss of antifreeze activity. There are multiple causes including positional change and steric blockage of some putative ice-binding residues.

Alanine↗
PubMed1998

Triterpenoid saponins from Vaccaria segetalis.

Four novel triterpenoid saponins were isolated from the seeds of Vaccaria segetalis. Their structures were established as vaccaroside A, gypsogenic acid-28-O-beta-D- glucopyranosyl-(1-->2)-beta-D-glucopyranosyl-(1-->6)-[beta-D- glucopyranosyl-(1-->3)]-beta-D-glucopyranoside; vaccaroside B, gypsogenic acid-28-O-beta-D-glucopyranosyl-(1-->2)-[3-hydroxyl-3- methylglutaroyl-(1-->6)]-beta-D-glucopyranosyl-(1-->6)-[beta-D- glucopyranosyl-(1-->3)]-beta-D-glucopyranoside; vaccaroside C, 23-O-beta-D-glucopyranosyl-gypsogenic acid-28-O-beta-D- glucopyranosyl-(1-->2)-beta-D-glucopyranosyl-(1-->6)- [beta-D-glucopyranosyl-(1-->3)]-beta-D-glucopyranoside and vaccaroside D, 3,4-secogypsogenic acid-28-O-beta-D-glucopyranosyl-(1-->3)- [beta-D-glucopyranosyl-(1-->6)]-beta-D-glucopyranoside by a combination of extensive NMR (DEPT, COSY, HOHAHA, HETCOR, HMBC and NOESY) studies and chemical degradation.

Carbohydrate Conformation↗
PubMed1998

Triterpenoid saponins and sapogenins from Vaccaria segetalis.

Four new triterpenoid saponins, vaccarosides E, F, G and H were isolated from the seeds of Vaccaria segatalis and were respectively defined to be 3-O-beta-D-galactopyranosyl(1-->2)-beta-D-glucuronopyranosyl] quillaic acid 28-O-beta-D-xylopyranosyl-(1-->4)-chi-L-rhamnopyranosyl-(1-->2)- [chi-L-arabinofuranosyl-(1-->3)]-beta-D-4-O-acetylfucopyranoside; 3-O-[beta-D-galactopyranosyl-(1-->2)-beta-D-glucuronopyranosyl] 3beta,4chi,16chi-trihydroxy-23-norolean-12-en-28-oic acid 28-O-beta-D-xylopyranosyl-(1-->4)-chi-L-rhamnopyranosyl-(1-->2)- [chi-L-arabinofuranosyl-(1-->3)]-beta-D-4-O-acetylfucopyranoside; 3-O-[beta-D-galactopyranosyl-(1-->2)-beta-D-glucuronopyranosyl] gypsogenin 28-O-beta-D-xylopyranosyl-(1-->4)-chi-L-rhamnopyranosyl-(1-->2)- [chi-L-arabinofuranosyl (1-->3)]-beta-D-4-O-acetylfucopyranoside; and 3-O-[beta-D-galactopyranosyl-(1-->2)-beta-D-glucuronopyranosyl] 3beta,4chi-dihydroxy-23norolean-12-en-28-oic acid 28-O-beta-D-xylopyranosyl-(1-->4)-chi-L-rhamnopyranosyl-(1-->2) -chi-L-arabinofuranosyl-(1-->3)]-beta-D-4-O-acetyfucopyranoside. Their structures were established on the basis of extensive NMR (DEPT, COSY, HOHAHA, HETCOR, HMBC AND NOESY), FAB-MS and ESI-MS studies as well as chemical strategies and enzymatic degradation. The new aglycones of two of the saponins have been designated as segetalic acid and vaccaric acid, respectively.

Carbohydrate Conformation↗
PubMed1998

Multiple behavioral anomalies in GluR2 mutant mice exhibiting enhanced LTP.

We have previously disrupted the ionotropic glutamate receptor type 2 gene (GluR2) using gene targeting in embryonic stem cells and generated mice which lacked the GluR2 gene product. Neurophysiological analyses of these mice showed a markedly enhanced long-term potentiation (LTP) and a 9-fold increase in kainate induced Ca2+ permeability in the hippocampus. Here, we analyze the behavioral and neuroanatomical consequences of GluR2 deficiency in homozygous null mutant and age-matched littermate control mice. We show that despite unaltered gross brain morphology, several aspects of behavior were abnormal in the mutants. Object exploration, rearing, grooming and locomotion were altered in the novel arena. Eye-closure reflex, motor performance on the rotating rod and spatial and non-spatial learning performance in the water maze were also abnormal in the mutants. These abnormalities together with the widespread expression pattern of GluR2 in most excitatory CNS pathways suggest that the absence of GluR2 leads to neurological phenotypes associated with not only the hippocampus but several other brain regions potentially including the cortex and cerebellum. We speculate that GluR2 mutant mice suffer from an overall non-specifically increased excitability that may alter cognitive functions ranging from stimulus processing to motivation and learning.

