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PubMedDate not supplied

Structural effects of carbohydrate-containing polycations on gene delivery. 1. Carbohydrate size and its distance from charge centers.

Cationic polymers have the ability to bind plasmid DNA (pDNA) through electrostatic interactions and condense it into particles that can be readily endocytosed by cultured cells. The effects that polycation structure has on toxicity and gene delivery efficiency are investigated here by synthesizing a series of amidine-based polycations that contain the carbohydrates d-trehalose and beta-cyclodextrin (CD) within the polycation backbone. The carbohydrate size (trehalose vs CD) and its distance from the charge centers affect the gene delivery behavior in BHK-21 cells. It is found that as the charge center is further removed from the carbohydrate unit, the toxicity is increased. Also, as the size of the carbohydrate moiety is enlarged from trehalose to beta-cyclodextrin, the toxicity is reduced. The absence of a carbohydrate in the polycation produces high toxicity. All carbohydrate polycations transfect BHK-21 cells to approximately the same level of gene expression.

Animals↗
PubMedDate not supplied

Structural effects of carbohydrate-containing polycations on gene delivery. 3. Cyclodextrin type and functionalization.

Linear cationic beta-cyclodextrin (beta-CD)-based polymers can form polyplexes with plasmid DNA and transfect cultured cells. The effectiveness of the gene delivery and the cellular toxicity has been related to structural features in these polycations. Previous beta-CD polycations were prepared from the cocondensation of 6(A),6(D)-dideoxy-6(A),6(D)-diamino-beta-CD monomers with other difunctionalized monomers such as dimethyl suberimidate (DMS). Here, the type of CD and its functionalization are varied by synthesizing numerous 3(A),3(B)-dideoxy-3(A),3(B)-diamino-beta- and gamma-CD monomers. Both alkyl- and alkoxydiamines are prepared in order to vary the nature of the spacing between the CD and the primary amines in the monomers. These diamino-CD-monomers are polymerized with DMS to yield amidine-based polycations. The nature of the spacer between the CD-ring and the primary amines of each monomer is found to influence both molecular weight and polydispersity of the polycations. When these polycations are used to form polyplexes with plasmid DNA, longer alkyl regions between the CD and the charge centers in the polycation backbone increase transfection efficiency and toxicity in BHK-21 cells, while increasing hydrophilicity of the spacer (alkoxy versus alkyl) provides for lower toxicity. Further, gamma-CD-based polycations are shown to be less toxic than otherwise identical beta-CD-based polycations.

Animals↗
PubMedDate not supplied

Sequence-recognition and cleavage of DNA by a netropsin-phenazine-di-N-oxide conjugate.

We report the synthesis, DNA-binding and cleaving properties, and cytotoxic activities of R-128, a hybrid molecule in which a bis-pyrrolecarboxamide-amidine element related to the antibiotic netropsin is covalently tethered to a phenazine-di-N-oxide chromophore. The affinity and mode of interaction of the conjugate with DNA were investigated by a combination of absorption spectroscopy, circular dichroism, and electric linear dichroism. This hybrid molecule binds to AT-rich sequences of DNA via a bimodal process involving minor groove binding of the netropsin moiety and intercalation of the phenazine moiety. The bidentate mode of binding was evidenced by linear dichroism using calf thymus DNA and poly(dA-dT).(dA-dT). In contrast, the drug fails to bind to poly(dG-dC).poly(dG-dC), because of the obstructive effect of the guanine 2-amino group exposed in the minor groove of this polynucleotide. DNase I footprinting studies indicated that the conjugate interacts preferentially with AT-rich sequences, but the cleavage of DNA in the presence of a reducing agent can occur at different sequences not restricted to the AT sites. The main cleavage sites were detected with a periodicity of about 10 base pairs corresponding to approximately one turn of the double helix. This suggests that the cleavage may be dictated by the structure of the double helix rather than the primary nucleotide sequence. The conjugate which is moderately toxic to cancer cells complements the tool box of reagents which can be utilized to produce DNA strand scission. The DNA cleaving properties of R-128 entreat further exploration into the use of phenazine-di-N-oxides as tools for investigating DNA structure.

