Papillae collected from the rumens of freshly killed cows were used to estimate the most appropriate methods for enzyme extraction from rumen epithelium and the amount of enzymes in extracts which might be of bacterial origin. Extractions of enzymes from fresh and frozen papillae were compared for the Polytron homogenizer (PT), the Potter-Elvehjem homogenizer (PE), the Waring blender, sonication and acetone powdering plus PE. PE extraction yielded solutions with the highest specific activity for each enzyme. PT extraction released the most protein and total enzyme activity into solution. PT extraction was chosen for the remaining tests because of the high total activity released. Mixed rumen bacteria were homogenized by sonication. Electrophoretic examination of epithelial and bacterial extracts showed differential migration for malate dehydrogenase. Lactate dehydrogenase from the epithelium showed four distinct isozymes whereas the bacterial enzyme showed little distinct band development. Contamination of epithelial extracts by bacterial protein was estimated to be less than 5%. The specific activities of 10 enzymes were found to be similar in epithelial and bacterial extracts so that a small amount of protein contamination would result in only a small contribution to total enzyme activity. The presence of the enzymes assayed in this study plus a number reported in the literature showed that rumen epithelial metabolism is more diverse than previously recognized. Key words: Rumen epithelium, enzymes, extraction
Methods for the measurement of a bacterial enzyme activity in cell lysates and extracts
