Changes in inositol-1,4,5-trisphosphate binding to hepatic plasma membranes caused by temperature, N-ethylmaleimide and menadione.
We investigated the effects of the sulfhydryl-alkylating agent N-ethylmaleimide (NEM) and menadione--a sulfhydryl-arylating agent, which can undergo redox cycling--on the [3H]inositol-1,4,5-trisphosphate ([3H]IP3) binding properties of rat plasma membranes. Rat liver plasma membranes were incubated for 15 min at 37 degrees with 0.1 mM, 0.2 mM, 0.5 mM NEM or 0.3 mM menadione and subsequently diluted for use in [3H]IP3 binding studies. An incubation as such (15 min at 37 degrees) already caused the dissociation constant (Kd) of [3H]IP3 binding to increase from 1.9 +/- 0.2 nM to 3.4 +/- 0.2 nM, with only a small change in the maximal number of IP3 binding sites (Bmax-values of 401 +/- 32 and 349 +/- 13 fmol/mg protein, respectively). Incubation with NEM (0.1, 0.2 and 0.5 mM) resulted in a dose dependent decrease in the Bmax with 41, 87 and 99%, respectively, without a significant change in the Kd compared to the time matched controls. Menadione (0.3 mM) decreased the Bmax with 54% without affecting the Kd. In contrast to our findings at 37 degrees, incubation of the plasma membranes with NEM (0.5 mM) at 0 degrees for 30 min did not affect [3H]IP3 binding. In order to account for this discrepancy, the reaction rate of NEM with glutathione was examined at both 0 degrees and 37 degrees by recording the changes in the UV-spectrum of NEM (lambda max = 302 nm) after addition of 1 mM NEM to 1 mM glutathione. A similar reaction rate was observed at both temperatures. These data suggest that alkylation of a sulfhydryl-moiety in the IP3-receptor molecule causes inactivation of the receptor function. Since at 0 degrees NEM is still able to react with sulfhydryl groups, but not able to inactivate the IP3-receptor, it can be suggested that the sulfhydryl-moiety of the IP3-receptor is masked and cannot be reached by a sulfhydryl-alkylating agent at 0 degrees.
Pruijn
F B
FB
Department of Pharmacochemistry, Faculty of Chemistry, Vrije Universiteit, Amsterdam, The Netherlands.
Sibeijn
J P
JP
Bast
A
A
D000818:Animals; D015220:Calcium Channels; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology*; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008099:Liver / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D011956:Receptors, Cell Surface / Q000187:drug effects* / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear; D013696:Temperature; D014812:Vitamin K / Q000494:pharmacology*
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D014812:Vitamin K; D015544:Inositol 1,4,5-Trisphosphate; D005033:Ethylmaleimide