Phosphorylation of lipids tightly bound to secretory immunoglobulin A in antibody fractions from human breast milk possessing protein kinase activity.
The fractions of antibodies (Abs) containing only sIgA and IgG were purified from human breast milk by Protein A-Sepharose chromatography and they catalyzed phosphorylation of casein in the presence of [gamma-32P]ATP. Also, 32P-labeled low-molecular-weight non-protein products are formed which are visible as radioactive background on the polyacrylamide gel lanes during electrophoresis of Abs under denaturing conditions. Separation of sIgA from IgG using a DEAE-sorbent with subsequent gel-filtration in 0.05 M NaOH indicates that the low-molecular-weight substances partially remain tightly bound to sIgA and are separated only by gel-filtration in a buffer containing 5% dioxane (non-denaturing resolution) or by extraction of the sIgA pellet with the chloroform--methanol mixture (2:1). The 32P-labeled substances were separated by TLC in the system used for phospholipid chromatography forming two fractions (Rf 0.83 and 0.66) that were stained with iodine. The data suggest that the substances co-isolated with sIgA are phospholipids. At 25 nM ATP, casein and lipids are 32P-labeled. At 1 microM ATP, the sIgA polypeptides are also phosphorylated. Gentle removal of the lipids from Ab preparation enhanced 32P incorporation into casein and sIgA polypeptides. Considering the heterogeneity of polyclonal sIgA in protein and ATP affinity, it is suggested that phosphorylation of casein and sIgA polypeptides is catalyzed by abzymes of different clonal origin.
Kit
Y Y
YY
Novosibirsk Institute of Bioorganic Chemistry, Novosibirsk, 630090, Russia. kit@modul.bioch.nsk.su
Shipitsin
M V
MV
Semenov
D V
DV
Richter
V A
VA
Nevinsky
G A
GA
D002853:Chromatography, Liquid / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007071:Immunoglobulin A, Secretory / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D007074:Immunoglobulin G / Q000302:isolation & purification; D050356:Lipid Metabolism; D008895:Milk, Human / Q000276:immunology*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*
D007071:Immunoglobulin A, Secretory; D007074:Immunoglobulin G; D011494:Protein Kinases