Animals↗
PubMed1998

Major triterpenoid saponins from saponaria officinalis

Two major triterpenoid saponins, named saponariosides A and B, were isolated from the whole plants of Saponaria officinalis and were respectively defined to be 3-O-beta-D-galactopyranosyl-(1-->2)-[beta-D-xylopyranosyl-(1-->3)]-be ta-D-glucuronopyranosyl quillaic acid 28-O-beta-D-xylopyranosyl-(1-->3)-beta-D-xylopyranosyl-(1-->4)-alpha- L-rhamnopyranosyl-(1-->2)-[beta-D-xylopyranosyl-(1-->3)-beta-D-4-O-ac etylquinovopyranosyl-(1-->4)]-beta-D-fucopyranoside (1); 3-O-beta-D-galactopyranosyl-(1-->2)-[beta-D-xylopyranosyl-(1-->3)]-be ta-D-glucuronopyranosyl quillaic acid 28-O-beta-D-xylopyranosyl-(1-->3)-beta-D-xylopyranosyl-(1-->4)-alpha- L-rhamnopyranosyl-(1-->2)-[beta-D-4-O-acetylquinovopyranosyl-(1-->4)] -beta-D-fucopyranoside (2). Their structures were established on the basis of extensive NMR (DEPT, DQF-COSY, HOHAHA, HETCOR, HMBC, and NOESY) and MS studies as well as chemical degradation.

Journal Article↗
PubMed1998

Expression of apolipoprotein(a) kringle IV type 9 in Escherichia coli: demonstration of a specific interaction between kringle IV type 9 and apolipoproteinB-100.

A number of studies have provided evidence that lipoprotein(a) [Lp(a)] assembly is a two-step process in which initial non-covalent interactions between apolipoprotein(a) [apo(a)] and apolipoproteinB-100 (apoB-100) precede specific disulfide bond formation. We have designed a construct encoding apo(a) kringle IV type 9 (KIV9) in which the unpaired cysteine at position 67 in this kringle is replaced with a tyrosine. The single kringle was expressed in bacteria and purified to homogeneity from cell homogenates. The purified derivative (designated KIV9deltaCys) was assessed for its ability to bind to purified human LDL. This interaction was detected either by ELISA using immobilized LDL or by column chromatography in which LDL binding to KIV9deltaCys immobilized on Ni2+-Sepharose was determined. In both cases, the interaction of KIV9deltaCys and LDL was observed. Further, we demonstrated that the binding interaction was sensitive to the addition of amino acids including lysine, the lysine analogue epsilon-aminocaproic acid, arginine, phenylalanine and proline, with arginine and lysine having the greatest inhibitory effect. Binding of KIV9deltaCys to an immobilized apoB peptide spanning residues 3732-3745 of apoB was also demonstrated by ELISA. As was the case for LDL, this binding interaction was sensitive to the addition of arginine and lysine. Computer modeling of KIV9 demonstrated an excellent fit with residues 3732-3738 (PSCKLDF) of the apoB peptide. The modeling predicts the presence of overlapping lysine and phenylalanine-binding pockets in KIV9 which explains the inhibitory effects of lysine, arginine and phenylalanine which were observed in the binding assays. In summary, this study represents the first demonstration that KIV9 can interact directly with LDL through non-covalent interactions which may contribute to the first step of Lp(a) formation.

Amino Acid Sequence↗
PubMed1997

Mice lacking metabotropic glutamate receptor 5 show impaired learning and reduced CA1 long-term potentiation (LTP) but normal CA3 LTP.

Class I metabotropic glutamate receptors (mGluRs) have been postulated to play a role in synaptic plasticity. To test the involvement of one member of this class, we have recently generated mutant mice that express no mGluR5 but normal levels of other glutamate receptors. The CNS revealed normal development of gross anatomical features. To examine synaptic functions we measured evoked field EPSPs in the hippocampal slice. Measures of presynaptic function, such as paired pulse facilitation in mutant CA1 neurons, were normal. The response of mutant CA1 neurons to low concentrations of (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylic acid (ACPD) was missing, which suggests that mGluR5 may be the primary high affinity ACPD receptor in these neurons. Long-term potentiation (LTP) in mGluR5 mutants was significantly reduced in the NMDA receptor (NMDAR)-dependent pathways such as the CA1 region and dentate gyrus of the hippocampus, whereas LTP remained intact in the mossy fiber synapses on the CA3 region, an NMDAR-independent pathway. Some of the difference in CA1 LTP could lie at the level of expression, because the reduction of LTP in the mutants was no longer observed 20 min after tetanus in the presence of 2-amino-5-phosphonopentanoate. We propose that mGluR5 plays a key regulatory role in NMDAR-dependent LTP. These mutant mice were also impaired in the acquisition and use of spatial information in both the Morris water maze and contextual information in the fear-conditioning test. This is consistent with the hypothesis that LTP in the CA1 region may underlie spatial learning and memory.

Animals↗
PubMed1997

Tolerance of low pH in Schizosaccharomyces pombe requires a functioning pub1 ubiquitin ligase.

