Alkaline Hydroxylamine Research Articles

The octameric structure of β-glucosidase from Botryodiplodia theobromae Pat

1. Whereas only beta-glucosidase A (beta-D-glucoside glucohydrolase, EC 3.2.1.21) was produced by the tropical fungus Botryodiplodia theobromae Pat. (I.M.I. 115626; A.T.C.C. 26123) in young cultures containing D-cellobiose as carbon source, lower-Mr forms (B, C and D) were found in older cultures when the pH had drifted from the initial value of pH 6.2 to pH 7.9. 2. The Michaelis constants (Km) of the various molecular forms of the enzyme were 0.30 +/- 0.03 mM-, 0.26 +/- 0.01 mM-, 0.20 +/- 0.02 mM- and 0.16 +/- 0.01 mM-o-nitrophenyl beta-D-glucopyranoside for beta-glucosidase forms A (Mr 320,000), B (Mr 160,000), C (Mr 80,000) and D (Mr 40,000) respectively. 3. Only beta-glucosidase D showed substrate inhibition. 4. Only L-arginine was found as the N-terminal residue, and beta-glucosidase A contained 31.7 +/- 0.6 mol of N-terminal L-arginine/mol of the enzyme. 5. Storage of purified beta-glucosidase A under mildly alkaline conditions caused its dissociation into the lower-Mr forms, whereas adjustment of the pH of a solution of beta-glucosidase A to pH 12.0 with 1 M-NaOH led to complete inactivation on incubation at 40 degrees C for 1 h and to the release of 25.2 +/- 1.5 mol of inorganic phosphate/mol of the enzyme. 6. O-Phospho-L-serine was isolated from the acid-hydrolysis product of beta-glucosidase A but not from that of beta-glucosidase D. 7. Reduction and carboxamidomethylation of the various forms of beta-glucosidase gave only one enzymically inactive protein with an Mr of 10,000-11,000. 8. After partial succinylation (3-carboxypropionylation) of beta-glucosidase D at pH 5.0 and removal of the precipitated protein formed, the supernatant solution contained beta-glucosidase components similar to the other molecular forms (A, B and C) and an aggregate (beta-glucosidase Xs) that gave a positive result in the alkaline hydroxylamine test, whereas N-succinylated beta-glucosidase D, an aggregate (form Xp) that behaved like beta-glucosidase Xs and traces of forms A, B and C were found by gel filtration of the solution of the precipitate solubilized at neutral pH (7.0-7.7). 9. These observations are discussed in terms of the proposed octameric structure of beta-glucosidase A based on the result of electron microscopy [Umezurike (1975) Biochem. J. 145, 361-368].

Polyhydroxamic serum albumin microcapsules: preparation and chelating properties

Polyhydroxamic serum albumin microcapsules were prepared through interfacial cross-linking with terephthaloyl chloride and further treatment of microcapsules with alkaline hydroxylamine. They exhibited iron-binding properties which were increased by esterification of the free carboxylic groups of the membrane with benzylic alcohol using a carbodiimide, prior to the hydroxylamine treatment.

Molecular forms of acetylcholinesterase in two sublines of human erythroleukemia K562 cells

Acetylcholinesterase (AChE) in K562 cells exists in two molecular forms. The major form, an amphiphilic dimer (G2a) which sediments at 5.3 S, and the minor form, an amphiphilic monomer (G1a) which sediments at 3.5 S. Extraction in the presence of the sulfhydryl alkylating agent N-ethylmaleimide was required to preserve the G2a form. In Triton X-100 extracts of the subline K562-243, phosphatidylinositol-specific phospholipase C (PtdIns-PLC) from Bacillus thuringiensis converted most of the G2a AChE into a hydrophilic dimer (G2h), indicating that the G2a form possessed a hydrophobic glycoinositol phospholipid that mediated its attachment to the membrane. Treatment of intact K562-243 cells with PtdIns-PLC released approximately 60% of the total AChE activity and provided an estimate of the externally exposed AChE. The direct conversion from an amphiphilic to a hydrophilic dimeric form by PtdIns-PLC was not obtained in extracts or intact cells of the subline K562-48. Instead, pretreatment with alkaline hydroxylamine was necessary to render the amphiphilic G2 form of this subline susceptible to digestion by the phospholipase. In this respect, the amphiphilic dimer of K562-48 AChE resembles the G2a form of human erythrocyte AChE, which is resistant to PtdIns-PLC because of the direct palmitoylation of an inositol hydroxyl group in the anchor [Roberts et al. (1988) J. Biol. Chem. 263, 18766-18775]. Release of this acyl chain by hydroxylamine renders the enzyme susceptible to PtdIns-PLC [Toutant et al. (1989) Eur. J. Biochem. 180, 503-508]. In both K562 sublines, sialidase decreased the migration of the G2a form but not of the G1a form of AChE. G1a forms thus appear to represent an intracellular pool of newly synthesized molecules residing in a compartment proximal to the trans-Golgi apparatus. The sialidase-resistant G1a molecules were also resistant to PtdIns-PLC digestion; possible explanations for this resistance are presented.

