Expression of the Streptococcus pneumoniae type 3 synthase in Escherichia coli. Assembly of type 3 polysaccharide on a lipid primer.

Synthesis of the type 3 capsular polysaccharide of Streptococcus pneumoniae is catalyzed by the membrane-localized type 3 synthase, which utilizes UDP-Glc and UDP-GlcUA to form high molecular mass [3-beta-d-GlcUA-(1-->4)-beta-d-Glc-(1-->](n). Expression of the synthase in Escherichia coli resulted in synthesis of a 40-kDa protein that was reactive with antibody directed against the C terminus of the synthase and was the same size as the native enzyme. Membranes isolated from E. coli contained active synthase, as demonstrated by the ability to incorporate Glc and GlcUA into a high molecular mass polymer that could be degraded by type 3 polysaccharide-specific depolymerase. As in S. pneumoniae, the membrane-bound synthase from E. coli catalyzed a rapid release of enzyme-bound polysaccharide when incubated with either UDP-Glc or UDP-GlcUA alone. The recombinant enzyme expressed in E. coli was capable of releasing all of the polysaccharide from the enzyme, although the chains remained associated with the membrane. The recombinant enzyme was also able to reinitiate polysaccharide synthesis following polymer release by utilizing a lipid primer present in the membranes. At low concentrations of UDP-Glc and UDP-GlcUA (1 microm in the presence of Mg(2+) and 0.2 microm in Mn(2+)), novel glycolipids composed of repeating disaccharides with linkages consistent with type 3 polysaccharide were synthesized. As the concentration of the UDP-sugars was increased, there was a marked transition from glycolipid to polymer formation. At UDP-sugar concentrations of either 5 microm (with Mg(2+)) or 1.5 microm (with Mn(2+)), 80% of the incorporated sugar was in polymer form, and the size of the polymer increased dramatically as the concentration of UDP-sugars was increased. These results suggest a cooperative interaction between the UDP-precursor-binding site(s) and the nascent polysaccharide-binding site, resulting in a non-processive addition of sugars at the lower UDP-sugar concentrations and a processive reaction as the substrate concentrations increase.

Effect of nitrendipine on renal function and on hormonal parameters after intravascular iopromide.

PURPOSE: To evaluate the effect of the low-molecular nonionic radiographic contrast agent iopromide (Ultravist) on renal function, vasoactive peptides (angiotensin II, aldosterone, arginine vasopressin, and atrial natriuretic factor (ANF)), and blood pressure, and to evaluate the influence of the calcium antagonist nitrendipine on these parameters. The findings were evaluated in a prospective double-blind and placebo-controlled randomized study. MATERIAL AND METHODS: Twenty-six patients undergoing routine aortofemoral arteriography for peripheral atherosclerotic disease were treated with nitrendipine tablets (10 mg) or placebo twice daily for a week. Angiography was performed on the fifth day of medication. Efficacy variables were determined on the day before and 2 days after arteriography. The glomerular filtration rate and renal plasma flow were measured by the constant infusion technique. Renal tubular function was estimated from the clearance of lithium. Hormones were measured by radioimmunoassays. RESULTS: Arteriography with iopromide did not change renal function. No differences between the nitrendipine and placebo groups were found in renal hemodynamics, tubular sodium handling, or blood pressure. Nitrendipine changed ANF (26.1%) compared to placebo (1.5%), whereas the other hormones were not affected. CONCLUSION: The use of iopromide for angiography did not affect renal function in normotensive patients with peripheral atherosclerotic disease. Short-term treatment with nitrendipine may lower the plasma levels of ANF but it had no effect on renal function or blood pressure. Treatment with calcium antagonists prior to arteriography with iopromide is not indicated in these patients.

Porous-coated metal-backed patellar components in total knee replacement. A postmortem retrieval analysis.

The use of porous-coated metal-backed patellar components to achieve consistent fixation by bone ingrowth and to provide relief of pain warrants serious scrutiny. We conducted a quantitative postmortem investigation of eleven consecutively retrieved components with use of high-resolution contact radiographs, electron microscopy, and histological analysis. The implants had been in situ for a mean (and standard deviation) of 45+/-36 months (range, one to eighty-four months). Analysis of the high-resolution contact radiographs revealed that a mean of 86+/-12 per cent (range, 61 to 100 per cent) of the porous coating was in contact with the host bone. Backscattered electron imaging showed that the mean volume fraction of bone ingrowth was 13+/-9 per cent (range, 0 to 30 per cent). No significant difference was detected, with the numbers available, between the volume fraction of the bone ingrowth measured in the porous coating and that of the host cancellous bone in the patellae.

