Optical spectroscopy of nicotinoprotein alcohol dehydrogenase from Amycolatopsis methanolica: a comparison with horse liver alcohol dehydrogenase and UDP-galactose epimerase

The NADH absorbance spectrum of nicotinoprotein (NADH-containing) alcohol dehydrogenase from Amycolatopsis methanolica has a maximum at 326 nm. Reduced enzyme-bound pyridine dinucleotide could be reversibly oxidized by acetaldehyde. The fluorescence excitation spectrum for NADH bound to the enzyme has a maximum at 325 nm. Upon excitation at 290 nm, energy transfer from tryptophan to enzyme-bound NADH was negligible. The fluorescence emission spectrum (excitation at 325 nm) for NADH bound to the enzyme has a maximum at 422 nm. The fluorescence intensity is enhanced by a factor of 3 upon binding of isobutyramide (Kd = 59 microM). Isobutyramide acts as competitive inhibitor (Ki = 46 microM) with respect to the electron acceptor NDMA (N,N-dimethyl-p-nitrosoaniline), which binds to the enzyme containing the reduced cofactor. The nonreactive substrate analogue trifluoroethanol acts as a competitive inhibitor with respect to the substrate ethanol (Ki = 1.6 microM), which binds to the enzyme containing the oxidized cofactor. Far-UV circular dichroism spectra of the enzyme containing NADH and the enzyme containing NAD+ were identical, indicating that no major conformational changes occur upon oxidation or reduction of the cofactor. Near-UV circular dichroism spectra of NADH bound to the enzyme have a minimum at 323 nm (Deltaepsilon = -8.6 M-1 cm-1). The fluorescence anisotropy decay of enzyme-bound NADH showed no rotational freedom of the NADH cofactor. This implies a rigid environment as well as lack of motion of the fluorophore. The average fluorescence lifetime of NADH bound to the enzyme is 0.29 ns at 20 degreesC and could be resolved into at least three components (in the range 0.13-0.96 ns). Upon binding of isobutyramide to the enzyme-containing NADH, the average excited-state lifetime increased to 1.02 ns and could be resolved into two components (0.37 and 1.11 ns). The optical spectra of NADH bound to nicotinoprotein alcohol dehydrogenase have blue-shifted maxima compared to other NADH-dehydrogenase complexes, but comparable to that observed for NADH bound to horse liver alcohol dehydrogenase. The fluorescence lifetime of NADH bound to the nicotinoprotein is very short compared to enzyme-bound NADH complexes, also compared to NADH bound to horse liver alcohol dehydrogenase. The cofactor-protein interaction in the nicotinoprotein alcohol dehydrogenase active site is more rigid and apolar than that in horse liver alcohol dehydrogenase. The optical properties of NADH bound to nicotinoprotein alcohol dehydrogenase differ considerably from NADH (tightly) bound to UDP-galactose epimerase from Escherichia coli. This indicates that although both enzymes have NAD(H) as nonexchangeable cofactor, the NADH binding sites are quite different.     

Horse liver alcohol dehydrogenase-catalyzed oxidation of aldehydes: dismutation precedes net production of reduced nicotinamide adenine dinucleotide

The oxidation of aldehydes by horse liver alcohol dehydrogenase (HL-ADH) is more complex than previously recognized. At low enzyme concentrations and/or high aldehyde concentrations, a pronounced lag in the assay progress curve is observed when the reaction is monitored for NADH production at 340 nm. When the progress of the reaction is followed by 1H NMR spectroscopy, rapid dismutation of the aldehyde substrate into the corresponding acid and alcohol is observed during the lag phase. Steady-state production of NADH commences only after aldehyde concentrations drop below 5% of their initial value; thereafter, NADH production occurs with continuous adjustment of the equilibrium between aldehyde, alcohol, NADH, and NAD+. The steady-state NADH production exhibits normal Michaelis-Menten kinetics and is in accord with earlier studies using much higher enzyme concentrations where no lag phase was reported. These results establish that the ability of HL-ADH to oxidize aldehydes is much greater than previously thought. The relationship between aldehyde dismutase and aldehyde dehydrogenase activities of HL-ADH is discussed.     

