We describe anaerobic stopped-flow monitored interactions of D-B-chloroalanine with D-amino acid oxidase and show that the kinetics of absorbance changes at 550 nm due to enzyme-bound intermediates can not be correlated with steady state turnover behavior unless it is assumed that only a small fraction of the enzyme directly participates in the α-β elimination process.
{"title":"Comparison of stopped flow and steady state kinetics in the reaction of D-amino-acid oxidase with -chloroalanine.","authors":"J G Voet, D J Porter, H J Bright","doi":"10.1515/znb-1972-0915","DOIUrl":"https://doi.org/10.1515/znb-1972-0915","url":null,"abstract":"We describe anaerobic stopped-flow monitored interactions of D-B-chloroalanine with D-amino acid oxidase and show that the kinetics of absorbance changes at 550 nm due to enzyme-bound intermediates can not be correlated with steady state turnover behavior unless it is assumed that only a small fraction of the enzyme directly participates in the α-β elimination process.","PeriodicalId":78857,"journal":{"name":"Zeitschrift fur Naturforschung. Teil B. Anorganische Chemie, organische Chemie, Biochemie, Biophysik, Biologie","volume":"27 9","pages":"1054-5"},"PeriodicalIF":0.0,"publicationDate":"1972-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1515/znb-1972-0915","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"15507569","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
D-6-hydroxynicotine oxidase contains 1 mole of FAD covalently bound to one mole of enzyme. To identify the covalent linkage between FAD and protein, an amino acid derivative of riboflavin (HNO-flavin) was isolated and purified. It was obtained from flavin peptides by hydrolysis with 6 N HCl at 95°C or with aminopeptidase M. The riboflavin derivative had the spectral characteristics of 8α-substituted flavins. It showed a pH-dependence of fluorescence with a pK of 4.65 and 86% quenching at pH 7. In thin layer chromatography it was identical with 8α-(N-3-histidyl)-riboflavin. Hydrolysis of HNO-flavin in 6 N HCl at 125°C liberated 1 mole of histidine per mole of flavin as shown by amino acid analysis. Since FAD is the coenzyme of D-6-hydroxynicotine oxidase, these results are taken as evidence that this enzyme contains 8a- (N-3-histidyl) -flavin-adenine-dinucleotide in the active center.
{"title":"Covalently bound flavin in D-6-hydroxynicotine oxidase from Arthrobacter oxidans.","authors":"M Brühmüller, H Möhler, K Decker","doi":"10.1515/znb-1972-0922","DOIUrl":"https://doi.org/10.1515/znb-1972-0922","url":null,"abstract":"D-6-hydroxynicotine oxidase contains 1 mole of FAD covalently bound to one mole of enzyme. To identify the covalent linkage between FAD and protein, an amino acid derivative of riboflavin (HNO-flavin) was isolated and purified. It was obtained from flavin peptides by hydrolysis with 6 N HCl at 95°C or with aminopeptidase M. The riboflavin derivative had the spectral characteristics of 8α-substituted flavins. It showed a pH-dependence of fluorescence with a pK of 4.65 and 86% quenching at pH 7. In thin layer chromatography it was identical with 8α-(N-3-histidyl)-riboflavin. Hydrolysis of HNO-flavin in 6 N HCl at 125°C liberated 1 mole of histidine per mole of flavin as shown by amino acid analysis. Since FAD is the coenzyme of D-6-hydroxynicotine oxidase, these results are taken as evidence that this enzyme contains 8a- (N-3-histidyl) -flavin-adenine-dinucleotide in the active center.","PeriodicalId":78857,"journal":{"name":"Zeitschrift fur Naturforschung. Teil B. Anorganische Chemie, organische Chemie, Biochemie, Biophysik, Biologie","volume":"27 9","pages":"1073-4"},"PeriodicalIF":0.0,"publicationDate":"1972-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1515/znb-1972-0922","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"15507576","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Ubiquinone-free succinate dehydrogenase may be activated by reduction by NADH (in the presence of an enzyme catalysing the transfer of reducing equivalents to succinate dehydrogenase) or Na2S2O4.
{"title":"Activation of soluble succinate dehydrogenase by reduction.","authors":"A D Klaasse, E C Slater","doi":"10.1515/znb-1972-0924","DOIUrl":"https://doi.org/10.1515/znb-1972-0924","url":null,"abstract":"Ubiquinone-free succinate dehydrogenase may be activated by reduction by NADH (in the presence of an enzyme catalysing the transfer of reducing equivalents to succinate dehydrogenase) or Na2S2O4.","PeriodicalId":78857,"journal":{"name":"Zeitschrift fur Naturforschung. Teil B. Anorganische Chemie, organische Chemie, Biochemie, Biophysik, Biologie","volume":"27 9","pages":"1077-8"},"PeriodicalIF":0.0,"publicationDate":"1972-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1515/znb-1972-0924","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"15507578","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The formation of a species with an absorption maximum around 410 nm, observed during the neutral photolysis of 10-(2'-hydroxyalkyl) flavins by earlier authors, is shown to depend on the presence of divalent anions. The structure of this new type of compounds is presented and a possible mechanism for the reaction is discussed.
