Continuous regeneration of NAD(P)+ by flavins covalently bound to sepharose.

Various flavins, FMN, FAD, and acriflavin, were immobilized to Sepharose using several different coupling methods. The only product stable enough to permit extended studies was acriflavin coupled to epoxy-substituted Sepharose. The nonenzymic oxidizing capacity towards NAD(P) H was investigated and a 25% specific activity, compared to that of free acriflavin, was observed. The reduced acriflavin was immediately auto-reoxidized in air and could thus be reused. It was shown that acriflavin-Sepharose preparations function as NAD(P)H oxidizing agents in a number of different dehydrogenase systems including lactate dehydrogenase (LDH), alcohol dehydrogenase (ADH), malate dehydrogenase (MDH), alanine dehydrogenase (alaDH), and glutamate dehydrogenase (GDH). The amount of expensive coenzyme necessary for high product formation of such systems was thereby markedly reduced.     

Protease covalently coupled to porous glass: Preparation and characterization

A microbial protease was immobilized by covalent attachment, to porous glass. This material was characterized for pH optimum, thermal stability, and operational half–life using casein as substrate. The immobilized enzyme was used for preparation of soya hydrolysates, low in free amino acids with high solubility.     

Invertase covalently coupled to porous glass: preparation and characterization

The enzyme invertase has been covalently coupled to porous glass particles. The product is extremely stable over a long period of time. Kinetic values for the immobilized enzyme are similar to the native enzyme. Excellent enzymatic activity for the immobilized enzyme was exhibited over a broad pH range. The immobilized enzyme when continuously operated for one month was found to have an operational half-life of over 40 days.     

Identification and characterization of a protein covalently bound to DNA of minute virus of mice.

We identified a protein which is covalently linked to a fraction of the DNA synthesized in cells infected with minute virus of mice. This protein is specifically bound to the 5' terminus of the extended terminal conformers of the minute virus of mice replicative-form DNA species and of a variable fraction of single-stranded viral DNA. The chemical stability of the protein-DNA linkage is characteristic of a phosphodiester bond between a tyrosine residue in the protein and the 5' end of the DNA. The terminal protein (TP) bound on all DNA forms has a relative molecular weight of 60,000; it is also seen free in extracts from infected cells. Immunologic comparison of the TP with the other known viral proteins suggests that the TP is not related to the capsid proteins or NS-1.     

Phosphorus 31 solid state NMR characterization of oligonucleotides covalently bound to a solid support.

31P cross polarization (CP) magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra were acquired for various linear and branched di- and tri-nucleotides attached to a controlled pore glass (CPG) solid support. The technique readily distinguishes the oxidation state of the phosphorus atom (phosphate versus phosphate), the presence or absence of a protecting group attached directly to phosphorus (cyanoethyl), and other large changes in the phosphorus chemistry (phosphate versus phosphorothioate). However, differences in configurational details remote from the phosphorus atom, such as the attachment position of the ribose sugar (2'5' versus 3'5'), or the particulars of the nucleotide bases (adenine versus uridine versus thymine), could not be resolved. When different stages of the oligonucleotide synthetic cycle were examined, 31P CPMAS NMR revealed that the cyanoethyl protecting group is removed during the course of chain assembly.     

Studies on acetylcholinesterase and cholinesterase covalently bound to polymaleinic anhydride.

Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) and cholinesterase (acylcholine acylhydrolase, EC 3.1.1.8), respectively, were covalently attached to a cross-linked copolymerisate of maleinic anhydride and butanediol-divinylether. Based on the coupling procedure reported by Brümmer et al. (Brummer, W., Hennrich, N., Klockow, M., Lang, H. and Orth, H.D. (1972) Eur. J. Biochem. 2k, 129--135), a simple method is described which requires only 24 h for completion and provides a sufficient yield. Although a polyanionic carrier was used the Km and k2 values as well as the substrate and pH optima of the bound acetylcholinesterase and bound cholinesterase did not differ considerably from the corresponding values of the free enzymes. Bound acetylcholinesterase and to some extent also bound cholinesterase did not lose any enzymatic activity after storage in saline at 4 degrees C for 140 days.     

