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D J Uhlinger

Publications and source records attributed to D J Uhlinger.

36 records · Page 2Linked to original sources
PubMed1993

Requirement for posttranslational processing of Rac GTP-binding proteins for activation of human neutrophil NADPH oxidase.

Rac1 and Rac2 are closely related, low molecular weight GTP-binding proteins that have both been implicated in regulation of phagocyte NADPH oxidase. This enzyme system is composed of multiple membrane-bound and cytosolic subunits and when activated catalyzes the one-electron reduction of oxygen to superoxide. Superoxide and its highly reactive derivatives are essential for killing microorganisms. Rac proteins undergo posttranslational processing, primarily the addition of an isoprenyl group to a carboxyl-terminal cysteine residue. We directly compared recombinant Rac1 and Rac2 in a human neutrophil cell-free NADPH oxidase system in which cytosol was replaced by purified recombinant cytosolic components (p47-phox and p67-phox). Processed Rac1 and Rac2 were both highly active in this system and supported comparable rates of superoxide production. Under different cell-free conditions, however, in which suboptimal amounts of cytosol were present in the assay mixture, processed Rac2 worked much better than Rac1 at all but the lowest concentrations. This suggests that a factor in the cytosol may suppress the activity of Rac1 but not of Rac2. Unprocessed Rac proteins were only weakly able to support superoxide generation in either system, but preloading of Rac1 or Rac2 with guanosine 5'-O-(3-thio-triphosphate) (GTP gamma S) restored activity. These results indicate that processing is required for nucleotide exchange but not for interaction with oxidase components.

Amino Acid Sequence↗
PubMed1992

Expression of recombinant myeloperoxidase using a baculovirus expression system.

Myeloperoxidase (MPO) is a glycosylated heme-containing enzyme present in the azurophilic granules of normal human polymorphonuclear neutrophils. This enzyme plays a major role in the microbicidal activity of the host defense system by catalyzing the formation of the potent oxidant, hypochlorous acid. Although the amino acid sequence of MPO has been deduced from the cDNA, the structural basis for the observed heterogeneity of this enzyme is not known. Furthermore, the nature of the prosthetic group and its mode of linkage to the apoprotein has not been determined. To address questions regarding the structural features of MPO, which arise during the complex posttranslational processing of this enzyme, we utilized a baculovirus system to express MPO in Sf9 insect cells. Two glycosylated, single-chain precursor species of MPO were observed: an 84 kDa species that was secreted and a 74 kDa species that was cell-associated. This is the first report of an expression system in which a cell-associated MPO precursor undergoes posttranslational proteolytic processing.

Animals↗
PubMed1992

A carboxy-terminal peptide from p47-phox is a substrate for phosphorylation by protein kinase C and by a neutrophil protein kinase.

Agonist-activated phosphorylation of neutrophil proteins including p47-phox, a cytosolic component of the respiratory burst oxidase, has been implicated in the signal transduction cascade which leads to activation of the superoxide generating respiratory burst. We have previously reported (J. Biol. Chem. 265, 17550-59) that in a cell-free activation system consisting of cytosol plus plasma membrane from human neutrophils, diacylglycerol acts synergistically with an anionic amphiphile such as sodium dodecyl sulfate (SDS) to augment superoxide generation and assembly of the oxidase, and that p47 phosphorylation can occur under these conditions. Herein, we show that a peptide corresponding to a carboxy terminal sequence of p47-phox is a substrate for phosphorylation both by purified protein kinase C (a mixture of alpha, beta, and gamma forms) and by a distinct kinase or kinases present in neutrophil cytosol. Based on its activator requirements, the neutrophil kinase differs from classical protein kinase C, but may be a protein kinase C variant, based on inhibition by a protein kinase C peptide. Although in the cell-free system phosphorylation occurs under conditions which are similar to those for activation of superoxide generation, phosphorylation is not required for activation (1). Rather, protein assembly or aggregation which occurs under activation conditions may also promote phosphorylation.

Adenosine Triphosphate↗
PubMed1992

Reconstitution and characterization of the human neutrophil respiratory burst oxidase using recombinant p47-phox, p67-phox and plasma membrane.