{"title":"Intermediates in the neutral photolysis of riboflavin.","authors":"M S Jorns, P Hemmerich","doi":"10.1515/znb-1972-0910","DOIUrl":"https://doi.org/10.1515/znb-1972-0910","url":null,"abstract":"The formation of a species with an absorption maximum around 410 nm, observed during the neutral photolysis of 10-(2'-hydroxyalkyl) flavins by earlier authors, is shown to depend on the presence of divalent anions. The structure of this new type of compounds is presented and a possible mechanism for the reaction is discussed.","PeriodicalId":78857,"journal":{"name":"Zeitschrift fur Naturforschung. Teil B. Anorganische Chemie, organische Chemie, Biochemie, Biophysik, Biologie","volume":"27 9","pages":"1040-4"},"PeriodicalIF":0.0,"publicationDate":"1972-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1515/znb-1972-0910","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"15507564","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Lipoamide dehydrogenase (EC 1.6.4.3) has been succesfully linked to a CNBr-activated polysaccharide matrix, Sepharose-4B, under different reaction conditions. The enzyme is probably bound more homogeneously at lower pH values (pH 7.5) than at pH 8.5. Such immobilized preparations yield V values 8-30% of the value of the V of the free enzyme (18,600 mole/min/mole of flavin). A low level of CNBr-activation in combination with substrate protection and a pH of 7.5 during the coupling reaction leads to the most active preparations. The Km values for both substrates increase considerably. The overall kinetic pattern of the matrix-bound enzyme preparations is not different from that of the free enzyme. Both activation by high concentration of lip (SH)2NH2 at low NAD+ levels and a stimulation of the V by increased phosphate concentration in the assay buffer is observed with free and some immobilized enzymes.
{"title":"Comparative kinetics between matrix-bound lipoamide dehydrogenase and the free enzyme.","authors":"J Visser, L Havekes, C Veeger","doi":"10.1515/znb-1972-0918","DOIUrl":"https://doi.org/10.1515/znb-1972-0918","url":null,"abstract":"Lipoamide dehydrogenase (EC 1.6.4.3) has been succesfully linked to a CNBr-activated polysaccharide matrix, Sepharose-4B, under different reaction conditions. The enzyme is probably bound more homogeneously at lower pH values (pH 7.5) than at pH 8.5. Such immobilized preparations yield V values 8-30% of the value of the V of the free enzyme (18,600 mole/min/mole of flavin). A low level of CNBr-activation in combination with substrate protection and a pH of 7.5 during the coupling reaction leads to the most active preparations. The Km values for both substrates increase considerably. The overall kinetic pattern of the matrix-bound enzyme preparations is not different from that of the free enzyme. Both activation by high concentration of lip (SH)2NH2 at low NAD+ levels and a stimulation of the V by increased phosphate concentration in the assay buffer is observed with free and some immobilized enzymes.","PeriodicalId":78857,"journal":{"name":"Zeitschrift fur Naturforschung. Teil B. Anorganische Chemie, organische Chemie, Biochemie, Biophysik, Biologie","volume":"27 9","pages":"1063-6"},"PeriodicalIF":0.0,"publicationDate":"1972-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1515/znb-1972-0918","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"15507572","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
D- and ʟ-hydroxynicotine oxidase were obtained in homogeneous form and compared with regard to molecular weight, subunit structure, reaction mechanism, and specificity. The most striking differences between these enantiozymes are the type of coenzyme binding and the reactivity towards artificial two-electron acceptors.
