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At least 19 recordsLinked to original sources
PubMed1979

Enzymic cis-trans isomerization of nitrofuran derivatives: isomerizing activity of xanthine oxidase, lipoyl dehydrogenase, DT-diaphorase and liver microsomes.

Xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2) supplemented with an electron donor could catalyze the cis-trans isomerization of 3-(5-nitro-2-furyl)-2-(2-furyl)acrylamide, 3-(5-nitro-2-furyl)-2-phenylacrylamide and 3-(5-nitro-2-furyl)-2-(2-furyl)acrylonitrile. The direction of isomerization (cis leads to trans, cis in equilibrium trans or trans leads to cis) is dependent on the chemical structure of these nitrofuran derivatives. Lipoyl dehydrogenase (NADH:lipoamide oxidereductase, EC 1.6.4.3), DT-diaphorase (NAD(P)H:(quinone-acceptor) oxidoreductase, EC 1.6.99.2) and liver microsomes could also catalyze the conversion of cis-3-(5-nitro-2-furyl)-2-(2-furyl)acrylamide to its trans isomer in the presence of an appropriate electron donor. Such isomerizing activity of these enzymes is much higher than their nitro-reducing activity. In addition, the cis-trans isomerization of some nitrofuran derivatives was demonstrated with the liver slices and the small intestines of rats. A new cis-trans isomerization mechanism which is based on transfer of a single electron by an enzyme system to a nitrofuran derivative to give the radical-anion was proposed. This postulated mechanism was supported by the preliminary experiments using pulse radiolysis technique.

Animals↗
PubMedDate not supplied

[Gel-permeating chromatography of interacting multi-component systems. I. Study of protein isomerization by the method of gel-permeation chromatography. II. Method of determining the kinetic constant of isomerization].

A technique is suggested of determining the isomerization constants by means of gel-permeation chromatography. Chromatograms of proteins and expressions for statistical moments of their distribution along the chromatographic column obtained in the authors previous work [1], are used. A necessary condition for optimal setup of the GPCh experiments is found. Fulfilment of this condition provides a maximal accuracy of determination of the isomerization constants.

Chromatography, Gel↗
PubMed1979

Role of proline isomerization in folding of ribonuclease A at low temperatures.

In unfolded RNase A there is an interconversion between slow-folding and fast-folding forms (U(S) right harpoon over left harpoon U(F)) that is known to show properties characteristic of proline isomerization in model peptides. Here, we accept the evidence that U(S) molecules contain nonnative proline isomers and we ask about the isomerization of these proline residues during folding. The U(S) right harpoon over left harpoon U(F) reaction in unfolded RNase A is used both to provide data on the kinetics of proline isomerization in the unfolded protein and as the basis of an assay for measuring proline isomerization during folding.The tyrosine-detected folding kinetics at low temperatures have been compared to those of proline isomerization in unfolded RNase A. The comparison is based on the recent observation that the U(S) right harpoon over left harpoon U(F) kinetics are independent of guanidinium chloride concentration, so that they can be extrapolated to low guanidinium chloride concentrations, at which folding takes place. At 0 degrees C the tyrosine-detected folding reaction is 100-fold faster than the conversion of U(S) to U(F) in unfolded RNase A. Consequently, the folding reaction is not rate-limited by proline isomerization as it occurs in unfolded RNase A. An assay is given for proline isomerization during folding. The principle is that native RNase A yields U(F) on unfolding, whereas protein molecules that still contain nonnative proline isomers yield U(S). Unfolding takes place at 0 degrees C, at which proline isomerization is slow compared to unfolding. This assay yields two important results: (i) The kinetics of proline isomerization during folding are substantially faster than in unfolded RNase A-e.g., 40-fold at 0 degrees C. The mechanism of the rate enhancement is unknown. (ii) At low temperatures (0-10 degrees C), and also in the presence of (NH(4))(2)SO(4), the tyrosine-detected folding reaction occurs before proline isomerization and yields a folded intermediate I(N) that is able to bind the specific inhibitor 2'-CMP. The results demonstrate that a folding intermediate is spectrally detectable when folding occurs at low temperatures. They suggest that low temperatures provide suitable conditions for determining the kinetic pathway of folding by characterizing folding intermediates.

Ammonium Sulfate↗
PubMed1978

Acid catalysis of the formation of the slow-folding species of RNase A: evidence that the reaction is proline isomerization.