Human neutrophil respiratory burst oxidase (NADPH-oxidase) activity can be reconstituted in a cell-free system consisting of plasma membrane, cytosol and an anionic amphiphile [e.g., sodium dodecyl sulfate (SDS) or arachidonate]. Herein, we report reconstitution of oxidase activity using isolated neutrophil plasma membrane together with purified recombinant p47-phox and p67-phox which had been produced using a baculovirus expression system. Activity required an anionic amphiphile (SDS or arachidonate) and was potentiated by diacylglycerol and GTP gamma S. Serial washes of the plasma membrane failed to affect its ability to reconstitute activity, indicating that a dissociable membrane component was not present. The Km for NADPH, 43 microM, was the same as that determined using cytosol in place of recombinant factors. The EC50 values for p47-phox and p67-phox under optimal activation conditions were 220 nM and 80 nM, respectively, indicating a relatively high affinity of these components in an activation complex. Since neither cytosolic component contains a nucleotide binding consensus sequence, these data indicate that the NADPH binding component of the oxidase resides in the plasma membrane.

Animals↗
PubMed1992

Stabilization of human neutrophil NADPH oxidase activated in a cell-free system by cytosolic proteins and by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide.

The superoxide-generating respiratory burst oxidase (NADPH oxidase) from human neutrophils can be activated in a cell-free system consisting of plasma membrane and cytosol by anionic amphiphiles such as sodium dodecyl sulfate and arachidonate (McPhail, L. C., Shirley, P. S., Clayton, C. C., and Snyderman, R. (1985) J. Clin. Invest. 75, 1735-1739; Curnutte, J. T. (1985) J. Clin. Invest. 75, 1740-1743; Bromberg, Y., and Pick, E. (1984) Cell. Immunol. 88, 213-221). Herein, the activity thus obtained is shown to be very labile at 37 degrees C. The rate of inactivation varied inversely with cytosol concentration. The stabilizing factor(s) was destroyed by heat and trypsin, indicating that it is protein in nature. Whereas cytosol from normal cells and from a chronic granulomatous disease patient lacking p67phox stabilized the oxidase activity, that from a chronic granulomatous disease patient lacking p47phox did not. Also, dialdehyde NADPH-treated cytosol showed no stabilizing effect, indicating that p47phox and a putative NADPH-binding component both participate in stabilization. The mechanism of inactivation was further explored by examining the stabilizing effect of agents that can act as chemical cross-linkers. Of several tested, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was the most effective, but others that utilize different chemical mechanisms were also partially effective. EDC extended the half-life at 37 degrees C from 2 to 120 min, protected against the inactivating effects of Triton X-100 and high salt, and did not affect the Km for NADPH. Stabilization required prior activation in the presence of both cytosol and membrane; and EDC treatment of cytosol, membrane, or a mixture of the two prior to the addition of sodium dodecyl sulfate failed to induce stabilization. EDC eliminated the requirement for the continuous presence of cytosol and activator. Dialysis did not cause a loss in activity, whereas control activity was diminished with dialysis and was largely restored with added sodium dodecyl sulfate. In the absence of EDC, the separation of cytosol from the membrane fraction resulted in a significant loss of activity, which was largely restored by the addition of cytosol. However, EDC treatment allowed the isolation of a nearly fully active oxidase in the membrane fraction, the activity of which was not influenced by added cytosol. These results support a model in which the active NADPH oxidase consists of a dissociable complex among membrane and cytosolic components and indicate that the longevity of the activated state requires continuous association of these components.

Cell Membrane↗
PubMed1992

Cell-free translocation of recombinant p47-phox, a component of the neutrophil NADPH oxidase: effects of guanosine 5'-O-(3-thiotriphosphate), diacylglycerol, and an anionic amphiphile.