{"title":"D- and L-6-hydroxynicotine oxidase, enantiozymes of Arthrobacter oxidans.","authors":"K Decker, V D Dai, H Möhler, M Brühmüller","doi":"10.1515/znb-1972-0921","DOIUrl":"https://doi.org/10.1515/znb-1972-0921","url":null,"abstract":"D- and ʟ-hydroxynicotine oxidase were obtained in homogeneous form and compared with regard to molecular weight, subunit structure, reaction mechanism, and specificity. The most striking differences between these enantiozymes are the type of coenzyme binding and the reactivity towards artificial two-electron acceptors.","PeriodicalId":78857,"journal":{"name":"Zeitschrift fur Naturforschung. Teil B. Anorganische Chemie, organische Chemie, Biochemie, Biophysik, Biologie","volume":"27 9","pages":"1072-3"},"PeriodicalIF":0.0,"publicationDate":"1972-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1515/znb-1972-0921","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"15507575","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The effects of experimental geometry on the theoretical polarizations of the S1 ← S0 and S2 ← S0 bands of π→π* type in riboflavin have been examined. Polarizations of these two bands are characterized by an angle between them in the range of 20—28° and are relatively insensitive to the input geometry. Thus the predicted polarizations are generally in agreement with fluorescence polarization spectrum of riboflavin at 77°K. Alloxazine forms a strong complex with KI, and the fluorescence and phosphorescence from the charge transfer states have been characterized by means of luminescence and photoselection measurements. Riboflavin did not form a strong complex with KI, but it forms aggregates (dimer) more readily than alloxazine. The excited states of flavins can be populated by the weak dipole-dipole coupling mechanism of energy transfer from 1La states of indoles to the S2 state of flavins. The measured critical distances estimated from the fluorescence depolarization experiments range from 31 Å for indole to 40 A for indole-2-carboxylic acid in glycerol-methanol mixture (9:1) at 263°K.
{"title":"Molecular luminescence studies of flavins. II. Interactions involving the excited states.","authors":"P S Song, T A Moore, W E Kurtin","doi":"10.1515/znb-1972-0901","DOIUrl":"https://doi.org/10.1515/znb-1972-0901","url":null,"abstract":"The effects of experimental geometry on the theoretical polarizations of the S1 ← S0 and S2 ← S0 bands of π→π* type in riboflavin have been examined. Polarizations of these two bands are characterized by an angle between them in the range of 20—28° and are relatively insensitive to the input geometry. Thus the predicted polarizations are generally in agreement with fluorescence polarization spectrum of riboflavin at 77°K. Alloxazine forms a strong complex with KI, and the fluorescence and phosphorescence from the charge transfer states have been characterized by means of luminescence and photoselection measurements. Riboflavin did not form a strong complex with KI, but it forms aggregates (dimer) more readily than alloxazine. The excited states of flavins can be populated by the weak dipole-dipole coupling mechanism of energy transfer from 1La states of indoles to the S2 state of flavins. The measured critical distances estimated from the fluorescence depolarization experiments range from 31 Å for indole to 40 A for indole-2-carboxylic acid in glycerol-methanol mixture (9:1) at 263°K.","PeriodicalId":78857,"journal":{"name":"Zeitschrift fur Naturforschung. Teil B. Anorganische Chemie, organische Chemie, Biochemie, Biophysik, Biologie","volume":"27 9","pages":"1011-5"},"PeriodicalIF":0.0,"publicationDate":"1972-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1515/znb-1972-0901","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"15510261","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
In previous communications it has been demonstrated that monogalactosyl diglyceride and the anionic chloroplasts lipids can be detected on the thylakoid membrane by specific antisera 1 _ 3 . The antigen determinants are of carbohydrate nature as was shown by specific agglutination inhibition tests. They are located on the outer surface of the thylakoid membrane and are directly accessible to the antibodies. The latter has been proven for the monogalactolipid 1, whereas the determinants having the carbohydrate structure like that of sulphoquinovosyl diglyceride2 stick out of the surface like phosphatidyl glycerol too 3, but are topographically rather arranged in gaps or pores of the membrane. Fatty acids are not involved in this antigenantibody reactions as precipitation studies with hydrated lipids and with lipids of different fatty acid compositions have revealed. On the other side, if sugar components represent the immunologically determinant groups, then it should be possible to confirm the results obtained with vertebrate antisera by using heterophilic agglutinins from plants (so-called lectins) 4, invertebrates and fish eggs. These are known to be excellent tools for the specific detection of terminal and innerchain carbohydrate structures of different configurations and conformations 5. As D-galactose is the main determinant sugar in plant glycolipids, we tested some of the galactose specific lectins in our system. It could be found, that the agglutinin from Ricinus communis, the antigalactosyl specificity of which is well established4, agglutinated chloroplasts and thylakoidfragments in a very specific way. This agglutination was inhibited by 0.03 M D-galactose, 0.06 M lactose, raffinose, stachyose, monogalactosyl glycerol (/9-glycosidic linkage), and digalactosyl glycerol (a-glycosidic linkage of D-galactose), but not by D-glucose, and L-arabinose. In addition, the specific anti-a-galactosyl agglutinin6 ("anti-B") from Salmo trutta (trout) also reacts well, especially after protease treatment. It is inhibited by carbohydrates with terminal a-glycosidic bound D-galactose (raffinose, stachyose, digalactosyl glycerol 0.03 M) and not by monogalactosyl glycerol, lactose, D-glucose, Dand Larabinose. We got weaker reactions with agglutinins from fungal origin like Fomes fomentarius, a lectin known as anti-B (a-galactosyl) reagent4'7, whereas other agglutinins (Arachis hypogoea "anti-T") directed to D-galactose-like structures 8 gave very weak aggluti-
