Differential activation of phospholipases by mitogenic EGF and neurogenic PDGF in immortalized hippocampal stem cell lines.

In several neuronal systems, nerve growth factor (NGF) and platelet-derived growth factor (PDGF) act as neurogenic agents, whereas epidermal growth factor (EGF) acts as a mitogenic agent. Hippocampal stem cell lines (HiB5) immortalized by the expression of a temperature-sensitive SV40 large T antigen also respond differentially to EGF and PDGF. While EGF treatment at the permissive temperature induces proliferation, the addition of PDGF induces differentiation at the non-permissive temperature. However, the mechanism responsible for these different cellular fates has not been clearly elucidated. In order to clarify possible critical signaling events leading to these distinct cellular outcomes, we examined whether either EGF or PDGF differentially induces the activation of phospholipases, such as phospholipase A(2) (PLA(2)), C (PLC), or D (PLD). Although EGF stimulation did not induce phospholipases, PDGF caused a rapid and transient activation of PLC and PLD, but not PLA(2). When the activation of PLC or PLD was blocked, the neurite outgrowth induced by PDGF was significantly inhibited. Although the activation of PLD occurred faster than PLC, blocking of PLD activity by transient expression of lipase-inactive mutants did not inhibit the induction of PLC activity by PDGF. These results suggest that the differential activation of phospholipases may play an important role in signal transduction by mitogenic EGF and neurotrophic PDGF in HiB5 neuronal hippocampal stem cells. In particular, the activation of phospholipase C and D may contribute to neuronal differentiation by neurogenic PDGF in the HiB5 cells.

Structure of aspergillopepsin I from Aspergillus phoenicis: variations of the S1'-S2 subsite in aspartic proteinases.

The crystal structure of aspergillopepsin I (AP) from Aspergillus phoenicis has been determined at 2.18 A resolution and refined to R and R(free) factors of 21.5 and 26.0%, respectively. AP has the typical two beta-barrel domain structure of aspartic proteinases. The structures of the two independent molecules are partly different, exemplifying the flexible nature of the aspartic proteinase structure. Notably, the 'flap' in one molecule is closer, with a largest separation of 4.0 A, to the active site than in the other molecule. AP is most structurally homologous to penicillopepsin (PP) and then to endothiapepsin (EP), which share sequence identities of 68 and 56%, respectively. However, AP is similar to EP but differs from PP in the combined S1'-S2 subsite that is delineated by a flexible psi-loop in the C-terminal domain. The S1' and S2 subsites are well defined and small in AP, while there is no definite border between S1' and S2 and the open space for the S2 subsite is larger in PP. Comparison of the structures indicates that the two amino-acid residues equivalent to Leu295 and Leu297 of AP are the major determining factors in shaping the S1'-S2 subsite in the fungal aspartic proteinases.

Mutational and kinetic evaluation of conserved His-123 in dual specificity protein-tyrosine phosphatase vaccinia H1-related phosphatase: participation of Tyr-78 and Thr-73 residues in tuning the orientation of His-123.

Active-site cysteine strategically positioned in the P-loop of protein-tyrosine phosphatases has been suggested to be further stabilized by hydrogen bonding arrays radiating out from the P-loop to neighboring residues. In this work, we investigated the structural role of histidine array in HC(X)(5)RS motif of the vaccinia H1-related protein phosphatase (VHR), using site-directed mutagenesis in conjunction with an extensive kinetic analysis. Conserved His-123 was mutated along with neighboring residues Tyr-78 and Thr-73. The increased pK(a) values of active-site Cys-124 found in Y78F and T73A mutants (6.51 and 6.75, respectively) were comparable to those of H123A and H123F mutants. Kinetic evaluation of Y78F and T73A mutants further implicates that the mutations perturb the relative position of Cys-124 within the P-loop. These results imply that Tyr-78 and Thr-73 make up an essential part of the His-123 array and structurally tune the Cys-124 position. Tyr-78 of VHR turns out to be the invariant Tyr reported in several protein-tyrosine phosphatases by a structure-based sequence alignment. Therefore, orientation of the imidazole ring of His-123 by the invariant Tyr-78 is crucial for maintaining the proper position of Cys-124 in the P-loop.

Melittin exerts multiple effects on the release of free fatty acids from L1210 cells: lack of selective activation of phospholipase A2 by melittin.

