Intracellular localization of inositol-phospholipid-metabolizing enzymes in rabbit fast-twitch skeletal muscle. Can D-myo-inositol 1,4,5-trisphosphate play a role in excitation-contraction coupling?
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 [Varsányi 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. 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'-[gamma-thio]triphosphate does not influence phosphatidylinositol-4,5-bisphosphate phosphodiesterase activity. It correlates well with the observation that neither alpha 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).
Varsányi
M
M
Institut für Physiologische Chemie, Abteilung für Biochemie Supramolekularer Systeme, Ruhr-Universität Bochum, Federal Republic of Germany.
Messer
M
M
Brandt
N R
NR
D000818:Animals; D002499:Centrifugation, Density Gradient; D015544:Inositol 1,4,5-Trisphosphate; D007295:Inositol Phosphates / Q000502:physiology*; D008861:Microsomes / Q000201:enzymology; D009119:Muscle Contraction; D009132:Muscles / Q000201:enzymology* / Q000648:ultrastructure; D010716:Phosphatidylinositols / Q000378:metabolism*; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D010770:Phosphotransferases / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor); D011817:Rabbits; D051381:Rats; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D013347:Subcellular Fractions / Q000201:enzymology*; D013403:Sugar Phosphates / Q000502:physiology*
D007295:Inositol Phosphates; D010716:Phosphatidylinositols; D013403:Sugar Phosphates; D015544:Inositol 1,4,5-Trisphosphate; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C