Daniel, J. T.

Templeton, George E.

English

UARK (University of Arkansas )

Journal of the Arkansas Academy of Science Volume 24 Article 19 1970 Enzymatic Evidence that Leucine from Tentoxin in Levorotatory J. T. Daniel University of Arkansas, Fayetteville George E. Templeton University of Arkansas, Fayetteville Follow this and additional works at: https://scholarworks.uark.edu/jaas  Part of the Plant Pathology Commons Recommended Citation Daniel, J. T. and Templeton, George E. (1970) "Enzymatic Evidence that Leucine from Tentoxin in Levorotatory," Journal of the Arkansas Academy of Science: Vol. 24 , Article 19. Available at: https://scholarworks.uark.edu/jaas/vol24/iss1/19 This article is available for use under the Creative Commons license: Attribution-NoDerivatives 4.0 International (CC BY-ND 4.0). Users are able to read, download, copy, print, distribute, search, link to the full texts of these articles, or use them for any other lawful purpose, without asking prior permission from the publisher or the author. This Article is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Journal of the Arkansas Academy of Science by an authorized editor of ScholarWorks@UARK. For more information, please contact scholar@uark.edu. Glyptostrobus europaeus (Brongn.) Heer in ArkansasTraverse, A. and Barghoorn, E. E. 1953. Micropaleonto- cox (Lower Eocene) of Arkansas. Am. MidNat. Vol. 80, No. 2. pp. 543-547.logy of the Brandon Lignite, an early Ter-tiary Coal in Central Vermont. Journ. Pal-eontology. 27:289-295. Wodehouse, R. P. 1935. Pollen Grains. Hafner Pub. CoWittlake, E. B. 1968. Fossil Mosses from the Upper Wil- pp. 270-271.Enzymatic Evidence That LeucineFrom Tentoxin Is LevorotatoryJ. T. Daniel and G. E. Templeton1Department of Plant Pathology, University of Arkansas, Fayetteville, Arkansas 72701Introduction Materials and MethodsHydrolysis of the peptideTentoxin is a highly specific, biologically active pep-tide from Atternaria tenuis Auct., a common seed andsoil inhabiting fungus. Grossly, its biological activityconsists solely of irreversibly blocking the developmentof chlorophyll in cotyledons of certain plants when ap-plied to seeds during imbibition or to young seedlingsThe crystalline toxin was hydrolyzed in 6 normal HC]at 110°C for 24 hours according to the procedure oSpackman (5). After removal of the HC1 in vacuo thehydrolyzate was taken up in 10% 2-propanol for immediate spotting of preparative thin layer plates.Iprior to their emergence from the soil. Most dicotstested, with the exception of tomato and members ofthe cruciferae, have been found to be sensitive whilemost monocots tested were resistant with the exceptionsof sorghum and crabgrass (1, 2, 6, 7).Separation of Leucine from the Hydrolyzate.Leucine was separated from the hydrolyzate by pre-parative thin layer chromatography on 2mm thick platesof Silica gel PF (Merck) with a propanol: water (80:36)solvent in one dimension. Leucine was located on thedeveloped chromatogram by reacting one edge of theplate with ninhydrin. The surface of the plate Containingleucine (unreacted) was scraped into a Millipore filterand the leucine was leached from it with distilled water.The concentration of leucine recovered was determinedon an aliquot from this solution using a standard colonmetric procedure.Halloin and associates (4) have shown that tentoxindisrupts chloroplast development in sensitive specieswithout noticeably affecting the ultrastructure of otherorganelles. They also demonstrated that it does notblock the conversion of protochlorophyll to chlorophyllin either sensitive or insensitive species. In fact, theyshowed a slight but consistent increase in chlorophyll inresistant species.Enzymatic Oxidation of LeucineThe peptide was purified by Grable (3) from culturefiltrates or mycelial mats of Alternaria tenuis grown onRichards