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The polarization of post-mitotic neurons is poorly understood. Preexisting spatially asymmetric cues, distributed within the neuron or as extracellular gradients, could be required for neurons to polarize. Alternatively, neurons might have the intrinsic ability to polarize without any preestablished asymmetric cues. In Caenorhabditis elegans, the UNC-40 (DCC) receptor mediates responses to the extracellular UNC-6 (netrin) guidance cue. For the HSN neuron, an UNC-6 ventral-dorsal gradient asymmetrically localizes UNC-40 to the ventral HSN surface. There an axon forms, which is ventrally directed by UNC-6. In the absence of UNC-6, UNC-40 is equally distributed and the HSN axon travels anteriorly in response to other cues. However, we find that a single amino acid change in the UNC-40 ectodomain causes randomly oriented asymmetric UNC-40 localization and a wandering axon phenotype. With UNC-6, there is normal UNC-40 localization and axon migration. A single UNC-6 amino acid substitution enhances the mutant phenotypes, whereas UNC-6 second-site amino acid substitutions suppress the phenotypes. We propose that UNC-40 mediates multiple signals to polarize and orient asymmetry. One signal triggers the intrinsic ability of HSN to polarize and causes randomly oriented asymmetry. Concurrently, another signal biases the orientation of the asymmetry relative to the UNC-6 gradient. The UNC-40 ectodomain mutation activates the polarization signal, whereas different forms of the UNC-6 ligand produce UNC-40 conformational changes that allow or prohibit the orientation signal.

The cornerstone of autotrophy, the C[O.sub.2]-fixing enzyme, D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), is hamstrung by slow catalysis and confusion between C[O.sub.2] and [O.sub.2] as substrates, an 'abominably perplexing' puzzle, in Darwin's parlance. Here we argue that these characteristics stem from difficulty in binding the featureless C[O.sub.2] molecule, which forces specificity for the gaseous substrate to be determined largely or completely in the transition state. We hypothesize that natural selection for greater C[O.sub.2]/[O.sub.2] specificity, in response to reducing atmospheric C[O.sub.2]:[O.sub.2] ratios, has resulted in a transition state for C[O.sub.2] addition in which the C[O.sub.2] moiety closely resembles a carboxylate group. This maximizes the structural difference between the transition states for carboxylation and the competing oxygenation, allowing better differentiation between them. However, increasing structural similarity between the carboxylation transition state and its carboxyketone product exposes the carboxyketone to the strong binding required to stabilize the transition state and causes the carboxyketone intermediate to bind so tightly that its cleavage to products is slowed. We assert that all Rubiscos may be nearly perfectly adapted to the differing C[O.sub.2], [O.sub.2], and thermal conditions in their subcellular environments, optimizing this compromise between C[O.sub.2]/[O.sub.2] specificity and the maximum rate of catalytic turnover. Our hypothesis explains the feeble rate enhancement displayed by Rubisco in processing the exogenously supplied carboxyketone intermediate, compared with its nonenzymatic hydrolysis, and the positive correlation between C[O.sub.2]/[O.sub.2] specificity and [sup.12]C/[sup.13]C fractionation. It further predicts that, because a more product-like transition state is more ordered (decreased entropy), the effectiveness of this strategy will deteriorate with increasing temperature. enzyme mechanisms | isotope fractionation | transition states

