Sidney Shifrin

Charge transfer and excitation-energy transfer in a model for enzyme-coenzyme interactions

Abstract 1. 1. The properties of indolylethynicotinamide are exanmined as a model for interactions between the tryptophan side chain of dehydrogenases and the nicotinamide moiety of NAD+. 2. 2. The long, diffuse absorption band in the spectrum of indolethylnicotinamide has been assigned to a charge-transfer transition. Absorption in the visible region is reminiscent of the spectrum of the glyceraldehyde-3-phosphate dehydrogenase-NAD+ complex. 3. 3. Indole emission is completely quenched by the pyridinium ion in this model compound by a mechanism which operates in other charge-transfer complexes. 4. 4. Although the absorption spectrum of indolylethyldihydronicotinamide gives no evidence of interaction between the two nitrogen heterocyclics, the fluorescence properties indicate that the transfer of energy of excitation from indole to dihydronicotinamide is quantitative. 5. 5. The importance of donor-acceptor complexes in biological systems is reemphasized.

Purification and characterization of the multiple forms of β-galactosidase of Escherichia coli

Abstract 1. 1. In constitutive or fully induced lactose fermenting strains of Escherichia coli , the β-galactosidase (EC 3.2.1.23) activity may be separated into at least ten well-defined zones of activity on polyacrylamide disc gels. A method is presented for the purification of these multiple forms of β-galactosidase exclusive of the major (95%) tetrameric species of this enzyme. 2. 2. Ultracentrifugation of a purified mixture of all isoenzymes yields six species with sedimentation values of approx. 16, 23, 27, 32, 36, and 41–45 S which are estimated to be aggregates of 4N, 6N, 8N, 10N, 12N, and 16N, respectively. 3. 3. The 16-S species has previously been shown to be a tetramer composed of identical subunits. The monomeric units of the 16-S and the 23–45-S species produced in urea appear to be identical by ultracentrifugation, electrophoresis in polyacrylamide disc gels, and amino acid composition. 4. 4. Mixtures of the 23–45-S species were found to be more sensitive than tetramer to thermal inactivation and denaturation. Dissociation of the tetramer in urea has previously been shown to proceed directly to monomer. Dissociation of the higher molecular weight forms proceeded through intermediate forms with sedimentation coefficients of approx. 16 and 10, presumably representing tetramer and dimer. 5. 5. Renaturation of urea-denatured heavy isoenzymes results in the formation of tetramers but not the heavier forms. This renatured tetramer is indistinguishable from the 16-S renatured tetramer in its sensitivity to urea and heat. 6. 6. The isoenzymes do not represent macromolecular complexes of β-galactosidase with other cell proteins or nucleic acids.

Influence of glycerol and other polyhydric alcohols on the quaternary structure of an oligomeric protein

Abstract Dissociation of tetrameric l -asparaginase from Escherichia coli B was examined in the presence of urea containing one of the following polyhydric alcohols: ethylene glycol, 1,2-propanediol, 1,3-propanediol, glycerol, erythritol, arabitol, adonitol, mannitol, sorbitol, inositol, glucose, sucrose, and polyethylene glycol. Low concentrations of these compounds accelerate the rate of subunit dissociation, and, with the exception of the propanediols and polyethylene glycol, higher concentrations decrease the rate at which the oligomeric enzyme dissociates. The specific concentration at which this transition occurs is related to the length of the carbon chain of the polyhydric alcohols and to the steric configuration of the hydroxyl groups about the asymmetric carbon atoms. In addition, the rate at which the oligomeric enzyme dissociates decreases as the number of hydroxymethyl groups per molecule polyol increases and reaches a maximum with the six carbon members. Low concentrations (1% by volume) of methanol, ethanol, ethylene glycol, propylene glycol, or glycerol contained in the renaturation buffer slightly accelerate the rate of reassembly of denatured subunits. The rate at which reassociation to the tetramer occurs also increases as the number of hydroxymethyl groups per molecule of polyhydric alcohol increases.

Effect of alcohols on the enzymatic activity and subunit association of β-galactosidase

Abstract The catalytic activity of β-galactosidase from Escherichia coli K 12 and the associative properties of its subunits have been studied in solutions of methanol, ethanol, i -propyl- and n -propyl alcohol. All of the alcohols at low concentrations (5%) stimulate the rate at which o -nitrophenyl-β- d -galactopyranoside is cleaved. The transferase activity of the enzyme was demonstrated by identification of methyl β-galactoside as one of the reaction products. Neither methanol nor ethanol dissociate the subunits of the active tetramer nor do they induce conformational changes in the protein as measured by sedimentation velocity, ultraviolet absorption spectroscopy, and fluorescence. Low concentrations of n -propyl alcohol in the absence of added magnesium ions, however, causes the tetramer to dissociate into inactive dimers.

