THE METABOLISM OF D-MYO-INOSITOL 1,4,5-TRISPHOSPHATE AND D-MYO-INOSITOL 1,3,4,5-TETRAKISPHOSPHATE BY PORCINE SKELETAL-MUSCLE

In soluble and particulate extracts from muscle D-myo-inositol 1,4,5-t risphosphate [Ins(l,4,5)P-3] and D-myo-inositol 1,3,4,5-tetrakisphosph ate [Ins(1,3,4,5)P-4] are metabolised stepwise to inositol. Ins(1,4,5) P-3 is rapidly dephosphorylated to D-myo-inositol 1,4-bisphosphate the n to D-myo-inositol 4-phosphate and finally inositol. In soluble extra cts Ins(1,3,4,5)P-4 is dephosphorylated to D-myo-nositol 1,3,4-trispho sphate then sequentially to D-myo-inositol 3,4-bisphosphate, D-myo-ino sitol 3-phosphate and inositol, while in particulate extracts D-myo-in ositol 1,3-bisphosphate is the predominant inositol bisphosphate forme d. Dephosphorylation of these inositol polyphosphates is Mg2+ dependen t and inhibited by D-2,3-bisphosphoglyceric acid. Ins(1,4,5)P-3 is als o phosphorylated to form Ins(1,3,4,5)P-4 in soluble extracts by Ins(1, 4,5)P-3 3-kinase. Ins(1,4,5)P-3 3-kinase activity is Mg2+ and ATP depe ndent and is stimulated by Ca2+ and calmodulin. Particulate (sarcotubu lar) inositol polyphosphate S-phosphatase (5-phosphatase) is found in membranes which are intimately involved in excitation-contraction coup ling and the generation of the primary Ca2+ signal of muscle cells. Pa rticulate 5-phosphatase had the highest specific activity in the trans verse-tubule membrane, when compared to the terminal cisternae and lon gitudinal-tubule membranes of the sarcoplasmic reticulum. Particulate Ins(1,3,4,5)P-4-3-phosphatase activity was also detected after fractio nation of solubilised sarcotubular membranes by DEAE-Sephacel. Particu late 5-phosphatase activity was purified 25600-fold to a specific acti vity of 25.6 mu mol Ins(1,4,5)P-3 hydrolysed . min(-1) . mg protein(-1 ), after DEAE-Sephacel and novel affinity chromatography using D-2,3-b isphosphoglycerate/agarose and Sepharose-4B-immobilised Ins(l,4,5)P-3- analog matrices. Purified particulate 5-phosphatase had apparent K-m o f 46.3 mu M and 1.9 mu M and V,, of 115 and 0.046 mu mol substrate hyd rolysed min(-1) . mg protein(-1), for Ins(1,4,5)P-3 and Ins(1,3,4,5)P- 4, respectively. In contrast, purified soluble type I . 5-phosphatase had apparent K, of 8.9 mu M and l.l mu M, and V-max of 3.55 and 0.13 m u mol substrate hydrolysed . min(-1) . mg protein(-)1, for Ins(1,4,5)P -3 and Ins(1,3,4,5)P-4, respectively. As in other cells, muscle 5-phos phatases have a lower affinity, but a higher capacity to metabolise In s(1,4,5)P-3 than Ins(1,3,4,5)P-4. Soluble type I 5-phosphatase may hav e a functional role in the metabolism of both inositol polyphosphates, while particulate 5-phosphatase may primarily metabolise Ins(1,4,5)P- 3. Purified Ins(1,4,5)P-3 3-kinase had an apparent K-m, of 0.42 mu M a nd a V-max of 4.12 nmol Ins(1,4,5)P-3 phosphorylated . min(-1) . mg pr otein(-1). The profile of inositol polyphosphate metabolism in muscle is similar to that reported in other tissues. The presence of enzymes involved in the metabolism of Ins(1,4,5)P-3/Ins(1,3,4,5)P-4, is consis tent with a role for the phosphoinositol-lipid-signalling pathway in C a2+ homeostasis of skeletal muscle.