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PubMed2003

Analogs of diadenosine tetraphosphate (Ap4A).

This review summarizes our knowledge of analogs and derivatives of diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A), the most extensively studied member of the dinucleoside 5',5"'-P1,Pn-polyphosphate (NpnN) family. After a short discussion of enzymes that may be responsible for the accumulation and degradation of Np4)N's in the cell, this review focuses on chemically and/or enzymatically produced analogs and their practical applications. Particular attention is paid to compounds that have aided the study of enzymes involved in the metabolism of Ap4A (Np4N'). Certain Ap4A analogs were alternative substrates of Ap4A-degrading enzymes and/or acted as enzyme inhibitors, some other helped to establish enzyme mechanisms, increased the sensitivity of certain enzyme assays or produced stable enzyme:ligand complexes for structural analysis.

Chromogenic Compounds↗
PubMed1996

A specific chromophoric substrate for activity assays of 1,3-1,4-beta-D-glucan 4-glucanohydrolases.

The synthesis of 4-methylumbelliferyl 3-beta-O-cellobiosyl-beta-D-glucopyranoside (3a) and its use as specific substrate to monitor enzyme activity of 1,3-1,4-beta-D-glucan 4-glucanohydrolases are described. The chromophoric substrate 3a is prepared by a chemoenzymatic approach starting from barley grain, whose beta-D-glucan polysaccharide is degraded down to a tri- and tetrasaccharide by an extracellular extract of recombinant E. coli expressing and secreting Bacillus licheniformis 1,3-1,4-beta-glucanase. The trisaccharide 1 is further chemically transformed into the title compound. Its use as substrate for an enzyme activity assay, the specificity of cleavage, and kinetic parameters are reported. As it undergoes a single glycosidic bond hydrolysis with release of 4-methylumbelliferone, direct UV monitoring of the reaction provides a sensitive kinetic assay of the enzyme action.

Biotechnology↗
PubMed1997

Mechanism of the oxidation of 3,5,3',5'-tetramethylbenzidine by myeloperoxidase determined by transient- and steady-state kinetics.

Earlier investigations of the oxidation of 3,5,3',5'-tetramethylbenzidine (TMB) using horseradish peroxidase and prostaglandin H-synthase have shown the formation of a cation free radical of TMB in equilibrium with a charge-transfer complex, consistent with either a two- or a one-electron initial oxidation. In this work, we exploited the distinct spectroscopic properties of myeloperoxidase and its oxidized intermediates, compounds I and II, to establish two successive one-electron oxidations of TMB. By employing stopped-flow techniques under transient-state and steady-state conditions, we also determined the rate constants for the elementary steps of the myeloperoxidase-catalyzed oxidation of TMB at pH 5.4 and 20 degrees C. The second-order rate constant for compound I formation from the reaction of native enzyme with H2O2 is 2.6 x 10(7) M-1 s-1. Compound I undergoes a one-electron reduction to compound II in the presence of TMB, and the rate constant for this reaction was determined to be (3.6 +/- 0.1) x 10(6) M-1 s-1. The spectral scans show that compound II accumulates in the steady state. The rate constant for compound II reduction to native enzyme by TMB obtained under steady-state conditions is (9.4 +/- 0.6) x 10(5) M-1 s-1. The results are applied to a new, more accurate assay for myeloperoxidase based upon the formation of the charge-transfer complex between TMB and its diimine final product.

Animals↗
PubMed2000

A new type of fluorogenic substrates for determination of cellular dipeptidyl peptidase IV (DP IV/CD26) activity.