The nuclear receptor PXR (pregnane X receptor) is a broad-specificity sensor that recognizes a wide variety of synthetic drugs and xenobiotic agents. On activation by these compounds, PXR coordinately induces a network of transporters, cytochrome P450 enzymes, and other genes that effectively clear xenobiotics from the liver and intestine. Like PXR, the majority of its target genes also possess a broad specificity for exogenous compounds. Thus, PXR is both a sensor and effector in a well integrated and generalized pathway for chemical immunity. Although it is clear that PXR responds to numerous foreign compounds, it is unclear whether it possesses an endogenous ligand. To address this issue, we noted that there is substantial overlap in the substrate specificities of PXR and its critical CYP3A target gene. This prompted us to ask whether endogenous CYP3A substrates also serve as PXR ligands. We demonstrate that 5[beta]-cholestane-3[alpha], 7[alpha], 12[alpha]-triol (triol), a cholesterol-derived CYP3A substrate, is a potent PXR agonist that effectively induces cyp3a expression in mice. This defines a critical salvage pathway that can be autoinduced to minimize triol accumulation. In contrast, triol can accumulate to very high levels in humans, and unlike mice, these people develop the severe clinical manifestations of cerebrotendinous xanthomatosis. The reason for these dramatic species differences has remained unclear. We now demonstrate that triol fails to activate human PXR or induce the CYP3A-salvage pathway. This explains why humans are more susceptible to sterol accumulation and suggests that synthetic ligands for human PXR could be used to treat cerebrotendinous xanthomatosis and other disorders of cholesterol excess.

Results discusses the monastrol inhibition of different human kinesin Eg5 constructs in terms of monastrol activation of ATPase, mantADP release, and motility. Data indicate that monastrol is very effective in arresting mitotic cells in vivo but not as effective in in vitro.

Results point out that mutation of the D444N domain of human cystathionine beta-synthase reduces the steady-state concentrations of the protein 4-fold with approximately 100-fold increase in the activation rate for S-adenosylmethionine leading to reduced enzyme activity in the fibroblast extracts.

Results reveal that the GTP-bound Rac proteins sustain the phagocytic NADPH oxidase activity, while membrane-bound GTPase activating proteins mediate the deactivation of NADPH oxidase. Data suggest that the deactivation causes termination of the production of the superoxide.

Results point out that the presence of the heme b and the absence of a metal histidine bond are involved in the the activation of soluble guanylyl cyclase. Further, the metalloprotein porphyrins serve as activators and are bound 4-coordinate to the enzyme, suggesting that the tetrapyrrole conformation is important.

Results show that reaction of dihydroiso-L-tryptophan with the alpha-glycerol phosphate-aldimine complex leads to a transient formation and decay of quinonoid during the catalysis of tryptophan synthase using dihydroiso-L-tryptophan. Data indicate that the transient trapping of quinonoid species is due to the allosteric interactions within the activated tryptophan bienzyme complex.

The fluorescence analysis reveals an activation-dependent conformational change in the G protein alpha leading to increased hydrophobic environment of the N-terminal region. The conformational changes occur upon G protein betagamma binding and activated to either the GDP-AlF(sub)4(sup)- or the GTP-gammaS bound conformation.

Results indicate that the lipid rafts inhibit the phospholipaseCbeta(sub)2-Gbetagamma association and activation. Data suggest that the inhibition of the interaction of phospholipaseCbeta(sub)2 and Gbetagamma is due to changes in the Gbetagamma membrane orientation brought about by the lipid raft domains.

Research demonstrates that lysophosphatidic acid activates the endothelial isoform of nitric oxide synthase of bovine aortic endothelial cells via a receptor-mediated pathway. Further, platelet-derived phospholipid, sphingosine-1-phosphate, also activates the enzyme via EDG-1 receptor, different from that of lysophosphatidic acid activation receptor.

Research demonstrates that sterols within caveolae are mobile and selectively transferred to apolipoprotein A-1 as shown by labeling experiments using a free cholesterol analogue containing a photoreactive benzophenone group. Data further indicate that sterol efflux enhances platelet-derived growth factor-mediated signal transduction.

Results demonstrate that lysine 125 of antithrombin is the main heparin-binding residue and aids in the initial recognition of heparin by antithrombin and enhances the binding affinity by 10-17 fold. Further, lysine 125 is involved in the stabilization of the heparin-induced, conformationally altered state of the inhibitor.

