Recognizing the pleckstrin homology domain fold in mammalian phospholipase D using hidden Markov models.

Phospholipase D was first described in plant tissue but has recently been shown to occur in mammalian cells where it is activated by cell surface receptors. Its mode of activation by receptors in unclear. Biochemical studies suggest that it may occur downstream of other effector proteins and that small GTP-dependent regulatory proteins may be involved. The sequence in a non-designated region of mammalian phospholipase D1 and 2 shows similarity to a structural domain that is present in signalling proteins that are regulated by protein kinases or heterotrimeric G-proteins. Mammalian phospholipase D has structural similarities with other lipid signalling phospholipases and thus may be regulated by receptors in an analogous fashion.

Molecular species analysis of a product of phospholipase D activation. Phosphatidylethanol is formed from phosphatidylcholine in phorbol ester- and bradykinin-stimulated PC12 cells.

Tumor-promoting phorbol esters or calcium-mobilizing receptor ligands stimulate phosphatidylcholine breakdown and in many cells this is accompanied by phospholipase D (PLD) activation. We tested whether or not a direct relationship exists between these two phenomena. Pheochromocytoma (PC12) cells were stimulated with the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate or with the calcium-mobilizing receptor ligand bradykinin in media containing 1% ethanol. The fatty acid composition of the molecular species of phosphatidylethanol (PEt), a product of PLD activation, formed in stimulated cells was compared with the molecular species of endogenous phospholipids isolated from unstimulated PC12 cells. PEt was isolated and analyzed by fast atom bombardment-mass spectrometry (FAB-MS) in the negative ion mode. Fatty acid composition and headgroup structure of the major PEt molecular ions were confirmed by linked scan analysis. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol were isolated from unstimulated cells and converted into phosphatidic acids using PLD. Mass spectra of the respective phosphatidic acids were obtained by fast atom bombardment-mass spectrometry as described above. The molecular species of PEt formed in 12-O-tetradecanoylphorbol-13-acetate- and bradykinin-stimulated PC12 cell were identical to those of phosphatidylcholine isolated from untreated cells.

Bis(monoacylglycero)phosphate from PC12 cells, a phospholipid that can comigrate with phosphatidic acid: molecular species analysis by fast atom bombardment mass spectrometry.

Phospholipids from pheochromocytoma (PC12) cells were purified by one-dimensional thin-layer chromatography (TLC). Material corresponding in RF to phosphatidic acid (PA) was analyzed by fast atom bombardment mass spectrometry (FAB). The molecular ions of the major constituents corresponded in mass to phosphatidylglycerols (PG), which, however, have a lower RF value. Analysis of the mass spectra demonstrated that this material consists of bis(monoacylglycero)phosphates (BMP, lysobisphosphatidic acid), a structural isomers of PG. Linked scans of individual molecular ions indicate that BMP from PC12 cells is esterified almost exclusively with monounsaturated (16:1 and 18:1) and polyunsaturated (20:4 and 22:6) fatty acids. One of the two major molecular species contains two monounsaturated (18:1/18:1), while the other contains both a monounsaturated (18:1) and a polyunsaturated (22:6) fatty acid ester. FAB in combination with TLC is ideally suited for analysis of molecular species of phospholipids.

Phospholipase D-catalyzed hydrolysis of phosphatidylcholine occurs with P-O bond cleavage.

Mammalian phospholipase D has been implicated in signal-transduction mechanisms, most recently in association with stimuli that enhance phosphatidylcholine (PC) turnover. It was previously unknown whether hydrolysis of PC by phospholipase D proceeds via P-O or C-O bond cleavage. Commercially available phospholipase D isolated from Streptomyces chromofuscus was used to hydrolyse distearoyl phosphatidylcholine (PC) in a detergent-containing buffer consisting of 90% 18O-water. The product of hydrolysis, phosphatidic acid (PA), was purified by thin-layer chromatography and analyzed using californium-252 plasma desorption mass spectrometry. An increase of two mass units was observed, compared to a distearoyl PA control, consistent with a reaction mechanism involving cleavage of the P-O bond.