Unfolded RNase A is known to contain an equilibrium mixture of two forms, a slow-folding form (U(1)) and a fast-folding form (U(2)). If U(1) is produced after unfolding by the slow cis-trans isomerization of proline residues about X-Pro imide bonds, then the formation of U(1) should be catalyzed by strong acids. Therefore, the rate of formation of U(1) has been measured at different HClO(4) concentrations. After rapid unfolding of the native protein in concentrated HClO(4) at 0 degrees , the slow formation of U(1) was measured by use of refolding assays. Catalysis of its formation was found at HClO(4) concentrations above 5 M. The uncatalyzed reaction follows apparent first-order kinetics but, in the acid-catalyzed range, two reactions are found. The faster reaction produces two-thirds of the slow-folding species and shows acid catalysis above 5 M HClO(4). Catalysis of the slower reaction begins at 8 M HClO(4). The faster reaction shows a 100-fold increase in rate at 10.6 M HClO(4) over the rate of the uncatalyzed reaction of 5 M. The activation enthalpy of the uncatalyzed reaction has been measured in two sets of unfolding conditions: DeltaH(double dagger) is 21.5 kcal/mol (1 kcal = 4.2 x 10(3) J) in 3.3 M HClO(4) and 21.0 kcal/mol in 5 M guanidine HCl, pH 2.5.Both acid catalysis of the formation of U(1) and its high activation enthalpy are consistent with the rate-limiting step being cis-trans isomerization either of X-Pro imide bonds or of peptide bond. The rate of the uncatalyzed reaction is in the range expected for proline isomerization and is 0.1% of that of peptide bond isomerization; thus, the simplest explanation for the formation of U(1) is proline isomerization. Earlier data, showing that the kinetic properties of the U(1) right arrow over left arrow U(2) reaction in refolding conditions differ from those of proline isomerization, can be explained if there is kinetic coupling between early steps in the folding of U(1) and its conversion to U(2).The existence of two acid-catalyzed reactions that are distinguished by the HClO(4) concentration at which catalysis begins suggests that at least two essential proline residues produce slow-folding species of RNase A by isomerization after unfolding. Because protonation of imide bonds is responsible for acid catalysis of proline isomerization, the slower reaction probably involves an imide bond with a low pK. It may be the bond connecting Lys-41 and Pro-42, because the positive charge on Lys-41 could make this bond more difficult to protonate.

Catalysis↗
PubMed1979

s-Cis and s-trans isomerism of the His-Pro peptide bond in angiotensin and thyroliberin analogues.

The dipeptide His-Pro isomerizes from all-s-trans to partly s-cis when titrated in D2O from acidic to neutral pD as observed by 13C and 1H nuclear magnetic resonance of the proline side chain. This isomerization is reported by the His C-2 and C-4 protons and carbons which show distinct, well-resolved resonances for each isomer. The influence of the His-Pro peptide bond rotational state on the histidine protons far removed from the bond has not been previously observed in model compounds or peptides. The peptides thyroliberin (TRH), [3-MeHis2]-TRH, and [3-MeHis6]-, [Sar1,Al8]-, and Nalpha-acetylangiotensin II were found to similarly isomerize from all-s-trans to partly s-cis as reported by their His C-2 and C-4 proton resonances. The His C-2 and C-4 protons in the peptides [1,3-diMeHis2]-TRH and [1-MeHis6]-, and [homoHis6]-angiotensin do not report this isomerization. Angiotensin II has previously been found to exhibit the same isomerization. The reporting of the s-trans to s-cis isomerization by the His C-2 proton appears to be correlated with the known potencies of the five angiotensin peptides in rat uterine strips and of the three TRH peptides by radioimmunoassay of released thyrotropin.

Angiotensin II↗
PubMed1978

Alkaline isomerization of thermoresistant cytochrome c-552 and horse heart cytochrome c studied by absorption and resonance Raman spectroscopy.

The structure of the thermoresistant cytochrome c (552, Thermus thermophilus) has been investigated at neutral and alkaline pH by absorption and resonance Raman spectroscopy and compared with that of horse heart cytochrome c. The ligands of the ferricytochrome c-552 at neutral pH are considered to be histidine and methionine, whereas the ligands of ferrocytochrome c-552 are histidine and another nitrogen base, histidine or lysine. Ferric cytochrome c-552 undergoes an alkaline isomerization with a pK of 12.3 (25 degrees C), accompanied by a ligand exchange. Horse heart cytochrome c has at least three isomerization states at alkaline pH (pK 9.3, 12.9 and greater than 13.5 at 25 degrees C). The replacement of the sixth ligand may not be involved in the second isomerization. The thermodynamic parameters for the isomerization were also estimated. The entropy change upon isomerization of cytochrome c-552 is negative, whereas for that of horse heart cytochrome c the entropy change is positive.