We reported previously that diacylglycerol (diC8) and GTP gamma S synergize with an anionic amphiphile such as sodium dodecyl sulfate (SDS) to produce high rates of superoxide generation in a cell-free system consisting of neutrophil plasma membrane plus cytosol [Burnham, D. N., Uhlinger, D. J., & Lambeth, J. D. (1990) J. Biol. Chem. 265, 17550-17559]. Here we investigate the effects of these activating factors on the plasma membrane association in an in vitro translated radiolabeled recombinant p47-phox protein. Apparent translocation, assayed by cosedimentation with plasma membranes, required the presence of excess cytosol and an anionic amphiphile, was enhanced by both GTP gamma S and diC8, and was inhibited by high salt, correlating qualitatively with activation; up to 70% cosedimentation was observed with the combination of activators (compared with less than 20% in their absence). Similar results were obtained using heat-inactivated cytosol, wherein another oxidase component, p67-phox, has been inactivated. Unexpectedly, from 50 to 80% of the apparent translocation occurred in the absence of membranes, indicating that protein aggregation accounted for a significant part of the observed translocation. Nevertheless, the percent translocation was increased in all cases by the presence of membranes, indicating some degree of protein-membrane interaction. While a control in vitro translated protein failed to translocate, cosedimentation of p47-phox occurred equally well when red blood cell or neutrophil plasma membranes lacking cytochrome b558 were used. Also, the peptide RGVHFIF, which is contained within the C-terminus of the large subunit of cytochrome b558, failed to inhibit translocation/aggregation of p47-phox, despite its ability to inhibit cell-free activation of the oxidase. The data are consistent with the following: (a) SDS, diC8, and GTP gamma S all act on cytosolic components to alter protein-protein and/or protein-membrane associations, and these changes are necessary (but not sufficient) for activation; (b) these altered associations are likely to function by increasing the local concentration of p47-phox and other components at the plasma membrane; (c) a high background of nonspecific associations in the cell-free activation system is likely to obscure any specific, functionally relevant associations (e.g., with cytochrome b558); and (d) the mechanism of translocation in the cell-free system differs from that seen in intact neutrophils.

Amino Acid Sequence↗
PubMed1992

Altered diacylglycerol level and metabolism in neutrophils from patients with localized juvenile periodontitis.

Diacylglycerol, a physiological activator of protein kinase C, was elevated nearly twofold in unstimulated peripheral blood neutrophils from patients with localized juvenile periodontitis compared with cells from normal individuals. These cells also showed an enhanced and prolonged elevation of diglyceride in response to N-formylmethionylleucylphenylalanine. The metabolism of a cell-permeant diacylglycerol by diglyceride kinase was significantly decreased, because of a fivefold or higher elevation in the apparent Km of cellular diglyceride kinase.

Adult↗
PubMed1991

Nucleoside triphosphate requirements for superoxide generation and phosphorylation in a cell-free system from human neutrophils. Sodium dodecyl sulfate and diacylglycerol activate independently of protein kinase C.

The NADPH-oxidase of human neutrophils can be activated in a cell-free system comprised of plasma membrane, cytosol, and an anionic amphiphile such as arachidonate or sodium dodecyl sulfate (SDS). Recently, we showed that diacylglycerol acts synergistically with SDS in the cell-free system to stimulate superoxide generation, with concurrent phosphorylation of a 47-kDa cytosolic protein which is thought to be a component of the oxidase (Burnham, D. N., Uhlinger, D. J., and Lambeth, J. D. (1990) J. Biol. Chem. 265, 17550-17559). We report herein that when undialyzed cytosol is used along with either SDS alone or SDS plus diacylglycerol as activators, adenosine 5'-(gamma-thio)triphosphate (ATP gamma S) and guanosine 5'-(gamma-thio)triphosphate (GTP gamma S) both stimulated superoxide generation several fold, yielding about the same maximal velocity. ATP and GTP showed lower levels of stimulation. Stimulation by ATP gamma S and GTP gamma S was nonadditive, and showed a 5-7-fold greater specificity for GTP gamma S. ATP gamma S stimulation was inhibited by the nucleoside diphosphate (NDP) kinase inhibitor UDP. In contrast, when extensively dialyzed cytosol was used, most of the stimulation by ATP gamma S was lost, while most of that by GTP gamma S was retained. Addition of GDP restored the ability of ATP gamma S to stimulate, consistent with NDP kinase-catalyzed formation of GTP gamma S from ATP gamma S plus GDP. This activity was demonstrated directly in both cytosol and plasma membrane. Using undialyzed cytosol, phosphorylation of p47 showed a similar nonspecificity for nucleoside triphosphates, due to NDP kinase activity, but revealed the expected ATP specificity when dialyzed cytosol was used. Neither ATP gamma S nor GTP gamma S were good substrates for protein phosphorylation. Under a variety of conditions, phosphorylation of p47 or other neutrophil proteins failed to correlate with oxidase activation. The present studies indicate that SDS and diacylglycerol stimulation of superoxide generation in the cell-free system is independent of protein kinase C or other protein kinase activity, and suggest a novel role for diacylglycerol in cell regulation.