Amino acid sequence alignment studies identify a new phosphatidylinositol 4,5-bisphosphate binding region, PBR1, near the first HxKxxxxD motif in the plant phosplipase Dbeta. Data indicate that PBR1 is an independent structural entity with binding properties and is important for the enzyme activity.

Target gene activation by nuclear hormone receptors, including estrogen receptors (ERs), is thought to be mediated by a variety of interacting cofactors. Here we identify a number of nuclear extract-derived proteins that interact with immobilized ER ligand binding domains in a 17/[beta]-estradiol-dependent manner. The most prominent of these are components of the thyroid hormone receptor-associated protein (TRAP)/Mediator coactivator complex, which interacts with ER[alpha] and ER[beta] in both unfractionated nuclear extracts and purified form. Studies with extracts from [TRAP220.sup.-/-] fibroblasts reveal that these interactions depend on TRAP220, a TRAP/Mediator subunit previously shown to interact with ER and other nuclear receptors in a ligand-dependent manner. The physiological relevance of the in vitro interaction is documented further by the isolation of an ER[alpha]-TRAP/Mediator complex from cultured cells expressing an epitope-tagged ER[alpha]. Finally, the complete TRAP/Mediator complex is shown to enhance ER function directly in a highly purified cell-free transcription system. These studies firmly establish a direct role for TRAP/Mediator, through TRAP220, in ER function.

Results reveal that the binding site of the rescue agent 4-methylimidazole at Trp 5 in the carbonic anhydrase II mutant is a nonproductive binding site, but changes on replacement of Trp 5. In the carbonic anhydrase III, the chemical rescue during catalysis is via binding of imidazole at Trp 5.

Mycobacterium tuberculosis (Mtb) infection induces the expression of matrix metalloproteinase-9 (MMP-9) in mouse lungs. In cultured human monocytic cells, Mtb bacilli and the cell wall glycolipid lipoarabinomannan (LAM) stimulate high levels of MMP-9 activity. Here, we explore the cellular mechanisms involved in the induction of MMP-9 by Mtb. We show that infection of THP-1 cells with Mtb caused a fivefold increase in MMP-9 mRNA that was associated with increased MMP-9 activity. MMP-9 induction was dependent on microtubule polymerization and protein kinase activation and was associated with increased DNA binding by the transcription factor activator protein-1 (AP-1), which appeared to be important for MMP-9 expression. We then explored the surface molecules potentially involved in Mtb induction of MMP-9, focusing on ligands of the mannose and [beta]-glucan receptors. MMP-9 activity was induced by the mannose receptor ligands mannan, zymosan, and LAM, whereas the [beta]-glucan receptor ligand laminarin was not effective. The most active inducers of MMP-9 activity were the particulate ligand zymosan and LAM. Pretreatment of cells with an anti-mannose receptor monoclonal antibody, but not anti-complement receptor 3, decreased the induction of MMP-9 activity by Mtb bacilli. Together, these results suggest that MMP-9 induction by Mtb occurs by receptor-mediated signaling mechanisms involving the binding of mannosylated ligands to mannose receptors, the modulation by cytoskeletal elements such as microtubules, the activation of protein kinases, and transcriptional activation by AP-1. matrix metalloproteinases; mannose receptor; protein kinases; cytoskeleton; activator protein-1; Mycobacterium

Results indicate that a positively charged epsilon-amino group of Lys166 lowers the activation energy for enolization of D-ribulose-1,5-bisphosphate carboxylase/oxygenase. Further, Lys166 plays an important role in the partial carboxylation reactions besides its role in the deprotonation and protonation reactions.