Fluorescence properties of reconstituted forms of UDP-glucose 4-epimerase from Saccharomyces fragilis

UDP-glucose 4-epimerase from Saccharomyces fragilis exhibits a very characteristic intense fluorescence with an excitation maximum at 360 nm and an emission maximum at 433 nm. The fluorescence spectrum resembles the fluorescence of free NADH with an apparent blue shift and, although the exact nature of the fluorophore is not known, the protein-bound NAD, which is a coenzyme for this reaction, or its reduced form is obviously involved in the emission of the fluorescence. The fluorphore therefore constitutes part of the active site. The inactivation of epimerase with diazinedicarboxylic acid bis(N,N-diethylamide), a reaction shown in the previous paper to form a disulfide linkage across the subunits, results in a simultaneous and correlated loss of the characteristic fluorescence of the enzyme. Reaction with mercaptoethanol restores the native fluorescence with a 2 nm blue shift in emission maximum. These epxeriments provide additional evidence that the two conformationally vicinal sulfhydryl groups are located at the active site. Unlike the reconstituted enzyme obtained from the diamide-inactivated enzyme, the partialy active enzymes reconstituted from p-chloromercuribenzoate-inactivated and heat-inactivated enzymes fail to show the reappearance of the characteristic native fluorescence. Treatment with N-ethylmaleamide, on the other hand, leads to a form of the inactive enzyme that fully retains its fluorescent properties. A model depicting the minimal changes at the active site during the process of inactivation and reconstitution by these various treatments is presented.     

Enzymatic, fluorometric assay of alpha-amylase in serum

An enzymatic, fluorometric method for kinetic assay of serum alpha-amylase (1,4alpha-D-glucan 4-glucanohydrolase, EC 3.2.1.1) is described. A soluble starch is used as substrate, in tris(hydroxymethyl)methylamine buffer. All measurements are made in Pyrex cuvettes at 37 degrees C, with a reaction volume of 1.16 ml. The assay is based on the following reaction sequence: (see article) The rate of appearance of fluorescence of NADH (lambdaex = 365 nm, lambdaem = 460 nm), developed in the indicator reaction (4), is measured and equated to the activity of alpha-amylase in serum. A calibration plot of the change of fluorescence per min vs. enzyme concentration shows a good proportionality in the range of 0.50-5.0 kU/liter.     

Interaction of thymidylate synthase with pyridoxal 5'-phosphate as studied by UV/visible difference spectroscopy and molecular modeling

Pyridoxal 5'-phosphate (PLP) is an effective inhibitor of Lactobacillus casei thymidylate synthase (TS), competitive with respect to the nucleotide substrate dUMP (Chen et al., 1989). The UV/vis difference spectra of TS-PLP complexes show lambda max at 328 nm due to the specific interaction between Cys 198 of TS and PLP to form a thiohemiacetal, and lambda min at 388 nm due to depletion of free PLP. At high concentrations of PLP a new absorbance at 430 nm forms due to nonspecific Schiff base formation between PLP and lysine residues of the enzyme. Using spectral titration at 328 nm, the binding constant of the specific TS-PLP complex was determined to be 0.5 microM, and the stoichiometry was 2 mol of PLP/mol of TS dimer. The 328-nm absorbance of the TS-PLP complex can be competitively and completely eliminated by addition of dUMP or dTMP; this serves as a convenient binding assay for molecules which bind to the active site of TS. Analogs of PLP which do not contain the phosphate or the aldehyde moieties of PLP bound poorly to the enzyme, thus demonstrating the importance of these functional groups for binding. When treated with PLP, C244T TS, which contains the active site Cys 198 as the sole cysteine residue, showed the same properties as the wild-type enzyme. Treatment of the C198A and C198S mutants with PLP did not produce the absorbance at 328 nm assigned to thiohemiacetal formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Liposome immunoassay by long-lived fluorescence detection

Immunoassay by fluorescence energy transfer from a europium chelate in liposome to allophycocyanin (APC) was demonstrated. Streptavidin or antibody to biotin was bonded to the liposome containing the europium chelate of 2-naphthoyltrifluoroacetone in the bilayer. When the biotin bonded to APC (APC-BT) was added to the prepared liposomes and the long-lived fluorescence (lambda ex 336 nm, lambda em 665 nm, delay time 0.05 ms, gate time 3.9 ms) was measured by a flow system, the fluorescence energy of the europium chelate was found to be transferred to APC-BT and the long-lived fluorescence intensity to increase linearly as the concentration of APC-BT (1-10 micrograms ml-1) increased. Further, the intensity decreased competitively with the concentration of biotin (0.1-100 microM) when biotin was added to the liposome solution containing a constant concentration of APC-BT.     