Animals↗
PubMed1977

Relaxation spectra of yeast hexokinases. Isomerization of the enzyme.

Yeast hexokinase isozymes P1 and P11 exhibit a pH dependent, rapid relaxation process at 15 degrees C at enzyme concentrations of 100-474 muM and over a pH range of 6-8. The process was detected by equilibrium temperature jump spectroscopy using the indicator probe phenol red. The value of 1/tau varies from about 6 ms-1 at pH 8 for both isozymes to 50 ms-1 for P1 and 85 ms-1 for P11 at pH 6. The data are consistent with a mechanism involving an enzyme isomerization coupled to an ionization. The forward rate constant for the isomerization of the proposed mechanism varies between 3 and 7 ms-1; the ratio of the reverse rate constant to the ionization Ka is between 0.5 and 2 X 10(11) M-1 S-1; the estimated pKa varies between 5.5 and 6.1. The ranges of values in rate constants and pKa represent variations observed between preparations of the same isozyme and between isozymes. The isomerization rate is at least 50 times faster than catalysis under all conditions and the pKa is lower than that controlling activity. The rate of isomerization is unchanged by addition of sugar and nucleotide ligands, but the amplitude of the process is perturbed. These data imply that isomerizing and ionizing forms are sensitive to events at the active site. These equilibria between forms of hexokinase are fast enough, and have the right properties, to be important to the mechanism and regulation of the enzyme.

Adenosine Triphosphate↗
PubMed1979

Isomerization of the double bonds of a conjugated fatty acid during beta-oxidation.

The beta-oxidation of an unsaturated fatty acid containing conjugated double bonds at odd-numbered carbon atoms has not previously been studied. It is, therefore, not clear whether, during the beta-oxidation of such an acid, the double bonds will be isomerized by enoyl-CoA isomerase (delta 3 - delta 2-enoyl-CoA isomerase) with the loss or retention of its conjugated nature. To investigate the problem, (E,E)-3,5-octadienoyl-CoA was synthesized for use as a model substrate, and enoyl-CoA isomerase was partially purified from bovine liver. The isomerization was followed by spectrophotometric and gas liquid chromatographic methods, and the results suggested that the isomerization of the model substrate proceeded with retention of a conjugated double bond system. It is, therefore, proposed that the beta-oxidation intermediate of alpha-eleostearic acid (delta 9,11,13 fatty acid) will also isomerize with retention of the conjugated double bond system.

Acyl Coenzyme A↗
PubMed1979

Voltage transients from photo-isomerizing azo dye in bilayer membranes.

Voltage transients are induced by brief light flashed on bilayer membranes with absorbed 3,3'-bis(alpha-(trimethylammonium)methyl)azobenzene (Bis-Q). The voltages are positive for trans-to-cis photo-isomerization, and negative for cis-to-trans photo-isomerization. The risetimes in phosphatidylethanolamine-decane bilayer membranes indicate that absorbed trans-Bis-Q is photo-isomerized to cis within 2 microseconds, and that cis is photo-isomerized to trans within 15 microseconds.

Azo Compounds↗
PubMed1975

Hepatoma, host liver, and normal rat liver lipids: distribution of isomeric monoene fatty acids in individual lipid classes.

Monoenoic acid fractions were isolated from phosphatidycholine, phosphatidylethanolamine, triglycerides, and cholesteryl esters of hepatoma 7288CTC, host liver, and normal liver from animals maintained on chow and fat free diets. Hexadecanoate (16:1), octadecenoate (18:1), and eisosenoate (20:1) fractions were analyzed quantitatively for their isomeric composition. The fat free diet had little or no effect relative to the chow diet on the isomeric composition of 16:1, 18:1, and 20:1 from any lipid class in either heptoma, host liver, or normal liver. Host livers were reduced in palmitoleic acid, and oleic and eicos-11-enoic acids were increased relative to normal liver. The 16:1 fraction from triglyceride of normal liver, host liver, and hepatoma contained 90, 80, and 75% palmitoleic acid, respectively. The 20:1 fraction from triglycerides of normal liver, host liver, and hepatoma contained ca. 55, 70, and 60% eicos-11-enoic acid, respectively, with the remainder consisting of eicos-13-enoic acid. The proportion of vaccenic acid in the 18:1 fraction was 60, 50, 20, and 25% for phosphatidylethanolamine, phosphatidylcholine, triglycerides, and cholesteryl esters, respectively, with oleic acid making up the balance. In contrast, all hepatoma lipid classes exhibited the same proportion of oleic (70%) and vaccenic (30%) acids. These data appear to be the first to demonstrate lipid class specificity for isomeric octadecenoic acids in normal liver and the loss of this specificity in a neoplasm.

