Glutaraldehyde pretreatment blocks phospholipase A2 modulation of adrenergic receptors

Treatment of rat cerebral cortical membranes with phospholipase A2 affects, in a parallel fashion, beta-, alpha 1- and alpha 2-adrenergic receptor binding, but not the affinity of these receptors for their respective ligands. Pretreatment of membranes with 0.1 percent glutaraldehyde blocks the effects of phospholipase A2 on adrenergic receptor binding. The results support the hypothesis that desensitization or "masking" of adrenergic receptors may involve changes in membrane lipid composition. Furthermore, glutaraldehyde may prove a useful tool in the investigation of the dynamic roles of lipids in receptor function and more specifically, their regulation and coupling to physiological events.     

Endothelin-3 stimulates production of endothelium-derived nitric oxide via phosphoinositide breakdown.

Cultured bovine endothelial cells (EC) have specific receptors for endothelin (ET)-3 functionally coupled to phosphoinositide breakdown. We studied whether ET-3 stimulates synthesis of nitric oxide (NO), an endothelium-derived relaxing factor that activates soluble guanylate cyclase in EC, and whether the ET-3-induced NO formation involves G-proteins. ET-3 dose-dependently stimulated production of intracellular cGMP in EC, of which effects were abolished by pretreatment with NG-monomethyl L-arginine, an inhibitor of NO synthesis, and methylene blue, an inhibitor of soluble guanylate cyclase. The stimulatory effects of ET-3 on cGMP production, inositol trisphosphate formation and increase in cytosolic free Ca2+ concentration were similarly blocked by pretreatment with pertussis toxin (PTX). These data suggest that ET-3 induces synthesis of NO mediated by phosphoinositide breakdown via PTX-sensitive G-protein in EC.     

Importance of the phosphocholine linkage on sphingomyelin molecular properties and interactions with cholesterol; a study with phosphate oxygen modified sphingomyelin-analogues

We have characterized the molecular properties and membrane behavior of synthetically modified sphingomyelin analogues, modified on the oxygen connecting the phosphocholine group to the ceramide backbone. The oxygen was replaced with an S-atom (S-PSM), an NH-group (NH-PSM) or a CH(2)-group (CH(2)-PSM). Diphenylhexatriene and Laurdan anisotropy experiments showed that an S-linkage increased and NH- and CH(2)-linkages decreased the stability of PSM-analogue bilayer membranes as compared to PSM. When the polarity of the interface was probed using Laurdan, S-PSM appeared to have a lower polarity as compared to PSM whereas NH-PSM and CH(2)-PSM had higher polarities of their respective interfaces. Fluorescence quenching-studies with cholestatrienol showed that all compounds formed SM/cholesterol-rich domains. The S-PSM/cholesterol and PSM/cholesterol domains displayed a similar thermostability, whereas NH-PSM/cholesterol and CH(2)-PSM/cholesterol domains were less thermostable. DSC on vesicles containing the PSM-analogues showed a more complex melting behavior as compared to PSM, whereas equimolar mixtures of the PSM-analogues and PSM showed almost ideal mixing with PSM for NH- and S-PSM. Our data show that the properties of the bond linking the phosphocholine head group to the 1-hydroxyl on the ceramide molecule is important for the stability of SM/SM and SM/cholesterol interactions.     

Novel process for enzymatic hydrolysis of proteins in an aqueous two-phase system for the production of peptide mixture

A novel process for the production of peptide mixtures is proposed. Biologically active peptides were synthesized using a thermolysin-catalyzed hydrolysis of a corn protein (zein) in an aqueous two-phase system. The mixture of peptides which was selectively recovered from the dextran-rich bottom phase had a higher angiotensin-converting enzyme (ACE) inhibitory activity than native zein.     

Production of 2-phenylethanol in roses as the dominant floral scent compound from L-phenylalanine by two key enzymes, a PLP-dependent decarboxylase and a phenylacetaldehyde reductase

We investigated the biosynthetic pathway for 2-phenylethanol, the dominant floral scent compound in roses, using enzyme assays. L-[(2)H8] Phenylalanine was converted to [(2)H8] phenylacetaldehyde and [(2)H8]-2-phenylethanol by two enzymes derived from the flower petals of R. 'Hoh-Jun,' these being identified as pyridoxal-5'-phosphate-dependent L-aromatic amino acid decarboxylase (AADC) and phenylacetaldehyde reductase (PAR). The activity of rose petal AADC to yield phenylacetaldehyde was nine times higher toward L-phenylalanine than toward its D-isomer, and this conversion was not inhibited by iproniazid, a specific inhibitor of monoamine oxidase. Under aerobic conditions, rose petal AADC stoichiometrically produced NH3 together with phenylacetaldehyde during the course of decarboxylation and oxidation, followed by the hydrolysis of L-phenylalanine. Phenylacetaldehyde was subsequently converted to 2-phenylethanol by the action of PAR. PAR showed specificity toward several volatile aldehydes.     

