Intracellular localization of inositol‐phospholipid‐metabolizing enzymes in rabbit fast‐twitch skeletal muscle

Rabbit fast‐twitch skeletal muscle microsomes have been separated by isopycnic centrifugation on a linear sucrose gradient into triads and light sarcoplasmic reticulum. In both fractions phosphatidylinositol‐kinase activity is found [Varsànyi et al. (1986) Biochem. Biophys. Res. Commun. 138 , 1395]. In contrast, phosphatidylinositol‐4‐phosphate kinase is nearly exclusively associated with triads. The phosphatidylinositol‐4,5‐bisphosphate‐phosphodiesterase activity shows a biphasic distribution: approximately 50% of the activity is associated with triads and 50% appears in the overlay. Triads have been broken mechanically into transverse tubules and terminal cisternae, then separated by isopycnic sucrose‐gradient centrifugation. Both fractions exhibit phosphatidylinositol‐kinase activity; the activities of phosphatidylinositol‐4‐phosphate kinase and phosphatidylinositol‐4,5‐bisphosphate phosphodiesterase are associated mainly with the transverse tubules. Consequently, in rabbit fast‐twitch skeletal muscle all necessary enzymes for production of D‐ myo ‐inositol 1,4,5‐trisphosphate are associated with transverse tubules. Phosphatidylinositol‐4,5‐bisphosphate phosphodiesterase associated with triads shows a pH optimum at 6.8. The enzyme is maximally active between pCa 5 and pCa 4. Mg 2+ inhibits the enzyme activity half‐maximally at about 1 mM. Guanine‐nucleotide‐binding proteins seem not to be involved in the regulation of enzyme activity; guanosine 5′‐[γ‐thio]triphosphate does not influence phosphatidylinositol‐4,5‐bisphosphate phosphodiesterase activity. It correlates well with the observation that neither α 1 ‐adrenergic nor muscarinic receptors have been found in fast‐twitch rabbit skeletal muscle. On basis of the respective enzyme activities estimations on maximal phosphatidylinositol turnover were made and a possible involvement of this signal pathway in excitation‐contraction coupling has been discussed. Furthermore, calculations show that during a single twitch D‐ myo ‐inositol 1,4,5‐trisphosphate concentration does not reach more than 2 nM. However, during a 4‐s tetanus D‐ myo ‐inositol 1,4,5‐trisphosphate can accumulate to a level which could effect force generation [Thieleczek and Heilmeyer (1986) Biochem. Biophys. Res. Commun. 135 , 662] and aldolase distribution (Thieleczek et al., unpublished results).