Animals↗
PubMed1991

Kinetic characterization of phospholipase A2 modified by manoalogue.

Manoalogue, a synthetic analogue of the sea sponge-derived manoalide, has been previously shown to partially inactivate the phospholipase A2 from cobra venom (Reynolds, L. J., Morgan, B. P., Hite, E. D., Mihelich, E. D., & Dennis, E. A. (1988) J. Am. Chem. Soc. 110, 5172) by reacting with enzyme lysine residues. In the present study, the inactivation of the phospholipases A2 from pig pancreas, bee venom, and cobra (Naja naja naja) venom by manoalogue was studied in detail. Manoalogue-treated enzymes were examined in the scooting mode on vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol. Here the native enzymes bound irreversibly to the vesicles and hydrolyzed all of the phospholipids in the outer monolayer without leaving the surface of the interface. All three manoalogue-treated enzymes showed reduced catalytic turnover for substrate hydrolysis in the scooting mode, and the modified enzymes did not hop from one vesicle to another. Thus, inactivation by manoalogue is not due to the decrease in the fraction of enzyme bound to the substrate interface. This result was also confirmed by fluorescence studies that directly monitored the binding of phospholipase A2 to vesicles. A chemically modified form of the pig pancreatic phospholipase A2 in which all of the lysine epsilon-amino groups have been amidinated was not inactivated by manoalogue, indicating that the modification of lysine residues and not the amino-terminus is required for the inactivation. Several studies indicated that the manoalogue-modified enzymes contain a functional active site. For example, studies that monitored the protection by ligands of the active site from attack by a alkylating agent showed that manoalogue-modified pig phospholipase A2 was capable of binding calcium, a substrate analogue, lipolysis products, and a competitive inhibitor. Furthermore, relative to native enzymes, manoalogue-modified enzymes retained significantly higher catalytic activities when acting on water-soluble substrates than when acting on vesicles in the scooting mode. Intact manoalogue had no affinity for the catalytic site on the enzyme as it did not inhibit the enzyme in the scooting mode and it did not protect the active site from alkylation. Pig pancreatic phospholipase A2 bound to micelles of 2-hexadecyl-sn-glycero-3-phosphocholine was resistant to inactivation by manoalogue, suggesting that the modification of lysine residues on the interfacial recognition surface of the enzyme was required for inactivation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Binding of a distamycin-ellipticine hybrid molecule to DNA and chromatin: spectroscopic, biochemical, and molecular modeling investigations.

A bifunctional molecule in which an ellipticine chromophore is attached to a distamycin residue via a diaminopropyl tether has been designed and synthesized in the expectation of creating a hybrid molecule capable of bidentate binding to DNA by both intercalation and minor-groove interactions. The strength and mode of binding to DNA of this conjugate have been studied by means of circular and linear dichroism as well as by stopped-flow kinetics and measurements of reactivity toward a chemical probe. The results converge to reveal that the ellipticine moiety of the hybrid largely dominates the binding reaction with DNA. In the presence of chromatin, the hybrid molecule binds preferentially to the internucleosomal DNA, a preference dictated by its intercalating chromophore. Theoretical computations were performed on the comparative complexation energies of distamycin, the ellipticine derivative, and the hybrid ligand with a B-representative octanucleotide, d(GCATATGC)2. The best binding configuration of the ellipticine derivative locates its aminoalkyl side chain in the minor groove where distamycin is also present. The molecular modeling analysis fully supports the involvement of a bimodal binding process for the hybrid and reveals that the binding of the conjugate to DNA favors a pronounced bending toward the minor groove. This effect is attributed to intercalation of the ellipticine chromophore. An interesting link is established between the DEPC reactivity experiments and the theoretical computations, suggesting that DEPC can be used as a probe for drug-induced DNA bending. On the basis of these results, we propose the design of a new hybrid ligand bearing an additional positively-charged amidine side chain to confer higher DNA-binding affinity.