Lipid-modified surface protein antigens expressing size variation within the species Mycoplasma hyorhinis

Monoclonal antibodies (MAbs) previously shown to recognize distinct epitopes selectively expressed on the surface of some Mycoplasma hyorhinis strains were used to define two discrete sets of lipid-modified membrane surface proteins showing marked size variation within this species. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis of Triton X-114 phase-fractionated proteins from six isolates of M. hyorhinis defined a set of amphiphilic integral membrane proteins of 23, 50, and 55 kilodaltons (kDa) recognized on respective isolates by one MAb and a second set of integral proteins of 88, 120, and 100 to 150 kDa recognized by another MAb. The first group of proteins all contained a common, amphiphilic 18-kDa limit tryptic polypeptide bearing the epitope. The size- and strain-variant surface antigens identified by the MAbs were shown to be lipid-modified proteins. Phase fractionation of [3H]palmitate-labeled organisms revealed numerous 3H-labeled proteins in all isolates, which partitioned exclusively into the hydrophobic phase. These proteins generally showed pronounced size variation among isolates and included the antigen variants recognized by the two MAbs, as demonstrated directly by immunoprecipitation of correspondingly sized 3H-labeled proteins from each isolate. A third MAb recognized an invariant, lipid-associated surface protein of 70 kDa on all M. hyorhinis isolates. Covalent modification of lipid-associated proteins was confirmed by identifying 3H-labeled methyl palmitate after acid methanolysis of Triton X-114 phase proteins derived from [3H]palmitate-labeled organisms. However, removal of covalently bound lipid from chloroform-methanol-extracted proteins by alkaline hydroxylamine was selective; complete removal was observed with only a few proteins, possibly including the 120-kDa form of one antigen variant. This suggested potential differences in the nature of covalent linkage among lipid-modified M. hyorhinis surface antigens. Intraspecies antigen variants described here in M. hyorhinis share some characteristics with size-variant antigens reported in phylogenetically related gram-positive eubacteria and may contribute to phenotypic diversification and differences in pathogenicity of mycoplasmas.

Major membrane surface proteins of Mycoplasma hyopneumoniae selectively modified by covalently bound lipid.

Surface protein antigens of Mycoplasma hyopneumoniae were identified by direct antibody-surface binding or by radioimmunoprecipitation of surface 125I-labeled proteins with a series of monoclonal antibodies (MAbs). Surface proteins p70, p65, p50, and p44 were shown to be integral membrane components by selective partitioning into the hydrophobic phase during Triton X-114 (TX-114)-phase fractionation, whereas p41 was concomitantly identified as a surface protein exclusively partitioning into the aqueous phase. Radioimmunoprecipitation of TX-114-phase proteins from cells labeled with [35S]methionine, 14C-amino acids, or [3H] palmitic acid showed that proteins p65, p50, and p44 were abundant and (with one other hydrophobic protein, p60) were selectively labeled with lipid. Covalent lipid attachment was established by high-performance liquid chromatography identification of [3H]methyl palmitate after acid methanolysis of delipidated proteins. An additional, unidentified methanolysis product suggested conversion of palmitate to another form of lipid also attached to these proteins. Alkaline hydroxylamine treatment of labeled proteins indicated linkage of lipids by amide or stable O-linked ester bonds. Proteins p65, p50, and p44 were highly immunogenic in the natural host as measured by immunoblots of TX-114-phase proteins with antisera from swine inoculated with whole organisms. These proteins were antigenically and structurally unrelated, since hyperimmune mouse antibodies to individual gel-purified proteins were monospecific and gave distinct proteolytic epitope maps. Intraspecies size variants of one surface antigen of M. hyopneumoniae were revealed by a MAb to p70 (defined in strain J, ATCC 25934), which recognized a larger p73 component on strain VPP11 (ATCC 25617). In addition, MAb to internal, aqueous-phase protein p82 of strain J failed to bind an analogous antigen in strain VPP11. These studies establish that a highly restricted set of distinct, lipid-modified hydrophobic membrane proteins are major surface antigens of M. hyopneumoniae and that structural variants of surface antigens occur within this species.