Melittin is known as a phospholipase A2 (PLA2) activator, but the selectivity of its effect on PLA2 is uncertain. We examined the selectivity of melittin effect on the release of free fatty acids (FFAs) from L1210 cells using various inhibitors. A systemic lipid analysis by HPLC and GLC revealed that melittin induced release of various FFAs including saturated, monounsaturated, and polyunsaturated FFAs. Various PLA2 inhibitors examined exerted only minimal effects on the melittin-induced arachidonic acid (AA) and palmitic acid (PAL) releases. Specific inhibitors of phosphatidylinositol-phospholipase C (U73122) and diacylglycerol lipase (RHC80267) exerted significant inhibitory effects on both AA and PAL releases. These results suggest that melittin-induced FFA release is most likely due to multiple participations of various types of lipases. Since BAPTA/AM, an intracellular Ca2+ chelator, did not influence the FFA release, the Ca2+ influxed by melittin appeared not to be a key factor for the FFA release. The mimicking of the melittin-induced FFA release by digitonin, a membrane-permeabilizing agent, implies that the membrane-perturbing action of melittin is likely the cause of the FFA release. Melittin also induced release of multiple FFAs from other cell lines including P388D1 and HL60. The rapid melittin-stimulated phospholipase D (PLD) observed in L1210 cells appeared not directly related to the steady release of FFA, as indicated by the fact that the PLD was not blocked by RHC80267. In view of melittin's multiple effects on the composition of cellular lipids, we conclude that melittin does neither exclusively release any single FFA nor selectively activate PLA2 in L1210 cells. The problem of using melittin as a PLA2 activator is discussed.

Effects of metal ions on the activity of protein tyrosine phosphatase VHR: highly potent and reversible oxidative inactivation by Cu2+ ion.

The posttranslational regulation of protein tyrosine phosphatases (PTPs) has been suggested to have a crucial role in maintaining the phosphotyrosine level in cells. Here we examined the regulatory effects of metal ions on human dual-specificity vaccinia H1-related protein tyrosine phosphatase (VHR) in vitro. Among various metal ions examined, Fe3+, Cu2+, Zn2+, and Cd2+ exerted their inactivational effects on VHR, and Cu2+ is the most potent inactivator. The VHR activity inactivated by the metal ions except Cu2+ was significantly restored by EDTA. The efficacy of Cu2+ for the VHR inactivation was about 200-fold more potent than that of H2O2. Cu2+ also inactivated other PTPs including PTP1B and SHP-1. The Cu2+-mediated inactivation at the submicromolar range was eradicated by dithiothreitol treatment. The loss of VHR activity correlated with the decreased [14C]iodoacetate labeling of active-site cysteine, suggesting that Cu2+ brought about the oxidation of the active-site cysteine. On the contrary, Zn2+ that exerted an inactivational effect at millimolar concentrations appeared not directly linked to the active-site cysteine, as indicated by the fact that [14C]iodoacetate labeling was unaffected and that the effect of Zn2+ on the Y78F mutant was increased. The reduction potential of VHR was estimated to be -331 mV by utilizing the reversibility of the redox state of VHR. Thus, we conclude that the highly potent Cu2+ inactivation of VHR is a consequence of the oxidation of the active-site cysteine and the mode of Zn2+ inactivation is distinct from that of Cu2+.

Molecular cloning and functional expression of a phospholipase D from cabbage (Brassica oleracea var. capitata).

We cloned and expressed a full-length cDNA encoding a phospholipase D of type alpha (PLDalpha) from cabbage. Analysis of the cDNA predicted an 812-amino-acid protein of 92.0 kDa. The deduced amino acid sequence of cabbage PLD has 83% and 80% identity with Arabidopsis PLDalpha and castor bean PLD, respectively. Expression of this cDNA clone in E. coli shows a functional PLD activity similar to that of the natural PLD.

Effects of mastoparan B and its analogs on the phospholipase D activity in L1210 cells.