Involvement of different cysteines in the inactivation of octopine dehydrogenase by p-chloromercuricphenyl sulfonic acid and o-phthalaldehyde

Inactivation of octopine dehydrogenase by p-chloromercuricphenyl sulfonic acid (PCMS) and o-phthalaldehyde have been investigated. The activity loss due to the PCMS was faster than o-phthalaldehyde. PCMS associated inhibition was reversed by dithiothreitol completely which was not observed with o-phthalaldehyde inactivated enzyme. Fluorescence spectra of o-phthalaldehyde modified enzyme showed the formation of isoindole derivative with characteristic emission maximum at 410 nm. This derivative formation essentially involves cross-linking of proximal cysteine and lysine residues. Protection and selective reversible reaction studies have established that NADH prevents the enzyme against PCMS inactivation and the essential cysteine present at the NADH binding site is not involved in the o-phthalaldehyde reaction.     

High-performance liquid chromatographic separation of microcystins derivatized with a highly fluorescent dienophile

Microcystins are potent hepatotoxins produced by cyanobacteria, and are also tumor promoters as well as potent inhibitors of the catalytic subunits of protein phosphatases 1 and 2A. In order to establish a physicochemical method for individual detection and determination of trace amounts of microcystins, we developed a derivatization method for fluorescence (FL) and chemiluminescence (CL) detection, in which a highly fluorescent dienophile, DMEQ-TAD (4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalinyl) ethyl]-1,2,4-triazoline-3,5-dione), was used as the labeling reagent. DMEQ-TAD reacted smoothly with the conjugated diene of the Adda moiety to give 2 stereoisomers of the adducts. As a result of the extensive experiments, the following reaction conditions were optimized for the labeling: sample amount, 10 micrograms; reaction solvent, DMF:acetonitrile (1:1); reaction time, 15 minutes; reaction temperature, 70 degrees C; amount of DMEQ-TAD used relative to that of microcystin, 80 equivalent. The resulting 6 adducts from microcystins-LR, -YR, and -RR can be separated from one another using the following reversed phase HPLC conditions in combination with a clean-up using ODS silica gel: column, Cosmosil 5C18-AR (150 x 4.6 I.D. mm); mobile phase, methanol:0.05M phosphate buffer (pH 3) (1:1); flow rate, 1.0 ml/min; detection, FL lambda ex 370 nm, lambda em 440 nm. The detection limits of the DMEQ-TAD derivatives were estimated to be 100 and 500 pg for LR, and 65 and 2,500 pg for RR using FL and CL detections, respectively; and the detection behavior was different from that of the Dns-Cys derivatives, which were more sensitive to CL than FL.     

Near-infrared heavy-atom-modified fluorescent dyes for base-calling in DNA-sequencing applications using temporal discrimination

A series of near-IR fluorescent dyes were prepared which contained an intramolecular heavy atom for altering the fluorescence lifetimes to produce a set of probes appropriate for base-calling in a single-lane DNA sequencing format. The heavy-atom modification consisted of an intramolecular halogen situated on a remote section of the chromophore in order to minimize the perturbation on the lifetimes and fluorescence quantum yields. In addition, the dye series possessed an isothiocyanate functional group to allow facile attachment to sequencing primers. The unconjugated dyes showed similar absorption and emission maxima (lambda abs = 765-768 nm; lambda em = 794-798 nm) as well as fluorescence quantum yields that were invariant, within experimental error, with the heavy atom. However, the lifetimes of these dyes were found to vary with the identity of the halogen substitution (I, tau f = 947 ps; F, tau f = 843 ps, measured in methanol), with an average variation within the dye series of 35 ps. The spectroscopic properties of the free dyes and the dyes conjugated to sequencing primers on the 5'-end of the oligonucleotide were determined in a DNA-sequencing matrix (denaturing gels containing formamide). The results indicated slight differences in the fluorescence properties of the free dyes compared to those of the dye/ primer conjugates in this particular matrix. Inspection of the ground-state absorption spectra showed significant aggregation for the free dyes in this solution, but the conjugated dyes exhibited no sign of aggregation due to the highly anionic nature of the oligonucleotide. The fluorescence lifetimes of the dye/primer conjugates demonstrated lifetimes which ranged from 735 to 889 ps, with an average variation of 51 ps, an adequate difference to allow facile discrimination of these dyes in DNA-sequencing conditions. In addition, the free solution electrophoretic mobilities of the native heavy-atom-modified dyes were found to be very similar. When the dye/primer conjugates were electrophoresed in a cross-linked polyacrylamide gel electrophoresis capillary column, they comigrated, indicating that, in single-lane sequencing applications, when utilizing these dyes, no postrun corrections would be required to correct for dye-dependent mobility shifts.     

