Abstract
An inositol polyphosphate receptor has been purified from bovine cerebellum which consists of three different polypeptides with Mr of 111,000, 102,000, and 52,000. Negative staining electron microscopy reveals globular-like structures 10-13 nm in diameter. The receptor has a Stokes radius of 400,000 daltons as determined by molecular sieve high performance liquid chromatography. The receptor preparation binds inositol 1,3,4,5-tetrakisphosphate, inositol hexaphosphate (or phytol), and inositol 1,4,5-trisphosphate (IP4, IP6, and IP3, respectively) with submicromolar affinity (0.19, 0.15, and 0.54 microM, respectively) at conditions approximating physiological ionic strength and pH. The purified receptor preparation, when reconstituted into planar bilayers, displays ion channel activity, preferentially permeable to K+. Permeability ratios of the channel are PK+/PNa+ approximately 5 and PK+/PCl approximately 19. In symmetrical 100 mM KCl, the channel is characterized by long open times (minutes) with a conductance of 7.2 picosiemens. The channel is selectively modulated by IP4. That is, at 1 microM IP4, the mean open time decreased substantially to rapid flicker behavior and the channel is completely closed at 10 microM IP4. IP6 and IP3 did not modulate the channel under similar conditions. Thus, the channel appears to be an IP4-modulated K+ channel.
Highlights
D "The concentration of [3H]IP4used to determine binding was 10 fractionsobtained from the phenyl-Sepharose 4B column; lane 6
Lane 1, supernatant extract of cerebellum microsomes with 2% CHAPS; lane 2, pooled fractions from the first heparin-agarose column; lane 3, pooled fractions obtained from the DEAE-Sepharose 6B column; lane 4, pooled fractions obtained from the second heparin-agarose column; lane 5,pooled
We have purified an inositol polyphosphate receptor from bovine cerebellum which binds IP, IP3,and IPS.The receptor preparation, when reconstituted into planar bilayers, displays channel currents were estimated from events with open times longer than 50 ms
Summary
Sterling Drug Inc., 81 Columbia Turnpike, Rensselaer, NY 12144. ll Recipient of NRSA Postdoctoral Fellowship of ‘the National Institutes of Health. Sterling Drug Inc., 81 Columbia Turnpike, Rensselaer, NY 12144. Ll Recipient of NRSA Postdoctoral Fellowship of ‘the National Institutes of Health. The abbreviations used are: IP3,inositol 1,4,5-trisphosphate; IP,, inositol 1,3,4,5-tetrakisphosphate;IPS, phytol or inositol hexaphosphate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel elec-. Protein determination was performed by the method of Kaplan [16] or by scanning densitometry of protein bands on Coomassie Blue-R-250 stained SDS-polyacrylamide gels using an automated gel analysis plus image processing system (TechnologyResources, Nashville, TN). Bovine serum albumin was used as standard. Trophoresis; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-lpropanesulfonic acid; DTT, dithiothreitol; BSA, bovine serum albu-. Electron Microscopy min; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonicacid; Negative staining of the purified receptor was performed as de-.
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