Cell Membrane↗
PubMed1991

Functional differences in human neutrophils isolated pre- and post-prandially.

Activated polymorphonuclear leukocytes have been associated with neoplasia, atherogenesis and reperfusion injury. Since some of these conditions are also correlated with dietary fat, we examined the functional characteristics of leukocytes isolated from subjects before and after consumption of a lipid-rich meal. There was up to 2-fold greater superoxide generation in response to agonists in leukocytes obtained post-prandially; the maximum increase was observed about 4 h after eating and followed the peak (2-4 h) in serum triglycerides. Neutrophils isolated post-prandially also exhibited impaired chemotaxis and defective bacterial killing, but normal phagocytosis. These findings provide a new variable that should be considered in studies of leukocytes.

Bacteroides↗
PubMed1990

Diradylglycerol synergizes with an anionic amphiphile to activate superoxide generation and phosphorylation of p47phox in a cell-free system from human neutrophils.

The superoxide-generating respiratory burst oxidase (NADPH oxidase) from human neutrophils can be activated in a cell-free system consisting of plasma membranes, cytosol, and an anionic amphiphile such as sodium dodecyl sulfate (SDS) or arachidonate, and guanosine 5'-(3-O-thio)triphosphate (GTP(gamma)S) augments activation. We report herein that short-chain diacylglycerols (e.g. dioctanoylglycerol (diC8)) synergize with SDS in the activation of superoxide generation in a dose- and time-dependent manner, resulting in rates up to 1400 nmol/min/mg plasma membrane protein, or 250-700% higher than the rate seen with SDS alone. diC8 did not affect significantly the dose response for either cytosol or SDS, indicating that the activation was not due to increased sensitivity of the oxidase toward either of these components. At optimal concentrations of SDS and diC8, additional activation was observed in the presence of GTP(gamma)S, indicating that diC8 and GTP activate by separate mechanisms. In contrast to diC8, other known activators of protein kinase C (phorbol myristate acetate and mezerein) augmented SDS activation only minimally (typically 20-30%), and neither diacylglycerols nor tumor promoters activated in the absence of SDS. Activation by diC8 was calcium and phosphatidylserine independent, and the specificity for neutral lipids was atypical for protein kinase C. Inhibitors of protein kinase C (staurosporine and a peptide substrate analog) also failed to inhibit the response. Nevertheless, phosphorylation of several neutrophil proteins including p47phox was seen with both SDS and diC8, and synergistic phosphorylation of p47phox was seen when both activating factors were present. Thus, diacylglycerol synergizes with SDS in activating both superoxide generation and p47phox phosphorylation in the cell-free activation system, but the activation is atypical of a protein kinase C mechanism.

Adult↗
PubMed1989

Stabilization of the superoxide-generating respiratory burst oxidase of human neutrophil plasma membrane by crosslinking with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide.

The superoxide-generating respiratory burst oxidase (NADPH-oxidase) of neutrophil plasma membranes is known to be highly unstable. In an attempt to stabilize the enzyme, we investigated the effect of crosslinking with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). The stability of superoxide-generating activity of plasma membrane was significantly enhanced by crosslinking. The half-life (t1/2) of the activity at 37 degrees C in the absence of crosslinker was about 2 min. Crosslinking extended the t1/2 significantly. Crosslinked material exhibited a biphasic loss of activity: about half was lost in each phase with respective t1/2 values of 20 and 240 min. The lifetime of the crosslinked material at 37 degrees C was further extended (about sixfold) with 30% glycerol, and the crosslinked material was completely stable for more than 2 weeks if stored on ice. Crosslinking also stabilized the activity to the effects of high salt and detergent, both of which have inactivating effects on the oxidase. In addition, crosslinking stabilizes not only the Vm but also the Km of the enzyme, which was noted to increase upon storage in the absence of crosslinking. Unlike the native material, the crosslinked oxidase failed to be stimulated (and in fact was inhibited) by phosphatidylserine, recently reported to be an activator of the oxidase (Tamura et al. (1988) J. Biol. Chem. 263, 17,621-17,626). The crosslinked plasma membrane provides a useful stabilized system for kinetic studies. When the activated plasma membrane was treated with EDC, the stabilized oxidase could not be solubilized effectively using detergents, since greater than 95% of the activity remained with the pellet following centrifugation, perhaps due to crosslinking to the cytoskeleton. However, when the activity was first detergent-solubilized, the soluble activity was also stabilized by EDC. This solubilized, crosslinked material may provide useful starting material for subsequent isolation and characterization of a stabilized active NADPH-oxidase.