Cross-linking and N-(1-pyrenyl)maleimide labeling of cysteine mutants of proton-pumping pyridine nucleotide transhydrogenase of Escherichia coli

The pyridine nucleotide transhydrogenase of Escherichiacoli is a proton pump composed of two subunits (alpha and beta) organized as an alpha2beta2 tetramer. The enzyme contains seven cysteine residues, five in the alpha-subunit and two in the beta-subunit. The reaction of these residues with the cross-linking agent cupric 1, 10-phenanthrolinate and with the fluorescent thiol reagent N-(1-pyrenyl)maleimide was investigated in mutants in which one or more of these cysteine residues had been mutated to serine or threonine residues. Mutation of alphaCys395 and alphaCys397 prevented disulfide bond formation to give the cross-linked alpha2 dimer. We concluded that the two alpha-subunits of the holoenzyme interface in the region of these two cysteine residues. Pyrenylmaleimide reacted with detergent-washed cytoplasmic membrane vesicles containing high levels of transhydrogenase protein to show characteristic fluorescence emission bands at 378-379, 397-398, and 419-420 nm. At higher ratios of pyrenylmaleimide:transhydrogenase (>5:1) and longer times of reaction, an eximer band at 470 nm was formed. This was attributed to interaction between noncovalently bound molecules of pyrenylmaleimide. The cysteine residues of the beta-subunit (betaCys147 and betaCys260) were covalently modified by pyrenylmaleimide. betaCys147 reacted more strongly than betaCys260 with the fluorophore, and the pyrene derivative of betaCys147 was more accessible to quenching by 5-doxylstearate, suggesting a proximity to the surface of the membrane. Covalent modification of betaCys260 resulted in inhibition of enzyme activity. The inhibition was attributed to the introduction of the bulky pyrene group into the enzyme.     

Dopaminergic activity and the discriminative stimulus effects of mu opioids in pigeons: importance of training dose and attenuation by the D3 agonist (+/-)-7-OH-DPAT

Chlorophyll fluorescence measurements were performed on osmotically lysed potato chloroplasts in order to characterize the reactions involved in the dark reduction of photosynthetic inter-system chain electron carriers. Addition of NADH or NADPH to lysed chloroplasts increased the chlorophyll fluorescence level measured in the presence of a non-actinic light until reaching Fmax, thus indicating an increase in the redox state of the plastoquinone (PQ) pool. The fluorescence increase was more pronounced when the experiment was carried out under anaerobic conditions and was about 50% higher when NADH rather than NADPH was used as an electron donor. The NAD(P)H-PQ oxidoreductase reaction was inhibited by diphenylene iodonium, N-ethylmaleimide and dicoumarol, but insensitive to rotenone, antimycin A and piericidin A. By comparing the substrate specificity and the inhibitor sensitivity of this reaction to the properties of spinach ferredoxin-NADP+-reductase (FNR), we infer that FNR is not involved in the NAD(P)H-PQ oxidoreductase activity and conclude to the participation of rotenone-insensitive NAD(P)H-PQ oxidoreductase. By measuring light-dependent oxygen uptake in the presence of DCMU, methyl viologen and NADH or NADPH as an electron donors, the electron flow rate through the NAD(P)H-PQ oxidoreductase is estimated to about 160 nmol O2 min-1 mg-1 chlorophyll. The nature of this enzyme is discussed in relation to the existence of a thylakoidal NADH dehydrogenase complex encoded by plastidial ndh genes. Copyright 1998 Elsevier Science B.V.     

