Multiple forms of the enzyme glycerophosphodiesterase are present in human brain

Abstract

Brain levels of glycerophosphodiesters, including glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE), are altered in many human central nervous system disorders. Although much information is available on the enzymes responsible for the formation of these phospholipid metabolites, little information is known regarding their catabolism, by glycerophosphodiesterases, in human brain. In both autopsied and biopsied temporal cortex, a phosphocholine-producing glycerophosphodiesterase activity was observed. In the presence of 1 mM EDTA, the enzyme possessed a pH optimum of 9.0, while the addition of 5 mM zinc acetate shifted the pH optimum to 10.5. When assayed at pH 9.0 in the absence of zinc acetate, the Km and Vmax were 104 +/- 2 microM and 77 +/- 18 nmol/h/mg protein, respectively, while assaying at pH 10.5 in the presence of 5mM zinc acetate yielded a Km of 964 +/- 56 microM, and a Vmax of 534 +/- 114 nmol/h/mg protein. Furthermore, whereas submillimolar concentrations of zinc acetate stimulated the activity of the enzyme in a dose-dependent manner when assayed at pH 10.5 (EC50 =20.3 +/- 3.0 microM), this did not result in a reciprocal inhibition of glycerophosphocholine phosphodiesterase (GPC PD) activity when assayed at a more acidic pH. This may suggest that human brain contains two phosphocholine-producing GPC PD activities, differentiable by their sensitivity to zinc ions. An activity capable of hydrolyzing GPE to form phosphoethanolamine could not be detected in either biopsied or autopsied brain. However, a choline/ethanolamine-producing glycerophosphodiesterase activity could be readily detected in biopsied, but not autopsied brain. this novel enzyme possessed a neutral pH optimum and was dependent upon divalent cations for activity. In conclusion, human brain contains at least two different glycerophosphodiesterases, a phosphocholine, and a choline/ethanolamine-producing activity, only one of which can be detected in autopsied tissue. The results of previous studies measuring brain glycerophosphodiesterase activity in degenerative brain conditions may need to be reevaluated in the light of these observations.

MIR16, a putative membrane glycerophosphodiester phosphodiesterase, interacts with RGS16

“…However, GP-PDE enzymatic activity with GPC (GPC-PDE, EC 3.1.4.2), glycerophosphoethanolamine, or glycerophosphoinositol (glycerophosphoinositol PDE, EC 3.1.4.44) as substrates has been observed in a number of mammalian tissues including liver (25,26), brain (27), and kidney (28). Preliminary studies on the biochemical properties of the enzyme suggest that multiple enzymes may be responsible for the activity (29,30). The MIR16 protein identified in this study may represent one of these enzymes.…”

Phospholipid biosynthetic enzymes in human brain

“…This was carried out as for choline kinase except that 100 µg of cytosolic protein was used, and ethanolamine replaced choline. Isolation of phosphoethanolamine was accomplished by modifying the procedure used for isolating phosphocholine (19), namely, the column was washed first with 2 mL of water, and phosphoethanolamine was eluted with 8 mL of water, as determined by comparision with a radiolabeled phosphoethanolamine standard. This procedure resulted in a recovery of approximately 60% for phosphoethanolamine, which was corrected for by running radiolabeled phosphoethanolamine standards in each assay.…”

“…Approximately 50 µg cytosolic or 80 µg of particulate protein was incubated for 1 h at 37°C in a buffer (final volume 50 µL) containing 15 mM magnesium chloride, 10 mM CTP, 1 mM 3 H-phosphoethanolamine (2 nCi/nmol), and 100 mM HEPES, pH 7.5, with alterations as described in the text. Following incubation, water-soluble products were extracted by the sequential addition of 200 µL chloroform/methanol [1:2 (vol/vol)], 50 µL chloroform, and 50 µL water (19). A 150 µL aliquot of the aqueous phase was dried by vacuum centrifugation, the residue resuspended in 30 µL of water containing 5 µg of ethanolamine, 10 µg of phosphoethanolamine, and 10 µg of CDPethanolamine, and applied to a silica gel TLC plate.…”

“…Approximately 30 µg of cytosolic protein was incubated for 1 h at 37°C in a buffer (final volume 100 µL) containing 10 mM magnesium chloride, 10 mM ATP, 100 mM HEPES, pH 8.0, 200,000 dpm 14 C-choline, and varying choline concentration, and other modifications, as described in the text. Following incubation, water-soluble products were extracted, and phosphocholine quantified by ion exchange chromatography as described previously (19). Column recovery was corrected for by running radiolabeled phosphocholine standards in each assay.…”

Purification and Characterization of Lysophospholipase C from Pig Brain

“…Alternatively, it is also possible that the toxicity of tellurite in demyelination may be ascribed to the inhibition of phosphocholine supply for re-synthesis of phosphatidylcholine [8,34]. There are at least two phosphodiesterases, which may supply phosphocholine in the resynthesis of phosphatidylcholine in brain tissue, as suggested from the previous publication that there might be two phosphodiesterases responsible for the conversion of GPC to phosphocholine in human brain samples [35]. From the present study, it is suggested that one enzyme may be GPC phosphocholine phosphodiesterase and the other, lysoPLC.…”