Plasmin 3-casein

Proteolytic breakdown of the casein Bacterial or native plasmin enzymes that are resistant to heat treatment may lead to the formation of a gel. 

Hydrolysis of primary caseins by plasmin. In 1969, Groves and coworkers showed that the y-casein fraction, as isolated by Hipp et al., is very heterogeneous, containing at least four distinct proteins y-casein, temperature-sensitive casein (TS, which is soluble in the cold but precipitates above 20°C), R-casein and S-casein. These four proteins were shown to be C-terminal fragments of /3-casein. In 1976, the nomenclature of the y-casein group was revised, as shown in Figure 4.7 and Table 4.3. 

Figure 4.7 Principal products produced from / -casein by plasmin.

Isolated as2-casein in solution is also very susceptible to plasmin eight peptide bonds are hydrolysed with the production of 14 peptides. Plasmin also hydrolyses as2-casein in milk but the peptides formed have not been identified, although at least some are included in the proteose-peptone fraction. 

Although less susceptible than / - and as2-caseins, isolated xsl -casein in solution is also readily hydrolysed by plasmin. It has been suggested that a minor ill-defined fraction of casein, called A-casein, consists of plasmin-produced fragments of asl-casein, but the situation is unclear. 

Figure 4.11 Amino acid sequence of bovine /1-casein, showing the amino acid substitutions in the genetic variants and the principal plasmin cleavage sites ( ) (from Swaisgood, 1992).

About 20% of the total protein of bovine milk belongs to a group of proteins generally referred to as whey or serum proteins or non-casein nitrogen. Acid and rennet wheys also contain casein-derived peptides both contain proteose-peptones, produced by plasmin, mainly from /J-casein, and the latter also contains (glyco)macropeptides produced by rennets from K-casein. These peptides are excluded from the present discussion. 

Proteinase (plasmin) Hydrolysis of peptide bonds, particularly in -casein Reduced storage stability of UHT products cheese ripening... 

Although in vitro, the cell wall-associated proteinase of the Lactococcus starters is quite active on 8-casein (and that from some strains on asl-casein also), in cheese, they appear to act mainly on casein-derived peptides, produced by chymosin from asl-casein or by plasmin from / -casein. 

Figure 10.23 Water-insoluble and water-soluble peptides derived from asl-casein (A), as2-casein (B) or -casein (C) isolated from Cheddar cheese DF = diafiltration. The principal chymosin, plasmin and lactococcal cell-envelope proteinase cleavage sites are indicated by arrows (data from T.K. Singh...

Peptides extracted from casein with N, N-dimethyl formamide have complex electrophoretic patterns identical to those of the fraction first prepared by Long and co-workers and called X-casein (El-Negoumy 1973). These peptides are identical electrophoretically to those released by the action of plasmin, which is present in fresh raw milk, upon asr casein (Aimutis and Eigel 1982). Two of these peptides have tryptic peptide maps and molecular weights identical to those of a pair of the peptides produced by plasmin degradation of asl-casein. These peptides appear to be fragments of a8l-casein which are present in milk as the result of plasmin proteolysis. More definitive information on their primary structure is needed before nomenclature for these fragments can be established. 

Eigel, W. N. 1981. Identification of proteose-peptone component 5 as a plasmin derived fragment of /3-casein. Int. J. Biochem. 13, 1081-1086. 

Milk is clarified by high-speed centrifugation to remove extraneous matter held in suspension. Clarification occurs prior to heat treatment of the milk to prevent dissolution of the extraneous matter. Although clarification removes somatic cells, the elevated levels of lipoprotein lipase activators and plasmin that may be associated with increased numbers of white blood cells in the milk are not eliminated. Therefore, increased lipolysis of milk fat by lipoprotein lipase and proteolysis of casein by plasmin may not be deterred. 

