Reversible binding of heparin to the loop peptide of endotoxin neutralizing protein.

Endotoxin neutralizing protein (ENP) from Limulus polyphemus is an amphipathic, 11.8 kDa protein with an isoelectric point of 10.2. ENP neutralizes lipopolysaccharide (LPS) and possesses antibacterial activity against Gram-negative bacteria. Heparin binds to ENP and blocks its LPS-neutralizing activity. The relative blocking activity of heparin is equal to low molecular weight heparin and polyanetholsulfonic acid > heparan sulfate > chondroitin sulfate A > chondroitin sulfate C. Endoproteinase Glu-C hydrolysis of recombinant ENP results in four major peptides, three of which are seen following separation on reversed phase HPLC. Heparin binds to the loop peptide (31-72), which includes the heparin binding consensus sequence XBBXBX between the two cysteine residues of ENP. When heparin is added to the digest and then applied to a C18 column, the loop peptide is bound; however, it dissociates and elutes with either 5 M NaCl or 0.1 M sodium phosphate, demonstrating reversible binding to heparin. LPS and lipid A both bind to the loop peptide and remove it from digests of ENP; however, neither complex could be dissociated by salt or sodium phosphate. Heparin, LPS, and lipid A individually bind to the same site on ENP.

Assay of endotoxin by limulus amebocyte lysate.

Horseshoe crabs fight off infectious agents with a complex array of proteins present in amebocytes, the major cell type in their hemolymph. These amebocytes contain both large and small granules (1). When exposed to bacteria or other infectious agents the amebocytes release proteins into their surroundings by exocytosis. The small granules of Limulus amebocytes contain antibacterial proteins, including polyphemusins and the big defensins (2). The large granules contain the Limulus anti-lipopolysaccharide factor (LALF) and the clot-forming group of serine protease zymogens. Exocytosis is initiated by the reaction of amebocytes with lipopolysaccharide (LPS) from Gram-negative bacteria or other microbial components. LPS is also called endotoxin because it is found in the outer membrane of the gram-negative bacterial cell wall. A solid clot forms in response to the lipid A portion of LPS, thereby walling off the infection site or preventing the loss of blood when the animal is damaged physically (3).

Limulus amebocyte lysate assay for detection of endotoxin in patients with sepsis syndrome. AMCC Sepsis Project Working Group.

Clinical predictions alone are insufficiently accurate to identify patients with specific types of bloodstream infection; laboratory assays might improve such predictions. Therefore, we performed a prospective cohort study of 356 episodes of sepsis syndrome and did Limulus amebocyte lysate (LAL) assays for endotoxin. The main outcome measures were bacteremia and infection due to gram-negative organisms; other types of infection were secondary outcomes. Assays were defined as positive if the result was > or = 0.4 enzyme-linked immunosorbent assay units per milliliter. There were positive assays in 119 (33%) of 356 episodes. Assay positivity correlated with the presence of fungal bloodstream infection (P < .003) but correlated negatively with the presence of gram-negative organisms in the bloodstream (P = .04). A trend toward higher rates of mortality in the LAL assay-positive episodes was no longer present after adjusting for severity. Thus, results of LAL assay did not correlate with the presence of bacteremia due to gram-negative organisms or with mortality after adjusting for severity but did correlate with the presence of fungal bloodstream infection.

Purification of Two Nitrate Reductases from Xanthomonas maltophilia Grown in Aerobic Cultures.

Xanthomonas maltophilia ATCC 17666 is an obligate aerobe that accumulates nitrite when grown on nitrate. Spectra of membranes from nitrate-grown cells exhibited b-type cytochrome peaks and A(615-630) indicative of d-type cytochrome but no absorption peaks corresponding to c-type cytochromes. The nitrate reductase (NR) activity was located in the membrane fraction. Triton X-100-extracted reduced methyl viologen-NRs were purified on DE-52, hydroxylapatite, and Sephacryl S-300 columns to specific activities of 52 to 67 mumol of nitrite formed per min per mg of protein. The cytochrome-containing NR(I) separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis into a 135-kDa alpha-subunit, a 64-kDa beta-subunit, and a 23-kDa gamma-subunit with relative band intensities indicative of a 1:1:1 alpha/beta/gamma subunit ratio and a M(r) of 222,000. The electronic spectrum of dithionite-reduced purified NR displayed peaks at 425, 528, and 558 nm, indicative of the presence of a cytochrome b, an interpretation consistent with the pyridine hemochrome spectrum formed. The cytochrome b of the NR was reduced under anaerobic conditions by menadiol and oxidized by nitrate with the production of nitrite. This NR contained 0.96 Mo, 12.5 nonheme iron, and 1 heme per 222 kDa: molybdopterin was detected with the Neurospora crassa nit-1 assay. A smaller reduced methyl viologen-NR (169 kDa), present in various concentrations in the Triton X-100 preparations, lacked a cytochrome spectrum and did not oxidize menadiol. The characteristics of the NRs and the absence of c-type cytochromes provide insights into why X. maltophilia accumulates nitrite.

