The metabolism of d‐myo‐inositol 1,4,5‐trisphosphate and d‐myo‐inositol 1,3,4,5‐tetrakisphosphate by porcine skeletal muscle
Authors: FOSTER Paul S. , HOGAN Simon P. , HANSBRO Philip M. , O'BRIEN Robert , POTTER Barry V. L. , OZAKI Shoichiro , DENBOROUGH Michael A. 
Published: March 2005

In soluble and particulate extracts from muscle d‐myo‐inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3] and d‐myo‐inositol 1,3,4,5‐tetrakisphosphate [Ins(1,3,4,5)P4] are metabolised stepwise to inositol. Ins(1,4,5)P3 is rapidly dephosphorylated to d‐myo‐inositol 1,4‐bisphosphate then to d‐myo‐inositol 4‐phosphate and finally inositol. In soluble extracts Ins(1,3,4,5)P4 is dephosphorylated to d‐myo‐inositol 1,3,4‐trisphosphate then sequentially to d‐myo‐inositol 3,4‐bisphosphate, d‐myo‐inositol 3‐phosphate and inositol, while in particulate extracts d‐myo‐inositol 1,3‐bisphosphate is the predominant inositol bisphosphate formed. Dephosphorylation of these inositol polyphosphates is Mg2+ dependent and inhibited by d‐2,3‐bisphosphoglyceric acid. Ins(1,4,5)P3 is also phosphorylated to form Ins(1,3,4,5)P4 in soluble extracts by Ins(1,4,5)P3 3‐kinase. Ins(1,4,5)P3 3‐kinase activity is Mg2+ and ATP dependent and is stimulated by Ca2+ and calmodulin. Particulate (sarcotubular) inositol polyphosphate 5‐phosphatase (5‐phosphatase) is found in membranes which are intimately involved in excitation‐contraction coupling and the generation of the primary Ca2+ signal of muscle cells. Particulate 5‐phosphatase had the highest specific activity in the transverse‐tubule membrane, when compared to the terminal cisternae and longitudinal‐tubule membranes of the sarcoplasmic reticulum. Particulate Ins(1,3,4,5)P4‐3‐phosphatase activity was also detected after fractionation of solubilised sarcotubular membranes by DEAE‐Sephacel. Particulate 5‐phosphatase activity was purified 25600‐fold to a specific activity of 25.6 μmol Ins(1,4,5)P3 hydrolysed min−1 mg protein−1, after DEAE‐Sephacel and novel affinity chromatography using d‐2,3‐bisphosphoglycerate/agarose and Sepharose‐4B‐immobilised Ins(1,4,5)P3‐analog matrices. Purified particulate 5‐phosphatase had apparent Km of 46.3 μM and 1.9 μM and Vmax of 115 and 0.046 μmol substrate hydrolysed min−1 mg protein−1, for Ins(1,4,5)P3 and Ins(1,3,4,5)P4, respectively. In contrast, purified soluble type I 5‐phosphatase had apparent Km of 8.9 μM and 1.1 μM and Vmax of 3.55 and 0.13 μmol substrate hydrolysed min−1 mg protein−1, for Ins(1,4,5)P3 and Ins(1,3,4,5)P4, respectively. As in other cells, muscle 5‐phosphatases have a lower affinity, but a higher capacity to metabolise Ins(1,4,5)P3 than Ins(1,3,4,5)P4. Soluble type I 5‐phosphatase may have a functional role in the metabolism of both inositol polyphosphates, while particulate 5‐phosphatase may primarily metabolise Ins(1,4,5)P3. Purified Ins(1,4,5)P3 3‐kinase had an apparent Km of 0.42 μM and a Vmax of 4.12 nmol Ins(1,4,5)P3 phosphorylated min−1 mg protein−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)P3/Ins(1,3,4,5)P4, is consistent with a role for the phosphoinositol‐lipidsignalling pathway in Ca2+ homeostasis of skeletal muscle.