The attachment of glycolytic enzymes to muscle ultrastructure

The glycolytic enzymes, lactic dehydrogenase and aldolase, usually thought to be freely dissolved in the sarcoplasmic matrix, are in good part attached to the muscle ultrastructure. This attachment becomes manifest when the enzyme activities and specific activities of the press juices of whole skeletal muscles (rabbit) are compared with those of minced muscles, all obtained by ultracentrifugation of the tissues at 40,000 xpm for 16 to 20 hours. Mincing causes a great increase in the activities, associated with a rise in the volume and protein concentration of the press juices. We interpret these increases to be due to the solution in the matrix of enzymes previously attached to the ultrastructure. The same conclusion is reached by a different method, which we call “washing the ultrastructure.” It consists in multiple centrifugations of whole skeletal muscles, and removal of press juices, alternating with periods of imbibition of a buffer (0.1 M phosphate at pH 7.5) too dilute to dissolve out the fibrous proteins. During the imbibitions enzymes diffuse out into the buffer not imbibed, which becomes an extract. After four centrifugation-imbibition sequences in as many days nearly all of the fluid matrix has been replaced by buffer. Enzyme activities fall steeply in press juices and extracts until nearly all freely dissolved enzymes have been washed away. Homogenates of the pressed muscles then show activities which are about half of those found in the homogenates of unpressed control muscles. We conclude that the enzymes found in the homogenates of the pressed muscles have previously been attached to the ultrastructure. Similar experiments with heart muscle indicate that nearly all of these enzymes are normally attached to the ultrastructure. Press juices contain only traces of activity, even after the heart has been minced. A fraction of the enzymes is slowly detached during the centrifugation-imbibition sequences, appearing mainly in the extracts.