Physiological roles of milk enzymes: an evolving picture

1. Physiological roles of milk enzymes: an evolving picture

2. Nissim Silanikove Department of Food Science Agricultural Research Organization, The Volcani Center, Israel

7. Milk phases

8. Role Of Milk In Regulation Of Milk Secretion: Negative Feed Back Mechanism Induced By Milk Components

9. Occasional changes in gland emptying

10. Daily changes in breast volume

11. Daily changes in breast milk synthesis

12. Plasminogen/Plasmin System

13. Milk plasminogen and plasmin concentrations (throughout lactation)

14. Milk plasmin x Milk yield x bST(throughout lactation)

15. Stress and the plasmin system The effect of dexamethasone on the PPS system in cows Values are mean ± SE; *P < 0.05 by t-test Silanikove et al, Life Sci., 2000

16. The Negative Feedback Mechanism Flow-Chart: The ARO View. Blue arrows denote flow of signal along the feedback loop, red arrows denote positive effect and black arrows denote suppressive effect

17. Milk yield (half) of sheep or goat infected with CNS specie in one gland and the contra-lateral being free. Sheep – open bars Goats – hatched bars Leitner et al., JDS, 2004

18. Plasmin activity:sheep or goat with one gland infected with CNS specie and the contra-lateral being free Sheep + 73.7%, P < 0.0007 Goat + 195%, P < 0.0003 PL activity, units/mL Leitner et al, JDS, 2004

19. Proteose-peptone concentration:sheep or goat with one gland infected with CNS specie and the contra-lateral being free Sheep + 247%, P < 0.0001 Goat +151%, P < 0.0001 P-p, g/L Leitner et al, JDS, 2004

20. Conclusions • The basal level of PL activity is higher in sheep than in goats, which explains the higher basal level of proteose-peptone • PL activity in infected glands is higher in sheep than in goats, which explains the higher increase in proteose-peptone • The higher increase in proteose-peptone concentration in sheep than in goats explains the more acute reduction in milk yield in sheep

21. Role Of The Plasmin System In Induction Of Active Involution

22. What happens in case of surplus?

23. Involution Definition

24. Involution

25. - + The model: Each goat or cow were Injected with casein hydrolyzate in the experimental gland (+) whereas the control gland (-) was treated with intact casein

26. CNH in Goats:Multiple treatments Silanikove et al, Life Sci., 2002

27. Histology of section in the alveolus control CNH treated glands. Most of the cells stained in the CNH treated gland are leukocytes Silanikove et al, unpublished data

28. Milk Enzymes As Components Of The Innate Immune System: Formation Of Free Radicals And Bacterocidic Bacteristatic Environment During Active Involution

29. The many faces of XOR Nitrate Nitrite Or2H2O2 SOD

30. 1. LPO compound I + H2O2 LPO compound 1 2. LPO compound I + NO2- LPO compound II + ●NO2 3. LPO compound I + NO2- LPO + ●NO2 Reaction of Lactoperoxidase with Hydrogen peroxide and Nitrite

31. Silanikove et al, FRBM, 2005

32. Silanikove et al, FRBM, 2005

34. Silanikove et al, FRBM, 2005

35. Scenario of NO cycling and metabolism in mammary secretion

36. Glutathione cycle in milk NO Nitrite LPO ** * 2 2 Supply to the young

37. Question Number 1 • In the mammary gland, XOR has an essential, non-enzymatic, structural role in fat secretion (Vorbach et al. Genes Dev 2002, 16:3223) • It is well established that XOR associated with fat secretion is located within the inner side of MFGM ) e.g. J. Physiol 2002, 545:567) Do we have sufficient XO to support its role in innate immunity? (Free radicals biol Med 2005, 38: 1139 )

38. Distribution of xanthine oxidase, alkaline phosphatase and acid phosphatase in milk fractions Xanthine oxidase Alkaline phosphataseAcid phosphatase % of total % of total % of total Whoe milk100 100 100 Fat* (MFGM) 33 45 48 WMP 21 39 34 Phos.lipids54 84 82 Casein 3 - - Truly soluble43 16 18

39. XORis mostly associated with milk serum as XO, though its activity is highest on membranes and where it is distributed between a.Physiologically inactive XDin theinner sideof MFGM and vesicles, and b.Physiologically activein the form ofXO, on theoutside surfaceof the vesicles.

40. QuestionNumber2 As mature fresh milk do not contains measurable amount of xanthine, but contains uric acid in the range of 30 40 -micro-molar, it is important to know whether it derived from milk xanthine or secreted as uric acid? Fresh milk (i.e., milk secreted into the alveoli within 5 to 10 min before sampling) was obtained at the end of noon-milking following injection of oxytocine.

41. Xanthine + hypoxanthine and uric acid concentration in oxytocin-induced and mature milk

42. Disappearance of hypoxanthine and appearance of uric acid in fresh milk

44. Scenario of NO cycling and metabolism in mammary secretion

45. Question Number 3 Does Catalase really plays a critical role in protecting the gland from oxidative stress by converting active nitrite to less active nitrate?

46. Xanthine dose-dependently enhance the conversion of nitrite to nitrate. Under the experimental conditions, approximately 40 mM of xanthine are converted to urate via XO within 4 h Silanikove et al, unpublished data

48. Catalase inhibitor, amino triazole, prevent the conversion of nitrite to nitrate. reaction conditions Nitrite-50 mM, Xanthine-200 mM, incubation time - 30 min. Silanikove et al, unpublished data

50. Relative changes in lipid oxidation in milk milk stored for 6 hours in the dark at 40C (A), Effects of catalase inhibitor (B), nitrite (10 mM) (C) and nitrite + catalase (D) inhibitor Silanikove et al, unpublished data