A strain of Schizosaccharomyces pombe carrying a disrupted Na+/H+ antiporter gene (sod2::sup3-5), in addition to the common auxotrophic mutations, ade6-216, ura4-D18 and leu1-32, is highly sensitive to media adjusted to pH 6.9. Reversion analysis of this strain yielded a group of revertants capable of growth at pH 6.9. Two of the revertants elongated and failed to form colonies at pH 3.5. Genetic characterization of one of the pH-sensitive elongated strains, J227, showed the presence of two independently segregating mutations. One, pub1 (protein ubiquitin ligase 1), has recently been reported as an E3 protein ubiquitin ligase involved in cdc25 turnover. The second has been named elp3-1 (elongated at low pH). Genetic dissection of the original strain revealed that poor growth at high pH was due to the presence of the auxotrophic markers, suggesting a possible inhibitory effect of high pH on the function of permeases responsible for uptake of the necessary nutrients. Suppression of the high pH sensitivity required the presence of both the pub1-1 and elp3-1 mutations. While the pub1-1 mutation reduced the capacity of cells to tolerate relatively moderate concentrations of LiCl (3 mM) in liquid culture, it was capable of partially suppressing the extreme Li+ sensitivity caused by the sod2 disruption. Under these conditions, the growth of pub1-1 sod2::ura4 double mutant cells was improved over that of either pub1-1 or sod2::ura4 cells. The elp3-1 mutation had no effect on the Li+ tolerance in either wild-type or sod2::ura4 backgrounds. pub1-1 cells are elongated and incapable of colony formation at pH 3.5. In contrast, elp3-1 cells are elongated at pH 3.5 and pH 5.5 (the normal pH of minimal medium) but can form colonies under both conditions. J227 cells are significantly longer than either single mutant at pH 3.5 and do not form colonies but are visually similar to elp3-1 cells at pH 5.5. Complementation cloning in the J227 background yielded a genomic clone of pub1, allowing us to define the intron-exon structure of the gene. Sequences with high homology to the predicted amino acid sequence of pub1 have been identified in Saccharomyces cerevisiae (RSP5/NPI1), human (hRPF1), mouse (mNedd4), and rat (rNedd4). Based on the nature of our mutant selection, the pH-sensitive phenotype of the strains selected, and the known involvement of RSP5/ NPI1 in membrane permease turnover in S. cerevisiae, we hypothesize a role for pub1, either directly or indirectly, in regulating membrane transport processes. This is further supported by the broad range of effects that the pub1-1 mutation exerts on overall performance of cells at high and low external pH, and in the presence of toxic levels of Li+.

Amino Acid Sequence↗
PubMed1997

Antifungal sterol biosynthesis inhibitors.

During the course of the last decade, the development of SBIs, and particularly sterol biomethylation inhibitors, has been based on the rational design approach. Successful though this approach has been in elucidating sterol biomethylation enzymology, its limitations are becoming apparent from the findings that: (i) 24,25-double bond metabolism gives rise to cholesterol and ergosterol in a mechanistically similar manner, (ii) 25-azasterols are harmful to human physiology, and (iii) side-chain modified sterols designed to inhibit the SMT enzyme in S. cerevisiae may be ineffective or operate by another kinetic mechanism in a related organism, rendering it therapeutically nonuseful. Nevertheless, it may be possible to ultimately capitalize on the unique aspects of sterol biomethylation chemistry and enzymology to design taxa-specific inhibitors. With increased understanding of the structure and function of SMT enzymes in different fungi, it should be possible to prepare novel mechanism-based inactivators to control SMT activity uniquely and with high specific activity.

Amino Acid Sequence↗
PubMed1997

Triterpenoid saponins from Mimusops elengi.

A novel minor triterpenoid saponin mimusin (3-O-[beta-D-glucopyranosyl- (1-->6)-beta-D-glucopyranosyl]-2 beta,3 beta,6 beta,23-tetrahydroxyolean- 12-en-28-oic acid 28-O-alpha-L-rhamnopyranosyl-(1-->3)-beta-D-xylopyranosyl-(1-->4)-alpha- L- rhamnopyranosyl-(1-->2)-alpha-L-arabinopyranoside was isolated from the seeds of Mimusops elengi, in addition to two known triterpenoid saponins, Mi-saponin A and 16 alpha-hydroxy Mi-saponin A. The structure of the minor saponin was established by comparing its 13C NMR and LS-MS linked-scan, ESI-MS data with FAB-MS of the mimusopsin isolated earlier from the same source.

Carbohydrate Conformation↗
PubMed1997

Protein phosphatases: structures and implications.

Protein phosphatases are signal transducing enzymes that dephosphorylate intracellular proteins phosphorylated on serine, threonine, and tyrosine residues. This brief review surveys recently determined structures of members of the protein tyrosine phosphatase and protein serine/threonine phosphatase families. In each family, characteristic and distinct structures confer different enzymatic mechanisms in catalyzing dephosphorylation reactions. Within each family, however, there exists remarkable similarity in active-site conformation and catalytic mechanism despite, in some cases, little or no sequence homology. The crystal structures also provide the basis for understanding the substrate binding specificity, inhibition by physiologically relevant compounds, and regulation of the protein phosphatases.

Animals↗