Hayabuchi, Y., N. B. Standen, and N. W. Davies. Angiotensin II inhibits and alters kinetics of voltage-gated [K.sup.+] channels of rat arterial smooth muscle. Am J Physiol Heart Circ Physiol 281: H2480-H2489, 2001.--The vasoconstrictor angiotensin II (ANG II) inhibits several types of [K.sup+] channels. We examined the inhibitory mechanism of ANG II on voltage-gated [K.sup.+] ([K.sub.v) currents (I.sub.Kv]) recorded from isolated rat arterial smooth muscle using patch-clamp techniques. Application of 100 nM ANG II accelerated the activation of [I.sub.Kv] but also caused inactivation. These effects were abolished by the A[T.sub.1] receptor antagonist losartan. The protein kinase A (PKA) inhibitor Rp-cyclic 3',5'-hydrogen phosphothioate adenosine (100 [micro]M) and an analog of diacylglycerol, 1,2dioctanyoyl-rac-glycerol (2 [micro]M), caused a significant reduction of [I.sub.Kv] Furthermore, the combination of 5 [micro]M PKA inhibitor peptide 5-24 (PKA-IP) and 100 [micro]M protein kinase C (PKC) inhibitor peptide 19-27 (PKC-IP) prevented the inhibition by ANG II, although neither alone was effective. The ANG II effect seen in the presence of PKA-IP remained during addition of the [Ca.sup.2+]-dependent PKC inhibitor Go6976 (1 [micro]M) but was abolished in the presence of 40 [micro]M PKC-[epsilon] translocation inhibitor peptide. These results demonstrate that ANG II inhibits Ky channels through both activation of PKC-[epsilon] and inhibition of PKA. potassium channel; activation; inactivation; protein kinase A; protein kinase C Received 22 May 2001; Accepted in Final Form 7 August 2001

Phosphopeptide map analyses of the monomeric and dimeric kinases demonstrate that dimerization increases phosphorylation of the regulatory activation loop of the insulin/IGF-1 receptor kinase. Data indicate that dimerization of the receptor kinases significantly increases the catalytic activity of the enzymes.

Results show that amino acid residues N189, T251, R216, and R249A located near the calcium binding site play a critical role in the phosphatidylserine-dependent activation of PKCalpha. Residue N189 particularly active in the activaion process, interacts with the lipid and its mutation to alanine abolishes activation.

The activation of the acetogenic bacterial acetyl-CoA synthase is brought about by the rapid binding of carbon dioxide and a two-electron reduction at one of the active sites. Data indicate that enzyme mediated carbon dioxide reduction rates and acetyl-CoA synthesis are synchronized by a catalytic mechanism.

Results demonstrate that the nature of nitrogen monoxide, released or produced under various redox environments, significantly influences modulation of prostaglandin H synthase-2 enzyme activity. Data indicate that nitric monoxide or nitric oxide enhance whereas peroxynitrite form inhibits the enzyme production.

Results reveal that activation of human factor VII by Xa factor and catalytic interaction of factor VII with factor X involve different regions of the catalytic domain. Data show that H101 and Y179 residues of the aromatic region of factor VII are involved in factor Xa activation of VII factor.

Results demonstrate that CTP: phosphocholine cytidylyltransferase binding and activation correlate with the curvature strain energy of class II lipids such as diacylglycerols and unsaturated phosphatidylethanaolamines. Data indicate that surface hydrophobicity of class II lipid membranes mediates the interaction between the enzyme and the membranes containing class II lipids.

The amino acid residues Glu-76 in the N-terminal tetratricopeptide repeat domain and Gln-495 at the C-terminus of the protein phosphatase 5 cause low basal activity of the enzyme. The fatty acid activation of the enzyme is primarily mediated by the tetratricopeptide repeat domain although implication of the C-terminal Asn-491is not ruled out.

Research describes a new reaction of RNAs in which RNAs perform amino acid activation analogous to phosphate anhydride synthesis by aminoacyl-transfer RNA synthetases. Results indicate that RNA KK13 activates leucine at acidic pH in the presence of calcium

Results demonstrate that mammalian protein kinase C-alpha is activated by RhoA and Cdc42 and to a lesser extent by Rac. Other forms of protein kinase C exhibit reduced activity suggesting a direct interaction between protein kinase C and Rho GTPases.

