Specificity of gamma-glutamyl cyclotransferase.

Using the phthaloyl method, 18 gamma-L-glutamyl peptides labelled with 14-C in the N-terminal position have been synthesized. The products were isolated by simple procedures using a Dowex-1 column or high voltage electrophoresis. The synthetic peptides contain minor impurities of the corresponding D-glutamyl isomers. The proportion of D-isomer was determined by the use of glutamic decarboxylase, or by a new method using digestion with purified gamma-glutamyl cyclotransferase and determination of the resulting 2-pyrrolidone-5-carboxylic acid (5-oxoproline). Evidence was obtained that gamma-glutamyl cyclotransferase acts only on the L-form of gamma-glutamyl substrates; the enzyme could, therefore, be used for preparation of gamma-D-glutamyl peptides from their racemic mixtures. The specificity of gamma-glutamyl cyclotransferase has been examined using pure enzyme prepared from pig liver, and extracts from tissues of rat and man. The basic structural requirement in substrates may be represented as gamma-L-glutamyl-NH--CHR--COOH. The amino acid linked to the gamma-glutamyl group must be in the L configuration.     

Discovery of a potent, selective, and less flexible selective norepinephrine reuptake inhibitor (sNRI)

The design, synthesis, and SAR of a series of ring-constrained norepinephrine reuptake inhibitors are described. A substantially rigid inhibitor with potent functional activity at the transporter (IC₅₀ = 8 nM) was used to develop a model for the distance and orientation of key features necessary for interaction with the norepinephrine transporter (NET).     

Use of benzoyl-L-phenylalanyl-L-valyl-L-arginine (3H) methyl ester as a sensitive and selective substrate for the human lymphokine, leukocyte migration inhibitory factor (LIF)

Human leukocyte migration inhibitory factor (LIF) appears to be a serine esterase and protease exhibiting specific affinity towards arginine esters and amides. On the basis of indirect evidence that an amide of the oligopeptide benzoyl-phenylalanyl-valyl-arginine might have high and selective affinity for LIF, we prepared an ester of this oligopeptide, benzoyl-phenylalanyl-valyl-arginine (3H) methyl ester (3H-BPVAME) for the direct measurement of LIF activity in a double-phase radio-enzyme assay. The hydrolysis of 3H-BPVAME followed enzyme-substrate kinetics in that the reaction was time-, temperature-, pH-, and concentration-dependent. 3H-BPVAME rpoved to be more selective and approximately 20 times more sensitive as a substrate for LIF than previously used radiolabeled substrates such as 3H-BAEE and 3H-TAME. Moreover, hydrolysis of 3H-BPVAME by partially purified LIF preparations was significantly inhibited by 10(-8) to 10 (-5) M of guanosine 3',5'-cyclic monophosphate (cGMP), further supporting the hypothesis that cGMP acts as a regulator of LIF activity. Inhibition of LIF-induced esterolysis was also provided by dibutyryl cGMP, but only at concentrations 10(-7) to 10(-5) M; 8-bromo cGMP and adenosine 3',5'-cyclic monophosphate were both ineffective. These results provide support for the use of 3H-BPVAME as a more selective substrate to detect esterase activity in LIF preparations than heretofore described and the possible development of a biochemical assay for the measurement of this lymphokine.     

The direct measurement of protein kinase C (PKC) activity in isolated membranes using a selective peptide substrate

A protein kinase C (PKC)-selective peptide substrate was used to develop a method for measuring PKC activity directly and quantitatively in isolated cell membranes without prior detergent extraction and reconstitution of the enzyme with phosphatidylserine and TPA in the presence of excess Ca2+. This simple and rapid method can reliably measure changes in membrane-associated PKC activity induced by various bioactive compounds such as hormones and growth factors. Also, this method, which measures PKC activity in its native membrane-associated state, has the advantage of being able to distinguish between active and inactive PKC associated with cell membranes.     