Properties of trout hemoglobin covalently bound to a solid matrix.

This paper reports the ligand binding properties of the major hemoglobin component from trout (Salmo irideus) covalently bound to a solid matrix (Sepharose or Sephadex). A comparison between the functional properties of this protein in solution and of the protein-matrix complex shows significant changes although the basic properties of the molecule are maintained on covalent binding to Sepharose (or Sephadex). Thus the Root effect, characteristic of Hb trout IV, is still present while the heme-heme interactions are, on the average, smaller in the matrix bound protein as compared to the soluble form. No differences in the O2 binding properties were observed when the protein was coupled to the resin, as the ligand bound or as the ligand free derivative. Although an unequivocal interpretation of the data is made difficult by the lack of information on the number and identity of the groups involved in the coupling, the main changes in the protein functional properties may be related to the chemical modifications "per se" more than to the immobilization imposed to the macromolecule by coupling to the matrix. Structural changes which mainly involve perturbation of the tertiary structure of the molecule may qualitatively rationalize the data.     

Activation of the P2X7 ion channel by soluble and covalently bound ligands

The homotrimeric P2X7 purinergic receptor has sparked interest because of its capacity to sense adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD) released from cells and to induce calcium signaling and cell death. Here, we examine the response of arginine mutants of P2X7 to soluble and covalently bound ligands. High concentrations of ecto-ATP gate P2X7 by acting as a soluble ligand and low concentrations of ecto-NAD gate P2X7 following ADP-ribosylation at R125 catalyzed by toxin-related ecto-ADP-ribosyltransferase ART2.2. R125 lies on a prominent cysteine-rich finger at the interface of adjacent receptor subunits, and ADP-ribosylation at this site likely places the common adenine nucleotide moiety into the ligand-binding pocket of P2X7.     

Analysis of ligand-binding data without knowledge of bound or free ligand molar concentration

A method is proposed to set the parameters in a nonlinear regression procedure to determine the equilibrium dissociation constant (Kd) and the high affinity receptor concentration (Bmax) of systems consisting of one ligand, one high affinity receptor, and n low affinity binding sites. This method is suitable when neither bound or free ligand formal concentrations nor the maximum of the binding signal can be deduced from the experimental data. The method makes use of (i) the abscissa of the first inflection point of the plot of any signal proportional to the binding of ligand to receptors versus the logarithm of the total ligand concentration, and (ii) the initial slope of the saturation curve plotted in direct coordinates. We first demonstrate that when such an inflection point exists, its abscissa lies between Bmax/2 + Kd(1 + d) and Bmax + 2Kd(1 + d), where d is a parameter representative of the binding to the low affinity sites. Second, we demonstrate that the initial slopes of two saturation curves in direct coordinates, where Bmax varies by a known factor, allows an estimation of the Bmax/Kd ratio, within certain limits. From these two sets of data it is subsequently possible to define a precise window for the values of both Bmax and Kd. The performance of the method has been evaluated in representative cases using Monte Carlo studies. The results establish conditions for the existence of an inflection point as well as the influence of low affinity binding, whether or not proportional to Bmax.     

Stability and biological activity of catecholamines and 5-hydroxytryptamine immobilized to sepharose and glass beads.

[14C]Noradrenaline, [14C]adrenaline, and 5-[14C]hydroxytryptamine were complexed to several types of Sepharose derivatives through an amide or a diazo linkage. Complexes were also made with arylamine glass beads through a diazo linkage. After the reaction the complexes were extensively washed with distilled water or dilute HCl or both, plus other solvents. Radioactivity appeared in the storage solutions and the amount increased with time. When initial low levels of release were achieved by extensive washing, the addition of a different solvent produced a marked increase in release. The biological activity of these complexes was tested on isolated rabbit aortic strips and rat fundus strips. All complexes caused the tissues to contract. The biological activity, however, closely paralleled the release of the ligands from the complexes and the release of ligand was demonstrated through the use of oil immersion and dialysis sac. These data do not support the recent claims that catecholamines bound to glass beads exert their pharmacological effects as covalently bound complexes on isolated tissue. The lack of stability of these complexes indicates the need for caution in drawing conclusions as to the site or the mechanism of action of immobilized catecholamines and other drugs.     