The stability of cell associated fluorescence is an essential requirement for measurements of cellular enzymatic activity via enzyme catalyzed liberation of fluorophores. Rhodamine 110 (R110), a highly fluorescent xanthene dye, was used to synthesize nonfluorescent dipeptidyl peptidase IV (DP IV) substrates Xaa-Pro-R110-Y allowing the stable covalent binding of the enzymatically released fluorescent R110-Y on cells. All compounds have been characterized as substrates of isolated DP IV with kcat/Km values of about 10(6) M-1.s-1. The hydrophobicity of the residue Y affects the affinity of the substrate to the catalytic site of DP IV. The compounds are characterized as sensitive substrates of cell surface associated DP IV of DP IV rich U-937 cells. The binding of the enzymatically released R110-Y on cells results in a stable cellular fluorescence. This way, the quantitative determination of cell surface associated DP IV activity is possible.

Amino Acids↗
PubMed1993

Syntheses of subtractively modified 2-chloro-4-nitrophenyl beta-maltopentaosides and their application to the differential assay of human alpha-amylases.

Three novel maltopentaosides, 2-chloro-4-nitrophenyl O-(6-deoxy-alpha-D-xylo-hex-5-enopyranosyl)-(1-->4)-tris[O-alpha-D - glucopyranosyl-(1-->4)]-beta-D-glucopyranoside (3), 2-chloro-4-nitrophenyl O-(6-deoxy-alpha-D-glucopyranosyl)-(1-->4)-tris[O- alpha-D-glucopyranosyl-(1-->4)]-beta-D-glucopyranoside (10), and 2-chloro-4-nitrophenyl O-(3,6-anhydro-alpha-D-glucopyranosyl)-(1-->4)-tris[O-alpha-D-glucopyran osyl- (1-->4)]-beta-D-glucopyranoside (26) were synthesized by chemical and enzymatic reactions. Two human alpha-amylases, salivary alpha-amylase (HSA) and pancreatic alpha-amylase (HPA), hydrolyzed 3 and 10 with the same specificity, almost entirely at a single D-glucosidic linkage, but had no hydrolytic activity for 26. Compound 3 was hydrolyzed by each of these amylases at an approximately equal rate, while 10 was hydrolyzed by HSA 4-fold faster than by HPA. Taking advantage of the difference in the hydrolytic rate of 10, we developed a new method for the differential assay of these two human alpha-amylases.

Carbohydrate Sequence↗
PubMed2002

Identification and characterization of allophenylnorstatine-based inhibitors of plasmepsin II, an antimalarial target.

Plasmepsin II is a key enzyme in the life cycle of the Plasmodium parasites responsible for malaria, a disease that afflicts more than 300 million individuals annually. Since plasmepsin II inhibition leads to starvation of the parasite, it has been acknowledged as an important target for the development of new antimalarials. In this paper, we identify and characterize high-affinity inhibitors of plasmepsin II based upon the allophenylnorstatine scaffold. The best compound, KNI-727, inhibits plasmepsin II with a K(i) of 70 nM and a 22-fold selectivity with respect to the highly homologous human enzyme cathepsin D. KNI-727 binds to plasmepsin II in a process favored both enthalpically and entropically. At 25 degrees C, the binding enthalpy (DeltaH) is -4.4 kcal/mol and the entropic contribution (-TDeltaS) to the Gibbs energy is -5.56 kcal/mol. Structural stability measurements of plasmepsin II were also utilized to characterize inhibitor binding. High-sensitivity differential scanning calorimetry experiments performed in the absence of inhibitors indicate that, at pH 4.0, plasmepsin II undergoes thermal denaturation at 63.3 degrees C. The structural stability of the enzyme increases with inhibitor concentration in a manner for which the binding energetics of the inhibitor can quantitatively account. The effectiveness of the best compounds in killing the malaria parasite was validated by performing cytotoxicity assays in red blood cells infected with Plasmodium falciparum. EC50s ranging between 6 and 10 microM (3-6 microg/mL) were obtained. These experiments demonstrate the viability of the allophenylnorstatine scaffold in the design of powerful and selective plasmepsin inhibitors.

Amides↗
PubMed1984

Improved version of the Kean partition assay for cerebroside sulfate.