{"title":"Use of heterophilic agglutinins in plant serology.","authors":"G Uhlenbruck, A Radunz","doi":"10.1515/znb-1972-0943","DOIUrl":"https://doi.org/10.1515/znb-1972-0943","url":null,"abstract":"In previous communications it has been demonstrated that monogalactosyl diglyceride and the anionic chloroplasts lipids can be detected on the thylakoid membrane by specific antisera 1 _ 3 . The antigen determinants are of carbohydrate nature as was shown by specific agglutination inhibition tests. They are located on the outer surface of the thylakoid membrane and are directly accessible to the antibodies. The latter has been proven for the monogalactolipid 1, whereas the determinants having the carbohydrate structure like that of sulphoquinovosyl diglyceride2 stick out of the surface like phosphatidyl glycerol too 3, but are topographically rather arranged in gaps or pores of the membrane. Fatty acids are not involved in this antigenantibody reactions as precipitation studies with hydrated lipids and with lipids of different fatty acid compositions have revealed. On the other side, if sugar components represent the immunologically determinant groups, then it should be possible to confirm the results obtained with vertebrate antisera by using heterophilic agglutinins from plants (so-called lectins) 4, invertebrates and fish eggs. These are known to be excellent tools for the specific detection of terminal and innerchain carbohydrate structures of different configurations and conformations 5. As D-galactose is the main determinant sugar in plant glycolipids, we tested some of the galactose specific lectins in our system. It could be found, that the agglutinin from Ricinus communis, the antigalactosyl specificity of which is well established4, agglutinated chloroplasts and thylakoidfragments in a very specific way. This agglutination was inhibited by 0.03 M D-galactose, 0.06 M lactose, raffinose, stachyose, monogalactosyl glycerol (/9-glycosidic linkage), and digalactosyl glycerol (a-glycosidic linkage of D-galactose), but not by D-glucose, and L-arabinose. In addition, the specific anti-a-galactosyl agglutinin6 (\"anti-B\") from Salmo trutta (trout) also reacts well, especially after protease treatment. It is inhibited by carbohydrates with terminal a-glycosidic bound D-galactose (raffinose, stachyose, digalactosyl glycerol 0.03 M) and not by monogalactosyl glycerol, lactose, D-glucose, Dand Larabinose. We got weaker reactions with agglutinins from fungal origin like Fomes fomentarius, a lectin known as anti-B (a-galactosyl) reagent4'7, whereas other agglutinins (Arachis hypogoea \"anti-T\") directed to D-galactose-like structures 8 gave very weak aggluti-","PeriodicalId":78857,"journal":{"name":"Zeitschrift fur Naturforschung. Teil B. Anorganische Chemie, organische Chemie, Biochemie, Biophysik, Biologie","volume":"27 9","pages":"1113-4"},"PeriodicalIF":0.0,"publicationDate":"1972-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1515/znb-1972-0943","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"15228840","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The CD-spectra of riboflavin and riboflavin analogues in aqueous solutions differ very little depending on pH and ionic strength, but are extremely sensitive upon solvent changes. The two bands in the region of 300 —500 nm seen in aqueous solutions are split into seven bands in less polar solvents, which can be assigned to seven vibronic transitions. The spectra may be interpreted by “through space” and “through chain” interactions of the sidechain centers of chirality with the flavin chromophore, which influence the two first π —π* transitions in different manner.
{"title":"Circular dichroism, self interaction and side chain conformation of riboflavin and riboflavin analogues.","authors":"G Scola-Nagelschneider, P Hemmerich","doi":"10.1515/znb-1972-0911","DOIUrl":"https://doi.org/10.1515/znb-1972-0911","url":null,"abstract":"The CD-spectra of riboflavin and riboflavin analogues in aqueous solutions differ very little depending on pH and ionic strength, but are extremely sensitive upon solvent changes. The two bands in the region of 300 —500 nm seen in aqueous solutions are split into seven bands in less polar solvents, which can be assigned to seven vibronic transitions. The spectra may be interpreted by “through space” and “through chain” interactions of the sidechain centers of chirality with the flavin chromophore, which influence the two first π —π* transitions in different manner.","PeriodicalId":78857,"journal":{"name":"Zeitschrift fur Naturforschung. Teil B. Anorganische Chemie, organische Chemie, Biochemie, Biophysik, Biologie","volume":"27 9","pages":"1044-6"},"PeriodicalIF":0.0,"publicationDate":"1972-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1515/znb-1972-0911","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"15507565","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
京公网安备 11010802042870号
京ICP备2023020795号-1
扫码关注我们
求助内容：
应助结果提醒方式：