Transient-state kinetics of the reaction of aspartate aminotransferase with aspartate at low pH reveals dual routes in the enzyme-substrate association process

In aspartate aminotransferase, the coenzyme pyridoxal 5'-phosphate forms a Schiff base with the epsilon-amino group of Lys258. The pH dependency of the steady-state kinetics of the overall reaction had indirectly suggested that the Schiff-base-unprotonated form of the enzyme (EL) is the active species that binds the monoanionic form of aspartate (SH+), the predominant species of the substrate in solution. In order to obtain direct information on the association process, we carried out transient-phase kinetics of the first half-reaction of the enzyme with aspartate at various pH. The disappearance of EL (lambdamax = 358 nm) was fast and independent of pH, but the disappearance of ELH+ (Schiff-base-protonated form, lambdamax = 430 nm) was slow and dependent on pH. At pH values below 6.8 and low concentrations of aspartate, the results could be interpreted to indicate that EL reacts rapidly with SH+ to form the pyridoxamine 5'-phosphate form of the enzyme (EM), and the reaction of ELH+ proceeds via the route ELH+ right arrow over left arrow EL right arrow over left arrow EM, where the first step was found to be rate limiting from the pH jump/drop study of the enzyme. At higher pH values, the rate of disappearance of ELH+ became larger than expected from the above scheme. This deviation became apparent with increasing pH, and could be excellently explained if we consider that it is due to the reaction of ELH+ with the dianionic form of aspartate (S). Thus, the formation of the Michaelis complex of aspartate aminotransferase and aspartate can proceed via two routes; route A is the association of EL with SH+ to form EL.SH+, which converts intramolecularly to ELH+.S, and route B is the association of ELH+ with S to form ELH+.S directly. ELH+.S is the prerequisite structure for further processing of the substrate by the enzyme. The reactions of EM and oxo acids yielded almost exclusively EL and SH+, and therefore route B does not seem to play an essential role in the overall reactions of the enzyme. Route B, however, may be important in the reaction mechanisms of other pyridoxal 5'-phosphate enzymes which have only the ELH+ form.     

Can energy transfer be an indicator for DNA intercalation?

Contact energy transfer from DNA bases to various ligands, which can be represented by the ratio of the fluorescence intensity Q(lambda)/Q310nm, is measured by conventional fluorometer. 4',6-Diamidino-2-phenylindole and 2'-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5'-bi-1H-benzimidazole can accept energy from DNA bases and exhibit the ratio Q(lambda)/Q310nm, similar to that of intercalators, although these molecules are known to bind preferentially to the minor groove of the adenine-thymine rich region of DNA. When porphyrin is intercalated in DNA or poly[d(G-C)2], the shape of the ratio Q(lambda)/Q310nm is distinct from that of the ethidium-DNA complex with two maxima at 250 nm and 280 nm. The ratio Q(lambda)/Q310nm of the porphyrin-poly[d(A-T)2] complex, in which porphyrin is known to bind 'outside' of the DNA stem, is similar to that of intercalators. Therefore, energy from excited DNA bases can be transferred not only to an intercalated ligand through direct contact but also to that bound in the minor groove. It follows from this observation that using energy transfer as a criterion for DNA intercalation requires extreme caution.     

Incorporation of ethidium bromide in the Drosophila salivary gland approached by microspectrofluorometry: evidence for the presence of both free and bound dye in the nuclei of cells in viable conditions

The incorporation of 10(-6) M ethidium bromide (EB) was studied in viable Drosophila melanogaster salivary glands with a spatial resolution reaching a few microns3, using a confocal laser microspectrofluorometer designed for spectral analysis. Spectra were recorded with the 514 nm Argon laser line during excitation times of 1 second (20 microW on the preparation) at 5 min intervals for 30 or 60 min, either at points in determined cell sites or serially throughout the cells. The fluorescence intensity time-course indicated that the EB intake was not an all-or-none process, but rather a graded, sensitive indicator of the functional state of the cell. On the micrometer scale, the cytoplasm behaved as an homogeneous substrate with the fluorescence intensity depending on EB intake and intracellular diffusion. In the nucleus, however, localized enhancement of the emission intensity was observed. Spectral analysis allowed us to characterize the interactions. The mean values of lambda max in the cytoplasm (600 nm), in the nucleus (601 nm) and outside the glands (602 nm) were less than for free EB in aqueous solution (630 nm); values of full width at half maximum were between 92 and 96 nm, which is much lower than the 120 nm observed for free EB. The recorded spectra were analyzed using a linear combination of two spectral models, namely free and DNA intercalated EB. In the nucleus, the free EB model spectra was found to represent up to 10% of the recorded spectra whereas it was near zero in the cytoplasm. The present data suggest that the intranuclear concentration of free EB (allowing for its lower fluorescence quantum yield) might be at least equal to that of the bound EB.     