In the Kean method for the colorimetric determination of sulfatide (1968. J. Lipid Res. 9: 319-327), the lipid is partitioned together with a blue cationic compound between two phases formed from chloroform, methanol, and water. The blue cation enters the chloroform-rich phase only as an ion pair with the lipid. This method has been improved by the use of a new mixture of solvents in which the desired layer floats above the excess dye. The lower volatility of the new solvent system improves the reproducibility of the technique.

Brain Chemistry↗
PubMedDate not supplied

["External chromophores" and intensive bands in circular dichroism spectra of liquid-crystalline microphases of nucleic acids].

From the analysis of CD-spectra of liquid crystalline microphases formed from DNA molecules in complexes with "external chromophores" requirements are formulated for the compounds to be used in revealing peculiarities of spatial structure of nucleic acids liquid crystalline microphases. These requirements satisfied it is possible to record the CD-spectra having intensive bands in the regions of chromophores absorption. These bands prove the helical pattern of the spatial structure of nucleic acid liquid crystalline microphase.

Chemical Phenomena↗
PubMed1991

The possible involvement of trypsin-like enzymes in germination of spores of Bacillus cereus T and Bacillus subtilis 168.

Germination of spores of Bacillus cereus T and Bacillus subtilis 168 was inhibited by the trypsin inhibitors leupeptin and tosyllysine chloromethyl ketone (TLCK) and by the substrates tosylarginine methyl ester (TAME), benzoyl-L-arginine-p-nitroanilide (L-BAPNA) and D-BAPNA. Potencies of these inhibitory compounds were estimated by finding the concentration which inhibited 50% germination (ID50), as measured by events occurring early (loss of heat resistance), at an intermediate stage [dipicolinic acid (DPA) release], and late in germination (decrease in optical density). In B. cereus T, all the compounds inhibited early and late events with the same ID50. In B. subtilis, TAME inhibited early and late events at the same ID50, but all other inhibitors had a lower ID50 for late events than for early events. This suggests that a trypsin-like enzyme activity is involved at two sequential stages in the germination of B. subtilis spores, one occurring at or before the loss of heat resistance and one at or before the decrease in optical density. Different trypsin-like activities were detected in broken dormant spores and germinated spores of B. cereus T and in germinated spores of B. subtilis by means of three chromogenic substrates: benzoyl-L-phenylalanyl-L-valyl-L-arginine-p-nitroanilide (L-PheVA), L-BAPNA and D-BAPNA. Separation of extracts of germinated spores on non-denaturing polyacrylamide gels showed that in both species the substrates were hydrolysed by three distinct enzymes with different electrophoretic mobilities. The three enzymes had different Ki values for the above inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

Arginine↗
PubMed1997

Estrone sulfatase: probing structural requirements for substrate and inhibitor recognition.

The enzyme-catalyzed desulfation of steroids is a transformation that plays an important role in steroid biosynthesis. Conversion of steroid sulfates to unconjugated steroids may provide a source of steroids for processes such as steroid transport and the growth and proliferation of breast cancer. Steroid sulfatase catalyzes the hydrolysis of 3beta-hydroxysteroid sulfates. To identify structural features important in enzyme-inhibitor interaction, a variety of steroidal and non-steroidal phosphate esters were synthesized and tested as inhibitors of steroid sulfatase activity. We report that the basic structure for enzyme-inhibitor binding does not include the steroid nucleus. Furthermore, the hydrophobicity of the non-steroidal phosphates was determined to be an important factor for optimal inhibition. The monoanionic form of the phosphorylated compounds was found to be the inhibitory species. The best non-steroidal inhibitor of steroid sulfatase activity was n-lauroyl tryamine phosphate with a Ki of 3.6 microM and 520 nM at pH 7.5 and 7.0. The poorest non-steroidal based inhibitor of sulfatase activity was tetrahydronaphthyl phosphate with a Ki of 870 and 360 microM at pH 7.5 and 7.0.

