Regional Distribution and Quantitative Measurement of the Phosphoinositidase C-Linked Guanine Nucleotide Binding Proteins G1α and Gqα in Rat Brain

Abstract: Levels of the guanine nucleotide binding proteins G₁₁α and G_qα, which produce receptor regulation of phosphoinositidase C., were measured immunologically in 13 regions of rat central nervous system. This was achieved by immunoblotting membranes from these regions with antisera (CQ series) that identify these two polypeptides equally, following separation of the membranes using sodium dodecyl sulphate-polyacrylamide gel electrophoresis conditions that can resolve G_qα and G₁₁α. In all regions examined, G_qα was more highly expressed than G₁₁α. Ratios of levels of G_qα to G₁₁α varied between the regions from 5:1 to 2:1. Quantitative measurements of the levels of G_qα and G₁₁α in each region were obtained by comparison with known amounts of purified liver G_qα and G₁₁α and with E. coli expressed recombinant G_qα. Areas that expressed G_qα highly included olfactory bulb (930 ng/ mg of membrane protein), frontal cortex (700 ng/mg of membrane protein), parietal occipital cortex (670 ng/mg of membrane protein), caudate putamen (1,003 ng/mg of membrane protein), hippocampus (1,045 ng/mg of membrane protein), hypothalamus (790 ng/mg of membrane protein), and cerebellum (950 ng/mg of membrane protein). More modest levels were observed in thalamus (450 ng/mg of membrane protein), pituitary (480 ng/mg of membrane protein), optic chiasma (330 ng/mg of membrane protein), and spinal cord (350 ng/mg of membrane protein). Gna was more evenly expressed with values ranging from about 170 ng/mg of membrane protein in spinal cord and optic chiasma to close to 300 ng/mg of membrane protein in regions expressing high levels of G_qα. A third polypeptide could be identified by the CQ antisera in all brain regions. The possibility that this polypeptide is the α subunit of G₁₄ is discussed.     

Support for Radiolabeling of a Glycine Recognition Domain on the N-Methyl-d-Aspartate Receptor Ionophore Complex by 5,7-[3H]Dichlorokynurenate in Rat Brain

Abstract: Pretreatment with Triton X-100 more than doubled the binding of radiolabeled 5,7-dichlorokynurenic acid (DCKA), a proposed antagonist at a glycine (Gly) recognition domain on the N-methyl-d -aspartate (NMDA) receptor ionophore complex, in rat brain synaptic membranes. The binding exhibited an inverse temperature dependency, reversibility, and saturability, the binding sites consisting of a single component with a high affinity (27.5 nM) and a relatively low density (2.87 pmol/mg of protein). The binding of both [³H]DCKA and [³H]Gly was similarly displaced by numerous putative agonists and antagonists at the Gly domain in a concentration-dependent manner at a concentration range of 100 nM to 0.1 mM. Among the 24 putative ligands tested, DCKA was the second most potent displacer of the binding of both radioligands with no intrinsic affinity for the binding of [³H]kainic acid and α-amino-3-hydroxy-5-[³H]methylisoxazole-4-propionic acid (AMPA) to the non-NMDA receptors. In contrast, the other proposed potent Gly antagonist, 5,7-dinitroquinoxaline-2,3-dione, was active in displacing the binding of [³H]glutamic ([³H]Glu) and D,L-(E)-2-amino-4-[³H]propyl-5-phosphono-3-pentenoic acids to the NMDA recognition domain with a relatively high affinity for the non-NMDA receptors. In addition, the proposed antagonist at the AMPA-sensitive receptor, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline, not only displaced weakly the binding of both [³H]- Gly and [³H]DCKA, but also inhibited the binding of (+)-5-[³H]methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine ([³H]MK-801) to an ion channel associated with the NMDA-sensitive receptor in the presence of added Glu alone in a manner sensitive to antagonism by further added Gly. Clear correlations were seen between potencies of the displacers to displace [³H]DCKA binding and [³H]Gly binding, in addition to between the potencies to displace [³H]-DCKA or [³H]Gly binding and to potentiate or inhibit [³H]MK-801 binding. All quinoxalines tested were invariably more potent displacers of [³H]DCKA binding than [³H]Gly binding, whereas kynurenines were similarly effective in displacing the binding of both [³H]Gly and [³H]-DCKA. These results undoubtedly give support to the proposal that [³H]DCKA is one useful radioligand available in terms of its high selectivity and affinity for the Gly domain in the brain. Possible multiplicity of the Gly domain is suggested by the differential pharmacological profiles between the binding of [³H]Gly and [³H]DCKA.     