solution supplemented with V-8 juice. Struct-ural determinations (7) have revealed it to be a cyclictetrapeptide consisting of leucine, N-methylalanine, gly-cine and N-methyldehydrophenylalanine. The sequenceof these amino acids and the stereoisomerism of all butglycine must be established to complete the determina-tion of the primary structure of this peptide.Snake venom L amino acid oxidase was used in aWarburg manometric procedure as outlined by Wellnerand Meister (9) to determine the susceptibility of ten-toxin-leucine to this enzyme. The concentration of tentoxin-leucine was 12 millimoles per ml. The enzyme wasobtained from Nutritional Biochemicals Corporation,Cleveland, Ohio and was used at a concentration of 100mg/ml. At this enzyme level oxygen uptake with L leu-cine was 3.6 microliters per minute. Commercially pre-pared D and L leucine were used as controls. Threereplications were employed.This study was undertaken to determine which opti-cal antipode of leucine is present in tentoxin.ResultsL amino acid oxidase oxidized leucine from tentoxin(1) Undergraduate Research Participant and Professor°f Plant Pathology, respectively.Arkansas Academy of Science Proceedings, Vol. XXIV,1970 47Journal of the Arkansas Academy of Science, Vol. 24 [1970], Art. 19Published by Arkansas Academy of Science, 1970J. T. Daniel and G. E. Templetonat a rate comparable to that of known L leucine (Figure Literature Cited1). The D leucine control was not oxidized.1. Fulton, N. D., Katharina Bollenbacher and B. j.Moore. 1960. A chlorosis of cotton seedlingsml. caused by Alternaria sp. Phytopathology 50:.1 / 575. (Abst.).i«_ o / 2- Fulton, N. D., Katharina Bollenbacher, and G. E.26 7 / Templeton. 1965. A metabolite from Alter-i / naria tenuis that inhibits chlorophyll produc-i / *^l-leuc,ne t'on- Phytopathology 55:49-51.C/> 2o—/J±f'yS 3. Grable, C. I. 1967. Purification and characterizationH' y/ of a chlorosis toxin from Alternaria tenuis. M.iB'toxin-leucine S. Thesis Univ. of Arkansas.I .Kr 4. Halloin, J. M., G. A. deZoeten, G. Gaard and J. C.,y Walker. 1970. The effects of tentoxin onkIO , t/ chlorophyll synthesis and plastid structure in// cucumber and cabbage. Plant Physiology. 45:/ 310-314.I /* 5. Spackman, D. H. 1962. Instruction manual AIM 2,iS Model 120B Amino Acid Analyzer. Spinco Di- |S % . . . . . ¦ . . . . . vision, Beckman Instruments Inc., Stanfordo io 20 30 40 60 60 70 80 90 ioo mo i2o Industrial Park, Palo Alto, Calif.M.CROL.TERS OF OXYGEN uptake 6 Templeton, G. E., C. I. Grable, N. D. Fulton and Ka-tharina Bollenbacher. 1967. Factors affect-Figure 1. Enzymatic Oxidation of Leucine from Tentoxin. ing the amount and pattern of chlorosis caus-ed by an Alternaria tenuis metabolite. Phyto-pathology 57:516-518.Summary and Conclusion 7. Templeton, G. E., W. L. Meyer, C. I. Grable and C. W.Sigel. 1967. The chlorosis toxin from Alter-ternaria tenuis is a cyclic tetrapeptide. Phyto-These data indicate that the leucine in tentoxin isevorotatory. Supporting data with higher concentrationof this antipode subjected to D amino acid oxidase wouldbe highly desirable but is currently considered too ex-ravagant with the limited supply of peptide available.pathology 57:873 (Abst.).Templeton, G. E., C. I. Grable, N. D. Fulton and W. L.Meyer. 1967. Tentoxin from Alternaria tenuis:its isolation and characterization. Proc. 1967Mycotoxin Res. Seminar held at Washington,D. C, June 8-9, 1967. USDA. pp. 27-29.8.tThese data further suggest that the optical isomerismleucine is not the basis for tentoxins inhibition oflorophyll or plastid formation in sensitive plant spe-9. Wellner, D. and A. Meister. 1960. Crystalline L-AminoAcid Oxidase of Crotalus adamanteus. TheJour, of Biological Chem. 235:2013-2018.48Journal of the Arkansas Academy of Science, Vol. 24 [1970], Art. 19https://scholarworks.uark.edu/jaas/vol24/iss1/19