The transport and utilization of acetyl coenzyme A by rat liver Golgi vesicles. O-acetylated sialic acids are a major product.

When intact rat liver Golgi vesicles were incubated with [acetyl-3H]acetyl coenzyme A, radioactivity was incorporated into the vesicles in a manner dependent upon temperature, time, protein, and acetyl-CoA concentration. The vesicles concentrated the label 121-fold relative to the medium within 20 min, suggesting an active transport mechanism operating in intact vesicles, and incorporated more than 50% of this label into acid-insoluble materials. This was supported by the finding that incorporation was markedly reduced by Triton X-100 at levels above its critical micellar concentration. While the intravesicular low molecular weight fraction was predominantly free acetate, acetate ions themselves were not permeant to the vesicles. Double-label experiments suggested that the transport process involved the entire acetyl-CoA molecule. This was further supported by the fact that coenzyme ASH, palmitoyl-CoA and butyryl-CoA were markedly inhibitory. Incorporation was optimal at 22 degrees C at pH 7.0, and was moderately stimulated by ATP. However, compounds known to abolish proton gradients or to inhibit the Golgi proton pump had no effect. The apparent Km for the utilization process was 0.61 microM with a Vmax of 21.3 pmol/mg of protein/min. Oligomycin and 4,4'-diisothiocyanostilbene-2,2'disulfonic acid were inhibitory, whereas CMP-NeuAc, UDP-GlcNAc, adenosine 3'-phosphate, 5'-phosphosulfate, atractylosides, tunicamycin, 2'5'-ADP, and 3',5'-ADP were not, showing that this transport process is distinct from other nucleotide transporters previously described in rat liver Golgi. 75-85% of the radioactivity incorporated was shown to be in O-acetylated sialic acids, by neuraminidase release, purification, and high pressure liquid chromatography. The majority of the neuraminidase-resistant radioactivity was released by alkaline hydroxylamine as [3H]acetylhydroxamate, but a significant fraction was resistant to this treatment. The nature of the non-sialic acid radioactivity remains unknown. The existence of this transport mechanism provides yet another level at which the O-acetylation of sialic acids could be regulated.

Activated Pectin Stained with Heavy Metals

Pectin was visualized in carrot tissue culture with a stain utilizing hydroxylamine and iron. Actively growing cells were fixed in glutaraldehyde or paraformaldehyde. The pectin was activated by an alkaline hydroxylamine treatment. When the so formed pectic hydroxamic acids were reacted with ferric chloride, a reddish-brown color was visible en masse, but the cells were not sufficiently stained for either light or electron microscopy.We hoped to intensify the stain by bridging iron to osmium. Since one Fe (III) complexes three gallocyanins, we tested this dye as a bridge in an attempt to amplify the staining. Thiocarbohydrazide (TCH) was tested as an alternate bridge. After excess iron was washed out, the cells were treated with either 1% gallocyanin for one hour, or 0.5% TCH for one minute. After excess bridge was removed by a second wash, a portion of each treatment was placed in 1% osmium tetroxide for one hour.

A reliable method for demonstrating axonal degeneration shortly after axotomy.