Autoradiography↗
PubMed1982

Quaternary structure of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus studied by a new reversible cross-linking procedure with bis(imidoesters).

The bis(amidine) cross-links formed between protein subunits by treating them with bis(imidoesters) were found to be rapidly broken by exposing the cross-linked proteins to methylamine buffers containing the aprotic solvent acetonitrile. This cleavage step could be introduced between the two dimensions of a diagonal gel electrophoretic separation of cross-linked proteins to facilitate identification of the contributors to a cross-linked species. Tests with the tetrameric enzyme glyceraldehyde-3-phosphate dehydrogenase demonstrated the simplicity and effectiveness of the technique. When the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus was treated with a range of bis(imidoesters), from dimethyl succinimidate to dimethyl suberimidate, the most informative set of products was obtained with dimethyl glutarimidate. The longer bis(imidoesters) caused too extensive cross-linking of the enzyme subunits, although the beta chain of the pyruvate decarboxylase component always appeared to be the most resistant. Almost all the cross-linked species up to pentamers of the lipoate acetyltransferase polypeptide chain (apparent Mr approximately 280 000) were identified by means of the diagonal gel electrophoretic procedure after cleavage of the cross-links. The introduction of the methylamine cleavage step enables the bis(imidoester) for such experiments to be selected purely for the efficacity of its cross-linking reaction with the protein and dispenses with the need to incorporate a specially cleavable bond in the reagent.

Chemical Phenomena↗
PubMed1984

Specificity of chicken liver carbohydrate binding protein.

Chicken hepatic lectin was isolated with affinity chromatography by using neoglycoproteins of bovine serum albumin (BSA) to which n moles of glycosides has been attached by amidination (Glycn-AI-BSA) [Lee, Y. C., Stowell, C. P., & Krantz, M. J. (1976) Biochemistry 15, 3956-3963] attached to Sepharose 4B. The same protein could be isolated from Man-, GlcNAc-, and Glc-AI-BSA-Sepharose columns and was identical with the protein previously reported [Kawasaki, T., & Ashwell, G. (1977) J. Biol. Chem. 252, 6536-6543]. The sugar specificity for binding to the isolated chicken hepatic lectin examined with Glycn-AI-BSA showed the order of potency for binding Glycn-AI-BSA to be D-GlcNAc greater than D-Glc, D-Man, L-Fuc greater than D-Gal, and the estimated Ki's for binding GlcNAc36-AI-BSA, Glc37-AI-BSA, Man33-AI-BSA, and L-Fuc28-AI-BSA were (6-20) X 10(-11), (2-3) X 10(-8), (3-9) X 10(-8), and 5 X 10(-8) M, respectively. The binding requirements of the binding protein were studied with a wide variety of Glycn-BSA's with different sugars and aglyconic linkages, as well as simple sugars and glycosides. It was concluded that (1) GlcNAc is the most potent sugar for binding, (2) the requirement for C-2 substituents is flexible, (3) an equatorial OH group at C-3 and C-4 must be present, (4) the 5-CH2OH group is not required for binding, (5) the lectin cannot accommodate a negative charge at C-6, and (6) D-Man and L-Fuc bind equally well.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1985

Reaction of electron-transfer flavoprotein with electron-transfer flavoprotein-ubiquinone oxidoreductase.

The oxidative half-reaction of electron-transfer flavoprotein (ETF), electron transfer from ETF to electron-transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO), is dependent on complementary surface charges on the two proteins. ETF is the positively charged member of the redox pair. The evidence is based on the pH and ionic strength dependencies of the comproportionation of oxidized ETF and ETF hydroquinone catalyzed by ETF-QO and on the effects of chemical modification of ETF on the comproportionation reaction. Acetylation of one and five epsilon-amino groups of lysyl residues results in 3- and 13-fold increases, respectively, in the Km of ETF-QO for ETF but no change in Vmax. Amidination, which maintains positive charge at modified loci, has no effect on steady-state kinetic constants. These chemical modifications have no effect on the equilibrium constant for equilibration of ETF redox states. The Km of ETF-QO for ETF is pH dependent above pH 8.5, suggesting titration of lysyl residues as previously observed in studies of the reductive half-reaction of ETF [Beckmann, J. D., & Frerman, F. E. (1983) J. Biol. Chem. 258, 7563-7569]. The ionic strength dependence of TN/KmETF for the reaction follows the limiting Brønsted equation ln (TN/Km) = ln k0 + 2 alpha Z1Z2I1/2, and Z1Z2, the product of charges on the reacting proteins, is similar to the value of Z1Z2 for the reductive half-reaction of ETF by the general acyl-CoA dehydrogenase. The ETF-QO-catalyzed comproportionation reaction exhibits a primary deuterium isotope effect in D2O, perhaps indicating the participation of solvent water in the electron-transfer reaction.