Chromogenic Compounds↗
PubMed2003

Chemical crosslinking of urokinase to pulmonary surfactant protein B for targeting alveolar fibrin.

Intraalveolar fibrin formation is a consistent finding in acute inflammatory and chronic interstitial lung disease. Polymerization of fibrin in the presence of pulmonary surfactant results in far-reaching incorporation of the hydrophobic surfactant compounds into the growing fibrin matrix, with loss of surface activity, altered fibrin structure and reduced susceptibility of the clot to fibrinolysis. For specific targeting of such alveolar fibrin, we designed a hybrid molecule consisting of the catalytic domain of urokinase (B-chain) and the hydrophobic surfactant protein B (SP-B), termed SPUC. The urokinase B-chain, obtained by limited reduction of human two-chain-urokinase (u-PA) and subsequent affinity purification, was chemically coupled to SP-B in a semi-organic solvent system using a hetero-bifunctional crosslinker. Purification of the chimeric proteins included reversed phase and cation exchange chromatography. SDS-PAGE and Western Blotting with immunostaining were employed for biochemical characterization of the conjugate. Chromogenic substrate assays, (125)I-based fibrin plate assays, active site titration and surface tension measurements (pulsating bubble surfactometer) were performed to analyze the specific fibrinolytic activity of the conjugate and its surface activity. SPUC was found i) to be assembled stoichiometrically in a 1: 1 fashion (SP-B: u-PA), ii) to fully retain the biophysical activity as compared to native SP-B and iii) to also retain the fibrinolytic activity. SPUC was 2-3 fold more effective in lysis of surfactant containing clots and 5-fold more resistant against plasminogen activator 1 (PAI-1) as compared to the native u-PA. We conclude that urokinase and SP-B can be chemically crosslinked, thereby yielding a fibrinolytic enzyme suitable for targeting alveolar fibrin.

Chromogenic Compounds↗
PubMed1996

Chemical nature of the light emitter of the Aequorea green fluorescent protein.

The jellyfish Aequorea victoria possesses in the margin of its umbrella a green fluorescent protein (GFP, 27 kDa) that serves as the ultimate light emitter in the bioluminescence reaction of the animal. The protein is made up of 238 amino acid residues in a single polypeptide chain and produces a greenish fluorescence (lambda max = 508 nm) when irradiated with long ultraviolet light. The fluorescence is due to the presence of a chromophore consisting of an imidazolone ring, formed by a post-translational modification of the tripeptide -Ser65-Tyr66-Gly67-. GFP has been used extensively as a reporter protein for monitoring gene expression in eukaryotic and prokaryotic cells, but relatively little is known about the chemical mechanism by which fluorescence is produced. To obtain a better understanding of this problem, we studied a peptide fragment of GFP bearing the chromophore and a synthetic model compound of the chromophore. The results indicate that the GFP chromophore consists of an imidazolone ring structure and that the light emitter is the singlet excited state of the phenolate anion of the chromophore. Further, the light emission is highly dependent on the microenvironment around the chromophore and that inhibition of isomerization of the exo-methylene double bond of the chromophore accounts for its efficient light emission.

Amino Acid Sequence↗
PubMed1994

[ANSA analysis. II. Aminonaphthalenesulfonamides--detecting groups for polysubstrate analysis of proteases].

The properties and synthetic methods of aminonaphthalenesulfonamides (ANSA) used as detectable groups of protease substrates are described. A list of chemical and physical properties of seventeen 5.1-ANSA with simple substituents is presented. A comparison of condition for the introduction and removal of acyl protecting groups (acetyl, trifluoroacetyl, phthaloyl, carbobenzoxy) used in ANSA synthesis is given. Examples of applicability of nitronaphthalenesulfonamides as intermediate compounds are given. The possibility of ANSA alkylation at both N(C) and N(S) is demonstrated. Substituted ANSA--sulfonylaziridenes--are used for the production of water-soluble derivatives containing the alcoxy group in the sulfonamide fragment. Criteria for the selection of detectable groups for polysubstrate analysis are discussed. Eighteen typical procedures for ANSA synthesis according to the schemes discussed are presented.

