TY - JOUR
T1 - Characterization of a phosphatidylinositol 4-phosphate-specific phosphomonoesterase in rat liver nuclear envelopes
AU - Smith, Charles D.
AU - Wells, William W.
N1 - Funding Information:
‘This work was supported by Grant AM 32930 from the United States Public Health Service. * To whom inquiries should be addressed. 3 Abbreviations used: PIP, phosphatidylinositol phosphate; PIPa, phosphatidylinositol 4,5-bisphosphate; PI, phosphatidylinositol; PA, phosphatidic acid; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; Mes, I-morpholineethanesulfonic Tricine, N-[2-hydroxy-l,l-bis(hydroxymethyl)ethyl]- glycine.
PY - 1984/12
Y1 - 1984/12
N2 - Incubation of rat liver nuclear envelopes with [γ-32P]ATP resulted in the synthesis of phosphatidylinositol-[4-32P]phosphate (PIP). Degradation of endogenously labeled PIP was observed upon the dilution of the labeled ATP with an excess of unlabeled ATP. This degradation was most rapid in the presence of EDTA, and was inhibited by MgCl2 and CaCl2. To further characterize the degradative activity, phosphatidyl-inositol[4-32P]phosphate and phosphatidylinositol [4,5-32P]bisphosphate (PIP2) were synthesized and isolated from erythrocyte plasma membranes. The 32P-labeled phospholipids were then resuspended in 0.4% Tween 80, a detergent that did not inhibit degradation of endogenously labeled PIP, and mixed with nuclear envelopes. [32P]PIP and [32P]PIP2 were degraded at rates of 2.25 and 0.04 nmol min-1 mg nuclear envelope protein-1, respectively. Only 32P was released from phosphatidyl[2-3H]inositol-[4-32P]phosphate, indicating that hydrolysis of PIP was due to a phosphomonoesterase activity (EC 3.1.3.36) in nuclear envelopes. Similarly, anion-exchange chromatographic analysis of the water-soluble products released from [32P]PIP indicated that inorganic phosphate was the sole 32P-labeled product. Hydrolysis of PIP was most rapid at neutral pH, and was not affected by inhibitors of acid phosphatase or alkaline phosphatase. Hydrolysis of PIP was also not inhibited by nonspecific phosphatase substrates, such as glycerophosphate, p-nitrophenylphosphate, AMP, or glucose 6-phosphate. Hydrolysis was stimulated by putrescine, and was inhibited by inositol 2-phosphate, spermidine, spermine, and neomycin.
AB - Incubation of rat liver nuclear envelopes with [γ-32P]ATP resulted in the synthesis of phosphatidylinositol-[4-32P]phosphate (PIP). Degradation of endogenously labeled PIP was observed upon the dilution of the labeled ATP with an excess of unlabeled ATP. This degradation was most rapid in the presence of EDTA, and was inhibited by MgCl2 and CaCl2. To further characterize the degradative activity, phosphatidyl-inositol[4-32P]phosphate and phosphatidylinositol [4,5-32P]bisphosphate (PIP2) were synthesized and isolated from erythrocyte plasma membranes. The 32P-labeled phospholipids were then resuspended in 0.4% Tween 80, a detergent that did not inhibit degradation of endogenously labeled PIP, and mixed with nuclear envelopes. [32P]PIP and [32P]PIP2 were degraded at rates of 2.25 and 0.04 nmol min-1 mg nuclear envelope protein-1, respectively. Only 32P was released from phosphatidyl[2-3H]inositol-[4-32P]phosphate, indicating that hydrolysis of PIP was due to a phosphomonoesterase activity (EC 3.1.3.36) in nuclear envelopes. Similarly, anion-exchange chromatographic analysis of the water-soluble products released from [32P]PIP indicated that inorganic phosphate was the sole 32P-labeled product. Hydrolysis of PIP was most rapid at neutral pH, and was not affected by inhibitors of acid phosphatase or alkaline phosphatase. Hydrolysis of PIP was also not inhibited by nonspecific phosphatase substrates, such as glycerophosphate, p-nitrophenylphosphate, AMP, or glucose 6-phosphate. Hydrolysis was stimulated by putrescine, and was inhibited by inositol 2-phosphate, spermidine, spermine, and neomycin.
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U2 - 10.1016/0003-9861(84)90226-1
DO - 10.1016/0003-9861(84)90226-1
M3 - Article
C2 - 6097190
AN - SCOPUS:0021733246
SN - 0003-9861
VL - 235
SP - 529
EP - 537
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 2
ER -