Ca2+-Guanine Nucleotide Interactions in Brain Membranes. II. Characteristics of [3H]Guanosine Triphosphate and [3H]β, γ-Imidoguanosine 5'-Triphosphate Binding and Catabolism in the Rat Hippocampus and Striatum

Abstract: With [³H]guanosine triphosphate ([³H]GTP) and [³H]β, γ -imidoguanosine 5′-triphosphate ([³H]GppNHp) as the labelled substrates, both the binding and the catabolism of guanine nucleotides have been studied in various brain membrane preparations. Both labelled nucleotides bound to a single class of noninteracting sites (K_D= 0.1-0.5 μm ) in membranes from various brain regions (hippocampus, striatum, cerebral cortex). Unlabelled GTP, GppNHp, and guanosine diphosphate (GDP) but not guanosine monophosphate (GMP) and guanosine competitively inhibited the specific binding of [³H]guanine nucleotides. Calcium (0.1–5 mm ) partially prevented the binding of [³H]GTP and [³H]GppNHp to hippocampal and striatal membranes. This resulted from both an increased catabolism of [³H]GTP (into [³H]guanosine) and the likely formation of Ca-guanine nucleotide^2- complexes. The blockade of guanine nucleotide catabolism was responsible for the enhanced binding of [³H]GTP to hippocampal membranes in the presence of 0.1 mm -ATP or 0.1 mm -GMP. Striatal lesions with kainic acid produced both a 50% reduction of the number of specific guanine nucleotide binding sites and an acceleration of [³H]GTP and [³H]GppNHp catabolism (into [³H]guanosine) in membranes from the lesioned striatum. This suggests that guanine nucleotide binding sites were associated (at least in part) with intrinsic neurones whereas the catabolising enzyme(s) would be (mainly) located to glial cells (which proliferate after kainic acid lesion). The characteristics of the [³H]guanine nucleotide binding sites strongly suggest that they may correspond to the GTP subunits regulating neurotransmitter receptors including those labelled with [³H]5-hydroxytryptamine ([³H]5-HT) in the rat brain.     

A small cadmium resistance plasmid isolated from Staphylococcus aureus

We have isolated from Staphylococcus aureus a plasmid named pIP983, which measures 3.2 kb and specifies resistance to cadmium. The cad gene it carries is of the B type, as indicated by the level of resistance it confers on S. aureus and the sequence homology with the known cadB gene. Sequences homologous to pIP983 were found on several large S. aureus plasmids. They were localized close to the mcr region of pI/258 and pII147, and, at least in the case of the latter plasmid, were not contiguous, but interrupted by nonhomologous DNA.     

Studies on aroyl- and aryl-hydrazide derivatives from d-glycero-d-gulo-heptono-1,4-lactone

A series of aroyl- and aryl-hydrazide derivatives was prepared from d-glycero-d-gulo-heptono-1,4-lactone (1). The reactivity of the NH proton in these hydrazides, in terms of their dissociation constants (pK_a), was determined from their electronic spectra, and correlated to the Hammett σ values of the substituents. Comparable reactivities of the NH protons for the compounds, and the effect of the substituent, were studied by n.m.r. spectroscopy. Decomposition of the aroylhydrazides with copper(II) sulfate or nitrous acid resulted in the regeneration of 1.     

Myeloperoxidase-catalyzed inactivation of alpha 1-protease inhibitor by human neutrophils

We have examined the effect of the myeloperoxidase-hydrogen peroxide-halide system and of activated human neutrophils on the ability of serum alpha 1-protease inhibitor (alpha 1-PI) to bind and inhibit porcine pancreatic elastase. Exposure to the isolated myeloperoxidase system resulted in nearly complete inactivation of alpha 1-PI. Inactivation was rapid (10 to 20 s); required active myeloperoxidase, micromolar concentrations of H2O2 (or glucose oxidase as a peroxide generator), and a halide cofactor (Cl- or I-); and was blocked by azide, cyanide, and catalase. Intact neutrophils similarly inactivated alpha 1-PI over the course of 5 to 10 min. Inactivation required the neutrophils, a halide (Cl-), and a phorbol ester to activate secretory and metabolic activity. It was inhibited by azide, cyanide, and catalase, but not by superoxide dismutase. Neutrophils with absent myeloperoxidase or impaired oxidative metabolism (chronic granulomatous disease) failed to inactivate alpha 1-PI, and these defects were specifically corrected by the addition of myeloperoxidase or H2O2, respectively. Thus, stimulated neutrophils secrete myeloperoxidase and H2O2 which combine with a halide to inactivate alpha 1-PI. We suggest that leukocyte-derived oxidants, especially the myeloperoxidase system, may contribute to proteolytic tissue injury, for example in elastase-induced pulmonary emphysema, by oxidative inactivation of protective antiproteases.     