Acyl-CoA Dehydrogenases↗
PubMed1986

Substrate specificity of formylglycinamidine synthetase.

Formylglycinamidine ribonucleotide (FGAM) synthetase, which catalyzes the conversion of formylglycinamide ribonucleotide (FGAR), glutamine, and ATP to FGAM, ADP, glutamate, and Pi, has been purified to homogeneity (sp act. 0.20 mumol min-1 mg-1) from chicken liver by an alternative procedure to that of Buchanan et al. [Buchanan, J. M., Ohnoki, S., & Hong, B. S. (1978) Methods Enzymol. 51, 193-201] (sp act. 0.12 mumol min-1 mg-1). A variety of new analogues of formylglycinamide ribonucleotide have been prepared in which the formylglycinamide arm (R = CH2NHCHO) has been replaced by R = CH3, CH2OH, CH2Cl, CH2NH3, CH2NHCOCH3, CH2NHCOCH2Cl, CH2NHCO2CH2Ph, and L-CHC-H3NHCHO. These compounds have been characterized by 1H and 13C NMR spectroscopy. With compounds R = CH3, CH2OH, and CH2NHCOCH3 and ATP, in the presence or absence of glutamine, FGAM synthetase catalyzes the production of Pi at 4.5, 48, and 20%, respectively, the rate of production of Pi from formylglycinamide ribonucleotide. Only R = CH2NHCOCH3 causes glutaminase activity as well as ATPase activity and has been shown to be converted to the amidine analogue. Both FGAR (R = CH2NHCHO) and the FGAR analogue (R = CH2NHCHOCH3) in the presence of ATP and FGAM synthetase and in the absence of glutamine form a complex isolable by Sephadex G-50 chromatography. FGAM synthetase is thus highly specific for its formylglycine side chain. [18O]-beta-FGAR was prepared biosynthetically, and FGAM synthetase was shown by 31P NMR spectroscopy to catalyze the transfer of amide 18O to inorganic phosphate.

Adenosine Triphosphatases↗
PubMed1986

Kinetics of native and modified liver alcohol dehydrogenase with coenzyme analogues: isomerization of enzyme-nicotinamide adenine dinucleotide complex.

Coenzyme analogues with the adenosine ribose replaced with n-propyl, n-butyl, and n-pentyl groups; coenzyme analogues with the adenosine replaced with 3-(4-acetylanilino)propyl and 6-(4-acetylanilino)hexyl moieties; and nicotinamide mononucleotide, nicotinamide hypoxanthine dinucleotide, and 3-acetylpyridine adenine dinucleotide were used in steady-state kinetic studies with native and activated, amidinated enzymes. The Michaelis and inhibition constants increased up to 100-fold upon modification of coenzyme or enzyme. Turnover numbers with NAD+ and ethanol increased in some cases up to 10-fold due to increased rates of dissociation of enzyme-reduced coenzyme complexes. Rates of dissociation of oxidized coenzyme appeared to be mostly unaffected, but the values calculated (10-60 s-1) were significantly less than the turnover numbers with acetaldehyde and reduced coenzyme (20-900 s-1, at pH 8, 25 degrees C). Rates of association of coenzyme analogues also decreased up to 100-fold. When Lys-228 in the adenosine binding site was picolinimidylated, turnover numbers increased about 10-fold with NAD(H). Furthermore, the pH dependencies for association and dissociation of NAD+ and turnover number with NAD+ and ethanol showed the fastest rates above a pK value of 8.0. Turnover with NADH and acetaldehyde was fastest below a pK value of 8.1. These results can be explained by a mechanism in which isomerization of the enzyme-NAD+ complex (110 s-1) is partially rate limiting in turnover with NAD+ and ethanol (60 s-1) and is controlled by ionization of the hydrogen-bonded system that includes the water ligated to the catalytic zinc and the imidazole group of His-51.