Synthesis of p-nitrophenyl 6(5)-O-benzyl-alpha-maltopentaoside, a substrate for alpha amylases

p-Nitrophenyl alpha-maltopentaoside, having a benzyl group on O-6 of the terminal (nonreducing) D-glucosyl group was prepared by use of a reductive ring-opening reaction. Highly regioselective reduction of p-nitrophenyl O-(2,3-di-O-benzoyl-4,6-O-benzylidene-alpha-D-glucopyranosyl)-(1----4)- tris[O-(2,3,6-tri-O-benzoyl-alpha-D-glucopyranosyl)-(1----4)]-2,3,6-tri- O- benzoyl-alpha-D-glucopyranoside by dimethylamine-borane and p-toluenesulfonic acid, followed by debenzoylation, gave p-nitrophenyl O-(6-O-benzyl-alpha-D-glucopyranosyl)-(1----4)-tris[O-alpha-D-glucopyran osyl- (1----4)]-alpha-D-glucopyranoside. An experiment was done on the mode of action of human pancreatic and salivary alpha amylases on this derivative. The compound is suitable as a substrate for the assay of alpha amylase when used with glucoamylase and alpha-D-glucosidase as coupling enzymes.     

Solid-phase synthesis of Biotin-S-Farnesyl-l-Cysteine,a surrogate substrate for isoprenylcysteine Carboxylmethyltransferase (ICMT)

Inhibition of isoprenylcysteine Carboxylmethyltransferase (ICMT) is of particular interest as a potential target for the development of cancer chemotherapeutic agents. Screening for inhibitors of ICMT utilises a scintillation proximity assay (SPA) in which Biotin-S-Farnesyl-l-Cysteine (BFC) acts as a surrogate substrate. A solid-phase synthesis protocol for the preparation of BFC using 2-chlorotrityl chloride resin as a solid support has been developed to provide sufficient supply of BFC for high throughput screening (HTS) and subsequent chemistry campaigns to target inhibitors of ICMT. The BFC prepared by this method can be produced quickly on large scale and is stable when stored at ?20 °C as a solid, in solution, or on the resin.     

Discovery and characterization of a substrate selective p38alpha inhibitor

A novel inhibitor of p38 mitogen-activated protein kinase (p38), CMPD1, identified by high-throughput screening, is characterized herein. Unlike the p38 inhibitors described previously, this inhibitor is substrate selective and noncompetitive with ATP. In steady-state kinetics experiments, CMPD1 was observed to prevent the p38alpha-dependent phosphorylation (K(i)(app) = 330 nM) of the splice variant of mitogen-activated protein kinase-activated protein kinase 2 (MK2a) that contains a docking domain for p38alpha and p38beta, but it did not prevent the phosphorylation of ATF-2 (K(i)(app) > 20 microM). In addition to kinetic studies, isothermal titration calorimetry and surface plasmon resonance experiments were performed to elucidate the mechanism of inhibition. While isothermal titration calorimetry analysis indicated that CMPD1 binds to p38alpha, CMPD1 was not observed to compete with ATP for p38alpha, nor was it able to interrupt the binding of p38alpha to MK2a observed by surface plasmon resonance. Therefore, deuterium exchange mass spectrometry (DXMS) was employed to study the p38alpha.CMPD1 inhibitory complex, to provide new insight into the mechanism of substrate selective inhibition. The DXMS data obtained for the p38alpha.CMPD1 complex were compared to the data obtained for the p38alpha.MK2a complex and a p38alpha.active site binding inhibitor complex. Alterations in the DXMS behavior of both p38alpha and MK2a were observed upon complex formation, including but not limited to the interaction between the carboxy-terminal docking domain of MK2a and its binding groove on p38alpha. Alterations in the D(2)O exchange of p38alpha produced by CMPD1 suggest that the substrate selective inhibitor binds in the vicinity of the active site of p38alpha, resulting in perturbations to regions containing nucleotide binding pocket residues, docking groove residues (E160 and D161), and a Mg(2+) ion cofactor binding residue (D168). Although the exact mechanism of substrate selective inhibition by this novel inhibitor has not yet been disclosed, the results suggest that CMPD1 binding in the active site region of p38alpha induces perturbations that may result in the suboptimal positioning of substrates and cofactors in the transition state, resulting in selective inhibition of p38alpha activity.     

Nitroxyl (NO-): a substrate for superoxide dismutase

The interactions of Cu, Zn superoxide dismutase (SOD) with nitroxyl (NO-) and nitric oxide (NO), both of which are thought to be biologically significant, have been studied but remain undefined. Having previously noted that NO- can reduce Cu (II), Zn SOD aerobically, we now report that it also can do so anaerobically and that Cu, Zn SOD can catalyze the elimination of NO(-) in the absence of O2.NO- acts as a reductant of ferricytochrome c anaerobically, but in the presence of O2 causes the oxidation of ferrocytochrome c and NADPH. Equivalent fluxes of NO-, and NO + O2- were able to comparably oxidize NADPH, but the oxidation by NO + O2- was more than fivefold more sensitive to inhibition by Cu, Zn SOD than was the oxidation by NO-. Thus Cu, Zn SOD inhibited NADPH oxidation by NO- by a route independent of catalyzing the dismutation of O2. Plausible mechanisms for those observations are offered and rate constants are estimated.     