Postmortem analysis of bone growth into porous-coated acetabular components.

Microradiography, backscattered electron microscopy, and histological analysis were used to conduct a quantitative postmortem study of seven consecutively retrieved anatomical porous replacement acetabular components that had been inserted during total hip arthroplasties. Screws had been used for the initial fixation of six components. The microradiographic analysis of all seven components showed that an average (and standard deviation) of 84 +/- 9 per cent (range, 72 to 93 per cent) of the porous coating was in direct apposition to the periprosthetic bone. The backscattered electron images demonstrated that an average of 12 +/- 6 per cent (range, 4 to 21 per cent) of the space available in the porous coating was occupied by ingrown bone. The amount of bone ingrowth was not significantly different among the three zones delineated by DeLee and Charnley. Uniformity of bone growth into the porous coating suggests that the preferential loading that occurs in the superior region did not differentially affect the bone ingrowth. The present study showed that consistent bone growth into anatomical porous replacement acetabular components can be achieved.

Postmortem analysis of consecutively retrieved asymmetric porous-coated tibial components.

The objective of this investigation was to conduct a postmortem analysis of 8 porous-coated asymmetric tibial components to measure the extent of radiolucencies and bone ingrowth. With the use of radiographic, electron microscope, and histologic analysis techniques, a quantitative postmortem study of 8 consecutively retrieved porous-coated tibial components was conducted. Time in situ averaged 47+/-36 months. The components were secured with 4 pegs and 2 screws. Autograft bone chips were applied to the resected tibia during implantation. Contact radiographs of an average of 8 3-mm sections from each implant revealed that 73%+/-17% of the porous coating had no apparent radiolucencies present between the host bone and porous coating for the series. Backscattered electron imaging showed that the bone ingrowth averaged 6%+/-2%. Histologic analysis was unable to demonstrate any adverse cellular response. The analysis suggested that this asymmetric implant design is stable and biocompatible and has potential for long-term clinical durability.

Bilayer membrane destabilization induced by dolichylphosphate.

Small vesicles containing the fluorescent probe calcein were used to investigate the effect of dolichyl phosphate (Dol-P) on phospholipid bilayer stability. In the absence of Dol-P, phospholipid vesicles retained the fluorescent probe upon the addition of divalent cations. Small vesicles containing Dol-P, however, exhibited calcein leakage when incubated in the presence of divalent cations. This effect was observed in liposomes composed of a mixture of phosphatidylethanolamine (PE), phosphatidylcholine (PC) and Dol-P, but not in PC/Dol-P liposomes. The rate of calcein leakage was proportional to divalent cation concentration and to temperature, but was independent of vesicle concentration. These results demonstrate that Dol-P has significant effects on the stability of PE containing phospholipid bilayers. Vesicle leakage was also promoted by the addition of rat liver Dol-P-mannose synthase (EC 2.4.1.83) to intact PE/PC/Dol-P vesicles. Enzyme induced leakage from phospholipid vesicles required the presence of both unsaturated PE and Dol-P. The phospholipid composition of leaky vesicles could be correlated with the lipid matrix required for maximal transferase activity of the rat liver synthase. The destabilizing effects of Dol-P on phospholipid bilayers may therefore be involved in the translocation of activated sugars across biological membranes.

Phospholipase-induced modulation of dolichyl-phosphomannose synthase activity.

Rat liver dolichyl-phosphomannose synthase is optimally active when the enzyme is reconstituted with lipids that prefer a nonlamellar macroscopic organization in isolation, such as phosphatidylethanolamine (PE), but the enzyme is only negligibly active in the presence of lipids that normally form stable bilayers, such as phosphatidylcholine (PC) [Jensen, J.W., & Schutzbach, J.S. (1985) Eur. J. Biochem. 153, 41-48]. We now report that the activity of the synthase can be modulated by incorporating diacylglycerol and lysophosphatidylcholine into the lipid matrix. Enzyme activity in PC bilayers was stimulated by the presence of diacylglycerol, a lipid that has a conical dynamic molecular shape and disrupts bilayer stability. In PC/diacylglycerol mixtures the apparent Km for dolichyl-P was 30-fold lower than the apparent Km for the polyprenol acceptor in PC membranes. Enzyme activity was also stimulated when diacylglycerol was generated in situ by incubation of PC vesicles with phospholipase C. In contrast, the activity of enzyme reconstituted in PE dispersions, or in PE/PC bilayers, was markedly inhibited by the presence of lysophospholipids. Enzyme activity was also reduced by the in situ generation of lysophospholipids in PE/PC vesicles by incubation with phospholipase A2. Since lysophospholipids and diacylglycerols arise in vivo as products of phospholipid metabolism, modulation of enzyme activity by these compounds may represent a potential regulatory mechanism for the synthesis of oligosaccharide lipids.