Alcohol Dehydrogenase↗
PubMed1986

Thrombospondin is a substrate for blood coagulation factor XIIIa.

Thrombospondin (TSP) is released from alpha granules of activated platelets, binds to platelet surfaces, and copolymerizes with fibrin. In the present experiments, we investigated the action of factor XIIIa (plasma transglutaminase) on TSP. Factor XIIIa catalyzed incorporation of [14C]putrescine into soluble TSP and ligation of TSP to itself and to fibrin intermediates. Proteolytic digestion of [14C]putrescine-labeled TSP with trypsin or thrombin yielded a labeled disulfide-bonded core of 90 or 120-130 kilodalton (kDa) subunits, labeled fragments of less than 10 kDa, and an unlabeled 30-kDa heparin-binding fragment, indicating the presence of multiple factor XIIIa reactive glutaminyl residues located in several domains of the molecule. TSP became ligated in fibrin clots formed from amidinated fibrinogen, i.e., fibrin that could not contribute lysyl residues to factor IIIa catalyzed cross-links. The disulfide-bonded core of TSP formed upon thrombin digestion copolymerized with fibrin as efficiently as intact TSP. However, a lower proportion of the disulfide-bonded core became ligated. These results indicate that TSP, both in clots and in solution, contributes glutaminyl and lysyl residues to factor XIIIa catalyzed ligation. Cross-linking may be important in stabilizing interactions among TSP, fibrinogen, or fibrin and other molecules in hemostatic plugs.

Blood Platelets↗
PubMed1989

Contribution of the N-terminal region of hirudin to its interaction with thrombin.

Hirudin is a 65-residue polypeptide that specifically inhibits thrombin by forming a tight, noncovalent complex with the enzyme. The role of the two amino-terminal valine residues and the N-terminal alpha-amino group of hirudin in the formation of the complex has been investigated by site-directed mutagenesis and chemical modification. Replacement of the two N-terminal valyl residues of recombinant hirudin by polar amino acids resulted in an increase in the inhibition constant (KI). In contrast, replacement of these residues by hydrophobic amino acids had little effect on the value for KI. These results demonstrated that the hydrophobic nature of the N-terminal residues of hirudin was important for its interaction with thrombin. Addition of a single amino acid to the N-terminus of hirudin resulted in a marked increase in the value of KI. A similar effect was observed when the positive charge of the alpha-amino group was removed by acetylation. In contrast, amidination of this group, which preserves the positive charge, resulted in a less pronounced increase in the value of KI. Thus, it appears that a positive charge immediately adjacent to the N-terminal hydrophobic residue is required for optimal binding to thrombin.

Amino Acid Sequence↗
PubMed1990

Bacterial siderophores: structures of pyoverdins Pt, siderophores of Pseudomonas tolaasii NCPPB 2192, and pyoverdins Pf, siderophores of Pseudomonas fluorescens CCM 2798. Identification of an unusual natural amino acid.