Osteopontin, a novel substrate for matrix metalloproteinase-3 (stromelysin-1) and matrix metalloproteinase-7 (matrilysin)

Osteopontin (OPN) is a secreted phosphoprotein shown to function in wound healing, inflammation, and tumor progression. Expression of OPN is often co-localized with members of the matrix metalloproteinase (MMP) family. We report that OPN is a novel substrate for two MMPs, MMP-3 (stromelysin-1) and MMP-7 (matrilysin). Three cleavage sites were identified for MMP-3 in human OPN, and two of those sites were also cleaved by MMP-7. These include hydrolysis of the human Gly166-Leu167, Ala201-Tyr202 (MMP-3 only), and Asp210-Leu211 peptide bonds. Only the N-terminal Gly-Leu cleavage site is conserved in rat OPN (Gly151-Leu152). These sites are distinct from previously reported cleavage sites in OPN for the proteases thrombin or enterokinase. We found evidence for the predicted MMP cleavage fragments of OPN in vitro in tumor cell lines, and in vivo in remodeling tissues such as the postpartum uterus, where OPN and MMPs are co-expressed. Furthermore, cleavage of OPN by MMP-3 or MMP-7 potentiated the function of OPN as an adhesive and migratory stimulus in vitro through cell surface integrins. We predict that interaction of MMPs with OPN at tumor and wound healing sites in vivo may be a mechanism of regulation of OPN bioactivity.     

Mode of action of bacterial collagenase on a synthetic substrate, (Pro-Pro-Gly)5.

Clostridial collagenase (EC 3.4.24.3) catalyzes the hydrolysis of (Pro-Pro-Gly)5 at minimum of three different rates, producing Pro-Pro, Gly-Pro-Pro and Gly-Pro-Pro-Gly, and various intermediate peptides. The intermediate and final products were separated by cation-exchange column chromatogrphy and identified, and their rates of formation were measured. Pro-Pro was released most rapidly with formation of the tridecapeptide. After the initial release of the N-terminal Pro-Pro, hexa- and heptapeptides were formed in larger amounts than tri-, tetra-, nona- and decapeptides from the tridecapeptide. The rates of disappearance of the intermediates decreased in the order trideca- greater than deca- and nona- greater than heptapeptide. The results indicate that the enzyme hydrolyzes inner linkages of the tridecapeptide having N- and C-terminal Gly residues, forming large peptides, preferentially to outer linkages, forming the tri- and tetrapeptides.     

DT56a (Femarelle): a natural selective estrogen receptor modulator (SERM)

A selective estrogen receptor modulator (SERM) is defined as a substance with dissimilar effects on different tissues: agonist in some and antagonists in others. The natural compound DT56a (Femarelle) was shown to activate estrogen receptors in human cultured female derived osteoblasts. It was also shown to relieve menopausal symptoms and to increase bone mineral density with no effect on sex steroid hormone levels and on the endometrial thickness. DT56a, similarly to estradiol-17β (E₂), stimulated the specific activity of creatine kinase (CK) in skeletal and vascular tissues of female rats, as a marker of estrogen receptor (ER) activation. However, in the uterus, CK was activated only by E₂ but not by DT56a. In order to prove that DT56a is a SERM, we examined the mutual interaction between DT56a and E₂, at supra physiological doses, in different tissues in both intact and ovariectomized female rats, as well as in human cultured vascular and bone cells. Administration of DT56a or E₂ stimulated CK in all tissues tested, but when given simultaneously, in intact immature female rats, DT56a completely abolished E₂ stimulation of CK in all organs except in the diaphyseal bone where the inhibition was partial. In ovariectomized female rats, DT56a abolished E₂'s stimulation of CK in diaphyseal bone, thymus, uterus and pituitary but caused a partial inhibition in aorta, left ventricle and epiphyseal cartilage. In human bone cells E₂ stimulation of CK, of alkaline phosphatase (AP) activity and of DNA synthesis was completely abolished by DT56a in post-menopausal cells and partially inhibited in pre-menopausal cells. In human vascular cells, inhibition of DNA synthesis by E₂ was completely abolished by DT56a and E₂-induced CK was partially inhibited by DT56a. The results support the finding that DT56a is a SERM; it stimulated different parameters similar to E₂, but when given simultaneously, at supra physiological doses, inhibited these E₂'s effects. Further investigations regarding intra and extra cellular mechanism of action of DT56a are currently performed.     