Modulation of dolichyl-phosphomannose synthase activity by changes in the lipid environment of the enzyme.

Rat liver dolichyl-phosphomannose synthase (GDP mannose-dolicholphosphate mannosyltransferase; EC 2.4.1.83) was previously shown to catalyze optimal rates of mannosyl transfer to dolichyl-P when the polyprenol acceptor was incorporated into a phosphatidylethanolamine (PE) matrix that has a tendency to adopt a nonbilayer (hexagonal HII) phase [Jensen, J. W., & Schutzbach, J. S. (1985) Eur. J. Biochem. 153, 41-48]. The present investigations now further define the properties of the lipid environment that are essential for mannosyltransferase activity. Monogalactosyl diglyceride (MGDG), a glycoglycerolipid that prefers a nonbilayer-phase organization in isolation, was shown to provide a suitable lipid matrix for synthase activity. By comparison, the enzyme was not activated by digalactosyl diglyceride (DGDG), which forms stable bilayer structures upon hydration. Enzyme activity in MGDG/DGDG mixtures decreased as the proportion of DGDG in the dispersion was increased. Although bilayer-forming phospholipids supported low rates of mannosyl transfer, enzyme activity was stimulated by the addition of MGDG to either phosphatidylcholine (PC) or PE/PC (1:1) membranes. The incorporation of agents known to destabilize bilayer structures including dolichols, ubiquinone, dodecane, and cholesterol into PE/PC (1:1) membranes also increased the rate of mannosyl transfer. Enzyme activity in PC membranes was stimulated by the presence of gramicidin and also by greatly increased concentrations of the substrate, dolichyl-P. The results demonstrate that the enzyme does not have a requirement for PE and suggest that the physical state of the lipid matrix is an important determinant for reconstitution of the synthase and polyprenol phosphate substrate in a productive complex. The formation of an enzyme/lipid complex was demonstrated by sucrose density gradient centrifugation and could be correlated with the lipid requirements for enzyme activity.

Characterization of mannosyl-transfer reactions catalyzed by dolichyl-mannosyl-phosphate-synthase.

Evidence suggesting that a single enzyme catalyzes mannosyl transfer from GDP-mannose to both dolichyl phosphate and to phenyl phosphate was obtained as follows: (a) The two activities were coeluted from columns of DEAE-cellulose and Sepharose CL-6B, (b) both reactions demonstrated similar kinetic constants for the glycosyl donor and for guanosine nucleoside inhibitors, (c) both reactions were sensitive to inhibition by low concentrations of nonionic detergents, and (d) both activities were found to be thermally inactivated at similar rates upon incubation at 55 degrees. The reaction conditions required for optimal mannosyl transfer by the purified enzyme preparation to the hydrophobic and water soluble acceptors, however, were found to be quite different. Whereas mannosyl transfer from GDP-mannose to dolichyl phosphate occurred at maximal rates only in the presence of specific phospholipids, the rate of mannosyl transfer to phenyl phosphate was essentially unaffected by the addition of phospholipid. These results indicate that dolichyl-mannosyl-phosphate-synthase, which has some of the properties of an intrinsic membrane protein, does not have an absolute requirement for phospholipid for catalytic activity, but rather that phospholipid is required for interaction of the enzyme with the long chain polyisoprenol substrate dolichyl phosphate.

Activation of dolichyl-phospho-mannose synthase by phospholipids.