Alkylation↗
PubMed1994

Binding and activation of plasminogen on the surface of osteosarcoma cells.

Plasmin (Pm) is a broad action serine protease implicated in numerous physiological functions. In bone, Pm may play a role in growth, resorption, metastasis, and the activation of growth factors. The various components of the Pm system are known to bind and function on the cell surface of various cell types, but no pertinent data are available describing membrane-bound Pm or its zymogen, plasminogen (Pg), in either normal or neoplastic bone cells. We report here that Pg binds to the surface of the human osteosarcoma cell line MG-63 and is activated to Pm by endogenous urokinase plasminogen activator (uPA). These conclusions are based on experiments utilizing radiolabeled compounds and a cell surface proteolytic assay measuring amidolytic activity of Pm. 125I-Pg binding to cells was time dependent, saturable, reversible, and specific. Binding was characterized by a relatively low affinity (Kd approximately 0.9 microM) and a high capacity (approximately 7.5 x 10(6) sites/cell). The binding of 125I-Pg was associated with lysine binding sites of the plasminogen molecule. Activation of 125I-Pg to 125I-Pm occurred on the cell surface and was dependent upon cell bound uPA, as determined by inhibitory antibodies. Binding of Pg to MG-63 monolayers represented approximately 80% bound specifically to the cell surface and the remainder to the surrounding extra-cellular matrix. Either co-incubation with uPA or pre-incubation with Pm resulted in increased 125I-Pg binding to osteosarcoma cells. Cell surface Pm proteolytic activity was confirmed by an amidolytic chromogenic assay. Both Pm and Pg bound to cells with Pg being activated by endogenous uPA. Plasmin activated on the cell surface was partially protected from inhibition by alpha 2-antiPm (requiring Pm lysine binding site interaction) but inhibited by aprotinin, (interacting directly with the Pm catalytic site). Resistance of cell bound Pm to alpha 2-antiPm inhibition suggests that cell surface proteolysis can occur in the presence of a soluble Pm inhibitor known to exist in the extracellular space. Based on these results, we speculate that the various bone physiological processes implicating Pm may occur at or near the bone cell surface.

Antibodies↗
PubMed1986

10-Acetyl-10-hydroxyxantho[2,3-f]tetralin 8-glycosides as angular chromophore analogues of anthracyclines: synthesis, redox properties, microsomal oxygen consumption, and antileukemic evaluation.

10-Acetyl-7,8-dihydroxyxantho[2,3-f]tetralin is obtained by photo-Fries rearrangement of an acylated and double ketal protected tetralin followed by sodium thiocresylate catalyzed rearrangement of the resulting benzoyltetralin. Introduction of the 10-hydroxy function with base, triethyl phosphite, and molecular oxygen affords six products. These include the desired epimeric 10-acetyl-7,8,10-trihydroxyxantho[2,3-f]tetralins in addition to products resulting from novel valence tautomerism and cycloreversion reactions in the oxidation reaction. Glycosidic coupling to the fully functionalized cis-8,10-dihydroxy epimer of the aglycon to protected chlorodaunosamine by a modified Koenigs-Knorr method proceeded satisfactorily. By contrast the epimeric trans-8,10-dihydroxy compound failed to undergo coupling under these conditions. This is attributed to facile competing intramolecular hemiketal formation in the latter case. The new angular glycosides are very resistant to electrochemical reduction and display very low (3-10%) augmentation of hepatic microsomal oxygen consumption relative to doxorubicin. The observed, albeit low, cytotoxicity against leukemia L1210 in cell culture provides an additional example where the presence of the quinone moiety in the parent anthracyclines, which is implicated in the clinical cardiotoxicity, may not be necessary for the expression of anticancer properties.