Carcinoma, Hepatocellular↗
PubMed1975

Consideration of the Possibility that the slow step in protein denaturation reactions is due to cis-trans isomerism of proline residues.

A model is proposed to account for the observation that the denaturation of small proteins apparently occurs in two kinetic phases. It is suggested that only one of these phases--the fast one--is actually an unfolding process. The slow phase is assumed to arise from the cis-trans isomerism of proline residues in the denaturated protein. From model compound data, it is shown that the expected rate for isomerism is in satisfactory agreement with the rates actually observed for protein folding. It is also shown that a simple model of protein unfolding based on the isomerism concept is very successful in accounting for many known experimental characteristics of the kinetics and thermodynamic of protein denaturation. Thus, the model is able to predict that two kinetic phases will be seen in the transition region while none are seen in the base-line regions, that both the fast and slow refolding phases lead to the native protein as the product, that the fast phase becomes the only observable phase for jumps ending far in the denatured base-line region, that most or all small proteins show a limiting low-temperature activation energy of ca. 20,000 cal, and that the relaxtion time for the slow phase seen in cytochrome c denaturation is much shorter than for all other small proteins. By utilizing "double-jump" experiments, it is shown directly that the slow phase is not part of the unfolding process but that it corresponds to a transition among two or more denatured forms which have identical spectroscopic (286.5 nm) properties. Thus, the slow relaxation is "invisible" except in the transition region where it couples to the fast unfolding equilibrium. Finally, since the present model assumes that only one of the major kinetic phases seen in denaturation reactions is concerned with the denaturation process per se, it is in agreement with numerous thermodynamic studies which show consistency with the two-state model for unfolding.

Calorimetry↗
PubMed1978

Photochemistry of visual pigments: an interpretation of spectral changes in terms of molecular associations and isomerization.

A unified view of the photochemical part of the visual process is presented. It is proposed that both conformational changes and changes in intermolecular interactions in the sequence that leads from rhodopsin through batho-lumi- and meta-I to meta-II- rhodopsin have to be considered in order to elucidate the mechanism of the visual process. The main intermolocular associations are assumed to be the hydrogen bond involving the nitrogen atom of the Schiff base and the interaction between a negative group and the beta-ionone ring. The two together can be used to explain the absorption wavelength of rhodopsin without actual protonation. The main line of thought is as follows: when light is absorbed the basicity of the Schiff base increases significantly. This triggers proton transfer in the H-bond. At the same time cis-trans isomerization begins but it only reaches the coplanar all-trans stage at metarhodopsin-II. Lumi-, meta-I and meta-II are way stations in the stepwise isomerization whereby the energy of the photon is used together with thermal energy. Batho- is probably still close to 11-cis which then becomes successively strained 13-cis and 15-cis. In vertebrate rhodopsins at the meta-II stage both the H-bond and the beta-ionone interaction are severed and meta-II becomes exposed to attack by water molecules. The importance of syn-anti isomerization on the C=N bond is emphasized. The irreversibility necessary for the production of a signal requires that the proton does not return to its original donor. The possible identity of the donor is discussed: it might be an amino acid or the polar part of a lipid. Relevant observations made on bacteriorhodopsin, squid rhodopsin and chicken iodopsin are discussed.

Amino Acids↗
PubMed1978

13C nuclear magnetic resonance study of the cis-trans isomerism in X-Pro-Pro tripeptides.

