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Transglutaminase activity

Transglutaminase activity. S. Beninati Dept. of Biology, Lab. of Cellular Biochemistry A. Lentini B.Provenzano T.Chiacchio G.Valle P.Mattioli. Putrescine. Spermidine. Spermine. Control. Sucrose. Sucrose Silica gel. Sucrose Silica gel Liq. N 2. Sucrose Silica gel Liq. N 2.

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Transglutaminase activity

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  1. Transglutaminase activity S. Beninati Dept. of Biology, Lab. of Cellular Biochemistry A. Lentini B.Provenzano T.Chiacchio G.Valle P.Mattioli

  2. Putrescine Spermidine Spermine Control Sucrose Sucrose Silica gel Sucrose Silica gel Liq. N2 Sucrose Silica gel Liq. N2 Control Sucrose Sucrose Silica gel Polyamine levels in shoot apex from Apple cv.Annurca and Apple cv. Gala dehydrated with sucrose Annurca pmol polyamine /mg protein pmol polyamine /mg protein Gala

  3. Putrescine Spermidine Spermine Control Control Mannitol Mannitol Mannitol Silica gel Mannitol Silica gel Mannitol Silica gel Liq. N2 Mannitol Silica gel Liq. N2 Polyamine levels in shoot apex from Apple cv.Annurca and Apple cv. Gala dehydrated with mannitol Annurca pmol polyamine /mg protein pmol polyamine /mg protein Gala

  4. Comparison of polyamine levels between control and frozen shoot apex dehydrated with sucrose ormannitol Apple cv.Annurca SUCROSE Apple cv.Gala Apple cv.Annurca MANNITOL Apple cv.Gala

  5. Ornithine ODC ? Putrescine PAO SPERMIDINE SYNTHASE N-acetylspermidine SSAT ? Spermidine PAO SPERMINE SYNTHASE N-acetylspermine SSAT Spermine

  6. Putrescine Spermidine Spermine Control Sucrose Sucrose Silica gel Sucrose Silica gel Liq. N2 Polyamine levels in shoot apex from Apple cv.Annurca dehydrated with sucrose Annurca pmol polyamine /mg protein

  7. Control Mannitol Mannitol Silica gel Mannitol Silica gel Liq. N2 Sucrose Silica gel Liq. N2 Control Sucrose Sucrose Silica gel Polyamine levels in shoot apex from Apple cv. Gala dehydrated with sucrose and mannitol pmol polyamine /mg protein pmol polyamine /mg protein

  8. Transglutaminase Shoot apex from: Apple cv. Annurca, Apple cv. Gala Almond cv. Supernova Transglutaminase activity has been revealed by means of an in vivo method, based on the uptake of [3H]-putrescine and its covalent incorporation into cell substrate protein.

  9. “in vivo” TGase assay Incubation of shoot apices with [3H]-putrescine in culture medium (20h at 25 °C). (Blank: apices boiled before incubation) Uptake and covalent incorporation into endogenous substrate proteins Washings Lysate preparation 6% PCA Washings Measurement of the radioactivity of [3H]-putrescine covalently bound to proteins [3H]-put [3H]-put [3H]-put [3H]-put

  10. In vivo TGase activity in shoot apex from Apple cv.Annurca and Apple cv. Gala dehydrated with sucrose Annurca pmol [3H]-putrescine / mg protein / 20h Blank Control Sucrose Sucrose Sucrose Silica Gel Silica Gel Liq. N2 pmol [3H]-putrescine / mg protein / 20h Gala Blank Control Sucrose Sucrose Sucrose Silica Gel Silica Gel Liq. N2

  11. In vivo TGase activity in shoot apex from Apple cv.Annurca and Apple cv. Gala dehydrated with mannitol pmol [3H]-putrescine / mg protein / 20h Annurca Blank Control Mannitol Mannitol Mannitol Silica Gel Silica Gel Liq. N2 pmol [3H]-putrescine / mg protein / 20h Gala Blank Control Mannitol Mannitol Mannitol Silica Gel Silica Gel Liq. N2

  12. In vivo TGase activity in shoot apex from Almond cv.Supernova dehydrated with sucrose pmol [3H]-putrescine / mg protein / 20h Blank Control Sucrose Sucrose Sucrose Silica Gel Silica Gel Liq. N2

  13. Transglutaminase catalyzed reactions CO NH e (g-Gln)Lys NH-R-NH2 Gln CO polyamine mono-derivative CO CO Gln Gln NH-R-NH polyamine bis-derivative

  14. Ornithine endogenous ODC exogenous Putrescine Put PAO * N-acetylspermidine 3H-Put SSAT * Spermidine Put 3H-Put PAO * N-acetylspermine 3H-Put Put SSAT TGase Spermine protein

  15. Putrescine Spermidine Spermine Control Sucrose Sucrose Silica gel Sucrose Silica gel Liq. N2 Sucrose Silica gel Liq. N2 Control Sucrose Sucrose Silica gel Polyamine levels in shoot apex from Apple cv.Annurca and Apple cv. Gala dehydrated with sucrose Annurca pmol polyamine /mg protein pmol polyamine /mg protein Gala

  16. Dehydration with sucrose Putrescine increase • Spermine decrease - polyamine oxidase (PAO) activity - changes in amine acetylation • Dehydration with mannitol • Putrescine/Spermidine decrease • Spermine unchanged - No PAO activity, polyamine leakage - Catabolic pathways and/or cell damage - Control of enzyme activities and metabolites of polyamine bisynthesis (PAO, SPM synthase, acetylpolyamines, g-glutamyl-derivatives of polyamines) - Use of enzyme inhibitors as a future strategy TGase • Gradual decrease of TGase activity during dehydration/cryopreservation procedure • Proteolytic events activated by sample treatment • Endogenous putrescine may inhibit TGase catalysis conclusion • - How artifacts due to sample handling may affect results? • The answer will improve the reliability of experimental observations

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