Menaquinol-nitrate oxidoreductase of Bacillus halodenitrificans.

When grown anaerobically on nitrate-containing medium, Bacillus halodenitrificans exhibited a membrane-bound nitrate reductase (NR) that was solubilized by 2% Triton X-100 but not by 1% cholate or deoxycholate. Purification on columns of DE-52, hydroxylapatite, and Sephacryl S-300 yielded reduced methyl viologen NR (MVH-NR) with specific activities of 20 to 35 U/mg of protein that was stable when stored in 40% sucrose at -20 degrees C for 6 weeks. 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxypropone-1-sulfonat e (CHAPSO) and dodecyl-beta-D-maltoside stimulated enzyme activity three- to fourfold. Membrane extractions yielded purified NR that separated after electrophoresis into a 145-kDa alpha subunit, a 58-kDa beta subunit, and a 23-kDa gamma subunit. The electronic spectrum of dithionite-reduced, purified NR displayed peaks at 424.6, 527, and 557 nm, indicative of the presence of a cytochrome b, an interpretation consistent with the pyridine hemochrome spectrum formed. Analyses revealed a molybdenum-heme-non-heme iron ratio of 1:1:8 for the NR and the presence of molybdopterin. Electron paramagnetic resonance (EPR) signals characteristic of iron-sulfur centers were detected at low temperature. EPR also revealed a minor signal centered in the g = 2 region of the spectra. Upon reduction with dithionite, the enzyme displayed signals at g = 2.064, 2.026, 1.906, and 1.888, indicative of the presence of low-potential iron-sulfur centers, which resolve most probably as two [4Fe-4S]+1 clusters. With menadiol as the substrate for nitrate reduction, the Km for nitrate was 50-fold less than that seen when MVH was the electron donor. The cytochrome b557-containing enzyme from B. halodenitrificans is characterized as a menaquinol-nitrate:oxidoreductase.

Occurrence of Nitrate Reductase and Molybdopterin in Xanthomonas maltophilia.

Fifteen of 23 ATCC strains and 2 of 9 clinical isolates of Xanthomonas maltophilia, all of which grew aerobically on ammonia, but not nitrate, as a sole nitrogen source, reduced nitrate to nitrite. X. maltophilia failed to grow anaerobically on complex medium with or without nitrate, so it is considered an obligate aerobe. Nitrate-reducing strains contained reduced methyl viologen nitrate reductase (MVH-NR) with specific activities ranging from 49.2 to 192 U mg of protein. Strain ATCC 17666 doubled its cell mass after 3 h of growth on nitrate broth under low aeration, possessed maximal MVH-NR activity, and converted the added nitrate to nitrite, which accumulated. Dissolved oxygen above 15% saturation greatly suppressed nitrite formation. All strains, except ATCC 14535, possessed between 0.25 and 5.05 pmol of molybdopterin mg of protein as measured by the Neurospora crassa nit-1 assay. The molybdopterin activity in the soluble fraction sedimented as a single symmetrical peak with an s(20,w) of 5.1. Studies identified MVH-NR in selected strains as a membrane-bound protein. The deoxycholate-solubilized MVH-NR sedimented as a single peak in sucrose density gradients with an s(20,w) of 8.8. The MVH-NR of X. maltophilia has the physical characteristics of a respiratory nitrate reductase and may enable cells to use nitrate as an electron sink under semiaerobic conditions.

Biosynthesis of galactogen: identification of a beta-(1----6)-D-galactosyltransferase in Helix pomatia albumen glands.