A method has been elaborated by which degenerating axons can be selectively impregnated with silver. Based on reconsideration of the physicochemical mechanisms of the degeneration methods it takes advantage of physical developers over the chemical ones. The staining procedure is applied to frozen sections of brains fixed with formol. It consists of 6 steps: (1) pretreatment with alkaline hydroxylamine, (2) washing in acetic acid, (3) impregnation in silver nitrate in the presence of ferric ions, (4) washing in citric acid, (5) physical development, and (6) washing in acetic acid. By electron microscopy silver precipitates by this method are almost entirely restricted to the cytoplasm of dense, degenerating axons, sparing mitochondria and myelin sheaths. No special expertise is required to achieve reproducible results. Large numbers of sections treated simultaneously, and large sections, can be stained uniformly. Light microscopic criteria are described which help diagnose the source of possible failures. Low background staining allows dark field illumination and television image analysis to be applied. The method works at survival times of only 3 to 5 days after axotomy. Hence, degenerating axons and axon terminals can be stained in alternating sections from the same brain using this method and another being described separately, which, using different conditions, demonstrates degenerating axon terminals.

Intraperiplasmic growth of Bdellovibrio bacteriovorus 109J: attachment of long-chain fatty acids to escherichia coli peptidoglycan

During the initial stages of intraperiplasmic growth of Bdellovibrio bacteriovorus on Escherichia coli, the peptidoglycan of the E. coli becomes acylated with long-chain fatty acids, primarily palmitic acid (60%) and oleic acid (20%). The attachment of the fatty acids to the peptidoglycan involves a carboxylic-ester bond, i.e., they were removed by treatment with alkaline hydroxylamine. Their linkage to the peptidoglycan does not involve a protein molecule. When the bdelloplast peptidoglycan was digested with lysozyme, the fatty acid-containing split products behaved as lipopeptidoglycan, i.e., they were extracted into the organic phase of 1-butanol:acetic acid:water (4:15) two-phase system; all of the lysozyme split products generated from normal E. coli peptidoglycan were extracted into the water phase. It is suggested that the function of the acylation reaction is to help stabilize the bdelloplast outer membrane against osmotic forces. In addition, a model is presented to explain how a bdellovibrio penetrates, stabilizes, and lyses a substrate cell.

Spectrophotometric determination of parathion and paraoxon using alkaline hydroxylamine solution for the liberation of 4-nitrophenol.

The half-time for the liberation of 4-nitrophenol from parathion (diethyl 4-nitrophenyl phosphorothionate) at 20 °C in the presence of 0.5 M sodium hydroxide solution is several hours, but this time can be reduced nearly a 1000-fold when the alkali is replaced with a mixture of 0.5 M sodium hydroxide solution and 0.67 M hydroxylamine solution. The reaction is almost instantaneous when the system also contains 33% of ethanol. A procedure is described, based on these findings, for the spectrophotometric determination of parathion and paraoxon via the 4-nitrophenol liberated. The method is simple and quantitative and does not involve the use of drastic chemical procedures such as those required for methods that are currently available. The sensitivity of the method is about 0.1 µmol of parathion. Mixtures of parathion and paraoxon can be determined by using a selective extraction procedure.

Determination of EPN, Its Oxygen Analog, and p-Nitrophenol in Aqueous Suspensions and Enzymic Digests

Abstract O-Ethyl O-p-nitrophenyl phenylphosphonothioate (EPN), its oxygen analog O-ethyl O-p-nitrophenyl phenylphosphonate (EPNO), and p-nitrophenol (PNP) are completely extractable from slightly acidic aqueous suspensions or enzymic digests by a mixture of equal volumes of benzene and isobutanol. In shaking the extract with 0.05N NaOH, PNP is quantitatively partitioned into the aqueous phase and can be determined spectrophotometrically at 410 nm. Aliquots of the solvent phase which now contains EPN and EPNO are evaporated to dryness at 120°C. One portion of the residue is treated with ethanol and alkaline hydroxylamine. The PNP liberated is equivalent to the total EPN -f EPNO. Another portion of the residue is dissolved in cyclohexane and shaken with alkaline hydroxylamine. EPNO, which has a greater reactivity and is more soluble in water, is rapidly decomposed to PNP and can be measured as above. EPN remains unaffected in the cyclohexane phase under these conditions. The value for EPN is calculated either by difference or, when necessary, by evaporating the cyclohexane and decomposing the residue with ethanol and hydroxylamine. The method is sensitive to 0.1 μmole EPN, EPNO, or PNP and can be used in the study of enzyme systems converting EPN and EPNO to PNP or EPN to EPNO. It can also be used in monitoring the preparation of EPNO from EPN by bromine water.