Pyoverdins were isolated and characterized respectively from the cultures of Pseudomonas tolaasii NCPPB 2192 (pyoverdins Pt, Pt A, and Pt B) and Pseudomonas fluorescens CCM 2798 (Pyoverdins Pf/1, Pf/2, Pf, Pf/3/1, and Pf/3/2) each grown in iron-deficient conditions. Their structures were established by using FAB-MS, NMR, and CD techniques. These siderophores are chromopeptides, and all but one (pyoverdin Pf/3/3) possess at the N-terminal end of their peptide chain the same chromophore that has been reported in pyoverdin Pa from Pseudomonas aeruginosa ATCC 15692 [Wendenbaum, S., Demange, P., Dell, A., Meyer, J. M., & Abdallah, M. A. (1983) Tetrahedron Lett. 24, 4877-4880] and pseudobactin B 10 from Pseudomonas B10 [Teintze, M., Hossain, M. B., Barnes, C. L., Leong, J., & Van der Helm, D. (1981) Biochemistry 20, 6446-6457] which is derived from 2,3-diamino-6,7-dihydroxyquinoline. In pyoverdins Pt this chromophore is bound to a linear peptide chain D-Ser-L-Lys-L-Ser-D-Ser-L-Thr-D-Ser-L-OHOrn-L-Thr-D-Ser-D-OHOrn (cyclic) which has its C-terminal end blocked by cyclic D-N delta-hydroxyornithine. In pyoverdins Pf, the peptide chain is also linear, SerCTHPMD-Gly-L-Ser-D-threo-OHAsp-L-Ala-Gly-D-Ala-Gly-L-O HOrn(cyclic), and contains an unusual natural amino acid which is the result of the condensation of 1 mol of serine and 1 mol of 2,4-diaminobutyric acid, forming a cyclic amidine. The pyoverdins Pt differ only in substituent bound to the nitrogen on C-3 of the chromophore, which is succinic acid in pyoverdin Pt A, succinamide in pyoverdin Pt, and alpha-ketoglutaric acid bound to the chromophore by its C-5 carbon atom in pyoverdin Pt B. Similarly, pyoverdin Pf/1, pyoverdin Pf/2, pyoverdin Pf (the major compound), and pyoverdin Pf/3/2 are substituted respectively by L-malic acid, succinic acid, L-malic amide, and succinamide. Pyoverdin Pf/3/3 has the same chromophore as azotobactin, the peptidic siderophore of Azotobacter vinelandii. These pyoverdins are very similar to pseudobactin B 10, the siderophore of Pseudomonas B10: they are linear peptides containing three bidentate groups strongly chelating Fe(III) and blocked at their N-terminal end by the catecholic chromophore and at their C-terminal end by cyclic N delta-hydroxyornithine. They differ therefore from other pyoverdins such as those from P. aeruginosa ATCC 15692 which contain a partly cyclic peptide [Briskot, G., Taraz, K., & Budzikiewicz, H. (1989) Liebigs Ann. Chem., 375-384].

Amino Acid Sequence↗
PubMed1982

Binding site of the rabbit liver lectin specific for galactose/N-acetylgalactosamine.

Neoglycoproteins containing various monosaccharides and disaccharides were prepared by modifying bovine serum albumin (BSA) with thioglycosides using amidination or reductive alkylation [Lee, Y. C., Stowell, C. P., & Krantz, M. J. (1976) Biochemistry 15, 3956-3963; Lee, R. T., & Lee, Y. C. (1980) Biochemistry 19, 156-163]. The binding specificity of the rabbit liver galactose/N-acetylgalactosamine specific lectin was studied with these neoglycoproteins by assaying their ability to inhibit 125I-labeled asialoorosomucoid binding to the isolated lectin or to the crude plasma membrane. The following observations were made: (1) All the galactose-containing neoglycoproteins (except two) had a similar inhibitory effect. (2) BSA derivatives containing glucose, with the sole exception of [Glc--S--CH2C(= NH)--NH]n-BSA, were poorer inhibitors than the galactose-containing BSA's, and the axial 4-OH of galactose appears to participate in the binding. (3) The binding site of the lectin for the C-6 region of galactose is apparently spacious, since a galactose substituted at the 6-OH with another monosaccharide can still be bound. (4) A negatively charged group at C-6 of galactose interfered with the binding of the lectin. (5) A bulky aglycon in an alpha-D-galactopyranoside considerably decreased the inhibitory power. For explanation of these results, it is proposed that the lectin possesses at least one negatively charged group at the binding site and the presence of this group is directly responsible for inability of the sialylated (negatively charged), native glycoproteins to bind to the mammalian hepatic lectin.