The feeding ecology of a selective browser, the giraffe (Giraffa camelopardalis tippelskirchi)

A herbivore can manipulate certain factors of its feeding behaviour in order to achieve the metabolic requirements for reproduction. These factors include the choice of habitat in which to feed, the selection criteria for choosing food items, and the time allocated to feeding or devoted to other energy-consuming activities. The manipulation of these behavioural factors by giraffe in the Serengeti National Park, Tanzania is analysed in relation to the seasonal changes in the quantity and quality of the food resource.
The pattern of habitat choice demonstrates seasonal movements by giraffe across the drainage catena in response to differences in the rates of browse production between woodland types.
Giraffe show a positive inter-specific selection for the minor food items with a low available biomass, and utilize the dominant species in approximate proportion to their availability. Plant part selection is shown for the flushing shoots with very high protein contents.
Selection criteria include a significant selection for phosphorus, and in the dry season a selection for high energy material by breeding females. With the high nutritional qualities of the diets, negative criteria such as secondary chemical compounds are important. Patterns of browse selection are correlated with inter-specific seasonal changes in plant phenology.
Giraffe devote more time to foraging as the biomass and quality of the food declines in the dry season. Energy is conserved by minimizing energy-consuming activities at the most demanding times of day.     

Functionalization of poly(methyl methacrylate) (PMMA) as a substrate for DNA microarrays

A chemical procedure was developed to functionalize poly(methyl methacrylate) (PMMA) substrates. PMMA is reacted with hexamethylene diamine to yield an aminated surface for immobilizing DNA in microarrays. The density of primary NH₂ groups was 0.29 nmol/cm². The availability of these primary amines was confirmed by the immobilization of DNA probes and hybridization with a complementary DNA strand. The hybridization signal and the hybridization efficiency of the chemically aminated PMMA slides were comparable to the hybridization signal and the hybridization efficiency obtained from differently chemically modified PMMA slides, silanized glass, commercial silylated glass and commercial plastic Euray™ slides. Immobilized and hybridized densities of 10 and 0.75 pmol/cm², respectively, were observed for microarrays on chemically aminated PMMA. The immobilized probes were heat stable since the hybridization performance of microarrays subjected to 20 PCR heat cycles was only reduced by 4%. In conclusion, this new strategy to modify PMMA provides a robust procedure to immobilize DNA, which is a very useful substrate for fabricating single use diagnostics devices with integrated functions, like sample preparation, treatment and detection using microfabrication and microelectronic techniques.     

Jararhagin,a snake venom metalloproteinase,induces a specialized form of apoptosis (anoikis) selective to endothelial cells

Jararhagin is a snake venom metalloproteinase (SVMP) from Bothrops jararaca involved in several hemostatic and inflammatory disorders that occur in human envenomings. In this study, we evaluated the effect of jararhagin on endothelial cells (tEnd). The exposure of tEnd to jararhagin (20 and 40μg/ml) resulted in apoptosis with activation of pro-caspase-3 and alterations in the ratio between Bax/Bcl-x_L. We observed that apoptosis was followed by decrease of cell viability and the loss of cell adhesion. Jararhagin induced changes in cell shape with a decrease in cell spreading, rounding up and detachment. This was accompanied by a rearrangement of actin network and a decrease in FAK association to actin and in tyrosine phosphorylated proteins. Morphological alterations and apoptosis were abolished when jararhagin catalytic activity was inhibited, indicating the importance of catalysis. Treatment of murine peritoneal adherent cells or fibroblasts with jararhagin did not result in apoptosis. The data indicate that the pro-apoptotic effect of jararhagin is selective to endothelial cells, interfering with the adhesion mechanisms and inducing anoikis. The present model might be useful for the study of the relationships between the architectural changes in the cytoskeleton and the complex phenomenon named anoikis.     

