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

Abstract

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 [Varsanyi et al. (1986) Biochem. Biophys. Rex Commun. 138, 13951. In contrast, phosphatidylinositol-4-phosphate kinase is nearly exclusively associated with triads. The phosphatidylinositol-4,5-bisphosphatephosphodiesterase 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.Mg2+ 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'-[y-thio]triphosphate does not influence phosphatidylinositol-4,5-bisphosphate phosphodiesterase activity. It correlates well with the observation that neither cr,-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 (1 986) Biochem. Biophys. Res. Commun. 135, 6621 and aldolase distribution (Thieleczek et al., unpublished results).

Masses of inositol phosphates in resting and tetanically stimulated vertebrate skeletal muscles

“…Although a putative voltage sensor of the T-tubule membrane is involved in the process of ECC[251, at present there is no evidence for activation of PIC by this protein or any other receptor-coupling mechanism. Activation of PIC in isolated triads by Ca2+ [6] and a recent finding of a Ca2+-activated PIC in bovine rod outer segments [26] argue for this activation mechanism of PIC in muscle, although the alternative, i.e. activation by increased substrate access, cannot be ruled out.…”

“…There is some experimental evidence that skeletal muscle depolarization leads to a transient increase in Ins(1,4,5)P3 turnover, as measured by means of radioisotope techniques [4,5]. On the other hand, the generation of Ins(1,4,5)P3 by PIC appears to be Ca2+-dependent [6], which is not in agreement with a direct activation of PIC by membrane depolarization. Ins(1,4,5)P3 has been described as the initiator of Ca2+ release from the sarcoplasmic reticulum (SR) during excitation-contraction coupling (ECC); however, the available data are controversial (for review, see [7]).…”

“…Ins(1,4,5)P3 and other inositol phosphates are potent competitive inhibitors of the glycolytic enzyme aldolase A [12]. We have shown that a fraction of the sarcoplasmic aldolase that is bound to cellular structures such as myofilaments and triads is released upon binding of inositol phosphates and fructose 1,6-bisphosphate [Fru (1,6)P2] to the enzyme [13]. This repartitioning of aldolase might also be responsible for the reported Ins(1,4,5)P3-induced enhancement of the Ca2+ sensitivity of the contractile mechanism [14].…”

Anionic phospholipids decrease the rate of slippage on the Ca2+-ATPase of sarcoplasmic reticulum

“…The observation that the optimal concentration of anionic phospholipid (10 mol %) equals the mole fraction of anionic phospholipid in the native SR membrane [29,30] suggests that reducing slippage might be important for the physiological function of the Ca# + -ATPase. The observation that PtdIns(4)P is more effective in reducing slippage than is PtdIns suggests a possible mechanism for control of the Ca# + accumulation by the ATPase ; PtdIns-4-kinase has been detected in SR, as has a Ca# + -dependent phosphomonoesterase capable of catalysing the breakdown of PtdIns(4)P [43,44].…”

Human phosphatidylinositol 4‐kinase isoform PI4K92

“…PtdIns kinase activity was assayed at 37 °C in an optimized test as described previously [16,17]. The phospholipids produced were extracted with chloroform and washed with 1 m HCl/methanol (1 : 1) as described previously [18].…”