Characteristics of the rumen proteolysis of fraction I (18S) leaf protein from lucerne (Medicago sativa L)

Abstract

1. The rate of proteolysis of fraction 1 (18S) leaf protein in the rumen of sheep and cattle was affected by diet and the rate on fresh lucerne (Medicago sativa L) was three to nine times the rate on a hay + concentrate diet.2. Simultaneous rumen fermentations in vivo and in an artificial rumen showed that the rates of proteolysis of fraction I in vitro was approximately 80% of the rates in sheep.3. Using 14C uniformly-labelled fraction I protein at low concentrations, proteolysis exhibited 1st-order kinetics. Over a wide range of protein concentrations the velocity v. substrate concentration curve showed Michaelis–Menten characteristics typical of an enzyme-catalysed reaction. With rumen fluid from a hay + concentrate-fed sheep the maximum velocity was 2.6 mg protein nitrogen/1 per min and the Michaelis constant was 75 mg nitrogen/1.4. Rapid adsorption of 14C-labelled fraction I protein onto bacterial cells preceded proteolysis.5. Sucrose-density-gradient analysis showed initial incorporation of 14C from protein into rumen bacteria followed by partial transfer to rumen protozoa.6. No peptides were detected during proteoiysis showing that the rate-limiting step occurred during the initial stages of proteolysis. Only small amounts of free amino acids were released except for leucine, isoleucine, valine and ornithine, which showed significantly increased levels.7. Volatile fatty acids were the main 14C-labelied end products and were rapidly produced in descending concentrations: acetate > propionate > 3-methyl + 2-methyl butyrate > butyrate > isobutyrate > valerate.