Effects of protease inhibitors on degradation of H3[14C]-casein by ruminal microorganisms

Effects of various chemicals on proteolytic activity of mixed ruminal microbes was evaluated as the rate of release of trichloroacetic soluble 14C (TCAS-14C) during 15 min incubation of H3[14C]-casein with ruminal fluid with or without 0.1 – 1 mm of the chemical. That TCAS-14C, presumed to be small peptides of H3[14C]-lysine or H3[14C]-lysine, was not further metabolized was confirmed by quantitative recovery of TCAS-14C on continued incubation. The viability of the microbial ecosystem was indicated by the rapid disappearance of TCAS-14C when 1-[14C]-leucine was similarly incubated. Synthetic protease inhibitors of serine and cysteine proteases (n-tosyl-1-lysine, chloromethyl ketone, phenylmethylsulfonyl fluoride, p-chloromercuribenzoate and iodoacetate) caused significant (P < .01) inhibition of proteolysis at all concentrations tested. Diphenyliodonium chloride, at 0.8 mM, resulted in a similar degree of inhibition as the most effective protease inhibitors. Inhibitors of microbial origin that inhibit cysteine and cysteine-serine protease, E-64 and leupeptin, respectively, also showed significant (P < .05) inhibitory effects at all concentrations, whereas inhibitors of aspartic proteases (pepstatin) had no inhibitory effect. Inhibitors of serine proteases, soybean trypsin inhibitor type II, also had 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 microbial protease inhibitors E-64 and leupeptin appear potentially most useful for limiting excessive degradation of intake proteins by the ruminal microbial ecosystem.