A beta-(1----6)-D-galactosyltransferase has been purified over 2000-fold by affinity chromatography on UDP-p-aminophenyl-Sepharose. The enzyme, from a pellet fraction (8000 x g) of Helix pomatia albumen gland, catalyzes transfer of D-galactose from UDP-galactose to a (1----6) linkage on acceptor H. pomatia galactogen. Three other polymers served as acceptors: beef lung galactan, Lymnaea stagnalis galactogen and arabinogalactan from larch wood. To determine the linkage specificity of the enzyme, it was incubated with UDP-D-galactose and acceptor galactogen that had been tritiated previously by treatment with galactose oxidase and [3H]KBH4. The [3H]galactogen reaction product was recovered, methylated, hydrolyzed and acetylated; tritiated derivatives were identified by mass spectroscopy of effluent fractions separated by gas chromatography. This analysis revealed that (1----6)-linked galactosyl groups had been added to the enzyme-treated acceptor galactogen. Also identified was a hydrolytic enzyme that removed terminal alpha 1,2-linked L-galactosyl residues from H. pomatia galactogen.

Neutralization of bacterial lipopolysaccharides by human plasma.

To quantify the neutralization of bacterial lipopolysaccharide (LPS) by human plasma, dilutions of Escherichia coli O113 LPS were incubated with plasma, followed by the addition of Limulus amebocyte lysate (LAL). The reaction between the LPS and LAL was monitored spectrophotometrically, and the concentration of LPS resulting in 50% lysate response (LR50) was determined. Analysis of 145 outdated plasma samples yielded a range of LR50 between 6 and 1,500 ng/ml. Pools of plasma with high and low LR50 were prepared. The pool with high LR50 neutralized 166-fold more E. coli 0113 LPS, 190-fold more E. coli 0111B4 LPS, 42-fold more Klebsiella pneumoniae LPS, and 29-fold more Salmonella typhimurium LPS than did the pool with low LR50. Each pool had similar immunoglobulin G (IgG) and IgM antibody levels to homologous LPS, measured by an enzyme-linked immunosorbent assay. Analysis of 212 fresh-frozen plasma units revealed a range of LR50 between 48 and 6,000 ng/ml. Incubation of LPS in a pool of fresh-frozen plasma with high LR50 elicited significantly less fever in the rabbit pyrogen test than did LPS incubated in plasma with low LR50 (fever index, 2.68 +/- 0.61 degrees C X h and 3.52 +/- 0.66 degrees C X h, respectively; P = 0.003). We conclude that there is a 100-fold range in the endotoxin-neutralizing capacity of human plasma and that this variation is not due to LPS-specific IgG or IgM antibodies. Further investigations are needed to determine whether differing susceptibility of patients to the effects of LPS is due to differences in the endotoxin-neutralizing capacity of their plasma and whether plasma screened for high endotoxin-neutralizing capacity may be therapeutically useful in endotoxemia.

Regulation of the Neurospora crassa assimilatory nitrate reductase.

Reduced nicotinamide adenine dinucleotide phosphate (NADPH)-nitrate reductase from Neurospora crassa was purified and found to be stimulated by certain amino acids, citrate, and ethylenediaminetetraacetic acid (EDTA). Stimulation by citrate and the amino acids was dependent upon the prior removal of EDTA from the enzyme preparations, since low quantities of EDTA resulted in maximal stimulation. Removal of EDTA from enzyme preparations by dialysis against Chelex-containing buffer resulted in a loss of nitrate reductase activity. Addition of alanine, arginine, glycine, glutamine, glutamate, histidine, tryptophan, and citrate restored and stimulated nitrate reductase activity from 29- to 46-fold. The amino acids tested altered the Km of NADPH-nitrate reductase for NADPH but did not significantly change that for nitrate. The Km of nitrate reductase for NADPH increased with increasing concentrations of histidine but decreased with increasing concentrations of glutamine. Amino acid modulation of NADPH-nitrate reductase activity is discussed in relation to the conservation of energy (NADPH) by Neurospora when nitrate is the nitrogen source.

Production of molybdenum-coordinating compound by Bacillus thuringiensis.

