Summary: Analysis of periplasmic enzymes of or biovar was hampered by the fact that only small amounts of marker enzyme activities were released from cells by osmotic shock or by lysozyme/EDTA treatment. However, up to 95 % of total activity of certain periplasmic marker enzymes could be measured in intact cells by including 0·03% Triton X-100 in enzyme assays. Less than 5% of the cytoplasmic marker enzyme β-hydroxybutyrate dehydrogenase could be measured under these conditions, indicating that treatment with dilute detergent does not significantly perturb the cytoplasmic membrane. Various lines of evidence indicate that the dilute detergent treatment does not significantly alter wall structure but, instead, permits the assay of periplasmic enzymes by facilitating diffusion of substrates and products to and from the periplasmic space. For pyrophosphatase and phosphodiesterase, the proportion of total enzyme activity which could be measured in intact cells was higher in bacteroids than in bacteria at all Triton concentrations, suggesting that the outer membrane of bacteroids may be more permeable than the outer membrane of cultured bacteria. The effect of detergent on the hydrolysis of four disaccharides — sucrose, α,α-trehalose, maltose and lactose — was studied using intact cultured biovar The response of lactose hydrolysis to the addition of detergent was four-fold greater than the response of the other disaccharidase activities, indicating that a rhizobial lactase may be present in the periplasmic space.


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