Preparation and characterization of 'heparinocytes': erythrocytes with covalently bound low molecular weight heparin

In an attempt to create the possibility of stable, long acting, intravascular anticoagulation, low molecular weight heparin was modified by introducing a sulfhydryl group into the molecule (LMWH-SH). Human erythrocytes were covalently grafted with LMWH-SH by the use of a heterobifunctional coupling reagent which reacts with the SH group of LMWH-SH and surface exposed amino groups of erythrocytes now called 'heparinocytes' (HC). HC were morphologically indistinguishable from untreated erythrocytes and displayed identical osmotic resistance. The functionality of HC was analyzed by classical coagulation tests in which they dose dependently inhibited clot formation. HC were also functional in recalcified whole blood inhibiting thrombin formation as assessed by the cleavage of the chromogenic substrate S-2238. The system appears applicable as a potential autologous, long-term anticoagulant treatment or prophylaxis.     

Growth hormone release inhibiting hormone: actions on thyrotrophin and prolactin secretion after thyrotrophinreleasing hormone.

The hypothalamic tetradecapeptide growth hormone release inhibiting hormone (GH-RIH) blocked the thyrotrophin response to thyrotrophin-releasing hormone (TRH) in normal people and in patients with primary hypothyroidism. This inhibition was dose related. The TRH-induced prolactin release was not affected by GH-RIH. This dissociation of the thyrotrophin and prolactin responses to TRH by GH-RIH suggests that there are different mechanisms for release of thyrotrophin and prolactin and that only the former is affected by GH-RIH.     

Protein covalently bound to minus-strand DNA intermediates of duck hepatitis B virus.

Analysis of duck hepatitis B viral DNA by gel electrophoresis, Southern blotting, and binding to benzoylated naphthoylated DEAE-cellulose showed that a protein is bound to the minus-strand virion DNA as well as to the full-length single strand, minus-strand species, and minus-strand DNA intermediates isolated from replicating complexes present in infected duck liver. By utilizing a modified dideoxynucleotidyl sequencing method, it was shown that the protein is covalently bound to the smallest detectable growing strands (ca. 30 bases) and that minus-strand synthesis begins at a unique site. These results support the notion that the protein may function as a primer for synthesis of the minus-strand DNA.     

Purification and characterization of vanillyl-alcohol oxidase from Penicillium simplicissimum. A novel aromatic alcohol oxidase containing covalently bound FAD.

Vanillyl-alcohol oxidase was purified 32-fold from Penicillium simplicissimum, grown on veratryl alcohol as its sole source of carbon and energy. SDS/PAGE of the purified enzyme reveals a single fluorescent band of 65 kDa. Gel filtration and sedimentation-velocity experiments indicate that the purified enzyme exists in solution as an octamer, containing 1 molecule flavin/subunit. The covalently bound prosthetic group of the enzyme was identified as 8 alpha-(N3-histidyl)-FAD from pH-dependent fluorescence quenching (pKa = 4.85) and no decrease in fluorescence upon reduction with sodium borohydride. The enzyme shows a narrow substrate specificity, only vanillyl alcohol and 4-hydroxybenzyl alcohol are substrates for the enzyme. Cinnamyl alcohol is a strong competitive inhibitor of vanillyl-alcohol oxidation. The visible absorption spectrum of the oxidized enzyme shows maxima at 354 nm and 439 nm, and shoulders at 370, 417 and 461 nm. Under anaerobic conditions, the enzyme is easily reduced by vanillyl alcohol to the two-electron reduced form. Upon mixing with air, rapid reoxidation of the flavin occurs. Both with dithionite reduction and photoreduction in the presence of EDTA and 5-deazaflavin the red semiquinone flavin radical is transiently stabilized. Opposite to most flavoprotein oxidases, vanillyl-alcohol oxidase does not form a flavin N5-sulfite adduct. Photoreduction of the enzyme in the presence of the competitive inhibitor cinnamyl alcohol gives rise to a complete, irreversible bleaching of the flavin spectrum.