Characterization and Expression of l-Amino Acid Oxidase of Mouse Milk

Abstract

L-Amino acid oxidase (LAO) was purified from mouse milk. LAO reacted with L-amino acids in an apparent order of Phe > Met, Tyr > Cys, Leu > His > > other 11 amino acids tested and produced H 2 O 2 in a dose-and time-dependent manner. LAO in milk had a molecular mass of about 113 kDa and was converted to a 60-kDa protein by SDS-PAGE. LAO consisted of two subunits. The N-and C-terminal amino acid sequence determination followed by cDNA cloning showed that the 60-kDa protein consisted of 497 amino acids. LAO mRNA spanned about 2.0 kb, and its expression was found only in the mammary epithelial cells. Glucocorticoid was essential for LAO gene expression. Thus, the LAO gene is expressed acutely upon the onset of milk synthesis. LAO mRNA increased 1 day before parturition, peaked during early to mid-lactation, and decreased at the end of lactation. This is the first demonstration showing that LAO is present in milk. Mastitis is caused by an intramammary bacterial infection. As mouse milk produced H 2 O 2 using endogenous free amino acids, we suggest that LAO, together with free amino acids, is responsible for killing bacteria in the mammary gland.

Human IL4I1 is a secreted l-phenylalanine oxidase expressed by mature dendritic cells that inhibits T-lymphocyte proliferation

“…Thus, like all LAAO enzymes described thus far, IL4I1 is secreted. 9,17 Second, IL4I1 preferentially hydrolyzed L-phenylalanine and its activity was not restricted to acidic pH, being also active at a pH approaching neutrality. Surprisingly, some of these results differed from data published by Mason et al for the mouse enzyme, although we used the same experimental conditions to test the enzymatic activity.…”

“…4 The IL4I1 protein shares the highest similarity with proteins presenting L-amino acid oxidase (LAAO) activity, such as snake venom LAAO, 7,8 and mouse milk LAAO. 9 Recently, Mason et al have demonstrated that mouse IL4I1 (mIL4I1) possesses an LAAO activity toward aromatic amino acids. The enzyme was mostly active at acidic pH, in contrast to other known members of the LAAO family, and its expression was restricted primarily to lysosomes.…”

L-amino acid oxidases: properties and molecular mechanisms of action

“…У млекопитающих LAAO выделены из печени, почек, мозга [42,43], секрета молочных желёз (у мышей) [44], и из полиморфноядерных лейкоцитов [45]. В зависимости от источника LAAO различаются по своим физико-химическим и биохимическим свойствам: молекулярной массе, степени гликозилирования молекулы, субстратной специфичности и по тому, каким образом осуществляется регуляции их синтеза в организме.…”

Notch4 intracellular domain binding to Smad3 and inhibition of the TGF-β signaling

“…Cells were lysed as previously described (Bianco et al, 2002;Sun et al, 2002). Protein (50 mg) was subjected to SDSpolyacrylamide gel electrophoresis (Invitrogen, Carlsbad, CA, USA) as described (Bianco et al, 2002;Sun et al, 2002).…”

“…Protein (50 mg) was subjected to SDSpolyacrylamide gel electrophoresis (Invitrogen, Carlsbad, CA, USA) as described (Bianco et al, 2002;Sun et al, 2002). The antibodies used were anti-V5 (Invitrogen, 1 : 5000), anti-Flag-HRP (Sigma, St Louis, MO, USA; 1 : 1000), anti-Notch4 (Upstate, Charlottesville, VA, USA; 1 : 1000), anti-P-Smad2 (Upstate, 1 : 1000), anti-Smad2 (Zymed, 1 : 1000), anti-Smad3 (Zymed, South San Francisco, CA, USA; 1 : 1000), anti-PSmad3 (kindly provided by Dr Michael Reiss), anti-Smad2/ 3(Santa Cruz, 1 : 250), anti-Lamin A/C (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA; 1 : 250) and anti-PAI-1 (American Diagnostica Inc., 1 : 2000).…”