Carbodiimides↗
PubMed1989

Diacylglycerol generation and phosphoinositide turnover in human neutrophils: effects of particulate versus soluble stimuli.

Serum-treated, or "opsonized" zymosan (OZ), a particulate material which can be phagocytized by polymorphonuclear leukocytes, activates the superoxide-generating respiratory burst in these cells. The use of dual wavelength spectroscopy in the present studies has allowed accurate continuous monitoring of superoxide generation (cytochrome c reduction) upon cellular activation by this turbid material; activation occurs after a short lag period (about 20 s) which is similar to the lag seen after activation with the chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP). Unlike the fMLP response which terminates after about 90 s, superoxide generation in response to OZ continues beyond 10 min, and is similar in this regard to the response seen with the protein kinase C activator phorbol myristate acetate (PMA). OZ and fMLP, but not PMA, also activate receptor-linked phospholipase C mechanisms as judged by the appearance of inositol trisphosphate (IP3) (as well as other inositol phosphates) and diacylglycerol (DAG), with the latter measured by a mass assay. The appearance of these potential mediators corresponded to the loss of phosphoinositides, in particular phosphatidylinositol 4,5-bisphosphate (PIP2). The magnitude of DAG and inositol sugar generation as well as the breakdown of PIP2 was considerably greater using OZ than with fMLP. In addition, while fMLP resulted in a transient increase in IP3 and DAG, OZ resulted in a sustained elevation of these molecules. With both agonists, the onset and duration of generation of putative mediators corresponded to the period of generation of O2-, consistent with a role for DAG and/or IP3 in the activation of the respiratory burst.

Diglycerides↗
PubMed1988

Modulation of the free sphingosine levels in human neutrophils by phorbol esters and other factors.

Because free long-chain bases have been recently found to have potent pharmacological effects when added to neutrophils (Wilson, E., Olcott, M. C., Bell, R. M., Merrill, A. H., Jr., and Lambeth, J. D. (1986) J. Biol. Chem. 261, 12616-12623) and other cell types, the levels in human neutrophils were measured by high-performance liquid chromatography. Sphingosine was the major free long-chain base in freshly isolated cells and ranged from 13 to 101 pmol/10(7) cells for different donors (mean +/- S.E. of 50 +/- 5, n = 17). Upon incubation at 37 degrees C, there was a time-dependent increase in free sphingosine (57 +/- 8% in 1 h, n = 17), but no change was seen at 4 or 25 degrees C. The sphingosine was apparently derived from more complex sphingolipids because little (less than 1%) could be accounted for by new synthesis from [14C]serine. Greater increases in free sphingosine were obtained when neutrophils were incubated with serum, plasma, or serum lipoproteins (about 2-fold higher than for cells incubated alone). In contrast, agonists such as phorbol 12-myristate 13-acetate, A23187, arachidonic acid, low concentrations (10 nM) of N-formyl-methionyl-leucyl-phenylalanine, and opsonized zymosan either decreased the amount of free sphingosine or blunted the time-dependent increase. This may be due to enhanced removal of free sphingosine because phorbol 12-myristate 13-acetate-treated cells exhibited an increased conversion of exogenously added [3H]sphinganine to ceramides. Endogenous sphingosine was approximately one-tenth the level found in neutrophils when exogenous long-chain bases were added to inhibit protein kinase C. Hence, depending on the subcellular localization of the endogenous versus exogenous long-chain bases, the amounts of free sphingosine in neutrophils might be sufficient to affect the function of these cells.

Calcimycin↗
PubMed1988

Nucleotide sequence for yeast dihydrolipoamide dehydrogenase.