Estimation of Malaoxon in the Presence of Malathion by the Ferric Hydroxamate Method

Abstract Both malathion and malaoxon are completely extractable from aqueous suspensions by benzene- isobutanol (1 + 1). Aliquots of the extract are evaporated at 110– 120°C, the residue is dissolved in methanol, and the total ferric hydroxamate color is determined. Extraction of a second aliquot with cyclohexane removes malathion and some malaoxon. Malaoxon is selectively partitioned from cyclohexane into alkaline hydroxylamine, leaving only malathion in the cyclohexane phase. After the cyclohexane phase is evaporated, the amount of malathion can be estimated by the ferric hydroxamate method. The difference in the color for the benzene-isobutanol extract and the cyclohexane extract represents that due to malaoxon; the amount of malaoxon may be calculated from a standard curve. The method, sensitive to about 0.2 μmole malaoxon, is useful for monitoring the laboratory preparation of malaoxon from malathion.

Comparative studies on Pseudomonas aeruginosa endotoxin

AbstractAttempts to obtain toxic preparations of endotoxin from Pseudomonas aeruginosa (C2) were initiated by employing five different extraction procedures which were subsequently assayed for mouse lethality using a variety of routes. The standard methods of extraction were the aqueous phenol, trichloracetic acid, ethylenediaminetetraacetate‐lysozyme, ethyl ether, and hot water procedures. The aqueous phenol preparation was found to be the most toxic and exhibited an LD50 value of 450 μg dry weight when administered intravenously and 840 μg intraperitoneally. No lethality was observed when endotoxin was administered intranasally. The second most lethal preparation was obtained by the trichloroacetic acid extraction and yielded LD50 values of 589 μg intravenously and 947 μg intraperitoneally. The other three preparations were considerably less lethal. The aqueous phenol and trichloroacetic acid preparation tended to have a higher content of those carbohydrates associated with the core region of the lipopolyssacharide molecule than did the lesser toxic preparations. Some correlation between lethality and lipid content as determined by the alkaline hydroxylamine procedure was also observed.

Detection of esters as hydroxamates after separation by paper electrophoresis

Paper electrophoresis lends itself to the study of several classes of compound containing a carboxylic ester function. These compounds can be located on pherograms after reaction with alkaline hydroxylamine, the resulting hydroxamates being revealed as coloured ferric iron complexes using a ferric chloride spray reagent. Wide variation is evident in the response of esters to the hydroxylamine reagent, and the differences in reactivity could be diagnostically useful. Although acetates and lactones react in mildly alkaline medium at room temperature, many esters such as acid phthalates, benzoates, and some tropane and pyrrolizidine alkaloids require a strongly alkaline reagent. Other pyrrolizidien alkaloids and many esters of o-sulphobenzoic acid require forcing conditions—elevated temperatures (up to 110°) in strongly alkaline medium—to form the corresponding hydroxamates. Some esters are not detectable using even more energetic conditions.

The effect of endotoxin on thin lipid bilayer membranes

The effect of endotoxin fromSalmonella typhimurium orEscherichia coli was studied on bilayer lipid membranes (1% lecithin-1% cholesterol in n-decane) formed in buffered 0.1M NaCl solution (pH 6.8). Endotoxin was added to the buffered solution either prior to membrane formation or after stable membranes were formed. In both cases, concentrations of 110 to 720 μg/ml endotoxin initiated a decrease in the electrical resistance of the membranes followed by their rupture. A 50 μg/ml concentration of the agent was unable to elicit any response. Also, the addition of an equal volume of buffer solution, serving as a control, caused no decrease in membrane resistance or survival time. Treatment of the endotoxin with alkaline hydroxylamine to remove esterand amide-bound fatty acids likewise abolished the membrane effect. This is the first report of an endotoxin effect on lipid bilayer membranes. The potential correlation of this interaction of bilayer and endotoxin with the diverse biologic effects of endotoxin is discussed.