Mastoparan B (MP-B), an amphiphilic alpha-helical peptide isolated from hornet venom, and its Ala-substituted analogs were examined for their effectiveness on phospholipase D (PLD) activity in L1210 cells. PLD activity was determined by measuring phosphatidylethanol produced from [3H]myristate-labelled cells in the presence of ethanol. PLD activity was stimulated by MP-B, 4MP-B (Lys4-->Ala), and 12MP-B (Lys12-->Ala), but not by 3MP-B (Leu3-->Ala) and 9MP-B (Trp9-->Ala). Other MPs including mastoparan 7 also stimulated the PLD activity, but inactive mastoparan 17 did not. The stimulatory effect of various MP analogs could be correlated with their alpha-helical contents. The PLD activity stimulated by MP-B was not affected by G-protein blocking chemicals. The extent of PLD stimulation by various MP-Bs, as well as by digitonin and beta-escin, correlated with the permeability of the membrane to ethidium bromide. These results suggest that the stimulation of PLD activity by MP-B in L1210 cells is probably coupled with membrane perturbation brought about by the peptide.

Phospholipase D activity in L1210 cells: a model for oleate-activated phospholipase D in intact mammalian cells.

Phospholipase D (PLD) in lymphocytic mouse leukemic L1210 cells has been found to be activated by oleate both in vitro and in intact cells. The PLD activity was measured by phosphatidylethanol produced from radiolabeled phosphatidylcholine or myristic acid in the presence of ethanol. This oleate-activated PLD was further characterized in intact cells and compared with that in HL60 cells. Unlike PLD in HL60 cells, the PLD in L1210 cells was activated by unsaturated fatty acids, stimulated by melittin, insensitive to guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), ADP-ribosylation factor (ARF) and phosphatidylinositol 4,5-bisphosphate (PIP2), independent of phorbol 12-myristate 13-acetate (PMA) and staurosporine, and inhibited by pervanadate. These observations indicate that the PLD present in L1210 cells is distinct from that in HL60 cells. Key PLD properties of L1210 cells such as insensitivity to GTP gamma S, ARF, PIP2, or PMA were in good agreement with currently known in vitro properties of the oleate-activated PLD found in mammalian sources. Therefore, the L1210 cells could be used as an intact-cell source for an oleate-activated PLD.

The G protein beta subunit Gpb1 of Schizosaccharomyces pombe is a negative regulator of sexual development.

A Schizosaccharomyces pombe homolog of mammalian genes encoding G protein beta subunits, gpb1+, was cloned by the polymerase chain reaction using primer pairs that correspond to sequences conserved in several G beta genes of other species followed by screening of genomic and cDNA libraries. The gpb1 gene encodes 317 amino acids that show 47% homology with human G beta 1 and G beta 2 and 40% homology with Saccharomyces cerevisiae G beta protein. Disruption of the gpb1 gene indicated that this gene is not required for vegetative cell growth. However, gpb1-disrupted haploid cells mated and sporulated faster than wild-type cells, both in sporulation (MEA) and in complex medium (YE): when examined 23 h after transfer to sporulation medium, 35% of gpb1-disrupted haploid pairs had undergone conjugation and sporulation, whereas only 3-5% of wild-type haploid pairs had done so. Overexpression of the gpb1 gene suppressed this facilitated conjugation and sporulation phenotype of gpb1-disrupted cells but did not cause any obvious effect in wild-type cells. Co-disruption of one of the two S. pombe G alpha-subunit genes, gpa2, in the gpb1-disrupted cells did not change the accelerated conjugation and sporulation phenotype of the gpb1- cells. However, co-disruption of the ras1 gene abolished the gpb1- phenotype. These results suggest that Gpb1 is a negative regulator of conjugation and sporulation that apparently works upstream of Ras1 function in S. pombe. The possible relationship of Gpb1 to two previously identified, putative G alpha proteins of S. pombe is discussed.

GERI-BP001 compounds, new inhibitors of acyl-CoA: cholesterol acyltransferase from Aspergillus fumigatus F37. I. Production, isolation, and physico-chemical and biological properties.

GERI-BP001 compounds, new inhibitors of acyl-CoA:cholesterol acyltransferase (ACAT), were isolated from a culture broth of Aspergillus fumigatus F37 by acetone extraction, EtOAc extraction, SiO2 column chromatography, and reverse phase HPLC. GERI-BP001 M, A, and B inhibit ACAT activity in an enzyme assay system using rat liver microsomes by 50% at concentrations of 42, 94, and 40 microM, respectively.

Crystallization and preliminary X-ray crystallographic analysis of carboxylesterase from pseudomonas fluorescens.