Time-resolved charge translocation by the Ca-ATPase from sarcoplasmic reticulum after an ATP concentration jump

Time-resolved measurements of currents generated by Ca-ATPase from fragmented sarcoplasmic reticulum (SR) are described. SR vesicles spontaneously adsorb to a black lipid membrane acting as a capacitive electrode. Charge translocation by the enzyme is initiated by an ATP concentration jump performed by the light-induced conversion of an inactive precursor (caged ATP) to ATP with a time constant of 2.0 ms at pH 6.2 and 24 degrees C. The shape of the current signal is triphasic, an initial current flow into the vesicle lumen is followed by an outward current and a second slow inward current. The time course of the current signal can be described by five relaxation rate constants, lambda1 to lambda5 plus a fixed delay D approximately 1-3 ms. The electrical signal shows that 1) the reaction cycle of the Ca-ATPase contains two electrogenic steps; 2) positive charge is moved toward the luminal side in the first rapid step and toward the cytoplasmic side in the second slow step; 3) at least one electroneutral reaction precedes the electrogenic steps. Relaxation rate constant lambda3 reflects ATP binding, with lambda(3,max) approximately 100 s(-1). This step is electroneutral. Comparison with the kinetics of the reaction cycle shows that the first electrogenic step (inward current) occurs before the decay of E2P. Candidates are the formation of phosphoenzyme from E1ATP (lambda2 approximately 200 s[-1]) and the E1P --> E2P transition (D approximately 1 ms or lambda1 approximately 300 s[-1]). The second electrogenic transition (outward current) follows the formation of E2P (lambda4 approximately 3 s[-1]) and is tentatively assigned to H+ countertransport after the dissociation of Ca2+. Quenched flow experiments performed under the conditions of the electrical measurements 1) demonstrate competition by caged ATP for ATP-dependent phosphoenzyme formation and 2) yield a rate constant for phosphoenzyme formation of 200 s(-1). These results indicate that ATP and caged ATP compete for the substrate binding site, as suggested by the ATP dependence of lambda3 and favor correlation of lambda2 with phosphoenzyme formation.     

Beta2 integrin/ICAM expression in Crohn''s disease

The kinetics of inactivation of Leuconostoc mesenteroides NRRL B-512F dextransucrase by o-phthalaldehyde showed that the reaction followed pseudo-first order reaction. The loss of enzyme activity was concomitant with an increase in fluorescence at 417 nm indicating that the inhibition involved the reaction of an epsilon-amino and a thiol group of the enzyme leading to the formation of an isoindole derivative. The stoichiometry of inactivation showed that one isoindole derivative was formed per enzyme molecule. The substrates sucrose and glucose provided protection against o-phthalaldehyde inactivation which was also corroborated by fluorescence studies. Dextransucrase was not inactivated by 5,5'-dithiobis(2-nitrobenzoic acid), showing that the cysteine present in close proximity to the lysine is not essential for enzyme activity. Denaturation of dextransucrase by urea or heat treatment prior to o-phthalaldehyde addition resulted in a decrease of fluorescence intensity indicating that the native conformation of the enzyme is essential for isoindole derivative formation. These results established that a lysine residue is present at the active site and is essential for the activity of dextransucrase.     

Reduced HIV-1 infectability of CD4+ lymphocytes from exposed-uninfected individuals: association with low expression of CCR5 and high production of beta-chemokines

Escherichia coli leader peptidase, an integral membrane protein, is responsible for the cleavage of the signal sequence of many exported proteins. Recent studies suggest that it is a novel serine protease that utilizes a serine-lysine catalytic dyad. In an effort to further understand the mechanism of this enzyme, an internally quenched fluorescent peptide substrate incorporating the leader peptidase cleavage site of maltose binding protein signal peptide, Y(NO2)-F-S-A-S-A-L-A-K-I-K(Abz) (anthraniloyl), was designed and synthesized. In the intact peptide, the fluorescence of the anthraniloyl group is quenched by the 3-nitrotyrosine. This quenched fluorescence is liberated upon cleavage of the peptide by the leader peptidase, resulting in increased fluorescence that could then be monitored fluorometrically. The designed substrate can be cleaved effectively by E. coli leader peptidase as detected by both HPLC and fluorescent spectroscopy. Mass spectra of cleavage products demonstrated that the cleavage occurs at the predicted site (A-K). The cleavage of the peptide substrate has a linear dependence on the enzyme concentration (0.1 to 1.9 microM) and the kcat/K(m) was calculated to be 71.1 M-1 s-1. These data are comparable with the unmodified peptide substrate. This report represents the first direct continuous assay based on fluorescence resonance energy transfer for E. coli leader peptidase.     

