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Purification and characterization of human erythrocyte phosphatidylinositol 4-kinase. Phosphatidylinositol 4-kinase and phosphatidylinositol 3-monophosphate 4-kinase are distinct enzymes.

ABSTRACT: PtdIns 4-kinase has been purified 83,000-fold from human erythrocyte membranes. The major protein detected by SDS/PAGE is of molecular mass 56 kDa, and enzymic activity can be renatured from this band of the gel. The characteristics of this enzyme are similar to other type II PtdIns kinases previously described: PtdIns presented in Triton X-100 micelles is preferred as a substrate over PtdIns vesicles, the enzyme possesses a relatively low Km for ATP (20 microM), and adenosine is an effective inhibitor. A monoclonal antibody raised against bovine brain type II PtdIns 4-kinase is an effective inhibitor of the purified enzyme. PtdIns(4,5)P2 inhibits by approx. 50% when added in equimolar amounts with PtdIns; PtdIns4P has little effect on activity. A PtdIns3P 4-kinase activity has also been detected in erythrocyte lysates. Approximately two-thirds of this activity is in the cytosolic fraction and one-third in the membrane fraction. No PtdIns3P 4-kinase activity could be detected in the purified type II PtdIns 4-kinase preparation, nor could this activity be detected in a bovine brain type III PtdIns 4-kinase preparation. The monoclonal antibody that inhibits the type II PtdIns 4-kinase does not affect the PtdIns3P 4-kinase activity in the membrane fraction. The cytosolic PtdIns3P 4-kinase can be efficiently recovered from a 60%-satd.-(NH4)2SO4 precipitate that is virtually free of PtdIns 4-kinase activity. We conclude that PtdIns3P 4-kinase is a new enzyme distinct from previously characterized PtdIns 4-kinases, and that this enzyme prefers PtdIns3P over PtdIns as a substrate.

SUBMITTER: Graziani A

PROVIDER: S-EPMC1132694 | biostudies-other | 1992 May

REPOSITORIES: biostudies-other

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Project description:Phosphate acetyltransferase (PTA) and acetate kinase (AK) of the hyperthermophilic eubacterium Thermotoga maritima have been purified 1,500- and 250-fold, respectively, to apparent homogeneity. PTA had an apparent molecular mass of 170 kDa and was composed of one subunit with a molecular mass of 34 kDa, suggesting a homotetramer (alpha4) structure. The N-terminal amino acid sequence showed significant identity to that of phosphate butyryltransferases from Clostridium acetobutylicum rather than to those of known phosphate acetyltransferases. The kinetic constants of the reversible enzyme reaction (acetyl-CoA + Pi -->/<-- acetyl phosphate + CoA) were determined at the pH optimum of pH 6.5. The apparent Km values for acetyl-CoA, Pi, acetyl phosphate, and coenzyme A (CoA) were 23, 110, 24, and 30 microM, respectively; the apparent Vmax values (at 55 degrees C) were 260 U/mg (acetyl phosphate formation) and 570 U/mg (acetyl-CoA formation). In addition to acetyl-CoA (100%), the enzyme accepted propionyl-CoA (60%) and butyryl-CoA (30%). The enzyme had a temperature optimum at 90 degrees C and was not inactivated by heat upon incubation at 80 degrees C for more than 2 h. AK had an apparent molecular mass of 90 kDa and consisted of one 44-kDa subunit, indicating a homodimer (alpha2) structure. The N-terminal amino acid sequence showed significant similarity to those of all known acetate kinases from eubacteria as well that of the archaeon Methanosarcina thermophila. The kinetic constants of the reversible enzyme reaction (acetyl phosphate + ADP -->/<-- acetate + ATP) were determined at the pH optimum of pH 7.0. The apparent Km values for acetyl phosphate, ADP, acetate, and ATP were 0.44, 3, 40, and 0.7 mM, respectively; the apparent Vmax values (at 50 degrees C) were 2,600 U/mg (acetate formation) and 1,800 U/mg (acetyl phosphate formation). AK phosphorylated propionate (54%) in addition to acetate (100%) and used GTP (100%), ITP (163%), UTP (56%), and CTP (21%) as phosphoryl donors in addition to ATP (100%). Divalent cations were required for activity, with Mn2+ and Mg2+ being most effective. The enzyme had a temperature optimum at 90 degrees C and was stabilized against heat inactivation by salts. In the presence of (NH4)2SO4 (1 M), which was most effective, the enzyme did not lose activity upon incubation at 100 degrees C for 3 h. The temperature optimum at 90 degrees C and the high thermostability of both PTA and AK are in accordance with their physiological function under hyperthermophilic conditions.

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Purification and characterization of human erythrocyte phosphatidylinositol 4-kinase. Phosphatidylinositol 4-kinase and phosphatidylinositol 3-monophosphate 4-kinase are distinct enzymes.

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