'Crystal lattice engineering,' an approach to engineer protein crystal contacts by creating intermolecular symmetry: crystallization and structure determination of a mutant human RNase 1 with a hydrophobic interface of leucines

A protein crystal lattice consists of surface contact regions, where the interactions of specific groups play a key role in stabilizing the regular arrangement of the protein molecules. In an attempt to control protein incorporation in a crystal lattice, a leucine zipper-like hydrophobic interface (comprising four leucine residues) was introduced into a helical region (helix 2) of the human pancreatic ribonuclease 1 (RNase 1) that was predicted to form a suitable crystallization interface. Although crystallization of wild-type RNase 1 has not yet been reported, the RNase 1 mutant having four leucines (4L-RNase 1) was successfully crystallized under several different conditions. The crystal structures were subsequently determined by X-ray crystallography by molecular replacement using the structure of bovine RNase A. The overall structure of 4L-RNase 1 is quite similar to that of the bovine RNase A, and the introduced leucine residues formed the designed crystal interface. To characterize the role of the introduced leucine residues in crystallization of RNase 1 further, the number of leucines was reduced to three or two (3L- and 2L-RNase 1, respectively). Both mutants crystallized and a similar hydrophobic interface as in 4L-RNase 1 was observed. A related approach to engineer crystal contacts at helix 3 of RNase 1 (N4L-RNase 1) was also evaluated. N4L-RNase 1 also successfully crystallized and formed the expected hydrophobic packing interface. These results suggest that appropriate introduction of a leucine zipper-like hydrophobic interface can promote intermolecular symmetry for more efficient protein crystallization in crystal lattice engineering efforts.     

Presence of immunoreactive endothelin in human plasma

A highly specific and sensitive radioimmunoassay has been established for measurement of human endothelin (hET) in human plasma. After extraction of plasma with an octyl-silica column, this assay allowed for detection of immunoreactive (IR) hET as low as 0.2 fmol/ml. In 16 healthy subjects, the mean concentration of plasma IR-hET was 0.6 fmol/ml. Reverse-phase HPLC coupled with radioimmunoassay revealed two major IR-hET components, one corresponding to authentic hET(1-21) and another with more hydrophilicity than hET(1-21). These data indicate that ET is a circulating vasoconstrictor hormone in man.     

Development of a mouse monoclonal antibody against the chondroitin sulfate-protein linkage region derived from shark cartilage

Glycosaminoglycans (GAGs) like chondroitin sulfate (CS) and heparan sulfate (HS) are synthesized on the tetrasaccharide linkage region, GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser, of proteoglycans. The Xyl can be modified by 2-O-phosphate in both CS and HS, whereas the Gal residues can be sulfated at C-4 and/or C-6 in CS but not in HS. To study the roles of these modifications, monoclonal antibodies were developed against linkage glycopeptides of shark cartilage CS proteoglycans, and one was characterized in detail. This antibody bound hexa- and pentasaccharide-peptides more strongly than unsaturated tetrasaccharide-peptides with the unnatural fourth sugar residue (unsaturated hexuronic acid), suggesting the importance of the fifth and/or fourth saccharide residue GalNAc-5 and/or GlcA-4. Its reactivity was not affected by treatment with chondro-4-sulfatase or alkaline phosphatase, suggesting that 4-O-sulfate on the Gal residues and 2-O-phosphate on the Xyl residue were not recognized. Treatment with weak alkali to cleave the Xyl-Ser linkage completely abolished the binding activity, suggesting the importance of the peptide moiety of the hexasaccharide-peptide for the binding. Based on the amino acid composition and matrix-assisted laser desorption ionization time-of-flight mass spectrometry analyses, it was revealed that the peptide moiety is composed of four amino acids, Ser, Pro, Gly, and Glu. Furthermore, the antibody stained wild-type CHO cells significantly, but much weakly mutant cells deficient in xylosyl- or galactosyltransferase-I required for the biosynthesis of the linkage region. These results suggest that the antibody recognizes the structure GalNAc(±6-O-sulfate)-GlcA-Gal-Gal-Xyl-Ser-(Pro, Gly, Glu). The antibody will be a useful tool for investigating the significance of the linkage region in the biosynthesis and/or intracellular transport of different GAG chains especially since such tools to study the linkage region are lacking.     

Generation of reactive oxygen species undergoing redox cycle of nostocine A: a cytotoxic violet pigment produced by freshwater cyanobacterium Nostoc spongiaeforme

Nostocine A (1) is an extracellular cytotoxic violet pigment produced by the freshwater cyanobacterium, Nostoc spongiaeforme TISTR 8169. Treatment with 1 was found to accelerate the generation of reactive oxygen species (ROS) in the green alga, Chlamydomonas reinhardtii, in the light. In vitro analysis revealed that 1 specifically eliminated superoxide radical anion (O(2)(-)) among several ROS tested. During the course of the reaction, oxygen (O(2)) was simultaneously synthesized and the O(2) synthesizing rate increased with the amount of 1 added. In contrast, O(2)(-) generation occurred when NADPH or NADH was added to a solution of 1 under aerobic condition. The reduction potential of 1 is very similar to that of O(2) indicating that 1 and O(2) can easily exchange electrons depending on the mass balance between their oxidized and reduced forms. Based on these results, the following hypothesis is formulated for the mechanism of intracellular ROS generation by treatment with 1: 1 taken into the target cells is reduced specifically by intracellular reductants such as NAD(P)H. When the O(2) level is sufficiently higher than that of 1, the reduced product of 1 is immediately oxidized by O(2). This is accompanied by the synthesis of O(2)(-) from O(2). The generation of O(2)(-) successively occurs, undergoing repeated redox cycles of 1, when the levels of the reductant and O(2) are still dominant to promote these reactions. This similar intracellular ROS generation mechanism to that of paraquat may cause the cytotoxicity.