Research examines the effect of the activator extracellular regulated kinase (ERK) on the transition state of the phosphoryl transfer reaction mediated by phosphatase MP3. Data show that Asp-262 is a general acid catalyst of ERK-dependent and -independent catalytic activation as well as a general base for the thiol-phosphate intermediate hydrolysis.

Results show that mutations in coactivator asiA and sigma factor rpoD suppress the transcription factor motA mutant activation defect. Data reveal that the mutant proteins can not recapitulate the suppression but exhibit properties of sigma(sup)70 mutants.

The affinity of the enzyme for calcium increases by three orders of magnitude upon activation of phospholipase A(sub)2 by the bound lipid. Data suggest that the calcium affinity increase may be mediated by a tertiary complex of calcium, enzyme, and the substrate subsequent to enzyme's activation.

Results show that S-nitrosylation inhibits the transcriptional activator NF-kB in vivo within the nucleus. Data further indicate that cellular NF-kB activity is regulated by p50 nitrosylation via inhibition of p50 binding to DNA .

Results show that the recombinant form of the human prostate serine protease prostinogen yields prostin upon trypsin activation. Data reveal that prostin is a physiological activator of the precursor of prostate specific antigen.

Results show that cathepsin L efficiently activates the pro-dipeptidyl peptidase I whereas the S form is less active. Cathepsin B has no effect on the proenzyme.

Results show that substrate activation and inhibition by tubocurarine of acetylcholinesterase activity are two manifestations of the same homotropic phenomenon of the inhibitor. Data point out that tubocurarine acts as an activator when heterotropic inhibition of the enzyme is less effective.

Results demonstrate that increased activity of protein kinase-zeta is mediated by insulin activation of the phosphatidylinositol phosphate, 3-phosphoinositide-dependent protein kinase-1 threonine-410 loop phosphorylation, threonine-560 autophosphorylation, and pseudosubstrate autoinhibition during insulin action in rat adipocytes.

Amino acid residue substitutions in and near ligand-binding sites of the chemoreceptor Trg result in either insensitive receptor incapable of signaling or pseudosensitive receptors that mimick ligand binding and capable of signaling. Data indicate that the former activate kinase similar to wild-type receptors whereas the latter exhibits low kinase activity.

Research demonstrates that the allosteric regulation of yeast pyruvate kinase is mediated by a diphospho sugar and two hydrophobic compounds as revealed by mutational and kinetic analyses combined with structure-based screening.

Research reveals that combined mutations in both single chain interleukin-5 and one-domain GM1 form backgrounds, involving glutamate 110 replacements along with other mutations, lead to partial antagonist interleukin-5 muteins. Data indicate that the receptor binding and activation epitopes are separate in interleukin-5.

Results demonstrate that the degree of gamma-carboxylation is dependent on the affinity of the gamma-carboxylase for the prothrombin propeptide. Data indicate that gamma-decarboxylation, directed either by factor X or prothrombin, leads to the production of uncarboxylated and fully gama-carboxylated pools of factor X.

Results demonstrate that an 13 to 16 fold enhancement in streptokinase binding to plasminogen and plasmin is observed when catalytic sites of the enzyme contain lysine in its extended conformation. Data indicate that plasminogen transition from compact to extended conformation is due to lysine binding interactions in the streptokinase-plasminogen complex.

Research reveals that a dimeric ubiquitin-conjugating enzyme complex Ubc13/Uev1A takes part not only in the assembly of polyubiquitin chains but also supports IkB kinase activation by the signal transducer TRAF6. Data further show that ubiquitin plays a regulatory role in activating the kinase through polyubiquitin chains.

Crystallographic analysis of native and mutant forms of cellulase Cel48F and substrate-inhibitor complex crystals reveal seven sub-sites located at two different positions in the enzyme tunnel. Data also indicate that in the processive endo-cellulase, the two sites exhibit different patterns of aromatic residue stacking whereas nonprocessive enzyme show one type of sugar.