Mutant aspartate aminotransferase (K258H) without pyridoxal-5'-phosphate-binding lysine residue. Structural and catalytic properties

If the pyridoxal-phosphate-binding lysine residue 258 of aspartate aminotransferase is exchanged for a histidine residue, the enzyme retains partial catalytic competence [Ziak, M., Jaussi, R., Gehring, H. and Christen, P. (1990) Eur. J. Biochem. 187, 329-333]. The three-dimensional structures of the mutant enzymes of both chicken mitochondria and Escherichia coli were determined at high resolution. The folding patterns of the polypeptide chains proved to be identical to those of the wild-type enzymes, small conformational differences being restricted to parts of the active site. If aspartate or glutamate was added to the pyridoxal form of the mutant enzyme [lambda max 392 nm and 330 nm (weak); negative CD at 420 nm, positive CD at 370 nm and 330 nm], the external aldimine (lambda max = 430 nm; negative CD at 360 nm and 430 nm) transiently accumulated. Upon addition of 2-oxoglutarate to the pyridoxamine form (lambda max 330 nm, positive CD), a putative ketamine intermediate could be detected; however, with oxalacetate, an equilibrium between external aldimine and the pyridoxal form, which was strongly in favour of the former, was established within seconds. The transamination cycle with glutamate and oxalacetate proceeds only three orders of magnitude more slowly than the overall reaction of the wild-type enzyme. The specific activity of the mutant enzyme is 0.1 U/mg at 25 degrees C and constant from pH 6.0 to 8.5. Reconstitution of the mutant apoenzyme with [4'-3H]pyridoxamine 5'-phosphate resulted in rapid release of 3H with a first-order rate constant kappa' = 5 x 10(-4) s-1 similar to that of the wild-type enzyme. Apparently, in aspartate aminotransferase, histidine can to some extent substitute for the active-site lysine residue. The imidazole ring of H258, however, seems too distant from C alpha and C4' to act efficiently as proton donor/acceptor in the aldimine-ketamine tautomerization, suggesting that the prototropic shift might be mediated by an intervening water molecule. Transmination of the internal to the external aldimine apparently can be replaced by de novo formation of the latter, and by its hydrolysis in the reverse direction.     

Calculation of diffusion-limited kinetics for the reactions in collision coupling and receptor cross-linking

Both enzyme (e.g., G-protein) activation via a collision coupling model and the formation of cross-linked receptors by a multivalent ligand involve reactions between two molecules diffusing in the plasma membrane. The diffusion of these molecules is thought to play a critical role in these two early signal transduction events. In reduced dimensions, however, diffusion is not an effective mixing mechanism; consequently, zones in which the concentration of particular molecules (e.g., enzymes, receptors) becomes depleted or enriched may form. To examine the formation of these depletion/ accumulation zones and their effect on reaction rates and ultimately the cellular response, Monte Carlo techniques are used to simulate the reaction and diffusion of molecules in the plasma membrane. The effective reaction rate at steady state is determined in terms of the physical properties of the tissue and ligand for both enzyme activation via collision coupling and the generation of cross-linked receptors. The diffusion-limited reaction rate constant is shown to scale with the mean square displacement of a receptor-ligand complex. The rate constants determined in the simulation are compared with other theoretical predictions as well as experimental data.     

7-OH-DPAT has d-amphetamine-like discriminative stimulus properties

A fluorometric procedure has been developed for detection and estimation of laccase activity in fungal broth cultures. Laccase solution was pretreated with catalase for 1 h at 37 degrees C and pH 5. Homovanillic acid was then added and the reaction mixture incubated for a further hour at 37 degrees C. The fluorescence was then developed by addition of 0.1 M glycine buffer at pH 10. Laccase preparations from Pyricularia oryzae, Coriolus hirsutus and Pycnoporus cinnabarinus catalysed formation of a fluorescent product of HVA but the optimum pH values of enzyme activities varied. The culture fluids of several other fungi also catalysed development of fluorescence in solutions containing HVA. p-Hydroxyphenylacetic acid was a poor substrate for all laccases in vivo except that produced by Perennipora tephropora.