Enzymatic Evidence that Leucine from Tentoxin in Levorotatory

ScholarWorks@UARK

Journal of the Arkansas Academy of ScienceVolume 24 Article 191970Enzymatic Evidence that Leucine from Tentoxin inLevorotatoryJ. T. DanielUniversity of Arkansas, FayettevilleGeorge E. TempletonUniversity of Arkansas, FayettevilleFollow this and additional works at: http://scholarworks.uark.edu/jaasPart of the Plant Pathology CommonsThis article is available for use under the Creative Commons license: Attribution-NoDerivatives 4.0 International (CC BY-ND 4.0). Users are able toread, download, copy, print, distribute, search, link to the full texts of these articles, or use them for any other lawful purpose, without asking priorpermission from the publisher or the author.This Article is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Journal of the Arkansas Academyof Science by an authorized editor of ScholarWorks@UARK. For more information, please contact scholar@uark.edu, ccmiddle@uark.edu.Recommended CitationDaniel, J. T. and Templeton, George E. (1970) "Enzymatic Evidence that Leucine from Tentoxin in Levorotatory," Journal of theArkansas Academy of Science: Vol. 24 , Article 19.Available at: http://scholarworks.uark.edu/jaas/vol24/iss1/19Glyptostrobus europaeus (Brongn.) Heer in ArkansasTraverse, A. and Barghoorn, E. E. 1953. Micropaleonto- cox (Lower Eocene) of Arkansas. Am. MidNat. Vol. 80, No. 2. pp. 543-547.logy of the Brandon Lignite, an early Ter-tiary Coal in Central Vermont. Journ. Pal-eontology. 27:289-295. Wodehouse, R. P. 1935. Pollen Grains. Hafner Pub. CoWittlake, E. B. 1968. Fossil Mosses from the Upper Wil- pp. 270-271.Enzymatic Evidence That LeucineFrom Tentoxin Is LevorotatoryJ. T. Daniel and G. E. Templeton1Department of Plant Pathology, University of Arkansas, Fayetteville, Arkansas 72701Introduction Materials and MethodsHydrolysis of the peptideTentoxin is a highly specific, biologically active pep-tide from Atternaria tenuis Auct., a common seed andsoil inhabiting fungus. Grossly, its biological activityconsists solely of irreversibly blocking the developmentof chlorophyll in cotyledons of certain plants when ap-plied to seeds during imbibition or to young seedlingsThe crystalline toxin was hydrolyzed in 6 normal HC]at 110°C for 24 hours according to the procedure oSpackman (5). After removal of the HC1 in vacuo thehydrolyzate was taken up in 10% 2-propanol for immediate spotting of preparative thin layer plates.Iprior to their emergence from the soil. Most dicotstested, with the exception of tomato and members ofthe cruciferae, have been found to be sensitive whilemost monocots tested were resistant with the exceptionsof sorghum and crabgrass (1, 2, 6, 7).Separation of Leucine from the Hydrolyzate.Leucine was separated from the hydrolyzate by pre-parative thin layer chromatography on 2mm thick platesof Silica gel PF (Merck) with a propanol: water (80:36)solvent in one dimension. Leucine was located on thedeveloped chromatogram by reacting one edge of theplate with ninhydrin. The surface of the plate Containingleucine (unreacted) was scraped into a Millipore filterand the leucine was leached from it with distilled water.The concentration of leucine recovered was determinedon an aliquot from this solution using a standard colonmetric procedure.Halloin and associates (4) have shown that tentoxindisrupts chloroplast development in sensitive specieswithout noticeably affecting the ultrastructure of otherorganelles. They also demonstrated that it does notblock the conversion of protochlorophyll to chlorophyllin either sensitive or insensitive species. In fact, theyshowed a slight but consistent increase in chlorophyll inresistant species.Enzymatic Oxidation of LeucineThe peptide was purified by Grable (3) from culturefiltrates or mycelial mats of Alternaria tenuis grown onRichards solution supplemented with V-8 juice. Struct-ural determinations (7) have revealed it