Biomedical subjects

E D Bastian

Publications and source records attributed to E D Bastian.

3 recordsLinked to original sources

PubMed1996

A method for isolating beta-casein.

A new method was developed for obtaining pure beta-CN. Calcium caseinate (3%) was reconstituted, renneted to form a gel, cooled (4 degrees C) to allow beta-CN dissociation from the caseinate gel, and centrifuged. The supernatant was warmed to 30 degrees C, precipitating pure beta-CN from solution. Large quantities of beta-CN were recovered by scaling-up this procedure, but these beta-CN preparations were less pure than the beta-CN that was prepared on a smaller scale. Chromatography (FPLC) and urea-PAGE showed beta-CN to be the main component in the precipitate. Chymosin, used to form the caseinate gel, did not extensively hydrolyze beta-CN under the conditions of these experiments. Calcium concentration, cooling time, and caseinate concentration influenced the recovery of beta-CN. Maximum recovery of beta-CN, under the experimental conditions used, occurred at 10 mM calcium, 48 h of cooling, and 3% caseinate concentration.

Calcium↗

PubMed1993

Inhibition of plasmin by beta-lactoglobulin using casein and a synthetic substrate.

Bovine plasmin (EC 3.4.21.7) activity was measured on H-D-valyl-L-leucyl-L-lysyl-4-nitroanilide and acid casein in the presence of native and heat-denatured beta-lactoglobulin (denatured at 100 degrees C for 15 min before being mixed with plasmin solutions). Native or denatured beta-lactoglobulin was then heated with plasmin at 60 degrees C for 15 min. Enzyme activity again was estimated after this mild heat treatment. Native and denatured beta-lactoglobulin inhibited the action of plasmin on H-D-valyl-L-leucyl-L-lysyl-4-nitroanilide and casein. The mild heat treatment (60 degrees C for 15 min) caused stronger inhibition of the activity of plasmin against casein and the synthetic substrate. For H-D-valyl-L-leucyl-L-lysyl-4-nitroanilide, inhibition was competitive in unheated mixtures, but heating beta-lactoglobulin with plasmin changed inhibition type to mixed. This change suggests a heat-dependent interaction between plasmin and beta-lactoglobulin. Native beta-lactoglobulin was more inhibitory of plasmin's action against casein than was denatured beta-lactoglobulin. The converse was observed when plasmin activity was measured with the synthetic substrate.

Animals↗

PubMed1991

Casein interference in bovine plasmin assays using a synthetic substrate.

Bovine plasmin (EC 3.4.21.7) activity on H-D-valyl-L-leucyl-L-lysyl-4-nitroanilide was measured by determining the change in absorbance at 405 nm. Initial rates of reactions were estimated at all combinations of the following substrate concentrations [.4, 4, and 40 times the substrate concentration at one-half maximum velocity (Vmax) (Km)] and casein concentrations [.068, .68, and 6.8 times the inhibitor constant for competitive inhibition (KI)]. By nonlinear least squares fitting of the data to an equation that described reversible enzyme kinetics, steady state kinetic parameters, maximum velocity (Vmax), substrate concentration at one-half maximum velocity (Vmax) (Km), inhibitor constant for competitive inhibition (KI), and inhibitor constant for uncompetitive inhibition (KI) were estimated. Casein fit the equation as a competitive inhibitor of bovine plasmin. This enzyme has a catalytic constant (Kcat) of .0158 change in absorbance at 405 nm/min per nM, substrate concentration at one-half maximum velocity (Vmax) (Km) of .107 mM substrate, and inhibitor constant for competitive inhibition (KI) of .86 mg/ml of casein. Bovine plasmin activity can be measured directly in bovine milk without interference from casein.

Animals↗