A histidine binding protein ofEscherichia coli: a component of cystine binding protein ofEscherichia coli

Summary Commercially obtained cystine binding protein (CBP), an osmotic shock protein ofEscherichia coli, was studied in an effort to determine its binding characteristics. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) analysis of commercially obtained CBP showed three protein bands. N-terminal amino acid microsequencing and subsequent computer search revealed that the sequence of one of these proteins (25-kDa) was nearly identical to histidine binding protein (HisJ) ofSalmonella typhimurium. Purification of CBP by HPLC yielded four protein peaks, of which one bound histidine exclusively. Binding was maximal at pH 5.0 to 6.0, at 4°C, did not require calcium or magnesium ions and was not inhibited by reduction of CBP disulfide bonds. Amino acids other than histidine or cystine did not bind to CBP. These data show that commercially available CBP is not a homogenous protein; it contains a histidine as well as a cystine binding component.     

The CD95 (Fas/APO-1) receptor is phosphorylatedin vitro andin vivo and constitutively associates with several cellular proteins

Different CD95 (Fas/APO-1) isoforms and phosphory lated CD95 species were identified in human T and B cell lines. We had shown previously that the CD95 intracellular domain (IC), expressed as a glutathione S-transferase (GST) fusion protein in murine L929 fibroblasts, was phosphorylatedin vivo. GST-CD95IC was phosphorylatedin vitro by a kinase present in extracts from the human lymphocytic cell lines Jurkat and MP-1 and from murine L929 cells. Phosphoamino acid analysis indicated that phosphorylation occurred at multiple threonine residues and also at tyrosine (Tyr232 and Tyr291) and serine. Amino acids 191 to 275 of CD95 were sufficient for phosphorylation at threonine, tyrosine and serine and also mediated interaction with a 35 kDa cellular protein. Immuno-precipitation of CD95 and chemical cross-linking revealed CD95-associated proteins of approximately 35, 45 and 75 kDa. GST-CD95IC affinity chromatography detected binding of the 35 and 75 kDa protein species. The 75 kDa species may correspond to the CD95-associated proteins RIP or FAF1 and the 35 kDa protein may represent a TRADD analogue. These data indicate that several cellular proteins interact with CD95, possibly in a multi-protein complex, and that a kinase activity is associated with CD95 not onlyin vitro but alsoin vivo. Therefore, receptor phosphorylation may play a role in CD95 signal transduction. This work was in part supported by a grant from the Health Research Council of New Zealand (to JW).     

Effect of ligand binding and conformational changes in proteins on oxygen quenching and fluorescence depolarization of tryptophan residues

The rotational freedom of tryptophan residues in protein-ligand complexes was studied by measuring steady-state fluorescence anisotropies under conditions of oxygen quenching. There was a decrease in the oxygen bimolecular quenching constant upon complexation of trypsin and alpha-chymotrypsin with proteinaceous trypsin inhibitors, of lysozyme with N-acetylglucosamine (NAG) and di(N-acetyl-D-glucosamine) ((NAG)2) and of hexokinase with glucose. Binding of the bisubstrate analogue N-phosphonacetyl-L-aspartate (PALA) to aspartate transcarbamylase (ATCase) and binding of biotin to avidin resulted in increased oxygen quenching constants. The tryptophan of human serum albumin (HSA) in the F state was more accessible to oxygen quenching than that in the N state. With the exception of ATCase, the presence of subnanosecond motions of the tryptophan residues in all the proteins is suggested by the short apparent correlation times for fluorescence depolarization and by the low apparent anisotropies obtained by extrapolation to a lifetime of zero. Complex formation evidently resulted in more rigid structures in the case of trypsin, alpha-chymotrypsin and lysozyme. The effects of glucose binding on hexokinase were not significant. Binding of biotin to avidin resulted in a shorter correlation time for the tryptophan residues. The N --> F transition in HSA resulted in a more rigid environment for the tryptophan residue. Overall, these changes in the dynamics of the protein matrix and motional freedom of tryptophan residues due to complex formation and subsequent conformational changes are in the same direction as those observed by other techniques, especially hydrogen exchange. Significantly, the effects of complex formation on protein dynamics are variable. Among the limited number of cases we examined, the effects of complex formation were to increase, decrease or leave unchanged the apparent dynamics of the protein matrix.     