Calcium-dependent incorporation of choline into phosphatidylcholine (PC) by base-exchange in rat brain membranes occurs preferentially with phospholipid substrates containing docosahexaenoic acid (22:6(n-3)).

The fatty acid composition of phosphatidylcholine (PC) formed by base-exchange was examined in rat brain membranes in vitro. The free choline incorporated into subspecies of PC by this phospholipase-D type activity can be distinguished from that which might enter indirectly via the last enzyme of the de novo pathway for phospholipid biosynthesis, cholinephosphotransferase, by its ionic requirements. Choline base-exchange in lysed synaptosomes is optimal when assayed at extracellular (mM) calcium concentrations and is blocked by magnesium. As much as 40% of the choline incorporated by base-exchange into rat brain membranes was recovered in subspecies of PC, representing no more than 10% of the total PC pool, which contained docosahexaenoic acid (22:6(n-3)). Docosahexaenoic acid is enriched in electrically-excitable membranes and its content in phospholipids of rat and human brain change during early development and increase with age.

Presence of base-exchange activity in rat brain nerve endings: dependence on soluble substrate concentrations and effect of cations.

The calcium-dependent, energy-independent incorporations of 14C-labeled bases, choline, ethanolamine, and serine, into their corresponding membrane phospholipids, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine, were compared in microsomes and in subcellular fractions prepared from a lysed crude mitochondrial (P2) pellet of whole rat brain. When activities were measured in the presence of an extracellular (1.25 mM) concentration of Ca2+, recovered activities were highest in the microsomal fraction, although substantial activity remained associated with the P2 homogenate even after repeated washing of the pellet. When this washed P2 homogenate was subfractionated, enrichment of all three exchange activities was obtained only in a fraction that was fivefold enriched over the homogenate and sevenfold enriched over the microsomal fraction in Na+, K+-ATPase, a plasma membrane marker. This strongly suggests that the base-exchange enzymes are normal constituents of synaptosomal plasma membranes. The three exchange activities were measured in synaptosomes prepared from whole rat brain in the presence of various substrate (base) concentrations, and kinetic constants were calculated. The Vmax values for choline, ethanolamine, and serine exchange were, respectively, 1.27 +/- 0.09, 1.60 +/- 0.17, and 0.56 +/- 0.06 nmol/mg of protein/h; the respective Km (apparent) values were 241 +/- 29, 65 +/- 18, and 77 +/- 22 microM. Endogenous levels of the three bases, choline, ethanolamine, and serine, in whole (microwaved) rat brains were 20 +/- 8, 78 +/- 28, and 639 +/- 106 nmol, respectively. That ethanolamine and serine incorporations had lower Km values than choline incorporation suggests that these bases are preferentially incorporated into their respective phospholipids.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of estradiol receptors in brain cytosols from perinatal ferrets.

Studies were undertaken to characterize the binding of [3H]estradiol ([3H]E2) to blood plasma and to brain cytosols collected from perinatal ferrets of both sexes. A dialysis experiment showed that the binding capacity of plasma for [3H]E2 was low in neonatal ferrets. Saturable, high-affinity binding of [3H]E2 to cytosols prepared from ferret hypothalamus + preoptic area (H + POA), basal temporal lobe, and midbrain + brainstem (sexes pooled) was demonstrated 5 days prior to the date of expected parturition (42-day gestation), as well as on the day of birth and on postnatal days 5 and 10. The binding of E2 to cytosols prepared from H + POA and temporal lobe of newborn ferrets met established criteria for estrogen receptors: (a) it was saturable; (b) it had a high affinity (Kd approximately 10(-10) M); (c) it was apparently macromolecular since it migrated in front of a 4.7 S dansyl-bovine serum albumin marker on sucrose gradients of low ionic strength and eluted in the void volume of Sephadex LH-20 columns; (d) it was apparently proteinaceous since binding was destroyed by preincubation with pronase; and (e) it was steroid-specific. Previous research suggests that perinatal exposure to E2 causes neither behavioral defeminization nor masculinization in the developing ferret. The present findings raise the question of whether estrogens, acting via neural receptors, normally affect brain development in ferrets of either sex.