Synthesis of daunosamine-containing disaccharides

Treatment of 2,3,6-trideoxy-1,4-di-O-(p-nitrobenzoyl)-3-(trifluoroacetamido)-l-lyxo-hexopyranose (1) with benzyl 2,3-dideoxy-d-glycero-pentopyranoside and p-toluenesulfonic acid gave a mixture of benzyl 2,3,6-trideoxy-4-O-p-nitrobenzoyl-3- (trifluoroacetamido)-l-lyxo-hexopyranoside (49%) and benzyl 2,3-dideoxy-4-O-[2,3,6-trideoxy-4-O-(p-nitrobenzoyl)-3-(trifluoroacetamido)-α-l-lyxo-hexopyranosyl]-d-glycero-pentopyranoside (4, 20 %). The structure of the disaccharide 4 was confirmed by a detailed, mass-spectrometric analysis in three modes, namely, negative- and positive-ion, chemical ionization, and electron impact. Similar treatment of the bis(p-nitrobenzoate) 1 with ethyl 2,3-dideoxy-d-glycero-pentopyranoside gave the ethyl glycoside and the desired disaccharide, showing that the transglycosylation is not restricted to benzyl glycosides. Removal of the p-nitrobenzoyl and the benzyl groups from 4 gave the disaccharide 2,3-dideoxy-4-O-(2,3,6-trideoxy-3-trifluoroacetamido-α-l-lyxo-hexopyranosyl)-d-glycero-pentopyranose.     

Synthesis of 7-(tetrahydropyran-2-yl)- and 7-(4-acetoxytetrahydropyran-2-yl)-ε-rhodomycinone and 7-(tetrahydropyran-2-yl)-ε-pyrromycinone

The ¹³C.n.m.r spectra of water-soluble and -insoluble glucans synthesized by enzymes isolated from six strains of Streptococcus mutans are interpreted. The glucans are shown to be composed primarily of α(1→3)- and α-(1→6)-linked glucosyl residues, and the relative abundance of each linkage is estimated from peak areas. Treatment of water-insoluble glucans with dextranase is found to result in water-soluble and -insoluble products, the former enriched in α-(1→6)-linkages and the latter in α-(1→3)-linkages. The structural conclusions arrived at by ¹³C-n.m.r. spectroscopy are consistent with data from methylation analysis and ¹H-n.m.r. spectroscopy.     

The reduction of aldehydes by broad bean and maize alcohol dehydrogenases and a study of the substrate binding to the enzyme protein

Alcohol dehydrogenase was prepared from 2-day germinating maize and 3-day germinating broad-bean seeds by ammonium sulphate fractionation of sodium phosphate extracts, chromatography onDEAE cellulose and Sephadex G-200. The activity of the broad beanADH amounted to182 800 units per mg protein, that of maizeADH 79 000 units per mg protein. Besides oxidation of a series of alcohols at pH optimum in the alkaline region and with KM equalling 10-2M, alcohol dehydrogenases isolated from both plants catalyze the reduction of acetaldehyde, n-propanal, n-butanal, isobutanal and crotonal at pH optimum in the neutral region with KM equalling 10-3M. The inhibition studies using fatty acids and chloride ions revealed that the oxidation of alcohols is inhibited competitively by both types of inhibitors, with inhibition constants of 10-2M and 10-1M, respectively. The inhibition in the presence of acetaldehyde is non-competitive since the inhibitors do not compete with acetaldehyde and do not form an enzyme-NADH-inhibitor complex, yet they obviously react with the enzyme-NAD product only, thus giving rise to an enzyme-NAD-inhibitor complex. These differences in the behaviour of inhibitors may be interpreted in the sense that the binding sites of ethanol and acetaldehyde as substrates for broad bean and maize alcohol dehydrogenases are non equivalent. The nonequivalency discussed in the text.     

Reactions of 3-(1-arylhydrazono-L-threo-2,3,4-trihydroxybutyl)-1-methyl-2-Quinoxalinones

The 1-methyl derivatives (3 and 4) of 3-(1-phenyl- (1) and 3-(1-p-bromophenylhydrazono-L-threo-2,3,4-trihydroxybutyl)-2-quinoxalinone (2) were prepared by methylation. Periodate oxidation of 3 gave 1-methyl-3-[1-(phenylhydrazono)glyoxal-1-yl]-2-quinoxalinone (5), which, on reduction with sodium borohydride, gave the corresponding 3-[2-hydroxy-1-(phenylhydrazono)ethyl] derivative (8). Reaction of 5 with hydroxylamine or benzoylhydrazine gave the corresponding 2-oxime (6) and 2-(benzoylhydrazone) (7), respectively. Acetic anhydride causes one molecule of 3 or 4 to undergo elimination of two molecules of water, with simultaneous acetylation and ring closure to afford pyrazoles 9 and 10, respectively. Pyrolysis of the triacetate of 3 led to the elimination of acetic acid from the sugar and the hydrazone residue, to give the 3-[5-(acetoxymethyl)-1-phenylpyrazol-3-yl] derivatives (9). Acetic acid was found to effect the same rearrangement, but without acetylation, of 1, 2, and 3 to give the 3-[5-(hydroxymethyl)] derivatives 11, 12, and 13, respectively. The structure of these pyrazoles was confirmed by a series of reactions, including methylation and acetylation. The n.m.r. and i.r. spectra of the compounds were investigated.