Activity and Nature of Plasminogen Activators Associated with the Casein Micelle

Plasminogen activator isolated according to the method of DeHarveng and Nielsen (1991). DDW = deionized distilled water.

Plasminogen activator isolated by the method of White et al. (1995). DDW = deionized distilled water.

Units of activity of urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) as measured using the chromogenic substrate (A) SpecPL (Spectrozyme PL; H-d-norleucyl-hexahydrotyrosol-lysine-p-nitroanilide diacetate; American Diagnostica, Greenwich, CT) or (B) S-2251 (d-valyl-l-leucyl-l-lysine 4-nitroanilide dihydrochloride; Sigma Chemical Co., St. Louis, MO), averaged from fractions extracted (in triplicate) from both milk samples. Different uppercase letters above the bars indicate differences (P≤0.05) between uPA and tPA activities within each isolated fraction and not across (refer to Table 5 for a description of each fraction). Different lowercase letters indicate differences (P≤0.05) between different fractions independently for each plasminogen activator type.

Units of activity of (A) urokinase-type plasminogen activator (uPA) and (B) tissue-type plasminogen activator (tPA) in fractions isolated (in triplicate) from 2 milk samples, as measured using the chromogenic substrate SpecPL (Spectrozyme PL; H-d-nor-leucyl-hexahydrotyrosol-lysine-p-nitroanilide diacetate; American Diagnostica, Greenwich, CT). Milk 1=milk with SCC of 609,000/mL; milk 2 = milk with SCC of 21,000/mL. Different uppercase letters above the bars indicate differences (P≤0.05) in activities between the 2 milk samples, within each isolated fraction and not across (refer to Table 5 for a description of each fraction).

Units of activity of plasmin (PL) and plasminogen (PG) as measured using the chromogenic substrate SpecPL (Spectrozyme PL; H-d-norleucyl-hexahydrotyrosol-lysine-p-nitroanilide diacetate; American Diagnostica, Greenwich, CT), averaged from fractions extracted (in triplicate) from both milk samples. Note that the concentration of SpecPL used for the PL assay was 1.6mM, whereas that for the PG assay was 3.2mM. Different lowercase letters indicate significant differences (P≤0.05) between different fractions independently for PL and PG (refer to Table 5 for a description of each fraction).

Sodium dodecyl sulfate-polyacrylamide gel electrophoretic visualization of the different proteins present in the isolated plasminogen activator fractions. Lane 1, molecular weight standards; lane 2, β-CN; lane 3, bovine plasmin; lane 4, bovine plasminogen; lane 5, urokinase-type plasminogen activator; lane 6, tissue-type plasminogen activator; lanes 7 to 9, Sup2 fractions (1 to 3, where Sup2 is the plasminogen activator supernatant isolated according to the method of DeHarveng and Nielsen, 1991); lane 10, White Sup (where White Sup is the plasminogen activator supernatant isolated according to the method of White et al., 1995); Lane 11, white casein. The gel presented is one replicate of 3.

Casein–plasminogen SDS-PAGE visualization of plasmin and plasminogen activator activities present in the isolated plasminogen activator fractions. Lane 1, molecular weight standards; lane 2, bovine plasmin; lane 3, urokinase-type plasminogen activator; lane 4, tissue-type plasminogen activator; lane 5, White Sup (where White Sup is the plasminogen activator supernatant isolated according to the method of White et al., 1995); lane 6, White CN (where White CN is the CN pellet isolated according to the method of White et al., 1995); lanes 7 to 9, Sup2 fractions (1 to 3; where Sup2 is the plasminogen activator supernatant isolated according to the method of DeHarveng and Nielsen, 1991). Arrows numbered 1 to 6 refer to zones of clearance in each lane. The gel presented is one replicate of 3.