to be a cyclictetrapeptide consisting of leucine, N-methylalanine, gly-cine and N-methyldehydrophenylalanine. The sequenceof these amino acids and the stereoisomerism of all butglycine must be established to complete the determina-tion of the primary structure of this peptide.Snake venom L amino acid oxidase was used in aWarburg manometric procedure as outlined by Wellnerand Meister (9) to determine the susceptibility of ten-toxin-leucine to this enzyme. The concentration of tentoxin-leucine was 12 millimoles per ml. The enzyme wasobtained from Nutritional Biochemicals Corporation,Cleveland, Ohio and was used at a concentration of 100mg/ml. At this enzyme level oxygen uptake with L leu-cine was 3.6 microliters per minute. Commercially pre-pared D and L leucine were used as controls. Threereplications were employed.This study was undertaken to determine which opti-cal antipode of leucine is present in tentoxin.ResultsL amino acid oxidase oxidized leucine from tentoxin(1) Undergraduate Research Participant and Professor°f Plant Pathology, respectively.Arkansas Academy of Science Proceedings, Vol. XXIV,1970 47Journal of the Arkansas Academy of Science, Vol. 24 [1970], Art. 19Published by Arkansas Academy of Science, 1970J. T. Daniel and G. E. Templetonat a rate comparable to that of known L leucine (Figure Literature Cited1). The D leucine control was not oxidized.1. Fulton, N. D., Katharina Bollenbacher and B. j.Moore. 1960. A chlorosis of cotton seedlingsml. caused by Alternaria sp. Phytopathology 50:.1 / 575. (Abst.).i«_ o / 2- Fulton, N. D., Katharina Bollenbacher, and G. E.26 7 / Templeton. 1965. A metabolite from Alter-i / naria tenuis that inhibits chlorophyll produc-i / *^l-leuc,ne t'on- Phytopathology 55:49-51.C/> 2o—/J±f'yS 3. Grable, C. I. 1967. Purification and characterizationH' y/ of a chlorosis toxin from Alternaria tenuis. M.iB'toxin-leucine S. Thesis Univ. of Arkansas.I .Kr 4. Halloin, J. M., G. A. deZoeten, G. Gaard and J. C.,y Walker. 1970. The effects of tentoxin onkIO , t/ chlorophyll synthesis and plastid structure in// cucumber and cabbage. Plant Physiology. 45:/ 310-314.I /* 5. Spackman, D. H. 1962. Instruction manual AIM 2,iS Model 120B Amino Acid Analyzer. Spinco Di- |S % . . . . . ¦ . . . . . vision, Beckman Instruments Inc., Stanfordo io 20 30 40 60 60 70 80 90 ioo mo i2o Industrial Park, Palo Alto, Calif.M.CROL.TERS OF OXYGEN uptake 6 Templeton, G. E., C. I. Grable, N. D. Fulton and Ka-tharina Bollenbacher. 1967. Factors affect-Figure 1. Enzymatic Oxidation of Leucine from Tentoxin. ing the amount and pattern of chlorosis caus-ed by an Alternaria tenuis metabolite. Phyto-pathology 57:516-518.Summary and Conclusion 7. Templeton, G. E., W. L. Meyer, C. I. Grable and C. W.Sigel. 1967. The chlorosis toxin from Alter-ternaria tenuis is a cyclic tetrapeptide. Phyto-These data indicate that the leucine in tentoxin isevorotatory. Supporting data with higher concentrationof this antipode subjected to D amino acid oxidase wouldbe highly desirable but is currently considered too ex-ravagant with the limited supply of peptide available.pathology 57:873 (Abst.).Templeton, G. E., C. I. Grable, N. D. Fulton and W. L.Meyer. 1967. Tentoxin from Alternaria tenuis:its isolation and characterization. Proc. 1967Mycotoxin Res. Seminar held at Washington,D. C, June 8-9, 1967. USDA. pp. 27-29.8.tThese data further suggest that the optical isomerismleucine is not the basis for tentoxins inhibition oflorophyll or plastid formation in sensitive plant spe-9. Wellner, D. and A. Meister. 1960. Crystalline L-AminoAcid Oxidase of Crotalus adamanteus. TheJour, of Biological Chem. 235:2013-2018.48Journal of the Arkansas Academy of Science, Vol. 24 [1970], Art. 19http://scholarworks.uark.edu/jaas/vol24/iss1/19

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Enzymatic Evidence that Leucine from Tentoxin in Levorotatory

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