Rabbit antiserum to the dihydrolipoamide dehydrogenase (dihydrolipoamide:NAD+ oxidoreductase, EC 1.8.1.4) component of the pyruvate dehydrogenase complex from bakers' yeast was used to screen plaques produced by a lambda gt11 yeast cDNA library. A 2.1-kilobase insert was isolated that also hybridized to a 17-base mixed oligonucleotide probe corresponding to the amino-terminal sequence of the yeast dihydrolipoamide dehydrogenase. The cDNA has a coding sequence of 499 amino acids that corresponds to a 21-residue signal peptide and a 478-residue mature protein (Mr = 51,558). Computer analysis shows that yeast dihydrolipoamide dehydrogenase has about 41% amino acid identity with Escherichia coli dihydrolipoamide dehydrogenase. Particularly striking is the conservation of sequence in the active site region of the dihydrolipoamide dehydrogenases from E. coli, yeast, and pig heart.

Amino Acid Sequence↗
PubMed1986

Phosphorylation-dephosphorylation of pyruvate dehydrogenase from bakers' yeast.

The pyruvate dehydrogenase complex was purified to homogeneity from bakers' yeast (Saccharomyces cerevisiae). No pyruvate dehydrogenase kinase activity was detected at any stage of the purification. However, the purified pyruvate dehydrogenase complex was phosphorylated and inactivated with purified pyruvate dehydrogenase kinase from bovine kidney. The protein-bound radioactivity was localized in the pyruvate dehydrogenase alpha subunit. The phosphorylated, inactive pyruvate dehydrogenase complex was dephosphorylated and reactivated with purified pyruvate dehydrogenase phosphatase from bovine heart. Tryptic digestion of the 32P-labeled complex yielded a single phosphopeptide, which was purified to homogeneity. The sequence of the phosphopeptide was established to be Tyr-Gly-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Thr-Thr-Tyr-Arg. This sequence is very similar to the sequence of a tryptic phosphotetradecapeptide derived from the alpha subunit of bovine kidney and heart pyruvate dehydrogenase: Tyr-His-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Val-Ser-Tyr-Arg.

Adenosine Triphosphate↗
PubMed1983

Relationship Between Physiological Status and Formation of Extracellular Polysaccharide Glycocalyx in Pseudomonas atlantica.

Marine pseudomonads, such as Pseudomonas atlantica, are readily isolated from sediments. These organisms form extracellular polysaccharide polymers (glycocalyx). The factors affecting the composition and amount of glycocalyx in batch culture of these organisms were examined. The formation of glycocalyx was stimulated by the inclusion of galactose as the carbon source and by increased surface area resulting from addition of sand to the medium. The composition of the glycocalyx changed during the growth cycle, with a marked increase in the proportions and absolute amounts of uronic acids as the rate of synthesis increased. In estuarine sediments, the glycocalyx contained a carbon content at least as great as in the microbes themselves. The greatest accumulation of these polymers occurred late in the stationary phase when the physiological status of the cells, as measured by the adenylate energy charge, showed maximal stress. Maximal formation of glycocalyx possibly could be used as an estimate of the nutritional status of these microbes.

Journal Article↗
PubMed1982

Estimations of uronic acids as quantitative measures of extracellular and cell wall polysaccharide polymers from environmental samples.

The extracellular polysaccharide polymers can bind microbes to surfaces and can cause physical modification of the microenvironment. Since uronic acids appear to be the components of these extracellular films that are most concentrated in a location outside the cell membrane, a quantitative assay for uronic acids was developed. Polymers containing uronic acids are resistant to quantitative hydrolysis, and the uronic acids, once released, form lactones irreproducibly and are difficult to separate from the neutral sugars. These problems were obviated by the methylation of the uronic acids and their subsequent reduction with sodium borodeuteride to the corresponding alcohol while they were in the polymer and could not form lactones. This caused the polymers to lose the ability to adhere to their substrates, so they could be quantitatively recovered. The hydrolysis of the dideuterated sugars was reproducible and could be performed under conditions that were mild enough that other cellular and extracellular polymers were not affected. The resulting neutral sugars were readily derivatized and then were separated and assayed by glass capillary gas-liquid chromatography. The dideuterated portion of each pentose, hexose, or heptose, identified by combined capillary gas-liquid chromatography and mass spectrometry, accurately provided the proportion of each uronic acid in each carbohydrate of the polymer. Examples of the applications of this methodology include the composition of extracellular polymers in marine bacteria, invertebrate feeding tubes and fecal structures, and the microfouling films formed on titanium and aluminum surfaces exposed to seawater.

Journal Article↗