Bacillus thuringiensis (ATCC 10792) produces a molybdenum reactive compound (given the trivial name chelin) during growth on iron-deficient medium. This compound accumulates in the culture medium in direct relation to the amount of L-arginine added and reaches a maximum concentration 24 to 48 h after the stationary phase of growth. Chelin absorbs light in the ultraviolet region with absorption maxima at 315 and 248 nm and minima at 284 and 240 nm. Chelin reacts with Na2MoO4, but not with Mo2O4(H2O)6-2+, to form a bright yellow molybdo-chelin complex which absorbs light with an absorption maximum at 330 nm, a minimum at 288 nm, and shoulders at 255 and 400 nm. The differential absorption of molybdo-chelin versus chelin at 425 nm can be used to quantify chelin. This differential absorbance is linear with increasing concentrations of Na2MoO4 and was used to calculate the molar extinction coefficient of molybdochelin at 425 nm (epsilon similar to 6,200). Chelin binds MoO4-2 minus to form a complex (molybdochelin) which migrates as a single band and elutes as a single peak, during acrylamide gel electrophoresis and Sephadex G-15 gel filtration. Molecular weight determinations using Sephadex G-15 gel filtration resulted in an estimated molecular weight of 550 for chelin and an estimated molecular weight of 760 for molybdo-chelin. The peptide nature of chelin is indicated by its positive ninhydrin reaction on thin-layer chromatography plates and by the presence of amino acids in acid-hydrolyzed samples. The major amino acid residues detected were threonine, glycine, and alanine.

In vitro restoration of nitrate reductase: investigation of Aspergillus nidulans and Neurospora crassa nitrate reductase mutants.

Extracts of Aspergillus nidulans wild type (bi-1) and the nitrate reductase mutant niaD-17 were active in the in vitro restoration of NADPH-dependent nitrate reductase when mixed with extracts of Neurospora crassa, nit-1. Among the A. nidulans cnx nitrate reductase mutants tested, only the molybdenum repair mutant, cnxE-14 grown in the presence of 10-minus 3 M Na2 MoO4 was active in the restoration assay. Aspergillus extracts contained an inhibitor(s) which was measured by the decrease in NADPH-dependent nitrate reductase formed when extracts of Rhodospirillum rubrum and N. crassa, nit-1 were incubated at room temperature. The inhibition by extracts of A. nidulans, bi-1, cnxE-14, cnxG-4 and cnxH-3 was a linear function of time and a logarithmic function of the protein concentration in the extract. The molybdenum content of N. crassa wild type and nit-1 mycelia were found to be similar, containing approx. 10 mu g molybdenum/mg dry mycelium. The NADPH-dependent cytochrome c reductase associated with nitrate reductase was purified from both strains. The NADPH-dependent cytochrome c reductase associated with nitrate reductase was purified from both strains. The enzyme purified from wild-type N. crassa contained more than 1 mol of molybdenum per mol of enzyme, whereas the enzyme purified from nit-1 contained negligible amounts of molybdenum.

In vitro formation of nitrate reductase using extracts of the nitrate reductase mutant of Neurospora crassa, nit-1, and Rhodospirillum rubrum.

In vitro formation of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-nitrate reductase (NADPH: nitrate oxido-reductase, EC 1.6.6.2) has been attained by using extracts of the nitrate reductase mutant of Neurospora crassa, nit-1, and extracts of either photosynthetically or heterotrophically grown Rhodospirillum rubrum, which contribute the constitutive component. The in vitro formation of NADPH-nitrate reductase is characterized by the conversion of the flavin adenine dinucleotide (FAD) stimulated NADPH-cytochrome c reductase, contributed by the N. crassa nit-1 extract from a slower sedimenting form (4.5S) to a faster sedimenting form (7.8S). The 7.8S NADPH-cytochrome c reductase peak coincides in sucrose density gradient profiles with the NADPH-nitrate reductase, FADH(2)-nitrate reductase and reduced methyl viologen (MVH)-nitrate reductase activities which are also formed in vitro. The constitutive component from R. rubrum is soluble (both in heterotrophically and photosynthetically grown cells), is stimulated by the addition of 10(-4) M Na(2)MoO(4) and 10(-2) M NaNO(3) to cell-free preparations, and has variable activity over the pH range from 3.0 to 9.5. The activity of the constitutive component in some extracts showed a threefold stimulation when the pH was lowered from 6.5 to 4.0. The constitutive activity appears to be associated with a large molecular weight component which sediments as a single peak in sucrose density gradients. However, the constitutive component from R. rubrum is dialyzable and is insensitive to trypsin and protease. These results demonstrate that R. rubrum contains the constitutive component and suggests that it is a low molecular weight, trypsin- and protease-insensitive factor which participates in the in vitro formation of NADPH nitrate reductase.