The effect of urea on subunit interactions of ß-galactosidase from Escherichia coli K12

Abstract The effect of increasing concentrations of urea on sedimentation velocity, enzymatic activity, antigenicity, and protein fluorescence of s-galactosidase (s- d -galactoside galactohydrolase, EC 3.2.1.23) indicates that the enzymatically active tetramer is completely dissociated into inactive monomer in 6 M urea. Removal of urea by dialysis results in reaggregation to the active tetramer together with a return to normal values of those properties which have been studied. Protein which is immunologically related to s-galactosidase but incapable of forming an enzymatically active tetramer was added to wild-type enzyme in the presence of 8 M urea. After renaturation, the enzymatically active tetramer was found to be a hybrid consisting of mutant and wild-type subunits containing full enzymatic activity.

A systematic examination of charge transfer interactions of a coenzyme model

Abstract 1. 1. The electron acceptor properties of the 3-carbamoylpyridinium ion (1-substituted nicotinamide) were examined in the presence of a series of para -substituted phenyl derivatives. 2. 2. Interaction was measured spectrophotometrically between the two moieties of N -( β - p -x-phenylethyl)-3-carbamoylpyridinium chlorides in which x was either hydrogen or one of the following groups: amino-, methoxy-, hydroxy-, methyl-, and chloro-. 3. 3. The wavelength of the intramolecular charge transfer transition was found to be related linearly to the ionization potential of the electron donor.

Specificity of thyroglobulin interactions with thyroid cells and membranes

Homologous species specificity is demonstrated with bovine and human thyroglobulin in which the two terminal sugars of the B carbohydrate chain, sialic acid and galactose have been removed by enzymatic hydrolysis. The species specificity is demonstrated by measuring the ability of the deglycosylated thyroglobulin derivatives to inhibit thyrotropin-induced increases in cAMP in human, rat and bovine thyroid cells in culture. Thus human-human or bovine-bovine interactions have higher activity coefficients by at least an order of magnitude than their heterologous counterparts. The homologous interactions are confirmed in binding studies and shown to be associated with negligible degradation of the bound ligand over a 24 hour period.

Coenzyme analogs: I. Preparation and properties of thionicotinamide methiodide

Abstract 1. 1. Thionicotinamide methiodide, a “model” for thionicotinamide adenine dinucleotide, was prepared from nicotinamide methiodide and P 2 S 5 . 2. 2. The absorption spectra of thionicotinamide, thionicotinamide methiodide and 1-methyl-1,4-dihydrothionicotinamide show maxima expected for the substitution of sulfur for oxygen when these are part of the chromophore. 3. 3. 1-Methyl-1,4-dihydrothionicotinamide undergoes a photochemical reaction in visible light in the presence or absence of oxygen. 4. 4. 1-Methyl-1,4-dihydrothionicotinamide combines with silver nitrate; however, a 10% excess of the metal appears to be consumed when the reaction is followed spectrophotometrically and a 2-fold excess when followed amperometrically.

Characterization of the ß-galactosidase from a lactose-negative, complementing mutant of Escherichia coli K12

Abstract The enzyme s-galactosidase (s- d -galactoside galactohydrolase, EC 3.2.1.23) synthesized by a lactose-negative, cross-reacting material (CRM) positive mutant of Escherichia coli K12, was purified and characterized by several physicochemical procedures. The basal level of enzymatic activity associated with extracts from this mutant are the result of low levels (5%) of naturally occurring tetramer which has a specific activity on the order of 10 −3 that of the wild-type 3300 strain and is more labile to thermal inactivation. The major CRM-positive, complementing protein has been shown, however, to be a dimer with a molecular weight of approx. 250 000. The complementing dimer shows complete immunological identity with the wild-type protein demonstrating that the dissociation of tetramer to dimer does not qualitatively effect the immunochemical properties of the enzyme. Finally, the mutational effect on the enzyme which prohibits the formation of the normal tetramer from dimers also prohibits the normal polymerization of monomers into more highly aggregated isozymes such as those which occur in the wild-type strain.

Coenzyme analogs: II. Photochemical inactivation of reduced thionicotinamide adenine dinucleotide☆

Abstract 1. 1. Reduced thionicotinamide adenine dinucleotide was inactivated by exposure to visible light. The dinucleotide photoproduct was a competitive inhibitor of dehydrogenase-catalyzed oxidation of NADH. 2. 2. The photochemical reaction appeared to take place in two consecutive steps resulting in the formation of a single final product. 3. 3. Amperometric titration of reduced thionicotinamide adenine dinucleotide demonstrated that the thiocarbonyl group was not as readily available for complexation with silver ions as in 1-methyl-1,4-dihydrothionicotinamide.