Reaction of nitric oxide with alkaline hydroxylamine

Reaction of nitric oxide with alkaline hydroxylamine

Leukocyte adenosine triphosphatases and the effect of endotoxin on their activity

Intact rabbit leukocytes from acute peritoneal exudates and leukocytes separated from rabbit and human blood catalyzed the hydrolysis of adenosine triphosphate. The pH optima and Michaelis constants for the adenosine triphosphatases of the three types of leukocytes were the same. The activity required Mg 2+ and was stimulated by added K +. Efforts to demonstrate Na + activation of the enzyme system were unsuccessful. Ouabain, as well as Ca 2+ and sulfhydryl reagents, inhibited the hydrolysis reaction. Greater percentage of inhibition with ouabain was obtained with blood or buffy coat adenosine triphosphatase than with exudate leukocyte adenosine triphosphatase. Similar results were obtained with cell-free leukocyte adenosine triphosphatase preparations. Endotoxin, bacterial lipopolysaccharide, inhibited both the intact cell and cell-free adenosine triphosphatases to about the same extent as ouabain. Treatment of the endotoxin with alkaline hydroxylamine to remove ester- and amide-bound fatty acids abolished its inhibitory activity for the adenosine triphosphatases.

Fluorescent Lipid from Brain

A SUBSTANCE has been obtained from the brain of rabbits killed with diisopropyl fluorophosphate that appears to have interesting properties and chemical structure. The substance can be obtained in amounts of 1.0–1.3 mg/g of brain by extraction with cold aqueous 5 per cent perchloric acid, followed by precipitation with phosphotungstic acid, and chromatography of the portion of the precipitate soluble in chloroform on silica with chloroform–methanol mixtures as eluant. The substance, obtained as a wax, gave a single fluorescent spot of RF 0.95 by thin layer chromatography on alumina with a pyridine–amyl alcohol–water (3 : 3 : 2) solvent system. The same areas also gave positive reactions with acidified permanganate, diazotized p-nitroaniline, Folin's phenol reagent, Schiff's fuchsin reagent, 2,4-dinitrophenylhydrazine, and with alkaline hydroxylamine followed by acidified ferric chloride solution. Elementary analysis showed some variation from lot to lot, with an average of 75.5 per cent carbon and 12.0 per cent hydrogen, and a range of ± 1 per cent for carbon and ±0.5 per cent for hydrogen.

Thin‐layer chromatography of aliphatic े 舒 and ै ‐lactones

AbstractHomologous aliphatic े‐ or ै‐lactones are separated by thin‐layer chromatography on a mixture of kieselguhr G and silica gel G (1:8). The thin‐layer plates are chromatographically impregnated with methanol as the stationary phase and developed 3 times with light petroleum (bp 80舑100C) saturated with methanol. े‐ And ै‐lactones can be separated from each other on the same adsorbent with a 1:1 mixture of light petroleum (bp 30舑40C) and isopropyl ether. The systems can be combined 2‐dimensionally. The lactones are detected by spraying the chromatoplates either with a 2% solution of iodine in methanol or with alkaline hydroxylamine followed by ferric chloride and acetic acid. These methods were used for the tentative identification of ै‐C10舑16 lactones in commercial Australian butteroil.

Phosdrin Residues in Fruits and Vegetables

Abstract The enzyme inhibition-spectrophotometric procedure for Phosdrin residues submitted by the manufacturer has been slightly modified and studied collaboratively on apples, tomatoes, and cabbages. This method is based on the ability of Phosdrin to inhibit cholinesterase. Phosdrin is extracted from crops with chloroform, transferred into water, purified to remove organic impurities, and determined quantitatively by the acetylcholinesterase inhibition procedure. The uninhibited cholinesterase hydrolyzes acetylcholine to acetic acid and choline. The acetylcholine not hydrolyzed is reacted with alkaline hydroxylamine and a ferric salt to form a red complex, the absorbance of which is measured spectrophotometrically at 540 mμ. The recoveries at the 0.164 and 0.328 ppm levels of addition ranged from 73.2 to 109.8% for apples, 70.1 to 111.0% for tomatoes, and 68.9 to 117.0% for cabbages. The mean recoveries for apples, tomatoes, arid cabbages were 95.9, 94.9, and 91.6%, respectively; the over-all recovery was 94.1%. The method is recommended as official, first action for quantitative determination of Phosdrin residues in fruits and vegetables.