A reassessment of the binding of napthaleneacetic acid by membrane preparations from maize

Naphthalene-1-acetic acid (NAA) binding by membrane fractions derived from maize has been re-evaluated. Using a computer curve-fitting procedure only one major type of NAA binding, in terms of binding affinity, could be identified. Auxins, antiauxins and structurally related compounds have been tested for their competitive effect on NAA binding and the inhibitor constants for a number of these have been determined. Extracts from various plant species have been examined for their NAA binding ability, but all showed much less binding than maize leaf or coleoptile preparations. The possibility of the NAA binding by maize extracts being due to a true hormone receptor is discussed.Abbreviations BA benzoic acid - CPIB p-chlorophenoxyisobutyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - DCB 2,4-dichlorobenzoic acid - IAA indolyl-3-acetic acid - NAA napthalene-1-acetic acid - 2-NAA napthalene-2-acetic acid - NAOA napthalene-2-oxyacetic acid - PA phenylacetic acid - PU phenylurea - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - TIBA 2,3,5-triiodobenzoic acid     

Isolation of a high molecular weight glycoconjugate derived from the surface of S purpuratus eggs that is implicated in sperm adhesion

Sea urchin sperm–egg adhesion is mediated by bindin, a sperm surface protein that has lectin-like activity. Bindin agglutinates eggs, and this interaction has been shown to be inhibited by glycopeptides released from the egg surface by protease treatment. In this study, we report the purification and properties of such an egg surface glycoconjugate that may be involved in sperm adhesion. The glycoconjugate was partially purified by gel filtration and affinity chromatography on bindin particles. Upon gel filtration on Sepharose CL 4-B, the glycoconjugate elutes near the void volume, suggesting that it has a molecular weight in excess of one million. In addition, we have found that the egg surface glycoconjugate agglutinates bindin particles, indicating that it is multivalent. Carbohydrate analysis indicates that the glycoconjugate is composed primarily of fucosc, xylose, galactose, and glucose. This purified egg surface component is the most potent inhibitor of bindin-mediated egg agglutination yet described.     

Abscisic acid binding to subcellular fractions from leaves of Vicia faba

A centrifugation binding assay has been used to demonstrate the binding of [³H] (±) abscisic acid to membrane-rich fractions prepared from leaves of Vicia faba L. Kinetic analysis of this binding shows evidence of saturation of binding sites with increasing concentration of ligand. Scatchard analysis of these data yields a biphasic plot possibly indicating the presence of two types of binding sites. The dissocation constant for the high affinity site has been calculated to be 3.5×10^-8 mol 1^-1.     

Mannosephilic adhesins produced by some strains of motile Aeromonas reveal structural details of Salmonella lipopolysaccharide through the phenomenon of co-agglutination

Abstract Some strains of motile Aeromonas produce lectin-like adhesins, whose activity can be inhibited by d -mannose. Such strains can co-agglutinate with some strains of Salmonella . Whether or not co-agglutination occurs is dependent upon both the properties of the Aeromonas adhesin and the structure of the Salmonella lipopolysaccharide (LPS). These studies have enabled new structural information for Salmonella LPS to be deduced and have confirmed previous studies regarding the nature of the Aeromonas adhesin binding site. It is possible that the observed in vitro co-agglutination between Aeromonas and Salmonella is a reflection of an in vivo situation which could modify the virulence of either or both bacteria.     

The NAM8 gene in Saccharomyces cerevisiae encodes a protein with putative RNA binding motifs and acts as a suppressor of mitochondrial splicing deficiencies when overexpressed.

Summary We have characterized the nuclear geneNAM8 inSaccharomyces cerevisiae. It acts as a suppressor of mitochondrial splicing deficiencies when present on a multicopy plasmid. The suppressed mutations affect RNA folding and are located in both group I and group II introns. The gene is weakly transcribed in wildtype strains, its overexpression is a prerequisite for the suppressor action. Inactivation of theNAM8 gene does not affect cell viability, mitochondrial function or mitochondrial genome stability. TheNAM8 gene encodes a protein of 523 amino acids which includes two conserved (RNP) motifs common to RNA-binding proteins from widely different organisms. This homology with RNA-binding proteins, together with the intronic location of the suppressed mitochondrial mutations, suggests that the NAM8 protein could be a non-essential component of the mitochondrial splicing machinery and, when present in increased amounts, it could convert a deficient intron RNA folding pattern into a productive one.