Expression of group IA phospholipase A2 in Pichia pastoris: identification of a phosphatidylcholine activator site using site-directed mutagenesis

Site-directed mutants of the group IA phospholipase A(2) from cobra venom were constructed and expressed in the methylotrophic yeast Pichia pastoris to probe for the proposed phosphatidylcholine (PC) activator site. Previous crystallographic and molecular modeling studies have identified two regions of the enzyme as likely candidates for this site. Residues Glu-55, Trp-61, Tyr-63, Phe-64, and Lys-65 were mutated to test the site advanced by Ortiz et al. [(1992) Biochemistry 31, 2887-2896] while Asp-23 and Arg-30 were mutated to assess the site proposed by Segelke et al. [(1998) J. Mol. Biol. 279, 223-232]. Expressed enzymes were purified by affinity chromatography and analyzed by SDS-PAGE, Western blotting, electrospray ionization mass spectroscopy, and circular dichroism. Both phospholipid headgroup specificity and rates of hydrolysis on monomeric PC substrates were determined and found to be similar for native, wild-type, and all of the mutant enzymes. These results suggest that all of the expressed enzymes were properly folded and contained functional catalytic sites. Mutations of the aromatic residues in the Ortiz site generally had little effect on PC activation, arguing against the importance of this region of the enzyme for PC activation; however, these aromatic amino acids appeared to be important for interfacial activation. In contrast, the D23N mutant in the Segelke site reduced PC activation by 10-fold without affecting activity toward micellar phosphatidylethanolamine substrates. Similar results were found with the D23N/R30M double mutant, suggesting that this region is critical for PC activation. These results provide evidence for the Segelke site as a PC activator site that is distinct from the catalytic site.     

Structure of human phenylethanolamine n-methyltransferase gene: existence of two types of mRNA with different transcription initiation sites

The chromosomal gene for human phenylethanolamine N-methyltransferase (PNMT; EC 2.1.1.28) was isolated from a human genomic library using a cloned human PNMT cDNA as a probe, and the nucleotide sequence was determined. PNMT is encoded in a single gene which consists of three exons. We observed newly the presence of minor PNMT mRNA (type B) besides the major mRNA (type A) as reported previously (Kaneda et al., J. Biol. Chem. 263, 7672–7677, 1988) by Northern hybridization. Type B mRNA carries an approximately 700 nucleotide-long untranslated region in the 5′ terminus. This suggests that two types of mRNA are produced from a single gene through the use of two alternative promoters. A TATA-like sequence locates 30 base pair upstream from the cap site of type A mRNA. Upstream of the cap site, there are several sequences resembling Spl binding sites and glucocorticoid responsive elements, with the latter also found in the first intron.     

A simple, rapid, and sensitive method of determining CSF IgG, using latex

A simple, rapid and sensitive method of determining cerebrospinal fluid IgG is presented. The procedure depends upon the fact that spinal fluid gamma globulin, and two of its components (IgG and IgM) will precipitate latex particles in proportion to their concentration, and that the optical density of the supernatant solution containing unprecipitated latex particles after centrifugation is inversely proportional to the concentration of these immunoglobulins. The method is sensitive to 50 nanograms, is inexpensive, requires 0.1 ml. or less of spinal fluid, and can be performed in minutes. Preliminary studies show that it compares favorably with results obtained by radial immunodiffusion.     

A simple and sensitive assay for determining plasma tipranavir concentration in the clinical setting by new HPLC method

A simple method for the quantification of tipranavir, the first non-peptidic HIV protease inhibitor, was developed and validated. Quinoxaline, as internal standard, was added to 50 microl of plasma before a liquid-liquid extraction by 600 microl of protein precipitation solution. The extracts were diluted before being injected in the chromatographic system. Chromatographic separation was made on a C18 column using potassium phosphate buffer (pH 3.2) and acetonitrile with gradient. Detection was performed by an UV detector at 260 nm. Relative error at three control quality concentrations ranged from -1.81 to 1.72%. Intra-day (CV%) and inter-day (CV%) precision ranged from 0.94 to 2.55% and from 3.07 to 4.24%, respectively. LOQ and LOD were 0.090 microg/ml and 0.035 microg/ml, respectively. Mean recovery was 87.1%+/-2.4%. Calibration curve was linear up to 180 microg/ml. Concentration range when optimized (0.703-180 microg/ml) proved to be adequate to measure tipranavir concentration in HIV-1-positive patients, therefore this method could be suitable for therapeutic drug monitoring of this drug.     

N49 phospholipase A2, a unique subgroup of snake venom group II phospholipase A2

A novel phospholipase A2 (PLA2) with Asn at its site 49 was purified from the snake venom of Protobothrops mucrosquamatus by using SP-Sephadex C25, Superdex 75, Heparin-Sepharose (FF) and HPLC reverse-phage C18 chromatography and designated as TM-N49. It showed a molecular mass of 13.875 kDa on MALDI-TOF. TM-N49 does not possess enzymatic, hemolytic and hemorrhagic activities. It fails to induce platelet aggregation by itself, and does not inhibit the platelet aggregation induced by ADP. However, it exhibits potent myotoxic activity causing inflammatory cell infiltration, severe myoedema, myonecrosis and myolysis in the gastrocnemius muscles of BALB/c mice. Phylogenetic analysis found that that TM-N49 combined with two phospholipase A2s from Trimeresurus stejnegeri, TsR6 and CTs-R6 cluster into one group. Structural and functional analysis indicated that these phospholipase A2s are distinct from the other subgroups (D49 PLA2, S49 PLA2 and K49 PLA2) and represent a unique subgroup of snake venom group II PLA2, named N49 PLA2 subgroup.     

Inflammatory factors stimulate expression of group II phospholipase A2 in rat cultured astrocytes. Two distinct pathways of the gene expression

Inflammatory factors such as tumor necrosis factor (TNF), interleukin 1 (IL-1), and lipopolysaccharide (LPS) greatly enhance the expression of group II phospholipase A2 (PLA2-II) mRNA, leading to increased secretion of PLA2-II enzyme from rat-cultured astrocytes. The potent antiinflammatory agent dexamethasone suppressed the PLA2-II expression induced by LPS. In vivo studies also demonstrated that the level of PLA2-II mRNA in the brain increased with intravenous injection of LPS. These results suggest that PLA2-II in the brain plays important roles in the inflammatory response. Agents which increase intracellular cAMP concentration did not stimulate PLA2-II expression by themselves but selectively enhanced TNF-induced PLA2-II expression about 5-fold. Phorbol ester, a well known protein kinase C activator, increased the PLA2-II expression. H-7, a protein kinase C inhibitor, inhibited the LPS-induced PLA2-II expression, but did not inhibit the TNF-induced one. Therefore, we conclude that the TNF-activated pathway differs from the LPS-activated one: the former is enhanced by cAMP and the latter involves protein kinase C.     

A sensitive method for determining quinolinic acid in animal tissues

A sensitive chromatographic method for isolation and measurement of quinolinic acid from rat liver and kidney is described. The method is based on the isolation of quinolinic acid by ion-exchange chromatography. The extraction of quinolinic acid consisted of the freeze clamping of the organ in liquid nitrogen, followed by deproteinization in perchloric acid. The neutralized extract was concentrated by freeze-drying and submitted to the action of concentrated perchloric acid to hydrolyze the nucleotides which interfered in the chromatographic separation of quinolinic acid. The sample was applied to a column of Dowex (HCOO^?) and eluted with a linear gradient of formic acid. The eluted fraction containing quinolinic acid was quantitatively measured by its absorbance at pH 2 and 268 nm in a spectrophotometer.