The other major casein in cheese is /3-casein, but it is generally not hydrolyzed by rennet in low-pH cheeses. Alkaline milk protease (plas-min) plays the major role in the hydrolysis of /3-casein (Richardson and Pearce 1981). The plasmin level in cheese is related to the pH of the curd at whey drainage, since plasmin dissociates from casein micelles as the pH is decreased. Richardson and Pearce (1981) found two or three times more plasmin activity in Swiss cheese than in Cheddar cheese. Swiss cheese curds are drained at pH 6.4 or higher, while Cheddar cheese curds are drained at pH 6.3 or lower. Proteolysis of /3-casein is significantly inhibited by 5% sodium chloride. The inhibitory influence of sodium chloride is most likely due to alteration of /3-casein or a reduction in the attractive forces between enzyme and substrate (Fox and Walley 1971). 

The gross proteolysis of casein is probably due solely to rennet and plasmin activity (O Keeffe et al. 1978). Bacterial proteases and peptides are responsible for subsequent breakdown of the large peptides produced by rennet and plasmin into successively smaller peptides and finally amino acids (O Keeffe et al. 1978). If the relative rate of proteinase activity by rennet, plasmin, and bacterial proteases exceeds that of the bacterial peptidase system, bitterness in the cheese could result. Bitter peptides can be produced from a,-,- or /3-casein by the action of rennet or the activity of bacterial proteinase on /3-casein (Visser et al. 1983). The proteolytic breakdown of /3-casein and the subsequent development of bitterness are strongly retarded by the presence of salt (Fox and Walley 1971 Stadhouders et al. 1983). The principal source of bitter peptides in Gouda cheese is 3-casein, and more particularly the C-terminal region, i.e., 3(193-209) and 3(193-207) (Visser et al. 1983). In model systems, bitter peptides are completely debittered by a peptidases system of S. cremoris (Visser et al. 1983). 

C-Methylated -Casein as a Substrate for Plasmin and Its Application to the Study of Milk Protein Transformations... 

Plasmin Hydrolysis of /3-Casein. Studies on the susceptibility of partially methylated /3-casein to cleavage by trypsin-like enzymes were carried out using the enzyme porcine plasmin. In preliminary investigations, we confirmed that the y-caseins produced by plasmin hydrolysis of native /3-casein were identical with those occurring naturally (28). These are designated yi-, y2-, and y3-casein according to the nomenclature recommendations of Whitney et al. (29) and correspond to residues 29-109, 106-209, and 198-209 of -casein. Presumably the proteose peptone products of plasmin hydrolysis are identical with their natural counterparts, but the latter were not available for comparison. 

Plasmin Hydrolysis of 14C-Methyl-/3-Casein. On the basis of a study by Ottesen and Svensson (2), which described the relative amounts of mono- and dimethyllysinyl derivatives produced by reductive methyla-... 

Figure 6. DEAE-cellulose chromatography of plasmin-treated H-/3-C admixed with whole casein.

P-Casein (13 mg) containing 0.036 fid M-P-C was subjected to plasmin hydrolysis for 45 min and the reaction mixture was dissolved in 7 mL column buffer (5mM Tris—3mM NaCl—urea, pH 8.55) together with 100 mg whole casein that had been alkylated with iodoacetamide. The sample solution was applied to a column (1.6 X 50 cm) of DEAE-cellulose equilibrated with column buffer. Elution was with a NaCl gradient (3.0-155mM) in column buffer (gradient volume, 1.0 L) 5.0 mL fractions were collected. Under the conditions used as as-caseins remained adsorbed to the column K-caseins were eluted in Fractions 35-56 Am measurement (----------) radioactivity (--) (28). 

Figure 7. PAGE of reaction mixture after plasmin hydrolysis of B-casein for 0, 20, 30, 40, and 70 min (Slots 1-5) and Fractions 1 ana 2 from hydroxyapatite chromatography of hydrolysis products after plasmin treatment of fi-casein for 70 min (Slots 6 and 7). Fraction 2 contains a further phosphopeptide that is not visible in Slot 7 but appears on disc gels in the expected position for proteose peptone component 8F (cf. Ref. 32) (28).

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