Dolichyl-phospho-mannose synthase, or GDPmannose:dolichyl-phosphate mannosyltransferase (EC 2.4.1.83), was solubilized from rat liver microsomes with 1.0% Nonidet P-40 and the enzyme was further purified by column chromatography on DEAE-cellulose in the presence of 0.1% Nonidet P-40. The purified enzyme preparation (880-fold over microsomes) was unstable in the presence of detergent and had no activity in the presence of Nonidet P-40, Triton X-100, octyl beta-glucoside, or deoxycholate. Detergent-free enzyme was active in the presence of phosphatidylethanolamine (PtdEtn) and in the presence of phospholipid mixtures of PtdEtn and phosphatidylcholine (PtdCho) when the molar proportion of PtdCho was 70% or less. The enzyme was inactive in the presence of PtdCho alone. Unsaturated species of PtdEtn have a tendency to destabilize membrane bilayers [Cullis, P. R. & de Kruijff, B. (1978) Biochim. Biophys. Acta 507, 207-218] and we have shown that dolichol promotes the destabilizing effect of PtdEtn on membranes composed of PtdCho and PtdEtn [Jensen, J. W. & Schutzbach, J. S. (1984) Biochemistry 23, 115-1119]. These results suggest that dolichyl-P-mannose synthase is optimally active in a phospholipid matrix that contains some component phospholipids that prefer non-bilayer structural organization in isolation. Heat-inactivation and sedimentation experiments demonstrated that the synthase associated with PtdEtn in the presence of dolichyl-P. The PtdEtn-reconstituted enzyme catalyzed the reversible transfer of mannose from GDP-mannose to dolichyl-P. The Km for GDP-mannose was found to be 0.69 microM and the apparent Km for dolichyl-P was 0.3 microM. GMP, GDP, and GTP inhibited mannosyl transfer 50% at concentrations of 16 microM, 1.3 microM and 3 microM respectively.

Induction of cellular efflux by a galactosamine polymer from Neurospora crassa.

A cationic polymer of D-galactosamine was isolated from culture filtrates of a colonial temperature-sensitive strain of Neurospora crassa. Adsorption of the polymer to the cell surface initiated immediate efflux of low molecular weight metabolites and subsequent loss of viability. The polymer appeared to bind to those sites on the cell surface that normally bind calcium ions. Chemical analysis of the polymer showed it to be partially N-acetylated. The polymer had an isoelectric point of 8.4. Thirty percent of the D-galactosamine residues contained free amino groups. A rapid assay that has potential application for monitoring the effect of a variety of other membrane-active factors on membrane permeability has been developed.

Biosynthesis of heparin. Substrate specificity of heparosan N-sulfate D-glucuronosyl 5-epimerase.

The substrate specificity of heparosan N-sulfate D-glucuronosyl 5-epimerase from a mouse mastocytoma was examined to determine the effects of N-acetyl and O-sulfate groups on substrate recognition by the enzyme. [5-3H]Glucuronosyl-labeled heparosan N-sulfate was prepared enzymatically and was modified chemically by partial N-desulfation and N-acetylation. After enzymatic release of tritium, the location of remaining label was determined by deaminative cleavage and analysis of resulting di-, tetra-, and higher oligosaccharides. This analysis indicated that a D-glucuronosyl residue is recognized as a substrate if it is linked at C-1 to an N-acetylated glucosamine residue and at C-4 to an N-sulfated unit. However, the reverse structure, in which the D-glucuronosyl moiety is bound at C-1 to an N-sulfated residue and at C-4 to N-acetylated glucosamine, is not a substrate. Similar studies with O-sulfated heparin intermediates showed that O-sulfate groups either at C-2 of the L-iduronosyl moieties or at C-6 of vicinal D-glucosaminyl moieties prevent 5-epimerization. These findings were confirmed by studies of the reverse reaction, in which tritium was incorporated from 3H2O into partially O-desulfated heparin and the location of incorporated radioactivity was determined. These and more direct experiments corroborated the previous conclusion that the L-iduronosyl moieties are formed after N-sulfation but before O-sulfation. Assessment of the influence of substrate size on the reaction further showed that a large substrate is preferred; an octasaccharide released tritium at a rate approximately 10% of that observed for the parent polysaccharide, and some release occurred also with smaller oligosaccharides.

New assay for uronosyl 5-epimerases.

Simple assays have been developed for the two uronosyl 5-epimerases which participate in the biosynthesis of heparin and dermatan sulfate (heparosan N-sulfate D-glucuronosyl 5-epimerase and chondroitin D-glucuronosyl 5-epimerase, respectively). Following previously published procedures, substrates labeled with tritium in the C-5 positions of the D-glucuronosyl and L-iduronosyl residues were prepared enzymatically by incubation of O-desulfated heparin and dermatan with 3H2O and crude epimerase preparations from bovine liver and human skin fibroblasts, respectively. In the new assays, 3H2O generated from these substrates during the epimerase reactions was quantitated by the method of Pollard et al. (Anal. Biochem. (1981) 110, 424-430). In this procedure, 3H2O in the aqueous reaction mixture is extracted into a toluene-based organic phase containing 25% isoamyl alcohol, while the polysaccharide substrate remains in the aqueous phase and does not generate scintillations. This procedure is much simpler than that used previously which involves distillation of each reaction mixture and quantitation of the radioactivity in the distillate. The new assays have been validated by the demonstration that conditions of linearity with time and enzyme concentration can be established for both epimerase reactions. Assays of this type should be useful in the study of any enzymatic reaction where 3H2O is formed from a 3H-labeled substrate and the unreacted substrate is not appreciably soluble in the organic phase.