Papaya glutamine cyclotransferase shows a singular five-fold beta-propeller architecture that suggests a novel reaction mechanism

Cyclisation of N-terminal glutamine and/or glutamate to yield pyroglutamate is an essential posttranslational event affecting a plethora of bioactive peptides and proteins. It is directly linked with pathologies ranging from neurodegenerative diseases to inflammation and several types of cancers. The reaction is catalysed by ubiquitous glutaminyl cyclotransferases (QCs), which present two distinct prototypes. Mammalian QCs are zinc-dependent enzymes with an alpha/beta-hydrolase fold. Here we present the 1.6-A-resolution structure of the other prototype, the plant analogue from Carica papaya (PQC). The hatbox-shaped molecule consists of an unusual five-fold beta-propeller traversed by a central channel, a topology that has hitherto been described only for some sugar-binding proteins and an extracellular nucleotidase. The high resistance of the enzyme to denaturation and proteolytic degradation is explained by its architecture, which is uniquely stabilised by a series of tethering elements that confer rigidity. Strikingly, the N-terminus of PQC specifically interacts with residues around the entrance to the central channel of a symmetry-related molecule, suggesting that this location is the putative active site. Cyclisation would follow a novel general-acid/base working mechanism, pivoting around a strictly conserved glutamate. This study provides a lead structure not only for plant QC orthologues, but also for bacteria, including potential human pathogens causing diphtheria, plague and malaria.     

Simultaneous estimation ofV max,Km, and the rate of endogenous substrate production (R) from substrate depletion data

The nonlinear and 3 linearized forms of the integrated Michaelis-Menten equation were evaluated for their ability to provide reliable estimates of uptake kinetic parameters, when the initial substrate concentration (S₀) is not error-free. Of the 3 linearized forms, the one where t/(S₀–S) is regressed against ln(S₀/S)/(S₀–S) gave estimates ofV _max and K_m closest to the true population means of these parameters. Further, this linearization was the least sensitive of the 3 to errors (±1%) in S₀. Our results illustrate the danger of relying on r² values for choosing among the 3 linearized forms of the integrated Michaelis-Menten equation. Nonlinear regression analysis of progress curve data, when S₀ is not free of error, was superior to even the best of the 3 linearized forms. The integrated Michaelis-Menten equation should not be used to estimateV _max and K_m when substrate production occurs concomitant with consumption of added substrate. We propose the use of a new equation for estimation of these parameters along with a parameter describing endogenous substrate production (R) for kinetic studies done with samples from natural habitats, in which the substrate of interest is an intermediate. The application of this new equation was illustrated for both simulated data and previously obtained H₂ depletion data. The only means by whichV _max, K_m, and R may be evaluated from progress curve data using this new equation is via nonlinear regression, since a linearized form of this equation could not be derived. Mathematical components of computer programs written for fitting data to either of the above nonlinear models using nonlinear least squares analysis are presented.     

5-(Pentafluorobenzoylamino)fluorescein: A selective substrate for the determination of glutathione concentration and glutathione S-transferase activity

5-(Pentafluorobenzoylamino)fluorescein (PFB-F), a new thiol-reactive molecule was synthesized to improve the detection limits and specificity of the assays for glutathione S-transferase (GST) activity and glutathione (GSH). A rapid assay method to measure GSH concentration or GST activity and the simultaneous analysis of multiple samples is possible because the glutathione adduct, GS-TFB-F, is separated from PFB-F by thin-layer chromatography (TLC) and can be quantitated by a fluorescence scanner. The detection limits for GSH and for GST activity using TLC were found to be as low as 10 pmol/microl and 1 ng/microl using equine liver GST, respectively. Determination of GSH concentration or GST activity in bovine pulmonary artery endothelial (BPAE) cell lysates gave a linear response for samples corresponding to 500-2500 cells. PFB-F could also measure GST activities of GST fusion proteins and prove to be a suitable substrate for determining the activities of human GST isozymes and other sources of mammalian GST. The selectivity of PFB-F with GSH was proven by comparing trace amount of the adducts that formed with cysteine and beta-galactosidase to that formed with GSH. The HPLC profile of a reaction mixture where cell lysate was used in place of purified GST, also shows only two main peaks, corresponding to GS-TFB-F and unreacted PFB-F. The selectivity of PFB-F for GSH was further confirmed by exposing BPAE cells to dl-buthionine-[S,R]-sulfoximine (BSO). Our results of GS-TFB-F determination indicate that 12-, 24-, or 36-h incubations with BSO caused 2-, 6-, or 7.6-fold reductions in GSH levels, respectively.