Bovine testis and human erythrocytes contain different subtypes of membrane-associated Ins(1,4,5)P3/Ins(1,3,4,5)P4 5-phosphomonoesterases.
1. We have purified membrane-associated Ins(1,4,5)P3/Ins(1,3,4,5)P4 5-phosphatases from bovine testis and human erythrocytes by chromatography on several media, including a novel 2,3-bisphosphoglycerate affinity column. 2. The enzymes have apparent molecular masses of 42 kDa (testis) and 70 kDa (erythrocyte), as determined by SDS/PAGE, and affinities for Ins(1,4,5)P3 of 14 microM and 22 microM respectively. 3. The two enzymes hydrolyse both Ins(1,4,5)P3 and Ins(1,3,4,5)P4 and are therefore type I Ins(1,4,5)P3 5-phosphatases [nomenclature of Hansen, Johanson, Williamson and Williamson (1987) J. Biol. Chem. 262, 17319-17326]. 4. On chromatofocusing, the partially purified testicular enzyme migrates as two peaks of activity, with pI values of about 5.8 and 5.5. The erythrocyte enzyme exhibits only the latter peak. 5. The testis 5-phosphatase is labile at 37 degrees C, but its activity can be maintained in the presence of 50 mM phorbol dibutyrate (PdBu). After PdBu treatment, a third form of the enzyme, with pI about 6.2, appears on chromatofocusing, but without change in its Km or Vmax. 6. Consideration of the properties of these enzymes and of the 5-phosphatases from other tissues suggests that type I Ins(1,4,5)P3 5-phosphatases are of two well-defined subtypes. We propose that these be termed type Ia [typified by the testis enzyme: approximately 40 kDa, higher affinity for Ins(1,4,5)P3] and Type Ib [typified by the erythrocyte enzyme: approximately 70 kDa, lower affinity for Ins(1,4,5)P3].
Hodgkin
M
M
Centre for Clinical Research in Immunology, School of Biochemistry, University of Birmingham, Edgbaston, U.K.
Craxton
A
A
Parry
J B
JB
Hughes
P J
PJ
Potter
B V
BV
Michell
R H
RH
Kirk
C J
CJ
D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D002850:Chromatography, Gel; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D000072181:Inositol Polyphosphate 5-Phosphatases; D007526:Isoelectric Point; D007527:Isoenzymes / Q000378:metabolism*; D008297:Male; D008970:Molecular Weight; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D013045:Species Specificity; D013737:Testis / Q000201:enzymology*
D003902:Detergents; D007527:Isoenzymes; D015240:Phorbol 12,13-Dibutyrate; D010744:Phosphoric Monoester Hydrolases; D000072181:Inositol Polyphosphate 5-Phosphatases