EFFECTS OF PROTEASE INHIBITORS ON DEGRADATION OF H-3[C-14]-CASEIN BY RUMINAL MICROORGANISMS

Effects of various chemicals on proteolytic activity of mixed ruminal microbes was evaluated as the rate of release of trichloroacetic solub le C-14 (TCAS-C-14) during 15 min incubation of H-3[C-14]-casein with ruminal fluid with or without 0.1-1 mM of the chemical. That TCAS-C-14 , presumed to be small peptides of H-3[C-14]-lysine or H-3[C-14]-lysin e, was not further metabolized was confirmed by quantitative recovery of TCAS-C-14 on continued incubation. The viability of the microbial e cosystem was indicated by the rapid disappearance of TCAS-C-14 when 1- [C-14]-leucine was similarly incubated. Synthetic protease inhibitors of serine and cysteine proteases (n-tosyl-1-lysine, chloromethyl keton e, phenylmethylsulfonyl fluoride, p-chloromercuribenzoate and iodoacet ate) caused significant (P < .01) inhibition of proteolysis at all con centrations tested. Diphenyliodonium chloride, at 0.8 mM, resulted in a similar degree of inhibition as the most effective protease inhibito rs. Inhibitors of microbial origin that inhibit cysteine and cysteine- serine protease, E-64 and leupeptin, respectively, also showed signifi cant (P < .05) inhibitory effects at all concentrations, whereas inhib itors of aspartic proteases (pepstatin) had no inhibitory effect. Inhi bitors of serine proteases, soybean trypsin inhibitor type II, also ha d significant (P < .05) inhibitory effects. These results confirm that protease of mixed ruminal microbes are predominantly of the cysteine and serine types. Such inhibition occurs within 2 minutes or less and, therefore, is likely a specific effect of protease inhibition. The mi crobial protease inhibitors E-64 and leupeptin appear potentially most useful for limiting excessive degradation of intake proteins by the r uminal microbial ecosystem.