Leakage of Aldolase from Rat Diaphragm Induced by EDTA

Abstract

Increased enzyme activity in serum is found in several nondisease states such as exercise (7) as well as under pathological conditions (5) including delirium tremens (19). The cellular mechanism of this elevation is obscure but may be related to the control, by divalent cations, of the passive permeability properties of several membrane types. Examples of this mode of regulation have been observed as an increased passage of macromolecules across mammalian intestine (17), the leakage of protein from rat liver slices (10), and the uptake of actinomycin by bacteria (12). Accordingly this study was designed to elucidate cellular enzyme loss from muscle with the intent of demonstrating the role of calcium and magnesium in the maintenance of membrane permeability and the functional integrity of skeletal muscle. The ionic permeability status of the preparation was monitored by assessing the transmembrane ionic gradient for sodium and potassium under the experimental conditions. Disodium dihydrogen ethylenediamine-tetraacetate (Na₂H₂EDTA) was the chelating agent used to promote enzyme loss from rat diaphragm muscle in vitro. Aldolase was selected for study because it is present in high concentration in muscle; it is believed to be a soluble intracellular enzyme; its leakage from rat diaphragm has been studied under other circumstances (18); and, unlike the metalloenzymes, the assay procedure for aldolase may be undertaken successfully in the presence of chelating agents (9).

Methods. Entire diaphragms of adult, 300–400 g, Sprague-Dawley rats maintained on commercial chow were removed under ether anesthesia and washed in ice-cold, physiologic saline solution. Each diaphragm was cut into segments of approximately 50 mg and placed in fresh, ice-cold saline to remove blood clots from the surface. The segments were blotted, weighed, and placed immediately into the appropriate incubation medium.