Animals↗
PubMed1985

[A new fluorogenic substrate for human galactocerebroside-beta-D-galactosidase].

A new fluorogenic compound--6-hexadecanoylamino-4-methyl-umbelliferyl-beta-D-gala cto pyranoside (HMGal), a substrate for human galactocerebroside beta-D-galactosidase (HG), has been synthesized. A method for determining the HG activity based on the use of HMGal as a fluorogenic substrate has been developed. The specificity of HMGal hydrolysis by HG has been demonstrated in experiments with enzyme preparations from human skin fibroblasts and leukocytes in normally and in hereditary glycolipidosis (GM1-gangliosidosis and Krabbe's disease). The use of HMGal permits to markedly increase the sensitivity of the method used for determining the HG activity.

Chemical Phenomena↗
PubMed1989

Reevaluation of the phenol-sulfuric acid reaction for the estimation of hexoses and pentoses.

Evidence is provided to show that in the conventional phenol-sulfuric acid reaction procedure, phenol underwent sulfonation in situ and the phenolsulfonic acid formed decreased the color intensity for hydroxymethyl furfural (HMF), furfural, and many hexoses and pentoses tested. A modified method is described to overcome this problem in which phenol was added after the dehydration of carbohydrates by sulfuric acid and after cooling the system. The color intensity around 475-485 nm for different compounds was fairly proportional to the amount of furfural derivatives (absorption at 310-320 nm) formed from the sugars in the modified method unlike in the conventional procedure. The studies also show that for condensation of HMF derivatives with phenol, heat is not necessary. The color intensity in the modified method also increased compared to that in the conventional method. The increase in the modified method compared to that in the conventional method was 6.0-fold for furfural, 9.1-fold for hydroxymethyl furfural, 3.7-fold for fructose, 2.3-fold for xylose, and 2.0-fold for glucose and arabinose. The possible reasons for this differential increase are discussed.

Chemical Phenomena↗
PubMed1983

Quantitative analysis of DNA-cleaving activities of macromomycin, auromomycin, and their free chromophores, and some DNA-binding characteristics.

The in vitro DNA-cutting activities of macromomycin (MCR), auromomycin (AUR), and their free chromophores were quantitatively analyzed by agarose gel electrophoresis of radioactive supercoiled pBR322 DNA. Methanol extract (chromophore) of MCR inhibited growth of cultured L5178Y cells and caused DNA strand scission without addition of reducing agents, whereas MCR required dithiothreitol (DTT) for its DNA cleavage. In the absence of DTT, concentrations of MCR chromophore, AUR and AUR chromophore to induce 50% degradation of form I DNA were 320 micrograms eq./ml, 44 micrograms/ml and 60 micrograms eq./ml, respectively. DTT stimulated DNA breakage by the drugs to a similar degree (ca. 4-fold). Since both chromophores showed the same biological activity, further studies were performed with AUR and its free chromophore. DNA strand breakage by AUR occurred rapidly and completed in 5 minutes. The rate of strand scission did not significantly change in a range of pH 7.0 to 10.5, but gradually declined below pH 7.0. Of 4 sulfhydryl compounds tested, DTT and cysteine exhibited potent stimulation, but glutathione and 2-mercaptoethanol weak or no enhancement. The DNA-cleaving activity of AUR and its free chromophore is blocked by addition of ethidium bromide, suggesting that the chromophore binds to DNA by an intercalation mechanism. Addition of poly[dG], poly [dG] X poly[dC], poly [dG-dC] X poly[dG-dC] or poly[dA-dT] X poly[dA-dT] protected DNA against degradation by AUR. Differential protecting effects of synthetic polynucleotides suggest that the chromophore shows higher affinity for guanine than for adenine, cytosine and thymine, and binds preferentially to a purine-pyrimidine neighboring sequence.

Animals↗