Invitro formation of assimilatory reduced nicotinamide adenine dinucleotide phosphate: nitrate reductase from a Neurospora mutant and a component of molybdenum-enzymes.

An active Neurospora-like assimilatory NADPH-nitrate reductase (EC 1.6.6.2), which can be formed in vitro by incubation of extracts of nitrate-induced Neurospora crassa mutant nit-1 with extracts of (a) certain other nonallelic nitrate reductase mutants, (b) uninduced wild type, or (c) xanthine oxidizing and liver aldehyde-oxidase systems was also formed by combination of the nit-1 extract with other acid-treated enzymes known to contain molybdenum. These molybdenum enzymes included (a) nitrogenase, or its molybdenum-iron protein, from Clostridium, Azotobacter, and soybeannodule bacteroids, (b) bovine liver sulfite oxidase, (c) respiratory formate-nitrate reductase from Escherichia coli, (d) NADH-nitrate reductase from foxtail grass (Setaria faberii), and (e) FADH(2)- and reduced methyl viologennitrate reductase preparations from certain Neurospora mutants. Several molybdenum-amino-acid complexes, as possible catalytic models of nitrogenase, were inactive (as were some previously tested 20 nonmolybdenum enzymes) in place of the acid-treated molybdenum-containing enzymes. The results imply the existence of a molybdenum-containing component shared by the known molybdenum-enzymes.

In vitro assembly of Neurospora assimilatory nitrate reductase from protein subunits of a Neurospora mutant and the xanthine oxidizing or aldehyde oxidase systems of higher animals.

In vitro assembly or complementation of a hybrid assimilatory nitrate reductase was attained by mixing a preparation of nitrate-induced N. crassa mutant nit-1 specifically with acid-treated (pH 2.5) bovine milk or intestinal xanthine oxidase, rabbit liver aldehyde oxidase, or chicken liver xanthine dehydrogenase. The complementation reaction specifically required induced nit-1, the only nitrate reductase mutant of Neurospora that lacked xanthine dehydrogenase and was unable to use hypoxathine or nitrate as a sole nitrogen source. The complementing activities of the above acid-treated enzymes correspond to their xanthine or aldehyde oxidizing activity profiles on sucrose density gradients. The resulting soluble, reduced nicotinamide adenine dinucleotide phosphate (NADPH)-nitrate reductases are the same as the Neurospora wild type enzyme in sucrose density gradient profile, molecular weight, substrate affinities, and sensitivity to inhibitors and temperature. By analogy to a similar in vitro complementation of nitrate reductase in mixtures of induced nit-1 and individual nonalleic Neurospora mutants, or uninduced wild type, the complemented nitrate apparently consists of an inducible protein subunit (possessing inducible NADPH-cytochrome c reductase) furnished by nit-1 and a subunit from the acid-treated xanthine or aldehyde oxidizing system which can substitute for the constitutive component furnished by the other mutants or uninduced wild type. The data suggest that Neurospora nitrate reductase and the xanthine oxidizing system and aldehyde oxidase of animals, all of which are molybdenum-containing enzymes catalyzing the reduction of nitrate to nitrite, share a highly similar protein subunit.

Formation of assimilatory nitrate reductase by in vitro inter-cistronic complementation in Neurospora crassa.

In vitro complementation of the soluble assimilatory nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-nitrate reductase was attained by mixing cell-free preparations of certain Neurospora nitrate reductase mutants: induced nit-1 (uniquely possessing inducible NADPH-cytochrome c reductase) with (a) uninduced or induced nit-2 or nit-3, or (b) uninduced wild type. The complementing activity of induced nit-1 is soluble while that of nit-2, nit-3, and wild type is particulate but not of mitochondrial origin. All fractions are inactivated by heat or trypsin. The NADPH-nitrate reductase enzymes formed in the above three complementing mixtures are similar to the wild-type enzyme in sucrose density gradient profiles, molecular weight, substrate affinity, sensitivity to inhibitors and temperature, but show different ratios of associated enzyme activities. The data suggest that nitrate reductase consists of at least two protein subunits: a nitrate-inductible subunit as reflected by inductible NADPH-cytochrome c reductase, and a constitutive protein which is activated (as indicated by the appearance of flavine adenine dinucleotide, reduced form (FADH(2))- and reduced methyl viologen-nitrate reductase activities) when it combines with the inductible subunit.