Acetylgalactosamine↗
PubMed1982

Direct transfer of one-carbon units in the transformylations of de novo purine biosynthesis.

It is shown that the transfer of formyl units in the de novo purine biosynthetic pathway as catalyzed by glycinamide ribonucleotide (GAR) transformylase and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase probably proceeds through a direct displacement mechanism involving only formyl donor (10-CHO-H4folate) and formyl acceptor (GAR or AICAR). The inability to observe enzyme-catalyzed solvent oxygen incorporation or uncoupling by hydroxylamine of 1:1 stoichiometry between formylated acceptor [formylglycinamide ribonucleotide or 5-(formylamino)imidazole-4-carboxamide ribonucleotide] and deformylated donor implies the absence of an amidine intermediate and suggests that either a formylated enzyme-bound intermediate is not formed or such an intermediate is not accessible to hydroxylamine.

Acyltransferases↗
PubMed1980

Activation of polysaccharides with 2-iminothiolane and its uses.

2-Iminothiolane, a cyclic thioimidate, is known to react readily with amino groups of proteins to give amidinated derivatives containing reactive sulfhydryl groups. In this report it will be shown that 2-iminothiolane also reacts with hydroxyl and sulfhydryl groups. Such a reaction with hydroxyl groups can be used to introduce sulfhydryl groups into polysaccharides. When this reaction is carried out in the presence of 4,4'-dithiodipyridine, polysaccharides containing 4-dithiopyridyl groups can be prepared. These activated polysaccharides couple easily via intermolecular disulfide bond formation with proteins containing thiol or 4-dithiopyridyl groups. The resulting polysaccharide--protein conjugates have good stability, and they are useful reagents for different biochemical applications such as the purification of proteins containing thiol groups and affinity chromatography.

Allergens↗
PubMed1979

Regulation of phospholipase A2 activity by the lipid-water interface: a monolayer approach.

Interfacial regulation of phospholipase A2 activity on lecithin monolayers was investigated by using radioactively labeled enzyme. Labeling of the protein with 125I did not produce a change of the enzyme and protein properties as compared to the 3H fully amidinated phospholipase A2. The induction time observed during pre-steady-state kinetics reflects the rate-limiting step of the penetration of the enzyme in the interface. This penetration is reversible. However, in the surface pressure range where the enzyme is able to hydrolyze the lecithin films, the desorption of the protein from the film is slow as compared to the adsorption. Below a surface pressure of 10 dyn/cm nonspecific adsorption occurs. Using lecithins with fatty acids of different chain lengths, we have shown that the kinetics of the penetration process is governed by the packing density of the substrate molecules independent of the surface pressure. However, the steady-state surface concentration of the enzyme increases with the fatty acyl chain length of the lecithin, indicating that hydrophobic interaction occurs between phospholipase A2 and the lipid molecules at the interface. From the lecithins used pancreatic phospholipase A2 preferentially splits substrate molecules with nine carbon atoms in the acyl chain.

Adsorption↗
PubMed1978

Effect of pH on the cross-bridge arrangement in synthetic myosin filaments.

Synthetic thick filaments were cross-linked with dimethyl suberimidate at various pH values over the range pH 6.8---8.3. The rate of cross-linking myosin heads to the thick filament surface decreases significantly over a narrow pH range (7.4--8.0) despite the fact that the rate of the chemical reaction (amidination of lysine side chains) shows a positive pH dependence. The fall in rate cannot be ascribed to dissociation of the filament during the cross-linking reaction since the sedimentation boundary of the cross-linked filament (pH 8.3) remains unaltered in the presence of high salt (0.5 M). The decreased rate of cross-linking is also not caused by a shift in reactivity of a small number of highly reactive lysine groups, since the time course of cross-linking (pH 7.2) is unaffected by preincubation with a monofunctional imidate ester. Our results suggest that the heads of the myosin molecules move away from the thick filament surface at alkaline pH but are held close to the surface at neutral pH.

Dimethyl Suberimidate↗