13C nuclear magnetic resonance has been used to characterize quantitatively the cis-trans isomerism about both peptide bonds in the tripeptides Ser-Pro-Pro and Arg-Pro-Pro. Detailed pH titration data indicate that the configuration about both peptide bonds is closely linked to titration of the terminal carboxyl group and, to a lesser extent, to titration of the terminal amino group. The Pro2 C-3 resonance has been found particularly useful for interpretation due to its sensitivity to the isomerization about both peptide bonds. Analysis of the probabilities of the trans-trans, cic-cis, cis-trans, and trans-cis isomers in aqueous solution indicates a stability decrease in the order given. Similarities in the isomerization behavior of the two peptides indicate that side chain interactions involving the first residue have very little effect on the observed cis/trans ratios. The sensitivity of the cis/trans ratio to titration of the terminal amino group is most readily explained on the basis of an indirect effect on carbonyl-carbonyl repulsion.

Arginine↗
PubMed1979

Mass-transfer effects on the rate of isomerization of D-glucose into D-fructose, catalyzed by whole-cell immobilized glucose isomerase.

The investigated catalyst system consists of immobilized Arthrobacter cells containing the enzyme glucose isomerase, which catalyzes the isomerization of glucose into fructose. The internal structure of the catalyst was determined from electrom microscope photographs of replicas of freeze-etched catalyst. On the basis of the photographs a model for the internal structure of the catalyst was proposed. This structure was subsequently used to describe the reaction including mass-transfer effects. It appeared that under normal operating conditions the external mass-transfer rate does not influence the overall rate of reaction. The effect of internal mass-transfer resistances on the overall reaction rate can well be accounted for by the so-called porous sphere model. The intrinsic kinetics of the isomerization catalyzed by the present catalyst system can be represented by a modified Michaelis-Menten equation for a reversible one-substrate reaction.

Arthrobacter↗
PubMed1976

High-pressure liquid chromatographic determination of chlorphenesin carbamate and the beta-isomeric carbamate.

A high-pressure liquid chromatographic assay was developed for the determination of chlorphenesin carbamate and its beta-isomeric carbamate. A single 4-mm i.d. X 30-cm column, prepacked with 10 micrometer fully porous silica gel particles, is used with 3% methanol in 50% water-saturated butyl chloride as the mobile phase. The procedure separates chlorphenesin carbamate from several possible impurities in addition to the beta-isomeric carbamate. The assay was applied to bulk drug and compressed tablets. The relative standard deviations for the assays of chlorphenesin carbamate and the beta-isomer are approximately 1 and 2%, respectively.

Carbamates↗
PubMed1979

syn-anti Isomerization in testosterone 17-enanthate 3-benzilic acid hydrazone.

UV spectrophotometry, high-pressure liquid chromatography, and NMR were used to investigate syn-anti isomerization in testosterone 17-enanthate 3-benzilic acid hydrazone. In the crystalline state, the isomeric ratio is dependent on the crystallization solvent. In solutions, including injectable pharmaceutical formulations, an equilibrium isomer mixture (2:1) is formed.

Chromatography, High Pressure Liquid↗
PubMed1978

Rapid and sensitive determination of enzymatic degradation products of isomeric chondroitin sulfates by high-performance liquid chromatography.

The separation and quantitative analysis of enzymatic degradation products of isomeric chondroitin sulfates by high-performance liquid chromatography (HPLC) are described. The substituted unsaturated disaccharides which result from digestion of chondroitin sulfates with chondroitinase are quickly separated on polar absorbents such as silica gel. The UV absorption properties of these unsaturated disaccharides permit UV measurement with detection limits of approximately 100 ng. Their separation by HPLC facilitates the use of enzymatic methods for the determination of chondroitin sulfates A, B and C. The potential of this method in clinical application is demonstrated by quantitative assays of glycosaminoglycans from a normal urine and urine from a patient with Hunter syndrome. The results are consistent with amount of isomeric chondroitin sulfates found in comparable urines by others.

Autoanalysis↗
PubMed1979

Relaxation studies of enzymes: rapid isomerization in deoxyribonuclease I.

Temperature-jump relaxation studies in deoxy-ribonuclease I were carried out at 10 degrees C and [I] = 0.1 M. The single observed relaxation time, which varied from 10(-4) to 10(-5) s, was characterized as a function of enzyme concentration, pH, and indicator concentration. The concentration and pH dependences of the relaxation time are in quantitative agreement with a mechanism involving an isomerization of the enzyme coupled to a rapid proton ionization process. The best fit forward and reverse isomerization rate constants are 6.5 X 10(3) and 7.2 X 10(4) s-1, respectively; the apparent pK is 5.7. The addition of urea brought about reductions in both the amplitude of the relaxation effect and the enzyme activity.

Animals↗