The biosynthesis of oligosaccharide-lipids. Activation of mannosyltransferase II by specific phospholipids.

Mannosyltransferase II catalyzes transfer directly from GDP-mannose to an oligosaccharide-lipid resulting in the formation of the alpha-1,3-mannosyl linkage in Man alpha 1-3(Man alpha 1-6)Man beta-GlcNAc beta-GlcNac-P-P-lipid. The enzyme has been solubilized and purified 660-fold from rabbit liver microsomes (Jensen, J. W., and Schutzbach, J. S. (1981) J. Biol. Chem. 256, 12899-12904). Enzyme activity was reconstituted in the presence of specific phospholipids and evidence for the formation of an enzyme-phospholipid complex was obtained from kinetic studies, by the stabilization of the enzyme by phosphatidylethanolamine, and by co-sedimentation of the enzyme with phospholipid. Maximal activity was restored only when the enzyme was reconstituted in the presence of synthetic or naturally occurring phosphatidylethanolamine which contained unsaturated acyl chains. Investigation of the phospholipid specificity required for activation of the enzyme and results obtained with phospholipid vesicles of mixed composition suggest that the enzyme may have a requirement for phospholipids that can associate to form nonbilayer lipid structures in aqueous environments.

The biosynthesis of oligosaccharide-lipids. Partial purification and characterization of mannosyltransferase II.

A mannosyltransferase that catalyzes transfer from GDP-mannose to tetrasaccharide-pyrophosphoryl-lipid with the formation of the alpha-1,3-mannosyl-mannose linkage in Man alpha 1-3(Man alpha 1-6)Man beta-GlcNAc beta-GlcNAc-P-P-lipid has ben purified 660-fold from rabbit liver microsomes. The enzyme was completely separated from mannosyltransferases that synthesize alpha-1,2-mannosyl-mannose linkages, but the purified preparation still contained some activity which synthesized alpha-1,6-mannosyl linkages. The enzyme has a requirement for divalent cations and a pH optimum between 6.8 and 7.3, and the purified enzyme was very sensitive to detergent concentration with optimal activity at 0.0225% Nonidet P-40. The extent of purification of the enzyme and its resistance to inhibition by amphomycin strongly suggest that the enzyme catalyzes direct transfer from nucleotide-sugar to the oligosaccharide-lipid acceptor.

The biosynthesis of oligosaccharide-lipids. Isolation of an oligosaccharide-P-P-lipid acceptor.

An oligosaccharide-P-P-lipid has been isolated from porcine liver by extraction with organic solvents and purified by chromatography on silica gel and DEAE-cellulose. The purified oligosaccharide-lipid was shown to contain mannose and N-acetylglucosamine in an approximate ratio of 1:1 and our results suggest that the major oligosaccharide component in the preparation was a tetrasaccharide with the composition (Man)2 (GlcNAc)2. When the oligosaccharide-lipid was incubated with GDP-[14C]mannose and a solubilized enzyme preparation from rabbit liver in the presence of MgCl2, three radioactive products could be isolated. The oligosaccharides in the products could be identified as a penta-, a hexa-, and a heptasaccharide. These products were formed by the stepwise addition of mannose to the growing oligosaccharide chain and GDP-mannose was indicated as the glycosyl donor in each reaction.

The biosynthesis of oligosaccharide-lipids. Formation of an alpha-1,2-mannosyl-mannose linkage.

An enzyme present in rabbit liver microsomes has been found to catalyze mannosyltransfer from GDP-mannose to exogenously added oligosaccharide-lipid acceptor resulting in the formation of an alpha-1,2-mannosyl-mannose linkage. Several lines of evidence suggest that the product is a heptasaccharide containing a single 1,6-linked branched glycosyl unit with 65% of the newly added mannosyl units in a terminal nonreducing position. The enzyme has been solubilized with non-ionic detergents and was partially purified on DEAE-cellulose and hydroxylapatite. The partially purified enzyme no longer catalyzed the formation of dolichol-P-mannose indicating a direct transfer from GDP-mannose to oligosaccharide-lipid acceptor.