Large crystals of carboxylesterase from Pseudomonas fluorescens have been grown in the presence of dioxane using ammonium sulfate and lithium sulfate as precipitant. They belong to the tetragonal space group P4(1)2(1)2 (or P4(3)2(1)2), with unit cell dimensions of a = 82.01 (+/-0.06) A and c = 145.44 (+/- 0.08) A. The presence of one carboxylesterase dimer in the asymmetric unit gives the crystal volume per protein mass (VM) of 2.56 A3/Da and solvent fraction of 52.0% by volume. The crystals diffract to at least 2.3 A Bragg spacing when exposed to CuK alpha X rays from a rotating anode generator. X-ray data (nearly complete to 2.6 A Bragg spacing) have been collected from a native crystal.

Crystallization and preliminary X-ray crystallographic analysis of alpha-amylase from Bacillus subtilis.

Large crystals of alpha-amylase from Bacillus subtilis have been obtained at room temperature using polyethylene glycol 6000 as precipitant. They grow to typical dimensions of 0.25 mm x 0.3 mm x 2.0 mm in five days. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions of a = 85.46 A, b = 166.5 A and c = 332.7 A. The asymmetric unit seems to contain eight molecules of alpha-amylase, with crystal volume per protein mass (Vm) of 2.69 A3/Da and solvent content of 54.3% by volume. Despite a very long c-axis, the crystals diffracted to about 2.2 A Bragg spacing using the rotating anode X-rays and were resistant to damage by X-rays. Thus they are suitable for structure determination by X-ray methods at high resolution. X-ray diffraction data have been collected to 3.4 A Bragg spacing from a native crystal.

Characterization of bovine aromatic L-amino acid decarboxylase expressed in a mouse cell line: comparison with native enzyme.

Bovine aromatic L-amino acid decarboxylase (AADC) was expressed in a mouse cell line, using a bovine papilloma virus-derived expression vector containing the full coding region of bovine AADC. The recombinant bovine AADC was characterized biochemically and immunochemically and compared with the native bovine AADC. The specific activity of crude recombinant bovine AADC was 30-fold higher than that of crude native AADC. With regard to optimal pH, effects of pyridoxal phosphate concentration and Km for 3,4-dihydroxyphenylalanine as a substrate, both native and recombinant enzymes were essentially identical. Rabbit polyclonal antiserum directed against bovine adrenal AADC recognized on Western blot a single protein band (molecular mass = 55,000 Dalton) in both native and recombinant bovine AADC crude extracts. Furthermore, double immunodiffusion analysis showed a single precipitin line of confluence with both enzyme preparations, indicating immunological identity of native and recombinant bovine AADC. Northern blot analysis identified a single mRNA species (2.2 kb) from native and recombinant bovine AADC preparations. The recombinant bovine AADC has two charge isozymes corresponding to those of the native bovine enzyme, although their relative abundances are different between native and recombinant enzymes. Taken together, our results show that recombinant bovine AADC, expressed from bovine AADC cDNA in a mouse cell line is not only enzymatically active, but also shares many biochemical and immunochemical common features with native bovine AADC.

Effects of pH changes and charge characteristics in the uptake of norepinephrine by synaptosomes of rat brain.

The pH dependence of the initial uptake of norepinephrine by rat whole brain synaptosomes was studied using short incubation times at 37 degrees C in order to examine the possible involvement of the phenolic OH group. The pH vs. uptake profile exhibits a maximum near pH 8.2 in H2O medium. When the medium was changed to 2H2O, the profile showed a shift of maximum corresponding to the pKa change of the phenolic OH group. The pH vs. uptake profile of tyramine was quite different from that of norepinephrine. These pH effects on uptake were explained as manifestations of the involvement of the phenolic OH group in the process. The amine and phenolic hydroxyl groups in norepinephrine were studied separately by employing two series of compounds structurally related to catecholamines, amphetamine-like and catechol-like, for their inhibitory effects on the uptake. The inhibitions were affected by changes in pH with changes in opposite directions found for the two series indicating the need for a positive charge in the side chain and suggesting an effect of the negative charge on the ring. These charge characteristics agreed with the pH profile observed in uptake. Consequently, the two groups with opposite charge characteristics in norepinephrine both appear to function in the uptake process.