Plasminogen-Activator in Human Early Milk: Its Partial Purification and Characterization

Abstract

SummaryMilk plasminogen-activator was partially purified from human transitional milk collected at about 10 days after delivery, by a five-step procedure involving chloroform treatment, ammonium sulfate precipitation, and column chromatography on Sephadex G-150, CM Sephadex C-50 and DEAE Sephadex A-50. This gave milk-activator with a maximum purification factor of about 2,400-fold with respect to the skimmed milk. The CM Sephadex-step preparation showed, on polyacrylamide gel electrophoresis, a single plasminogen-activator activity band located between the bands of albumin and prealbumin of human serum. This preparation exhibited no kinin forming activity. The activator hydrolyzed acetyl-glycyl-L-lysine methyl ester with similar order kinetic constants to urokinase, and was inhibited strongly by diisopropyl-fluorophosphate. The molecular weight of the activator as estimated by gel filtration was approximately 86,000, the isoelectric points as estimated by gel isoelectric focusing were pH 7.2, 6.9 and 6.6, and the activator activity was not quenched by antiurokinase globulin, indicating that the milk-activator is a different entity from urokinase.

Proteins and proteolysis in pre-term and term human milk and possible implications for infant formulae

“…1). Milk proteases present in breast milk, including plasmin, are capable of hydrolysing b-casein in milk (Ferranti et al, 2004;Greenberg & Groves, 1984;Okamoto, Horie, Nagamatsu, & Yamamoto, 1981) and could contribute to the complement-inhibitory activity of human milk (Ogundele, 1999). This study contributes new information about the protein composition of human milk, underlining the key role of plasmin: its higher level in milk for premature infants results in more extensive hydrolysis of protein, leading to the production of small peptides which could facilitate the digestion process.…”

Proteolytic Systems in Milk: Perspectives on the Evolutionary Function within the Mammary Gland and the Infant

“…In order to create active plasmin, its inactive form plasminogen must be cleaved at a specific peptide bond by plasmin activators (38). Two serine proteases in both bovine and human milk perform this task: urokinase-type plasminogen activator and tissue-type plasminogen activator (16, 20, 22, 23).…”

Determination of t-PA activity and t-PA antigen in human milk

“…Most reports of t-PA activity assay in human milk have used an assay on fibrin plates [1,2,3,4]. We determined the t-PA activity levels in human milk by a bioimmunoassay using a specific and sensitive monoclonal antibody (SP-322) against an epitope non-intervening the active site of t-PA. Alterations of the post-delivery t-PA activity and t-PA antigen in human milk were investigated from the first to the two hundred and tenth day.…”

“…Although a considerable number of publications on tissue plasminogen activator (t-PA) in human milk have appeared, most investigators have determined t-PA activity using an assay on fibrin plates [1,2,3,4]. We measured t-PA activity by a bioimmunoassay and t-PA antigen by ELISA using a monoclonal antibody (SP-322) [6], and estimated t-PA index ((t-PA activity, ng/ml) / (t-PA antigen, ng/ml) χ 100) in human colostrum and milk to evaluate the fluctuations of postdelivery t-PA.…”