The determination of the initial velocities of enzyme-catalysed reactions from stopped-assay data [proceedings].

the gradient fractions for L-glutamine y-2-naphthylamide hydrolase activity, i.e. in the absence of glycylglycine, three peaks of activity were again observed. The activities a t densities of 1.24 and 1.15 were only 10% of the transpeptidase activity. However, the peak corresponding to soluble enzyme gave the same activity as that obtained in the presence of acceptor. This indicates that this activity is not due to a soluble form of y-glutamyl transpeptidase, but is the activity of a hydrolytic enzyme acting on Lglutamine y-2-naphthylaniide. Determination of the apparent Michaelis constant for L-glutamine y-2-naphthylamide for each gradient fraction revealed that the K , of the particulate activity was 0 . 3 m ~ and that of the soluble activity was 2 0 ~ ~ . This confirms that the particulate and soluble activities are due to different enzymes. Comparison of the distribution of y-glutaniyl transpeptidase with that of marker enzymes for various organelles indicates that the particulate y-glutamyl transpeptidase activity is associated with the plasma membrane. However, although the distribution of y-glutamyl transpeptidase activity is similar to that of other plasma-membrane enzymes (Figs. lale , --), there are differences in the distributions not only between y-glutamyl transpeptidases and the marker enzymes but also between the marker enzymes themselves. This suggests a heterogeneity of the plasma membrane. Preparation of a rat liver homogenate in iso-osmotic sucrose containing digitonin ( I mg/ml) and subjected to isopycnic centrifugation as before gave the results shown in Figs. I (a)-(e) (----). The particulate y-glutamyl transpeptidase activity and all the plasma-membrane marker activities, except leucine-2-naphthylamidase, have shifted to higher densities. Although there is again a marked similarity between the distributions, they are not identical. The membrane perturbant digitonin solubilizes a large amount of particulate leucine-2-naphthylamidase (Fig. Id) . This enzyme has been reported (Emmelot & Visser, 1971) to be associated with the small globular units found on the outer aspect of plasma membrane forming the biliary canuliculi, and can be removed by detergents. The loss of leucine-2-naphthylamidase activity from the 1.24-density region of the sucrose gradient suggests that enzyme activities occurring in this region may be located in the biliary canulicular plasma membrane. The results indicate that, in rat liver, y-glutamyl transpeptidase is a particulate enzyme and is associated with the plasma membrane. The differences in the equilibrium densities of y-glutamyl transpeptidase and the plasma-membrane marker enzymes suggest a distinct heterogeneity of this membrane. At the present it is uncertain whether this is a reflection of different cell types or of these enzymes being located in different zones of the plasma membrane.